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WO2025137293A2 - Amélioration de la polymérisation d'acide nucléique par des composés aromatiques - Google Patents

Amélioration de la polymérisation d'acide nucléique par des composés aromatiques Download PDF

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WO2025137293A2
WO2025137293A2 PCT/US2024/061051 US2024061051W WO2025137293A2 WO 2025137293 A2 WO2025137293 A2 WO 2025137293A2 US 2024061051 W US2024061051 W US 2024061051W WO 2025137293 A2 WO2025137293 A2 WO 2025137293A2
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independently selected
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WO2025137293A3 (fr
Inventor
Brent BANASIK
Kendall BERG
Ali Marie CARROLL
Jagadeeswaran CHANDRASEKAR
Ajay Kumar CHINNAM
Peter CRISALLI
Abdul Rouf DAR
Grant Christopher DERDEYN-BLACKWELL
Grisha ETKIN
Aaron Jacobs
Omid KHAKSHOOR
Mark Stamatios Kokoris
Melud Nabavi
Dylan O'CONNELL
Tavis William PRICE
Cameron John SZCZESNY
Meng C. Taing
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Roche Sequencing Solutions Inc
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Roche Sequencing Solutions Inc
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
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    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C07D249/06—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D421/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms
    • C07D421/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present disclosure relates to new chemical entities, more specifically to new organic molecules optionally having inorganic components, including compositions thereof, and methods for the manufacture and utilization thereof, particularly in influencing enzyme performance.
  • An individual's unique DNA sequence provides valuable information concerning their susceptibility to certain diseases. It also provides patients with the opportunity to screen for early detection and/or to receive preventative treatment. Furthermore, given a patient's individual blueprint, clinicians will be able to administer personalized therapy to maximize drug efficacy and/or to minimize the risk of an adverse drug response. Similarly, determining the blueprint of pathogenic organisms can lead to new treatments for infectious diseases and more robust pathogen surveillance. Low cost, whole genome DNA sequencing will provide the foundation for modern medicine. To achieve this goal, sequencing technologies must continue to advance with respect to throughput, accuracy, and read length.
  • SBS sequencing by synthesis
  • Illumina, Inc. 454 Life Sciences, Ion Torrent, Pacific Biosciences
  • analogous ligation-based platforms Complete Genomics, Life Technologies Corporation
  • GnuBio, Inc. uses picoliter reaction vessels to control millions of discreet probe sequencing reactions
  • Halcyon Molecular was attempting to develop technology for direct DNA measurement using a transmission electron microscope.
  • Nanopore based nucleic acid sequencing is a compelling approach that has been widely studied.
  • Kasianowicz et al. Proc. Natl. Acad. Sci. USA 93: 13770-13773, 1996) characterized single-stranded polynucleotides as they were electrically translocated through an alpha hemolysin nanopore embedded in a lipid bilayer. It was demonstrated that during polynucleotide translocation partial blockage of the nanopore aperture could be measured as a decrease in ionic current.
  • Polynucleotide sequencing in nanopores is burdened by having to resolve tightly spaced bases (0.34 nm) with small signal differences immersed in significant background noise.
  • Translocation speed can be reduced by adjusting run parameters such as voltage, salt composition, pH, temperature, and viscosity, to name a few. However, such adjustments have been unable to reduce translocation speed to a level that allows for single base resolution.
  • Stratos Genomics has developed a method called Sequencing by Expansion (“SBX”) that uses a biochemical process to transcribe the sequence of DNA onto a measurable polymer called an "Xpandomer” (Kokoris et al., U.S. Pat. No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion”).
  • the transcribed sequence is encoded along the Xpandomer backbone in high signal -to-noise reporters that are separated by about 10 nm and are designed for high-signal-to-noise, well-differentiated responses. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to native DNA.
  • Xpandomers can enable several next generation DNA sequencing detection technologies and are well suited to nanopore sequencing.
  • Xpandomers are generated from non-natural nucleotide analogs, termed XNTPs, characterized by lengthy substituents that enable the Xpandomer backbone to be expanded following synthesis (see Published PCT Appl. No. W02016/081871 to Kokoris et al., herein incorporated by reference in its entirety). Because of their atypical structures, XNTPs, as well as other nucleotide analogs (e.g., nucleotide analogs modified with detectable label moieties) introduce novel challenges as substrates for currently available DNA polymerases. Published PCT Appl. Nos.
  • WO2017/087281 and WO2018/204717 to Kokoris et al. herein incorporated by reference in their entirety, describes engineered DP04 polymerase variants with enhanced primer extension activity utilizing non-natural, bulky nucleotide analogues as substrates.
  • Replication slippage is thought to encompass the following steps: (i) copying of the first repeat by the replication machinery, (ii) replication pausing and dissociation of the polymerase from the newly synthesized end, (iii) unpairing of the newly synthesized strand and its pairing with the second repeat, and (iv) resumption of DNA synthesis. Arrest of the replication machinery within a repeated region thus results in misalignment of primer and template.
  • the present disclosure provides compounds, compositions and uses thereof that enhance nucleic acid polymerase activity.
  • polymerase activity is enhanced in polymerization reactions under conditions that introduce one or more challenges to the polymerase, e.g., conditions that include non-natural nucleotide analog substrates or template motifs that impair polymerase processivity.
  • Such enhancement is achieved by supplementing a polymerization reaction with one or more compounds of the present disclosure, which may be referred to herein as "Polymerase Enhancing Molecules" (“PEMs").
  • PEMs Polymerase Enhancing Molecules
  • One aspect of the present disclosure is a compound, such as a PEM, having Formula (I): or a solvate, hydrate, tautomer, chelate or salt thereof, wherein a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4; m is 1, 2 or 3; m' is 1, 2 or 3; n is 0, 1 or 2; p is 0, 1 or 2;
  • Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S;
  • L is a linking group
  • M is, at each occurrence, independently selected from hydrogen, halogen and C 1 -C 4 alkyl;
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, wherein substituents for Ari are, at each occurrence, independently selected from halogen, -OH, -CN, -NO2, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl , C 1 -C 6 heteroalkyl, C 1 - C 6 cycloloalkyl, -OR 0 , -CONH 2 , -C(O)NR 1 R 1 ', -C(O)(CH
  • R 2 is, at each occurrence, independently selected from C 2 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;
  • R 3 is, at each occurrence, independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, -C 1 -C 6 -OH, -C 1 -C 6 -C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroary
  • R 4 is, at each occurrence, independently selected from one or more heteroatom interrupted alkylene, wherein the heteroatom is O, S, NH, or a combination thereof;
  • Y is, at each occurrence, independently selected from Ar2, -C(O)-Ar2, - (CH2) a Ar2, -(CH 2 ) 3 PO(OEt) 2 , or -CH 2 CO 2 Me;
  • Ar2 is, at each occurrence, independently selected from (i) a substituted or unsubstituted 5- and 6-membered monocyclic aromatic or heteroaromatic ring; (ii) a substituted or unsubstituted 9- and 10-membered fused bicyclic rings comprising two monocyclic rings together, where at least one of the two monocyclic rings is an aromatic or a heteroaromatic ring; and (iii) a substituted or unsubstituted 13- and 14-membered fused tricyclic rings comprising three monocyclic rings together, where at least one of the three monocyclic rings is an aromatic or a heteroaromatic ring; wherein each Ar2 is independently substituted with G 1 , G 2 , G 3 , G 4 and G 5 , wherein: when Ar2 is monosubstituted, G 1 is, at each occurrence, independently selected from oxo, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -
  • G 2 , G 3 , G 4 and G 5 are, at each occurrence, independently selected from absent or selected from the groups comprising, halogen, -CN, -NO 2 , -SO3R 3 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -E-O-R 3 , — E-(CH 2 ) a C(O)R 3 , -E-CO2H, -E-CHO, -E-C(O)R 3 , -E- C(0)NH(0H), -E-C(0)NHR 1 , -E-C(O)N(H)C(H)(R 1 )(R 1 '), -E-
  • the compounds of Formula (I) are in the form of a salt.
  • Z is not a triazole.
  • Z is not a
  • Z is a diazole (e.g., imidazole, pyrazole, pyrimidine, pyridazine, pyrazine).
  • Z is pyrrole, thiophene, or triazole (e.g., 1,2,3-triazole;
  • Z is thiazole. In yet other embodiments, Z is isoxazole. In even further embodiments, Z is a tetrazole.
  • each Z is independently substituted with one "M” moiety. In other embodiments, each Z is independently substituted with two "M” moieties. In yet other embodiments, each Z is independently substituted with three "M” moieties.
  • L is a heteroalkylene group of 2 to 10 carbon atoms in length. In other embodiments, L is a heteroalkylene group of 2 to 10 carbon atoms in length, wherein one or more carbon atoms is replaced with at least one heteroatom selected from oxygen, nitrogen, and sulfur.
  • Non-limiting examples of suitable L groups bridging two Ari groups are set forth below:
  • Another aspect of the present disclosure is a polymer, copolymer, or metallogel of any of the compounds having Formula (I).
  • Another aspect of the present disclosure is a composition including two or more molecules of any one of the compounds having Formula (I).
  • Another aspect of the present disclosure is a compound having Formula (IC): or a solvate, hydrate, tautomer, chelate or salt thereof, wherein: a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4; m is 1, 2 or 3; m' is 1, 2 or 3; n is 0, 1 or 2; p is 0, 1 or 2;
  • W is N when X is C or W is C when X is N; is a single or double bond, wherein the double bond begins at whichever of W or X is carbon;
  • L is a linking group
  • M is, at each occurrence, independently selected from hydrogen, halogen and C 1 - C4 alkyl;
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, wherein substituents for Ari are, at each occurrence, independently selected from halogen, -OH, -CN, -NO2, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl , C 1 -C 6 heteroalkyl, C 1 -C 6 cycloloalkyl, -OR 0 , -CONH2, -C(O)NR 1 R 1 , -C(O)(CH 2 )
  • R 2 is, at each occurrence, independently selected from C 2 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;
  • R 3 is, at each occurrence, independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, -C 1 -C 6 -OH, -C 1 -C 6 -C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;
  • R 4 is, at each occurrence, independently selected from one or more heteroatom interrupted alkylene wherein the heteroatom is O, S, NH, or a combination thereof;
  • Y is, at each occurrence, independently selected from Ar2, -C(O)-Ar2, - (CH2) a Ar2, -(CH 2 ) 3 PO(OEt)2, or -CH 2 CO 2 Me;
  • Ar2 is, at each occurrence, independently selected from (i) a substituted or unsubstituted 5- and 6-membered monocyclic aromatic or heteroaromatic ring; (ii) a substituted or unsubstituted 9- and 10-membered fused bicyclic rings comprising two monocyclic rings together, where at least one of the two monocyclic rings is an aromatic or a heteroaromatic ring; and (iii) a substituted or unsubstituted 13- and 14-membered fused tricyclic rings comprising three monocyclic rings together, where at least one of the three monocyclic rings is an aromatic or a heteroaromatic ring; wherein each Ar2 is independently substituted with G 1 , G 2 , G 3 , G 4 and G 5 , wherein: when Ar2 is monosubstituted, G 1 is, at each occurrence, independently selected from oxo, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -
  • G, G 3 , G 4 and G 5 are, at each occurrence, independently selected from absent or selected from the groups comprising, halogen, -CN, -NO 2 , -SO3R 3 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -E-O-R 3 , — E-(CH 2 ) a C(O)R 3 , -E-CO2H, -E-CHO, -E-C(O)R 3 , -E-C(0)NH(0H), -E- C(0)NHR 1 , -E-C(O)N(H)C(H)(R 1 )(R 1 ), -E-C(O)N(R 1 )((CH 2 ) a P(O)(O)OH) 2 , -E-
  • Another aspect of the present disclosure is a polymer, copolymer, or metallogel of any of the compounds having Formula (IC).
  • composition comprising two or more molecules of any one of the compounds having Formula (IC).
  • Another aspect of the present disclosure is a method of enhancing a nucleic acid polymerase reaction, the method including the steps of forming a nucleic acid polymerase reaction composition including a template nucleic acid, a nucleic acid polymerase, a mixture of nucleotides and/or nucleotide analogs, at least one PEM (such as those compounds having any one of Formulas (I), (IA), (IB), (IC), and/or (ID) as set forth herein); and incubating the nucleic acid polymerase reaction composition under conditions allowing a nucleic acid polymerization reaction. It is believed that the at least one PEM increases the processivity, rate, and/or fidelity of the nucleic acid polymerase reaction. In some embodiments, the at least one PEM increases the length of a resulting nucleic acid product compared to a nucleic acid polymerase reaction lacking the at least one PEM.
  • the nucleic acid polymerase is a DNA polymerase.
  • the DNA polymerase is DPO4 or a variant thereof.
  • the mixture of nucleotides or nucleotide analogs is a mixture of nucleotide analogs comprising nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates includes a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric tether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphoramidate bond.
  • the nucleic acid polymerization reaction produces an expandable polymer of nucleotide analogs, wherein the expandable polymer encodes the nucleobase sequence information of the template nucleic acid.
  • the conditions for allowing a nucleic acid polymerization reaction includes a suitable polymerization buffer and an oligonucleotide primer.
  • the suitable buffer includes one or more components selected from MnCE, a buffer, a salt, a sugar, a single-strand binding protein (SSB), imidazole, pyrazole, triazole, betaine, a molecular crowding agent, dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, and/or urea.
  • the reaction mixture further includes a nucleic acid intercalating agent.
  • the reaction mixture further includes a polyanion recognition moiety.
  • the mixture of nucleotides or nucleotide analogs includes nucleotide analogs comprising a detectable label.
  • the detectable label is an optically detectable label selected from the group consisting of luminescent, chemiluminescent, fluorescent, fluorogenic, chromophoric or chromogenic labels.
  • PEM including any of the compounds disclosed herein having any one of Formulas (I), (IA), (IB), (IC), and/or (ID) set forth herein
  • a PEM including any of the compounds disclosed herein having any one of Formulas (I), (IA), (IB), (IC), and/or (ID) set forth herein
  • compositions including at least one PEM (including any of the compounds disclosed herein having any one of Formulas (I), (IA), (IB), (IC), and/or (ID) set forth herein), and a mixture of nucleotide analogs. It is believed that this composition is useful, e.g., when combined with a polymerase, wherein the at least one PEM increases the number and accuracy of nucleotide analogs incorporated into a daughter strand during a template-dependent polymerization reaction relative to an identical polymerization reaction absent the present of the at least one PEM.
  • the at least one PEM comprises a plurality of PEMs.
  • the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric ether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphorami date bond.
  • each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of
  • the composition further includes a buffer which includes one or more components selected from MnCE, a buffer, a salt, a sugar, a single-strand binding protein (SSB), imidazole, pyrazole, triazole, betaine, a molecular crowding agent, dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N- methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, and/or urea.
  • the composition further includes a single-strand binding protein (SSB).
  • the composition further includes urea.
  • the mixture of nucleotide analogs includes nucleotide analogs including a detectable label.
  • the detectable label is an optically detectable label selected from the group consisting of luminescent, chemiluminescent, fluorescent, fluorogenic, chromophoric or chromogenic labels.
  • Another aspect of the present disclosure is a method of sequencing a DNA or RNA template, the method including the steps of forming a DNA polymerase reaction composition including the DNA or RNA template, a replication primer that complexes with the template, a DNA polymerase, a mixture of nucleotides or nucleotide analogs, and at least one PEM (including any of the compounds disclosed herein having any one of Formulas (I), (IA), (IB), (IC), and/or (ID) as set forth herein), and incubating the DNA polymerase reaction composition under conditions allowing a DNA polymerization reaction, wherein the at least one PEM increases the rate, fidelity or processivity of the DNA polymerase reaction.
  • a DNA polymerase reaction composition including the DNA or RNA template, a replication primer that complexes with the template, a DNA polymerase, a mixture of nucleotides or nucleotide analogs, and at least one PEM (including any of the compounds disclosed herein having any one of Formulas (I
  • the method may further include determining the sequence of the nucleotides or nucleotide analogs in the resulting polymer of nucleotides or nucleotide analogs.
  • the at least one PEM is a compound having any one of Formulas (I), (IA), (IB), (IC), and (ID). In other embodiments, the at least one PEM is any one of the compounds set forth herein in Table 1.
  • the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric ether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphoramidate bond.
  • the DNA polymerase is DPO4 or a variant thereof.
  • the resulting polymer of nucleotide analogs is an expandable polymer.
  • the method further includes the step of contacting the expandable polymer with a phosphoramidate cleavage agent to produce an expanded polymer of nucleotide analogs.
  • the polymeric tether moiety of each of the nucleotide analogs comprises a reporter moiety unique to the nucleobase of the analog.
  • the reporter moieties produce a characteristic electronic signal.
  • the step of determining the sequence of the nucleotide analogs includes the step of translocating the expanded polymer of nucleotide analogs through a nanopore.
  • PEM as those compounds having any one of Formulas (I), (IA), (IB), (IC), and/or (ID) as set forth herein
  • composition comprising a PEM (as those compounds having any one of Formulas (I), (IA), (IB), (IC), and/or (ID) as set forth herein) and a polynucleotide.
  • composition comprising a PEM (as those compounds having any one of Formulas (I), (IA), (IB), (IC), and/or (ID) as set forth herein) a polypeptide, e.g., a polypeptide such as an enzyme, where the enzyme may be a nucleic acid polymerase.
  • a PEM as those compounds having any one of Formulas (I), (IA), (IB), (IC), and/or (ID) as set forth herein
  • a polypeptide e.g., a polypeptide such as an enzyme, where the enzyme may be a nucleic acid polymerase.
  • each atom identified in a chemical formula may be any of the isotopes of that atom.
  • C carbon
  • H hydrogen
  • O oxygen
  • N nitrogen
  • Embodiment 1 A or a solvate, hydrate, tautomer, chelate or salt thereof, wherein a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4; m is 1, 2 or 3; m' is 1, 2 or 3; n is 0, 1 or 2; p is 0, 1 or 2; Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S, where each Z may be independently substituted with one or more halogens and/or one or more C1-C4 alkyl groups;
  • L is a linking group
  • M is, at each occurrence, independently selected from hydrogen, halogen and C1-C4 alkyl
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, wherein substituents for Ari are, at each occurrence, independently selected from halogen, -OH, -CN, -NO2, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl , C 1 -C 6 heteroalkyl, C 1 - C 6 cycloloalkyl, -OR 0 , -CONH 2 , -C(O)NR 1 R 1 ', -C(O)(CH
  • R 3 is, at each occurrence, independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, -C 1 -C 6 -OH, -C 1 -C 6 -C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;
  • R 4 is, at each occurrence, independently selected from one or more heteroatom interrupted alkylene wherein the heteroatom is O, S, NH, or a combination thereof;
  • Y is, at each occurrence, independently selected from Ar2, -C(O)-Ar2, - (CH2) a Ar2, -(CH 2 ) 3 PO(OEt)2, or -CH 2 CO 2 Me;
  • Ar2 is, at each occurrence, independently selected from (i) a substituted or unsubstituted 5- and 6-membered monocyclic aromatic or heteroaromatic ring; (ii) a substituted or unsubstituted 9- and 10-membered fused bicyclic rings comprising two monocyclic rings together, where at least one of the two monocyclic rings is an aromatic or a heteroaromatic ring; and (iii) a substituted or unsubstituted 13- and 14-membered fused tricyclic rings comprising three monocyclic rings together, where at least one of the three monocyclic rings is an aromatic or a heteroaromatic ring; wherein each Ar2 is independently substituted with G 1 , G 2 , G 3 , G 4 and G 5 , wherein: when Ar2 is monosubstituted, G 1 is, at each occurrence, independently selected from oxo, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -
  • G 2 , G 3 , G 4 and G 5 are, at each occurrence, independently selected from absent or selected from the groups comprising, halogen, -CN, -NO 2 , -SO3R 3 , C 1 -C 6 alkyl, C 1 - C 6 haloalkyl, -E-O-R 3 , — E-(CH 2 ) a C(O)R 3 , -E-CO2H, -E-CHO, -E-C(O)R 3 , -E- C(0)NH(0H), -E-C(0)NHR 1 , -E-C(O)N(H)C(H)(R 1 )(R 1 '), -E-
  • Embodiment 3 The compound of embodiment 1, wherein the compound of Formula (I) has Formula (IC): or a solvate, hydrate, tautomer, chelate or salt thereof, wherein
  • W is N when X is C, or W is C when X is N; is a single or double bond, wherein the double bond begins at whichever of W or X is carbon; and m, m', n, p, L, and Y are as defined above.
