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WO2025072834A2 - Method of sequencing and synthesizing l-polynucleotide - Google Patents

Method of sequencing and synthesizing l-polynucleotide Download PDF

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WO2025072834A2
WO2025072834A2 PCT/US2024/049074 US2024049074W WO2025072834A2 WO 2025072834 A2 WO2025072834 A2 WO 2025072834A2 US 2024049074 W US2024049074 W US 2024049074W WO 2025072834 A2 WO2025072834 A2 WO 2025072834A2
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dntp
polymerase
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WO2025072834A3 (en
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Dae Hyun Kim
Bo Shun HUANG
Jun Hyuk CHUNG
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Dxome Co Ltd
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Dxome Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/14—Pyrrolo-pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • L-form polymerase for example, can synthesize a polynucleotide using D-form nucleotide, but not L-form nucleotide.
  • the present disclosure provides a novel method of sequencing or synthesizing L-form polynucleotide.
  • Compositions for the sequencing or synthesis methods such as novel L-form nucleotides and D-form enzymes, are also provided. The methods are expected to enable high-throughput sequencing and synthesis of L-polynucleotides.
  • the present disclosure provides a mirror image nucleotide comprising: a. a pentose sugar selected from a (3R,4S)-3,4,5-trihydroxypentanal and a (4R)-4,5- dihydroxypentanal; wherein the H of the 5’ hydroxyl is substituted by one or more phosphate group; and wherein H of the 3' hydroxyl group, if present, is optionally substituted by a cleavable protecting group; b. a nitrogenous base, and c.
  • a cleavable label comprising a cleavable linker and a label; wherein at least one of a cleavable protecting group and a cleavable label is present.
  • the present disclosure provides a nucleotide comprising: a.
  • a pentose sugar comprising a cyclic form of a (3R,4S)-3,4,5- trihydroxypentanal or (4R)-4,5-dihydroxypentanal; wherein the H of the 5-hydroxyl group of the pentose sugar is substituted by one or more phosphate group; and wherein the H of the 3-hydroxyl group of the (3R,4S)-3,4,5- trihydroxypentanal is optionally substituted by a cleavable protecting group; b. a nitrogenous base linked to the 2 position of the pentose sugar, and c.
  • a cleavable label comprising a cleavable linker and a label; wherein at least one of a cleavable protecting group and a cleavable label is present.
  • the cleavable label is present.
  • the cleavable label is linked to the nitrogenous base, the 3’ O, or the 5’ phosphate group.
  • the mirror image nucleotide has a structure according to Formula I: Formula I wherein BASE is a nitrogenous base, and R' is a cleavable protecting group.
  • the label is selected from the group consisting of: [13] In certain embodiments of the mirror image nucleotide of Formula I or II, the cleavable protecting group is selected from an allyl, a dimethyl disulfide, a nitrobenzyl, and an azido protecting group. [14] In certain embodiments of the mirror image nucleotide of Formula I or II, the cleavable protecting group is selected from: .
  • the cleavable linker is photocleavable, is cleaved by contact with water-soluble phosphines, or is cleaved by water-soluble transition metal-containing catalysts.
  • the cleavable linker comprises an allyl, a disulfide, or an azido group.
  • the cleavable linker comprises: .
  • the compound has a structure selected from:
  • tributylammonium pyrophosphate and tributylamine in anhydrous DMF is added in one portion at room temperature and stirred for 30 min.
  • Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0) is then added and the mixture is stirred for 1 h at room temperature.
  • concentrated NH 4 OH is added and stirred overnight at room temperature.
  • the resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water.
  • the crude mixture is then purified with anion exchange chromatography on DEAE-Sephadex A-25 at 4°C using a gradient of TEAB (pH 8.0; 0.1–1.0 M).
  • the 9°N DNA polymerase was split into two fragments (a 466-aa 9°N-N fragment and a 310-aa 9°N-C fragment) at the split site between K466 and M467.
  • the synthesis of 466-aa 9°N-N fragment was designed as nine synthetic peptides (D-9°N-N-1 to D-9°N-N-9) ( Figure 2A) via solid phase peptide synthesis which ligate at the certain cysteine residue as shown in Figure 2B.
  • guanidine hydrochloride (Gn ⁇ HCl), Na 2 HPO 4 ⁇ 12H 2 O, NaH 2 PO 4 ⁇ 2H 2 O, sodium nitrite (NaNO2), Sodium hydroxide (NaOH), hydrochloric acid sodium 2-mercaptoethanesulfonate, 4-Mercaptophenylacetic acid (MPAA), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP ⁇ HCl), DL-1,4-dithiothreitol (DTT), 2,2′-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride (VA-044), Glutathione (reduced form), and palladium chloride (PdCl2) were purchased commercially from Sigma-aldrich, Alfa aesar, TCI, Duksan, etc.
  • Fmoc-based SPPS Fmoc-based SPPS. All peptides were synthesized by Fmoc-based SPPS on the Liberty Blue automated microwave peptide synthesizer (CEM) and PurePep® Chorus automated peptide synthesizer. All the peptide hydrazides were synthesized on hydrazine-2- chlorotrityl chloride resin. For each peptide hydrazide, the first residue was attached to the hydrazine-2-chlorotrityl chloride resin by a double coupling method using 5 equiv. amino acid, 10 equiv. DIC, and 5 equiv. Oxymapure. All resins were swelled in DMF for 30 min before coupling.
  • CEM Liberty Blue automated microwave peptide synthesizer
  • PurePep® Chorus automated peptide synthesizer All the peptide hydrazides were synthesized on hydrazine-2- chlorotrityl chlor
  • the Fmoc groups of assembled amino acids were removed by treatment with 20% piperidine and 0.1 M Oxyma in DMF at 85 °C. Coupling of amino acids except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH was carried out at 85 °C using 5 equiv. amino acid, 5 equiv. Oxymapure and 10 equiv. DIC for 2 min. The coupling reactions for Fmoc- Cys(Trt)-OH and Fmoc-His(Trt)-OH were carried out at 50 °C for 10 min to avoid side reactions at high temperature.
  • Trifluoroacetyl thiazolidine-4-carboxylic acid-OH was coupled using 5 equiv. Oxymapure and 10 equiv. DIC at room temperature overnight. Double coupling strategy was used for the peptides beyond 20 amino acids.
  • peptides were cleaved from resin using H 2 O/thioanisole/triisopropylsilane/1,2-ethanedithiol/trifluoroacetic acid (0.5/0.5/0.5/0.25/8.25) (vol/vol). The cleavage reaction took 2.5 h under agitation at 27 °C. Cold ether was added to precipitate the crude peptide.
  • Method B General approach. Peptide hydrazides were dissolved at a specified concentration (usually 1-20 mg/mL) in 6 M Gn.HCl containing 200 mM MPAA in a 1.5 mL Eppendorf tube. This forms a heterogeneous suspension and mixing sonication/vortexing should break any large pieces of solid MPAA. The pH can be adjusted to pH 3 if needed. A stock solution of acetyl acetone (acac) was made in water (10x-20x) and 1 eq to 5 eq acac were added to the peptide mixture.
  • acac acetyl acetone
  • Tfa-D-Thz-OH synthesis [347] To a solution of D-4-thiazolidinecarboxylic acid (1 g, 7.509 mmol) suspended in MeOH (40 mL) was added triethylamine (2.62 mL, 18.772 mmol) and the suspension was stirred for 10 min. Ethyltrifluroacetate (0.98 mL, 8.2599 mmol) was then added drop-wise and the mixture was stirred for 48 h at RT.
  • Step 8 Add 4 ml of DMF to the resin, gently agitate it for 10 s and then drain it. 10) Repeat Step 8. 11) Wash the resin by repeating Steps 2–6 twice. 12) Add 4 ml of 5% (vol/vol) MeOH/DMF to the resin, gently agitate it for 10 min and then drain it to unreacted sites on the resin to be capped. 13) Repeat Steps 2–6 to wash the resin thoroughly. 14) Add 4 ml of DCM to the resin, agitate it gently for 10 s and then drain it. Directly use the resin for the next coupling step.
  • D-9°N-N- 6@24-mer was synthesized on PurePep® Chorus automated peptide synthesizer by following the conditions mentioned in experimental methods.
  • D-9°N-N-6@24-mer synthesized by the process was analyzed by HPLC chromatogram and the results are provided in Figures 8B-8C.
  • Solid Phase Peptide Synthesis of D-9°N-C-3@9-mer CDTDGLHAT-NHNH2 (SEQ ID NO: 14) ( Figure 9A)
  • D-9°N-C-3@9-mer was synthesized on the Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in experimental methods.
  • D- 9oN-C-3@21-mer was synthesized on the Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in experimental methods. The product was observed as the TFA-deprotected D-9°N-C-3@21-mer as provided in Figures 12B and 12C.
  • Solid phase peptide synthesis of D-9°N-C-3@Cys35-mer CKEFLKYINP KLPGLLELEY EGFYVRGFFV TKKKY-NHNH 2.
  • SEQ ID NO: 16 D-9 o N-C-3@Cys35- mer was synthesized on the Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in experimental methods.
  • D-9°N-C-3@35-mer AKEFLKYINP KLPGLLELEY EGFYVRGFFV TKKKY- NHNH2.
  • Figure 15A D-9 o N-C-3@35-mer was synthesized on the Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in experimental methods. The product was observed as expected mass. as provided in Figures 15B.
  • CEM automated microwave peptide synthesizer
  • Figure 17D is ESI-MS of D-9°N-C-7@72-mer with MPAA attachment and Figure 17E shows MPAA-attached ligated.
  • the following peptides were produced by solid phase peptide synthesis on PurePep® Chorus automated peptide synthesizer by following the conditions mentioned in experimental methods. The product was analyzed by HPLC chromatogram and production of the peptide was confirmed.
  • NCL Native Chemical Ligation
  • Na 2 HPO 4 ⁇ 12H 2 O (0.1 M) containing 6 M Gn ⁇ HCl (pH 5.7-6.0) also prepared as same manner.
  • VA-044 Adjust the pH to 6.0–7.0, and filter the solution by using a 13 mm ⁇ 0.22 ⁇ m microporous membrane filter. VA-044, 0.1 M. Weight 9.7 mg of VA-044 into a 2-ml Eppendorf reaction tube and add 0.3 ml of 0.2 M phosphate solution (pH 6.9–7.0) containing 6 M Gn ⁇ HCl. Completely dissolve VA-044 by using a vortex and an ultrasonic cleaning bath.
  • reaction was kept in ice-salt bath under stirring for 20 min, after which 70 uL of 0.2 M MPAA (in 6 M Gn ⁇ HCl and 0.1 M Na2HPO4, pH 6.5) with D-9 o N-N-2@62-mer (2.58 mg, 1 eq) was added and the pH of the reaction mixture was adjusted to 6.6-6.8 with NaOH solution at room temperature. After 14 h, the reaction mixture was reduced by 0.15 M TCEP and purified by HPLC (purification conditions: 5-95% CH3CN (with 0.1% TFA) gradient in H2O (with 0.1% TFA) over 30 min on a Welch C4 column).
  • D-9°N-C-12 is generated by desulfurizing one or more cysteine residues of D- 9°N-C-11 to alanine residues at one or more positions Cys 500 , Cys 539 , Cys 595 , Cys 651 and Cys 714 .
  • D-9°N-C-12 is generated from D-9°N-C-11 by Acm deprotection at Cys 506 and Cys 509 . 6.14.
  • Figure 20A provides the amino-acid sequence of (D)-form terminal deoxynucleotidyl transferase, where all the amino acids are D-form amino acids.
  • the (D)-form terminal deoxynucleotidyl transferase is a 381-aa protein.
  • Figure 20B provides the synthetic chemical ligation route of the terminal deoxynucleotidyl transferase.
  • D-TdT-WT-1@His6+46-mer was synthesized on PurePep® Chorus automated peptide synthesizer by following the conditions mentioned in experimental methods.
  • D-TdT-WT-1@His6+46-mer synthesized by the process was analyzed by HPLC chromatogram and the results are provided in Figures 21B.
  • Figure 21A is the peptide sequence of D-TdT-WT-1@(His) 6 +46-mer.
  • fragment D-TdT-WT-1@(His)6+46-mer (3.0 mg) was dissolved in 300 ⁇ L of 6 M Gn.HCl that contained 200 mM MPAA in a 1.5 mL Eppendorf tube. Acac was dissolved to 150 mM in water and 2.5 eq were added to the dissolved solution of D-TdT-WT-1@(His)6+46-mer. A small stir bar was added to the Eppendorf tube and the reaction was allowed to stir for 4 hours.
  • Fragment D-TdT-WT-2@61- mer (2.74 mg, 0.8 eq) was dissolved in 300 ⁇ L of 6 M Gn.HCl that contained 200 mM Na2HPO4.12H2O at pH 8.5.
  • Figure 22A This solution was then added to the thioesterification solution, which resulted in a 600 ⁇ L solution at pH 5.3. The pH of this solution was adjusted to pH 7.0 with the addition of 1 M NaOH and the reaction was left to stir at room temperature. After 48 hours, 50 mM TCEP was added and analyzed by HPLC after 30 min stirring.
  • the reaction is then stopped by heating at 70 ⁇ C for 10 minutes or by adding 2 ⁇ L 0.5M EDTA.
  • the collected fractions was concentrated and the resulting residue was purified by SAX column (0%B to 80%B over 50 min, A:15% ACN in water; B: 15%ACN in 1M TEAB). After purification, the collected fractions were concentrated under reduced pressure and the resulting residue was redissolved with water and lyophilized. The lyophilized product (12.7 mg) was obtained as colorless syrup.
  • the mixture was stirred for for 1 h, then the mixture was added 0.1M TEAB buffer (pH 8.5, 15 mL). The resulting mixture was stirred for 2 h, then the mixture was added NH3 solution (10 mL). The resulting mixture was stirred for 16 h, then concentrated under reduced pressure. The resulting residue was diluted with H 2 O, and washed with CH 2 Cl 2 . The aqueous layer was concentrated under reduced pressure. The resulting residue was purified by C18 column (0%B to 90%B over 50 min, A: 0.1M TEAB in water/B: ACN). The resulting residue was purified by SAX column (0%B to 80%B over 50 min, A:15% ACN in water/15%ACN in 1M TEAB).
  • dTTP-NB-3 dTTP-NB-2 (1.6 g, 4.49 mmol, 1.0 eq.) was solubilized in dry pyridine (16 mL) at 0 o C under nitrogen atmosphere. To it, TMSCl (1.7 ml, 13.4 mmol, 3 eq.) was added. The reaction mixture was allowed to room temperature slowly and stirred for 3 h. After completion of reaction, evaporated under reduced pressure and immediately sealed under nitrogen.
  • reaction mixture 15 ml of DMA added to reaction mixture and followed by the addition of triethyl amine (5.2 ml, 35.9 mmol, 8 eq) at 0°C followed by PhCOCl (1.05 ml, 8.98 mmol, 2 eq).
  • triethyl amine 5.2 ml, 35.9 mmol, 8 eq
  • PhCOCl 1.05 ml, 8.98 mmol, 2 eq
  • reaction mixture was allowed to stirred for 3 h.
  • the reaction mixture was diluted with water and then extracted with CH 2 Cl 2 (3 x 50 mL). The combined organic layers were washed with brine and dried over anhydrous Na 2 SO 4 .
  • the resulting crude product was further purified with flash column chromatography (15% ethyl acetate in hexane) to afford 800 mg of yellow sticky material of dTTP-NB-4 and dTTP-NB-5 (in 2:1) mixture as indicated by LC-MS. Without further purification, the mixture was directly used as starting material in the next step without further purification.
  • dTTP-NB-8 To a solution of dTTP-NB-7 (330 mg; 0.67 mmol) in 8 mL of anhydrous THF at 0 °C, TBAF in THF solution (1.0 M; 2.78 ml; 2.78 mmol) was added. The reaction mixture was allowed to warm to room temperature and continue stirring for 2 h with exclusion of air and light. The mixture was poured into cold water (50 ml), and the resulting mixture was extracted 10% MeOH/DCM (3 x 50 mL).
  • the collected fractions were concentrated and the resulting residue was purified by SAX column (0%B to 80%B over 50 min, A:15% ACN in water; B: 15%ACN in 1M TEAB). After purification, the collected fractions were concentrated under reduced pressure and the resulting residue was re-dissolved with water and lyophilized. The lyophilized product (33 mg) was obtained as white foam.
  • NB-dCTP-4 To a stirred solution of NB-dCTP-3 (1.1 g, 2.7 mmol) in CH2Cl2 (100 ml), 2-nitrobenzyl bromide (689 mg, 1.15 mmol) and 20% aq. NaOH solution (50 ml) were added sequentially at RT with the protection from light and air. Later, tetrabutylammonium bromide (TBAB) (447 mg, 0.5 mmol) was added and stirred for 16 h at RT.
  • TBAB tetrabutylammonium bromide
  • NB-dCTP-5 To a stirred solution of NB-dCTP-4 (0.15 g, 0.31 mmol) in THF (5 ml) was added 0.62 ml of 1 M TBAF in THF at 0 °C with the protection from light and air. The reaction mixture was stirred for 3 h at 0 °C and volatiles were evaporated on rotavapor.
  • D-TdT-WT-1@His 6 +60-mer was synthesized on CEM Liberty Blue automated peptide synthesizer by following the conditions mentioned in experimental methods.
  • D-TdT-WT-1@His 6 +60-mer synthesized analyzed by HPLC and ESI-MS. The results are provided in Figures. 24A and 24B.
  • D-TdT-WT-2 to D-TdT-WT-7 were synthesized and analyzed. The products were analyzed by HPLC chromatogram and production of the peptide was confirmed.
  • This disclosure provides mirror image polypeptides including mirror-image nucleic acid polymerases or terminal deoxynucleotidyl transferases, and precursor fragments thereof, that are suitable for condensation via native chemical ligation.
  • sequences depicted herein can refer to a D-form polypeptide sequence, even though in general the sequences are depicted using capital letter one-letter codes for the individual amino acid residues.

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Abstract

The present disclosure provides a novel method of amplifying, sequencing or synthesizing L- form polynucleotide. Further provided include L-form nucleotides and D-form enzymes that can be used in the method. The method of making the L-form nucleotides and D-form enzymes is also provided.

Description

METHOD OF SEQUENCING AND SYNTHESIZING L-POLYNUCLEOTIDE 1. SEQUENCE LISTING [1] The instant application contains a Sequence Listing. 2. BACKGROUND [2] Chirality is a geometric property of molecules. A molecule is chiral if it cannot be superposed on its mirror image by combination of rotations, translations, and some conformational changes. [3] Many natural biomolecules, such as proteins, DNA and RNA, are chiral. Amino acids except glycine have a chiral carbon atom adjacent to the carboxyl group. This chiral center allows the amino acids to exist as stereoisomers, also referred to as enantiomers, that can be characterized as either L or D based on optical activity. However, most amino acids found in nature have L-form, except glycine which has no chirality. Nucleic acids have their chiral centers on their backbones. Unlike amino acids, nucleic acids are dominantly in D- form in nature. Due to chiral specificity, biomolecules can interact and make use of other molecules or substrates only when they have certain chirality. L-form polymerase, for example, can synthesize a polynucleotide using D-form nucleotide, but not L-form nucleotide. [4] Recently, researchers have generated mirror-image forms of some biomolecules as an attempt to create a mirror-image artificial system based on D-form amino acids, L-form nucleic acids and D-form proteins. Further, mirror-image therapeutic proteins or polynucleotides have been developed, e.g., with advantageous such slower biodegradation in the body. However, development of mirror-image biomolecules and systems faces a crucial barrier by lacking an efficient and reliable technology for sequencing or synthesizing the molecules. 3. SUMxARY [5] The present disclosure provides a novel method of sequencing or synthesizing L-form polynucleotide. Compositions for the sequencing or synthesis methods, such as novel L-form nucleotides and D-form enzymes, are also provided. The methods are expected to enable high-throughput sequencing and synthesis of L-polynucleotides. [6] Accordingly, in a first aspect, the present disclosure provides a mirror image nucleotide comprising: a. a pentose sugar selected from a (3R,4S)-3,4,5-trihydroxypentanal and a (4R)-4,5- dihydroxypentanal; wherein the H of the 5’ hydroxyl is substituted by one or more phosphate group; and wherein H of the 3' hydroxyl group, if present, is optionally substituted by a cleavable protecting group; b. a nitrogenous base, and c. optionally a cleavable label comprising a cleavable linker and a label; wherein at least one of a cleavable protecting group and a cleavable label is present. [7] In some embodiments, the present disclosure provides a nucleotide comprising: a. a pentose sugar comprising a cyclic form of a (3R,4S)-3,4,5- trihydroxypentanal or (4R)-4,5-dihydroxypentanal; wherein the H of the 5-hydroxyl group of the pentose sugar is substituted by one or more phosphate group; and wherein the H of the 3-hydroxyl group of the (3R,4S)-3,4,5- trihydroxypentanal is optionally substituted by a cleavable protecting group; b. a nitrogenous base linked to the 2 position of the pentose sugar, and c. optionally a cleavable label comprising a cleavable linker and a label; wherein at least one of a cleavable protecting group and a cleavable label is present. [8] In some embodiments, the cleavable label is present. In certain embodiments, the cleavable label is linked to the nitrogenous base, the 3’ O, or the 5’ phosphate group. [9] In certain embodiments the mirror image nucleotide has a structure according to Formula I:
Figure imgf000004_0001
Formula I wherein BASE is a nitrogenous base, and R' is a cleavable protecting group. [10] In certain embodiments the mirror image nucleotide has a structure according to Formula II
Figure imgf000005_0003
Formula II wherein BASE is a nitrogenous base; R' is a cleavable protecting group or H; R2—Label is a cleavable label comprising a cleavable linker R2 and a label. [11] In certain embodiments the mirror image nucleotide has structure according to Formula III:
Figure imgf000005_0002
Formula III wherein BASE is a nitrogenous base; R2—Label is a cleavable label comprising a cleavable linker R2 and a label. [12] In certain embodiments of the mirror image nucleotide of Formula II or III, the label is selected from the group consisting of:
Figure imgf000005_0001
[13] In certain embodiments of the mirror image nucleotide of Formula I or II, the cleavable protecting group is selected from an allyl, a dimethyl disulfide, a nitrobenzyl, and an azido protecting group. [14] In certain embodiments of the mirror image nucleotide of Formula I or II, the cleavable protecting group is selected from:
Figure imgf000006_0001
. [15] In certain embodiments of the mirror image nucleotide of Formula II or III, wherein the cleavable linker is photocleavable, is cleaved by contact with water-soluble phosphines, or is cleaved by water-soluble transition metal-containing catalysts. [16] In certain embodiments of the mirror image nucleotide of Formula II or III, wherein the cleavable linker comprises an allyl, a disulfide, or an azido group. [17] In certain embodiments of the mirror image nucleotide of Formula II or III, wherein the cleavable linker comprises:
Figure imgf000006_0002
. [18] In certain embodiments of the mirror image nucleotide of Formula I, the compound has a structure selected from:
Figure imgf000007_0001
[19] In certain embodiments, R’ comprises a methyl disulfide, an allyl, an azide, or nitrobenzyl moiety. In certain embodiments, R’ is selected from:
Figure imgf000007_0002
. [20] In certain embodiments of the mirror image nucleotide of Formula II, the compound has a structure selected from:
Figure imgf000008_0001
. [21] In certain embodiments, R’ comprises a methyl disulfide, an allyl, an azide, or nitrobenzyl moiety. In certain embodiments, R’ is selected from:
Figure imgf000009_0001
[22] In certain embodiments, R2 comprises:
Figure imgf000009_0002
. [23] In certain embodiments of the mirror image nucleotide of Formula III, the compound has a structure selected from:
Figure imgf000009_0003
Figure imgf000010_0001
. [24] In certain embodiments, R2 comprises:
Figure imgf000010_0002
[25] In certain embodiments of the mirror image nucleotide of Formula II, the compound has a structure selected from:
Figure imgf000011_0001
Figure imgf000012_0001
. wherein the R’ is selected from:
Figure imgf000012_0002
. [26] In certain embodiments of the mirror image nucleotide of Formula III, the compound has a structure selected from:
Figure imgf000012_0003
Figure imgf000013_0001
Figure imgf000014_0001
. [27] In certain embodiments, R’ comprises a methyl disulfide, an allyl, an azide, or nitrobenzyl moiety. In certain embodiments, R’ is selected from:
Figure imgf000014_0002
. [28] In a second aspect, the present disclosure provides a mirror-image nucleic acid polymerase comprising a sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the polymerase comprises D-form amino acids. [29] In certain embodiments, the polymerase consists of D-form amino acids. [30] In certain embodiments, the polymerase comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1. In further embodiments, the polymerase comprises a sequence having at least 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. [31] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid sites selected from E276, K317, N424, and S651 compared to SEQ ID NO: 1. [32] In certain embodiments, the polymerase comprises one or more substitutions selected from E276A, K317G, N424A, and S651A compared to SEQ ID NO: 1. [33] In certain embodiments, the polymerase comprises E276A, K317G, N424A, and S651A substitutions compared to SEQ ID NO: 1. [34] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744 compared to SEQ ID NO: 1. [35] In certain embodiments, the polymerase comprises one or more substitutions from Ile to Ala, Val, Leu, or Tyr at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744 compared to SEQ ID NO: 1. [36] In certain embodiments, the polymerase comprises one or more substitutions selected from I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO: 1. [37] In certain embodiments, the polymerase comprises substitutions of I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO: 1. [38] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid sites selected from M129, I130, G131, D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1. [39] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid sites selected from D141, E143, Y409, and A485 compared to SEQ ID NO: 1. [40] In certain embodiments, the polymerase comprises one or more substitutions selected from D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1. [41] In certain embodiments, the polymerase comprises substitutions of D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1. [42] In certain embodiments, the polymerase comprises one or more modifications at one or more amino acid sites selected from D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1. [43] In certain embodiments, the polymerase comprises one or more substitutions selected from D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1. [44] In certain embodiments, the polymerase comprises substitutions of D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1. [45] In certain embodiments, the polymerase comprises one or more modifications selected from substitution of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, I521L or addition of D between I130 and G131 compared to SEQ ID NO: 1. [46] In certain embodiments, the polymerase comprises substitutions of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L and addition of D between I130 and G131 compared to SEQ ID NO: 1. [47] In certain embodiments, the polymerase comprises the sequence selected from SEQ ID NOs: 2-7. [48] In certain embodiments, the polymerase consists of the sequence selected from SEQ ID NOs: 2-7. [49] In a third aspect of the present disclosure, a method of replicating an L-polynucleotide is provided, the method comprising the step of: incubating a mixture comprising (i) the L- polynucleotide, (ii) an L-primer, (iii) L-dNTP, (iv) the polymerase (e.g., as described herein), and (v) a buffer, thereby inducing replication of the L-polymerase. [50] In certain embodiments, the L-polynucleotide is DNA or RNA. [51] In certain embodiments, wherein the mixture comprises L-dATP, L-dGTP, L-dCTP, and L-dTTP. [52] In certain embodiments, the buffer comprises 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl. [53] In certain embodiments, the incubation step comprises PCR. [54] In a fourth aspect of the present disclosure, a method of sequencing an L- polynucleotide is provided, the method comprising the cycle of: a. incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-3’-O-R’-dNTP-R2-Label, (iv) the polymerase (e.g., as described herein), and (v) a buffer, thereby obtaining a replication product; b. detecting a signal from the L-3’-O-R’-dNTP-R2-Label incorporated into the replication product; and c. inducing cleavage of the R’ group and R2 group of the L-3’-O-R’-dNTP-R2- Label incorporated into the replication product. [55] In certain embodiments, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times. [56] In certain embodiments, the L-3’-O-R’-dNTP-R2-Label comprises L-3’-O-R’-dATP- R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, and L-3’-O-R’-dCTP-R2- Label, wherein each label is different. In certain embodiments, the L-3’-O-R’-dNTP-R2- Label has a structure according to Formula II as defined in Section 5.2.2.1.2. In certain embodiments, the signal is a fluorescent signal. [57] In a fifth aspect of the present disclosure, a method of sequencing an L-polynucleotide is provided, the method comprising the steps of: a. incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-dNTP, (iv) an L-ddNTP-R2-Label or L-3’-O-R’-dNTP-R2-Label, (v) the polymerase (e.g., as described herein), and (vi) a buffer, thereby obtaining a replication product; b. separating the replication product; and c. detecting a signal from the L-ddNTP-R2-Label or L-3’-O-R’-dNTP-R2-Label incorporated into the replication product. [58] In certain embodiments, the L-dNTP comprises L-dATP, L-dTTP, L-dGTP, and L- dCTP. In certain embodiments, the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L- ddTTP-R2-Label, L-ddGTP-R2-Label, and L-ddCTP-R2-Label, wherein each label is different. [59] In certain embodiments, the L-ddNTP-R2-Label has Formula III as defined section 5.2.2.2.1. In certain embodiments, the L-3’-O-R’-dNTP-R2-Label comprises L-3’-O-R’- dATP-R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, and L-3’-O-R’- dCTP-R2-Label, wherein each label is different. In certain embodiments, the L-3’-O-R’- dNTP-R2-Label has Formula II as defined in section 5.2.2.1.2. In certain of these embodiments, the signal is a fluorescent signal. [60] In certain of these embodiments, the incubation step comprises PCR. In certain of these embodiments, the separation step comprises separating the replication product by size. [61] In a sixth aspect of the present disclosure, a method of sequencing an L- polynucleotide is provided, the method comprising the cycle of: a. incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-3’-O-R’-dNTP, (iv) L-ddNTPs-R2-Label or L-dNTPs-R2-Label , (v) the polymerase (e.g., as described herein), and (vi) a buffer, thereby obtaining a replication product; b. detecting a signal from the L-ddNTP-R2-Label or L-3’-O-R’-dNTP-R2-Label incorporated into the replication product; and c. inducing cleavage of i) the R’ group of the L-3’-O-R’-dNTP and R2 group of L-ddNTPs-R2-Label incorporated into the replication product; or ii) the R’ group of the L-3’- O-R’-dNTP, R’ and R2 group of the L-3’-O-R’-dNTP-R2-Label incorporated into the replication product. [62] In certain embodiments, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times. [63] In certain embodiments, the L-3’-O-R’-dNTP comprises L-3’-O-R’-dATP, L-3’-O- R’-dTTP, L-3’-O-R’-dGTP, and L-3’-O-R’-dCTP. [64] In certain embodiments, the L-3’-O-R’-dNTP has a structure according to Formula I as defined in Section 5.2.2.1.1. [65] In certain embodiments, the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L- ddTTP-R2-Label, L-ddGTP-R2-Label, and L-ddCTP-R2-Label, wherein each label is different. [66] In certain embodiments, the L-ddNTP-R2-Label has a structure according to Formula III as defined in Section 5.2.2.2.1. In certain embodiments, the L-3’-O-R’-dNTP-R2-Label comprises L-3’-O-R’-dATP-R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2- Label, and L-3’-O-R’-dCTP-R2-Label, wherein each label is different. In certain embodiments, the L-3’-O-R’-dNTP-R2-Label has a structure according to has Formula II as defined in Section 5.2.2.1.2. In certain embodiments, the signal is a fluorescent signal. [67] A seventh aspect of the present disclosure provides a mirror-image terminal deoxynucleotidyl transferase comprising a sequence having at least 90% sequence identity to SEQ ID NO: 36, wherein the polymerase comprises D-form amino acids. [68] In some embodiments, the transferase consists of D-form amino acids. In some embodiments, the transferase comprises a sequence having at least 95% sequence identity to SEQ ID NO: 36. In some embodiments, the transferase comprises a sequence having at least 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 36. In some embodiments, the transferase comprises one or more modifications at one or more amino acid sites of SEQ ID NO: 36. [69] In a seventh aspect, the present disclosure provides a method of synthesizing L- polynucleotide, comprising the step of: incubating a mixture comprising (i) an L-primer, (ii) L-dNTP or L-ddNTP, (iii) the transferase disclosed herein and (iv) a buffer, thereby inducing synthesis of the L- polynucleotide. [70] In some embodiments, the L-polynucleotide is DNA or RNA. In some embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP or L-dTTP. In some embodiments, the L- dNTP comprises (i) L-dATP or L-dTTP and (ii) L-dGTP or L-dCTP. In some embodiments, the L-dNTP comprises L-dATP, L-dGTP, L-dCTP and L-dTTP. [71] In some embodiments, the mixture comprises a radio-labeled L-ddNTP. In some embodiments, the L-ddNTP is a radio-labeled L-ddATP, L-ddGTP, L-ddCTP or L-ddTTP. In some embodiments, the L-ddNTP comprises L-ddNTP-R2-Label or L-3’-O-R’-dNTP-R2- Label. [72] In some embodiments, the method further comprises the step of stopping reaction by heating or by adding a chelating agent. In some embodiments, the chelating agent is EDTA. [73] In an eight aspect of the present disclosure, a kit is provided for replication or synthesis of an L-polynucleotide, the kit comprising (i) the polymerase or the transferase of any one of the preceding embodiments and (ii) optionally, a buffer. [74] In certain embodiments, the kit comprises L-dNTP. In certain embodiments, the L- dNTP comprises L-dATP, L-dGTP, L-dCTP, L-dTTP or L-UTP. [75] In certain embodiments, the kit comprises L-3’-O-R’-dNTP-R2-Label. In certain embodiments, the kit comprises L-3’-O-R’-dNTP-R2-Label comprises L-3’-O-R’-dATP-R2- Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, L-3’-O-R’-dCTP-R2-Label or L-3’-O-R’-dUTP-R2-Label. In certain embodiments, the kit comprises the L-3’-O-R’-dNTP- R2-Label has Formula II as defined in section 5.2.2.1.2. [76] In certain embodiments, the kit comprises L-ddNTP-R2-Label. In certain embodiments, the kit comprises the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L- ddTTP-R2-Label, L-ddGTP-R2-Label, L-ddCTP-R2-Label or L-ddUTP-R2-Label. In certain embodiments, the L-ddNTP-R2-Label has Formula III as defined in Section 5.2.2.2.1. [77] In certain embodiments, the kit comprises L-3’-O-R’-dNTP. In certain embodiments, the L-3’-O-R’-dNTP comprises L-3’-O-R’-dATP, L-3’-O-R’-dTTP, L-3’-O-R’-dGTP, and L- 3’-O-R’-dCTP. In certain embodiments, the L-3’-O-R’-dNTP has Formula I as defined in Section 5.2.2.1.1. 4. BRIEF DESCRIPTION OF THE DRAWINGS [78] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where: [79] Figure 1 provides the amino-acid sequence of (D)-form mutant 9◦N DNA polymerase (candidate #1-1) (SEQ ID NO: 8). All the amino acids are D-form amino acids. Diamond = mutation introduced for NCL; Circle = NCL site (start of the ‘second’ strand); Square = potential substitution sites for isoleucines; Triangle= mutation introduced for modified nucleotides. [80] Figure 2A shows the sequence of D-form 9°N DNA polymerase mutant 1-I, 9°N-N fragment (SEQ ID NO: 9), where sequences of nine synthetic peptides are presented in separate lines. Figure 2B shows synthetic route for the D-form 9°N DNA polymerase mutant 1-I, 9°N-N fragment. [81] Figure 3A shows the sequence of D-form 9°N DNA polymerase mutant 1-I, 9°N-C fragment (SEQ ID NO: 10), where sequences of six synthetic peptides are presented in separate lines. Figure 3B shows synthetic route for the D-form 9°N DNA polymerase mutant 1-I, 9°N-C fragment. [82] Figure 4A provides TLC staining of Tfa-(D)-Thz-OH, i.e. (S)-3-(2,2,2- trifluoroacetyl)thiazolidine-4-carboxylic acid). Figure 4B provides 1H NMR (500 MHz, CDCl3) result of Tfa-(D)-Thz-OH, i.e., (S)-3-(2,2,2-trifluoroacetyl)thiazolidine-4-carboxylic acid). [83] Figure 5 illustrates synthesis of 2-Cl-(Trt)-NHNH2 as detailed in Example 13. [84] Figure 6A is the peptide sequence of D-9°N-N-6@5-mer (SEQ ID NO: 11). Figure 6B provides analytical HPLC chromatogram of the crude D-9°N-N-6@5-mer (λ=214 nm). Column: Welch C4. Gradient: 0-20 min 100-80% A in B, 20-40 min 80-50% A in B, 40-60 min 50-20% A in B, and 60-70 min 20-0% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Figure 6C provides analytical HPLC chromatogram of the pure D-9oN- N-6@5-mer. Gradient: 95-5% CH3CN (0.1% TFA) in H2O (0.1% TFA) over 30 min. Purification conditions: Column: Welch C4 semipreparative column. Gradient: 1-20 min 100- 80% A in B, 20-40 min 80-50% A in B, 40-50 min 50-20% A in B, and 50-55 min 20-0% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). [85] Figure 7A is the peptide sequence of D- 9°N-N-6@11-mer (SEQ ID NO: 12); Figure 7B provides analytical HPLC chromatogram of the crude D-9°N-N-6@11-mer (λ=214 nm). Column: Welch C4. Gradient: 95-5% CH3CN (0.1% TFA) in H2O (0.1% TFA) over 30 min; Figure 7C provides analytical HPLC chromatogram of the pure D-Pfu-9°oN-N-6@11-mer. Gradient: 95-5% CH3CN (0.1% TFA) in H2O (0.1% TFA) over 30 min. Purification conditions: Column: Welch C4 semipreparative column. Gradient: 1-20 min 100-80% A in B, 20-40 min 80-50% A in B, 40-50 min 50-20% A in B, and 50-55 min 20-0% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA); Figure 7D provides MALDI-TOF mass spectrum of D-9°oN-N-6@11-mer. [86] Figure 8A is the peptide sequence of D-9°N-N-6@24-mer (SEQ ID NO: 13); Figure 8B provides analytical HPLC chromatogram of the crude D-9°N-N-6@24-mer (λ=214 nm). Column: Welch C4. Gradient: 0-20 min 100-80% A in B, 20-40 min 80-50% A in B, 40-60 min 50-20% A in B, and 60-70 min 20-0% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA); Figure 8C provides analytical HPLC chromatogram of the pure D-Pfu- 9°N-N-6@5-mer. Gradient: 95-5% CH3CN (0.1% TFA) in H2O (0.1% TFA) over 30 min. Purification conditions: Column: Welch C4 semipreparative column. Gradient: 1-20 min 100- 80% A in B, 20-40 min 80-50% A in B, 40-50 min 50-20% A in B, and 50-55 min 20-0% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). [87] Figure 9A is the peptide sequence of D-9°N-C-3@9-mer (SEQ ID NO: 14); Figure 9B provides analytical HPLC chromatogram of the crude D-9°N-C-3@9-mer (λ=214 nm). Column: Welch C4. Gradient: 0-20 min 100-80% A in B, 20-40 min 80-50% A in B, 40-60 min 50-20% A in B, and 60-70 min 20-0% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semipreparative column. Gradient: 1-20 min 100-80% A in B, 20-40 min 80-50% A in B, 40-60 min 50-20% A in B, and 60-70 min 20-0% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA); Figure 9C provides HRMS (ESI) spectrum of D-9°N-C-3@9-mer: [M+H]+ Calcd for C36H59N13O15S 946.4053; found 946.4064. [88] Figure 10A provides analytical HPLC chromatogram of the purified D-9°N-C-1@33- mer (λ=214 nm). Column: Welch C4. Gradient: 0-20 min 75-35% A in B, 20-23 min 35-0% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). b) ESI-MS spectrum of D-9oN-C-1@33-mer: Observed 3985.5, calculated 3985.6. Figure 10B provides ESI-MS spectrum of D-9oN-C-1@33-mer. [89] Figure 11A provides analytical HPLC chromatogram of the purified D-9°N-C-2@39- mer (λ=214 nm). Column: Welch C4. Gradient: 0-20 min 75-35% A in B, 20-23 min 35-0% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Figure 11B provides ESI-MS spectrum of D-9oN-C-2@39-mer: Observed 4921.5, calculated 4921.6. [90] Figure 12A provides peptide sequence of D-9°N-C-3@21-mer (SEQ ID NO: 15); Figure 12B provides analytical HPLC chromatogram of the crude D-9°N-C-3@21-mer (λ=214 nm). Column: Welch C4. Gradient: 0-30 min 90-50% A in B, 30-40 min 50-5% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semipreparative column. Gradient: 0-30 min 90-50% A in B, 30-40 min 50-5% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Figure 12C provides ESI-MS spectrum of D-9°N-C-3@21-mer. Figure 12D provides the MS spectrum and observed mass 2196.1 of D-9°N-C-3@21-mer without TFA protection. [91] Figure 13A is the peptide sequence of D-9°N-C-3@ Cys35-mer (SEQ ID NO: 16); Figure 13B provides analytical HPLC chromatogram of the crude D-9oN-C-3@ Cys35-mer (λ=214 nm). Column: Welch C4. Gradient: 0-30 min 70-20% A in B, 30-40 min 20-5% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semipreparative column. Gradient: 0-30 min 70-20% A in B, 30-40 min 20-5% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Figure 13C provides ESI-MS spectrum of D-9°N-C-3@21-mer; Figure 13D provides observed mass of 4276 of D-9°N-C-3@Cys35-mer. [92] Figure 14A is the peptide sequence of D-9°N-C-3@56-mer (SEQ ID NO: 17); Figure 14B provides analytical HPLC chromatogram of the crude D-9oN-C-3@56-mer (λ=214 nm). Column: Welch C4. Gradient: 0-30 min 80-30% A in B, 30-40 min 30-5% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semipreparative column. Gradient: 0-30 min 80-30% A in B, 30-40 min 30-5% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). [93] Figure 15A is the peptide sequence of D-Pfu-9°N-C-3@35-mer (SEQ ID NO: 18); Figure 15B provides analytical HPLC chromatogram of the crude D-Pfu-9°N-C-3@35-mer (λ=214 nm). Column: Welch C4. Gradient: 0-30 min 80-30% A in B, 30-40 min 30-5% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semipreparative column. Gradient: 0-30 min 80-30% A in B, 30-40 min 30-5% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). [94] Figure 16A is the peptide sequence of D-9°N-N-6@5-mer and D-9°N-N-6@11-mer (SEQ ID NO 19 and SEQ ID NO: 20); Figure 16B provides synthetic route for the preparation of D- 9°N-N-6@16-mer. D-9°N-N-6@5-mer (4 mg, 1 equiv.) was dissolved in 0.4 mL acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in ice-salt bath (-15 °C), and 40 μl 0.5 M NaNO2 (in acidified ligation buffer) was added. The reaction was kept in ice-salt bath under stirring for 15 min, after which 0.4 ml 0.2 M MPAA (in 6 M Gn·HCl and 0.1 M Na2HPO4, pH 5.7) was added. After the addition of D-Pfu-9°N-N-6@11-mer (7.5 mg), the pH of the reaction mixture was adjusted to 6.6-6.8 with NaOH solution at room temperature. After 12 h, the reaction mixture was reduced by TCEP and purified by HPLC (purification conditions: 5-95% CH3CN (with 0.1% TFA) gradient in H2O (with 0.1% TFA) over 30 min on a Welch C4 column). The ligation product was obtained with a yield of 80% (9.0 mg); Figure 16C provides analytical HPLC chromatogram of the peaks transformation in progressing the NCL reaction (λ=214 nm). Column: Welch C4. Gradient: 5-95% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 30 min. [95] Figure 17A is the peptide sequence of D-9°N-C-1 and D-9oN-C-2 (SEQ ID NO: 21, 22, 23); Figure 17B provides the synthetic route for the preparation of D-9°N-C-7@72-mer. D-9°N-C-1@33-mer (5.4 mg) was dissolved in 0.27 mL acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in ice-salt bath (-15 °C), and 27 μl 0.5 M NaNO2 (in acidified ligation buffer) was added. The reaction was kept in ice-salt bath under stirring for 20 min, after which 0.14 ml 0.4 M MPAA (in 8 M Gn·HCl and 0.1 M Na2HPO4, pH 5.7) was added. After the addition of D-9°N-C-2@39-mer (5.8 mg), the pH of the reaction mixture was adjusted to 6.6-6.8 with NaOH solution at room temperature. After 16 h the mixture was purified by HPLC (purification conditions: 20-70% CH3CN (with 0.1% TFA) gradient in H2O (with 0.1% TFA) over 30 min on a Polaris C18 column. The ligation product was obtained. Figure 17C is the analytical HPLC chromatogram of the peaks transformation in progressing the NCL reaction (λ=214 nm). Column: Welch C4. Gradient: 20-70% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 30 min. Figure 17D is ESI-MS of D-9°N-C-7@72-mer with MPAA attachment; Figure 17E shows MPAA-attached ligated product (Observed mass: 9042; calculated mass: 9042). [96] Figure 18A is the peptide sequence of D-9°N-N-1@(His)6+39-mer and D-9°N-N- 2@62-mer. Figure 18B illustrates synthetic route for the preparation of D-9°N-N- 10@(His)6+101-mer. Figure 18C shows analytical HPLC chromatogram of the peaks transformation in progressing the NCL reaction (λ=214 nm). Column: Welch C4. Gradient: 5-95% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 30 min [√ = confirmed peak]. Figure 18D is deconvoluted MS (ESI-MS) spectrum of purified D-9°N-N- 10@(His)6+101-mer [Calculated: 12875.8, observed: 12876.8]. [97] Figure 19A is the peptide sequence of D-9°N-N-7@52-mer and D-9°N-N-8@56-mer. Figure 19B illustrates synthetic route for the preparation of D-9°N-N-13@108-mer. Figure 19C provides analytical HPLC chromatogram of the peaks transformation in progressing the NCL reaction (λ=214 nm). Column: Welch C4. Gradient: 5-95% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 30 min [√ = confirmed peak]. Figure 19D is deconvoluted MS (ESI-MS) spectrum of purified D-9oN-N-13@108-mer [Calculated: 12467.52, observed: 12467.39]. [98] Figure 20 provides the synthetic chemical ligation route of the terminal deoxynucleotidyl transferase. [99] Figure 21A is the peptide sequence of D-TdT-WT-1@(His)6+46-mer. Figure 21B provides analytical HPLC chromatogram of the crude D-TdT-WT-1@(His)6+46-mer (λ=214 nm). Figure 21C is deconvoluted MS (ESI-MS) spectrum of purified D-TdT-WT- 1@(His)6+46-mer [Calculated: 5922.97, observed: 5922.97]. [100] Figure 22A is the peptide sequence of D-TdT-WT-1@(His)6+46-mer and D-TdT- WT-2@61-mer. Figure 22B illustrates synthetic route for the preparation of D-TdT-WT- 8@(His)6+107-mer. Figure 22C provides analytical HPLC chromatogram of the crude D- TdT-WT-1@(His)6+46-mer, D-TdT-WT-2@61-mer and D-TdT-WT-8@(His)6+107-mer. Figure 22D is deconvoluted MS (ESI-MS) spectrum of purified D-TdT-WT-1@(His)6+107- mer [Calculated: 12663.21, observed: 12663.23]. [101] Figure 23 shows synthetic route including a convergent fragment assembly strategy for the (D)-form mutant terminal deoxynucleotidyl transferase provided in Example 18. [102] Figure 24A provides results from analytical HPLC chromatogram of the crude D- TdT-WT-1@66-mer (λ=214 nm). Column: Welch C4. Gradient: 1-30 min 80-30% water (0.1% TFA) in ACN (0.1% TFA), 30-35 min 30-0% water (0.1% TFA) in ACN (0.1% TFA) and 35-47 min 0-80% water (0.1% TFA). Figure 24B shows deconvoluted MS (ESI-MS) spectrum of purified D-TdT-WT-1@66-mer. Purification conditions: 1-5 min 100-80% water (0.1% TFA) in ACN (0.1% TFA), 5-55 min 80-30% water (0.1% TFA) in ACN (0.1% TFA) and 55-60 min 30-0% water (0.1% TFA) using (C18 semipreparative column). 5. DETAILED DESCRIPTION 5.1. Definitions [103] “L-Nucleic acid” or “L-nucleotide” shall mean any nucleic acid or nucleotide molecule having L chirality, including, without limitation, L-DNA, L-RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR systems, reagents and consumables (Perkin Elmer Catalogue 1996- 1997, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA). [104] The term “D-polymerase” as used herein refers to a protein having a mirror form of an original polymerase having L-chirality. The original polymerase can be a polymerase obtained or modified from nature or synthetically created. The original polymerase can be a DNA or RNA polymerase or transferase, such as terminal deoxynucleotidyl transferase. [105] Although the base is usually referred to as a purine or pyrimidine, the skilled person will appreciate that derivatives and analogs are available which do not alter the capability of the nucleotide or nucleoside to undergo Watson-Crick base pairing. As used herein, the term “base” includes a compound or molecule whose core structure is the same as, or closely resembles that of, a natural base, but which has a chemical or physical modification, such as a different or additional side groups, which allows the derivative nucleotide or nucleoside to be linked to another molecule. For example, the base can be a deazapurine. [106] "Hybridize" shall mean the annealing of one single-stranded nucleic acid to another nucleic acid based on sequence complementarity. The propensity for hybridization between nucleic acids depends on the temperature and ionic strength of their milieu, the length of the nucleic acids and the degree of complementarity. The effect of these parameters on hybridization is well known in the art (see Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.) [107] Dehybridize is understood by those skilled in the art to mean to disassociate the hybridized primer (or extended strand thereof) from the target nucleic acid without destroying the target nucleic acid and thus permitting further hybridization of a second primer to the target nucleic acid. Hybridization as used herein in one embodiment means stringent hybridization, for examples as described in Sambrook, J., Russell, D. W., (2000) Molecular Cloning: A Laboratory Manual: Third Edition. [108] The term “polynucleotide” as used herein refers to a linear polymer comprising more than one nucleotide monomers. In some embodiments, a polynucleotide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotide monomers. It can be DNA, RNA or a modification thereof. [109] As used herein, hybridization of a primer sequence shall mean annealing sufficient such that the primer is extendable by creation of a phosphodiester bond. [110] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit (if appropriate) of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. 5.2. Mirror image nucleotides for use in sequencing or synthesizing L- polynucleotide 5.2.1. L-Nucleotide and L-Nucleosides [111] In one aspect, the disclosure provides mirror image nucleosides or nucleotides. The mirror image nucleosides or nucleotides can be used for synthesis of L-polynucleotide or to sequence a L-polynucleotide. A L-polynucleotide can include a pentose sugar, a nitrogenous base and a phosphate containing group. [112] Pentose sugars have linear and cyclic forms, and it is understood that the L- nucleosides and L-nucleotides of this disclosure include pentose sugar units which can be referred to via their linear or cyclic form. In some embodiments, the pentose sugar is a L- ribose or L-deoxyribose. In some embodiments, the pentose sugar is a (3R,4S)-3,4,5- trihydroxypentanal and a (4R)-4,5-dihydroxypentanal. The nitrogenous base can be attached to the C1-position of the L-ribose or L-deoxyribose, and the phosphate containing group can be attached to the C5-position. [113] In some embodiments, the mirror image nucleosides or nucleotides include at least one of (i) a cleavable protecting group on a 3' O of the pentose ring (if present) and (ii) a cleavable label. In some embodiments, the mirror image nucleosides or nucleotides include neither (i) a cleavable protecting group on a 3' O of the pentose ring (if present) nor (ii) a cleavable label. It is understood that when referring to the pentose ring of a pentose sugar, the 3’ position or 5’ position of the ring can alternatively be referred to as the 3 position or 5 position. [114] The mirror image nucleosides or nucleotides can be used for sequencing reactions, polynucleotide synthesis, nucleic acid amplification, nucleic acid hybridization assays, single nucleotide polymorphism studies, techniques using enzymes such as polymerase, reverse transcriptase, terminal transferase, techniques that use Labelled dNTPs (e.g., nick translation, random primer labeling, end-labeling (e.g., with terminal deoxynucleotidyl-transferase), reverse transcription, or nucleic acid amplification. [115] In some embodiments of the invention, a chemically modified mirror image nucleoside or nucleotide is provided that comprises at least one of (i) a cleavable protecting group on a 3' O of the pentose sugar if present and (ii) a cleavable label. In embodiments where the mirror image nucleotide comprises a cleavable label, the label is not particularly limited, provided that the label provides means for detection and R’ includes H. 5.2.1.1 Base [116] In certain embodiments, the nitrogenous base is a purine, or a pyrimidine. In certain embodiments, the nitrogenous base is a deazapurine. In certain embodiments, the nitrogenous base is selected from thymine (5-methyl-2,4-dioxipyrimidine), cytosine (2-oxo-4- aminopyrimidine), 5-methyl-cytosine (2-oxo-5-methyl-4-aminopyrimidine), and uracil (2,4- dioxoypyrimidine), adenine (6-aminopurine), 7-deaza adenine (7H-Pyrrolo[2,3-d]pyrimidin- 4-amine; 6-Amino-7-deazapurine), guanine (2-amino-6-oxypurine), 7-deaza guanine (2- Amino-4-hydroxy-pyrrolo-[2,3-d]-pyrimidine; 2-amino-7deaza-6-oxypurine), hypoxanthine (1,9-Dihydro-6H-purin-6-one), Xanthine (3,7-Dihydro-1H-purine-2,6-dione) and 5- nitroindole. In certain embodiments, the nitrogenous base is thymine, uracil, cytosine, adenine, guanine, 7-deaza adenine or 7-deaza guanine. 5.2.1.2 R’ - Cleavable Protecting Groups [117] In certain embodiments, R’ is selected from H and a cleavable protecting group. The cleavable protecting groups of the invention are not particularly limited, as long as the resulting nucleotides are efficient substrates for the mirror image DNA polymerase. [118] The skilled person will appreciate how to attach a suitable protecting group to the pentose ring to block interactions with the 3'-OH. The protecting group can be attached directly at the 3' position or can be attached at the 2' position (the protecting group being of sufficient size or charge to block interactions at the 3' position). Alternatively, the protecting group can be attached at both the 3' and 2' positions and can be cleaved to expose the 3' OH group. [119] Suitable protecting groups will be apparent to the skilled person and can be formed from any suitable protecting group disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. The protecting group should be removable (or modifiable) to produce a 3' OH group. The process used to obtain the 3' OH group can be any suitable chemical or enzymic reaction. [120] In some embodiments, a cleavable protecting group comprises an allyl, nitrobenzyl, 1- methyl-2-alkyl disulfide or methyl azido moiety. [121] In particular embodiments, the cleavable protecting group is selected from:
