JPH07145201 | PHOSPHORYLATION OF SACCHARIDES |
WO/1983/002616 | COMPOSITION AND METHOD FOR FORMING A5'p5'pn3'p (AppNp) |
WO/2015/054465 | SUBSTITUTED NUCLEOSIDES, NUCLEOTIDES AND ANALOGS THEREOF |
ZHOU WEI (US)
US20180274025A1 | 2018-09-27 | |||
US20160355541A1 | 2016-12-08 | |||
US20180274024A1 | 2018-09-27 | |||
US20190144482A1 | 2019-05-16 | |||
US20130158249A1 | 2013-06-20 |
CLAIMS WHAT IS CLAIMED IS: 1. A nucleoside 5’-triphosphate analog according to formula (II) or formula (IV): or a salt or protonated form thereof, or a salt or protonated form thereof, wherein: n is independently 0, 1, or 2; and base B is independently selected from the group consisting , 2. A composition comprising a first and a second nucleoside 5’triphosphate analog, each of the first and the second nucleoside 5’triphosphate analog is defined according to claim 1, wherein: the base is different for the first and the second nucleoside 5’-triphosphate analogs; the first nucleoside 5’triphosphate analog comprises an azide; and the second nucleoside 5’triphosphate analog comprises a terminal alkyne. 3. A method of sequencing a polynucleotide comprising performing a polymerization reaction in a reaction system comprising a target polynucleotide to be sequenced, one or more polynucleotide primers which hybridize with the target polynucleotide to be sequenced, a catalytic amount of a polymerase enzyme, and one or more nucleoside 5’-triphosphate analogs of any one of claim 1 or claim 2, thereby generating one or more sequencing products complementary to the target polynucleotide, wherein the one or more sequencing products comprises one incorporated nucleotide derived from the one or more nucleoside 5’-triphosphate analogs. 4. The method of claim 3, wherein the one or more 5’-triphosphate analogs are at a concentration of no more than 2 μM, 3 μM 5 μM, 10 μM, 50 μM, 100 μM 400 μM. 5. The method of claim 3, further comprising: treating the one or more sequencing products with one or more reagents, each of the one or more reagents comprises a detectable label and a reactive group; wherein the reactive group is an azide or a terminal alkyne; and covalently attaching the detectable label with the incorporated nucleotide. 6. The method of claim 5, further comprising: detecting the presence of the detectable label attached to the incorporated nucleotide. 7. The method of claim 6, further comprising: treating the one or more sequencing products with (i) a reducing reagent of dithiothreitol (DTT), 2-mercaptoethanol, trialkylphosphine, triarylphosphine, tris(3-hydroxypropyl)phosphine (THPP) or tris(2- carboxyethyl)phosphine; or (ii) a basic reagent. 8. The method of claim 7, wherein the reducing reagent is trialkylphosphine, triarylphosphine, tris(3-hydroxypropyl)phosphine (THPP) or tris(2-carboxyethyl)phosphine. 9. The method of claim 7, wherein the basic reagent is a buffer having a pH from about 10 to about 11. 10. The method of claim 7, wherein the basic reagent is a sodium carbonate/sodium bicarbonate buffer. 11. A nucleoside 5’-triphosphate analog according to formula (VII) or formula (VIII): or a salt or protonated form thereof, or a salt and/or protonated form thereof, wherein: XX is independently ^N3 or ethynyl; base B is independently selected from the group consisting , Linker is independently , wherein p is 0-3, q is 0-12, and r is 1-3. 12. A composition comprising a first and a second nucleoside 5’triphosphate analog, each of the first and the second nucleoside 5’triphosphate analog is defined according to claim 11, wherein: the base is different for the first and the second nucleoside 5’-triphosphate analogs; the first nucleoside 5’triphosphate analog comprises an azide; and the second nucleoside 5’triphosphate analog comprises a terminal alkyne. 13. A method of sequencing a polynucleotide comprising performing a polymerization reaction in a reaction system comprising a target polynucleotide to be sequenced, one or more polynucleotide primers which hybridize with the target polynucleotide to be sequenced, a catalytic amount of a polymerase enzyme, and one or more nucleoside 5’-triphosphate analogs of any one of claim 11 or claim 12, thereby generating one or more sequencing products complementary to the target polynucleotide, wherein the one or more sequencing products comprises one incorporated nucleotide derived from the one or more nucleoside 5’-triphosphate analogs. 14. The method of claim 13, further comprising: treating the one or more sequencing products with one or more reagents, each of the one or more reagents comprises a detectable label and a reactive group; wherein the reactive group is an azide or a terminal alkyne; and covalently attaching the detectable label with the incorporated nucleotide. 15. The method of claim 14, further comprising: detecting the presence of the detectable label attached to the incorporated nucleotide. 16. The method of claim 15, further comprising: treating the one or more sequencing products with (i) a reducing reagent of dithiothreitol (DTT), 2-mercaptoethanol, trialkylphosphine, triarylphosphine, tris(3-hydroxypropyl)phosphine (THPP) or tris(2- carboxyethyl)phosphine; or (ii) a basic reagent. 17. The method of claim 16, wherein the reducing reagent is trialkylphosphine, triarylphosphine, tris(3-hydroxypropyl)phosphine (THPP) or tris(2-carboxyethyl)phosphine. 18. The method of claim 16, wherein the basic reagent is a buffer having a pH from about 10 to about 11. 19. The method of claim 16, wherein basic reagent is a sodium carbonate/sodium bicarbonate buffer. 20. A method for determining the sequence of an immobilized target polynucleotide, comprising: (a) monitoring the sequential incorporation of nucleotides complementary to the immobilized target polynucleotide, wherein each of the nucleotides independently is a nucleoside 5’-triphosphate analog of claim 1 or claim 11, and wherein the identity of each nucleotide incorporated is determined by detection of a detectable label linked to 3’ oxygen of the nucleotide incorporated; and (b) removing the detectable label from the 3’ oxygen by cleavage a covalent linker between the 3’ oxygen and the detectable linker; wherein non-incorporated nucleotides are removed prior to detection and the detectable label is removed subsequent to detection. 21. The method of claim 20, further comprising a first step and a second step, wherein in the first step, a first composition comprising two different nucleotides is brought into contact with the target polynucleotide, non-incorporated nucleotides are removed prior to detection and the detectable label is removed subsequent to detection, and wherein in the second step, a second composition comprising two different nucleotides not included in the first composition is brought into contact with the target polynucleotide, and non-incorporated nucleotides are removed prior to detection and subsequent to removal of the label, and wherein the first step and the second step are optionally repeated one or more times. 22. The method of claim 20, wherein the removing produced a 3’-OH group on the nucleotide incorporated. 23. The method of claim 20, wherein the nucleotides are incorporated using a polymerase. 24. The method of claim 23, wherein the polymerase is an engineered polymerase. 25. The method of claim 20, wherein the detectable label is a fluorophore. 26. The method of claim 20, wherein the detectable label linked to 3’ oxygen of the nucleotide incorporated is via a 1,2,3-triazole moiety. 27. The method of claim 20, further comprising a click chemistry step, wherein in the click chemistry step a first reactive group covalently attached to the 3’ oxygen of the nucleotide incorporated reacts with a second reactive group covalently attached to the detectable label. 28. The method of claim 27, wherein the click chemistry step forms a 1,2,3-triazole between the first reactive group and the second reactive group. 29. The method of claim 27, wherein the first reactive group is an azido group and the second reactive group is an ethynyl group. 30. The method of claim 27, wherein the first reactive group is an ethynyl group and the second reactive group is an azido group. 31. A method for determining the sequence of an immobilized target polynucleotide, comprising: (a) providing one or two nucleotides, wherein each of the nucleotides is independently a nucleotide of claim 1 or claim 11; (b) incorporating a nucleotide into a complement of the immobilized target polynucleotide and removing non-incorporated one or more nucleotides; (c) attaching label to 3’ oxygen of the nucleotide incorporated in (b) using a click chemistry reaction; (d) after (c), detecting the label attached to the 3’ oxygen of the nucleotide incorporated, thereby determining the type of nucleotide incorporated; (e) after (d), removing the label attached to the 3’ oxygen of the nucleotide; and (f) repeating steps (b)-(e) one or more times; thereby determining the sequence of the immobilized target polynucleotide. 32. A method for determining the sequence of an immobilized target single-stranded polynucleotide, comprising: monitoring the sequential incorporation of complementary nucleotides, wherein each of the complementary nucleotide has a base that is not linked to a detectable label, wherein each of the complementary nucleotides has a deoxyribose sugar moiety and the deoxyribose sugar moiety comprises a first reactive group attached via the 3’ oxygen atom, and wherein the identity of each nucleotide incorporated is determined by detection of a label covalently linked to the 3’ oxygen atom via a click chemistry reaction with the first reactive group after the nucleotide is incorporated, and subsequent removal of the label to form a free 3’-OH on the nucleotide incorporated. 