  • Embodiment 4 The compound of any one of embodiments 1 - 3, wherein Ari is monocyclic heterocyclic aryl.
  • Embodiment 5 The compound of any one of embodiments 1 - 3, wherein Ari is selected from: wherein each group "Z" of Formulas (I), (IA), or (IB) is located at positions "k” on Ari; or wherein each triazole of Formula (IC) is located at positions "k” on Ari.
  • Embodiment 6 The compound of any one of embodiments 1 - 3, wherein Ari is bicyclic aryl.
  • Embodiment 7 The compound of any one of embodiments 1 - 3, wherein Ari is a bicyclic carbocyclic aryl selected from: wherein each group "Z" of Formulas (I), (IA), or (IB) is located at positions "k” on Ari; or wherein each triazole of Formula (IC) is located at positions "k” on Ari.
  • Ari is a bicyclic carbocyclic aryl selected from: wherein each group "Z" of Formulas (I), (IA), or (IB) is located at positions "k” on Ari; or wherein each triazole of Formula (IC) is located at positions "k” on Ari.
  • Embodiment 8 The compound of any one of embodiments 1 - 3, wherein Ari is a bicyclic heterocyclic aryl selected from: wherein each group "Z" of Formulas (I), (IA), or (IB) is located at positions "k” on Ari; or wherein each triazole of Formula (IC) is located at positions "k” on Ari.
  • Embodiment 9 The compound of any one of embodiments 1 - 3, wherein Ari is tricyclic aryl.
  • Embodiment 10 The compound of embodiment 9, wherein the tricyclic aryl is selected from:
  • Embodiment 11 The compound of embodiment 9, wherein the tricyclic aryl selected from:
  • each group "Z” of Formulas (I), (IA), or (IB) is located at positions "k” on Ari; or wherein each triazole of Formula (IC) is located at positions "k” on Ari.
  • Embodiment 12 The compound of embodiment 1, wherein Ari is a tricyclic heteroaryl selected from wherein each group "Z" of Formulas (I), (IA), or (IB) is located at positions "k” on Ari; or wherein each triazole of Formula (IC) is located at positions "k” on Ari.
  • Embodiment 13 The compound of any one of the preceding embodiments, wherein Ar2 is a substituted or unsubstituted 5-membered monocyclic aromatic ring selected from the group consisting of thiophene, 1,2-thiazole, 1,3-thiazole, furan, 1,2-oxazole, 1,3-oxazole, IH-pyrrole, IH-pyrazole, oxadiazole, thiadiazole, 1,2,4-triazole, 1,2,3-triazole and 1H- imidazole.
  • Embodiment 14 The compound of any one of embodiments 1 - 12, wherein Ar2 is a substituted or unsubstituted 6-membered monocyclic aromatic ring selected from the group consisting of benzene, pyridine, pyridazine, pyrimidine and pyrazine.
  • Embodiment 15 The compound of any one of embodiments 1 - 12, wherein Ar2 is a substituted or unsubstituted 9-membered fused bicyclic aromatic ring system selected from the group consisting of benzofuran, 1,3-benzoxazole, furo[3,2-b]pyridine, furo[3,2- c]pyridine, furo[2,3-c]pyridine, furo[2,3-b]pyridine, indole, IH-benzimidazole, 1H- pyrrolo[3,2-b]pyridine, lH-pyrrolo[3,2-c]pyridine, lH-pyrrolo[2,3-c]pyridine, 1H- pyrrolo[2,3-b]pyridine, benzothiophene, 1,3 -benzothiazole, thienol[3,2-b]pyridine, thieno[3,2-c]pyridine, thieno[2,3-c]pyridine, benzoxadi azo
  • Embodiment 16 The compound of any one of embodiments 1 - 12, wherein Ar2 is a substituted or unsubstituted 10-membered fused bicyclic aromatic ring system selected from the group consisting of naphthylene, quinoline, quinazoline, quinoxaline, 1,5- naphthyridine, 1,6-naphthyridine, 1,7-naphthyridine, 1,8-naphthyridine, isoquinoline, phthalazine, 2,6-naphthyridine and 2,7-naphthyridine.
  • Ar2 is a substituted or unsubstituted 10-membered fused bicyclic aromatic ring system selected from the group consisting of naphthylene, quinoline, quinazoline, quinoxaline, 1,5- naphthyridine, 1,6-naphthyridine, 1,7-naphthyridine, 1,8-naphthyridine,
  • Embodiment 17 The compound of any one of embodiments 1 - 12, wherein Ar2 is a substituted or unsubstituted pyridinyl ring selected from wherein the substituent G (where G may be any one of G 1 , G 2 , G 3 , G 4 and G 3 as defined above) is present 0, 1, or 2 times on the pyridinyl ring.
  • G may be any one of G 1 , G 2 , G 3 , G 4 and G 3 as defined above
  • Embodiment 18 The compound of any one of embodiments 1 - 12, wherein Ar2 is a phenyl ring having the formula wherein the substituent G (or G 1 , G 2 , G 3 , G 4 and G 5 as defined herein) is present 0, 1 or 2 times on the phenyl ring.
  • G is aryl (such as phenyl) substituted with G 2 , G 3 , G 4 and G5
  • Embodiment 19 The compound of any one of embodiments 1 - 12, wherein Ar2 is a phenyl ring selected from
  • Embodiment 20 The compound of embodiments 1 - 18, wherein Ar2 includes one or more amino substituents.
  • Embodiment 21 The compound of embodiments 1 - 18, wherein Ar2 includes one or more methoxy substituents.
  • Embodiment 22 The compound of embodiments 1 - 18, wherein Ar2 includes one or more carboxylic acid substituents.
  • Embodiment 23 The compound of embodiments 1 - 18, wherein Ar2 includes one or more -CH2-CO2-CH3 substituents.
  • Embodiment 24 The compound of embodiments 1 - 18, wherein Ar2 includes one or more trifluoromethyl substituents.
  • Embodiment 25 The compound of embodiments 1 - 18, wherein Ar2 includes one or more hydroxyl substituents.
  • Embodiment 26 The compound of embodiments 1 - 18, wherein Ar2 includes at least two substituents, where a first substituent is carboxylic acid, and second substituent is hydroxyl.
  • Embodiment 28 The compound of embodiments 1 - 18, wherein Ar2 includes at least one sulfonic acid group (-SO3H) or a salt thereof (-SO 2 O ).
  • Embodiment 30 The compound of any one of the preceding embodiments where the compound is in the form of a chelate.
  • Embodiment 31 The compound of embodiment 30, wherein the chelate is a copper chelate.
  • Embodiment 32 The compound of any one of the preceding embodiments where the compound is in the form of a salt.
  • Embodiment 33 The compound of any one of the preceding embodiments, wherein the compound has a logP of at least 4.9.
  • Embodiment 34 The compound of embodiment 1, wherein the compound has one of the formulas: or a salt or hydrate thereof.
  • Embodiment 35 The compound of embodiment 1, wherein the compound has one of the formulas: or a salt or hydrate thereof.
  • Embodiment 36 The compound of embodiment 1, wherein Ar2 includes at least two substituents selected from hydroxyl, carboxylic acid carboxamide, and trifluorom ethyl.
  • Embodiment 38 The compound of embodiment 1, wherein the compound has one of the formulas:
  • Embodiment 39 The compound of embodiment 1, wherein the compound has one of the formulas: or a salt or hydrate thereof.
  • Embodiment 40 The compound of embodiment 1, wherein the compound has one of the formulas:
  • Embodiment 41 The compound of embodiment 1, wherein the compound has one of the formulas:
  • Embodiment 42 The compound of embodiment 1, wherein the compound has one of the formulas:
  • Embodiment 43 The compound of embodiment 1, wherein the compound has one of the formulas:
  • Embodiment 44 The compound of embodiment 1, wherein the compound has one of the formulas:
  • Embodiment 45 The compound of embodiment 1, wherein the compound has one of the formulas:
  • Embodiment 46 The compound of embodiment 1, wherein the compound has one of the or a salt or hydrate thereof.
  • Embodiment 47 The compound of embodiment 1, wherein the compound has one of the formulas:
  • Embodiment 48 A composition comprising a compound of any one of embodiments 1 - 47, and a molecular crowding agent.
  • Embodiment 49 The composition of embodiment 48, wherein the molecular crowding agent is a polyalkylene glycol.
  • Embodiment 50 A composition comprising a compound of any one of embodiments 1 - 47, and an aqueous buffer.
  • Embodiment 51 The composition of embodiment 50, wherein the aqueous buffer is Tris
  • Embodiment 52 A composition comprising a compound of any one of embodiments 1 - 47, and a polynucleotide.
  • Embodiment 53 The composition of embodiment 52, wherein the polynucleotide is a 20- 60 mer oligonucleotide.
  • Embodiment 54 The composition of embodiment 52, wherein the polynucleotide is a 25- 50 mer oligonucleotide.
  • Embodiment 55 A composition comprising a compound of any one of embodiments 1 - 47, and a protein.
  • Embodiment 56 The composition of embodiment 55, wherein the protein is a DNA polymerase.
  • Embodiment 57 A composition comprising a compound of any one of embodiments 1 - 47, and a mixture of nucleotides or nucleotide analogs.
  • Embodiment 58 A composition for enhancing the processivity, fidelity, or rate of a DNA polymerase reaction comprising at least one compound of any one of embodiments 1 - 47, and a mixture of nucleotide analogs.
  • Embodiment 59 A composition comprising at least one compound of any one of embodiments 1 - 47 and a mixture of nucleotide analogs, wherein the at least one compound of any one of embodiments 1 - 47, increases the number and accuracy of nucleotide analogs incorporated into a daughter strand during a template-dependent polymerization reaction relative to an identical polymerization reaction absent the at least one compound of any one of embodiments 1 - 47.
  • Embodiment 60 The composition of embodiment 59, wherein the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric ether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphoramidate bond.
  • each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety
  • Embodiment 61 The composition of embodiment 59, further comprising a buffer which includes one or more components selected from MnCh, a buffer, a salt, a sugar, a singlestrand binding protein (SSB), imidazole, pyrazole, triazole, betaine, a molecular crowding agent, dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, and/or urea.
  • a buffer which includes one or more components selected from MnCh, a buffer, a salt, a sugar, a singlestrand binding protein (SSB), imidazole, pyrazole, triazole, betaine, a molecular crowding agent, dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP),
  • Embodiment 62 The composition of embodiment 59, further comprising a single-strand binding protein.
  • Embodiment 63 The composition of embodiment 59, further comprising urea.
  • Embodiment 64 The composition of embodiment 59, wherein the mixture of nucleotide analogs comprises nucleotide analogs comprising a detectable label.
  • Embodiment 65 The composition of 64, wherein the detectable label is an optically detectable label selected from the group consisting of luminescent, chemiluminescent, fluorescent, fluorogenic, chromophoric or chromogenic labels.
  • Embodiment 67 A method of enhancing a nucleic acid polymerase reaction, the method comprising: a. forming a nucleic acid polymerase reaction composition comprising: i. a template nucleic acid, ii. a nucleic acid polymerase, iii. a mixture of nucleotides or nucleotide analogs, and iv. at least one compound of any of embodiments 1-47; and b. incubating the nucleic acid polymerase reaction composition under conditions allowing a nucleic acid polymerization reaction, wherein the at least one compound of any one of embodiments 1 - 47 increases the processivity, rate, or fidelity of the nucleic acid polymerase reaction.
  • Embodiment 71 The method of embodiment 67, wherein the nucleic acid polymerase is a DNA polymerase.
  • Embodiment 72 The method of embodiment 71, wherein the DNA polymerase is DPO4 or a variant thereof.
  • Embodiment 73 The method of embodiment 67, wherein the mixture of nucleotides or nucleotide analogs is a mixture of nucleotide analogs comprising nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric tether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphorami date bond.
  • Embodiment 74 The method of embodiment 67, wherein the nucleic acid polymerization reaction produces an expandable polymer of nucleotide analogs, wherein the expandable polymer encodes the nucleobase sequence information of the template nucleic acid.
  • Embodiment 75 The method of embodiment 67, wherein the conditions for facilitating a nucleic acid polymerization reaction comprise a suitable polymerization buffer and an oligonucleotide primer.
  • Embodiment 76 The method of embodiment 67, wherein the suitable buffer includes one or more components selected from MnCh, a buffer, a salt, a sugar, a single-strand binding protein (SSB), imidazole, pyrazole, triazole, betaine, a molecular crowding agent, dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, and/or urea.
  • MnCh MnCh
  • a buffer a salt
  • a sugar a single-strand binding protein (SSB)
  • SSB single-strand binding protein
  • imidazole imidazole
  • pyrazole imidazole
  • triazole betaine
  • betaine a molecular crowding agent
  • DMSO dimethyl sulfoxide
  • NMP N-methyl-2-pyrrolidon
  • Embodiment 77 The method of embodiment 67, wherein the reaction mixture further comprises a single-strand binding protein.
  • Embodiment 78 The method of embodiment 67, wherein the reaction mixture further comprises urea.
  • Embodiment 79 The method of embodiment 67, wherein the mixture of nucleotides or nucleotide analogs comprises nucleotide analogs comprising a detectable label.
  • Embodiment 80 The method of embodiment 73, wherein the detectable label is an optically detectable label selected from the group consisting of luminescent, chemiluminescent, fluorescent, fluorogenic, chromophoric or chromogenic labels.
  • Embodiment 81 A method of sequencing a DNA or RNA template, the method comprising the steps of: a. forming a DNA polymerase reaction composition comprising: i. a DNA or RNA template, ii. a replication primer that complexes with the template, iii. a DNA polymerase, iv. a mixture of nucleotides or nucleotide analogs, v. at least one compound of any of embodiments 1-43, b. incubating the DNA polymerase reaction composition under conditions allowing a DNA polymerization reaction, wherein the at least one compound of any one of embodiments 1 - 47 increases the rate, fidelity or processivity of the DNA polymerase reaction; and c. determining the sequence of the nucleotides or nucleotide analogs in the resulting polymer of nucleotides or nucleotide analogs.
  • Embodiment 82 The method of embodiment 81, wherein the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric ether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphoramidate bond.
  • each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether mo
  • Embodiment 83 The method of any one of embodiments 81 or 82, wherein the DNA polymerase is DPO4 or a variant thereof.
  • Embodiment 84 The method of any one of embodiments 81 or 82, wherein the resulting polymer of nucleotide analogs is an expandable polymer.
  • Embodiment 85 The method of any one of embodiment 81, further including the step of contacting the expandable polymer with a phosphoramidate cleavage agent to produce an expanded polymer of nucleotide analogs.
  • Embodiment 86 The method of any one of embodiments 81 or 82, wherein the polymeric tether moiety of each of the nucleotide analogs comprises a reporter moiety unique to the nucleobase of the analog.
  • Embodiment 87 The method of embodiment 86, wherein the reporter moieties produce a characteristic electronic signal.
  • Embodiment 88 The method of embodiment 81, wherein the step of determining the sequence of the nucleotide analogs comprises the step of translocating the expanded polymer of nucleotide analogs through a nanopore.
  • Embodiment 89 A polymer, copolymer, or oligomer derived from one or more of the compounds of any one of embodiments 1 - 47.
  • Embodiment 90 An composition comprising one or more of the compounds of any one of embodiments 1 - 47.
  • Embodiment 91 The composition of embodiment 90, wherein the composition comprises at least two molecules of a compound having any one of embodiments 1 - 47.