Figure imgf000027_0001
where the squiggly line demarks the point of attachment to the 3’O. 5.2.1.3 Cleavable Label Constructs (R2-Label) 5.2.1.3.1 Labels [122] The present invention can make use of conventional detectable labels that can be modified to covalently attach to a nucleotide via a cleavable linker. [123] Detection can be carried out by any suitable method, including fluorescence spectroscopy or by other optical means. A particularly contemplated label is a fluorophore, which, after absorption of energy, emits radiation at a defined wavelength. Many suitable fluorescent labels are known. For example, Welch et al. (Chem. Eur. J. 5(3):951–960, 1999) discloses dansyl-functionalised fluorescent moieties that can be used in the present invention. Zhu et al. (Cytometry 28:206–211, 1997) describes the use of the fluorescent labels Cy3 and Cy5, which can also be used in the present invention. Labels suitable for use are also disclosed in Prober et al. (Science 238:336–341, 1987); Connell et al. (BioTechniques 5(4):342–384, 1987), Ansorge et al. (Nucl. Acids Res. 15(11):4593–4602, 1987) and Smith et al. (Nature 321:674, 1986). Other commercially available labels include, but are not limited to: ATTO-dyes (e.g., Atto 655 and Atto 647N), Quasar-dyes, CF-dyes, fluorescein, rhodamine (including TMR, texas red and Rox), Alexa Fluor® 647, 488, 532, 594, 633;, Dyomics-dyes,, bodipy, acridine, R6G, Cy3, Cy3.5, Cy5, Cy5.5, , coumarin, pyrene, benzanthracene and the cyanins. [124] Although fluorescent labels are particularly contemplated, other forms of detectable labels will be apparent as useful to those of ordinary skill. For example, microparticles, including quantum dots (Empodocles, et al., Nature 399:126–130, 1999), gold nanoparticles (Reichert et al., Anal. Chem. 72:6025–6029, 2000), microbeads (Lacoste et al., Proc. Natl. Acad. Sci USA 97(17):9461–9466, 2000), and tags detectable by changes in pH and voltage, spectrometry such as mass spectroscopy, Raman spectroscopy and surface plasmon resonance (SPR) sensing can all be used. i. Fluorophores [125] In certain embodiments of the L-nucleotides of the present disclosure, the label is a fluorophore selected from: Rox, bodipy, bodipy-FL-510, R6G and Cy5 and functional derivatives thereof. [126] In particular embodiments, the fluorophore is selected from:
Figure imgf000028_0001
5.2.1.3.2 Cleavable Linkers (R2) [127] Certain embodiments of the L-nucleotides of the present disclosure comprise a cleavable label linked to, e.g., the nitrogenous base, the 3’ O, or the 5’ phosphate group through a cleavable linker. In certain embodiments, the linker is attached to the nitrogenous base. In certain embodiments, the linker is attached to C8 of a purine base, the C7 of a 7- deaza purine base, or to the C5 of a pyrimidine base. [128] In certain embodiments, the cleavable linker is photocleavable, is cleaved by contact with water-soluble phosphines, or is cleaved by water-soluble transition metal-containing catalysts. [129] Suitable linkers known to those of skill in the art include, but are not limited to, disulfide linkers, acid labile linkers (including dialkoxybenzyl linkers, Sieber linkers, indole linkers, t-butyl Sieber linkers, electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, cleavage under reductive conditions, oxidative conditions, cleavage via use of safety-catch linkers, and cleavage by elimination mechanisms. i. Electrophilically Cleaved Linkers [130] Electrophilically cleaved linkers are typically cleaved by protons and include cleavages sensitive to acids. Suitable linkers include the modified benzylic systems such as trityl, p-alkoxybenzyl esters and p-alkoxybenzyl amides. Other suitable linkers include tert- butyloxycarbonyl (Boc) groups and the acetal system. [131] The use of thiophilic metals, such as nickel, silver or mercury, in the cleavage of thioacetal or other sulphur-containing protecting groups can also be considered for the preparation of suitable linker molecules. ii. Nucleophilically Cleaved Linkers [132] Nucleophilic cleavage is also a well-recognized method in the preparation of linker molecules. Groups such as esters that are labile in water (i.e., can be cleaved simply at basic pH) and groups that are labile to non-aqueous nucleophiles, can be used. Fluoride ions can be used to cleave silicon-oxygen bonds in groups such as triisopropyl silane (TIPS) or t- butyldimethyl silane (TBDMS). iii. Photocleavable Linkers [133] Photocleavable linkers have been used widely in carbohydrate chemistry. It is preferable that the light required to activate cleavage does not affect the other components of the modified nucleotides. For example, if a fluorophore is used as the label, it is preferable if this absorbs light of a different wavelength to that required to cleave the linker molecule. Suitable linkers include those based on O-nitrobenzyl compounds and nitroveratryl compounds. Linkers based on benzoin chemistry can also be used (Lee et al., J. Org. Chem. 64:3454–3460, 1999). iv. Cleavage Under Reductive Conditions [134] There are many linkers known that are susceptible to reductive cleavage. Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups. Disulphide bond reduction is also known in the art. v. Cleavage Under Oxidative Conditions [135] Oxidation-based approaches are well known in the art. These include oxidation of p- alkoxybenzyl groups and the oxidation of sulphur and selenium linkers. The use of aqueous iodine to cleave disulphides and other sulphur or selenium-based linkers is also within the scope of the invention. vi. Safety-catch Linkers [136] Safety-catch linkers are those that cleave in two steps. In a preferred system the first step is the generation of a reactive nucleophilic center followed by a second step involving an intra-molecular cyclization that results in cleavage. For example, levulinic ester linkages can be treated with hydrazine or photochemistry to release an active amine, which can then be cyclized to cleave an ester elsewhere in the molecule (Burgess et al., J. Org. Chem. 62:5165– 5168, 1997). vii. Cleavage by Elimination Mechanisms [137] Elimination reactions can also be used. For example, the base-catalyzed elimination of groups such as Fmoc and cyanoethyl, and palladium-catalyzed reductive elimination of allylic systems, can be used. 5.2.1.3.3 Spacers [138] As well as the cleavage site, the linker can comprise one or more a spacer moiety. A spacers (linkers and bridges) can be any typically used in, e.g., the synthesis of bioconjugates. The spacer distances the nucleotide base from the cleavage site or label. The length of the linker is unimportant provided that the label is held a sufficient distance from the nucleotide so as not to interfere with any interaction between the nucleotide and an enzyme. [139] In particular embodiments, the cleavable linker comprises an allyl, disulfide or azido group along with additional spacers. In certain embodiments, the cleavable linker comprises:
Figure imgf000030_0001
Figure imgf000031_0001
. 5.2.1.3.4 Cleavable Label Constructs [140] In certain embodiments of the mirror image nucleosides or nucleotides provided herein, the cleavable label comprises a label as described in section 5.2.1.3.1 and a cleavable linker in more detail in section 5.2.1.3.2. [141] In particular embodiments, the cleavable label construct is selected from:
Figure imgf000031_0002
; wherein the squiggly line demarks the point of attachment to the mirror image nucleotide, including attachment to a linker/spacer attached to a nucleotide for purposes of enabling attachment, e.g., an amine moiety. 5.2.2. Embodiments of the present disclosure [142] In certain embodiments, an L- nucleoside or L-nucleotide are provided comprising: a pentose sugar selected from a (3R,4S)-3,4,5-trihydroxypentanal and a (4R)-4,5- dihydroxypentanal; wherein the C-5 of the sugar is substituted by a hydroxyl, monophosphate, diphosphate or triphosphate; and the C-3 of the sugar is substituted by H, or O-R’ where R’ is H or a cleavable protecting group; a nitrogenous base; and optionally a cleavable label; wherein the L- nucleoside or L-nucleotide comprises at least one selected from (i) a cleavable protecting group and (ii) a cleavable label. [143] In some embodiments, the present disclosure provides a nucleotide comprising: a pentose sugar comprising a cyclic form of a (3R,4S)-3,4,5-trihydroxypentanal or (4R)-4,5-dihydroxypentanal; wherein the H of the 5-hydroxyl group of the pentose sugar is substituted by one or more phosphate group; and wherein the H of the 3-hydroxyl group of the (3R,4S)-3,4,5- trihydroxypentanal is optionally substituted by a cleavable protecting group; a nitrogenous base linked to the 2 position of the pentose sugar, and optionally a cleavable label comprising a cleavable linker and a label; wherein at least one of a cleavable protecting group and a cleavable label is present. [144] Embodiments provided herein will be further described with reference to nucleotides. However, unless indicated otherwise, the reference to nucleotides is also intended to be applicable to nucleosides. [145] In certain embodiments, an L-nucleotide is provided having a structure according to Formula A:
Figure imgf000033_0001
Formula A wherein R’ is H or a cleavable protecting group; X is H or hydroxyl, BASE is a nitrogenous base, and wherein the nucleotide optionally comprises a cleavable label, and wherein at least one of a cleavable protecting group or a cleavable label is present. [146] In certain embodiments, the nitrogenous base is as described in Section 5.2.1.1 herein. In certain embodiments, the cleavable protecting group is as described in Section 5.2.1.2 herein. In certain embodiments, the cleavable label is as described in Section 5.2.1.3 herein. [147] In any of the described embodiments, the location of the cleavable label is not particularly limited and may be linked to the nucleotide through any position chemically feasible and/or commonly known in the art. In certain embodiments, the cleavable label is attached to the nitrogenous base, through a terminal phosphate, or at the 3’ O in which case the cleavable protecting group is a cleavable label. 5.2.2.1 (L) - Nucleotide Reversible Terminator (L-NRT) 5.2.2.1.1 [(L)-3’-O-R’-dNTPs] - Formula I [148] In an aspect of the present disclosure, an L-nucleotide is provided having a structure according to Formula I:
Figure imgf000033_0002
Formula I wherein Base is a nitrogenous base, and R' is a cleavable protecting group. [149] In certain embodiments, the nitrogenous base is as described in Section 5.2.1.1 herein. In certain embodiments, the cleavable protecting group is as described in Section 5.2.1.2 herein. [150] In certain embodiments, an L-nucleotide is provided having a structure according to:
Figure imgf000034_0001
. [151] In certain embodiments, R’ comprises a cleavable protecting group as described in Section 5.2.1.2 herein. In further embodiments, the cleavable protecting group, R’, is selected from:
Figure imgf000034_0002
where the squiggly line demarks the point of attachment to the 3’O. 5.2.2.1.2 [(L)-3’-O-R’-dNTP-R2-Label] - Formula II [152] In an aspect of the present disclosure, an L-nucleotide is provided having a structure according to Formula II:
Figure imgf000035_0001
Formula II wherein Base is a nitrogenous base; R' is a cleavable protecting group or H; and Label-R2 together comprise a cleavable label construct. [153] In certain embodiments, the nitrogenous base is as described in Section 5.2.1.1 herein. In certain embodiments, the cleavable protecting group is as described in Section 5.2.1.2 herein. In certain embodiments, the cleavable label construct is as described in Section 5.2.1.3 herein. [154] In certain embodiments, an L-nucleotide is provided having a structure selected from:
Figure imgf000035_0002
Figure imgf000036_0001
. [155] In certain embodiments, R’, the cleavable protecting group is as described in Section 5.2.1.2 herein. In certain embodiments, R2, the cleavable linker, is as described in Section 5.2.1.3.2 herein. [156] In particular embodiments, R’ is selected from:
Figure imgf000036_0002
where the squiggly line demarks the point of attachment to the 3’O. [157] In particular embodiments, R2 comprises:
Figure imgf000037_0001
. [158] In particular embodiments, an L-nucleotide is provided having a structure selected from:
Figure imgf000037_0002
Figure imgf000038_0001
wherein R’ is H or a cleavable protecting group is as described in Section 5.2.1.2 herein. [159] In certain embodiments, R’ is selected from:
Figure imgf000038_0002
. where the squiggly line demarks the point of attachment to the 3’O. 5.2.2.2 Labeled Terminator 5.2.2.2.1 [(L)-ddNTP-R2-Label] Formula III [160] In an aspect of the present disclosure, an L-nucleotide is provided having a structure according to Formula III:
Figure imgf000039_0002
Formula III wherein Base is a nitrogenous base; R2 is a cleavable linker; and Label-R2 together comprise a cleavable label construct. [161] In certain embodiments, the nitrogenous base is as described in Section 5.2.1.1 herein. In certain embodiments, Label-R2, the cleavable label construct, is as described in Section 5.2.1.3 herein. [162] In certain embodiments, an L-nucleotide is provided having a structure selected from:
Figure imgf000039_0001
Figure imgf000040_0001
. [163] In certain embodiments, R2, the cleavable linker is as described in Section 5.2.1.3.2 herein. In particular embodiments, R2 comprises:
Figure imgf000040_0002
[164] In particular embodiments, an L-nucleotide is provided having a structure selected from:
Figure imgf000040_0003
Figure imgf000041_0001
. 5.3. Mirror-image polymerase [165] In another aspect, the present disclosure provides a mirror-image polymerase. Specifically, the mirror-image polymerase comprises D-form amino acids. In some embodiments, the mirror-image polymerase consists of D-form amino acids. In some embodiments, the mirror-image polymerase comprises both D-form and L-form amino acids. In some embodiments, the mirror-image polymerase does not comprise L-form amino acids. [166] In some embodiments, the mirror-image nucleic acid is a mirror image of a DNA polymerase or an RNA polymerase. In some embodiments, the mirror-image nucleic acid is a mirror image of 9°N DNA polymerase or a modification thereof. [167] In some embodiments, the mirror-image polymerase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises a sequence having at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises a sequence having at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises a sequence having at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises a sequence having at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises a sequence having at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises the sequence of SEQ ID NO: 1. [168] In some embodiments, the mirror-image polymerase has a sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase has a sequence having at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase has a sequence having at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase has a sequence having at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase has a sequence having at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase has a sequence having at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase has the sequence of SEQ ID NO: 1. [169] In some embodiments, the mirror-image polymerase comprises one or more modifications at one or more amino acid positions disclosed in Table 1 compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises one or more amino acid substitutions disclosed in Table 1 compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises all the amino acid substitutions disclosed in Table 1.
Figure imgf000042_0001
Figure imgf000043_0001
[170] In some embodiments, the mirror-image polymerase comprises one or more modifications compared to SEQ ID NO: 1 for native chemical ligation (NCL). In some embodiments, the mirror-image polymerase comprises one or more modifications at one or more amino acid sites selected from E276, K317, N424, and S651 compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises one or more substitutions selected from E276A, K317G, N424A, and S651A compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises E276A, K317G, N424A, and S651A substitutions compared to SEQ ID NO: 1. [171] In some embodiments, the mirror-image polymerase comprises one or more substitution of isoleucine compared to SEQ ID NO: 1. In some embodiments, the mirror- image polymerase comprises one or more modifications at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744 compared to SEQ ID NO: 1. In some embodiments, the mirror- image polymerase comprises one or more substitutions from Ile to Ala, Val, Leu, or Tyr at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744 compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises one or more substitutions selected from I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO: 1. [172] In some embodiments, the mirror-image polymerase comprises one or more modifications compared to SEQ ID NO: 1 to improve its interaction with an L-nucleotide or a modification thereof disclosed herein. [173] In some embodiments, the mirror-image polymerase comprises one or more modifications at one or more amino acid sites selected from M129, I130, G131, D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises one or more modifications at one or more amino acid sites selected from D141, E143, Y409, and A485 compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises one or more substitutions selected from D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises substitutions of D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1. [174] In some embodiments, the mirror-image polymerase comprises one or more modifications at one or more amino acid sites selected from D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises one or more substitutions selected from D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises substitutions of D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1. [175] In some embodiments, the mirror-image polymerase comprises one or more modifications selected from substitution of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, I521L or addition of D between I130 and G131 compared to SEQ ID NO: 1. In some embodiments, the mirror-image polymerase comprises substitutions of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L and addition of D between I130 and G131 compared to SEQ ID NO: 1. [176] In some embodiments, the mirror-image polymerase comprises a sequence selected from SEQ ID Nos 1-7. In some embodiments, the polymerase has a sequence selected from SEQ ID Nos: 1-7. [177] The mirror-image polymerase can be obtained by chemical synthesis. In some embodiments, polypeptides or small peptides are synthesized by solid phase peptide synthesis and then ligated by chemical ligation to obtain the mirror-image polymerase. In some embodiments, the mirror-image polymerase has the same enzymatic activity as the original polymerase but acts on D-form nucleotides. 5.4. Mirror-image transferase [178] In one aspect, the present disclosure provides a mirror-image transferase. In some embodiments, the mirror-image transferase is (D)-form terminal deoxynucleotidyl transferase. The (D)-form terminal deoxynucleotidyl transferase can be a template independent polymerase that catalyzes the addition of L-form deoxynucleotides to the 3' hydroxyl terminus of a D-form DNA molecules. [179] Specifically, the mirror-image transferase comprises D-form amino acids. In some embodiments, the mirror-image transferase consists of D-form amino acids. In some embodiments, the mirror-image transferase comprises both D-form and L-form amino acids. In some embodiments, the mirror-image transferase does not comprise L-form amino acids. [180] In some embodiments, the mirror-image nucleic acid is a mirror image of a terminal deoxynucleotidyl transferase having the below sequence of SEQ ID NO: 36:
Figure imgf000045_0001
) [181] In some embodiments, the mirror-image transferase comprises a sequence having at least 90% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase comprises a sequence having at least 96% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase comprises a sequence having at least 96% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase comprises a sequence having at least 97% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase comprises a sequence having at least 98% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase comprises a sequence having at least 99% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase comprises the sequence of SEQ ID NO: 36. [182] In some embodiments, the mirror-image transferase has a sequence having at least 90% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase has a sequence having at least 96% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase has a sequence having at least 96% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase has a sequence having at least 97% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase has a sequence having at least 98% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase has a sequence having at least 99% sequence identity to SEQ ID NO: 36. In some embodiments, the mirror-image transferase has the sequence of SEQ ID NO: 36. [183] In some embodiments, the mirror-image polymerase comprises one or more modifications for NCL (native chemical ligation) sites. In some embodiments, the mirror- image polymerase comprises one or more isoleucine (I) to valine (V) substitutions. In some embodiments, the mirror-image polymerase comprises one or more modifications for interaction with modified nucleotides. [184] The mirror-image transferase can be obtained by chemical synthesis. In some embodiments, polypeptides or small peptides are synthesized by solid phase peptide synthesis and then ligated by chemical ligation to obtain the mirror-image transferase. In some embodiments, the mirror-image transferase has the same enzymatic activity as the original transferase but acts on D-form nucleotides. [185] In some embodiments, the mirror-image transferase is obtained by ligating more than one synthetic peptides. 5.5. Method of use [186] In one aspect, the present disclosure provides methods of using mirror-image nucleotides and mirror-image polymerase disclosed herein. The mirror-image compositions can be used for replicating or sequencing L-polynucleotide. In some embodiments, the compositions are used for a sequencing method adopted from sequencing-by-synthesis method, Sanger method, or a combination thereof. 5.5.1. Method of replicating L-form template [187] The present disclosure provides a method of replicating an L-polynucleotide. The method can comprise the step of: incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-dNTP, (iv) the mirror-form polymerase, and (v) a buffer, thereby inducing replication of the L- L-polynucleotide. In some embodiments, the L-polynucleotide is L-form DNA or RNA. In some embodiments, the L-polynucleotide comprises at least 10, 20, 30, 50, 100, 200, 500, or more nucleotides. [188] In some embodiments, the mixture comprises two or more L-dNTPs. In some embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, L-dTTP, or a modification thereof. In some embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, and L- dTTP. [189] In some embodiments, the mixture comprises a buffer developed for use with 9°N DNA polymerase. In some embodiments the buffer comprises Tris-HCl at a concentration of 1mM to 200mM, 5mM to 150mM, 10mM to 100mM, 20mM to 100mM or 30mM to 80mM. In some embodiments, the buffer comprises Tris-HCl at a concentration of about 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM. In some embodiments, the buffer comprises MgCl2 at a concentration of 1mM to 100mM, 5mM to 100mM, 10mM to 100mM, 15mM to 50mM, or 15mM to 30mM. In some embodiments, the buffer comprises MgCl2 at a concentration of about 10mM, 20mM, 30mM, 40mM or 50mM. In some embodiments, the buffer comprises MnCl2 at a concentration of 1mM to 100mM, 5mM to 100mM, 10mM to 100mM, 15mM to 50mM, or 15mM to 30mM. In some embodiments, the buffer comprises MnCl2 at a concentration of about 10mM, 20mM, 30mM, 40mM or 50mM. In some embodiments, the buffer comprises DTT at a concentration of 0.1mM to 10mM, 0.5mM to 5mM, 0.5mM to 3mM or 0.5mM to 2mM. In some embodiments, the buffer comprises DTT at a concentration of about 0.5mM, 1mM, 1.5mM or 2mM. In some embodiments, the buffer comprises KCl at a concentration of 10mM to 100mM, 20mM to 80mM, 25mM to 75mM or 30mM to 70mM. In some embodiments, the buffer comprises KCl at a concentration of about 25mM, 30mM, 40mM, 50mM, 60mM, 70mM or 100mM. In some embodiments, the buffer has a pH between 7 and 8. In some embodiments, the buffer has a pH at about 7, 7.5 or 8. [190] In some embodiments, the buffer comprises Tris-HCl, MgCl2, DTT and KCl. In some embodiments, the buffer comprises Tris-HCl, MnCl2, DTT and KCl. In some embodiments, the buffer comprises Tris-HCl, MnCl2, MgCl2, DTT and KCl. In some embodiments, the buffer comprises 50 mM Tris-HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl. 5.5.2. Method of sequencing [191] The present disclosure provides a method of sequencing an L-polynucleotide. The L- polynucleotide comprises L-form nucleotides. The L-polynucleotide can be DNA or RNA. [192] In some embodiments, the sequencing method comprises a cycle of: (a) incubating a mixture comprising (i) an L-polynucleotide, (ii) an L-primer, (iii) L-3’-O-R’-dNTP-R2-Label, (iv) a mirror-form polymerase, and (v) a buffer, thereby obtaining a replication product; (b) detecting a signal from the L-3’-O-R’-dNTP-R2-Label incorporated into the replication product; and (iii) inducing cleavage of the R’ group and R2 group of the L-3’-O-R’-dNTP-R2- Label incorporated into the replication product. The method involves (i) incorporation of L- 3’-O-R’-dNTP-R2-Label, (ii) identification of the incorporated nucleotide by signals from the incorporated L-3’-O-R’-dNTP-R2-Label, and (iii) cleavage of the Label, along with the reinitiation of the polymerase reaction for continuing sequence determination. The L-3’-O- R’-dNTP-R2-Label includes a chemical moiety (R’) capping the 3’-OH and a Label tethered to the base through a chemically cleavable linker (R2). The 3′-capping moiety (R’) and the Label on the reaction product are cleaved to reinitiate the polymerase reaction. [193] In some embodiments, the method comprises multiple cycles. In some embodiments, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times. In some embodiments, the cycle is repeated less than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 times. [194] In some embodiments, the L-polynucleotide comprises more than 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 nucleotides. In some embodiments, the L- polynucleotide comprises less than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 nucleotides. [195] In some embodiments, the L-3’-O-R’-dNTP-R2-Label comprises L-3’-O-R’-dATP- R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, or L-3’-O-R’-dCTP-R2- Label. In some embodiments, the L-3’-O-R’-dNTP-R2-Label comprises two or more of L-3’- O-R’-dATP-R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, and L-3’-O- R’-dCTP-R2-Label. In some embodiments, the L-3’-O-R’-dNTP-R2-Label comprises L-3’- O-R’-dATP-R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, and L-3’-O- R’-dCTP-R2-Label. Labels attached to each kind of dNTP can be unique and identifiable. [196] In some embodiments, the L-3’-O-R’-dNTP-R2-Label has a structure according to Formula I as described in 5.2.2.1.2. In some embodiments, the L-3’-O-R’-dNTP-R2-Label has a structure according to Formula II as described in 5.2.2.1.2. [197] In some embodiments, the L-3’-O-R’-dNTP-R2-Label includes a fluorescent label. In the case, the signal is a fluorescent signal. In some embodiments, each type of L-3’-O-R’- dNTP-R2-Label (e.g., dATP, dGTP, dTTP, dCTP) is tagged to a unique label. In some embodiments, each type of L-3’-O-R’-dNTP-R2-Label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal. The L-3’-O-R’-dNTP-R2-Label can include any of the labels disclosed in 5.2.1.3.1. In some embodiments, the L-3’-O-R’-dNTP-R2-Label includes a non-fluorescent label. [198] In some embodiments, the sequencing method comprises the steps of (a) incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-dNTP, (iv) an L-ddNTP- R2-Label, (v) a mirror-image polymerase disclosed herein, and (vi) a buffer, thereby obtaining a replication product; (b) separating the replication product; and (c) detecting a signal from the L-ddNTP-R2-Label incorporated into the replication product. The incubation step can comprise PCR reaction. [199] In preferred embodiments of the method, a low ratio of L-ddNTP-R2-Label is added to the mixture compared to L-dNTP. During incubation (e.g., PCR reaction), L-ddNTP-R2- Label lacking 3’-OH group can be incorporated at random by the mirror-image polymerase to the replication product. Incorporation of L-ddNTP-R2-Label terminates the replication process. The reaction can induce production of oligonucleotide copies of the replication products terminated at a random length by L-ddNTP-R2-Label. [200] In the method, the replication product can be separated by size. In some embodiments, the replication product is separated by size via gel electrophoresis. By detecting a signal from the L-ddNTP-R2-Label incorporated into the replication product, the identity of the terminal L-ddNTP-R2-Label (e.g., ddATP, ddGTP, ddTTP, or ddCTP) can be determined for the replication product. The data can show types of nucleotides along the length of the L- polynucleotide. This process can allow determination of the sequence of the L- polynucleotide. [201] In some embodiments, the L-dNTP comprises L-dATP, L-dTTP, L-dGTP, or L- dCTP. In some embodiments, the L-dNTP comprises one or more of L-dATP, L-dTTP, L- dGTP, and L-dCTP. In some embodiments, the L-dNTP comprises L-dATP, L-dTTP, L- dGTP, and L-dCTP. [202] In some embodiments, the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L- ddTTP-R2-Label, L-ddGTP-R2-Label, or L-ddCTP-R2-Label. In some embodiments, the L- ddNTP-R2-Label comprises one or more of L-ddATP-R2-Label, L-ddTTP-R2-Label, L- ddGTP-R2-Label, and L-ddCTP-R2-Label. In some embodiments, the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L-ddTTP-R2-Label, L-ddGTP-R2-Label, and L-ddCTP-R2- Label. Labels attached to each kind of ddNTP can be unique and identifiable. [203] In some embodiments, the L-ddNTP-R2-Label has a structure disclosed in 5.2.2.2.1. In some embodiments, the L-ddNTP-R2-Label has Formula III as defined in 5.2.2.2.1. [204] In some embodiments, the L-ddNTP-R2-Label includes a fluorescent label. In the case, the signal is a fluorescent signal. In some embodiments, each type of L-ddNTP-R2- Label (e.g., ddATP, ddGTP, ddTTP, ddCTP) is tagged to a unique label. In some embodiments, each type of L-ddNTP-R2-Label (e.g., ddATP, ddGTP, ddTTP, ddCTP) provides a unique fluorescent signal. The L-ddNTP-R2-Label can include any of the labels disclosed in 5.2.1.3.1. In some embodiments, the L-ddNTP-R2-Label includes a non- fluorescent label. [205] In some embodiments, the sequencing method comprises a cycle of (a) incubating a mixture comprising (i) an L-polynucleotide, (ii) an L-primer, (iii) L-3’-O-R’-dNTP, (iv) L- ddNTPs-R2-Label, (v) a mirror-image polymerase provided herein, and (vi) a buffer, thereby obtaining a replication product; (b) detecting a signal from the L-ddNTP-R2-Label incorporated into the replication product; and (c) inducing cleavage of the R’ group of the L- 3’-O-R’-dNTP and R2 group of L-ddNTPs-R2-Label incorporated into the replication product. In this method, the polymerase reaction is performed with the combination of 3′- capped nucleotide reversible terminators (L-3’-O-R’-dNTP) and cleavable fluorescent dideoxynucleotides (L-ddNTPs-R2-Label). In this method, sequences are determined by the signal of each Label on the reaction products terminated by ddNTPs (L-ddNTPs-R2-Label). Upon removing the 3′-OH capping group (R’ group) from the incorporated nucleotide reversible terminators (L-3’-O-R’-dNTP) and the Label from the DNA products terminated by ddNTPs (L-ddNTPs-R2-Label), the polymerase reaction reinitiates to continue the sequence determination. [206] In some embodiments, the method comprises multiple cycles. In some embodiments, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times. In some embodiments, the cycle is repeated less than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 times. [207] In some embodiments, the L-polynucleotide comprises more than 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 nucleotides. In some embodiments, the L- polynucleotide comprises less than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 nucleotides. [208] In some embodiments, the L-3’-O-R’-dNTP comprises L-3’-O-R’-dATP, L-3’-O-R’- dTTP, L-3’-O-R’-dGTP, or L-3’-O-R’-dCTP. In some embodiments, the L-3’-O-R’-dNTP comprises one or more of L-3’-O-R’-dATP, L-3’-O-R’-dTTP, L-3’-O-R’-dGTP, and L-3’-O- R’-dCTP. In some embodiments, the L-3’-O-R’-dNTP comprises L-3’-O-R’-dATP, L-3’-O- R’-dTTP, L-3’-O-R’-dGTP, and L-3’-O-R’-dCTP. [209] In some embodiments, the L-3’-O-R’-dNTP has any of the structure provided in 5.2.2.1.1. In some embodiments, the L-3’-O-R’-dNTP has the Formula I as defined in 5.2.2.1.1 [210] In some embodiments, the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L- ddTTP-R2-Label, L-ddGTP-R2-Label, or L-ddCTP-R2-Label. In some embodiments, the L- ddNTP-R2-Label comprises one or more of L-ddATP-R2-Label, L-ddTTP-R2-Label, L- ddGTP-R2-Label, and L-ddCTP-R2-Label. In some embodiments, the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L-ddTTP-R2-Label, L-ddGTP-R2-Label, and L-ddCTP-R2- Label. Labels attached to each kind of dNTP can be unique and identifiable. [211] In some embodiments, the L-ddNTP-R2-Label has any of the structure provided in 5.2.2.2.1. In some embodiments, the L-ddNTP-R2-Label has a formula III as defined in 5.2.2.2.1. In some embodiments, each type of L-3’-O-R’-dNTP-R2-Label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal. The L-ddNTP-R2-Label can include any of the labels disclosed in 5.2.1.3.1. In some embodiments, the L-ddNTP-R2-Label includes a non-fluorescent label. [212] In some embodiments, the sequencing method comprises a cycle of: (a) incubating a mixture comprising (i) an L-polynucleotide, (ii) an L-primer, (iii) L-dNTP, (iv) a mirror-form polymerase, and (v) a buffer, thereby obtaining a reaction product; and (b) detecting release of PPi molecule in the reaction product. In some embodiments, the sequencing method comprises a plurality of the cycles, wherein in each cycle, the mixture comprises a single type of L-dNTP selected from L-dATP, L-dTTP, L-dGTP and L-dCTP. In some embodiments, the sequencing method comprises a plurality of cycles, wherein each cycle is performed by changing the type of L-dNTP to one of L-dATP, L-dTTP, L-dGTP and L-dCTP stepwise. [213] In some embodiments, the step of detecting release of PPi molecule comprises the steps of (i) processing the reaction product to convert PPi molecule ATP if present and to generate a detectable signal from the ATP if present; and (ii) detecting presence or absence of the detectable signal. In some embodiments, the ATP is processed with luciferase to generate the detectable signal. In some embodiments, the detectable signal is light. In some embodiments, the PPi molecule is converted into ATP by ATP-sulfurylase. 5.5.3. Method of synthesizing L-polynucleotide [214] In one aspect, the present disclosure provides a method of synthesizing an L- polynucleotide using a mirror-image transferase (e.g., terminal deoxynucleotidyl transferase). In some embodiments, the mirror-image transferase is used to catalyze the repetitive addition of deoxyribonucleotides to the 3’-OH of oligodeoxyribonucleotides and single-stranded or double-stranded DNA. In some embodiments, the mirror-image transferase is used for production of synthetic homo-and heteropolymers, homopolymeric tailing of linear duplex DNA with any type of 3’-OH terminus, labeling of oligonucleotide, DNA or RNA, 5’-RACE (rapid amplification of cDNA ends) or in situ localization of apoptosis. The mirror-image transferase can be used for any other known applications of transferases. [215] In some embodiments, the method of synthesizing L-polynucleotide comprises the step of incubating a mixture comprising (i) an L-primer, (ii) L-dNTP or L-ddNTP, (iii) a mirror-image transferase disclosed herein and (iv) a buffer, thereby inducing synthesis of the L- polynucleotide. In some embodiments, the L-polynucleotide is DNA or RNA. The L- dNTP or L-ddNTP can be any of the mirror-image nucleotide or combination thereof disclosed herein. [216] In some embodiments, the mixture comprises L-dNTP. In some embodiments, the mixture comprises L-ddNTP. In some embodiments, the mixture comprises L-dATP, L- dGTP, L-dCTP or L-dTTP. In some embodiments, the L-dNTP comprises (i) L-dATP or L- dTTP and (ii) L-dGTP or L-dCTP. In some embodiments, the L-dNTP comprises L-dATP, L-dGTP, L-dCTP and L-dTTP. [217] In some embodiments, the mixture comprises L-3’-O-R-dNTP. In some embodiments, the mixture comprises L-3’-O-R-dATP, L-3’-O-R-dGTP, L-3’-O-R-dCTP or L-3’-O-R-dTTP. In some embodiments, the mixture comprises L-3’-O-R-dATP, L-3’-O-R- dGTP, L-3’-O-R-dCTP and L-3’-O-R-dTTP. [218] In some embodiments, the mixture comprises a radio-labeled L-ddNTP. In some embodiments, the L-ddNTP is a radio-labeled L-ddATP, L-ddGTP, L-ddCTP or L-ddTTP. In some embodiments, the L-ddNTP comprises L-ddNTP-R2-Label or L-3’-O-R’-dNTP-R2- Label disclosed herein. [219] In some embodiments, the method further comprises the step of removing 3’-O-R group by a deprotection reaction. In some embodiments, the deprotection reaction comprises incubation of the mixture with TCEP buffer. In some embodiments, the deprotection reaction comprises incubation with 50mM TCEP buffer at 37-55^C for 5-15 minutes. In some embodiments, R is azidomethyl. [220] In some embodiments, the method further comprises the step of stopping reaction by heating or by adding a chelating agent. In some embodiments, the chelating agent is EDTA. [221] In some embodiments, incubation is performed at 20 to 40 ^C. In some embodiments, incubation is performed at 37 to 55 ^C. In some embodiments, incubation is performed at 22 to 37 ^C. In some embodiments, incubation is performed at 37 ^C. In some embodiments, incubation is performed at 30-37 ^C. [222] In some embodiments, the heating is performed at a temperature above 50 ^C. In some embodiments, the heating is performed at a temperature above 55 ^C. In some embodiments, the heating is performed at a temperature above 60 ^C. In some embodiments, the heating is performed at a temperature above 60 ^C. In some embodiments, the heating is performed at 70 ^C. [223] In some embodiments, the method comprises repeating the steps provided above. In some embodiments, the method comprises repeating the step of incubating a mixture for the next nucleotide addition. In some embodiments, the method comprises repeating the steps of incubating the mixture and the deprotection reaction. [224] In some embodiments the method further comprises the step of sequencing the synthesized L-polynucleotide. 5.6. Kit [225] The present disclosure provides a kit for replicating, sequencing or synthesizing L- polymerase. The kit comprises a mirror-image polymerase or a mirror-image transferase disclosed herein and optionally, a buffer. [226] In some embodiments, the kit further comprises L-dNTP. In some embodiments, the L-NTP comprises L-dATP, L-dGTP, L-dCTP, L-dTTP or L-UTP. In some embodiments, the L-NTP comprises one or more of L-dATP, L-dGTP, L-dCTP, L-dTTP and L-UTP. In some embodiments, the L-NTP comprises L-dATP, L-dGTP, L-dCTP, and L-dTTP. In some embodiments, the L-NTP comprises L-dATP, L-dGTP, L-dCTP, and L-UTP. In some embodiments, the L-NTP comprises L-dATP, L-dGTP, L-dCTP, L-dTTP and L-UTP. [227] In some embodiments, the kit comprises L-3’-O-R’-dNTP-R2-Label. In some embodiments, the L-3’-O-R’-dNTP-R2-Label comprises L-3’-O-R’-dATP-R2-Label, L-3’-O- R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, L-3’-O-R’-dCTP-R2-Label or L-3’-O-R’- dUTP-R2-Label. In some embodiments, the L-3’-O-R’-dNTP-R2-Label comprises one or more of L-3’-O-R’-dATP-R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, L-3’-O-R’-dCTP-R2-Label and L-3’-O-R’-dUTP-R2-Label. In some embodiments, the L-3’- O-R’-dNTP-R2-Label comprises one or more of L-3’-O-R’-dATP-R2-Label, L-3’-O-R’- dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, and L-3’-O-R’-dCTP-R2-Label. In some embodiments, the L-3’-O-R’-dNTP-R2-Label comprises one or more of L-3’-O-R’-dATP-R2- Label, L-3’-O-R’-dGTP-R2-Label, L-3’-O-R’-dCTP-R2-Label and L-3’-O-R’-dUTP-R2- Label. In some embodiments, each type of L-3’-O-R’-dNTP-R2-Label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal. [228] In some embodiments, the L-3’-O-R’-dNTP-R2-Label has Formula II as defined in 5.2.2.1.2. In some embodiments, the L-3’-O-R’-dNTP-R2-Label has any of the structures disclosed in 5.2.2.1.2. [229] In some embodiments, the kit comprises L-ddNTP-R2-Label. In some embodiments, the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L-ddTTP-R2-Label, L-ddGTP-R2- Label, L-ddCTP-R2-Label or L-ddUTP-R2-Label. L-ddNTP-R2-Label. In some embodiments, the L-ddNTP-R2-Label comprises one or more of L-ddATP-R2-Label, L- ddTTP-R2-Label, L-ddGTP-R2-Label, L-ddCTP-R2-Label and L-ddUTP-R2-Label. In some embodiments, the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L-ddTTP-R2-Label, L- ddGTP-R2-Label, and L-ddCTP-R2-Label. In some embodiments, the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L-ddGTP-R2-Label, L-ddCTP-R2-Label and L-ddUTP-R2- Label. [230] In some embodiments, the L-ddNTP-R2-Label has Formula III as defined in 5.2.2.2.1. In some embodiments, the L-ddNTP-R2-Label has any of the structures disclosed in 5.2.2.2.1. [231] In some embodiments, the kit further comprises L-3’-O-R’-dNTP. The L-3’-O-R’- dNTP can comprise L-3’-O-R’-dATP, L-3’-O-R’-dTTP, L-3’-O-R’-dGTP, or L-3’-O-R’- dCTP. In some embodiments, the L-3’-O-R’-dNTP comprises one or more of L-3’-O-R’- dATP, L-3’-O-R’-dTTP, L-3’-O-R’-dGTP, and L-3’-O-R’-dCTP. In some embodiments, the L-3’-O-R’-dNTP comprises L-3’-O-R’-dATP, L-3’-O-R’-dTTP, L-3’-O-R’-dGTP, and L-3’- O-R’-dCTP. [232] In some embodiments, the L-3’-O-R’-dNTP has Formula I as defined in 5.2.2.1.1. In some embodiments, the L-3’-O-R’-dNTP has any of the structures disclosed in 5.2.2.1.1. [233] In some embodiments, each of the L-nucleotides-Label in the kit has a unique label. In some embodiments, some of the L-nucleotides-Label in the kit has the same label. In some embodiment, the label is a fluorescent label. In some embodiments, each of the L- nucleotides-Label in the kit provides a unique fluorescent signal. 6. EXAMPLES 6.1. Example 1. Synthesis of (L)-3’-O-N3-dNTPs [234] Materials and Methods [235] Starting material beta-L-deoxy adenosine, beta-L-deoxy guanosine, beta-L-deoxy cytidine, beta-L-deoxy thymidine are available for purchase, e.g., at Chemgenes. All solvents and reagents are reagent grades, purchased commercially, and used without further purification unless specified.
