ENZYMATIC SYNTHESIS OF 4'-ETHYNYL NUCLEOSIDE ANALOGUES

Abstract

The present invention relates to an enzymatic synthesis of 4'-ethynyl-2'-deoxy nucleosides and analogues thereof, for example, EFdA, which eliminates the use of protecting groups in the intermediates, improves the stereoselectivity of glycosylation, and reduces the number of process steps required to manufacture the compounds. It also relates to the novel intermediates employed in the process.

Description

ENZYMATIC SYNTHESIS OF 4'-ETHYNYL NUCLEOSIDE ANALOGUES Reference to the list of sequences sent electronically The sequence list for this application is submitted electronically via EFS-Web as an ASCII sequence list with the filename “24608WOPCT-SEQLIST02JUI2019.txt”, created on July 2, 2019, and with a size of 80.5 kb. This sequence list submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background of the invention 4'-Ethin-2'-deoxynucleoside analogues are known for their activity against HIV, AIDS and related diseases. qj znnn / Lznz / E / Yi ONzbasé' An example of a 4'-ethynyl nucleoside analogue is 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA, also known as MK-8591), which is a nucleoside reverse transcriptase translocation inhibitor that blocks viral replication of HIV-1 and SIV in vitro (Kawamoto, A., Kodama, E., Sarafianos SF et al., Int. J. Biochem. Cell Biol.; 40(11):2410-20

[2008] ; Ohrui, H., Kohgo, S., Hayakawa, H. et al., Nucleosides, Nucleotides ​EFdA is metabolized in cells to its active triphosphate anabolite, which inhibits HIV reverse transcriptase. In contrast to currently available nucleoside reverse transcriptase inhibitors (NsRTIs) and nucleotide reverse transcriptase inhibitors (NtRTIs) for the treatment of HIV infection, which lack a 3'-OH group to block the incorporation of the incoming nucleotide, EFdA retains a 3'-OH group and acts as a chain terminator by preventing primer:template translocation to the reverse transcriptase (RT) active site and preventing the binding of incoming deoxyribonucleotide triphosphates (dNTPs). Furthermore, the modified ribose ring fold of EFdA is thought to contribute to reverse transcriptase inhibition by positioning the 3'-OH group where phosphotransfer of the incoming nucleotide is inefficient. (Michailidis E, et al.), Mechanism of inhibition of HIV-1 reverse transcriptase by 4’-ethynyl-2-fluoro-2’-desoxyadenos¡ne triphosphate, J Biol. Chem 284:35681-35691

[2009] ; Michailidis E, y colaboradores., 4’-Etin¡l-2-fluoro-2’-desox¡adenosine (EFdA) inhibits HIV-1 reverse transcriptase with múltiple mechanisms, J Biol Chem 289:24533-24548