  • Embodiment 92 A compound having the structure: wherein a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4;
  • Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S;
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, wherein substituents for Ari are, at each occurrence, independently selected from halogen, -OH, -CN, -NO2, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl , C 1 -C 6 heteroalkyl, C 1 - C 6 cycl
  • R 3 is, at each occurrence, independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, -C 1 -C 6 -OH, -C 1 -C 6 -C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;
  • R 4 is, at each occurrence, independently selected from one or more heteroatom interrupted alkylene, wherein the heteroatom is O, S, NH, or a combination thereof;
  • G 1 is, at each occurrence, independently selected from oxo, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -E-O-R 3 , -E-C(R 1 )(R 1 ')(R 3 ), -NH 2 , -NO 2 , -SO3R 3 , -SChCr, -
  • G 2 , G 3 , G 4 and G 5 are, at each occurrence, independently selected from absent or selected from the groups comprising, halogen, -CN, -NO2, -SO3R 3 , C 1 -C 6 alkyl, C 1 - C 6 haloalkyl, -E-O-R 3 , -E-(CH 2 ) a C(O)R 3 , -E-CO 2 H, -E-CHO, -E-C(O)R 3 , -E- C(O)NH(OH), -E-C(O)NHR 1 , -E-C(O)N(H)C(H)(R 1 )(R 1 '), -E-
  • Embodiment 93 The compound of embodiment 92, wherein each substituent of Ari is independently selected from-C(O)-CH3, -C(O)-NH 2 , -C(O)O-CH 2 CH3, -CF 3 , -C(O)O- t-butyl, -C(O)-N(CH 3 ) 2 , -C(O)-N(H)CH 3 , -C(O)-N(H)CH 2 CH 3 , -C(O)-N(H)(cycloalkyl), -C(O)-N(H)(CH 2 ) 4 , -C(O)-N(CH 2 CH 3 ) 2 , -C(O)-N(H)(t-butyl), -C(O)-N(H)(C6- cycloalkyl), -C(O)-N(H)(CH 2 ) 3 , -C(O)-N(H)(H)(H
  • Embodiment 94 A compound having the structure: a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4;
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, wherein substituents for Ari are, at each occurrence, independently selected from from C(O)-CH 3 , C(O)-NH 2 , C(O)O-CH 2 CH 3 , -CF 3 , C(O)O-t-butyl, C(O)-
  • R 0 is, at each occurrence, independently selected from C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;
  • R 3 is, at each occurrence, independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, -C 1 -C 6 -OH, -CI-C6-C(0)0H, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;
  • R 4 is, at each occurrence, independently selected from one or more heteroatom interrupted alkylene, wherein the heteroatom is O, S, NH, or a combination thereof;
  • G 1 is, at each occurrence, independently selected from oxo, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -E-O-R 3 , -E-C(R 1 )(R 1 ')(R 3 ), -NH 2 , -NO 2 , -SO3R 3 , -SO3O; -
  • G 2 , G 3 , G 4 and G 3 are, at each occurrence, independently selected from absent or selected from the groups comprising, halogen, -CN, -NO 2 , -SO3R 3 , C 1 -C 6 alkyl, C 1 - C 6 haloalkyl, -E-O-R 3 , -E-(CH 2 ) a C(O)R 3 , -E-CO 2 H, -E-CHO, -E-C(O)R 3 , -E- C(O)NH(OH), -E-C(O)NHR 1 , E C(O)N(H)C(H)(R 1 )(R 1 '), E
  • Embodiment 95 A compound having any one of Formulas (IB) or (ID): wherein wherein a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4;
  • Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S;
  • M is, at each occurrence, independently selected from hydrogen, halogen and C1-C4 alkyl
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, wherein substituents for Ari are, at each occurrence, independently selected from halogen, -OH, -CN, -NO2, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl , C 1 -C 6 heteroalkyl, C 1 - C 6 cycloloalkyl, -OR 0 , -CONH2, -C(O)NR 1 R 1 ', -C(O)(CH 2
  • Embodiment 96 A composition comprising two or more molecules of a compound having any one of the following formulas: wherein a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4;
  • Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S;
  • Embodiment 100 A compound having Formula (I): or a solvate, hydrate, tautomer, chelate or salt thereof, wherein a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4; m is 1, 2 or 3; m' is 1, 2 or 3; n is 0, 1 or 2; p is 0, 1 or 2;
  • Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S;
  • Y is, at each occurrence, independently selected from Ar2, -C(O)-Ar2, -(CH2) a Ar2, -(CH 2 ) 3 PO(OEt) 2 , or -CH 2 CO 2 Me;
  • L is a linking group
  • M is, at each occurrence, independently selected from hydrogen, halogen and C 1 - C4 alkyl;
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, wherein substituents for Ari are, at each occurrence, independently selected from halogen, -OH, -CN, -NO 2 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl , C 1 -C 6 heteroalkyl, C 1 - C 6 cycloalkyl, -OR 0 , -CONH 2 , -C(O)NR 1 R 1 ', -C(O)(CH
  • R 2 is, at each occurrence, independently selected from C 2 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;
  • R 3 is, at each occurrence, independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, -C 1 -C 6 -OH, -C 1 -C 6 -C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;
  • R 4 is, at each occurrence, independently selected from one or more heteroatom interrupted alkylene, wherein the heteroatom is O, S, NH, or a combination thereof;
  • Ar2 is, at each occurrence, independently selected from (i) a substituted or unsubstituted 5- and 6-membered monocyclic aromatic or heteroaromatic ring; (ii) a substituted or unsubstituted 9- and 10-membered fused bicyclic rings comprising two monocyclic rings together, where at least one of the two monocyclic rings is an aromatic or a heteroaromatic ring; and (iii) a substituted or unsubstituted 13- and 14-membered fused tricyclic rings comprising three monocyclic rings together, where at least one of the three monocyclic rings is an aromatic or a heteroaromatic ring; wherein each Ar2 is independently substituted with G 1 , G 2 , G 3 , G 4 and G 5 , wherein: when Ar2 is monosubstituted, G 1 is, at each occurrence, independently selected from oxo, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -
  • G 2 , G 3 , G 4 and G 5 are, at each occurrence, independently selected from absent or selected from the groups comprising, halogen, -CN, -NO 2 , -SO3R 3 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -E-O-R 3 , -E-(CH 2 ) a C(O)R 3 , -E-CO2H, -E-CHO, -E-C(O)R 3 , -E- C(0)NH(0H), -E-C(0)NHR 1 , -E-C(O)N(H)C(H)(R 1 )(R 1 '), -E-
  • Embodiment 101 The compound of embodiment 100, wherein Z includes two heteroatoms.
  • Embodiment 102 The compound of embodiment 100, wherein Z is a diazole.
  • Embodiment 103 The compound of embodiment 102, wherein the diazole is selected from the group consisting of imidazole, pyrazole, pyrimidine, pyridazine, and pyrazine.
  • Embodiment 10 The compound of embodiment 100, wherein Z includes three heteroatoms.
  • Embodiment 105) The compound of embodiment 100, wherein Z includes four heteroatoms.
  • Embodiment 106 The compound of embodiment 100, where Z is selected from the group consisting of pyrrole, thiophene, thiazole, isoxazole, and tetrazole.
  • Embodiment 107) The compound of any one of embodiments 100 - 106, wherein Ari is phenyl.
  • Embodiment 1078 The compound of any one of embodiments 100 - 106, wherein Ari is pyridine.
  • Embodiment 109) The compound of any one of embodiments 100 - 106, wherein Ari is furan.
  • Embodiment 110 The compound of any one of embodiments 100 - 106, wherein Ari is carbazole.
  • Embodiment 111) The compound of any one of embodiments 100 - 106, wherein Ari is naphthyl.
  • Embodiment 112 The compound of any one of embodiments 100 - 106, wherein Ari is pyridazine.
  • Embodiment 113 The compound of any one of embodiments 100 - 106, wherein Ari is thiophene.
  • Embodiment 114) The compound of any one of embodiments 100 - 106, wherein Ari is pyrrole.
  • Embodiment 115 The compound of any one of embodiments 100 - 106, wherein Ari is dibenzofuran.
  • Embodiment 116) The compound of any one of embodiments 100 - 106, wherein Ari is naphthyridine.
  • Embodiment 117 The compound of any one of embodiments 100 - 116, wherein each substituent of Ari is independently selected from -C(O)-CH3, -C(O)-NH 2 , -C(O)O- CH2CH3, -CF 3 , -C(O)O-t-butyl, -C(O)-N(CH 3 ) 2 , -C(O)-N(H)CH 3 , -C(O)- N(H)CH 2 CH 3 , -C(O)-N(H)(cycloalkyl), -C(O)-N(H)(CH 2 ) 4 , -C(O)-N(CH 2 CH 3 ) 2 , - C(O)-N(H)(t-butyl), -C(O)-N(H)(C6-cycloalkyl), -C(O)-N(H)(CH 2 )3, -C(O)-
  • Embodiment 118 The compound of any one of embodiments 100 - 116, wherein Ari is substituted with at least one -SO2-N(H)(R 1 ) group.
  • Embodiment 119 The compound of embodiment 118, wherein R 1 is a Ci - Cr, alkyl.
  • Embodiment 120 The compound of embodiment 119, wherein the Ci - C 6 alkyl is methyl or ethyl.
  • Embodiment 121) The compound of any one of embodiments 100 - 120, wherein Ar2 is selected from the group consisting of benzene, pyridine, pyridazine, pyrimidine and pyrazine.
  • Embodiment 122) The compound of any one of embodiments 100 - 121, wherein each Ar2 is substituted with one or more moieties selected from the group consisting of -NH 2 , -C(O)OH, -OH, -OCH3, -C(O)NHOH, -C(O)NH 2 , -(CH 2 ) 3 C(O)OH, -CF 3 , -Cl, - P(O)(OH) 2 , -F, -C(O)N(H)CH 2 CH 3 , -C(O)N(H)CH 2 C(O)OCH 3 ,
  • Embodiment 123 The compound of any one of embodiments 100 - 121, wherein each Ar2 is substituted with at least one -SO3H moiety or at least one -SO 3 ‘ moiety.
  • Embodiment 124) The compound of any one of embodiments 100 - 121, wherein each
  • Ar2 is substituted with at least two -SO3H moieties or at least two SO 3 moieties.
  • Embodiment 125 The compound of any one of embodiments 100 - 121, wherein each
  • Ar2 is substituted with at least two moieties, wherein a first of the at least two moieties comprises one of a -SO3H moiety or a - SO 3 moiety.
  • Embodiment 126) The compound of embodiment 125, wherein a second of the at least two moieties is selected from the group consisting of -NO2, -CF3, -F, -Cl, -I, methyl, ethyl, propyl, and butyl.
  • Embodiment 127) The compound of any one of embodiments 100 - 121, wherein the compound has any one of Formulas (IA) or (IB):
  • Embodiment 1278 The compound of any one of embodiments 100 - 121, wherein the compound has the formula:
  • Embodiment 129) The compound of any one of embodiments 100 - 121, wherein the compound has any one of the formulas:
  • Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S;
  • Y is, at each occurrence, independently selected from Ar2, -C(O)-Ar2, -(CH2) a Ar2, -(CH 2 ) 3 PO(OEt)2, or -CH 2 CO 2 Me;
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, wherein Ari is substituted with at least one of -OH, -C(O)NR 1 R 1 , - C(O)(CH 2 ) a NR 1 R 1 , -CO(CH 2 ) a OC(O)R 3 , -OC(O)R 3 , -S(O) 2 R 1 , -S(O) 2 NR 1 R 1 ', -CH 2 - NR 1 S(O) 2 R 3
  • G 2 , G 3 , G 4 and G 5 are, at each occurrence, independently selected from absent or selected from the groups comprising, halogen, -CN, -NO2, -SO3R 3 , C 1 -C 6 alkyl, C 1 - C 6 haloalkyl, -E-O-R 3 , — E-(CH 2 ) a C(O)R 3 , -E-CO2H, -E-CHO, -E-C(O)R 3 , -E- C(0)NH(0H), -E-C(0)NHR 1 , -E-C(O)N(H)C(H)(R 1 )(R 1 '), -E-
  • Embodiment 135) The compound of embodiment 134, wherein R 1 'is C 1 -C 6 alkyl.
  • Embodiment 137 The compound of embodiment 135, wherein the C 1 -C 6 alkyl comprises at least one substituent.
  • Embodiment 138 The compound of embodiment 136, wherein the at least one substituent is hydroxyl.
  • Embodiment 141) The compound of embodiment 140, wherein R 1 is H.
  • Embodiment 143 The compound of embodiment 142, wherein the C 1 -C 6 alkyl is branched.
  • Embodiment 145) The compound of embodiment 144, wherein the at least one substituent is hydroxyl.
  • Embodiment 146) The compound of embodiment 140, wherein R 1 and R 1 together form a cycloalkyl group or a heterocycloalkyl group.
  • Embodiment 147) The compound of any one of embodiments 131 - 146, wherein each Ar2 is substituted with at least one -SO3H moiety or at least one -SO 3 ' moiety.
  • Embodiment 150 The compound of embodiment 149, wherein a second of the at least two moieties is selected from the group consisting of -NO2, -CF3, -F, -Cl, -I, methyl, ethyl, propyl, and butyl.
  • Embodiment 151) The compound of any one of embodiments 131 - 145, wherein the compound has any one of the formulas:
  • Embodiment 153 The compound of embodiment 131, wherein the compound has the formula: [0191] Embodiment 154) The compound of embodiment 131, wherein the compound has the formula:
  • Embodiment 156) The compound of any one of embodiments 131 - 154, wherein Z is selected from the group consisting of imidazole, pyrazole, pyrimidine, pyridazine, and pyrazine.
  • Embodiment 157) The compound of any one of embodiments 131 - 154, wherein Z is not a triazole.
  • Embodiment 159) A compound having any one of the formulas: or a solvate, hydrate, tautomer, chelate or salt thereof, wherein
  • Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S;
  • Y is, at each occurrence, independently selected from Ar2, -C(O)-Ar2, -(CH2) a Ar2, - (CH 2 ) 3 PO(OEt) 2 , or -CH 2 CO 2 Me;
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole;
  • Ar2 is, at each occurrence, independently selected from (i) a substituted or unsubstituted 5- and 6-membered monocyclic aromatic or heteroaromatic ring; (ii) a substituted or unsubstituted 9- and 10-membered fused bicyclic rings comprising two monocyclic rings together, where at least one of the two monocyclic rings is an aromatic or a heteroaromatic ring; and (iii) a substituted or unsubstituted 13- and 14-membered fused tricyclic rings comprising three monocyclic rings together, where at least one of the three monocyclic rings is an aromatic or a heteroaromatic ring; wherein each Ar2 is independently substituted with G 1 , G 2 , G 3 , G 4 and G 5 , wherein: when Ar2 is monosubstituted, G 1 is, at each occurrence, independently selected from oxo, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -
  • R 1 and R 1 ' together form a substituted or unsubstituted heterocyclic ring, including, but not limited to, azetidine, pyrrolidine, piperidine, piperazine, morpholine,
  • R 2 is, at each occurrence, independently selected from C 2 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 - C 6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy; and
  • Embodiment 160 The compound of embodiment 159, wherein Z includes two heteroatoms.
  • Embodiment 161 The compound of embodiment 160, wherein Z is a diazole.
  • Embodiment 162 The compound of embodiment 161, wherein the diazole is selected from the group consisting of imidazole, pyrazole, pyrimidine, pyridazine, and pyrazine.
  • Embodiment 163 The compound of embodiment 159, wherein Z includes three heteroatoms.
  • Embodiment 164) The compound of embodiment 159, wherein Z includes four heteroatoms.
  • Embodiment 165) The compound of embodiment 159, where Z is selected from the group consisting of pyrrole, thiophene, thiazole, isoxazole, and tetrazole.
  • Embodiment 166) The compound of embodiment 159, wherein Z is a triazole.
  • Embodiment 167) The compound of embodiment 159, wherein Z is not a triazole.
  • Embodiment 1678 The compound of any one of embodiments 159- 167, wherein Ari is phenyl.
  • Embodiment 169) The compound of any one of embodiments 159- 167, wherein Ari is pyridine.
  • Embodiment 170 The compound of any one of embodiments 159- 167, wherein Ari is furan.
  • Embodiment 171) The compound of any one of embodiments 159- 167, wherein Ari is carbazole.
  • Embodiment 172 The compound of any one of embodiments 159- 167, wherein Ari is naphthyl.
  • Embodiment 173 The compound of any one of embodiments 159- 167, wherein Ari is pyridazine.
  • Embodiment 174) The compound of any one of embodiments 159- 167, wherein Ari is thiophene.
  • Embodiment 176) The compound of any one of embodiments 159- 167, wherein Ari is dibenzofuran.
  • Embodiment 177) The compound of any one of embodiments 159- 167, wherein Ari is naphthyridine.
  • Embodiment 1778 The compound of any one of embodiments 159- 167, wherein each
  • Ar2 is substituted with one or more moieties selected from the group consisting of -NH?, -C(O)OH, -OH, -OCH 3 , -C(O)NHOH, -C(O)NH 2 , -(CH 2 ) 3 C(O)OH, -CF 3 , -Cl, - P(O)(OH) 2 , -F, -C(O)N(H)CH 2 CH 3 , -C(O)N(H)CH 2 C(O)OCH 3 ,
  • Embodiment 179) The compound of any one of embodiments 159 - 177, wherein each Ar2 is substituted with at least one -SO 3 H moiety or at least one -SO 3 ‘ moiety.
  • Embodiment 180 The compound of any one of embodiments 159 - 177, wherein each Ar2 is substituted with at least two -SO3H moieties or at least two SO 3 moieties.
  • Embodiment 181) The compound of any one of embodiments 159 - 177, wherein each
  • Ar2 is substituted with at least two moieties, wherein a first of the at least two moieties comprises one of a -SO3H moiety or a -SOs’ moiety.
  • Embodiment 182 The compound of embodiment 181, wherein a second of the at least two moieties is selected from the group consisting of -NO2, -CF3, -F, -Cl, -I, methyl, ethyl, propyl, and butyl.
  • Embodiment 183) A compound having any one of the formulas:
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole;
  • R 2 is, at each occurrence, independently selected from C 2 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 - C6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;
  • R 3 is, at each occurrence, independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, -C 1 -C 6 -OH, -C 1 -C 6 -C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine; and
  • G 2 and G 3 are, at each occurrence, independently selected from absent or selected from the groups comprising, halogen, -CN, -NO 2 , -SO3R 3 , C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -E-O-R 3 , — E-(CH 2 ) a C(O)R 3 , -E-CO2H, -E-CHO, -E-C(O)R 3 , -E-
  • Embodiment 184) The compound of embodiment 183, wherein the compound has any one of the formulas:
  • Embodiment 185) The compound of embodiment 183, wherein the compound has any one of the formulas:
  • Embodiment 1878 The compound of embodiment 187, wherein the C 1 -C 6 alkyl is branched.
  • Embodiment 189) The compound of embodiment 187, wherein the C 1 -C 6 alkyl comprises at least one substituent.
  • Embodiment 191) The compound of any one of embodiments 183 - 184, wherein R 1 and R 1 ' together form a cycloalkyl group or a heterocycloalkyl group.
  • Embodiment 192 The compound of any one of embodiments 185 - 191, wherein G 2 and G 3 are each H.
  • Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S;
  • Y is, at each occurrence, independently selected from Ar2, -C(O)-Ar2, -(CH2) a Ar2, - (CH 2 ) 3 PO(OEt) 2 , or -CH 2 CO 2 Me;
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole;
  • Ar2 is, at each occurrence, independently selected from (i) a substituted or unsubstituted 5- and 6-membered monocyclic aromatic or heteroaromatic ring; (ii) a substituted or unsubstituted 9- and 10-membered fused bicyclic rings comprising two monocyclic rings together, where at least one of the two monocyclic rings is an aromatic or a heteroaromatic ring; and (iii) a substituted or unsubstituted 13- and 14-membered fused tricyclic rings comprising three monocyclic rings together, where at least one of the three monocyclic rings is an aromatic or a heteroaromatic ring; wherein each Ar2 is independently substituted with -C(O)OR 3 , -C 1 -C 6 haloalkyl, - NO 2 , and -SO3H;
  • R 3 is, at each occurrence, independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, -C 1 -C 6 -OH, -CI-C6-C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine.
  • Embodiment 195) The compound of embodiment 194, wherein the -C 1 -C 6 haloalkyl is -CF 3 .
  • Embodiment 19-7 The compound of embodiment 194, wherein Ar2 comprises two substituents.
  • Embodiment 198 The compound of embodiment 194, wherein Ar2 comprises three substituents.
  • Embodiment 199 The compound of embodiment 194, wherein Ar2 comprises four substituents.
  • Embodiment 200 The compound of embodiment 194, wherein the compound includes one -C(O)OR 3 moiety and one -C 1 -C 6 haloalkyl moiety.
  • Embodiment 201) The compound of embodiment 194, wherein the compound includes one -NO2 moiety and one -SO3H moiety.
  • Embodiment 202) The compound of embodiment 194, wherein the compound includes - SO3H moiety and one -C 1 -C 6 haloalkyl moiety.
  • Embodiment 203 The compound of embodiment 194, wherein the compound includes one -C(O)OR 3 moiety and one -NO2 moiety.
  • Embodiment 204) The compound of embodiment 194, wherein the compound includes at least one -SO3H moiety.
  • Embodiment 205) A polymer, copolymer, or metallogel comprising any one of the compounds of embodiments 100 - 204.
  • Embodiment 206) A composition comprising at least two molecules of any one of the compounds of embodiments 100 - 204.
  • Embodiment 207) A method of enhancing a nucleic acid polymerase reaction, the method comprising: a. forming a nucleic acid polymerase reaction composition comprising: i. a template nucleic acid, ii. a nucleic acid polymerase, iii. a mixture of nucleotides or nucleotide analogs, and iv. at least one of the compounds of any one of embodiments 1 - 105; and b. incubating the nucleic acid polymerase reaction composition under conditions allowing a nucleic acid polymerization reaction, wherein the at least one compound of any one of embodiments 100 - 204 increases the process! vity, rate, or fidelity of the nucleic acid polymerase reaction.
