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WO2021197371A1 - Conjugué biologique contenant un lieur rigide - Google Patents

Conjugué biologique contenant un lieur rigide Download PDF

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WO2021197371A1
WO2021197371A1 PCT/CN2021/084341 CN2021084341W WO2021197371A1 WO 2021197371 A1 WO2021197371 A1 WO 2021197371A1 CN 2021084341 W CN2021084341 W CN 2021084341W WO 2021197371 A1 WO2021197371 A1 WO 2021197371A1
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bond
bioconjugate
linker
biotin
oligonucleotide
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Chinese (zh)
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陈锦森
黄静
王建鹏
李竑
吴政宪
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Nanjing Jinsirui Science and Technology Biology Corp
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Nanjing Jinsirui Science and Technology Biology Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the invention belongs to the field of bioconjugate coupling, and specifically relates to the application field of a fixed-length rigid linker between conjugated biomolecules.
  • Bioconjugation refers to the joining of two molecules, at least one of which is a biomolecule, by a covalent bond through chemical means.
  • Biomolecules synthesized and modified through biological coupling can have multiple functions, such as tracking cellular events, revealing enzyme functions, determining protein biodistribution, imaging specific biomarkers, and delivering drugs to target cells.
  • a linker spacer
  • Linkers are divided into two types, flexible and rigid in molecular structure.
  • the substructure of flexible linker unit is generally composed of carbon-carbon single bonds, carbon-oxygen single bonds, and carbon-nitrogen single bonds.
  • Rigid linkers are generally composed of benzene in structure. Groups, pyridine groups, conjugated heterocycles, alkenes, alkynes, and three-dimensional rigid structures (such as ⁇ helices and ⁇ sheets) formed by hydrogen bonds. This difference in molecular structure makes them different in aqueous solutions in that the flexible linker unit forms a worm-like coiled chain due to the free rotation of the covalent bond; while the rigid linker is restricted due to the limited rotation of the covalent bond. Make it into a rod-like linear chain.
  • oligoproline was used as a rigid linker in the early days, but because the peptide bond belongs to the sp 2 hybrid property , Due to the type of solvent and the difference of the substituents, the normal/trans isomerism will occur, which will cause the spacer linkers composed of the same unit number to have different lengths, resulting in poor data reproducibility.
  • the biological coupling modification of oligonucleotides is helpful to solve the problem of how to improve the delivery of oligonucleotides to the cytosol or nucleus.
  • This method has enabled many such drug precursors to successfully enter the clinic in the past 5 years.
  • Experimental research stage At present, the market can provide spacer linkers for oligonucleotide coupling modification with linear alkanes with different carbon numbers, linear PEG structures, and dSpacers with tetrahydrofuran five-membered sugar ring structures. They all It is a flexible linker; for bioconjugates containing rigid linkers, there is no product on the market, and there is no similar research in the literature.
  • the present invention provides a formula
  • n 1 and n 2 are each independently selected from O, S or N;
  • X is selected from molecules containing fluorescent dyes, biotin, N-acetylgalactosamine, cholesterol, enzymes or antibodies;
  • Y is selected from nucleic acid sequences containing any base of 1-200 nt;
  • P 1 and P 2 are each independently selected from a phosphodiester bond, a phosphorothioate bond, a phosphorothioate bond, an alkyl phosphonate bond,
  • R1 and R2 each independently have the structure shown in formula (II),
  • Z is O, NH or CONH, and n is selected from any integer value of 1-10.
  • both n 1 and n 2 are oxygen.
  • the rigid linker structure is Length is
  • the X is selected from molecules containing fluorescent dyes, biotin, N-acetylgalactosamine or cholesterol. In some embodiments of the present invention, the X is selected from molecules containing fluorescent dyes, biotin, or cholesterol. In some embodiments of the present invention, the X is selected from molecules containing fluorescent dyes or biotin. In some embodiments of the present invention, the X is selected from molecules containing fluorescent dyes. In some embodiments of the invention, a biotin molecule. In some embodiments of the invention, the cholesterol molecule.