[2014] ). In in-vitro HIV replication assays, EFdA is a potent antiretroviral and exhibits comparable antiviral activity against clinical isolates across all subtypes evaluated. It is rapidly anabolicized in both lymphoid-derived cell lines and peripheral blood mononuclear cells to the in vitro active triphosphate, and the intracellular half-life of EFdA triphosphate (EFdA-TP) exceeds 72 hours. (Stoddart, CA, Galkina, et al., Oral Administration of the Nucleoside EFdA (4'-Ethynyl-2-Fluoro-2'-Desoxyadenosine) Provides Rapid Suppression of HIV Viremia in Humanized Mice and Favorable Pharmacokinetic Properties in Mice and the Rhesus Macaque, Antimicrob Agents Chemother, 2015 Jul; 59(7): 4190-4198, Published online, May 4, 2015). EFdA has shown efficacy in animal models of HIV infection, including humanized mouse models and a rhesus macaque model infected with SIV. Pharmacokinetic studies of orally administered EFdA in mice and rhesus monkeys have shown rapid absorption and high plasma concentrations. A long intracellular half-life was demonstrated by the fact that peripheral blood mononuclear cells isolated from rhesus macaques were refractory to SIV infection 24 h after drug administration. (Ibid.) Previous syntheses of 4'-ethynyl nucleoside analogs, including EFdA, suffer from modest stereoselectivity in the formation of the CN bond between the ethynyl-deoxyribose sugar and the 2-fluoroadenine nucleobase (also referred to as 2-fluoro-9H-purin-6-amine). The previous synthesis also requires protecting groups to carry out the glycosylation reaction, which reduces the efficiency of the synthesis. The synthesis described by Kei Fukuyama et al. (Synthesis of EFdA via a Diastereoselective Aldol Reaction of a Protected 3-Keto Furanose, Organic Letters 2015, 17(4), pp. 828-831; DOI: 10.1021 / ol5036535) is a 14-step synthesis of D-glucose diacetonide that uses diastereoselective reactions to adjust the three stereocenters. The stereochemistry of the anomeric center is controlled by having a 2'-acetoxy targeting group that is subsequently removed by hydrolysis and deoxygenation. This pathway requires four chromatographic purifications and the stoichiometric use of a toxic organotin reagent for the late-step deoxygenation. In another route (see Mark McLaughlin, et al., Enantioselective Synthesis of 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) via Enzymatic Desymmetrization, Organic Letters 2017, 19 (4), pp. 926-929), the fully substituted 4'-carbinol is stereoselectively generated by enzymatic desymmetrization. The 3'-stereocenter is fitted with catalytic asymmetric transfer hydrogenation, and the 1' anomeric linkage is established at modest stereoselectivity using substrate control, with an upgrade in stereochemical purity achieved by crystallization of an intermediate. This process requires 15 steps, the use of several protecting groups, and generates the glycosyl linkage between the nucleobase and the sugar fragments at low stereoselectivity (1.8:1). A 12-step synthesis to make EFdA from R-glyceraldehyde acetonide is described in α / ζηηη / ίζηζ / Β / γι Kageyama, M., et al., Concise Synthesis of the Anti-HIV Nucleosides EFdA, Biosci. Biotechnol. Biochem, 2012, 76, pp. 1219-1225; and Enantioselective Total Synthesis of the Potent Anti-HIV Nucleosides EFdA, Masayuki Kageyama, et al., Organic Letters 2011 13 (19), pp. 5264-5266 [DOI: 10.1021 / ol202116k]. The synthesis uses the chiral starting material to establish the 3' stereocenter with moderate diastereoselectivity. After chromatographic separation of the stereoisomers, the new stereocenter is used to guide a diastereoselective alkyne addition to establish the fully substituted 4' stereocenter. The T anomeric position is established with little stereocontrol and requires chromatography to separate the anomers. The pathway requires chromatographic separation of diastereomers in two different stages and starts from an expensive chiral starting material. Kohgo, S., et al., Design, Efficient Synthesis, and Anti-HIV Activity of 4'-C-Cyano- and 4'-C-Ethynyl-2'-deoxy Purine Nucleosides, Nucleosides, Nucleotides and Nucleic Acids, 2004, 23, pp. 671-690 [DOI: 10.1081 / NCN-120037508] describes a synthetic pathway that starts from an existing nucleoside and modifies both the sugar and nucleobase portions. It is an 18-step synthesis that begins with 2-amino-2'-deoxyadenosine with a low overall yield of 2.5%. It is known that enzymes such as purine nucleoside phosphorylase (PNP, EC 2.4.2.1) can form the glycosyl linkage on a nucleoside if it uses nucleoside analogs with high stereoselectivity and without the use of protecting groups. See, for example, the review: New Trends in Nucleoside Biotechnology, Mikhailopulo, L.A., Miroshnikov, A.L., Acta Naturae 2010, 2, pp. 36–58. However, the current range of sugar fragments capable of undergoing the PNP-catalyzed reaction has been limited to the α-1-phosphates of natural ribose and deoxyribose, along with a small number of analogs with small H, NH2, or F substituents at the C2' and C3' positions and C5' OH group replacements. There have been no reports of successful PNP-catalyzed glycosylation using sugars with carbon substituents on the ring or any substitution at the C4' position. Access to ribose and deoxyribose α-1-phosphate substrates for PNP-catalyzed glycosylation has been shown by the translocation of the phosphate group from the 5'-hydroxyl to the 1'-hydroxyl position with the enzyme phosphopentomutase (PPM, EC 5.4.2.7) (see Mikhailopulo, L.A., et al., supra). However, the range of sugars for which PPM is capable of catalyzing this reaction has been limited to ribose, arabinose, 2-deoxyribose, and 2,3-dideoxyribose. No examples of successful reaction with sugar phosphates containing any of the additional substituents have been reported. Deoxyribose phosphate aldolase enzymes (DERA, EC 4.1.2.4) are known to catalyze the aldol addition of acetaldehyde to other short-chain aldehydes (see review: Stephen M. Dean, et al., Recent Advances in Aldolase-Catalyzed Asymmetric Synthesis, Adv. Synth. Catal. 2007, 349, pp. 1308-1320; DOI: 10.1002 / adsc.200700115). However, no examples have been reported with aldehydes that carry a carbon atom completely substituted to the aldehyde group. U.S. Patent 7,229,797 describes the formation of deoxyribonucleosides from unnaturally unsubstituted deoxyribose 1-phosphate using purine nucleoside phosphorylase (PNP) and additionally using enzymes such as sucrose phosphorylase to remove the inorganic phosphate byproduct and drive equilibrium. It does not describe enzyme engineering to create PNP enzymes that can generate nucleosides from unnaturally unnatural 4-ethynyl-D-2-deoxyribose 1-phosphate, nor that by engineering the enzymes PPM and DERA to act on unnatural substrates, 4-ethynyl-D-2-deoxyribose 1-phosphate can be generated. Given the difficulty and lengthy synthetic options developed to date for producing 4'-ethynyl nucleoside analogues, it would be desirable to develop an improved enzymatic synthesis for 4'-ethynyl nucleoside analogues such as EFdA that reduces the number of process steps, minimizes the use of protecting groups, improves the stereoselectivity of glycosylation, and avoids the use of toxic materials. Surprisingly, PPM enzymes have been found to have some activity with the 3-atom ethynyl substituent at the 4' position on ribose and that the activity of the PPM enzyme could be improved by introducing mutations into the enzymes to successfully develop a reaction for the isomerization of 4-ethynyl-D-2-deoxyribose 5-phosphate (6) to 1-phosphate 4-ethynyl-D-2-deoxyribose (6.5) catalyzed by PPM to allow a more efficient method for the production of 4'-ethynyl-2'-deoxynucleosides. Additionally, it has also been found that PNP enzymes have some activity with the 3-atom ethynyl substituent at position 4 on deoxyribose and that the activity of the PNP enzyme could be improved by introducing mutations into the enzymes to successfully develop a PNP-catalyzed glycosylation reaction to allow a more efficient method for the production of 4'-ethynyl-2'-deoxynucleosides. A further improvement to the general synthetic method resulted from the discovery that DERA enzymes, particularly the DERA from Shewanella halifaxensis, have activity for the aldol reaction with 2-ethynyl-glyceraldehyde 3-phosphate having a fully substituted α-carbon. This discovery enabled the efficient synthesis of 4-ethynyl-2-deoxyribose 5-phosphate, a precursor for 4'-ethynyl-2'-deoxynucleoside analogues, including EFdA. Brief description of the invention The present invention involves the use of engineered enzymes in a novel enzymatic synthesis of 4'-ethynyl-2'-deoxynucleoside analogs, including EFdA, which eliminates the use of intermediate protecting groups, improves the stereoselectivity of glycosylation, and significantly reduces the number of process steps required to manufacture the compounds compared to prior methods, among other process improvements. It further relates to novel intermediates that are an integral part of the enzymatic process. The overall process is summarized in the following Reaction Scheme 1 and Reaction Scheme 2; the latter Reaction Scheme provides an alternative method for manufacturing compound 5: ο / ζηηη / ίζηζ / Β / γι Reaction Scheme 1 - deoxyribose aldolase galactose oxidase ο / ζηηη / ίζηζ / Β / γι phosphopentomutase The acidic form or salts of the phosphate intermediates may be employed in the process described herein and are not limited to the specific acid or salt forms provided in the process step examples herein. For all phosphate intermediates described herein, 2X+ represents any combination of two protons, a proton with another monovalent cation, two monovalent cations (the same or different), or a divalent cation. The phosphate intermediates described herein with -HO3PO- may likewise have any combination of two protons, a proton with another monovalent cation, two monovalent cations (the same or different), or a divalent cation associated with the phosphate group.Examples include, but are not limited to, calcium, magnesium, or zinc salts; mono- or disodium salts, mono- or dipotassium salts, mono- or dilithium salts; mono- or diammonium salts; or mono- or divalent salts with primary, secondary, or tertiary amines. As will be understood in the art, intermediate compounds shown or named herein as aldehydes or hydrates in the synthetic steps herein may exist in any form or a mixture of these forms in the reactions described herein. For example, compounds (4) and (5) are represented in Reaction Scheme 1 as a hydrate and an aldehyde, respectively, but each may exist as a hydrate or aldehyde or a mixture thereof in the reaction steps where each is present. Each form is encompassed by reference to the numbers of compound (4) or (5) within the process steps herein. hydrate or aldehyde hydrate or aldehyde The Composite (3) is achiral and can be shown at this point as any of the following: Compound (6) may exist in its ring form or as an open-chain aldehyde or hydrate, each as an acid or a salt thereof, in the reaction steps where it is present: open-chain aldehyde or hydrate Detailed description of the invention 4'-Ethynyl-2'-deoxynucleosides and analogues thereof Nucleosides with an anomeric CN linkage have been explored for activity against HIV, AIDS, and related diseases. 4'-Ethynyl-2'-deoxynucleosides and their analogues comprise a 4'-ethynyl-2'-deoxyribose linked via an anomeric CN linkage to a purine or pyrimidine nucleobase (adenine, guanine, cytosine, thymine, or uracil) or a modified purine or pyrimidine nucleobase. It has been found that analogues of 4'-ethynyl-2'-deoxynucleosides such as EFdA can be synthesized using a single-pot final-step process by combining 4-ethynyl-2'-deoxyribose 5-phosphate (6) with two enzymes, phosphopentomutase (PPM) [e.g., but not limited to SEQ ID NO.: 8] and purine nucleoside phosphorylase (PNP) [e.g., but not limited to SEQ ID NO.: 9, SEQ ID NO.: 15], as shown in Reaction Scheme 2. Reaction Scheme 2 Reaction Scheme 2A ο / ζηηη / ίζηζ / Β / γι nh2 (EFdA) sucrose phosphorylase / / sucrose glucose-1-phosphate As shown in Reaction Scheme 2, the final step of the synthesis employs a two-enzyme reaction with an optional third enzyme to drive the reaction equilibrium toward the desired end product. The final step begins with compound (6) or a salt thereof, where (6) is either 4-ethynyl-2-deoxyribose 5-phosphate in ring form as shown above or the open-chain aldehyde or hydrate form thereof. Compound (6) is combined with phosphopentomutase (PPM), purine nucleoside phosphorylase (PNP), sucrose phosphorylase, sucrose, and a nucleobase, for example, unsubstituted or substituted adenine, in a buffered solution containing a manganese(II) salt and adjusted as necessary to a pH in the range of approximately 6.5 to 8.0, or more particularly from approximately 7.0 to 7.5. A molar ratio of sucrose:compound (6) may be, but is not limited to, from approximately 1:1 to 4:1. The components of this one-pot reaction may be combined in any order. The reaction is stirred within a temperature range that does not denature the enzymes, for example, from approximately 30 to 45°C, and more specifically from approximately 35 to 45°C. To a certain extent, cooler temperatures may work but slow down the reaction rate. Any buffer solution with a suitable pH and containing a manganese(II) salt can be used in the reaction. Examples of such buffer solutions include, but are not limited to: triethanolamine; PIPES, for example, piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, for example, 3-(N-morpholino)propanesulfonic acid or 3-morpholinopropane-1-sulfonic acid; HEPES, for example, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; TRIS, for example, tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol; and BIS-TRIS methane, for example, 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. More specifically, the buffer solution is triethanolamine. The manganese(II) salt in the buffer solution may be, for example, manganese chloride, manganese chloride hydrate, manganese bromide, manganese iodide, manganese nitrate, and / or manganese sulfate.The concentration of manganese in the buffer solution can vary from approximately 0.05 mM to approximately 10 mM, and is particularly approximately 5 mM. The equilibrium reaction can be driven to a high final product conversion by consuming the inorganic phosphate salt by phosphorolysis of sucrose to D-fructose and α-D-glucose-1-phosphate, catalyzed by sucrose phosphorylase (EC 2.4.1.7) added to the reaction mixture. However, any of the other options for removing phosphate during the reaction can be employed, for example, adding calcium, magnesium, or manganese to the reaction to precipitate a phosphate salt instead of using sucrose phosphorylase and sucrose. This highly efficient and environmentally friendly process has the advantage of forming the anomeric bond between the sugar and the nucleobase with very high stereoselectivity without the use of protecting groups or organic solvents and can be carried out as a one-pot reaction. Once the reaction is complete, the final product can be isolated using standard procedures known to people skilled in ordinary experience in the art, such as, but not limited to, isolation by crystallization of the final product and collection by filtration, or extraction in an appropriate solvent followed by crystallization. As shown in Reaction Scheme 2A, the final step of the synthesis can alternatively employ a 3-enzyme reaction with an optional 4th enzyme to drive the reaction equilibrium towards the desired end product. The final step begins with compound (5) or a salt thereof, wherein (5) is (R)-2-ethynyl-glyceraldehyde 3-phosphate, a hydrate form thereof. Compound (5) is combined with deoxyribose-phosphate aldolase (DERA), acetaldehyde, phosphopentomutase (PPM), purine nucleoside phosphorylase (PNP), sucrose phosphorylase, sucrose, and a nucleobase or an analogue thereof, e.g., unsubstituted or substituted adenine, in a buffered solution containing a manganese(II) salt and adjusted as necessary to a pH in the range of approximately 4 to 10, or particularly from approximately 6.5 to 8.0, or more particularly from approximately 7.0 to 7.5. A molar ratio of sucrose to compound (5) may be, but is not limited to, from approximately 1:1 to 4:1. The components of this one-pot reaction may be combined in any order. The reaction takes place within a temperature range that does not denature the enzymes, for example, from approximately 30 to 45°C, or particularly from approximately 35 to α / zenηη / izηζ / B / γι 45°C. To a certain extent, cooler temperatures can work but slow down the reaction rate. Acetaldehyde is added as a solution, and more specifically as a 40% by weight solution in isopropyl alcohol. Any suitable acetaldehyde solution or pure acetaldehyde may be used in the reaction. Examples of such solutions include, but are not limited to: a solution of acetaldehyde in isopropanol, a solution of acetaldehyde in ethanol, a solution of acetaldehyde in water, and a solution of acetaldehyde in THF. The molar ratio of aldehyde to compound (5) may be, but is not limited to, approximately 0.5:1 to 4:1, and more specifically 1.5:1. Any buffer solution with a suitable pH and containing a manganese(II) salt can be used in the reaction. Examples of such buffer solutions include, but are not limited to: triethanolamine; PIPES, for example, piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, for example, 3-(N-morpholino)propanesulfonic acid or 3-morpholinopropane-1-sulfonic acid; HEPES, for example, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; TRIS, for example, tris(hydroxymethyl)aminoethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol; and BIS-TRIS methane, for example, 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. More specifically, the buffer solution is triethanolamine. The manganese(II) salt in the buffer solution may be, for example, manganese chloride, manganese chloride hydrate, manganese bromide, manganese iodide, manganese nitrate, and / or manganese sulfate.The concentration of manganese in the buffer solution can vary from approximately 0.05 mM to approximately 10 mM, and is particularly approximately 5 mM. The equilibrium reaction can be driven to high conversion of the final product by consuming the byproduct inorganic phosphate salt through phosphorolysis of sucrose to D-fructose and D-glucose-1-phosphate, catalyzed by sucrose phosphorylase (EC 2.4.1.7) added to the reaction mixture. However, any of the other options for removing phosphate during the reaction can be employed; for example, adding calcium, magnesium, or manganese to the reaction to precipitate a phosphate salt instead of using sucrose phosphorylase and sucrose. This highly efficient and environmentally friendly process has the advantage of forming the anomeric bond between the sugar and the nucleobase with very high stereoselectivity without the use of protecting groups or organic solvents and can be carried out as a one-pot reaction. Once the reaction is complete, the final product can be isolated using standard procedures known to persons of ordinary experience in the art, such as, but not limited to, isolation by crystallization of the final product and collection by filtration, or extraction in an appropriate solvent followed by crystallization. Several upstream intermediates, used in the present process for the synthesis of the final product 4'-ethynyl-2'-deoxynucleosides and analogues thereof, are also manufactured using enzymatic reaction methods as shown in Reaction Scheme 