  • Embodiment 2078 The method of embodiment 207, wherein the nucleic acid polymerase is a DNA polymerase.
  • Embodiment 210) The method of embodiment 207, wherein the mixture of nucleotides or nucleotide analogs is a mixture of nucleotide analogs comprising nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric tether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphoramidate bond.
  • each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric
  • Embodiment 211) The method of embodiment 207, wherein the nucleic acid polymerization reaction produces an expandable polymer of nucleotide analogs, wherein the expandable polymer encodes the nucleobase sequence information of the template nucleic acid.
  • Embodiment 213) The method of embodiment 212, wherein the suitable polymerization buffer comprises one or more components selected from MnCh, a buffer, a salt, a sugar, a single-strand binding protein (SSB), imidazole, pyrazole, triazole, betaine, a molecular crowding agent, dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2- pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, and/or urea.
  • MnCh MnCh
  • a buffer a salt
  • a sugar a single-strand binding protein
  • SSB single-strand binding protein
  • imidazole imidazole
  • pyrazole imidazole
  • triazole betaine
  • betaine a molecular crowding agent
  • DMSO dimethyl sulfoxide
  • NMP N-methyl-2- pyrroli
  • Embodiment 214) The method of embodiment 207, wherein the nucleic acid polymerase reaction composition further comprises a single-strand binding protein.
  • Embodiment 215) The method of embodiment 207, wherein the nucleic acid polymerase reaction composition further comprises urea.
  • Embodiment 216) The method of embodiment 207, wherein the mixture of nucleotides or nucleotide analogs comprises nucleotide analogs comprising a detectable label.
  • Embodiment 217) The method of embodiment 216, wherein the detectable label is an optically detectable label selected from the group consisting of luminescent, chemiluminescent, fluorescent, fluorogenic, chromophoric or chromogenic labels.
  • Embodiment 2178 Use of the compounds of any one of embodiments 100 - 204 in a nucleic acid polymerase reaction.
  • Embodiment 219) A method of sequencing a DNA or RNA template, the method comprising the steps of: a. forming a DNA polymerase reaction composition comprising: i. a DNA or RNA template, ii. a replication primer that complexes with the template, iii. a DNA polymerase, iv. a mixture of nucleotides or nucleotide analogs, v. at least one of the compounds of any one of embodiments 100 - 204; b. incubating the DNA polymerase reaction composition under conditions allowing a DNA polymerization reaction, wherein the at least one compound of any one of embodiments 100 - 204 increases the rate, fidelity or processivity of the DNA polymerase reaction; and c.
  • a DNA polymerase reaction composition comprising: i. a DNA or RNA template, ii. a replication primer that complexes with the template, iii. a DNA polymerase, iv. a mixture of nucleotides or
  • Embodiment 220 The method of embodiment 219, wherein the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric ether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphoramidate bond.
  • the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and
  • Embodiment 221) The method of embodiment 220, wherein the DNA polymerase is DPO4 or a variant thereof.
  • Embodiment 222) The method of embodiment 219, wherein the resulting polymer of nucleotide analogs is an expandable polymer.
  • Embodiment 223 The method of any one of embodiments 219 - 222, further comprising the step of contacting the expandable polymer with a phosphoramidate cleavage agent to produce an expanded polymer of nucleotide analogs.
  • Embodiment 224) The method of embodiment 220, wherein the polymeric tether moiety of each of the nucleotide analogs comprises a reporter moiety unique to the nucleobase of the analog.
  • Embodiment 225 The method of embodiment 224, wherein each of the reporter moieties produce a characteristic electronic signal.
  • Embodiment 226) The method of embodiment 219, wherein the step of determining the sequence of the nucleotide analogs comprises the step of translocating the expanded polymer of nucleotide analogs through a nanopore.
  • Embodiment 227) Use of the compounds of any one of embodiments 100 - 204 in sequencing a DNA or RNA template.
  • Embodiment 2278 A composition comprising any one of the compounds of embodiments 1 - 105, and a molecular crowding agent.
  • Embodiment 229) The composition of embodiment 228, wherein the molecular crowding agent is a polyalkylene glycol.
  • Embodiment 230 A composition comprising any one of the compounds of embodiments 100 - 204, and a buffer.
  • the buffer comprises one or more component selected from MnCE, a buffer, a salt, a sugar, a single-strand binding protein (SSB), imidazole, pyrazole, triazole, betaine, a molecular crowding agent, dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, and/or urea.
  • MnCE dimethyl sulfoxide
  • NMP N-methyl-2-pyrrolidone
  • BHA butylated hydroxyanisole
  • Embodiment 232 The composition of embodiment 231, wherein the salt is selected from the group consisting of NaCl, NaBr, NaOAc, NaF, sodium formate, sodium phosphate monobasic, sodium phosphate dibasic, NaSO4, sodium carbonate, sodium bicarbonate, sodium hexanoate, sodium glutamate, sodium perchlorate, CsCl, LiCl, LiOAc, LiF, lithium carbonate, LiPC>4, KC1, KOAc, KF, KSO4, potassium phosphate monobasic, potassium phosphate dibasic, potassium carbonate, potassium bicarbonate, potassium glutamate, NH4CI, NH4F, NH4OAC, NH4SO4, NFEBr, ammonium citrate, ammonium carbonate, ammonium bicarbonate, ammonium sulfite, ammonium glutamate, ammonium phosphate monobasic, tetramethylammonium chloride (TMAC1), trimethylamine N-oxide
  • Embodiment 233 The composition of embodiment 231, wherein the salt is an inorganic salt.
  • Embodiment 235 The composition of embodiment 231, wherein the imidazole is a derivative or analog of imidazole selected from the consisting of imidazole chloride, imidazole acetate, 1 -methylimidazole, 2-m ethylimidazole, 1 -ethylimidazole, l-ethyl-3- methylimidazolium chloride, 2-methyl-2-imidazoline, l-butyl-3-methylimidazolium chloride, 1-methylimidazolium chloride, l-hexyl-3-methylimidazolium, 3 -octyl- 1- methylimidazolium, and l-decyl-3-methylimidazolium.
  • imidazole is a derivative or analog of imidazole selected from the consisting of imidazole chloride, imidazole acetate, 1 -methylimidazole, 2-m ethylimidazole, 1 -eth
  • Embodiment 242 A composition comprising any one of the compounds of embodiments 100 - 204, and a protein.
  • Embodiment 244 A composition comprising any one of the compounds of embodiments 100 - 204, and a mixture of nucleotides or nucleotide analogs.
  • Embodiment 245) A composition for enhancing the processivity, fidelity, or rate of a DNA polymerase reaction comprising at least one of the compounds of any one of embodiments 100 - 204, and a mixture of nucleotide analogs.
  • Embodiment 247) The composition of embodiment 246, wherein the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric ether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphoramidate bond.
  • each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety
  • Embodiment 2478 The composition of embodiment 247, further comprising a buffer component selected from at least one of Tris OAc, NH4OAc, PEG, a water-miscible organic solvent, polyphosphate 60, NMS, and MnC12.
  • a buffer component selected from at least one of Tris OAc, NH4OAc, PEG, a water-miscible organic solvent, polyphosphate 60, NMS, and MnC12.
  • Embodiment 250 The composition of embodiment 247, further comprising urea.
  • Embodiment 251 The composition of embodiment 247, wherein the mixture of nucleotide analogs comprises nucleotide analogs comprising a detectable label.
  • FIG. 2 is a schematic illustrating more details of one embodiment of an XNTP.
  • FIG. 5 is a gel showing primer extension products.
  • FIGS. 6A and 6B are histogram displays of populations of aligned reads of nanopore- derived sequences.
  • FIGS. 7A and 7B are histogram displays of populations of aligned reads of nanopore- derived sequences.
  • FIG. 8 is a gel showing primer extension products.
  • FIG. 9 is a gel showing primer extension products.
  • FIG. 10 is a gel showing primer extension products.
  • FIG. 11 is a gel showing primer extension products.
  • FIG. 13 is a gel showing primer extension products.
  • FIG. 14 is a gel showing primer extension products.
  • FIG. 15 illustrates a series of gels which show primer extension products from various exemplary PEM compounds and, in particular, PEM compounds 277 (Lane 2), 51 (Lane 3), 73 (Lane 4), 114 (Lane 5), 311 (Lane 6), 301 (Lane 7), 331 (Lane 8), 273 (Lane 9), and 367 (Lane 10) (according to the compound numbers set forth within Table 1, herein).
  • a method involving steps a, b, and c means that the method includes at least steps a, b, and c.
  • steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • the present disclosure provides compounds, such as PEM compound or a solvate, hydrate, tautomer, chelate or salt thereof, wherein a is 0 or an integer ranging from 1 - 4; a' is 0 or an integer ranging from 1 - 4; m is 1, 2 or 3; m' is 1, 2 or 3; n is 0, 1 or 2; p is 0, 1 or 2;
  • Z is a 5-membered or 6-membered heteroaromatic ring including one, two, three, or four heteroatoms selected from O, N, or S, where each Z may be independently substituted with one or more halogens and/or one or more C1-C4 alkyl groups;
  • L is a linking group
  • M is, at each occurrence, independently selected from hydrogen, halogen and C 1 - C4 alkyl;
  • Ari is, at each occurrence, independently selected from optionally substituted phenyl, pyridine, bipyridine, tripyridine, pyrazine, pyridazine, furan, dibenzofuran, thiophene, pyrrole, selenophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, wherein substituents for Ari are, at each occurrence, independently selected from halogen, -OH, -CN, -NO2, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl , C 1 -C 6 heteroalkyl, C 1 -C 6 cycloloalkyl, -OR 0
  • R 2 is, at each occurrence, independently selected from C2-C 6alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;
  • R 3 is, at each occurrence, independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, -C 1 -C 6 -OH, -C 1 -C 6 -C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine;
  • R 4 is, at each occurrence, independently selected from one or more heteroatom interrupted alkylene wherein the heteroatom is O, S, NH, or a combination thereof;
  • Y is, at each occurrence, independently selected from Ar2, -C(O)-Ar2, -(CH2) a Ar2, - (CH 2 ) 3 PO(OEt) 2 , or -CH 2 CO 2 Me;
  • Ar2 is, at each occurrence, independently selected from (i) a substituted or unsubstituted 5- and 6-membered monocyclic aromatic or heteroaromatic ring; (ii) a substituted or unsubstituted 9- and 10-membered fused bicyclic rings comprising two monocyclic rings together, where at least one of the two monocyclic rings is an aromatic or a heteroaromatic ring; and (iii) a substituted or unsubstituted 13- and 14-membered fused tricyclic rings comprising three monocyclic rings together, where at least one of the three monocyclic rings is an aromatic or a heteroaromatic ring; wherein each Ar2 is independently substituted with G 1 , G 2 , G 3 , G 4 and G 5 , wherein: when Ar2 is monosubstituted, G 1 is, at each occurrence, independently selected from oxo, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, -
  • Z is not a triazole. In some embodiments, in some embodiments, Z is not a 1,2,3-triazole or a 1,2,4-triazole.
  • Z is a diazole (e.g., imidazole, pyrazole, pyrimidine, pyridazine, pyrazine).
  • Z is pyrrole, thiophene, or triazole (e.g., 1,2,3-triazole; 1,2,4-triazole).
  • Z is thiazole.
  • Z is isoxazole.
  • Z is a tetrazole.
  • Z is selected from imidazole, thiazole, isoxazole.
  • each Z is independently substituted with one "M” moiety. In other embodiments, each Z is independently substituted with two "M” moieties. In yet other embodiments, each Z is independently substituted with three “M” moieties.
  • one or more of the compounds having Formula (I) may be polymerized (e.g., via cationic polymerization, such as by incubating one or more PEMs with KC1). In some embodiments, the present disclosure provides a composition including two or more molecules of the compounds having any one of Formulas (I), (IA), (IB), (IC), and/or (ID).
  • the compounds of Formula (I) have any one of Formulas (IA) or (IB): or a solvate, hydrate, tautomer, chelate or salt thereof, wherein Ari, Ar2, M, Z, and Y are as defined above.
  • Z is not a triazole.
  • Z is not 1,2, 3 -triazole or 1,2,4-triazole.
  • Z is a triazole.
  • one or more of the compounds having any one of Formulas (IA) or (IB) may be polymerized (e.g., via cationic polymerization, such as by incubating one or more PEMs with KC1).
  • the present disclosure provides a composition including two or more molecules of the compounds having any one of Formulas (I A) or (IB).
  • the compounds of Formula (I) have Formula (IC): or a solvate, hydrate, tautomer, chelate or salt thereof, wherein:
  • W is N when X is C or W is C when X is N; is a single or double bond, wherein the double bond begins at whichever of W or X is carbon; and m, m', n, p, Ari, L, and Y are as defined above.
  • one or more of the compounds having Formula (IC) may be polymerized (e.g., via cationic polymerization, such as by incubating one or more PEMs with KC1).
  • the present disclosure provides for a composition including two or more molecules of the compounds having Formula (IC).
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n- pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-butadienyl, 2,4- pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen atom (-O-).
  • Halo refers to bromo, chloro, fluoro or iodo.
  • Haloalkyl refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, l-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, 1- bromomethyl-2-bromoethyl, and the like.
  • alkenylene or "alkenylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, containing at least one double bond, and optionally having an indicated number of carbon atoms, e.g., from two to twelve carbon atoms.
  • alkenylene groups are ethenylene, propenylene, n-butenylene, and the like.
  • the alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a double bond or a single bond.
  • the points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain.
  • Aryl refers to a ring system radical comprising at least 5 ring atoms, optionally comprising 1-6 hetero ring atoms selected from O, S and N, and at least one aromatic ring.
  • a 5-membered monocyclic aromatic ring contains 5 ring atoms selected from carbon and heteroatoms, while a 6-membered monocyclic aromatic ring contains 6 ring atoms selected from carbon and heteroatoms.
  • Exemplary monocyclic aromatic rings having 5 members is pyrrole and having six-members is pyridine.
  • the aryl radical may be, e.g., a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems.
  • the aryl radical when the aryl radical includes non-carbon ring atoms, e.g., oxygen, sulfur, and nitrogen, the aryl group may be referred to as a heteroaryl group.
  • the heteroaryl radical may be, e.g., a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems.
  • the nitrogen, carbon, or sulfur atoms in the heteroaryl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized.
  • Heterocyclyl refers to a stable 3- to 18-membered aromatic or non-aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur.
  • the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quatemized; and the heterocyclyl radical may be partially or fully saturated.
  • R is alkyl, aralkyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl .
  • R is methyl
  • the acyl group may be referred to as acetyl.
  • Alkoxy refers to a radical of the formula -OR where R is an alkyl or haloalkyl radical. In one embodiment, the alkoxy radical contains up to six carbon atoms. Representative alkoxy groups include methoxy and ethoxy. An alkoxy that is substituted with halo may be called herein a haloalkoxy, which includes for example trifluoromethoxy, trichloromethoxy and the like.
  • Heterocyclylalkyl refers to a radical of the formula -RbRh where Rb is an alkylene chain as defined above and Rh is a heterocyclyl radical as defined above, and if the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl may be attached to the alkyl radical at the nitrogen atom.
  • the alkylene chain of the heterocyclylalkyl radical may be optionally substituted as defined above for an alkylene chain.
  • the heterocyclyl part of the heterocyclylalkyl radical may be optionally substituted as defined above for a heterocyclyl group.
  • N-heteroaryl refers to a heteroaryl radical as defined above containing at least one nitrogen and where the point of attachment of the heteroaryl radical to the rest of the molecule is through a nitrogen atom in the heteroaryl radical.
  • An N-heteroaryl radical may be optionally substituted as described above for heteroaryl radicals.
  • Heteroarylalkyl refers to a radical of the formula -RbRi where Rb is an alkylene chain as defined above and Ri is a heteroaryl radical as defined herein.
  • the heteroaryl part of the heteroarylalkyl radical may be optionally substituted as defined herein for a heteroaryl group.
  • the alkylene chain part of the heteroarylalkyl radical may be optionally substituted as defined herein for an alkylene chain.
  • an arylalkyl group refers to a heteroarylalkyl group wherein the heteroaryl portion is replaced with the corresponding carbocyclic aryl group, i.e., heteroatoms are replaced with carbon, with adjustment as necessary for hydrogen substitution.
  • Hydroxy alkyl refers to a radical of the formula -RbOH where Rb is an alkylene chain as defined herein.
  • the -OH (hydroxyl a.k.a. hydroxy) group can be attached to any carbon in the alkylene chain.
  • the alkylene chain part of the heteroarylalkyl radical may additionally be optionally substituted as defined above for an alkylene chain.
  • C carbon
  • H hydrogen
  • O oxygen
  • N nitrogen
  • Isotopically labeled PEM compounds may be useful in tracking PEM compounds or portions thereof during their use in assays etc.
  • the PEM compounds of any one of Formulas (I), (IA), (IB), (IC), and (ID) include an Ari moiety, where, in some embodiments, Ari is an aryl group, also referred to as an aromatic moiety.
  • the aromatic moiety may be a carbocyclic or heterocyclic aromatic moiety, where each of the aromatic ring atoms are carbon in a carbocyclic aromatic moiety, while at least one of the aromatic ring atoms is nitrogen, oxygen, or sulfur in a heterocyclic aromatic moiety.
  • Ari includes 1 - 6 rings, where up to six of the ring atoms may be selected from oxygen, sulfur, and nitrogen, with the remainder being carbon atoms.
  • the Ari moiety may comprise 1 - 5 rings, where up to five of the ring atoms may be selected from oxygen, sulfur, and nitrogen.
  • the Ari group may comprise 1 - 4 rings, where up to four of the ring atoms may be selected from oxygen, sulfur, and nitrogen.
  • the Ari moiety may comprise 1 - 3 rings, where up to three of the ring atoms may be selected from oxygen, sulfur, and nitrogen.
  • Ari may comprise 1 - 2 rings, where up to three of the ring atoms may be selected from oxygen, sulfur, and nitrogen.
  • each ring may independently be a five-membered ring, i.e., five ring atoms form the ring, or a sixmembered ring, or a seven-membered ring, while in some embodiments each of the rings is either a five- or six-membered ring.
  • Non-limiting examples of aromatic Ari moieties include heterocyclic aromatic moieties, which may also be referred to as a heteroaryl group.
  • any heterocyclic moiety may contain one or two or three or four or five or six aromatic rings, in addition to containing 1 or 2 or 3 or 4 or 5 or 6 heteroatoms, i.e., atoms other than carbon, selected from nitrogen, sulfur and oxygen atoms.
  • the heteroatom if present, is nitrogen, oxygen, sulfur, or selenium.
  • the aromatic moiety may be a monocyclic heterocyclic moiety, e.g., pyridinyl, which is a six-membered C5 aromatic moiety, or pyrazinyl, which is a six-membered C4 aromatic moiety.
  • the aromatic moiety may be a bicyclic heterocyclic moiety, e.g., quinolinyl or isoquinolinyl, which are ten-membered C9 aromatic moieties, or 1,5-naphthylidinyl, 2,6- naphthylidinyl or 2,7-naphthylidinyl, which are exemplary ten-membered CL aromatic moieties.
  • the heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, S, and Se.