6.1.1. Synthesis of (L) 3’-O-N3-dATP 6.1.1.1 Scheme 1. Synthesis of (L) 3’-O-N3-dATP
Figure imgf000055_0001
[236] To a stirred solution of the ‘starting material’ (beta-L-deoxy adenosine) (1.0 eq, 2.00 g, 7.48 mmol) is co-evaporated with anhydrous pyridine, dissolved in 15 ml of anhydrous pyridine and sealed with septum. After stirring for 5 minutes under argon, trimethylsilyl chloride (TMS-Cl, 5.0 eq, 4.06 g, 37.4 mmol) is added via syringe. Benzoyl chloride (1.2 eq, 1.26 g, 8.98 mmol) is added dropwise via syringe over a period of 20 minutes and after 30 minutes and stirring is continued for 2.5 h, while a clear yellow solution is formed. Then, 4 ml H2O is added at once and after 5 minutes, 8 ml of aqueous ammonia solution (28-30%) is added at once stirring for additional 15 minutes. The mixture is evaporated to dryness and the oily residue co-evaporated twice with toluene to give a yellow solid (compound dA-1). To a stirred mixture of compound dA-1 (1.50 g; 3.96 mmol) and imidazole (693 mg; 9.51 mmol) in anhydrous DMF (21.0 mL), tert-butyldimethylsilyl chloride (TBDMSCl) (765 mg; 4.92 mmol) is added. The reaction mixture is stirred at room temperature for 20 h. After evaporation, the residue is purified by flash column chromatography using CH3OH-CH2Cl2 (1:20) as the eluent to afford compound dA-2 as white solid. To a stirred solution of compound dA-2 (3.0 g; 6.38 mmol) in DMSO (12 ml), acetic acid (5.5 ml) and acetic anhydride (17.6 ml) are added. The reaction mixture is stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) is added and the aqueous layer is extracted with CH2Cl2 (3 x 100 ml). The combined organic extract is washed with saturated NaHCO3 solution (100 ml) and dried over Na2SO4. After concentration, the residue is purified by flash column chromatography (hexane/ethyl acetate, 1:1 to 1:4) to afford compound dA-3 as a white powder. To a stirred solution of compound dA-3 (400 mg; 0.76 mmol) in dry CH2Cl2 (7 ml) under nitrogen, cyclohexene (400 ml), and SO2Cl2 (155 ml; 1.91 mmol, redistilled) are added. The reaction mixture is stirred at 0°C for 2 h. The solvent is first removed under reduced pressure and then under a high-vacuum pump for 10 min. The residue is dissolved in dry DMF (5 ml) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture is dispersed in distilled water (50 ml) and extracted with CH2Cl2 (3 x 50 ml). The combined organic layer is dried over Na2SO4 and concentrated under reduced pressure. The residue is dissolved in MeOH (5 ml) and stirred with NH4F (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent is removed under reduced pressure. The reaction mixture is concentrated under reduced pressure and partitioned between H2O and CH2Cl2. The organic layer is separated and dried over Na2SO4. After concentration, the crude product is purified by flash column chromatography (ethyl acetate/methanol, 100:0 to 98:2) to afford compound dA-4 as a white powder. Compound dA-4 (123 mg; 0.3 mmol) and proton sponge (75.8 mg; 0.35 mmol) are dried in a vacuum desiccator over P2O5 overnight before dissolving in trimethyl phosphate (600 ml). Then freshly distilled POCl3 (40 ml; 0.35 mmol) is added dropwise at 0°C and the mixture is stirred at 0°C for 2 h. Subsequently, a well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) is added in one portion at room temperature and stirred for 30 min. Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) is then added and the mixture is stirred for 1 h at room temperature. Then concentrated NH4OH (15 ml) is added and stirred overnight at room temperature. The resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water. The crude mixture is then purified with anion exchange chromatography on DEAE-Sephadex A-25 at 4°C using a gradient of TEAB (pH 8.0; 0.1–1.0 M). The crude product is further purified by reverse- phase HPLC to afford (L) 3’-O-N3-dATP (compound dA-5). 6.1.2. Synthesis of (L) 3’-O-N3-dGTP 6.1.2.1 Scheme 2. Synthesis of (L) 3’-O-N3-dGTP
Figure imgf000057_0001
Beta-L-deoxy guanosine dG-1
Figure imgf000057_0002
dG-6 [237] To a stirred solution of the ‘starting material’ (beta-L-deoxy guanosine) (1.0 eq, 2.00 g, 7.13 mmol) is co-evaporated with anhydrous pyridine (3 × 4 mL) and then dissolved in anhydrous pyridine (2 mL). The resulting solution is protected from moisture (drying tube), purged with argon and placed on ice. To the ice cold solution, TMS-Cl (4.51 mL, 58.4 mmol, 8.2 eq.) is added dropwise via a syringe. The ice bath is then removed and the mixture is stirred for 2 hours. The solution is cooled on ice and isobutyric anhydride (0.29 mL, 15.69 mmol, 2.2 eq.) is added dropwise via a syringe and the ice bath is removed. After stirring for another 2 hours at room temperature, the reaction is placed again on ice and ice cold water (20 mL) is slowly added, followed after 15 minutes by concentrated ammonia solution (1.5 mL) to get a final 2.5 M concentration of ammonia. The mixture is kept on ice for 30 minutes, and then evaporated to dryness. The residue is co-evaporated with toluene (3 × 5 mL) to remove traces of water, resuspended in MeOH and filtered to remove the precipitate. The filtrate is then concentrated, dissolved in a small amount of MeOH, absorbed on silica gel and purified by column chromatography (DCM/MeOH 95 : 5 to 91 : 9 (v/v)) to give compound dG-1 as a yellow solid. Compound dG-1 (495 mg, 1.07 mmol) is co- evaporated three times with dry pyridine, dried under high vacuum, and dissolved in 2 cm3 dry N,N-dimethylformamide in an ice bath. Di-tert-butylsilyl bis(trifluoromethanesulfonate) (590 mg, 1.34 mmol) is added dropwise over a period of 15 min and the reaction mixture is stirred at 0oC for 30 min. Imidazole (419 mg, 6.15 mmol) is added and the mixture is stirred for 15 min at 0oC and for 15 min at room temperature. Then, 241 mg tert-butyldimethlsiliyl chloride (1.59 mmol) is added and the solution is stirred at 60 oC for another 2h. The mixture is diluted with dichloromethane, washed with brine, dried over sodium sulfate, and evaporated. The crude product is purified by column chromatography on silica gel (methanol:dichloromethane 0:100–2:98) as white foam (compound dG-2). To a stirred solution of compound dG-2 (3.0 g; 6.08 mmol) in DMSO (12 ml), acetic acid (5.5 ml) and acetic anhydride (17.6 ml) are added. The reaction mixture is stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) is added and the aqueous layer is extracted with CH2Cl2 (3 x 100 ml). The combined organic extract is washed with saturated NaHCO3 solution (100 ml) and dried over Na2SO4. After concentration, the residue is purified by flash column chromatography (hexane/ethyl acetate, 1:1 to 1:4) to afford compound dG-3 as a white powder. To a stirred solution of compound dG-3 (1.0 g; 2.0 mmol) in dry pyridine (22 ml), diphenylcarbamoyl chloride (677 mg; 2.92 mmol) and DIEA (N, N- diisopropylethylamine) (1.02 ml; 5.9 mmol) are added. The reaction mixture is stirred under nitrogen atmosphere at room temperature for 3 h. The solvent is removed under high vacuum. The crude product is purified by flash column chromatography (ethyl acetate/hexane, 1:1 to 7:3) to afford compound dG-4 as a yellowish powder. To a stirred solution of compound dG- 4 (400 mg; 0.71 mmol) in dry CH2Cl2 (7 ml) under nitrogen, cyclohexene (400 ml), and SO2Cl2 (155 ml; 1.91 mmol, redistilled) are added. The reaction mixture is stirred at 0 °C for 2 h. The solvent is first removed under reduced pressure and then under a high-vacuum pump for 10 min. The residue is dissolved in dry DMF (5 ml) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture is dispersed in distilled water (50 ml) and extracted with CH2Cl2 (3 x 50 ml). The combined organic layer is dried over Na2SO4 and concentrated under reduced pressure. The residue is dissolved in MeOH (5 ml) and stirred with NH4F (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent is removed under reduced pressure. The reaction mixture is concentrated under reduced pressure and partitioned between H2O and CH2Cl2. The organic layer is separated and dried over Na2SO4. After concentration, the crude product is purified by flash column chromatography (ethyl acetate/methanol, 100:0 to 98:2) to afford compound dG-5 as a white powder. Compound dG-5 (123 mg; 0.28 mmol) and proton sponge (75.8 mg; 0.35 mmol) are dried in a vacuum desiccator over P2O5 overnight before dissolving in trimethyl phosphate (600 ml). Then freshly distilled POCl3 (40 ml; 0.35 mmol) is added dropwise at 0°C and the mixture is stirred at 0 °C for 2 h. Subsequently, a well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) is added in one portion at room temperature and stirred for 30 min. Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) is then added and the mixture is stirred for 1 h at room temperature. Then concentrated NH4OH (15 ml) is added and stirred overnight at room temperature. The resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water. The crude mixture is then purified with anion exchange chromatography on DEAE-Sephadex A-25 at 4 °C using a gradient of TEAB (pH 8.0; 0.1– 1.0 M). The crude product is further purified by reverse-phase HPLC to afford (L) 3’-O-N3- dGTP (compound dG-6).
6.1.3. Synthesis of (L) 3’-O-N3-dCTP 6.1.3.1 Scheme 3. Synthesis of (L) 3’-O-N3-dCTP
Figure imgf000060_0001
[238] The preparation procedure for (L) 3’-O-N3-dCTP (compound dC-5) is similar to the synthesis protocol (5 steps) outlined above for (L) 3’-O-N3-dATP. Starting material for synthesis of (L) 3’-O-N3-dCTP is beta-L-deoxy cytidine (Chemgenes).
6.1.4. Synthesis of (L) 3’-O-N3-dTTP 6.1.4.1 Scheme 4. Synthesis of (L) 3’-O-N3-dTTP
Figure imgf000061_0001
Beta-L-deoxy thymidine dT-1 dT-2
Figure imgf000061_0002
- [239] To a stirred mixture of the ‘starting material’ (beta-L-deoxy thymidine) (1.3 g; 3.87 mmol) and imidazole (693 mg; 9.51 mmol) in anhydrous DMF (21.0 mL), tert- butyldimethylsilyl chloride (TBDMSCl) (765 mg; 4.92 mmol) is added. The reaction mixture is stirred at room temperature for 20 h. After evaporation, the residue is purified by flash column chromatography using CH3OH-CH2Cl2 (1:20) as the eluent to afford compound dT-1 as white solid. To a stirred solution of compound dT-1 (3.0 g; 6.38 mmol) in DMSO (12 ml), acetic acid (5.5 ml) and acetic anhydride (17.6 ml) are added. The reaction mixture is stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) is added and the aqueous layer is extracted with CH2Cl2 (3 x 100 ml). The combined organic extract is washed with saturated NaHCO3 solution (100 ml) and dried over Na2SO4. After concentration, the residue is purified by flash column chromatography (hexane/ethyl acetate, 1:1 to 1:4) to afford compound dT-2 as a white powder. To a stirred solution of compound dT-2 (400 mg; 0.76 mmol) in dry CH2Cl2 (7 ml) under nitrogen, cyclohexene (400 ml), and SO2Cl2 (155 ml; 1.91 mmol, redistilled) are added. The reaction mixture is stirred at 0 °C for 2 h. The solvent is first removed under reduced pressure and then under a high-vacuum pump for 10 min. The residue is dissolved in dry DMF (5 ml) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture is dispersed in distilled water (50 ml) and extracted with CH2Cl2 (3 x 50 ml). The combined organic layer is dried over Na2SO4 and concentrated under reduced pressure. The residue is dissolved in MeOH (5 ml) and stirred with NH4F (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent is removed under reduced pressure. The reaction mixture is concentrated under reduced pressure and partitioned between H2O and CH2Cl2. The organic layer is separated and dried over Na2SO4. After concentration, the crude product is purified by flash column chromatography (ethyl acetate/methanol, 100:0 to 98:2) to afford compound dT-3 as a white powder. Compound dT-3 (123 mg; 0.3 mmol) and proton sponge (75.8 mg; 0.35 mmol) are dried in a vacuum desiccator over P2O5 overnight before dissolving in trimethyl phosphate (600 ml). Then freshly distilled POCl3 (40 ml; 0.35 mmol) is added dropwise at 0 °C and the mixture is stirred at 0 °C for 2 h. Subsequently, a well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) is added in one portion at room temperature and stirred for 30 min. Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) is then added and the mixture is stirred for 1 h at room temperature. Then concentrated NH4OH (15 ml) is added and stirred overnight at room temperature. The resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water. The crude mixture is then purified with anion exchange chromatography on DEAE-Sephadex A-25 at 4 °C using a gradient of TEAB (pH 8.0; 0.1–1.0 M). The crude product is further purified by reverse- phase HPLC to afford (L) 3’-O-N3-dTTP (compound dT-4). 6.2. Example 2. Synthesis of (L) 3’-O-R’-dNTP [240] A person of skill in the art, having reviewed the exemplified methods provided herein would readily be able to adapt these methods to produce compounds with various R’ groups protecting the 3’O. Specifically, the allyl and the nitrobenzyl analogs of the described (L)-3’- O-N3-dNTPs are contemplated utilizing allyl bromide and 2-nitrobenzyl bromide respectively, instead of the acetic acid / acetic anyhydride step in the above reaction schemes 1-4. Specific reference is made to the methods provided in Ju J. et al. 2006, PNAS, vol. 103, No. 52 and Wu J. et al. 2007, PNAS, vol. 104, No. 42 respectively, the entire contents of each of which, including the supporting information, are incorporated herein by reference. 6.3. Example 3. Synthesis of (L) 3’-O-N3-dNTP-Label 6.3.1. Synthesis of (L) 3’-O-N3-dATP-ROX 6.3.1.1 Scheme 5. Synthesis of NH2 -(L) 3’-O-N3-dATP
Figure imgf000063_0001
6.3.1.2 Scheme 6. Synthesis of (L) 3’-O-N3-dATP-ROX
Figure imgf000064_0001
[241] Azido-ROX compound (ROX-N3-Linker). (2-{2-[3-(2-Amino-ethylcarbamoyl)- phenoxy]- 1-azido-ethoxy}-ethoxy)-acetic acid Linker-6 (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006) US Patent Appl US20060160081A1) is dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of ROX NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF(400µl) is added slowly to the above reaction mixture and then stirred at room temperature for 5 h with exclusion of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3/CH3OH, 1:4). [242] L-3’-O-N3-dATP-ROX compound (L-3’-O’-N3-dATP-ROX). To a stirred solution of ROX-N3-Linker in dry DMF (2 ml), DSC (N,N’-disuccinimidyl carbonate) (3.4mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol) is added. The reaction mixture is stirred at room temperature for 2 h. TLC indicated that ROX-N3-Linker is completely converted to compound ROX-N3-Linker NHS ester, which is directly used to couple with L-amino-dATP (13 µmol) in NaHCO3/Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture is stirred at room temperature for 3 h with exclusion of light. The reaction mixture is purified by a preparative silica gel TLC plate (CH3OH/CH2Cl2,1:1). The crude product is further purified on reverse-phase HPLC to afford L-3’-O-N3-dATP-ROX (L- 3’-O-N3-dATP-ROX).
6.3.2. Synthesis of (L) 3’-O-N3-dGTP-Cy5 6.3.2.1 Scheme 7. Synthesis of NH2 -(L) 3’-O-N3-dGTP
Figure imgf000066_0001
6.3.2.2 Scheme 8. (L) 3’-O-N3-dGTP-Cy5
Figure imgf000067_0001
[243] Azido-Cy5 compound (Cy5-N3-Linker). (2-{2-[3-(2-Amino-ethylcarbamoyl)- phenoxy]- 1-azido-ethoxy}-ethoxy)-acetic acid Linker-6 (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006) US Patent Appl US20060160081A1) is dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of Cy5 NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF(400µl) is added slowly to the above reaction mixture and then stirred at room temperature for 5 h with exclusion of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3/CH3OH, 1:4). [244] L-3’-O-N3-dGTP-Cy5 compound (L-3’-O-N3-dGTP-Cy5). To a stirred solution of Cy5-N3-Linker in dry DMF (2 ml), DSC (N,N’-disuccinimidyl carbonate) (3.4mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol) is added. The reaction mixture is stirred at room temperature for 2 h. TLC indicated that Cy5-N3-Linker is completely converted to compound Cy5-N3-Linker NHS ester, which is directly used to couple with L-amino-dGTP (13 µmol) in NaHCO3/Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture is stirred at room temperature for 3 h with exclusion of light. The reaction mixture is purified by a preparative silica gel TLC plate (CH3OH/CH2Cl2,1:1). The crude product is further purified on reverse-phase HPLC to afford L-3’-O-N3-dGTP-Cy5 (L- 3’-O-N3-dGTP-Cy5). 6.3.3. Synthesis of (L) 3’-O-N3-dCTP-Bodipy-FL-510 6.3.3.1 Scheme 9. Synthesis of NH2 -(L) 3’-O-N3-dCTP
Figure imgf000068_0001
6.3.4. Scheme 10. Synthesis of (L) 3’-O-N3-dCTP-Bodipy-FL-510
Figure imgf000069_0001
[245] Azido-Bodipy-FL-510 (Compound BODIPY-FL-510-N3-Linker). (2-{2-[3-(2-Amino- ethylcarbamoyl)-phenoxy]- 1-azido-ethoxy}-ethoxy)-acetic acid Linker-6 (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006) US Patent Appl US20060160081A1) is dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of Bodipy-FL-510 NHS (N-hydroxysuccinimide) ester (Invitrogen) (5.0 mg, 0.013 mmol) in DMF(400µl) is added slowly to the above reaction mixture and then stirred at room temperature for 5 h with exclusion of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3/CH3OH, 1:4) to afford BODIPY-FL-510-N3-Linker. [246] L-3’-O-N3-dCTP-Bodipy-FL-510 (compound L-3’-O-N3-dCTP-Bodipy-FL-510). To a stirred solution of BODIPY-FL-510-N3-Linker in dry DMF (2 ml), DSC (N,N’- disuccinimidyl carbonate) (3.4mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol) is added. The reaction mixture is stirred at room temperature for 2 h. TLC indicated that BODIPY-FL-510-N3-Linker is completely converted to compound BODIPY- FL-510-N3-Linker NHS ester, which is directly used to couple with L-amino-dCTP (13 µmol) in NaHCO3/Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture is stirred at room temperature for 3 h with exclusion of light. The reaction mixture is purified by a preparative silica gel TLC plate (CH3OH/CH2Cl2,1:1). The crude product is further purified on reverse-phase HPLC to afford L-3’-O-N3-dCTP-Bodipy-FL-510. 6.3.5. Synthesis of (L) 3’-O-N3-dUTP-R6G 6.3.5.1 Scheme 11. Synthesis of NH2 -(L) 3’-O-N3-dUTP
Figure imgf000070_0001
6.3.5.2 Scheme 12. Synthesis of (L) 3’-O-N3-dUTP-R6G
Figure imgf000071_0001
[247] Azido-R6G (Compound R6G-N3-Linker). (2-{2-[3-(2-Amino-ethylcarbamoyl)- phenoxy]- 1-azido-ethoxy}-ethoxy)-acetic acid (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006) US Patent Appl US20060160081A1) is dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of R6G NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF(400µl) is added slowly to the above reaction mixture and then stirred at room temperature for 5 h with exclusion of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3/CH3OH, 1:4). [248] L-3’-O-N3-dUTP-R6G. To a stirred solution of R6G-N3-Linker in dry DMF (2 ml), DSC (N,N’-disuccinimidyl carbonate) (3.4 mg, 13.2 µmol) and DMAP (4- dimethylaminopyridine) (1.6 mg, 13.2 µmol) is added. The reaction mixture is stirred at room temperature for 2 h. TLC indicated that R6G-N3-Linker is completely converted to compound R6G-N3-Linker NHS ester, which is directly used to couple with L-amino-dUTP (13 µmol) in NaHCO3/Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture is stirred at room temperature for 3 h with exclusion of light. The reaction mixture is purified by a preparative silica gel TLC plate (CH3OH/CH2Cl2,1:1). The crude product is further purified on reverse- phase HPLC to afford L-3’-O-N3-dUTP-R6G (L-3’-O-N3-dUTP-R6G). 6.4. Example 4. Synthesis of (L) 3’-O-allyl-dNTP-allyl-Label [249] A person of skill in the art, having reviewed the exemplified methods provided herein would readily be able to adapt the provided methods to produce compounds with various R’ groups protecting the 3’O and alternative cleavable linkers, for example incorporating the allyl-fluorophore linkers reported in Ju J. et al. 2006, PNAS, vol. 103, No. 52, in accordance with the schemes below. Specifically, the allyl analogs of the described (L)-3’-O-N3-dNTP- NH2 compounds are contemplated utilizing allyl bromide instead of the acetic acid / acetic anyhydride step in the above reaction schemes 5, 7, 9, and 11. Specific reference is made to the methods provided in Ju J. et al. 2006, PNAS, vol. 103, No. 52, the entire contents of which, including the supporting information, are incorporated herein by reference.
Figure imgf000072_0001
Figure imgf000073_0002
Figure imgf000073_0001
6.5. Example 5. Synthesis of mirror-image polymerase [250] The amino acid sequence of 9°N polymerase is divided into multiple segments. Each polypeptide segment is synthesized using a solid phase peptide synthesis method (Fmoc- SPPS) based on a strategy of using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group. Once polypeptide segments are synthesized, the polypeptide segments are separated and purified using a semi-preparative grade reversed-phase high performance liquid chromatography (RP-HPLC). The separated polypeptide segments are ligated using a natural chemical ligation method from the C-terminus to the N-terminus. After the chemical ligation reaction is completed, the target product is isolated using semi-preparative grade RP-HPLC. [251] The synthesized mirror-image polymerase is processed for folding renaturation. Using circular dichroism and mass spectrometry, it is confirmed that the mirror-image polymerase is correctly folded. 6.6. Example 6. Activity of mirror-image polymerase [252] The mirror-image polymerase is mixed with (i) a polymerase reaction buffer; (ii) L- primer; (iii) L-polynucleotide template and (iv) four kinds of 0.4 mM (L) dNTPs. The reaction mixture is placed at 37 °C. for 4 hours, and the reaction is terminated by adding 1 μl of 0.5 M EDTA. Activity of the mirror-image polymerase is measured based on yields of a polynucleotide fragment complementary to the template or using double stranded DNA intercalating dyes (e.g. EvaGreen dyes) and measuring the increase in fluorescence. 6.7. Example 7. Mirror-image polymerase chain reaction [253] The mirror-image polymerase is mixed with (i) a polymerase reaction buffer; (ii) 2.5 μM L-primer; (iii) 2.5 μM L-polynucleotide template and (iv) four kinds of 0.4 mM (L) dNTPs. The initial cycle consisted of 5 min at 95 °C., 5 min at 50 °C. (during which polymerase and BSA additions are made) and 5 min at 70 °C. The segments of each subsequent PCR cycle are the following: 1 min at 93 °C., 1 min at 50 °C. and 5 min at 70 °C. After 0, 13, 23 and 40 cycles, 20 μl amounts of 100 μl volumes are removed and subjected to agarose gel electrophoresis with ethidium bromide present to quantitate the amplification of the template sequence. The reaction yields polynucleotide fragments complementary to the template. 6.8. Example 8. A general procedure of specific single base extension, deprotection and re-initiation of extension of L-primer using (L)-3’-O-R- dNTPs, (L)-3’-O-R-dNTPs-R-Label, (L)-ddNTPs-R2-Label and -D- polymerase to demonstrate sequencing by synthesis (SBS) [254] To obtain de novo DNA sequencing data on a L-Primer/L-DNA template immobilized on a solid surface, first, verification of accurate and specific single base extension is performed using a combination mixture of solution A consisting of 3’-O-N3- dCTP (3 mM), 3’-O-N3-dTTP (3 mM), 3’-O-N3-dATP (3 mM) and 3’-O-N3-dGTP (0.5 mM) and solution B consisting of ddCTP-N3-Bodipy-FL-510 (50 nM), ddUTP-N3-R6G (100 nM), ddATP-N3-ROX (200 nM), and ddGTP-N3-Cy5 (100 nM) in each polymerase extension reaction. For example, along with the modified L-nucleotide reversible terminators, 60 pmol of the self-priming DNA template, 1X Thermopol II reaction buffer, 40 nmol of MnCl2 and 1 unit of D-polymerase is added together in a total reaction volume of 20 ml. The reaction consisted of incubations at 94 °C for 5 min, 4 °C for 5 min, and 65 °C for 20 min. Subsequently, the extension product is analyzed by fluorescence based gel electrophoresis (and additionally, MALDI-TOF MS can be used as an alternative option) to confirm specific incorporation of the correct nucleotide. For cleavage of the DNA extension product bearing the 3’-O-N3-dNTP and ddNTP-N3-fluorophores, the DNA product is resuspended in 50 ml of 100 mM TCEP solution (pH 9.0) at 65 °C for 15 min and then analyzed by either gel electrophoresis or MALDI-TOF MS to confirm cleavage of the R-label/protection group, thereby allowing re-initiation of extension of the next base. The above describes a complete single cycle of SBS (extension, label detection, and cleavage). [255] Generally, separate solutions, ‘Solution A’ consisting of four kinds of L- 3’-O-R’- dNTP (each with dATP, dTTP, dGTP or dCTP) and ‘Solution B’ consisting of four kinds of L-ddNTP-R2-Label (each with ddATP, ddTTP, ddGTP or ddCTP) are used in the polymerase extension reaction. Solution A and Solution B are mixed in a specific ratio (i.e. 7:3 v/v, 9:1 v/v) with a mirror-image polymerase, a L-primer, buffer and L-polynucleotide template and the mixture is incubated over multiple cycles of sequence by synthesis (SBS). During the SBS cycles, the mirror-image polymerase synthesizes a complementary sequence to the L- polynucleotide using the combination of 3'-capped nucleotide reversible terminators (L-3’-O- R’-dNTP) and cleavable fluorescent dideoxynucleotides (L-ddNTPs-R2-Label). Replication products terminated by ddNTPs (L-ddNTPs-R2-Label) are detected using the signal from the Label specific to ddATP, ddTTP, ddGTP or ddCTP. After detection of the signal, the 3'-OH capping group (R’ group) from the incorporated nucleotide reversible terminators (L-3’-O- R’-dNTP) and the Label from the DNA products terminated by ddNTPs (L-ddNTPs-R2- Label) are cleaved and the polymerase reaction reinitiates. Since each Label conjugated to dATP, dTTP, dGTP or dCTP is unique, the fluorescent signals from the Label indicate the nucleotide corresponding to the termination site. Based on the signals, the sequence of the L- polynucleotide template is determined. [256] The L-polynucleotide template is also sequenced using four kinds of L-3’-O-R’- dNTP-R2-Label (each with dATP, dTTP, dGTP or dCTP). The L-polynucleotide template is mixed with the four kinds of L-3’-O-R’-dNTP-R2-Label (each with dATP, dTTP, dGTP or dCTP), L-primer, D-polymerase and buffer in a similar reaction condition described above. The mixture is incubated over multiple cycles of sequence by synthesis (SBS). During the SBS step, L-3’-O-R’-dNTP-R2-Label is incorporated to the synthesized product. The L-3’-O- R’-dNTP-R2-Label includes a chemical moiety (R’) capping the 3’-OH and a Label tethered to the base through a chemically cleavable linker (R2). Following the incorporation, fluorescent signals from the Label is detected and then the 3'-capping moiety (R’) and the Label on the reaction product are cleaved to reinitiate the polymerase reaction. Since each Label conjugated to dATP, dTTP, dGTP or dCTP is unique, the fluorescent signals from the Label indicate the nucleotide corresponding to the termination site. Based on the signals, the sequence of the L-polynucleotide template is determined. 6.9. Example 9. Sequencing L-polynucleotide by a method analogous to Sanger sequencing [257] The L-polynucleotide template is sequenced (analogous to sanger sequencing) using four kinds of L-ddNTP-R2-Label (each with ddATP, ddTTP, ddGTP or ddCTP). The four kinds of L-ddNTP-R2-Label are mixed with an L-polynucleotide template, an L-primer, four kinds of L-dNTP (L-dATP, L-dGTP, L-dCTP and L-dTTP), D-polymerase and buffer in a similar reaction condition described above. In the mixture, L-ddNTP-R2-Label is added to the mixture in a much smaller amount compared to L-dNTP. PCR (cycle sequening – generation of DNA fragment ladder) is performed with the mixture. The replication product is separated by size using gel electrophoresis and signals from the L-ddNTPs-R2-Label incorporated into the replication product are detected. Since each Label conjugated to dATP, dTTP, dGTP or dCTP is unique, the fluorescent signals from the Label indicate the nucleotide corresponding to the termination site. Based on the fluorescence signals and the fragment mobility (based on size), the sequence of the L-polynucleotide template is determined. 6.10. Example 10. Completed Synthesis of (L)-3’-O-azidomethyl-dNTPs [258] Four target molecules and their synthetic routes were designed as shown in Scheme 13–16. The L-3'-O-N3-dTTP (also referred to as L-3'-O-azidomethyl-dTTP) (dT-4) and L-3'- O-N3-dCTP (dC-5) were prepared and characterized by 1H, 31P NMR and HRMS. For the synthesis of L-3'-O-N3-dATP (dA-5) and L-3'-O-N3-dGTP (dG-6), we have synthesized the intermediates dA-3 and dG-5, respectively. [259] Materials and Methods [260] All solvents and reagents were reagent grades, purchased commercially, and used without further purification unless specified. All chemicals were purchased from Sigma- Aldrich, Fisher Scientific, TCI etc. 1H NMR spectra were recorded on a Bruker AscendTM (400 MHz) spectrometer from Chapman University and reported in parts per million (ppm) from a CDCl3 (7.26 ppm) or D2O. Data were reported as follows: (s = singlet, d = doublet, t = triplet, td = triplet of doublets, q = quartet, m = multiplet, dt = doublet of triplets, dd = doublet of doublets, J = coupling constant in Hz, integration). Proton-decoupled 31P NMR spectra were recorded on a Bruker AscendTM (121.4 MHz) spectrometer from Chapman University. High-resolution mass spectra (HRMS) were obtained from School of Pharmacy, Chapman University and Analytical Chemistry Instrumentation Facility at University California of Riverside. Starting materials beta-L-deoxythymidine, beta-L-deoxycytidine, beta-L-deoxyadenosine, and beta-L-deoxyguanosine were purchased from Chemgenes. Analytical (Polaris 180A C18-A, 4.6 x 250 mm, 5 um) and semi-prep (Polaris 180A C18-A, 4.6 x 250 mm, 5 um) HPLC columns were purchased from Agilent. The 3’-O-modified nucleotides were purified with reverse-phase HPLC on a 4.6 x 250 mm C18 column (Polaris), mobile phase: A, 25 mM TEAB buffer in water; B, 25 mM TEAB buffer in acetonitrile. Elution was performed isocratic conditions as described in each procedure. 6.10.1. Synthesis of (L) 3’-O-azidomethyl-dTTP 6.10.1.1 Scheme 13. Synthesis of (L) 3’-O-N3-dTTP (dT-4)
Figure imgf000077_0001
6.10.1.1.1 Experimental procedure: [261] dT-1 synthesis: To a solution of β-L-deoxy Thymidine (1.5 g, 6.18 mmol) in anhydrous N,N-dimethylformamide (DMF) (37.5 mL) was added imidazole (633 mg, 9.30 mmol) and tert-butyldimethylsilyl chloride (1.02 g, 6.81 mmol) were added at 0 oC under nitrogen. After stirred at room temperature for 3 h, the solution was added iced clod water and extracted with EtOAc (2 x 50 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated. The resulting residue was dissolved in CH3OH and added silica gel. The solution was dried under reduced pressure until dryness. The resulting residue was purified by column chromatography (2:98 to 5:95, CH3OH–CH2Cl2) to afford dT-1 (1.8 g, 82%) as a white solid. Rf 0.35 (1:19 CH3OH–CH2Cl2). Product was confirmed by TLC. [262] dT-2 synthesis: To a stirred solution of dT-1 (1.9 g; 5,33 mmol) in DMSO (18 ml) was added acetic acid (9 ml) and acetic anhydride (27 ml) at room temperature. The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution was added at 0 oC and stirred for 30 min, and the aqueous layer was extracted with CH2Cl2 (2 x 100 ml). The combined organic layers were dried over Na2SO4, filtered and concentrated. The resulting residue was purified by flash column chromatography (1:1, Hexanes–EtOAc) to afford dT-2 as a yellowish syrup (2.0 g, 90%). Rf 0.55 (1:1 hexanes–EtOAc); 1H NMR (400 MHz, CDCl3): δ 9.36 (s, 1H, NH), 7.48 (d, 1H, 4J = 1.1 Hz, H-6), 6.31 (dd, 1H, J1’,2’a = 5.6 Hz, J1’,2’b = 8.6 Hz, H-1’), 4.68 (d, 1H, Jgem = 11.8 Hz, CH2S), 4.61 (d, 1H, Jgem = 11.8 Hz, CH2S), 4.47 (app dt, 1H, J3’,2’b = 5.9 Hz, J3’,2’a = 1.9 Hz, H-3’), 4.12–4.08 (m, 1H, H-4’), 3.89 (dd, 1H, J5’a,4’ = 2.6 Hz, Jgem =11.3 Hz, H-5’a), 3.80 (dd, 1H, J5’a,4’ = 2.9 Hz, Jgem =11.3 Hz, H-5’b), 2.41 (ddd, 1H, J2’a,3’ = 1.9 Hz, J2’a,1’ = 5.6 Hz, Jgem = 13.6 Hz, H-2’a), 2.16 (s, 3H, SCH3), 1.99 (ddd, 1H, J2’b,3’ = 5.9 Hz, J2’b,1’ = 8.6 Hz, Jgem = 13.6 Hz, H-2’b), 1.92 (d, 3H, 4J = 1.1 Hz, CH3), 0.93 (s, 9H, (CH3)3CSi), 0.12 (s, 6H, (CH3)2Si)); HRMS (ESI) m/z [M + H]+ calcd for C18H33N2O5SSi 417.1879; Found 417.1881.
Figure imgf000078_0001
[263] dT-3 synthesis: To a stirred solution of dT-2 (2.0 g, 4.80 mmol) in dry CH2Cl2 (45 mL), cyclohexene (2.1 mL) and SO2Cl2 (1.0 M in DCM) (6.48 mL, 6.48 mmol) were added. The reaction mixture was stirred at 0 °C for 3 h. The volatiles were removed under reduced pressure. The residue was dissolved in dry DMF (30 mL) and reacted with NaN3 (1.88 g, 28.8 mmol) at room temperature for 2 h. The reaction mixture was dispersed in cold distilled water (100 mL) and extracted with EtOAc (2 x 200 mL). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was dissolved in CH3CN (10 mL) and reacted with 2M HCl (2-3 mL) at 0 °C for 5 h. Saturated Na2CO3 solution was added and extracted with CH2Cl2 (2 x 50 mL), dried on Na2SO4, and concentrated. The organic layer was washed with water and the organic layer was dried on Na2SO4, and concentrated. The resulting residue was purified by flash column chromatography (hexane/ethyl acetate, 3:7 to 1:4) to afford dT-3 as a white powder. Rf 0.20 (1:4 hexanes–EtOAc); 1H NMR (400 MHz, CDCl3): δ 8.63 (s, 1H, NH), 7.38 (d, 1H, 4J = 1.1 Hz, H-6), 6.13 (app t, 1H, J1’,2’a = J1’,2’b = 7.0 Hz, H-1’), 4.77 (d, 1H, Jgem = 9.0 Hz, CH2N3), 4.70 (d, 1H, Jgem = 9.0 Hz, CH2N3), 4.50 (app dt, 1H, J3’,2’b = 6.0 Hz, J3’,2’a = 3.5 Hz, H-3’), 4.16–4.12 (m, 1H, H-4’), 3.98 (dd, 1H, J5’a,4’ = 2.7 Hz, Jgem =12.0 Hz, H-5’a), 3.84 (dd, 1H, J5’a,4’ = 2.8 Hz, Jgem = 12.0 Hz, H-5’b), 2.51–2.38 (m, 3H, H-2’a, H-2’b, 5’-OH), 1.94 (d, 3H, 4J = 1.1 Hz, CH3); HRMS (ESI) m/z [M + H]+ calcd for C11H16N5O5S 298.1151; Found 298.1144
Figure imgf000079_0001
[264] dT-4 synthesis: dT-3 (120 mg, 0.403 mmol) was dried in a vacuum desiccator over P2O5 overnight. To a solution of dT-3 in trimethyl phosphate (5 mL) was added POCl3 (94.3 uL, 1.00 mmol) dropwisely at 0 °C. The mixture was stirred at 0 °C for 2 hours and then was added a well-vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (738 uL, 3.10 mmol) in anhydrous DMF (2 mL). The mixture was stirred for 1 hour at room temperature and 0.1M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 20 ml) was then added and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 10 ml of water. The crude mixture was extracted with CH2Cl2 (2 x 10 mL) and the aqueous layerwas concentrated under reduced pressure. The residue was then purified with anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.5; 0.1–0.8 M). The fractions with products (0.3M – 0.4M) was collected and concentrated under reduced pressure. The residue was diluted with ddH2O and subjected to C18 HPLC (4% isocratic, 25 mM TEAB in acetonitrile: 25 mM TEAB in water). The product (retention time: 19 min) was collected and concentrated under reduced pressure to afford dT- 4 (14 mg, 6.5%) as syrup. Rf 0.28 (1:2 TEAB–ACN); 1H NMR (400 MHz, D2O): δ 7.61 (d, 1H, 4J = 1.0 Hz, H-6), 6.19 (dd, 1H, J1’,2’a = 5.8 Hz, J1’,2’b = 8.6 Hz, H-1’), 4.74 (d, 1H, Jgem = 8.8 Hz, CH2N3), 4.69 (d, 1H, Jgem = 8.8 Hz, CH2N3), 4.53–4.48 (m, 1H, H-3’), 4.25–4.20 (m, 1H, H-4’), 4.12–4.00 (m, 2H, H-5’a, H-5’b), 2.37 (ddd, 1H, J2’a,1’ = 5.8 Hz, J2’a,3’ = 2.0 Hz, Jgem = 14.4 Hz, H-2’a), 2.25 (ddd, 1H, J2’a,1’ = 8.8 Hz, J2’a,3’ = 5.9 Hz, Jgem = 14.4 Hz, H-2’b), 1.78 (d, 3H, 4J = 1.0 Hz, CH3); 31P NMR (121.4 MHz, D2O): δ –10.95 (bs, 1P), –11.75 (d, J = 20.0 Hz, 1P), –23.36 (bs, 1P); HRMS (ESI) m/z [M – H]– calcd for C11H17N5O14P3 – 535.9990; Found 535.9993.
