Enhancement of Nucleic Acid Polymerization by Aromatic Compounds

Abstract

To provide compounds, methods and compositions for improving nucleic acid polymerization, including DNA replication by in vitro primer extension to generate polymers for nanopore-based single molecule sequencing of a DNA template.SOLUTION: A method of enhancing a nucleic acid polymerase reaction, comprises: 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; and b. incubating the nucleic acid polymerase reaction composition under conditions allowing a nucleic acid polymerization reaction, wherein the at least one compound increases the processivity, rate, or fidelity of the nucleic acid polymerase reaction.SELECTED DRAWING: Figure 6A

Description

Technical Field

[0001] The present invention generally relates to novel chemical entities, and more specifically to novel organic molecules optionally having inorganic components (including their compositions), and methods for their manufacture and use, particularly in affecting the performance of enzymes. Description Regarding the Sequence Listing

[0002] The sequence listing associated with this application is provided in text format instead of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 870225_425WO_SEQUENCE_LISTING.txt. The text file is 15 KB, was created on June 19, 2020, and was electronically submitted using EFS-Web.

Background Art

[0003] The measurement of biomolecules is fundamental to modern medicine and is widely used in medical research, more specifically in diagnosis and treatment, and drug development. Nucleic acids encode the information necessary for an organism to function and reproduce and are essentially the blueprint of life. Determining such a blueprint is useful in pure research and applied science. In medicine, sequencing can be used in the diagnosis and treatment development of various medical conditions including cancer, heart disease, autoimmune disorders, multiple sclerosis, and obesity. In the industry, sequencing can be used to design improved enzyme processes or synthetic organisms. In biology, this tool can be used, for example, to study the health of ecosystems and thus has a wide range of usefulness. Similarly, the measurement of proteins and other biomolecules provides markers and understanding of disease and pathogenic transmission.

[0004] An individual's unique DNA sequence provides valuable information regarding susceptibility to specific diseases. It also provides patients with the opportunity to undergo screening for early detection and / or preventive measures. Furthermore, taking into account the patient's individual blueprint, clinicians will be able to administer personalized treatment in order to maximize drug efficacy and / or minimize the risk of drug adverse reactions. 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 of modern medicine. To achieve this goal, sequencing technologies must continue to advance with respect to throughput, accuracy, and read length.

[0005] Over the past decade, a number of next-generation DNA sequencing technologies have become commercially available, dramatically reducing the cost of whole-genome sequencing. These include sequencing-by-synthesis ("SBS") platforms (Illumina, Inc., 454 Life Sciences, Ion Torrent, Pacific Biosciences) and ligation-based platforms (Complete Genomics, Life Technologies Corporation). Many other technologies that utilize a variety of sample processing and detection methods have been developed. For example, GnuBio, Inc. (Cambridge, Massachusetts) controls millions of unobtrusive probe sequencing reactions using picoliter reaction vessels, while Halcyon Molecular (Redwood City, California) has attempted to develop a technology for direct DNA measurement using transmission electron microscopy.

[0006] Nanopore-based nucleic acid sequencing is a well-studied and compelling approach. Kasianowicz et al. (Proc. Natl. Acad. Sci. USA 93:13770-13773, 1996) described alpha-hemolysin nano embedded in a lipid bilayer. Single-stranded polynucleotides that have migrated electrically through pores were characterized. During polynucleotide translocation, it was demonstrated that partial blockage of the nanopore aperture can be measured as a decrease in the ionic current. However, polynucleotide sequencing in nanopores is challenging because it has to resolve closely spaced bases (0.34 nm) with small signal differences submerged in significant background noise. The measurement challenge of single-base resolution in nanopores is made more demanding by the rapid translocation rates typically on the order of 1 base per microsecond that are observed for polynucleotides. For example, the translocation rate can be decreased by adjusting the operating parameters such as voltage, salt composition, pH, temperature, and viscosity. However, such adjustments have not been able to decrease the translocation rate to a level that enables single-base resolution.

[0007] Stratos Genomics developed a method called Sequencing by Expansion (SBX) that uses a biochemical process to transcribe the sequence of DNA onto a measurable polymer called "Xpandomer" (Kokoris et al., U.S. Patent No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion"). The transcribed sequences are encoded along the Xpandomer backbone at high signal-to-noise reporters separated by approximately 10 nm and are designed for high signal-to-noise, high differential response. These differences provide a significant performance improvement in the sequence read efficiency and accuracy of Xpandomer compared to native DNA. Xpandomer enables several next-generation DNA sequencing detection techniques and is well-suited for nanopore sequencing.

[0008] Xpandomers are generated from unnatural nucleotide analogs called XNTPs, which are characterized by long substituents that allow for the expansion of the Xpandomer backbone after synthesis (see PCT Publication No. WO 2016 / 081871 by Kokoris et al., which is hereby incorporated by reference in its entirety). Due to their non-canonical structures, XNTPs as well as other nucleotide analogs (e.g., nucleotide analogs modified with a detectable label moiety) introduce novel challenges as substrates for currently available DNA polymerases. Kokoris et al.'s WO 2017 / 087281 and WO 2018 / 204717, which are hereby incorporated by reference in their entirety, describe engineered DP04 polymerase variants with improved primer extension activity using unnatural bulky nucleotide analogs as substrates.

[0009] Within the DNA template itself, certain nucleotide sequence motifs are known to present additional replication challenges to DNA polymerases. Of particular importance are homopolymer runs, or short repetitive DNA sequences, which can cause slippage strand mispairing, i.e., "replication slippage". Replication slippage is thought to involve the following steps: (i) copying of the first repeat by the replication machinery, (ii) pausing of replication and dissociation of the polymerase from the newly synthesized terminus, (iii) unpairing of the newly synthesized strand and pairing with the second repeat, and (iv) resumption of DNA synthesis. Thus, when the replication machinery stalls within the repetitive region, misalignment of the primer and template occurs. In vivo, misalignment of the two DNA strands during replication can lead to DNA rearrangements such as deletions or duplications of various lengths. In vitro, replication slippage results in replication errors at the site of the slippage event. Such a decrease in polymerase processivity or accuracy can significantly impair specific applications or desired genetic manipulations.

[0010] Accordingly, there is a need for new methods and compositions for improving polymerase reactions under conditions comprising one or more reagents having non-conventional structures (e.g., sequencing by extension (SBX) as well as DNA amplification, conventional sequencing, labeling, detection, cloning, and other applications in biotechnology and other uses in biomedicine), which would find value in the art. The present invention meets these needs and provides further related advantages.

[0011] Not all of the subject matter described in the Background section is necessarily prior art, and it should not be assumed that it is prior art simply as a result of its description in the Background section. Along these lines, the recognition of a problem of the prior art described in the Background section or related to such subject matter should not be treated as prior art unless explicitly stated to be so. Instead, any discussion of any subject matter in the Background section should be treated as part of the inventors' approach to a particular problem that may itself also be inventive.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0013]

Non-Patent Document 1

Summary of the Invention

[0014] Briefly stated, the present disclosure provides compounds, compositions and uses thereof that enhance nucleic acid polymerase activity. In certain embodiments, polymerase activity is enhanced in a polymerization reaction under conditions that introduce one or more challenges to the polymerase, such as conditions that include non-natural nucleotide analog substrates or template motifs that impair the polymerase's processing ability. Such enhancement is achieved by supplementing the polymerization reaction with one or more compounds of the present disclosure, optionally referred to herein as polymerase enhancing molecules or PEMs.

[0015] In one aspect, the PEM has the formula (I)

Chemical formula

Chemical formula

[0016] In one aspect, the present disclosure provides a method of enhancing a nucleic acid polymerase reaction, the method comprising forming a nucleic acid polymerase reaction composition comprising a template nucleic acid, a nucleic acid polymerase, a mixture of nucleotides and / or nucleotide analogs, and at least one PEM; and incubating the nucleic acid polymerase reaction composition under conditions that permit a nucleic acid polymerization reaction. The PEM increases the throughput, speed, and / or fidelity of the nucleic acid polymerase reaction. In one embodiment, the at least one PEM increases the length of the resulting nucleic acid product as compared to a nucleic acid polymerase reaction lacking the PEM.

[0017] In further embodiments, the nucleic acid polymerase is a DNA polymerase. In certain embodiments, the DNA polymerase is DPO4 or a variant thereof. In other embodiments, the mixture of nucleotides or nucleotide analogs is a mixture of nucleotide analogs comprising nucleoside triphosphoramidates, each of the nucleoside triphosphoramidates comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, and a polymeric linker moiety, wherein a first end of the polymeric linker moiety is attached to the nucleobase and a second end of the polymeric linker moiety is attached to the α-phosphate of the nucleoside triphosphoramidate to provide for extension of the nucleotide analog upon cleavage of the phosphoramidate bond. In some embodiments, the nucleic acid polymerization reaction produces a polymerizable polymer, the polymerizable polymer encoding the nucleobase sequence information of the template nucleic acid. In other embodiments, the conditions that permit the nucleic acid polymerization reaction include a suitable polymerization buffer and an oligonucleotide primer. In further embodiments, the suitable buffer includes, for example, one or more of TrisOAc, NH4OAc, PEG, water-miscible organic solvents (such as DMF, NMP, and acetone), polyphosphoric acid 60, and MnCl2. In other embodiments, the reaction mixture further comprises a nucleic acid intercalating agent. In other embodiments, the reaction mixture further comprises a polyanion recognition moiety. In further embodiments, the mixture of nucleotides or nucleotide analogs comprises nucleotide analogs comprising a detectable label. In still other embodiments, the detectable label is an optically detectable label selected from the group consisting of luminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, or chromogenic labels.

[0018] In another aspect, the disclosure provides a composition comprising at least one PEM and a mixture of nucleotide analogs. The composition is useful, for example, when combined with a polymerase, and the at least one PEM increases the number and accuracy of nucleotide analogs incorporated into the daughter strand during a template-dependent polymerization reaction as compared to the same polymerization reaction in the absence of the at least one PEM. In other embodiments, the at least one PEM comprises a plurality of PEMs.

[0019] Optionally, the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, each of the nucleoside triphosphoramidates comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymeric linker moiety, wherein a first end of the polymeric linker moiety is attached to the nucleobase and a second end of the polymeric linker moiety is attached to the α-phosphate of the nucleoside triphosphoramidate to provide for extension of the nucleotide analog by cleavage of the phosphoramidate bond. In other embodiments, the composition further comprises at least one of TrisOAc, NH4OAc, PEG, water-miscible organic solvents (such as DMF and NMP), polyphosphoric acid 60, N-methylsuccinimide (NMS), and MnCl2, e.g., two, three, four, etc., or each, in a buffer solution. In other embodiments, the composition further comprises a single-stranded binding protein (SSB). In other embodiments, the composition further comprises urea. In certain embodiments, the mixture of nucleotide analogs comprises a nucleotide analog comprising a detectable label. In some embodiments, the detectable label is an optically detectable label selected from the group consisting of luminescent, chemiluminescent, fluorescent, fluorogenic, chromophore, or chromogenic labels.

[0020] In another aspect, the present invention provides a method for sequencing a DNA or RNA template, the method comprising forming a DNA polymerase reaction composition comprising a DNA or RNA template, a replication primer that forms a complex with the template, a DNA polymerase, a mixture of nucleotides or nucleotide analogs, and at least one PEM, and incubating the DNA polymerase reaction composition under conditions that allow a DNA polymerization reaction, wherein the at least one PEM increases the rate, fidelity, or throughput of the DNA polymerase reaction. The method may further comprise determining the sequence of the nucleotides or nucleotide analogs in the resulting polymer of nucleotides or nucleotide analogs. The PEM may be described as a compound of formula (I). In some embodiments, the at least one PEM is selected from compounds of formula (II). In other embodiments, the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, each of the nucleoside triphosphoramidates comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, and a polymer linker moiety, wherein a first end of the polymer linker moiety is attached to the nucleobase and a second end of the polymer linker moiety is attached to the α-phosphate of the nucleoside triphosphoramidate to provide extension of the nucleotide analog by cleavage of the phosphoramidate bond. In other embodiments, the DNA polymerase is DPO4 or a variant thereof. In other embodiments, the resulting polymer of nucleotide analogs is an extendable polymer. In other embodiments, the method further comprises contacting the extendable polymer with a phosphoramidate cleavage agent to generate an extended polymer of nucleotide analogs. In certain embodiments, each polymer linker moiety of the nucleotide analogs comprises a reporter moiety unique to the nucleobase of the analog. In other embodiments, the reporter moiety generates a characteristic electronic signal. In yet other embodiments, the step of determining the sequence of the nucleotide analogs comprises translocating the extended polymer of nucleotide analogs through a nanopore.

[0021] Accordingly, in one embodiment, the present disclosure provides a composition comprising a PEM and a polynucleotide. In another embodiment, the present disclosure provides a composition comprising a PEM and a polypeptide, such as a polypeptide such as an enzyme, which may be a nucleic acid polymerase.

[0022] The following are some exemplary specific and numbered embodiments of the present disclosure. Also, unless otherwise specified, each atom specified by a chemical formula may be any of the isotopes of that atom. For example, the name C (carbon) includes 12 C, 13 C or 14 C and mixtures thereof, particularly naturally occurring isotope mixtures, while H (hydrogen) includes 1 H, 2 H and 3 H and mixtures thereof, O (oxygen) includes 16 O and 18 O and mixtures thereof, N (nitrogen) includes 14 N and 15 N and mixtures thereof, etc., and for other atoms:

[0023] 1) Formula (I)

Chemical formula

Chemical formula

Chem.

Chem.

[0024] 2) The compound of Embodiment 1, wherein Ar1 is monocyclic heteroaryl.

[0025] 3) The compound of Embodiment 2, wherein Ar1 is selected from

Chemical formula

[0026] 4) The compound of Embodiment 1, wherein Ar1 is bicyclic aryl.

[0027] 5) Ar1 is

Chemical formula

[0028] 6) Ar1 is

Chemical formula

[0029] 7) The compound of embodiment 1, wherein Ar1 is a tricyclic aryl.

[0030] 8) Ar1 is

Chemical formula

[0031] 9) Ar1 is

Chemical formula

[0032] 10) Ar1 is

Chemical formula

[0033] 11) Ar2 is a substituted 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, 1H-pyrrole, 1H-pyrazole, oxadiazole, thiadiazole, 1,2,4-triazole, 1,2,3-triazole and 1H-imidazole, the compound of embodiment 1.

[0034] 12) The compound of Embodiment 1, wherein Ar2 is a 6-membered monocyclic aromatic ring selected from the group consisting of benzene, pyridine, pyridazine, pyrimidine, and pyrazine.

[0035] 13) The compound of Embodiment 1, wherein Ar2 is a 9-membered condensed 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, 1H-benzimidazole, 1H-pyrrolo[3,2-b]pyridine, 1H-pyrrolo[3,2-c]pyridine, 1H-pyrrolo[2,3-c]pyridine, 1H-pyrrolo[2,3-b]pyridine, benzothiophene, 1,3-benzothiazole, thieno[3,2-b]pyridine, thieno[3,2-c]pyridine, thieno[2,3-c]pyridine, benzoxadiazole, benzothiadiazole, benzisoxazole, benzotriazole, and thieno[2,3-b]pyridine.

[0036] 14) The compound of Embodiment 1, wherein Ar2 is a 10-membered condensed 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.

[0037] 15) Ar2 is

Chemical formula

[0038] 16) Ar2 is of the formula

Chemical formula

[0039] 17) Ar2 is

Chemical Formula

[0040] 18) The substitution on Ar2 contains an amino group, and is a compound of Embodiment 1.

[0041] 19) The substitution on Ar2 contains a methoxy group, and is a compound of Embodiment 1.

[0042] 20) The substitution on Ar2 contains a carboxylic acid, and is a compound of Embodiment 1.

[0043] 21) The substitution on Ar2 contains -CH2-CO2-CH3, and is a compound of Embodiment 1.

[0044] 22) The substitution on Ar2 contains a trifluoromethyl group, and is a compound of Embodiment 1.

[0045] 23) The substitution on Ar2 contains a hydroxyl group, and is a compound of Embodiment 1.

[0046] 24) The substitution on Ar2 is one carboxylic acid and one hydroxyl group, and is a compound of Embodiment 1.

[0047] 25) The substitution on Ar2 is one carboxylic acid and one trifluoromethyl group, and is a compound of Embodiment 1.

[0048] 26) The compound of Embodiment 1 in the form of a chelate.

[0049] 27) The compound of Embodiment 26, wherein the chelate is a copper chelate.

[0050] 28) The compound of Embodiment 1 having a logP of at least 4.9.

[0051] 29) n is 0, m is 2, and having one of the following structures (III) or (IV):

Chemical formula

[0052] 30) The compound of Embodiment 1 having a substitution on Ar2, including at least two of hydroxyl, carboxylic acid carboxamide, and trifluoromethyl.

[0053] 31) Having one of the following structures (V), (VI), (VII), (VIII), or (IX):

Chemical formula

[0054] 32) Having one of the following structures (X), (XI), or (XII):

Chemical formula

[0055] 33) Having one of the following structures (XIII), (XIV), or (XV):

Chemical formula

[0056] 34) The following structure (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) or (XXII): [Chemical formula] A compound of Embodiment 1 having one of the following:

[0057] 35) The following structure (XXIII), (XXIV) or (XXV): [Chemical formula] A compound of Embodiment 1 having one of the following:

[0058] 36) The following structure (XXVI), (XXVII) or (XXVIII): [Chemical formula] A compound of Embodiment 1 having one of the following:

[0059] 37) The following structure (XXIX), (XXX), (XXXI) or (XXXII) [Chemical formula] A compound of Embodiment 1 having one of the following:

[0060] 38) 4,4'-(Pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-(Pyridine-3,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((9H-Carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((9H-Carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid; 4,4'-((4-Methoxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-Carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-Nitropyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 5,5'-((4-Cyanopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-Methylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 5,5'-((4-(Ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Methoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Methylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-Carbamoylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-(Pyrazine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-(1,3-Phenylenebis(1H-1,2,3-triazole-4,1-diyl))dianiline; 4,4'-(1,3-Phenylenebis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid; 4-(4-(3-(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid; 4,4'-(Pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid; 4-(4-(3-(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid; 4,4'-((3,5-Dimethylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4,3-Pyridine-2,6-diyl)bis(5-iodo-1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-Acetamidopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((9-Acetyl-9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-(Pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(N,2-dihydroxybenzamide); 4,4'-(Pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzamide); 4,4'-((4-Carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((1,10-Phenanthroline-2,9-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Trifluoromethyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((3-Cyanopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((3-Nitropyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 3,3'-((4-Cyanopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(tert-Butoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4-(4-(4-Cyanopyridin-2-yl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid; 5-(4-(6-(4-(3-Carboxy-4-hydroxy-5-methylphenyl)-1H-1,2,3-triazol-1-yl)-4-(methoxycarbonyl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-3-methylbenzoic acid; 4,4'-((4-(Dimethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Cyclopropylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(But-3-yn-1-ylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(tert-Butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Propylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Phenylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-((2-Acetamidoethyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(4-Cyclopropylpiperazine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2- hydroxybenzoic acid); 4,4'-((4-(Carbamimidoylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Piperidine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Cyclobutylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((1,10-Phenanthroline-3,8-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Cyclopentylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Dipropylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Di-sec-butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-(Naphthalene-2,7-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-(Naphthalene-2,3-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Dibutylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-((2-Hydroxyethyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Cyclohexylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Benzylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(4-Methylpiperazine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4-(4-(3-(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid; 4,4',4'',4''' -((((Butane-1,4-diylbis(azanediyl))bis(carbonyl))bis(pyridine-4,2,6-triyl))tetrakis(1H-1,2,3-triazole-4,1-diyl))tetrakis(2-hydroxybenzoic acid); 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3,5,6-trichloropicolinic acid); 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 7,7'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H- 1,2,3-triazole-4,1-diyl))bis(2-hydroxy-1,8-naphthyridine-4-carboxylic acid); 5,5'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-fluorobenzoic acid); 5,5'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-fluorobenzoic acid); 4,4'-((4-(Methylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(Morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(Diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-Carbamoylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(Ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(Azetidine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(Ethyl(methyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); N-Ethyl-2,6-bis(1-(4-(2,2,2-trifluoroacetyl)phenyl)-1H-1,2,3-triazol-4-yl)isonicotinamide; 4,4'-(Pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(Cyclopropylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(Butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 5,5'-((4-(Diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 5,5'-((4-(Morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-(Pyridazine-3,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 5,5'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-(trifluoromethyl)benzoic acid); 3,3'-(((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(4,1-phenylene))dipropionic acid; 4,4'-(((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(4,1-phenylene))dibutyric acid; 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diphthalic acid; 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-methoxybenzoic acid); 5,5'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diisophthalic acid; 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-hydroxybenzoic acid); Diethyl (3-(4-(6-(1-(3-(diethoxyphosphoryl)propyl)-1H-1,2,3-triazol-4-yl)-4-(ethylcarbamoyl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)propyl)phosphonate; 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-methylbenzoic acid); 2,2'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-(trifluoromethyl)benzoic acid); 4,4'-((4-((2-hydroxyethyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-nitrobenzoic acid); 4,4'-((4-((3,3,3-trifluoropropyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); (4-(4-(4-(ethylcarbamoyl)-6-(1-(4-phosphonophenyl)-1H-1,2,3-triazol-4-yl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)phenyl)phosphonic acid; 4,4',4'',4''' -((((butane-1,4-diylbis(azanediyl))bis(carbonyl))bis(pyridine-4,2,6-triyl))tetrakis(1H-1,2,3-triazole-4,1-diyl))tetrakis(2-(trifluoromethyl)benzoic acid); 2,2'-((4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))diacetic acid; Dimethyl 2,2'-((4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))diacetate; (2S,2'S)-2,2'-((4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))disuccinic acid; 2,2'-((2,2'-((4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl)bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))bis(acetyl))bis(azanediyl))diacetic acid; 2,6-Bis(1-(4-cyano-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-N-ethylisonicotinamide; 4,4'-(Thiophene-2,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); and 4,4'-(Furan-2,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid) The compound of Embodiment 1 selected from any one of the above.

[0061] 39) 4,4'-(1,3-Phenylenebis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((9H-Carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dianiline; 3,6-Bis(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)-9H-carbazole; 4,4'-(1,4-Phenylenebis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-(1,3-Phenylenebis(1H-1,2,3-triazole-4,1-diyl))dianiline; 4,4'-(1,3-Phenylenebis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid; 1,3-Bis(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)benzene; 4,4'-(Pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))dianiline; 4-(4-(3-(1-(4-carboxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid; 4-(4-(3-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid; 4,4'-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 2-(1-(1H-benzo[d]imidazol-4-yl)-1H-1,2,3-triazol-4-yl)-6-(1-(1H-benzo[d]imidazol-7-yl)-1H-1,2,3-triazol-4-yl)-N-ethylisonicotinamide; 4,4'-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((5-carboxy-1,3-phenylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((5-carboxy-1,3-phenylene)bis(1H-1,2,3-triazole-1,4-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((5-(Ethylcarbamoyl)-1,3-phenylene)bis(1H-1,2,3-triazole-1,4-diyl))bis(2-(trifluoromethyl)benzoic acid); 3'-(4-(4-(Ethylcarbamoyl)-6-(1-(3'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)-1H-1,2,3-triazol-4-yl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-3-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid; 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-cyanobenzoic acid); and 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-chlorobenzoic acid) A compound selected from any one of the above.

[0062] 40) A composition comprising a compound according to any one of Embodiments 1 to 39 and a molecular crowding agent.

[0063] 41) The composition of Embodiment 41, wherein the molecular crowding agent is a polyalkylene glycol.

[0064] 42) A composition comprising a compound according to any one of Embodiments 1 to 39 and an aqueous buffer.

[0065] 43) The composition of Embodiment 43, wherein the aqueous buffer is Tris HCl.

[0066] 44) A composition comprising a compound according to any one of Embodiments 1 to 39 and a polynucleotide.

[0067] 45) The composition of Embodiment 44, wherein the polynucleotide is an oligonucleotide of 20 to 60 mer.

[0068] 46) A composition comprising a compound according to any one of Embodiments 1 to 39 and a protein.