  • the X further includes a flexible linker structure.
  • Flexible linkers refer to covalent bonds that can rotate freely to form a worm-like coiled chain structure.
  • Common flexible linkers include, but are not limited to, linear alkanes, linear PEG, branched alkanes, and tetrahydrofuran five-membered rings Structure.
  • the flexible linker is selected from alkanes, linear PEG, branched alkanes, tetrahydrofuran five-membered rings, or combinations thereof.
  • the flexible connector structure is or
  • the Y is selected from a nucleic acid sequence of any base of 5-150 nt, preferably a nucleic acid sequence of any base of 10-100 nt, and more preferably a nucleic acid sequence of any base of 15-50 nt.
  • the sequence is most preferably a nucleic acid sequence of any base of 21 nt.
  • the P 1 and P 2 are both phosphodiester bonds.
  • the Z is oxygen, and n is any integer value of 2-8. In some embodiments of the present invention, the Z is oxygen, and n is 2, 3, 4, 5, 6, 7, or 8. In some specific embodiments of the present invention, said Z is oxygen and n is 2.
  • the X is selected from fluorescent dyes containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2 -. In a specific embodiment of the present invention, said X is comprising For the fluorescent dye of the structure, P1 and P2 are both phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
  • the X is selected from biotin containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2 -.
  • said X is selected from the group comprising In the structure of biotin, P1 and P2 are both phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
  • the X is selected from N-acetylgalactosamine containing a flexible linker structure, P 1 and P 2 are both phosphodiester bonds, and R 1 and R 2 are both -O-( CH 2 ) 2 -.
  • said X is selected from the group comprising In the structure of N-acetylgalactosamine, P1 and P2 are both phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
  • the X is selected from cholesterol containing a flexible linker structure, P 1 and P 2 are selected from phosphodiester bonds, and R 1 and R 2 are both -O-(CH 2 ) 2- .
  • said X is selected from the group comprising In the structure of cholesterol, P1 and P2 are selected from phosphodiester bonds, and R1 and R2 are both -O-(CH 2 ) 2 -.
  • the biological conjugate of the present invention is used in nucleic acid diagnosis, sequencing, genome analysis, detection of genetic variation, identification of single nucleotide polymorphism, oligonucleotide enrichment and purification, solid-phase difference display scheme, and protein interaction process , Peptide modification, drug delivery system or mass spectrometry analysis and detection applications.
  • the present invention in order to obtain a rigid linker that can be used for oligonucleotide modification, the present invention selects 4,4'-biphenol with a rigid structure as the main body, and the two ends are connected with ethylene glycol to form L-1, this structure can improve the water solubility of the rigid linker.
  • the rigid linker phosphoramidite monomer can be used for solid-phase synthesis.
  • the fluorescent dyes of the present invention include many common commercial dyes, such as Rhodamine series, BODIPY series, fluorescein (FITC) series, Coumarin series, cyanine series, Oxazine, ATTO series, AleaxFluor, LightCycler series, etc.
  • the fluorescent dye includes, but is not limited to, 4-acetylamino-4'-isothiocyanatostilbene (isothiocyanatostilbene)- 2,2'Disulfonic acid, acridine, derivatives of acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino- N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide Amine, anthranilamide, Brilliant Yellow, coumarin, coumarin derivatives, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-trifluoromethyl Coumaran 151, cyanosine; 4',6-d
  • rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123 , Rhodamine isothiocyanate X, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivatives of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and Lanthanide chelate derivatives, cyanine, pyrelium dyes,
  • ROX 6-carboxy
  • nucleic acid refers to a polymer of nucleotides (such as ribonucleotides, deoxyribonucleotides, nucleotide analogs, etc.), and includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA-RNA Hybrids, oligonucleotides, polynucleotides, aptamers, peptide nucleic acids (PNA), PNA-DNA conjugates, PNA-RNA conjugates, locked nucleic acids (LNA), etc., which include linear or branched co- Nucleotides linked together.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • DNA-RNA Hybrids DNA-RNA Hybrids
  • oligonucleotides polynucleotides
  • PNA peptide nucleic acids
  • PNA PNA-DNA conjugates
  • PNA-RNA conjugates locked nucleic acids
  • Nucleic acids are usually single-stranded or double-stranded, and generally contain phosphodiester bonds, although in some cases, nucleic acid analogs that may have alternative backbones are included, including, for example, phosphoramidites (Beaucage et al. (1993) Tetrahedron 49 (10 ): 1925) phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc.
  • nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids (see Jenkins et al. (1995) Chem. Soc. Rev. pages 169-176), and analogs such as Rawls, C&E News Jun. 2 , 1997, described on page 35.
  • the nucleic acid is deoxyribonucleic acid or ribonucleic acid.
  • the nucleic acid is a primer or a probe. In some specific embodiments of the invention, the nucleic acid is a primer.
  • the bioconjugate with a rigid linker of fixed length provided by the present invention can fix the spacing between the biomolecules connected at the two ends of the rigid linker, avoid the mutual interference between the two biomolecules, and thus can better Complete the coupling process of biomolecules, and in the process of using coupled molecules, better performance of the connected biomolecules.
  • the synthesis efficiency of the coupling structure is higher, and the fluorescence intensity of the dye is basically The same; in addition, the present invention also found that when the two ends of the rigid linker are connected to biotin and oligonucleotide chains, the rigid linker can extend the biotin, making it easier to be coupled by streptavidin Obtained; and the use of rigid linkers to connect cholesterol is more flexible than flexible linkers (such as C6), which can better improve the lipophilicity of oligonucleotides.
  • Figure 1 Synthetic route diagram of the rigid linker phosphoramidite monomer L.
  • Figure 2 Structure diagram of oligonucleotide modified by rigid linker L'.
  • Figure 3 Structure diagram of oligonucleotide modified with rigid linker L'and dye FAM.
  • Figure 4 Structure diagram of oligonucleotide modified by rigid linker L'and Biotin.
  • Figure 5 Structure diagram of oligonucleotide modified by rigid linker L'and GalNAc.
  • Figure 6 Structure diagram of oligonucleotide modified with rigid linker L'and cholesterol.
  • Figure 7 LC-MS image of crude oligonucleotide structure modified by rigid linker L'.
  • Figure 8 The crude quality spectrum of the oligonucleotide structure modified by the rigid linker L'.
  • Figure 9 Crude HPLC chart of oligonucleotide modified with rigid linker L'and dye FAM.
  • Figure 10 HPLC chart after purification of oligonucleotide modified with rigid linker L'and dye FAM.
  • Figure 11 Mass spectrum of oligonucleotide modified with rigid linker L'and dye FAM.
  • Figure 12 Fluorescence spectrum of the dye FAM after the rigid linker L'and the flexible linkers C6 and C18 are connected.
  • Figure 13 HPLC chart of crude oligonucleotide modified by rigid linker L'and Biotion.
  • Figure 14 HPLC chart after purification of oligonucleotide modified with rigid linker L'and Biotin.
  • Figure 15 Mass spectrum of oligonucleotide modified with rigid linker L'and Biotin.
  • Figure 16 HPLC chart of crude oligonucleotide modified with rigid linker L'and GalNAc.
  • Figure 17 HPLC chart after purification of oligonucleotide modified with rigid linker L'and GalNAc.
  • Figure 18 Mass spectrum of oligonucleotide modified with rigid linker L'and GalNAc.
  • Figure 19 HPLC chart of crude oligonucleotide modified with rigid linker L'and cholesterol.
  • Figure 20 HPLC chart after purification of oligonucleotide modified with rigid linker L'and cholesterol.
  • Figure 21 Mass spectrum of oligonucleotide modified with rigid linker L'and cholesterol.