3; Reaction Scheme 3A and Reaction Scheme 3B. Reaction Scheme 3 peroxidase catalase uz galactose oxidase kinase o / znnn / Lznz / E / Yii deoxyribose aldolase Reaction Scheme 3A catalase peroxidase O2galactose oxidase Bn-NHHN'Bl1 pTsOH HO ^Bn deoxyribose aldolase kinase Reaction Scheme 3B Compound 4: Oxidase Reaction As shown in Reaction Scheme 3, (R)-2-ethynyl-glyceraldehyde (4) is prepared by reacting galactose oxidase with 2-ethynyl-propane-1,2,3-triol (3) in a buffered solution adjusted as necessary to a pH in a range of about 3 to 10, or more particularly about 6 to 8.Any buffer solution having a suitable pH range may be used, for example but not limited to, sodium phosphate; sodium acetate; PIPES, for example piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, for example 3-(N-morpholino)propanesulfonic acid or 3-morpholinopropane-1-sulfonic acid; HEPES, for example 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; TRIS, for example tris(hydroxymethyl)aminomethane or 2-Amino-2-(hydroxymethyl)propane-1,3-diol; and BIS-TRIS methane, for example, 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borate; CAPS, for example, N-cyclohexyl-3-aminopropanesulfonic acid; MES, for example, 2-(N-morpholino)ethanesulfonic acid; CHES, for example, N-cyclohexyl-2-aminoethanesulfonic acid; glycine; or bicine (N,N-Bis(2-hydroxyethyl)glycine); with sodium phosphate being preferred. Copper and a peroxidase are both used in the reaction to activate galactose oxidase (GOase). Copper can be supplied to the reaction mixture by adding CuSO4, Cu(OAc)2, CuCl2, or other Cu(II) or Cu(I) salts. The peroxidase can be horseradish peroxidase, a peroxidase derived from other organisms, or it can be replaced by an oxidant such as ferricyanide, iridate, manganese(III) salts, persulfate salts, and other one- or two-electron oxidants, or inorganic or organic oxidants. Preferably, the peroxidase is horseradish peroxidase. Catalase is also added to help prevent GOase deactivation. The catalase can be from a mammalian (bovine) source or from a bacterial or fungal source such as Corynebacterium, Aspergillus, or other organisms known in the art for this purpose. The reaction proceeds in the presence of oxygen. A convenient method is to spray the reaction with air. Alternatively, other systems can be used to generate oxygen, such as hydrogen peroxidase / catalase, superoxide, or other methods known in the art for this purpose. The reaction can be carried out with a substrate concentration of approximately 10 to 180 g / L, and particularly from 20 to 50 g / L. The reaction can be run at a temperature of approximately 0 to 40°C, and particularly from approximately 10 to 30°C. Compound 8: Aminal formation As exemplified in Reaction Scheme 3A, (R)-2-ethynylglyceraldehyde (4) can be isolated in its aminal form (e.g., compound 8) by reacting it with any amine, diamine, or amino alcohol that forms a stable Λ / , / V-acetal or Λ / ,O-acetal, for example, but not limited to, N,N'-dibenzylethane-1,2-diamine, Λ / ,Λ / '-dimethylethane-1,2-diamine, Λ / ,Λ / '-diphenylethane-1,2-diamine, and N-benzylethanolamine; with Λ / ,Λ / '-dibenzylethane-1,2-diamine being preferred. The reaction is carried out in an organic solvent at a temperature at or below approximately 50°C, preferably 20 to 30°C, to avoid aminal decomposition. Any solvent that is not miscible with water may be used, for example, but not limited to, MTBE, 2-MeTHF, CPME, diethyl ether, diisopropyl ether, ethyl acetate, isopropyl acetate, toluene, DCM, or a mixture thereof, with MTBE being preferred.The reaction can be carried out with a substrate concentration of approximately 10 to 100 g / L, and particularly 20 to 50 g / L. Optionally, the aminal can be further purified by crystallization from an organic solvent, for example, but not limited to, MTBE, 2-MeTHF, CPME, diethyl ether, diisopropyl ether, ethyl acetate, isopropyl acetate, toluene, DCM, or a mixture thereof, with MTBE being preferred. Crystallization is carried out at or below 50°C, for example, at approximately 40°C, to avoid decomposition of the aminal. The reaction proceeds in the absence of oxygen. A convenient method is to spray the reaction with N2. Alternatively, other systems can be employed to exclude oxygen, such as argon, helium, or other methods known in the art for this purpose. Compound 4: Regeneration of aldehyde 4 from the aminal 8 (R)-2-Ethynyl-glyceraldehyde (4) can be regenerated from its respective aminal by reacting it with an organic or inorganic acid in the presence of an organic solvent that is not visible with water, and at a temperature at or below 50°C, for example, approximately 0 to 15°C, to avoid decomposition of the aminal. Any organic or inorganic acid can be used, for example, but not limited to, p-toluenesulfonic acid, methanesulfonic acid, camphorsulfonic acid, acetic acid, hydrochloric acid, phosphoric acid, sulfuric acid.p-Toluenesulfonic acid is preferred in the reaction with the amino acid 8 due to the low solubility of the salt / V, / V'-dibenzylethane-1,2-diamine bis p-toluenesulfonate in water. Any solvent that is not miscible with water may be used, for example, but not limited to, MTBE, 2-MeTHF, CPME, diethyl ether, diisopropyl ether, ethyl acetate, isopropyl acetate, toluene, DCM, or a mixture thereof. With MTBE and 2-MeTHF being preferred, the reaction can be carried out with a substrate concentration of approximately 5 to 100 g / L, and particularly 20 to 50 g / L. Optionally, the aldehyde 4 solution can be further treated with a resin to remove excess organic or inorganic acid. Resin treatment can be carried out using basic resins such as DOWEXMRMARATHONMRA resin (hydroxide form) and AMBERLYSTMR15 resin (hydrogen form), or a mixture of these, preferably a mixture of DOWEXMRMARATHONMRA resin (hydroxide form) and AMBERLYSTMR15 resin. Optionally, the aldehyde 4 solution can be further evaporated under vacuum or purified with a gas to remove excess organic solvent. Compound 5: Kinase reaction ο / ζηηη / ίζηζ / Β / γι As shown in Reaction Scheme 3 and Reaction Scheme 3A, the (R)2-ethynyl-glyceraldehyde 3-phosphate hydrate (5) is prepared by reacting pantothenate kinase (PanK) of the natural type from E. coli or a variant thereof, with compound (4) in a buffered solution as required to a pH in a range of approximately 4 to 10, or particularly from approximately 6.5 to 8.5 or more particularly 5.5 to 8.5.Any buffer solution having a suitable pH range may be used, for example but not limited to, sodium phosphate, PIPES, for example piperazine-N,N'-bis(2-ethanesulfonic acid); BIS-TRIS methane, for example, 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borate; HEPES, for example, 4-(2-hydroxyethyl)-1-piperazinathanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; triethanolamine and TRIS, for example, tris(hydroxymethyl)aminomethane or 2-Amino-2-(hydroxymethyl)propane-1,3-diol; with sodium phosphate being preferred. The reaction can be carried out in the presence of any suitable divalent metal salt, for example, but not limited to a magnesium salt, e.g., magnesium chloride, and salts of cobalt, manganese, zinc, or calcium. This reaction uses adenosine 5'-diphosphate (ADP) as the phosphate source required for regeneration to 5'-triphosphate (ATP). ATP can be generated in situ and subsequently regenerated by any known method using ADP, adenosine 5'-monophosphate (AMP), or adenosine. For example, a combination of acetyl phosphate and acetate kinase can be used to regenerate ADP to ATP. Similarly, in the presence of pyruvate, phosphate, and oxygen, a combination of pyruvate oxidase and catalase generates acetyl phosphate, which, in the presence of acetate kinase, can be used to regenerate ADP to ATP. The reaction can be carried out with a substrate concentration of approximately 10 to 100 g / L, and particularly from approximately 20 to 40 g / L. The reaction can be run at a temperature of approximately 0 to 40°C, and particularly from approximately 10 to 25°C. The reaction can also be carried out with pantothenate kinase (PanK) immobilized on a resin, or with both PanK and acetate immobilized on the kinase resin. Any suitable enzyme immobilization method known in the art may be used, for example, but not limited to, Immobilized Metal Ion Affinity Chromatography (IMAC) resin, or affinity resin immobilization using other biological tags, covalent immobilization, immobilization on ionic resins, adsorption immobilization, encapsulation, and / or crosslinked enzymes. For example, Metal Ion Affinity Chromatography (IMAC) resin may be used, or any suitable combination of IMAC resin and a divalent cation, where the cation may be, for example, but not limited to, nickel, cobalt, copper, zinc, iron, and / or aluminum. In particular, nickel-loaded IMAC resin may be used.Preferably, both acetate kinase and pantothenate kinase (PanK) are immobilized in the resin. Compound 9: Kinase Reaction _ OH 703P°0 < / OH 2X As shown in Reaction Scheme 3B, (S)-2-ethynylpropane-1,2,3-triol 1-phosphate (9) is prepared by reacting pantothenate kinase (PanK) of the natural type from E. cali or a variant thereof, with compound (3) in a buffered solution adjusted as required to a pH in a range of approximately 4 to 10, or particularly from approximately 6.5 to 8.5 or more particularly 5.5 to 8.5.Any buffer solution having a suitable pH range may be used, for example, but not limited to, sodium phosphate, PIPES, for example, piperazine-N,N'bis(2-ethanesulfonic acid); BIS-TRIS methane, for example, 2-[Bis(2-hydroxyethyl)amino]-2(hydroxymethyl)propane-1,3-diol; borate; HEPES, for example, 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; triethanolamine and TRIS, for example, tris(hydroxymethyl)aminomethane or 2-Amino-2-(hydroxymethyl)propane-1,3-diol, with sodium phosphate being preferred. The reaction can be carried out in the presence of any suitable bivalent metal salt, for example, but not limited to a magnesium salt, for example, magnesium chloride, and salts of cobalt, manganese, zinc, or calcium. This reaction uses adenosine 5'-diphosphate (ADP) as the phosphate source, which is regenerated to 5'-triphosphate (ATP). ATP can be generated in situ and subsequently regenerated by any known method from ADP, adenosine 5'-monophosphate (AMP), or adenosine. For example, a combination of acetyl phosphate and acetate kinase can be used to regenerate ADP to ATP. Alternatively, (a) a combination of pyruvate oxidase, catalase, and acetate kinase can be used in the presence of pyruvate, phosphate, and oxygen to regenerate ADP to ATP, or (b) a combination of pyruvate oxidase, catalase, and acetate kinase can be used in the presence of pyruvate, phosphate, and oxygen in combination with acetyl phosphate and acetate kinase for the regeneration of ATP from ADP. The reaction can be carried out with a substrate concentration of approximately 10 to 100 g / L, and specifically from approximately 20 to 40 g / L. The reaction can be run at a temperature of approximately 0 to 40°C, and specifically from approximately 10 to 25°C. The reaction can also be carried out with pantothenate kinase (PanK) immobilized on a resin, or with both PanK and acetate kinase immobilized on the resin. Any suitable enzyme immobilization method can be used in the technique, for example, but not limited to, immobilized metal ion affinity chromatography (IMAC) resin, or any affinity resin immobilization using other biological tags, covalent immobilization, immobilization on ionic resins, adsorption immobilization, encapsulation, and / or crosslinked enzymes. For example, metal ion affinity chromatography (IMAC) resin, or any suitable combination of IMAC resin and a divalent cation, can be used, where the cation can be, for example, but not limited to, nickel, cobalt, copper, zinc, iron, and / or aluminum. In particular, nickel-loaded IMAC resin can be used.Preferably, both acetate kinase and pantothenate kinase (PanK) are immobilized in the resin. Compound 5: ο / ζηηη / ίζηζ / Β / γι Oxidase Reaction As shown in Reaction Scheme 3B, (R)-2-ethynyl-glyceraldehyde hydrate 3-phosphate (5) is prepared by reacting galactose oxidase with (S)-2-ethynyl-propane-1,2,3-triol 1-phosphate (9) in a buffered solution adjusted as required to a pH in a range of about 3 to 10, or more particularly about 6 to 8.Any buffer solution having a suitable pH range may be used, for example, but not limited to, sodium phosphate; sodium acetate; PIPES, for example, piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, for example, 3-(N-morpholino)propanesulfonic acid or 3-morpholinopropane-1-sulfonic acid; HEPES, for example, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-1]ethanesulfonic acid; TRIS, for example, tris(hydroxymethyl)aminomethane or 2-Amino-2-(hydroxymethyl)propane-1,3-diol; and BIS-TRIS methane, for example, 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borate; CAPS, for example, N-cyclohexyl-3-aminopropanesulfonic acid; MES, for example, 2-(N-morpholino)ethanesulfonic acid; CHES, for example, N-cyclohexyl-2-aminoethanesulfonic acid; glycine; or bicine (N,N-Bis(2-hydroxyethyl)glycine); with sodium phosphate being preferred. Copper and a peroxidase are both used in the reaction to activate galactose oxidase (GOase). Copper can be supplied to the reaction mixture by adding CuSO4, Cu(OAc)2, CuCb, and other Cu(11) or Cu(1) salts. The peroxidase can be horseradish peroxidase, a peroxidase derived from other organisms, or it can be replaced by an oxidant such as ferricyanide, iridate, manganese(III) salts, persulfate salts, and other one- or two-electron oxidants, or inorganic or organic oxidants. Preferably, the peroxidase is horseradish peroxidase. Catalase can also be added to help prevent GOase inactivation. The catalase can be from a mammalian (bovine) source or from a bacterial or fungal source such as Corynebacterium, Aspergillus, or other organisms known in the art for this purpose. The reaction proceeds in the presence of oxygen. A convenient method is to spray the reaction with air. Alternatively, other systems can be used to generate oxygen, such as hydrogen peroxide / catalase, superoxide, or other methods known in the art for this purpose. The reaction can be carried out with a substrate concentration of approximately 10 to 180 g / L, and particularly 20 to 50 g / L. The reaction can be run at a temperature of approximately 0 to 40°C, and particularly from approximately 10 to 30°C. Compound 6: Deoxyribose-Phosphate Aldolase Reaction (PERA) An important advantage of this new route for producing compound (6) over previously known processes is that it creates the sugar structure in the correct oxidation state without the use of protecting groups. 4-Ethynyl-D-2-deoxyribose 5-phosphate (6) is prepared by reacting deoxyribose phosphate aldolase (DERA) with (R)-2-ethynylglyceraldehyde 3-phosphate (5) as an acid or salt thereof, and acetaldehyde from an aqueous solution adjusted as necessary to a pH in the range of approximately 5 to 9, or more particularly approximately 6 to 8. Examples of salts of (5) include, but are not limited to, calcium, magnesium, zinc, mono- or di-Na salts, mono- or di-K salts, or mono- or di-Li salts; mono- or di-ammonium salts; or monovalent or divalent salts with primary, secondary, or tertiary amines. The reaction can be carried out in an open vessel or is preferably carried out in a sealed vessel to prevent evaporation of the acetaldehyde. The reaction can be carried out with a substrate concentration of approximately 10 to 100 g / L, specifically approximately 30 to 60 g / L. It can be run at a temperature of approximately 0 to 40°C, and specifically approximately 25 to 35°C. The reaction can be run without any buffer solution. Alternatively, buffer solutions can be used, for example, but not limited to, triethanolamine; phosphate; MOPS, for example, 3-(N-morpholino)propanesulfonic acid or 3-morpholinopropane-1-sulfonic acid; HEPES, for example, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; BIS-TRIS methane, for example, 2-[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borate; PIPES, for example, piperazine-N,N'-bis(2-ethanesulfonic acid); MES, for example, 2(N-morpholino)ethanesulfonic acid; and borate; or other buffer solutions having a suitable pH range that do not have any of the primary amine groups. Each step and method of the processes described herein involving the use of one or more enzymes is carried out at a temperature that does not denature the enzyme(s). Each step and method of the processes described herein involving the use of one or more enzymes can be carried out at a pH in the range of approximately 3 to 10 or approximately 4 to 10. A “nucleobase” (or “nitrogenous base” or “base”) is a pyrimidine or purine heterocycle of nucleic acids such as DNA and RNA. As used herein, nucleobases include adenine, guanine, cytosine, thymine, or uracil, as well as nucleobases with non-natural modifications, for example, where the base has one or more non-natural substituents, or a modification affecting the heteroatom(s) in a base that precludes any change to anomeric CN linkage. A 4'-ethynyl-2'-deoxy nucleoside contains a nucleobase. As used herein, an analogue of a 4'-ethynyl-2'-deoxy nucleoside means an unnatural modification to the nucleoside base, for example, where the base has one or more unnatural substituents, or a modification affecting the heteroatom(s) in the base that excludes any change to the anomeric C-N bonding. As used herein, “phosphopentomutase” (“PPM”) enzymes (e.g., EC 5.4.2.7) are enzymes that catalyze the reversible isomerization of ribose 1-phosphate to ribose 5-phosphate and related compounds such as deoxyribose phosphate and analogues of ribose phosphate and deoxyribose phosphate. As used herein, “purine nucleoside phosphorylase” (“PNP”) enzymes (EC 2.4.2.2) are enzymes that catalyze the reversible phosphorolysis of purine ribonucleosides and related compounds (e.g., deoxyribonucleosides and ribonucleoside and deoxyribonucleoside analogues) to the free purine base and ribose-1-phosphate (and analogues thereof). As used herein, “sucrose phosphorylase” (“SP”) enzymes (EC 2.4.1.7) are enzymes that catalyze the reversible phosphorolysis of sucrose to the base D-fructose and glucose-1-phosphate (and analogues thereof). Sucrose phosphorylase (SP) in combination with sucrose is employed in combination with purine nucleoside phosphorylase (PNP) and phosphomutase (PPM) to remove free phosphate ions from the reaction, where the combination of the enzymes catalyzes the formation of nucleoside MK-8591 (EFdA), while in some embodiments it could be replaced by other methods known in the art. As used herein, “deoxyribose-phosphate aldolase” (“DERA”) (e.g., EC 4.1.2.4) refers to an enzyme in a family of lyases that reversibly cleave or create carbon-carbon bonds. Deoxyribose-phosphate aldolases as used herein include naturally occurring (natural type) deoxyribose-phosphate aldolase as well as non-naturally occurring modified polypeptides generated by human manipulation. Natural type deoxyribose-phosphate aldolase catalyzes the reversible reaction of 2-deoxy-D-ribose 5-phosphate to D-glyceraldehyde 3-phosphate and acetaldehyde. As used herein, “pantothenate kinase” (“PanK”) refers to enzymes (EC 2.7.1.33) that naturally phosphorylate pantothenate to form 4’-phosphopantothenate. Variant enzymes derived from these PanK enzymes may exhibit enhanced activity and stereoselectivity toward the 3OH group of D-ethynylglyceraldehyde, regardless of whether these variants retain their natural function toward pantothenate. As used herein, galactose oxidase enzymes (“GOase”; EC 1.1.3.9) are copper-dependent