33. The method of claim 32, further comprising: (a) providing said nucleotides; and wherein said monitoring comprises: (b) incorporating a nucleotide into a complement of the immobilized target single- stranded polynucleotide; (c) covalently attaching the label to the 3’ oxygen; (d) detecting the label covalently attached to the 3’ oxygen of the nucleotide, thereby determining the type of nucleotide incorporated; (e) removing the label covalently attached to the 3’ oxygen of the nucleotide; and (f) optionally repeating steps (b)-(e) one or more times; thereby determining the sequence of the immobilized target single-stranded polynucleotide. 34. The method of claim 32, wherein each of the nucleotides are brought into contact with the immobilized target single-stranded polynucleotide sequentially, with removal of non- incorporated nucleotides prior to addition of the next nucleotide, and wherein detection and removal of the label is carried out either after addition of each nucleotide, or after addition of two nucleotides in a composition. 35. The method of claim 32, wherein each of the nucleotides is a deoxyribonucleotide triphosphate. 36. The method of claim 32, wherein the label is a fluorophore. 37. The method of claim 32, wherein first reactive group attached via the 3’ oxygen atom limits the incorporation of further nucleotides into a nucleic acid template strand. 38. The method of claim 32, wherein the immobilized target single-stranded polynucleotide is immobilized on a solid support. 39. The method of claim 38, wherein the solid support is a bead or microsphere. 40. The method of claim 38, wherein the solid support is a glass slide. 41. The method of claim 38, wherein the solid support is a flow cell. |
[0095] Scheme 1: [0096] Reagents and conditions for Scheme 1: (i) tert-butyldiphenylsilyl chloride, pyridine, RT, 12 h; (ii) 3-bromopropionyl chloride, 4-N,N-dimethylaminopyridine (DMAP), 0 °C to RT, 12 h; (iii) sodium azide, DMF, RT, 72h; (iv) Et3N·HF complex, THF, 55 °C, 12 h; (v) (a) 2-chloro- 1H-1,3,2-benzodioxaphosphorin-4-one, pyridine, THF, RT, 1.5 h, (b) tributylamine, tributylammonium pyrophosphate (0.5 M in DMF), RT, 2 h; (c) tert-butyl hydrogen peroxide (5.0 M in hexane), RT, 1 h; (d) water, RT, 2 h. [0097] Using an acetylene-containing acyl chloride or acid in the coupling reaction (ii) may provide an intermediate that can be further processed according to Scheme 1 to provide a nucleotide triphosphate analog with an alkynylalkanoate blocking group on the 3’ oxygen. Step (iii) would be omitted in such a transformation. Other ways of introducing the acetylene moiety are available. [0098] Although Scheme 1 only shows the reactions leading to the thymidine analog of the triphosphate, similar reaction routes can be used to lead to other nucleotide trisphosphate analogs by the appropriate protection/deprotection strategies. [0099] General synthetic route leading to azidoalkanoate or alkynylalkanoate blocking group on the 3’-OH group of the ribose or deoxyribose are available. See, for example, Schemes A and B. [00100] Scheme A: [00101] Reagents and conditions for Scheme A: (i) tert-butyldiphenylsilyl chloride, pyridine; (ii) 3-bromopropionyl chloride, 4-N,N-dimethylaminopyridine (DMAP); (iii) sodium azide, DMF; (iv) Et 3 N·HF complex, THF; (v) (a) 2-chloro-1H-1,3,2-benzodioxaphosphorin-4- one, pyridine, THF, (b) tributylamine, tributylammonium pyrophosphate (0.5 M in DMF); (c) tert-butyl hydrogen peroxide (5.0 M in hexane); (d) water. Base in Scheme A is a nucleobase with or without protecting group(s). When Base is a nucleobase with protecting group(s), additional steps to add or remove the protecting group(s) may be added to the steps described in Scheme A. [00102] Scheme B: [00103] Reagents and conditions for Scheme B: (i) tert-butyldiphenylsilyl chloride, pyridine; (ii) pent-4-ynoyl chloride, 4-N,N-dimethylaminopyridine (DMAP); (iii) Et3N·HF complex, THF; (iv) (a) 2-chloro-1H-1,3,2-benzodioxaphosphorin-4-one, pyridine, THF, (b) tributylamine, tributylammonium pyrophosphate (0.5 M in DMF); (c) tert-butyl hydrogen peroxide (5.0 M in hexane); (d) water. Base is a nucleobase with or without protecting group(s). When base is a nucleobase with protecting group(s), additional steps to add or remove the protecting group(s) may be added to the steps described in Scheme B. Base in Scheme B is a nucleobase with or without protecting group(s). When Base is a nucleobase with protecting group(s), additional steps to add or remove the protecting group(s) may be added to the steps described in Scheme B. [00104] General synthetic route leading to azido-alkyl-disulfide-methylene or alkynyl- alkyl-disulfide-methylene blocking group on the 3’-OH group of the ribose or deoxyribose are available. See, for example, Schemes C and D. [00105] Scheme C: [00106] Reagents and conditions for Scheme C:(i).(a) sulfuryl chloride, DCM; (b) potassium p-toluenethiosulfonate , Ceric ammonium nitrate (CAN), (c) 4-pentynyl-thiol triethylammonium salt 12; (ii) triethylamine-trihydrofluoride; (iii). Et3N.HF complex, THF; (iv). (a) 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one, pyridine, THF; (b) tributylamine, tributylammonium pyrophsphate (0.5 M in DMF), tributylamine; (c) tert-butylhydrogenperoxide (5.0M solution in Hexanes), (d) water. Base in Scheme C is a nucleobase with or without protecting group(s). When Base is a nucleobase with protecting group(s), additional steps to add or remove the protecting group(s) may be added to the steps described in Scheme C. [00107] Scheme D: [00108] Reagents and conditions for Scheme C:(i).(a) sulfuryl chloride, DCM; (b) potassium p-toluenethiosulfonate , Ceric ammonium nitrate (CAN), (c) 3-azidopropyl-thiol triethylammonium salt; (ii) triethylamine-trihydrofluoride; (iii). Et3N.HF complex, THF; (iv). (a) 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one, pyridine, THF; (b) tributylamine, tributylammonium pyrophsphate (0.5 M in DMF), tributylamine; (c) tert-butylhydrogenperoxide (5.0M solution in Hexanes), (d) water. Base in Scheme D is a nucleobase with or without protecting group(s). When Base is a nucleobase with protecting group(s), additional steps to add or remove the protecting group(s) may be added to the steps described in Scheme D. [00109] There may be many different routes leading to the synthesis of a reversible terminator of the general formula (I): wherein w is 1-5; X is O, S, or BH 3 ; n is 0, 1 or 2; w is 1, 2, 3, 4, or 5; and B is a nucleotide base or an analog thereof. [00110] Although the present disclosure only present a few synthetic routes leading to the reversible terminator, other similar or different synthetic routes may be possible when taken into consideration of the particular structure of the targeted reversible terminator. Such synthetic methods to connect two intermediates may be used similar to what have been disclosed herein. [00111] To prepare reversible terminators according to the present disclosure, the conversion of nucleosides to the corresponding nucleoside 5´-triphosphates may use any one of the many published protocols for carrying out this purpose. (See, for instance, Caton-Williams J, et al., “Use of a Novel 5´-Regioselective Phosphitylating Reagent for One-Pot Synthesis of Nucleoside 5´-Triphosphates from Unprotected Nucleosides,” Current Protocols in Nucleic Acid Chemistry, 2013, 1.30.1-1.30.21; Nagata S, et al., “Improved method for the solid-phase synthesis of oligoribonucleotide 5´-triphosphates,” Chem. Pharm. Bull., 2012, 60(9):1212-15; Abramova et al., “A facile and effective synthesis of dinucleotide 5´ triphosphates,” Bioorg. Med. Chem., 15:6549-6555, 2007; Abramova et al., “Synthesis of morpholine nucleoside triphosphates,” Tet. Lett., 45:4361, 2004; Lebedev et al., “Preparation of oligodeoxyribonucleotide 5'-triphosphates using solid support approach,” Nucleos. Nucleot. Nucleic. Acids, 20: 1403, 2001; Hamel et al., “Synthesis of deoxyguanosine polyphosphates and their interactions with the guanosine 5´-triphosphate requiring protein synthetic enzymes of Escherichia coli,” Biochemistry, 1975, 14(23):5055-5060; Vaghefi M., “Chemical synthesis of nucleoside 5′-triphosphates,” In: Nucleoside Triphosphates and their Analogs, pp.1-22, Taylor & Francis, 2005; Burgess et al., “Synthesis of nucleoside triphosphates,” Chem. Rev., 100:2047- 2059, 2000). [00112] Reversible terminators in the present disclosure comprise an azidoalkanoate group at the 3’ oxygen of the sugar moiety. Reversible terminator nucleotides of this type may be useful in methodologies for determining the sequence of polynucleotides. The methodologies in which these reversible terminator nucleotides are useful may include, but are not limited to, automated Sanger sequencing, NGS methods including, but not limited to, sequencing by synthesis, and the like. Many method of analyzing or detecting a polynucleotide may optionally employ the presently disclosed reversible terminator nucleotides. Such methods may optionally employ a solid substrate to which the template is covalently bound. The solid substrate may be a particle or microparticle or flat, solid surface of the type used in current instrumentation for sequencing of nucleic acids. (See, for example, Ruparel et al., Proc. Natl. Acad. Sci., 102:5932- 5937, 2005; EP 1,974,057; WO 93/21340 and U.S. Patent Nos.5,302,509 and 5,547,839, and references cited