Figure imgf000080_0001
[265] dC-1 synthesis: β-L-Deoxycytidine (1 g, 4.40 mmol) was co-evaporated with anhydrous pyridine (2 x 10 mL) and then dissolved in anhydrous pyridine (15 mL) under nitrogen. Trimethylsilyl chloride (TMSCl, 2.79 mL, 22.0 mmol) was slowly added and the mixture was stirred at room temperature for 1 hour, after which benzoyl chloride (2.56 mL, 22.0 mmol) was added and the solution was stirred at room temperature for another 24 h. After cooling to 0 °C, water (10 mL) was added and the mixture was stirred at 0 °C for 20 min. Then, concentrated ammonia solution (15 mL) was added and the solution was stirred for a further 1 hour while warming to rt. The solvent was evaporated under reduced pressure and the resultant crude product was purified by column chromatography (19:1, CH2Cl2– CH3OH) to afford dC-1 (520 mg, 36%) as white solid. Rf 0.35 (1:19 CH3OH–CH2Cl2). [266] dC-2 synthesis: To a solution of dC-1 (280 mg, 0.84 mmol) in anhydrous DMF (5.0 mL) with imidazole (172 mg, 2.53 mmol) and tert-butyldimethylsilyl chloride (204 mg, 1.35 mmol) at 0 °C. The solution was stirred under nitrogen at 0 °C for 3 hours. After completion of the reaction (TLC monitoring), cold water (10 mL) was added and extracted with EtOAc (3 x 20 mL). The combined organic layers were dried over Na2SO4, fitered and concentrated. The resulting residue was purified by column chromatography (19:1 to 9:1, CH2Cl2–CH3OH) afforded dC-2 (280 mg, 74%) as colorless oil. Rf 0.32 (1:19 CH3OH–CH2Cl2). [267] dC-3 synthesis. To a stirred solution of dC-2 (240 mg, 0.538 mmol) in DMSO (6 ml) was added acetic acid (3 ml) and acetic anhydride (9 ml) at room temperature. The reaction mixture was stirred at room temperature for 72 h. A saturated NaHCO3 solution was added at 0 oC and stirred for 30 min, and the aqueous layer was extracted with CH2Cl2 (2 x 30 ml). The combined organic extract was dried over Na2SO4, filtered and concentrated. The crude product was purified by flash column chromatography (1:1, hexanes–EtOAc) to afford dC-3 (163 mg, 60%) as a white powder. Rf 0.22 (1:1 hexanes–EtOAc); 1H NMR (400 MHz, CDCl3): δ 8.42 (d, 1H, J = 7.5 Hz), 7.92 (d, 2H, J = 7.5 Hz, ArH), 7.67–7.58 (m, 1H, ArH), 7.56–7.38 (m, 3H, ArH), 6.29 (app t, 1H, J1’,2’a = J1’,2’b = 6.0 Hz, H-1’), 4.69 (d, 1H, Jgem = 11.7 Hz, CH2S), 4.61 (d, 1H, Jgem = 11.7 Hz, CH2S), 4.50 (app dt, 1H, J3’,2’a = J3’,4’ = 4.0 Hz, J3’,2’b = 6.0 Hz, H-3’), 4.21–4.16 (m, 1H, H-4’), 4.00 (dd, 1H, J5’a,4’ = 3.2 Hz, Jgem =11.8 Hz, H-5’a), 3.84 (dd, 1H, J5’a,4’ = 2.6 Hz, Jgem = 11.8 Hz, H-5’b), 2.79 (ddd, 1H, J2’a,1’ = 6.0 Hz, J2’a,3’ = 4.0 Hz, Jgem = 13.7 Hz, H-2’a), 2.21–2.13 (m, 4H, H-2’b, SCH3), 0.95 (s, 9H, (CH3)3CSi); 0.15 (s, 3H, CH3Si), 0.14 (s, 3H, CH3Si); HRMS (ESI) m/z [M + H]+ calcd for C24H36N3O5SSi 506.2145; Found 506.2150. [268] dC-4 synthesis. To a stirred solution of dC-3 (220 mg, 0.67 mmol) in dry CH2Cl2 (6 mL) was added cyclohexene (200 uL) and SO2Cl2 in CH2Cl2 (1.0 M, 0.6 mL) were added. After stirred at 0°C for 1.5 hours, the volatiles were removed under reduced pressure. To a solution of the residue in dry DMF (3 mL) was added NaN3 (169 mg, 2.61 mmol) at room temperature for 3 hours. The reaction mixture was dispersed in cold distilled water (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was performed de- TBDMS reaction by 2M HCl, the reaction was over 1-2 h at room temperature. The mixture was neutralized by saturated NaHCO3 solution and diluted with EtOAc. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (1:2, Hexanes–EtOAc to 100% EtOAc) to afford dC-4 (73 mg, 56%) as a colorless oil. Rf 0.3 (1:4 hexanes–EtOAc); 1H NMR (400 MHz, CDCl3): δ 8.99 (bs, 1H, NH), 8.32 (d, 1H, J = 7.5 Hz), 7.87 (d, 2H, J = 7.5 Hz, ArH), 7.65–7.53 (m, 2H), 7.52–7.45 (m, 2H, ArH), 6.18 (app t, 1H, J1’,2’a = J1’,2’b = 6.3 Hz, H-1’), 4.79 (d, 1H, Jgem = 9.2 Hz, CH2N3), 4.68 (d, 1H, Jgem = 9.2 Hz, CH2N3), 4.52 (app dt, 1H, J3’,2’a = J3’,4’ = 3.9 Hz, J3’,2’b = 6.3 Hz, H-3’), 4.26–4.22 (m, 1H, H-4’), 4.04 (dd, 1H, J5’a,4’ = 2.8 Hz, Jgem =12.0 Hz, H-5’a), 3.89 (dd, 1H, J5’a,4’ = 2.8 Hz, Jgem = 12.0 Hz, H-5’b), 3.45 (bs, 1H, 5’-OH), 2.68 (ddd, 1H, J2’a,1’ = 6.3 Hz, J2’a,3’ = 3.9 Hz, Jgem = 13.5 Hz, H-2’a), 2.44 (app dt, 1H, J2’b,1’ = J2’b,3’ = 6.3 Hz, Jgem = 13.5 Hz, H-2’b); HRMS (ESI) m/z [M + H]+ calcd for C17H19N6O5387.1417; Found 387.1426. [269] dC-5 synthesis: dC-4 (100 mg, 0.259 mmol) was dried in a vacuum desiccator over P2O5 overnight. To a solution of dC-4 in trimethyl phosphate (5 mL) was added POCl3 (60.5 uL, 0.647 mmol) dropwisely at 0 °C. The mixture was stirred at 0 °C for 2 hours and then was added a well-vortexed mixture of tributylammonium pyrophosphate (355 mg, 0.647 mmol) and tributylamine (473.8 uL, 1.99 mmol) in anhydrous DMF (2 mL). The mixture was stirred for 1.5 hours at room temperature and 0.1M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 0.1 M, 15 ml) was then added and the mixture was stirred for 1 hour at room temperature. The mixture was then added concentrated ammonium hydroxide (10 mL) and stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 30 ml of water. The crude mixture was extracted with CH2Cl2 (2 x 20 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified with anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.5; 0.2–0.8 M). The fractions with products (0.3M – 0.4M) was collected and concentrated under reduced pressure. The residue was diluted with ddH2O and subjected to C18 HPLC (2% isocratic, 25 mM TEAB in ACN: 25 mM TEAB in water). The product (retention time: 13.2 min) was collected and concentrated under reduced pressure to afford dC-5 (18 mg, 13.4%) as syrup. Rf 0.43 (1:2 TEAB–ACN); 1H NMR (400 MHz, D2O): δ 8.00–7.88 (m, 1H), 6.23 (dd, 1H, J1’,2’a = 5.6 Hz, J1’,2’b = 7.9 Hz, H-1’), 6.18–6.06 (m, 1H) 4.85–4.74 (m, 2H, CH2N3), 4.56– 4.51 (m, 1H, H-3’), 4.35–4.29 (m, 1H, H-4’), 4.18–4.09 (m, 2H, H-5’a, H-5’b), 2.55–2.46 (m, 1H, H-2’a), 2.31–2.19 (m, 1H, H-2’b); HRMS (ESI) m/z [M – H]– calcd for C10H16N5O13P3– 520.9994; Found 520.9990. 6.10.3. Synthesis of L-3'-O-azidomethyl-dATP: 6.10.3.1 Scheme 15. Synthesis of L-3'-O- N3-dATP (dA-5)
Figure imgf000083_0001
Experimental procedure: [270] dA-1 synthesis: β-L-deoxy Adenosine (2.0 g, 7.96 mmol) was co-evaporated with anhydrous pyridine (2 times: 10 mL + 10 mL) and dissolved in anhydrous pyridine  (20 mL). The resulting solution was cooled to 0 oC and added chlorotrimethylsilane (TMSCl, 5.06 mL, 39.8 mmol) dropwisely via a syringe. The mixture was stirred for 1 hours at 0 oC. The solution was added benzoyl chloride (4.62 mL, 39.8 mmol) dropwisely via a syringe and ice bath was removed. After stirred at room temperature for 3 hours, the flask was placed on ice bath and concentrated ammonia solution (15 mL) was added. The solution was stirred for 30 min at 0 °C, and then evaporated to dryness. The residue was dissolved in CH3OH and silica gel was added. The mixture was dried under reduced pressure. The residue was purified by column chromatography (49:1 to 19:1, CH2Cl2–CH3OH) to afford dA-1 (2.12 g, 75%) as white solid. [271] dA-2 synthesis: To a solution of dA-1 (2.12 g, 5.98 mmol) in anhydrous DMF (30 mL) was added imidazole (608 mg, 8.95 mmol) and tert-butyldimethylsilyl chloride (989 mg, 6.56 mmol) at 0 oC under nitrogen. The reaction mixture was stirred for 3 hours at 0 °C. After the completion of reaction, cold water (50 mL) was added and the mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated The resulting residue was purified by column chromatography (1:2, Hexanes–EtOAc to 100% EtOAc) to afford compound dA-2 (1.92 g, 69%) as colorless oil. [272] dA-3 synthesis: To a stirred solution of dA-2 (1.8 g; 3.83 mmol) in DMSO (24 ml) was added acetic acid (12 ml) and acetic anhydride (36 ml) at room temperature. After stirred at room temperature for 72 hours. The solution was added saturated NaHCO3 solution at 0 oC and stirred for 30 min. The mixture was extracted with CH2Cl2 (2 x 100 ml). The combined organic layers wer dried over Na2SO4, filtered and concentrated. The residue was purified by flash column chromatography (1:1, Hexanes–EtOAc) to afford dA-3 (1.27 g, 63%) as a white foam. Rf 0.28 (1:7 hexanes–EtOAc); 1H NMR (400 MHz, CDCL3): δ 9.28 (bs, 1H, NH), 8.77 (s, 1H), 8.33 (s, 1H), 8.02 (d, 2H, J = 7.5 Hz, ArH), 7.62–7.55 (m, 1H, ArH), 7.53–7.47 (m, 2H, ArH), 6.51 (dd, 1H, J1’,2’a = 7.2 Hz, J1’,2’b = 6.0 Hz, H-1’), 4.74–4.64 (m, 3H, CH2S, H- 3’), 4.25–4.18 (m, 1H, H-4’), 3.89 (dd, 1H, J5’a,4’ = 4.4 Hz, Jgem =11.0 Hz, H-5’a), 3.82 (dd, 1H, J5’a,4’ = 3.3 Hz, Jgem = 11.0 Hz, H-5’b), 2.79 (ddd, 1H, J2’a,1’ = 7.2 Hz, J2’a,3’ = 6.0 Hz, Jgem = 13.7 Hz, H-2’a), 2.63 (ddd, 1H, J2’b,1’ = 6.0 Hz, J2’b,3’ = 3.0 Hz, Jgem = 13.7 Hz, H-2’b), 2.18 (s, 3H, SCH3), 0.91 (s, 9H, (CH3)3CSi); 0.10 (s, 6H, (CH3)2Si); HRMS (ESI) m/z [M + H]+ calcd for C25H36N5O4SSi 530.2157; Found 530.2132. [273] dA-4 synthesis: To a stirred solution of dA-3 (300 mg; 1.0 eq., 0.566 mmol) in dry CH2Cl2 (10 mL) add SO2Cl2 (1.0 M in DCM) (0.84 mL, 0.849 mmol, 1.5 equiv.) at – 30 oC. The reaction mixture was stirred at –30 °C for 20-30 min. After the starting material was finished (TLC), add cyclohexene (400 uL) before it is bringing to room temperature. The volatiles were removed under reduced pressure (water bath temperature <10 oC) and dried for 10 min under high pressure. The residue was dissolved in dry DMF (8 mL) and reacted with NaN3 (320 mg, 4.924 mmol, 8.7 equiv.) at room temperature for 3 h. The reaction mixture was dispersed in cold distilled water (10 mL) and extracted with EtOAc (2x10 mL). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was used for next reaction without further purification. The crude (300 mg) was dissolved in dry THF (6 mL) and reacted with 1M TBAF (0.84 mL, 0.849 mmol, 1.5 equiv.) at 0 °C for 1 h. The reaction was quenched with the addition of MeOH (5 mL) and solvent was removed under reduced pressure. The residue was suspended in water (5 mL) and extracted with CH2Cl2 (2 x 5 mL). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash column chromatography (hexane/ethyl acetate, 1:4 to 100% ethyl acetate) to afford dA-4 (203 mg, 87.5%) as a white foam Rf 0.40 (2:98 MeOH–EtOAc); 1H NMR (500 MHz, CDCl3): δ 9.09 (s, 1H), 8.79 (s, 1H), 8.08 (s, 1H), 8.03 (d, 2H, J = 7.5 Hz), 7.65 – 7.59 (m, 1H), 7.54 (t, 1H, J = 7.7 Hz), 6.34 (dd, 1H, J = 9.5, 5.5 Hz), 5.83 (d, 1H, J = 10.2 Hz), 4.78 (s, 1H), 4.68 (d, 1H, J = 5.4 Hz), 4.37 (s, 1H), 4.02 (d, 1H, J = 12.9 Hz), 3.86 – 3.78 (m, 1H), 3.16 – 3.06 (m, 1H), 2.52 (dd,1H, J = 13.7, 5.5 Hz). [274] dA-5 synthesis: dA-4 (60 mg, 0.146 mmol) with proton sponge (37.59 mg, 0.175 mmol) was dried in a vacuum desiccator over P2O5 overnight. To a solution of dC-4 in trimethyl phosphate (0.49 mL) was added POCl3 (21 uL, 0.219 mmol) dropwisely at 0 °C. The mixture was stirred at 0 °C for 1 hour and then was added a well-vortexed mixture of tributylammonium pyrophosphate (304 mg) and tributylamine (0.27 ml, 2.31 mmol) in anhydrous DMF (1.2 mL). The mixture was stirred for 20 min at room temperature and 0.1M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.0, 0.1 M, 4 ml) was then added and the mixture was stirred for 3 hours at room temperature. The mixture was then added concentrated ammonium hydroxide (15 mL) and stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 30 ml of water. The crude mixture was extracted with CH2Cl2 (2 x 20 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified by Prep HPLC (C18 column) followed by anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.0; 0.05–1.0 M). The fractions with products (0.3M – 0.4M) was collected and concentrated under reduced pressure and subjected to lyophilize using ddH2O to afford dA-5 (50 mg, 62%) as a foam type solid.1H NMR (500 MHz, D2O): δ 8.36 (s, 1H), 8.08 (s, 1H), 6.34 (m, 1H), 4.82-4.73 (m, 1H), 4.64-4.61 (m, 1H), 4.35–4.30 (m, 1H), 4.12- 3.96 (m, 1H), 2.77-2.67 (m, 1H), 2.65-2.55 (m, 1H).; 31P NMR (202 MHz, D2O): δ -9.76 (brs, 1P), -10.82 (d, 1P, J = 18.4 Hz), -22.56 (brs, 1P). HRMS (ESI) m/z [M – H]– calcd for C11H16N8O12P3– 545.0106; Found 545.0105. 6.10.4. Synthesis of L-3'-O-azidomethyl-dGTP: 6.10.4.1 Scheme 16. Synthesis of L-3’-O-N3-dGTP (dG-6)
Figure imgf000086_0001
[275] Experimental procedure: [276] dG-1 synthesis: β-L-deoxyguanosine (2 g, 7.48 mmol) was co-evaporated (2 x 12 mL), and then added pyridine (30 mL) and cool the suspension to 0 °C. Trimethylsilyl chloride (4.8 mL, 37.4 mmol) was added dropwise via syringe. The ice bath was then removed and the mixture was stirred for 1 hour. The solution was cooled to 0 °C and isobutyric anhydride (6.2 mL, 37.4 mmol) was added dropwise via a syringe. The ice bath was removed and the resulting solution was stirred at room temperature for 3 hours. After stirred for 3 hours, the mixture was cooled to 0 °C and ice cold water (5 mL) was slowly added and stirred for 15 min, followed by concentrated ammonia solution (10 mL) to a final 2.5 M concentration of ammonia. The mixture was stirred on ice bath for 1 hour and then evaporated to dryness. The residue was redissolved in CH3OH and was added silica gel and concentrated until dryness. The mixture was concentrated until dryness. The resulting residue was purified by column chromatography (19:1 to 9:1, CH2Cl2–CH3OH) to give dG-1 (1.85 g, 73%) as a white solid. [277] dG-2 synthesis: To a solution of dG-1 (1.8 g, 5.33 mmol) in anhydrous DMF (30 mL) was added imidazole (544 mg, 8.0 mmol) and tert-butyldimethylsilyl chloride (885 mg, 5.87 mmo) at 0 °C under nitrogen, and warmed to room temperature. After stirred for 8 h at room temperature, the solution was added iced cold water and extracted with EtOAc (3 x 100 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (19:1 to 9:1, CH2Cl2–CH3OH) to afford compound dG-2 (1.9 g, 79%) as a white foam. [278] dG-3 synthesis: To a stirred solution of dG-2 (1.7 g; 3.76 mmol) in DMSO (12 ml) was added acetic acid (6 ml) and acetic anhydride (18 ml). After stirred at room temperature for 48 h, the solution was added saturated NaHCO3 solution at 0 oC and stirred for 30 min, and the aqueous layer was extracted with EtOAc (2 x 100 ml). The combined organic layers were dried over Na2SO4, filtered and concentrated. The crude product was purified by flash column chromatography (1:1 to 1:3, Hexanes–EtOAc) to afford dG-3 (1.2 g; 65%) as a light yellowish foam Rf 0.26 (1:4 hexanes–EtOAc); 1H NMR (400 MHz, CDCL3): δ 12.1 (s, 1H, NH), 9.40 (s, 1H, NH), 7.98 (s, 1H), 6.18 (app t, 1H, J1’,2’a = J1’,2’b = 6.9 Hz, H-1’), 4.71–4.61 (m, 3H, CH2S, H-3’), 4.15–4.09 (m, 1H, H-4’), 3.78 (d, 2H, J = 3.9 Hz, H-5’a, H-5’b), 2.74 (sep, 1H, J = 7.0 Hz, COCH(CH3)2), 2.57–2.46 (m, 2H, H-2’a, H-2’b), 2.15 (s, 3H, SCH3), 1.25 (app t, 6H, J = 7.0 Hz, COCH(CH3)2), 0.89 (s, 9H, (CH3)3CSi); 0.08 (s, 3H, (CH3)2Si), 0.07 (s, 3H, (CH3)2Si); HRMS (ESI) m/z [M + H]+ calcd for C22H38N5O5SSi 512.2363; Found 512.2360. [279] dG-4 synthesis. To a stirred solution of dG-3 (1.20 g; 2.35 mmol) in dry pyridine (25 ml) was added diphenylcarbamoyl chloride (814 mg; 3.52 mmol) and DIPEA (N,N- diisopropylethylamine) (1.23 ml; 7.03 mmol) at room temperature. After stirred at room temperature for 3 hours, the solvent was removed under high vacuum. The residue was diluted with EtOAc and washed with 2M HCl and saturated NaHCO3. The organic layer was dried with Na2SO4, filtered and concentrated. The residue was purified by flash column chromatography (1:1 to 1:3, Hexanes–EtOAc) to afford dG-4 (1.5 g, 90%) as a foam-type yellowish powder. Rf 0.5 (1:1 hexanes–EtOAc); 1H NMR (400 MHz, CDCL3): δ 8.25 (s, 1H, NH), 8.05 (s, 1H), 7.50–7.33 (m, 8H, ArH), 7.29–7.21 (m, 2H, ArH), 6.41 (app t, 1H, J1’,2’a = J1’,2’b = 6.7 Hz, H-1’), 4.76–4.66 (m, 3H, CH2S, H-3’), 4.19–4.13 (m, 1H, H-4’), 3.88 (dd, 1H, J5’a,4’ = 4.5 Hz, Jgem = 11.1 Hz, H-5’a), 3.81 (dd, 1H, J5’b,4’ = 3.6 Hz, Jgem = 11.1 Hz, H- 5’b), 2.96 (bs, 1H, COCH(CH3)2), 2.75 (ddd, 1H, J2’b,1 = 6.7 Hz, J2’a,3’ = 7.2 Hz, Jgem = 13.6 Hz, H-2’a), 2.57 (ddd, 1H, J2’a,1 = 6.7 Hz, J2’a,3’ = 3.2 Hz, Jgem = 13.6 Hz, H-2’b), 2.18 (s, 3H, SCH3), 1.28 (d, 6H, J = 6.8 Hz, COCH(CH3)2), 0.92 (s, 9H, (CH3)3CSi); 0.11 (s, 3H, (CH3)2Si), 0.10 (s, 3H, (CH3)2Si); HRMS (ESI) m/z [M + H]+ calcd for C35H47N6O6SSi 707.3047; Found 707.3048. [280] dG-5 synthesis: To a stirred solution of dG-4 (490 mg, 0.694 mmol) in dry CH2Cl2 (14 mL) was added cyclohexene (2.1 mL) and 1.0 M SO2Cl2 in CH2Cl2 (1.39 mL, 1.39 mmol) at 0 °C. After stirred at 0 °C for 1 hour, the volatiles were removed under reduced pressure. To a solution of the residue in dry DMF (14 mL) was added NaN3 (271 mg, 4.61 mmol) at room temperature. After stirred at room temperature for 2 h, the reaction mixture was dispersed in distilled water (200 mL) and extracted with EtOAc (2 x 200 mL). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. To a solution of the residue win acetonitrile (14 mL) was added 2M HCl (7 mL) at 0 °C). The reaction mixture was stirred for 30 min. The solution was diluted with EtOAc (100 mL) and washed with saturated NaHCO3. The organic layer was washed with water (2 x 50 mL) and the organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (1:2 to 0:1, Hexanes–EtOAc) to afford dG-4 as colorless oil. Rf 0.32 (1:4 hexanes–EtOAc); 1H NMR (400 MHz, CDCL3): δ 8.38 (s, 1H, NH), 8.08 (s, 1H), 7.46–7.39 (m, 4H, ArH), 7.37– 7.32 (m, 4H, ArH), 7.27–7.20 (m, 2H, ArH), 6.22 (app t, 1H, J1’,2’a = J1’,2’b = 6.6 Hz, H-1’), 5.04–4.98 (m, 1H, H-3’), 4.77 (s, 2H, CH2N3), 4.19–4.14 (m, 1H, H-4’), 3.88 (dd, 1H, J5’a,4’ = 2.9 Hz, Jgem = 12.6 Hz, H-5’a), 3.80 (dd, 1H, J5’b,4’ = 2.9 Hz, Jgem = 12.6 Hz, H-5’b), 2.99 (app dt, 1H, J2’b,1 = J2’a,3’ = 6.6 Hz, Jgem = 13.7 Hz, H-2’a), 2.79–2.66 (m, 1H, COCH(CH3)2), 2.48 (ddd, 1H, J2’a,1 = 6.6 Hz, J2’a,3’ = 3.8 Hz, Jgem = 13.7 Hz, H-2’b); HRMS (ESI) m/z [M + H]+ calcd for C28H30N9O6588.2219; Found 588.2177. [281] dG-6 synthesis: dG-5 (40 mg, 0.146 mmol) with proton sponge (37.59 mg, 0.175 mmol) was dried in a vacuum desiccator over P2O5 overnight. To a solution of dG-5 in trimethyl phosphate (0.49 mL) was added POCl3 (21 uL, 0.219 mmol) dropwisely at 0 °C. The mixture was stirred at 0 °C for 1 hour and then was added a well-vortexed mixture of tributylammonium pyrophosphate (304 mg) and tributylamine (0.27 ml, 2.31 mmol) in anhydrous DMF (1.2 mL). The mixture was stirred for 20 min at room temperature and 0.1M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.0, 0.1 M, 4 ml) was then added and the mixture was stirred for 3 hours at room temperature. The mixture was then added concentrated ammonium hydroxide (15 mL) and stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 30 ml of water. The crude mixture was extracted with CH2Cl2 (2 x 20 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified by Prep HPLC (C18 column) followed by anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.0; 0.05–1.0 M). The fractions with products (0.3M – 0.4M) was collected and concentrated under reduced pressure and subjected to lyophilize using ddH2O to afford dG-6 (2.7 mg, 4.5%) as a foam type solid.1H NMR (400 MHz, D2O): δ 8.09 (s, 1H), 6.21 (m, 1H, H-1’), 4.83 (d, 1H, J = 11.5 Hz, CH2N3), 4.77 (d, 1H, J = 11.5 Hz, CH2N3), 4.64–4.61 (m, 1H), 4.37–4.31 (m, 1H), 4.16–4.03 (m, 2H, H-5’a, H-5’b), 2.81–2.72 (m, 1H, H-2’a), 2.61-2.50 (m, 1H, H-2’b); 31P NMR (121.4 MHz, D2O): δ –10.8 (bs, 1P), -11.5 (d, 1P, J = 18.4 Hz), -23.2 (bs, 1P). HRMS (ESI) m/z calcd for [M] C11H17N8O13P3562.0128; Found [M – H]– 561.0072. 6.11. Example 11. Synthesis of (L) 3'-O-azidomethyl-dNTP-Label intermediates [282] The synthesis of L-3'-O-azidomethy-dNTP-FLs was designed and split into three two parts, synthesis of linker (Scheme 17), synthesis of the NH2-L-3’-O-N3-dNTP intermediates (Schemes 18-23) and then coupling of the intermediate with the fluorophore, as described in Example 3, above. [283] Currently, the synthesis of linker (Scheme 17) afforded compound 6, referred to as intermediate Linker-6, which was characterized by 1H NMR and HRMS. The iFluor488 was attached to Linker-6, which gave Linker-7 for the coupling reaction of the NH2-L-3’-O-N3- dCTP intermediate. The first NH2-L-3’-O-N3-dNTP we focused on is the synthesis of NH2-L- 3’-O-N3-dUTP (Scheme 18). Currently, the synthesis of NH2-L-3’-O-N3-dUTP was completed to the step 3 and the intermediate “dUTP-FL-3” was obtained. The synthetic plans of other L-3'-O-azidomethy-dNTP-FL intermediates are also attached below (Schemes 19-21). [284] In view of the compatibility of fluorophore attached dNTP with 9°N DNA polymerase, we also synthesized NH2-(L)3'-O-N3-7-deaza-dGTP (Scheme 26) and NH2-(L) 3'-O-N3-7-deaza-dATP (Scheme 23). The starting materials of nucleotide bases (6-chloro-7- deazaguanine and 6-chloro-7-deaza-7-iodopurine) and 2-deoxy sugar (1-chloro-3,5-di-O-p- toluoyl-2-L-deoxyribofuranose) are commercially available. deaza-dATP-FL-8 and deaza- dGTP-FL-9 were synthesized and characterized by NMR and HRMS. 6.11.1. Scheme 17. Synthesis of Linker
Figure imgf000090_0001
[285] Synthesis of Linker-1
Figure imgf000091_0002
[286] To a solution of ethyl-3-hydroxybenzoate (3.32 g, 20 mmol) in anhydrous DMF (8 ML) was added potassium carbonate (5.53 g, 40 mmol), sodium iodide (1.2 g, 0.4 mmol) and 2-bromomethyl-1,3-dioxolane (8.3 mL, 80 mmol) at room temperature. The mixture was heated to 120 °C and stirred overnight. The mixture was warmed to room temperature and evaporated under reduced pressure. The residue was diluted with CH2Cl2 (250 mL) and washed with water. The aqueous layer was washed with CH2Cl2 twice (2x50 mL). The combined organic layer was dried with MgSO4, filtered and concentrated. The residue was purified by column chromatography (hexanes–EtOAc, 19:1 to 2:1) to obtain Linker-1 (4.6 g, 91%). Rf 0.54 (100% CH2Cl2); 1H NMR (400 MHz, CDCL3): δ 7.67 (dt, 1H, J = 1.1, 7.1 Hz, ArH), 7.61 (dd, 1H, J = 1.5, 2.6 Hz, ArH), 7.35 (t, 1H, J = 7.8 Hz, ArH), 7.15 (ddd, 1H, J = 1.0, 2.7, 8.6 Hz, ArH), 5.32 (t, 1H, J = 4.0 Hz, CH), 4.38 (q, 2H, J = 7.2 Hz, OCH2CH3), 4.12–3.96 (m, 6H, OCH2CH2O, ArOCH2), 1.40 (t, 3H, Jgem = 7.2 Hz, OCH2CH3). [287] Synthesis of Linker-2
Figure imgf000091_0001
[288] To a solution of Linker-1 (2.70 g, 10.7 mmol) in azidotrimethylsilane (1.55 mL, 11.8 mmol) was added tin (IV) chloride (80 uL) at room temperature. The mixture was stirred at room temperature for 2 hours, then 2% aqueous methanol (10 mL) was added. The mixture was stirred at room temperature for 30 min, then concentrated under reduced pressure. The residue was co-evaporated with EtOH (2x30 mL) and the resulting residue was purified by column chromatography (hexanes–EtOAc, 2:1 to 1:1) to obtain Linker-2 (1.42 g, 45%). Rf 0.32 (2:1 hexanes–EtOAc); 1H NMR (400 MHz, CDCl3): δ 7.70 (dt, 1H, J = 1.1, 7.6 Hz, ArH), 7.61 (dd, 1H, J = 1.4, 2.5 Hz, ArH), 7.38 (t, 1H, J = 7.8 Hz, ArH), 7.15 (ddd, 1H, J = 0.9, 2.5, 8.1 Hz, ArH), 4.90 (t, 1H, J = 5.3 Hz, CHN3), 4.40 (q, 2H, J = 7.2 Hz, OCH2CH3), 4.25 (dd, 1H, J = 5.3, 10.2 Hz, ArOCH2), 4.17 (dd, 1H, J = 5.3, 10.2 Hz, ArOCH2), 4.06– 4.00 (m, 1H, OCH2), 3.91–3.73 (m, 3H, OCH2CH2OH), 1.40 (t, 3H, Jgem = 7.2 Hz, OCH2CH3); HRMS (ESI) m/z [M – H]– calcd for C15H18N3O7352.1236; Found 352.1165. [289] Synthesis of Linker-3
Figure imgf000092_0001
[290] To a solution of Linker-2 (1.42 g, 4.8 mmol) in EtOH (5 mL) was added 4N NaOH (5 mL) at room temperature. The mixture was stirred at room temperature for 3 hours, then the mixture was concentrated under reduced pressure and acidified by 2N HCl (20 mL) and extracted with CH2Cl2 twice (2 x 50 mL). The combined organic layer was dried over MgSO4 and filtered and concentrated to obtain Linker-3 (1.21 g, 94%).Rf 0.41 (19:1 EtOAc– CH3OH); 1H NMR (400 MHz, CDCL3): δ 7.78 (dt, 1H, J = 1.1, 7.6 Hz, ArH), 7.67 (dd, 1H, J = 1.4, 2.5 Hz, ArH), 7.43 (t, 1H, J = 7.8 Hz, ArH), 7.21 (ddd, 1H, J = 0.9, 2.5, 8.1 Hz, ArH), 4.92 (t, 1H, J = 5.2 Hz, CHN3), 4.26 (dd, 1H, J = 5.2, 10.1 Hz, ArOCH2), 4.17 (dd, 1H, J = 5.2, 10.1 Hz, ArOCH2), 4.08–4.01 (m, 1H, OCH2), 3.89–3.77 (m, 3H, OCH2CH2OH); HRMS (ESI) m/z [M – H]– calcd for C11H12N3O5266,0867; Found 266.0794. [291] Synthesis of Linker-4
Figure imgf000092_0002
[292] To a solution of Linker-3 (320 mg, 1.57 mmol) in THF (5 mL) was added 60% NaH (188 mg, 4.71 mmol) at 0 °C. The mixture was stirred at 0 °C for 10 min, then mixture was added ethyl 2-bromoacetate (382 µL, 3.45 mmol). The mixture was warmed to room temperature and stirred for 4 hours. After stirring 4 hours, cold water (50 mL) was poured into reaction mixture and the resulting mixture was extracted with CH2Cl2 (50 mL). The organic layer was discarded and the aqueous layer was acidified by addition of 2N HCl (25 mL). The resulting mixture was extracted with CH2Cl2 (2 x 50 mL) and the collecting organic layers were dried over Na2SO4 and filtered and concentrated. The resulting residue was purified by column chromatography (98:2, CH2Cl2–CH3OH) to obtain the Linker-4 (120 mg, 22%) as light yellowish oil. Rf 0.67 (1:1:0.5 EtOAc–Hexanes–AcOH); 1H NMR (400 MHz, CDCl3): δ 7.75 (app dt, 1H, J = 1.1, 7.5 Hz, ArH), 7.64 (dd, 1H, J = 1.4, 2.5 Hz, ArH), 7.39 (t, 1H, J = 7.8 Hz, ArH), 7.19 (ddd, 1H, J = 0.8, 2.6, 8.2 Hz, ArH), 4.96 (app t, 1H, J = 5.0 Hz, CHN3), 4.28–4.12 (m, 6H, ArOCH2, OCH2CH3, OCH2C=O), 4.05 (app dt, 1H, J = 4.1, 11.4 Hz, OCH2), 3.97–3.87 (m, 1H, OCH2), 3.82 (appt, 2H, J = 4.8 Hz, OCH2), 1.29 (t, 3H, J = 7.2 Hz, OCH2CH3) HRMS (ESI) m/z [M – H]– calcd for C15H18N3O7352.1236; Found 352.1165. [293] Synthesis of Linker-5
Figure imgf000093_0001
[294] To a solution of Linker-4 (120 mg, 0.339 mmol) in DMF (1 mL) was added DSC (104 mg, 0.407 mmol) and DMAP (49.8 mg, 0.407 mmol) at room temperature. The mixture was stirred at room temperature for 10 min, then mixture was added N-(2-aminoethyl)-2,2,2- trifluoroacetamide (78.5 mg, 0.407 mmol) and DIPEA (142 µL, 0.815 mmol) at room temperature. The mixture was stirred at room temperature for overnight, and quenched with 1N Na2HPO4 solution (25 mL). The mixture was extracted with CH2Cl2 until no product were observed in aqueous layer. The combined organic layers were dried over Na2SO4 and filtered and concentrated. The resulting residue was purified by column chromatography (1:1, Hexanes–EtOAc) to obtain Linker-5 (42 mg, 25%) as colorless oil. Rf 0.62 (1:1:0.5 EtOAc– Hexanes–AcOH); 1H NMR (400 MHz, CDCl3): δ 8.25 (bs, 1H, NH), 7.42–7.30 (m, 4H, ArH, NH), 7.07 (ddd, 1H, J = 0.9, 2.5, 8.0 Hz, ArH), 4.90 (app t, 1H, J = 4.9 Hz, CHN3), 4.25–4.07 (m, 6H, ArOCH2, OCH2CH3, OCH2C=O), 4.05–3.99 (m, 1H, OCH2), 3.89–3.82 (m, 1H, OCH2), 3.79 (appt, 2H, J = 4.2 Hz, OCH2), 3.70–3.62 (m, 2H, NCH2CH2N), 3.61–3.54 (m, 2H, NCH2CH2N), 1.28 (t, 3H, J = 7.2 Hz, OCH2CH3); HRMS: [M]: C19H24F3N5O7, calc. 491.1646; Found [M – H]– 490.1574. [295] Synthesis of Linker-6
Figure imgf000094_0001
[296] To a solution of Linker-5 (42 mg, 0.113 mmol) in EtOH (2 mL) was added 4N NaOH (2mL) at room temperature. The mixture was stirred at room temperature for 2 hours, then the mixture was evaporated under reduced pressure and the residue was re-dissolved in 15 mL water. The mixture was extracted with CH2Cl2 (2x15mL). The aqueous layer was acidified by addition of 2N HCl to pH 2. Then the solution was extracted with CH2Cl2 (3x15mL). The aqueous layer was neutralized by addition of 1N NaOH to pH 8, and then evaporated to dryness under reduced pressure. The white solid was triturated with CH2Cl2/CH3OH (1:1, 2x15 mL). The solids were filtered and the filtrated solution was evaporated to the dryness. The resulting gum was added 10% CH3OH in CH2Cl2 (15 mL) and the white solid was filtered off. The filtered solution was evaporated to dryness to give Linker-6 (32 mg, 83%) as colorless oil; 1H NMR (400 MHz, CDCl3): δ 8.25 (bs, 1H, NH), 7.41–7.32 (m, 2H, ArH), 7.31–7.27 (m, 1H, ArH), 7.17–7.10 (m, 1H, ArH), 5.02 (app t, 1H, J = 4.4 Hz, CHN3), 4.19 (d, 2H, J = 4.4 Hz, ArOCH2), 4.01–3.95 (m, 1H, OCH2), 3.87–3.78 (m, 3H, OCH2C=O, OCH2), 3.67–3.63 (m, 2H, OCH2), 3.60 (app t, 2H, J = 6.0 Hz, NCH2CH2N), 3.01 (app t, 2H, J = 6.0 Hz, NCH2CH2N); HRMS: M: C15H20N3NaO6, calc. 389.1312; Found [M + H]+ 390.1386. [297] Synthesis of Linker-7
Figure imgf000094_0002
Figure imgf000095_0002
[298] Linker-7 was (1.1 mg,0.0030 mmol) was dissolved in DMF (50ul) and 1 M NaHCO3 aqueous solution (15 ul). A solution of fluorophore (1.98 mg, 0.0021 mmol) in DMF (60ul) was added slowly to the reaction mixture and then was stirred at room temperature for 5 h with exclusion of light. The crude product was purified on a preparative HPLC. (HPLC gradient: A, 100% 0.1 M TEAB; B 100% MeCN: 0-2 min, 5% B (flow 2-10 ml/min); 2-19 min, 5-45% B (flow 10 ml/min); 19-21 min, 45-95% B (flow 10 ml/min); 21-24 min, 95% B (flow 10 ml/min); 24-26 min, 95-5% B (flow 10 compound with retention time of 17.85 min was obtained as ml/min); 26-30 min, 5% B (flow 10-2 ml/min). The title compound with retention time of 17.85 min was obtained as water). HRMS (ESI): m/z 993.2051 [M – Na]–. 6.11.2. Scheme 18. Synthesis of NH2-(L)-3’-O-azidomethyl-dUTP
Figure imgf000095_0001
Experimental procedure: [299] Synthesis of dUTP-FL-1: Suspended Beta-L-deoxyuridine (2.4 g, 10.51 mmol, 1.0 eq.) in methanol (30 mL) and stirred for 10 min. Later added Iodine (8.0 g, 31.55 mmol, 3 eq) and Silver nitrate (5.3 g, 31.55 mmol, 3eq) and stirred the reaction mixture for 3hr at 40°C. After the completion of the reaction, the reaction mixture was filtered and the filtrate was subjected to column chromatography on silica gel using MeOH/CH2Cl21:19 to 1:9 to obtain compound dUTP-FL-1 as a white solid (1.8g, 48%). Rf 0.65 (9:1 DCM–MeOH), 1HNMR (500 MHz, DMSO-d6) δ 11.63 (s, 1H), 8.36 (s, 1H), 6.05 (t, J = 6.5 Hz, 1H), 4.24 – 4.12 (m, 2H), 3.79 – 3.69 (m, 2H), 3.61 – 3.46 (m, 2H), 2.15 – 1.96 (m, 2H).
Figure imgf000096_0001
[300] Synthesis of dUTP-FL-2: Compound 1 (400 g, 1.12 mmol, 1.0 eq.) was solubilized in anhydrous DMF (20 mL). To it, imidazole (114.3 mg, 1.68 mmol, 1.5 eq.) and tert- butyldimethylsilyl chloride (185.6 mg, 1.23 mmol, 1.1 eq.) were added at 0 oC under nitrogen. The reaction mixture was stirred for 12 h at room temperature. Then add cold water and extracted with EtOAc (3 x 50 mL). The combined organic layers were dried on anhydrous Na2SO4, concentrated, and the resulting residue was subjected to column chromatography using 2-5% MeOH in CH2Cl2 as eluent to afford compound dUTP-FL-2 as a foam type white solid (348 mg, 63%). Rf 0.45 (19:1 DCM–MeOH)1HNMR (500 MHz, ) δ 8.38 (s, 1H), 8.10 (s, J = 4.0 Hz, 1H), 6.29 (dd, J = 8.1, 5.6 Hz, 1H), 4.47 (dd, J = 5.4, 3.1 Hz, 1H), 4.08 (q, J = 2.3 Hz, 1H), 3.86 (ddd, J = 40.5, 11.4, 2.5 Hz, 2H), 2.41 (ddd, J = 13.4, 5.6, 2.1 Hz, 1H), 2.09 (ddd, J = 13.6, 8.1, 5.7 Hz, 2H), 1.98 (d, J = 3.5 Hz, 1H), 0.92 (s, J = 2.9 Hz, 9H), 0.15(s, 3H) – 0.13 (s, 3H).
Figure imgf000096_0002
[301] Synthesis of dUTP-FL-3: To a stirred solution of 2 (108 mg; 0.23mmol) in DMSO (8 ml), acetic acid (4 ml) and acetic anhydride (12 ml) were added. The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (50 ml) was added at 0 oC and stirred for 30 min, and the aqueous layer was extracted with EtOAc (3 x 100 ml). The combined organic extract was dried over Na2SO4 and concentrated. The crude product was purified by flash column chromatography (ethyl acetate/hexane, 1:1 to 7:3) to afford dUTP- FL-3 as a foam-type white solid (106 mg, 87%)). Rf 0.85 (1:1 Hexane–EtOAc) 1HNMR (500 MHz, ) δ 8.86 (s), 8.09 (s), 7.27 (s), 6.23 (dd, J = 8.3, 5.5 Hz), 5.42 – 5.25 (m), 4.36 (d, J = 5.9 Hz), 4.17 (d, J = 2.1 Hz), 3.87 (ddd, J = 56.2, 11.5, 2.3 Hz), 2.54 (ddd, J = 13.6, 5.5, 1.7 Hz), 2.12 (s), 2.04 (ddd, J = 14.0, 8.3, 5.9 Hz), 1.36 – 1.21 (m, 1H), 0.95 (s, 9H), 0.17(s, 3H), 0.16(s, 3H).
Figure imgf000097_0001
Figure imgf000098_0001
[302] Experimental procedure:
Figure imgf000098_0002
[303] Synthesis of dUTP-FL-1: To a solution of beta-L-deoxy uridine (1 g, 4.38 mmol) in H2O (10 mL) was added sodium azide (854 mg, 13.14 mmol) and N-iodosuccinimide (1.48 g, 6. 57 mmol) then stirred at room temperature for 19 hours. After complete reaction, the reaction was filtered out and washed with water. The remaining filtrate was extracted with DCM x2 and the combined organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (1:10 to 1:3, CH3OH:CH2Cl2) to give dUTL-FL-1 as white solid (1.27 g, 80%). 1HNMR (400 MHz, CD3OD) δ 8.54 (s, 1H), 6.24 (app t, J = 6.5 Hz, 1H), 4.42 (app dt, J =3.4 Hz, J =6.1 Hz, 1H), 3.97–3.93 (m, 1H), 3.84 (dd, J = 12.0 Hz, J = 3.0 Hz, 1H), 3.75 (dd, J = 12 Hz, J = 3.3 Hz, 1H), 2.37–2.18 (m, 2H). HRMS (ESI): m/z [M] calcd for C9H11IN2O5353.9717; Found [M – H]– 352.9644.
Figure imgf000099_0001
[304] Synthesis of dUTP-FL-2: To a solution of dUTP-FL-1 (60 mg, 0.17 mmol) in N,N- dimethylformamide (3 mL) was added CuI (6.5 mg, 0.34 mmol) and triethylamine (50 uL) and then stirred at room temperature for few minutes for CuI to be activated. Then, 2,2,2- trifluoro-N-(prop-2-ynyl) acetamide (76.8 mg, 0.51 mmole) and Tetrakis(triphenylphosphine)palladium (0) (19.6 mg, 0.0017 mmol) were added. The reaction was run for 24 h at the room temperature and was concentrated. The resulting residue was purified by column chromatography (1:10:10 to 1:4:4, CH3OH: CH2Cl2: EtOAc) to give dUTP-FL-2 as yellow oil (32 mg, 50%).1HNMR (400 MHz, CD3OD ) δ 8.34 (s, 1H), 6.25 (app t, J = 6.5 Hz, 1H), 4.6 (s, 1H), 4.43-4.39 (m, 1H), 4.29 (s, 2H), 4.42 (app dt, J = 7.0 Hz, J = 7.1 Hz, 1H), 3.97–3.94 (m, 1H), 3.82 (dd, J = 12.0 Hz, J = 3.0 Hz, 1H), 3.75 (dd, J = 12 Hz, J = 3.5 Hz, 1H); HRMS (ESI): m/z [M] calcd for C14H14F3N3O6377.0825; Found [M + H]+ 378.0893.
Figure imgf000099_0002
[305] Synthesis of dUTP-FL-3: To a solution of dUTP-FL-2 (590 mg, 1.57 mmol) in N,N-dimethylformamide (10 mL) was added imidazole (160.32 mg, 2.35 mmol) and tert- butyldimethyl chloride (260.29 mg, 1.73 mmol) at 0 °C and then warmed to room temperature. After stirred at room temperature for 10 hours, the solution was added iced water and extracted with ethyl acetate x 2. The combined organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (1:3 to 1:1, Hexanes:EtOAc) to give dUTL-FL-3 as yellow oil (210 mg, 43%). 1HNMR (400 MHz, CDCl3) δ 8.69 (s,1H), 8.11 (s, 1H), 6.19–6.24 (m, 1H), 4.64 (app dt, J =11.9 Hz, J =14.6 Hz, 1H), 4.47 (d, 1H), 4.16–4.05 (m, 1H), 3.92 (dd, J = 2.5 Hz, J = 11.4 Hz, 1H), 3.80 (dd, J = 2.4 Hz, J = 11.5 Hz, 1H), 2.15 (s, 3H), 0.95 (s, 9H), 0.17 (d, 6H); HRMS (ESI): m/z [M] calcd for C20H28F3N3O6Si 491.1699; Found [M + H]+ 492.1778.
Figure imgf000100_0001
[306] Synthesis of dUTP-FL-4: To a solution of dUTP-FL-3 (164 mg, 0.46 mmol) in DMSO (1 mL) was added AcOH (200 uL) and acetic anhydride (200 uL) at room temperature. After stirred at room temperature for 24 hours, the solution was added iced water and solid sodium bicarbonate. The water layer was extracted with ethyl acetate x 2. The combined organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (3:1 to 1:1, Hexanes:EtOAc) to give dUTP- FL-4 as colorless oil (70 mg, 27%). 1HNMR (400 MHz, CDCl3) δ 9.35(s,1H), 7.96 (s, 1H), 7.49 (s, 1H), 6.10 (dd, J = 5.7 Hz, 1H), 4.33–3.32 (m, 1H), 4.24–4.13 (m, 2H), 4.02 (d, 1H), 3.83 (dd, J = 2.4 Hz, J = 11.5 Hz, 1H), 3.75 (dd, J = 2.0 Hz, J = 11.4 Hz, 1H), 2.38–2.33 (m, 1H), 2.01 (s, 3H), 0.78 (s, 9H), 0.01 (d, 6H); HRMS (ESI): m/z [M] calcd for C22H32F3N3O6SSi 551.1733; Found [M + H]+ 552.1825.
Figure imgf000100_0002
[307] Synthesis of dUTP-FL-5: To a solution of dUTP-FL-4 (50 mg, 0.63 mmol) in dry CH2Cl2 was added cyclohexene (30 uL, 0.3 mmol) at 4 °C, then sulfurylchoride (1M in CH2Cl2, 0.11 ml, 0.11 mmol) was added dropwise under N2. After 40 min TLC indicated the full consumption of dUTP-FL-4, the solvent was evaporated and the residue was subjected to high vacuum for 20 min. It was then redissolved in dry DMF (1 ml) and treated with NaN3 (30 mg, 0.45 mmol). The resulting suspension was stirred under room temperature for 2 h. The reaction was quenched with CH2Cl2 and the organic layers were washed with sat aq. NaCl solution. After removing the solvent, the resulting yellow gum was redissolved in 2M HCl:Acetonitrile (2:1) and was stirred at room temperature for 30 min. The solvent was removed and the reaction worked up with CH2Cl2 and sat. aq. NaHCO3 solution. The aqueous layer was extracted three times with CH2Cl2. Purification by chromatography on silica (EtOAc:Heptane 1:1 to 100% EtOAc) gave dUTP-FL-5 as a pale yellow foam. 1HNMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 6.22 (dd, J = 6.2, 7.4 Hz, 1H, H-1’), 4.83–4.76 (m, 2H, CH2N3), 4.62 (bs, 1H), 4.47–4.42 (m, 1H), 4.29 (s, 2H, CH2N), 4.14–4.09 (m, 1H), 3.84 (dd, J = 3.3 Hz, J = 12.0 Hz, 1H, H-5’a), 3.77 (dd, J = 3.3 Hz, J = 12.0 Hz, 1H, H-5’b), 2.50 (ddd, 1H, J = 3.0, 6.1, 13.8 Hz, H-2’a), 2.35–2.25 (m, 1H, H-2’b); HRMS (ESI): m/z [M] calcd for C15H15F3N6O6 432.1005; Found [M + Na]+ 455.0887. [308] Synthesis of dUTP-FL-6: The dUTP-FL-6 (174 mg, 0.402 mmol) and Proton sponge (103 mg, 0.483 mmol) was dried over weekend. A solution of dUTP-FL-6 (174 mg, 0.402 rnmol) and proton sponge (103 mg, 0.483 rnmol) in trimethylphosphate (1 mL) was stirred for 30 min. To a solution of the mixture was added phosphoryl oxychloride (56.4 uL, 0.604 mmol) directly at 0 °C. The mixture was stirred at 0 °C for two hours. Then, the mixture was added a well-vortexed mixture of tributylammonium pyrophosphate (834 mg) and tributylamine (834 uL, 3.10 mmol) in anhydrous DMF (2 mL) at room temperature. The mixture was stirred for 1 hour at room temperature. To the mixture was added triethylammonium bicarbonate buffer (TEAB buffer, 20 mL, 0.1 M, pH 8.5) and stirred for 1h at room temperature. Ammonium solution was added and stirred for overnight at room temperature. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 30 ml of water. The crude mixture was extracted with CH2Cl2 (2 x 20 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified by Prep HPLC (C18 column) followed by anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.0; 0.05–1.0 M). The fractions with products (0.3M – 0.4M) was collected and concentrated under reduced pressure and subjected to lyophilize using ddH2O to afford dUTP-FL-6 (27 mg, 21%) as a foam type solid. 1HNMR (400 MHz, D2O) δ 8.32 (s, 1H), 6.20 (t, J = 6.6 Hz, 1H, H-1’), 4.81–4.73 (m, 2H, CH2N3), 4.57–4.52 (m, 1H), 4.34–4.29 (m, 1H), 4.24–4.08 (m, 2H, H-5’a, H-5’b), 3.91(s, 2H, CH2N), 2.52 (ddd, 1H, J = 3.0, 6.1, 13.8 Hz, H-2’a), 2.38–2.25 (m, 1H, H-2’b); 31P NMR (121.4 MHz, D2O) δ –10.8 (bs, 1P), –11.7 (d, 1P, J = 19.2 Hz), –23.1 (bs, 1P); HRMS (ESI): m/z [M] calcd for C13H19N6O14P3 576.0172; Found [M – Na]– 575.0108. 6.11.4. Scheme 19. Synthesis of NH2-L-3’-O-azidomethyl-dCTP
Figure imgf000102_0001
6.11.5. Scheme 19b. Synthesis of NH2-L-3’-O-azidomethyl-dCTP
Figure imgf000103_0001
[309] Experimental procedure: [310] Synthesis of dCTP-FL-1: To a dried round bottom flask β-L-deoxycytidine (1g, 4.40 mmol), iodine (1.67g, 60 mmol) and mCPBA (0.75g, 4.40 mmol) were dissolved in 15 mL DMF. The reaction was stirred for 2h at room temperature and afterwards evaporated to dryness under reduced pressure. The resultant crude product was purified by column chromatography (1:9, DCM-MeOH) to afford dCTP-FL-1 (0.83g, yield 53%) as an orange solid.1H NMR (500 MHz, CD3OD) δ 8.49 (s, 1H), 6.17 (t, J = 6.3 Hz, 1H), 4.36 (dt, J = 6.3, 4.1 Hz, 1H), 3.92 (dd, J = 6.9, 3.4 Hz, 1H), 3.80 (dt, J = 15.6, 4.9 Hz, 1H), 3.71 (dd, J = 12.0, 3.4 Hz, 1H), 2.34 (ddd, J = 13.6, 6.2, 4.2 Hz, 1H), 2.20–2.07 (m, 1H). HRMS (ESI): m/z [M] calcd for C9H12IN3O4352.9900; Found [M + Na]+ 375.9765. [311] Synthesis of dCTP-FL-2: To a light protected round bottom flask dCTP-FL-1 (650 mg, 1.84 mmol) was added and dissolved in DMF (10 ml). Later, to the above mixture CuI (70 mg, 0.36 mmol), triethylamine (0.5 ml), 2,2,2- trifluoro-N-prop-2-ynyl-acetamide (833.7 mg, 5.52 mmol) and at last Pd (PPh3)4 (212.5 mg, 0.184 mmol) were added and stirred under argon atmosphere for 18 h at room temperature. A bicarbonate resin (100 mg) was added, and the mixture was stirred for a further 1 h. The reaction mixture was filtered through the celite, and filtrate was evaporated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (CH2Cl2: EtOAc: MeOH 4.5:4.5:1,) to afford the desired product as beige solid (490 mg, yield 71%). 1H NMR (500 MHz, D2O) δ 8.04 (s, 1H), 6.08 (t, J = 6.5 Hz, 1H), 4.37 – 4.23 (m, 1H), 4.23 (s, 2H), 3.94 (d, J = 4.3 Hz, 1H), 3.73 (dd, J = 12.6, 3.0 Hz, 1H), 3.64 (dd, J = 12.3, 5.0 Hz, 1H), 2.39–2.28 (m, 1H), 2.19–2.13 (m, 1H). HRMS (ESI): m/z [M] calcd for C14H15F3N4O5376.1000; Found [M + H]+ 377.1066. [312] Synthesis of dCTP-FL-3: To a solution of dCTP-FL-2 (300 mg, 0.797 mmol) in anhydrous DMF (5.0 mL) was added with imidazole (82.12 mg, 1.195 mmol) and tert- butyldimethylsilyl chloride (144.14 mg, 0.956 mmol) at 0 °C. The solution was stirred under nitrogen at 0 °C for 2 h. After completion of the reaction (TLC monitoring), cold water (10 mL) was added and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by column chromatography (100%EtOAc to EtOAc:MeOH 9.5:0.5) to afford dCTP-FL-3 (210 mg, 54%) as beige solid. 1H NMR (500 MHz, DMSO-d6) δ 9.92 (t, J = 5.3 Hz, 1H), 7.97 (s, 1H), 7.83 (s, 1H), 6.87 (d, J = 22.9 Hz, 1H), 6.11 (dd, J = 7.2, 6.1 Hz, 1H), 5.27 (d, J = 4.1 Hz, 1H), 4.24 (d, J = 5.3 Hz, 2H), 4.17 (td, J = 6.0, 3.1 Hz, 1H), 3.88 (q, J = 2.8 Hz, 1H), 3.81 (dd, J = 11.5, 2.6 Hz, 1H), 3.72 (dd, J = 11.6, 3.1 Hz, 1H), 2.19–2.17 (m, 1H), 1.96–1.88 (m, 1H), 0.86 (s, 9H), 0.07 (s, 6H). HRMS (ESI): m/z [M] calcd for C20H29F3N4O5Si 490.1900; Found [M + Na]+ 513.1751. [313] Synthesis of dCTP-FL-4: To a stirred solution of dCTP-FL-3 (210 mg, 0.428 mmol) in DMSO (6 ml) was added acetic acid (2 ml) and acetic anhydride (2 ml) and stirred at room temperature for 48h. After completion of the reaction, a saturated NaHCO3 solution was added at 0 °C and stirred for 30 min, and the aqueous layer was extracted with EtOAc (3 x 30 ml). The combined organic extract was dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography (1:1, hexanes–EtOAc) to afford dCTP-FL-4 (130 mg, 52%) as a colorless oil.1H NMR (500 MHz, CDCl3) δ 8.32 (s, 1H), 8.14 (s, 1H), 6.15 (t, J = 6.4 Hz,1H), 4.62 (dd, J = 32.9, 11.7 Hz, 2H), 4.42 (dt, J = 5.7, 2.8 Hz, 1H), 4.37 (t, J = 4.2 Hz, 2H), 4.18 (dd, J = 4.8, 2.3 Hz, 1H), 3.94 (dd, J = 11.5, 2.6 Hz, 1H), 3.79 (dd, J = 11.5, 2.2 Hz, 1H), 2.66 (ddd, J = 13.7, 5.9, 2.9 Hz, 1H), 2.57 (s, 3H), 2.13 (s, 3H), 2.02 (s, 1H), 0.88 (s, 9H), 0.10 (s, 6H). HRMS (ESI): m/z [M] calcd for C24H35F3N4O6SSi 592.2000; Found [M + H]+ 593.2071. [314] Synthesis of dCTP-FL-5: To a stirred solution of dCTP-FL-4 (130 mg, 0.67 mmol) in anhydrous CH2Cl2 (6 mL) was added cyclohexene (1 mL) and SO2Cl2 in CH2Cl2 (1.0 M, 0.4 mL) were added. After stirring at 0 °C for 1 h, the volatiles were removed under reduced pressure. To the residue dry DMF (3 mL) and NaN3 (169 mg, 2.61 mmol) were added and stirred at room temperature for 6 h. The reaction mixture was dispersed in cold distilled water (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. To the resulting residue acetonitrile (10 mL) and 2M HCl (3 drops) were added, and the reaction was stirred for 1-2 h at room temperature. The mixture was neutralized by saturated NaHCO3 solution and diluted with EtOAc. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (Hexanes–EtOAc (1:1) to 100% EtOAc to EtOAc-MeOH (19:1) to afford dCTP-FL-5 (25mg, 28%) as an orange oil. 1H NMR (500 MHz, DMSO-d6) δ 9.89 (t, J = 4.8 Hz, 1H), 8.06 (s, 1H), 7.80 (s, 1H), 6.84 (s, 1H), 6.00 (dd, J = 7.3, 6.0 Hz, 1H), 5.11 (s, 1H), 4.76 (s, 2H), 4.25 (dt, J = 6.0, 3.0 Hz, 1H), 4.20 (d, J = 5.1 Hz, 2H), 3.93–3.88 (m, 1H), 3.59–3.47 (m, 1H), 2.32–2.23 (m,1H), 2.14–2.01 (m, 1H). HRMS (ESI): m/z [M] calcd for C15H16F3N7O5431.1200; Found [M + H]+ 432.1237.