  • an heteroaryl group designated as a C2-heteroaryl can be a 5-membered ring with two carbon atoms and three heteroatoms, a 6-membered ring with two carbon atoms and four heteroatoms and so forth.
  • a C4-heteroaryl can be a 5-membered ring with one heteroatom, a 6-membered ring with two heteroatoms, and so forth.
  • Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthal enyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, iso quinolinyl, tetrahydroquinol
  • heteroaryl and “heteroaryl groups” include fused ring compounds such as wherein at least one ring, but not necessarily all rings, are aromatic, including tetrahydroquinolinyl, tetrahydroisoquinolinyl, indolyl and 2,3-dihydro indolyl.
  • any two carbons of the Ari moiety may be substituted with one of these two Z-Ar2 moieties or triazole-Ar2 moieties.
  • Ari when Ari is substituted benzene, Ari may be substituted in the ortho, meta or para positions, as shown below, where k designates where the substitution may occur on the aromatic moiety:
  • Ari when Ari is substituted naphthalene and m is 2, Ari may be substituted at any two naphthyl carbon atoms, where the following structures show the substitution options, with k showing where the "Z" group substitution (e.g., Z-Ar2_or triazol e-Ar2) may occur on the aromatic moiety:
  • Ari is a monocyclic heteroaromatic structure selected from wherein the rings of the "Z" group (e.g., triazole rings) are substituted at positions k on Ari.
  • Ari is a monocyclic carbocyclic structure selected from: wherein the rings of the "Z" group (e.g., triazole rings) are substituted at positions k on Ari.
  • Ari is a bicyclic carbocyclic structure selected from: wherein the rings of the "Z" group (e.g., triazole rings) are substituted at positions k on Ari.
  • Ari is a polycyclic heterocyclic structure having two sixmembered rings and one five-membered ring, and one nitrogen ring atom and selected from:
  • rings of the "Z" group e.g., triazole rings
  • Ari is a polycyclic heterocyclic structure having three six-membered rings and two nitrogen ring atoms and being selected from: wherein the rings of the "Z" group (e.g., triazole rings) are substituted at positions k on Ari.
  • Ari includes both substituted and unsubstituted moieties as described herein.
  • Ari is a substituted aromatic moiety.
  • Ari is unsubstituted aromatic moiety.
  • a substituted Ari moiety one or more hydrogen atoms that would have been bonded to a ring atom has been replaced with a substituent, for example, optionally 1, or 2, or 3, or 4, or 5, or 6 of the hydrogen atoms may be replaced with a substituent (or moiety).
  • a substituent on Ari does not refer to the Z-Ar2 (e.g., triazole- Ar2) moiety that is necessarily present when m equals 1, or the two Z-Ar2 (e.g., triazole-Ar2) moieties that are necessarily present when m equals 2, or the three Z-Ar2 (e.g., triazol e-Ar2) moieties that are necessarily present when m equals 3.
  • Z-Ar2 e.g., triazole- Ar2
  • two Z-Ar2 e.g., triazole-Ar2
  • the three Z-Ar2 e.g., triazol e-Ar2
  • a substituent on Ari includes atoms selected from deuterium, halogen (F, Cl, Br, I), carbon, nitrogen, oxygen and sulfur, and optionally will also contain hydrogen, and also will contain additional atoms that form a counterion, if present.
  • Deuterium and halide are considered monovalent atoms, while carbon, nitrogen, oxygen, and sulfur, because they are capable of simultaneously forming more than one covalent bond, are considered multivalent atoms.
  • a substituent on Ari may have multiple multivalent atoms, e.g., 1 - 25 multivalent atoms, or 1 - 22 multivalent atoms, or 1 - 15 multivalent atoms, or 1 - 10 multivalent atoms, or 1 - 5 multivalent atoms, the atoms being optionally selected from carbon, nitrogen, oxygen, and sulfur. Illustrations of substituents with up to 10 multivalent atoms are provided below. Other substituents, including substituents with up to 25 multivalent atoms, are known by analogy to one of ordinary skill in the art.
  • a substituent on Ari contains 0 multivalent atoms.
  • a hydrogen bonded to a ring atom is replaced with another monovalent atom, such as deuterium, fluorine, chlorine, bromine, or iodine.
  • a substituent on Ari contains 1 multivalent atom.
  • a substituent on Ari contains 2 multivalent atoms.
  • one or more hydrogen atoms bonded to a ring atom of Ari are replaced with a first multivalent atom which, in turn, is bonded to a second multivalent atom, thus providing a substituent formed from two multivalent atoms, where open valencies on the multivalent atoms are filled with one or more monovalent atoms. Examples of these substituents are well known to one of ordinary skill in the art.
  • a substituent on Ari contains 3 multivalent atoms.
  • one or more hydrogen atoms bonded to a ring atom of Ari are replaced with a first multivalent atom which, in turn, is bonded directly or indirectly to each of a second and third multivalent atom; thus, the first multivalent atom is bonded to a second multivalent atom, and a third multivalent atom is bonded to either or both of the first and second multivalent atoms, thus providing a substituent formed from three multivalent atoms, where open valencies on the multivalent atoms are filled with one or more monovalent atoms.
  • substituents are well known to one of ordinary skill in the art and are provided herein, e.g., nitro, methylketone, carboxyl.
  • a substituent on Ari contains 4 multivalent atoms.
  • one or more hydrogen atoms bonded to a ring atom of Ari are replaced with a first multivalent atom which, in turn, is bonded directly or indirectly to each of a second, third and fourth multivalent atom, thus providing a substituent formed from four multivalent atoms, where open valencies on the multivalent atoms are filled with one or more monovalent atoms.
  • substituents are well known to one of ordinary skill in the art and are provided herein, e.g., methylester (CO2CH3), N-methylcarboxamide (C(O)NHCH 3 ) and acetamide (NHC(O)CH 3 ).
  • substituents are well known to one of ordinary skill in the art and are provided herein, e.g., ethylester (CO2CH2CH3), S-ethylcarbothioate (C(O)SCH2CH3), A-ethylcarboxamide (C(O)NHCH2CH3) and A,A-dimethylcarboxamide (C(O)N(CH 3 ) 2 ).
  • ethylester CO2CH2CH3
  • S-ethylcarbothioate C(O)SCH2CH3
  • A-ethylcarboxamide C(O)NHCH2CH3
  • A,A-dimethylcarboxamide C(O)N(CH 3 ) 2
  • a substituent on Ari contains 6 multivalent atoms.
  • one or more hydrogen atoms bonded to a ring atom of Ari are replaced with a first multivalent atom which, in turn, is bonded directly or indirectly to each of a second, third, fourth, fifth and sixth multivalent atom, thus providing a substituent formed from six multivalent atoms, where open valencies on the multivalent atoms are filled with one or more monovalent atoms.
  • A'-cyclopropyl carboxamide C(O)NH- cyclopropyl
  • .V-propyl carb oxami de C(O)NHCH2CH2CH3
  • N-(2- hydroxyethyl)carboxamide C(O)NHCH2CH2OH
  • a substituent on Ari contains 7 multivalent atoms.
  • one or more hydrogen atoms bonded to a ring atom of Ari are replaced with a first multivalent atom which, in turn, is bonded directly or indirectly to each of a second, third, fourth, fifth sixth and seventh multivalent atom, thus providing a substituent formed from seven multivalent atoms, where open valencies on the multivalent atoms are filled with one or more monovalent atoms.
  • substituents are well known to one of ordinary skill in the art and are provided herein, e.g., N-(n-butyl)carboxamide (C(O)NHCH 2 CH 2 CH2CH3), N-(t-butyl)carboxamide (C(O)NHC(CH 3 )3), N,N- diethylcarboxamide (C(O)N(CH2CH 3 )2), and N-cyclobutylcarboxamide (C(O)NH(cy cl obuty 1)) .
  • a substituent on Ari contains 8 multivalent atoms.
  • one or more hydrogen atoms bonded to a ring atom of Ari are replaced with a first multivalent atom which, in turn, is bonded directly or indirectly to each of a second, third, fourth, fifth sixth, seventh and eighth multivalent atom, thus providing a substituent formed from eight multivalent atoms, where open valencies on the multivalent atoms are filled with one or more monovalent atoms.
  • substituents are well known to one of ordinary skill in the art and are provided herein, e.g., N-cyclopentylcarboxamide (C(O)NH(cyclopentyl)), (piperidin-l-yl)methanone (C(O)-piperidin-l-yl) and (morpholin- 4-yl)methanone (C(O)-morpholin-4-yl).
  • a substituent on Ari contains 9 multivalent atoms.
  • one or more hydrogen atoms bonded to a ring atom of Ari are replaced with a first multivalent atom which, in turn, is bonded directly or indirectly to each of a second, third, fourth, fifth sixth, seventh, eighth and ninth multivalent atom, thus providing a substituent formed from nine multivalent atoms, where open valencies on the multivalent atoms are filled with one or more monovalent atoms.
  • a substituent on Ari contains 10 multivalent atoms.
  • one or more hydrogen atoms bonded to a ring atom of Ari are replaced with a first multivalent atom which, in turn, is bonded directly or indirectly to each of a second, third, fourth, fifth sixth, seventh, eighth, ninth and tenth multivalent atom, thus providing a substituent formed from ten multivalent atoms, where open valencies on the multivalent atoms are filled with one or more monovalent atoms.
  • substituents are well known to one of ordinary skill in the art and are provided herein, e.g., N- benzylcarboxamide (C(O)NHCH2(phenyl)).
  • Ari is substituted aryl wherein at least one substituent on Ari is selected from halogen, hydroxyl, mercaptan, nitro, and nitrile.
  • Ari is substituted with at least one substituent is selected from halogen, -OH, -CN, -NO2, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl , C 1 -C 6 heteroalkyl, C 1 -C 6 cycloloalkyl, -OR 0 , -CONH 2 , -C(O)NR 1 R 1 ', -C(O)(CH 2 ) a NR 1 R 1 ', -NR 1 R 1 ', -NR 1 C(O)R 3 , -C(O)SR 3 , -COR 3 , -CO(CH 2 ) a OC(O)R 3 , -OC(O)R 3 , -C(O)OR 3 , -C-O-R 3 , mercaptan, - R 4 -H, -SOR 1 , -S(O) 2 R 1 ,
  • R 2 is, at each occurrence, independently selected from C 2 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy;
  • R 4 is, at each occurrence, independently selected from one or more heteroatom interrupted alkylene wherein the heteroatom is O, S, NH, or a combination thereof.
  • Ari is substituted with at least one substituent selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted haloalkyl, and substituted or unsubstituted haloalkoxy.
  • Ari is substituted with at least one moiety selected from -O-(C1 6 alkyl), C 1 -6 alkyl, C 1 -6 haloalkyl, -CO2- C 1 -6 alkyl, -CONH- C 1 -6 alkyl, -CONH 2 , CN; and -NO 2 .
  • the compounds of the present disclosure include a linker, L.
  • the linker L may be a direct bond.
  • the linker is not a direct bond, but is instead one or more atoms, particularly atoms selected from carbon, nitrogen, oxygen, sulfur.
  • the linker may be an alkylene group (e.g., C 1 -C 6 alkylene), or a substituted alkylene.
  • the linker may be a heteroalkylene linker, which refers to a substituted or non- substituted alkylene which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within and/or placed at one or more terminal position(s) of the parent chain.
  • L is a heteroalkylene group of 2 to 10 carbon atoms in length, wherein one or more carbon atoms is replaced with at least one heteroatom selected from oxygen, nitrogen, and sulfur.
  • L may be a heteroalkylene linker having at least one N, O or S heteroatom, wherein the heteroalkylene may be a straight chain or cyclized and optionally substituted, where exemplary substituents include oxo, -OH, C 1 -4alkyl and C 1 -4alkoxy.
  • heteroalkylene linker groups include amide-containing heteroalkylene groups such as -C(O)NH-alkylene- and -C(O)NH-alkylene-NHC(O)-, where alkylene is optionally C 1 -C 6 alkylene.
  • heteroalkylene groups include ester-containing heteroalkylene groups such as -C(O)O-alkylene- and -C(O)O- alkylene-OC(O)-, where in one embodiment alkylene is unsubstituted C 1 -C 6 alkylene, and in another embodiment alkylene is substituted C 1 -C 6 alkylene.
  • the linker is hydrolytically stable, so that it does not decompose, degrade, or otherwise break when the PEM is placed into water.
  • the linker L includes from 1 to about 25 atoms excluding hydrogen and halogen from that atomic count, where the linker may optionally be composed of atoms selected from carbon, nitrogen, oxygen and sulfur, in addition to hydrogen and halogen.
  • the linker has fewer than 25 atoms (excluding hydrogen and halogen), e.g., it includes 1 to about 20 atoms, or 1 to about 15 atoms, or 1 to about 10 atoms, or 1 to about 5 atoms, in each case excluding hydrogen and halogen from that atomic count, where the counted atoms may optionally be selected from carbon, oxygen, nitrogen and sulfur.
  • a group "Z" (e.g., a triazole) in a PEM compound of Formula (I) may be substituted in addition to being directly bonded to Ari and Ar2.
  • compounds of the present disclosure may optionally be described as including the chemical formula are as defined herein.
  • Z may be substituted with one or more "M” moieties, such as 1 "M” moiety, 2 "M” moieties, 3 "M” moieties, 4 "M” moieties, etc.
  • "Z” is a triazole, and the triazole ring is substituted only by Ari and Ar2, i.e., M is hydrogen.
  • the Ar2 moieties may optionally have the same chemical structure at each occurrence.
  • those Ar2 moieties are not necessarily identical to one another, and in fact they may be non -identical.
  • each Ar2 moiety may differ from one another in terms of the Ar2 ring atoms and/or in terms of the substitution on the Ar2 ring atoms.
  • both Ar2 groups are phenyl, but one phenyl is substituted with carboxyl while the other phenyl is substituted with methoxy, as in, e.g., 4-(4-(3-(l-(4- methoxyphenyl)-lH-l, 2,3-triazol-4-yl)phenyl)-lH-l,2,3-triazol-l-yl)benzoic acid, then the compound is considered to have two different Ar2 groups.
  • the two Ar2 groups may be positional isomers of one another, as in when both Ar2 groups are phenyl, and both phenyl rings are substituted with hydroxyl and carboxyl, but the locations of the hydroxyl and/or carboxyl groups are different on the two phenyl rings, e g., if on one phenyl ring the triazole is located at the 3 position (meta) relative to the carboxyl group while on the other phenyl ring the triazole is located at the 4 position (para) relative to the carboxyl group, then the two Ar2 groups are considered to be positional isomers and non-identical.
  • the Ar2 rings are identical in all respects at each occurrence in a compound of the present disclosure.
  • the Ar2 ring atoms are identical at each occurrence of Ar2, but the substitution on the Ar2 rings is non-identical at each occurrence of Ar2.
  • the Ar2 ring atoms are non-identical at each occurrence of Ar2, and the substitution on the Ar2 rings may or may not be identical.
  • the compounds of Formula (1) include at least one Ar2 moiety.
  • Ar2 is a monocyclic 6-membered aromatic ring, non-limiting examples of which include phenyl, pyridinyl and pyrazinyl, and where the Ar2 group optionally includes substituents on the ring atoms.
  • Ar2 is a 5- membered monocyclic aromatic ring, which may optionally be substituted.
  • Ar2 is a 5- or 6-membered aromatic ring, which may optionally be substituted.
  • Ar2 is a 9- or 10-membered fused bicyclic ring comprising two 5- and/or 6-membered monocyclic rings fused together, where at least one of the two monocyclic rings is an aromatic ring.
  • Ar2 is a 9- or 10-membered fused bicyclic ring comprising two 5- and/or 6-membered monocyclic rings fused together, where both of the two monocyclic rings are an aromatic ring.
  • Ar2 is a 13- and 14-membered fused tricyclic ring comprising three monocyclic rings together, where at least one of the three monocyclic rings is an aromatic or a heteroaromatic ring.
  • Ar2 may be any of these options, that is, Ar2 is selected from (a) 5- membered monocyclic aromatic rings, (b) 6-membered monocyclic aromatic rings, (c) 9- membered fused bicyclic rings comprising one 5-membered and one 6-membered monocyclic ring fused together, where at least one of the two monocyclic rings, and optionally both of the monocyclic rings, is an aromatic ring, and (d) 10-membered fused bicyclic rings comprising two 6-membered monocyclic rings fused together, where at least one of the two monocyclic rings, and optionally both of the monocyclic rings, is an aromatic ring,
  • Ar2 is a 5- membered monocyclic aromatic ring selected from the group consisting of thiophene, 1,2- thiazole, 1,3-thiazole, furan, 1,2-oxazole, 1,3-oxazole, IH-pyrrole, IH-pyrazole, oxadiazole, thiadiazole, 1,2,4-triazole, 1,2, 3 -triazole and IH-imidazole.
  • Ar2 is a 6- membered monocyclic aromatic ring selected from the group consisting of benzene, pyridine, pyridazine, pyrimidine and pyrazine.
  • Ar2 is a 9- membered fused bicyclic aromatic ring system selected from the group consisting of benzofuran, 1,3 -benzoxazole, furo[3,2-b]pyridine, furo[3,2-c]pyridine, furo[2,3- c]pyridine, furo[2,3-b]pyridine, indole, IH-benzimidazole, lH-pyrrolo[3,2-b]pyridine, lH-pyrrolo[3,2-c]pyridine, lH-pyrrolo[2,3-c]pyridine, lH-pyrrolo[2,3-b]pyridine, benzothiophene, 1,3 -benzothiazole, thienol[3,2-b]pyridine, thieno[3,2-c]pyridine, thieno[2,3-c]pyridine, benzoxadi azo
  • Ar2 is a 10- membered fused bicyclic aromatic ring system selected from the group consisting of naphthalene, quinoline, quinazoline, quinoxaline, 1,5-naphthyridine, 1,6-naphthyridine,
  • a compound of the present disclosure includes at least one Ar2 group, where the Ar2 group includes at least one aromatic ring and optionally includes one or more substituents on the aromatic ring, such as two or more substituents, such as three or more substituents, such as four or more substituents, where any of the one or more substituents may be the same or different.
  • Ar2 includes at least one, i.e., one or more, substituent on the aromatic ring, such as 1 to 5, or 1 to 4, or 1 to 3, or 1 - 2 substituents.
  • Ar2 includes exactly one substituent on the aromatic ring.
  • Ar2 includes exactly two substituents on the aromatic ring.
  • Ar2 includes exactly three substituents on the aromatic ring.
  • Ar2 includes exactly four substituents on the aromatic ring.
  • Ar2 includes two or more substituents on the aromatic ring.
  • the one or more substituents on the ring atoms of Ar2 are selected from substituents optionally named "G", where the substituents are selected from halogen, -CN, -NO 2 , -SO 3 R 3 , CI-C 6 alkyl, C 1 -C 6 haloalkyl, -E-O-R 3 , -E-(CH 2 ) a C(O)R 3 , -E- CO 2 H, -E-CHO, -E-C(O)R 3 , -E-C(O)NH(OH), -E-C(O)NHR 1 , -E- C(O)N(H)C(H)(R 1 )(R 1 '), -E-NR 1 R 1 ', -E-OR 2 , -E-C(O)N(R 1 )((CH 2 ) a P(O)(O)OH) 2 , -E- C
  • R 0 is, at each occurrence, independently selected from C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;
  • R 1 and R 1 together form a substituted or unsubstituted heterocyclic ring, including, but not limited to, azetidine, pyrrolidine, piperidine, piperazine, morpholine,
  • R 2 is, at each occurrence, independently selected from C 2 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 - C 6 heteroalkyl, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted haloalkoxy; and
  • R 3 is, at each occurrence, independently selected from H, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 heteroalkyl, -C 1 -C 6 -OH, -C 1 -C 6 -C(O)OH, or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, substituted or unsubstituted haloalkoxy, and guanidine.
