Enzymatic synthesis of 4'-ethyl nucleoside analogues

Abstract

The present invention relates to an enzymatic synthesis of 4'-ethynyl 2'-deoxynucleosides and analogs thereof, such as EFdA, that eliminates the use of protecting groups on intermediates, improves the stereoselectivity of glycosylation, and reduces the number of process steps required to produce the compounds. It also relates to novel intermediates used in the process.

Description

[Background technology]

[0001] Background of the Invention

[0002] 4'-ethynyl 2'-deoxynucleoside analogs are known for their activity against HIV, AIDS, and related diseases. [Formula 1]

[0003] An example of a 4'-ethinyl nucleoside analog is 4'-ethinyl 2'-deoxyadenosine (EFdA, also known as MK-8591), a nucleoside reverse transcriptase translocation inhibitor that blocks HIV-1 and SIV virus replication in vitro (Kawamoto, E., Sarafianos SF et al., Int. J. Biochem. Cell Biol.; 40(11):2410-20

[2008] ; Ohrui, H., H. et al., Nucleosides & Nucleic Acids, 26, 1543-1546

[2007] ) and in vivo (Hattori, S., K., Nakata, H. et al, Antimicrobial. Agents and Chemotherapy, 53, 3887-3893

[2009] ). EFdA is claimed in U.S. Patent No. 7,339,053 (referred to as 2'-deoxy-4'-C-ethynyl 2-fluoroadenosine in Patent No. 053). EFdA has the following chemical structure: [chemical 2]

[0004] EFdA is metabolized intracellularly to an active triphosphate anabolite that inhibits HIV reverse transcriptase. In contrast to nucleoside reverse transcriptase inhibitors (NsRTIs) and nucleotide-based reverse transcriptase inhibitors (NtRTIs) currently available for the treatment of HIV infection, which lack a 3'-OH group that inhibits the binding of incoming nucleotides, EFdA retains a 3'-OH group and acts as a chain terminator by preventing primer / template translocation at the reverse transcriptase (RT) active site and inhibiting the binding of incoming deoxyribonucleotide triphosphates (dNTPs). Furthermore, the packer of the modified ribose ring in EFdA is thought to contribute to the inhibition of reverse transcriptase by positioning the 3'-OH group in a vector where phosphate group transfer from incoming nucleotides is inefficient. (Michailidis E, et al., Mechanism of inhibition of HIV-1 reverse transcriptase by 4'-ethynyl-2-fluoro-2'-deoxyadenosine triphospate, J Biol Chem 284:35681-35691

[2009] ; Michailidis E, et al., 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) inhibits HIV-1 reverse transcriptase with multiple mechanisms, J Biol Chem 289:24533-24548

[2014] ).

[0005] In in vitro HIV replication assays, EFdA is a potent antiretroviral agent, exhibiting comparable antiviral activity against clinical isolates across all evaluated subtypes. It is rapidly assimilated into active triphosphate in vitro in both lymphoid cell lines and peripheral blood mononuclear cells, with an intracellular half-life of EFdA triphosphate (EFdA-TP) exceeding 72 hours. (Stoddart, CA, Galkina, et al., Oral Administration of the Nucleoside EFdA (4'-ethinyl-2-Fluoro-2'-Deoxyadenosine) 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, online May 4, 2015).

[0006] EFdA has been shown to be effective in animal models of HIV infection, including humanized mouse models and SIV-infected rhesus monkey models. Pharmacokinetic studies of EFdA administered orally to mice and rhesus monkeys showed rapid absorption and high plasma concentrations. Peripheral blood mononuclear cells isolated from rhesus monkeys were inresponsive to SIV infection 24 hours after drug administration, indicating a long intracellular half-life. (Ibid.)

[0007] Conventional synthesis of 4'-ethynyl nucleoside analogs, including EFdA, suffers from low stereoselectivity in the formation of the CN bond between the ethynyldeoxyribose sugar and the 2-fluoroadenine (also known as 2-fluoro-9H-purine-6-amine) nucleobase. Conventional synthesis also requires protecting groups to carry out glycosylation reactions, which reduces synthetic efficiency.

[0008] The synthesis described in (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 from D-glucose diacetonide, establishing three stereocenters using a diastereoselective reaction. The stereochemistry of the anomalous centers is controlled by the presence of a 2′-acetoxy-directing group, which is subsequently removed by hydrolysis and deoxygenation. This pathway requires four chromatographic purifications and the stoichiometric use of toxic organotin reagents for late deoxygenation.

[0009] In an alternative pathway (see Mark McLaughlin, et al., Enantioselective Synthesis of 4′-ethyny.-2-fluoro-2′-deoxyadenosine (EFdA) via Enzymatic Desymmetrization, Organic Letters 2017, 19 (4), pp. 926-929), the completely substituted 4′ carbinol is stereoselectively generated by enzymatic desymmetrization. The 3'-steric center is established by catalytic asymmetric transfer hydrogenation, the anomal-1'-bond is established with moderate stereoselectivity using substrate control, and improved stereochemical purity is achieved by crystallization of the intermediate. This process requires 15 steps, necessitates the use of protecting groups, and generates the glycosyl bond between the nucleobase and sugar fragment with low stereoselectivity (1.8:1).

[0010] A 12-step synthesis for producing EFdA from R-glyceraldehyde acetonide is described in Kageyama, M., et al., Concise Synthesis of the Anti-HIV Nucleoside EFdA, Biosci. Biotechnol. Biochem, 2012, 76, pp. 1219–1225; and Enantioselective Total Synthesis of the Potent Anti-HIV Nucleoside EFdA, Masayuki Kageyama, et al., Organic Letters 2011 13 (19), pp. 5264–5266 [DOI: 10.1021 / ol202116k]. This synthesis uses chiral starting materials to establish a 3' stereocenter with moderate diastereoselectivity. After separating the stereoisomers by chromatography, diastereoselective alkyne addition is induced using the new stereocenter to establish a completely substituted 4' stereocenter. The anomal-1' position has limited stereocontrol, and chromatography is required to separate the anomalous region. This pathway requires chromatographic separation of diastereoisomers at two different stages, and starts with expensive chiral starting materials.

[0011] 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 route that starts with an existing nucleoside and modifies both the sugar and nucleobase moieties. The synthesis involves 18 steps starting with 2-amino-2′-deoxyadenosine, and the overall yield is low at 2.5%.

[0012] Enzymes such as purine nucleoside phosphorylase (PNP, EC 2.4.2.1) are known to form glycosylation bonds in nucleosides and nucleoside analogs with high stereoselectivity and without the use of protecting groups. See, for example, the review *New Trends in Nucleoside Biotechnology*, Mikhailopulo, IA, Miroshnikov, AI, Acta Naturae 2010, 2, pp. 36-58. However, the current range of sugar fragments that can undergo PNP-catalyzed reactions is limited to the α-1-phosphate forms of natural ribose and deoxyribose and a few analogs having small H, NH2, or F substituents at the C2' and C3' positions and substitutions of a C5' OH group. There have been no reports of successful PNP-catalyzed glycosylation of sugars with carbon substituents on the ring or with any substitution at the C4' position.

[0013] Access to ribose and deoxyribose α-1-phosphate substrates for PNP-catalyzed glycosylation has been demonstrated by the transfer of the phosphate group from the 5'-hydroxyl position to the 1'-hydroxyl position by the enzyme phosphopentumutase (PPM, EC 5.4.2.7) (see Mikhailopulo, IA, previously cited). However, the range of sugars that can catalyze this reaction is limited to ribose, arabinose, 2-deoxyribose, and 2,3-dideoxyribose. No successful reactions with sugar phosphates containing other substituents have been reported.

[0014] The enzyme deoxyriboside phosphate aldolase (DERA, EC 4.1.2.4) is known to catalyze the aldol addition of acetaldehyde to other short-chain aldehydes (see review: Stephen M. Dean, et al., Recent Advances in Aldolase-Catal-Cath. Synth. Catal. 2007, 349, pp. 1308 - 1320; DOI: 10.1002 / adsc.200700115). However, no examples have been reported for aldehydes with a completely substituted carbon α.

[0015] U.S. Patent No. 7,229,797 describes the production of a deoxyribonucleoside from natural unsubstituted deoxyribose 1-phosphate by using a purine nucleoside phosphorylase (PNP), further by removing inorganic phosphate byproducts using an enzyme such as a sucrose phosphorylase and driving equilibrium. This document does not disclose enzymatic engineering for the creation of a PNP enzyme capable of producing a nucleoside from unnatural 4-ethynyl D-2-deoxyribose 1-phosphate, nor does it disclose a PNP enzyme capable of producing 4-ethynyl D-2-deoxyribose 1-phosphate to act on an unnatural substrate via engineering of PPM and DERA enzymes. [Prior art documents] [Patent Documents]

[0016] [Patent Document 1] U.S. Patent No. 7,339,053 [Patent Document 2] U.S. Patent No. 7,229,797 [Non-patent literature]

[0017] [Non-Patent Document 1] Kawamoto, E., Sarafianos SF et al., Int. J. Biochem. Cell Biol.; 40(11):2410-20

[2008] ; [Non-Patent Document 2] Ohrui, H., H. et al., Nucleosides & Nucleic Acids, 26, 1543 - 1546

[2007] )

Non - Patent Document 3

[2009]

Non - Patent Document 4

[2009]

Non - Patent Document 5

[2014] )

Non - Patent Document 6

Non - Patent Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 12

[0018] Given the difficult and time-consuming synthetic options developed to date for producing 4'-ethynyl nucleoside analogs, it would be desirable to develop improved enzymatic synthesis methods for 4'-ethynyl nucleoside analogs such as EFdA, which reduce the number of process steps, minimize the use of protecting groups, improve the stereoselectivity of glycosylation, and avoid the use of toxic substances. [Means for solving the problem of the invention]

[0019] Surprisingly, the PPM enzyme is active at the 3-atom ethynyl substituent at the 4' position on ribose, and it was found that by introducing a mutation into this enzyme, the PPM enzyme activity could be improved, and the isomerization reaction of 4-ethynyl D-2-deoxyribose 5-phosphate (6.) catalyzed by PPM could be successfully developed, enabling a more efficient method for producing 4'-ethynyl 2-deoxynucleoside.

[0020] Furthermore, it was discovered that the PNP enzyme has activity at the tri-atom ethynyl substituent at the 4th position of deoxyribose, and that introducing mutations into this enzyme improves PPM enzyme activity, enhances the sugar modification reaction catalyzed by PPM, and enables a more efficient method for producing 4'-ethynyl 2-deoxynucleoside.

[0021] Further improvements to the overall synthetic method stemmed from the discovery that the DERA enzyme, particularly DERA from Shewanella halifaxensis, possesses activity in the aldol reaction with 2-ethynyl-glyceraldehyde 3-phosphate having a fully substituted α-carbon. This discovery enabled the efficient synthesis of 4-ethynyl D-2-deoxyribose 5-phosphate, a precursor of 4'-ethynyl 2'-deoxynucleoside analogs, including EFdA.