6.11.6. Scheme 20. Synthesis of NH2-L-3’-O-N3-dATP
Figure imgf000106_0001
6.11.7. Scheme 20a. Alternative Synthesis of NH2-L-3’-O-N3-dATP
Figure imgf000107_0001
6.11.8. Scheme 21. Synthesis of NH2-L-3’-O-N3-dGTP
Figure imgf000108_0001
6.11.9. Scheme 21a. Alternative Synthesis of NH2-L-3’-O-N3-dGTP
Figure imgf000109_0001
Figure imgf000110_0001
6.12.1.1 Experimental procedure [315] To a solution of 4-chloro-5-iodo-7H-pyrrolo[2.3-d]pyrimidine (1.0 g, 3.58 mmol) in CH3CH (60 mL), powdered KOH (85%, 0.5 g, 7.57 mmol) and TDA-1 (0.075 mL, 0.24 mmol) were added at room temperature. After stirring for 10 min, halogenose (1.7 g, 4.37 mmol) was introduced and the stirring was continued for another 10 min. Insoluble material was filtered off and washed several times with hot acetone. The combined filtrates were evaporated to dryness. The residue was applied onto flash chromatography (silical gel, column 5 x 15 cm, elution with pertroleum ether–EtOAc, 4:1). The combined fractions containing the product were evaporated to yiled 4-chloro-7-[2-deoxy-3,5-di-O-(4- methylbenzoyl-β-L-erythro-pentofuranosyl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidine as a colorless solid (2.02 g, 89%). A solution of intermediate (1.8 g, 2.85 mmol) in NH3/CH3OH (saturated at 0 °C, 145 mL) was stirred at room temperature for 24 hours and the solvent was evaporated. Flash chromatography (silica gel, column 4 x 16 cm, CH2Cl2/CH3OH, 9:1) yielded compound 7-deaza-dA-2 as colorless solid (0.45 g, 40%. Rf 0.45 (CH2Cl2/CH3OH, 9:1). To a solution of 7deaza-dA-2 (236 mg, 0.6298 mmol) in N,N-dimethylformamide (5 mL) was added CuI (24 mg, 0.1260 mmol) and triethylamine (250 uL) and then stirred at room temperature for few minutes. Then, 2,2,2-trifluoro-N-(prop-2-ynyl) acetamide (285 mg, 1.89 mmol) and Tetrakis(triphenylphosphine)palladium(0) (72.78 mg, 0.06 mmol) were added. The reaction was run for 24 h at the room temperature and was concentrated. The resulting residue was purified by column chromatography (1:10:10 to 1:4:4, CH3OH:CH2Cl2:EtOAc) to give 7-deaza-dA-3 as yellow solid (164.2 mg, 65.3%). To a stirred mixture of 7-deaza-dA-3 (1.00 g, 2.83 mmol) and imidazole (462 mg, 6.79 mmol) in anhydrous DMF (14.0 mL), tert-butyldimethylsilyl chloride (TBDPSCl) (510 mg, 3.28 mmol) was added. The reaction mixture was stirred at room temperature for 20 h. After evaporation, the residue was purified by flash column chromatography (CH2Cl2–CH3OH, 20:1) to afford 7-deaza-dA-4 as white solid (1.18 g, 89%). A solution of the 7-deaza-dA-4 (128 mg, 0.2492 mmol) was dissolved in a mixture of CH3OH:N,N-dimethylacetal (10:1) and stirred at 40 °C. The reaction monitored by TLC, was completed after 2 h. The solvent was removed under vacuum. Purification by chromatography on silica (EtOAc: CH3OH=15:1 to 10:1) gave 7-deaza-dA-5 as a clear yellow oil (99.5mg, 70.2%). To a stirred solution of compound 7-deaza-dA-5 in DMSO, acetic acid and acetic anhydride are added. The reaction mixture is stirred at room temperature for 48 h. A saturated NaHCO3 solution is added and the aqueous layer is extracted with CH2Cl2. The combined organic extract is washed with saturated NaHCO3 solution and dried over Na2SO4. After concentration, the residue is purified by flash column chromatography (hexane/ethyl acetate, 1:1 to 1:4) to afford compound 7-deaza-dA-6. To a stirred solution of compound 7-deaza-dA-6 in dry CH2Cl2 under nitrogen, cyclohexene, and SO2Cl2 are added. The reaction mixture is stirred at 0°C for 2 h. The solvent is first removed under reduced pressure and then under a high-vacuum pump for 10 min. The residue is dissolved in dry DMF and reacted with NaN3 at room temperature for 3 h. The reaction mixture is dispersed in distilled water (50 ml) and extracted with CH2Cl2 (3 x 50 ml). The combined organic layer is dried over Na2SO4 and concentrated under reduced pressure. The residue is dissolved in MeOH and stirred with NH4F at room temperature for 24 h. The solvent is removed under reduced pressure. The reaction mixture is concentrated under reduced pressure and partitioned between H2O and CH2Cl2. The organic layer is separated and dried over Na2SO4. After concentration, the crude product is purified by flash column chromatography (ethyl acetate/methanol, 100:0 to 98:2) to afford compound 7-deaza-dA-7. Compound 7-deaza-dA-7 and proton sponge are dried in a vacuum desiccator over P2O5 overnight before dissolving in trimethyl phosphate. Then freshly distilled POCl3 is added dropwise at 0°C and the mixture is stirred at 0°C for 2 h. Subsequently, a well- vortexed mixture of tributylammonium pyrophosphate and tributylamine in anhydrous DMF is added in one portion at room temperature and stirred for 30 min. Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0) is then added and the mixture is stirred for 1 h at room temperature. Then concentrated NH4OH is added and stirred overnight at room temperature. The resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water. The crude mixture is then purified with anion exchange chromatography on DEAE-Sephadex A-25 at 4°C using a gradient of TEAB (pH 8.0; 0.1–1.0 M). The crude product is further purified by reverse-phase HPLC to afford (L) 3’-O-N3-7-deaza-dATP (compound 7-deaza-dA-8). 6.12.2. Scheme 23a. Alternative Synthesis of NH2 -(L) 3’-O-azidomethyl-7- deaza-dATP
Figure imgf000112_0001
Figure imgf000113_0001
6.12.3. Scheme 23b. Alternative Synthesis of NH2 -(L) 3’-O-azidomethyl- 7-deaza-dATP N
Figure imgf000113_0002
Figure imgf000114_0001
[316] Synthesis of 7dN-dATP-FL-2: To a solution of 4-chloro-5-iodo-7H-pyrrolo [2.3-d] pyrimidine (4.0 g, 14.4 mmol) in MeCN (200ml), powdered KOH (1.8g, 7.57 mmol) and TDA-1 (0.3 ml, 0.86 mmol) were added at room temperature. After stirring for 30 min, 1-Cl- 2-deoxysugar (8.6 g, 18.7mmol) was introduced and the stirring was continued for another 30 min. Insoluble material was filtered off and washed several times with hot acetone. The combined filtrates were evaporated to dryness. The residue was applied onto purification by chromatography on silica (EtOAc:Hexanes 1:1 to 100% EtOAc) to give intermediate as a yellow solid (4.22 g, 89%). A suspension of intermediate (1.2 g, 1.89 mmol) in a mixture of 28% aq. NH3/dioxane (1:1, 100 ml) was stirred for 17 h in a reflux condition. The residue was applied onto purification by chromatography on silica (EtOAc:Hexanes:CH3OH 1:1:0.1) to give 7-dN-dATP-FL-2 as a yellow solid (296mg, 41.4%). HRMS (ESI): m/z [M] calcd for C11H13IN4O3376.0039; Found [M + H]+ 377.0113.
Figure imgf000114_0002
[317] Synthesis of 7dN-dATP-FL-3: To a solution of 7-dN-dATP-FL-2 (236 mg,0.6298 mmol) in N,N-dimethylformamide (5 mL) was added CuI (24mg, 0.1260 mmol) and triethylamine (250uL) and then stirred at room temperature for few minutes. Then, 2,2,2- trifluoro-N-(prop-2-ynyl) acetamide (285mg, 1.89 mmole) and Tetrakis(triphenylphosphine)palladium(0) (72.78mg, 0.06mmol) were added. The reaction was run for 24 h at the room temperature and was concentrated. The resulting residue was purified by column chromatography (1:10:10 to 1:4:4, CH3OH:CH2Cl2:EtOAc) to give 7-dN- dATP-FL-3 as yellow solid (164.2 mg, 65.3%).1HNMR (400 MHz, MeOH-d6) δ 10.10 (s, 1H), 8.12 (s, 1H), 7.76 (s, 1H), 6.53-6.46 (m, 1H), 5.26 (d, 1H), 5.08 (t, J = 5.6 Hz, 1H), 4.35 – 4.26 (m, 2H), 3.84 – 3.81(m, 1H), 2.59(dd, J = 4.8Hz, J = 0.8Hz, 1H), 2.49-2.43 (m, 1H), 2.21-2.16 (m, 1H); HRMS (ESI): m/z [M] calcd for C16H16F3N5O4399.1154; Found [M+ H]+ 400.1234
Figure imgf000115_0001
[318] Synthesis of 7dN-dATP-FL-4: To a solution of 7dN-dATP-FL-3 (156 mg, 0.3907 mmol) in N,N-dimethylformamide (4 mL) was added imidazole (39.8 mg,0.586mmol) and tert -Butyl(chloro)diphenylsilane (118.12 mg,3.25 mmol) at 0 °C and then was warmed to room temperature. After stirred at room temperature for 10 hours, the solution was added to iced water and extracted with ethyl acetate x 2. The combined organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (1:2 to 100%, Hexanes:EtOAc) to give 7dN-dATP-FL-4 as yellow oil (128mg, 63.8%). 1HNMR (400 MHz, MeOH-d6) δ 8.11 (s, 1H), 7.66 (d, 4H), 7.51 (s, 1H), 7.42-7.33 (m, 6H), 6.56 (t, J= 6.5 Hz, 1H), 4.63-4.59 (m, 1H), 4.28 (s, 2H), 4.01-4.00 (m, 1H), 3.90 (dd, J = 11.5 Hz, J = 3.15 Hz, 1H), 3.81 (dd, J =11.4 Hz, J = 3.82 Hz, 1H), 2.57- 2.38 (m, 2H), 1.05 (s, 9H); HRMS (ESI): m/z [M] calcd for C32H34F3N5O4Si 637.2362; Found [M + H]+ 638.2438
Figure imgf000116_0001
[319] Synthesis of 7dN-dATP-FL-5: A solution of the 7dN-dATP-FL-4 (128mg 0.2492mmol) was dissolved in a mixture of CH3OH:N,N-dimethy- lacetal (10:1) and stirred at 40° C. The reaction monitored by TLC, was completed after 2 h. The solvent was removed under vacuum. Purification by chromatography on silica (EtOAc: CH3OH=15:1 to 10:1) gave 7dN-dATP-FL-4 as a clear yellow oil (99.5mg, 70.2%). 1H NMR (400 MHz, CDCl3) δ 8.77 (s, 1H CH=N), 8.42 (s, 1H), 7.70–7.60 (m, 4H, ArH), 7.54 (s, 1H), 7.48–7.32 (m, 6H, ArH), 6.90 (bs, 1H, CH2NH), 6.67 (t, 1H, J= 6.5 Hz, H-1’), 4.69-4.62 (m, 1H), 4.41–4.29 (m, 2H, CH2NH), 4.05-3.98 (m, 1H), 3.91–3.80 (m, 2H, H-5’a, H-5’b), 3.20 (s, 3H, (CH3)2N), 3.18 (s, 3H, (CH3)2N), 2.56-2.39 (m, 2H, H-2’a, H-2’b), 1.07 (s, 9H, tBu of TBDPS); HRMS (ESI): m/z [M] calcd for C35H39F3N6O4Si 692.2788; Found [M + H]+ 693.2865.
Figure imgf000116_0002
[320] Synthesis of 7dN-dATP-FL-6: To a solution of 7dN-dATP-FL-5 (99.5mg, 0.175mmol) in DMSO 4 mL) was added AcOH (800ul) and acetic anhydride (800 ul) at room temperature. After stirred at room temperature for 18 hours, the solution was added iced cold water and solid sodium bicarbonate. The water layer was extracted with ethyl acetate x2. The combined organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (Hexanes: EtOAc =3:1 to 1:1,) to give 7dN- dATP-FL-6 (48mg, 40%) as white solid.1H NMR (400 MHz, CDCl3) δ 9.57 (bs, 1H), 8.30 (s, 1H), 7.67 (s, 1H), 7.60–7.50 (m, 4H), 7.42-7.33 (m, 6H), 6.56 (t, J= 6.5 Hz, 1H), 4.65- 4.62 (m, 1H), 4.61–4.54 (m, 2H, CH2N), 4.28–4.23 (m, 2H, CH2S), 4.15-4.10 (m, 1H), 3.92– 385 (m, 1H, H-5’a), 3.83–3.73 (m, 1H, H-5’b), 2.68–2.57 (m, 1H, H-2’a), 2.51-2.38 (m, 1H, H-2’b), 2.10 (s, 3H, SCH3), 1.05 (s, 9H, tBu of TBDPS); HRMS (ESI): m/z [M] calcd for C35H39F3N6O4Si 752.2821; Found [M + H]+ 753.2897.
Figure imgf000117_0001
[321] Synthesis of 7dN-dATP-FL-7: To a solution of 7dN-dATP-FL-6 (20 mg, 0.032 mmol) in dry CH2Cl2 was added cyclohexene (0.03ml, 0.16 mmol) at 4°C., then sulfurylchoride (1M in CH2Cl2, 0.03 ml, 0.031mmol) was added drop wise under N2. After 40 min TLC indicated the full consumption of the compound 6, the solvent was evaporated and the residue was subjected to high vacuum for 20 min. It was then redissolved in dry DMF (1 ml) and treated with NaN3 (30 mg, 0.45 mmol). The resulting suspension was stirred under room temperature for 2 h. The reaction was quenched with CH2Cl2 and the organic layers were washed with sat aq. NaCl solution. After removing the solvent, the resulting yellow gum was redissolved in 2M HCl:Acetonitrile (2:1) and was stirred at room temperature for 30 min. The solvent was removed and the reaction worked up with CH2Cl2 and sat. aq. NaHCO3 solution. The aqueous layer was extracted three times with CH2Cl2. Purification by chromatography on silica (EtOAc:Heptane 1:1 to 100% EtOAc) gave 7dN- dATP-FL-7 as a pale yellow foam (15 mg, 73%). 1HNMR (400 MHz, MeOH-d6 ) δ 8.86 (s), 8.09 (s), 6.44 (app dt, J =2.5 Hz, J =6.0 Hz, 1H) 4.86-4.80 (m, 2H), 4.57-4.54 (m, 1H),4.19- 4.16 (m, 1H), 3.81 (dd, J=3.6 Hz, 12 Hz, 1H), 3.75 (dd, J = 12.1 Hz, J = 3.3 Hz, 1H), 2.73- 2.66 (m, 1H), 2.53 (dd, J=2.3 Hz, J = 5.7 Hz, 1H), 2.49 (dd, J=2.31 Hz, J = 5.9 Hz, 1H); HRMS (ESI): m/z [M] calcd for C17H17F3N8O4 454.1341; Found [M + H]+ 455.1415.
Figure imgf000118_0001
[322] Synthesis of 7dN-dATP-FL-8: 7-dN-dATP-FL-7 (71 mg, 0.156 mmol) was dried in a vacuum desiccator over P2O5 overnight. To a solution of 7dN-dATP-FL-7 in trimethyl phosphate (700 µl) was added POCl3 (22.4 µl, 0.22 mmol) dropwisely at 0 °C. The mixture was stirred at 0 °C for 2 hours and then was added a well-vortexed mixture of tributylammonium pyrophosphate (343.1 mg) and tributylamine (286 µl) in anhydrous DMF (1 mL). The mixture was stirred for 1 hour at room temperature and 0.1M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 20 ml) was then added and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 10 ml of water. The crude mixture was extracted with CH2Cl2 (2 x 10 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified with anion exchange chromatography on DEAE-Sephadex A- 25 using a gradient of TEAB (pH 8.5; 0.1–0.8 M). The fractions with products (0.3M – 0.4M) was collected and concentrated under reduced pressure. The residue was resuspended in H2O (5 mL) and lyophilized to achieve 7dN-dATP-FL-8 (22 mg, 23.7%). 1H NMR (400 MHz, D2O) δ 7.88 (s, 1H), 7.77 (bs, 1H), 6.46 (app t, 1H, J = 7.6 Hz, H-1’), 4.88–4.77 (m, 2H, CH2N3), 4.63–4.57 (m, 1H), 4.36–4.28 (m, 1H), 4.14–4.01 (m, 2H, H-5’a, H-5’b), 3.97 (s, 2H, CH2NH2), 2.60–2.43 (m, 2H, H-2’a, H-2’b); 31P NMR (121.4 MHz, D2O) δ –10.6 (d, 1P, J = 18.9 Hz), –11.3 (d, 1P, J = 18.9 Hz), –23.1 (t, 1P, J = 18.9 Hz); HRMS (ESI): m/z [M] calcd for C17H17F3N8O4598.0492; Found [M – H]– 597.0460.
6.12.4. Scheme 25. Synthesis of (L) 3’-O-N3-7-deaza-dATP-ROX
Figure imgf000119_0001
[323] Azido-ROX compound (ROX-N3-Linker). (2-{2-[3-(2-Amino-ethylcarbamoyl)- phenoxy]- 1-azido-ethoxy}-ethoxy)-acetic acid Linker-6 (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006) US Patent Appl US20060160081A1) is dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of ROX NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF(400µl) is added slowly to the above reaction mixture and then stirred at room temperature for 5 h with exclusion of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3/CH3OH, 1:4). [324] L-3’-O-N3-7-deaza-dATP-ROX compound (L-3’-O-N3-7-deaza-dATP-ROX). To a stirred solution of ROX-N3-Linker in dry DMF (2 ml), DSC (N,N’-disuccinimidyl carbonate) (3.4mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol) is added. The reaction mixture is stirred at room temperature for 2 h. TLC indicated that ROX-N3- Linker is completely converted to compound ROX-N3-Linker NHS ester, which is directly used to couple with L-amino-7-deaza-dATP (13 µmol) in NaHCO3/Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture is stirred at room temperature for 3 h with exclusion of light. The reaction mixture is purified by a preparative silica gel TLC plate (CH3OH/CH2Cl2,1:1). The crude product is further purified on reverse-phase HPLC to afford L-3’-O-N3-dATP-ROX (L-3’-O-N3-dATP-ROX). 6.12.5. Scheme 26. Synthesis of NH2 -(L) 3’-O-azidomethyl-7-deaza-dGTP
Figure imgf000120_0001
6.12.5.1 Experimental procedure [325] To a suspension of powdered KOH (85%, 1.15 g, 17.42 mmol) and TDA-1 (0.2 mL, 0.63 mmol) in CH3CN (60 mL), 2-amino-4-chloro-7H-pyrrolo[2.3-d]pyrimidine (842 mg, 4.99 mmol) was added at room temperature. After stirring for 5 min, halogenose (2.53 g, 6.51 mmol) was introduced within 15 min, and the stirring was continued for 30 min. Insoluble material was filtered off and washed several times with CH3CN. The combined filtrates were evaporated to dryness. The residue was applied onto flash chromatography (silical gel, column 6 x 12 cm, elution with CH2Cl2). The combined fractions containing the product were evaporated to yield a colorless solid (2.21 g, 85%). A solution of intermediate (1.04 g, 2.00 mmol) in 0.5M NaOCH3/CH3OH (60 mL) was stirred under reflux for 3 hours. The mixture was neutralized with AcOH and evaporated. The residue was applied onto flash chromatography (silica gel, CH2Cl2/CH3OH, 95:5) yielded compound 7-deaza-dG-1 as colorless solid (400 mg, 71%). To a stirred solution of the 7-deoxy-dG-1 is co-evaporated with anhydrous pyridine (3 × 4 mL) and then dissolved in anhydrous pyridine (2 mL). The resulting solution is protected from moisture (drying tube), purged with argon and placed on ice. To the ice cold solution, TMS-Cl (4.51 mL, 58.4 mmol, 8.2 eq.) is added dropwise via a syringe. The ice bath is then removed and the mixture is stirred for 2 hours. The solution is cooled on ice and isobutyric chloride (0.29 mL, 15.69 mmol, 2.2 eq.) is added dropwise via a syringe and the ice bath is removed. After stirring for another 2 hours at room temperature, the reaction is placed again on ice and ice cold water (20 mL) is slowly added, followed after 15 minutes by concentrated ammonia solution (1.5 mL) to get a final 2.5 M concentration of ammonia. The mixture is kept on ice for 30 minutes, and then evaporated to dryness. The residue is co-evaporated with toluene (3 × 5 mL) to remove traces of water, resuspended in MeOH and filtered to remove the precipitate. The filtrate is then concentrated, dissolved in a small amount of MeOH, absorbed on silica gel and purified by column chromatography (DCM/MeOH 95 : 5 to 91 : 9 (v/v)) to give compound 7-deaza-dG-2. Compound 7-deaza-dG- 2 is co-evaporated three times with dry pyridine, dried under high vacuum, and dissolved in 2 cm3 dry N,N-dimethylformamide in an ice bath. Imidazole (419 mg, 6.15 mmol) is added and the mixture is stirred for 15 min at 0 °C and for 15 min at room temperature. Then, 241 mg tert-butyldimethlsiliyl chloride (1.59 mmol) is added and the solution is stirred at 60 °C for another 2h. The mixture is diluted with dichloromethane, washed with brine, dried over sodium sulfate, and evaporated. The crude product is purified by column chromatography on silica gel (methanol:dichloromethane 0:100–2:98) as white foam (compound 7-deaza-dG-3). To a vigorously stirred solution of 7-deaza-dG-3 in anhydrous DMF was added NIS. The reaction mixture was stirred at room temperature for 22 h, and then most solvent was removed under vacuum. Diethyl ether and saturated NaHCO3 were added. The organic layer was washed with saturated NaCl, and dried over Na2SO4. After evaporation, the residue was purified by flash column chromatography to afford 7-deaza-dG-4. To a stirred solution of compound 7-deaza-dG-4 in DMSO (12 ml), acetic acid (5.5 ml) and acetic anhydride (17.6 ml) are added. The reaction mixture is stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) is added and the aqueous layer is extracted with CH2Cl2 (3 x 100 ml). The combined organic extract is washed with saturated NaHCO3 solution (100 ml) and dried over Na2SO4. After concentration, the residue is purified by flash column chromatography to afford compound 7-deaza-dG-5. To a stirred solution of compound 7- deaza-dG-5 in dry CH2Cl2 under nitrogen, cyclohexene, and SO2Cl2 are added. The reaction mixture is stirred at 0 °C for 2 h. The solvent is first removed under reduced pressure and then under a high-vacuum pump for 10 min. The residue is dissolved in dry DMF and reacted with NaN3 at room temperature for 3 h. The reaction mixture is dispersed in distilled water (50 ml) and extracted with CH2Cl2 (3 x 50 ml). The combined organic layer is dried over Na2SO4 and concentrated under reduced pressure. The residue is purified by flash column chromatography (ethyl acetate/methanol, 100:0 to 98:2) to afford compound 7-deaza-dG-6. To a solution of 7-deaza-dG-6 in anhydrous DMF, tetrakis(triphenylphosphine)palladium(0) and CuI were added. The reaction mixture was stirred at room temperature for 10 min. Then N-propargyltrifluoroacetamide and Et3N were added into the above reaction mixture. The reaction was stirred at room temperature for 1.5 h with exclusion of iar and light. After evaporation, the residue was dissolved in ethyl acetate. The mixture was washed with saturated aqueous NaHCO3, NaCl and dried over anhydrous Na2SO4. After evaporation, the residue was purified by flash column chromatography to7-deaza-dG-7. To a stirred solution of 7-deaza-dG-7 in anhydrous CH3CN were added NaI and chlorotrimethylsilane. The reaction was stirred at room temperature for 1 h and then at 50 °C for 12 h. The solvent was evaporated, and the residue was dissolved in THF. Tetrabutylammonium fluoride (TBAF) in THF solution was added, and the reaction was stirred at room temperature for 1h. The solvent was evaporated, and the residue was dissolved in EtOAc. The solution was washed with saturated aqueous NaCl and drived over anhydrous Na2SO4. After evaporation of the solvent, the residue was purified by flash column chromatography (EtOAc–CH3OH) to afford 7- deaza-dG-8. Compound 7-deaza-dG-8 and proton sponge are dried in a vacuum desiccator over P2O5 overnight before dissolving in trimethyl phosphate. Then freshly distilled POCl3 is added dropwise at 0°C and the mixture is stirred at 0°C for 2 h. Subsequently, a well- vortexed mixture of tributylammonium pyrophosphate and tributylamine in anhydrous DMF is added in one portion at room temperature and stirred for 30 min. Triethyl ammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0) is then added and the mixture is stirred for 1 h at room temperature. Then concentrated NH4OH is added and stirred overnight at room temperature. The resulting mixture is concentrated under vacuum and the residue is diluted with 5 ml of water. The crude mixture is then purified with anion exchange chromatography on DEAE-Sephadex A-25 at 4°C using a gradient of TEAB (pH 8.0; 0.1–1.0 M). The crude product is further purified by reverse-phase HPLC to afford (L) 3’-O-N3-7-deaza-dATP (compound 7-deaza-dG-9). 6.12.6. Scheme 26a. Alternative Synthesis of NH2-(L) 3’-O-azidomethyl-7- deaza-dGTP
Figure imgf000123_0001
Figure imgf000124_0001
[327] Synthesis of 7dN-dGTP-FL-1: To a suspension of KOH (0.68 g, 8.71 mmol) and TDA-1 (0.1 mL, 0.32 mmol) in CH3CN (30 mL), 2-amino-4-chloro-7H-Pyrrolo[2,3- d]pyrimidine (421 mg, 2.50 mmol) was added at room temperature. After stirring for 5 min, 1-Cl sugar (1.27 g, 3.25 mmol) was added, and continue stirring for 30 min. The insoluble material was filtered off, the precipitate was washed with CH3CN, and the filtrate was evaporated to dryness. The residue was purified by column chromatography (Hexanes– EtOAc, 4:1 to 1:1) to obtain product (1.10 g, 82%) as white foam. The starting material (1.10 g, 2.00 mmol) was added NaOCH3 in CH3OH (0.5 M, 30 mL) at room temperature. The solution was heated to reflux, and stirred for 3 hours. The mixture was cooled to room temperature and neutralized by addition of AcOH. The mixture was then evaporated to dryness. The residue was purified by column chromatography (Hexanes–EtOAc, 1:1 to 100% EtOAc) to obtain 7-dN-dGTP-FL-1 (420 mg, 75%) as yellowish foam. Rf 0.21 (100% EtOAc), 1HNMR (400 MHz, CD3OD) δ 7.00 (d, J = 3.7 Hz, 1H), 6.37 (dd, J1’,2’b = 5.9 Hz, J1’,2’a = 8.6 Hz, 1H, H-1’), 6.32 (d, J = 3.7 Hz, 1H), 4.51 (app dt, J3’,2’a = 5.8 Hz, J3’,2’b = J3’,4’ = 2.3 Hz, 1H, H-3’), 4.05–3.97 (m, 4H, OCH3, H-4’), 3.81 (dd, J5’a,4’ = 3.4 Hz, Jgem = 12.1 Hz, 1H, H-5’a), 3.72 (dd, J5’b,4’ = 3.5 Hz, Jgem = 12.1 Hz, 1H, H-5’b), 2.70 (ddd, J2’a,3’ = 5.9 Hz, J2’a,1’ = 8.6 Hz, Jgem = 14.5 Hz, 1H, H-2’a), 2.24 (ddd, J2’b,3’ = 2.3 Hz, J2’b,1’= 5.9 Hz, Jgem = 14.5 Hz, 1H, H-2’b).
Figure imgf000125_0001
[328] Synthesis of 7dN-dGTP-FL-2: To a solution of 7dN-dGTP-FL-1 (420 mg, 1.50 mmol) in pyridine (5 mL) was added butyric anhydride (1.22 mL, 7.50 mmol) at room temperature. After stirring for 24 h, the reaction mixture was quenched with CH3OH. The mixture was then evaporated and extracted with EtOAc, washed with 2N HCl and saturated aqueous NaHCO3. The organic layer was dried over Na2SO4, and then filtered and concentrated. The residue was purified by column chromatography (1:1, Hexanes–EtOAc) to obtain intermediate (412 mg, 81%). To a solution of the intermediate (412 mg, 1.21 mmol) in pyridine was added benzoyl chloride (705 uL, 6.07 mmol) at room temperature. After stirring for 2 hours, the mixture was quenched with CH3OH, and evaporated. The residue diluted with EtOAc, washed with 2N HCl and saturated aqueous NaHCO3. The organic layer was dried over Na2SO4, and then filtered and concentrated. The residue was purified by column chromatography (3:1, Hexanes–EtOAc) to obtain 7dN-dGTP-FL-2 (425 mg, 67%) as white foam. Rf 0.53 (3:1, Hexanes–EtOAc), Rf 0.52 (3:1 Hexanes–EtOAc), 1H NMR (400 MHz, CDCl3) δ7.89–7.82 (m, 4H, ArH), 7.54–7.46 (m, 2H, ArH), 7.41–7.35 (m, 4H, ArH), 7.17 (d, J = 3.7 Hz, 1H), 6.50 (d, J = 3.7 Hz, 1H), 6.39 (dd, J1’,2’b = 6.8 Hz, J1’,2’a = 8.5 Hz, 1H, H-1’), 5.25 (app dt, J3’,2’a = 6.2 Hz, J3’,2’b = J3’,4’ = 2.2 Hz, 1H, H-3’), 4.35–4.29 (m, 2H, H-5’a, H- 5’b), 4.24–4.19 (m, 1H, H-4’), 3.79 (s, 3H, OCH3), 2.67–2.53 (m, 2H, (CH3)2CH), 2.48–2.39 (m, 1H, H-2’a), 2.33–2.24 (m, 1H, H-2’b), 1.25–1.16 (m, 12H, (CH3)2CH).
Figure imgf000126_0001
[329] Synthesis of 7dN-dGTP-FL-3: To a solution of 7dN-dGTP-FL-2 (425 mg, 0.81 mmol) in DMF (8.0 mL) was added NIS (200 mg, 0.892 mmol) at room temperature. After stirring overnight, the mixture was diluted with EtOAc, and washed with saturated NaHCO3(aq). The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (Hexanes–EtOAc, 10:1 to 3:1) to obtain 7-dN- dGTP-FL-3 (330 mg, 63%) as yellowish foam. Rf 0.42 (3:1, Hexanes–EtOAc), 1HNMR (400 MHz, CDCl3) δ 7.84–7.72 (m, 4H, ArH), 7.52–7.43 (m, 2H, ArH), 7.41–7.31 (m, 4H, ArH), 7.28 (s, 1H), 6.31 (dd, J = 6.2 Hz, 8.0 Hz, 1H, H-1’), 5.24–5.19 (m, 1H, H-3’), 4.33– 4.27 (m, 2H, H-5’a, H-5’b), 4.22–4.17 (m, 1H, H-4’), 3.77 (s, 3H, OCH3), 2.59 (heptet, J = 6.9 Hz, 2H, (CH3)2CH), 2.35–2.19 (m, 2H, H-2’a, H-2’b), 1.21–1.19 (m, 6H, (CH3)2CH), 1.19–1.16 (m, 6H, (CH3)2CH); HRMS (ESI) m/z [M] calcd for C34H35IN4O8754.1518; Found [M + H]+ 755.1591.
Figure imgf000126_0002
[330] Synthesis of 7dN-dGTP-FL-4: To a solution of 7dN-dGTP-FL-3 (330 mg, 0.508 mmol) in CH3OH (8.0 mL) was added 0.5M CH3ONa in CH3OH (2 mL) at room temperature. After stirring 1.5 hours, the mixture was neutralized by addition of AcOH (100 uL) and then concentrated. The residue was purified by column chromatography (Hexanes– EtOAc, 3:1 to 100% EtOAc) to obtain 7dN-dGTP-FL-4 (240 mg, 75%) as white solid. Rf 0.23 (100% EtOAc); 1HNMR (400 MHz, CD3OD) δ 8.01–7.95 (m, 2H, ArH), 7.66–7.59 (m, 2H, ArH, H-6), 7.57–7.50 (m, 2H, ArH), 6.66 (dd, J = 6.7, 6.6 Hz, 1H, H-1’), 4.59–4.51 (m, 1H, H-3’), 4.10 (s, 3H, OCH3), 3.99–3.94 (m, 1H, H-4’), 3.79 (dd, J = 3.8, 12.0 Hz, 1H, H- 5’a), 3.73(dd, J = 4.4, 12.0 Hz, H-5’b), 2.68–2.57 (m, 1H, H-2’a), 2.38(ddd, J = 3.4, 6.1, 13.4 Hz, H-2’b); HRMS (ESI) m/z [M] calcd for C19H19IN4O5510.0409; Found [M + H]+ 511.0482.
Figure imgf000127_0001
[331] Synthesis of 7dN-dGTP-FL-5: To a solution of 7dN-dGTP-FL-4 (200 mg, 0.392 mmol) in DMF (5.0 mL) was CuI (15 mg, 0.095 mmol) and Et3N (0.5 mL) at room temperature. After stirring at room temperture for 5 min under N2, the mixture was added Alkyne (177.5 mg, 1.18 mmol) and Pd(PPh3)4 (45 mg, 0.039 mmol) at room temperature. After stirring 24 hours, the mixture was concentrated and the residue was purified by column chromatography (Hexanes–EtOAc, 1:1 to Hexanes–EtOAc–CH3OH, 2:2:1) to obtain 7dN- dGTP-FL-5 (185 mg, 88%) as yellowish oil. Rf 0.11 (2:2:1, Hexanes–EtOAc–CH3OH); 1HNMR (400 MHz, CD3OD) δ 8.03–7.91 (m, 2H, ArH), 7.71–7.50 (m, 4H, ArH, H-6), 6.64 (dd, J = 6.7, 6.6 Hz, 1H, H-1’), 4.58–4.52 (m, 1H, H-3’), 4.33 (s, 2H, CH2N), 4.10 (s, 3H, OCH3), 3.99–3.94 (m, 1H, H-4’), 3.79 (dd, J = 3.8, 12.0 Hz, 1H, H-5’a), 3.73 (dd, J = 4.4, 12.0 Hz, H-5’b), 2.67–2.57 (m, 1H, H-2’a), 2.39 (ddd, J = 3.4, 6.1, 13.4 Hz, H-2’b); HRMS (ESI) m/z [M] calcd for C24H22F3N5O6533.1534; Found [M + Na]+ 556.1428.
Figure imgf000127_0002
[332] Synthesis of 7dN-dGTP-FL-6: To a solution of 7dN-GTP-FL-5 (220 mg, 0.412 mmol) in Pyridine (2.0 mL) was added TBDPSCl (118 uL, 0.454 mmol) at 0 °C, and slowly warmed to room temperature. After stirring 72 hours, the mixture was quenched by addition of CH3OH (5 mL) and then concentrated. The residue was diluted with EtOAc, and then washed with 2N HCl and saturated NaHCO3. The organic layer was dried over Na2SO4 and then filtered and concentrated. The residue was purified by column chromatography (Hexanes–EtOAc, 4:1 to 1:1) to obtain 7dN-dGTP-FL-6 (235 mg, 74%) as white foam. Rf 0.34 (1:1, Hexanes–EtOAc); 1HNMR (400 MHz, CD3OD) δ 7.98–7.90 (m, 2H, ArH), 7.74– 7.31 (m, 14H, ArH, H-6), 6.65 (dd, J = 6.7, 6.6 Hz, 1H, H-1’), 4.71–4.64 (m, 1H, H-3’), 4.33 (s, 2H, CH2N), 4.10 (s, 3H, OCH3), 4.05–3.98 (m, 1H, H-4’), 3.96–3.83 (m, 2H, H-5’a, H- 5’b), 2.70–2.54 (m, 1H, H-2’a), 2.49–2.37 (m, 1H, H-2’b), 1.06 (s, 9H, (CH3)3C); HRMS (ESI) m/z [M] calcd for C40H40F3N5O6Si 771.2713; Found [M + H]+ 772.2789.
Figure imgf000128_0001
[333] Synthesis of 7dN-dGTP-FL-7: To a solution of 7dN-dGTP-6 (180 mg, 0.233 mmol) in DMSO (6.0 mL) was added AcOH (1.5 mL) and Ac2O (3 mL) at room temperature. After stirring 24 hours, the mixture was quenched by addition of Sat. NaHCO3(aq). The mixture was stirred for 30 min and was diluted with EtOAc, and then washed with saturated NaHCO3. The organic layer was dried over Na2SO4 and then filtered and concentrated. The residue was purified by column chromatography (Hexanes–EtOAc, 4:1 to 1:1) to obtain 7dN-dGTP-FL- 7 (154 mg, 79%) as white foam. Rf 0.67 (1:1, Hexanes–EtOAc); 1HNMR (400 MHz, CD3OD) δ 7.97–7.90 (m, 2H, ArH), 7.70–7.32 (m, 14H, ArH, H-6), 6.58 (dd, J = 6.2, 7.3 Hz, 1H, H- 1’), 4.83–4.78 (m, 1H, H-3’), 4.77–4.67 (m, 2H, CH2S), 4.30 (s, 2H, CH2N), 4.12–4.04 (m, 4H, H-4’, OCH3), 3.88 (dd, J = 4.2, 11.9 Hz, 1H, H-5’a), 3.81 (dd, J = 4.2, 11.9 Hz, 1H, H- 5’b), 2.69–2.58 (m, 1H, H-2’a), 2.50 (ddd, J = 3.1, 6.2, 13.7 Hz, 1H, H-2’b), 2.12 (s, 3H, SCH3), 1.06 (s, 9H, (CH3)3C); HRMS (ESI) m/z [M] calcd for C42H44F3N5O6SSi 831.2751; Found [M + H]+ 832.2826.
Figure imgf000129_0001
[334] Synthesis of 7dN-dGTP-FL-8: To a solution of 7dN-dGTP-FL-7 (154 mg, 0.185 mmol) in CH2Cl2 (2.0 mL) was added cyclohexene (500 uL) and 1M sulfuryl chloride (555 uL, 0.555 mmol) at 0 °. After stirring 30 min, the mixture was concentrated. The residue was then dissolved in DMF (2.0 mL) and the mixture was added NaN3 (120 mg, 1.85 mmol) at room temperature. After stirring for 1 hour, the mixture was diluted with EtOAc, and then washed with H2O. The organic layer was dried over Na2SO4 and then filtered and concentrated. The residue was purified by column chromatography (Hexanes–EtOAc, 4:1 to 1:1) to obtain 7dN-dGTP-FL-8 (142 mg, 93%) as colorless oil. Rf 0.67 (1:1, Hexanes– EtOAc); 1HNMR (400 MHz, CD3OD) δ 7.96–7.89 (m, 2H, ArH), 7.69–7.29 (m, 14H, ArH, H-6), 6.55 (app t, J = 6.7 Hz, 1H, H-1’), 4.83 (d, J = 9.0 Hz, 1H, CH2N3), 4.78–4.73 (m, 2H, H-3’, CH2N3), 4.30 (s, 2H, CH2N), 4.15–4.06 (m, 4H, H-4’, OCH3), 3.90 (dd, J = 4.2, 11.3 Hz, 1H, H-5’a), 3.84 (dd, J = 4.4, 11.3 Hz, 1H, H-5’b), 2.79–2.69 (m, 1H, H-2’a), 2.55 (ddd, J = 3.7, 6.3, 13.6 Hz, 1H, H-2’b), 1.06 (s, 9H, (CH3)3C); HRMS (ESI) m/z [M] calcd for C41H41F3N8O6Si 826.2897; Found [M + H]+ 827.2973.