therein). Optionally, the sequencing reaction employing the presently disclosed reversible terminator nucleotides may be performed in solution or the reaction is performed on a solid phase, such as a microarray or on a microbead, in which the DNA template is associated with a solid support. Solid supports may include, but are not limited to, plates, beads, microbeads, whiskers, fibers, combs, hybridization chips, membranes, single crystals, ceramics, and self-assembling monolayers and the like. Template polynucleic acids may be attached to the solid support by covalent binding such as by conjugation with a coupling agent or by non- covalent binding such as electrostatic interactions, hydrogen bonds or antibody-antigen coupling, or by combinations thereof. There are a wide variety of methods of attaching nucleic acids to solid supports. Linkers [00113] Linkers or contemplated herein are of sufficient length and stability to allow efficient hydrolysis or removal by chemical or enzymatic means. Useful linkers may be readily available and may be capable of reacting with a hydroxyl moiety (or base or nucleophile) on one end of the linker or in the middle of the linker. The number of carbons or atom in a linker, optionally derivatized by other functional groups, must be of sufficient length to allow either chemical or enzymatic cleavage of the blocking group, if the linker is attached to a blocking group or if the linker is attached to the detectable label. [00114] While precise distances or separation may be varied for different reaction systems to obtain optimal results, in some cases, a linkage that maintains the bulky label moiety at some distance away from the nucleotide may be provided, e.g., a linker of 1 to 20 nm in length, to reduce steric crowding in enzyme binding sites. Therefore, the length of the linker may be, for example, 1-50 atoms in length, or 1-40 atoms in length, or 2-35 atoms in length, or 3 to 30 atoms in length, or 5 to 25 atoms in length, or 10 to 20 atoms in length, etc. [00115] Linkers may be comprised of any number of basic chemical starting blocks. For example, linkers may comprise linear or branched alkyl, alkenyl, or alkynyl chains, or combinations thereof, For instance, amino-alkyl linkers, e.g., amino-hexyl linkers, have been used, and are generally sufficiently rigid to maintain such distances. The longest chain of such linkers may include as many as 2 atoms, 3 atoms, 4 atoms, 5 atoms, 6 atoms, 7 atoms, 8 atoms, 9 atoms, 10 atoms, or even 11-35 atoms, or even 35-50 atoms. The linear or branched linker may also contain heteroatoms other than carbon, including, but not limited to, oxygen, sulfur, phosphate, and nitrogen. A polyoxyethylene chain (also commonly referred to as polyethyleneglycol, or PEG) is a preferred linker constituent due to the hydrophilic properties associated with polyoxyethylene. Insertion of heteroatom such as nitrogen and oxygen into the linkers may affect the solubility and stability of the linkers. [00116] In some cases, a linker may be selected from a group selected from alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heteroarylalkylene, heterocycloalkylene, arylene, heteroarylene, or [R2-K-R2]n, or combinations thereof; and each linker group may be substituted with 0-6 R3; each R2 is independently alkylene, alkenylene, alkynylene, heteroarylalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylalkylene; K is a bond, –O–, –S–, –S(O) –, –S(O 2 ) –, –C(O) –, –C(O)O –, –C(O)N(R3)–, or each R3 is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, substituted with 0-6 R 5 ; each R 5 is independently halogen, alkyl, –OR 6 , –N(R 6 ) 2 , –SR 6 , –S(O)R 6 , –SO 2 R 6 , or –C(O)OR 6 ; each R 6 is independently –H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, or heterocycloalkyl; and n is an integer from 1-4 [00117] The linker may be rigid in nature or flexible. Rigid structures include laterally rigid chemical groups, e.g., ring structures such as aromatic compounds, multiple chemical bonds between adjacent groups, e.g., double or triple bonds, in order to prevent rotation of groups relative to each other, and the consequent flexibility that imparts to the overall linker. Thus, the degree of desired rigidity may be modified depending on the content of the linker, or the number of bonds between the individual atoms comprising the linker. Further, addition of ringed structures along the linker may impart rigidity. Ringed structures may include aromatic or non-aromatic rings. Rings may be anywhere from 3 carbons, to 4 carbons, to 5 carbons or even 6 carbons in size. Rings may also optionally include heteroatoms such as oxygen or nitrogen and also be aromatic or non-aromatic. Rings may additionally optionally be substituted by other alkyl groups and/or substituted alkyl groups. [00118] Linkers that comprise ring or aromatic structures can include, for example aryl alkynes and aryl amides. Other examples of the linkers of the disclosure include oligopeptide linkers that also may optionally include ring structures within their structure. [00119] For example, in some cases, polypeptide linkers may be employed that have helical or other rigid structures. Such polypeptides may be comprised of rigid monomers, which derive rigidity both from their primary structure, as well as from their helical secondary structures, or may be comprised of other amino acids or amino acid combinations or sequences that impart rigid secondary or tertiary structures, such as helices, fibrils, sheets, or the like. By way of example, polypeptide fragments of structured rigid proteins, such as fibrin, collagen, tubulin, and the like may be employed as rigid linker molecules. Labels & Dyes [00120] A label or detectable label that associated with the present reversible terminators, may be any moiety that comprises one or more appropriate chemical substances or enzymes that directly or indirectly generate a detectable signal in a chemical, physical or enzymatic reaction. A large variety of labels are well known in the art. (See, for instance, PCT/GB2007/001770). [00121] For instance, one class of such labels is fluorescent labels. Fluorescent labels have the advantage of coming in several different wavelengths (colors) allowing distinguishably labeling each different terminator molecule. (See, for example, Welch et al., Chem. Eur. J.,5(3):951-960, 1999). One example of such labels is dansyl-functionalized fluorescent moieties. Another example is the fluorescent cyanine-based labels Cy3 and Cy5, which can also be used in the present disclosure. (See, Zhu et al., Cytometry, 28:206-211, 1997). Labels suitable for use are also disclosed in Prober et al., Science, 238:336-341, 1987; Connell et al., BioTechniques, 5(4):342-384, 1987; Ansorge et al., Nucl. Acids Res., 15(11):4593-4602, 1987; and Smith et al., Nature, 321:674, 1986. Other commercially available fluorescent labels include, but are not limited to, fluorescein and related derivatives such as isothiocyanate derivatives, e.g. FITC and TRITC, rhodamine, including TMR, texas red and Rox, bodipy, acridine, coumarin, pyrene, benzanthracene, the cyanins, succinimidyl esters such as NHS- fluorescein, maleimide activated fluorophores such as fluorescein-5-maleimide, phosphoramidite reagents containing protected fluorescein, boron-dipyrromethene (BODIPY) dyes, and other fluorophores, e.g.6-FAM phosphoramidite 2. All of these types of fluorescent labels may be used in combination, in mixtures and in groups, as desired and depending on the application. [00122] Various commercially available fluorescent labels are known in the art, such as Alexa Fluor Dyes, e.g., Alexa 488, 555, 568, 660, 532, 647, and 700 (Invitrogen-Life Technologies, Inc., California, USA, available in a wide variety of wavelengths, see for instance, Panchuk, et al., J. Hist. Cyto., 47:1179-1188, 1999). Also commercially available are a large group of fluorescent labels called ATTO dyes (available from ATTO-TEC GmbH in Siegen, Germany). These fluorescent labels may be used in combinations or mixtures to provide distinguishable emission patterns for all terminator molecules used in the assay since so many different absorbance and emission spectra are commercially available. [00123] In various exemplary embodiments, a label comprises a fluorescent dye, such as, but not limited to, a rhodamine dye, e.g., R6G, R110, TAMRA, and ROX, a fluorescein dye, e.g., JOE, VIC, TET, HEX, FAM, etc., a halo-fluorescein dye, a cyanine dye. e.g., CY3, CY3.5, CY5, CY5.5, etc., a BODIPY® dye, e.g., FL, 530/550, TR, TMR, etc., a dichlororhodamine dye, an energy transfer dye, e.g., BIGD YE™ v 1 dyes, BIGD YE™ v 2 dyes, BIGD YE™ v 3 dyes, etc., Lucifer dyes, e.g., Lucifer yellow, etc., CASCADE BLUE®, Oregon Green, and the like. Other exemplary dyes are provided in Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Products, Ninth Ed. (2003) and the updates thereto. Non-limiting exemplary labels also include, e.g., biotin, weakly fluorescent labels (see, for instance, Yin et al., Appl Environ Microbiol.,69(7):3938, 2003; Babendure et al., Anal. Biochem., 317(1):1, 2003; and Jankowiak et al., Chem. Res. Toxicol., 16(3):304, 2003), non-fluorescent labels, colorimetric labels, chemiluminescent labels (see, Wilson et al., Analyst, 128(5):480, 2003; Roda et al., Luminescence,18(2):72, 2003), Raman labels, electrochemical labels, bioluminescent labels (Kitayama et al., Photochem. Photobiol., 77(3):333, 2003; Arakawa et al., Anal. Biochem., 314(2):206, 2003; and Maeda, J. Pharm. Biomed. Anal., 30(6): 1725, 2003), and the like. [00124] Multiple labels