  • Non-limiting examples of substituents for Ar2 include, but are not limited to, H, -NH 2 , - C(O)OH, -OH, -OCH3, -C(0)NH0H, -C(O)NH 2 , -(CH 2 )3C(O)OH, -CF 3 , -Cl, - P(O)(OH) 2 , -F, -C(O)N(H)CH 2 CH 3 , -C(O)N(H)CH 2 C(O)OCH 3 ,
  • the substitution on Ar2 includes amino (-NH 2 ). In some embodiments, the substitution on Ar2 includes alkoxy, e.g., C 1 -C 6 alkoxy. For example, in one embodiment, the substitution on Ar2 includes methoxy. In some embodiments, the substitution on Ar2 includes carboxylic acid or alkylene-carboxylic acid. For example, in some embodiments, the substitution on Ar2 of PEM compounds of Formula (I) includes carboxylic acid. In some embodiments, the substitution on Ar2 includes carboxylic acid ester, or alkylene-carboxylic acid ester. For example, in one embodiment the substitution on Ar2 of PEM compounds of the Formula (I) includes -CH 2 -CO 2 -CH3.
  • the substitution on Ar2 includes a haloalkyl group, e.g., a C 1 -C 6 haloalkyl group.
  • the substitution on Ar2 of a PEM compounds of Formula (I) includes trifluorom ethyl.
  • the substitution on Ar2 includes hydroxyl or hydroxyl-substituted alkyl, e.g., hydroxyl-substituted C 1 -C 6 alkyl.
  • the substitution on Ar2 of a compound of Formula (I) includes hydroxyl (-OH).
  • the substitution on Ar2 includes one group selected from carboxylic acid and alkylene-carboxylic acid, e.g., C 1 -C 6 alkylene-carboxylic acid, and another group selected from hydroxyl and hydroxyl-substituted alkyl, e.g., C 1 -C 6 alkyl substituted with one hydroxyl.
  • the substitution on Ar2 is, or includes one carboxylic acid and one hydroxyl.
  • the substitution on Ar2 includes one group selected from carboxylic acid and alkylene-carboxylic acid, e.g., C 1 -C 6 alkylene-carboxylic acid, and one group selected from haloalkyl, e.g., C 1 -C 6 haloalkyl.
  • the substitution on Ar2 is, or includes one carboxylic acid group and one trifluoromethyl group.
  • the substitution on Ar2 includes one group selected from hydroxyl and hydroxyl-substituted alkyl, e.g., C 1 -C 6 alkyl substituted with one hydroxyl, and another group selected from haloalkyl, e.g., C 1 -C 6 haloalkyl.
  • the substitution on Ar2 is, or includes one hydroxyl group and one trifluoromethyl group.
  • the substitution on the Ar2 ring of Formula (I) includes at least of one of a) carboxylic acid and alkylene-carboxylic acid, e.g., C 1 -C 6 alkylene-carboxylic acid; b) hydroxyl and hydroxyl-substituted alkyl, e.g., C 1 -C 6 alkyl substituted with one hydroxyl; and c) haloalkyl, e.g., C 1 -C 6 haloalkyl.
  • carboxylic acid e.g., C 1 -C 6 alkylene-carboxylic acid
  • b) hydroxyl and hydroxyl-substituted alkyl e.g., C 1 -C 6 alkyl substituted with one hydroxyl
  • haloalkyl e.g., C 1 -C 6 haloalkyl.
  • at least one of carboxylic acid, hydroxyl and trifluoromethyl e.g.
  • the substitution on the Ar2 ring of Formula (I) includes at least two of a) carboxylic acid and alkylene-carboxylic acid, e.g., C 1 -C 6 alkylene-carboxylic acid; b) hydroxyl and hydroxyl-substituted alkyl, e.g., C 1 -C 6 alkyl substituted with one hydroxyl; and c) haloalkyl, e.g., C 1 -C 6 haloalkyl.
  • carboxylic acid and alkylene-carboxylic acid e.g., C 1 -C 6 alkylene-carboxylic acid
  • b) hydroxyl and hydroxyl-substituted alkyl e.g., C 1 -C 6 alkyl substituted with one hydroxyl
  • haloalkyl e.g., C 1 -C 6 haloalkyl.
  • the substitution on the Ar2 ring of Formula (I) includes at least one -SO3H moiety, or the salt or hydrate thereof. In some embodiments, the substitution on the Ar2 ring of Formula (I) includes at least two -SO3H moi eties (at any positions relative to each other on the Ar2 ring), or the salt or hydrate thereof. [0430] In some embodiments, the Ar2 ring of Formula (I) includes at least two substituents, where a first substituent of the at least two substituents is a -SO3H moiety or a -SCh’ moiety; and wherein a second substituent of the at least two substituents is selected from any of the "G" substituents described herein.
  • the Ar2 ring of Formula (I) includes at least three substituents, where a first substituent of the at least two substituents is a -SO3H moiety or a -SCh' moiety; and wherein a second and a third substituent of the at least three substituents is selected from - NO2, -CF3, -F, -Cl, -I, methyl, ethyl, propyl, and butyl.
  • the Ar2 ring of Formula (I) includes at least three substituents, where a first substituent of the at least two substituents is a -SO3H moiety or a -SC ’ moiety; and wherein a second substituent of the at least three substituents is substituents is a -SO3H moiety; and wherein a third substituent of the at least three substituents is selected from -NO2, -CF3, -F, methyl, ethyl, propyl, and butyl.
  • the substitution on the Ar2 ring of Formula (I) includes at least one -S(O)(O)N(H)CH2C(O)OH moiety, at least one -C(O)N(H)CH2CH2SO3H moiety, or at least one -C(O)N(H)CH2PO3H2.
  • an Ar2 moiety is substituted with at least two substituents, where a first substituent of the at least two substituents is one of -S(O)(O)N(H)CH 2 C(O)OH, -C(O)N(H)CH 2 CH 2 SO3H, or -C(O)N(H)CH 2 PO 3 H 2 .
  • the substitution on the Ar2 ring of Formula (I) includes at least an -SO 2 O’ moiety. In other embodiments, the substitution on the Ar2 ring of Formula (I) includes at least an -SO 2 O' moiety and at least one other moiety, including any of those moi eties described herein for "G.”
  • the Ar2 group may be substituted with an -SO 2 O' moiety, and may be further substituted with G 2 , G 3 , G 4 and G 5 .
  • phenyl further substituted with G 2 or with G 2 and G 3 .
  • Z is a triazole
  • the compound of Formula (I) has the structure:
  • Z is a triazole
  • the compound of Formula (I) has the structure:
  • the PEM compounds of the present disclosure have at least hydroxyl and carboxylic substitution on Ar2 and may have other substitution on Ar2.
  • Ar2 may be substituted with hydroxyl, carboxylic acid, and alkyl, e.g., C 1 -C 6 alkyl, to provide, e.g., a compound of the formula:
  • Z is selected from imidazole, pyrazole, pyrimidine, pyridazine, pyrazine, pyrrole, thiophene, triazole (e.g., 1,2,3-triazole; 1,2,4-triazole, thiazole, and isoxazole. In some embodiments, Z is not a triazole. In some embodiments, in some embodiments, Z is not 1,2,3-triazole or 1,2,4-triazole. In some embodiments, Ari is selected from pyridine, bipyridine, phenyl, and carbazole.
  • Ari may be substituted with -C(O)-CH 3 , -C(O)-NH2, -C(O)O-CH 2 CH3, -CF3, -C(O)O-t-butyl, -C(O)-N(CH 3 ) 2 , -C(O)-N(H)CH 3 , -C(O)-N(H)CH 2 CH 3 , -C(O)-N(H)(cycloalkyl), - C(0) -N(H)(CH 2 ) 4 , -C(O)-N(CH 2 CH 3 ) 2 , C(O) N(H)(t butyl), C(0) N(H)(C6 cycloalkyl), -C(O)-N(H)(CH 2 ) 3 , -C(O)-N(H)(C5-cycloalkyl), -C(0)-N(H)(C4- cycloalkyl), -
  • Z is a triazole
  • the compound of Formula (I) has the structure:
  • the PEM compounds of the present disclosure may have haloalkyl and carboxylic acid substitution on Ar2 rather than hydroxyl and carboxylic acid as illustrated in the structures above.
  • the PEM compounds of the present disclosure may be described by the formula: [0451]
  • Z is selected from imidazole, pyrazole, pyrimidine, pyridazine, pyrazine, pyrrole, thiophene, triazole (e.g., 1,2,3-triazole; 1,2,4-triazole, thiazole, and isoxazole.
  • Z is not a triazole.
  • Z is not 1,2,3-triazole or 1,2,4-triazole.
  • Ari is selected from pyridine, bipyridine, phenyl, and carbazole.
  • Ari may be substituted with -C(O)-CH 3 , -C(O)-NH 2 , -C(O)O-CH 2 CH 3 , -CF 3 , -C(O)O-t-butyl, -C(O)-N(CH 3 ) 2 , -C(O)-N(H)CH 3 , -C(O)-N(H)CH 2 CH 3 , -C(O)-N(H)(cycloalkyl), - C(O)-N(H)(CH 2 ) 4 , -C(O)-N(CH 2 CH 3 ) 2 , -C(O)-N(H)(t-butyl), -C(O)-N(H)(C6- cycloalkyl), -C(O)-N(H)(CH 2 ) 3 , -C(O)-N(H)(C5-cycloalkyl), -C(O)-CH 3
  • Z is a triazole
  • the compound of Formula (I) has the structure:
  • the PEM compounds of the present disclosure include one or more -SO 3 H moieties or -SO 2 O' moieties, and optionally one or more additional substituents on Ar2 (such as any one of G 2 , G 3 , G 4 , or G 5 as described herein.
  • additional substituents on Ar2 such as any one of G 2 , G 3 , G 4 , or G 5 as described herein. Examples of such PEM compounds include, but are not limited to, those recited below:
  • Z is selected from imidazole, pyrazole, pyrimidine, pyridazine, pyrazine, pyrrole, thiophene, triazole (e g., 1,2,3-triazole; 1,2,4-triazole, thiazole, and isoxazole. In some embodiments, Z is not a triazole. In some embodiments, in some embodiments, Z is not 1,2,3-triazole or 1,2,4-triazole. In some embodiments, Ari is selected from pyridine, bipyridine, phenyl, and carbazole.
  • the group Ari may be substituted (such as with any of the substituents noted herein), or unsubstituted.
  • Ari may be substituted with -C(O)-CH 3 , -C(O)-NH2, -C(O)O-CH2CH 3 , - CF 3 , -C(O)O-t-butyl, -C(O)-N(CH 3 ) 2 , C(O)-N(H)CH 3 , -C(O)-N(H)CH 2 CH 3 , C(O)- N(H)(cycloalkyl), -C(O)-N(H)(CH 2 ) 4 , -C(O)-N(CH 2 CH 3 ) 2 , -C(O)-N(H)(t-butyl), -C(O)- N(H)(C6-cycloalkyl), -C(O)-N(H)(CH 2 ) 3 ,
  • Z is a triazole, and the above-recited compounds of Formula (I) have the structure: or the salt or hydrate thereof.
  • Ari may be substituted with -C(O)-CH 3 , -C(O)-NH 2 , -C(O)O- CH 2 CH 3 , -CF 3 , -C(O)O-t-butyl, -C(O)-N(CH 3 ) 2 , -C(O)-N(H)CH 3 , -C(O)-N(H)CH 2 CH 3 , -C(O)-N(H)(cycloalkyl), -C(O)-N(H)(CH 2 ) 4 , -C(O)-N(CH 2 CH 3 ) 2 , -C(O)-N(H)(t-butyl), -C(O)-N(H)(C6-cycloalkyl), -C(O)-N(H)(CH 2 ) 3 , -C(O)-N(H)(C5 -cycloalkyl),
  • PEM compounds in accordance with the present disclosure that include an Ar2 moiety substituted with -SO 3 H (or the salt thereof) include, but are not limited to, those compounds recited below:
  • the -SO3H group (or the salt thereof, namely -SO3 ) may be provided at any ring position, such as para to the group "Z,” meta to the group “Z,” or ortho to the group “Z.”.
  • Z is selected from imidazole, pyrazole, pyrimidine, pyridazine, pyrazine, pyrrole, thiophene, triazole (e.g., 1,2,3-triazole; 1,2,4-triazole, thiazole, tetrazole, and isoxazole.
  • Z is not a triazole.
  • Z is not 1,2,3-triazole or 1,2,4-triazole.
  • Ari is selected from pyridine, bipyridine, phenyl, and carbazole.
  • PEM compounds in accordance with the present disclosure that include an Ar2 moiety substituted with -SO3H (or the salt thereof) include, but are not limited to, those compounds recited below:
  • Ari is selected from pyridine, bipyridine, phenyl, and carbazole.
  • PEM compounds including substituents on Ar2 include those having the following structures:
  • Z is selected from imidazole, pyrazole, pyrimidine, pyridazine, pyrazine, pyrrole, thiophene, tetrazole, triazole (e.g., 1,2,3-triazole; 1,2,4-triazole, thiazole, and isoxazole.
  • Z is not a triazole.
  • Z is not 1,2,3-triazole or 1,2,4-triazole.
  • Ari is selected from pyridine, bipyridine, phenyl, and carbazole.
  • the group Ari may be substituted (such as with any of the substituents noted herein), or unsubstituted.
  • Ari may be substituted with -C(O)-CH 3 , -C(O)-NH 2 , -C(O)O-CH2CH3, - CF 3 , -C(O)O-t-butyl, -C(O)-N(CH 3 ) 2 , -C(O)-N(H)CH 3 , -C(O)-N(H)CH 2 CH 3 , -C(O)- N(H)(cycloalkyl), -C(O)-N(H)(CH 2 ) 4 , -C(O)-N(CH 2 CH 3 ) 2 , -C(O)-N(H)(t-butyl), -C(O)- N(H)(C6-cycloalkyl), -C(O)-N(H)(CH 2 )
  • the PEM compounds of Formula (I) may be in the form of a chelate, such as a copper chelate.
  • a copper chelate may be formed by combining a PEM compound of the present disclosure with copper sulfate.
  • the PEM compounds of Formula (I) may be in the form of a salt, either an acid addition salt or a base addition salt, depending on the substituents on the Ari and Ar2 groups.
  • the PEM structures include all stable stereoisomeric forms thereof.
  • the PEM compounds described herein may have one or more chiral (or asymmetric) centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-.
  • the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers (e.g., cis or trans).
  • the present disclosure provides for polymers of the PEM compounds described herein.
  • the PEM compounds disclosed herein serve as monomers which may be polymerized into polymers (e.g., via cationic polymerization, such as by incubating a PEM with KC1).
  • the term "polymer” is defined as being inclusive of homopolymers, copolymers, and oligomers.
  • homopolymer is defined as a polymer derived from a single species of monomer, e.g., a single PEM monomer species.
  • copolymer is defined as a polymer derived from more than one species of monomer, including copolymers that are obtained by copolymerization of two monomer species (such as two different PEM monomer species).
  • oligomer is defined as a low molecular weight polymer in which the number of repeating units does not exceed twenty.
  • the polymers of the present disclosure include two repeating units, such as three repeating units, such as four repeating units, such as five repeating units, such as six repeating units, etc.
  • the polymers, copolymers, and/or oligomers are formed through covalent, ionic, or weak interactions.
  • the present disclosure provides for compositions including two or more molecules of any one of the compounds disclosed herein (such as two or more compounds having any one of Formulas (I), (IA), (IB), (IC), and/or (ID)).
  • the compositions include 10 or more molecules of any of the compounds disclosed herein, such as 20 or more molecules of any of the compounds disclosed herein, such as 50 or more molecules of any of the compounds disclosed herein, such as 100 or more molecules of any of the compounds disclosed herein, such as 200 or more molecules of any of the compounds disclosed herein, etc.
  • the PEM compounds of the present disclosure generally are water soluble.
  • One measure of water solubility is the logP value of a compound.
  • LogP values may be calculated using commercial software, based on the chemical structure of the compound. For instance, the CHEMDRAW chemical drawing software (Cambridgesoft Limited, a subsidiary of PerkinElmer Holdings) can calculate a logP value for a drawn chemical structure.
  • a PEM compound of the present disclosure has a logP of at least 4.9.
  • the compounds of the present disclosure may typically be synthesized by the reaction of diethynyl compounds of the formula Ar l(OCH)2 with azide compounds of the formula Ar2-N3, such as in the presence of Cu(I) catalyst. See also Crowley J.D., McMorran D.A. (2012) “Click-Triazole” Coordination Chemistry: Exploiting 1,4-Disubstituted-1,2,3-Triazoles as Ligands. In: Kosmrlj J. (eds.) Click Triazoles. Topics in Heterocyclic Chemistry, vol. 28. Springer, Berlin, Heidelberg doi.org/10.1007/7081_2011_67.
  • non-limiting examples of suitable compounds of the formula Arl (C ⁇ CH)2 include:
  • Each of these reaction products may function as the precursor to Ari in preparing PEMs of the present disclosure.
  • the reactions (I), (II) and (111) illustrate the preparation of a precursor to a substituted Ari moiety of the present disclosure.
  • nucleic acids also referred to as polynucleotides, are covalently linked series of nucleotides in which the 3' position of the pentose of one nucleotide is joined by a phosphodiester group to the 5' position of the next.
  • a nucleic acid molecule can be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination of both.
  • DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are biologically occurring polynucleotides in which the nucleotide residues are linked in a specific sequence by phosphodiester linkages.
  • variant polymerase examples are the variants of Sulfolobus sulfataricus DPO4 described in published PCT patent application WO2017/087281 Al and PCT patent applications nos. PCTUS2018/030972 and PCTUS2018/64794 which are hereby incorporated by reference in their entirety.
  • enhancing a nucleic acid polymerase reaction refers to the ability of an additive, e.g., a PEM to enable a nucleic acid polymerase to synthesize a primer extension product at least one subunit longer in length than it would in the absence of the PEM.
  • the rate of a nucleic acid polymerase reaction as used herein refers to the average speed at which a nucleic acid polymerase extends a polymer chain. As used herein, the terms “speed” and “elongation rate” are used inter-changeably.
  • the nucleotide incorporation assay of Hogrefe et al. (Methods in Enzymol. Vol. 334, pp. 91-116 (2001)) can be used to measure the rate of polymerization. Briefly, polymerase activity can be measured as the rate of incorporation of 32 P-dCTP into activated salmon sperm DNA (purchased from Pharmacia; for activation protocol see C. C. Richardson, Procedures in Nucl. Acid Res.
  • Nucleotide substrates and DNA are used in large excess, typically at least 10 times the Km for the polymerase being assayed, e.g., 200 ⁇ M each of dATP, dTTP, and dGTP, 195 ⁇ M of dCTP plus 5 ⁇ IM of labeled dCTP, and 250 ⁇ g/ml of activated DNA.