[0069] 47) The composition of embodiment 46, wherein the protein is DNA polymerase.

[0070] 48) A composition comprising any one compound of embodiments 1 to 39 and a mixture of nucleotides or nucleotide analogs.

[0071] 49) A composition for improving the processing ability, fidelity, or rate of a DNA polymerase reaction, comprising at least one compound of any one of embodiments 1 to 39 and a mixture of nucleotide analogs.

[0072] 50) A composition comprising at least one compound of any one of embodiments 1 to 39 and a mixture of nucleotide analogs, wherein at least one compound of any one of embodiments 1 to 39 increases the number and accuracy of nucleotide analogs incorporated into the daughter strand during a template-dependent polymerization reaction as compared to the same polymerization reaction in the absence of at least one compound of any one of embodiments 1 to 39.

[0073] 51) The composition of embodiment 50, wherein the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, each of the nucleoside triphosphoramidates comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymer bridging moiety, wherein a first end of the polymer bridging moiety is attached to the nucleobase and a second end of the polymer bridging moiety is attached to the α-phosphate of the nucleoside triphosphoramidate to provide an extension of the nucleotide analog upon cleavage of the phosphoramidate bond.

[0074] 52) The composition of embodiment 51, further comprising a buffering component selected from at least one of TrisOAc, NH4OAc, PEG, a water-miscible organic solvent, polyphosphoric acid 60, NMS, and MnCl2.

[0075] 53) The composition of embodiment 51, further comprising a single-stranded binding protein.

[0076] 54) The composition of embodiment 51, further comprising urea.

[0077] 55) The composition of embodiment 51, wherein the mixture of nucleotide analogs comprises a nucleotide analog comprising a detectable label.

[0078] 56) The composition of embodiment 55, wherein the detectable label is an optically detectable label selected from the group consisting of luminescence, chemiluminescence, fluorescence, phosphorescence, chromophore or chromogenic label.

[0079] 57) A kit for sequencing a nucleic acid template, comprising at least one composition of any one of embodiments 40 to 56.

[0080] 58) A method for enhancing a nucleic acid polymerase reaction, comprising: a. i. a template nucleic acid, ii. a nucleic acid polymerase, iii. a mixture of nucleotides or nucleotide analogs, and iv. forming a nucleic acid polymerase reaction composition comprising at least one compound of any one of embodiments 1 to 39; and b. incubating the nucleic acid polymerase reaction composition under conditions that allow nucleic acid polymerization, wherein at least one compound of any one of embodiments 1 to 39 increases the processing ability, rate or fidelity of the nucleic acid polymerase reaction.

[0081] 59) The method of embodiment 58, wherein at least one compound of any one of embodiments 1 to 39 increases the length of the resulting nucleic acid product as compared to a nucleic acid polymerase reaction lacking at least one compound of any one of embodiments 1 to 39.

[0082] 60) The method of embodiment 58, wherein at least one compound of any one of embodiments 1 to 39 comprises a plurality of compounds of any one of embodiments 1 to 39.

[0083] 61) The method of embodiment 58, wherein the nucleic acid polymerase is a DNA polymerase.

[0084] 62) The method of embodiment 61, wherein the DNA polymerase is DPO4 or a variant thereof.

[0085] 63) The method of embodiment 58, wherein the mixture of nucleotides or nucleotide analogs is a mixture of nucleotide analogs containing nucleoside triphosphoramidates, each of the nucleoside triphosphoramidates comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymer linker moiety, wherein a first end of the polymer linker moiety is attached to the nucleobase and a second end of the polymer linker moiety is attached to the α-phosphate of the nucleoside triphosphoramidate to provide extension of the nucleotide analog by cleavage of the phosphoramidate bond.

[0086] 64) The method of embodiment 58, wherein the nucleic acid polymerization reaction produces a polymer that is extendable with nucleotide analogs, and the extendable polymer encodes the nucleic acid base sequence information of the template nucleic acid.

[0087] 65) The method of embodiment 58, wherein the conditions that enable the nucleic acid polymerization reaction include a suitable polymerization buffer and an oligonucleotide primer.

[0088] 66) The method of embodiment 58, wherein the suitable buffer comprises components selected from the group consisting of TrisOAc, NH4OAc, PEG, water-miscible organic solvents, polyphosphoric acid 60, NMS, and MnCl2. method.

[0089] 67) The method of embodiment 58, wherein the reaction mixture further comprises a single-stranded binding protein.

[0090] 68) The method of embodiment 58, wherein the reaction mixture further comprises urea.

[0091] 69) The method of embodiment 58, wherein the mixture of nucleotides or nucleotide analogs comprises nucleotide analogs containing a detectable label.

[0092] 70) The method of embodiment 69, wherein the detectable label is an optically detectable label selected from the group consisting of luminescence, chemiluminescence, fluorescence, phosphorescence, chromophore or chromogenic label.

[0093] 71) A method for sequencing a DNA or RNA template, comprising: a. i. A DNA or RNA template, ii. A replication primer that forms a complex with the template, iii. A DNA polymerase, iv. A mixture of nucleotides or nucleotide analogs, v. Forming a DNA polymerase reaction composition comprising at least one compound of any one of embodiments 1 to 39; b. Incubating the DNA polymerase reaction composition under conditions that allow a DNA polymerization reaction, wherein at least one compound of any one of embodiments 1 to 39 increases the rate, fidelity or throughput of the DNA polymerase reaction; and c. Determining the sequence of nucleotides or nucleotide analogs in the resulting polymer of nucleotides or nucleotide analogs.

[0094] 72) The method of embodiment 71, wherein the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, each of the nucleoside triphosphoramidates comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine and a polymer linker moiety, wherein a first end of the polymer linker moiety is attached to the nucleobase and a second end of the polymer linker moiety is attached to the α phosphate of the nucleoside triphosphoramidate to provide extension of the nucleotide analog upon cleavage of the phosphoramidate bond.

[0095] 73) The method of embodiment 71 or 72, wherein the DNA polymerase is DPO4 or a variant thereof.

[0096] 74) The method of embodiment 71 or 72, wherein the obtained polymer of the nucleotide analog is an extendable polymer.

[0097] 75) The method of embodiment 74, further comprising the step of contacting the extendable polymer with a phosphoramidate cleaving agent to generate an extended polymer of the nucleotide analog.

[0098] 76) The method of embodiment 71 or 72, wherein each polymer linking chain portion of the nucleotide analog comprises a reporter portion specific to the nucleobase of the analog.

[0099] 77) The method of embodiment 72, wherein the reporter portion produces a characteristic electronic signal.

[0100] 78) The method of embodiment 72, wherein the step of determining the sequence of the nucleotide analog comprises the step of translocating the extended polymer of the nucleotide analog through a nanopore.

[0101] The above and additional features of the present invention and methods for obtaining them will become apparent, and the present invention will be best understood by reference to the following more detailed description. All references disclosed herein are incorporated herein by reference in their entirety as if each were individually incorporated.

[0102] This brief summary is provided to introduce specific concepts in a simplified form that will be further described in more detail in the following detailed description. Except where specifically stated otherwise, this brief summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter.

[0103] Details of one or more embodiments are set forth in the following description. Features illustrated or described in connection with one exemplary embodiment can be combined with features of other embodiments. Accordingly, various combinations of the embodiments described herein can be made to provide further embodiments. Aspects of the embodiments can be modified, as needed, to provide yet further embodiments using the concepts of various patents, applications, and publications identified herein. Other features, objects, and advantages will be apparent from the description, drawings, and claims.

Brief Description of the Drawings

[0104] Exemplary features of the present disclosure, their nature, and various advantages will become apparent from the accompanying drawings and the following detailed description of various embodiments. Non-limiting and non-exhaustive embodiments are described with reference to the accompanying drawings, and like labels or reference numerals refer to like parts throughout the various figures unless otherwise specified. The sizes and relative positions of the elements of the drawings are not necessarily drawn to scale. For example, the shapes of the various elements are selected, enlarged, and positioned to improve the visibility of the drawing. The particular shapes of the elements depicted are selected to facilitate recognition in the drawings.

[0105]

Figure 1A

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Figure 7A

Figure 7B

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Mode for Carrying Out the Invention

[0106] The present invention can be more easily understood by referring to the following detailed description of the preferred embodiments of the present invention and the examples included in this specification. Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0107] In one aspect, the PEM of the present disclosure has the formula (I) [Chemical formula] [wherein: m is 1, 2, or 3; m' is 1, 2, or 3; n is 0, 1, or 2; p is 0, 1, or 2; When X is C, W is N, or when X is N, W is C; [Chemical formula] is a single bond or a double bond, and the double bond starts from where either W or X is carbon; L is a linking group; M is independently selected from hydrogen, halogen, and C1-C4 alkyl each time it appears; Ar1 is independently selected from pyridine, pyrazine, pyridazine, furan, thiophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole each time it appears, which may be substituted, The substituents of Ar1 are each time it appears halogen, -OH, -CN, -NO2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C1-C6 cycloalkyl, -OR 0 , -CONH2, -C(O)NR 1 R 1’ , -NR 1 R 1’ , -NR 1 C(O)R 3 , -C(O)SR 3 , -COR 3 , -OC(O)R 3 , -C(O)OR 3 , mercaptan, -R 4 -H, -SOR 1 , -S(O)2R 1 , -S(O)2NR 1 R 1’ , and -NS(O)2R 3selected independently therefrom; R 0 is, for each occurrence, independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; R 1 and R 1’ is, for each occurrence, H, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2- C 6 alkynyl, arylalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroaryl-C(=NH)NH2, -CH2CO2R 0 -CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R 3 -CH2C(O)NH CH2CO2H,

Chemical formula

Chemical formula

[0108] As used herein and in the appended claims, unless otherwise specified, the following terms have the indicated meanings.

[0109] Before the specific chemical groups named herein, there is an abbreviated notation indicating the total number of carbon atoms found in the indicated chemical group. For example; C 1-4 C1-C4 alkyl, which may be written instead of alkyl, represents an alkyl group having from at least 1 to 4 carbon atoms, and C4-C 12 cycloalkylalkyl (similarly C 4-12 written as cycloalkylalkyl may also) represents a cycloalkylalkyl group having a total of 4 to 12 carbon atoms. The total number of carbons in the abbreviated notation does not include carbons that may be present in substituents of the described group. As an example, C1-C6 alkyl refers to an alkyl radical containing 1 to 6 carbon atoms; C1-C6 haloalkyl refers to a haloalkyl radical containing 1 to 6 carbon atoms; C1-C6 alkylene refers to an alkylene diradical containing 1 to 6 carbon atoms.

[0110] In addition to the above, as used herein and in the appended claims, unless otherwise specified, the following terms have the indicated meanings.

[0111] "Alkyl" consists of only carbon and hydrogen atoms, contains no unsaturation, and optionally has the indicated number of carbon atoms, for example, 1 to 12 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms, and refers to a straight-chain or branched hydrocarbon chain radical bonded to the rest of the molecule by a single bond. Examples are methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. When unsaturation is introduced into the alkyl group, the resulting group may be called an unsaturated alkyl group, and unsaturated alkyl groups are generally known as alkenyl groups (having at least one carbon-carbon double bond) and alkynyl groups (having at least one carbon-carbon triple bond). In one embodiment, when specified, the alkyl group in the compounds of the present disclosure may be an unsaturated alkyl group or may contain an unsaturated alkyl group.

[0112] "Alkenyl" consists of only carbon and hydrogen atoms, contains at least one double bond, and optionally has the indicated number of carbons, for example, 2 to 12 carbon atoms, or 2 to 8 carbon atoms, or 2 to 6 carbon atoms, or 2 to 4 carbon atoms, and refers to a straight-chain or branched hydrocarbon chain radical group bonded to the rest of the molecule by a single bond, for example, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, and the like.

[0113] "Alkynyl" consists of only carbon and hydrogen atoms, contains at least one triple bond, and optionally has the indicated number of carbons, for example, 2 to 12 carbon atoms, or 2 to 8 carbon atoms, or 2 to 6 carbon atoms, or 2 to 4 carbon atoms, and refers to a straight-chain or branched hydrocarbon chain radical group bonded to the rest of the molecule by a single bond, for example, ethynyl, prop-1-ynyl, but-1-ynyl, pent-1-ynyl, pent-1,4-diyne, and the like.

[0114] "Halo" refers to bromo, chloro, fluoro, or iodo.

[0115] "Haloalkyl" refers to an alkyl radical as defined above substituted by one or more halo radicals as defined above, for example, trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, 1-bromomethyl-2-bromoethyl, and the like. Similarly, "haloalkenyl" refers to an alkenyl radical as defined herein substituted by one or more halo radicals as defined herein, and "haloalkynyl" refers to an alkynyl radical as defined herein substituted by one or more halo radicals as defined herein.

[0116] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain consisting only of carbon and hydrogen, containing no unsaturation, and optionally having the indicated number of carbon atoms, connecting the remainder of the molecule to a radical group. Examples are methylene, ethylene, propylene, n-butylene, and the like is. The alkylene chain is bonded to the remainder of the molecule via a single bond and to the radical group via a single bond. The points of attachment of the alkylene chain to the remainder of the molecule and the radical group can be through one carbon or any two carbons within the chain. Similar to alkyl groups, unsaturation can be introduced into the alkylene chain to provide an unsaturated alkylene chain. When unsaturation is introduced into the alkylene chain, the resulting group may be called an unsaturated alkylene group or chain, and unsaturated alkylene chains are generally known as alkenylene groups (having at least one carbon-carbon double bond) and alkynylene groups (having at least one carbon-carbon triple bond). In one embodiment, when specified, the alkylene chain in the compounds of the present disclosure may be an unsaturated alkyl group or may contain an unsaturated alkylene chain.

[0117] "Alkenylene" or "alkenylene chain" refers to a straight-chain or branched divalent hydrocarbon chain consisting only of carbon and hydrogen, containing at least one double bond, and optionally having the indicated number of carbon atoms, for example, 2 to 12 carbon atoms, connecting the rest of the molecule to a radical group. Examples of alkenylene groups are ethenylene, propenylene, n-butenylene, etc. The alkenylene chain is bonded to the rest of the molecule via a single bond and to the radical group via a double bond or a single bond. The bonding points of the alkenylene chain to the rest of the molecule and the radical group can be through one carbon or any two carbons within the chain.

[0118] "Aryl" refers to a cyclic radical containing at least 5 ring atoms, optionally containing 1 to 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, and a 6-membered monocyclic aromatic ring contains 6 ring atoms selected from carbon and heteroatoms. An exemplary monocyclic aromatic ring having 5 members is pyrrole, and an exemplary monocyclic aromatic ring having 6 members is pyridine. The aryl radical can be, for example, a monocyclic, bicyclic, tricyclic, or tetracyclic ring system that may include a fused or bridged ring system. A carbocyclic aryl radical contains only carbon in the ring atoms, and examples include aryl radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, preiadene, pyrene, and triphenylene, but are not limited thereto. In one embodiment, aryl is phenyl or naphthyl, and in another embodiment, it is phenyl. When the aryl radical contains non-carbon ring atoms, such as oxygen, sulfur, and nitrogen, the aryl group can be referred to as a heteroaryl group. The heteroaryl radical can be, for example, a monocyclic, bicyclic, tricyclic, or tetracyclic ring system that may include a fused or bridged ring system. The nitrogen, carbon, or sulfur atoms in the heteroaryl radical may optionally be oxidized, and the nitrogen atoms may optionally be quaternized.

[0119] An "arylalkyl" group is an alkyl, alkenyl or alkynyl group as defined above, wherein a hydrogen atom thereof is replaced by an aryl group as defined above. Representative aralkyl groups include benzyl (-CH2phenyl), phenylethyl (-CH2CH2phenyl) and phenylethylene (-CH=CHphenyl) groups and condensed (cycloalkylaryl) alkyl groups such as 4-ethyl-indanyl. The aralkyl group may be substituted by an aryl moiety, an alkyl, alkenyl or alkynyl moiety, or both.

[0120] "Condensed" refers to a ring system including ring fusion between rings, and ring fusion refers to a ring that shares two adjacent ring atoms. A condensed ring containing two 5- and / or 6-membered monocyclic rings fused to each other refers to a bicyclic ring system in which each ring is monocyclic, independently has 5 or 6 ring atoms, and the two rings are fused in that they share two ring atoms. For example, naphthalene is a 10-membered condensed ring system formed from two 6-membered monocyclic rings (benzene) fused to each other. Naphthalene is bicyclic in that it contains two (bi = 2) rings. As another example, 1,3-benzothiazole, which is a 9-membered condensed ring system formed by fusing one 6-membered ring (benzene) and one 5-membered ring (1,3-thiazole) to each other. 1,3-benzothiazole is bicyclic in that it contains two rings.

[0121] "Carbocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring radical consisting of 3 to 18 carbon atoms. Unless specifically indicated otherwise herein, a carbocyclic radical may be monocyclic, bicyclic, tricyclic or tetracyclic ring system, may include a condensed or bridged ring system, and may be partially or fully saturated. Non-aromatic carbocyclic radicals include cycloalkyl, and aromatic carbocyclic radicals include aryl.

[0122] "Cycloalkyl" consists of only carbon and hydrogen atoms, may include a fused or bridged ring system, has 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, is saturated or unsaturated, and refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical bonded to the rest of the molecule by a single bond. Examples of monocyclic radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like.

[0123] "Heterocyclyl" refers to a stable 3- to 18-membered aromatic or non-aromatic ring radical consisting of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless specifically indicated otherwise herein, a heterocyclyl radical may be monocyclic, bicyclic, tricyclic, or tetracyclic, may include a fused or bridged ring system, the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical may be optionally oxidized, the nitrogen atoms may be optionally quaternized, and the heterocyclyl radical may be partially or completely saturated. Examples of non-aromatic heterocyclyl radicals include dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl, but are not limited thereto.

[0124] Optionally, but only when specifically identified, each of the alkyl, alkenyl, alkylene, alkenylene, carbocyclyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl in the PEM compounds of the present disclosure is alkyl, alkenyl, halo, haloalkyl, haloalkenyl, cyano, oxo, thioxo, nitro, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, -R b -OR a 、-R b -OC(O)-R a 、-R b -N(R a )2、-R b -C(O)R a 、-R b -C(O)OR a 、-R b -C(O)N(R a )2、-R b -N(R a )C(O)OR c 、-R b -N(R a )C(O)R c 、-R b -N(R a )S(O) t R c (t is 1 to 2), -R b -N=C(OR a )R a 、-R b -S(O) t OR c (t is 1 to 2), -R b -S(O) s R c (s is 0 to 2), and -R b -S(O) t N(R a )2(t is 1 to 2) (wherein each R a is independently hydrogen, alkyl, alkenyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl, and each R bis independently, a direct bond or a linear or branched alkylene or alkenylene chain, each R c is optionally substituted by one or more unsubstituted (e.g., the alkyl substituents on the alkyl group are not further substituted, i.e., the alkyl substituents are unsubstituted alkyl) substituents selected from the group consisting of alkyl, alkenyl, haloalkyl, cyclo alkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl).

[0125] "Amino" refers to the -NH2 radical. "Cyano" refers to the -CN radical. "Hydroxy" refers to the -OH radical. "Nitro" refers to the -NO2 radical. "Oxo" refers to the =O substituent. "Thioxo" refers to the =S substituent. "Trifluoromethyl" refers to the -CF3 radical. "Trifluoromethoxy" refers to the -OCF3 radical. Mercaptan, also known as thiol, refers to the -SH radical.

[0126] "Acyl" refers to the radical -C(O)R, which can also be written as -C(=O)R, wherein R is alkyl, aralkyl, carbocyclic, aryl, heteroaryl or heterocyclyl. For example, when R is methyl, the acyl group may be referred to as acetyl.

[0127] "Alkoxy" refers to the radical of formula -OR where R is an alkyl or haloalkyl radical. In one embodiment, the alkoxy radical contains up to 6 carbon atoms. Representative alkoxy groups include methoxy and ethoxy. Alkoxy substituted with halo may be referred to herein as haloalkoxy and includes, for example, trifluoromethoxy, trichloromethoxy, etc.

[0128] "Heteroalkenylene" or "heteroalkenylene chain" refers to a linear or branched divalent hydrocarbon chain consisting of carbon and hydrogen and at least one heteroatom selected from N, O and S, connecting the remainder of the molecule to a radical group.

[0129] "Haloalkoxy" refers to an alkoxy radical substituted by one or more halo radicals as defined above, for example, trifluoromethoxy, difluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy, 3-bromo-2-fluoropropyloxy and the like. The alkoxy portion of the haloalkoxy radical may be substituted as defined above for alkoxy groups.

[0130] "N-Heterocyclyl" refers to a heterocyclyl radical containing at least one nitrogen. The N-heterocyclyl radical may be substituted as defined above for heterocyclyl radicals.

[0131] "Heterocyclylalkyl" has the formula -R b R h wherein R b is an alkylene chain as defined above, and R h is a heterocyclyl radical as defined above. When the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl may be bonded to the alkyl radical by a nitrogen atom. The alkylene chain of the heterocyclylalkyl radical may be substituted as defined above for alkylene chains. The heterocyclyl portion of the heterocyclylalkyl radical may be substituted as defined above for heterocyclyl groups.

[0132] "N-Heteroaryl" refers to a heteroaryl radical as defined above containing at least one nitrogen and having the point of attachment to the remainder of the molecule of the heteroaryl radical through a nitrogen atom in the heteroaryl radical. The N-heteroaryl radical may be substituted as defined above for heteroaryl radicals.

[0133] "Heteroarylalkyl" has the formula -R b R i wherein R b is an alkylene chain as defined above, and Ri is a heteroaryl radical as defined herein. The heteroaryl moiety of the heteroarylalkyl radical may be substituted as defined herein for heteroaryl groups. The alkylene chain moiety of the heteroarylalkyl radical may be substituted as defined herein for alkylene chains. Similarly, an arylalkyl group refers to a heteroarylalkyl group in which the heteroaryl moiety is replaced by the corresponding carbocyclic aryl group, i.e., the heteroatoms are replaced by carbon and adjusted as necessary with respect to hydrogen substitution.

[0134] "Hydroxyalkyl" has the formula -R b OH, where R b is an alkylene chain as defined herein. The -OH (hydroxyl, also known as hydroxy) group can be attached to any carbon in the alkylene chain. The alkylene chain moiety of the heteroarylalkyl radical may optionally be further substituted as defined above for alkylene chains.

[0135] The PEM compounds described herein having acidic or basic groups can generally be used as free acids or free bases. Alternatively, the PEM compounds having acidic or basic groups may be used in the form of salts, such as acid addition salts or base addition salts. Acid addition salts of free amino acid compounds can be prepared by methods well known in the art and can be formed from organic acids and inorganic acids. Suitable organic acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, cinnamic acid, aspartic acid, stearic acid, palmitic acid, glycolic acid, glutamic acid, and benzenesulfonic acid. Suitable inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid. Base addition salts include salts formed with carboxylate anions and include organic and inorganic cations selected from alkali and alkaline earth metals (e.g., lithium, sodium, potassium, magnesium, barium, and calcium) and ammonium ions and their substituted derivatives (e.g., dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, etc.). Thus, the term "salt" of the PEM compounds described herein is intended to encompass all salt forms.

[0136] The PEM compounds of the present disclosure may be in the form of a chelate. A chelate refers to a compound containing an organic ligand (such as a triazole-Ar group) bonded to a central metal atom at two or more points.

[0137] Regarding stereoisomers, the PEM compounds described herein may have one or more chiral (or asymmetric) centers and, accordingly, can give rise to enantiomers, diastereomers, and other stereoisomers that can be defined as (R)- or (S)- from the perspective of absolute stereochemistry. If the compounds described herein contain an olefinic double bond or other centers due to geometric asymmetry and unless otherwise specified, the compounds are intended to contain both E and Z geometric isomers (e.g., cis or trans). Similarly, unless otherwise indicated, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are intended to be included. Thus, various stereoisomers and their mixtures are considered to include "enantiomers," which refer to two stereoisomers that are mirror images and cannot be superimposed on each other. Accordingly, the compounds can occur in any isomeric form, including racemates, racemic mixtures, and as individual enantiomers or diastereomers.