can also be used in the disclosure. For example, bi-fluorophore FRET cassettes (Tet. Letts., 46:8867-8871, 2000) are well known in the art and can be utilized in the disclosed methods. Multi-fluor dendrimeric systems (J. Amer. Chem. Soc., 123:8101-8108, 2001) can also be used. Other forms of detectable labels are also available. For example, microparticles, including quantum dots (Empodocles, et al., Nature,399:126-130, 1999), gold nanoparticles (Reichert et al., Anal. Chem., 72:6025-6029, 2000), microbeads (Lacoste et al., Proc. Natl. Acad. Sci. USA, 97(17):9461-9466, 2000), and tags detectable by mass spectrometry can all be used. [00125] Multi-component labels can also be used in the disclosure. A multi-component label is one which is dependent on the interaction with a further compound for detection. The most common multi-component label used in biology is the biotin-streptavidin system. Biotin is used as the label attached to the nucleotide base. Streptavidin is then added separately to enable detection to occur. Other multi-component systems are available. For example, dinitrophenol has a commercially available fluorescent antibody that can be used for detection. [00126] Thus, a “label” as presently defined is a moiety that facilitates detection of a molecule. Common labels in the context of the present disclosure include fluorescent, luminescent, light-scattering, and/or colorimetric labels. Suitable labels may also include radionuclides, substrates, cofactors, inhibitors, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. As other non-limiting examples, the label can be a luminescent label, a light-scattering label (e.g., colloidal gold particles), or an enzyme (e.g., Horse Radish Peroxidase (HRP)). [00127] Fluorescence energy transfer (FRET) dyes may also be employed, such as DY- 630/DY-675 from Dyomics GmbH of Germany, which also commercially supplies many different types of dyes including enzyme-based labels, fluorescent labels, etc. (See, for instance, Dohm et al., “Substantial biases in ultra-short read data sets from high-throughput DNA sequencing,” Nucleic Acids Res., 36:e105, 2008). Other donor/acceptor FRET labels include, but are not limited to: (See also, Johansen, M. K., “Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers,” Methods in Molecular Biology, vol.335: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols, Edited by: V. V. Didenko, Humana Press Inc., Totowa, N.J.). Other dye quenchers are commercially available, including dabcyl, QSY quenchers and the like. (See also, Black Hole Quencher Dyes from Biosearch Technologies, Inc., Novato, Calif.; Iowa Black Dark Quenchers from Integrated DNA Technologies, Inc. of Coralville, Iowa; and other dye quenchers sold by Santa Cruz Biotechnology, Inc. of Dallas, Tex.). [00128] The label and linker construct can be of a size or structure sufficient to act as a block to the incorporation of a further nucleotide onto the nucleotide of the disclosure. This permits controlled polymerization to be carried out. The block can be due to steric hindrance, or can be due to a combination of size, charge and structure. Polymerase Enzymes used in SBS/SBE Sequencing [00129] As already commented upon, one of the key challenges facing SBS or SBE technology is finding reversible terminator molecules capable of being incorporated by polymerase enzymes efficiently and which provide a blocking group that can be removed readily after incorporation. Thus, to achieve the presently claimed methods, polymerase enzymes must be selected which are tolerant of modifications at the 3´ and 5´ ends of the sugar moiety of the nucleoside analog molecule. Such tolerant polymerases are known and commercially available. [00130] BB Preferred polymerases lack 3´-exonuclease or other editing activities. As reported elsewhere, mutant forms of 9°N-7(exo-) DNA polymerase can further improve tolerance for such modifications (WO 2005024010; WO 2006120433), while maintaining high activity and specificity. An example of a suitable polymerase is THERMINATOR™ DNA polymerase (New England Biolabs, Inc., Ipswich, MA), a Family B DNA polymerase, derived from Thermococcus species 9°N-7. The 9°N-7(exo-) DNA polymerase contains the D141A and E143A variants causing 3´-5´ exonuclease deficiency. (See, Southworth et al., “Cloning of thermostable DNA polymerase from hyperthermophilic marine Archaea with emphasis on Thermococcus species 9°N-7 and mutations affecting 3´-5´ exonuclease activity,” Proc. Natl. Acad. Sci. USA, 93(11): 5281-5285, 1996). THERMINATOR™ I DNA polymerase is 9°N- 7(exo-) that also contains the A485L variant. (See, Gardner et al., “Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases,” Nucl. Acids Res., 30:605-613, 2002). THERMINATOR™ III DNA polymerase is a 9°N-7(exo-) enzyme that also holds the L408S, Y409A and P410V mutations. These latter variants exhibit improved tolerance for nucleotides that are modified on the base and 3´ position. Another polymerase enzyme useful in the present methods and kits is the exo- mutant of KOD DNA polymerase, a recombinant form of Thermococcus kodakaraensis KOD1 DNA polymerase. (See, Nishioka et al., “Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme,” J. Biotech., 88:141-149, 2001). The thermostable KOD polymerase is capable of amplifying target DNA up to 6 kbp with high accuracy and yield. (See, Takagi et al., “Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR,” App. Env. Microbiol., 63(11):4504-4510, 1997). Others are Vent (exo-), Tth Polymerase (exo-), and Pyrophage (exo-) (available from Lucigen Corp., Middletown, WI, US). Another non-limiting exemplary DNA polymerase is the enhanced DNA polymerase, or EDP. (See, WO 2005/024010). [00131] When sequencing using SBE, suitable DNA polymerases include, but are not limited to, the Klenow fragment of DNA polymerase I, SEQUENASE™ 1.0 and SEQUENASE™ 2.0 (U.S. Biochemical), T5 DNA polymerase, Phi29 DNA polymerase, THERMOSEQUENASE™ (Taq polymerase with the Tabor-Richardson mutation, see Tabor et al., Proc. Natl. Acad. Sci. USA, 92:6339-6343, 1995) and others known in the art or described herein. Modified versions of these polymerases that have improved ability to incorporate a nucleotide analog of the disclosure can also be used. [00132] Further, it has been reported that altering the reaction conditions of polymerase enzymes can impact their promiscuity, allowing incorporation of modified bases and reversible terminator molecules. For instance, it has been reported that addition of specific metal ions, e.g., Mn 2+ , to polymerase reaction buffers yield improved tolerance for modified nucleotides, although at some cost to specificity (error rate). Additional alterations in reactions may include conducting the reactions at higher or lower temperature, higher or lower pH, higher or lower ionic strength, inclusion of co-solvents or polymers in the reaction, and the like. [00133] Random or directed mutagenesis may also be used to generate libraries of mutant polymerases derived from native species; and the libraries can be screened to select mutants with optimal characteristics, such as improved efficiency, specificity and stability, pH and temperature optimums, etc. Polymerases useful in sequencing methods are typically polymerase enzymes derived from natural sources. Polymerase enzymes can be modified to alter their specificity for modified nucleotides as described, for example, in WO 01/23411, U.S. Patent No. 5,939,292, and WO 05/024010. Furthermore, polymerases need not be derived from biological systems. De-Blocking: Removal of the 3’ Blocking Group and the Detectable Label [00134] After incorporation, the 3’ blocking group or derivative thereof (e.g., the label attached to the 3’-O after the click chemistry reaction) can be removed from the reversible terminator molecules by various means including, but not limited to, chemical means. Removal of the blocking group reactivates or releases the growing polynucleotide strand, freeing it to be available for subsequent extension by the polymerase enzyme. This enables the controlled extension of the primers by a single nucleotide in a sequential manner. The reversible terminators disclosed herein are designed to allow such removal by chemical means, and, in some cases, by enzymatic means. [00135] In one embodiment, the reducing reagents to carry out the disulfide cleavage may be THPP, DTT or 2-mercaptoethanol. In another embodiment, the reducing reagent to carry out the disulfide cleavage may be DTT. In still another embodiment, the reducing reagent to carry out the disulfide cleavage may be 2-mercaptoethanol. In one embodiment, the reducing reagents may be trialkylphosphine and triarylphosphine. In another embodiment, the reducing reagent to carry out the disulfide cleavage is trialkylphosphine. In another embodiment, the reducing reagent may be THPP. In one embodiment, the reducing reagent to carry out the disulfide cleave is tris(2-carboxyethyl)phosphine. [00136] DTT may be used to reduce the disulfide bonds. DTT may reduce solvent- accessible disulfide bonds, for example, the disulfide bonds of the novel reversible terminators disclosed herein. The pH of the reaction may be controlled such that DTT can cleave the disulfide bond. For example, at pH above 7. [00137] Trialkylphosphine can reduce organic disulfides to thiols in water. Since trialkylphosphines are kinetically stable in aqueous solution, selective for the reduction of the disulfide linkage, and unreactive toward many other functional groups other than disulfides, they may be reducing agents in biochemical applications, including reactions with nucleotides such as DNA and RNA molecules. [00138] One advantage to use trialkylphosphines over triarylphosphines (e.g., Ph 3 P) is that the former are more likely to be liquids, which can be more easily kept from exposing to air. Another advantage of using trialkylphosphines is the fact that the resulting trialkylphosphine oxide can be water soluble and thus, are readily removed from the water-insoluble products by a simple wash with aqueous solutions. [00139] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” can be intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof can be used in either the detailed description and/or the claims, such terms can be intended to be inclusive in a manner similar to the term “comprising”. [00140] The term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which may depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, the term “about” as used herein indicates the value of a given quantity varies by +/−10% of the value, or optionally +/−5% of the value, or in some embodiments, by +/−1% of the value so described. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values may be described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. Also, where ranges and/or subranges of values are provided, the ranges and/or subranges can include the endpoints of the ranges and/or subranges. [00141] The term “substantially” as used herein can refer to a value approaching 100% of a given value. For example, an active agent that is “substantially localized” in an organ can indicate that about 90% by weight of an active agent, salt, or metabolite can be present in an organ relative to a total amount of an active agent, salt, or metabolite. In some cases, the term can refer to an amount that can be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some cases, the term can refer to an amount that can be about 100% of a total amount. [00142] As used herein, nucleotides are abbreviated with 3 letters. The first letter indicates the identity of the nitrogenous base (e.g. A for adenine, G for guanine), the second letter indicates the number of phosphates (mono, di, tri), and the third letter is P, standing for phosphate. Nucleoside triphosphates that contain ribose as the sugar, ribonucleoside triphosphates, are conventionally abbreviated as NTPs, while nucleoside triphosphates containing deoxyribose as the sugar, deoxyribonucleoside triphosphates, are abbreviated as dNTPs. For example, dATP stands for deoxyribose adenine triphosphate. NTPs are the building blocks of RNA, and dNTPs are the building blocks of DNA. [00143] The term “immobilization” as used herein generally refers to forming a covalent bond between two reactive groups. For example, polymerization of reactive groups is a form of immobilization. A Carbon to Carbon covalent bond formation is an example of immobilization. [00144] The term “label” or “detectable label” as used herein generally refers to any moiety or property that is detectable, or allows the detection of an entity which is associated with the label. For example, a nucleotide, oligo- or polynucleotide that comprises a fluorescent label may be detectable. In some cases, a labeled oligo- or polynucleotide permits the detection of a hybridization complex, for example, after a labeled nucleotide has been incorporated by enzymatic means into the hybridization complex of a primer and a template nucleic acid. A label may be attached covalently or non-covalently to a nucleotide, oligo- or polynucleotide. In some cases, a label can, alternatively or in combination: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the second label, e.g., FRET; (iii) stabilize hybridization, e.g., duplex formation; (iv) confer a capture function, e.g., hydrophobic affinity, antibody/antigen, ionic complexation, or (v) change a physical property, such as electrophoretic mobility, hydrophobicity, hydrophilicity, solubility, or chromatographic behavior. Labels may vary widely in their structures and their mechanisms of action. Examples of labels may include, but are not limited to, fluorescent labels, non-fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, radioactive labels, mass- modifying groups, antibodies, antigens, biotin, haptens, enzymes (including, e.g., peroxidase, phosphatase, etc.), and the like. Fluorescent labels may include dyes of the fluorescein family, dyes of the rhodamine family, dyes of the cyanine family, or a coumarine, an oxazine, a boradiazaindacene or any derivative thereof. Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NAN and ZOE. Dyes of the rhodamine family include, e.g., Texas Red, ROX, R110, R6G, and TAMRA. FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, and TAMRA are commercially available from, e.g., Perkin-Elmer, Inc. (Wellesley, Mass., USA), Texas Red is commercially available from, e.g., Thermo Fisher Scientific, Inc. (Grand Island, N.Y., USA). Dyes of the cyanine family include, e.g., CY2, CY3, CY5, CY5.5 and CY7, and are commercially available from, e.g., GE Healthcare Life Sciences (Piscataway, N.J., USA). [00145] The term “different detectable label” or “differently labeled” as used herein generally refers to the detectable label being a different chemical entity or being differentiated among the different bases to which the labels are attached to. [00146] As used herein, the solid substrate used can be biological, non-biological, organic, inorganic, or a combination of any of these. The substrate can exist as one or more particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, or semiconductor integrated chips, for example. The solid substrate can be flat or can take on alternative surface configurations. For example, the solid substrate can contain raised or depressed regions on which synthesis or deposition takes place. In some examples, the solid substrate can be chosen to provide appropriate light-absorbing characteristics. For example, the substrate can be a polymerized Langmuir Blodgett film, functionalized glass (e.g., controlled pore glass), silica, titanium oxide, aluminum oxide, indium tin oxide (ITO), Si, Ge, GaAs, GaP, SiO 2 , SiN 4 , modified silicon, the top dielectric layer of a semiconductor integrated circuit (IC) chip, or any one of a variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycyclicolefins, or combinations thereof. [00147] Solid substrates can comprise polymer coatings or gels, such as a polyacrylamide gel or a PDMS gel. Gels and coatings can additionally comprise components to modify their physicochemical properties, for example, hydrophobicity. For example, a polyacrylamide gel or coating can comprise modified acrylamide monomers in its polymer structure such as ethoxylated acrylamide monomers, phosphorylcholine acrylamide monomers, betaine acrylamide monomers, and combinations thereof. [00148] The term “hydroxyl protective group” as used herein generally refers to any group which forms a derivative of the hydroxyl group that is stable to the projected reactions wherein said hydroxyl protective group subsequently optionally can be selectively removed. Said hydroxyl derivative can be obtained by selective reaction of a hydroxyl protecting agent with a hydroxyl group. [00149] The term “complementary” as used herein generally refers to a polynucleotide that forms a stable duplex with its “complement,” e.g., under relevant assay conditions. Typically, two polynucleotide sequences that are complementary to each other have mismatches at less than about 20% of the bases, at less than about 10% of the bases, preferably at less than about 5% of the bases, and more preferably have no mismatches. [00150] A “polynucleotide sequence” or “nucleotide sequence” as used herein generally refers to a polymer of nucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or a character string representing a nucleotide polymer, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence (e.g., the complementary nucleic acid) can be determined. [00151] A “linker group” or a “linker” as used herein generally refers to a cleavable linker as described in this disclosure or a group selected from alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heteroarylalkylene, heterocycloalkylene, arylene, heteroarylene, or [R2-K-R2]n, or combinations thereof; and each linker group may be substituted with 0-6 R3; each R 2 is independently alkylene, alkenylene, alkynylene, heteroarylalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylalkylene; K is a bond, –O–, –S–, –S(O) –, –S(O 2 ) –, –C(O) –, –C(O)O –, –C(O)N(R 3 )–, or each R 3 is independently hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, substituted with 0-6 R 5 ; each R 5 is independently halogen, alkyl, –OR 6 , –N(R 6 ) 2 , –SR 6 , –S(O)R 6 , –SO 2 R 6 , or –C(O)OR 6 ; each R 6 is independently –H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, or heterocycloalkyl; and n is an integer from 1-4. [00152] A “sugar moiety” as used herein generally refers to both ribose and deoxyribose and their derivatives/analogs. [00153] Two polynucleotides “hybridize” when they associate to form a stable duplex, e.g., under relevant assay conditions. Nucleic acids hybridize due to a variety of well characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays” (Elsevier, New York), as well as in Ausubel, infra. [00154] The term “polynucleotide” (and the equivalent term “nucleic acid”) encompasses any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides, e.g., a typical DNA or RNA polymer, peptide nucleic acids (PNAs), modified oligonucleotides, e.g., oligonucleotides