[0022] Summary of the Invention

[0023] The present invention involves the use of a modified enzyme in the novel enzymatic synthesis of 4'-ethynyl 2'-deoxynucleoside analogs, including EFdA, which eliminates the use of protecting groups on the intermediate, improves the stereoselectivity of glycosylation, and, among other process improvements, significantly reduces the number of process steps required to produce the compound compared to previous methods. The present invention further relates to a novel intermediate that is an integral part of the enzymatic process.

[0024] The overall process is summarized in the following reaction equations 1 and 2. The latter reaction equation provides an alternative method for preparing compound 5.

[0025] [C3]

[0026] [C4]

[0027] Acid forms or salts of phosphate intermediates may be used in the processes described herein, and are not limited to the specific acid forms or salt forms provided in the examples of process steps herein. For all phosphate intermediates described herein, 2X + This represents any combination of two protons, one proton and one other monovalent cation, two monovalent cations (identical or different), or one divalent cation.

[0028] 同様に、-HO 3 POで示されたリン酸中間体は、2つのプロトン、1つのプロトンと1つの他の一価のカチオン、2つの一価のカチオン(同一または異なった)または1つの二価のカチオンの任意の組合せを有することができる。例としては、カルシウム、マグネシウム、または亜鉛の塩;モノまたはジナトリウム塩、モノまたはジカリウム塩、モノまたはジリチウム塩;モノまたはジアンモニウム塩;または第一級、第二級または第三級アミンを有する一価または二価の塩が挙げられるが、これらに限定されない。

[0029] As is well understood in the art, intermediate compounds shown or named herein as aldehydes or hydrates in the synthesis steps herein may exist in any of those forms or as mixtures thereof in the reactions described herein. For example, compounds (4) and (5) are depicted as a hydrate and an aldehyde, respectively, in reaction formula 1, but may exist in the reaction steps in which each exists in the form of a hydrate or an aldehyde or as a mixture thereof. Each of these forms is encompassed by referring to compound number (4) or (5) in the steps herein.

[0030] [C5]

[0031] Compound (3) is achiral and may be represented herein by any of the following: [C6]

[0032] Compound (6) can exist as a cyclic or open-chain aldehyde or hydrate, and as an acid or a salt thereof, in the reaction steps in which each exists. [C7]

[0033] Detailed description of the invention

[0034] 4-ethynyl 2-deoxynucleic acid and analogs having anoma-CN bond [C8] Its activity against HIV, AIDS, and related diseases is being explored. 4'-Ethynyl 2'-deoxyribose and its analogues include 4'-ethynyl 2'-deoxyribose linked via anoma-CN bonds to a purine or pyrimidine nucleobase (adenine, guanine, cytosine, thymine, or uracil) or a modified purine or pyrimidine nucleobase.

[0035] Reaction Equation 2 shows that 4-ethynyl 2'-deoxynucleoside analogs, such as EFdA, can be synthesized using a final-step one-pot method by combining 4-ethynyl D-2-deoxyribose 5-phosphate (6) with two enzymes, phosphopentummutase (PPM) [e.g., SEQ ID NO: 8, but not limited to this] and purine nucleoside phosphorylase (PNP) [e.g., SEQ ID NO: 9, SEQ ID NO: 15]. [C9] [C10]

[0036] As shown in reaction equation 2, the final step of the synthesis involves a two-enzyme reaction (which may also involve a third enzyme) to bring the reaction equilibrium toward the desired final product. The final step begins with compound (6) or a salt thereof, where (6) is the cyclic 4-ethynyl-2-deoxyribo-5-phosphate or its open-chain aldehyde or hydrate form as described above.

[0037] Compound (6) is combined with nucleobases such as phosphopentummutase (PPM), purine nucleoside phosphorylase (PNP), sucrose phosphorylase, sucrose, and unsubstituted or substituted adenine in a buffer solution containing manganese(II) salt, appropriately adjusted to a pH of about 6.5–8.0 or higher, particularly in the range of about 7.0–7.5. The molar ratio of sucrose to compound (6) may be, but is not limited to, about 1:1 to 4:1. The components of this one-pot reaction can be combined in any order.

[0038] The reaction should be carried out with stirring at a temperature range that does not denature the enzyme, for example, around 30–45°C, or more specifically, around 35–45°C. While the reaction may proceed at lower temperatures to some extent, this will likely slow down the reaction rate.

[0039] Any buffer containing a manganese(II) salt and having an appropriate pH can be used in the reaction. Examples of such buffers include triethanolamine; PIPES, e.g., piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, e.g., 3-(N-morpholino)propanesulfonic acid or 3-morpholinopropane-1-sulfonic acid; HEPES, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; TRIS, e.g., tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol; and BIS-TRIS methane, e.g., 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. In particular, triethanolamine is a suitable buffer. Manganese(II) salts in buffers include, for example, manganese chloride, manganese chloride hydrate, manganese bromide, manganese iodide, manganese nitrate, and / or manganese sulfate. The manganese concentration in the buffer ranges from approximately 0.05 mM to approximately 10 mM, and is particularly close to approximately 5 mM.

[0040] The equilibrium reaction can proceed to the high conversion of the final product by consuming the by-product inorganic phosphate through the phosphorylation and decomposition of sucrose to D-fructose and α-D-glucose-1-phosphate. This is catalyzed by sucrose phosphorylase (EC 2.4.1.7) added to the reaction mixture. However, instead of using sucrose phosphorylase and sucrose, the phosphate can be precipitated by adding calcium, magnesium, or manganese to the reaction, using any other option for phosphate removal. This highly efficient and ecological process has the advantage of forming an anomalous bond between the sugar and nucleobase with very high stereoselectivity without the use of protecting groups or organic solvents, and can be carried out as a one-pot reaction.

[0041] Once the reaction is complete, the final product can be isolated using standard procedures known to those skilled in the art, including, but not limited to, isolation by crystallization and collection by filtration, or crystallization after extraction in a suitable solvent.

[0042] As shown in reaction equation 2A, the final step of the synthesis can be driven towards the desired final product using a trienzyme reaction (which may also involve a fourth enzyme). The final step begins with compound (5) or a salt thereof, where (5) is in the form of (R)-2-ethynylglyceraldehyde 3-phosphate or its hydrate.

[0043] Compound (5) is combined with deoxyribose phosphate aldolase (DERA), acetaldehyde, phosphopentumutase (PPM), purine nucleoside phosphorylase (PNP), sucrose phosphorylase, sucrose, and their nucleobases or analogues, such as unsubstituted or substituted adenines, in a buffer solution containing a manganese(II) salt, with the pH adjusted as appropriate to a range of about 4 to 10, or particularly about 6.5 to 8.0, or higher, particularly about 7.0 to 7.5. The molar ratio of sucrose to compound (5) may be, but is not limited to, about 1:1 to 4:1. The components of this one-pot reaction can be combined in any order.

[0044] The reaction takes place within a temperature range that does not denature the enzyme, for example, around 30–45°C, or especially around 35–45°C. While colder temperatures may work to some extent, they will slow down the reaction rate.

[0045] Acetaldehyde is added as a solution, particularly as a 40% by weight solution in isopropyl alcohol. Any suitable solution of acetaldehyde or neat acetaldehyde can be used in the reaction. Examples of such solutions include, but are not limited to, acetaldehyde solutions in isopropanol, acetaldehyde solutions in ethanol, acetaldehyde solutions in water, and acetaldehyde solutions in THF. The molar ratio of aldehyde to compound (5) is not limited, but can be approximately 0.5:1 to 4:1, and more specifically 1.5:1.

[0046] Any buffer having an appropriate pH and containing a manganese(II) salt can be used in the reaction. Examples of such buffers include triethanolamine; PIPES, e.g., piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, e.g., 3-(N-morpholino)propanesulfonic acid or 3-morpholinopropane-1-sulfonic acid; HEPES, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; TRIS, e.g., tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol; and BIS-TRIS methane, e.g., 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. In particular, triethanolamine is a buffer. Manganese(II) salts in buffers include, for example, manganese chloride, manganese chloride hydrate, manganese bromide, manganese iodide, manganese nitrate, and / or manganese sulfate. The manganese concentration in the buffer ranges from approximately 0.05 mM to approximately 10 mM, and is particularly close to approximately 5 mM.

[0047] The equilibrium reaction can proceed to the high conversion of the final product by consuming the by-product inorganic phosphate through the phosphorylation and decomposition of sucrose to D-fructose and α-D-glucose-1-phosphate. This is catalyzed by sucrose phosphorylase (EC 2.4.1.7) added to the reaction mixture. However, instead of using sucrose phosphorylase and sucrose, the phosphate can be precipitated by adding calcium, magnesium, or manganese to the reaction, using any other option for phosphate removal. This highly efficient and ecological process has the advantage of forming an anomalous bond between the sugar and nucleobase with very high stereoselectivity without the use of protecting groups or organic solvents, and can be carried out as a one-pot reaction.

[0048] Once the reaction is complete, the final product can be isolated using standard procedures known to those skilled in the art, including, but not limited to, isolation by crystallization and collection by filtration, or crystallization after extraction in a suitable solvent.

[0049] Several upstream intermediates used in this step for the synthesis of the final product 4'-ethynyl 2'-deoxynucleoside and its analogs are also prepared using enzymatic reactions as shown in reaction formula 3 (reaction formulas 3A and 3B). [C11] [C12] [C13]

[0050] Compound 4: Oxidase reaction

[0051] As shown in reaction equation 3, (R)-2-ethynylglyceraldehyde (4) is prepared by reacting galactosoxidase with 2-ethynylpropane-1,2,3-triol (3) in a buffer solution whose pH is appropriately adjusted to approximately 3 to 10 or higher, particularly in the range of approximately 6 to 8. Any buffer having a suitable pH range, e.g., sodium phosphate; sodium acetate; PIPES, e.g., piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, e.g., 3-(N-morpholino)propanesulfonic acid or 3-piperazineethanesulfonic acid; HEPES, e.g., 4-(2-hydroxyethyl)-1-piperazinesulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; TRIS, e.g., tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol; and TRISmethane, e.g., 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; boric acid; CAPS, e.g., N-cyclohexyl-3-aminopropanesulfonic acid; MES, e.g., 2-(N-morpholino)ethanesulfonic acid; CHES, for example, N-cyclohexyl-2-aminoethanesulfonic acid, glycine, or bicine (N,N-bis(2-hydroxyethyl)glycine); sodium phosphate is preferred.

[0052] Both copper and peroxidase are used in the reaction to activate galactosoxidase (GOase). Copper is supplied to the reaction mixture by adding CuSO4, Cu(OAc)2, CuCl2, Cu(II), or other salts of Cu(I). The peroxidase may be horseradish peroxidase or a peroxidase derived from another organism, and may be substituted with an oxidizing agent such as ferricyanide, iridetes, manganese(III) salts, persulfates, and one or two other electron oxidizing agents, or inorganic or organic oxidizing agents. Preferably, the peroxidase is horseradish peroxidase. Catalase is also added to prevent inactivation of GOase. The catalase may be from a mammalian source (cattle) or from a bacterial or fungal source such as Corynebacterium, Aspergillus, or other organisms known in the art for this purpose.