[0138] Furthermore, some of the crystalline forms of the PEM compounds can exist as polymorphs contemplated herein. Additionally, some of the PEM compounds can form solvates with water or other organic solvents as well. Such solvates are similarly included within the scope of the compounds described herein.

[0139] As will be understood by those skilled in the art, any of the foregoing compounds can incorporate radioisotopes. Accordingly, the use of the same isotope-labeled compounds described herein, in which one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number normally found in nature, is also contemplated. Examples of isotopes that can be incorporated into these compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine. Accordingly, references to elements such as hydrogen (H) or carbon (C) are intended to encompass all of their isotopes. For example, for other atoms, the name C (carbon) is 12 C, 13 C or 14comprising C and their mixtures, while H (hydrogen) is, 1 H, 2 H and 3 comprising H and their mixtures, O (oxygen) is, 16 O and 18 comprising O and their mixtures, N (nitrogen) is, 14 N and 15 including N and their mixtures, etc. Isotope-labeled PEM compounds can be useful for tracking PEM compounds or parts thereof during use in assays, etc.

[0140] In the PEM compound of formula (I), Ar1 is an aryl group, also called the aromatic moiety. The aromatic moiety may be a carbocyclic or heterocyclic aromatic moiety, and each of the aromatic ring atoms is a carbon of the carbocyclic aromatic moiety, and at least one of the aromatic ring atoms is nitrogen, oxygen or sulfur of the heterocyclic aromatic moiety.

[0141] In one embodiment, Ar1 may contain 1 to 6 rings, up to 6 of the ring atoms may be selected from oxygen, sulfur and nitrogen, and the rest are carbon atoms. Optionally, the Ar1 moiety may contain 1 to 5 rings, and up to 5 of the ring atoms may be selected from oxygen, sulfur and nitrogen. As another option, the Ar1 group may contain 1 to 4 rings, and up to 4 of the ring atoms may be selected from oxygen, sulfur and nitrogen. As yet another option, the Ar1 moiety may contain 1 to 3 rings, and up to 3 of the ring atoms may be selected from oxygen, sulfur and nitrogen. As a further example, Ar1 may contain 1 to 2 rings, and up to 3 of the ring atoms may be selected from oxygen, sulfur and nitrogen. In any case, each ring can independently be a 5-membered ring (i.e., 5 ring atoms form the ring), or a 6-membered ring, or a 7-membered ring, but in one option, each ring is either a 5-membered ring or a 6-membered ring.

[0142] Exemplary aromatic moieties are carbocyclic aromatic moieties. The carbocyclic moiety may contain 1 (e.g., benzene), or 2 (e.g., naphthalene, azulene), or 3 (e.g., acenaphthylene, fluorene), or 4 (e.g., fluoranthene, aceanthrylene), or 5 (e.g., pentacene, picene), or 6 (e.g., hexacene) aromatic rings. For convenience, the Ar1 group may be exemplified herein by naming its unsubstituted form (e.g., benzene), but in the compounds of the present disclosure, the Ar1 group is the corresponding radical. For example, when m is 2 and Ar1 is otherwise unsubstituted, two ring hydrogens are substituted with triazole groups. For example, the aromatic moiety may be a monocyclic carbocyclic moiety, i.e., phenyl, also referred to as a C6 aromatic moiety. As another example, the aromatic moiety may be a bicyclic carbocyclic moiety, e.g., C 10 It may be naphthyl, which is an aromatic moiety.

[0143] Exemplary Ar1 aromatic moieties are heterocyclic aromatic moieties, which may also be referred to as heteroaryl groups. 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 heterocyclic moiety may contain 1 or 2 or 3 or 4 or 5 or 6 aromatic rings. Optionally, the heteroatom is nitrogen if present. For example, the aromatic moiety may be a monocyclic heterocyclic moiety, e.g., pyridinyl, which is a 6-membered C5 aromatic moiety, or pyrazinyl, which is a 6-membered C4 aromatic moiety. As another example, the aromatic moiety may be a bicyclic heterocyclic moiety, e.g., quinolin yl or isoquinolinyl, which is a 10-membered C9 aromatic moiety, or 1,5-naphthyridinyl, 2,6-naphthyridinyl, or 2,7-naphthyridinyl, which are exemplary 10-membered C8 aromatic moieties.

[0144] Accordingly, a heteroaryl group is an aromatic ring compound containing 5 or more ring members, one or more of which are heteroatoms such as, but not limited to, N, O, and S. A heteroaryl group called C2-heteroaryl can be a 5-membered ring having 2 carbon atoms and 3 heteroatoms, a 6-membered ring having 2 carbon atoms and 4 heteroatoms, and the like. Similarly, C4-heteroaryl can be a 5-membered ring having 1 heteroatom, a 6-membered ring having 2 heteroatoms, and the like. The number of carbon atoms + the number of heteroatoms is equal to the total number of ring atoms. Examples of heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, and quinazolinyl groups. Accordingly, the terms "heteroaryl" and "heteroaryl group" include condensed ring compounds in which at least one ring is aromatic but not necessarily all rings are aromatic, including tetrahydroquinolinyl, tetrahydroisoquinolinyl, indolyl, and 2,3-dihydroindolyl.

[0145] When m is 2 such that Ar1 is necessarily substituted with two triazole-Ar2 moieties, any two carbons of the Ar1 aromatic moiety may be substituted with one of these two triazole-Ar2 moieties. For example, when Ar1 is a substituted benzene, Ar1 may be substituted at the ortho, meta, or para positions as shown below, and k indicates the location where substitution can occur in the aromatic moiety.

Chemical formula

[0146] As another example, when Ar1 is a substituted naphthalene and m is 2, Ar1 may be substituted at any two naphthyl carbon atoms, the following structures show substitution options, and k indicates the location where triazole substitution provided by (triazole - Ar2) can occur on the aromatic moiety.

Chem.

[0147] The previous example showed triazole substitution on Ar1 using a carbocyclic aromatic Ar1 group as an exemplary Ar1 moiety. However, the same principle applies to triazole substitution on a heterocyclic aromatic Ar1 group. For example, when Ar1 is a substituted pyridine and m is 2, the two triazole groups of (triazole - Ar2) may be located at any of the following positions on the pyridine ring, and k is used to indicate the positions where the triazole groups can be located.

Chem.

[0148] Therefore, in one exemplary embodiment, Ar1 is

Chem.

Chem.

Chem.

Chem.

Chem.

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chem.

Chem.

[0149] Ar1 includes both substituted and unsubstituted aromatic moieties described herein. In one embodiment, Ar1 is a substituted aromatic moiety. In one embodiment, Ar1 is an unsubstituted aromatic moiety, which may also be referred to as an unsubstituted aromatic moiety. In the substituted aromatic moiety, one or more hydrogen atoms that would have been bonded to the ring atoms are substituted with substituents. For example, optionally, 1 or 2 or 3 or 4 or 5 or 6 of the hydrogen atoms may be substituted with substituents. The substituents on Ar1 do not refer to the triazole-Ar2 moiety that necessarily exists when m is equal to 1, or the two triazole-Ar2 moieties that necessarily exist when m is equal to 2, or the three triazole Ar2 moieties that necessarily exist when m is equal to 3.

[0150] In one embodiment, the substituents on Ar1 consist of atoms selected from deuterium, halogen (F, Cl, Br, I), carbon, nitrogen, oxygen, and sulfur, optionally contain hydrogen, and, if present, also contain additional atoms that form counterions. Deuterium and halides are considered monovalent atoms, and carbon, nitrogen, oxygen, and sulfur are considered polyvalent atoms because they can form two or more covalent bonds simultaneously. In addition to monovalent atoms, the substituents on Ar1 may have a plurality of polyvalent atoms, for example, 1 to 25 polyvalent atoms, or 1 to 22 polyvalent atoms, or 1 to 15 polyvalent atoms, or 1 to 10 polyvalent atoms, or 1 to 5 polyvalent atoms, and the atoms are optionally selected from carbon, nitrogen, oxygen, and sulfur. Examples of substituents having up to 10 polyvalent atoms are provided below. Other substituents including substituents having up to 25 polyvalent atoms are known to those skilled in the art as well.

[0151] In one embodiment, the substituent on Ar1 contains 0 polyvalent atoms. In this embodiment, the hydrogen bonded to the ring atom is replaced with another monovalent atom such as deuterium, fluorine, chlorine, bromine, or iodine. The hydrogen bonded to the ring atom is replaced with another monovalent atom such as deuterium, fluorine, chlorine, bromine, or iodine.

[0152] In one embodiment, the substituent on Ar1 contains 1 polyvalent atom. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced with a single polyvalent atom, and the open valence on the polyvalent atom is satisfied with one or more monovalent atoms. Examples include hydroxyl (OH), thiol (SH), amino (NH2), methyl (CH3), and methylene (=CH2) (fully or partially halogenated and deuterated forms thereof, such as CF3).

[0153] In one embodiment, the substituent on Ar1 contains 2 polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced with a first polyvalent atom, and the first polyvalent atom is bonded to a second polyvalent atom, thus providing a substituent formed from two polyvalent atoms, and the open valence on the polyvalent atom is satisfied with one or more monovalent atoms. Examples of these substituents are well known to those skilled in the art. Specific examples include ethyl (CH2CH3), ethylene (CH=CH2), ethynyl (C≡CH), ethylidene (=CHCH3), aminomethyl (CH2NH2), aminomethylene (=CHNH2), thiomethylene (=CHSH), hydroxymethylene (=CHOH), hydroxymethyl (CH2OH), thiomethyl (CH2SH), N-methylamine (NHCH3), methyl sulfide (SCH3), methoxy (OCH3), nitrile (CN), formyl (C(O)H), thioformyl (C(S)H), N-hydroxy (N-OH), hydroxylamine (ONH2), hydrazine (NH2NH2), diazine (N=NH), diazonium (N≡N) (fully or partially halogenated and deuterated forms thereof, such as OCF3 and CH2CD3).

[0154] In one embodiment, the substituent on Ar1 contains three polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a first polyvalent atom, and the first polyvalent atom is directly or indirectly bonded to each of the second and third polyvalent atoms. Thus, the first polyvalent atom is bonded to the second polyvalent atom, and the third polyvalent atom is bonded to either or both of the first and second polyvalent atoms, thus providing a substituent formed from three polyvalent atoms, and the open valence on the polyvalent atom is filled with one or more monovalent atoms. Examples of these substituents are well known to those skilled in the art and are provided herein, for example, nitro, methyl ketone, carboxyl.

[0155] In one embodiment, the substituent on Ar1 contains four polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a first polyvalent atom, and the first polyvalent atom is directly or indirectly bonded to each of the second, third, and fourth polyvalent atoms. Thus, a substituent formed from four polyvalent atoms is provided, and the open valence on the polyvalent atom is filled with one or more monovalent atoms. Examples of these substituents are well known to those skilled in the art and are provided herein, for example, methyl ester (CO2CH3), N-methylcarboxamide (C(O)NHCH3), and acetamide (NHC(O)CH3).

[0156] In one embodiment, the substituent on Ar1 contains five polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a first polyvalent atom, and the first polyvalent atom is directly or indirectly bonded to each of the second, third, fourth, and fifth polyvalent atoms. Thus, a substituent formed from five polyvalent atoms is provided, and the open valence on the polyvalent atom is filled with one or more monovalent atoms. Examples of these substituents are well known to those skilled in the art and are provided herein, for example, ethyl ester (CO2CH2CH3), S-ethylthiocarbonate (C(O)SCH2CH3), N-ethylcarboxamide (C(O)NHCH2CH3), and N,N-dimethylcarboxamide (C(O)N(CH3)2).

[0157] In one embodiment, the substituent on Ar1 contains six polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar 1 are replaced by a first polyvalent atom, and the first polyvalent atom is directly or indirectly bonded to each of the second, third, fourth, fifth, and sixth polyvalent atoms, thus providing a substituent formed from six polyvalent atoms, and the open valence on the polyvalent atom is satisfied by one or more monovalent atoms. Examples of these substituents are well known to those skilled in the art and are provided herein, for example, N-cyclopropylcarboxamide (C(O)NH-cyclopropyl), N-propylcarboxamide (C(O)NHCH2CH2CH3), N-(2-hydroxyethyl)carboxamide (C(O)NHCH2CH2OH), and N-carbamimidocarboxamide (C(O)NHC(=NH)NH2).

[0158] In one embodiment, the substituent on Ar1 contains seven polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a first polyvalent atom, and the first polyvalent atom is directly or indirectly bonded to each of the second, third, fourth, fifth, sixth, and seventh polyvalent atoms, thus providing a substituent formed from seven polyvalent atoms, and the open valence on the polyvalent atom is satisfied by one or more monovalent atoms. Examples of these substituents are well known to those skilled in the art and are provided herein, for example, N-(n-butyl)carboxamide (C(O)NHCH2CH2CH2CH3), N-(t-butyl)carboxamide (C(O)NHC(CH3)3), N,N-diethylcarboxamide (C(O)N(CH2CH3)2), and N-cyclobutylcarboxamide (C(O)NH(cyclobutyl)).

[0159] In one embodiment, the substituent on Ar1 contains eight polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a first polyvalent atom, and the first polyvalent atom is directly or indirectly bonded to each of the second, third, fourth, fifth, sixth, seventh, and eighth polyvalent atoms, thus providing a substituent formed from eight polyvalent atoms, and the open valence bonds on the polyvalent atoms are satisfied by one or more monovalent atoms. Examples of these substituents are well known to those skilled in the art and are provided herein, for example, N-cyclopentylcarboxamide (C(O)NH(cyclopentyl)), (piperidin-1-yl)methanone (C(O)-piperidin-1-yl), and (morpholin-4-yl)methanone (C(O)-morpholin-4-yl).

[0160] In one embodiment, the substituent on Ar1 contains nine polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a first polyvalent atom, and the first polyvalent atom is directly or indirectly bonded to each of the second, third, fourth, fifth, sixth, seventh, eighth, and ninth polyvalent atoms, thus providing a substituent formed from nine polyvalent atoms, and the open valence bonds on the polyvalent atoms are satisfied by one or more monovalent atoms. Examples of these substituents are well known to those skilled in the art and are provided herein, for example, di(isopropyl)ester (C(O)O(CH(CH3)2)2), di(n-propyl)ester (C(O)O(CH2CH2CH3)2), N-cyclohexylcarboxamide (C(O)NH(cyclohexyl)), (4-methylpiperazin-1-yl)methanone (C(O)(4-methylpiperazin-1-yl)), 2-(acetylamino)ethylcarboxamide (C(O)NHCH2CH2NHC(O)CH3), and N-phenylcarboxamide (C(O)NH(phenyl)).

[0161] In one embodiment, the substituent on Ar1 contains 10 polyvalent atoms. In this embodiment, one or more hydrogen atoms bonded to the ring atoms of Ar1 are replaced by a first polyvalent atom, and the first polyvalent atom is directly or indirectly bonded to each of the second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth polyvalent atoms, thus providing a substituent formed from 10 polyvalent atoms, and the open valence on the polyvalent atom is satisfied by one or more monovalent atoms. Examples of these substituents are well known to those skilled in the art and are provided herein, for example, N-benzylcarboxamide (C(O)NHCH2(phenyl)).

[0162] In one embodiment, Ar1 is a substituted aryl in which at least one substituent on Ar1 is selected from the group consisting of halogen, hydroxyl, mercaptan, nitro and nitrile.

[0163] In one embodiment, Ar1 is a substituted aryl in which at least one substituent on Ar1 is halogen, -OH, -CN, -NO2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C1-C6 cycloalkyl, -OR 0 、-CONH2、-C(O)NR 1 R 1’ 、-NR 1 R 1’ 、-NR 1 C(O)R 3 、-C(O)SR 3 、-COR 3 、-OC(O)R 3 、-C(O)OR 3 、mercaptan, -R 4 -H、-SOR 1 、-S(O)2R 1 、-S(O)2NR 1 R 1’ and -NS(O)2R 3 and is a substituted aryl selected from; (a) R 0is, for each occurrence, independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; (b) R 1 and R 1’ is, for each occurrence, H, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroaryl, -C(=NH)NH2, -CH2CO2R 0 , -CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R 3 , -CH2C(O)NHCH2CO2H,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0164] In one embodiment, Ar1 is substituted aryl, and at least one substituent on Ar1 is 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.

[0165] The Ar1 group contains an aromatic moiety as described herein, which aromatic moiety may also be optionally substituted as described herein, and the substitution is (triazole - Ar2) mIn addition to being substituted with a base. In one embodiment, exemplary substituents of Ar1 are halides such as fluoride, chloride, and bromide, alkyl groups having 1 to 6 carbon atoms such as methyl and ethyl, haloalkyl groups having 1 to 6 carbon atoms such as trifluoromethyl, cyano, formyl, and carboxamide.In another embodiment, exemplary substituents of Ar1 include nitro (-NO2), cyano (-CN), carboxylic acid (-COOH or its salt), carboxamide (-C(O)NH2), C1-C6 alkoxy including methoxy, C1-C6 alkyl including methyl, C1-C6 haloalkyl including trifluoromethyl, C1-C6 heteroalkyl including amide, for example, -NHC(O)(C1-C6 alkyl), -NHC(O)(C1-C6 heteroalkyl)-C(O)NH(C1-C6 alkyl), -C(O)NH(C1-C6 heteroalkyl), -C(O)N(C1-C6 alkyl)(C1-C6 alkyl), -C(O)N(C1-C6 alkyl)(C1-C6 heteroalkyl) and -C(O)N(C1-C6 heteroalkyl)(C1-C6 heteroalkyl) (including the following: -NHC(O)CH3, C(O)NHCH3, -C(O)N(CH3)2, -NHC(O)CH2CH3, C(O)NHCH2CH3, -C(O)N(CH3)CH2CH3, -C(O)N(CH2CH3)2, -C(O)NH(C1-C6 cycloalkyl) and -NHC(O)(C1-C6 cycloalkyl)) (for example, C(O)NH(cyclopropyl), -NHC(O)-cyclopropyl, C(O)NH(cyclohexyl), NHC(O)-cyclohexyl), C(O)NHCH2CH2CH2CH3, -C(O)NH(C(CH3)3), -C(O)NH(CH2CH2OH), ketone, for example, -C(O)(C1-C6 alkyl) including -C(O)CH3, -C(O)(cycloalkyl) including -C(O)-cyclohexyl, and C(O)-(heterocycloalkyl) where heterocycloalkyl can be, for example, morpholinyl, piperidinyl, piperazinyl, N-methylpiperazinyl, esters such as -CO2CH3, -CO2CH2CH3, -CO2CH2CH2CH3, -CO2CH2(CH3)2 including -CO2-(C1-C6 alkyl), and thioesters such as C(O)-S-(C1-C6 alkyl) including -C(O)-S-CH3 and -C(O)-S-CH2CH3.

[0166] In one embodiment, Ar1 has at least one substituent on Ar1 that is -O-(C 1-6 alkyl), C 1-6 alkyl, C1-6 haloalkyl, -CO2-C 1-6 alkyl, -CONH-C 1-6 alkyl, -CONH2, CN; and a substituted aryl selected from the group consisting of -NO2.

[0167] When n is 1 or 2, the compound of formula (I) will include a linker L. In one embodiment, the linker L can be a direct bond. In another embodiment, the linker is not a direct bond, but instead is one or more atoms, particularly atoms selected from carbon, nitrogen, oxygen, sulfur. In another embodiment, the linker can be an alkylene group (e.g., C1-C6 alkylene) or a substituted alkylene. The linker may be a heteroalkylene linker, which further includes at least one heteroatom selected from oxygen, nitrogen, or sulfur (e.g., 1, 2, 3, or 4 heteroatoms) within the parent chain and / or a substituted or unsubstituted alkylene disposed at one or more terminal positions of the parent chain. In one embodiment, L is a heteroalkylene group having a length of 2 to 10 carbon atoms, wherein one or more carbon atoms are replaced by at least one heteroatom selected from oxygen, nitrogen, and sulfur. In one embodiment, L may be a heteroalkylene linker having at least one N, O, or S heteroatom, and the heteroalkylene may be linear, or cyclized, and optionally substituted, and exemplary substituents include oxo, --OH, C 1-4 alkyl and C 1-4Examples of alkoxy groups are provided. Examples of 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 C1-C6 alkylene. Other examples of 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 C1-C6 alkylene and in another embodiment, alkylene is substituted C1-C6 alkylene. In one embodiment, the linker is hydrolytically stable such that the PEM does not decompose or degrade or otherwise break down when placed in water.

[0168] Linker L typically need not be overly long and in one embodiment contains from 1 to about 25 atoms excluding hydrogen and halogen from its atomic count, and the linker may optionally be composed of atoms selected from carbon, nitrogen, oxygen, and sulfur in addition to hydrogen and halogen. In various other embodiments, the linker has less than 25 atoms (excluding hydrogen and halogen), for example, the linker contains from 1 to about 20 atoms, or from 1 to about 15 atoms, or from 1 to about 10 atoms, or from 1 to about 5 atoms, and in each case, excluding hydrogen and halogen from its atomic count, the counted atoms may optionally be selected from carbon, oxygen, nitrogen, and sulfur.

[0169] In one embodiment, the triazole ring in the compound of formula (I) may be substituted in addition to being directly bonded to Ar1 and Ar2. Generally, the compounds of the present disclosure have the chemical formula

Chemical formula

[0170] Thus, in one embodiment, the present disclosure provides a formula wherein Ar1 and Ar2 are defined elsewhere in this specification and M is selected from hydrogen, C1-C4 alkyl, and a halide [Chemical formula] of the compound. Optionally, as described above, M may be hydrogen, or in another option, M may be a halide such as iodide, exemplified by the compound 4,4'-((pyridine-2,6-diyl)bis(5-iodo-1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid). As in the above structure, when the compounds of the present disclosure have two or more M-substituted triazole rings, M is independently selected each time it appears. However, in one embodiment, M is the same atom each time it appears in the compounds of the present disclosure. For example, the present disclosure provides compounds wherein M is hydrogen each time M appears. In another example, the present disclosure provides compounds wherein M is iodide each time M appears.

[0171] The compound of formula (I) contains at least one triazole-Ar2 moiety. In one embodiment, the compounds of the present disclosure contain two or more triazole-Ar2 moieties, for example of the formula [Chemical Formula] When the compounds of the present disclosure contain two or more triazole-Ar2 moieties, the Ar2 moieties may optionally have the same chemical structure each time they appear. However, when the PEM compounds of the present disclosure contain multiple triazole-Ar2 moieties, in one embodiment, those Ar2 moieties are not necessarily identical to each other, and in fact, they may not be identical. The Ar2 moieties may differ from each other with respect to the Ar2 ring atoms and / or with respect to the substitutions on the Ar2 ring atoms. For example, if one Ar2 group is phenyl and the other Ar2 group is pyridinyl, the two Ar2 groups are different with respect to the ring atoms constituting the Ar2 group. As another example, for example, 4-(4-(3-(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)benzoic acid, although both Ar2 groups are phenyl, when one phenyl is substituted with carboxyl and the other phenyl is substituted with methoxy, the compound is considered to have two different Ar2 groups. In yet another example, the two Ar2 groups can be positional isomers of each other, as in the case where both Ar2 groups are phenyl and both phenyl rings are substituted with hydroxyl and carboxyl, but the positions of the hydroxyl and / or carboxyl groups are different on the two phenyl rings. For example, when the triazole is located at the 3-position (meta) relative to the carboxyl group on one phenyl ring and the triazole is located at the 4-position (para) relative to the carboxyl group on the other phenyl ring, the two Ar2 groups are considered to be positional isomers and non-identical. In one embodiment, the Ar2 ring is identical in all respects each time it appears in the compounds of the present disclosure. In one embodiment, the Ar2 ring atoms are identical each time Ar2 appears, but the substitutions on the Ar2 ring are not identical each time Ar2 appears. In another embodiment, the Ar2 ring atoms are not identical each time Ar2 appears, and the substitutions on the Ar2 ring may or may not be identical.