enzymes that, in the presence of biomolecular oxygen, catalyze the oxidation of primary alcohols to the corresponding aldehydes. They act in both a regiospecific and enantiospecific manner, allowing for synthetic procedures that require little or no functional group protection and yield the desired stereoisomer. The mode of oxidation is mild and controlled, such that the activity does not lead to over-oxidation of the alcohol to its corresponding carboxylic acid. As used herein, horseradish peroxidase (HRP, EC 1.11.1.7) is an iron-dependent enzyme that activates and maintains the catalytic activity of GOase by oxidizing a redox-inactive state of the active site that occurs during normal GOase catalytic cycling. Type I HRP is used catalytically in the examples included herein; however, it is not intended to be exclusive to this function, as other electron-transfer enzymes belonging to these enzyme classes, as well as chemical reagents, can fulfill this role. As used herein, “catalase” refers to the heme-dependent enzyme (EC 1.11.1.6) that acts on hydrogen peroxide, a byproduct of galactose oxidase or pyruvate oxidase reactions, which can render the enzymes inactive above certain levels of hydrogen peroxide. Catalase is employed as a catalytic maintenance enzyme in the examples herein to convert hydrogen peroxide to water and oxygen, whereas in some embodiments it could be replaced by other methods, such as electrochemical decomposition of hydrogen peroxide. A heme-dependent catalase is employed in a catalytic manner in the examples herein; however, it is not intended to be exclusive in this function, as other enzymes belonging to this class can fulfill this role. As used herein, “acetate kinase (“AcK”)” refers to an enzyme (EC 2.7.2.1) that catalyzes the formation of acetate phosphate from adenosine triphosphate (ATP). It can also catalyze the reverse reaction, phosphorylating adenosine 5’-diphosphate (ADP) to adenosine 5’-triphosphate (ATP) in the presence of acetyl phosphate. Acetate kinase is used to recycle ATP required by pantothenate kinase (PanK) in the examples herein, whereas in some embodiments, the combination of acetyl phosphate and acetate kinase recycling could be replaced by other methods known in the art. As used herein, “pyruvate oxidase” (“PO”) refers to an enzyme (EC 1.2.3.3) dependent on the flavin adenine (FAD) and thiamine diphosphate nucleotides. Pyruvate oxidase is an enzyme belonging to the oxidoreductase family, specifically those that act on the aldehyde or oxo group of a donor with oxygen as an acceptor and catalyze the chemical reaction between pyruvate phosphate ion and biomolecular oxygen to form acetyl phosphate, carbon dioxide, and hydrogen peroxide. Pyruvate oxidase (PO) is employed in combination with acetate kinase (AcK) and catalase as a catalytic ATP regeneration combination in the examples herein, where the combination of enzymes catalyzes the formation of ATP from ADP in the presence of oxygen, pyruvate and phosphate ions, whereas in some modalities it could be replaced by other methods known in the technique. As used herein, “natural-type” and “naturally sourced” enzymes refer to the form found in nature. For example, a natural-type polypeptide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation. As used herein, “engineered,” “variant,” “mutant,” and “non-naturally occurring,” when used with reference to an enzyme that includes a polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a way that would not otherwise exist in nature. In some embodiments, the polypeptide is identical to a naturally occurring polypeptide but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. “Percent sequence identity”, “percent identity”, and “percent identical” with respect to the enzymes used herein to refer to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., spacing) compared to the reference sequences for optimal alignment of the two sequences.The percentage is calculated by determining the number of positions where either an identical nucleic acid base or amino acid residue occurs in both sequences, or where a nucleic acid base or amino acid residue aligns with a space to produce the number of matching positions. This number of matching positions is then divided by the total number of positions in the comparison window, and the result is multiplied by 100 to produce the percentage of sequence identity. Determination of optimal alignment and the percentage of sequence identity is performed using the BLAST and BLAST 2.0 algorithms (see, for example, Altschul et al., 1990, J. Mol. Biol. 215: 403–410 and Altschul et al., 1977, Nucleic Acids Res. 3389–3402). The software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. Briefly, BLAST analyses involve first identifying high-score sequence pairs (HSPs) by identifying short words of length W in the construct sequence that either match or meet some positive-value threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbor-word score threshold (Altschul et al., supra). These initial neighbor-word hits Q / znnn / Lznz / E / Yii act as seeds to initiate searches for the longest HSPs containing them. Word hits are then extended in both directions along each sequence until the cumulative alignment score increases. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a matching residue pair; always >0) and N (penalty score for non-matching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word hits in each direction stops when: the cumulative alignment score falls by X% of its maximum achieved value; the cumulative score goes below zero, due to the accumulation of one or more negative-scoring residue alignments; or the end of any sequence is reached.The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following omissions: a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses the following omissions: a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Nati Acad Sci USA 89:10915). Numerous other algorithms are available that function similarly to BLAST in providing the percentage of identity for two sequences. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, or by the similarity search method of Pearson and Lipman, 1988, Proc. Nati. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see in general, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a co-investment between Greene Publishing Associates, Inc. and John Wiley and Sons, Inc., (1995 Supplement) (Ausubel)).Additionally, the determination of sequence alignment and sequence identity percentage can be done using the BESTFIT or GAP programs in the GCG Wisconsin Software Package (Accelerys, Madison Wl), using provided default parameters. “Substantial identity” refers to a polynucleotide or polypeptide sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity, when compared to the reference sequence over a comparison window of at least 20 residue positions, frequently over a window of at least 3050 residues, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions totaling 20 percent or less of the reference sequence over the comparison window.In the specific modalities applied to polypeptides, the term “substantial identity” indicates that two polypeptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs using default space weights, share at least 80 percent sequence identity, preferably at least 89 percent sequence identity, and more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, residue positions that are not identical differ by conservative amino acid substitutions. Stereoselectivity refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, where the formation of one stereoisomer is favored over the other, or complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is referred to as enantioselectivity, the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is commonly reported alternatively in the technique (typically as a percentage) as the enantiomeric excess (ee), calculated according to the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer].Where stereoisomers are diastereomers, stereoselectivity is referred to as a diastereoselectivity, the fraction (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, commonly reported alternatively as the diastereomeric excess (de). Enantiomeric excess and diastereomeric excess are types of stereomeric excess. The phrase “suitable reaction conditions” refers to those conditions in the enzyme conversion reaction solution (e.g., enzyme loading ranges, substrate loading, temperature, pH, buffer solutions, cosolvents, etc.) under which each polypeptide used in the present invention is capable of converting a substrate to the desired product compound. Some exemplary suitable reaction conditions are provided herein. As used herein, “substrate” in the context of an enzymatic conversion reaction process refers to the compound or molecule acted upon by the modified enzymes used herein. As used herein, “product” in the context of an enzymatic conversion process refers to the compound or molecule that results from the action of an enzymatic polypeptide on a substrate. As used herein, “increasing” the yield of a product (e.g., a 4'-ethynyl-2'-deoxyribose phosphate analogue or 4'-ethynyl-2'-deoxynucleoside analogue) from a reaction occurs when a particular component present during the reaction (e.g., an enzyme) causes more product to be produced, compared to a reaction conducted under the same conditions with the same substrate, but in the absence of the component of interest. As used herein, “equilibrium” or “equilibrium” as used herein refers to the process that results in a steady-state concentration of the chemical species in a chemical or enzymatic reaction (e.g., interconversion of two species A and B), including interconversion of stereoisomers, as determined by the forward association constant and the reverse association constant of the chemical or enzymatic reaction. “Enantiomeric excess” (ee) is a measure of purity used for chital substances. It reflects the degree to which a sample contains one enantiomer in greater amounts than the other. For example, a racemic mixture has an ee of 0%, while a single, completely pure enantiomer has an ee of 100%; and a sample containing 70% of one enantiomer and 30% of the other has an ee of 40% (70% - 30%). Diastereomeric excess (de) is calculated in the same way as ee when only two diastereomers are present in the mixture. Protein, “enzyme”, polypeptide, and peptide are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, without regard to length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). Included within this definition are D- and L-amino acids and mixtures of D- and L-amino acids. As used herein, the term “approximately” means an acceptable error for a particular value. In some cases, “approximately” means within 0.05%, 0.5%, 1.0%, or 2.0% at the lower and upper ends of the given value range. With respect to pH, “approximately” means plus or minus 0.5. As used herein, “substantially pure” polypeptide or “purified” protein refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis, it is more abundant than any other individual macromolecular species in the composition), and is generally considered substantially purified when the target species comprises at least approximately 50 percent of the macromolecular species present on a molar or weight percent basis. However, in some embodiments, the composition comprises polypeptide that is less than 50% pure (e.g., approximately 10%, approximately 20%, approximately 30%, approximately 40%, or approximately 50%).In general, a substantially pure polypeptide composition comprises approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, approximately 95% or more, and approximately 98% or more of all macromolecular species (mole or weight percent) present in the composition. In some embodiments, the polypeptide is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) where the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, isolated polypeptides are substantially pure polypeptide compositions. As used herein, “enhanced property” of an enzyme refers to at least one improved property of an enzyme. In some embodiments, the present invention employs modified polypeptides PPM, PNP, DERA, PanK, AcK, SP, and / or GOase that exhibit an improvement in any enzymatic property as compared to a reference polypeptide PPM, PNP, DERA, PanK, AcK, SP, or GOase, respectively, and / or a natural-type polypeptide PPM, PNP, DERA, PanK, AcK, SP, or GOase, respectively, and / or another modified polypeptide PPM, PNP, DERA, PanK, AcK, SP, or GOase, respectively. In this way, the level of “enhancement” can be determined and compared among the various polypeptides, including natural-type as well as modified polypeptides. The properties Improved Q / znnn / Lznz / E / Yii properties include, but are not limited to, these properties such as increased protein expression, increased yield of proposed product, increased substrate specificity or affinity (i.e., increased activity on the substrate), increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzyme activity, increased specific activity, increased substrate resistance or inhibition of the final product, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, and altered temperature profile.In additional embodiments, the term is used with reference to at least one improved property of the enzymes PPM, PNP, DERA, PanK, AcK, SP, and / or GOase. In some embodiments, the present invention employs modified PPM, PNP, DERA, PanK, AcK, SP, and / or GOase polypeptides that exhibit an improvement in any enzymatic property as compared to a reference PPM, PNP, DERA, PanK, AcK, SP, and / or GOase polypeptide, respectively; and / or a natural-type polypeptide; and / or another improved PPM, PNP, DERA, PanK, AcK, SP, and / or GOase polypeptide, respectively. In this way, the level of “improvement” can be determined and compared among the various polypeptides, including both natural-type and modified polypeptides. As used herein, “conversion” (“conv” or “conv.”) refers to the enzymatic conversion (or biotransformation) of a substrate(s) to the corresponding product(s). “Percentage” conversion refers to the percentage of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzyme activity” or “activity” of a polypeptide can be expressed as the percentage conversion of substrate to product over a specific period of time. As used herein, “stereoselectivity” refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, where the formation of one stereoisomer is favored over the other, or complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is referred to as enantioselectivity, the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is commonly reported alternatively in the art (typically as a percentage) as the enantiomeric excess (“ee”) calculated according to the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer].Where stereoisomers are diastereomers, stereoselectivity is referred to as diastereoselectivity, the fraction (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, commonly reported alternatively as the diastereomeric excess (“of”). Enantiomeric excess and diastereomeric excess are types of stereomeric excess. The present process invention encompasses the use of modified polypeptides PPM, PNP, DERA, PanK, AcK, SP, and GOase, particularly those having SEQ ID NO.s 1 to 21, and sequences comprising one or more conservative amino acid substitutions that may be referred to as α / ζηηη / ίζηζ / Β / γι conservatively modified variants of each of SEQ ID NO.s 1 to 21. As used herein, “conservative” amino acid substitution refers to the substitution of amino acids in a protein with other amino acids that have similar characteristics (e.g., acidic, basic, charged, polar or nonpolar, side chain size, hydrophobicity / hydrophilicity, backbone conformation, or rigidity, etc.) such that the changes can be made frequently without altering the biological activity of the protein. This includes one or more substitutions of an amino acid in the polypeptide with a different amino acid within the same or a similar defined class of amino acids. Those experienced in the technique recognize that, in general, substitutions of individual amino acids in the non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)).Furthermore, substitutions of structurally or functionally similar amino acids are less likely to alter biological activity.By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.Additional exemplary conservative amino acid substitutions are set forth in Table 1. TABLE 1. Exemplary Conservative Amino Acid Substitutions Q / znn / Lznz / E / Yii Original residue Conservative substitution Wing (A) Gly; Ser Arg (R) Lys; His Assn (N) Gln; His Asp (D) Glu; Asn Cys (C) Ser; Only Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln lle(l) Leu; Val Leu (L) lie; Val Lys (K) Arg; His Original residue Conservative substitution Met (M) Leu; Hey; Tyr Phe (F) Tyr; Master; Leu Pro (P) Ser (S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) won; Soon α / ζηηη / ίζηζ / Β / γι The term amino acid substitution set, or substitution set, refers to a group of amino acid substitutions in a polypeptide sequence, compared to a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. A “functional fragment” refers to a polypeptide having an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding portions in the sequence to which it is being compared (e.g., a full-length engineered PPM, PNP, DERA, PanK, AcK, SP or GOase enzyme used in the present invention) and which retains substantially all the full-length polypeptide activity. As used herein, “deletion” refers to the modification of a polypeptide by the removal of one or more amino acids from the reference polypeptide. Deletions may comprise the removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids constituting the reference enzyme while retaining enzymatic activity and / or retaining the enhanced properties of an engineered PPM, PNP, DERA, PanK, AcK, SP, or GOase enzyme. Deletions may target internal and / or terminal portions of the polypeptide. In various forms, the deletion may comprise a continuous segment or may be discontinuous. Deletions are typically indicated by a symbol in the amino acid sequences. As used herein, “insertion” refers to the modification of the polypeptide by the addition of one or more amino acids from the reference polypeptide. Insertions may be internal portions of the polypeptide, or at the carboxy or amino-terminus. Insertions as used herein include fusion proteins as known in the art. The insertion may be a contiguous segment of amino acids or separated by one or more amino acids in the naturally occurring polypeptide. The additional acronyms and abbreviations used herein are as follows: LC-MS liquid chromatography-mass spectrometry g / L gram(s) per liter THF tetrahydrofuran mL milliliter(s) NMR nuclear magnetic resonance spectroscopy mmol millimole RT 0 rt room temperature (ambient, approximately 25°C) mg milligram sccm cubic centimeter standard per minute kg kilogram rpm revolutions per minute N Normal M mol / molarity conv conversion mM millimolar NMR nuclear magnetic resonance ML microliter(s) aqueous aq DMSO dimethyl sulfoxide hr, h hour(s) TsOH p-toluenesulfonic acid HPLC high-performance liquid chromatography Bn benzyl DCM dichloromethane CPME cyclopentyl methyl ether 2-MeTHF 2-Methyltetrahydrofuran MTBE methyl tert-butyl ether ESI Electrospray ionization HR-MS High-resolution mass spectrometry Experimental Procedures Preparation of 2-ethynyl-2-hydroxypropane-1,3-diyl diacetate (2) Method A: ο / ζηηη / ίζηζ / Β / γι 2 To a solution of diacetoxyacetone (1) (1) (159 g, 914.0 mmol) in THF (1000 mL) 1600 mL of a 0.5 M solution of ethynyl magnesium chloride in THF was added at -35°C, maintaining the temperature below -20°C. After the reaction reached completion, acetic acid (78 mL) in 400 mL of methyl tert-butyl ether (MTBE) was added dropwise, maintaining the temperature below -20°C. MTBE (800 mL) was then added, and the mixture was warmed to room temperature. Water-saturated NaCl (1000 mL) was added, followed by water-saturated NH4Cl solution (1050 mL). The organic layer was separated, dried over Na2SO4, and evaporated to yield compound (2) as an oil (160 g, 88%). 