  • the present invention selects four widely used biomolecules, namely fluorescent dyes, biotin, cholesterol and N-acetylgalactosamine, all of which can perform efficient biological coupling reactions with linkers.
  • Fluorescent labeling of oligonucleotides is the main detection method in DNA diagnosis, sequencing and genome analysis. Fluorescent probes have a wide range of applications in detecting genetic variation and identifying single nucleotide polymorphisms. Biotin-modified oligonucleotides have been widely used in enrichment, purification, solid-phase difference display schemes, and solid-phase genome and plasmid sequencing schemes.
  • GalNAc N-acetylgalactosamine
  • ASGPR ASialoGlycoProtein receptor
  • Cholesterol-modified oligonucleotides are recognized by high-density lipoproteins and low-density lipoproteins (HDL and LDL) in vivo, and are internalized through cholesterol-binding receptors. After intravenous or intraperitoneal injection, these oligonucleotide conjugates escape renal clearance, thereby greatly affecting their pharmacokinetics by extending the retention time of nucleic acids in plasma.
  • the rigid linker phosphoramidite monomer L was used as a raw material to connect to the 5 of an oligonucleotide with a length of 21 bases.
  • 'End (5'-HO-CTCTATGGGCAGTCGGTGAAT-OH-3', the structure is shown in Figure 2).
  • CTCTATGGGCAGTCGGTGAAT After editing the position information containing the rigid linker phosphoramidite monomer L and the oligonucleotide sequence (CTCTATGGGCAGTCGGTGAAT), upload it to the automated Dr. Oligo48 DNA synthesizer.
  • Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, respectively placed in the corresponding synthesis channel of the synthesizer, solid-phase synthesis and
  • the method of cleavage and deprotection is the same as that of conventional primer solid-phase synthesis, see (Beaucage et al., J. Tetrahedron Letters. 22.20:1859-1862 (1981)), where the coupling time of compound L is 320s ⁇ 4 times, and the coupling time is 3min. After obtaining the primer containing the rigid linker L', it was separated, purified and recovered by HPLC.
  • the product was analyzed by HPLC.
  • the HPLC gradient elution condition for crude product analysis was 0-6min, and the concentration of acetonitrile was 5%-20%; 6.01-8min, The acetonitrile concentration is 88%; 8.01-10min, the acetonitrile concentration is 5%.
  • the pure product analysis HPLC gradient elution conditions are 0-6min, the acetonitrile concentration is 5%-30%, 6.01-8min, the acetonitrile concentration is 88%, 8.01 10min, the concentration of acetonitrile was 5%.
  • the terminal hydroxyl group of the linker can be further modified, for example, by coupling with the phosphoramidite monomer of the dye (Carboxyfluorescein FAM) to obtain the dye and the oligonucleotide by the rigid linker L'
  • the spaced-apart structure is shown as Probe 1 in Figure 3.
  • Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, dye FAM phosphoramidite monomers with a concentration of 0.057M; respectively placed in the synthesis After the corresponding synthesis channel of the instrument alone, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis.
  • the coupling time is 320s ⁇ 4 times, and the dye coupling time is 3min. After the primer containing the dye donor and linker is obtained, it is separated and purified by HPLC. The product is analyzed by HPLC.
  • the crude product is analyzed by HPLC gradient elution conditions of 0-6min , Acetonitrile concentration is 5%-20%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%.
  • the main peak with retention time of 6.065min is the pure product further modified by dye
  • Analytical HPLC gradient elution conditions are 0-6min, acetonitrile concentration is 5%-30%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%.
  • the purity is 95.4%.
  • the yield after synthesis and purification is shown in Table 1. It can be seen that the synthesis yield from the rigid linker is 76.9% and 72.6% higher than the yield of the flexible linker, respectively.
  • the terminal hydroxyl group of the linker can also be modified by biotin, which is a biomolecule that is an important part of an enzyme that affects cell growth and affects amino acids involved in protein synthesis, as shown in Probe A in Figure 4.