[0172] The compounds of formula (I) include at least one Ar2 moiety, in one embodiment, Ar2 is a monocyclic aromatic ring selected from phenyl and pyridinyl, optionally substituted. In one embodiment, Ar2 is a monocyclic 6-membered aromatic ring, examples being phenyl, pyridinyl and pyrazinyl, where the Ar2 group also optionally includes substituents on the ring atoms. In another embodiment, Ar2 is an optionally substituted 5-membered monocyclic aromatic ring. In another embodiment, Ar2 is an optionally substituted 5- or 6-membered aromatic ring. In another embodiment, Ar2 are each independently selected from the group consisting of phenyl, pyridinyl, pyrazinyl, and phenyl. In another embodiment, 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. In another embodiment, 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 aromatic rings. In one embodiment, Ar2 may be any of these options, i.e., Ar2 is selected from (a) a 5-membered monocyclic aromatic ring, (b) a 6-membered monocyclic aromatic ring, (c) a 9-membered fused bicyclic ring 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 are aromatic rings, and (d) a 10-membered fused bicyclic ring comprising two 6-membered monocyclic rings fused together, where at least one of the two monocyclic rings and optionally both of the monocyclic rings are aromatic rings;

[0173] In the compound of formula (I), optionally, 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, 1H-pyrrole, 1H-pyrazole, oxadiazole, thiadiazole, 1,2,4-triazole, 1,2,3-triazole, and 1H-imidazole.

[0174] In the compounds of formula (I), optionally, Ar2 is a six-membered monocyclic aromatic ring selected from the group consisting of benzene, pyridine, pyridazine, pyrimidine and pyrazine.

[0175] In the compound of formula (I), optionally, 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, 1H-benzimidazole, 1H-pyrrolo[3,2-b]pyridine, 1H-pyrrolo[3,2-c]pyridine, 1H-pyrrolo[2,3-c]pyridine, 1H-pyrrolo[2,3-b]pyridine, benzothiophene, 1,3-benzothiazole, thieno[3,2-b]pyridine, thieno[3,2-c]pyridine, thieno[2,3-c]pyridine, benzoxadiazole, benzothiadiazole, benzisoxazole, benzotriazole and thieno[2,3-b]pyridine.

[0176] In the compound of formula (I), optionally, Ar2 is a 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.

[0177] As described above, the compounds of the present disclosure include at least one Ar2 group, the Ar2 group includes at least one aromatic ring, and optionally includes one or more substituents on the aromatic ring. In one embodiment, Ar2 includes at least one, i.e., one or more substituents, such as 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2 substituents on the aromatic ring. Optionally, Ar2 includes exactly one substituent on the aromatic ring. In another option, Ar2 includes exactly two substituents on the aromatic ring. In yet another option, Ar2 includes exactly three substituents on the aromatic ring. In a further option, Ar2 includes exactly four substituents on the aromatic ring. In any one embodiment, Ar2 includes two or more substituents on the aromatic ring.

[0178] In one embodiment, one or more substituents on the ring atoms of Ar2 are selected from substituents arbitrarily named "G", and the substituents are halogen, C1-C6 alkyl, C1-C6 haloalkyl, -E-CO2H, -E-CHO, -E-C(O)R 3 , -E-C(O)NH(OH), -E-C(O)NHR 1 , -E-CONR 1 R 1’ , -E-NR 1 R 1’ , and -E-OR 2 selected from; (a) E is selected from a direct bond and C1-C6 alkylene; (b) R 0 is, each occurrence, independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted hete rocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; (c) R 1 and R 1’ are, each occurrence, independently H, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroaryl, -C(=NH)NH2, -CH2CO2R 0 , -CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R 3 , -CH2C(O)NHCH2CO2H,

Chem.

Chem.

Chem.

[0179] In one embodiment, the substitution on Ar2 contains amino (-NH2). In one embodiment, the substitution on Ar2 contains alkoxy, such as C1-C6 alkoxy. For example, in one embodiment, the substitution on Ar2 contains methoxy. In one embodiment, the substitution on Ar2 contains carboxylic acid or alkylene carboxylic acid. For example, in one embodiment, the substitution on Ar2 of the PEM compound of formula (I) contains carboxylic acid. In one embodiment, the substitution on Ar2 contains carboxylic acid ester or alkylene-carboxylic acid ester. For example, in one embodiment, the substitution on Ar2 of the PEM compound of formula (I) contains -CH2-CO2-CH3. In one embodiment, the substitution on Ar2 contains a haloalkyl group, such as a C1-C6 haloalkyl group. For example, in one embodiment, the substitution on Ar2 of the PEM compound of formula (I) contains trifluoromethyl. In one embodiment, the substitution on Ar2 contains hydroxyl or hydroxyl-substituted alkyl, such as hydroxyl-substituted C1-C6 alkyl. For example, in one embodiment the substitution on Ar2 of the compound of formula (I) contains hydroxyl (-OH).

[0180] In one embodiment, the substitution on Ar2 includes one group selected from carboxylic acids and alkylene carboxylic acids, such as C1-C6 alkylene carboxylic acids, and another group selected from hydroxyl and hydroxyl-substituted alkyls, such as C1-C6 alkyl substituted with one hydroxyl. For example, in one embodiment, the substitution on Ar2 is one carboxylic acid and one hydroxyl, or includes them.

[0181] In one embodiment, the substitution on Ar2 includes one group selected from carboxylic acids and alkylene carboxylic acids, such as C1-C6 alkylene carboxylic acids, and one group selected from haloalkyls, such as C1-C6 haloalkyls. For example, in one embodiment, the substitution on Ar2 is one carboxylic acid group and one trifluoromethyl group, or includes them.

[0182] In one embodiment, the substitution on Ar2 includes one group selected from hydroxyl and hydroxyl-substituted alkyls, such as C1-C6 alkyl substituted with one hydroxyl, and another group selected from haloalkyls, such as C1-C6 haloalkyls. For example, in one embodiment, the substitution on Ar2 is one hydroxyl group and one trifluoromethyl group, or includes them.

[0183] In one embodiment, the substitution on the Ar2 ring of formula (I) includes at least one of a) carboxylic acids and alkylene carboxylic acids, such as C1-C6 alkylene carboxylic acids; b) hydroxyl and hydroxyl-substituted alkyls, such as C1-C6 alkyl substituted with one hydroxyl; and c) haloalkyls, such as C1-C6 haloalkyls. For example, it is at least one of carboxylic acid, hydroxyl, and trifluoromethyl.

[0184] In one embodiment, the substitution on the Ar2 ring of formula (I) includes at least two of: a) carboxylic acid and alkylene carboxylic acid, such as C1-C6 alkylene carboxylic acid; b) hydroxyl and hydroxyl-substituted alkyl, such as C1-C6 alkyl substituted with one hydroxyl; and c) haloalkyl, such as C1-C6 haloalkyl. For example, at least two of carboxylic acid, hydroxyl and trifluoromethyl.

[0185] In one embodiment, the substitution on the Ar2 ring of formula (I) includes at least three of: a) carboxylic acid and alkylene carboxylic acid, such as C1-C6 alkylene carboxylic acid; b) hydroxyl and hydroxyl-substituted alkyl, such as C1-C6 alkyl substituted with one hydroxyl; and c) haloalkyl, such as C1-C6 haloalkyl. That is, Ar2 may be substituted with carboxylic acid, hydroxyl and trifluoromethyl.

[0186] For example, in one embodiment, the Ar2 group is

Chemical formula

[0187] In one embodiment, the Ar2 group is a substituted phenyl group, and the substituent of the phenyl group is an aryl further substituted with G 2 、G 3 、G 4 and G 5 ; in a more specific embodiment, the substituent of the phenyl group is a phenyl further substituted with G 2 、G 3 、G 4 and G 5 ; for example, a phenyl further substituted with G 、 such as G 2 or G 2 and G 3 。

[0188] As described above, in one embodiment, the PEM compounds of the present disclosure may have hydroxyl and carboxylic acid substitutions on Ar2. These two groups may be located at various positions on the Ar2 ring. For example, in one embodiment, the present disclosure provides a PEM compound of formula (I) described by the following formula:

Chemical formula

[0189] In another embodiment, the PEM compound of formula (I) of the present disclosure has hydroxyl and carboxylic acid substitutions on Ar2 as provided in the following formula:

Chemical formula

[0190] In yet another embodiment, the PEM compound of formula (I) of the present disclosure has hydroxyl and carboxylic acid substitutions on Ar2 as shown in the following formula:

Chemical formula

[0191] In one embodiment, the PEM compound of formula (I) of the present disclosure has at least hydroxyl and carboxylic acid substitutions on Ar2 and may have other substitutions on Ar2. For example, Ar2 is substituted with hydroxyl, carboxylic acid, and alkyl, such as C1-C6 alkyl, to provide, for example, a compound of the following formula:

Chemical formula

[0192] As described above, in one embodiment, the PEM compound of formula (I) of the present disclosure may have haloalkyl and carboxylic acid substitutions on Ar2 instead of hydroxyl and carboxylic acid as shown in the above structure. As an example, the PEM compound of the present disclosure may be described by the following formula:

Chemical formula

[0193] The PEM compounds of formula (I) include solvates including their hydrates, chelates, and salt forms. In some examples, the PEM compounds can be amorphous, while in other examples, the PEM compounds can be crystalline. Furthermore, some of the crystalline forms of the compounds can exist as polymorphs contemplated herein. Additionally, some of the compounds may form solvates with water or other organic solvents. Such solvates are likewise included within the scope of the compounds described herein.

[0194] The PEM compounds of formula (I) may be in the form of chelates such as copper chelates. The copper chelates can be formed by combining the PEM compounds of the present disclosure with copper sulfate. The PEM compounds of formula (I) can be in the form of either acid addition salts or base addition salts depending on the substituents on the Ar1 and Ar2 groups.

[0195] The PEM structure includes all of its stable stereoisomeric forms. Thus, the PEM compounds described herein may have one or more chiral (or asymmetric) centers and, accordingly, can give rise to enantiomers, diastereomers, and other stereoisomers that can be defined as (R)- or (S)- from the perspective of absolute stereochemistry. When the compounds described herein contain olefinic double bonds or other centers due to geometric asymmetry and unless otherwise specified, the compounds are intended to contain both E and Z geometric isomers (e.g., cis or trans). Similarly, unless otherwise indicated, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are intended to be included. Thus, various stereoisomers and mixtures thereof are considered to include "enantiomers" which refer to two stereoisomers that are mirror images that cannot be superimposed on each other. Thus, the compounds can occur in any isomeric form including racemates, racemic mixtures, and as individual enantiomers or diastereomers.

[0196] The PEM compounds of the present disclosure are generally water-soluble. One measure of water solubility is the logP value of the compound. The LogP value can be calculated using commercially available software based on the chemical structure of the compound. For example, CHEMDRAW chemical drawing software (Cambridgesoft Limited, a subsidiary of PerkinElmer Holdings) can calculate the logP value of the drawn chemical structure. In one embodiment, the PEM compounds of the present disclosure have a logP of at least 4.9.

[0197] The compounds of the present disclosure, such as the PEM compounds of formula (I) above, can typically be synthesized by the reaction of a diethynyl compound of formula Ar1(C≡CH)2 with an azide compound of formula Ar2-N3 in the presence of a 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.

[0198] Certain and similar reactants can also be identified through indexes of known chemical substances prepared by the Chemical Abstracts Service of the American Chemical Society, which are available in most public and university libraries and online databases (contact the American Chemical Society in Washington, D.C. for further details). Chemical substances that are known but not commercially available in catalogs can be prepared by custom chemical synthesis houses, and many of the standard chemical supply houses (such as those listed above) offer custom synthesis services. A reference for the preparation and selection of the pharmaceutical salts of the present disclosure is P.H. Stahl & C.G. Wermuth, "Handbook of Pharmaceutical Salts," Verlag Helvetica Chimica Acta, Zurich, 2002.

[0199] Compounds of the formula Ar1(C≡CH) are commercially available, for example, from TCI America (Portland, Oregon, United States of America), which sells 1,3 - diethynylbenzene, 1,4 - diethynylbenzene, 2,6 - diethynylpyridine, and 3,6 - diethynylcarbazole.

[0200] Generally, ethynyl aromatic compounds can be prepared by Seyferth - Gilbert homologation from aryl aldehydes using dimethyl(diazomethyl)phosphonate, which is available from MilliporeSigma Corp. (St. Louis, Missouri, United States). Alternatively, dimethyl(diazomethyl)phosphonate can be generated in situ from dimethyl - 1 - diazo - 2 - oxopropylphosphonate (Ohira - Bestmann reagent). See, for example, Seyforth et al., J. Org. Chem. 36(10):1379 - 1386(1971). doi:10.1021 / jo00809a014 and Bestman et al., Synlett. 1996(06):521 - 522(1996). doi:10.1055 / s - 1996 - 5474.

[0201] Another route to ethynyl aromatic compounds involves a Sonogashira coupling of a haloaromatic compound with (t-butyldimethylsilyl)acetylene in the presence of a palladium catalyst. The ethynyl aromatic forms the silyl group upon subsequent deprotection. See, for example, Sonogashira, Organomet. Chem., 653:46-49 (2002). doi:10.1016 / s0022-328x(02)01158-0.

[0202] The following reactions (I), (II), and (III) illustrate exemplary preparations of diethynyl aromatic compounds. In reaction (I), 2,6-dibromopyridin-4-amine is converted to the corresponding 2,6-diethynylpyridin-4-amine compound. In reaction (II), 2,6-diiodo-4-nitroaniline is converted to the corresponding 2,6-diethynyl-4-nitroaniline. In reaction (III), 2-hydroxy-3,5-diiodobenzoic acid is converted to the corresponding 3,5-diethynyl-2-hydroxybenzoic acid. In each case, the conversion proceeds through an intermediate di-trimethylsilyl (TMS) compound as shown.

Chemical formula

[0203] Each of these reaction products, namely 2,6-diethynylpyridin-4-amine, and 2,6-diethynyl-4-nitroaniline, and 3,5-diethynyl-2-hydroxybenzoic acid, can function as a precursor to Ar1 in the preparation of the PEMs of the present disclosure. Thus, each of them represents an Ar1(C≡CH)2 compound that can react with an azide compound of formula Ar2-N3 in the presence of a Cu(I) catalyst to provide a PEM. Reactions (I), (II), and (III) illustrate the preparation of precursors of the substituted Ar1 moieties of the present disclosure.

[0204] Compounds of the formula Ar2-N3 are similarly commercially available, for example, from TCI America (Portland, Oregon, United States of America), Synthonix (Wake Forest, North Carolina, United States), SigmaAldrich (St. Louis, Missouri, United States), Toronto Research Chemicals (Toronto, Canada), and AnaSpec (Fremont, California, United States). Generally, azides of the formula Ar2-N3 can be prepared by nucleophilic substitution of electrophilic compounds such as alkyl, benzyl, or allyl iodides or bromides with sodium azide.

[0205] Generally, the compounds used in the reactions described herein can be prepared from commercially available chemical substances and / or compounds described in the chemical literature according to organic synthesis techniques known to those skilled in the art. "Commercially available chemical substances" include Across Organics (Pittsburgh, Pennsylvania), Aldrich Chemical (Milwaukee, Wisconsin, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester, Pennsylvania), Crescent Chemical Co. (Hauppauge N.Y.), Eastman Organic Chemicals, Eastman Kodak Company (Rochester N.Y.), Fisher Scientific Co. (Pittsburgh, Pennsylvania), Fisons Chemicals (Lancashire UK), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, California), Key Organics (Cornwall U.K.), Lancas ter Synthesis (Windham N.H.), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem, Utah), Pfaltz & Bauer, Inc. (Waterbury, Connecticut), Polyorganix (Houston, Texas), Pierce Chemical Co. (Rockford Ill.), Riedel de Haen AG (Hanover, Germany), Spectrum Quality Product, Inc. (New Brunswick, N.J.), TCI America (Portland, Oregon), Trans World Chemicals, Inc. (Rockville, Maryland), and Wako Chemicals USA, Inc. (Richmond, Virginia). are available from standard commercial sources including these.

[0206] In one embodiment, the PEM compounds of the present disclosure, such as the PEM compounds of formula (I), are present in a composition. For example, the PEM compounds of the present disclosure may be present in a composition that also includes an aqueous buffer. In one embodiment, the PEM compounds of the present disclosure are present in a composition that includes a biomolecule such as a polypeptide and / or polynucleotide. The polypeptide may be an enzyme such as DNA polymerase. The following definitions may be useful for understanding these compositions and their specific uses.

[0207] As used herein, "nucleic acid", also referred to as polynucleotide, is a covalently linked series of nucleotides in which the 3'-position of the pentose of one nucleotide is linked to the next 5'-position by a phosphodiester group. Nucleic acid molecules 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 nucleotide residues are linked in a specific sequence by phosphodiester bonds. As used herein, the terms "nucleic acid", "polynucleotide", or "oligonucleotide" include any polymeric compound having a straight-chain backbone of nucleotides. Oligonucleotides, also called oligomers, are generally polynucleotides of shorter chains. Nucleic acids are generally referred to as "target nucleic acids" or "target sequences" when for the purpose of sequencing.

[0208] As used herein, the term "template-dependent manner" is intended to refer to a process involving template-dependent extension of a primer molecule (e.g., DNA synthesis by DNA polymerase). The term "template-dependent manner" refers to the polynucleotide synthesis of RNA or DNA, and the sequence of the newly synthesized strand of the polynucleotide is determined by well-known complementary base pairing rules (see, for example, Watson, J.D. et al., In: Molecular Biology of the Gene, 4th Ed., W.A. Benjamin, Inc., Menlo Park, Calif. (1987)).

[0209] As used herein, "nucleic acid polymerase" generally refers to an enzyme for ligating 3'-OH 5'-triphosphate nucleotides, oligomers, and their analogs. Polymerases include DNA-dependent DNA polymerase, DNA-dependent RNA polymerase, RNA-dependent DNA polymerase, RNA-dependent RNA polymerase, T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase 1, Klenow fragment, Thermophilus aquaticus DNA polymerase, Tth DNA polymerase, VentR® DNA polymerase (New England Biolabs), Deep VentR® DNA polymerase (New England Biolabs), Bst DNA Polymerase Large Fragment, Stoe ffel Fragment, 9° N DNA Polymerase, 9° N DNA polymerase, Pfu DNA Polymerase, Tfl DNA Polymerase, Tth DNA Polymerase, Phi29 Polymerase, Tli DNA polymerase, eukaryotic DNA polymerase beta, telomerase, Therminator™ polymerase (New England Biolabs), KOD HiFi™ DNA polymerase (Novagen), KOD1 DNA polymerase, Q-beta replicase, terminal transferase, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase. The polymerase according to the present invention can be a variant, mutant variant, or chimeric polymerase.

[0210] As used herein, "DPO4 DNA polymerase" is a DNA polymerase that is naturally expressed by archaea, Sulfolobus solfataricus, or related Y-family DNA polymerases and functions in the replication of damaged DNA by a process generally known as translesion synthesis (TLS). Y-family DNA polymerases are homologous to DPO4 polymerase. Examples include prokaryotic enzymes, PolII, PolIV, PolV, archaeal enzymes, Dbh, and eukaryotic enzymes, Rev3p, Rev1p, Pol η, REV3, REV1, Pol Ι, and Pol κ DNA polymerases, and chimeras thereof. Modified recombinant DPO4 DNA polymerase contains one or more mutations compared to the naturally occurring wild-type DPO4 DNA polymerase, for example, one or more mutations that enhance the ability to utilize bulky nucleotide analogs as substrates or as another polymerase property, and may include additional changes or modifications to the wild-type DPO4 DNA polymerase, for example, one or more deletions, insertions, and / or fusions of additional peptide or protein sequences (e.g., for immobilizing the polymerase on a surface or otherwise tagging the polymerase enzyme). Examples of variant polymerases according to the present invention are variants of Sulfolobus sulfataricus DPO4 described in published PCT patent application International Publication No. WO 2017 / 087281A1 and PCT patent application numbers PCTUS2018 / 030972 and PCTUS2018 / 64794, which are hereby incorporated by reference in their entirety.

[0211] As used herein, "nucleic acid polymerase reaction" refers to an in vitro method for making a new strand of nucleic acid in a template-dependent manner or extending an existing nucleic acid (e.g., DNA or RNA). The nucleic acid polymerase reaction according to the present invention includes a primer extension reaction, which results in the incorporation of nucleotides or nucleotide analogs at the 3'-end of the primer such that the incorporated nucleotides or nucleotide analogs are complementary to the corresponding nucleotides of the target polynucleotide. The primer extension product of the nucleic acid polymerase reaction can be further used for single molecule sequencing or as a template for synthesizing additional nucleic acid molecules.

[0212] Primer extension reaction reagents typically include (i) a polymerase enzyme; (ii) a buffer; and (iii) one or more extendable nucleotides or nucleotide analogs. The primer extension reaction can be used to measure the length of nucleic acid products obtained under specific experimental conditions and to determine the effect of various polymerase reaction additives (e.g., PEM) on polymerase activity by comparing, for example, the lengths of the extended primer products by gel electrophoresis.

[0213] As used herein, "enhancing a nucleic acid polymerase reaction" refers to the ability of an additive, such as PEM, to enable a nucleic acid polymerase to synthesize a primer extension product that is at least one subunit longer than in the absence of PEM.

[0214] The rate of the nucleic acid polymerase reaction as used herein refers to the average rate at which the nucleic acid polymerase extends the polymer chain. As used herein, the terms "rate" and "extension rate" are used interchangeably. The polymerization rate can be measured using the nucleotide incorporation assay of Hogrefe et al. (method of Enzymol. Vol. 334, pp. 91-116 (2001)). Briefly, polymerase activity is assayed on activated salmon sperm DNA 32A, which can be measured as the rate of incorporation of P-dCTP (purchased from Pharmacia; for the activation protocol, see C. C. Richardson, Procedures, Nucl. Acid Res. (Cantoni and Davies, eds.), p. 263-276 (1966), p. 264). The reaction buffer can be, for example, 50 mM Tris HCl (pH 8.0), 5 mM MgCl2, 1 mM dithiothreitol (DTT), 50 μg / ml bovine serum albumin (BSA), and 4% (v / v) glycerol. Nucleotide substrates and DNA are in large excess, typically at least 10-fold the Km of the polymerase being assayed. For example, 200 μM dATP, 195 μM dCTP, 5 μM labeled dCTP, and 250 μg / ml activated DNA are used. The reaction is quenched on ice and an aliquot of the reaction mixture is spotted onto an ion exchange filter (e.g., Whatman DE81). Unincorporated nucleotides are washed away and then the incorporated radioactivity is measured by scintillation counting.

[0215] As used herein, "increasing the rate" refers to an increase of 5-10%, 10-50%, or 50-100% or more compared to a polymerization reaction lacking the PEM that increases the rate as defined herein.

[0216] As used 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 of continuing to act on the substrate rather than 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 the template, the structure of the nucleotide or nucleotide analog substrate, and reaction conditions such as salt concentration, temperature, or the presence of specific additives.

[0217] As used herein, "increasing the processing capacity" refers to an increase of 5-10%, 10-50%, or 50-100% or more compared to a polymerization reaction lacking the PEM that increases the processing capacity as defined herein. Methods for measuring the processing capacity of a nucleic acid polymerase are generally known in the art, as described, for example, in Sambrook et al., 1989, In Molecular Cloning, 2nd Edition, CSH Press, 7.79-7.83 and 13.8, and are described in U.S. Patent Application Publication No. 2002 / 0119467, Published PCT Application International Publication No. 01 / 92501, and U.S. Patent No. 5,972,603, the entireties of which are incorporated herein by reference.

[0218] As used herein, the term "fidelity" refers to the accuracy of nucleic acid polymerization by a template-dependent nucleic acid polymerase. The fidelity of a DNA polymerase is measured by the error rate (the frequency of incorporating incorrect nucleotides, i.e., nucleotides that are not incorporated in a template-dependent manner). The fidelity or error rate of a DNA polymerase can be measured using assays known in the art (see, for example, Lundburg et al., 1991 Gene, 108:1-6). As used herein, "increasing the fidelity" refers to an increase of 5-10%, 10-50%, or 50-100% or more compared to a polymerization reaction lacking the additive that increases the fidelity as defined herein.

[0219] As used herein, the term "plurality" refers to "at least two."