comprising nucleotides that are not typical to biological RNA or DNA, such as 2′-O-methylated oligonucleotides, and the like. The nucleotides of the polynucleotide can be deoxyribonucleotides, ribonucleotides or nucleotide analogs, can be natural or non-natural, and can be unsubstituted, unmodified, substituted or modified. The nucleotides can be linked by phosphodiester bonds, or by phosphorothioate linkages, methylphosphonate linkages, boranophosphate linkages, or the like. The polynucleotide can additionally comprise non-nucleotide elements such as labels, quenchers, blocking groups, or the like. The polynucleotide can be, e.g., single-stranded or double-stranded. [00155] The term “oligonucleotide” as used herein generally refers to a nucleotide chain. In some cases, an oligonucleotide is less than 200 residues long, e.g., between 15 and 100 nucleotides long. The oligonucleotide can comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 bases. The oligonucleotides can be from about 3 to about 5 bases, from about 1 to about 50 bases, from about 8 to about 12 bases, from about 15 to about 25 bases, from about 25 to about 35 bases, from about 35 to about 45 bases, or from about 45 to about 55 bases. The oligonucleotide (also referred to as “oligo”) can be any type of oligonucleotide (e.g., a primer). Oligonucleotides can comprise natural nucleotides, non-natural nucleotides, or combinations thereof. [00156] The term “analog” in the context of nucleic acid analog is meant to denote any of a number of known nucleic acid analogs such as, but not limited to, LNA, PNA, etc. Further, a “nucleoside triphosphate analog” may contain 3-7 phosphate groups, wherein one of the oxygen (-O-) on the phosphate may be replaced with sulfur (-S-) or borane (-BH 3 -). Still further, a “nucleoside triphosphate analog” may contain a base which is an analog of adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). For example, the bases are included: oc et o. - wherein Y is CH or N. One nitrogen atom of the purines and pyrimidines base, or analogs thereof, is connected to the ribose or deoxyribose C-1 position. As shown above, one carbon atom of the purines and pyrimidines base, or analogs thereof, is connected to a linker to a label. [00157] The term “aromatic” used in the present application means an aromatic group which has at least one ring having a conjugated pi electron system, i.e., aromatic carbon molecules having 4n+2 delocalized electrons, according to Hückel’s rule, and includes both carbocyclic aryl, e.g., phenyl, and heterocyclic aryl groups, e.g., pyridine. The term includes monocyclic or fused-ring polycyclic, i.e., rings which share adjacent pairs of carbon atoms, groups. [00158] The term “heterocyclic nucleic acid base” used herein means the nitrogenous bases of DNA or RNA. These bases can be divided into two classes: purines and pyrimidines. The former includes guanine and adenine and the latter includes cytosine, thymine, and uracil. [00159] The term “aromatic” when used in the context of “aromatic solvent” as used in the present disclosure means any of the known and/or commercially available aromatic solvents, such as, but not limited to, toluene, benzene, xylenes, any of the Kesols, and/or GaroSOLs, and derivatives and mixtures thereof. [00160] The term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated, i.e. C 1 -C 10 means one to ten carbon atoms in a chain. Non-limiting examples of saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n- pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4- pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl.” [00161] The term “alkylene” by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by —CH 2 CH 2 CH 2 CH 2 —, and further includes those groups described below as “heteroalkylene.” Typically, an alkyl (or alkylene) group may have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present disclosure. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. [00162] The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively. [00163] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, —CH 2 —CH 2 —O—CH 3 , —CH 2 —CH 2 — NH—CH 3 , —CH 2 —CH 2 —N(CH 3 )—CH 3 , —CH 2 —S—CH 2 —CH 3 , —CH 2 —CH 2 , —S(O)— CH 2 , —CH 2 —CH 2 —S(O) 2 —CH 3 , —CHCH—O—CH 3 , —Si(CH 3 )3, —CH 2 —CHN—OCH 3 , and —CHCH—N(CH 3 )—CH 3 . Up to two heteroatoms may be consecutive, such as, for example, —CH 2 —NH—OCH 3 and —CH 2 —O—Si(CH 3 ) 3 . Similarly, the term “heteroalkylene” by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, —CH 2 —CH 2 —S—CH 2 —CH 2 — and —CH 2 —S—CH 2 — CH 2 —NH—CH 2 —. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini, e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like. Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula —C(O) 2 R′— represents both —C(O) 2 R′— and —R′C(O) 2 —. [00164] The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. [00165] The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-C4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2- trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. [00166] The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, substituent that can be a single ring, such as those that follow Hückel's rule (4n+2, where n is any integer), or multiple rings (preferably from 1 to 5 rings), which are fused together or linked covalently and including those which obey Clar's Rule. The term “heteroaryl” refers to aryl groups (or rings) that contain from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2- naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5- indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl, 2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. [00167] For brevity, the term “aryl” when used in combination with other terms, e.g., aryloxy, arylthioxy, arylalkyl, includes both aryl and heteroaryl rings as defined above. Thus, the term “arylalkyl” is meant to include those radicals in which an aryl group is attached to an alkyl group, e.g., benzyl, phenethyl, pyridylmethyl and the like, including those alkyl groups in which a carbon atom, e.g., a methylene group, has been replaced by, for example, an oxygen atom, e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like. [00168] Each of the above terms, e.g., “alkyl,” “heteroalkyl,” “aryl” and “heteroaryl,” is meant to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below. [00169] Substituents for the alkyl and heteroalkyl radicals, including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl, are generically referred to as “alkyl group substituents,” and they can be one or more of a variety of groups selected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO 2 R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, — NR″C(O) 2 R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O) 2 R′, — S(O) 2 NR′R″, —NRSO 2 R′, —CN and —NO 2 in a number ranging from zero to (2M′+1), where M′ is the total number of carbon atoms in such radical. R′, R″, R′″ and R″″ each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of the disclosure includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″ and R″″ groups when more than one of these groups is present. When R′ and R″ are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include, but not be limited to, 1- pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl, e.g., —CF 3 and —CH 2 CF 3 ) and acyl, e.g., —C(O)CH 3 , —C(O)CF 3 , — C(O)CH 2 OCH 3 , and the like). [00170] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are generically referred to as “aryl group substituents.” The substituents are selected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, - halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO 2 R′, —CONR′R″, —OC(O)NR′R″, — NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O) 2 R′, —NR—C(NR′R″R′″)═NR′″, —NR— C(NR′R″)═NR′″, —S(O)R′, —S(O) 2 R′, —S(O) 2 NR′R″, —NRSO 2 R′, —CN and —NO 2 , —R′, —N 3 , —CH(Ph) 2 , fluoro(C 1 -C 4 )alkoxy, and fluoro(C 1 -C 4 )alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R′, R″, R′″ and R″″ are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound of the disclosure includes more than one R group, for example, each of the R groups is independently selected as are each R′, R″, R′″ and R″″ groups when more than one of these groups is present. In the schemes that follow, the symbol X represents “R” as described above. [00171] As used herein, the term “click chemistry,” generally refers to reactions that are modular, wide in scope, give high yields, generate only inoffensive byproducts, such as those that can be removed by nonchromatographic methods, and are stereospecific (but not necessarily enantioselective). See, e.g., Angew. Chem. Int. Ed., 2001, 40(11):2004-2021, which is entirely incorporated herein by reference for all purposes. In some cases, click chemistry can describe a pair of functional groups that can selectively react with each other in mild, aqueous conditions. [00172] An example of click chemistry reaction can be the Huisgen 1,3-dipolar cycloaddition of an azide and an alkyne, or a Copper-catalyzed reaction of an azide with an alkyne, to form a 5-membered heteroatom ring called 1,2,3-triazole. The reaction can also be known as a Cu(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC), a Cu(I) click chemistry or a Cu + click chemistry. Catalyst for the click chemistry can be Cu(I) salts, or Cu(I) salts made in situ by reducing Cu(II) reagent to Cu(I) reagent with a reducing reagent (Pharm Res.2008, 25(10): 2216–2230). Known Cu(II) reagents for the click chemistry can include, but are not