[0053] The reaction proceeds in the presence of oxygen. One convenient method is to sparge the reaction with air. Alternatively, other systems for generating oxygen can be used, such as hydrogen peroxide / catalase, superoxide, or other methods known in the art for this purpose.

[0054] The reaction can be carried out at substrate concentrations of approximately 10–180 g / L, particularly 20–50 g / L. This reaction can be carried out at temperatures of approximately 0–40°C, especially 10–30°C.

[0055] Compound 8: Aminal formation

[0056] As illustrated in reaction formula 3A, (R)-2-ethynylglyceraldehyde (4) can be isolated in its amine form (e.g., compound 8) by reacting it with any amine, diamine, or amino alcohol that forms a stable N,N-acetal or N,O-acetal, such as N,N'-dibenzylethan-1,2-diamine, N,N'-dimethylethane-1,2-diamine, N,N'-diphenylethane-1,2-diamine, and N-benzylethanolamine, but N,N'-dibenzylethan-1,2-diamine is preferred. The reaction is carried out in an organic solvent at a temperature of about 50°C or less, preferably 20-30°C, to avoid the decomposition of the amine. Any solvent immiscible with water, such as MTBE, 2-MeTHF, CPME, diethyl ether, diisopropyl ether, ethyl acetate, isopropyl acetate, toluene, DCM, or mixtures thereof, can be used, but are not limited to these. The reaction can be carried out at substrate concentrations of approximately 10–100 g / L, particularly 20–50 g / L.

[0057] The aminal can be further purified by crystallization from an organic solvent, such as 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 a temperature of approximately 40°C or less to avoid decomposition of the aminal.

[0058] The reaction proceeds in the absence of oxygen. One simple method is to inject and sparge the reaction with N2. Alternatively, other systems for eliminating oxygen can be used, such as the use of argon, helium, or other methods known in the art for this purpose.

[0059] Compound 4: Regeneration of Aldehyde 4 from Aminal 8

[0060] (R)-2-ethynylglyceraldehyde (4) can be regenerated from each aminal by reacting it with an organic or inorganic acid at a temperature of 50°C or less, for example, about 0 to 15°C, in the presence of an organic solvent that is immiscible with water, in order to avoid the decomposition of the aminal. Any organic or inorganic acid may be used, but is not limited to, p-toluenesulfonic acid, methanesulfonic acid, camphoresulfonic acid, acetic acid, hydrochloric acid, phosphoric acid, or sulfuric acid. Due to the low solubility in water of N,N'-dibenzylethane-1,2-diaminebis-p-toluenesulfonate, p-toluenesulfonic acid is preferred in the reaction with aminal 8. Any solvent that is immiscible with water, for example, MTBE, 2-MeTHF, CPME, diethyl ether, diisopropyl ether, ethyl acetate, isopropyl acetate, toluene, DCM, or mixtures thereof; MTBE and 2-MeTHF are preferred. The reaction can be carried out at substrate concentrations of approximately 5–100 g / L, particularly 20–50 g / L.

[0061] Optionally, the aldehyde 4 solution can be further treated with a resin to remove excess organic or inorganic acids. The resin treatment can be carried out with a basic resin such as DOWEX® MARATHON® A resin (hydroxide type) or AMBERLYST® 15 resin (hydrogen type), or a mixture thereof, preferably a mixture of DOWEX® MARATHON® A resin (hydroxide type) and AMBERLYST® 15 resin.

[0062] Optionally, the aldehyde 4 solution can be further evaporated under vacuum or swept with gas to remove excess organic solvent.

[0063] Compound 5: Kinase reaction [C14] As shown in Figures 3 and 3A, (R)-2-ethynylglyceraldehyde 3-phosphate hydrate (5) is prepared by reacting wild-type or variant pantothenate quinase (PanK) derived from Escherichia coli with compound (4) in a buffer solution whose pH is appropriately adjusted to a range of about 4 to 10, preferably about 6.5 to 8.5, and more preferably 5.5 to 8.5. A buffer having a suitable pH range can be used, for example (but not limited to), sodium phosphate, PIPES, e.g., piperazine-N,N′-bis(2-ethanesulfonic acid); Bis-TRIS methane, e.g., 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borate, HEPES, e.g., 4-(2-hydroxyethyl)-1-piperazinesulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; triethanolamine and TRIS, e.g., tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol, with sodium phosphate being preferred. For example, sodium phosphate is preferred. The reaction can be carried out in the presence of any suitable divalent metal salt, e.g., magnesium salt, e.g., magnesium chloride, and salts of cobalt, manganese, zinc, or calcium, but is not limited to these.

[0064] This reaction utilizes adenosine 5'-diphosphate (ADP) as a phosphate source that requires regeneration to 5'-triphosphate (ATP). ATP is produced in situ and can then be regenerated from ADP, adenosine 5'-monophosphate (AMP), or adenosine by any known method. For example, a combination of acetyl phosphate and acetate kinase can be used to regenerate ADP into ATP. For example, a combination of pyruvate oxidase and catalase produces acetyl phosphate in the presence of pyruvate, phosphate, and oxygen, and can therefore be used to regenerate ADP into ATP in the presence of acetate kinase.

[0065] The reaction can be carried out at substrate concentrations of approximately 10–100 g / L, particularly 20–40 g / L. This reaction can be carried out at temperatures of approximately 0–40°C, particularly 10–25°C.

[0066] The reaction can be carried out with pantothenate quinase (PanK) immobilized on the resin, or with both PanK and acetate quinase immobilized on the resin. Any suitable enzyme immobilization method known in the art, such as immobilized metal ion affinity chromatography (IMAC) resin, or affinity resin immobilization using other biological tags, covalence immobilization, immobilization on ionic resin, immobilization by adsorption, encapsulation, and / or crosslinking enzymes, can be used, but are not limited to these. For example, metal ion affinity chromatography (IMAC) resin can be used, or any suitable combination of IMAC resin and divalent cations can be used, where the cations may be, for example, nickel, cobalt, copper, zinc, iron, and / or aluminum, but are not limited to these. Nickel-containing IMAC resins can be used in particular. Preferably, both acetate quinase and pantothenate quinase (PanK) are immobilized on the resin.

[0067] Compound 9: Kinase reaction [C15]

[0068] As shown in reaction formula 3B, (S)-2-ethynylpropane-1,3-triol-1-phosphate (9) is prepared by reacting wild-type or variant pantothenate quinase (PanK) derived from Escherichia coli with compound (3) in a buffer solution adjusted to a pH of approximately 4 to 10, preferably approximately 6.5 to 8.5, and more preferably 5.5 to 8.5. A buffer having a suitable pH range can be used, for example (but not limited to), sodium phosphate, PIPES, e.g., piperazine-N,N′-bis(2-ethanesulfonic acid); Bis-TRIS methane, e.g., 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borate, HEPES, e.g., 4-(2-hydroxyethyl)-1-piperazinesulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; and other olamines and TRIS, e.g., tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol, with sodium phosphate being preferred. For example, sodium phosphate is preferred. The reaction can be carried out in the presence of any suitable divalent metal salt, e.g., magnesium salt, e.g., magnesium chloride, and salts of cobalt, manganese, zinc, or calcium, but is not limited to these.

[0069] This reaction utilizes adenosine 5'-diphosphate (ADP) as a phosphate source that requires regeneration to 5'-triphosphate (ATP). ATP is produced in situ and can then be regenerated from ADP, adenosine 5'-monophosphate (AMP), or adenosine by any known method. For example, a combination of acetyl phosphate and acetate quinase can be used to regenerate ADP into ATP. Alternatively, (a) a combination of pyruvate oxidase, catalase, and acetate quinase in the presence of pyruvate, phosphate, and oxygen can be used to regenerate ADP into ATP, or (b) a combination of pyruvate oxidase, catalase, and acetate quinase in the presence of pyruvate, phosphate, and acetate quinase, along with a combination of acetyl phosphate and acetate quinase, can be used to regenerate ATP from ADP.

[0070] The reaction can be carried out at substrate concentrations of approximately 10–100 g / L, particularly 20–40 g / L. This reaction can be carried out at temperatures of approximately 0–40°C, particularly 10–25°C.

[0071] The reaction can be carried out with pantothenate quinase (PanK) immobilized on the resin, or with both PanK and acetate quinase immobilized on the resin. Any suitable enzyme immobilization method known in the art, such as immobilized metal ion affinity chromatography (IMAC) resin, or affinity resin immobilization using other biological tags, covalence immobilization, immobilization on ionic resin, immobilization by adsorption, encapsulation, and / or crosslinking enzymes, can be used, but are not limited to these. For example, metal ion affinity chromatography (IMAC) resin can be used, or any suitable combination of IMAC resin and divalent cations can be used, where the cations may be, for example, nickel, cobalt, copper, zinc, iron, and / or aluminum, but are not limited to these. Nickel-containing IMAC resins can be used in particular. Preferably, both acetate quinase and pantothenate quinase (PanK) are immobilized on the resin.

[0072] Compound 5: Oxidase reaction [C16]

[0073] As shown in reaction equation 3B, (R)-2-ethynylglyceraldehyde hydrate 3-phosphate (5) is prepared by reacting galactosoxidase with (S)-2-ethynylpropane-1,2,3-triol 1-phosphate (9) in a buffer adjusted to a pH of approximately 3 to 10, or particularly approximately 6 to 8. Any buffer with a suitable pH range can be used, for example (but not limited to), sodium phosphate; sodium acetate; PIPES, e.g., piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, e.g., 3-(N-morpholino)propanesulfonic acid or 3-piperazineethanesulfonic acid; HEPES, e.g., 4-(2-hydroxyethyl)-1-piperazinesulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; TRIS, for example tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol; Bis-TRISmethane, for example 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borate; CAPS, for example N-cycloexyl-3-aminopropanesulfonic acid; glycine; or bicine(N,N-bis(2-hydroxyethyl)glycine); sodium phosphate can be used, and sodium phosphate is preferred.

[0074] Both copper and peroxidase are used in the reaction to activate galactosoxidase (GOase). Copper is supplied to the reaction mixture by adding CuSO4, Cu(OAc)2, CuCl2, or other salts of Cu(II) or Cu(I). The peroxidase may be horseradish peroxidase or a peroxidase derived from another organism, and can be substituted with an oxidizing agent such as fericyanide, iridetes, manganese(III) salts, persulfates and one or two other electron oxidizing agents, or inorganic or organic oxidizing agents. Preferably, the peroxidase is horseradish peroxidase. Catalase is also added to prevent inactivation of GOase. The catalase may be from a mammalian source (cattle) or from a bacterial or fungal source such as Corynebacterium, Aspergillus, or other organisms known in the art for this purpose.