[0220] "XNTP" is an expandable 5'-triphosphate modified nucleotide substrate that is compatible with template-dependent enzymatic polymerization. XNTP has two different functional components. That is, a nucleobase 5'-triphosphoramidate and, within each nucleoside triphosphoramidate, a linker chain that is attached at a position that allows for controlled extension by intranucleotide cleavage of the phosphoramidate bond. As used herein, XNTP is an exemplary "non-natural highly substituted nucleotide analog substrate". Exemplary XNTPs and methods for making them are described, for example, in PCT International Publication No. WO 2016 / 081871, which is hereby incorporated by reference in its entirety.

[0221] An "Xpandomer intermediate" is an intermediate product (also referred to herein as a "daughter strand") collected from XNTPs and formed by polymerase-mediated template-directed assembly of XNTPs using a target nucleic acid template. The newly synthesized Xpandomer intermediate is a constrained Xpandomer. Under a process step in which the phosphoramidate bond provided by XNTP is cleaved, the constrained Xpandomer is no longer constrained and is an Xpandomer product that extends as the linker chain is extended.

[0222] "Xpandomer" or "Xpandomer product" is a synthetic molecular construct generated by the growth of a constrained Xpandomer synthesized by a template-directed assembly of XNTP substrates. The Xpandomer extends relative to the target template from which it is generated. It is composed of the ligation of subunits, each subunit being a motif, and each motif being a member of a library that includes sequence information, a linker chain, and optionally a portion, or all, of the substrate, all of which are derived from a formative substrate construct. The Xpandomer is designed to extend to be longer than the target template, thereby reducing the linear density of the sequence information of the target template along its length. Further, the Xpandomer optionally provides a platform for increasing the size and abundance of a reporter, which then improves the signal-to-noise for detection. The lower linear information density and stronger signal enhance the resolution and reduce the sensitivity requirements for detecting and multiplexing the sequence of the template strand.

[0223] "Linker chain" or "linker chain member" refers to a polymer or molecular construct having a substantially linear dimension and having terminal portions at each of two opposing ends. The linker chain binds to a nucleoside triphosphoramidate having a bond at the terminal portion to form an XNTP. The bond serves to constrain the linker chain in a "constrained configuration". The linker chain has a "constrained configuration" and an "extended configuration". The constrained configuration is found in the XNTP and the daughter strand or Xpandomer intermediate. The constrained configuration of the linker chain is a precursor to the extended configuration as seen in the Xpandomer product. The transition from the constrained configuration to the extended configuration results in the cleavage of a phosphoramidate bond that is selectively cleavable. The linker chain includes one or more reporters or reporter constructs capable of encoding the sequence information of the substrate along its length. The linker chain provides a means for extending the length of the Xpandomer, thereby reducing the linear density of the sequence information.

[0224] A "linking chain element" or "linking chain segment" is a polymer having a substantially linear dimension with two ends, and the ends form end linkages for connecting the linking chain elements. A linking chain element is a segment of a linking chain. Such polymers include, but are not limited to, polyethylene glycol, polyglycol, polypyridine, polyisocyanate, polyisocyanate, poly(triarylmethyl) methacrylate, polyaldehyde, polypyrrolinone, polyurea, polyglycol phosphodiester, polyacrylate, polymethacrylate, polyacrylamide, polyvinyl ester, polystyrene, polyamide, polyurethane, polycarbonate, polybutyrate, polybutadiene, polybutyrolactone, polypyrrolidinone, polyvinyl phosphonate, polyacetamide, polysaccharide, polyhyaluronate, polyamide, polyimide, polyester, polyethylene, polypropylene, polystyrene, polycarbonate, polyterephthalate, polysilane, polyurethane, polyether, polyamino acid, polyglycine, polyproline, N-substituted polylysine, polypeptide, side-chain N-substituted peptide, poly-N-substituted glycine, peptoid, side-chain carboxyl-substituted peptide, homopeptide, oligonucleotide, ribonucleic acid oligonucleotide, deoxyribonucleic acid oligonucleotide, oligonucleotide modified to prevent Watson-Crick base pairing, oligonucleotide analog, polycytidylic acid, polyadenylic acid, polyuridylic acid, poly thymidine, polyphosphate, polynucleotide, polyribonucleotide, polyethylene glycol-phosphodiester, peptide polynucleotide analog, threosyl-polynucleotide analog, glycol-polynucleotide analog, morpholino-polynucleotide analog, locked nucleotide oligomer analog, polypeptide analog, branched polymer, comb polymer, star polymer, dendritic polymer, random, gradient and block copolymers, anionic polymer, cationic polymer, polymer forming a base loop, a polymer containing rigid segments and mobile segments. ne, polypyrrolidinone, polyvinyl phosphonate, polyacetamide, polysaccharide, polyhyaluronate, polyamide, polyimide, polyester, polyethylene, polypropylene, polystyrene, polycarbonate, polyterephthalate, polysilane, polyurethane, polyether, polyamino acid, polyglycine, polyproline, N-substituted polylysine, polypeptide, side-chain N-substituted peptide, poly-N-substituted glycine, peptoid, side-chain carboxyl-substituted peptide, homopeptide, oligonucleotide, ribonucleic acid oligonucleotide, deoxyribonucleic acid oligonucleotide, oligonucleotide modified to prevent Watson-Crick base pairing, oligonucleotide analog, polycytidylic acid, polyadenylic acid, polyuridylic acid, poly thymidine, polyphosphate, polynucleotide, polyribonucleotide, polyethylene glycol-phosphodiester, peptide polynucleotide analog, threosyl-polynucleotide analog, glycol-polynucleotide analog, morpholino-polynucleotide analog, locked nucleotide oligomer analog, polypeptide analog, branched polymer, comb polymer, star polymer, dendritic polymer, random, gradient and block copolymers, anionic polymer, cationic polymer, polymer forming a base loop, a polymer containing rigid segments and mobile segments may be included, but are not limited thereto.

[0225] A "reporter" is composed of one or more reporter elements. The reporter serves to analyze the genetic information of a target nucleic acid.

[0226] A "reporter construct" includes one or more reporters capable of generating a detectable signal, which generally includes sequence information. This signal information is called the "reporter code" and is subsequently decoded into genetic sequence data. The reporter construct may also include other structural components such as linker segments or polymers, graft copolymers, block copolymers, affinity ligands, oligomers, haptens, aptamers, dendrimers, linkers or affinity binding groups (e.g., biotin).

[0227] The "reporter code" is the genetic information from the measured signal of the reporter construct. The reporter code is decoded to provide sequence-specific genetic information data.

[0228] Accordingly, in one embodiment, the present disclosure provides a composition comprising the PEM disclosed herein and a buffer. In another embodiment, the present disclosure provides a composition comprising the PEM disclosed herein and a plurality of nucleotides and / or nucleotide analogs. In another embodiment, the present disclosure provides a composition comprising the PEM disclosed herein and a polynucleotide. In another embodiment, the present disclosure provides a composition comprising the PEM disclosed herein and a protein, optionally, the protein is a polymerase comprising any of the above polymerases.

[0229] In one embodiment, the present disclosure provides a composition comprising a PEM compound of the present disclosure, e.g., a PEM compound of formula (I), and a molecular crowding agent. Generally, the molecular crowding agent comprises a series of large neutral polymers. Examples of 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 described in U.S. Patent No. 7,399,590, which is incorporated herein by reference. In one embodiment, the molecular crowding agent is a polyalkylene glycol optionally having a number average molecular weight of from 4,000 to 10,000. In one embodiment, the molecular crowding agent is a derivative of a polyalkylene glycol, e.g., one or both of the terminal hydroxyl groups of the polyalkylene glycol are in the form of ester or ether groups. In one embodiment, the molecular crowding agent is an inert water-soluble polymer.

[0230] In one embodiment, the present disclosure provides a composition comprising a PEM compound of the present disclosure and an aqueous buffer. In one embodiment, the PEM compound has formula (I). In one option, the composition has a pH of about 6 to 8.5 and the buffer serves to stabilize the pH of the composition. An exemplary buffer is Tris HCl. Other suitable buffers include buffers known in the art, such as phosphate buffers, citrate buffers, sodium acetate buffers, sodium carbonate buffers, and the like.

[0231] In one embodiment, the present disclosure provides a composition comprising a PEM compound of the present disclosure, for example, a PEM compound of formula (I), and a polynucleotide. In one option, the polynucleotide is single-stranded, for example, single-stranded DNA or single-stranded RNA. When the polynucleotide is intended to function as a primer, the polynucleotide is a single-stranded DNA molecule. When intended to function as a primer, the polynucleotide can have a length of about 10 to 60 mers, for example, 20 to 30 oligonucleotides. Alternatively, the polynucleotide may function as a template, in which case it may be single-stranded DNA or single-stranded RNA and may have a length of 30 bases to kilobases and values exceeding that, for example, 10 k bases and lengths exceeding that.

[0232] In one embodiment, the present disclosure provides a composition comprising a PEM compound of the present disclosure, for example, a PEM compound of formula (I), and a protein. For example, the protein can be an enzyme, a nucleic acid polymerase, a DNA polymerase. An example of a suitable DNA polymerase is a variant of DPO4 polymerase, as discussed herein.

[0233] In one embodiment, the present disclosure provides a composition comprising at least one PEM compound of the present disclosure, e.g., a PEM compound of formula (I), and a mixture of nucleotides or nucleotide analogs, wherein the at least one compound increases the number and accuracy of nucleotide analogs incorporated into the daughter strand during a template-dependent polymerization reaction as compared to the same polymerization reaction in the absence of the at least one compound. Optionally, the mixture of nucleotide analogs comprises nucleoside triphosphoramidates, each of the nucleoside triphosphoramidates having a nucleobase selected from adenine, guanine, thymine, and cytosine and a polymer linker moiety, wherein a first end of the polymer linker moiety is attached to the nucleobase and a second end of the polymer linker moiety is attached to the α-phosphate of the nucleoside triphosphoramidate to provide extension of the nucleotide analog by cleavage of the phosphoramidate bond. Optionally, the composition further comprises a buffer comprising one or more of TrisOAc, NH4OAc, PEG, a water-miscible organic solvent (such as dimethylformamide (DMF), N-methylpyrrolidone (NMP), or acetone), polyphosphoric acid 60, NMS, and MnCl2. Optionally, the composition also comprises a single-stranded binding protein. Optionally, the composition comprises urea. Optionally, the mixture of nucleotide analogs comprises a nucleotide analog comprising a detectable label, which is optionally one of luminescent, chemiluminescent, fluorescent, fluorogenic, chromophore, or chromogenic. In one embodiment, the composition comprises two or more of these options, e.g., all of these options.

[0234] In one aspect of the present disclosure, the PEMs and their compositions disclosed herein can be used to enhance nucleic acid polymerization reactions or to improve the properties of the resulting nucleic acids, such as the length or accuracy of the reaction products. Polymerization reactions include, for example, primer extension reactions, PCR, mutagenesis, isothermal amplification, DNA sequencing, and probe labeling. Such methods are well known in the art. Enhancement can be provided by stimulating nucleotide incorporation through mechanisms such as an increase in the processivity of the polymerase (i.e., reduction in dissociation of the polymerase from the template), an increase in substrate binding or enzyme catalysis rate, and an increase in the accuracy or fidelity of nucleotide incorporation. Additionally, enhancement can be provided by reducing obstacles to nucleic acid templates such as secondary structure and double-stranded DNA. Overcoming or modifying such obstacles by the addition of PEMs allows for more accurate or efficient polymerization reactions, or the use of lower denaturation / extension temperatures or isothermal temperatures. By overcoming or modifying such obstacles, polymerization reactions can be performed more accurately or efficiently, or lower denaturation / extension temperatures or isothermal temperatures can be used.

[0235] In some embodiments, PEMs can be used in combination with another class of additives to enhance polymerase reactions. One exemplary class of additives is minor groove binding proteins (MGBs). In one embodiment, the MGB is selected from the group consisting of distamycin A and its synthetic analogs, netropsin, (+)-CC-1065, duocarmycin, pyrrolobenzodiazepine, trabectedin and its analogs, hext dyes and their derivatives, lexitropsin, thiazotropsin A, diamidines, and polyamides. In certain embodiments, at least one minor groove binding moiety is a Hoechst dye. Further information regarding the use of MGBs to enhance polymerase reactions can be found in the applicant's co-pending application entitled ENHANCEMENT OF NUCLEIC ACID POLYMERIZATION BY MGBS.

[0236] One exemplary polymerase reaction that can be enhanced by PEM is the polymerization of unnatural nucleotide analogs known as "XNTPs" that form the basis of the "Sequencing by Expansion" (SBX) protocol developed by Stratos Genomics (see, e.g., Kokoris et al., U.S. Patent No. 7,939,259, "High Throughput Nucleic Acid Sequencing by Expansion"). Generally, 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 at high signal-to-noise reporters separated by approximately 10 nm and is designed for high signal-to-noise, high discrimination response. These differences provide a significant performance improvement in the sequence read efficiency and accuracy of Xpandomers compared to native DNA. A generalized overview of the SBX process is shown in FIGS. 1A, 1B, 1C, and 1D.

[0237] XNTP is an extendable 5’-triphosphate modified nucleotide substrate that is compatible with template-dependent enzymatic polymerization. A highly simplified XNTP is shown in FIG. 1A, which highlights the unique features of these nucleotide analogs. XNTP100 has two distinct functional regions. Namely, a selectively cleavable phosphoramidate bond 110 that links the 5’ α-phosphate 115 to the nucleobase 105, and a linker chain 120 that is attached within the nucleoside triphosphoramidate at a position that allows for controlled extension by intranucleotide cleavage of the phosphoramidate bond. The linker chain of XNTP consists of linker arm portions 125A and 125B separated by a selectively cleavable phosphoramidate bond. Each linker is attached to one end of a reporter 130 via a linking group (LG) as disclosed in U.S. Patent No. 8,324,360 to Kokoris et al., which is hereby incorporated by reference in its entirety. XNTP100 is shown in a “constrained configuration” characteristic of the XNTP substrate and daughter strand after polymerization. The constrained configuration of the polymerized XNTP is a precursor to the extended configuration as seen in the Xpandomer product. The transition from the constrained configuration to the extended configuration occurs upon cleavage of the P--N bond of the phosphoramidate within the primary backbone of the daughter strand.

[0238] The synthesis of Xpandomer is summarized in FIGS. 1B and 1C. During assembly, monomer XNTP substrates 145 (XATP, XCTP, XGTP, and XTTP) are polymerized on the extendable end of a nascent daughter strand 150 by a process of template-directed polymerization using a single-stranded template 140 as a guide. Generally, this process starts from a primer and proceeds in the 5’ to 3’ direction. Generally, DNA polymerase or other polymerases are used to form the daughter strand, and the conditions are selected such that a complementary copy of the template strand is obtained. After the daughter strand is synthesized, the coupled linker chains include a constrained Xpandomer that further includes the daughter strand . The linker chains in the daughter strand have a “constrained configuration” of the XNTP substrate. The constrained configuration of the linker chains is a precursor to the extended configuration as seen in the Xpandomer product.

[0239] As shown in Figure 1C, the transition from the constrained configuration 160 to the extended configuration 165 is due to the cleavage of a selectively cleavable phosphoramidate bond (shown by the non-shaded ellipse for simplicity) within the primary backbone of the daughter strand. In this embodiment, the bridging strands include one or more reporter constructs or reporter constructs 130A, 130C, 130G, or 130T specific to the nucleobases to which they are linked, thereby encoding the sequence information of the template. In this way, the bridging strands provide a means of extending the length of the Xpandomer and reducing the linear density of the sequence information of the parental strand.

[0240] Figure 1D shows the Xpandomer 165 moving from the cis reservoir 175 through the nanopore 180 to the trans reservoir 185. As it passes through the nanopore, each of the reporters of the linearized Xpandomer (labeled "G", "C", and "T" in this figure) generates a distinct and reproducible electronic signal (shown by the superimposed trace 190) specific to the nucleobase to which it is linked.

[0241] Figure 2 shows the generalized structure of XNTP in more detail. The XNTP 200 is composed of a nucleobase triphosphoramidate 210 separated by selectively cleavable phosphoramidate bonds 230 into linker arm portions 220A and 220B. The bridging strands are linked to the nucleoside triphosphoramidate by linking groups 250A and 250B, and the first bridging strand terminus is linked to a heterocycle 260 (represented here by cytosine, although the heterocycle may be any one of the four standard nucleobases, A, C, G, or T). The second bridging strand terminus is linked to the alpha phosphate 270 of the nucleobase backbone. Those skilled in the art will understand that many suitable coupling chemistries known in the art can be used to form the final XNTP substrate product, for example, that the bridging strand linkage can be achieved by a triazole bond.

[0242] In this embodiment, the linker chain 275 is composed 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 plays a unique function during the translocation of Xpandomer through the nanopore and the generation of a unique and reproducible electronic signal. The linker chain 275 is designed for translocation control by hybridization (TCH). As shown, the TCE can hybridize to a complementary oligomer (CO) and provides a region of hybridization located adjacent to the reporter code. Different reporter codes are sized to block the ion current passing through the nanopore at different measurable levels. Using phosphoramidite chemistry typically used in oligonucleotide synthesis, specific reporter codes can be efficiently synthesized. The reporter can be designed by selecting a specific phosphoramidite sequence from a commercially available library. Such a library includes, but is not limited to, polyethylene glycol having a length of 1 to 12 or more ethylene glycol units, aliphatics having a length of 1 to 12 or more carbon units, deoxyadenosine (A), deoxycytosine (C), deoxyguanosine (G), deoxythymine (T), and abasic (Q). Since the duplexed TCE associated with the reporter code also contributes to ion current blocking, the combination of the reporter code and the TCE can be referred to as a "reporter". Following the reporter code, in one embodiment, there is an enhancer containing a spermine polymer.

[0243] Figure 3 shows the cleaved Xpandom in the process of moving the α-hemolysin nanopore An embodiment of er is shown. This biological nanopore is embedded in a lipid bilayer membrane that separates and electrically isolates two reservoirs of electrolyte. A typical electrolyte has a 1 molar concentration of KCl buffered to pH 7.0. When a small voltage, typically 100 mV, is applied across the bilayer, the nanopore restricts the flow of ionic current and is the primary resistance in the circuit. The Xpandomer reporter is designed to give a specific ionic current blockade level, and the sequence information can be read by measuring the sequence of the ionic current levels as the reporter translocates through the nanopore.

[0244] The α-hemolysin nanopore is typically oriented such that translocation occurs by entering on the vestibule side and exiting from the base side. As shown in Figure 3, the nanopore is oriented to first capture the Xpandomer from the base side. This orientation is advantageous because it results in fewer blockade artifacts when using the TCH method and entering the vestibule first. Unless otherwise indicated, the base side will be the assumed translocation direction first. As the Xpandomer translocates, the reporter enters the base until its duplexed TCE stops at the base entrance. Since the duplex has a diameter of about 2.4 nm while the base entrance is about 2.2 nm, the reporter is held in the base until the complementary strand 395 of the duplex dissociates (is released), after which translocation proceeds to the next reporter. Since the Xpandomer is still translocating and diffusing out of the pore, it is highly undesirable for the free complementary strand to enter the nanopore.

[0245] In one embodiment, each member of the reporter code (following the double strand) is formed by an ordered selection of phosphoramidites that can be selected from many commercially available libraries. Each constituent phosphoramidite contributes to the net ionic resistance according to its position within the nanopore (which is located after double-strand arrest), its displacement, its charge, its interaction with the nanopore, its chemical and thermal environment, and other factors. The charge on each phosphoramidite is, in part, due to phosphate ions that have a nominal charge of -1 but are effectively reduced by counterion shielding. The force pulling on the double strand is due to these effective charges along the reporter that are acted on by the local electric field. Since each reporter can have a different charge distribution, it can exert a different force on the double strand for a given applied voltage. The force transmitted along the reporter backbone also helps to stretch the reporter to give a repeatable blocking response.

[0246] The sequencing-by-expansion (SBX) methodology developed by the inventors provides a significant performance improvement in the sequence read efficiency and accuracy of Xpandomer compared to native DNA. However, the first transcription of the sequence of a natural DNA template into a measurable Xpandomer depends on the ability of a DNA polymerase that utilizes XNTPs as substrates (the generalized structure of XNTPs is discussed herein with reference to FIGS. 1A and 2). The inventors have found that most DNA polymerases do not efficiently polymerize XNTPs. However, by including appropriate additives such as the PEMs of the present disclosure, the efficiency and accuracy of XNTP polymerization into Xpandomer are improved. Thus, the PEMs disclosed herein can be used in the context of the SBX methodology to enhance DNA polymerase primer extension reactions using XNTPs as substrates.

[0247] Representative primer extension reactions may include the following reagents: 2 pmol of primer, 2.2 pmol of 45-mer oligonucleotide template, 50 pmol of each XNTP (XATP, XCTP, XGTP, and XTTP), 50 mM Tris HCl (pH 6.79), 200 mM NaCl, 20% PEG, 5% NMS, 0.5 nmol of polyphosphate 60.19, 0.3 mM MnCl2, and 0.6 μg of purified recombinant DNA polymerase protein. PEM is typically added to this mixture at concentrations ranging from micro- to millimolar. The reaction may also include additional additives such as single-stranded binding protein (SSB), urea, and NMS. The reaction is carried out at 23°C for 1 hour. The reaction product (i.e., the constrained Xpandomer) is treated to cleave the phosphoramidate bond, thereby generating a linearized Xpandomer. The reaction products are analyzed using gel electrophoresis on a 4-12% acrylamide gel to resolve and visualize Xpandomer products of different lengths.

[0248] Accordingly, in one embodiment, the present disclosure provides an aqueous (water-containing) composition comprising PEM and a buffer, particularly a buffer suitable for performing a DNA polymerization reaction, and Tris HCl is an exemplary buffer of this type. In one embodiment, the present disclosure provides a composition comprising PEM and a DNA polymerase protein. In one embodiment, the present disclosure provides a composition comprising PEM and a polynucleotide, such as an oligonucleotide of 20-90 mer, 20-60 mer, 30-90 mer, or 30-60 mer. In one embodiment, the present disclosure provides a composition comprising each of these components, i.e., an aqueous composition comprising PEM, a buffer, a DNA polymerase protein, and a polynucleotide.

[0249] To examine the accuracy of enhanced XNTP polymerization, primer extension products can be sequenced using the SBX protocol. Briefly, the constrained Xpandomer products of XNTP polymerization are cleaved to generate linearized Xpandomer. This is achieved by first quenching the extension reaction with a solution containing 100 mM EDTA, 2 mM THPTA, and 2% Tween-20. The sample is then amine-modified with a solution of 1 M NaHCO3 and 1 M succinic anhydride in DMF. Cleavage of the phosphoramidate bond is carried out with 37% HCl, and the linearized Xpandomer is purified with a QIAquick column (QIAGEN, Inc.).

[0250] For sequencing, protein nanopores are prepared by inserting α-hemolysin into a DPhPE / hexadecane bilayer member in buffer B1 containing 2 M NH4Cl and 100 mM HEPES (pH 7.4). The cis well is perfused with buffer B2 containing 0.4 M NH4Cl, 0.6 M GuCl, and 100 mM HEPES, pH 7.4. The Xpandomer sample is heated to 70 °C for 2 minutes, cooled completely, and then 2 μL of the sample is added to the cis well. A voltage pulse of 90 mV / 390 mV / 10 μs is then applied, and data are acquired using Labview acquisition software.

[0251] Sequence data are analyzed by histogram display of a population of sequence reads from a single SBX reaction. The analysis software aligns each sequence read to the template sequence and trims the range of the sequence at the end of the reads that do not align to the correct template sequence.

[0252] In one embodiment, the present disclosure provides a method for enhancing the accuracy of XNTP polymerization, the method comprising adding the PEM disclosed herein to the DNA polymerization reaction described above.

[0253] In one embodiment, the present disclosure provides a kit that can be used in the methods described herein. The kit includes 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 that can function as a primer, for example, and / or a polynucleotide that can function as a template, for example.