limited to, Cu(II) ^(TBTA) complex and Cu(II) (THPTA) complex. TBTA, which is tris-[(1- benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known as tris-(benzyltriazolylmethyl)amine, can be a stabilizing ligand for Cu(I) salts. THPTA, which is tris- (hydroxypropyltriazolylmethyl)amine, can be another example of stabilizing agent for Cu(I). Other conditions can also be accomplished to construct the 1,2,3-triazole ring from an azide and an alkyne using copper-free click chemistry, such as by the Strain-promoted Azide-Alkyne Click chemistry reaction (SPAAC, see, e.g., Chem. Commun., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90), each of which is entirely incorporated herein by reference for all purposes. [00173] Unless otherwise noted, the term “catalytic amount,” as used herein, includes that amount of the reactant that is sufficient for a reaction of the process of the disclosure to occur. Accordingly, the quantity that constitutes a catalytic amount is any quantity that serves to allow or to increase the rate of reaction, with larger quantities typically providing a greater increase. The quantity used in any particular application may be determined in large part by the individual needs of the manufacturing facility. Factors which enter into such a determination include the catalyst cost, recovery costs, desired reaction time, and system capacity. An amount of reactant may be used in the range from about 0.001 to about 0.5 equivalents, from about 0.001 to about 0.25 equivalents, from about 0.01 to about 0.25 equivalents, from about 0.001 to about 0.1, from about 0.01 to about 0.1 equivalents, including about 0.005, about 0.05 or about 0.08 equivalents of the reactant/substrate, or in the range from about 0.001 to about 1 equivalents, from about 0.001 to about 0.5 equivalents, from about 0.001 to about 0.25 equivalents, from about 0.001 to about 0.1 equivalents, from about 0.01 to about 0.5 equivalents or from about 0.05 to about 0.1 equivalents, including about 0.005, about 0.02 or about 0.04 equivalents. [00174] Unless otherwise noted, the term “cleavable chemical group,” as used herein, includes chemical group that caps the —OH group at the 3′-position of the ribose or deoxyribose in the nucleotide analogue. The cleavable chemical group may be any chemical group that 1) is stable during the polymerase reaction, 2) does not interfere with the recognition of the nucleotide analogue by polymerase as a substrate, and 3) is cleavable by a reducing reagent or under the reduction conditions. [00175] Applicants are aware that there are many conventions and systems by which organic compounds may be named and otherwise described, including common names as well as systems, such as the IUPAC system. Abbreviations [00176] Abbreviations used throughout the present application have the meanings provided below. The meanings provided below are not meant to be limiting, but are meant to also encompass any equivalent common or systematic names understood by one of skill in the art. The meaning commonly understood by one of skill in the art should be ascribed to any other abbreviated names not listed below. I2=iodine TBDMS=tert-butyldimethylsilyl TBDPS= tert-butyldiphenylsilyl BOC=tert-butyloxycarbonyl Pyr=pyridine base THF=tetrahydrofuran TsOH=p-toluene sulfonic acid DCA=dichloroacetic acid Bu 3 N=tributyl amine DMF=dimethylformamide Py=pyridine TEAB=triethylammonium bicarbonate DMTO=4,4′-dimethoxytriphenylmethoxy CEO=2-cyanoethoxy TIPSCl=triisopropylsilyl ether chloride Et=ethyl EtOAc=ethyl acetate Ph=phenyl (PhO) 2 P(O)Cl=diphenylphosphoryl chloride CEO-P(NiPr 2 ) 2 =O-(2-cyanoethyl)-N,N,N,N-tetraisopropylphosphorodiami dite iPr2NH=diisopropylamine DBU=1,8-diazabicycloundec-7-ene FMOC=fluorenylmethyloxycarbonyl TCEP=(tris(2-carboxyethyl)phosphine) CDI=1,1′-carbonyldiimidazole RT=room temperature MeOH=methanol TBA=tert-butyl alcohol or 2-methyl-2-propanol TEA=triethanolamine TFP=tetrafluoropropanol or 2,2,3,3-tetrafluoro-1-propanol BSA=bovine serum albumin DTT=dithiothreitol ACN=acetonitrile NaOH=sodium hydroxide IE HPLC=ion-exchange high performance liquid chromatography TLC=thin-layer chromatography TCEP=tris(2-carboxyethyl)phosphine Synthetic Methods [00177] The size and scale of the synthetic methods may vary depending on the desired amount of end product. It is understood that while specific reactants and amounts are provided in the Examples, one of skill in the art knows other alternative and equally feasible sets of reactants that may also yield the same compounds. Thus, where general oxidizers, reducers, solvents of various nature (aprotic, apolar, polar, etc.) are utilized, equivalents may be contemplated for use in the present methods. [00178] For instance, in all instances, where a drying agent is used, contemplated drying agents include all those reported in the literature and known to one of skill, such as, but not limited to, magnesium sulfate, sodium sulfate, calcium sulfate, calcium chloride, potassium chloride, potassium hydroxide, sulfuric acid, quicklime, phosphorous pentoxide, potassium carbonate, sodium, silica gel, aluminum oxide, calcium hydride, lithium aluminum hydride (LAH), potassium hydroxide, and the like. (See, Burfield et al., “Desiccant Efficiency in Solvent Drying. A Reappraisal by Application of a Novel Method for Solvent Water Assay,” J. Org. Chem., 42(18):3060-3065, 1977). The amount of drying agent to add in each work up may be optimized by one of skill in the art and is not particularly limited. Further, although general guidance is provided for work-up of the intermediates in each step, it is generally understood by one of skill that other optional solvents and reagents may be equally substituted during the work- up steps. However, in some exceptional instances, it was found the very specific work-up conditions are required to maintain an unstable intermediate. Those instances are indicated below in the steps in which they occur. [00179] Many of the steps below indicate various work-ups following termination of the reaction. A work-up involves generally quenching of a reaction to terminate any remaining catalytic activity and starting reagents. This is generally followed by addition of an organic solvent and separation of the aqueous layer from the organic layer. The product is typically obtained from the organic layer and unused reactants and other spurious side products and unwanted chemicals are generally trapped in the aqueous layer and discarded. The work-up in standard organic synthetic procedures found throughout the literature is generally followed by drying the product by exposure to a drying agent to remove any excess water or aqueous byproducts remaining partially dissolved in the organic layer and concentration of the remaining organic layer. Concentration of product dissolved in solvent may be achieved by any known means, such as evaporation under pressure, evaporation under increased temperature and pressure, and the like. Such concentrating may be achieved by use of standard laboratory equipment such as rotary-evaporator distillation, and the like. This is optionally followed by one or more purification steps which may include, but is not limited to, flash column chromatography, filtration through various media and/or other preparative methods known in the art and/or crystallization/recrystallization. (See, for instance, Addison Ault, “Techniques and Experiments for Organic Chemistry,” 6 th Ed., University Science Books, Sausalito, Calif., 1998, Ann B. McGuire, Ed., pp.45-59). Though certain organic co-solvents and quenching agents may be indicated in the steps described below, other equivalent organic solvents and quenching agents known to one of skill may be employed equally as well and are fully contemplated herein. Further, most of the work-ups in most steps may be further altered according to preference and desired end use or end product. Drying and evaporation, routine steps at the organic synthetic chemist bench, need not be employed and may be considered in all steps to be optional. The number of extractions with organic solvent may be as many as one, two, three, four, five, or ten or more, depending on the desired result and scale of reaction. Except where specifically noted, the volume, amount of quenching agent, and volume of organic solvents used in the work-up may be varied depending on specific reaction conditions and optimized to yield the best results. [00180] Additionally, where inert gas or noble gas is indicated, any inert gas commonly used in the art may be substituted for the indicated inert gas, such as argon, nitrogen, helium, neon, etc. [00181] A number of patents and publications are cited herein in order to more fully describe and disclose the present methods, compounds, compositions and kits, and the state of the art to which they pertain. The references, publications, patents, books, manuals and other materials cited herein to illuminate the background, known methods, and in particular, to provide additional details with respect to the practice of the present methods, compositions and/or kits, are all incorporated herein by reference in their entirety for all purposes, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference. EXAMPLES [00182] It is understood that the examples and embodiments described herein are for illustrative purposes and that various modifications or changes in light thereof may be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the claims. Accordingly, the following examples are offered to illustrate, but not to limit, the claimed