  • biotin phosphoramidite monomer is coupled to obtain a structure in which the biotin and the oligonucleotide are separated by the L'between the rigid linkers.
  • sequence position information including Biotin (5'-Biotin phosphoramidite monomer), rigid linker phosphoramidite monomer L and oligonucleotide (CTCTATGGGCAGTCGGTGAAT), upload it to the automated Dr. Oligo48 DNA Synthesizer.
  • Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, and the concentration of biotin monomer was 0.067M; they were placed in separate synthesizers. After the corresponding synthesis channel, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis, see Beaucageetal., J. Tetrahedron Letters. 22.20:1859-1862 (1981), where the coupling time of compound L is 320s ⁇ 4 The coupling time of Biotin is 3min.
  • the primer containing the biotin monomer and the linker After obtaining the primer containing the biotin monomer and the linker, it is separated, purified and recovered by HPLC.
  • the product was analyzed by HPLC.
  • the crude product analysis conditions are 0-6min, acetonitrile concentration is 5%-20%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%.
  • the main peak with a retention time of 6.047 min is a product further modified by biotin, which has a high coupling efficiency.
  • the pure product analysis conditions are 0-6min, acetonitrile concentration is 5%-30%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%.
  • Oligonucleic acid chains connected to biotin are respectively marked as Probe B (5'-Biotin-C6-CTCTATGGGCAGTCGGTGAAT-OH-3'), Probe C (5'-Biotin-C18-CTCTATGGGCAGTCGGTGAAT-OH-3'); in the same amount
  • Probe B (5'-Biotin-C6-CTCTATGGGCAGTCGGTGAAT-OH-3')
  • Probe C (5'-Biotin-C18-CTCTATGGGCAGTCGGTGAAT-OH-3')
  • the amount of remaining oligo-nucleic acid strands in the supernatant solution after the magnetic bead grabbing is detected to obtain a comparison of grabbing efficiency.
  • the Dynabeads TM magnetic beads used in this experiment have a maximum adsorption value of 200 pmol for single-stranded oligonucleotides. Take 20 ⁇ L of Dynabeads TM magnetic beads, which can adsorb single-stranded oligonucleotides with a maximum adsorption value of 40pmol. Take 0.95 times of the maximum adsorption value (experiment 1, 38pmol single-stranded oligonucleotide), 1.30 times (experiment 2, 52pmol single-stranded oligonucleotide) and 1.70 times (experiment three, 68pmol single-stranded oligonucleotide).
  • Glycidyl oligonucleotides for experiments.
  • the supernatant was taken for concentration test using a Nanodrop instrument, and the measured results are shown in Table 2. From the concentration of oligonucleotides in the supernatant, the ratio of the remaining oligonucleotides in the supernatant to the total primers can be calculated.
  • the volume of the supernatant is 80 ⁇ L
  • the relative molecular mass of the oligonucleotide is 7218
  • the results obtained by referring to the above calculation method are shown in Table 3 (where the relative molecular mass of Probe B oligonucleotide is 7062.8, and Probe C oligonucleotide The relative molecular mass of nucleotides is 7227).
  • Probe B 0.0ng/ ⁇ L 0.6ng/ ⁇ L 1.9ng/ ⁇ L
  • Probe C 0.0ng/ ⁇ L 0.5ng/ ⁇ L 2.1ng/ ⁇ L
  • GalNAc N-Acetylgalactosamine
  • sequence information including N-acetylgalactosamine (5'-GalNAc C3 phosphoramidite monomer), rigid linker phosphoramidite monomer L, and oligonucleotide sequence position information (CTCTATGGGCAGTCGGTGAAT), upload In the automated Dr. Oligo48 DNA synthesizer.