[0254] For example, in one embodiment, 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 compounds of the present disclosure can be used to increase the number and accuracy of nucleotide analogs incorporated into daughter strands during a template-dependent polymerization reaction as compared to the same polymerization reaction in the absence of at least one compound of the present disclosure. Optionally, the mixture of nucleotide analogs includes nucleoside triphosphoramidates, each of the nucleoside triphosphoramidates includes a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, and a polymeric linker moiety, wherein a first end of the polymeric linker moiety is attached to the nucleobase and a second end of the polymeric linker moiety is attached to the α-phosphate of the nucleoside triphosphoramidate to provide extension of the nucleotide analog by cleavage of the phosphoramidate bond. Optionally, the mixture of nucleotide analogs includes nucleotide analogs comprising a detectable label, and the detectable label is an optically detectable label selected from the group consisting of a luminescent, chemiluminescent, fluorescent, phosphorescent, chromophore, or chromogenic label. Optionally, the kit includes an aqueous buffer including TrisOAc, NH4OAc, PEG, a water-miscible organic solvent (such as dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), acetone, etc.), polyphosphoric acid 60, NMS, and MnCl2. Optionally, the kit includes a single-stranded binding protein. Optionally, the kit includes urea. Optionally, the kit includes two or more of these components, for example, three or four, or all of the specified components.

[0255] The compounds can be prepared by methods known to those skilled in the art, such methods being identifiable by various reference books and databases. Suitable reference books and papers that detail the synthesis of reactants useful in the preparation of the compounds of the present disclosure, or provide references to papers describing the preparation, include, for example, "Synthetic Organic Chemistry" John Wiley & Sons, Inc., New York; S.R. Sandler et al., "Organic Functional Group Preparations" 2nd Ed., Academic Press, New York, 1983; H.O. House, "Modem Synthetic Reactions" 2nd Ed., W.A. Benjamin, Inc. Menlo Park, Calif 1972; T.L. Gilchrist, "Heterocyclic Chemistry" 2nd Ed., John Wiley & Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure" 4th Ed., Wiley-Interscience, New York, 1992.References to papers that detail the synthesis of reactants useful in the preparation of the compounds of the present disclosure, or describe the preparation, and further suitable reference books and papers include, for example, the following: Fuhrhop, J. and Penzlin G., "Organic Synthesis: Concepts, Methods, Starting Materials", Second, Revised and Enlarged Edition (1994), John Wiley & Sons, ISBN: 3-527-29074-5; Hoffman, R.V., "Organic Chemistry, An Intermediate Text" (1996), Oxford University Press, ISBN 0-19-509618-5; Larock, R.C., "Comprehensive Organic Transformations: A Guide to Functional Group Preparations", 2nd Edition (1999), Wiley-VCH, ISBN: 0-471-19031-4; March, J., "Advanced Organic Chemistry: Reactions, Mechanisms, and Structure", 4th Edition (1992), John Wiley & Sons, ISBN: 0-471-60180-2; Otera, J. (editor), "Modern Carbonyl Chemistry" (2000), Wiley-VCH, ISBN: 3-527-29871-1; Patai, S., "Patai’s 1992 Guide to the Chemistry of Functional Groups" (1992), Interscience, ISBN: 0-471-93022-9; Quin, L.D et al., "A Guide to Organophosphorus Chemistry" (2000), Wiley-Interscience, ISBN: 0-471-31824-8; Solomons, T.W.G., "Organic Chemistry", 7th Edition (200。 0) John Wiley & Sons, ISBN: 0-471-19095-0; Stowell, J.C., 「Intermediate Organic Chemistry」 2nd Edition (1993) Wiley-Interscience, ISBN: 0-471-57456-2; 「Industrial Organic Chemicals: Starting Materials and Intermediates: An Ullmann’s Encyclopedia」 (1999) John Wiley & Sons, ISBN: 3-527-29645-X (8 volumes); 「Organic Reactions」 (1942 - 2000) John Wiley & Sons (more than 55 volumes); and 「Chemistry of Functional Groups」 John Wiley & Sons (73 volumes).

Examples

[0256] The compounds shown in Table 1 were prepared according to the general examples disclosed in this specification.

[0257] Materials and Methods 4-Azidosalicylic acid and 2,6-dibromo-4-pyridinecarboxylic acid were obtained from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada). 4-Azido-2-(trifluoromethyl)benzoic acid, 3-amino-5-hydroxybenzoic acid, and 4-aminoisophthalic acid were obtained from Matrix Scientific (Columbia, South Carolina, USA). 3-Amino-6-(trifluoromethyl)benzoic acid hydrochloride, 1-(4-aminophenyl)-2,2,2-trifluoroethan-1-one, methylglycylglycinate hydrochloride, 3,3,3-trifluoropropan-1-amine, and diethyl 3-aminopropan-1-ylphosphonate were obtained from Enamine LLC (Monmouth Junction, New Jersey, USA). Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU), 1,3-diethynylbenzene, 2,6-diethynylpyridine, 3,5-diethynylpyridine, 3,6-diethynylcarbazole, 4-azidobenzoic acid, cyclopropylamine, 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-(aminophenyl)phosphonic acid, 2,6-dichloro-9H-purine, dimethyl aspartate hydrochloride, 4-amino-2-(trifluoromethyl)benzonitrile, 3,5-diaminobenzoic acid, and 2,5-dibromofuran were obtained from TCI America (Portland, Oregon, USA). 4-Methoxy-2,6-dibromopyridine, 4-nitro-2,6-dibromopyridine, 2,6-dibromo-4-pyridinecarboxylic acid, and methylglycinate hydrochloride were obtained from Chem-Impex International, Inc. (Wood Dale, Illinois).Tetrakis(triphenylphosphine)palladium(0), ethynyltrimethylsilane, DMSO, DMF, MeOH, EtOAc, sodium ascorbate, copper sulfate, diisopropylamine, EDTA, morpholine, diethylamine, ammonium hydroxide, ethylamine, ethanol, azetidine, n-ethyl-n-ethylamine, methyl 2,6-dichloropyridine-4-carboxylate, ethyl 2,6-dibromopyridine-4-carboxylate, 4-methyl-2,6-dichloropyridine, 2-chloro-4-cyanopyridine, 4-amino-2-(trifluoromethyl)benzoic acid, 2-bromo-4-cyanopyridine, methyl azidoacetate, 4-azidoaniline hydrochloride, 4-methoxyphenyl azide 4-amino-2-fluorobenzoic acid, n-butylamine, 3-amino-5-(trifluoromethyl)benzoic acid, 3-(4-aminophenyl)propionic acid, 4-(4-aminophenyl)-butyric acid, 4-amino-2-methoxybenzoic acid, 2-amino-3-(trifluoromethyl)benzoic acid, aminoethanol, 1,4-diaminobutane and 2,5-dibromothiophene were obtained from Sigma - Al. drich Corp. (St. Louis, Missouri, USA). TLC and flash chromatography solvents were obtained from Sigma - Aldrich or Thermo Fisher Scientific Inc. (Waltham, Massachusetts, USA).

[0258] Flash chromatography was performed on a Reveleris Prep Purification System manufactured by Buchi Corp. (New Castle, Delaware). This system was equipped with a hand-packed column (2.3 cm in diameter × 8 cm in height) filled with C18 spherical silica gel (catalog number 76646-01) from Sorbent Technologies, Inc. (Norcross, Georgia) and sealed with a polypropylene frit. 1-1.5 mL of the sample was directly loaded onto the head of the column. The mobile phases were water (A) and acetonitrile (B). A gradient of 0-2% B was run for 2 minutes, followed by a gradient of 2-100% B at a flow rate of 28 mL / min for 20 minutes. UV was monitored at 220 nm, 260 nm, and 280 nm. Fractions were collected at a UV threshold of 0.1 AU. Thin layer chromatography was performed using TLC silica gel 60 F254 (catalog number 1.05534.0001) backed with aluminum from EMD Millipore Corp. (Billerica, Massachusetts, USA). ESI Mass Spec was performed by Numega Resonance Lab (San Diego, California, USA) using a Perkin Elmer PE-SCIEX API-150 mass spectrometer in positive and negative modes.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Chemical formula

[0259] Compound 1 was prepared by mixing 4-azidosalicylic acid B (1.79 mg, 10 μmol) and 2,6-diethynylpyridine A (0.67 mg, 5 μmol) in DMSO (150 μL). This solution was mixed with a solution of TBTA (5.1 mg, 0.96 μmol) and sodium ascorbate (6.4 mg, 32 μmol) in DMSO (95 μL). The click reaction was initiated by adding 20 mM copper sulfate (5 μL) with stirring. The progress of the reaction was analyzed by TLC (94:5:1 ethyl acetate:methanol:acetic acid), and the reaction was completed in 5 minutes based on the consumption of azide and alkyne. The volume of the reaction mixture was adjusted to 1 mL using DMSO and 0.5 M EDTA (100 μL). The solid was 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 in 50 - 75% yield upon rotary evaporation. 11H NMR (300 MHz, DMSO-d6) δ ppm 3.29 (2H, br. S., (O(18)H and O(33)H)), 7.17 - 7.29 (4H, m, (C(7)H, C(11)H, C(28)H, C(32)H)) 7.85 (2H, d, J = 8.11 (C(10)H and C(31)H)), 8.06 (3H, s, (C(15)H, C(16)H, C(17)H)) 9.36 (2H, s, (C(5)H and C(25)H)). Example 2 Synthesis of 4,4'-(pyridine-3,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0260] According to the method of Example 1, compound 2 was prepared using 4-azidosalicylic acid B and 3,5-diethynylpyridine C. 1 1H NMR (300 MHz, DMSO-d6) δ ppm 3.29 (2H, br. S., (O(18)H and O(33)H)) 7.17 - 7.29 (4H, m, (C(7)H, C(11)H, C(28)H, C(31)H)) 7.85 (2H, d, J = 8.11 Hz, (C(10)H and C(31)H)) 8.06 (3H, s,, (C(13)H, C(15)H, C(17)H)) 9.36 (2H, s, (C(5)H and C(25)H)). Example 3 Synthesis of 4,4'-(1,3-phenylenebis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0261] According to the method of Example 1, compound 3 was prepared using 4-azidosalicylic acid B and 1,3-diethynylbenzene D. 1 1H NMR (300 MHz, DMSO-d6) δ ppm 3.29 (2H, br. S., (O(18)H and O(33)H)) 7.16 - 7. 27 (4H, m, (C(7)H, C(11)H, C(28)H, C(32)H)) 7.57 - 7.67 (1H, m, (C(16)H)) 7.80 - 7.88 (2H, m, (C(10)H and C(31)H)) 7.94 (2H, d, J = 7.63 Hz, (C(15)H and C(17)H) 8.57 (1H, s, (C(13)H)) 9.35 (2H, s, (C(5)H and C(25)H). Example 4 Synthesis of 4,4'-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0262] According to the method of Example 1, compound 4 was prepared using 4-azidosalicylic acid B and 3,6-diethynylcarbazole E. 1 H NMR (300 MHz, DMSO-d6) δ ppm 3.29 (2H, br.S., (O(25)H and O(40)H)) 7.16 - 7.27 (4H, m, (C(7)H, C(11)H, C(35)H, C(39)H)) 7.61 (2H, d, J = 8.34 Hz, (C(10)H and C(38)H)) 7.84 (2H, d, J = 7.87 Hz, (C(22)H and C(24)H)) 8.02 (2H, d, J = 8.34 Hz, (C(21)H and C(23)H)) 8.79 (2H, s, (C(13)H and C(19)H)) 9.28 (2H, s, (C(5)H and C(32)H)) 11.53 (1H, s, (N(16)H). Example 5 Synthesis of 4,4'-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dianiline

Chemical Structure

[0263] According to the method of Example 1, compound 5 was prepared using 4-azidoaniline hydrochloride F and 3,6-diethynylcarbazole E. Example 6 Synthesis of 4,4'-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))dibenzoic acid

Chem.

[0264] Compound 6 was prepared using 4-azidobenzoic acid G and 3,6-diethynylcarbazole E according to the method of Example 1. Example 7 Synthesis of 3,6-bis(1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)-9H-carbazole

Chem.

[0265] Compound 7 was prepared using 4-azidoanisole H and 3,6-diethynylcarbazole E according to the method of Example 1. Example 8 Synthesis of dimethyl 2,2'-((9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diacetate

Chem.

[0266] Compound 8 was prepared using methyl azidoacetate I and 3,6-diethynylcarbazole E according to the method of Example 1. Example 9 Synthesis of 4,4'-((4-methoxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chem.

[0267] The preparation of Compound 9 was initiated by synthesizing 4-methoxy-2,6-diethynylpyridine J from 4-methoxy-2,6-dibromopyridine and ethynyltrimethylsilane using the conditions described by Sonogashira (Organomet. Chem., 653:46-49 (2002). doi:10.1016 / s0022-328x(02)01158-0). The synthesis of Compound 9 was completed by clicking 4-azidosalicylic acid B and Compound J according to the method of Example 1. Example 10 Synthesis of 4,4'-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0268] The preparation of Compound 10 was initiated by synthesizing 4-cyano-2,6-diethynylpyridine K from 4-cyano-2,6-dibromopyridine and ethynyltrimethylsilane using the conditions described by Sonogashira (Organomet. Chem., 653:46-49 (2002). doi:10.1016 / s0022-328x(02)01158-0). The synthesis of Compound 10 was completed by clicking 4-azidosalicylic acid B and Compound K according to the method of Example 1. Example 11 Synthesis of 4,4'-((4-nitropyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0269] The preparation of Compound 11 was initiated by synthesizing 4-nitro-2,6-diethynylpyridine L from 4-nitro-2,6-dibromopyridine and ethynyltrimethylsilane using the conditions described by Sonogashira (Organomet. Chem., 653:46 - 49 (2002). doi:10.1016 / s0022-328x(02)01158-0). The synthesis of Compound 11 was completed by clicking 4-azidosalicylic acid B and Compound L according to the method of Example 1. By doing so, the synthesis of Compound 11 was completed. Example 12 Synthesis of 5,5'-((4-cyanopyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0270] Compound 12 was prepared using 5-azidosalicylic acid M and 4-cyano-2,6-diethynylpyridine K according to the method of Example 10. Example 13 Synthesis of 4,4'-((4-methylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0271] Compound 13 was initiated by synthesizing 4-methyl-2,6-diethynylpyridine N from 4-methyl-2,6-dichloropyridine and ethynyltrimethylsilane using the conditions described by Sonogashira (Organomet. Chem., 653:46 - 49 (2002). doi:10.1016 / s0022-328x(02)01158-0). The 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. By doing so, the synthesis of Compound 11 was completed. Example 14 Synthesis of 4,4'-((4-(Ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chem.

[0272] Compound 14 was initiated by synthesizing ethyl 2,6-diethynylpyridine-4-carboxylate O from ethyl 2,6-dibromopyridine-4-carboxylate and ethynyltrimethylsilane using the conditions described by Sonogashira (Organomet. Chem., 653:46 - 49 (2002). doi:10.1016 / s0022-328x(02)01158-0). The synthesis of Compound 14 was completed by clicking 4-azidosalicylic acid B and ethyl 2,6-diethynylpyridine-4-carboxylate O according to the method of Example 1. Example 15 Synthesis of 5,5'-((4-(Ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chem.

[0273] Compound 15 was prepared using 5-azidosalicylic acid M and ethyl 2,6-diethynylpyridine-4-carboxylate O clicked according to the method of Example 14. Example 16 Synthesis of 4,4'-((4-(Methoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chem.

[0274] Compound 16 was initiated by synthesizing methyl 2,6 - diethynylpyridine - 4 - carboxylate P from methyl 2,6 - dichloropyridine - 4 - carboxylate and ethynyltrimethylsilane using the conditions described by Sonogashira (Organomet. Chem., 653:46 - 49 (2002). doi:10.1016 / s0022 - 328x(02)01158 - 0). The synthesis of compound 16 was completed by clicking 4 - azidosalicylic acid B and ethyl 2,6 - diethynylpyridine - 4 - carboxylate P according to the method of Example 1. Example 17 Synthesis of 4,4’ - ((4 - (ethylcarbamoyl)pyridine - 2,6 - diyl)bis(1H - 1,2,3 - triazole - 4,1 - diyl))bis(2 - hydroxybenzoic acid)

Chemical formula

[0275] The synthesis of compound 17 was initiated by mixing 2,6 - dibromo - 4 - pyridinecarboxylic acid (0.2 g, 0.71 mmol), DIPEA (0.18 g, 1.42 mmol) and HATU (0.27 g, 0.71 mmol) in DMF (900 μl). Ethylamine (0.154 ml, 1.78 mmol) was added immediately and mixed for 1 hour. The reaction was completed by TLC and purified by flash chromatography on silica gel using an ethyl acetate / hexane gradient. N - Ethyl - 2,6 - dibromo - 4 - carboxamide was isolated as a yellow solid in 69% yield. Using the Sonogashira method described in Example 16, N - ethyl - 2,6 - diethynyl - 4 - carboxamide Q was prepared using ethyltrimethylsilane. The synthesis of compound 17 was completed by clicking 4 - azidosalicylic acid B and N - ethyl - 2,6 - diethynyl - 4 - carboxamide Q according to the method of Example 1. Example 18 Synthesis of 4,4’ - ((4 - (methylcarbamoyl)pyridine - 2,6 - diyl)bis(1H - 1,2,3 - triazole - 4,1 - diyl))bis(2 - hydroxybenzoic acid)

Chem.

[0276] Compound 18 was prepared using methylamine, and an amide was formed according to the method of Example 17. Example 19 Synthesis of 4,4’-((4-carbamoylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chem.

[0277] Compound 19 was prepared using ammonia, and an amide was formed according to the method of Example 17. Example 20 Synthesis of 4,4’-(pyrazine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chem.

[0278] Compound 20 was initiated by the synthesis of 2,6-diethynylpyrazine T from 2,6-dichloropyrazine and ethynyltrimethylsilane using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12):e3824(2017)). The synthesis of Compound 20 was completed by clicking 4-azidosalicylic acid B and 2,6-diethynylpyrazine T according to the method of Example 1. Example 21 Synthesis of 4,4’-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chem.

[0279] The synthesis of compound 73 was initiated from the diazotization of 4-amino-2-(trifluoromethyl)benzoic acid with sodium nitrite and sulfuric acid, followed by nucleophilic substitution with azide (Org. Synth. 1942, 22, 96) to form 4-azido-2-(trifluoromethyl)benzoic acid (U), which was purified by flash chromatography. The synthesis of compound 73 was completed by clicking 4-azido-2-(trifluoromethyl)benzoic acid U and N-ethyl-2,6-diethynyl-4-carboxamide Q according to Example 1. Example 22 Synthesis of 7,7'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxy-1,8-naphthyridine-4-carboxylic acid)

Chemical formula

[0280] The synthesis of compound 74 was initiated by diazotizing 7-amino-2-hydroxy-1,8-naphthyridine-4-carboxylic acid with sodium nitrite and sulfuric acid, followed by nucleophilic substitution with azide (Org. Synth. 1942, 22, 96) to form 7-azido-2-hydroxy-1,8-naphthyridine-4-carboxylic acid (V), which was purified by flash chromatography. The synthesis of compound 74 was completed by clicking 7-azido-2-hydroxy-1,8-naphthyridine-4-carboxylic acid V and N-ethyl-2,6-diethynyl-4-carboxamide Q according to Example 1. Example 23 Synthesis of 4-(4-(3-(1-(4-carboxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid

Chemical formula

[0281] The synthesis of compound 28 was completed in two steps. First, according to the method of Example 3, 2,6-diethynylbenzene D was clicked with half the amount of 4-azidosalicylic acid B in 140 iridium 140 etoric to prepare 4-(4-(3-ethynylphenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid. The second step was to click 4-azidobenzoic acid G according to the method of Example 1 to obtain 4-(4-(3-(1-(4-carboxyphenyl)-1H-1,2,3-triazol-4-yl)phenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid 28. Example 24 Synthesis of 4-(4-(4-cyanopyridin-2-yl)-1H-1,2,3-triazol-1-yl)-2-hydroxybenzoic acid

Chemical Structure

[0282] The preparation of compound 43 was started by synthesizing 4-cyano-2-ethynylpyridine W from 4-cyano-2-chloropyridine and ethynyltrimethylsilane using the conditions described by Sonogashira (Organomet. Chem., 653:46 - 49 (2002). doi:10.1016 / s0022-328x(02)01158-0). The synthesis of compound 43 was completed by clicking 4-azidosalicylic acid B and compound W according to the method of Example 1. Example 25 Synthesis of 4,4'-((pyridine-2,6-diyl)bis(5-iodo-1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0283] The preparation of compound 31 started with the synthesis of 2,6-bis(iodoethynyl)pyridine X from 2,6-diethynylpyridine A according to the method of Tepper et al. (Org. Lett., 2015, 17(23), pp 5740-574), including treatment with n-iodosuccinimide and silver nitrate and isolation by flash chromatography. The synthesis of compound 31 was completed by clicking 4-azidosalicylic acid B and compound X according to the method of Example 1. Example 26 Synthesis of 4,4'-((3,5-dimethylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical formula

[0284] Compound 30 was started by the synthesis of 2,6-diethynyl-3,5-dimethylpyridine Y from 2,6-dibromo-3,5-dimethylpyridine and ethynyltrimethylsilane using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12):e3824(2017)). The 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. Example 27 Synthesis of 4,4'-((9-acetyl-9H-carbazole-3,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical formula

[0285] Compound 33 was initiated by synthesizing 9-acetyl-3,6-diethynylcarbazole Z from 9-acetyl-3,6-diiodocarbazole and ethynyltrimethylsilane using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12):e3824(2017)). The synthesis of compound 33 was completed by clicking 4-azidosalicylic acid B and 9-acetyl-3,6-diethynylcarbazole Z according to the method of Example 1. Example 28 Synthesis of 4,4'-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(N,2-dihydroxybenzamide)

Chemical Structure

[0286] The synthesis of compound 34 was initiated by mixing N-hydroxysuccinimide 4-azidosalicylate (40 mg, 0.145 mmol) in DMF (72 μl), to which water (72 μl) containing hydroxylamine hydrochloride (30 mg, 0.43 mmol) was added and mixed overnight. The product was detected by TLC and the reactants were purified by flash chromatography on silica gel using a gradient of methylene chloride and methylene chloride 144 iridium-MeOH. 4-Azido-N,2-dihydroxybenzamide AA was isolated in 57% yield. The synthesis of compound 34 was completed by clicking 4-azido-N,2-dihydroxybenzamide AA and 2,6-diethynylpyridine A according to the method of Example 1. Example 29 Synthesis of 5-(4-(6-(4-(3-carboxy-4-hydroxy-5-methylphenyl)-1H-1,2,3-triazol-1-yl)-4-(methoxycarbonyl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-3-methylbenzoic acid

Chemical Structure

[0287] The synthesis of compound 44 was initiated from the diazotization of 5-amino-2-hydroxy-3-methylbenzoic acid with sodium nitrite and sulfuric acid, followed by nucleophilic substitution with azide (Org. Synth. 1942, 22, 96) to form 5-azido-2-hydroxy-3-methylbenzoic acid (BB), which was purified by flash chromatography. According to Example 1, the synthesis of compound 44 was completed by clicking 5-azido-2-hydroxy-3-methylbenzoic acid BB with 2,6-diethynylpyridine-4-carboxylate P. Example 30 Synthesis of 4,4'-((4-(but-3-yn-1-ylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0288] The synthesis of compound 47 was initiated by mixing 2,6-dibromo-4-pyridinecarboxylic acid (0.2 g, 0.7 1 mmol), DIPEA (0.18 g, 1.42 mmol), and HATU (0.27 g, 0.71 mmol) in DMF (900 μl). Butynylamine (0.154 ml, 1.78 mmol) was added immediately and the mixture was stirred for 1 hour. The reaction was completed by TLC and purified by flash chromatography on silica gel using an ethyl acetate / hexane gradient. N-(but-3-yn-1-yl)-2,6-dibromoisonicotinamide was isolated as a solid. Using the Sonogashira method described in Example 16, N-(but-3-yn-1-yl)-2,6-diethynylisonicotinamide CC was prepared using ethyltrimethylsilane. According to the method of Example 1, the synthesis of compound 47 was completed by clicking 4-azidosalicylic acid B and N-(but-3-yn-1-yl)-2,6-diethynylisonicotinamide CC. Example 31 Synthesis of 4,4'-(naphthalene-2,7-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid) [Chemical formula]

[0289] The synthesis of compound 63 was initiated from the synthesis of 2,7-diethynylnaphthalene DD from 2,7-dibromonaphthalene and ethynyltrimethylsilane using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12): e3824 (2017)). The synthesis of compound 63 was completed by clicking 4-azidosalicylic acid B and 2,7-diethynylnaphthalene DD according to the method of Example 1. Example 32 Synthesis of 4,4'-(naphthalene-2,3-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid) [Chemical formula]

[0290] The synthesis of compound 64 was initiated from the 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)). The synthesis of compound 64 was completed by clicking 4-azidosalicylic acid B and 2,3-diethynylnaphthalene EE according to the method of Example 1. Example 33 Synthesis of 4,4',4'',4''' - ((((butane-1,4-diylbis(azanediyl))bis(carbonyl))bis(pyridine-4,2,6-triyl))tetrakis(1H-1,2,3-triazole-4,1-diyl))tetrakis(2-hydroxybenzoic acid) [Chemical formula]

[0291] Compound 71 was initiated by the synthesis of 2,6 - diethynyl - 4 - pyridinecarboxylic acid from 2,6 - dibromo - 4 - pyridinecarboxylic acid and ethynyltrimethylsilane using the Sonogashira method described by Bhowmick, S. et al. (App. Organomet. Chem. 31(12):e3824(2017)). 2,6 - Diethynyl - 4 - pyridinecarboxylic acid was treated with HATU, DIPEA and 1,4 - diaminobutane to obtain N,N’-(butane - 1,4 - diyl)bis(2,6 - diethynylisonicotinamide) (FF) after isolation by flash chromatography. The synthesis of Compound 71 was completed by clicking 4 - azidosalicylic acid B and N,N’-(butane - 1,4 - diyl)bis(2,6 - diethynylisonicotinamide) (FF) according to the method of Example 1. Example 34 Synthesis of 4,4’-((4-(ethylcarbamoyl)pyridine - 2,6 - diyl)bis(1H - 1,2,3 - triazole - 4,1 - diyl))bis(3,5,6 - trichloropicolinic acid)

Chemical formula

[0292] The synthesis of Compound 72 was initiated by the diazotization of 4 - amino - 3,5,6 - trichloropyridine - 2 - carboxylic acid with sodium nitrite and sulfuric acid, followed by nucleophilic substitution with azide (Org. Synth. 1942, 22, 96) to form 4 - azido - 3,5,6 - trichloropyridine - 2 - carboxylic acid (GG), which was purified by flash chromatography. According to Example 1, the synthesis of Compound 72 was completed by clicking 4 - azido - 3,5,6 - trichloropyridine - 2 - carboxylic acid GG with N - ethyl - 2,6 - diethynyl - 4 - carboxamide Q. Example 35 Synthesis of 4,4’-((4-(methylcarbamoyl)pyridine - 2,6 - diyl)bis(1H - 1,2,3 - triazole - 4,1 - diyl))bis(2 - (trifluoromethyl)benzoic acid)

Chem.