[0075] The reaction proceeds in the presence of oxygen. One convenient method is to sparge the reaction with air. Alternatively, other systems for generating oxygen can be used, such as hydrogen peroxide / catalase, superoxide, or other methods known in the art for this purpose.

[0076] The reaction can be carried out at substrate concentrations of approximately 10–180 g / L, particularly 20–50 g / L. This reaction can be carried out at temperatures of approximately 0–40°C, especially 10–30°C.

[0077] Compound 6: Deoxyribo-phosphate-aldras (DERA) reaction

[0078] A key advantage of this new route for producing compound (6) over known processes is that it creates the sugar skeleton in the correct oxidative state without using protecting groups.

[0079] 4-Ethynyl D-2-deoxyribose 5-phosphate (6) is prepared by reacting acetaldehyde in an aqueous solution of deoxyribose phosphate aldorase (DERA) and (R)-2-ethynylglyceraldehyde 3-phosphate (5) as an acid or salt, with the pH adjusted as needed to about 5 to 9 or higher, particularly in the range of about 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 or salts; or monovalent or divalent salts having primary, secondary or tertiary amines. The reaction is preferably carried out in an open container or in a sealed container to prevent evaporation of acetaldehyde.

[0080] The reaction can be carried out at substrate concentrations of approximately 10–100 g / L, particularly 30–60 g / L. It can also be carried out at temperatures of approximately 0–40°C, particularly 25–35°C.

[0081] This reaction can be carried out without a buffer. Alternatively, the following buffers may be used, but are not limited to: triethanolamines; phosphates; MOPS, e.g., 3-(N-morpholino)propanesulfonic acid or 3-morpholinopropane-1-sulfonic acid; HEPES, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; BIS-TRIS methane, e.g., 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borates; PIPES, e.g., piperazine-N,N'-bis(2-ethanesulfonic acid); MES, e.g., 2-(N-morpholino)ethanesulfonic acid; and borates; or other buffers having a suitable pH range and not containing primary amine groups.

[0082] Each step and method of the processes described herein, which involve the use of one or more enzymes, is carried out at a temperature that does not denature the one or more enzymes. Each step and method of the processes described herein, which involve the use of one or more enzymes, can be carried out at a pH in the range of about 3 to 10 or about 4 to 10.

[0083] A “nucleobase” (or “nitrogen base” or “base”) is a pyrimidine or purine heterocycle of nucleic acids such as DNA and RNA. As used herein, a nucleobase includes adenine, guanine, cytosine, thymine, or uracil, as well as nucleobases with unnatural modifications, such as nucleobases in which the base has one or more unnatural substituents, or modifications that affect heteroatoms in the base, except for any changes to the anomalous-CN bond.

[0084] 4'-ethynyl 2'-deoxynucleosides contain a nucleobase. As used herein, analogues of 4'-ethynyl 2'-deoxynucleosides mean unnatural modifications to the base of the nucleoside, such as the base having one or more unnatural substituents, or modifications affecting heteroatoms in the base, other than conversion to an anomalous-CN bond.

[0085] As used herein, “phosphopentumutase” (“PPM”) enzyme (e.g., EC 5.4.2.7) is an enzyme that catalyzes the reversible isomerization of ribose 1-phosphate to ribose 5-phosphate and related compounds such as deoxyribose phosphate and analogs of ribose phosphate and deoxyribose phosphate.

[0086] As used herein, the “purine nucleoside phosphorylase” (“PNP”) enzyme (EC 2.4.2.2) is an enzyme that catalyzes the reversible phosphorylation of purine ribonucleosides and related compounds (e.g., deoxyribonucleosides and analogs of ribonucleosides and deoxyribonucleosides) to free purine bases and ribose-1-phosphate (and its analogues).

[0087] As used herein, “sucrose phosphorylase” ("SP") enzyme (EC 2.4.1.7) is an enzyme (and its analogues) that catalyzes the reversible phosphorylation of sucrose to the D-fructose base and glucose-1-phosphate. The combination of sucrose phosphorylase (SP) and sucrose is used in combination with purine nucleoside phosphorylase (PNP) and phosphomutase (PPM) to remove free phosphate ions from a reaction, where the combination of enzymes catalyzes the formation of the nucleoside MK-8591 (EFdA), although in some embodiments this can be replaced by other methods known in the art.

[0088] As used herein, “deoxyribose-phosphate aldolases” (“DERA”) (e.g., EC 4.1.2.4) refers to enzymes in the family of lyases that reversibly cleave or create carbon-carbon bonds. As used herein, deoxyribose-phosphate aldolases include naturally occurring (wild-type) deoxyribose-phosphate aldolases as well as unnaturally occurring engineered polypeptides produced by human engineering. Wild-type deoxyribose-phosphate aldolases catalyze the reversible reaction from 2-deoxy-D-ribose-5-phosphate to D-glyceraldehyde 3-phosphate and acetaldehyde.

[0089] As used herein, “pantothenate quinase” (“PanK”) refers to the enzyme (EC 2.7.1.33) that in its natural form phosphorylates pantothenic acid to produce 4'-phosphopantothenic acid. Mutant enzymes derived from such PanK enzymes may exhibit improved activity and stereoselectivity toward the 3'OH group of D-ethynylglyceraldehyde, regardless of whether such mutants retain their innate function toward pantothenic acid.

[0090] As used herein, “galactosoxidase” (”GOase”; EC 1.1.3.9) enzymes are copper-dependent enzymes that, in the presence of two molecules of oxygen, catalyze the oxidation of primary alcohols to their corresponding aldehydes. These enzymes act both regiospecifically and enantiospecifically, enabling synthetic approaches that require little to no functional group protection, yielding the desired stereoisomers. The mode of oxidation is mild, and the activity is controlled to prevent over-oxidation of the alcohol to its corresponding carboxylic acid.

[0091] As used herein, the horseradish peroxidase (HRP, EC 1.11.1.7) enzyme is an iron-dependent enzyme that activates and maintains GOase catalytic activity by oxidizing the inactive redox state of the active site that occurs during a normal GOase catalytic cycle. Type I HRP is used catalytically in the examples contained herein, but this is not intended to exclude other electron transport enzymes and chemical compounds belonging to this class or other enzyme classes that perform similar roles.

[0092] As used herein, “catalase” refers to a heme-dependent enzyme (EC 1.11.1.6) that acts on hydrogen peroxide, a byproduct of the galactosoxidase or pyruvate oxidase reaction, which inactivates these enzymes above a certain level. Catalase is used as a catalyst-maintaining enzyme in the examples herein to convert hydrogen peroxide to water and oxygen, whereas in some embodiments it may be replaced by other methods, such as the electrochemical decomposition of hydrogen peroxide. Although heme-dependent catalase is employed in a catalytic manner in the examples contained herein, it is not meant to be limited to this role, as other enzymes of this class may perform this role.

[0093] As used herein, “acetate quinase” (“AcK”) refers to the enzyme (EC 2.7.2.1) that catalyzes the production of acetyl phosphate from acetic acid and 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 quinase is used in the examples herein to recycle ATP required by pantothenate quinase (PanK), but in some embodiments, the acetyl phosphate acetate quinase recycling combination can be replaced by other methods known in the art.

[0094] As used herein, “pyruvate oxidase” (“PO”) refers to an enzyme (EC 1.2.3.3) that depends on flavin adenine dinucleotide (FAD) and thiamine diphosphate. Pyruvate oxidase is an enzyme belonging to the oxidoreductase family, and acts particularly on the aldehyde or oxo group of an oxygen-accepting donor, catalyzing the chemical reaction between pyruvate, phosphate ions, and two molecules of oxygen to produce acetyl phosphate, carbon dioxide, and hydrogen peroxide. In the examples herein, pyruvate oxidase (PO) is used in combination with acetate quinase (AcK) and catalase as a catalytic ATP regeneration combination, where this combination of enzymes catalyzes the production of ATP from ADP in the presence of oxygen, pyruvate, and phosphate ions, although in some embodiments it can be replaced by other methods known in the art.

[0095] As used herein, “wild-type” and “naturally occurring” enzymes refer to their forms found in nature. For example, a wild-type polypeptide sequence is a sequence found in organisms that can be isolated from a natural source and has not been intentionally modified by human intervention.

[0096] As used herein, “manipulated,” “mutant,” “mutant,” and “unnaturally occurring” refer to an enzyme containing a polypeptide, or a substance corresponding to the natural or natural form of a substance, that has been modified in a manner that would not occur naturally. In some embodiments, the polypeptide is identical to a naturally occurring polypeptide but is produced or derived by manipulation using synthetic materials and / or recombinant techniques.

[0097] In this specification, the terms “percentage of sequence identity,” “percentage of identity,” and “percentage of identity” relating to an enzyme are used to refer to a comparison between polynucleotide or polypeptide sequences, which is determined by comparing two optimally aligned sequences on a comparison window, where the portion of the polynucleotide or polypeptide sequence on the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where either the same nucleobase or amino acid residue occurs, or the number of positions where the nucleobase or amino acid residue aligns with a gap and matches, dividing the number of matching positions by the total number of positions on the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment and percent sequence identity are determined 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). Software for performing BLAST analysis is available through the National Center for Biotechnology Information website.

[0098] In short, BLAST analysis involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match a word of the same length in the database sequence or satisfy a certain positive threshold score T when matching a word of the same length. T is called the neighbor word score threshold (Altschul et al., previously mentioned). These initial neighbor word hits act as seeds to initiate a search for longer HSPs that contain them. Next, word hits are extended bidirectionally along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction is stopped if: the cumulative alignment score decreases by an amount X from its maximum achieved value; one or more negative-scoring residue alignments accumulate, resulting in a cumulative score of zero or less; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. In the BLASTN program (for nucleotide sequences), word length (W) 11, expected euro length 10, M=5, N=-4, and comparison of both strands are used as deletions. For amino acid sequences, the BLASTP program uses world length (W) 3, expected euro (E) 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915) as deletions.

[0099] Many other algorithms that function similarly to BLAST are available to provide percent identity between two sequences. Optimal alignment of sequences for comparison can be achieved, for example, by computer image or visual inspection of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package) such as Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J. Mol's homology algorithm; Pearson and Lipman, 1988, Proc's similarity search algorithm (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package) (generally, see Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)). Furthermore, sequence alignment and percent sequence identity determination can be performed using the BESTFIT or GAP programs from the GCG Wisconsin software package (Accelerys, Madison WI). In this case, default parameters are used.

[0100] "Substantially identical" refers to a polynucleotide or polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity compared to a reference sequence spanning a comparison window of at least 20 residue positions, often a window of at least 30 to 50 residues, where the percentage of sequence identity is calculated by comparing it to a sequence containing no more than 20 percent of deletions or additions to the reference sequence across the comparison window. In specific embodiments applied to polypeptides, the term "substantially identical" means that two polypeptide sequences share at least 80% sequence identity, preferably at least 89%, and more preferably at least 95% sequence identity (e.g., 99% sequence identity) when optimally aligned by a programmed GAP or BESTFIT with default gap weights. Preferably, the positions of non-identical residues differ by conservative amino acid substitutions.