  • the reactions are quenched on ice, and aliquots of the reaction mixture are spotted onto ion exchange filters (e.g., Whatman DE81). Unincorporated nucleotide is washed through, followed by scintillation counting to measure incorporated radioactivity.
  • increasing the rate refers to an increase of 5 - 10%, 5 - 20%, 10 - 50%, or 50 - 100% or more, as compared to a polymerization reaction that lacks a PEM that increases rate as defined herein.
  • processivity refers to the extent of polymerization by a nucleic acid polymerase during a single contact between the polymerase and its template, i.e., its property to continue to act on a substrate instead of dissociating therefrom.
  • the extent of polymerization refers to the number of nucleotides or nucleotide analogs added by the polymerase during a single contact between the polymerase and its template.
  • Processivity can depend on the nature of the polymerase, the sequence of a template, the structure of the nucleotide or nucleotide analog substrates, and the reaction conditions, for example, salt concentration, temperature, or the presence of specific additives.
  • increasing the processivity refers to an increase of 5-10%, 5 - 20%, 10- 50%, or 50-100% or more, as compared to a polymerization reaction that lacks a PEM that increases processivity as defined herein.
  • Methods for measuring processivity of a nucleic acid polymerase are generally known in the art, e.g., as described in Sambrook et al. 1989, In Molecular Cloning, 2nd Edition, CSH Press, 7.79-7.83 and 13.8, as described in U.S. published patent application no. 2002/0119467, published PCT application no. W001/92501 and in U.S. Pat. No. 5,972,603, the entireties of which are incorporated herein by reference.
  • fidelity refers to the accuracy of nucleic acid polymerization by template-dependent nucleic acid polymerase.
  • the fidelity of a DNA polymerase is measured by the error rate (the frequency of incorporating an inaccurate nucleotide, i.e., a nucleotide that is not incorporated at a template-dependent manner).
  • the fidelity or error rate of a DNA polymerase may be measured using assays known to the art (see for example, Lundburg et al., 1991 Gene, 108: 1-6).
  • “increasing the fidelity” refers to an increase of 5-10%, 10-50%, or 50-100% or more, as compared to a polymerization reaction that lacks an additive that increases fidelity as defined herein.
  • XNTP is an expandable, 5' triphosphate modified nucleotide substrate compatible with template dependent enzymatic polymerization.
  • An XNTP has two distinct functional components; namely, a nucleobase 5'-triphosphoramidate and a tether that is attached within each nucleoside triphosphoramidate at positions that allow for controlled expansion by intra-nucleotide cleavage of the phosphoramidate bond.
  • XNTPs are exemplary "nonnatural, highly substituted nucleotide analog substrates", as used herein. Exemplary XNTPs and methods of making the same are described, e.g., in Applicants' published PCT application no. W02016/081871, herein incorporated by reference in its entirety.
  • Xpandomer or "Xpandomer product” is a synthetic molecular construct produced by expansion of a constrained Xpandomer, which is itself synthesized by template-directed assembly of XNTP substrates.
  • the Xpandomer is elongated relative to the target template it was produced from. It is composed of a concatenation of subunits, each subunit a motif, each motif a member of a library, comprising sequence information, a tether and optionally, a portion, or all of the substrate, all of which are derived from the formative substrate construct.
  • the Xpandomer is designed to expand to be longer than the target template thereby lowering the linear density of the sequence information of the target template along its length.
  • the Xpandomer optionally provides a platform for increasing the size and abundance of reporters which in turn improves signal to noise for detection. Lower linear information density and stronger signals increase the resolution and reduce sensitivity requirements to detect and decode the sequence of the template strand.
  • Tether or "tether member” refers to a polymer or molecular construct having a generally linear dimension and with an end moiety at each of two opposing ends.
  • a tether is attached to a nucleoside triphosphoramidate with a linkage at end moiety to form an XNTP.
  • the linkages serve to constrain the tether in a "constrained configuration”.
  • Tethers have a "constrained configuration” and an "expanded configuration”.
  • the constrained configuration is found in XNTPs and in the daughter strand, or Xpandomer intermediate.
  • the constrained configuration of the tether is the precursor to the expanded configuration, as found in Xpandomer products.
  • Tethers comprise one or more reporters or reporter constructs along its length that can encode sequence information of substrates.
  • the tether provides a means to expand the length of the Xpandomer and thereby lower the sequence information linear density.
  • Tether element or "tether segment” is a polymer having a generally linear dimension with two terminal ends, where the ends form end-linkages for concatenating the tether elements.
  • Tether elements are segments of tether.
  • Such polymers can include, but are not limited to: polyethylene glycols, polyglycols, polypyridines, polyisocyanides, polyisocyanates, poly(triarylmethyl)methacrylates, polyaldehydes, polypyrrolinones, polyureas, polyglycol phosphodiesters, polyacrylates, polymethacrylates, polyacrylamides, polyvinyl esters, polystyrenes, polyamides, polyurethanes, polycarbonates, polybutyrates, polybutadienes, polybutyrolactones, polypyrrolidinones, polyvinylphosphonates, polyacetamides, polysaccharides, polyhyaluranates, polyamides, polyimides, polyesters, polyethylenes,
  • Reporter construct comprises one or more reporters that can produce a detectable signal(s), wherein the detectable signal(s) generally contain sequence information. This signal information is termed the "reporter code” and is subsequently decoded into genetic sequence data.
  • a reporter construct may also comprise tether segments or other architectural components including polymers, graft copolymers, block copolymers, affinity ligands, oligomers, haptens, aptamers, dendrimers, linkage groups or affinity binding group (e.g., biotin).
  • Reporter Code is the genetic information from a measured signal of a reporter construct.
  • the reporter code is decoded to provide sequence-specific genetic information data.
  • the present disclosure provides a composition comprising a PEM as disclosed herein and a buffer.
  • the present disclosure provides a composition comprising a PEM as disclosed herein and a plurality of nucleotides and/or nucleotide analogs.
  • the present disclosure provides a composition comprising a PEM as disclosed herein and a polynucleotide.
  • the present disclosure provides a composition comprising a PEM as disclosed herein and a protein, where optionally the protein is a polymerase including any of the polymerases described above.
  • the composition further comprises one or more component selected from MnCE, a buffer, a salt, a sugar, a single-strand binding protein (SSB), imidazole, pyrazole, triazole, betaine, a molecular crowding agent, dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, urea, and a mixture of nucleotides or nucleotide analogs.
  • DMSO dimethyl sulfoxide
  • NMP N-methyl-2-pyrrolidone
  • BHA butylated hydroxyanisole
  • the salt is salt is selected from the group consisting of NaCl, NaBr, NaOAc, NaF, sodium formate, sodium phosphate monobasic, sodium phosphate dibasic, NaSC>4, sodium carbonate, sodium bicarbonate, sodium hexanoate, sodium glutamate, sodium perchlorate, CsCl, LiCl, LiOAc, LiF, lithium carbonate, LiPCE, KC1, KOAc, KF, KSO4, potassium phosphate monobasic, potassium phosphate dibasic, potassium carbonate, potassium bicarbonate, potassium glutamate, NH4CI, NH4F, NH4OAC, NH4SO4, NFUBr, ammonium citrate, ammonium carbonate, ammonium bicarbonate, ammonium sulfite, ammonium glutamate, ammonium phosphate monobasic, tetramethylammonium chloride (TMAC1), trimethylamine N-oxide (TMAO), te
  • TMAC1 trimethylamine
  • the sugar is maltose, trehalose, cellobiose or sucrose.
  • the SSB is selected from the group consisting of KOD (SEQ ID NO: 6), Gp32 (SEQ ID NO: 7), TTH (SEQ ID NO: 8), SSB1 (SEQ ID NO: 9), RecA (SEQ ID NO: 10), RPA (SEQ ID NO: 11), and NCp7 (SEQ ID NO: 12).
  • the imidazole includes (apart from imidazole as such) imidazole derivatives, such as imidazole chloride, imidazole acetate, 1 -methylimidazole, 2- methylimidazole, 1 -ethylimidazole, l-ethyl-3-methylimidazolium chloride, 2-methyl-2- imidazoline, l-butyl-3-methylimidazolium chloride, 1-methylimidazolium chloride, 1- hexyl-3-methylimidazolium, 3 -octyl- 1-methylimidazolium, or l-decyl-3- methylimidazolium.
  • imidazole derivatives such as imidazole chloride, imidazole acetate, 1 -methylimidazole, 2- methylimidazole, 1 -ethylimidazole, l-ethyl-3-methylimidazolium chloride, 2-methyl-2- imidazoline
  • the alkanediol is ethylenglycol, a propanediol or a butanediol, such as a propanediol.
  • the propanediol is 1,2-propanediol or 1,3-propanediol, such as 1,2 propanediol.
  • the butanediol is 1,2-butanediol, 1,3 -butanediol, 1,4- butanediol, 2,3-butanediol, 2,4-butanediol, or 3,4-butanediol, such as 1,2-butanediol.
  • the molecular crowding agent is PEG, such as PEG4k to PEG25k or PEG4k to PEG 10k, such as PEG5k, PEG8k, or PEGlOk.
  • the polyphosphate is tripolyphosphate, tetrapolyphosphate, pentapolyphosphate, hexapolyphosphate, trimetaphosphate (TMP), hexametaphosphate (HMP), or polyphosphate 60.
  • the present disclosure provides a composition comprising a PEM compound of the present disclosure, e.g., a PEM compound of any one of Formulas (I), (IA), (IB), (IC), and (ID) and a molecular crowding agent.
  • molecular crowding agents include a range of large, neutral polymers.
  • useful molecular crowding reagents include, but are not limited to, polyethylene glycol (PEG), ficoll, dextran, or polyvinyl alcohol. Exemplary molecular crowding reagents and formulations are set forth in U.S. Pat. No. 7,399,590, which is incorporated herein by reference.
  • the molecular crowding agent is a polyalkylene glycol, optionally having a number average molecular weight of 4,000-10,000.
  • the molecular crowing agent is a derivative of a polyalkylene glycol, e.g., one or both of the terminal hydroxyl groups of a polyalkylene glycol is in the form of an ester or ether group.
  • the polyalkylene glycol is polyethylene glycol.
  • the molecular crowding agent is an inert, water-soluble polymer.
  • the present disclosure provides a composition comprising a PEM compound of the present disclosure and an aqueous buffer.
  • the PEM compound has any one of Formulas (I), (IA), (IB), (IC), and (ID).
  • the composition has a pH of about 6 to 8.5, and the buffer helps to stabilize the pH of the composition.
  • An exemplary buffer is Tris HC1.
  • suitable buffers include those known in the art, e.g., phosphate buffers, citric acid buffers, sodium acetate buffers, sodium carbonate buffers, and the like.
  • the buffer is selected from the group consisting of TrisCi, TrisOAc, NH4OAC, MES, and HEPES.
  • the present disclosure provides a composition comprising a PEM compound of the present disclosure, e.g., a PEM compound of any one of Formulas (I), (IA), (IB), (IC), and (ID), and a polynucleotide.
  • a PEM compound of the present disclosure e.g., a PEM compound of any one of Formulas (I), (IA), (IB), (IC), and (ID)
  • the polynucleotide is single stranded, e g., single stranded DNA or a single stranded RNA.
  • the polynucleotide is intended to function as a primer, the polynucleotide is a single stranded DNA molecule.
  • the polynucleotide When intended to function as a primer, the polynucleotide may have a length of about 10- 60 mer oligonucleotide, e.g., 20-30 oligonucleotides.
  • the polynucleotide may alternatively function as a template, in which case it may be a single stranded DNA or a single stranded RNA and may have a length of from 30 bases to kilobase and above values, e.g., 10k bases and above.
  • the present disclosure provides a composition comprising a PEM compound of the present disclosure, e.g., a PEM compound of any one of Formulas (I), (1A), (IB), (IC), and (ID), and a protein.
  • the protein may be an enzyme, a nucleic acid polymerase, a DNA polymerase.
  • a suitable DNA polymerase is a variant of DP04 polymerase, as discussed herein.
  • the present disclosure provides a composition comprising at least one PEM compound of the present disclosure, e.g., a PEM compound having any one of Formulas (I), (IA), (IB), (IC), and (ID), and a mixture of nucleotides or nucleotide analogs wherein the at least one compound increases the number and accuracy of nucleotide analogs incorporated into a daughter strand during a template-dependent polymerization reaction relative to an identical polymerization reaction absent the at least one compound.
  • a PEM compound of the present disclosure e.g., a PEM compound having any one of Formulas (I), (IA), (IB), (IC), and (ID), and a mixture of nucleotides or nucleotide analogs wherein the at least one compound increases the number and accuracy of nucleotide analogs incorporated into a daughter strand during a template-dependent polymerization reaction relative to an identical polymerization reaction absent the at least one compound.
  • the mixture of nucleotide analogs includes nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates has a nucleobase selected from adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric ether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphoramidate bond.
  • nucleobase selected from adenine, guanine, thymine, and cytosine
  • polymeric tether moiety wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric ether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to
  • the composition further includes a buffer which includes one or more components selected from MnCh, a buffer, a salt, a sugar, a singlestrand binding protein (SSB), imidazole, pyrazole, triazole, betaine, a molecular crowding agent, dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, and/or urea.
  • the composition also includes a single-strand binding protein.
  • the composition includes urea.
  • the mixture of nucleotide analogs includes nucleotide analogs that comprise a detectable label, where the detectable label is optionally one of luminescent, chemiluminescent, fluorescent, fluorogenic, chromophoric or chromogenic.
  • the composition includes two or more of these options, e.g., all, of these options.
  • PEMs and compositions thereof as disclosed herein may be used to enhance a nucleic acid polymerization reaction or improve the properties of the resulting nucleic acid, e.g., the length or accuracy of the reaction product.
  • Polymerization reactions include, e.g., primer extension reactions, PCR, mutagenesis, isothermal amplification, DNA sequencing, and probe labeling. Such methods are well known in the art.
  • Enhancement may be provided by stimulating nucleotide incorporation through mechanisms such as increasing processivity of the polymerase (i.e., reducing dissociation of the polymerase from the template), increasing the rate of substrate binding or enzymatic catalysis, and increasing the accuracy or fidelity of nucleotide incorporation.
  • enhancement may be provided by reducing impediments in the nucleic acid template, such as secondary structure and duplex DNA.
  • impediments in the nucleic acid template such as secondary structure and duplex DNA.
  • PEMs can allow polymerization reactions to occur more accurately or efficiently or allow the use of lower denaturation/extension temperatures or isothermal temperatures.
  • a PEM may be used in combination with another additive classes to enhance a polymerase reaction.
  • One exemplary class of additives is minor groove binding proteins (MGBs).
  • MGB is selected from the group consisting of distamycin A and synthetic analogs thereof, netropsin, (+)-CC-1065, duocarmycins, pyrrolobenzodiazepines, trabectin and analogs thereof, Hoechst dyes and derivatives thereof, lexitropsin, thiazotropsin A, diamidines, and polyamides.
  • the at least one minor groove binding moiety is a Hoechst dye. More information about the use of MGBs to enhance a polymerase reaction may be found in applicants' co-filed application titled ENHANCEMENT OF NUCLEIC ACID POLYMERIZATION BY MGBS.
  • One exemplary polymerase reaction that can be enhanced with PEMs is the polymerization of the non-natural nucleotide analogs known as "XNTPs", which forms the basis of the "Sequencing by Expansion” (SBX) protocol, developed by Stratos Genomics (see, e.g., Kokoris et al., U.S. Pat. No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion”).
  • SBX uses this biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an "Xpandomer".
  • the transcribed sequence is encoded along the Xpandomer backbone in high signal-to-noise reporters that are separated by about 10 nm and are designed for high-signal -to-noise, well- differentiated responses. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to native DNA.
  • FIGS. 1A, IB, 1C and ID A generalized overview of the SBX process is depicted in FIGS. 1A, IB, 1C and ID.
  • XNTPs are expandable, 5' triphosphate modified nucleotide substrates compatible with template dependent enzymatic polymerization.
  • a highly simplified XNTP is illustrated in FIG. 1A, which emphasizes the unique features of these nucleotide analogs:
  • XNTP 100 has two distinct functional regions; namely, a selectively cleavable phosphorami date bond 110, linking the 5' a-phosphate 115 to the nucleobase 105, and a tether 120 that is attached within the nucleoside triphosphoramidate at positions that allow for controlled expansion by intra-nucleotide cleavage of the phosphoramidate bond.
  • the tether of the XNTP is comprised of linker arm moieties 125A and 125B separated by the selectively cleavable phosphoramidate bond.
  • Each linker attaches to one end of a reporter 130 via a linking group (LG), as disclosed in U.S. Pat. No. 8,324,360 to Kokoris et al., which is herein incorporated by reference in its entirety.
  • XNTP 100 is illustrated in the "constrained configuration", characteristic of the XNTP substrates and the daughter strand following polymerization.
  • the constrained configuration of polymerized XNTPs is the precursor to the expanded configuration, as found in Xpandomer products. The transition from the constrained configuration to the expanded configuration occurs upon scission of the P-N bond of the phosphoramidate within the primary backbone of the daughter strand.
  • FIGS. IB and 1C Synthesis of an Xpandomer is summarized in FIGS. IB and 1C.
  • the monomeric XNTP substrates 145 XATP, XCTP, XGTP and XTTP
  • XATP, XCTP, XGTP and XTTP are polymerized on the extendable terminus of a nascent daughter strand 150 by a process of template-directed polymerization using single-stranded template 140 as a guide.
  • this process is initiated from a primer and proceeds in the 5' to 3' direction.
  • a DNA polymerase or other polymerase is used to form the daughter strand, and conditions are selected so that a complimentary copy of the template strand is obtained.
  • the coupled tethers comprise the constrained Xpandomer that further comprises the daughter strand.
  • Tethers in the daughter strand have the "constrained configuration" of the XNTP substrates.
  • the constrained configuration of the tether is the precursor to the expanded configuration, as found the Xpandomer product.
  • the transition from the constrained configuration 160 to the expanded configuration 165 results from cleavage of the selectively cleavable phosphoramidate bonds (illustrated for simplicity by the unshaded ovals) within the primary backbone of the daughter strand.
  • the tethers comprise one or more reporters or reporter constructs, 130A, 130C, 130G, or 130T, specific for the nucleobase to which they are linked, thereby encoding the sequence information of the template.
  • the tethers provide a means to expand the length of the Xpandomer and lower the linear density of the sequence information of the parent strand.
  • FIG. ID illustrates an Xpandomer 165 translocating through a nanopore 180, from the cis reservoir 175 to the trans reservoir 185.
  • each of the reporters of the linearized Xpandomer (in this illustration, labeled “G”, “C” and “T”) generates a distinct and reproducible electronic signal (illustrated by superimposed trace 190), specific for the nucleobase to which it is linked.
  • FIG. 2 depicts the generalized structure of an XNTP in more detail.
  • XNTP 200 is comprised of nucleobase triphosphoramidate 210 with linker arm moieties 220A and 220B separated by selectively cleavable phosphoramidate bond 230.
  • Tethers are joined to the nucleoside triphosphoramidate at linking groups 250A and 250B, wherein a first tether end is joined to the heterocycle 260 (represented here by cytosine, though the heterocycle may be any one of the four standard nucleobases, A, C, G, or T) and the second tether end is joined to the alpha phosphate 270 of the nucleobase backbone.
  • the heterocycle 260 represented here by cytosine, though the heterocycle may be any one of the four standard nucleobases, A, C, G, or T
  • the second tether end is joined to the alpha phosphate 270 of the nucleobase backbone.