[0293] N-Methyl-2,6-diethynylpyridine-4-carboxamide HH was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and methylamine. Compound 79 was prepared using the copper click method of Example 1 with diethynyl HH and azide U. Mass spectrometry (ESI negative mode) of Compound 79: C 27 H 16 Calculated value of F6N8O5 646.47; Measured value: 645 [M-H + . Example 36 Synthesis of 4,4'-((4-(Morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chem.

[0294] N-Morpholino-2,6-diethynylpyridine-4-carboxamide II was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and morpholine. Compound 80 was prepared using the copper click method of Example 1 with diethynyl II and azide U. Mass spectrometry (ESI negative mode) of Compound 80: Calculated value of C30H20F6N8O6 702.53; Measured value: 701.1 [M-H+] Example 37 Synthesis of 4,4'-((4-(Diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chem.

[0295] N,N-Diethylamino-2,6-diethynylpyridine-4-carboxamide JJ was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and N,N-diethylamine. Compound 81 was prepared using the copper click method of Example 1 with diethynyl JJ and azide U. Mass spectrometry (ESI negative mode) of Compound 81: C 30 H 22 Calculated value for F6N8O5 688.55; Measured value: 687 [M+H] + 。 Example 38 Synthesis of 4,4'-((4-carbamoylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chemical formula

[0296] 2,6-Diethynylpyridine-4-carboxamide KK was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and ammonia. Compound 82 was prepared using the copper click method of Example 1 with diethynyl KK and azide U. Mass spectrometry (ESI negative mode) of Compound 82: C 26 H 14 Calculated value for F6N8O5 632.44; Measured value: 631.1 [M+H] + 。 Example 39 Synthesis of 4,4'-((4-(ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chemical formula

[0297] Compound 83 was prepared using the copper click method of Example 1 with ethyl-2,6-diethynylpyridine-4-carboxylate O and azide U. Mass spectrometry (ESI negative mode) of Compound 83: C28 H 17 Calculated value of F6N7O6: 661.48; Measured value: 659.9 [M-H + . Example 40 Synthesis of 4,4'-((4-(azetidine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0298] N-azetidinyl-2,6-diethynylpyridine-4-carboxamide LL was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and azetidine. Compound 84 was prepared using the copper click method of Example 1 with diethynyl LL and azide B. Mass spectrometry (ESI negative mode) of Compound 84: C 27 H 20 Calculated value of N8O7: 568.51; Measured value: 567 [M-H + . Example 41 Synthesis of 4,4'-((4-(ethyl(methyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical Structure

[0299] N-methyl-N-ethyl-2,6-diethynylpyridine-4-carboxamide MM was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and N-methyl-N-ethylamine. Compound 85 was prepared using the copper click method of Example 1 with diethynyl MM and azide B. Mass spectrometry (ESI negative mode) of Compound 85: C 27 H 22 Calculated value of N8O7: 570.52; Measured value: 569.1 [M-H + . Example 42 Synthesis of N-Ethyl-2,6-bis(1-(4-(2,2,2-trifluoroacetyl)phenyl)-1H-1,2,3-triazol-4-yl)isonicotinamide

Chemical formula

[0300] 1-(4-Aminophenyl)-2,2,2-trifluoroethan-1-one was diazotized with sodium nitrite and sulfuric acid and subsequently substituted with azide (Org. Synth. 1942, 22, 96 DOI: 10.15227 / orgsyn.022.0096) to form 1-(4-azidophenyl)-2,2,2-trifluoroethan-1-one NN, which was purified by flash chromatography. Compound 86 was prepared using the copper click method of Example 1 with azide NN and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 86: C 28 H 18 Calculated value for C + H Example 43 Synthesis of 4,4'-(Pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chemical formula

[0301] Compound 87 was prepared using the copper click method of Example 1 with azide U and 2,6-diethynylpyridine A. Mass spectrometry (ESI negative mode) of Compound 87: C 25 H 13 Calculated value for C + H Example 44 Synthesis of 4,4'-((4-(Cyclopropylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid) [ka]

[0302] N-cyclopropylamido-2,6-diethynylpyridine-4-carboxamide OO was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid with cyclopropylamine. Compound 88 was made using the copper click method of Example 1 with diethinyl OO and azide U. Mass spectrometry of compound 88 (ESI negative mode): C 29 H 18 Calculated value of F6N8O5: 672.50; Measured value: 671.1 [MH + ]. Example 45 Synthesis of 4,4'-((4-(butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid) [ka]

[0303] N-Butylamide-2,6-diethynylpyridine-4-carboxamide PP was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid with 1-aminobutane. Compound 89 was made using the copper click method of Example 1 with diethinyl PP and azide U. Mass spectrometry of compound 89 (ESI negative mode): C 30 H 22 Calculated value of F6N8O5: 688.55; Measured value: 687.1 [MH + ]. Example 46 Synthesis of 5,5'-((4-(diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid) [ka]

[0304] The diazotization of 5-amino-2-(trifluoromethyl)benzoic acid was carried out according to Example 22 to prepare 5-azido-2-(trifluoromethyl)benzoic acid QQ. Compound 90 was prepared using the copper click method of Example 1 with azide QQ and diethynyl JJ. Mass spectrometry (ESI negative mode) of Compound 90: C 30 H 22 Calculated value of F6N8O5 688.55; Measured value: 687.1 [M-H + . Example 47 Synthesis of 5,5'-((4-(morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chemical formula

[0305] Compound 91 was prepared using the copper click method of Example 1 with azide QQ and diethynyl II. Mass spectrometry (ESI negative mode) of Compound 91: C 30 H 20 Calculated value of F6N8O6 702.53; Measured value: 701 [M-H + . Example 48 Synthesis of 4,4'-(pyridazine-3,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid)

Chemical formula

[0306] Using 3,6-dibromopyridazine, 3,6-diethynylpyridazine was prepared according to the Sonogashira method described in Example 9. Compound 92 was prepared using the copper click method of Example 1 with azide B and 3,6-diethynylpyridazine. Mass spectrometry (ESI negative mode) of Compound 92: C 22 H 14 Calculated value of N8O6 486.1; Measured value: 485 [M-H+ . Example 49 Synthesis of 5,5'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-(trifluoromethyl)benzoic acid) [Chem.]

[0307] The diazotization of 3-amino-5-(trifluoromethyl)benzoic acid was carried out according to Example 22 to prepare 3-azido-5-(trifluoromethyl)benzoic acid RR. Compound 93 was prepared using the copper click method of Example 1 with azide RR and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 93: C 28 H 18 Calculated value of C16H6F6N8O5: 660.49; Measured value: 659 [M-H + . Example 50 Synthesis of 4,4'-((4-carboxypyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid) [Chem.]

[0308] Compound 94 was prepared using the copper click method of Example 1 with azide U and 2,6-diethynylpyridine-4-carboxylic acid. Mass spectrometry (ESI negative mode) of Compound 94: C 26 H 13 Calculated value of C16H5F6N7O6: 633.42; Measured value: 632 [M-H + . Example 51 Synthesis of 3,3'-(((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(4,1-phenylene))dipropionic acid [Chem.]

[0309] The diazotization of 3-(4-aminophenyl)propanoic acid was carried out according to Example 22 to prepare 3-(4-azidophenyl)propanoic acid SS. Compound 95 was prepared using the copper click method of Example 1 with azide SS and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 95: C 30 H 28 Calculated value for N8O5 580.61; Measured value: 579 [M-H + . Example 52 Synthesis of 4,4'-(((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(4,1-phenylene))dibutyric acid

Chemical Structure

[0310] The diazotization of 4-(4-aminophenyl)butanoic acid was carried out according to Example 22 to prepare 4-(4-azidophenyl)butanoic acid TT. Compound 96 was prepared using the copper click method of Example 1 with azide TT and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 96: C 32 H 32 Calculated value for N8O5 608.66; Measured value: 607 [M-H + . Example 53 Synthesis of 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diphthalic acid

Chemical Structure

[0311] The diazotization of 4-aminophthalic acid was carried out according to Example 22 to prepare 4-azidophthalic acid UU. Compound 97 was prepared using the copper click method of Example 1 with azide UU and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 97: C 28 H 20Calculated value of N8O9: 612.52; Measured value: 611.1 [M-H + . Example 54 Synthesis of 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-methoxybenzoic acid)

Chemical Structure

[0312] Diazotization of 4-amino-2-methoxybenzoic acid was carried out according to Example 22 to prepare 4-azido-2-methoxybenzoic acid VV. Compound 98 was prepared using the copper click method of Example 1 with azide VV and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 98: C H 28 H 24 Calculated value of N8O7: 584.55; Measured value: 583 [M-H + . Example 55 Synthesis of 5,5'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))diisophthalic acid

Chemical Structure

[0313] Diazotization of 4-aminoisophthalic acid was carried out according to Example 22 to prepare 4-azidoisophthalic acid WW. Compound 99 was prepared using the copper click method of Example 1 with azide WW and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 99: C 28 H 20 Calculated value of N8O9: 612.52; Measured value: 611 [M-H + . Example 56 Synthesis of 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-hydroxybenzoic acid)

Chemical Structure

[0314] The diazotization of 4-amino-3-hydroxybenzoic acid was carried out according to Example 22 to prepare 4-azido-3-hydroxybenzoic acid XX. Compound 100 was prepared using the copper click method of Example 1 with azide XX and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 100: C H 26 H 20 The calculated value of N8O7 is 556.50; the measured value: 555.1 [M-H + . Example 57 Synthesis of diethyl (3-(4-(6-(1-(3-(diethoxyphosphoryl)propyl)-1H-1,2,3-triazol-4-yl)-4-(ethylcarbamoyl)167iridin-2-yl)-1H-1,2,3-triazol-1-yl)propyl)phosphonate

Chemical formula

[0315] The diazotization of O,O-diethyl (3-aminopropyl)phosphonate was carried out according to Example 22 to prepare O,O-diethyl (3-azidopropyl)phosphonate YY. Compound 101 was prepared using the copper click method of Example 1 with azide YY and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 101: C 26 H 42 The calculated value of N8O7P2 is 640.62; the measured value: 639.3 [M-H + . Example 58 Synthesis of 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-methylbenzoic acid)

Chemical formula

[0316] The diazotization of 4-amino-2-methylbenzoic acid was carried out according to Example 22 to prepare 4-amino-2-methylbenzoic acid ZZ. Compound 102 was prepared using the copper click method of Example 1 with azide ZZ and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 102: C 28 H 24 Calculated value of N8O5 is 552.55; Measured value: 551.1 [M-H + . Example 59 Synthesis of 4,4'-((5-carboxy-1,3-phenylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chemical Structure

[0317] Compound 103 was prepared using the copper click method of Example 1 with azide Q and 3,5-diethynylbenzoic acid. Mass spectrometry (ESI negative mode) of Compound 103: C 27 H 14 Calculated value of F6N6O6 is 632.44; Measured value: 631 [M-H + . Example 60 Synthesis of 2,2'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-(trifluoromethyl)benzoic acid)

Chemical Structure

[0318] The diazotization of 2-amino-3-(trifluoromethyl)benzoic acid was carried out according to Example 22 to prepare 2-azido-3-(trifluoromethyl)benzoic acid AAA. Compound 104 was prepared using the copper click method of Example 1 with azide AAA and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 104: C 28 H 18 Calculated value of F6N8O5 is 660.49; Measured value: 659 [M-H + . Example 61 Synthesis of 4,4'-((4-((2-Hydroxyethyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chemical formula

[0319] N-(2-Hydroxyethyl)amino-2,6-diethynylpyridine-4-carboxamide BBB was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and aminoethanol. Compound 105 was prepared using the copper click method of Example 1 with diethynyl BBB and azide U. Mass spectrometry (ESI negative mode) of Compound 1 05: Calculated for C 28 H 18 F6N8O6 676.49; Found: 675 [M-H + . Example 62 Synthesis of 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-nitrobenzoic acid)

Chemical formula

[0320] Diazotization of 4-amino-2-nitrobenzoic acid was carried out according to Example 22 to prepare 4-azido-2-nitrobenzoic acid CCC. Compound 106 was prepared using the copper click method of Example 1 with azide CCC and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 106: C 26 H 18 N 10 O9 Calculated 614.49; Found: 613 [M-H + . Example 63 Synthesis of 4,4'-((4-((3,3,3-trifluoropropyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chem.

[0321] N-(3,3,3-Trifluoropropyl)-2,6-diethynylpyridine-4-carboxamide DDD was prepared according to Example 17 using HATU coupling of 2,6-diethynylpyridine-4-carboxylic acid and 3,3,3-trifluoropropan-1-amine. Compound 107 was prepared using the copper click method of Example 1 with diethynyl DDD and azide U. Mass spectrometry (ESI negative mode) of Compound 107: C 29 H 17 Calculated value for F9N8O5: 728.49; Measured value: 727 [M-H + . Example 64 Synthesis of 4,4’’,4’’’-((((butane-1,4-diylbis(azanediyl))bis(carbonyl))bis(pyridine-4,2,6-triyl))tetrakis(1H-1,2,3-triazole-4,1-diyl))tetrakis(2-(trifluoromethyl)benzoic acid)

Chem.

[0322] Compound 108 was prepared using the copper click method of Example 1 with diethynyl FF and azide U. Mass spectrometry (ESI negative mode) of Compound 108: C 56 H 34 F 12 N 16 O 10 Calculated value: 1318.97; Measured value: 1316.9 [M-H + . Example 65 (Synthesis of (4-(4-(4-(Ethylcarbamoyl)-6-(1-(4-phosphonophenyl)-1H-1,2,3-triazol-4-yl)iridin-2-yl)-1H-1,2,3-triazol-1-yl)phenyl)phosphonic acid) [Chemical formula]

[0323] The diazotization of (4-aminophenyl)phosphonic acid was carried out according to Example 22 to prepare (4-azidophenyl)phosphonic acid EEE. Compound 109 was prepared using the copper click method of Example 1 with azide EEE and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 109: C 24 H 22 Calculated value of N8O7P2 is 596.44; Measured value: 595 [M-H + . Example 66 (Synthesis of 2,2'-((4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))diacetic acid) [Chemical formula]

[0324] Methyl (4-azido-2-(trifluoromethyl)benzoyl)glycine FFF was prepared according to the HATU condensation method described in Example 17 by coupling azide U with methyl glycinate. After saponifying the methyl ester FFF with 1N NaOH and neutralizing, (4-azido-2-(trifluoromethyl)benzoyl)glycine GGG was obtained. Compound 110 was prepared using the copper click method of Example 1 with diethynyl Q and azide GGG. Mass spectrometry (ESI negative mode) of Compound 110: C 32 H 24 F6N 10 Calculated value of O7 is 774.60; Measured value: 773 [M-H + . Example 67 Synthesis of dimethyl 2,2’-((4,4’-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))diacetate

Chemical Structure

[0325] Compound 111 was prepared using the copper click method of Example 1 with azide FFF and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 111: C 34 H 28 F6N 10 Calculated value for O7 is 802.65; Measured value: 801.1 [M-H + . Example 68 (2S,2’S)-2,2’-((4,4’-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))disuccinic acid synthesis

Chemical Structure

[0326] (4-Azido-2-(trifluoromethyl)benzoyl)aspartate HHH was prepared by coupling azide U with dimethyl aspartate according to the HATU condensation method described in Example 17 to obtain dimethyl (4-azido-2-(trifluoromethyl)benzoyl)aspartate. The dimethyl ester was saponified with 1N NaOH and then neutralized to obtain HHH. Compound 112 was prepared using the copper click method of Example 1 with diethynyl Q and azide HHH. Mass spectrometry (ESI negative mode) of Compound 112: C3 6H 28 F6N 10 O 11 Calculated value for is 890.67; Measured value: 889 [M-H + . Example 69 Synthesis of 2,2’-((2,2’-((4,4’-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl)bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))bis(acetyl))bis(azanediyl))diacetic acid

Chem.

[0327] (4-Azido-2-(trifluoromethyl)benzoyl)glycylglycine III was prepared according to the HATU condensation method described in Example 17 by coupling azide U with methyl-glycylglycinate. After saponifying the methyl ester with 1N NaOH and neutralizing it, (4-azido-2-(trifluoromethyl)benzoyl)-glycylglycine III was obtained. Compound 113 was prepared using the copper click method of Example 1 with diethynyl Q and azide III. Mass spectrometry (ESI negative mode) of compound 113: C 36 H 30 F6N 12 Calculated value for O9 is 888.70; Measured value: 887 [M-H + . Example 70 Synthesis of 2,6-bis(1-(4-cyano-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-N-ethylisonicotinamide

Chem.

[0328] Diazotization of 4-amino-2-(trifluoromethyl)benzonitrile was carried out according to Example 22 to prepare 4-azido-2-(trifluoromethyl)benzonitrile JJJ. Compound 114 was prepared using the copper click method of Example 1 with azide JJJ and diethynyl Q. Mass spectrometry (ESI negative mode) of compound 114: C 28 H 16 F6N 10 Calculated value for O is 622.50; Measured value: 621 [M-H + . Example 71 Synthesis of 4,4'-((5-carboxy-1,3-phenylene)bis(1H-1,2,3-triazole-1,4-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chemical formula

[0329] The diazotization of 3,5-diaminobenzoic acid was carried out according to Example 22 to obtain 3,5-diazidobenzoic acid KKK. Using 4-bromo-2-(trifluoromethyl)benzoic acid, 4-ethyl-2-(trifluoromethyl)benzoic acid LLL was prepared according to the Sonogashira method described in Example 9. Compound 115 was prepared using the copper click method of Example 1 with bis-azide KKK and mono-ethynyl LLL. Mass spectrometry (ESI negative mode) of Compound 115: C Prepared using the copper click method of Example 1. Mass spectrometry (ESI negative mode) of Compound 115: C 27 H 14 Calculated value of C18H6F6N6O6: 632.44; Measured value: 631 [M-H + . Example 72 Synthesis of 4,4'-((5-(ethylcarbamoyl)-1,3-phenylene)bis(1H-1,2,3-triazole-1,4-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chemical formula

[0330] 3,5-Diazido-N-ethylbenzamide MMM was prepared according to the HATU condensation method described in Example 17 by coupling bis-azide acid KKK with ethylamine. Compound 116 was prepared using the copper click method of Example 1 with mono-ethynyl LLL and bis-azide MMM. Mass spectrometry (ESI negative mode) of Compound 116: C 29 H 19 Calculated value of C20H7F6N7O5: 659.51; Measured value: 658.1 [M-H + . Example 73 Synthesis of 4,4'-(thiophene-2,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chemical formula

[0331] Using 2,5-dibromothiophene, 2,5-diethynylthiophene NNN was prepared according to the Sonogashira method described in Example 9. Compound 117 was prepared using the copper click method of Example 1 with azide U and diethynyl NNN. Mass spectrometry (ESI negative mode): C 24 H 12 Calculated value of F6N6O4 is 594.45; Measured value: 593 [M-H + . Example 74 Synthesis of 4,4'-(furan-2,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid)

Chemical formula

[0332] Using 2,5-dibromofuran, 2,5-diethynylfuran OOO was prepared according to the Sonogashira method described in Example 9. Compound 118 was prepared using the copper click method of Example 1 with azide U and diethynyl OOO. Mass spectrometry (ESI negative mode): C 24 H 12 Calculated value of F6N6O5 is 578.39; Measured value: 577 [M-H + . Example 75 Synthesis of 3'-(4-(4-(ethylcarbamoyl)-6-(1-(3'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)-1H-1,2,3-triazol-4-yl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-3-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid - carbon dioxide (1 / 1) [Chemical]

[0333] Methyl 4-bromo-2-(trifluoromethyl)benzoate (142 mg, 0.5 mmol), 3-aminophenylboronic acid (137 mg, 1 mmol), Pd(PPh3)2Cl2 (35 mg, 0.1 mmol) and K2CO3 (138 mg, 2 mmol) were mixed to initiate the synthesis of 3'-amino-3-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid, and methanol (2 ml) was added. The solution was heated at 45 °C for 1.5 hours. The methanol was removed by evaporation. The residual solid was dissolved in dimethylformamide (1 ml) and purified by silica chromatography using Reveleris Prep eluting with an EtOAc / hexane mobile phase. Appropriate fractions were collected based on UV and evaporated to give methyl 3'-amino-3-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylate (101 mg, 68% yield).

[0334] The diazotization of methyl 3'-amino-3-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylate was carried out according to Example 22 to prepare methyl 3'-azido-3-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylate. The methyl ester was saponified with KOH, neutralized with 0.25 HCl and recovered with EtOAc to give 3'-azido-3-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid PPP. Compound 119 was prepared using the copper click method of Example 1 with azido PPP and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 119: C 40 H 26 The calculated value for C8H8N8O5 is 812.69. Example 76 Synthesis of 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-cyanobenzoic acid) [Chemical]

[0335] The diazotization of 4-amino-2-cyanobenzoic acid was carried out according to Example 22 to prepare 4-azido-2-cyanobenzoic acid QQQ. Compound 120 was prepared using the copper click method of Example 1 with azide QQQ and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 120: C 28 H 18 N 10 The calculated value of O5 is 574.50. Example 77 Synthesis of 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-chlorobenzoic acid)

Chemical Structure

[0336] The diazotization of 4-amino-2-chlorobenzoic acid was carried out according to Example 22 to prepare 4-azido-2-chlorobenzoic acid RRR. Compound 121 was prepared using the copper click method of Example 1 with azide RRR and diethynyl Q. Mass spectrometry (ESI negative mode) of Compound 121: C 26 H 18 The calculated value of Cl2N8O5 is 593.38. Example 78 Screening of PEMs for Enhancement of XNTP Polymerization

[0337] The Sequence By eXpansion (SBX) methodology developed by the inventors provides a significant performance improvement in the sequence read efficiency and accuracy of Xpandomer compared to native DNA. However, the initial transcription of the sequence of natural DNA templates into measurable Xpandomer depends on the ability of DNA polymerases that utilize XNTPs as substrates (the generalized structure of XNTPs is discussed herein with reference to FIGS. 1A and 2). The inventors have found that most DNA polymerases do not efficiently polymerize XNTPs. To improve the efficiency and accuracy of XNTP polymerization into Xpandomer, several PEMs were screened for their ability to enhance DNA polymerase primer extension reactions using XNTPs as substrates.