Figure imgf000129_0002
[335] Synthesis of 7dN-dGTP-FL-9: To a solution of 7dN-GTP-8 (98 mg, 0.121 mmol) in MeOH (2.0 mL) was added NH4F (13.4 mg, 0.363 mmol) at room temperature. After stirring 24 hours, the mixture was concentrated and the residue was diluted with EtOAc, and then washed with H2O. The organic layer was dried over Na2SO4 and then filtered and concentrated. The residue was purified by column chromatography (Hexanes–EtOAc, 4:1 to 1:1) to obtain 7dN-dGTP-FL-9 (46 mg, 67%) as colorless oil. Rf 0.27 (1:1, Hexanes– EtOAc); 1HNMR (400 MHz, CD3OD) δ 8.00–7.94 (m, 2H, ArH), 7.67 (s, 1H, H-6), 7.65– 7.60 (m, 1H, ArH), 7.57–7.51 (m, 2H, ArH), 6.58 (dd, J = 6.2, 7.4 Hz, 1H, H-1’), 4.86–4.79 (m, 2H, CH2N3), 4.70–4.64 (m, 1H, H-3’), 4.33 (s, 2H, CH2N), 4.15–4.04 (m, 4H, H-4’, OCH3), 3.83–3.71 (m, 2H, H-5’a, H-5’b), 2.77–2.68 (m, 1H, H-2’a), 2.55 (ddd, J = 3.0, 6.3, 13.6 Hz, 1H, H-2’b); HRMS (ESI) m/z [M] calcd for C25H23F3N8O6588.1705; Found [M + H]+ 589.1776. 6.12.8. Scheme 27. Synthesis of (L) 3’-O-azidomethyl-7-deaza-dGTP-Cy5
Figure imgf000130_0001
[336] Azido-Cy5 compound (Cy5-N3-Linker). (2-{2-[3-(2-Amino-ethylcarbamoyl)- phenoxy]- 1-azido-ethoxy}-ethoxy)-acetic acid Linker-6 (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006) US Patent Appl US20060160081A1) is dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of Cy5 NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF(400µl) is added slowly to the above reaction mixture and then stirred at room temperature for 5 h with exclusion of light. The crude product is purified on a preparative silica gel TLC plate (CHCl3/CH3OH, 1:4). [337] L-3’-O-N3-7-deaza-dGTP-Cy5 compound (L-3’-O-N3-7-deaza-dGTP-Cy5). To a stirred solution of Cy5-N3-Linker in dry DMF (2 ml), DSC (N,N’-disuccinimidyl carbonate) (3.4mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol) is added. The reaction mixture is stirred at room temperature for 2 h. TLC indicated that Cy5-N3- Linker is completely converted to compound Cy5-N3-Linker NHS ester, which is directly used to couple with L-amino-7-deaza-dGTP (13 µmol) in NaHCO3/Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture is stirred at room temperature for 3 h with exclusion of light. The reaction mixture is purified by a preparative silica gel TLC plate (CH3OH/CH2Cl2,1:1). The crude product is further purified on reverse-phase HPLC to afford L-3’-O-N3-dGTP-Cy5 (L-3’-O-N3-dGTP-Cy5). 6.13. Example 13. Chemical Synthesis of (D)-form 9°N DNA polymerase via Solid Phase Peptide Synthesis and Native Chemical Ligation [338] Figure 1 provides the amino-acid sequence of (D)-form mutant 9°N DNA polymerase (candidate #1-1), where all the amino acids are D-form amino acids. Diamond = mutation introduced for NCL; Circle = NCL site (start of the ‘second’ strand); Square = potential substitution sites for isoleucines; Triangle= mutation introduced for modified nucleotides.
Figure imgf000131_0001
Figure imgf000132_0001
[339] The 9°N DNA polymerase was split into two fragments (a 466-aa 9°N-N fragment and a 310-aa 9°N-C fragment) at the split site between K466 and M467. The synthesis of 466-aa 9°N-N fragment was designed as nine synthetic peptides (D-9°N-N-1 to D-9°N-N-9) (Figure 2A) via solid phase peptide synthesis which ligate at the certain cysteine residue as shown in Figure 2B. Similarly, the 310-aa 9°N-C fragment was split into six synthetic peptides (Figure 3A), which are synthesized via solid phase peptide synthesis, and then ligate at the certain cysteine residues as shown in Figure 3B. Experimental Methods: [340] Materials. 2-Chlorotrityl chloride resin (loading=0.98 mmol g−1) was purchased from Purepep. Fmoc-D-amino acids, D-4-thiazolidinecarboxylic acid, hydrazine hydrate, ethyl cyanoglyoxylate-2-oxime (Oxyma), N,N’-diisopropylcarbodiimide (DIC), trifuoroacetic acid, N,N-dimethylformamide (DMF), dichloromethane, Piperidine, thioanisole, triisopropylsilane, 1,2-ethanedithiol, and trifuoroacetic acid for peptide synthesis were purchased commercially from Chempep, Sigma-aldrich, Alfa aesar, TCI, etc. The reagents for NCL reaction, i.e, guanidine hydrochloride (Gn·HCl), Na2HPO4·12H2O, NaH2PO4·2H2O, sodium nitrite (NaNO2), Sodium hydroxide (NaOH), hydrochloric acid sodium 2-mercaptoethanesulfonate, 4-Mercaptophenylacetic acid (MPAA), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl), DL-1,4-dithiothreitol (DTT), 2,2′-azobis[2-(2-imidazolin-2-yl) propane] dihydrochloride (VA-044), Glutathione (reduced form), and palladium chloride (PdCl2) were purchased commercially from Sigma-aldrich, Alfa aesar, TCI, Duksan, etc. [341] Fmoc-based SPPS. All peptides were synthesized by Fmoc-based SPPS on the Liberty Blue automated microwave peptide synthesizer (CEM) and PurePep® Chorus automated peptide synthesizer. All the peptide hydrazides were synthesized on hydrazine-2- chlorotrityl chloride resin. For each peptide hydrazide, the first residue was attached to the hydrazine-2-chlorotrityl chloride resin by a double coupling method using 5 equiv. amino acid, 10 equiv. DIC, and 5 equiv. Oxymapure. All resins were swelled in DMF for 30 min before coupling. The Fmoc groups of assembled amino acids were removed by treatment with 20% piperidine and 0.1 M Oxyma in DMF at 85 °C. Coupling of amino acids except Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH was carried out at 85 °C using 5 equiv. amino acid, 5 equiv. Oxymapure and 10 equiv. DIC for 2 min. The coupling reactions for Fmoc- Cys(Trt)-OH and Fmoc-His(Trt)-OH were carried out at 50 °C for 10 min to avoid side reactions at high temperature. Trifluoroacetyl thiazolidine-4-carboxylic acid-OH was coupled using 5 equiv. Oxymapure and 10 equiv. DIC at room temperature overnight. Double coupling strategy was used for the peptides beyond 20 amino acids. After the completion of peptide chain assembly, peptides were cleaved from resin using H2O/thioanisole/triisopropylsilane/1,2-ethanedithiol/trifluoroacetic acid (0.5/0.5/0.5/0.25/8.25) (vol/vol). The cleavage reaction took 2.5 h under agitation at 27 °C. Cold ether was added to precipitate the crude peptide. After centrifugation, the supernatant was discarded and the precipitates were washed twice with ether. The crude peptides were dissolved in CH3CN/H2O, analyzed by RP-HPLC and purified by semi-preparative HPLC. Collected peptide fractions were analyzed by electrospray ionization mass spectrometry (ESI- MS). [342] Native Chemical Ligation (NCL). [343] Method A: The C-terminal peptide hydrazide segment was dissolved in acidified ligation buffer (aqueous solution of 6M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in an ice–salt bath (−15 °C), and 10 equiv. NaNO2 in acidified ligation buffer (pH 3.0) was added. The activation reaction system was kept in an ice–salt bath under stirring for 20 min, after which 40 equiv. MPAA in ligation buffer and 1 equiv. N-terminal cysteine peptide were added, and the pH of the solution was adjusted to 6.6-6.8 at room temperature. After overnight reaction, 150 mM TCEP in ligation buffer (pH adjusted to 7.0) was added to dilute the system twice and the reaction system was kept at room temperature for 30 min with stirring. Finally, the ligation product was analyzed by HPLC and purified by semi-preparative HPLC. Purified ligation fractions were analyzed by ESI-MS. [344] Method B: General approach. Peptide hydrazides were dissolved at a specified concentration (usually 1-20 mg/mL) in 6 M Gn.HCl containing 200 mM MPAA in a 1.5 mL Eppendorf tube. This forms a heterogeneous suspension and mixing sonication/vortexing should break any large pieces of solid MPAA. The pH can be adjusted to pH 3 if needed. A stock solution of acetyl acetone (acac) was made in water (10x-20x) and 1 eq to 5 eq acac were added to the peptide mixture. Finally, a small stir bar was added to the tube and the mixture is allowed to stir for 2-4 hours. [345] After 2-4 hours, an equimolar amount of the Cys-fragment peptide (the C-terminal fragment of the ligated product) was dissolved in 6 M Gn.HCl with 200 mM Na2HPO4 and 50 mM TCEP (TCEP is used to reduce disulfides between the Cys fragments and is not necessary) (pH 8.5) to a volume equal to that which the thioesterification reaction was occurring. These two solutions were mixed together upon which the resultant pH will be around 5-7 and the MPAA emulsion dissolves. The solution should be clear or slightly yellow. The pH of the combined solution was then adjusted to pH 7-7.4 by the addition of 1 M NaOH. The ligation reaction can then be left to stir for 4-18 hours. [346] Tfa-D-Thz-OH synthesis:
Figure imgf000134_0001
[347] To a solution of D-4-thiazolidinecarboxylic acid (1 g, 7.509 mmol) suspended in MeOH (40 mL) was added triethylamine (2.62 mL, 18.772 mmol) and the suspension was stirred for 10 min. Ethyltrifluroacetate (0.98 mL, 8.2599 mmol) was then added drop-wise and the mixture was stirred for 48 h at RT. Then the mixture was concentrated and subjected to silica gel chromatography using MeOH/CH2Cl2 (1:5) to give Tfa-(D)-Thz-OH, i.e. (S)-3- (2,2,2-trifluoroacetyl)thiazolidine-4-carboxylic acid) as a pale-yellow oil (0.9 g, 3.92 mmol, 52.3%) (Figure 4A).1H NMR (500 MHz, CDCl3): δ(ppm) 9.35 (s, 1H), 5.09 (dd, J = 6.9, 4.2 Hz, 1H), 4.86-4.77 (m, 1H, rotamers), 4.75-4.61 (m, 1H, rotamers), 3.52-3.28 (m, 2H, rotamers) (Figure 4B). It is understood that Thz can also be referred to as Cys(Thz), since it is a protected cysteine residue. [348] Fmoc-based SPPS (Figure 5): 1) Weigh 102 mg of 2-Cl-(Trt)-Cl resin (0.98 mmol g−1) into a peptide synthesis vessel. 2) Add 5 ml of DMF to the resin from the top of the reaction vessel, gently agitate it for 10 s and then drain it. 3) Repeat Step 2 two more times. 4) Add 5 ml of DCM to the resin, gently agitate it for 10 s and then drain it. 5) Repeat Step 4 two more times. 6) Repeat Step 2 three more times. 7) Swell the resin in 4 ml of 50% (vol/vol) DMF/DCM for 30 min, and then drain it. 8) Add 4 ml of 5% (vol/vol) NH2NH2.H2O to the resin for hydrazination. Gently agitate the mixture for 30 min in a constant-temperature shaker at 30 °C and then drain the solution by vacuum filtration. 9) Add 4 ml of DMF to the resin, gently agitate it for 10 s and then drain it. 10) Repeat Step 8. 11) Wash the resin by repeating Steps 2–6 twice. 12) Add 4 ml of 5% (vol/vol) MeOH/DMF to the resin, gently agitate it for 10 min and then drain it to unreacted sites on the resin to be capped. 13) Repeat Steps 2–6 to wash the resin thoroughly. 14) Add 4 ml of DCM to the resin, agitate it gently for 10 s and then drain it. Directly use the resin for the next coupling step. 15) For storage, dry the resin under high vacuum for 2 h and store dried 2-Cl-(Trt)- NHNH2 resin at –20 °C under argon gas for up to 2 weeks. [349] Solid Phase Peptide Synthesis of D-9°N-N-6@5-mer: Tfa-Thz-AVYE-NHNH2 (SEQ ID NO: 11) (Figure 6A). D-9°N-N-6@5-mer was synthesized on PurePep® Chorus automated peptide synthesizer by following the conditions mentioned in experimental methods. D-9°N-N-6@5-mer synthesized by the process was analyzed by HPLC chromatogram and the results are provided in Figures 6B-6C. [350] Solid Phase Peptide Synthesis of D-9°N-N-6@11-mer: CVFGKPKEKVY-NHNH2 (SEQ ID NO: 20): D-9°N-N-6@11-mer was synthesized on PurePep® Chorus automated peptide synthesizer by following the conditions mentioned in experimental methods. D-9°N- N-6@11-mer synthesized by the process was analyzed by HPLC chromatogram and the results are provided in Figures 7B-7D. [351] Solid Phase Peptide Synthesis of D-9°N-N-6@24-mer: CEEIAQAWESGEGLERVARYSMED-NHNH2 (SEQ ID NO: 13) (Figure 8A). D-9°N-N- 6@24-mer was synthesized on PurePep® Chorus automated peptide synthesizer by following the conditions mentioned in experimental methods. D-9°N-N-6@24-mer synthesized by the process was analyzed by HPLC chromatogram and the results are provided in Figures 8B-8C. [352] Solid Phase Peptide Synthesis of D-9°N-C-3@9-mer: CDTDGLHAT-NHNH2 (SEQ ID NO: 14) (Figure 9A) D-9°N-C-3@9-mer was synthesized on the Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in experimental methods. D-9°N-C-3@9-mer synthesized by the process was analyzed by HPLC chromatogram and the results are provided in Figures 9B-9C. [353] Solid Phase Peptide Synthesis of Customized D-9°N-C-1@33-mer: MKATVDPLEK KLLDYRQRLI KILANSFYGYYGY-NHNH2 (SEQ ID NO: 46). D-9°N-C-1@33-mer synthesized by the process was analyzed by HPLC chromatogram and the results are provided in Figures 10A-10B. [354] Solid Phase Peptide Synthesis of Customized D-9°N-C-2@39-mer: CKARWY- C(Acm)-KE-C(Acm) AESVTAWGRE YIEMVIRELE EKFGFKVLY-NHNH2 (SEQ ID NO: 47). D-9°N-C-2@39-mer synthesized by the process was analyzed by HPLC chromatogram and the results are provided in Figures 11A-11B. [355] Solid phase peptide synthesis of D-9°N-C-3@21-mer: TFA-Thz- DTDGLHATIPGADAETVKKK-NHNH2 (SEQ ID NO: 15). D- 9oN-C-3@21-mer was synthesized on the Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in experimental methods. The product was observed as the TFA-deprotected D-9°N-C-3@21-mer as provided in Figures 12B and 12C. [356] Solid phase peptide synthesis of D-9°N-C-3@Cys35-mer: CKEFLKYINP KLPGLLELEY EGFYVRGFFV TKKKY-NHNH2. (SEQ ID NO: 16) D-9oN-C-3@Cys35- mer was synthesized on the Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in experimental methods. The product was observed as expected mass. as provided in Figures 13B-13D. [357] Solid phase peptide synthesis of D-9°N-C-3@56-mer: CDTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEGFYVRGFFVTKKKY- NHNH2. (SEQ ID NO: 17) (Figure 14A) D- 9oN-C-3@56-mer was synthesized on the Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in experimental methods. The product was observed as expected mass. as provided in Figures 14B. [358] D-9°N-C-3@35-mer: AKEFLKYINP KLPGLLELEY EGFYVRGFFV TKKKY- NHNH2. (SEQ ID NO: 18) (Figure 15A) D-9oN-C-3@35-mer was synthesized on the Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions mentioned in experimental methods. The product was observed as expected mass. as provided in Figures 15B. [359] Synthesis of D-9oN-N-6@16-mer: Tfa-Thz-AVYECVFGKPKEKVY-NHNH2 (SEQ ID NO:25). The process for the preparation of D- 9oN-N-6@16-mer is illustrated in Figure 16B and the HPLC chromatogram analysis of the D- 9oN-N-6@16-mer is provided in Figure 16C. [360] Synthesis of D-9oN-C-7@72-mer: MKATVDPLEKKLLDYRQRLIKILANSFYGYYGYCKARWY-C(Acm)-KE-C(Acm) AESVTAWGRE YIEMVIRELE EKFGFKVLY-NHNH2 (SEQ ID NO: 23). The process for the preparation of D-9oN-C-7@72-mer is illustrated in Figure 17B. Figure 17C is the analytical HPLC chromatogram of the peaks transformation in progressing the NCL reaction (λ=214 nm). Figure 17D is ESI-MS of D-9°N-C-7@72-mer with MPAA attachment and Figure 17E shows MPAA-attached ligated. [361] The following peptides were produced by solid phase peptide synthesis on PurePep® Chorus automated peptide synthesizer by following the conditions mentioned in experimental methods. The product was analyzed by HPLC chromatogram and production of the peptide was confirmed.
Figure imgf000137_0001
Figure imgf000138_0001
[362] Native Chemical Ligation (NCL): Reagent Preparation: NaOH, 1 M and 6 M. Dissolve 40 mg or 240 mg of NaOH in 1 ml of deionized H2O. HCl, 6 M. Mix 1 ml of 12 M HCl with 1 ml of deionized H2O. Two different phosphate solutions (0.1 M) containing 6 M Gn·HCl (pH 3.0–3.1 and pH 5.7–6.0, respectively). For a 10-ml solution, mix 156 mg of NaH2PO4·2H2O and 5.74 g of Gn·HCl into a 10-ml volumetric flask and adjust it to the final volume with deionized H2O. Adjust the pH to 3.0–3.1 with 6 M NaOH and 6 M HCl. Filter the solution by using a 13 mm × 0.22 µm microporous membrane filter. The filtered solution can be stored at 4 °C for at least 1 month. Na2HPO4·12H2O (0.1 M) containing 6 M Gn·HCl (pH 5.7-6.0) also prepared as same manner. NaNO2, 0.5 M. Dissolve 17 mg of NaNO2 in 0.5 ml of deionized H2O. TCEP, 0.1 M. Dissolve 58 mg of TCEP·HCl into 1 ml of 0.2 M phosphate solution (pH 3.0) containing 6 M guanidine hydrochloride (Gn·HCl). Adjust the pH to 6.0–7.0, and filter the solution by using a 13 mm × 0.22 µm microporous membrane filter. VA-044, 0.1 M. Weight 9.7 mg of VA-044 into a 2-ml Eppendorf reaction tube and add 0.3 ml of 0.2 M phosphate solution (pH 6.9–7.0) containing 6 M Gn·HCl. Completely dissolve VA-044 by using a vortex and an ultrasonic cleaning bath. [363] Synthesis of D-9oN-N-10@(His)6+101-mer: [364] (His)6- MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYCLLKDDSAIEDVKKVTAKR HGTVVKVKRAEKVQKKFLGRPVEVWKLYFNHPQDVPAIRDRIR-NHNH2 (SEQ ID NO: 52). (Figure 18A) D-9oN-N-10@(His)6+101-mer was synthesized using Native Chemical Ligation Method A protocol. The process for the preparation of D-9oN-N- 10@(His)6+101-mer is illustrated in Figure 18B and the HPLC chromatogram analysis of the D-9oN-N-10@(His)6+101-mer is provided in Figure. 18C. A deconvoluted MS (ESI-MS) spectrum of purified D-9oN-N-10@(His)6+101-mer was calculated to be 12875.8 and measured to be 12876.8. As illustrated in Figure 18B, D-9oN-N-1@(His)6+39-mer (2 mg, 1 equiv.) was dissolved in 75 µL acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in ice-salt bath (−15 °C), and 7.2 μL of 0.5 M NaNO2 (in acidified ligation buffer) was added. The reaction was kept in ice-salt bath under stirring for 20 min, after which 70 uL of 0.2 M MPAA (in 6 M Gn·HCl and 0.1 M Na2HPO4, pH 6.5) with D-9oN-N-2@62-mer (2.58 mg, 1 eq) was added and the pH of the reaction mixture was adjusted to 6.6-6.8 with NaOH solution at room temperature. After 14 h, the reaction mixture was reduced by 0.15 M TCEP and purified by HPLC (purification conditions: 5-95% CH3CN (with 0.1% TFA) gradient in H2O (with 0.1% TFA) over 30 min on a Welch C4 column). [365] Synthesis of D-9oN-N-13@108-mer: [366] D-9oN-N-13@108-mer was synthesized using Native Chemical Ligation Method B protocol. The process for the preparation of D-9oN-N-13@108-mer is illustrated in Figure 19B and the HPLC chromatogram analysis of the D-9oN-N-13@108-mer is provided in Figure 19C. As illustrated in Figure 19B, fragment D-9oN-N-7@52-mer (2.2 mg) was dissolved in 300 µL of 6 M Gn.HCl that contained 200 mM MPAA in a 1.5 mL Eppendorf tube. Acac was dissolved to 150 mM in water and 2.5 eq were added to the dissolved solution of D-9oN-N-7@52-mer. A small stir bar was added to the Eppendorf tube and the reaction was allowed to stir for 4 hours. Fragment D-9oN-N-8@56-mer (2.29 mg, 1 eq) was dissolved in 300 µL of 6 M Gn.HCl that contained 200 mM Na2HPO4.12H2O at pH 8.5. This solution was then added to the thioesterification solution, which resulted in a 600 µL solution at pH 5.3. The pH of this solution was adjusted to pH 7.0 with the addition of 1 M NaOH and the reaction was left to stir overnight. After 18 hours, 50 mM TCEP was added and analyzed by HPLC after 30 min stirring. c) Analytical HPLC chromatogram of the peaks transformation in progressing the NCL reaction (λ=214 nm). Column: Welch C4. Gradient: 5-95% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 30 min [√ = confirmed peak]. Figure 19D shows Deconvoluted MS (ESI-MS) spectrum of purified D-9oN-N-13@108-mer [Calculated: 12467.52, observed: 12467.39]. [367] Using the method, the following peptides were synthesized.
Figure imgf000140_0001
Figure imgf000141_0001
[368] Using similar methods, D-9°N-C-11 is generated as illustrated in Figure 3B. Additionally, D-9°N-C-12 is generated by desulfurizing one or more cysteine residues of D- 9°N-C-11 to alanine residues at one or more positions Cys500, Cys539, Cys595, Cys651 and Cys714. D-9°N-C-12 is generated from D-9°N-C-11 by Acm deprotection at Cys506 and Cys509. 6.14. Example 14. Chemical Synthesis of (D)-form terminal deoxynucleotidyl transferase via Solid Phase Peptide Synthesis and Native Chemical Ligation (1st method) [369] Figure 20A provides the amino-acid sequence of (D)-form terminal deoxynucleotidyl transferase, where all the amino acids are D-form amino acids. The (D)-form terminal deoxynucleotidyl transferase is a 381-aa protein. Figure 20B provides the synthetic chemical ligation route of the terminal deoxynucleotidyl transferase. The synthesis of terminal deoxynucleotidyl transferase was designed as seven synthetic peptides (D-TdT-WT-1 to D- TdT-WT-7) via solid phase peptide synthesis and the seven synthetic peptides were ligated at the certain cysteine residue as shown. [370] For synthesis of the seven synthetic peptides, the experimental methods provided above related to the D-polymerase were used. For example, Figure 21A shows D-TdT-WT- 1@(His)6+46-mer: (His)6NSSPSPVPGSQNVPAPAVKKISQYAC(Acm)QRRTTLNNYNQLFTDALDIL- NHNH2 (SEQ ID NO: 57). D-TdT-WT-1@His6+46-mer was synthesized on PurePep® Chorus automated peptide synthesizer by following the conditions mentioned in experimental methods. D-TdT-WT-1@His6+46-mer synthesized by the process was analyzed by HPLC chromatogram and the results are provided in Figures 21B. [371] Figure 21A is the peptide sequence of D-TdT-WT-1@(His)6+46-mer. Figure 21B is analytical HPLC chromatogram of the crude D-TdT-WT-1@(His)6+46-mer (λ=214 nm). Column: Welch C4. Gradient: 1-20 min 100-80% water (0.1% TFA) in ACN (0.1% TFA), 20-40 min 80-50% water (0.1% TFA) in ACN (0.1% TFA), 40-60 min 50-20% water (0.1% TFA) and 60-70 min 20-0% water (0.1% TFA) in ACN (0.1% TFA) [√ = confirmed peak]. Figure 21C is deconvoluted MS (ESI-MS) spectrum of purified D-TdT-WT-1@His6+46-mer [Calculated: 5922.97, observed: 5922.97]. Purification conditions: 1-20 min 100-80% water (0.1% TFA) in ACN (0.1% TFA), 20-40 min 80-50% water (0.1% TFA) in ACN (0.1% TFA), 40-50 min 50-20% water (0.1% TFA) and 50-60 min 20-0% water (0.1% TFA) in ACN (0.1% TFA) (C18 semipreparative column). [372] Additionally, the following peptides were produced by solid phase peptide synthesis on PurePep® Chorus automated peptide synthesizer by following the conditions mentioned in experimental methods. The product was analyzed by HPLC chromatogram and production of the peptide was confirmed.
Figure imgf000142_0001
Figure imgf000143_0001
[373] The seven synthetic peptides were ligated at the certain cysteine residue following the protocol described above. For example, fragment D-TdT-WT-1@(His)6+46-mer (3.0 mg) was dissolved in 300 µL of 6 M Gn.HCl that contained 200 mM MPAA in a 1.5 mL Eppendorf tube. Acac was dissolved to 150 mM in water and 2.5 eq were added to the dissolved solution of D-TdT-WT-1@(His)6+46-mer. A small stir bar was added to the Eppendorf tube and the reaction was allowed to stir for 4 hours. Fragment D-TdT-WT-2@61- mer (2.74 mg, 0.8 eq) was dissolved in 300 µL of 6 M Gn.HCl that contained 200 mM Na2HPO4.12H2O at pH 8.5. (Figure 22A) This solution was then added to the thioesterification solution, which resulted in a 600 µL solution at pH 5.3. The pH of this solution was adjusted to pH 7.0 with the addition of 1 M NaOH and the reaction was left to stir at room temperature. After 48 hours, 50 mM TCEP was added and analyzed by HPLC after 30 min stirring. (Figure 22B) D-TdT-WT-8@(His)6+107-mer was purified by Preparative HPLC chromatography (Retention time: 13.8 min). (Figure 22C) Column: Welch C4. Gradient: 70-30% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 30 min [√ = confirmed peak]. Observed MS for D-TdT-WT-8@(His)6+107-mer was 12663.23 (calc. 12663.21). (Figure 22D) [374] Using the method, the following peptides were synthesized.
Figure imgf000143_0002
Figure imgf000144_0001
Figure imgf000145_0001
[375] Preparation of D-TdT-WT-14@(His)6+381-mer: [376] D-TdT-WT-13@(His)6+381-mer was dissolved in 200 mM TCEP solution (6M Gn.HCl and 0.1 M Na2HPO4, pH 7.0), containing 20 mM VA-044 and 40 mM reduced L- glutathione. The reaction was stirred at 37 °C for overnight. The desulfurization product D- TdT-WT-14@(His)6+381-mer was purified by semi-preparative HPLC (retention time: 17.2 min). Observed MS of D-TdT-WT-14@(His)6+381-mer was 44854 (calc. 44855.2). Column: Welch C4. Gradient: 20% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) in 10 mins, then 20-70% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 20 min. This reaction leads to desulfurization of one or more cysteine residues of D-TdT-WT-13 to alanine residues at one or more positions Cys47, Cys108, Cys162, Cys224, Cys268, and Cys317. [377] Synthesis of D-TdT-WT-15@(His)6+381-mer: [378] D-TdT-WT-14@(His)6+381-mer was dissolved in an aqueous solution of 6M Gn.HCl and 0.1 M Na2HPO4, 40 mM TCEP, pH 7.0. PdCl2 was dissolved in an aqueous solution of 6M Gn.HCl and added to peptide solution. The reaction was stirred at 30 °C. After 2 hours, 2M DTT (in an aqueous solution of 6M Gn.HCl) was added. The reaction mixture was stirred for 30 min and purified by HPLC to obtain D-TdT-15@(His)6+381-mer (retention time: 17.5 min). Observed MS of D-TdT-WT-15@(His)6+381-mer was 44365 (calc. 44367.2). Column: Welch C4. Gradient: 20% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) in 10 mins, then 20-70% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 20 min. This reaction leads to Acm deprotection at Cys26, Cys59, Cys87, Cys173, Cys249, Cys275, and Cys309. 6.15. Example 15: Synthesizing L-polynucleotide using D- terminal deoxynucleotidyl transferase. [379] The D- terminal deoxynucleotidyl transferase is mixed with a reaction buffer, a L- DNA fragment, L-dNTP (e.g., L-dATP, L-dTTP, L-dGTP or L-dCTP) and water. The mixture is incubated at 22-37^C for 15-30 minutes. The reaction is then stopped by heating at 70 ^C for 10 minutes or by adding 2µL 0.5M EDTA. [380] The D-terminal deoxynucleotidyl transferase is mixed with a reaction buffer, a L- DNA fragment, L-3’-O-R-dNTP (N=A or C or G or T) and water. The mixture is incubated at 22-37oC for 15-30 minutes. The 3’-O-R group is removed using a deprotection reaction (i.e. 50mM TCEP buffer incubation at 37-55oC for 5-15 minutes, if R = azidomethyl). The next nucleotide addition can be performed by repeating the reaction from the above. [381] A L-polynucleotide generated by the method is sequenced and confirmed that L- dNTP is incorporated into the 3’ end of the DNA fragment. The synthesized L-polynucleotide can be used to store information. [382] The D-terminal deoxynucleotidyl transferase is also used for DNA and oligonucleotide 3’-end labeling. The enzyme is mixed with a radiolabeled L-ddNTP, a buffer, a linear DNA and water. The mixture is incubated at 37 ^C for 15 minutes. The reaction is then stopped by heating at 70 ^C for 10 minutes or by adding 2µL 0.5M EDTA. [383] The L-polynucleotide generated by the method is sequenced and confirmed that L- ddNTP is incorporated into the 3’ end of the linear DNA. 6.16. Example 16: Synthesis of (L) 3’-O-azidomethyl-7-deaza-dNTP-Label intermediates Materials [384] All solvents and reagents were reagent grades, purchased commercially, and used without further purification unless specified. All chemicals were purchased from Sigma- Aldrich, Fisher Scientific, TCI etc.1H NMR spectra were recorded on a Bruker AscendTM (400 MHz) spectrometer from Chapman University and reported in parts per million (ppm) from a CDCl3 (7.26 ppm) or D2O. Data were reported as follows: (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, J = coupling constant in Hz, integration). Proton-decoupled 31P NMR spectra were recorded on a Bruker AscendTM (121.4 MHz) spectrometer from Chapman University. High-resolution mass spectra (HRMS) were obtained from School of Pharmacy, Chapman University and Analytical Chemistry Instrumentation Facility at University California of Riverside. Starting materials beta-L-deoxythymidine, beta- L-deoxycytidine, beta-L-deoxyadenosine, and beta-L-deoxyguanosine were purchased from Chemgenes. Analytical (Polaris 180A C18-A, 4.6 x 250 mm, 5 um) and semi-prep (Polaris 180A C18-A, 4.6 x 250 mm, 5 um) HPLC columns were purchased from Agilent. The 3’-O- modified nucleotides were purified with reverse-phase HPLC on a 4.6 x 250 mm C18 column (Polaris), mobile phase: A, 25 mM TEAB buffer in water; B, 25 mM TEAB buffer in acetonitrile. Elution was performed isocratic conditions as described in each procedure. 6.16.1. Scheme 29: Synthesis of NH2-L-3’-O-azidomethyl-7-deaza-dATP:
Figure imgf000147_0001
Figure imgf000148_0001
[385] Synthesis of 7dNATP-FL-1: To a solution of 4-chloro-5-iodo-7H-pyrrolo [2.3-d] pyrimidine (4.0 g, 14.4 mmol) in MeCN (200ml), powdered KOH (1.8g, 7.57 mmol) and TDA-1 (0.3 ml, 0.86 mmol) were added at room temperature. After stirring for 30 min, 1-Cl- 2-deoxysugar (8.6 g, 18.7mmol) was introduced and the stirring was continued for another 30 min. Insoluble material was filtered off and washed several times with hot acetone. The combined filtrates were evaporated to dryness. The residue was applied onto purification by chromatography on silica (EtOAc:Hexanes 1:1 to 100% EtOAc) to give 7-dNATP-FL-1 as a yellow solid (4.22 g, 89%). 1HNMR (400 MHz, CD3OD) δ 8.44 (s, 1H), 7.89 (d, 2H), 7.78 (d, 2H), 7.73 (s, 1H), 6.66 (dt, J = 1.61Hz, J = 7.85Hz, 1H), 4.51-4.49 (m, 1H), 3.56(s, 1H), 2.98-2.93 (m, 1H), 2.75–2.70 (m, 1H), 2.34 (s, 1H) 2.32 (s, 1H); HRMS (ESI): m/z [M] calcd for C27H23ClIN3O5631.0371; found [M + H]+ 632.0456
Figure imgf000149_0001
[386] Synthesis of 7dNATP-FL-2: A suspension of 7dNATP-FL-1 (1.2 g, 1.89 mmol) in a mixture of 28% aq. NH3/dioxane (1:1, 100 ml) was stirred for 17 h in a reflux condition. The residue was applied onto purification by chromatography on silica (EtOAc:Hexanes:CH3OH 1:1:0.1) to give 7-dNATP-FL-2 as a yellow solid (296mg, 41.4%). 1HNMR (400 MHz, CD3OD) δ 8.11 (s, 1H), 7.61 (s, 1H), 7.76 (s, 1H), 6.52 (dt, J = 2.1Hz, J = 6.0Hz,1H), 4.60 (s, 1H), 4.53-4.51 (m, 1H), 4.02-4.00 (m, 1H), 3.82–3.79(dd, J = 3.3Hz, J = 11.9Hz, 1H), 3.75- 3.71(dd, J = 3.5Hz, J = 12Hz, 1H), 2.66–2.59 (m, 1H), 2.36–2.30 (m, 1H); HRMS (ESI): m/z [M] calcd for C11H13IN4O3376.0039; Found [M + H]+ 377.0113
Figure imgf000149_0002
[387] Synthesis of 7dNATP-FL-3: To a solution of 7dNATP-FL-2 (236 mg,0.6298 mmol) in N,N-dimethylformamide (5 mL) was added CuI (24mg, 0.1260 mmol) and triethylamine (250uL) and then stirred at room temperature for few minutes. Then, 2,2,2-trifluoro-N-(prop- 2-ynyl) acetamide (285mg, 1.89 mmole) and Tetrakis(triphenylphosphine)palladium(0) (72.78mg, 0.06mmol) were added. The reaction was run for 24 h at the room temperature and was concentrated. The resulting residue was purified by column chromatography (1:10:10 to 1:4:4, CH3OH:CH2Cl2:EtOAc) to give 7dNATP-FL-3 as yellow solid (164.2 mg, 65.3%). 1HNMR (400 MHz, CD3OD) δ 10.10 (s, 1H), 8.12 (s, 1H), 7.76 (s, 1H), 6.53-6.46 (m, 1H), 5.26 (d, 1H), 5.08 (t, J = 5.6 Hz, 1H), 4.35 – 4.26 (m, 2H), 3.84–3.81(m, 1H), 2.59(dd, J = 4.8Hz, J = 0.8Hz, 1H), 2.49–2.43 (m, 1H), 2.21–2.16 (m, 1H); HRMS (ESI): m/z [M] calcd for C16H16F3N5O4399.1154; Found [M+ H]+ 400.1234
Figure imgf000150_0001
[388] Synthesis of 7dNATP-FL-4: To a solution of 7dNATP-FL-3 (156 mg, 0.3907 mmol) in N,N-dimethylformamide (4 mL) was added imidazole (39.8 mg, 0.586mmol) and tert-Butyl(chloro)diphenylsilane (118.12 mg,3.25 mmol) at 0 °C and then was warmed to room temperature. After stirred at room temperature for 10 hours, the solution was added to iced water and extracted with ethyl acetate x 2. The combined organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (1:2 to 100%, Hexanes:EtOAc) to give 7dNATP-FL-4 as yellow oil (128mg, 63.8%). 1H NMR (400 MHz, CD3OD) δ 8.11 (s, 1H), 7.66 (d, 4H), 7.51 (s, 1H), 7.42-7.33 (m, 6H), 6.56 (t, J = 6.5 Hz, 1H), 4.63–4.59 (m, 1H), 4.28 (s, 2H), 4.01–4.00 (m, 1H), 3.90 (dd, J = 11.5 Hz, J = 3.15 Hz, 1H), 3.81 (dd, J = 11.4 Hz, J = 3.82 Hz, 1H), 2.57– 2.38 (m, 2H), 1.05 (s, 9H); HRMS (ESI): m/z [M] calcd for C32H34F3N5O4Si 637.2362; Found [M + H]+ 638.2438
Figure imgf000151_0001
[389] Synthesis of 7dNATP-FL-5: A solution of the 7dNATP-FL-4 (128mg 0.2492mmol) was dissolved in a mixture of CH3OH:N,N-dimethyl-acetal (10:1) and stirred at 40 °C. The reaction monitored by TLC, was completed after 2 h. The solvent was removed under vacuum. Purification by chromatography on silica (EtOAc: CH3OH=15:1 to 10:1) gave 7dNATP-FL-5 as a clear yellow oil (99.5mg, 70.2%). HRMS (ESI): m/z [M] calcd for C35H39F3N6O4Si 692.2788; Found [M + H]+ 693.2865
Figure imgf000151_0002
[390] Synthesis of 7dNATP-FL-6: To a solution of 7dNATP-FL-5 (99.5mg, 0.175mmol) in DMSO 4 mL) was added AcOH (800 uL) and acetic anhydride (800 uL) at room temperature. After stirred at room temperature for 18 hours, the solution was added iced cold water and solid sodium bicarbonate. The water layer was extracted with ethyl acetate x2. The combined organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (Hexanes: EtOAc =3:1 to 1:1,) to give 7dNATP-FL-6 (48mg, 40%) as white solid. HRMS (ESI): m/z [M] calcd for C35H39F3N6O4Si 752.2821; Found [M + H]+ 753.2897.
Figure imgf000152_0001
[391] Synthesis of 7dNATP-FL-7: To a solution of 7dNATP-FL-6 (20 mg, 0.032 mmol) in dry CH2Cl2 was added cyclohexene (0.03 ml, 0.16 mmol) at 4 °C., then sulfurylchoride (1M in CH2Cl2, 0.03 ml, 0.031 mmol) was added drop wise under N2. After 40 min TLC indicated the full consumption of 7dNATP-FL-6, the solvent was evaporated and the residue was subjected to high vacuum for 20 min. It was then redissolved in dry DMF (1 ml) and treated with NaN3 (30 mg, 0.45 mmol). The resulting suspension was stirred under room temperature for 2 h. The reaction was quenched with CH2Cl2 and the organic layers were washed with sat aq. NaCl solution. After removing the solvent, the resulting yellow gum was redissolved in 2M HCl:Acetonitrile (2:1) and was stirred at room temperature for 30 min. The solvent was removed and the reaction worked up with CH2Cl2 and sat. aq. NaHCO3 solution. The aqueous layer was extracted three times with CH2Cl2. Purification by chromatography on silica (EtOAc:Hexanes 1:1 to 100% EtOAc) gave 7dNATP-FL-7 as a pale yellow foam (15 mg, 73%). 1HNMR (400 MHz, CD3OD) δ 8.86 (s), 8.09 (s), 6.44 (app dt, J =2 .5 Hz, J = 6.0 Hz, 1H) 4.86-4.80 (m, 2H), 4.57-4.54 (m, 1H),4.19–4.16 (m, 1H), 3.81 (dd, J = 3.6 Hz, 12 Hz, 1H), 3.75 (dd, J = 12.1 Hz, J = 3.3 Hz, 1H), 2.73–2.66 (m, 1H), 2.53 (dd, J = 2.3 Hz, J = 5.7 Hz, 1H), 2.49 (dd, J = 2.3 Hz, J = 5.9 Hz, 1H); HRMS (ESI): m/z [M] calcd for C17H17F3N8O4 454.1341; Found [M + H]+ 455.1415.
Figure imgf000152_0002
[392] Synthesis of 7dNATP-FL-8: 7-dNATP-FL-7 (40 mg, 0.088 mmol) was dried in a vacuum desiccator over P2O5 overnight. To a solution of 7dNATP-FL-7 in trimethyl phosphate (1 mL) was added POCl3 (20.5 uL, 0.22 mmol) dropwisely at 0 °C. The mixture was stirred at 0 °C for 2 hours and then was added a well-vortexed mixture of tributylammonium pyrophosphate (110 mg) and tributylamine (157 uL, 0.620 mmol) in anhydrous DMF (0.5 mL). The mixture was stirred for 1 hour at room temperature and 0.1M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 20 ml) was then added and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 10 ml of water. The crude mixture was extracted with CH2Cl2 (2 x 10 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified with anion exchange chromatography on DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.5; 0.1–0.8 M). The fractions with products (0.3M – 0.4M) was collected and concentrated under reduced pressure. [393] HRMS (ESI): m/z [M] calcd for C17H17F3N8O4598.0486; Found [M – H]– 597.0413. [394] Labeling of the 7dNATP-FL-8 proceeds according to the synthesis described in Scheme 25. 6.16.2. Scheme 30: Synthesis of NH2-L-3’-O-azidomethyl-7-deaza-dGTP:
Figure imgf000153_0001
Figure imgf000154_0001
[395] Synthesis of deaza-dGTP-FL-1: To a suspension of KOH (0.68 g, 8.71 mmol) and TDA-1 (0.1 mL, 0.32 mmol) in CH3CN (30 mL), 2-amino-4-chloro-7H-Pyrrolo[2,3- d]pyrimidine (421 mg, 2.50 mmol) was added at room temperature. After stirring for 5 min, 1-Cl sugar (1.27 g, 3.25 mmol) was added, and continue stirring for 30 min. The insoluble material was filtered off, the precipitate was washed with CH3CN, and the filtrate was evaporated to dryness. The residue was purified by column chromatography (Hexanes– EtOAc, 4:1 to 1:1) to obtain product (1.10 g, 82%) as white foam. The starting material (1.10 g, 2.00 mmol) was added NaOCH3 in CH3OH (0.5 M, 30 mL) at room temperature. The solution was heated to reflux, and stirred for 3 hours. The mixture was cooled to room temperature and neutralized by addition of AcOH. The mixture was then evaporated to dryness. The residue was purified by column chromatography (Hexanes–EtOAc, 1:1 to 100% EtOAc) to obtain deaza-dGTP-FL-1 (420 mg, 75%) as yellowish foam. Rf 0.21 (100% EtOAc), 1HNMR (400 MHz, CD3OD) δ 7.00 (d, J = 3.7 Hz, 1H), 6.37 (dd, J1’,2’b = 5.9 Hz, J1’,2’a = 8.6 Hz, 1H, H-1’), 6.32 (d, J = 3.7 Hz, 1H), 4.51 (app dt, J3’,2’a = 5.8 Hz, J3’,2’b = J3’,4’ = 2.3 Hz, 1H, H-3’), 4.05–3.97 (m, 4H, OCH3, H-4’), 3.81 (dd, J5’a,4’ = 3.4 Hz, Jgem = 12.1 Hz, 1H, H-5’a), 3.72 (dd, J5’b,4’ = 3.5 Hz, Jgem = 12.1 Hz, 1H, H-5’b), 2.70 (ddd, J2’a,3’ = 5.9 Hz, J2’a,1’ = 8.6 Hz, Jgem = 14.5 Hz, 1H, H-2’a), 2.24 (ddd, J2’b,3’ = 2.3 Hz, J2’b,1’= 5.9 Hz, Jgem = 14.5 Hz, 1H, H-2’b).
Figure imgf000155_0001
[396] Synthesis of deaza-dGTP-FL-2: To a solution of deaza-dGTP-FL-1 (420 mg, 1.50 mmol) in pyridine (5 mL) was added butyric anhydride (1.22 mL, 7.50 mmol) at room temperature. After stirring for 24 h, the reaction mixture was quenched with CH3OH. The mixture was then evaporated and extracted with EtOAc, washed with 2N HCl and saturated aqueous NaHCO3. The organic layer was dried over Na2SO4, and then filtered and concentrated. The residue was purified by column chromatography (1:1, Hexanes–EtOAc) to obtain intermediate (412 mg, 81%). To a solution of the intermediate (412 mg, 1.21 mmol) in pyridine was added benzoyl chloride (705 uL, 6.07 mmol) at room temperature. After stirring for 2 hours, the mixture was quenched with CH3OH, and evaporated. The residue diluted with EtOAc, washed with 2N HCl and saturated aqueous NaHCO3. The organic layer was dried over Na2SO4, and then filtered and concentrated. The residue was purified by column chromatography (3:1, Hexanes–EtOAc) to obtain deaza-dGTP-FL-2 (425 mg, 67%) as white foam. Rf 0.53 (3:1, Hexanes–EtOAc), Rf 0.52 (3:1 Hexanes–EtOAc), 1H NMR (400 MHz, CDCl3) δ7.89–7.82 (m, 4H, ArH), 7.54–7.46 (m, 2H, ArH), 7.41–7.35 (m, 4H, ArH), 7.17 (d, J = 3.7 Hz, 1H), 6.50 (d, J = 3.7 Hz, 1H), 6.39 (dd, J1’,2’b = 6.8 Hz, J1’,2’a = 8.5 Hz, 1H, H-1’), 5.25 (app dt, J3’,2’a = 6.2 Hz, J3’,2’b = J3’,4’ = 2.2 Hz, 1H, H-3’), 4.35–4.29 (m, 2H, H-5’a, H-5’b), 4.24–4.19 (m, 1H, H-4’), 3.79 (s, 3H, OCH3), 2.67–2.53 (m, 2H, (CH3)2CH), 2.48–2.39 (m, 1H, H-2’a), 2.33–2.24 (m, 1H, H-2’b), 1.25–1.16 (m, 12H, (CH3)2CH).
Figure imgf000156_0001
[397] Synthesis of deaza-dGTP-FL-3: To a solution of deaza-dGTP-FL-2 (425 mg, 0.81 mmol) in DMF (8.0 mL) was added NIS (200 mg, 0.892 mmol) at room temperature. After stirring overnight, the mixture was diluted with EtOAc, and washed with saturated NaHCO3(aq). The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (Hexanes–EtOAc, 10:1 to 3:1) to obtain 7-dN- dGTP-FL-3 (330 mg, 63%) as yellowish foam. Rf 0.42 (3:1, Hexanes–EtOAc), 1HNMR (400 MHz, CDCl3) δ 7.84–7.72 (m, 4H, ArH), 7.52–7.43 (m, 2H, ArH), 7.41–7.31 (m, 4H, ArH), 7.28 (s, 1H), 6.31 (dd, J = 6.2 Hz, 8.0 Hz, 1H, H-1’), 5.24–5.19 (m, 1H, H-3’), 4.33– 4.27 (m, 2H, H-5’a, H-5’b), 4.22–4.17 (m, 1H, H-4’), 3.77 (s, 3H, OCH3), 2.59 (heptet, J = 6.9 Hz, 2H, (CH3)2CH), 2.35–2.19 (m, 2H, H-2’a, H-2’b), 1.21–1.19 (m, 6H, (CH3)2CH), 1.19–1.16 (m, 6H, (CH3)2CH); HRMS (ESI) m/z [M] calcd for C34H35IN4O8754.1518; Found [M + H]+ 755.1591.