  • tether 275 is comprised of several functional elements, including enhancers 280A and 280B, reporter codes 285A and 285B, and translation control elements (TCEs) 290A and 290B. Each of these features performs a unique function during translocation of the Xpandomer through a nanopore and generation of a unique and reproducible electronic signal.
  • Tether 275 is designed for translocation control by hybridization (TCH). As depicted, the TCEs provide a region of hybridization which can be duplexed to a complementary oligomer (CO) and are positioned adjacent to the reporter codes. Different reporter codes are sized to block ion flow through a nanopore at different measurable levels.
  • reporter codes can be efficiently synthesized using phosphoramidite chemistry typically used for oligonucleotide synthesis.
  • Reporters can be designed by selecting a sequence of specific phosphoramidites from commercially available libraries. Such libraries include but are not limited to polyethylene glycol with lengths of 1 to 12 or more ethylene glycol units, aliphatic with lengths of 1 to 12 or more carbon units, deoxyadenosine (A), deoxycytosine (C), deoxy guanodine (G), deoxythymine (T), abasic (Q).
  • the duplexed TCEs associated with the reporter codes also contribute to the ion current blockage, thus the combination of the reporter code and the TCE can be referred to as a "reporter".
  • the enhancers which in one embodiment comprise spermine polymers.
  • FIG. 3 shows one embodiment of a cleaved Xpandomer in the process of translocating an a-hemolysin nanopore.
  • This biological nanopore is embedded into a lipid bilayer membrane which separates and electrically isolates two reservoirs of electrolytes.
  • a typical electrolyte has 1 molar KC1 buffered to a pH of 7.0.
  • a small voltage typically 100 mV
  • the nanopore constricts the flow of ion current and is the primary resistance in the circuit.
  • Xpandomer reporters are designed to give specific ion current blockage levels and sequence information can be read by measuring the sequence of ion current levels as the sequence of reporters translocate the nanopore.
  • each member of a reporter code (following the duplex) is formed by an ordered choice of phosphoramidites that can be selected from many commercial libraries.
  • Each constituent phosphorami di te contributes to the net ion resistance according to its position in the nanopore (located after the duplex stop), its displacement, its charge, its interaction with the nanopore, its chemical and thermal environment and other factors.
  • the charge on each phosphoramidite is due, in part, to the phosphate ion which has a nominal charge of -1 but is effectively reduced by counterion shielding.
  • the force pulling on the duplex is due to these effective charges along the reporter which are acted upon by the local electric fields. Since each reporter can have a different charge distribution, it can exert a different force on the duplex for a given applied voltage.
  • the force transmitted along the reporter backbone also serves to stretch the reporter out to give a repeatable blocking response.
  • a primer extension reaction may include an aqueous composition consisting of the following components in water: 0.02-0.2 ⁇ g/pl polymerase, 0.1-1 mM MnC12, 20-150 ⁇ M XNTPs, 20-100 mM buffer, 100-200 mM salt, 0.05-0.5 % (w/v) sugar, 5-50 mM PEM, 0.05-0.5 mM polyphosphate, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 2-8% (w/v) alkanediol, 2-10% (w/v) acetamide, 0.1-1% (w/v) glycerol, 200-400 mM betaine, 10-30% (w/v) PEG, 3-10 % (v/v) NMP, 0.5-1.5 mMBHA, and 1-5 % (v/v) DMSO, and further optionally 0.02-1 ⁇ g/pl SSB.
  • a primer extension reaction may include an aqueous composition consisting of the following components in water: 0.02-0.2 ⁇ g/pl polymerase, 0.1-1 mM MnC12, 20-150 ⁇ M XNTPs, 20-100 mM buffering agent, 100-200 mM salt, 0.05-0.5 % (w/v) sugar, 5-50 mM PEM, 0.05-0.5 mM HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 200-400 mM betaine, 10-30% (w/v) PEG, 3-10 % (v/v) NMP, 0.5- 1.5 mMBHA, and 1-5 % (v/v) DMSO, and further optionally 0.02- 1 ⁇ g/pl SSB.
  • a primer extension reaction may include an aqueous composition consisting of the following components in water: 0.02-0.2 ⁇ g/pl polymerase, 0.1-1 mM MnC12, 20-150 ⁇ M XNTPs, 20-100 mM buffering agent, 100-200 mM salt, 0.05-0.5 % (w/v) sugar, 5-50 mM PEM, 0.05-0.5 mM HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 10-30% (w/v) PEG, and 2-8% (w/v) alkanediol, and further optionally 0.02-1 ⁇ g/pl SSB.
  • a primer extension reaction may include an aqueous composition consisting of the following components in water: 0.02-0.2 ⁇ g/pl polymerase, 0.1-1 mM MnC12, 20-150 ⁇ M XNTPs, 20-100 mM buffering agent, 100-200 mM salt, 0.05-0.5 % (w/v) sugar, 5-50 mM PEM, 0.05-0.5 mM HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 10-30% (w/v) PEG, and 2-10% (w/v) acetamide, and further optionally 0.02-1 ⁇ g/pl SSB.
  • a primer extension reaction may include an aqueous composition consisting of the following components in water: 0.02-0.2 ⁇ g/pl polymerase, 0.1-1 mM MnC12, 20-150 ⁇ M XNTPs, 20-100 mM buffering agent, 100-200 mM salt, 0.05-0.5 % (w/v) sugar, 5-50 mM PEM, 0.05-0.5 mM HMP, and optionally one or more selected from the group consisting of 140-300 mM imidazole, 10-30% (w/v) PEG, 0.1-1% (w/v) glycerol, 2-8% (w/v) alkanediol, and 2-10% (w/v) acetamide, and further optionally 0.02-1 ⁇ g/pl SSB.
  • the primer extension reaction may also include a primer and a template, such as an oligonucleotide template.
  • a representative primer extension reaction may include the following reagents: 2 pmol primer, 2.2 pmol 45mer oligonucleotide template, 50 pmol of each XNTP (XATP, XCTP, XGTP, and XTTP), 50 mM Tris HC1, pH 6.79, 200 mM NaCl, 20% PEG, 5% NMS, 0.5 nmol polyphosphate 60.19, 0.3 mM MnCh, and 0.6 ⁇ g of purified recombinant DNA polymerase protein. PEMs are added to this mixture at a concentration typically in the micro to millimolar range. Reactions may also include additional additives, such as singlestrand binding protein (SSB), urea, and NMS.
  • SSB singlestrand binding protein
  • Reactions are run for 1 hour at 23°C.
  • Reaction products i.e., constrained Xpandomers
  • Reaction products are treated to cleave the phosphoramidate bonds, thereby to generating linearized Xpandomers.
  • Reaction products are analyzed using gel electrophoresis on 4-12% acrylamide gels to resolve and visualize Xpandomer products of different lengths.
  • the present disclose provides an aqueous (water containing) composition comprising a PEM and a buffer, particularly a buffer suitable for conducting a DNA polymerization reaction, where Tris HC1 is an exemplary buffer of this type.
  • the present disclosure provides a composition comprising a PEM and a DNA polymerase protein.
  • the present disclosure provides a composition comprising a PEM and a polynucleotide, e g., a 20-90 mer, 20-60 mer, 30-90 mer, or a 30-60 mer, oligonucleotide.
  • the present disclosure provides a composition that comprises each of these components, i.e., an aqueous composition comprising a PEM, a buffer, a DNA polymerase protein, and a polynucleotide.
  • primer extension products may be sequenced using the SBX protocol. Briefly, the constrained Xpandomer products of XNTP polymerization are cleaved to generate linearized Xpandomers. This is accomplished by first quenching the extension reaction with a solution containing 100 mM EDTA, 2 mM THPTA, and 2% Tween-20. Then the sample is subjected to amine modification with a solution of 1 M NaHCCE and 1 M succinic anhydride in DMF. Cleavage of the phosphoramidate bonds is carried out with 37% HC1 and linearized Xpandomers are purified with QIAquick columns (QIAGEN, Inc.).
  • protein nanopores are prepared by inserting a-hemolysin into a DPhPE/hexadecane bilayer member in buffer Bl, containing 2 M NH4CI and 100 mM HEPES, pH 7.4.
  • the cis well is perfused with buffer B2, containing 0.4 M NH4CI, 0.6 M GuCl, and 100 mM HEPES, pH 7.4.
  • the Xpandomer sample is heated to 70° C for 2 minutes, cooled completely, then a 2 pL sample is added to the cis well.
  • a voltage pulse of 90mV/390mV/10ps is then applied and data is acquired via Labview acquisition software.
  • Sequence data is analyzed by histogram display of the population of sequence reads from a single SBX reaction.
  • the analysis software aligns each sequence read to the sequence of the template and trims the extent of the sequence at the end of the reads that does not align with the correct template sequence.
  • the present disclosure provides a method of increasing the accuracy of enhancement of XNTP polymerization, where the method comprises adding a PEM as disclosed herein to the DNA polymerization reaction as described above.
  • the present disclosure provides a kit, where the kit may be used in a method as described herein.
  • the kit will include at least one compound of the present disclosure, and one or more of a) a molecular crowding agent, b) an aqueous buffer, c) a protein such as a polymerase, d) a polynucleotide which may function, for example, as a primer, and/or a polynucleotide which may function, for example, as a template.
  • the present disclosure provides a kit for sequencing a nucleic acid template.
  • the kit includes at least one compound of the present disclosure and a mixture of nucleotide analogs.
  • the compound of the present disclosure may be used to increases the number and accuracy of nucleotide analogs incorporated into a daughter strand during a template-dependent polymerization reaction relative to an identical polymerization reaction absent the at least one compound of the present disclosure.
  • the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, wherein each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of the polymeric ether moiety is attached to the alpha phosphate of the nucleoside triphosphoramidate to provide for expansion of the nucleotide analogs by cleavage of the phosphoramidate bond.
  • each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric tether moiety, wherein a first end of the polymeric tether moiety is attached to the nucleobase and a second end of
  • the mixture of nucleotide analogs comprises nucleotide analogs comprising a detectable label, where the detectable label is an optically detectable label selected from the group consisting of luminescent, chemiluminescent, fluorescent, fluorogenic, chromophoric or chromogenic labels.
  • the kit includes an aqueous buffer which includes one or more components selected from MnCE, a buffer, a salt, a sugar, a single-strand binding protein (SSB), imidazole, pyrazole, triazole, betaine, a molecular crowding agent, dimethyl sulfoxide (DMSO), an alkanediol, glycerol, N-methyl-2-pyrrolidone (NMP), acetamide, butylated hydroxyanisole (BHA), a polyphosphate, and/or urea.
  • the kit includes a singlestrand binding protein.
  • the kit includes urea.
  • the kit includes two or more of these components, e.g., 3, or 4, or all of the named components.
  • 3-amino-6- (trifluoromethyl)benzoic acid hydrochloride, l-(4-aminophenyl)-2,2,2-trifluoroethan-l- one, methyl glycylglycinate hydrochloride, 3,3,3-trifluoropropan-l-amine and diethyl 3- aminopropan-l-ylphosphonate were from Enamine LLC (Monmouth Junction, NJ, USA).
  • Tris[(l-benzyl-lH-l,2,3-triazol-4-yl)methyl]amine (TBTA), O-(7-Azabenzotriazol-l-yl)- N,N,N',N'-tetramethyluronium hexafluoro-phosphate (HATU), 1,3-diethynylbenzene, 2,6- diethynylpyridine, 3,5-diethynylpyridine, 3,6-diethynylcarbazole, 4-azidobenzoic acid, cyclopropyl amine, 6-amino-2-naphthoic acid, 4-aminophthalic acid, 4-amino-3- hydroxybenzoic acid, 4-amino-2-methylbenzoic acid, 4-amino-2,3,5,6-tetrafluorobenzoic acid, 4-amino-2-nitrobenzoic acid, n-(2-aminoethyl)acetamide, 4- (aminoph
  • Flash chromatography was performed on a Reveleris Prep Purification System from Buchi Corp. (New Castle, DE). The system was fitted with a hand packed column (2.3 cm diameter X 8 cm height) filled with C18 Spherical Silica Gel (Cat. No. 76646-01) from Sorbent Technologies, Inc (Norcross, GA) and sealed with polypropylene frits. Samples of 1 to 1.5 mL were loaded directly on the head of the column. Mobile phases were water (A) and acetonitrile (B). A gradient of 0 to 2% B in 2 minutes followed by 2 to 100% B in 20 minutes at a flow of 28 ml/min. UV was monitored at 220 nm, 260 nm and 280 nm.
  • each triazole may be substituted with an imidazole, a pyrazole, a pyrimidine, a pyridazine, a pyrazine, a pyrrole, a thiophene, a thiazole, an isoxazole, or a tetrazole.
  • the compounds set forth in Table 1 may be polymerized (e.g., via cationic polymerization, such as by incubating a PEM with KC1).
  • a composition may include two or more molecules of the compounds set forth in Table 1.
  • Compound 1 was prepared by mixing 4-azidosalicylic acid B (1.79 mg, 10 pmol) and 2,6- diethynylpyridine A (0.67 mg, 5 pmol) in DMSO (150 pL). This solution was mixed with a solution of TBTA (5.1 mg, 0.96 pmol) and sodium ascorbate (6.4 mg, 32 pmol) in DMSO (95 pL). The click reaction was initiated by the addition of 20 Mm copper sulfate (5 pL) with agitation.
  • reaction was analyzed by TLC (94:5:1 ethyl acetate: methanol : acetic acid) and the reaction was complete in 5 minutes based on the consumption of azide and alkyne.
  • the reaction mixture volume was brought to 1 Ml with DMSO and 0.5 M EDTA (100 pL). Solids were isolated and dissolved in additional DMSO. The DMSO solutions were combined and purified by flash chromatography as described above in Materials and Methods. The product formed a glassy solid upon rotary evaporation in a 50 to 75% yield. 3.29 (2H, br.
  • Compound 2 was prepared using 4-azidosalicylic acid B and 3,5-diethynylpyridine C according to the method of Example 1.
  • 1 H NMR 300 MHz, DMSO-d6) ⁇ ppm 3.29 (2H, br.
  • Compound 5 was prepared using 4-azidoaniline hydrochloride F and 3,6- diethynyl carb azole E according to the method of Example 1.
  • Compound 7 was prepared using 4-azidoanisole H and 3,6-diethynylcarbazole E according to the method of Example 1.
  • HYDROXYBENZOIC ACID [0586]
  • Compound 13 started with synthesis of 4-methyl-2,6-diethynylpyridine N from 4-methyl- 2,6-dichloropyridine and ethynyltrimethylsilane using conditions described by Sonogashira (Organomet. Chem., 653: 46-49 (2002). doi: 10.1016/s0022-328x(02)01158- 0).
  • Synthesis of compound 11 was completed by clicking 4-azidosalicylic acid B and 4- methyl-2,6-diethynylpyridine N according to the method of Example 1.
  • Compound 15 was prepared using 5-azidosalicylic acid M and ethyl 2,6- diethynylepyridine-4-carboxylate O clicked according to the method of Example 14.
  • N-ethyl-2,6-diethynl-4-carboxamide Q was made with ethyltrimethylsilane using the Sonogashira method described in Example 16. Synthesis of compound 17 was completed by clicking 4-azidosalicylic acid B and N-ethyl- 2,6-diethynl-4-carboxamide Q according to the method of Example 1.
  • Synthesis of compound 74 started with diazotization of 7-amino-2-hydroxy- l,8-naphthyridine-4-carboxylic acid with sodium nitrite and sulfuric acid followed by nucleophilic displacement with azide (Org. Synth. 1942, 22, 96) to form 7-azido- 2-hydroxy-l,8-naphthyridine-4-carboxylic acid (V) which was purified via flash chromatography.
  • Synthesis of compound 74 was completed by clicking 7-azido-2- hydroxy-l,8-naphthyridine-4-carboxylic acid V and N-ethyl-2,6-diethynl-4- carboxamide Q according to Example 1.
  • Compound 30 was started with synthesis of 2,6-diethynyl-3,5-dimethylpyridine Y from 2,6-dibromo-3,5-dimethylpyridine and ethynyltrimethylsilane using Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12):e3824 (2017)). Synthesis of compound 30 was completed by clicking 4-azidosalicylic acid B and 2,6- diethynyl-3,5-dimethylpyridine Y according to the method of Example 1.
  • N-(but-3-yn-l-yl)-2,6- diethynylisonicotinamide CC was made with ethyltrimethylsilane using the Sonogashira method described in Example 16. Synthesis of compound 47 was completed by clicking 4-azidosalicylic acid B and N-(but-3-yn-l-yl)-2,6-diethynylisonicotinamide CC according to the method of Example 1.
  • Compound 64 was started with synthesis of 2,3-diethynylnaphthalene EE from 2,3- dibromonaphthalene and ethynyltrimethylsilane using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12):e3824 (2017). Synthesis of compound 64 was completed by clicking 4-azidosalicylic acid B and 2,3- diethynylnaphthalene EE according to the method of Example 1.
  • N-Methyl-2,6-diethynylpyridine-4-carboxamide HH was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid with methylamine.
  • Compound 79 was made using the copper click method in Example 1 using diethynyl HH and azide U.
  • N-Morpholino-2,6-diethynylpyridine-4-carboxamide II was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid with morpholine.
  • Compound 80 was made using the copper click method in Example 1 using diethynyl II and azide U.
  • N,N-diethylamino-2,6-diethynylpyridine-4-carboxamide JJ was prepared according to Example 17 using the HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid with N,N-diethylamine.
  • Compound 81 was made using the copper click method in Example 1 using diethynyl JJ and azide U.
  • N-Azetidinyl-2,6-diethynylpyridine-4-carboxamide LL was prepared according to Example 17 using the HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid with azetidine.
  • Compound 84 was made using the copper click method in Example 1 using diethynyl LL and azide B.
  • N-Methyl-N-ethyl-2,6-diethynylpyridine-4-carboxamide MM was prepared according to Example 17 using the HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid with N- methyl-N-ethylamine.
  • Compound 85 was made using the copper click method in Example 1 using diethynyl MM and azide B.
  • N-Cyclopropylamido-2,6-diethynylpyridine-4-carboxamide OO was prepared according to Example 17 using the HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid with cyclopropylamine.
  • Compound 88 was made using the copper click method in Example 1 using diethynyl OO and azide U.
  • N-Butylamido-2,6-diethynylpyridine-4-carboxamide PP was prepared according to Example 17 using the HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid with 1- aminobutane.
  • Compound 89 was made using the copper click method in Example 1 using diethynyl PP and azide U.

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Abstract

La présente divulgation concerne des composés, des procédés et des compositions pour améliorer la polymérisation d'acide nucléique, comprenant la réplication d'ADN par extension d'amorce in vitro pour générer, par exemple, des polymères pour le séquençage de molécule unique à base de nanopores d'un modèle d'ADN. Une composition de réaction d'acide nucléique polymérase est pourvue de fractions d'amélioration de polymérisation, ce qui permet une activité améliorée de l'ADN polymérase avec des analogues nucléotidiques, ce qui permet d'obtenir une longueur améliorée de produits d'extension d'amorce pour des applications de séquençage.
PCT/US2024/061051 2023-12-22 2024-12-19 Amélioration de la polymérisation d'acide nucléique par des composés aromatiques Pending WO2025137293A2 (fr)

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