[0101] Stereoselectivity refers to the chemical or enzymatic preference for the formation of one stereoisomer over another. Stereoselectivity is partial; sometimes one stereoisomer is formed more favorably than the other, and sometimes only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is called enantioselectivity and is the fraction (typically reported as a percentage) of one enantiomer in the total of both. It is alternatively reported (typically as a percentage) in the art as an enantiomer excess (e.), calculated according to the formula [major-enantiomer--minor-enantiomer] / [major-enantiomer-+minor-enantiomer-]. When the stereoisomers are diastereoisomers, the stereoselectivity is called diastereoselectivity and is the proportion of one diastereomer in a mixture of two diastereomers (typically reported as a percentage), and is generally alternatively reported as diastereomer excess (de). Enantiomer excess and diastereomer excess are types of stereoisomer excess.

[0102] The term "appropriate reaction conditions" refers to the conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffer, co-solvent, etc.) that enable each polypeptide used in the present invention to convert a substrate into a desired product compound. Several exemplary appropriate reaction conditions are provided herein.

[0103] As used herein, “substrate” in the context of an enzymatic conversion reaction process refers to a compound or molecule acted upon by the manipulated enzyme used herein.

[0104] As used herein, the term "product" in relation to an enzymatic conversion process means a compound or molecule resulting from the action of an enzymatic polypeptide on a substrate.

[0105] As used herein, the yield of a product from a reaction (e.g., 4'-ethynyl 2'-deoxyriboth phosphate analog or 4'-ethynyl 2'-deoxynucleoside analog) is increased when a particular component (e.g., an enzyme) present in the reaction causes the product to be produced in greater quantities compared to when the component of interest is not present.

[0106] As used herein, “equilibrium” or “equilibrium” refers to a process in a chemical or enzymatic reaction that brings about a steady-state concentration of a chemical species (e.g., the interconversion of two species A and B), which includes the interconversion of stereoisomers determined by the forward and reverse rate constants of the chemical or enzymatic reaction.

[0107] "Enantiomer excess" (ee) is a measure of purity used for chiral substances. It reflects the degree to which one enantiomer is present in a sample in greater quantities than the other. For example, a racemic mixture has an enantiomer excess of 0%, while a sample with one perfectly pure enantiomer has an ee of 100%, and a sample with 70% of one enantiomer and 30% of the other has an ee of 40% (70%–30%). Diastereomer excess (de) is calculated in the same way as when only two diastereoisomers are present in a mixture.

[0108] "Protein," "enzyme," "polypeptide," and "peptide" are used interchangeably herein to mean a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). Included in this definition are D- and L-amino acids, as well as mixtures of D- and L-amino acids.

[0109] Here, "approximately" refers to an acceptable margin of error for a given value. In some cases, "approximately" means within 0.05%, 0.5%, 1.0%, or 2.0% at the lower and upper limits of a given value range. For pH, "approximately" means plus or minus 0.5.

[0110] As used herein, “substantially pure” polypeptide or “purified” protein means a composition in which the polypeptide species is the dominant species present (i.e., in moles or by weight, more than any other individual polymer species in the composition), and generally, a substantially purified composition is one in which the species of interest constitutes at least about 50% of the polymer species present in moles or by weight. However, in some embodiments, the polypeptide-containing composition contains polypeptides with a purity of less than 50% (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%). Generally, a substantially pure polypeptide composition contains about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all polymer species present in the composition in moles or by weight. In some embodiments, the polypeptide is purified to an essentially homogeneous degree (i.e., no contaminating species can be detected in the composition by conventional detection methods), where the composition consists of essentially a single polymer species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated polypeptide is substantially a pure polypeptide composition.

[0111] As used herein, “improved properties” of an enzyme refers to at least one improved property of the enzyme. In some embodiments, the present invention utilizes recombinant PPM, PNP, DERA, PanK, AcK, SP, and / or GOase polypeptides that exhibit improvement in any of the enzyme properties compared to a reference PPM, PNP, DERA, PanK, AcK, SP, or GOase polypeptide, and / or other modified PPM, PNP, DERA, PanK, AcK, SP, or GOase polypeptide, respectively. Thus, the level of “improvement” can be determined and compared among various polypeptides, including the wild type, as well as among the manipulated polypeptides. Improved properties include, but are not limited to, properties such as increased protein expression, increased production of the intended product, increased substrate specificity or affinity (i.e., increased activity for the substrate), increased thermal activity, increased thermal stability, increased pH activity, increased stability, increased enzyme activity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased resistance to proteolytic activity (i.e., decreased sensitivity to proteolysis), reduced aggregation, increased solubility, and changes in temperature profile. In additional embodiments, the term is used to refer to at least one improved property of PPM, PNP, DERA, PanK, AcK, SP, and / or GOase enzymes. In some embodiments, the present invention uses modified PPM, PNP, DERA, PanK, SP, and / or GOase polypeptides that exhibit improvements in any enzymatic properties compared to reference PPM, PNP, DERA, PanK, AcK, SP, and / or GOase polypeptides, respectively; and / or other modified PPM, PNP, DERA, PanK, AcK, SP, and / or GOase polypeptides. Thus, the level of "improvement" can be determined and compared among various polypeptides, including the wild type, as well as among the manipulated polypeptides.

[0112] Here, "conversion" refers to the enzymatic (or in vivo) conversion of a substrate to the corresponding product, and "percent conversion" refers to the percentage of a substrate converted to the product within a certain period under specific conditions. Therefore, the "enzymatic activity" or "activity" of a polypeptide can be expressed as the percentage conversion of the substrate to the product over a specific period.

[0113] As used herein, “stereoselectivity” means that one stereoisomer is preferentially formed in chemical or enzymatic reactions over another. Stereoselectivity is partial; sometimes the formation of one stereoisomer is more favorable than the other, and sometimes only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is called enantioselectivity and is the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is generally (typically as a percentage) alternatively reported in the art as an enantiomer excess ("ee") calculated from the formula [major-enantiomer--minor-enantiomer-] / [major-enantiomer-+minor-enantiomer-]. When the stereoisomers are diastereoisomers, the stereoselectivity is called diastereoselectivity and is the proportion of one diastereomer in a mixture of two diastereomers (typically reported as a percentage), and is generally alternatively reported as diastereomer excess ("de"). Enantiomer excess and diastereomer excess are types of stereoisomer excess.

[0114] The present invention encompasses the use of manipulated PPM, PNP, DERA, PanK, AcK, SP, and GOase polypeptides, particularly those having SEQ ID NOs: 1-21, and sequences containing one or more conservative amino acid substitutions, each of which is called a conservatively modified variant of SEQ ID NOs: 1-21.

[0115] As used herein, “conservative” amino acid substitution refers to the substitution of an amino acid in a protein that has similar properties (e.g., acidity, basicity, positive or negative charge, polarity or nonpolarity, side chain size, hydrophobicity / hydrophilicity, skeletal conformation and rigidity) so that it can be frequently changed without altering the biological activity of the protein. This includes the substitution of one or more amino acids in a polypeptide with different amino acids within the same or similar defined class of amino acids. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p.224 (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. For example, but not limited to, in some embodiments, amino acids having aliphatic side chains are substituted with other aliphatic amino acids (e.g., alanine, valine, leucine, and isoleucine); amino acids having hydroxyl side chains are substituted with other amino acids having hydroxyl side chains (e.g., serine and threonine); amino acids having aromatic side chains are substituted with other amino acids having aromatic side chains (e.g., phenylalanine, tyrosine, tryptophan, and histidine); amino acids having basic side chains are substituted with other amino acids (e.g., lysine and arginine); amino acids having acidic side chains are substituted with other amino acids having acidic side chains (e.g., aspartic acid or glutamic acid); and / or hydrophobic or hydrophilic amino acids are substituted with other hydrophobic or hydrophilic amino acids, respectively. Further exemplary conservative amino acid substitutions are shown in Table 1.

[0116] [Table 1]

[0117] 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.

[0118] A “functional fragment” refers to a polypeptide having an amino-terminal and / or carboxy-terminal deletion and / or an internal deletion, but whose remaining amino acid sequence is identical to the corresponding position in the sequence it is being compared to (e.g., the fully-length manipulated PPM, PNP, DERA, PanK, AcK, SP, or GOase enzymes used in this invention), and which retains substantially all of the activity of the full-length polypeptide.

[0119] As used herein, “deletion” means modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty 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 enzyme activity and / or retaining improved properties of the manipulated PPM, PNP, DERA, PanK, AcK, SP, or GOase enzyme. Deletions may be directed to the internal and / or terminal portions of the polypeptide. In various embodiments, deletions may include continuous segments or be discontinuous. Deletions are typically indicated by a “-” in the amino acid sequence.

[0120] As used herein, “insertion” means a polypeptide formed by the addition of one or more amino acids from a reference polypeptide. Insertions may be located within the polypeptide, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins known in the art. Insertions may be a contiguous segment of amino acids or separated by one or more amino acids from naturally occurring polypeptides.

[0121] The additional acronyms and abbreviations used here are as follows: [Table 2]

[0122] Experimental Procedure Synthesis of 2-ethynyl-2-hydroxypropane-1,3-diyldiacetate (2) Method A: [C17] To a solution of diacetoxyacetone (1) in THF (1000 mL) at -35°C (159 g, 914.0 mmol), 1600 mL of a 0.5 M solution of magnesium ethynyl chloride in THF, maintained at a temperature below -20°C, was added. After the reaction was complete, acetic acid (78 mL) in 400 mL of methyl tert-butyl ether (MTBE) was added dropwise while maintaining the temperature below -20°C. Then, MTBE (800 mL) was added and the mixture was heated to room temperature. Saturated NaCl in water (1000 mL) was added, followed by saturated NH4Cl solution in water (1050 mL). The organic layer was separated, dried on Na2SO4, and evaporated to obtain compound (2) as an oily substance (160 g, 88%). 1H NMR (CDCl3, 500 MHz): δ 4.26 (dd, 4H), 2.55 (s, 1H), 2.14 (s, 6H).

[0123] Synthesis of 2-ethynylpropane-1,2,3-triol (3) Method B: [C18] To an ethanol solution of 2-ethynyl 2-hydroxypropane-1,3-diyldiacetate (2) (70 g, 350 mmol), a 0.5 M solution of sodium methoxylate in methanol (69.9 mL, 35.0 mmol) was added at room temperature (rt). The reaction was stirred at rt for 2 hours (h) to complete. The solvent was evaporated, and the residue was redissolved in 100 mL of water and extracted with 3 × 50 mL of MTBE. The aqueous layer was sprayed with nitrogen to remove the residual solvent, and nuclear magnetic resonance (NMR) (maleic acid as an internal standard) was performed to obtain a 40.9% solution of 2-ethynylpropane-1,2,3-triol (3) (108 g, 100% yield). ¹H NMR (D₂O, 500 MHz): δ 3.60 (dd, 4 H), 2.85 (s, 1 H).