[0338] A representative primer extension reaction may contain the following reagents: 2 pmol of primer, 2.2 pmol of 45-mer oligonucleotide template, 50 pmol of each XNTP (XATP, XCTP, XGTP, and XTTP), 50 mM Tris HCl (pH 6.79), 200 mM NaCl, 20% PEG, 5% NMS, 0.5 nmol polyphosphate 60.19, 0.3 mM MnCl2, and 0.6 μg of purified recombinant DNA polymerase protein. The reaction may be carried out at 23° C. for 1 hour. The reaction products (i.e., the constrained Xpandomer) are treated to cleave the phosphoramidate bond, thereby generating linearized Xpandomer. To resolve and visualize Xpandomer products of different lengths, the reaction products may be analyzed using gel electrophoresis on a 4-12% acrylamide gel. For the PEM screening described above, PEMs were typically tested in the range from micro to millimolar.

[0339] Surprisingly, several PEMs were observed to significantly and reproducibly enhance DNA polymerase-mediated primer extension by XNTPs. Representative gels demonstrating this enhancement are shown in FIGS. 4 and 5. Referring to FIG. 4, as can be seen in lane 1 (without PEM additive), DNA polymerase incorporates only up to approximately 14 XNTPs under these conditions Extend the template-bound primer. However, by adding a specific PEM to the primer extension reaction, the polymerase is able to synthesize fairly long extension products, as seen, for example, in lanes 3 (compound 4), 7 (compound 3), and 9 (compound 1). In contrast, several different aromatic compounds have little or no effect on XNTP polymerization (see, for example, lanes 2, 4 - 6, and 8), indicating that the PEM activity is specific to compounds 1, 3, and 4.

[0340] Similarly, referring to FIG. 5, in the absence of the PEM additive, DNA polymerase exhibits moderate primer extension activity with XNTPs (lane 1, no PEM additive), but the addition of various concentrations of compound 2 (lanes 8 - 10) significantly enhances the primer extension activity. Again, this PEM activity is specific to compound 2 as other unrelated aromatic compounds had no effect (lanes 2 - 7). Example 79 PEM enhances sequencing by extension (SBX).

[0341] To examine the precision of the PEM-dependent enhancement of XNTP polymerization, the primer extension products were sequenced using the SBX protocol. Briefly, the restricted Xpandomer product of XNTP polymerization is cleaved to generate a linearized Xpandomer. This is achieved first by quenching the extension reaction with a solution containing 100 mM EDTA, 2 mM THPTA, and 2% Tween-20. The sample is then amine-modified with a solution of 1 M NaHCO3 and 1 M succinic anhydride in DMF. Cleavage of the phosphoramidate bond is performed with 37% HCl, and the linearized Xpandomer is purified on a QIAquick column (QIAGEN, Inc.).

[0342] For array determination, protein nanopores are prepared by inserting α-hemolysin into DphPE / hexadecane bilayer members in buffer B1 containing 2 M NH4Cl and 100 mM HEPES (pH 7.4). The cis well is perfused with buffer B2 containing 0.4 M NH4Cl, 0.6 M GuCl, and 100 mM HEPES, pH 7.4. The Xpandomer sample is heated to 70 °C for 2 minutes, cooled completely, and then 2 μL of the sample is added to the cis well. Then, a voltage pulse of 90 mV / 390 mV / 10 μs is applied, and data is acquired using Labview acquisition software.

[0343] Array data is analyzed by histogram display of a population of sequence reads from a single SBX reaction. The analysis software aligns each sequence read to the template sequence and trims the range of the sequence at the ends of reads that do not align to the correct template sequence. Representative histograms of SBX sequencing of a 45mer template are shown in FIGS. 6A (control without additive) and 6B (SBX in the presence of PEM compound 1). As can be seen from the figure, in the absence of compound 1, the sequence reads are not accurate beyond around base 18 of the template. Notably, the addition of compound 1 to the SBX reaction increased the accuracy of the sequence reads over the full length of the 45mer template.

[0344] These results prompted further experiments to test the ability of PEM compound 1 to enhance SBX of longer templates. FIGS. 7A and 7B show histograms of SBX sequencing of 60mer and 80mer templates, respectively. Surprisingly, compound 1 enabled accurate sequence reads all the way to the end of each of these longer templates. These results demonstrate robust and accurate enhancement of XNTP polymerization activity by a novel PEM that potently increases the ability of SBX to provide nanopore-based nucleic acid sequence information. Example 80 PEM enables the synthesis of long-chain Xpandomer products

[0345] We successfully replicated templates up to 80 nucleotides in length accurately with Xpandomer Subsequently, XNTP polymerization reactions were performed using four longer templates consisting of 88, 127, 227, and 277 nucleotides in length. A variant of DPO4 DNA polymerase called C4552 (SEQ ID NO: 1) was used in these polymerization reactions, and the reaction conditions were optimized for C4552 activity in the presence of PEM Compound 1. Other suitable DPO4 polymerase variants include, but are not limited to, those of SEQ ID NOs: 2 - 5. In addition to 1 mM of Compound 1, the reaction additives included 1 mM of urea and 2.75 μg of single-stranded binding protein (Eco SSB). The extension reactions were carried out in a final volume of 10 μL using 0.85 pmol of template, 0.5 pmol of oligonucleotide primer, and 1 nmol of each XNTP. The reactions were performed in a buffer consisting of 50 mM Tris Cl (pH 8.84), 200 mM NH4Oac, and 20% PEG8K supplemented with 5% NMS, 3 or 4 nmol of polyphosphate PP-60.20, and 2 mM MnCl2. 1.2 μg of purified recombinant DNA polymerase protein was used in each extension reaction, and the reactions were carried out at 23°C for 1 - 2 hours. The results of representative extension reactions using the longer templates are shown in Figure 8. In particular, in the presence of Compound 1, the polymerase was able to polymerize XNTPs to generate complete Xpandomer copies of the longer templates in the range of 88 (lanes 1 and 6) - 277 (lanes 5 and 10) nucleotides in length, respectively. Lanes 1 - 5 and 6 - 10 represent identical extension reactions except for the amount of the PP-60.20 additive, which was 3 nmol in lanes 1 - 5 and 4 nmol in lanes 6 - 10. These results highlight the surprising advantages afforded by Compound 1 in reactions that require polymerization of non-natural, highly substituted nucleotide analogs by DNA polymerase, suggesting that this compound, along with other PEMs, can significantly expand the possibilities of the SBX sequencing protocol. Example 81 Next-generation PEM enhances XNTP polymerization and generates long-chain Xpandomer products

[0346] Based on the advantageous properties observed in PEM Compound 1, a next-generation PEM compound was designed for the purpose of improving specific properties including, but not limited to, the water solubility of the molecule. Representative next-generation PEM structures are described in Examples 9-34 and Table 7.

[0347] The PEM activities of Compounds 9-11 were tested in a primer extension assay using three 100-mer templates derived from the HIV1, 2, and 3 genomes. The primer extension reaction contained the following reagents: 75 mM Tris Cl (pH 8.44), 175 mM NH4Oac, 20% PEG8K, 5% NMS, 0.8 nmol PP-60.20, 0.6 mM MnCl2, 2.3 μg Tth single-stranded binding protein (SSB), 0.5 M or 1 M urea, 200 pmol of each XNTP, 1.1 pmol of template, 1 pmol of oligonucleotide primer, 1.2 μg of purified recombinant C4552 DNA polymerase, and 0.5 mM PEM. A 10-μL primer extension reaction was performed at 23 °C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. A representative gel showing the primer extension products is shown in Figure 9. As shown in Lane 1 (HIV1 template), Lane 2 (HIV2 template), and Lane 4 (HIV3 template, no SSB and 1 M urea), Compound 1 enables the polymerization of XNTPs to full-length Xpandomer copies of the three different 100-mer templates (the gel migration positions of the 100-mers are indicated by arrows). Similarly, each of Compound 9 (Lanes 13-16), Compound 10 (Lanes 9-12), and Compound 11 (Lanes 5-8) enables XNTP polymerization at least as efficiently as Compound 1 for each of the three different 100-mer templates. These results suggest that PEM activity can be optimized by increasing various physicochemical properties of the compound such as water solubility.

[0348] The PEM activity of compound 12 was tested in a primer extension assay using a 100-mer template of HIV-2. The primer extension reaction contained the following reagents: 50 mM Tris-Cl, 200 mM NH4OAc, 20% PEG8K, 5% NMS, 0.6 nmol of PP-60.20, 0.6 mM MnCl2, 2.75 μg / μl of Eco single-stranded binding protein (SSB), 1 M urea, 50 pmol of each XNTP, 1.1 pmol of template, 1 pmol of oligonucleotide primer, 1.2 μg / μl of purified recombinant C4760 DNA polymerase (SEQ ID NO: 2), and 0.5, 1 or 1.5 mM of PEM. A 10 μL primer extension reaction was carried out at 23 °C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. A representative gel showing the primer extension products is shown in Figure 10. As shown in lanes 5 (0.5 mM of PEM), 6 (1 mM of PEM), and 7 (1.5 mM of PEM), compound 12 enables the polymerization of XNTPs to the full-length Xpandomer copy of the 100-mer template in a manner comparable to that of compound 10 (lane 1) (the gel migration position of the 100-mer is indicated by an arrow). Lanes 2-4 show the primer extension products from reactions with structurally related additives lacking robust PEM activity. These results suggest that PEM activity can be determined by a very specific chemical structure.

[0349] The PEM activities of compounds 13 and 14 were tested in a primer extension assay using a 100-mer template of HIV-2. The primer extension reaction contained the following reagents: 50 mM Tris-Cl, 200 mM NH4OAc, 20% PEG8K, 5% NMS, 0.6 nmol of PP-60.20, 0.6 mM MnCl2, 2.75 μg / μl of Eco single-stranded binding protein (SSB), 1 M urea, 50 pmol of each XNTP, 1.1 pmol of template, 1 pmol of oligonucleotide primer, 1.2 μg / μl of purified recombinant C4760 DNA polymerase (a variant of DPO4, see SEQ ID NO: 2), and 0.5, 1, 1.5, 2, or 2.5 mM of PEM. A 10-μL primer extension reaction was performed at 23 °C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. A representative gel showing the primer extension products is shown in FIG. 11 (the position of the full-length HIV-2 100-mer is indicated by an arrow). As shown in lanes 5-7 (various concentrations of compound 14) and 8-10 (various concentrations of compound 13), each of these next-generation PEMs enables the polymerization of XNTPs to full-length Xpandomer copies of the 100-mer template in a pattern comparable to that of compound 10 (lanes 1-4).

[0350] The PEM activity of compound 15 was tested in a primer extension assay using a 411-mer amplicon template. The primer extension reaction contained the following reagents: 50 mM Tris Cl, 200 mM NH4Oac, 20% PEG8K, 5% NMS, 3 nmol PP-60.20, 2 mM MnCl2, 2 μg of Kod single-stranded binding protein (SSB), 1 M urea, 250 pmol of each XNTP, 1 pmol of template, 1 pmol of oligonucleotide primer, 1.2 μg of purified recombinant C4760 DNA polymerase (a variant of DPO4, see SEQ ID NO: 2), and 2 (lane 2) or 3 (lane 3) mM of PEM. A 10-μL primer extension reaction was carried out at 37 °C for 20 minutes, and the reaction products were analyzed by gel electrophoresis. A representative gel showing the primer extension products is shown in Figure 12 (the position of the 277-mer is indicated by an arrow). As shown in lanes 2 and 3 (two different concentrations of compound 15), this next-generation PEM enables the polymerization of XNTPs onto long Xpandomer copies of the 411-mer template in a manner comparable to that of compound 14 (lane 1). Notably, the polymerase is completely dependent on the addition of PEM to the reaction to be able to synthesize these long Xpandomer products. Even longer extension products can be obtained by optimizing various reaction parameters, such as the extension time and / or the concentrations of various additives.

[0351] The PEM activities of compounds 16, 17, and 18 and their combinations were tested in a primer extension assay using a 100-mer template derived from HIV-2. The primer extension reaction contained the following reagents: 50 mM Tris Cl, 200 mM NH4Oac, 20% or 25% PEG8K, 5% NMS, 0.6 nmol PP-60.20, 0.6 mM MnCl2, 2 μg of Kod single-stranded binding protein (SSB), 1 M urea , 50 pmol of each XNTP, 1 pmol of template, 1 pmol of oligonucleotide primer, 1.2 μg of purified recombinant C4760 DNA polymerase (variant of DPO4, see SEQ ID NO: 2) and 0.5 - 2 mM of PEM. A 10 μL primer extension reaction was carried out at 37 °C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. A representative gel showing the primer extension products is shown in Figure 13 (the position of the 100mer product is indicated by an arrow). As shown in lane 2 (2 mM of compound 16) and lanes 3 - 6 (0.5, 1, 2, 3 mM of compound 17), these next-generation PEMs enable the polymerization of XNTPs to long Xpandomer copies of the 100mer template in a manner comparable to that of compound 14 (lane 1). Furthermore, the combination of 2 mM of compound 14 and 0.1 mM (lane 7) or 0.3 mM (lanes 8 and 9) of compound 17 also enables the polymerization of XNTPs to long Xpandomer copies of the 100mer template, indicating that combinations of PEMs may allow for the use of lower doses of each individual PEM. Similarly, the combination of 2 mM of compound 16 and 0.1 mM (lane 10) or 0.3 mM (lanes 11 and 12) of compound 18 also appears to allow for the use of lower doses of each individual PEM to enable the polymerization of XNTPs to full-length copies of the 100mer template.

[0352] The PEM activity of compound 19 was tested in a primer extension assay using a 411mer amplicon template. The primer extension reaction contained the following reagents: 50 mM Tris Cl, 200 mM NH4Oac, 20% PEG8K, 5% NMS, 3 nmol PP-60.20, 2 mM MnCl2, 2 μg of Kod single-stranded binding protein (SSB), 1 M urea, 250 pmol of each XNTP, 0.5 pmol of template, 0.5 pmol of oligonucleotide primer, 1.2 μg of purified recombinant C4760 DNA polymerase (a variant of DPO4, see SEQ ID NO: 2), and 0.5 (lane 2), 1 (lane 3), or 1.5 mM (lane 4) of PEM. A 10 μL primer extension reaction was carried out at 37 °C for 30 minutes, and the reaction products were analyzed by gel electrophoresis. A representative gel showing the primer extension products is shown in Figure 14 (the position of the 277mer product is indicated by an arrow). As shown in lanes 2 - 4, this next-generation PEM allows for the polymerization of XNTPs onto long Xpandomer copies of the 411mer template, although in a less efficient manner than compound 14 (lane 1). These results suggest that PEM activity can be specific to the structure of the PEM and / or the length of the template. Example 82 Third-generation PEM compounds

[0353] To further investigate the relationship between PEM structure and polymerase enhancing activity, and to optimize various physicochemical properties, a "third generation" of PEM compounds was synthesized as described herein, for example in Table 1 (compounds 79 - 118) and Examples 35 - 74. These PEMs were tested in primer extension reactions using DNA templates of the following lengths: A) 45mer; B) 100mer; C) 150mer; and D) 222mer. The primer extension reactions were carried out under conditions comparable to those described in Examples 77 and 78, and the extension products were similarly analyzed by gel electrophoresis. The preliminary functional characteristics of the third-generation compounds showing PEM activity are summarized in Table 2 below.

Table 2

[0354] including U.S. Provisional Patent Application No. 62 / 867,049, filed Jun. 26, 2019 U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet, including but not limited to these, are hereby incorporated by reference in their entirety. Such documents may be incorporated by reference, for example, for the purpose of explaining and disclosing materials and methodologies described in publications that may be used in connection with the invention described herein. The publications described above and throughout the text are provided only for the purpose of disclosure prior to the filing date of this application. Nothing in this specification should be construed as an admission that the present disclosure has no right to antedate such publications by virtue of prior invention. The following is the description of the claims at the time of filing. [Claim 1] A method for enhancing a nucleic acid polymerase reaction, comprising: a. i. a template nucleic acid, ii. a nucleic acid polymerase, iii. a mixture of nucleotides or nucleotide analogs, iv. at least one compound of formula (I), to form a nucleic acid polymerase reaction composition; and b. incubating the nucleic acid polymerase reaction composition under conditions that allow a nucleic acid polymerization reaction wherein at least one compound of formula (I) increases the processing ability, rate, or fidelity of the nucleic acid polymerase reaction; The compound of formula (I) is: [Chemical formula] represented by or a solvate, hydrate, tautomer, chelate or salt thereof, wherein m is 1, 2 or 3; m' is 1, 2 or 3; n is 0, 1 or 2; p is 0, 1 or 2; when X is C, W is N, or when X is N, W is C; [Chemical formula] is a single bond or a double bond, and the double bond starts from where either W or X is carbon; L is a linking group; M is independently selected, each occurrence, from hydrogen, halogen, and C1-C4 alkyl; Ar1 is independently selected, each occurrence, from pyridine, pyrazine, pyridazine, furan, thiophene, naphthalene, fluorene, phenanthrene, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, phenanthroline, purine, and carbazole, which may be substituted; The substituents of Ar1 are, each occurrence, halogen, -OH, -CN, -NO2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C1-C6 cycloalkyl, -OR 0 , -CONH2, -C(O)NR 1 R 1’ , -NR 1 R 1’ , -NR 1 C( O)R 3 , -C(O)SR 3 , -COR 3 , -OC(O)R 3 , -C(O)OR 3 , mercaptan, -R 4 -H, -SOR 1 , -S(O)2R 1 , -S(O)2NR 1 R 1’ , and -NS(O)2R 3 and are independently selected from; R 0 is independently selected, each occurrence, from C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; R 1 and R 1’For each occurrence, H, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroaryl, -C(=NH)NH2, -CH2CO2R 0 ,-CH2C(O)NHCH2CO2H, -CH2CH2OH, -CH2CH2NHC(O)R 3 ,-CH2C(O)NHCH2CO2H,

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Claims

1. A method for enhancing a nucleic acid polymerase reaction, comprising: a. i. A template nucleic acid, ii. A nucleic acid polymerase, iii. A mixture of nucleotides or nucleotide analogs, iv. At least one compound of formula (I), to form a nucleic acid polymerase reaction composition; and b. Incubating the nucleic acid polymerase reaction composition under conditions that allow a nucleic acid polymerization reaction wherein at least one compound of formula (I) increases the processing ability, rate or fidelity of the nucleic acid polymerase reaction; The compound of formula (I) is as follows: 4,4'-((4-(methylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-carbamoylpyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(ethoxycarbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(azetidine-1-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 4,4'-((4-(ethyl(methyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); N-ethyl-2,6-bis(1-(4-(2,2,2-trifluoroacetyl)phenyl)-1H-1,2,3-triazol-4-yl)isonicotinamide; 4,4'-(pyridine-2,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(Cyclopropylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(Butylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 5,5'-((4-(Diethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 5,5'-((4-(Morpholine-4-carbonyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-(Pyridazine-3,6-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-hydroxybenzoic acid); 5,5'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-(trifluoromethyl)benzoic acid); 3,3'-(((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(4,1-phenylene))dipropionic acid; 4,4'(((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(4,1-phenylene))dibutyric acid; 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))phthalic acid; 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-methoxybenzoic acid); 5,5'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))isophthalic acid; 4,4'-((4-(Ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-hydroxybenzoic acid); Diethyl (3-(4-(6-(1-(3-(diethoxyphosphoryl)propyl)-1H-1,2,3-triazol-4-yl)-4-(ethylcarbamoyl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)propyl)phosphonate; 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-methylbenzoic acid); 2,2'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(3-(trifluoromethyl)benzoic acid); 4,4'-((4-((2-hydroxyethyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-nitrobenzoic acid); 4,4'-((4-((3,3,3-trifluoropropyl)carbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); (4-(4-(4-(ethylcarbamoyl)-6-(1-(4-phosphonophenyl)-1H-1,2,3-triazol-4-yl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)phenyl)phosphonic acid; 4,4',4'',4''' -(((butane-1,4-diylbis(azanediyl))bis(carbonyl))bis(pyridine-4,2,6-triyl))tetrakis(1H-1,2,3-triazole-4,1-diyl))tetrakis(2-(trifluoromethyl)benzoic acid); 2,2'-((4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))diacetic acid; Dimethyl 2,2'-((4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))diacetate; (2S,2'S)-2,2'-(((4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))disuccinic acid; 2,2'-(((2,2'-((4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoyl))bis(azanediyl))bis(acetyl))bis(azanediyl))diacetic acid; 2,6-bis(1-(4-cyano-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-N-ethylisonicotinamide; 4,4'-(thiophene-2,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 4,4'-(furan-2,5-diylbis(1H-1,2,3-triazole-4,1-diyl))bis(2-(trifluoromethyl)benzoic acid); 3'-(4-(4-(ethylcarbamoyl)-6-(1-(3'-(trifluoromethyl)-[1,1'-biphenyl]-3-yl)-1H-1,2,3-triazol-4-yl)pyridin-2-yl)-1H-1,2,3-triazol-1-yl)-3-(trifluoromethyl)-[1,1'-biphenyl]-4-carboxylic acid; 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-cyanobenzoic acid); and selected from the group consisting of 4,4'-((4-(ethylcarbamoyl)pyridine-2,6-diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(2-chlorobenzoic acid), or a solvate, hydrate, tautomer, chelate or salt thereof, wherein the nucleic acid polymerase is a DNA polymerase, the method.

2. The method according to claim 1, wherein the compound of formula (I) increases the length of the nucleic acid product obtained as compared to a nucleic acid polymerase reaction lacking the compound of formula (I).

3. The method according to claim 1 or 2, wherein the at least one compound of formula (I) comprises a plurality of compounds of formula (I).

4. The method according to any one of claims 1 to 3, wherein the DNA polymerase is DPO4 or a variant thereof.

5. The method according to any one of claims 1 to 4, wherein the mixture of nucleotides or nucleotide analogs is a mixture of nucleotide analogs containing nucleoside triphosphoramidates, and each of the nucleoside triphosphoramidates comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, and a polymer bridging moiety, wherein a first end of the polymer bridging moiety (moiet0y) is bound to the nucleobase, and a second end of the polymer bridging moiety is bound to the α-phosphate of the nucleoside triphosphoramidate to provide extension of the nucleotide analog by cleavage of the phosphoramidate bond.

6. The method according to any one of claims 1 to 5, wherein the nucleic acid polymerization reaction produces a polymer capable of being extended with nucleotide analogs, and the polymer capable of being extended encodes the nucleobase sequence information of the template nucleic acid.

7. The method according to any one of claims 1 to 6, wherein the conditions enabling the nucleic acid polymerization reaction comprise a suitable polymerization buffer and an oligonucleotide primer.

8. The appropriate polymerization buffer is Tris OAc, NH ４ OAc, PEG, a water-miscible organic solvent, polyphosphoric acid 60, NMS, and MnCl ２ The method according to claim 7, comprising at least one of.

9. The method according to any one of claims 1 to 8, wherein the reaction mixture further comprises a single-stranded binding protein.

10. The method according to any one of claims 1 to 9, wherein the reaction mixture further comprises urea.

11. The method according to any one of claims 1 to 10, wherein the mixture of nucleotides or nucleotide analogs comprises nucleotide analogs containing a detectable label.

12. The method according to claim 11, wherein the detectable label is an optically detectable label selected from the group consisting of luminescence, chemiluminescence, fluorescence, phosphorescence, chromophore, or chromogenic label.

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