1H-NMR (CDCI3, 500 MHz): δ 4.26 (dd, 4H), 2.55 (s, 1H), 2.14 (s, 6H). Preparation of 2-ethynyl-propane-1,2,3-triol (3) Method B: Na+O o? znnn / lzoz / e / yia A 0.5M sodium methoxylate solution in methanol (69.9 mL, 35.0 mmol) was added to a solution of 2-ethynyl-2-hydroxypropane-1,3-diyl diacetate (2) (70 g, 350 mmol) in ethanol at room temperature (rt). The reaction was stirred at rt for 2 hours (h) to completion. The solvents were evaporated, and the residue was redissolved in 100 mL of water and extracted with 3 x 50 mL of MTBE. The aqueous layer was sprayed with nitrogen to remove residual solvents, yielding a 40.9% solution of 2-ethynylpropane-1,2,3-triol (3) (108 g, 100% yield) as determined by nuclear magnetic resonance (NMR) (maleic acid as internal standard). 1H-NMR (D2O, 500 MHz): δ 3.60 (dd, 4 H), 2.85 (s, 1H). Alternative Preparations of (R)-2-ethynyl-glyceraldehyde (4) Method C1: HO Galactose Oxidase In a stirred reactor, 2-ethynylpropane-1,2,3-triol (3) (1.1 g, 9.47 mmol) in sodium phosphate buffer (30 mL, 100 mM, pH 7.0) containing antifoam 204 (Sigma A6426, 1 drop ~ 20 pL) was heated to 30°C with air spray at 12.5 sccm. Galactose oxidase (GOase, SEQ ID NO.: 1) (250 mg), horseradish peroxidase* (Type I, 5 mg) and bovine catalase** (5 mg) dissolved in sodium phosphate buffer (5 mL 100 mM, pH 7.0) were added to the reactor, followed by the addition of aqueous CuSO4 solution (100 mM, 150 pL). The reaction mixture was stirred at 600 rpm with air spray for 47 h to provide (R)-2-ethynyl-glyceraldehyde (4) at 47% conversion (by NMR) and 72% ee. (The product was not isolated). 1H-NMR (D2O, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). * Horseradish Peroxidase: Type I natural horseradish peroxidase, commercially available from SIGMA (P8125), isolated from horseradish roots (Amoracia rusticana). ** Bovine catalase: commercially available bovine heme-dependent catalase from Sigma (C1345) Method C2: In a 100 L jacketed stirred reactor charged with deionized water (56.2 kg), sodium dihydrogen phosphate (1.212 kg, 10 moles) was added. The pH was adjusted to 7.02 using 10 N sodium hydroxide solution (852.6 g) at 25°C. The reactor was charged with Antifoam 204 (A6426, 10 mL), followed by CuSO₄·5H₂O (6.5 g). Galactose oxidase (451.2 g) (SEQ ID NO.: 10) was added, and the mixture was stirred for 15 min while being sprayed with air. Horseradish peroxidase* (200.2 g) and catalase** (502.6 g) were added, and the reactor was rinsed with water (2.0 kg). Then, 2-ethynylpropane-1,2,3-triol solution (3) in water (9.48%, 30.34 kg, 24.72 mol) was added, followed by an additional portion of Antifoam 204 (A6426, 10 mL). The reaction was air-sprayed and stirred overnight to provide 94.0 kg of (R)-2-ethynylglyceraldehyde (4) at 66% conversion (by NMR) and 84% ee. The assay yield was 60%: 1H-NMR (D2O, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). * Horseradish Peroxidase: Purified, commercially available, natural type horseradish peroxidase from Toyobo (PEO-301), isolated from horseradish roots (Amoracia rusticana). * * Bovine catalase: heme-dependent catalase of bovine origin, commercially available from Sigma (C1345). The above reaction was also carried out using galactose oxidase (SEQ ID NO.: 11) and product (4) was obtained at 67% conversion (by NMR) and 88% ee and assay yield 59%: 1H-NMR (D2O, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). Method C3: α / ζηηη / ίζηζ / Β / γι In a 100 mL EasyMax vessel equipped with a sprayer and flow controller, water (82 mL) and PIPES potassium buffer (5 mL, 0.5 M) were loaded. The pH was adjusted to 7.5 using 5 M KOH solution at 25°C. Antifoam 204 (200 µL) was added, followed by evolved galactose oxidase (SEQ ID NO.: 17,450 mg of enzyme powder) and copper(II) sulfate pentahydrate (100 µL, 100 mM). The reaction mixture was sprayed with air at 125 standard cubic centimeters per minute (sccm) for 15 min. Bovine catalase (C1345, Sigma-Aldrich, 150 mg, 2000-5000 U / mg, 0.75 MU) was loaded, followed by horseradish peroxidase (HRP, Toyobo PEO-301, 100 mg, 130 U / mg, 1.3 kU) and aqueous solution of 2-ethynyl-propane-1,2,3-triol (3) (25% wt, 12 mL, 25.8 mmol).The reaction mixture was stirred at 30°C with aeration at 125 sccm and sampled using EasySampler for 20 h to provide 70% conversion and form compound (4)(( / ?)-2-ethynylglyceraldehyde) in 58% assay yield and 99% ee 1H NMR (D2O, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). The crude reaction stream was fed directly to the subsequent phosphorylation step. Method C4: Oxidation with immobilized galactose oxidase immobilized galactose oxidase α / ζηηη / ίζηζ / Β / γι Enzyme immobilization procedure Ni Nuvia IMAC-loaded resin (16 mL based on the specified volume) was added to a filter funnel and washed with bonding buffer (10 column volumes, 160 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution. Lyophilized evolved galactose oxidase powder (SEQ ID NO.: 17, 2.00 g) was resuspended in copper(II) sulfate solution (100 pM; 5.00 mL) in a beaker, followed by the addition of bonding buffer (50 mL) and the resin. The solution was mixed using a rotary mixer at 20°C for 5 h. The resin was filtered and washed with bonding buffer (10 column volumes, 160 mL) and PIPES buffer (10 column volumes, 160 mL; 50 mM, pH 7.5) and used directly in a reaction. Reaction procedure: In a 100 mL EasyMax vessel equipped with a sprayer and flow controller, water (82 mL) and PIPES potassium buffer (5 mL, 1 M) were loaded. The pH was adjusted to 7.5 using 5 M KOH solution at 25°C. Antifoam 204 (200 µL) was added, followed by resin-immobilized evolved galactose oxidase (SEQ ID NO.: 17, 750 mg of enzyme powder per 6 mL of resin) and copper(II) sulfate pentahydrate (100 µL, 100 mM). The reaction mixture was sprayed with air at 125 standard cubic centimeters per minute (sccm) for 15 min. Bovine catalase (C1345, Sigma-Aldrich, 210 mg, 2000-5000 U / mg, 1.05 MU) was loaded, followed by horseradish peroxidase (HRP, Toyobo PEO-301, 100 mg, 130 U / mg, 1.3 kU) and aqueous 2-ethynylpropane-1,2,3-triol solution (3) (25 wt%, 13 mL, 29.4 mmol). The reaction mixture was stirred at 25°C with aeration at 125 sccm.After 22 h the reaction reached 91% conversion to provide a 200 mM (R)-2-ethynylglyceraldehyde solution (4) (100 mL, 68% assay yield, 97% ee 1H NMR (D2O, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). The crude reaction stream was taken directly into the subsequent phosphorylation step. Method C5: Optional isolation of aldehyde through aminal formation (8) Step 1: Preparation of (S)-2-(1,3-dibenzylimidazolidin-2-yl)but-3-yne-1,2-diol A 100 L jacketed cylindrical vessel equipped with a nitrogen bubble former, mechanical stirrer, and thermocouple was coupled with the crude oxidase reaction stream containing (R)-2-ethynylglyceraldehyde ((4), 26.0 kg, 1.85 wt. aldehyde, 3.64 mol) and was inert under a nitrogen atmosphere. The aqueous solution was heated to 20°C, and dimethyldodecane-1-amine oxide (DDAO) (30 wt. in water, 798 g, 0.96 mol) was added, followed by MTBE (55.3 kg, 76 L) and Λ / ,Λ / '-dibenzylethane-1,2-diamine (1.55 kg, 6.43 mol). The two-phase, brown mixture was stirred overnight at 20°C under a nitrogen atmosphere. After 17 hours, stirring was stopped and the organic phase was removed and discarded. A light brown MTBE solution of (S)-2-(1,3-dibenzylimidazolidine-2-yl)but-3-yne-1,2-diol (56.5 kg, 2.02% by animal weight, 3.39 mmol, 93% assay yield) was obtained. Six similar MTBE solutions were processed together in a single distillation and crystallization step (total 374.4 kg of solution, containing 7.91 kg of animal). A 50 L jacketed cylindrical vessel equipped with a mechanical stirrer, distillation head (condenser at -20°C), and thermocouple was charged with animal solution (45 L). A vacuum was applied to the vessel (65–95 torr), and the jacket was set to 40°C. The solvent was removed by distillation until a volume of 35 L was reached. At this point, the internal temperature was 6.1°C, and a whitish solid had begun to crystallize. The remaining MTBE solution was added slowly, maintaining a constant volume of 35–40 L and an internal temperature of 0–10°C. Once all the MTBE solution had been added, the volume was reduced to 25 L. Distillation was stopped, the vessel was inertized with nitrogen, and the jacket temperature was lowered to 10°C. The resulting pale yellow suspension was aged at this temperature for 2 hours, and the solids were collected by filtration. The filter thorax was washed with cold MTBE (-2°C) (12.7 kg) and then the low flow of nitrogen is dried for 7 hours. (S)-2-(1,3-dibencylimidazolidin-2-yl)-but-3-ino-1,2-diol is obtained as a white crystalline solid (5.75 kg). RMN-1H (500 MHz, DMSO-Ó6) δ 7.42 - 7.35 (m, 4H), 7.32 (td, J = 7.5, 1.6 Hz, 4H), 7.27 - 7.21 (m, 2H), 5.10 (t, J= 5.6 Hz, 1H), 5.03 (s, 1H), 4.28 (d, J= 13.3Hz, 1H), 4.16 (d, J = 13.3 Hz, 1H), 3.76 (s, 1H), 3.70-3.58 (m, 4H), 3.21 (d, J= 0.9 Hz, 1H), 2.90-2.80 (m, 2H), 2.60-2.51 (m, 2H). RMN-13C (126 MHz, DMSO-6) δ 140.0, 140.0, 128.5, 128.3, 128.2, 128.1, 126.8, 126.8, 88.6, 86.9, 75.0, 74.0, 66.4, 60.7, 60.5, 50.4, 50.3, 39.5. HR-MS (ESI) Aminal (Μ + H+) C21H25N2O2+ calculated 337.1911; encontrada 337.1922. Step 2: Preparation of (R)-2-ethinyl-glyceraldehyde (4) of aminal (8) α / ζηηη / ίζηζ / Β / γι A 4 L jacketed cylindrical vessel equipped with a nitrogen bubbler and mechanical stirrer was charged with TsOH*H2O (12.0 g, 63.1 mmol), water (60 mL), (S)-2-(1,3-dibenzylimidazolidine-2-11)but-3-eno-1,2-diol (110 g, 327 mmol), and MTBE (1700 mL). The two-phase mixture was placed under nitrogen, and the jacket temperature was set to 15°C. A solution of TsOH*H2O (114 g, 599.3 mmol) in water (600 mL) was added dropwise for 1.5 hours with overhead stirring (200 rpm). After the addition was complete, the jacket temperature was reduced to 5°C, and the resulting suspension was aged for 1 hour. The solids were removed by filtration and washed with cold water (270 mL). The two-phase solution was transferred to a separatory funnel and the organic phase was removed and discarded. The aqueous phase was treated with DOWEXMRMARATHONMRTA resin (hydroxide form, 11.0 g) and AMBERLYSTMR15 resin (hydrogen form, 11.0 g).0 g) while being sprayed with N2 at a rate of 200 sccm for 24 hours to remove residual MTBE. The resins were removed by filtration to provide a colorless aqueous solution of (R)-2-hydroxy-2-(hydroxymethyl)but-3-inal (774 g, 4.6 wt% aldehyde, 82% yield). 1H-NMR (500 MHz, D2O) δ 5.01 (s, 1H), 3.77 (d, J= 11.7 Hz, 1H), 3.73 (d, J= 11.7 Hz, 1H), 2.92 (s, 1H). NMR-13C (126 MHz, D2O) δ 129.4, 125.4, 90.3, 81.0, 76.0, 73.9, 65.3. HRMS (ESI) Aldehyde dimer (2M + Na+) Ci0Hi2NaO6+ calculated 251.0526; found 251.0530. Alternative Preparations of 3-phosphate ( / ?)-2-ethynyl-glyceraldehyde (5): Method D1: Acetate kinase: ATP regeneration system Pantothenate kinase Pank α / ζηηη / ίζηζ / Β / γι ATP Acetate kinase Acetate phosphate In a stirred reactor, a solution of adenosine diphosphate disodium salt (40 mg, 0.087 mmol) and magnesium chloride (38 mg, 0.400 mmol) in HEPES buffer (66 mM, pH 7.5, 30 mL) was added to (R)-2-ethynyl-glyceraldehyde (4) (1.9 mL, 210 g / L solution in water, 3.51 mmol), followed by acetate kinase (SEQ ID NO.: 3) (40 mg), and pantothenate kinase (SEQ ID NO.: 2) (120 mg). The reaction mixture was heated to 25°C and a potassium lithium salt solution of acetyl phosphate (1.3 g, 7.01 mmol) in HEPES buffer (50 mM, pH 7.5, 10 mL) was added dropwise for 4 hours, with the pH maintained at 7.5 using 5M sodium hydroxide. The reaction was stirred for 18 hours to give (R)-2-ethynyl-glyceraldehyde 3-phosphate (5) at 85% conversion (by HPLC) (the product was not isolated). 1H NMR (D2O, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): calculated for C5H7O6P (MH): 193.1; found 193.0. Method D2: Pyruvate Oxidase ATP Regeneration System Pantothenate kinase Pank ATP Acetate kinase Pyruvate kinase Pyruvate Phosphate U2 In a stirred reactor, a solution of sodium pyruvate (3.11 g, 28 mmol) and phosphoric acid (0.523 mL, 7.71 mmol) in 76 mL of water pH 7.5 was loaded with (R)-2-ethynyl-glyceraldehyde (4) (3.8 mL, 210 g / L solution in water, 7.01 mmol), disodium salt of adenosine diphosphate (80 mg, 0.174 mmol), thiamine pyrophosphate (40 mg, 0.086 mmol), flavin adenine dinucleotide disodium salt hydrate (64 mg, 0.077 mmol), and magnesium chloride (400 mL, 1 M solution in water, 0.4 mmol). The pH was adjusted to 7.5 with 5 M aqueous sodium hydroxide and the reaction volume was adjusted to 80 mL with water. Acetate kinase (SEQ ID NO.: 3) (80 mg), pyruvate oxidase (SEQ ID NO.: 4) (80 mg, lyophilized cell-free extracts), pantothenate kinase (SEQ ID NO.: 2) (400 mg), and catalase (800 mL, ammonium sulfate suspension CAT-101, Biocatalytics) were added. The reaction was stirred at 500 rpm and 30°C with air spray for 72 hours to provide (R)-2-ethynyl-glyceraldehyde 3-phosphate 5 at 95% conversion (by HPLC) (The product was not isolated). 1H-NMR (D2O, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): calculated for C5H7O6P (MH): 193.1; found 193.0. The above reaction was also carried out using pantothenate kinase (SEQ ID NO.: 13) and product 5 was obtained at 66% conversion. (The product was not isolated.) 1H-NMR (D2O, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). Method D3: Acetate kinase: ATP regeneration system using immobilized enzymes ο / ζηηη / ίζηζ / Β / γι Acetate phosphate Enzyme immobilization procedure: Nickel-loaded resin from NUVIAMR(IMAC) Immobilized Metal Ion Affinity Chromatography (168 mL based on the set volume) was added to a filter funnel and washed with bonding buffer (1.6 L; 500 mM sodium chloride, 50 mM sodium phosphate, pH 8.0). In a separate container, pantothenate kinase (8.4 g) (SEQ ID NO.: 12) and acetate kinase (2.8 g) (SEQ ID NO.: 3) were dissolved in bonding buffer (500 mL). The washed resin was loaded into the container, and the solution was stirred for 4 hours at 20°C. The resin was filtered and washed first with bonding buffer (1.6 L) followed by piperazine-N,N'-bis(2-ethanesulfonic acid) buffer (PIPES) (840 mL; 50 mM, pH 6.5). The washed resin was used directly in the next step. Reaction procedure: A 1 L reactor was charged with a solution of (R)-2-ethynyl-glyceraldehyde (4) in water (608.7 g, 4.6% wt, 212 mmol) and cooled to 5°C. To the cooled solution were added piperazine-N,N'-bis(2-ethanesulfonic acid) buffer (PIPES) (32.7 mL, 1 M, pH 6.5, 32.7 mmol), magnesium chloride (9.33 mL, 1 M, 9.33 mmol), diammonium acetyl phosphate salt (51.8 g, 265 mmol), disodium adenosine diphosphate salt hydrate (1.17 g, 2.12 mmol), and water (192 mL). The solution was stirred and the pH was adjusted to 6.4 using 5 N KOH. The reaction was heated to 20°C and 168 mL of resin with co-immobilized pantothenate kinase (SEQ ID NO.: 12) and acetate kinase (SEQ ID NO.: 3) were added. The reaction was stirred for 10 hours with 5 N KOH to maintain a pH of 6.4 to provide (R)-2-ethynylglyceraldehyde 3-phosphate (5) in 92% conversion (by HPLC) and 91% yield (by 31P NMR with tetraphenylphosphonium chloride as an internal standard) (the product was not isolated).1H NMR (D2O, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2 H), 2.88 (s, 1H). LC-MS: (ES, m / z): calculated for C5H7O6P (MH): 193.1; found 193.0. Preparation of 4-ethynyl-D-2-deoxyribose 5-phosphate (6) Method E: ο / ζηηη / ίζηζ / Β / γι To a solution of (R)-2-ethynyl-glyceraldehyde 3-phosphate (5) (5, 20 mL, 5.3 mmol) in water, an acetaldehyde solution in water (40% wt, 2.02 mL, 15.9 mmol) at room temperature was added, followed by the addition of Deoxyribose-phosphate (DERA) (SEQ ID NO.: 6), 25 mg of solution in triethanolamine hydrochloride buffer solution (1 mL, 1 M, pH 7.0). The reactor was sealed and the mixture was stirred overnight at 30°C and 600 rpm to provide 4-ethynyl-2-deoxyribose 5-phosphate (6) at 99% conversion and 99% ee, 99% as a 1:1 anomer mixture (Product not isolated), α-anomer: 1H-NMR (D2O, 600 MHz) δ 5.31 (t, 1H), 4.13 (t, 1H), 3.81-3.72 (m, 2H), 2.89 (s, 1H), 2.42-2.34 (m, 1H), 1.87-1.79 (m, 1H); 13C-NMR (D2O, 151 MHz) δ 97.7 (s), 81.4 (d), 79.4 (s), 78.9 (s), 71.1 (s), 67.7 (d), 39.6 (s). β-anomer: 1H-NMR (D2O, 600 MHz) δ 5.40 (dd, 1H), 4.28 (t, 1H), 3.88-3.80 (m, 2H), 2.87 (s, 1H), 2.13-2.06 (m, 1H), 2.04-1.97 (m, 1H); 13C-NMR (D2O, 151 MHz) δ 97.3 (s), 82.2 (d), 78.7 (s), 78.5 (s), 71.3 (s), 68.4 (d), 39.6 (s). LC-MS: (ES, m / z): calculated for C7H10O7P (MH): 237.0; found 237.0. Alternative preparations of (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2(hydroxymethyl)tetrahydrofuran-3-ol monohydrate (7) [alternative name 4'-ethynyl-2-fluoro-2'-deoxyadenosine or EFdA] Method F1: ((2R,3S)-2-ethynyl-3,5-dihydroxytetrahydrofuran-2-yl)methylammonium acid phosphate (1.00 g, 3.91 mmol) was dissolved in 10 mL of pH 7.5 buffer solution (100 mM triethanolamine HCl containing 5 mM MnCl2). The pH of the solution was adjusted to 7.3 with 5 N NaOH. 2-Fluoroadenine (0.599 g, 3.91 mmol) and sucrose (2.68 g, 7.82 mmol) were added to the solution. The enzyme solution was prepared by dissolving phosphopentomutase (SEQ ID NO.: 8) (100 mg), purine nucleoside phosphorylase (SEQ ID NO.: 9) (50 mg), and sucrose phosphorylase (SEQ ID NO.: 7) (10 mg) in 10 mL of pH 7.5 buffer. The enzyme solution was added to the reagent mixture, and the resulting suspension was stirred at 40°C. After 20 h, the suspension was cooled to 0°C and filtered, rinsing with cold water. The solid was dried. (5) (950 mL, 157 mmol) that ο / ζηηη / ίζηζ / Β / γι by suction to provide the title compound (1.12 g, 92%) as a single isomer. NMR-1H: (300 MHz, DMSO-d6, ppm): δ 7.68 (br s, 2H), 7.32 (d, J = 2.0 Hz, 1H), 6.44 (t, J = 5.8 Hz, 1H), 5.52 (d, J = 5.6 Hz, J = 17, t 6.0 Hz, 1H), 4.44 (q, J = 6.4 Hz, 1H), 3.60 (q, J = 6.0 Hz, 1H), 3.53 (q, J = 6.4 Hz, 1H), 3.48 (s, 1H), 2.48-2.41 (m, 1Hz, 2.37), 3.3 m. 1H). NMR-,3C (150.92 MHz, DMSO-d6, ppm) δ 158.5 (d, JCF = 203.5), 157.6 (d, JCF = 21.2), 150.2 (d, JCF = 20.2), 139.7 (d, JCF = 17.4), JCF = 17.4. 