[0124] Alternative preparation of (R)-2-ethynylglyceraldehyde (4) C1 method: [C19] 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 the defoaming agent 204 (Sigma A6426, 1 drop to 20 μL) was heated to 30°C while spagging at 12.5 sccm. Galactosoxidase (GOOase, SEQ ID NO: 1) (250 mg), hoseradish peroxidase (type I, 5 mg), and bovine catarase** (5 mg) were dissolved in sodium phosphate buffer (5 mL, 100 mM, pH 7.0), and then aqueous CuSO4 solution (100 mM, 150 μL) was added. The reaction mixture was stirred at 600 rpm for 47 hours while spraying with air to obtain (R)-2-ethynylglyceraldehyde (4) with a conversion rate of 47% (by NMR) and 72% ee. (This compound was not isolated.) ¹H NMR (D₂O, 500 MHz): δ 4.29 (s, ¹H), 3.65 (dd, ²H), 2.83 (s, ¹H). * Horseradish peroxidase: A wild-type peroxidase derived from commercially available horseradish type I, derived from SIGMA (P8125) isolated from horseradish root (Amoracia rusticana). ** Bovine Catalase: A heme-dependent catalase derived from bovine sources, commercially available from Sigma (C1345).**

[0125] C2 method: [C20] Sodium phosphate (1.212 kg, 10 moles) was added to a 100 L jacketed furnace containing deionized water (56.2 kg). The pH was adjusted to 7.02 using 10 N sodium hydroxide solution (852.6 g) at 25°C. Antifoam 204 (A6426, 10 mL) was added to the reactor, followed by CuSO4·5H2O (6.5 g). Galactosulfidase (451.2 g) (SEQ ID NO: 10) was added, and the mixture was stirred for 15 minutes while sparging with air. Horseradish peroxidase* (200.2 g) and catalase** (502.6 g) were added, and the reactor was washed with water (2.0 kg). Next, a solution of 2-ethynylpropane-1,2,3-triol (3) in water (9.48%, 30.34 kg, 24.72 mol) was added, followed by the addition of antifoaming agent 204 (A6426, 10 mL). The reaction was sparged with air and stirred overnight to obtain 94.0 kg of (R)-2-ethynylglyceraldehyde (4) with 66% conversion (by NMR) and 84% ee. Analysis of the 60% solution yielded the following results: 1H NMR (D2O, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). * Horseradish peroxidase: A purified, wild-type peroxidase derived from horseradish (Amoracia rusticana), commercially available from Toyobo (PEO-301), which was isolated from horseradish root (Amoracia rusticana). ** Bovine catalase: A heme-dependent catalase derived from bovine sources, commercially available from Sigma (C1345).

[0126] The above reaction was also carried out using galactosoxidase (SEQ ID NO: 11), and product (4) was obtained with 67% conversion (by NMR) and 88% ee, with an analytical yield of 59%: ¹H NMR (D₂O, 500 MHz): δ 4.29 (s, ¹H), 3.65 (dd, ²H), 2.83 (s, ¹H).

[0127] C3 method: [C21]

[0128] A 100 mL EasyMax container equipped with a sparger and flow controller was filled with water (82 mL) and PIPES potassium buffer (5 mL, 0.5 M). The pH was adjusted to 7.5 using 5 M KOH solution at 25 °C. Defoaming agent 204 (200 μL) was added, followed by evolved galactosoxidase (SEQ ID NO: 17, 450 mg enzyme powder) and copper(II) sulfate pentahydrate (100 μL, 100 mM). The reaction mixture was spagged with 125 standard cubic centimeters / minute (sccm) of air for 15 minutes. Bovine catarase (C1345, Sigma-Aldrich, 150 mg, 2000-5000 U / mg, 0.75 MU) was added, followed by horseradish peroxidase (HRP, Toyobo PEO-301, 100 mg, 130 U / mg, 1.3 kU) and an aqueous solution of 2-ethynylpropane-1,2,3-triol (3) (25 wt%, 12 mL, 25.8 mmol). The reaction mixture was stirred at 30°C with 125 sccm of aeration and sampled using an Easy Sampler over 20 hours, yielding 70% conversion, 58% assay yield, and 99% ee to produce compound (4) ((R)-2-ethynylglyceraldehyde). ¹H NMR (D₂O, 500 MHz): δ 4.29 (s, ¹H), 3.65 (dd, ²H), 2.83 (s, ¹H). The crude reaction flow was directly carried to the next phosphorylation step.

[0129] C4 method: Oxidation by immobilized galactosoxidase [C22]

[0130] Enzyme immobilization procedure: Nuvia IMAC Ni charged resin (16 mL based on sedimentation volume) was added to a filter-funnel and washed with binding buffer (10 column volume, 160 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin preservative. The lyophilized galactosoxidase (SEQ ID NO: 17, 2.00 g) powder, evolved in a container, was resuspended in copper(II) sulfate solution (100 μM; 5.00 mL), and binding buffer (50 mL) and the resin were added. The solution was mixed using a rotary mixer at 20°C for 5 hours. This resin was filtered and washed with binding buffer (10 column volume, 160 mL) and potassium PIPES buffer (10 column volume, 160 mL; 50 mM, pH 7.5) and used directly in the reaction.

[0131] Reaction procedure: Water (82 mL) and PIPES potassium buffer (5 mL, 1 M) were added to a 100 mL EasyMax container equipped with a sparger and flow controller. The pH was adjusted to 7.5 using 5 M KOH solution at 25°C. Defoaming agent 204 (200 μL) was added, followed by resin-immobilized evolved galactosoxidase (SEQ ID NO: 17, 750 mg enzyme powder per 6 mL of resin) and copper(II) sulfate pentahydrate (100 μL, 100 mM). The reaction mixture was sparged with 125 standard cubic centimeters / minute (sccm) of air for 15 minutes. Bovine catarase (C1345, Sigma-Aldrich, 210 mg, 2000-5000 U / mg, 1.05 MU) was added, followed by horseradish peroxidase (HRP, Toyobo PEO-301, 100 mg, 130 U / mg, 1.3 kU) and an aqueous solution of 2-ethynylpropane-1,2,3-triol (3) (25 wt%, 13 mL, 29.4 mmol). The reaction mixture was stirred at 25°C and aerated at 125 sccm. After 22 hours, the reaction reached 91%, and a solution of 200 mM (R)-2-ethynylglyceraldehyde (4) (100 mL, 68% assay yield, 97% ee) was added. 1H NMR (D2O, 500 MHz): The values ​​obtained were δ4.29(s,1H), 3.65(dd,2H), and 2.83(s,1H). The crude reaction flow was directly carried to the next phosphorylation step.

[0132] C5 Method: Arbitrary isolation of aldehydes via the formation of aminal(8) Step 1: Preparation of (S)-2-(1,3-dibenzylimidazolidine-2-yl)but-3-ene-1,2-diol [C23] A crude oxidase reaction stream containing (R)-2-ethynylglyceraldehyde (4), 26.0 kg (1.85 wt% aldehyde, 3.64 mol), was placed in a 100 L jacketed cylindrical vessel equipped with a nitrogen bubbler, mechanical stirrer, and thermocouple, and inactivated under an N2 atmosphere. The aqueous solution was heated to 20°C, and N,N-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 N,N'-dibenzylethan-1,2-diamine (1.55 kg, 6.43 mol). The brown two-phase mixture was stirred overnight at 20°C under a nitrogen atmosphere. After 17 hours, stirring was stopped, the organic phase was removed, and the mixture was discarded. A pale brown MTBE solution of (S)-2-(1,3-dibenzylimidazolidine-2-yl)-3-in-1,2-diol (56.5 kg, 2.02 wt% aminal, 3.39 mmol, 93% assay yield) was obtained.

[0133] Six similar MTBE solutions were processed together in a single distillation and crystallization step (totaling 374.4 kg of solution, containing 7.91 kg of aminal).

[0134] A 50 L jacketed cylindrical vessel equipped with a mechanical stirrer, a distillation head (-20°C condenser), and a thermocouple was loaded with 45 L of aminal solution. 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 the volume reached 35 L. At this point, the internal temperature was 6.1°C, and an off-white solid had begun to crystallize. The remaining MTBE solution was slowly added while 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 decreased to 25 L. Distillation was stopped, nitrogen was deactivated in the vessel, and the jacket temperature was reduced to 10°C. The resulting pale yellow suspension was left at this temperature for 2 hours, and the solid was recovered by filtration. The filter cake was washed with cold (-2°C) MTBE (12.7 kg) and then dried under a stream of nitrogen for 7 hours. (S)-2-(1,3-dibenzylimidazolidin-2-yl)-but-3-in-1,2-diol was obtained as an off-white crystalline solid (5.75 kg). 1H NMR (500 MHz, DMSO-d6) 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).13C NMR (126 MHz, DMSO-d6) 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) Amineral (M + H+) C21H25N2O2+ 337: Calculated value 1911; 337. Detected value 1922.

[0135] Step 2 Preparation of (R)-2-ethynyl-glyceraldehyde (4) from aminal (8) [C24] In a 4L jacketed cylindrical container equipped with a nitrogen bubbler and mechanical stirrer, TsOH·H2O (12.0g, 63.1 mmol), water (60 mL), (S)-2-(1,3-dibenzylimidazolidine-2-yl)buta-3-in-1,2-diol (110g, 327 mmol), and MTBE (1700 mL) were placed. The two-phase mixture was placed under nitrogen and the jacket temperature was set to 15°C. An aqueous solution of TsOH·H2O (114g, 599.3 mmol) (600 mL) was added dropwise over 1.5 hours with stirring (200 rpm). After the addition was complete, the jacket temperature was lowered to 5°C and the resulting slurry was allowed to stand for 1 hour. Solid matter was removed by filtration and washed with cold water (270 mL). The two-phase solution was transferred to a separation funnel, the organic phase was removed, and discarded. The aqueous phase was treated with DOWEX® MARATHON® A resin (hydroxide form, 11.0 g) and AMBERLYST® 15 resin (hydrogen form, 11.0 g), while simultaneously sparging with N2 at a rate of 200 sccm for 24 hours to remove residual MTBE. This resin was removed by filtration to obtain a colorless aqueous solution of (R)-2-hydroxy-2-(hydroxymethyl)buta-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).13C NMR (126MHz, D2O) δ 129.4, 125.4, 90.3, 81.0, 76.0, 73.9, 65.3. HRMS (ESI) Aldehyde dimer (2M + Na + ) C10H12NaO6 + Calculated value: 251.0526; Detected value: 251.0530.