  • sequence information including N-acetylgalactosamine (5'-GalNAc C3 phosphoramidite monomer), rigid linker phosphoramidite monomer L, and oligonucleotide sequence position information (CTCTATGGGCAGTCGGTGAAT)
  • Monomers A, C, G, T phosphoramidite monomers were dissolved in acetonitrile with a concentration of 0.06M; compound L was dissolved in acetonitrile with a concentration of 0.10M, and the concentration of GalNAc monomer was 0.054M; respectively placed in a separate corresponding synthesizer After synthesizing the channel, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis, see Beaucageetal., J. Tetrahedron Letters. 22.20:1859-1862 (1981), where the coupling time of compound L is 320s ⁇ 4 times , 5'-GalNAcC3 phosphoramidite monomer coupling time is 12min.
  • the primer containing 5'-GalNAc C3 and the linker After obtaining the primer containing 5'-GalNAc C3 and the linker, it is separated, purified and recovered by HPLC.
  • the product was analyzed by HPLC.
  • the analysis conditions are 0-6min, acetonitrile concentration is 5%-30%, 6.01-8min, acetonitrile concentration is 88%. 8.01-10min, acetonitrile concentration is 5%.
  • 16 the main peak with a retention time of 3.405 min is the product further modified by N-acetylgalactosamine, and the coupling efficiency is high.
  • the yield was 65.0%, and the purity of the obtained modification was 98.9%.
  • the addition of lipophilic groups such as cholesterol molecules to oligonucleotides is expected to enhance its cellular uptake and membrane permeability.
  • the L linker can also connect cholesterol molecules to oligonucleotides by solid-phase synthesis, as shown in Probe 4 in Figure 6. After editing the sequence information including cholesterol (5'-Cholesteryl-TEG phosphoramidite monomer) and linker sequence position information (CTCTATGGGCAGTCGGTGAAT), upload it to the automated Dr. Oligo48 DNA synthesizer.
  • Monomers A, C, G, T phosphoramidite monomers are dissolved in acetonitrile with a concentration of 0.06M; compound L is dissolved in acetonitrile with a concentration of 0.10M, and the concentration of cholesterol monomer is 0.050M; they are placed in separate corresponding synthesizers After synthesizing the channel, the methods of solid-phase synthesis and cleavage and deprotection are the same as those of conventional primer solid-phase synthesis, see (Beaucageetal., J. Tetrahedron Letters. 22.20:1859-1862 (1981)), where the coupling time of compound L is 320s ⁇ 4 The coupling time of cholesterol monomer is 3min.
  • the primer containing cholesterol and linker After obtaining the primer containing cholesterol and linker, it is separated, purified and recovered by HPLC. The product is analyzed by HPLC. The analysis conditions are 0-6min, the concentration of acetonitrile is 40%-80%, 6.01 -8min, the acetonitrile concentration is 88%. 8.01-10min, the acetonitrile concentration is 5%. As shown in Figure 19, the main peak with a retention time of 3.0157min is the product further modified by N-acetylgalactosamine, and the coupling efficiency is high. 20, after to give a clean product, retention time 3.189min; ESI mass spectrum having a molecular weight by a confirmation (FIG.
  • the partition coefficient (abbreviated as P) is defined as the specific ratio of the solute concentration between two solvents (liquid phase two phases), especially for unionized solutes, so the ratio
  • the logarithm of is logP.
  • the logP value is a measure of lipophilicity or hydrophobicity, and the theoretically calculated partition coefficient is recorded as clogP.

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Abstract

L'invention concerne un conjugué biologique contenant un lieur rigide dans le domaine du couplage de conjugués biologiques. Le conjugué biologique selon l'invention peut fixer un intervalle entre des biomolécules reliées aux deux extrémités du lieur rigide, et éviter une interférence mutuelle entre deux biomolécules, ce qui permet de mieux achever le couplage des biomolécules. Dans le procédé d'utilisation de la molécule conjuguée, les propriétés entre les biomolécules reliées sont meilleures.
PCT/CN2021/084341 2020-03-31 2021-03-31 Conjugué biologique contenant un lieur rigide Ceased WO2021197371A1 (fr)

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CN104244987A (zh) * 2011-12-15 2014-12-24 株式会社百奥尼 新型寡核苷酸缀合物及其应用
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