Figure imgf000156_0002
[398] Synthesis of deaza-dGTP-FL-4: To a solution of deaza-dGTP-FL-3 (330 mg, 0.508 mmol) in CH3OH (8.0 mL) was added 0.5M CH3Ona in CH3OH (2 mL) at room temperature. After stirring 1.5 hours, the mixture was neutralized by addition of AcOH (100 uL) and then concentrated. The residue was purified by column chromatography (Hexanes–EtOAc, 3:1 to 100% EtOAc) to obtain deaza-dGTP-FL-4 (240 mg, 75%) as white solid. Rf 0.23 (100% EtOAc); 1HNMR (400 MHz, CD3OD) δ 8.01–7.95 (m, 2H, ArH), 7.66–7.59 (m, 2H, ArH, H- 6), 7.57–7.50 (m, 2H, ArH), 6.66 (dd, J = 6.7, 6.6 Hz, 1H, H-1’), 4.59–4.51 (m, 1H, H-3’), 4.10 (s, 3H, OCH3), 3.99–3.94 (m, 1H, H-4’), 3.79 (dd, J = 3.8, 12.0 Hz, 1H, H-5’a), 3.73(dd, J = 4.4, 12.0 Hz, H-5’b), 2.68–2.57 (m, 1H, H-2’a), 2.38(ddd, J = 3.4, 6.1, 13.4 Hz, H-2’b); HRMS (ESI) m/z [M] calcd for C19H19IN4O5510.0409; Found [M + H]+ 511.0482.
Figure imgf000157_0001
[399] Synthesis of deaza-dGTP-FL-5: To a solution of deaza-dGTP-FL-4 (200 mg, 0.392 mmol) in DMF (5.0 mL) was CuI (15 mg, 0.095 mmol) and Et3N (0.5 mL) at room temperature. After stirring at room temperature for 5 min under N2, the mixture was added Alkyne (177.5 mg, 1.18 mmol) and Pd(PPh3)4 (45 mg, 0.039 mmol) at room temperature. After stirring 24 hours, the mixture was concentrated and the residue was purified by column chromatography (Hexanes–EtOAc, 1:1 to Hexanes–EtOAc–CH3OH, 2:2:1) to obtain deaza- dGTP-FL-5 (185 mg, 88%) as yellowish oil. Rf 0.11 (2:2:1, Hexanes–EtOAc–CH3OH); 1HNMR (400 MHz, CD3OD) δ 8.03–7.91 (m, 2H, ArH), 7.71–7.50 (m, 4H, ArH, H-6), 6.64 (dd, J = 6.7, 6.6 Hz, 1H, H-1’), 4.58–4.52 (m, 1H, H-3’), 4.33 (s, 2H, CH2N), 4.10 (s, 3H, OCH3), 3.99–3.94 (m, 1H, H-4’), 3.79 (dd, J = 3.8, 12.0 Hz, 1H, H-5’a), 3.73 (dd, J = 4.4, 12.0 Hz, H-5’b), 2.67–2.57 (m, 1H, H-2’a), 2.39 (ddd, J = 3.4, 6.1, 13.4 Hz, H-2’b); HRMS (ESI) m/z [M] calcd for C24H22F3N5O6533.1534; Found [M + Na]+ 556.1428.
Figure imgf000157_0002
[400] Synthesis of deaza-dGTP-FL-6: To a solution of deaza-GTP-FL-5 (220 mg, 0.412 mmol) in pyridine (2.0 mL) was added TBDPSCl (118 uL, 0.454 mmol) at 0 °C, and slowly warmed to room temperature. After stirring 72 hours, the mixture was quenched by addition of CH3OH (5 mL) and then concentrated. The residue was diluted with EtOAc, and then washed with 2N HCl and saturated NaHCO3. The organic layer was dried over Na2SO4 and then filtered and concentrated. The residue was purified by column chromatography (Hexanes–EtOAc, 4:1 to 1:1) to obtain deaza-dGTP-FL-6 (235 mg, 74%) as white foam. Rf 0.34 (1:1, Hexanes–EtOAc); 1HNMR (400 MHz, CD3OD) δ 7.98–7.90 (m, 2H, ArH), 7.74– 7.31 (m, 14H, ArH, H-6), 6.65 (dd, J = 6.7, 6.6 Hz, 1H, H-1’), 4.71–4.64 (m, 1H, H-3’), 4.33 (s, 2H, CH2N), 4.10 (s, 3H, OCH3), 4.05–3.98 (m, 1H, H-4’), 3.96–3.83 (m, 2H, H-5’a, H- 5’b), 2.70–2.54 (m, 1H, H-2’a), 2.49–2.37 (m, 1H, H-2’b), 1.06 (s, 9H, (CH3)3C); HRMS (ESI) m/z [M] calcd for C40H40F3N5O6Si 771.2713; Found [M + H]+ 772.2789.
Figure imgf000158_0001
[401] Synthesis of deaza-dGTP-FL-7: To a solution of deaza-dGTP-6 (180 mg, 0.233 mmol) in DMSO (6.0 mL) was added AcOH (1.5 mL) and Ac2O (3 mL) at room temperature. After stirring 24 hours, the mixture was quenched by addition of Sat. NaHCO3(aq). The mixture was stirred for 30 min and was diluted with EtOAc, and then washed with saturated NaHCO3. The organic layer was dried over Na2SO4 and then filtered and concentrated. The residue was purified by column chromatography (Hexanes–EtOAc, 4:1 to 1:1) to obtain deaza-dGTP-FL-7 (154 mg, 79%) as white foam. Rf 0.67 (1:1, Hexanes–EtOAc); 1HNMR (400 MHz, CD3OD) δ 7.97–7.90 (m, 2H, ArH), 7.70–7.32 (m, 14H, ArH, H-6), 6.58 (dd, J = 6.2, 7.3 Hz, 1H, H-1’), 4.83–4.78 (m, 1H, H-3’), 4.77–4.67 (m, 2H, CH2S), 4.30 (s, 2H, CH2N), 4.12–4.04 (m, 4H, H-4’, OCH3), 3.88 (dd, J = 4.2, 11.9 Hz, 1H, H-5’a), 3.81 (dd, J = 4.2, 11.9 Hz, 1H, H-5’b), 2.69–2.58 (m, 1H, H-2’a), 2.50 (ddd, J = 3.1, 6.2, 13.7 Hz, 1H, H- 2’b), 2.12 (s, 3H, SCH3), 1.06 (s, 9H, (CH3)3C); HRMS (ESI) m/z [M] calcd for C42H44F3N5O6Ssi 831.2751; Found [M + H]+ 832.2826.
Figure imgf000159_0001
[402] Synthesis of deaza-dGTP-FL-8: To a solution of deaza-dGTP-FL-7 (154 mg, 0.185 mmol) in CH2Cl2 (2.0 mL) was added cyclohexene (500 uL) and 1M sulfuryl chloride (555 uL, 0.555 mmol) at 0 °C. After stirring 30 min, the mixture was concentrated. The residue was then dissolved in DMF (2.0 mL) and the mixture was added NaN3 (120 mg, 1.85 mmol) at room temperature. After stirring for 1 hour, the mixture was diluted with EtOAc, and then washed with H2O. The organic layer was dried over Na2SO4 and then filtered and concentrated. The residue was purified by column chromatography (Hexanes–EtOAc, 4:1 to 1:1) to obtain deaza-dGTP-FL-8 (142 mg, 93%) as colorless oil. Rf 0.67 (1:1, Hexanes– EtOAc); 1HNMR (400 MHz, CD3OD) δ 7.96–7.89 (m, 2H, ArH), 7.69–7.29 (m, 14H, ArH, H-6), 6.55 (app t, J = 6.7 Hz, 1H, H-1’), 4.83 (d, J = 9.0 Hz, 1H, CH2N3), 4.78–4.73 (m, 2H, H-3’, CH2N3), 4.30 (s, 2H, CH2N), 4.15–4.06 (m, 4H, H-4’, OCH3), 3.90 (dd, J = 4.2, 11.3 Hz, 1H, H-5’a), 3.84 (dd, J = 4.4, 11.3 Hz, 1H, H-5’b), 2.79–2.69 (m, 1H, H-2’a), 2.55 (ddd, J = 3.7, 6.3, 13.6 Hz, 1H, H-2’b), 1.06 (s, 9H, (CH3)3C); HRMS (ESI) m/z [M] calcd for C41H41F3N8O6Si 826.2897; Found [M + H]+ 827.2973.
Figure imgf000159_0002
[403] Synthesis of deaza-dGTP-FL-9: To a solution of deaza-dGTP-FL-8 (98 mg, 0.121 mmol) in MeOH (2.0 mL) was added NH4F (13.4 mg, 0.363 mmol) at room temperature. After stirring 24 hours, the mixture was concentrated and the residue was diluted with EtOAc, and then washed with H2O. The organic layer was dried over Na2SO4 and then filtered and concentrated. The residue was purified by column chromatography (Hexanes– EtOAc, 4:1 to 1:1) to obtain deaza-dGTP-FL-9 (46 mg, 67%) as colorless oil. Rf 0.27 (1:1, Hexanes–EtOAc); 1HNMR (400 MHz, CD3OD) δ 8.00–7.94 (m, 2H, ArH), 7.67 (s, 1H, H-6), 7.65–7.60 (m, 1H, ArH), 7.57–7.51 (m, 2H, ArH), 6.58 (dd, J = 6.2, 7.4 Hz, 1H, H-1’), 4.86– 4.79 (m, 2H, CH2N3), 4.70–4.64 (m, 1H, H-3’), 4.33 (s, 2H, CH2N), 4.15–4.04 (m, 4H, H-4’, OCH3), 3.83–3.71 (m, 2H, H-5’a, H-5’b), 2.77–2.68 (m, 1H, H-2’a), 2.55 (ddd, J = 3.0, 6.3, 13.6 Hz, 1H, H-2’b); HRMS (ESI) m/z [M] calcd for C25H23F3N8O6588.1705; Found [M + H]+ 589.1776. 6.17. Scheme 30a: Synthesis of NH2-L-3’-O-azidomethyl-7-deaza-dGTP:
Figure imgf000160_0001
[404] Experimental procedure:
Figure imgf000161_0001
[405] Synthesis of 7-dN-dGTP-FL-2: To a suspension of KOH (0.68 g, 8.71 mmol) and TDA-1 (0.1 mL, 0.32 mmol) in CH3CN (30 mL), N-(4-Chloro-5-iodo-7h-pyrrolo[2,3- d]pyrimidin-2-yl)-2,2-dimethylpropionamide (756 mg, 2.00 mmol) was added at room temperature. After stirring for 10 min, 1-Cl sugar (1.02 g, 2.6 mmol) was added over 30 minutes, and continue stirring for 30 min. The insoluble material was filtered off, the precipitate was washed with 15 mL CH3CN, and the filtrate was evaporated to dryness. The residue was purified by column chromatography (dichloromethane) to obtain 7dN-dGTP- FL-2 (1.15 g, 80%) as brown foam. 1HNMR (400 MHz, CD3OD) δ 8.05 (s, 1H), 7.79 (s, 1H,), 7.33 (s, 1H), 7.92 ~ 7.80 (m, 4H),7.22 ~ 7.16 (m, 4H), 6.64 (dd, J = 1.4 Hz, J = 7.6 Hz, 1H), 5.70 (dt, 1H), 4.66 (dt, J = 5.8 Hz, J = 2.3 Hz, 1H, H-3’), 4.56 (dd, J5’a,4’ = 3.4 Hz, Jgem = 12.1 Hz, 1H, H-5’a), 4.48 (dd, J = 3.5 Hz, J = 12.1 Hz, 1H), 2.85 (ddd, J2’a,3’ = 5.9 Hz, J2’a,1’ = 8.6 Hz, Jgem = 14.5 Hz, 1H, H-2’a), 2.71 (ddd, J = 2.3 Hz, J = 5.9 Hz, Jgem = 14.5 Hz, 1H), 2.36 (s, 3H,), 2.34 (s, 3H,) 1.26 (s, 9H).
Figure imgf000161_0002
[406] 7dN-dGTP-FL-2 (0.73 g, 1.00 mmol) was added NaOH in water (2.0 M, 3.0 mL) at room temperature. The solution was heated to reflux, and stirred for 3 hours. TLC indicated that all starting materials consumed. The mixture was cooled to room temperature and neutralized by addition of 2.0 M HCl. The mixture was then evaporated to dryness. The residue was purified by column chromatography (DCM; DCM:MeOH, 95:5; DCM:MeOH, 90:10;) DCM:MeOH, 85:15; to obtain 7-dN-dGTP-FL-3 (220 mg, 55%) as yellowish foam. 1HNMR (400 MHz, CD3OD) δ 7.81 (s, 1H), 7.79 (s, 1H), 7.16 (d, J = 9.0 Hz, 1H), 6.26 (d, J = 3.7 Hz, 1H), 4.35 (dt, J = 5.8 Hz, J = 2.3 Hz, 1H, H-3’), 3.82 (dd, J5’a,4’ = 3.4 Hz, Jgem = 12.1 Hz, 1H, H-5’a), 3.62 (dd, J5’b,4’ = 3.5 Hz, Jgem = 12.1 Hz, 1H, H-5’b), 2.38 (ddd, J2’a,3’ = 5.9 Hz, J2’a,1’ = 8.6 Hz, Jgem = 14.5 Hz, 1H, H-2’a), 2.14 (ddd, J2’b,3’ = 2.3 Hz, J2’b,1’= 5.9 Hz, Jgem = 14.5 Hz, 1H, H-2’b). LRMS (ESI) m/z [M] calcd for C11H13IN4O4392.14; Found [M + NH]+ 393.15.
Figure imgf000162_0001
[407] Synthesis of 7dN-dGTP-FL-4: To a solution of 7dN-dGTP-FL-3(186 mg, 0.5 mmol) in DMF (4.0 mL) was CuI (15 mg, 0.095 mmol) and Et3N (0.5 mL) at room temperature. After stirring at room temperature for 5 min under N2, the mixture was added Alkyne (220.5 mg, 1.5 mmol) and Pd(PPh3)4 (45 mg, 0.039 mmol) at room temperature. After heating at 55 °C for 13 hours, HPLC indicated that the starting nucleoside consumed, the mixture was concentrated and the residue was purified by column chromatography (DCM; DCM:MeOH, 90:10; DCM:MeOH, 85:1)) to obtain 7dN-dGTP-FL-4 (170 mg, 82%) as brown foam. MS (ESI) m/z [M] calcd for C16H16F3N5O5415.11; Found [M + H]+ 416.15.
Figure imgf000162_0002
[408] Synthesis of 7dN-dGTP-FL-5: To a solution of 7dN-dGTP-FL-4(108 mg, 0.25 mmol) in ACN(5.0 mL) was added N,N-dimethylformamide dimethyl acetal 100 mg at room temperature. After stirring at room temperature for 45 min under N2, HPLC indicated that the starting nucleoside consumed, the mixture was concentrated and the residue was purified by column chromatography (DCM; DCM:MeOH, 90:10; DCM:MeOH, 85:1)) to achieved brown foam. 6.18. Example 17: Synthesis of NH2-L-3’-O-N3-dNTP via alternative pathway 6.18.1. Scheme 32: Synthesis of NH2-L-3’-O-N3-dUTP
Figure imgf000163_0001
6.18.1.1 Experimental procedure:
Figure imgf000164_0001
[409] Synthesis of dUTP-FL-1: To a solution of beta-L-deoxy uridine (1 g, 4.38 mmol) in H2O (10 mL) was added sodium azide (854 mg, 13.14 mmol) and N-iodosuccinimide (1.48 g, 6. 57 mmol) then stirred at room temperature for 19 hours. After complete reaction, the reaction was filtered out and washed with water. The remaining filtrate was extracted with DCM x2 and the combined organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (1:10 to 1:3, CH3OH:CH2Cl2) to give dUTL-FL-1 as white solid (1.27 g, 80%). 1HNMR (400 MHz, CD3OD) δ 8.54 (s, 1H), 6.24 (app t, J = 6.5 Hz, 1H), 4.42 (app dt, J =3.4 Hz, J =6.1 Hz, 1H), 3.97–3.93 (m, 1H), 3.84 (dd, J = 12.0 Hz, J = 3.0 Hz, 1H), 3.75 (dd, J = 12 Hz, J = 3.3 Hz, 1H), 2.37–2.18 (m, 2H). HRMS (ESI): m/z [M] calcd for C9H11IN2O5353.9717; Found [M – H]– 352.9644
Figure imgf000164_0002
[410] Synthesis of dUTP-FL-2: To a solution of dUTP-FL-1 (60 mg, 0.17 mmol) in N,N- dimethylformamide (3 mL) was added CuI (6.5 mg, 0.34 mmol) and triethylamine (50 uL) and then stirred at room temperature for few minutes for CuI to be activated. Then, 2,2,2- trifluoro-N-(prop-2-ynyl) acetamide (76.8 mg, 0.51 mmole) and Tetrakis(triphenylphosphine)palladium (0) (19.6 mg, 0.0017 mmol) were added. The reaction was run for 24 h at the room temperature and was concentrated. The resulting residue was purified by column chromatography (1:10:10 to 1:4:4, CH3OH: CH2Cl2: EtOAc) to give dUTP-FL-2 as yellow oil (32 mg, 50%).1HNMR (400 MHz, CD3OD ) δ 8.34 (s, 1H), 6.25 (app t, J = 6.5 Hz, 1H), 4.6 (s, 1H), 4.43-4.39 (m, 1H), 4.29 (s, 2H), 4.42 (app dt, J = 7.0 Hz, J = 7.1 Hz, 1H), 3.97–3.94 (m, 1H), 3.82 (dd, J = 12.0 Hz, J = 3.0 Hz, 1H), 3.75 (dd, J = 12 Hz, J = 3.5 Hz, 1H); HRMS (ESI): m/z [M] calcd for C14H14F3N3O6377.0825; Found [M + H]+ 378.0893
Figure imgf000165_0001
[411] Synthesis of dUTP-FL-3: To a solution of dUTP-FL-2 (590 mg, 1.57 mmol) in N,N-dimethylformamide (10 mL) was added imidazole (160.32 mg, 2.35 mmol) and tert- butyldimethyl chloride (260.29 mg, 1.73 mmol) at 0 °C and then warmed to room temperature. After stirred at room temperature for 10 hours, the solution was added iced water and extracted with ethyl acetate x 2. The combined organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (1:3 to 1:1, Hexanes:EtOAc) to give dUTL-FL-3 as yellow oil (210 mg, 43%). 1HNMR (400 MHz, CDCl3) δ 8.69 (s,1H), 8.11 (s, 1H), 6.19–6.24 (m, 1H), 4.64 (app dt, J =11.9 Hz, J =14.6 Hz, 1H), 4.47 (d, 1H), 4.16–4.05 (m, 1H), 3.92 (dd, J = 2.5 Hz, J = 11.4 Hz, 1H), 3.80 (dd, J = 2.4 Hz, J = 11.5 Hz, 1H), 2.15 (s, 3H), 0.95 (s, 9H), 0.17 (d, 6H); HRMS (ESI): m/z [M] calcd for C20H28F3N3O6Si 491.1699; Found [M + H]+ 492.1778
Figure imgf000165_0002
[412] Synthesis of dUTP-FL-4: To a solution of dUTP-FL-3 (164 mg, 0.46 mmol) in DMSO (1 mL) was added AcOH (200 uL) and acetic anhydride (200 uL) at room temperature. After stirred at room temperature for 24 hours, the solution was added iced water and solid sodium bicarbonate. The water layer was extracted with ethyl acetate x 2. The combined organic layer was dried with Na2SO4, filtered and concentrated. The resulting residue was purified by column chromatography (3:1 to 1:1, Hexanes:EtOAc) to give dUTP- FL-4 as colorless oil (70 mg, 27%). 1HNMR (400 MHz, CDCl3) δ 9.35(s,1H), 7.96 (s, 1H), 7.49 (s, 1H), 6.10 (dd, J = 5.7 Hz, 1H), 4.33–3.32 (m, 1H), 4.24–4.13 (m, 2H), 4.02 (d, 1H), 3.83 (dd, J = 2.4 Hz, J = 11.5 Hz, 1H), 3.75 (dd, J = 2.0 Hz, J = 11.4 Hz, 1H), 2.38–2.33 (m, 1H), 2.01 (s, 3H), 0.78 (s, 9H), 0.01 (d, 6H); HRMS (ESI): m/z [M] calcd for C22H32F3N3O6SSi 551.1733; Found [M + H]+ 552.1825
Figure imgf000166_0001
[413] Synthesis of dUTP-FL-5: To a solution of dUTP-FL-4 (50 mg, 0.63 mmol) in dry CH2Cl2 was added cyclohexene (30 uL, 0.3 mmol) at 4 °C, then sulfurylchoride (1M in CH2Cl2, 0.11 ml, 0.11 mmol) was added dropwise under N2. After 40 min TLC indicated the full consumption of dUTP-FL-4, the solvent was evaporated and the residue was subjected to high vacuum for 20 min. It was then redissolved in dry DMF (1 ml) and treated with NaN3 (30 mg, 0.45 mmol). The resulting suspension was stirred under room temperature for 2 h. The reaction was quenched with CH2Cl2 and the organic layers were washed with sat aq. NaCl solution. After removing the solvent, the resulting yellow gum was redissolved in 2M HCl:Acetonitrile (2:1) and was stirred at room temperature for 30 min. The solvent was removed and the reaction worked up with CH2Cl2 and sat. aq. NaHCO3 solution. The aqueous layer was extracted three times with CH2Cl2. Purification by chromatography on silica (EtOAc:Hexanes 1:1 to 100% EtOAc) gave dUTP-FL-5 as a pale yellow foam. HRMS (ESI): m/z [M] calcd for C15H15F3N6O6 432.1005; Found [M + Na]+ 455.0887 6.18.2. Scheme 33: Synthesis of NH2-L-3’-O-azidomethyl-dCTP
Figure imgf000167_0001
6.18.2.1 Experimental procedure: [414] Synthesis of dCTP-FL-1: To a dried round bottom flask β-L-deoxycytidine (1g, 4.40 mmol), iodine (1.67g, 60 mmol) and mCPBA (0.75g, 4.40 mmol) were dissolved in 15 mL DMF. The reaction was stirred for 2h at room temperature and afterwards evaporated to dryness under reduced pressure. The resultant crude product was purified by column chromatography (1:9, DCM-MeOH) to afford dCTP-FL-1 (0.83g, yield 53%) as an orange solid.1H NMR (500 MHz, CD3OD) δ 8.49 (s, 1H), 6.17 (t, J = 6.3 Hz, 1H), 4.36 (dt, J = 6.3, 4.1 Hz, 1H), 3.92 (dd, J = 6.9, 3.4 Hz, 1H), 3.80 (dt, J = 15.6, 4.9 Hz, 1H), 3.71 (dd, J = 12.0, 3.4 Hz, 1H), 2.34 (ddd, J = 13.6, 6.2, 4.2 Hz, 1H), 2.20–2.07 (m, 1H). HRMS (ESI): m/z [M] calcd for C9H12IN3O4352.9900; Found [M + Na]+ 375.9765. [415] Synthesis of dCTP-FL-2: To a light protected round bottom flask dCTP-FL-1 (650 mg, 1.84 mmol) was added and dissolved in DMF (10 ml). Later, to the above mixture CuI (70 mg, 0.36 mmol), triethylamine (0.5 ml), 2,2,2- trifluoro-N-prop-2-ynyl-acetamide (833.7 mg, 5.52 mmol) and at last Pd (PPh3)4 (212.5 mg, 0.184 mmol) were added and stirred under argon atmosphere for 18 h at room temperature. A bicarbonate resin (100 mg) was added, and the mixture was stirred for a further 1 h. The reaction mixture was filtered through the celite, and filtrate was evaporated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (CH2Cl2: EtOAc: MeOH 4.5:4.5:1,) to afford the desired product as beige solid (490 mg, yield 71%).1H NMR (500 MHz, D2O) δ 8.04 (s, 1H), 6.08 (t, J = 6.5 Hz, 1H), 4.37 – 4.23 (m, 1H), 4.23 (s, 2H), 3.94 (d, J = 4.3 Hz, 1H), 3.73 (dd, J = 12.6, 3.0 Hz, 1H), 3.64 (dd, J = 12.3, 5.0 Hz, 1H), 2.39–2.28 (m, 1H), 2.19–2.13 (m, 1H). HRMS (ESI): m/z [M] calcd for C14H15F3N4O5376.1000; Found [M + H]+ 377.1066. [416] Synthesis of dCTP-FL-3: To a solution of dCTP-FL-2 (300 mg, 0.797 mmol) in anhydrous DMF (5.0 mL) was added with imidazole (82.12 mg, 1.195 mmol) and tert- butyldimethylsilyl chloride (144.14 mg, 0.956 mmol) at 0 °C. The solution was stirred under nitrogen at 0 °C for 2 h. After completion of the reaction (TLC monitoring), cold water (10 mL) was added and extracted with EtOAc (3 x 30 mL). The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by column chromatography (100% EtOAc to EtOAc:MeOH 9.5:0.5) to afford dCTP-FL-3 (210 mg, 54%) as beige solid. 1H NMR (500 MHz, DMSO-d6) δ 9.92 (t, J = 5.3 Hz, 1H), 7.97 (s, 1H), 7.83 (s, 1H), 6.87 (d, J = 22.9 Hz, 1H), 6.11 (dd, J = 7.2, 6.1 Hz, 1H), 5.27 (d, J = 4.1 Hz, 1H), 4.24 (d, J = 5.3 Hz, 2H), 4.17 (td, J = 6.0, 3.1 Hz, 1H), 3.88 (q, J = 2.8 Hz, 1H), 3.81 (dd, J = 11.5, 2.6 Hz, 1H), 3.72 (dd, J = 11.6, 3.1 Hz, 1H), 2.19–2.17 (m, 1H), 1.96–1.88 (m, 1H), 0.86 (s, 9H), 0.07 (s, 6H). HRMS (ESI): m/z [M] calcd for C20H29F3N4O5Si 490.1900; Found [M + Na]+ 513.1751. [417] Synthesis of dCTP-FL-4: To a stirred solution of dCTP-FL-3 (210 mg, 0.428 mmol) in DMSO (6 ml) was added acetic acid (2 ml) and acetic anhydride (2 ml) and stirred at room temperature for 48h. After completion of the reaction, a saturated NaHCO3 solution was added at 0 °C and stirred for 30 min, and the aqueous layer was extracted with EtOAc (3 x 30 ml). The combined organic extract was dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography (1:1, hexanes–EtOAc) to afford dCTP-FL-4 (130 mg, 52%) as a colorless oil.1H NMR (500 MHz, CDCl3) δ 8.32 (s, 1H), 8.14 (s, 1H), 6.15 (t, J = 6.4 Hz,1H), 4.62 (dd, J = 32.9, 11.7 Hz, 2H), 4.42 (dt, J = 5.7, 2.8 Hz, 1H), 4.37 (t, J = 4.2 Hz, 2H), 4.18 (dd, J = 4.8, 2.3 Hz, 1H), 3.94 (dd, J = 11.5, 2.6 Hz, 1H), 3.79 (dd, J = 11.5, 2.2 Hz, 1H), 2.66 (ddd, J = 13.7, 5.9, 2.9 Hz, 1H), 2.57 (s, 3H), 2.13 (s, 3H), 2.02 (s, 1H), 0.88 (s, 9H), 0.10 (s, 6H). HRMS (ESI): m/z [M] calcd for C24H35F3N4O6SSi 592.2000; Found [M + H]+ 593.2071. [418] Synthesis of dCTP-FL-5: To a stirred solution of dCTP-FL-4 (130 mg, 0.67 mmol) in anhydrous CH2Cl2 (6 mL) was added cyclohexene (1 mL) and SO2Cl2 in CH2Cl2 (1.0 M, 0.4 mL) were added. After stirring at 0 °C for 1 h, the volatiles were removed under reduced pressure. To the residue dry DMF (3 mL) and NaN3 (169 mg, 2.61 mmol) were added and stirred at room temperature for 6 h. The reaction mixture was dispersed in cold distilled water (30 mL) and extracted with EtOAc (2 x 30 mL). The combined organic extract was dried over Na2SO4 and concentrated under reduced pressure. To the resulting residue acetonitrile (10 mL) and 2M HCl (3 drops) were added, and the reaction was stirred for 1-2 h at room temperature. The mixture was neutralized by saturated NaHCO3 solution and diluted with EtOAc. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (Hexanes–EtOAc (1:1) to 100% EtOAc to EtOAc-MeOH (19:1) to afford dCTP-FL-5 (25mg, 28%) as an orange oil. 1H NMR (500 MHz, DMSO-d6) δ 9.89 (t, J = 4.8 Hz, 1H), 8.06 (s, 1H), 7.80 (s, 1H), 6.84 (s, 1H), 6.00 (dd, J = 7.3, 6.0 Hz, 1H), 5.11 (s, 1H), 4.76 (s, 2H), 4.25 (dt, J = 6.0, 3.0 Hz, 1H), 4.20 (d, J = 5.1 Hz, 2H), 3.93–3.88 (m, 1H), 3.59–3.47 (m, 1H), 2.32–2.23 (m,1H), 2.14–2.01 (m, 1H). HRMS (ESI): m/z [M] calcd for C15H16F3N7O5431.1200; Found [M + H]+ 432.1237. [419] Synthesis of dCTP-FL-6: dCTP-FL-5 (68 mg, 0.157 mmol) and proton sponge (40 mg, 0.189 mmol) were dried in a vacuum desiccator over P2O5 overnight. To a solution of dCTP-FL-5 and proton sponge in trimethyl phosphate (1 mL) was added POCl3 (30 uL, 0.375 mmol) drop wise at 0 °C. The mixture was stirred at 0 °C for 2 hours and then was added a well-vortexed mixture of tributylammonium pyrophosphate (148 mg) and tributylamine (233.8 uL, 1.26 mmol) in anhydrous DMF (2 mL). The mixture was stirred for 1.5 hours at room temperature and 0.1M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 0.1 M, 5 ml) was then added and the resulting mixture was stirred for 1 hour at room temperature. To the mixture was then added concentrated ammonium hydroxide (5 mL) and stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 20 ml of water. The crude mixture was extracted with CH2Cl2 (2 x 20 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified with anion exchange chromatography on DEAE-Sephadex A- 25 using a gradient of TEAB (pH 8.5; 0.1–0.8 M). The fractions with products (0.3M – 0.6M) were collected and concentrated under reduced pressure. The residue was diluted with ddH2O and subjected to C18 HPLC (100 to 0% A in B over 60 min, where A= 25 mM TEAB in water, B= 25 mM TEAB in ACN). The product (retention time: 11.3 min) was collected and concentrated under reduced pressure to afford dCTP-FL-6 (6.7 mg, yield 7.4 %) as a syrup. 1H NMR (500 MHz, D2O) δ 7.64 (s, 1H), 6.22 (dd, J = 8.5, 5.9 Hz, 1H), 4.78 – 4.72 (m, 2H), 4.70 (s, 2H), 4.55 – 4.51 (m, 1H), 4.25 (t, J = 6.6 Hz, 1H), 4.14 – 4.04 (m, 2H), 2.44 – 2.37 (m, 1H), 2.31 – 2.22 (m, 1H).31P NMR (202 MHz, D2O): δ –10.47 (bs, 1P), –11.23 (d, J = 20.0 Hz, 1P), –22.96 (bs, 1P) 6.18.3. Scheme 34: Synthesis of NH2-L-3’-O-azidomethyl-dGTP
Figure imgf000170_0001
NH2-L-3'-O-azidomethyl-dGTP-FL 6.18.3.1 Experimental procedure: [420] Synthesis of dGTP-FL-1: [421] To a suspension of 2'-deoxyguanosine (2.0 g) in a mixture of acetonitrile (17.5 ml) and water (5 ml) was added N-bromosuccinimide (1.33 g, 7.2 mmol, 1eq) in several portions. The reaction mixture was stirred for 30 min at room temperature. The solvents were removed, and the precipitate was suspended in acetone (15 ml), stirred for 4 hours at room temperature and cooled 48 h at -20°C. The precipitate was collected by filtration, extensively washed with cold acetone, and dried under vacuum to give 2.1 g (82%) of a slightly yellow powder. NMR (500 MHz, ) δ 10.84 (s, 1H), 6.29 (s, 1H), 6.24 (dd, J = 8.2, 6.6 Hz, 1H), 4.53 – 4.48 (m, 1H), 3.95 (dd, J = 6.2, 3.7 Hz, 1H), 3.75 (dd, J = 11.9, 3.9 Hz, 1H), 3.62 (dd, J = 12.0, 4.2 Hz, 1H), 3.10 – 3.03 (m, 1H), 2.15 (dd, J = 13.2, 6.4 Hz, 1H). [422] Synthesis of dGTP-FL-2: [423] dGTP-FL-1 (1.0 g, 2.891 mmol) was dissolved in dry DMF (15 ml). 2,2,2- trifluoro- N-prop-2-ynyl-acetamide (1.56 g, 10.34 mmol), Pd(PPh3)4 (0.33 g, 0.289 mmol), CuI (0.29 g, 1.514 mmol), and Et3N (0.6 ml, 4.33 mmol) were added to the reaction mixture and was stirred at 55 oC under argon. After 3.5 h of stirring, the reaction mixture was evaporated and purified by silica gel column chromatography (CH2Cl2/ethyl acetate/MeOH; 4.5:4.5:1) to get pure product (730 mg, 60%) as a beige solid. NMR (500 MHz, ) δ 10.96 (s, 1H), 10.22 (t, J = 5.4 Hz, 1H), 6.70 (s, 2H), 6.28 – 6.11 (dd, 1H), 4.89 (s, 1H), 4.39 – 4.34 (m, 3H), 3.78 (td, J = 5.6, 3.2 Hz, 2H), 3.63 – 3.60 (m, 1H), 3.49 (d=m, J = 5.7 Hz,1H), 3.05 – 2.99 (m, 1H), 2.09 (ddd, J = 13.1, 6.6, 3.0 Hz,1H). 6.19. Example 18. Synthesis of L-3'-O-nitrobenzyl-dNTP 6.19.1. Synthesis of L-3’-O-nitrobenzyl-dATP
Figure imgf000171_0001
Figure imgf000172_0001
[425] Synthesis of dATP-NB-1: To a solution of beta-L-2’-deoxyadenosine (2.5 g, 10.0 mmol) in DMF (25 mL) was added imidazole (4.5 g, 66 mmol) and TBSCl (4.82 g, 32 mmol) at room temperature. The mixture was stirred at room temperature for overnight, and quenched with the addition of CH3OH (20 mL). The resulting mixture was concentrated in vacuo. The residue was diluted with CH2Cl2 and washed with saturated NaHCO3(aq), then the organic layer was dried over Na2SO4, filtered and concentrated in vacuo. To a solution of the crude residue in DMF (15 mL) was added (Boc)2O (6.55 g, 30 mmol) and DMAP (3.66 g, 30 mmol) at room temperature. The mixture was stirred at room temperature for overnight. The mixture was concentrated under reduced pressure, and the residue was diluted with CH2Cl2, and then washed with saturated NaHCO3(aq). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give dATP-NB-1 (5.6 g, 82%) as white foam. Rf 0.72 (100% EtOAc); 1HNMR (400 MHz, CDCl3) δ 8.85 (s, 1H, H-8), 8.43 (s, 1H, H-2), 6.53 (app t, 1H, J = 6.3 Hz, H-1’), 4.62 (app dt, 1H, J = 5.6, 3.7 Hz, H-3’), 4.05–4.01 (m, 1H, H-4’), 3.89 (dd, 1H, J = 4.2, 11.5 Hz, H-5’a), 3.79 (dd, 1H, J = 3.1, 11.5 Hz, H-5’b), 2.67–2.58 (m, 1H, H-2’a), 2.47 (ddd, 1H, J = 4.1, 6.3, 13.1 Hz, H-2’b), 1.44 (s, 18H, 2 x tBu of Boc), 0.92 (s, 18H, 2 x tBu of TBS), 0.11 (s, 6H, 2 x CH3 of TBS), 0.09 (s, 6H, 2 x CH3 of TBS); HRMS (ESI) m/z [M] calcd for C32H57N5O7Si2679.3797; Found [M + H]+ 680.3812.
Figure imgf000173_0001
[426] Synthesis of dATP-NB-2: To a solution of dATP-NB-1 (5.6 g, 8.24 mmol) in THF (30 mL) was added 1M TBAF (24.7 mL) at room temperature. The mixture was stirred at room temperature for 30 min, and was concentrated in vacuo. The residue was purified by silica gel column chromatography to give dATP-NB-2-diol (2.6 g, 70%) as white foam. Rf 0.35 (100% EtOAc); 1HNMR (400 MHz, CD3OD) δ 8.79 (s, 1H, H-8), 8.73 (s, 1H, H-2), 6.54 (app t, 1H, J = 6.7 Hz, H-1’), 4.65 (app dt, 1H, J = 5.9, 3.3 Hz, H-3’), 4.03–3.98 (m, 1H, H-4’), 3.78 (dd, 1H, J = 3.7, 12.0 Hz, H-5’a), 3.66 (dd, 1H, J = 4.0, 12.0 Hz, H-5’b), 2.83–2.75 (m, 1H, H-2’a), 2.45 (ddd, 1H, J = 3.4, 6.3, 13.5 Hz, H-2’b), 1.33 (s, 18H, 2 x tBu of Boc); To a solution of dATP-NB-2-diol (2.6 g, 4.65 mmol) in DMF (15 mL), was added imidazole (0.78 g, 11.5 mmol) and TBSCl (1.13 g, 7.49 mmol) at 0 °C. The mixture was gradually warmed to room temperature and stirred for two days. The reaction was quenched by water, and the mixture was extracted three times with EtOAc. The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give dATP-NB-2 (3.4 g, 73%) as white foam. Rf 0.27 (1:1, Hexanes–EtOAc); 1HNMR (400 MHz, CDCl3) δ 8.85 (s, 1H, H-8), 8.40 (s, 1H, H-2), 6.56 (app t, 1H, J = 6.4 Hz, H-1’), 4.74– 4.67 (m, 1H, H-3’), 4.11–4.07 (m, 1H, H-4’), 3.90–3.86 (m, 2H, H-5’a, H-5’b), 2.79–2.68 (m, 1H, H-2’a), 2.57 (ddd, 1H, J = 4.3, 6.4, 13.6 Hz, H-2’b), 1.45 (s, 18H, 2 x tBu of Boc), 0.91 (s, 9H, tBu of TBS), 0.11 (s, 3H, CH3 of TBS), 0.10 (s, 3H, CH3 of TBS); HRMS (ESI) m/z [M] calcd for C26H43N5O7Si 565.2932; Found [M + H]+ 566.3025.
Figure imgf000173_0002
[427] Synthesis of dATP-NB-3: To a solution of dATP-NB-2 (1.2 g, 2.21 mmol) in CH2Cl2 (5 mL) was added a solution of TBAOH (0.86 mL, 4.24 mmol, 55% aqueous solution) and NaI (20 mg) in NaOH (1M, 5 mL). To the mixture, a solution of 2-nitrobenzyl bromide (2.3 g, 10.6 mmol) in CH2Cl2 (3 mL) was added dropwise, and the reaction mixture was stirred at room temperature for two hours in the dark. The organic layer was separated and the aqueous layer was washed twice with CH2Cl2. The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (4:1 to 1:1, Hexanes–EtOAc) to give dATP-NB-3 (1.43 g, 96%) as white foam. Rf 0.55 (1:1, Hexanes–EtOAc).
Figure imgf000174_0001
[428] Synthesis of dATP-NB-4: To a solution of dATP-NB-3 (245 mg, 0.35 mmol) in CH2Cl2 (20 mL) was added silica gel 60 (10 g, 100–200 mesh) at room temperature. The mixture was evaporated under reduced pressure to dryness. The residue was heated to 70–80 °C under oil pump vacuum for 5 hours, then the residue was purified by silica gel column chromatography to give dATP-NB-4 (132 mg, 74%) as colorless syrup. Rf 0.46 (100% EtOAc); 1H NMR (400 MHz, CDCl3) δ 8.36 (s, 1H, H-8), 8.15 (s, 1H, H-2), 8.09 (dd, 1H, J = 1.2, 8.7 Hz, ArH), 7.82 (d, 1H, J = 8.0 Hz, ArH), 7.68 (dt, 1H, J = 1.2, 7.7 Hz, ArH), 7.50–7.45 (m, 1H, ArH), 6.50 (app t, 1H, J = 7.1 Hz, H-1’), 6.50 (bs, 2H, NH2), 5.01–4.91 (m, 2H, CH2Ar), 4.45–4.41 (m, 1H, H-3’), 4.32–4.28 (m, 1H, H-4’), 3.91 (dd, 1H, J = 4.6, 11.3 Hz, H-5’a), 3.84 (dd, 1H, J = 3.4, 11.3 Hz, H-5’b), 2.74–2.68 (m, 2H, H-2’a, H-2’b), 0.92 (s, 9H, tBu of TBS), 0.107 (s, 3H, CH3 of TBS), 0.103 (s, 3H, CH3 of TBS); HRMS (ESI) m/z [M] calcd for C23H33N6O5Si 500.2203; Found [M + H]+ 501.2631.
Figure imgf000174_0002
[429] Synthesis of dATP-NB-5: To a solution of dATP-NB-4 (145 mg, 0.28 mmol) in pyridine (2 mL) was added Benzoyl Chloride (43.8 µL, 0.38 mmol) at room temperature. The mixture was stirred for 2 hours. The mixture was diluted with EtOAc, and washed with 1N HCl, saturated NaHCO3 solution and water. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography to give dATP-NB-5 (131 mg, 75%) as colorless syrup. Rf 0.07 (1:2, Hexanes–EtOAc); 1HNMR (400 MHz, CDCl3) δ 9.03 (bs, 1H, NHBz), 8.81 (s, 1H, H-8), 8.35 (s, 1H, H-2), 8.10 (dd, 1H, J = 1.2, 8.3 Hz, ArH), 8.05–8.00 (m, 2H, ArH), 7.82 (dd, 1H, J = 0.9, 8.0 Hz, ArH), 7.69 (dt, 1H, J = 1.2, 7.5 Hz, ArH), 7.64–7.58 (m, 1H, ArH), 7.55–7.46 (m, 3H, ArH), 6.57 (app t, 1H, J = 6.4 Hz, H-1’), 5.04–4.90 (m, 2H, CH2Ar), 4.47–4.42 (m, 1H, H-3’), 4.35–4.31 (m, 1H, H- 4’), 3.93 (dd, 1H, J = 4.3, 11.6 Hz, H-5’a), 3.85 (dd, 1H, J = 3.3, 11.6 Hz, H-5’b), 2.77–2.72 (m, 2H, H-2’a, H-2’b), 0.91 (s, 9H, tBu of TBS), 0.10 (s, 6H, 2x CH3 of TBS; HRMS (ESI) m/z [M] calcd for C30H36N6O6Si 604.2466; Found [M + H]+ 604.2531.
Figure imgf000175_0001
[430] Synthesis of dATP-NB-6: To a solution of dATP-NB-5 (479 mg, 0.79 mmol) in THF (5 mL) was added 1M TBAF (1 mL, 1.0 mmol) at room temperature. The mixture was stirred for 2 hours. The mixture was concentrated and the residue was purified by silica gel column chromatography to give dATP-NB-6 (350 mg, 87%) as colorless syrup. Rf 0.54 (1:1, Hexanes–EtOAc); 1HNMR (400 MHz, CDCl3) δ 9.05 (bs, 1H, NHBz), 8.83 (s, 1H, H-8), 8.35 (s, 1H, H-2), 8.12–8.07 (m, 2H, H-2, ArH), 8.07–8.03 (m, 2H, ArH), 7.79–7.75 (m, 1H, ArH), 7.70 (dt, 1H, J = 1.3, 7.4 Hz, ArH), 7.68–7.62 (m, 1H, ArH), 7.60–7.50 (m, 3H, ArH), 6.37 (dd, 1H, J = 6.6, 9.8 Hz, H-1’), 5.93, dd, 1H, J = 1.9, 11.9 Hz), 5.04 (d, 1H, J = 13.8 Hz, CH2Ar), 4.93 (d, 1H, J = 13.8 Hz, CH2Ar), 4.59 (d, 1H, J = 5.1 Hz), 4.45 (bs, 1H), 4.10–4.03 (m, 1H, H-5’a), 3.89–3.80 (m, 1H, H-5’b), 3.11 (ddd, 1H, J = 5.1, 9.7, 13.8 Hz, H-2’a), 2.61 (dd, 1H, J = 5.2, 13.8 Hz, H-2’b); HRMS (ESI) m/z [M] calcd for C24H22N6O6490.1601; Found [M + H]+ 491.1652. [431] Synthesis of dATP-NB-7: A solution of starting material (40 mg, 0.067 mmol) and proton sponge (17.3 mg, 0.0806 mmol) in trimethyl phosphate (300 uL) was warmed to 50 °C for 10 min, then cooled to 0°C. phosphorous oxychloride (9.42 uL, 0.10 mmol) was added into the above solution. After 2 h, the reaction mixture was added a well-vortexed solution of tributylammonium pyrophosphate (184 mg, 0.336 mmol) and tributylamine (184 uL) in DMF (500 uL). The mixture was stirred for for 1 h, then the mixture was added 0.1M TEAB buffer (pH 8.5, 15 mL). The resulting mixture was stirred for 2 h, then the mixture was added NH3 solution (10 mL). The resulting mixture was stirred for 16 h, then concentrated under reduced pressure. The resulting residue was diluted with H2O, and washed with CH2Cl2. The aqueous layer was concentrated under reduced pressure. The resulting residue was purified by C18 column (0%B to 90%B over 50 min, A: 0.1M TEAB in water/B: ACN). The collected fractions was concentrated and the resulting residue was purified by SAX column (0%B to 80%B over 50 min, A:15% ACN in water; B: 15%ACN in 1M TEAB). After purification, the collected fractions were concentrated under reduced pressure and the resulting residue was redissolved with water and lyophilized. The lyophilized product (12.7 mg) was obtained as colorless syrup. 1H NMR (400 MHz, D2O) δ 8.45 (bs, 1H), 8.17 (bs, 1H), 7.95 (d, 1H, J = 7.9 Hz, ArH), 7.69–7.60 (m, 2H, ArH), 7.51–7.42 (m, 1H, ArH), 6.36 (app t, 1H, J = 7.1 Hz, H-1’), 4.96–4.84 (m, 2H, CH2Ar), 4.59–4.53 (m, 1H), 4.45–4.38 (m, 1H), 4.19–4.02 (m, 2H, H-5’a, H-5’b), 2.75–2.61 (m, 2H, H-2’a, H-2’b); 31P NMR (121.4 MHz, D2O) δ –10.8 (bs, 1P), –11.5 (bs, 1P), –23.3 (bs, 1P); HRMS (ESI) m/z [M] calcd for C17H21N6O14P3626.0329; Found [M – H]– 625.0897. 6.19.2. Synthesis of L-3’-O-nitrobenzyl-dGTP
Figure imgf000176_0001
Figure imgf000177_0001
[433] Synthesis of dGTP-NB-1: To a solution of beta-L-2’-deoxyguanosine (1.0 g, 3.74 mmol) in DMF (10 mL) was added imidazole (1.68 g, 24.7 mmol) and TBSCl (1.8 g, 12.0 mmol) at room temperature. The mixture was stirred at room temperature for overnight, and quenched with the addition of CH3OH (20 mL). The resulting mixture was extracted with EtOAc and water and the organic layer was dried over Na2SO4, filtered and concentrated in vacuo. To a solution of the crude residue in DMF (10 mL) was added (Boc)2O (4.08 g, 15.5 mmol) and DMAP (1.64 g, 15.5 mmol) at room temperature. The mixture was stirred at room temperature for overnight. The mixture was concentrated under reduced pressure, and the residue was diluted with CH2Cl2, and then washed with saturated NaHCO3(aq). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give dGTP-NB-1 (1.3 g, 44%) as white foam; 1HNMR (400 MHz, CDCl3) δ 8.24 (s, 1H, H-8), 6.43 (app t, 1H, J = 6.2 Hz, H-1’), 4.56 (app dt, 1H, J = 5.3, 4.0 Hz, H-3’), 4.01–3.96 (m, 1H, H-4’), 3.87 (dd, 1H, J = 4.0, 11.5 Hz, H-5’a), 3.76 (dd, 1H, J = 3.1, 11.5 Hz, H-5’b), 2.53–2.35 (m, 2H, H-2’a, H-2’b), 1.71 (s, 9H, tBu of Boc), 1.40 (s, 18H, 2 x tBu of Boc), 0.92 (s, 9H, tBu of TBS), 0.90 (s, 9H, tBu of TBS), 0.11–0.06 (m, 12H, 4 x CH3 of TBS).