[0136] Alternative preparation methods for (R)-2-ethynylglyceraldehyde 3-phosphate (5): D1 Method: Acetate-tokinase: ATP regeneration system [C25] In a stirring reactor, a solution of adenosine disodium diphosphate (40 mg, 0.087 mmol) and magnesium chloride (38 mg, 0.400 mmol) in HEPES buffer (66 mM, pH 7.5, 30 mL) was mixed with (R)-2-ethynylglyceraldehyde (4) (1.9 mL, 210 g / L solution in water, 3.51 mmol), followed by the addition of acetate quinase (SEQ ID NO: 3) (40 mg) and pantothenate quinase (SEQ ID NO: 2) (120 mg). The reaction mixture was heated to 25°C, and lithium potassium acetyl phosphate solution (1.3 g, 7.01 mmol) in HEPES buffer (50 mM, pH 7.5, 10 mL) was added dropwise over 4 hours, maintaining the pH at 7.5 using 5 M sodium hydroxide. The reaction was stirred for 18 hours to give (R)-2-ethynylglyceraldehyde 3-phosphate (5) by 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): C₅H₃O₆P (MH): Measured value 193.1; Detected value 193.0.

[0137] D2 method: Pyruvate oxidase ATP regeneration system [C26] 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 at pH 7.5 was mixed with (R)-2-ethynylglyceraldehyde (4) (3.8 mL, 210 g / L solution in water, 7.01 mmol), adenosine diphosphate disodium salt (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 μL, 1 M solution in water, 0.4 mmol). The pH was readjusted to 7.5 with 5 M sodium hydroxide, and the reaction volume was readjusted to 80 mL with water. Acetate quinase (SEQ ID NO: 3) (80 mg), pyruvate oxidase (SEQ ID NO: 4) (80 mg, lyophilized cell extract), pantothenate quinase (SEQ ID NO: 2) (400 mg), and catalase (800 μL, ammonium sulfate suspension CAT-101, Biocatalytics) were added. The reaction mixture was stirred at 500 rpm and 30°C for 72 hours while sparging with air, yielding (R)-2-ethynylglyceraldehyde 3-phosphate 5 with a 95% conversion rate (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 C₅H₃O₆P (MH): 193.1; detected value 193.0.

[0138] The above reaction was also carried out using pantothenate quinase (SEQ ID NO: 13), and product 5 was obtained with 66% conversion. (This product was not isolated.) ¹H NMR (D₂O, 400 MHz): δ 5.02 (s, ¹H), 4.00 (dq, ²H), 2.88 (s, ¹H).

[0139] D3 Method: Acetate-tokinase: ATP regeneration system using immobilized enzymes [C27]

[0140] Enzyme immobilization procedure: NUVIA® immobilized metal ion affinity chromatography (IMAC) nickel-charged resin (168 mL based on sedimentation volume) was added to a filter-funnel and washed with binding buffer (1.6 L; 500 mM sodium chloride, 50 mM sodium phosphate, pH 8.0). In a container, pantothenate quinase (8.4 g) (SEQ ID NO: 12) and acetate quinase (2.8 g) (SEQ ID NO: 3) were dissolved in binding buffer (500 mL). The washed resin was placed in a container and the solution was stirred at 20°C for 4 hours. The resin was filtered and washed first with binding buffer (1.6 L), followed by washing with piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (840 mL; 50 mM, pH 6.5). The washed resin was used directly in the next step.

[0141] Reaction procedure: A solution of (R)-2-ethynylglyceraldehyde (4) in water (608.7 g, 4.6 wt%, 212 mmol) was placed in a 1 L reactor and cooled to 5°C. The cooled solution consisted of piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (32.7 mL, 1 M, pH 6.5, 32.7 mmol), magnesium chloride (9.33 mL, 1 M, 9.33 mmol), diammonium acetyl phosphate (51.8 g, 265 mmol), adenosine disodium diphosphate 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 immobilized with pantothenate quinase (SEQ ID NO: 12) and acetate quinase (SEQ ID NO: 3) was added. The reaction was stirred for 10 hours with 5N KOH to maintain pH 6.4, and (R)-2-ethynylglyceraldehyde 3-phosphate (5) was given 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, ²H), 2.88 (s, ¹H). LC-MS: (ES, m / z): C₅H₃O₆P (MH): Calculated value 193.1; Detected value 193.0.

[0142] Synthesis of 4-ethynyl D-2-deoxyribose 5-phosphate (6) Method E: [C28] To an aqueous solution of (R)-2-ethynylglyceraldehyde 3-phosphate (5) (5, 20 mL, 5.3 mmol), an aqueous solution of acetaldehyde (40 wt.%, 2.02 mL, 15.9 mmol) was added at room temperature, followed by the addition of 25 mg of deoxyribose-phosphate aldorase (DERA) (SEQ ID NO: 6) in triethanolamine hydrochloride buffer (1 mL, 1 M, pH 7). The reactor was sealed, and the mixture was stirred overnight at 30°C and 600 rpm to obtain 4-ethynyl D-2-deoxyribose 5-phosphate (6) in 99% convolution. 99% deoxyribose was not isolated as a 1:1 anomalous mixture. α-anomal: 1H NMR (D2O, 600MHz) 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, 151MHz) δ 97.7 (s), 81.4 (d), 79.4 (s), 78.9 (s), 71.1 (s), 67.7 (d), 39.6 (s). β-anomalous cell: ¹H NMR (D₂O, 600MHz) 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, 151MHz) 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): CₙH₁₀OₙP (MH): 237.0; Detected value 237.0

[0143] Alternative preparations for (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purine-9-yl)-2-ethynyl 2-(hydroxymethyl)tetrahydrofuran-3-ol monohydrate (7) [also known as 4'-ethynyl 2-fluoro-2'-deoxyadenosine or EFdA]

[0144] F1 method [C29]

[0145] Ammonium hydrogen phosphate ((2R,3S)-2-ethynyl 3,5-dihydroxytetrahydrofuran-2-yl)methyl (1.00 g, 3.91 mmol) was dissolved in 10 mL of pH 7.5 buffer (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. Enzyme solutions were prepared by dissolving phosphopentumtase (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 solutions were added to the reagent mixture, and the resulting suspension was shaken at 40°C. After 20 hours, the suspension was cooled to 0°C, filtered, and washed with cold water. The solid was dried by vacuum, and the labeled compound (1.12 g, 92%) was obtained as a single isomer. 1H NMR: (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, 1H), 5.27 (t, 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, 1H), 2.37-2.30 (m, 1H).13C NMR (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 = 2.4), 117.4 (d, JCF = 4.0), 85.1, 82.0, 81.4, 78.7, 70.1, 64.2, 38.1.LC-MS: (ES, m / z): C12H12FN5O3 (M+Na): 316.0822; 計算値316.0818。

[0146] The PPM and PNP enzymes used in this step were derived from mutations in enzymes from Escherichia coli, respectively. The sucrose phosphorylase (SP) used at this stage was derived from Alloscardovia onnicolens; SPs from other organisms could also have been used.

[0147] F2 method: [C30] Triethanolamine (7.09 g, 47.5 mmol) was added to an aqueous solution of (R)-2-ethynylglyceraldehyde 3-phosphate (5) (950 mL, 157 mmol) containing piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer at a pH of approximately 5.5–6.0. ​​The pH of the solution was adjusted to 7.1–7.6 using potassium hydroxide (8 mL, 8 M). Manganese(II) chloride hydrate (0.592 g, 4.70 mmol) was added, followed by sucrose (161 g, 470 mmol) to obtain a pH of 7.5. The following enzymes were then added to the solution: deoxyribose phosphate aldolase (SEQ ID NO: 14) (461 mg), sucrose phosphorylase (SEQ ID NO: 7) (494 mg), phosphopentumutase (SEQ ID NO: 8) (2.63 g), and purine nucleoside phosphorylase (SEQ ID NO: 15) (659 mg). After the enzymes dissolved, 2-fluoroadenine (19.80 g, 125 mmol) was added. The reaction was heated to 35°C, and acetaldehyde was added (40 wt% in isopropyl alcohol, 29.8 mL, 235 mmol). After reacting for 2 hours, EFdA crystal product (0.96 g, 2 mol%) was seeded onto the mixture. After reacting at 35°C for 26 hours, the slurry was cooled to 0°C and the solid was collected by filtration and washing twice with water (40 mL ea.). The solid was dried under nitrogen sweep. Corrected values ​​of 43.2 g, 92 wt%, and 96.2% were obtained. 1 H NMR: (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, 1H), 5.27 (t, 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, 1H), 2.37-2.30 (m, 1H). 1313C Nuclear Magnetic Resonance (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 = 2.4), 117.4 (d, JCF = 4.0), 85.1, 82.0, 81.4, 78.7, 70.1, 64.2, 38.1. LC-MS: (ES, m / z): C12H12FN5O3 (M+Na): 316.0822; calculated value 316.0818.

[0148] (S)-2-Ethynyl-propan-1,2,3-triol 1-phosphate (9) alternative preparation method G1 method: Acetate kinase: ATP regeneration system using enzyme sequence numbers 2 and 3 [Chemical formula 31] Into a 50 mL reactor, an aqueous solution of 2-ethynylpropan-1,2,3-triol (3) (9.29 g, 9.46 wt%, 7.57 mmol), potassium PIPES buffer (1.02 mL, 1 M, pH 6.5, 1.02 mmol), magnesium chloride (292 μL, 1 M, 0.292 mmol), diammonium acetyl phosphate salt (1.851 g, 89 wt%, 9.46 mmol), disodium adenosine diphosphate salt hydrate (ADP, 42 mg, 0.076 mmol, 0.01 eq), and water (28 mL) were added. The pH was adjusted to 6.4 using 5 M KOH, the solution was warmed to 20 °C, and evolved pantothenate kinase PanK sequence number 2 (264 mg) and acetate kinase AcK sequence number 3 (88 mg) were added. The reaction was stirred for 16 hours while maintaining the pH at 6.4 using 5 N KOH. The final reaction content gave (S)-2-ethynylpropan-1,2,3-triol 1-phosphate (9) with >95% e.e. and 99% conversion ([ 31 P-NMR). The product was not isolated. 1 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).13 C 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): [M-1] - C5H8O6P: 195.0058; Detected value 195.0068 [MH] - : 195.0058.

[0149] G2 method: Acetate-tokinase: ATP regeneration system using enzymes SEQ ID NO: 20 and SEQ ID NO: 21. [C32] 2-ethynylpropane-1,2,3-triol(3) (11.47 kg, 8.7 wt%, 8.61 mol) and water (7.5 kg) were added to an aqueous jacket reactor, followed by 1 L of 1 M BIS-TRIS methane buffer pH 6.5 and magnesium chloride (41.4 g). ATP (48 g, 0.086 mol, 0.01 equivalent) and 2.021 kg of diammonium acetyl phosphate (89%, 10.33 mmol) were added, the mixture was heated to 20°C, and the pH was readjusted to 6.8 using KOH (270.4 g). Evolved pantothenate quinase SEQ ID NO: 20 (20.4 g) and evolved acetate quinase SEQ ID NO: 21 (3 g) were loaded as solids. The reaction was stirred at 20°C for 16 hours, after which the pH decreased to 5.5. Quantitative conversion of 2-ethynylpropane-1,2,3-triol(3) 1 The results were determined by H and 31P NMR. The prepared (S)-2-ethynylpropane-1,2,3-triol-1-phosphate (9) solution (397 mM, 22.5 kg, 98% yield) was used in the subsequent oxidation step without further purification. 1 H 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).