invention. [00183] The following examples describe the detail synthetic steps shown in Scheme 1. Specifically, reagents and conditions used in Scheme 1 are: (i) tert-butyldiphenylsilyl chloride, pyridine, RT, 12 h; (ii) 3-bromopropionyl chloride, 4-N,N-dimethylaminopyridine (DMAP), 0 °C to RT, 12 h; (iii) sodium azide, DMF, RT, 72h; (iv) Et3N·HF complex, THF, 55 °C, 12 h; (v) (a) 2-chloro-1H-1,3,2-benzodioxaphosphorin-4-one, pyridine, THF, 1.5 h, (b) tributylamine, tributylammonium pyrophosphate, 4 h; (c) tert-butyl hydrogen peroxide, 1 h. [00184] Synthesis of 5’O-tert-butydiphenylsilyl thymidine (1): A solution of thymidine (5.01 g, 20.6 mmol) in dry pyridine (50 mL) was cooled to 0 °C and tert- butyl(chloro)diphenylsilane (5.90 mL, 22.7 mmol) was added dropwise under nitrogen. The reaction mixture was further stirred at room temperature overnight. All the volatiles were removed on vacuum and the residue was dissolved in ethyl acetate and the organic layer was washed by water and brine. The organic layer was dried over Na 2 SO 4 , filtered and concentrated. The residue was purified by flash chromatography on silica gel using (MeOH: DCM, 0 to 3%) to yield the titled compound 9.81 g of 1 as white foam (90%). [00185] Synthesis of 3’O-(3-bromopropionyl)-5’O-tert-butydiphenylsilyl thymidine (2): 3-Bromopropionyl chloride (0.83 mL, 8.21 mmol) was added slowly to a mixture of 1 (1.50 g, 3.12 mmol) and DMAP (0.38 g, 3.12 mmol) in dry DCM (25 mL) at 0 °C. The reaction further stirred overnight at room temperature. All the volatiles were removed under vacuum and the residue was purified by flash chromatography on silica gel using (MeOH: DCM, 0-1.5%) to yield the titled compound 2 (1.32 g, 69%). 1 H-NMR (CDCl3): δ 8.24 (br s, 1h7.64-7.68 (m, 4H, Ar-H), 7.40-7.48 (m, 7H, Ar-H, and HC-6), 6.38-6.41 (dd, J = 4.0, 7.6 Hz, 1H, HC-1’), 5.50 (d, J = 4.8 Hz, 1H, HC-3’), 4.09-4.11 (m, 1H, HC-4’), 3.97-4.00 (m, 2H, OCH 2 -5’), 3.57-3.60 (t, J = 5.2 Hz, 2H, COOCH 2 ), 2.95-2.98 (t, J = 5.2 Hz, 2H, CH 2 Br), 2.45-2.49 (m, 1H, HC-2’), 2.26- 2.31 (m, 1H, HC-2’), 1.55 (s, 3H, CH 3 ), 1.10 (s, 9H, (CH 3 ) 3 . LCMS: calcd. for C 29 H 35 BrN 2 O 6 Si, 614.14; found (M+1) 615.14. [00186] Synthesis of 3’O-(3-azidopropionyl)-5’O-tert-butydiphenylsilyl thymidine (3): NaN 3 (0.61 g, 9.41 mmol) was added to the solution of compound 3 (1.16 g, 1.88 mmol) in DMF (14 mL). The reaction mixture was stirred at room temperature for 3 days. All the volatiles were removed on vacuum. The residue was purified by flash chromatography (EtOAc : Hexanes, 10 to 40%) to yield the titled compound 3 (0.55 g, 51 %). 1 H-NMR (CDCl3): δ 8.21 (br s, 1h, NH), 7.64-7.68 (m, 4H, Ar-H), 7.40-7.48 (m, 7H, Ar-H, and HC-6), 6.38-6.41 (dd, J = 4.0, 7.6 Hz, 1H, HC-1’), 5.50 (d, J = 4.8 Hz, 1H, HC-3’), 4.09 (d, J = 1.2Hz, 1H, HC-4’), 3.96-4.02 (m, 2H, OCH 2 -5’), 3.59-3.61 (t, J = 4.8 Hz, 2H, COOCH 2 ), 2.60-2.63 (t, J = 5.2 Hz, 2H, CH 2 Br), 2.44-2.47 (m, 1H, HC-2’), 2.27-2.31 (m, 1H, HC-2’), 1.55 (s, 3H, CH 3 ), 1.10 (s, 9H, (CH 3 )3. LCMS: calcd. for C 29 H 35 N 5 O 6 Si, 577.24; found (M-1) 576.24. [00187] Synthesis of 3’O-(3-azidopropionyl)-thymidine (4): To a solution of 3 (0.54 g, 0.94 mmol) in THF (14 mL) under N2 was added TEA(HF)3 (0.76 mL, 4.69 mmol). The reaction mixture was kept at 55 °C overnight. All the volatiles were removed on vacuum and the residue was purified by flash chromatography on silica gel (MeOH:DCM, 0-3%) to yield the desired product 4 (0.29 g, 90%). 1 H-NMR (CDCl3): δ 8.27 (br s, 1H, NH), 7.48 (s, 1H, HC-6), 6.22-6.25 (dd, J = 4.4, 6.8 Hz, 1H, HC-1’), 5.41-5.43 (m, 1H, HC-3’), 44.11-4.12 (m, 1H, HC-4’), 3.91- 3.97 (m, 2H, OCH 2 -5’), 3.59-3.62(t, J = 5.2 Hz, 2H, COOCH 2 ), 2.62-2.64 (t, J = 5.2 Hz, 2H, CH 2 N3), 2.39-2.47 (m, 2H, HC-2’), 1.93 (s, 3H, CH 3 ). LCMS: calcd. for C13H17N5O6, 339.12; found (M+Na) 362.10. [00188] Synthesis of 3’O-(3-azidopropionyl)-thymidine triphosphate (5): To a solution of 3 (0.26 g, 0.76 mmol) in pyridine (1mL) and THF (2 mL), a solution of 2-chloro-4h-1,3,2- benzodioxaphosphorin-4-one (0.195 g, 0.96 mmol) in THF (1mL) was added and stirred for 45 min under nitrogen. Tributylamine (0.72 ml) and tributylammonium pyrophosphate (2.3 mL, 0.5 M solution in DMF) was added to the reaction mixture and stirred further for 1.5 h. A solution of tert-butyl hydrogen peroxide (0.7 mL, 5.0 M solution in decane) was added to it and stirred for 1h. To the reaction mixture was then added water (1mL) and stirred for 2h. The crude reaction mixture was concentrated, and the residue was purified by RP HPLC using 50 mm TEAB and Acetonitrile, to afford the desired product 5. LCMS: calcd. for C13H 2 0N5O15P3, 579.02; found (M-1) 578.01. [00189] Cleavage of 3’O-(3-azidopropionyl)-thymidine triphosphate: Heating triphosphate 5 with trishydroxypropylphosphine (THPP) or tris(2-carboxyethyl) phosphine (TCEP) in 1× TE buffer at 55 ^C for 5 min, neatly cleaved the 3’ O-azidoalkanoate as shown in the Scheme 2. [00190] Scheme 2 [00191] Reagents and conditions: (i) Trishydroxyethylphosphine (THPP) or Dithiothretol (DTT), 1× TE buffer, 55 ^C, 5 min. [00192] Enzymatic Incorporation and Cleavage Studies: (2R,3S,5R)-2- (((hydroxy((hydroxy(phosphonooxy)phosphoryl)oxy)phosphoryl)o xy)methyl)-5-(5-methyl-2,4- dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl 3-azidopropanoate (5), a model compound, was synthesized similar to conditions of the relevant reactions disclosed in Scheme 1. [00193] FIG.2 shows that compound 5 can be used in enzymatic incorporation in the presence of DNA polymerase (“CENT1”) (lane 3), blockage of further extension after incorporation of the terminator (lane 4) by treating the enzymatic product thus obtained in a “runaway” reaction in the presence of all four unmodified dNTPs and a polymerase, cleavage of the label and the blocking group (lane 5), and further extension by the next base added (lane 6) after the cleavage. [00194] Synthesis of 3’O-[(dithio-1-butynyl)-methyl)-thymidine triphosphate (15) [00195] The key intermediate required for the synthesis of desired triphosphate (15), 4- pentynyl-thiol triethylammonium salt 12 was prepared from commercially available 4-pentyne- 1-ol as shown in the Scheme 3. [00196] Scheme 3 [00197] Thymidine was then converted to the desired 3’O-dithioalkyne thymidine triphosphate 15 as shown in the Scheme 4. The crude product was purified from reverse phase HPLC and analyzed from its LCMS data; Mass calcd: C16H25N2O14P3S2, 626.00; found (M- 1) 625.00. [00198] Scheme 4 [00199] Reagents and conditions for Scheme 4:(i).(a) sulfuryl chloride, DCM, -78°C; (b) potassium p-toluenethiosulfonate , ACN, RT, (c) 4-pentynyl-thiotributylammonium salt 12, RT; (ii) triethylamine-trihydrofluoride, THF, RT; (iii). Et3N.HF complex, THF, 55°C, 12 h; (iv). (a) 2-Chloro-4H-1,3,2-benzodioxaphosphorin-4-one, pyridine, THF, RT, 1h; (b) tributylamine, tributylammonium pyrophsphate (0.5M in DMF), tributylamine, RT, 2h; (c) Tert- butylhydrogenperoxide (5.0M solution in Hexanes), RT, 1h,(d) water, RT 2h. [00200] Cleavage of 3’O-[(dithio-1-butynyl)-methyl)-thymidine triphosphate (15): The 3’O-bolcking group, alkyne dithioalkynemethyl ether from triphosphate 15 was cleaved by heating with 1, 4-dithiothreitol or (DTT) or tris(2-carboxyethyl) phosphine (TCEP) in 1×TE buffer (pH 9.5) at 55 degree Celsius for 5 min to afford thymidine triphosphate as shown in the Scheme 5. [00201] Scheme 5 [00202] Enzymatic incorporation of 3’O-[(dithio-1-butynyl)-methyl)-thymidine triphosphate (15) [00203] The model compound, 3’O-[(dithio-1-butynyl)-methyl)-thymidine triphosphate (15) showed excellent enzymatic incorporation as evidenced from lane 3, 4, 6 and 7 in FIG.3. [00204] Click chemistry reaction experiment [00205] Click Reaction (Scheme 6): Thymidine azide 6 undergo unprecedented copper (I) catalyzed [3+2] cycloaddition (CuAAC) reaction with alkyne (13) at 60 degree Celsius within 5 minutes to afford cyclized triazole adduct 16. The reaction was monitored by LCMS and confirmed from its mass spectral data, mass calc for C61H75N7O11S2Si2, 1201.45; found (M- 1), 1200.45. [00206] Scheme 6 [00207] Click chemistry reaction of azido ester triphosphate (20) with alkyne- attached dye [00208] Procedure: [00209] 1) 25 µL 0.5 mM THPTA and 25 µL 0.25 mM CuSO 4 ·5H 2 O were mixed and incubated at room temperature for 30 min. [00210] 2) To the incubated solution obtained in step 1) were added 25 µL 5 mM compound 20 and 25 µL 1 mM compound 21, followed by adding 25 µL 2.5 mM sodium ascorbate. The reaction mixture was heated at 40 °C for 5 minutes. LC-MS showed the desire coupled product in 86% yields.; Calcd mass for C45H53N8O23P3, 1166.24; Found (M-1), 1165.23. [00211] As shown in FIG.4 displaying the LCMS spectrum of the reaction products, the product (retention time at 3.338 minute) and starting material (retention time at 3.005 minute) both showed double peaks in UV and −ESI. [00212] Click Reaction (Scheme 7): Thymidine azide 21 undergo unprecedented copper (I) catalyzed [3+2] cycloaddition (CuAAC) reaction with alkyne-attached label (20) at 40 degree Celsius within 5 minutes to afford cyclized triazole adduct 22. The reaction was monitored by LCMS and confirmed from its mass spectral data, mass calc for C45H53N8O23P3, 1166.24; Found (M-1), 1165.23. THPTA (tris-hydroxypropyltriazolylmethylamine) is a water-soluble, effective accelerating ligand for copper-catalyzed Alkyne-Azide click chemistry reactions (CuAAC). [00213] Summary of the reaction in Scheme 7: [00214] Scheme 7 [00215] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
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