4.0), 85.1, 82.0, 81.4, 78.7, 70.1, 64.2, 38.1. LC-MS: (ES, m / z): calculated for C12H12FN5O3 (M+Na): 316.0822; found 316.0818. The PPM and PNP enzymes used in this step were each derived from mutations starting from E. coli (Escherichia coli) enzymes. Sucrose phosphorylase (SP) used in this step was derived from Alloscardovia omnicolens; the SP derived from other organisms could also be used. Method F2: r' Deoxyribose phosphate aldolase OHnN A2O,PO OH . „ + / , ~~~< phosphopentomustase ase 'ry S \ JL Λ -——----2XOH hNpurine nucleoside5phosphorylase To an aqueous solution of (R)-2-ethynyl-glyceraldehyde 3-phosphate containing piperazine-N,N'-bis(2-ethanesulfonic acid) buffer (PIPES) at a pH of approximately 5.5 to 6.0, triethanolamine (7.09 g, 47.5 mmol) was added. The pH of the solution was adjusted from 7.1 to 7.6 using potassium hydroxide (8 mL, 8M). Manganese(II) chloride hydrate (0.592 g, 4.70 mmol) was added, followed by sucrose (161 g, 470 mmol), giving a pH of 7.5. The following enzymes were added to the solution: deoxyribose-phosphate aldolase (SEQ ID NO.: 14) (461 mg), sucrose phosphorylase (SEQ ID NO.: 7) (494 mg), phosphopentomutase (SEQ ID NO.: 8) (2.63 g), and purine nucleoside phosphorylase (SEQ ID NO.: 15) (659 mg). Once the enzymes dissolved, 2-fluoroadenine (19.80 g, 125 mmol) was added. The reaction was heated to 35°C, and acetaldehyde (40 wt% in isopropyl alcohol, 29.8 mL, 235 mmol) was added. After the reaction lasted 2 h, the mixture was seeded with the crystalline product EFdA (0.96 g, 2 mol%). After the reaction for 26 ha at 35°C, the suspension was cooled to 0°C, and the solids were collected by filtration, washed with water twice (40 mL each). The solids were dried under a nitrogen flush. Yield 43.2 g, 92% by weight, 96.2% corrected. NMR-1H: (300 MHz, DMSO-d6, ppm): δ 7.68 (brs, 2H), 7.32 (d, J = 2.0 Hz, 1H), 6.44 (t, J = 5.8 Hz, 1H), 5.52 (d, J = 5.6 Hz, J = 5.2Hz), J 6.0 Hz, 1H), 4.44 (q, J = 6.4 Hz, 1H), 3.60 (q, J = 6.0 Hz, 1H), 3.53 (q, J = 6.4 Hz, 1H), 3.48 (s, 1H), 2.48-2.41 (m, 1Hz, 2.37), 3.3 m. 1H). NMR-,3C (150.92 MHz, DMSO-d6, ppm) δ 158.5 (d, JCF = 203.5), 157.6 (d, JCF = 21.2), 150.2 (d, JCF = 20.2), 139.7 (d, JCF = 17.4), JCF = 17.4. 4.0), 85.1, 82.0, 81.4, 78.7, 70.1, 64.2, 38.1. LC-MS: (ES, m / z): calculated for C12H12FN5O3 (M+Na): 316.0822; found 316.0818. Alternative preparations of 1-phosphate (S)-2-ethynyl-propane-1,2,3-triol I (9): Method G1: Kinase acetate: ATP regeneration system using SEQ enzymes. ID No.: 2y SEQ. ID No.: 3 Acetate kinase 2X+ Phosphate acetate α / ζηηη / ίζηζ / Β / γι A 50 mL reactor was charged with a solution of 2-ethynylpropane-1,2,3-triol (3) in water (9.29 g, 9.46 wt%, 7.57 mmol), PIPES potassium buffer (1.02 mL, 1 M, pH 6.5, 1.02 mmol), magnesium chloride (292 pL, 1 M, 0.292 mmol), diammonium acetyl phosphate salt (1.851 g, 89 wt%, 9.46 mmol), adenosine diphosphate disodium hydrate (ADP, 42 mg, 0.076 mmol, 0.01 eq), and water (28 mL). The pH was adjusted to 6.4 using 5 M KOH, the solution was heated to 20°C, and pantothenate evolved kinase PanK SEQ was added. ID No.: 2 (264 mg) and acetate kinase AcK SEQ. ID No.: 3 (88 mg). The reaction was stirred for 16 hours with pH maintained at 6.4 using 5 N KOH. The contents of the final reaction yielded 1-phosphate (S)-2-ethynylpropane-1,2,3-triol (9) in >95% ee and 99% conversion (by 31P-NMR). The product was not isolated. 1H-NMR (D2O, 500 MHz) δ 3.89 (m, 2H), 3.72 (d, J = 11.6 Hz, 1H), 3.65 (d, J = 11.6 Hz, 1H), 2.93 (s, 1H). 13C-NMR (D2O, 126 MHz) δ 82.9 (s), 75.1 (s), 71.0 (d, J = 6.9 Hz), 67.0 (d, J = 4.5 Hz), 64.7 (s). 31P-NMR (D2O, 202 MHz) δ 3.39. HRMS: (ESI, m / z): calculated for [M-1 ] · CsHsOeP: 195.0058; Found 195.0068 [MH]-: 195.0058. Method G2: Acetate kinase: ATP regeneration system using enzyme SEQ. ID No.: 20 and enzyme SEQ. ID No.: 21 Acetate kinase 2X+ Acetate phosphate (S)-9 A jacketed reactor was charged with an aqueous solution of 2-ethynylpropane-1,2,3-triol (3) (11.47 kg, 8.7 wt%, 8.61 mol) and water (7.5 kg), followed by 1 M BIS-TRIS methane buffer solution pH 6.5 (1 L) and magnesium chloride (41.4 g). ATP (48 g, 0.086 mol, 0.01 equivalent) and diammonium acetyl phosphate (2.021 kg, 89%, 10.33 mmol) were added, the solution was heated to 20°C, and the pH was adjusted to 6.8 using KOH (270.4 g). Evolved pantothenate kinase SEQ. ID No.: 20 (20.4 g) and evolved acetate kinase SEQ. ID No.: 21 (3 g) were then charged as solids. The reaction was stirred at 20°C for 16 h, during which time the pH dropped to 5.5. The quantitative conversion of 2-ethynylpropane-1,2,3-triol (3) was obtained as evaluated by 1H and 31P NMR. This prepared 1-phosphate (S)-2-ethynylpropane-1,2,3-triol (9) solution (397 mM, 22.5 kg, 98% yield) was used in the subsequent oxidation step without any further purification.1H-NMR (D2O, 500 MHz) δ 3.89 (m, 2H), 3.72 (d, J = 11.6 Hz, 1 H), 3.65 (d, J = 11.6 Hz, 1H), 2.93 (s, 1H). Method G3: Acetate kinase: ATP regeneration system using enzyme SEQ. ID No.: 20 and enzyme SEQ. ID No.: 21 with deuterated compound (3) to assign absolute stereochemistry and show desymmetrization phosphorylation. 3-d2, 955 erPhosphate acetate' (S)-9-(3,3-d2) (5)-3-(1,1-d2) 5 ο / ζηηη / ίζηζ / Β / γι Pantothenate evolved kinase SEQ. ID No.: 20 (100 pL of 10 g / L solution in water) and acetate evolved kinase SEQ. ID No.: 21 (100 pL of 2 g / L solution in water) were added to a solution containing diammonium acetyl phosphate (41 mg), 2-ethynylpropane-1,1-O2-1,2,3-triol ((R)-3-d2, 20 mg, 170 pmol), manganese chloride (10 pL of 1 M solution in water), ADP (10 pL of 100 g / L solution in water), and sodium phosphate buffer solution (10 pL of 1 M solution in water) in water (800 pL) at pH 6.5. The reaction was incubated for 24 h at room temperature to provide deuterated 2-ethynylpropane-1,2,3-triol 1-phosphate analogs (S)-9-(3,3-d2) and (S)-9-(1,1-d2) in a 95:5 ratio and 99% overall yield. The ratio of phosphorylated compounds was determined by 31P-NMR, which is ~95:5, confirming the stereoselective phosphorylation of 2-ethynylpropane-1,2,3-triol (3) at the pro-(S) hydroxyl group (i.e., a desimerizing phosphorylation). 1H-NMR (D2O, 500 MHz) δ 3.89 (m, 2H), 3.72 (d, J = 11.6 Hz, 1 H), 3.65 (d, J= 11.6 Hz, 1H), 2.93 (s, 1H).13C NMR (D2O, 126 MHz) δ 82.9 (s), 75.1 (s), 71.0 (d, J = 6.9 Hz), 67.0 (d, J = 4.5 Hz), 64.7 (s). Method G4: Acetate kinase: ATP regeneration system using immobilized enzymes SEQ. ID No.: 20 and enzyme SEQ. ID No.: 21 Acetate kinase 2X Acetate phosphate (S)-9 Enzyme immobilization procedure: Ni Nuvia IMAC-loaded resin (75 mL based on the specified volume) was added to a filter funnel and washed with water (9 column volumes, 3 x 225 mL) and bonding buffer (1 column volume, 75 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). Lyophilized pantothenate kinase powder (SEQ ID NO.: 20, 6.0 g) was resuspended in bonding buffer (200 mL) in a separate container, and the washed resin was added. The solution was mixed using a rotary mixer at 25°C for 6 h. The resin was filtered and washed with bonding buffer (6 column volumes, 6 x 225 mL) and BIS-TRIS buffer (8 column volumes, 600 mL; 50 mM, pH 6.2). Reaction procedure: An aqueous solution of 2-ethynylpropane-1,2,3-triol (3) (574 g, 8.7 wt%, 0.430 mol) and water (350 mL) was loaded into a jacketed reactor, followed by 1 M BIS-TRIS methane buffer solution pH 6.5 (50 mL) and magnesium chloride (2.033 g, 0.01 mol). ATP (2.37 g, 0.0043 mol, 0.01 eq) and diammonium acetyl phosphate (101 g, 89%, 0.530 mmol, 1.2 eq) were added, the solution was heated to 20°C, and the pH was adjusted to 6.8 using 5 M KOH. The resin with immobilized pantothenate kinase SEQ. ID No.: 20 (25 mL) and evolved acetate kinase SEQ. ID No.: 21 (0.15 g) were then loaded as solids. The reaction was stirred at 20°C for 16 h during which the pH decreased to 5.5. The quantitative conversion of 2-ethynylpropane-1,2,3-triol (3) to (S)-2-ethynylpropane-1,2,3-triol 1-phosphate (9) was obtained as evaluated by 1H-NMR and 31P-NMR. 1H-NMR (DZO, 500 MHz) δ 3.89 (m, 2H), 3.72 (d, J = 11.6 Hz, 1 H), 3.65 (d, J = 11.6 Hz, 1H), 2.93 (s, 1H). Alternative preparations of (R)-2-ethynyl-glyceraldehyde 3-phosphate (5): Method H1: Immobilized Galactose Oxidase EQ ID No.: 16 ο / ζηηη / ίζηζ / Β / γι Enzyme immobilization procedure: Ni Nuvia IMAC-loaded resin (10 mL based on the specified volume) was added to a filter funnel and washed with bonding buffer (10 column volumes, 100 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution and yield 16 g of washed resin. In a separate container, lyophilized evolved galactose oxidase powder (SEQ ID NO.: 16, 750 mg) was resuspended in copper(II) sulfate solution (100 μM; 5.00 mL), followed by the addition of bonding buffer (20 mL) and the washed resin (3.0 g). The solution was mixed using a rotary mixer at 20°C for 5 h. The resin was filtered and washed with bonding buffer (10 column volumes, 100 mL) and BIS-TRIS buffer (10 column volumes, 100 mL; 50 mM, pH 7.5) and used directly in the glycosylation reaction. Reaction procedure: The resin with immobilized galactose oxidase SEQ ID NO.: 16 (3.0 g) was added to a solution of (S)-2-ethynyl-propane-1,2,3-triol 1-phosphate (9, 5.4 mmol, 270 mM, 20 mL) in BIS-TRIS methane buffer (35 mM, pH adjusted to 7.2), followed by the addition of copper(II) sulfate solution in water (30 pL, 100 mM) and horseradish peroxidase (PEO-301, 18 mg) and bovine catalase (C1345, 120 mg) resuspended in water (600 pL). The reaction was sealed with a gas-permeable membrane and vigorously shaken at 22°C for 4 days to achieve a final conversion of 77% and provide (R)-2-ethynylglyceraldehyde 3-phosphate (5) at 95% ee. The enzyme resin was filtered, and the (R)-2-ethynylglyceraldehyde 3-phosphate (5) solution was used directly in the glycosylation reaction. 1H-NMR (D2O, 400 MHz): 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): calculated for C5H7O6P (MH): 193.1; found 193.0. Method H2: Immobilized Galactose Oxidase SEQ ID No.: 17 Galactose Oxidase ο / ζηηη / ίζηζ / Β / γι Enzyme immobilization procedure: Ni Nuvia IMAC-loaded resin (10 mL based on the specified volume) was added to a filter funnel and washed with bonding buffer (10 column volumes, 100 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution and yield 16 g of washed resin. In a separate container, lyophilized evolved galactose oxidase powder (SEQ ID NO.: 16, 750 mg) was resuspended in copper(II) sulfate solution (100 μM; 5.00 mL), followed by the addition of bonding buffer (20 mL) and the washed resin (3.0 g). The solution was mixed using a rotary mixer at 20°C for 5 h. The resin was filtered and washed with bonding buffer (10 column volumes, 100 mL) and BIS-TRIS methane buffer (10 column volumes, 100 mL; 50 mM, pH 7.5) and used directly in the reaction. Reaction procedure: The resin with immobilized evolved galactose oxidase SEQ ID NO.: 17 (3.0 g) was added to a solution of (S)-2-ethynyl-propane-1,2,3-triol 1-phosphate (9, 5.4 mmol, 270 mM, 20 mL) in BIS-TRIS methane buffer (35 mM, pH adjusted to 7.2), followed by the addition of copper(II) sulfate solution in water (30 pL, 100 mM) and horseradish peroxidase (PEO-301, 18 mg) and bovine catalase (C1345, 120 mg) resuspended in water (600 pL). The reaction was sealed with a gas-permeable membrane and vigorously stirred at 22°C for 4 days to achieve a final conversion of 77% and provide 3-phosphate (R)-2-ethynylglyceraldehyde (5) at 95% ee. The enzyme resin was filtered, and the 3-phosphate (R)-2-ethynylglyceraldehyde (5) solution was used directly in the glycosylation reaction. NMR-Ή (DzO, 400 MHz): 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): calculated for C5H7O6P (MH): 193.1; found 193.0. Method H3: Immobilized Galactose Oxidase SEQ ID No.: 18 Enzyme immobilization procedure: Ni Nuvia IMAC-loaded resin (3 mL based on the specified volume) was added to a filter funnel and washed with bonding buffer (10 column volumes, 30 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution and yield 2.4 g of washed resin. Lyophilized evolved galactose oxidase powder (SEQ ID NO.: 18, 75 mg) was resuspended in copper(II) sulfate solution (100 μM; 1.00 mL) in a vial, followed by the addition of bonding buffer (5 mL) and the washed resin (400 mg). The solution was washed using a rotary mixer at 20°C for 5 h. The resin was filtered and washed with bonding buffer (10 column volumes, 4 mL) and BIS-TRIS methane buffer (10 column volumes, 4 mL; 50 mM, pH 7.5) and used directly in a reaction. Reaction procedure: Immobilized evolved GOase SEQ ID NO.: 18 (400 mg) was added to a solution of (S)-2-ethynylpropane-1,2,3-triol 1-phosphate ((9), 5.4 mmol, 270 mM, 1 mL) in BIS-TRIS methane buffer (35 mM, pH adjusted to 7.2), followed by the addition of horseradish peroxidase (PEO-301, 1 mg) and Corynebacterium glutamicum catalase (Roche, lyophilized, #11650645103, 3 mg) resuspended in water (100 pL). The reaction was sealed with a gas-permeable membrane and vigorously shaken at 30°C for 48 h. The final conversion after 2 days reached 90%, and the (R)-2-ethynylglyceraldehyde 3-phosphate (5) was >99% ee. The enzyme resin was filtered, and the (R)-2-ethynylglyceraldehyde 3-phosphate (5) solution was used directly without further purification. 1H-NMR (D2O, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): calculated for C5H7O6P (MH): 193.1; found 193.0. Method H4: Immobilized Galactose Oxidase SEQ ID No.: 19 Q / znnn / Lznz / E / Yii Enzyme immobilization procedure: Ni Nuvia IMAC-loaded resin (3 mL based on the specified volume) was added to a filter funnel and washed with bonding buffer (10 column volumes, 30 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution and yield 2.4 g of washed resin. Lyophilized evolved galactose oxidase powder (SEQ ID NO.: 19, 75 mg) was resuspended in copper(II) sulfate solution (100 μM; 1.00 mL) in a vial, followed by the addition of bonding buffer (5 mL) and the washed resin (400 mg). The solution was washed using a rotary mixer at 20°C for 5 h. The resin was filtered and washed with bonding buffer (10 column volumes, 4 mL) and BIS-TRIS methane buffer (10 column volumes, 4 mL; 50 mM, pH 7.5) and used directly in a reaction. Reaction procedure: Immobilized evolved GOase SEQ ID NO.:18 (400 mg) was added to a solution of 1-phosphate (S)-2-ethynylpropane-1,2,3-triol (9, 5.4 mmol, 270 mM, 1 mL) in BIS-TRIS methane buffer (35 mM, pH adjusted to 7.2), followed by the addition of horseradish peroxidase (PEO301, 1 mg) and Corynebacterium glutamicum catalase (Roche, lyophilized, #11650645103, 3 mg) resuspended in water (100 pL). The reaction was sealed with a gas-permeable membrane and vigorously shaken at 30°C for 48 h. The final conversion after 2 days reached 100%, and 3-phosphate (R)-2-ethynylglyceraldehyde (5) was obtained at >99%. The enzyme resin was filtered, and the 3-phosphate (R)-2-ethynylglyceraldehyde (5) solution was used directly without further purification. 1H-NMR (D2O, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): calculated for C5H7O6P (MH): 193.1; found 193.0. “Amino acids” are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-ILJB Commission on Biochemical Nomenclature. For the purposes of this description, the codes used for the genetically encoded amino acids for the enzymes used in the methods herein are conventional and are shown in Table 2: ο / ζηηη / ίζηζ / Β / γι TABLE 2 Amino Acid One-Letter Code Amino Acid One-Letter Code alanine A isoleucine I arginine R leucine L asparagine N lysine K aspartic acid D methionine M asparagine or aspartic acid B phenylalanine F cysteine ​​C proline P glutamic acid E serine S glutamine Q threonine T glutamine or glutamic acid Z tryptophan W glycine G tyrosine Y histidine H valine V The sequence ID numbers for the enzymes used, or that could be used, in the process for synthesizing EFdA described herein and in the process steps identified in the Experimental Procedures described herein are provided, but not limited to, those in Table 3. TABLE 3 SEQ ID NO: ENZYME AND AMINOACID SEQUENCE 1 Galactose oxidase (GOase) = Variant of Galactose oxidase of Fusarium qramínaarum (formerly known as Dactvlium dendroidea) IDMKTTQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGTNWGSPVASGSWFADSTTK YSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVP AAAAIEPTSGRVLMWSSYRNDAFEGSPGGITLTSSWDPGSTVSTGSTV PGISMDGNGQIWDETATGGNDAKKTSLYDSSSDSWIPGPDMQVARGYQSSATMSD GRVFTIGGSFSGGRVEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYRSDNHAW LFGWKKGSVFQAGPSTAMNWYTSGSGDVKQCGSAGVAGVA AVKGKILTFGGSPDYEDSDATTNAHIITLGEPGTSPNTVFASNGLYFARTFHTSVVLPD GSTFITGGQRRGIPTEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFN GGGGLCGDCTTNHFDAQIFTPNYLYDSNGSTQTQRTQTIKSTGST SKASLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNS AGVPSVASTIRVTQGGGGSWSHPQFEK 2 Pantothenate Cinase (PanK) = Variant of E. coli Pantothenate Cinase MSIKEQTLMTPYLQFDRNQWAALRDSVPMTLSEDEIARLKGINEDLSLEEVAEIYLPLSRLLNFYISSNLRRQAVLEQFLGTNGQRIPYIISIAGSVAVGKSTTARVLQALLSRWPEHR RVELITTDGFLHPNQVLKERGLMKKKGFPESYDMHRLVKFVSDLKSGVPNVTAPVYS HLIYDVIPDGDKTVVQPDILILEGLNVLQSGMDYPHDPHHVFVSDFVDFSIYVDAPEDLL QTWYINRFLKFREGAFTDPDSYFHNYAKLTKEEAIKTAMTIWKEMNWLNLKQNILPTR ERASLILTKSANHAVEEVRK 3 Aceta Chinasa (ApocetaKto natural). of Thermotoca marítima MGSHHHHHHGSRVLVINSGSSSIKYQLIEMEGEKVLCKGIAERIEGSRLVHRVGDEK HVIERELPDHEEALKLILNTLVDEKLGVIKDLKEIDAVGHRVVHGGERFKESVLVDEEVL KAIEEVSPLAPLHNPANLMGIKAAMKLLPGVPNVAVFDTAFHQTIPQKAYLYAIPYEYYE KYKIRRYGFHGTSHRYVSKRAAEILGKKLEELKIITCHIGNGGASVAAVKYGKCVDTSMG FTPLEGLVMGTRSGDLDPAIPFFIMKEGILSQYDKSQYDKGGGGGG EEAALKGDEWCKLVLEIYDYRIAKYIGAYAAAMNGVDAIVFTAGVGENSPITREDVCSY LEFLGVKLDKQKNEETIRGKEGIISTPDSRVKVLVVPTNEELMIARDTKEIVEKIGR 4 Pyruvate Oxidase (PO) = Pyruvate oxidase tipo of Streophiptolus natural MGSSHHHHHHSSGLVPRGSHMTVGKTKVSTASLKVLAGWGIDTIYGIPSGTLAPLME ALGEQEETDIKFLQVKHEEVGAMAAVMQWKFGGKLGVCVGSGGGPGASHLYDAAMDNTPVLAILGSPPQRELNMDAFQELNQNPMYDHIAVYNRRVAYAEQLPKLIDDAIR TAISKRGVAVLEVPGDFGYKEIANDAFYSSGHSYRDYVSSAINEVDIDAAVEVLNKSKR AVIYAGIGTMGHGPAVQELSRKIKAPIITTAKNFETFDYDFEGLTGSTYRVGWKPANEA VKEADTVLFVGSNPFFAEVEGTFSNVENFIQIDNNPTMLGKRHNADVAILGDAGEAVQ α / ζηηη / ίζηζ / Β / γι MLLEKVAPVEESAWWNANLKNIQNWRDYMTKLETKENGPLQLYQVYNAINKYADEDA IYSIDVGNTTQTSIRHLHMTPKNMWRTSPLFASMGIALPGGIGAKNVYPERQVFNLMG DGAFSMNYQDIVTNVRYNMPVINWFTNTEYGFIKNKYEDTNTNTFGTEFTDVDYAMI GEAQGAVGFTVSRIEDMDQVMAAAVKANKEGKTVVIDAKITKDRPIPVETLKLDPALYS EEEIKAYKERYEAEELVPFSEFLKAEGLESKVAK 5 Desoxiribosa-fosfato Aldolasa (DERA) = Desoxiribosa-fosfato Aldolasa tipo natural de Shewanella halifaxensis MSDLKKAAQQAISLMDLTTLNDDDTDQKVIELCHKAKTPAGDTAAICIYPRFIPIARKTL NEIGGDDIKIATVTNFPHGNDDIAIAVLETRAAVAYGADEVDWFPYRALMEGNETVGF ELVKACKEACGEDTILKVIESGVLADPALIRKASELSIDAGADFIKTSTGKVAVNATLEA AEIMMTVISEKNPKVGFKPAGGVKDAAAAAAEFLGVAARLLGDDWATPATRFRFGASSLL TNLLHTLELADAPQGAQGY 6 Desoxiribosa-fosfato Aldolasa (DERA) = Variante of Desoxiribosa-fosfato Aldolasa (DERA) of Shewanella halifaxensis MCDLKKAAQRAISLMDLTTLNDDDTDQKVIELCHKAKTPAGDTAAIVIYPRFIPIARKTL NEIGGLDIKIVTVTNFPHGNDDIAIAVLETRAAVAYGADEVDWFPYRALMEGNETVGF ELVKACKEACGEDTILKVIESGVLKDPALIRKASEISIDAGADFIKTSTGKVAVNATLEAAEIIMTVISEKNPKVGFKPAGGIKDAAAAAEFLGVAARLLGDDWATPATFRFGATDLLTN LLHTLELADPQGAQGY 7 Sucrose phosphorylase (SP) = Sucrose phosphorylase natural type of Alloscardovia omnicolens MKNKVQLITYADRLGDGTLKSMTETLRKHFEGVYEGVHILPFFTPFDGADAGFDPVDH TKVDPRLGSWDDVAELSTTHDIMVDTIVNHMSWESEQFQDVMAKGEDSEYYPMFLT MSSIFPDGVTEEDLTAIYRPRPGLPFTHYNWGGKTRLVWTTFTPQQVDIDTDSEMGW NYLLSILDQLSQSHVSQIRLDAVGYGAKEKNSSCFMTPKTFKLIERIKAEGEKRGLETLI EVHSYYKKQVEIASKVDRVYDFAIPGLLLHALEFGKTDALAQWIDVRPNNAVNVLDTH DGIGVIDIGSDQMDRSLAGLVPDEEVDALVESIHRNSKGESQEATGAAASNLDLYQVN CTYYAALGSDDQKYIAARAVQFFMPGVPQVYYVGALAGSNDMDLLKRTNVGRDINRH YYSAAEVASEVERPWQALNALGRFRNTLSAFDGEFSYSNADGVLTMTWADDATRAT LTFAPKANSNGASVARLEWTDAAGEHATDDLIANPPVVA 8 Phosphopentomutase (PPM) = Variant of Phosphopentomutase of E. coli MKRAFIMVLDSFGIGATEDAERFGDVGADTLGHIAECAKGEADNGRKGPLNLPNLTR LGLAKAHEGSTGFIPAGMDGNAEVIGAYAWAHEMSGKDSVSGHWEIAGVPVLFEW GYFSDHENSFPQELLDKLVERANLPGYLGNCRSSGTVILDQLGEEHMKTGKPIFYTSA ASVFQIACHEETFGLDKLYELCEIAREELTNGGYNIGRVIARPFIGDKAGNFQRTGNRRDLAVEPPAPTVLQKLVDEKHGQWSVGKIADIYANCGITKKVKATGLDALFDATIKEMK EAGDNTIVFTNFVDFDSSWGHRRDVAGYAAGLELFDRRLPELMSLLRDDDILILTADH GCDPTWTGTDHTREHIPVLVYGPKVKPGSLGHRETFADIGQTLAKYFGTSDMEYGKA ο / ζηηη / ίζηζ / Ε / γΐι MF 9 Purina nucleoside phosphorylase (PNP) = Variant of Purina nucleoside phosphorylase of E. coli MATPHINAEMGDFADVVLMPGDPLRAKYIAETFLEDAREVNNVRGMLGFTGTYKGRKI SVMGHGAGIPSCCSIYTKELITDFGVKKIIRVGSCGAVLPHKWGKVKL RIRFKDHDFAAIADFDMVRNAVDAAKALGIDARVGNLFSADLFYSPDGEMFDVMEKYG ILGVEMEAAGIYGVAAEFGAKALTICTVSDHIRTHEQTTAAERQTTFNDMIKIALESVLL GDKE 10 Galactose oxidase (GOase) = Variant of Galactose oxidase (GOase) como Dactvlium dendroides) MASAPIGVAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTQYGVNGDPKPPHTTITI DMKTVQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGVNWGSPVASGSWFADSTTK YSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVP SAAAIEPTSGRVLMWSSYRQDAFEGSPGGITLTSSWDPSTGIVSDRTSTVTKHDMFC PGISMDGNGQIWSGGGNDAKTSLYDSSSWIPGPQYFQGVGVGGVMSGRV TIGGSFSGGQVEKNGEVYSPSKTWTSLPNAKVNPMLTADKQGLYRSDNHAWLFGW KKGSVFQAGPSTAMNWYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKG KILTFGGSPDYEDSDATTNAHIITLGEPGTS TGGQQRGIPTEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSILLLPDGRVFNGGGGLCGDCTTNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSWVGGWITIWTDMSISAA SLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGV PS VASTIR VTQG QTG Η Η Η Η Η H 11 Galactosa oxidasa (GOasa) - Variant de Galactosa oxidasa de Fusaríum araminaarum (anteriormente conocida como Dactvlium dendroides) MASAPIGVAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTQYGVNGDPKPPHTITI DMKTVQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGVNWGSPVASGSWFADSTTK YSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVP SAAAIEPTSGRVLMWSSYRQDAFEGSPGGITLTSSWDPSTGIVSDRTSTVTGHDMFC PGISMDGNGQIWSGGNDAKKTSLYDSSSDSWIPGPDMQVARGYNSSATMSDGRVF TIGGSFSGGQVEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYRSDNHAWLFGW KKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSNRGVAPDAMCGNAVMYDAVKG KILTFGGSPDYQDSDATTNAHIITLGEPGTSPNTVFASNGLLFARTFHTSVVLPDGSTFI TGGQQRGIPTEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGG GLCGDCETNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVWGGWITIWTDMSISAA SLIRYGTATHTVNTDQRRIPLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGV PSVASTINVTQGQTGHHHHHH 12 Pantotenato Cinasa (PanK) = Variant of PantotenatoCinase of E. coli MSIKEQTLMTPYLQLDRNQWAALRDSNPMTLSEDEIARLKGINEDLSLEEVAEVYLPLS α / ζηηη / ίζηζ / Β / γι RLLNFYISSNLRRQAVLEQFLGTNGQRIPYIISIAGSVAVGKSTTARVLQALLSRWPEHR RVELITTDGFLHPNQVLKERGLMKKKGFPESYDMHRLMKFVKDLKSGVPNVTAPVYS HLIYDVIPDGDKTVVQPDILILEGLNVLQSGMDYPHDPHHVFVSDFVDFSIYVDAPEDLL QTWYINRFLKFREGAFTDPDSYFHGYAKLTKEEAIKTAMTIWKEMNHLNLKQNILPTRE RASLILTKSANHIVEEVRLRK 13 Pantothenate Kinase (PanK) = Variant of Pantothenate Kinase from E.coli MHHHHHHGGMSIKEQTLMTPYLQLDRNQWAALRDSNPMTLSEDEIARLKGINEDLSL EEVAEVYLPLSRLLNFYISSNLRRQAVLEQFLGTNGQRIPYIISIAGSVAVGKSTTARVL QALLSRWPEHRRVEHITTDGFLHPNQVLKERGLMGKKGFPESYDMHRLMKFVKDLKS GVPNVTAPVYSHLIYDVIPDGDKTWQPDILILEGLNVLQSGMDYPHDPHHVFVSDFVD FSIYVDAPEDLLQTWYINRFLKFREGAFTDPDSYFHGYAKLTKEEAIKTAMTIWKEMNH LNLKQNILPTRERASLILTKSANHIVEEVRLRK 14 Desoxiribosa-fosfato Aldolasa (DERA) = Variante de Desoxiribosa-fosfato Aldolasa (PERA) of Shewanella halifaxensis MHHHHHHCDLKKAAQRAISLMDLTTLNDDDTDQKVIELCHKAKTPAGDTAAIVIYPRFI PIARKTLNEIGGLDIKIVTVTNFPHGNDDIAIAVLETRAAVAYGADEVDVVFPYRALMEG NETVGFELVKACKEACGEDTILKVIESGVLKDPALIRKASEISIDAGADFIKTSTGKVAV NATLEAAEIIMTVISEKNPKVGFKPAGGIKDAAAAAAEFLGVAARLLGDDWATPATRFG ATDLLTNLLHTLELADAPQGAQGY 15 Purina nucleósido fosforilasa (PNP) = Variante of Purina nucleósido Fosforilase of E.coli MATPHINAEMGDFADVVLMPGDPLRAKYIAETFLEDAREVNNVRGMLGFTGTYKGRKI SVMGHGMGIPSCSIYTKELITDFGVKKIIRVGSCGAVLPHVKLRDVVIGMGACTDSKVN RIRFKDHDFAAIADFDMVRNAVDAAKALGIDARVGNLFSADLFYSPDGEMFDVMEKYG ILGVEMEAAGIYGVAAEFGAKALTICTVSDHIRTHEQTTAAERQTTFNDMIKIALESVLL GDKE 16 Galactosa oxidasa (GOasa) = Variant of Galactosa oxidasa of Fusaríum araminaarum (anteriormente conocida como Dactvlium dendroides} MASAPIGVAIPRNNWAVTCDSAQSGNECNKAIDGNKDTFWHTQYGVNGDPKPPHTITI DMKTVQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGVNWGSPVASGSWFADSTTK YSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVP SAAAIEPTSGRVLMWSSYRQDAFEPSPGGITLTSSWDPSTGIVSDRTSTVTGHDMFC PGISMDGNGQIWSGGNDAKKTSLYDSSSDSWIPGPDMQVARGYNSSATMSDGRVF TIGGSYSGGQVEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYRSDNHAWLFGW KKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSDRGVAPDAMCGNAVMYDAVKG KILTFGGSPDYQDSDATTNAHIITLGEPGTSPNTVFASNGLLFARTFHTSVVLPDGSVFI TGGQQRGVPLEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGG. α / ζηηη / ίζηζ / Β / γι Galactose oxidase (GOase) = Variant of Galactose oxidase from Fusarium qraminaarum (previously known as (Dactylium dendroides) SAAAIEPTSGRVLMWSSYRQDAFEDSPGGITLTSSWDPSTGIVSDRTSTVTGHDMFC PGISMDGNGQIWSGGNDAKKTSLYDSSSDSWIPGPDMQVARGYNSSATMSDGRVF TIGGSYSGGQVEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYRSDNHAWLFGW KKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSDRGVAPDAMCGNAVMYDAVKG KILTFGGSPDYQDSDATTNAHIITLGEPGTSPNTVFASNGLLFARTFHTSVVLPDGSVFI TGGQQRGVPLEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGG GLCGDCETNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVWGGWITIWTDMSISAA SLIRYGTATHTVNTDQRRIGLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGV PSVASTINVTQGQTGHHHHHH 18 Galactose oxidase (GOase) = Variant of Galactose oxidase ofFusarium qraminaarum (previously known as Dactylium dendroides) MASAPIGVAIPRNNWAVTCDSAQSGNECIKAIDGNKDTFWHTQYGVNGDPKPPHTITI DMKTVQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGVNWGSPVASGSWFADSTTK YSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVP SAAAIEPTSGRVLMWSSYRQDAFEDSPGGITLTSSWDPSTGIVSDRTSTVTGHDMFC PGISMDGNGQIWSGGNDAKKTSLYDSSSDSWIPGPDMQVARGYNSSATMSDGRVF TIGGSYSGGQVEKNGEVYSPSSKTWTSLPNAKVNPMLTADKRGLYRSDNHAWLFGW KKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSDRGVAPDAMCGNAVMYDAVKG KILTFGGSPDYQDSDATTNAHIITLGEPGTSPNTVFASNGLLFARTFHTSVVLPDGSVFI TGGQQRGVPLEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGG GLCGDCETNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVWGGWITIWTDMSISAA SLIRYGTATHTVNTDQRRIGLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGV PSVASTINVTQGQTGHHHHHH 19 Galactose oxidase (GOase) = Galactose oxidase variant from Fusarium quaminaarum (formerly known as Dactylium dendroides) MASAPIGVAIPRNNWAVTCDSAQSGNECIKAIDGNKDTFWHTQYGVNGDPKPPHTITI DMKTVQNVNGLSVLPRQDGNQNGWIGRHEVYLSSDGVNWGSPVASGSWFADSTTKYSNFETRPARYVRLVAITEANGQPWTSIAEINVFQASSYTAPQPGLGRWGPTIDLPIVP SAAAIEPTSGRVLMWSSYRQDAFRDSPGGITLTSSWDPSTGIVSDRTSTVTGHDMFC ο / ζηηη / ίζηζ / Β / γι PGISMDGNGQIWSGGNDAKKTSLYDSSDSWIPGPDMQVARGYNSSATMSDGRVF TIGGSYSGGQVEKNGEVYSPSSKTWTSLPNAKVNPMLTADKQGLYRSDNHAWLFGW KKGSVFQAGPSTAMNWYYTSGSGDVKSAGKRQSDRGVAPDAMCGNAVMYDAVKG KILTFGGSPDYQDSDATTNAHIITLGEPGTSPNTVFASNGLLFARTFHTSVVLPDGSVFI TGGQQRGVPLEDSTPVFTPEIYVPEQDTFYKQNPNSIVRAYHSISLLLPDGRVFNGGG GLCGDCETNHFDAQIFTPNYLYDSNGNLATRPKITRTSTQSVWGGWITIWTDMSISAA SLIRYGTATHTVNTDQRRIGLTLTNNGGNSYSFQVPSDSGVALPGYWMLFVMNSAGV PSVASTINVTQQGTGHHHHHH 20 Pantothenate Kinase (PanK) = Variant of Pantothenate Kinase of E.colí MHHHHHHGGSGSIKEQTLMTPYLQLDRNQWAALRDSNPMTLSEDEIARLKGINEDLS LEEVAEVYLPLSRLLNFYISSNLRRQAQLEQFLGTNGQRIPYIISIAGSVAVGKSTFARV LQALLSRWPEHRRVEHITTDGFLHPNQVLKERGLMGKKGFPESYDMHRLMKFVKDLK SGVPNVTAPVYSHLIYDVIPDGDKTVVQPDILILEGLNVLQSGMDYPHDPHHVFVSDFV DFSIYVDAPEDLLQTWYINRFLKFREGAFTDPDSYFHGYAKLTKEEAIKTAMTIWKEMN HVNLKQNILPTRERASLILTKSANHIVEEVRLRK 21 Acetato Cinasa (AcK) = Variant de Acetato Cinasa de Thermotoga maritime MGSHHHHHHGSRVLNINSGSSSIKYQLIEMEGEKVLCKGIAERIGIEGSRLVHRVGDEK HVIERELPDHEEALKLILNTLVDEKLGVIKDLKEIDAVGHRVVHGGERFKESVLVDEEVL KAIEEVSPLAPLHNPANLMGIKAAMKLLPGVPNVQVFDTAFHQTIPQKAYLYAIPYEYY EKYKIRRYGFHGISHRYVSKRAAEILGKKLEELKIITCHIGNGASVAAVKYGKCVDTSMG FTPLEGLVMGTRSGDLDPAIPFFIMEKEGISPQEMYDILNKKSGVYGLSKGFSSDMRD NLEAALKGDEWCKLVLEIYDYRIAKYIGAYAAAMNGVDAIVFTAGVGENSPITREDVCK YLEFLGVKLDKQKNEETIRGKEGIISTPDSRVKVLVVPTNEELMIARDTKEIVEKIGR. α / ζηηη / ίζηζ / Β / γι Horseradish peroxidase: Commercially available horseradish Type I natural type peroxidase from SIGMA (P8125), isolated from horseradish roots (Amoracia rusticana). Catalase: (1) natural type catalase from bovine liver, commercially available from SIGMA (C1345); or (2) CAT-101, Biocatalytics; or (3) from Corynebacterium glutamicum (Roche, #11650645103). Additional embodiments of this invention include, but are not limited to, the use of the following enzymes in the synthetic process steps described herein to produce a 4'-ethynyl 2'-deoxy nucleoside or an analogue thereof, for example, EFdA. A. A purine nucleoside phosphorylase. 1A. An engineered purine nucleoside phosphorylase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO.: 9 or SEQ ID NO.: 15, or a functional fragment thereof, wherein the engineered purine nucleoside phosphorylase polypeptide sequence comprises at least one amino acid substitution or an exposed amino acid substitution compared to SEQ ID NO.: 9 or SEQ ID NO.: 15. 2A. The designed purine nucleoside phosphorylase of 1A, wherein the designed purine nucleoside phosphorylase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 9 or SEQ ID NO: 15. 3A. An engineered purine nucleoside phosphorylase comprising the polypeptide sequence as set out in SEQ ID NO: 9 or SEQ ID NO: 15. A4. The purine nucleoside phosphorylase designed from any of 1A to 3A, comprising at least one improved property compared to the naturally occurring E. coli purine nucleoside phosphorylase. 5A. The designed purine nucleoside phosphorylase of 4A, wherein the improved property comprises enhanced activity on substrate compound 6.5 (in its ring form or as an open-chain aldehyde or hydrate, or a salt of any of the foregoing) compared to the naturally occurring E. coli purine nucleoside phosphorylase. 6A. The engineered purine nucleoside phosphorylase of 4A, wherein the improved property comprises enhanced production of EFdA (compound 7) compared to the naturally occurring E. coli purine nucleoside phosphorylase. 7A. The designed purine nucleoside phosphorylase from any of A1 to 6A, wherein the designed purine nucleoside phosphorylase is purified. 8A. The purine nucleoside phosphorylase designed from any of 1A to 7A, wherein at least one amino acid substitution (i.e., one or more amino acid substitution(s)) is conservative. B. A phosphopentomutase. 1B. An engineered phosphopentomutase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO.: 8, a functional fragment thereof, wherein the engineered phosphopentomutase polypeptide sequence comprises at least one amino acid substitution or an exposed amino acid substitution compared to SEQ ID NO.: 8. 2B. The engineered phosphopentomutase of 1B, wherein the engineered phosphopentomutase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO.: 8. 3B. An engineered phosphopentomutase comprising the polypeptide sequence as set out in SEQ ID NO.: 8. 4B. The engineered phosphopentomutase from any of 1B to 3B, comprising at least one improved property compared to the natural-type E. coli phosphopentomutase. 5B. The engineered phosphopentomutase of 4B, wherein the enhanced property comprises enhanced activity on substrate compound 6 (in its ring form or as an open-chain aldehyde or hydrate, or a salt of any of the foregoing) compared to the natural-type E. coli phosphopentomutase. 6B. The engineered phosphopentomutase of 4B, wherein the enhanced property comprises enhanced production of compound 6.5 or compound 7 (EFdA) compared to natural-type E. coli phosphopentomutase. 7B. The engineered phosphopentomutase from any of 1B to 6B, wherein the engineered phosphopentomutase is purified. 8B. The phosphopentomutase designed from any of 1B to 7B, wherein at least one amino acid substitution (i.e., one or more amino acid substitution(s)) is conservative. C. A deoxyribose-phosphate aldolase. 1C. A deoxyribose-phosphate aldolase comprising of the natural type of polypeptide sequence of Shewanella halifaxensis as set out in SEQ ID NO.: 5. 2C. An engineered deoxyribose-phosphate aldolase comprising the polypeptide sequence as set out in SEQ ID NO.: 6 or SEQ ID NO.: 14. 3C. An engineered deoxyribose-phosphate aldolase, wherein the engineered deoxyribose-phosphate aldolase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO.: 5, SEQ ID NO.: 6 or SEQ ID NO.: 14. 4C. An engineered deoxyribose-phosphate aldolase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO.: 5, SEQ ID NO.: 6 or SEQ ID NO.: 14, or a functional fragment thereof, wherein the engineered deoxyribose-phosphate aldolase polypeptide sequence comprises at least one amino acid substitution or an exposed amino acid substitution compared to SEQ ID NO.: 5, SEQ ID NO.: 6 or SEQ ID NO.: 14. 5C. The deoxyribose-phosphate aldolase of any 1C to 4C, having activity on substrate compound 5 (3-phosphate (R)-2-ethynyl-glyceraldehyde, the hydrate thereof, or a salt of any of the above). 6C. The deoxyribose-phosphate aldolase of any of C1 to C5, comprising the ability to produce compound 6 (4-ethynyl-2-deoxyribose 5-phosphate, or the open-chain aldehyde or hydrate form thereof, or a salt of any of the foregoing) without the need for protecting groups on substrate compound 5 (3-phosphate (R)-2-ethynyl-glyceraldehyde, the hydrate thereof, or a salt of any of the foregoing) during the reaction. 7C. The engineered deoxyribose-phosphate aldolase of any of 2C to 6C, wherein the deoxyribose-phosphate aldolase has an improved property comprising the improved production of compound 6 (4-ethynyl-D-2-deoxyribose 5-phosphate, or the open-chain aldehyde or hydrate form thereof, or a salt of any of the foregoing) compared to the naturally occurring Shewanella halifaxensis deoxyribose-phosphate aldolase. 8C. Deoxyribose-phosphate aldolase from any of 1C to 7C, wherein the deoxyribose-phosphate aldolase is purified. 9C. The deoxyribose-phosphate aldolase designed from any of 2C to 7C, wherein the al α / ζηηη / ίζηζ / Β / γι minus one amino acid substitution (i.e., one or more amino acid substitution(s)) are conservative amino acid substitution(s). D. A pantothenate kinase. 1D. An engineered pantothenate kinase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 20, a functional fragment thereof, wherein the engineered pantothenate kinase polypeptide sequence comprises at least one amino acid substitution or an exposed amino acid substitution compared to SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 20. 2D. The engineered pantothenate kinase of 1D, wherein the engineered pantothenate kinase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO.: 2, SEQ ID NO.: 12, SEQ ID NO.: 13 or SEQ ID NO.: 20. D. An engineered pantothenate kinase, comprising the polypeptide sequence as set out in SEQ ID NO.: 2, SEQ ID NO.: 12, SEQ ID NO.: 13 or SEQ ID NO.: 20. 4D. The pantothenate kinase designed from any of 1D to 3D, comprising at least one improved property compared to the natural-type E. coli pantothenate kinase. 5D. The engineered pantothenate kinase of 4D, wherein the enhanced property comprises the enhanced activity on substrate compound 4 ((R)-2-ethynyl-glyceraldehyde or hydrate thereof) compared to the naturally occurring E. coti pantothenate kinase. 6D. The engineered pantothenate kinase of 5D, wherein the improved property comprises enhanced production of compound 5 (3-phosphate (R)-2-ethynyl-glyceraldehyde), compared to natural-type pantothenate kinase. 7D. The engineered pantothenate kinase of 4D, wherein the enhanced property comprises enhanced activity on substrate compound 3 (2-ethynyl-propane-1,2,3-triol) compared to the naturally occurring E. cali pantothenate kinase. 8D. The engineered pantothenate kinase of 7D, wherein the improved property comprises enhanced production of compound 9 (1-phosphate (S)-2-ethynylpropane-1,2,3-triol), compared to natural-type pantothenate kinase. 9D. The pantothenate kinase engineered from any of 1D to 8D, wherein the pantothenate kinase is purified. 10D. The pantothenate kinase designed from any of 1D to 9D, wherein at least one amino acid substitution (i.e., one or more amino acid substitution(s)) are conservative amino acid substitution(s). E. A galactose oxidase. 1E. An engineered galactose oxidase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs.: 1, 10, 11, 16, 17, 18 or 19, or a functional fragment thereof, wherein the engineered galactose oxidase polypeptide sequence comprises at least one amino acid substitution or an exposed amino acid substitution compared to SEQ ID NOs.: 1, 10, 11, 16, 17, 18 or 19. 2E. The engineered galactose oxidase of 1E, wherein the engineered galactose oxidase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID Nos.: 1, 10, 11, 16, 17, 18 or 19. 3E. An engineered galactose oxidase comprising the polypeptide sequence as set out in SEQ ID Nos.: 1, 10, 11, 16, 17, 18 or 19. 4E. The designed galactose oxidase from any of 1E to 3E, comprising at least one improved property compared to natural-type F. graminaarum galactose oxidase. 5E. The designed galactose oxidase of 4E, wherein the improved property comprises enhanced activity on a substrate that is a primary alcohol compared to the naturally occurring F. graminaarum galactose oxidase. 6E. The designed galactose oxidase of 4E, wherein the improved property comprises enhanced activity on substrate compound 3 (2-ethynyl-propane-1,2,3-triol) compared to the naturally occurring F. graminaarum galactose oxidase. 7E. The engineered galactose oxidase of 6E, wherein the improved property comprises improved production of compound 4 ((R)-2-ethynyl-glyceraldehyde or hydrate thereof) compared to the naturally occurring F. graminaarum galactose oxidase. 8E. The engineered galactose oxidase of 4E, wherein the enhanced property comprises enhanced activity on substrate compound 9 (1-phosphate (S)-2-ethynylpropane-1,2,3-triol), compared to the naturally occurring F. graminaarum galactose oxidase. 9E. The engineered galactose oxidase of 8E, wherein the improved property comprises improved production of compound 5 (3-phosphate (R)-2-ethynyl-glyceraldehyde or hydrate thereof), compared to the naturally occurring F. graminaarum galactose oxidase. 10E. The galactose oxidase engineered from any of 1E to 9E, wherein the galactose oxidase is purified. 11E. The galactose oxidase designed from any of 1E to 10E, wherein at least one amino acid substitution (i.e., one or more amino acid substitution(s)) is conservative. F. An acetate kinase. 1F. An acetate kinase, comprising the polypeptide sequence of Thermotoga maritimuna as set out in SEQ ID NO.: 3 or SEQ ID NO.: 21. 2F. An engineered acetate kinase, wherein the engineered acetate kinase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO.: 3 or SEQ ID NO.: 21. 3F. An engineered acetate kinase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO.: 3 or SEQ ID NO.: 21, a functional fragment thereof, wherein the engineered acetate kinase polypeptide sequence comprises at least one amino acid substitution or one amino acid substitution exposed to SEQ ID NO.: 3 or SEQ ID NO.: 21. 4F. The 2F or 3F acetate kinase comprising at least one improved property compared to the naturally occurring T. maritima acetate kinase. 5F. The acetate kinase of 4F, wherein the improved property comprises enhanced activity for recycling the cofactor ATP in the phosphorylation reaction on substrate compound 4 ((R)-2-ethynyl-glyceraldehyde or hydrate thereof) compared to the naturally occurring Thermotoga maritima acetate kinase. 6F. The acetate kinase of 5F, wherein the improved property comprises improved production of compound 5 (3-phosphate (R)-2-ethynyl-glyceraldehyde, a hydrate form thereof, or a salt thereof) compared to the naturally occurring Thermotoga maritima acetate kinase. 7F. The acetate kinase of 4F, wherein the improved property comprises enhanced activity for recycling the co-factor ATP in the phosphorylation reaction on substrate compound 3 (2-ethynylpropane-1,2,3-triol) compared to the naturally occurring Thermotoga maritima acetate kinase. 8F. The acetate kinase of 7F, wherein the improved property comprises improved production of compound 9 1-phosphate ((S)-2-ethynyl-propane-1,2,3-triol or a salt of any of the foregoing) compared to the naturally occurring Thermotoga maritima acetate kinase. 9F. Acetate kinase from any of 1F to 8F, wherein the acetate kinase is purified. 10F. The acetate kinase designed from any of 2F to 7F, wherein at least one amino acid substitution (i.e., one or more amino acid substitution(s)) is a conservative amino acid substitution.