Figure imgf000178_0001
[434] Synthesis of dGTP-NB-2: To a solution of dGTP-NB-1 (530 mg, 0.67 mmol) in THF (5 mL) was added 1M TBAF (2 mL, 2.0 mmol) at room temperature. The mixture was stirred at room temperature for 30 min, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (100% EtOAc) to give dGTP-NB- 2-diol (340 mg, 90%) as white solid. 1HNMR (400 MHz, CDCl3) δ 7.94 (s, 1H, H-8), 6.31 (dd, 1H, J = 5.6, 9.1 Hz, H-1’), 4.96 (dd, 1H, J = 2.9, 10.8 Hz), 4.78 (bs, 1H), 4.16 (bs, 1H), 3.92–3.84 (m, 1H, H-5’a), 3.78–3.70 (m, 1H, H-5’b), 3.10–3.01 (m, 1H, H-2’a), 2.32 (ddd, 1H, J = 1.1, 5.4, 13.2 Hz, H-2’b), 1.92 (d, 1H, J = 3.1 Hz, OH), 1.71 (s, 9H, tBu of Boc), 1.43 (s, 18H, 2 x tBu of Boc). To a solution of dGTP-NB-2-diol (340 mg, 0.6 mmol) in DMF (5 mL) was added imidazole (61.2 mg, 0.9 mmol) and TBSCl (108 mg, 0.72 mmol) at 0°C. The mixture was stirred at 0°C and gradually warm to room temperature and stirred for 5 additional hours. The mixture was quenched with CH3OH, then concentrated under reduced pressure. The residue was diluted with EtOAc and washed with H2O. The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give dGTP-NB-2 (337 mg, 82%) as white foam; 1HNMR (400 MHz, CDCl3) δ 8.20 (s, 1H, H-8), 6.46 (app t, 1H, J = 6.4 Hz, H-1’), 4.68–4.61 (m, 1H, H-3’), 4.07–4.02 (m, 1H, H-4’), 3.90–3.84 (m, 2H, H-5’a, H-5’b), 2.66– 2.58 (m, 1H, H-2’a), 2.57 (ddd, 1H, J = 4.2, 6.4, 13.5 Hz, H-2’b), 1.70 (s, 9H, tBu of Boc), 1.40 (s, 18H, 2 x tBu of Boc), 0.91 (s, 9H, tBu of TBS), 0.11 (s, 3H, CH3 of TBS), 0.10 (s, 3H, CH3 of TBS).
Figure imgf000179_0001
[435] Synthesis of dGTP-NB-3: To a solution of dGTP-NB-2 (337 g, 0.494 mmol) in CH2Cl2 (5 mL) was added a solution of TBAOH (0.21 mL, 0.989 mmol, 55% aqueous solution) and NaI (4 mg) in NaOH (1M, 1 mL). To the mixture, a solution of 2-nitrobenzyl bromide (534 mg, 2.47 mmol) in CH2Cl2 (3 mL) was added dropwise, and the reaction mixture was stirred at room temperature for two hours in the dark. The organic layer was separated and the aqueous layer was washed twice with CH2Cl2. The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (4:1 to 1:1, Hexanes–EtOAc) to give dGTP-NB-3 (320 mg, 75%) as white foam. Rf 0.55 (1:1, Hexanes–EtOAc); 1HNMR (400 MHz, CDCl3) δ 8.24 (s, 1H, H-8), 8.10 (dd, 1H, J = 1.2, 8.1 Hz, ArH), 7.81 (dd, 1H, J = 0.8, 8.1 Hz, ArH), 7.69 (app dt, 1H, J = 1.2, 8.1 Hz, ArH), 7.51–7.45 (m, 1H, ArH), 6.48 (dd, 1H, J = 6.6, 7.9 Hz, H- 1’), 4.95 (s, 2H, CH2Ar), 4.41–4.37 (m, 1H, H-3’), 4.30–4.26 (m, 1H, H-4’), 3.90 (dd, 1H, J = 4.3, 11.3 Hz, H-5’a), 3.83 (dd, 1H, J = 3.1, 11.3 Hz, H-5’b), 2.69 (ddd, 1H, J = 2.3, 5.7, 13.4 Hz, H-2’a), 2.59 (ddd, 1H, J = 5.7, 8.0, 13.4 Hz, H-2’b), 1.71 (s, 9H, tBu of Boc), 1.40 (s, 18H, 2 x tBu of Boc), 0.90 (s, 9H, tBu of TBS), 0.11 (s, 3H, CH3 of TBS), 0.11 (s, 3H, CH3 of TBS).
Figure imgf000179_0002
[436] Synthesis of dGTP-NB-4: To a solution of dGTP-NB-3 (320 mg, 0.39 mmol) in CH2Cl2 (30 mL) was added silica gel 60 (10 g, 100–200 mesh) at room temperature. The mixture was evaporated under reduced pressure to dryness. The residue was heated to 70–80 °C under oil pump vacuum for 5 hours, then the residue was purified by silica gel column chromatography to give dGTP-NB-4 (150 mg, 74%) as white solid; 1HNMR (400 MHz, CDCl3) δ 8.02–7.97 (m, 1H, ArH), 7.75–7.68 (m, 2H, ArH, H-8), 7.62–7.54 (m, 1H, ArH), 7.42–7.35 (m, 1H, ArH), 6.17 (app t, 1H, J = 6.2 Hz, H-1’), 5.92 (bs, 2H, NH2), 4.92–4.80 (m, 2H, CH2Ar), 4.31–4.26 (m, 1H, H-3’), 4.19–4.13 (m, 1H, H-4’), 3.78–3.68 (m, 2H, H- 5’a, H-5’b), 2.58–2.43 (m, 2H, H-2’a, H-2’b), 0.90 (s, 9H, tBu of TBS), 0.11 (s, 6H, 2 x CH3 of TBS).
Figure imgf000180_0001
[437] Synthesis of dGTP-NB-6: To a solution of dGTP-NB-4 (150 mg, 0.29 mmol) in THF (2 mL) was added 1M TBAF (0.58 mL, 0.58 mmol) at room temperature. The mixture was stirred at room temperature for 30min. The mixture was evaporated and the residue was dissolved in CH3OH (5 mL). The mixture was added DMF–DMA (0.5 mL) at room temperature, and then heated to 50 °C. The mixture was stirred at 50 °C for 4 hours, then the evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give dGTP-NB-6 (60 mg, 45%) as white solid; 1HNMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 8.07 (s, 1H), 8.05–7.97 (m, 1H, ArH), 7.82–7.75 (m, 1H, ArH), 7.75– 7.69 (m, 1H, ArH), 7.59–7.52 (m, 1H, ArH), 6.34 (dd, 1H, J = 5.8, 8.3 Hz, H-1’), 5.00 (dd, 1H, J = 12.4 Hz, CH2Ar), 4.90 (dd, 1H, J = 12.4 Hz, CH2Ar), 4.46–4.41 (m, 1H, H-3’), 4.24– 4.19 (m, 1H, H-4’), 3.84–3.70 (m, 2H, H-5’a, H-5’b), 3.19 (s, 3H, (CH3)2N), 3.17 (s, 3H, (CH3)2N), 2.82–2.72 (m, 1H, H-2’a) 2.66–2.62 (m, 1H, H-2’b).
Figure imgf000180_0002
[438] Synthesis of dGTP-NB-7: A solution of starting material (60 mg, 0.131 mmol) and proton sponge (42.2 mg, 0.197 mmol) in trimethyl phosphate (300 uL) was warmed to 50 °C for 10 min, then cooled to 0°C. phosphorous oxychloride (9.42 uL, 0.10 mmol) was added into the above solution. After 2 h, the reaction mixture was added a well-vortexed solution of tributylammonium pyrophosphate (368 mg, 0.672 mmol) and tributylamine (368 uL) in DMF (1 mL). The mixture was stirred for for 1 h, then the mixture was added 0.1M TEAB buffer (pH 8.5, 15 mL). The resulting mixture was stirred for 2 h, then the mixture was added NH3 solution (10 mL). The resulting mixture was stirred for 16 h, then concentrated under reduced pressure. The resulting residue was diluted with H2O, and washed with CH2Cl2. The aqueous layer was concentrated under reduced pressure. The resulting residue was purified by C18 column (0%B to 90%B over 50 min, A: 0.1M TEAB in water/B: ACN). The resulting residue was purified by SAX column (0%B to 80%B over 50 min, A:15% ACN in water/15%ACN in 1M TEAB). After purification, the product (18 mg) was obtained as colorless syrup; 1H NMR (400 MHz, D2O) δ 8.10 (bs, 1H, H-8), 8.00–7.91 (m, 1H, ArH), 7.67–7.61 (m, 2H, ArH), 7.50–7.42 (m, 1H, ArH), 6.17 (dd, 1H, J = 5.9, 8.7 Hz, H-1’), 4.94–4.83 (m, 2H, CH2Ar), 4.56–4.51 (m, 1H), 4.40–4.33 (m, 1H), 4.15–4.00 (m, 2H, H-5’a, H-5’b), 2.74–2.65 (m, 1H, H-2’a) 2.62–2.54 (m, 1H, H-2’b); 31P NMR (121.4 MHz, D2O) δ –10.9 (bs, 1P), –11.5 (d, 1P, J = 19.4 Hz), –23.3 (bs, 1P). 6.19.3. Synthesis of L-3’-O-nitrobenzyl-dTTP
Figure imgf000181_0001
Figure imgf000182_0001
[440] Synthesis of dTTP-NB-2: β-L-deoxy thymidine (2.0 g, 8.256 mmol, 1.0 eq.) was solubilized in anhydrous DMF (30 mL). To it, imidazole (843 mg, 12.4 mmol, 1.5 eq.) and tert-butyldimethylsilyl chloride (1.368 g, 9.08 mmol, 1.1 eq.) were added at 0 oC under nitrogen. The reaction mixture was stirred for 4 h at room temperature. Then add cold water and extracted with EtOAc (2 x 20 mL). The combined organic layers were dried on anhydrous Na2SO4, concentrated and the resulting residue was subjected to column chromatography using 5-7% MeOH in CH2Cl2 as eluent to afford dTTP-NB-2 (2.21 g, 75%) as a white solid. NMR (500 MHz, CD3OD) δ 7.60 (q, J = 1.0 Hz, 1H), 6.24 (dd, J = 7.9, 5.9 Hz, 1H), 4.34 (dt, J = 5.5, 2.6 Hz, 1 H), 3.94 (q, J = 2.8 Hz, 1H), 3.90 – 3.86 (m, 1H), 3.83 – 3.79 (m, J = 11.4, 3.2 Hz, 1H), 2.26 – 2.21 (m, J = 13.4, 5.9, 2.6 Hz, 1H), 2.11 (ddd, J = 13.6, 8.0, 6.0 Hz, 1H), 1.85 (d, J = 1.2 Hz, 3H), 0.92 (s, 9H), 0.12 – 0.09 (m, 6 H). ES-MS (ESI) m/z [M + H]+ calcd for C16H29N2O5Si+ 357.1840; Found 357.1720
Figure imgf000182_0002
[441] Synthesis of dTTP-NB-3: dTTP-NB-2 (1.6 g, 4.49 mmol, 1.0 eq.) was solubilized in dry pyridine (16 mL) at 0 oC under nitrogen atmosphere. To it, TMSCl (1.7 ml, 13.4 mmol, 3 eq.) was added. The reaction mixture was allowed to room temperature slowly and stirred for 3 h. After completion of reaction, evaporated under reduced pressure and immediately sealed under nitrogen. 15 ml of DMA added to reaction mixture and followed by the addition of triethyl amine (5.2 ml, 35.9 mmol, 8 eq) at 0°C followed by PhCOCl (1.05 ml, 8.98 mmol, 2 eq). The reaction was stirred for 4 h. After completion of reaction (TLC monitored), reaction mixture was chilled to 0 °C, brine was added and stirred vigorously for another 3 hours. Reaction mixture was extracted with EtOAc thrice. The combined organic layers were dried on anhydrous Na2SO4, concentrated, and the resulting residue was subjected to column chromatography using 30% EtOAc/Hex as eluent to afford dTTP-NB-3 (1.2 g, 58%) after concentration as a white solid. NMR (500 MHz, ) δ 7.94 – 7.90 (m, 2H), 7.76 (q, J = 1.0 Hz, 1H), 7.72 – 7.67 (m, 1H), 7.55 – 7.51 (m, 2H), 6.22 (dd, J = 7.7, 6.0 Hz, 1H), 4.39 – 4.34 (m, 1H), 3.97 (q, J = 2.9 Hz, 1H), 3.94 – 3.89 (m, 1H), 3.87 – 3.81 (m, 1H), 2.33 – 2.27 (m, 1H), 2.24 – 2.17 (m, 1H), 1.92 (d, J = 1.2 Hz, 3H), 0.94 (s, 9H), 0.14-0.13 (m, 6H). ES-MS (ESI) m/z [M + H]+ calcd for C23H33N2O6Si+ 461.2103; Found 461.2145.
Figure imgf000183_0001
[442] Synthesis of dTTP-NB-4: To a stirred solution of dTTP-NB-3 (1.18 g, 2.56 mmol, 1.0 eq.) in DCM (24 mL) was added 2-nitrobenzylbromide (718 mg, 3.32 mmol, 1.3 eq) solubilized in dry DCM (20 mL) under nitrogen atmosphere at 0 °C with exclusion of light. To it, 1M aq. NaOH (10ml) and Bu4NOH (60%; 10 ml) were added with strictly maintaining the mentioned order of addition. The reaction mixture was allowed to stirred for 3 h. The reaction mixture was diluted with water and then extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4. After filtration and concentration, the resulting crude product was further purified with flash column chromatography (15% ethyl acetate in hexane) to afford 800 mg of yellow sticky material of dTTP-NB-4 and dTTP-NB-5 (in 2:1) mixture as indicated by LC-MS. Without further purification, the mixture was directly used as starting material in the next step without further purification. ES-MS (ESI) m/z [M + H]+ calcd for C30H38N3O8Si+ 596.2423; Found 596.2468.
Figure imgf000184_0001
[443] Synthesis of dTTP-NB-7: 30% Ammonium hydroxide solution (1.44 ml; 12.32 mmol) was added to the mixture of dTTP-NB-4 and dTTP-NB-5 (1.18 g) in ethanol (15 ml). The reaction mixture was stirred for 1 h at room temperature with exclusion of light, and then subjected to evaporation. The residue was extracted with CH2Cl2 (3 x 50 mL3). The organic layers were combined and washed with brine, dried over anhydrous Na2SO4. After concentration, the residue was further purified with flash column chromatography (40% to 60% ethyl acetate in hexane) to afford dTTP-NB-7 as yellow solid (330 mg, 90% yield). H- NMR (500 MHz, CDCl3) δ 8.64 (broad s, 1H ), 8.07 (dd, J = 8.2, 1.2 Hz, 1H), 7.78 (dd, J = 7.8, 0.9 Hz, 1H), 7.66 (td, J = 7.7, 1.3 Hz, 1H), 7.51 – 7.49 (m, 1H), 7.48 – 7.43 (m, 1H), 6.34 (dd, J = 8.8, 5.4 Hz, 1H), 4.94 – 4.86 (m, 2H), 4.25 – 4.19 (m, 2H), 3.93-3.88 (m, 1H), 3.83 – 3.79 (m, 1H), 2.51 (ddd, J = 13.5, 5.4, 1.4 Hz, 1H), 2.05 – 1.99 (m, 1H), 1.91 (d, J = 1.2 Hz, 3H), 0.91 (s, 1H), 0.11 – 0.10 (m, J = 2.4 Hz, 6H). ES-MS (ESI) m/z [M + H]+ calcd for C23H34N3O7Si+ 492.2161; Found 492.2323.
Figure imgf000184_0002
[444] Synthesis of dTTP-NB-8: To a solution of dTTP-NB-7 (330 mg; 0.67 mmol) in 8 mL of anhydrous THF at 0 °C, TBAF in THF solution (1.0 M; 2.78 ml; 2.78 mmol) was added. The reaction mixture was allowed to warm to room temperature and continue stirring for 2 h with exclusion of air and light. The mixture was poured into cold water (50 ml), and the resulting mixture was extracted 10% MeOH/DCM (3 x 50 mL). The organic layers were combined, washed with brine and dried over anhydrous Na2SO4. After concentration, the residue was purified with flash column chromatography (1-7% CH3OH in CH2Cl2) to afford dTTP-NB-8 (170 mg, 67% yield) as white solid: 1H NMR (500 MHz,, CDCl3) 8.64 (br s, 1H), 8.05 (dd, J = 8.2, 1.1 Hz, 1H), 7.76 – 7.70 (m, 1H), 7.65 (td, J = 7.6, 1.2 Hz, 1H), 7.49 – 7.43 (m, 1H), 7.40 (d, J = 1.2 Hz, 1H), 6.15 (dd, J = 7.9, 6.2 Hz), 4.90 (q, J = 14.1 Hz, 2H), 4.36 (dt, J = 5.8, 2.8 Hz, 1H), 4.18 (q, J = 2.8 Hz, 1H), 3.98-3.92 (m, 1H), 3.85-3.78 (m, 1H), 2.75 – 2.55 (m, 1H), 2.48 – 2.35 (m, 1H), 1.91 (d, J = 1.2 Hz, 3H). ES-MS (ESI) m/z [M + H]+ calcd for C17H20N3O7Si+ 378.1296; Found 378.1306.
Figure imgf000185_0001
[445] Synthesis of dTTP-NB-9: A solution of starting material (60 mg, 1.0 eq, 0.159 mmol) and proton sponge (41 mg, 1.2 eq) were dried in vacuum oven under P2O5 for overnight. Later, the reaction mixture was sealed under nitrogen and trimethyl phosphate (0.53 µL) was added and made the solution was completely dissolved, then cooled to 0°C. POCl3 (22.3 µL, 1.5 eq) was added into the above solution and stirred for 1 h at the same temperature. After 1 h, a well-vortexed solution of tributylammonium pyrophosphate (329 mg, 0.9 mmol) and tributylamine (0.29 µL) in DMF (1.2 mL) was added to the reaction mixture and stirred for 20 min at RT. Then the mixture was added 0.1M TEAB buffer (pH 8.0, 5 mL) and stirred for 2 h. After that NH3 solution (5 mL) was added and the resulting mixture was stirred for another 3 h, then concentrated under reduced pressure. The resulting residue was purified by C18 column (0%B to 90%B over 50 min, A: 0.1M TEAB in water/B: ACN). The collected fractions were concentrated and the resulting residue was purified by SAX column (0%B to 80%B over 50 min, A:15% ACN in water; B: 15%ACN in 1M TEAB). After purification, the collected fractions were concentrated under reduced pressure and the resulting residue was re-dissolved with water and lyophilized. The lyophilized product (33 mg) was obtained as white foam.1H NMR (400 MHz, D2O) δ 7.97 (d, J = 8.6 Hz, 1H), 7.70 – 7.62 (m, 3H), 7.51 – 7.46 (m, 1H), 6.23 (dd, J = 9.2, 5.6 Hz, 1H), 4.88 (q, J = 12.8 Hz, 2H), 4.47 (d, J = 5.5 Hz, 1H), 4.32 – 4.27 (m, 1H), 4.16 – 4.05 (m, 2H), 2.41 (dd, J = 13.8, 6.0 Hz, 1H), 2.29 – 2.18 (m, 1H), 1.83 (d, J = 0.8 Hz, 3H); 31P NMR (121.4 MHz, D2O) δ –7.33 (d, 1P), –10.89 (d, 1P), –21.77 (t, 1P); HRMS (ESI) m/z [M] calcd for C17H21N3O16P3 616.0140; Found [M – H]– 616.0145. 6.19.4. Synthesis of L-3’-O-nitrobenzyl-dCTP
Figure imgf000186_0001
[446] Experimental Procedure:
Figure imgf000187_0001
[447] Synthesis of dCTP-NB-1: To a stirred solution of beta-L-2’-deoxycytidine 1 (0.26 g; 1.40 mmol) in dry pyridine (9 mL), TBDMSCl (212mg; 1.28 mmol) was added and the mixture was stirred at room temperature for 20 h. After evaporation, the residue was purified by flash column chromatography using CH3OH–CH2Cl2 (1:10) as the eluent to afford dCTP- NB-2 as white solid (300 mg, 76% yield). 1H-NMR (500 MHz, CD3OD) δ 8.19 (d, J = 7.7 Hz, 1H), 6.19 (t, J = 6.2 Hz, 1H), 5.99 (d, J = 7.7 Hz, 1H), 4.36-4.33 (m, 1H), 4.05-3.99 (m, 1H), 3.94-3.90 (m, 1H), 3.85-3.80 (m, 1H), 2.42-2.36 (m, 1H), 2.19-2.11 (m, 1H), 0.91 (s, 9H), 0.14 – 0.09 (m, 6H). ES-MS (ESI) m/z [M + H]+ calcd for C15H28N3O4Si+ 342.1844; Found 342.1863.
Figure imgf000187_0002
[448] Synthesis of dCTP-NB-3: To a stirred solution of dCTP-NB-2 (0.3 g; 0.879 mmol) in MeOH (9 mL), HC(OMe2)NMe2 (0.7 mL, 5.27 mmol) was added and the mixture was stirred at room temperature for 30 min. After evaporation, the residue was purified by wet- column chromatography using CH3OH-CH2Cl2 (1:20) as the eluent to afford dCTP-NB-3 as viscous liquid (0.29 g, 83% yield). 1H NMR (500 MHz,CD3OD) δ 8.65 (s,1H), 8.20 (d, J = 7.3 Hz, 1H), 6.21 (t, J = 6.2 Hz, 1H), 6.10 (d, J = 7.3 Hz, 1H), 4.38 – 4.30 (m, 1H), 3.99-3.95 (dd, J = 6.3, 2.7 Hz, 1H), 3.96 – 3.92 (m, 1H), 3.85 – 3.81 (m, 1H), 3.20 (s, 3H), 3.13-3.07 (m, 1H), 2.49-2.41 (m, 1H), 2.15-2.06 (m, 1H), 0.90 (s, 9H), 0.14-0.09 (m, 6H). ES-MS (ESI) m/z [M + H]+ calcd for C18H33N4O4Si+ 397.2266; Found 397.2440.
Figure imgf000188_0001
[449] Synthesis of NB-dCTP-4: To a stirred solution of NB-dCTP-3 (1.1 g, 2.7 mmol) in CH2Cl2 (100 ml), 2-nitrobenzyl bromide (689 mg, 1.15 mmol) and 20% aq. NaOH solution (50 ml) were added sequentially at RT with the protection from light and air. Later, tetrabutylammonium bromide (TBAB) (447 mg, 0.5 mmol) was added and stirred for 16 h at RT. Then CH2Cl2 (100 mL) was added to the reaction mixture and the organic layer was separated. The aqueous layer was extracted with CH2Cl2 (2 X 100 ml). The combined organic layers were washed with brine solution and dried over anhyd Na2SO4. After evaporation, the resulting residue was purified by flash column chromatography (3% MeOH in CH2Cl2) to afford NB-dCTP-4 (600 mg, 45% yield).1H-NMR (500 MHz,CD3OD) δ 8.00 (dd, J = 8.2, 1.1 Hz, 1H), 7.94 (d, J = 7.5 Hz, 1H), 7.77 – 7.73 (m, 1H), 7.68 (td, J = 7.6, 1.2 Hz, 1H), 7.51 (td, J = 8.2, 1.5 Hz, 1H), 6.26 – 6.20 (m, 1H), 5.85 (d, J = 7.3 Hz, 1H), 4.89 (d, J = 7.1 Hz, 2H), 4.24 – 4.20 (m, 1H), 4.16 (q, J = 2.9 Hz, 1H), 3.91 – 3.87 (m, 1H), 3.85 – 3.80 (m, 1H), 2.57 (ddd, J = 13.7, 5.9, 2.9 Hz, 1H), 2.12 – 2.04 (m, 1H), 0.89 (s, 9H), 0.10 – 0.08 (m, 6H). ES-MS (ESI) m/z [M + H]+ calcd for C22H33N4O6Si+ 477.2164; Found 477.2175.
Figure imgf000188_0002
[450] Synthesis of NB-dCTP-5: To a stirred solution of NB-dCTP-4 (0.15 g, 0.31 mmol) in THF (5 ml) was added 0.62 ml of 1 M TBAF in THF at 0 °C with the protection from light and air. The reaction mixture was stirred for 3 h at 0 °C and volatiles were evaporated on rotavapor. The resulting crude residue was directly subjected to silica gel column chromatography (elution at 8% MeOH in CH2Cl2) to afford NB-dCTP-5 (90 mg, 78%).1H- NMR (500 MHz,CD3OD ) ) δ 8.00 (dd, J = 8.1, 1.2 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.78 – 7.75 (m, 1H), 7.68 (td, J = 7.7, 1.3 Hz, 1H), 7.51 (td, J = 8.2, 1.4 Hz, 1H), 6.23 (dd, J = 7.9, 5.8 Hz, 1H), 5.88 (d, J = 7.5 Hz, 1H), 4.90 (d, J = 4.1 Hz, 1H), 4.27 – 4.21 (m, 1H), 4.13 (q, J = 3.6 Hz, 1H), 3.74 (qd, J = 12.0, 3.7 Hz, 1H), 2.53 (ddd, J = 13.7, 5.8, 2.5 Hz, 1H), 2.11 (ddd, J = 13.9, 7.9, 6.1 Hz, 1H). ES-MS (ESI) m/z [M + H]+ calcd for C16H19N4O6 + 363.1299; Found 363.1311. 6.20. Example 19. Alternative chemical Synthesis of (D)-form terminal deoxynucleotidyl transferase via Solid Phase Peptide Synthesis and Native Chemical Ligation (2nd method) [451] An alternative way to synthesize a terminal deoxynucleotidyl transferase was developed. The method involve synthesis of seven peptides (D-TdT-WT-1 to D-TdT-WT-7) via solid phase peptide synthesis which are the ligated at the certain cysteine residue as bolded and underlined below.
Figure imgf000189_0001
[452] The (D)-form terminal deoxynucleotidyl transferase was synthesized via Solid Phase Peptide Synthesis and Native Chemical Ligation methods described above. Figure 23 shows the convergent fragment assembly strategy for the (D)-form mutant terminal deoxynucleotidyl transferase. [453] Using the methods, D-TdT-WT-1@His6+60-mer: (His)6NSSPSPVPGSQNVPAPAVKKISQYAC(Acm)QRRTTLNNYNQLFTDALDILAEND ELRENEGSCL-NHNH2 (SEQ ID NO: 72). D-TdT-WT-1@His6+60-mer was synthesized on CEM Liberty Blue automated peptide synthesizer by following the conditions mentioned in experimental methods. D-TdT-WT-1@His6+60-mer synthesized, analyzed by HPLC and ESI-MS. The results are provided in Figures. 24A and 24B. [454] Using similar process, D-TdT-WT-2 to D-TdT-WT-7 were synthesized and analyzed. The products were analyzed by HPLC chromatogram and production of the peptide was confirmed.
Figure imgf000190_0001
[455] The seven synthetic peptides were ligated at the certain cysteine residue following the protocol described above. For example, NCL reaction was performed with D-TdT-WT4 + D- TdT-WT5 using method B to yield D-TdT-WT-9@106-mer. [456] Using similar methods, following peptides are synthesized.
Figure imgf000191_0001
7. EQUIVALENTS AND INCORPORATION BY REFERENCE [457] While the various embodiments of the present disclosure has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. [458] All publications, patents, patent applications, and other documents cited within the body of the instant specification, including U.S. Provisional Appl. No. 63/585,885, are hereby incorporated by reference in their entireties, for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes. 8. SEQUENCES [459] This disclosure provides mirror image polypeptides including mirror-image nucleic acid polymerases or terminal deoxynucleotidyl transferases, and precursor fragments thereof, that are suitable for condensation via native chemical ligation. Thus, it is understood that the sequences depicted herein can refer to a D-form polypeptide sequence, even though in general the sequences are depicted using capital letter one-letter codes for the individual amino acid residues.
Figure imgf000191_0002
Figure imgf000192_0001
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001

Claims

WHAT IS CLAIMED IS: 1. A nucleotide or nucleoside comprising: a. a pentose sugar selected from a (3R,4S)-3,4,5-trihydroxypentanal and a (4R)- 4,5-dihydroxypentanal; wherein the H of the 5’ hydroxyl is substituted by one or more phosphate group; and wherein H of the 3' hydroxyl group, if present, is optionally substituted by a cleavable protecting group; b. a nitrogenous base, and c. optionally a cleavable label comprising a cleavable linker and a label; wherein at least one of a cleavable protecting group and a cleavable label is present.
2. The nucleotide of claim 1, wherein the cleavable label is linked to the nitrogenous base, the 3’ O, or the 5’ phosphate group.
3. The nucleotide of claim 1, having a structure according to Formula I:
Figure imgf000198_0001
Formula I wherein Base is a nitrogenous base, and R' is a cleavable protecting group.
4. The nucleotide of claim 1, having a structure according to Formula II
Figure imgf000198_0002
Formula II wherein Base is a nitrogenous base; R' is a cleavable protecting group or H; R2—Label is a cleavable label comprising a cleavable linker R2 and a label.
5. The nucleotide of claim 1, having a structure according to Formula III:
Figure imgf000199_0003
Formula III wherein Base is a nitrogenous base; R2—Label is a cleavable label comprising a cleavable linker R2 and a label.
6. The nucleotide of claim 1, 2, 4, or 5 wherein the label is selected from the group consisting of:
Figure imgf000199_0001
7. The nucleotide of any one of claims 1-4, wherein the cleavable protecting group is selected from an allyl, a dimethyl disulfide, a nitrobenzyl, and an azido protecting group.
8. The nucleotide of claim 7, wherein the cleavable protecting group is selected from:
Figure imgf000199_0002
.
9. The nucleotide of claim 1, 2, 4, or 5, wherein the cleavable linker is photocleavable, is cleaved by contact with water-soluble phosphines, or is cleaved by water-soluble transition metal-containing catalysts.
10. The nucleotide of claim 9, wherein the cleavable linker comprises an allyl or an azido group.
11. The nucleotide of claim [139], wherein the cleavable linker comprises:
Figure imgf000200_0001
.
12. The nucleotide of claim 3, selected from: - H2N -
Figure imgf000201_0001
and
Figure imgf000201_0002
wherein the R’ is selected from:
Figure imgf000202_0002
Figure imgf000202_0003
13. The nucleotide of claim 4, selected from:
Figure imgf000202_0001
Figure imgf000203_0001
and
Figure imgf000203_0002
; wherein the R’ is selected from:
Figure imgf000203_0003
, ; and R2 comprises:
Figure imgf000203_0004
, or
Figure imgf000203_0005
14. The nucleotide of claim 5, selected from:
Figure imgf000204_0001
Figure imgf000205_0001
and ;
Figure imgf000205_0002
wherein R2 comprises:
Figure imgf000206_0002
, or
Figure imgf000206_0003
15. The nucleotide of claim 4, selected from:
Figure imgf000206_0001
Figure imgf000207_0001
and
Figure imgf000207_0002
; wherein the R’ is selected from:
Figure imgf000208_0002
,
Figure imgf000208_0003
16. The nucleotide of claim 5, selected from:
Figure imgf000208_0001
Figure imgf000209_0001
and
Figure imgf000210_0001
; wherein the R’ is selected from:
Figure imgf000210_0002
,
Figure imgf000210_0003
17. A mirror-image nucleic acid polymerase comprising a sequence having at least 90% sequence identity to SEQ ID NO: 1, wherein the polymerase comprises D-form amino acids.
18. The polymerase of claim 17, wherein the polymerase consists of D-form amino acids.
19. The polymerase of claim 17 or 18, comprising a sequence having at least 95% sequence identity to SEQ ID NO: 1.
20. The polymerase of claim 19, comprising a sequence having at least 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1.
21. The polymerase of any one of claims 17-20, comprising one or more modifications at one or more amino acid sites selected from E276, K317, N424, and S651 compared to SEQ ID NO: 1.
22. The polymerase of claim 21, comprising one or more substitutions selected from E276A, K317G, N424A, and S651A compared to SEQ ID NO: 1.
23. The polymerase of claim 22, comprising E276A, K317G, N424A, and S651A substitutions compared to SEQ ID NO: 1.
24. The polymerase of any one of claims 17-23, comprising one or more modifications at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744 compared to SEQ ID NO: 1.
25. The polymerase of claim 24, wherein comprising one or more substitutions from Ile to Ala, Val, Leu, or Tyr at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744 compared to SEQ ID NO: 1.
26. The polymerase of claim 25, comprising one or more substitutions selected from I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO: 1.
27. The polymerase of claim 26, comprising substitutions of I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO: 1.
28. The polymerase of any one of claims 17-27, comprising one or more modifications at one or more amino acid sites selected from M129, I130, G131, D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1.
29. The polymerase of claim 28, comprising one or more modifications at one or more amino acid sites selected from D141, E143, Y409, and A485 compared to SEQ ID NO: 1.
30. The polymerase of claim 29, comprising one or more substitutions selected from D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1.
31. The polymerase of claim 30, comprising substitutions of D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1.
32. The polymerase of claim 28, comprising one or more modifications at one or more amino acid sites selected from D141, E143, L408, Y409, P410, A485, T514, and I521 compared to SEQ ID NO: 1.
33. The polymerase of claim 32, comprising one or more substitutions selected from D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1.
34. The polymerase of claim 33, comprising substitutions of D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1.
35. The polymerase of claim 28, comprising one or more modifications selected from substitution of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, I521L or addition of D between I130 and G131 compared to SEQ ID NO: 1.
36. The polymerase of claim 35, comprising substitutions of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L and addition of D between I130 and G131 compared to SEQ ID NO: 1.
37. The polymerase of any one of claims 17-36, comprising the sequence selected from SEQ ID NOs: 2-7.
38. The polymerase of claim 37, consisting of the sequence selected from SEQ ID NOs: 2-7.
39. A method of synthesizing a mirror-image nucleic acid polymerase comprising native chemical ligation (NCL) of two or more precursor fragments selected from D-polypeptides having any one of SEQ ID NOs: 37-56.
40. The method of claim 39, further comprising: desulfurization of one or more cysteine residues to one or more alanine residues, optionally wherein the one or more cysteine residues are selected from Cys500, Cys539, Cys595, Cys651 and Cys714.
41. The method of claim 39 or 40, further comprising: Acm deprotection at Cys506 and Cys509.
42. A mirror-image nucleic acid polymerase generated by the method of any one of claims 39-41.
43. A mirror-image nucleic acid polymerase generated by the method described in Example 13.
44. A method of replicating an L-polynucleotide, comprising the step of: incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-dNTP, (iv) the polymerase of any one of claims 17-38, 42 and 43, and (v) a buffer, thereby inducing replication of the L- polynucleotide.
45. The method of claim 44, wherein the L-polynucleotide is DNA or RNA.
46. The method of any one of claims 44-45, wherein the mixture comprises L-dATP, L- dGTP, L-dCTP, and L-dTTP.
47. The method of any one of claims 44-46, wherein the buffer comprises 50 mM Tris- HCl, pH 7.5, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl.
48. The method of any one of claims 44-47, wherein the incubation step comprises PCR.
49. A method of sequencing an L-polynucleotide, comprising the cycle of: a. incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-3’-O-R’-dNTP-R2-Label, (iv) the polymerase of any one of claims 17-38, 42 and 43, and (v) a buffer, thereby obtaining a replication product; b. detecting a signal from the L-3’-O-R’-dNTP-R2-Label incorporated into the replication product; and c. inducing cleavage of the R’ group and R2 group of the L-3’-O-R’-dNTP-R2- Label incorporated into the replication product. 50. The method of claim 49, wherein the cycle is repeated at least 3, 5, 10,
50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.
51. The method of any one of claims 49-50, wherein the L-3’-O-R’-dNTP-R2-Label comprises L-3’-O-R’-dATP-R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2- Label, and L-3’-O-R’-dCTP-R2-Label, wherein each label is different.
52. The method of any one of claims 49-51, wherein the L-3’-O-R’-dNTP-R2-Label has a structure according to Formula II as defined in any one of claims 4, 6-13, and 15.
53. The method of claim 52, wherein the L-3’-O-R’-dNTP-R2-Label is a nucleotide of claim 13 or 15.
54. The method of any one of claims 49-53, wherein the signal is a fluorescent signal.
55. A method of sequencing an L-polynucleotide, comprising the steps of: a. incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-dNTP, (iv) an L-ddNTP-R2-Label or L-3’-O-R’-dNTP-R2-Label, (v) the polymerase of any one of claims 17-38. 42 and 43, and (vi) a buffer, thereby obtaining a replication product; b. separating the replication product; and c. detecting a signal from the L-ddNTP-R2-Label or L-3’-O-R’-dNTP-R2-Label incorporated into the replication product.
56. The method of claim 55, wherein the L-dNTP comprises L-dATP, L-dTTP, L-dGTP, and L-dCTP.
57. The method of claim 55 or 56, wherein the L-ddNTP-R2-Label comprises L-ddATP- R2-Label, L-ddTTP-R2-Label, L-ddGTP-R2-Label, and L-ddCTP-R2-Label, wherein each label is different.
58. The method of any one of claims 55-57, wherein the L-ddNTP-R2-Label has Formula III as defined in any one of claims 5, 6, 9-11.
59. The method of claim 58, wherein the L-ddNTP-R2-Label is the nucleotide of claim 14 or 16.
60. The method of claim 55 or 56, wherein the L-3’-O-R’-dNTP-R2-Label comprises L- 3’-O-R’-dATP-R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, and L-3’- O-R’-dCTP-R2-Label, wherein each label is different.
61. The method of any one of claims 55-56, and 60, wherein the L-3’-O-R’-dNTP-R2- Label has Formula II as defined in any one of claims 4, 6-13, and 15.
62. The method of claim 61, wherein the L-3’-O-R’-dNTP-R2-Label is the nucleotide of claim 13 or 15.
63. The method of any one of claims 55-62, wherein the signal is a fluorescent signal.
64. The method of any one of claims 55-63, wherein the incubation step comprises PCR.
65. The method of any one of claims 55-64, wherein the separation step comprises separating the replication product by size.
66. A method of sequencing an L-polynucleotide, comprising the cycle of: a. incubating a mixture comprising (i) the L-polynucleotide, (ii) an L-primer, (iii) L-3’-O-R’-dNTP, (iv) L-ddNTPs-R2-Label or L-dNTPs-R2-Label , (v) the polymerase of any one of claims 17-38, 42 and 43, and (vi) a buffer, thereby obtaining a replication product; b. detecting a signal from the L-ddNTP-R2-Label or L-3’-O-R’-dNTP-R2-Label incorporated into the replication product; and c. inducing cleavage of i) the R’ group of the L-3’-O-R’-dNTP and R2 group of L-ddNTPs-R2-Label incorporated into the replication product; or ii) the R’ group of the L-3’-O-R’-dNTP, R’ and R2 group of the L-3’-O-R’-dNTP-R2- Label incorporated into the replication product.
67. The method of claim 66, wherein the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.
68. The method of any one of claims 66-67, wherein the L-3’-O-R’-dNTP comprises L- 3’-O-R’-dATP, L-3’-O-R’-dTTP, L-3’-O-R’-dGTP, and L-3’-O-R’-dCTP.
69. The method of any one of claims 66-68, wherein the L-3’-O-R’-dNTP has a structure according to Formula I as defined in any one of claim 3, 7, and 8.
70. The method of claim 69, wherein the L-3’-O-R’-dNTP is a nucleotide of claim 12.
71. The method of any one of claims 66-70, wherein the L-ddNTP-R2-Label comprises L- ddATP-R2-Label, L-ddTTP-R2-Label, L-ddGTP-R2-Label, and L-ddCTP-R2-Label, wherein each label is different.
72. The method of any one of claims 66-66, wherein the L-ddNTP-R2-Label has a structure according to Formula III as defined in any one of claim 5, 6, and 9-11.
73. The method of claim 72, wherein the L-ddNTP-R2-Label is a nucleotide of claim 14 or 16.
74. The method of any one of claims 66-70, wherein the L-3’-O-R’-dNTP-R2-Label comprises L-3’-O-R’-dATP-R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2- Label, and L-3’-O-R’-dCTP-R2-Label, wherein each label is different.
75. The method of any one of claims 66-71 and 74, wherein the L-3’-O-R’-dNTP-R2- Label has a structure according to has Formula II as defined in any one of claims 4, 6-13, and 15.
76. The method of claim 72, wherein the L-3’-O-R’-dNTP-R2-Label is a nucleotide of claim 13 or 15.
77. The method of any one of claims 66-76, wherein the signal is a fluorescent signal.
78. A mirror-image terminal deoxynucleotidyl transferase comprising a sequence having at least 90% sequence identity to SEQ ID NO: 36, wherein the transferase comprises D-form amino acids.
79. The transferase of claim 78, wherein the transferase consists of D-form amino acids.
80. The transferase of claim 78 or 79, comprising a sequence having at least 95% sequence identity to SEQ ID NO: 36.
81. The transferase of claim 80, comprising a sequence having at least 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 36.
82. The transferase of any one of claims 78-81, comprising one or more modifications at one or more amino acid sites of SEQ ID NO: 36.
83. The transferase of claim 82, comprising two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acid substitutions compared to SEQ ID NO: 36.
84. The transferase of any one of claims 78-83, comprising any one of precursor fragments having a sequence selected from SEQ ID NOs: 57-78 or a modification thereof.
85. A method of synthesizing a mirror-image terminal deoxynucleotidyl transferase comprising native chemical ligation (NCL) of two or more precursor fragments selected from D-polypeptides having any one of SEQ ID NOs: 57-78.
86. The method of claim 85, further comprising desulfurization of one or more cysteine residues to one or more alanine residues.
87. The method of claim 86, wherein the one or more cysteine residues are selected from Cys47, Cys108, Cys162, Cys224, Cys268, and Cys317.
88. The method of claim 85 or 86, further comprising Acm deprotection at Cys26, Cys59, Cys87, Cys173, Cys249, Cys275, and Cys309
89. A mirror-image terminal deoxynucleotidyl transferase generated by the method of any one of claims 85-88.
90. A mirror-image terminal deoxynucleotidyl transferase generated by the method described in Example 14 or 19.
91. A method of synthesizing L-polynucleotide, comprising the step of: incubating a mixture comprising (i) an L-primer, (ii) L-dNTP or L-ddNTP, (iii) the transferase of any one of claims 78-84, 89 and 90 and (iv) a buffer, thereby inducing synthesis of the L- polynucleotide.
92. The method of claim 91, wherein the L-polynucleotide is DNA or RNA.
93. The method of claim 91 or 92, wherein the mixture comprises L-dATP, L-dGTP, L- dCTP or L-dTTP.
94. The method of claim 93, wherein the L-dNTP comprises (i) L-dATP or L-dTTP and (ii) L-dGTP or L-dCTP.
95. The method of claim 93, wherein the L-dNTP comprises L-dATP, L-dGTP, L-dCTP and L-dTTP.
96. The method of claim 91 or 92, wherein the mixture comprises a radio-labeled L- ddNTP, optionally the L-ddNTP is a radio-labeled L-ddATP, L-ddGTP, L-ddCTP or L- ddTTP.
97. The method of claim 96, wherein the mixture comprises L-3’-O-R-dNTP, optionally wherein the L-3’-O-R-dNTP is L-3’-O-R-dATP, L-3’-O-R-dGTP, L-3’-O-R-dCTP or L-3’- O-R-dTTP.
98. The method of claim 91 or 92, wherein the L-ddNTP comprises L-ddNTP-R2-Label or L-3’-O-R’-dNTP-R2-Label.
99. The method of any one of claims 91-98, further comprising the step of stopping reaction by heating or by adding a chelating agent.
100. The method of claim 99, wherein the chelating agent is EDTA.
101. A kit for replication or synthesis of an L-polynucleotide, comprising (i) the polymerase of any one of claims 17-38, 42 and 43 or the transferase of any one of claims 78- 84, 89 and 90 and (ii) optionally, a buffer.
102. The kit of claim 101, further comprising L-dNTP.
103. The kit of claim 102, wherein the L-dNTP comprises L-dATP, L-dGTP, L-dCTP, L- dTTP or L-UTP.
104. The kit of any one of claims 101-103, further comprising L-3’-O-R’-dNTP-R2-Label.
105. The kit of claim 104, wherein the L-3’-O-R’-dNTP-R2-Label comprises L-3’-O-R’- dATP-R2-Label, L-3’-O-R’-dTTP-R2-Label, L-3’-O-R’-dGTP-R2-Label, L-3’-O-R’-dCTP- R2-Label or L-3’-O-R’-dUTP-R2-Label.
106. The kit of any one of claims 101-105, wherein the L-3’-O-R’-dNTP-R2-Label is of Formula II as defined in any one of claims 4-11.
107. The kit of claim 106, wherein the L-3’-O-R’-dNTP-R2-Label is a nucleotide of 13 or 15.
108. The kit of any one of claims 101-107, further comprising L-ddNTP-R2-Label.
109. The kit of claim 108, wherein the L-ddNTP-R2-Label comprises L-ddATP-R2-Label, L-ddTTP-R2-Label, L-ddGTP-R2-Label, L-ddCTP-R2-Label or L-ddUTP-R2-Label.
110. The kit of any one of claims 101-109, wherein the L-ddNTP-R2-Label is of Formula III as defined in any one of claims 5, 6, and 9-11.
111. The kit of claim 110, wherein the L-ddNTP-R2-Label is the nucleotide of claim 14 or 16.
112. The kit of any one of claims 101-111, further comprising L-3’-O-R’-dNTP.
113. The kit of claim 112, wherein the L-3’-O-R’-dNTP comprises L-3’-O-R’-dATP, L-3’- O-R’-dTTP, L-3’-O-R’-dGTP, and L-3’-O-R’-dCTP.
114. The kit of any one of claims 101-113, wherein the L-3’-O-R’-dNTP is of Formula I as defined in claim 3 or 12.
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