Claims

CLAIMS 1. A method for synthesizing a 4-ethynyl 2'-deoxy nucleoside or an analogue thereof, characterized in that it comprises combining compound 6.5: HO^\O (OPO32__ / 2X+ HO 6.5 with purine nucleoside phosphorylase and a nucleobase or an analogue thereof, in a buffered solution containing a manganese(II) salt, and wherein 2X+ is (a) two protons, (b) a proton and a monovalent cation, (c) two monovalent cations wherein each cation is the same or different, or (d) a divalent cation.

2. The method according to claim 1 characterized in that the 4'-ethynyl 2'-deoxy nucleoside or an analogue thereof is α / ζηηη / ίζηζ / Β / γι 3. The method according to claim 1 or 2, characterized in that it further comprises isolating 4. The method according to claim 1, characterized for synthesizing a 4'-ethynyl 2'-deoxynucleoside or an analogue thereof, further comprising combining compound 6 and phosphopentomutase with purine nucleoside phosphorylase and nucleobase in the buffered solution containing a manganese(II) salt.

5. The method according to claim 4, further comprising removing the inorganic phosphate byproduct from the reaction solution.

6. The method according to claim 5, characterized in that it comprises removing the inorganic phosphate byproduct from the reaction solution (a) by adding sucrose phosphorylase and sucrose to the reaction mixture or (b) by adding calcium, magnesium or manganese to the reaction mixture.

7. The method according to any of claims 4 to 6, characterized in that it further comprises isolating the 4'-ethynyl 2'-deoxy nucleoside or analogue thereof.

8. The method according to any of claims 4 to 6, characterized in that the 4'-ethynyl 2'-deoxy nucleoside or analogue thereof is α / ζηηη / ίζηζ / Β / γι 9. The method according to claim 8, characterized in that it further comprises isolating 10. The method according to claim 4, characterized in that it further comprises the step of synthesizing compound 6, wherein the synthesis comprises combining compound 5 with acetaldehyde and deoxyribose-phosphate aldolase in an aqueous solution to produce compound 6; wherein 2X+ is (a) two protons, (b) a proton and a monovalent cation, (c) two monovalent cations wherein each cation is the same or different, or (d) a divalent cation.

11. The method according to claim 10, characterized in that the reaction is carried out in a sealed container.

12. The method according to claim 10 or 11, characterized in that it further comprises the step of synthesizing compound 5, wherein the synthesis comprises combining compound q? znnn / Lznz / E / YiA with pantothenate kinase in a buffered solution, in the presence of a divalent metal salt, with ATP as a phosphate source wherein the ATP is regenerated in situ, to produce compound 5.

13. The method according to claim 12, characterized in that ATP is regenerated in situ using (a) acetyl phosphate and acetate kinase, or (b) pyruvate oxidase, catalase and acetate kinase in the presence of pyruvate, phosphate and oxygen or (c) a combination thereof.

14. The method according to claim 13, characterized in that (a) the pantothenate kinase is immobilized or (b) the pantothenate kinase and the acetate kinase are immobilized.

15. The method according to claim 12, characterized in that it further comprises the step of synthesizing compound 4, wherein the synthesis comprises combining compound 3 with galactose oxidase, copper, catalase and peroxidase or an oxidant, in the presence of oxygen, in a buffered solution to produce compound 4.

16. The method according to claim 15, characterized in that the galactose oxidase is immobilized.

17. A method for synthesizing a 4'-ethynyl 2'-deoxy nucleoside or an analogue thereof, characterized in that it comprises combining compound 5, acetaldehyde and a nucleobase or an analogue thereof, with deoxyribose-phosphate aldolase, phosphopentomutase and purine nucleoside phosphorylase, in a buffered solution containing a manganese(II) salt, wherein 2X+ is (a) two protons, (b) a proton and a monovalent cation, (c) two monovalent cations wherein each cation is the same or different, or (d) a divalent cation.

18. The method according to claim 17, characterized in that it further comprises removing the inorganic phosphate by-product from the reaction mixture.

19. The method according to claim 18, characterized in that it comprises removing the inorganic phosphate byproduct from the reaction mixture (a) by adding sucrose phosphorylase and sucrose to the reaction mixture or (b) by adding calcium, magnesium, or manganese to the reaction mixture.

20. The method according to any of claims 17 to 19, characterized in that it further comprises isolating the 4'-ethynyl 2'-deoxy nucleoside.

21. The method according to any of claims 17 to 19, characterized in that the 4'-ethynyl 2'-deoxy nucleoside is 22. The method according to claim 21, characterized in that it further comprises isolating.

23. A method for synthesizing the compound 6.5 OPO32' 2X HO 6 characterized in that it comprises combining the compound 6 OH with phosphopentomutase, in a buffered solution containing a manganese(II) salt, wherein 2X+ is (a) two protons, (b) a proton and a monovalent cation, (c) two monovalent cations wherein each cation is the same or different, or (d) a divalent cation.

24. A method for synthesizing compound 6 characterized in that it comprises combining compound 5 with acetaldehyde and deoxyribose-phosphate aldolase in aqueous solution to produce compound 6; wherein 2X+ is (a) two protons, (b) a proton and a monovalent cation, (c) monovalent cations wherein each cation is the same or different, or (d) a divalent cation.

25. A method for synthesizing compound 5 a two solution cations o / ζηηη / ίζηζ / B / γι characterized in that it comprises combining compound 4 with pantothenate kinase in a buffered solution, in the presence of a divalent metal salt, with ATP as a phosphate source wherein the ATP is regenerated in situ, wherein 2X+ is (a) two protons, (b) a proton and a monovalent cation, (c) two monovalent cations wherein each cation is the same or different, or (d) a divalent cation.

26. The method according to claim 25, characterized in that ATP is regenerated in situ using (a) acetyl phosphate and acetate kinase, or (b) pyruvate oxidase, catalase and acetate kinase in the presence of pyruvate, phosphate and oxygen or (c) a combination thereof.

27. The method according to claim 26, characterized in that (a) the pantothenate kinase is immobilized or (b) the pantothenate kinase and the acetate kinase are immobilized.

28. A method for synthesizing compound 4 characterized in that it comprises combining compound 3 q? znnn / Lznz / E / YiA with galactose oxidase, copper, catalase, and peroxidase or an oxidant, in the presence of oxygen, in a buffered solution to produce compound 4.

29. The method according to claim 28, characterized in that the galactose oxidase is immobilized.

30. A method for isolating compound 4 characterized in that it comprises (1) reacting compound 4 with an amine, diamine, or amino alcohol that forms a stable Λ / ,Λ / acetal or Λ / ,O-acetal, in an organic solvent that is not miscible with water, in the absence of oxygen to form an aminal; and (2) reacting the aminal with an organic or inorganic acid in the presence of an organic solvent that is not miscible with water to regenerate compound 4.

31. A method for synthesizing compound 5 characterized in that it comprises combining compound 9 2 >°H ;°3p0X <x0H 2X con galactosa oxidasa en una solución amortiguada, en presencia de oxígeno, catalasa y ya sea una peroxidasa o un oxidante químico, para producir el compuesto 5, en donde 2X+ es (a) dos protones, (b) un protón y un catión monovalente, (c) dos cationes monovalentes en donde cada catión es el mismo o diferente, o (d) un catión divalente.

32. A method for synthesizing compound 9 2X characterized in that it comprises combining compound 3 q? znnn / Lznz / E / YiA with pantothenate kinase in a buffered solution, in the presence of a divalent metal salt, with ATP as a phosphate source wherein the ATP is regenerated in situ, to produce compound (9), wherein 2X+ is (a) two protons, (b) a proton and a monovalent cation, (c) two monovalent cations wherein each cation is the same or different, or (d) a divalent cation.

33. The compound 34. The compound 35. The compound characterized by 2X+ being (a) two protons, (b) a proton and a monovalent cation, (c) two monovalent cations where each cation is the same or different, or (d) a divalent cation.

36. The compound 2X+ __J HO' 6 where 2X+ is (a) two protons, (b) a proton and a monovalent cation, (c) two monovalent cations where each cation is the same or different, or (d) a divalent cation.

37. The compound ο0Ρ032' __I 2X+ HO 6.5 α / ζηηη / ίζηζ / Β / γι characterized in that 2X+ is (a) two protons, (b) a proton and a monovalent cation, (c) two monovalent cations wherein each cation is the same or different, or (d) a divalent cation.