[0150] G3 Method: Acetate-tokinase: The absolute stereochemistry is assigned using the enzymes SEQ ID NO: 20 and SEQ ID NO: 2 and the deuterated compound (3), and desymmetric phosphorylation is demonstrated. [C33]

[0151] Advanced pantothenate quinase SEQ ID NO: 20 (100 μL aqueous solution of 10 g / L) and advanced acetate quinase SEQ ID NO: 21 (100 μL aqueous solution of 2 g / L) were added to a pH 6.5 solution containing diammonium acetyl phosphate (41 mg), 2-ethynylpropane-1,1-d2-1,2,3-triol ((R)-3-d2, 20 mg, 170 μmol), magnesium chloride (10 μL 1 M solution in water), ADP (100 μL 100 g / L solution in water), and sodium phosphate buffer (10 μL 1 M solution in water) in 800 μL of water. This reaction was incubated at rt for 24 hours to obtain 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% total yield. 31 By 1P NMR, the ratio of phosphorylated compounds was determined to be approximately 95:5, and stereoselective phosphorylation of 2-ethynylpropane-1,2,3-triol(3) at the pro-(S)hydroxyl group (i.e., desymmetric phosphorylation) was confirmed. 1 H NMR (D2O, 500MHz) δ3.89(m, 2H), 3.72(d, J = 11.6Hz, 1H), 3.65(d, J = 11.6Hz, 1H), 2.93(s, 1H). 13 C 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).

[0152] G4 method: Acetate-tokinase: ATP regeneration system using immobilized enzymes SEQ ID NO: 20 and SEQ ID NO: 21. [C34] Enzyme immobilization procedure: Nuvia IMAC Ni charged resin (75 mL based on sedimentation volume) was added to a filter funnel and washed with water (9 column volumes, 3 × 225 mL) and binding buffer (1 column volume, 75 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). Lyophilized pantothenate quinase (SEQ ID NO: 20, 6.0 g) powder was resuspended in binding buffer (200 mL) in a container and the washed resin was added. Mixing was performed using a rotary mixer at 25°C for 6 hours. The resin was filtered and washed with binding buffer (6 column volumes, 6 × 225 mL) and BIS-TRIS buffer (8 column volume, 600 mL; 50 mM, pH 6.2). Reaction procedure: An aqueous solution of 2-ethynylpropane-1,2,3-triol(3) (574 g, 8.7% by weight, 0.430 mol) and water (350 mL) was placed in a jacketed reactor, followed by the addition of 1 M BIS-TRIS methane buffer pH 6.5 (50 mL) and magnesium chloride (2.033 g, 0.01 mol). ATP (2.37 g, 0.0043 mol, 0.01 equivalent) and diamonium phosphate (101 g, 89%, 0.530 mmol, 1.2 equivalents) were added, the mixture was heated to 20°C, and the pH of the liquid was readjusted to 6.8 using 5 M KOH. A resin (25 mL) immobilized with pantothenate quinase SEQ ID NO: 20 and evolved acetate quinase SEQ ID NO: 21 (0.15 g) was loaded as a solid. The reaction was stirred at 20°C for 16 hours, during which time 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 performed. 1 H and 31 The results were determined by P NMR (D2O, 500 MHz) with δ values ​​of 3.89 (m, 2H), 3.72 (d, J = 11.6 Hz, 1H), 3.65 (d, J = 11.6 Hz, 1H), and 2.93 (s, 1H).

[0153] Alternative preparation methods for (R)-2-ethynylglyceraldehyde 3-phosphate (5): H1 Method: Immobilized galactosoxidase SEQ ID NO: 16 [C35] Enzyme immobilization procedure: Nuvia IMAC Ni charged resin (10 mL based on sedimentation volume) was added to a filter-funnel and washed with binding buffer (10 column volume, 100 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). The resin preservative was removed to obtain 16 g of washed resin. Galactosoxidase (SEQ ID NO: 16, 750 mg) lyophilized powder, evolved in a container, was resuspended in copper(II) sulfate solution (100 μM; 5.00 mL), and binding buffer (20 mL) and washed resin (3.0 g) were added. The mixture was mixed using a rotary mixer at 20°C for 5 hours. This resin was filtered and washed with binding buffer (10 column volume, 100 mL) and BIS-TRIS buffer (10 column volume, 100 mL; 50 mM, pH 7.5) and used directly for the glycosylation reaction.

[0154] Reaction procedure: The resin of immobilized galactosoxidase SEQ ID NO: 16 (3.0 g) was added to a solution of (S)-2-ethynylpropane-1,2,3-triol-1-phosphate (9, 5.4 mmol, 270 mM, 20 mL) in BIS-TRIS methane buffer (adjusted to 35 mM, pH 7.2), followed by the addition of copper(II) sulfate solution in water (30 μL, 100 mM), and horseradish peroxidase (PEO-301, 18 mg) and bovine catarase (C1345, 120 mg) resuspended in water (600 μL). The reaction mixture was sealed with a gas-permeable membrane and vigorously shaken at 22°C for 4 days until 77% final conversion was reached, yielding (R)-2-ethynylglyceraldehyde 3-phosphate (5) at 95% ee. The enzyme resin was filtered, and a solution of (R)-2-ethynylglyceraldehyde 3-phosphate (5) was used directly in the glycosylation reaction. ¹H NMR (D₂O, 400 MHz): δ 5.02 (s, ¹H), 4.00 (dq, ²H), 2.88 (s, ¹H). LC-MS (ES, m / z): C₅H₃O₆P (MH): Measured value 193.1; Detected value 193.0.

[0155] H2 method: Immobilized galactosoxidase SEQ ID NO: 17 [C36] Enzyme immobilization procedure: Nuvia IMAC Ni charged resin (10 mL based on sedimentation volume) was added to a filter-funnel and washed with binding buffer (10 column volume, 100 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). The resin preservative was removed to obtain 16 g of washed resin. In a container, the lyophilized powder of evolved galactosoxidase (SEQ ID NO: 16, 750 mg) was resuspended in copper(II) sulfate solution (100 μM; 5.00 mL), and binding buffer (20 mL) and washed resin (3.0 g) were added. The mixture was mixed using a rotary mixer at 20°C for 5 hours. This resin was filtered and washed with binding buffer (10 column volume, 100 mL) and BIS-TRIS methane buffer (10 column volume, 100 mL; 50 mM, pH 7.5) and used directly in the reaction. Reaction procedure: Immobilized galactosoxidase SEQ ID NO.:17 (3.0 g) was added to a solution of (S)-2-ethynylpropane-1,2,3-triol-1-phosphate (9, 5.4 mmol, 270 mM, 20 mL) in BIS-TRIS methane buffer (adjusted to 35 mM, pH 7.2), followed by the addition of copper(II) sulfate solution in water (30 μL, 100 mM), and horseradish peroxidase (PEO-301, 18 mg) and bovine catarase (C1345, 120 mg) resuspended in water (600 μL). This reaction was sealed with a gas-permeable membrane and vigorously shaken at 22°C for 4 days until 77% final conversion was reached, yielding (R)-2-ethynylglyceraldehyde 3-phosphate (5) at 95% ee. The enzyme resin was filtered off, and a solution of (R)-2-ethynylglyceraldehyde 3-phosphate (5) was used directly in the glycosylation reaction. ¹H NMR (D₂O, 400 MHz): δ 5.02 (s, ¹H), 4.00 (dq, ²H), 2.88 (s, ¹H). LC-MS: (ES, m / z): r C₅H₃O₆P (MH): Calculated value 193.1; Detected value 193.0.

[0156] H3 method: Immobilized galactosoxidase SEQ ID NO: 18 [C37] Enzyme immobilization procedure: Nuvia IMAC Ni charged resin (3 mL based on sedimentation volume) was added to a filter-funnel and washed with binding buffer (10 column volume, 30 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). The resin storage solution was removed to obtain 2.4 g of washed resin. Lyophilized vial-evolved galactosoxidase (SEQ ID NO: 18, 75 mg) powder was resuspended in copper(II) sulfate solution (100 μM; 1.00 mL), and binding buffer (5 mL) and washed resin (400 mg) were added. The mixture was mixed using a rotary mixer at 20°C for 5 hours. This resin was filtered and washed with binding buffer (10 column volume, 4 mL) and BIS-TRIS methane buffer (10 column volume, 4 mL; 50 mM, pH 7.5) and used directly in the reaction. Reaction procedure: Immobilized evolved GOase SEQ ID NO.:18 (400 mg) was added to (S)-2-ethynyl-propane-1,2,3-triol 1-phosphate solution ((9), 5.4 mmol, 270 mM, 1 mL) in BIS-TRIS methane buffer (adjusted to 35 mM, pH 7.2). Then, horseradish peroxidase (PEO-301, 1 mg) and catalase derived from Corynebacterium glutamicum (Roche, lyophilized, #11650645103, 3 mg), resuspended in water (100 μL), were added. This reaction was sealed with a gas-permeable membrane and vigorously shaken at 30°C for 48 hours. After 2 days, the final conversion reached 90%, and (R)-2-ethynylglyceraldehyde 3-phosphate (5) > 99% ee. The enzyme resin was filtered off, and the solution of (R)-2-ethynylglyceraldehyde 3-phosphate (5) 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): r C₅H₃O₆P (MH): Calculated value 193.1; Detected value 193.0.

[0157] H4 method: Immobilized galactosoxidase SEQ ID NO: 19 [C38] Enzyme immobilization procedure: Nuvia IMAC Ni charged resin (3 mL based on sedimentation volume) was added to a filter-funnel and washed with binding buffer (10 column volume, 30 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). The resin storage solution was removed to obtain 2.4 g of washed resin. In a container, lyophilized powder of evolved galactosoxidase (SEQ ID NO: 19, 75 mg) was resuspended in copper(II) sulfate solution (100 μM; 1.00 mL), and binding buffer (5 mL) and washed resin (400 mg) were added. The mixture was mixed using a rotary mixer at 20°C for 5 hours. This resin was filtered and washed with binding buffer (10 column volume, 4 mL) and BIS-TRIS methane buffer (10 column volume, 4 mL; 50 mM, pH 7.5) and used directly in the reaction. Reaction procedure: Immobilized GOase SEQ ID NO: 18 was added (400 mg) to a solution of (S)-2-ethynyl-propane-1,2,3-triol 1-phosphate (9, 5.4 mmol, 270 mM, 1 mL) in BIS-TRIS methane buffer (adjusted to 35 mM, pH 7.2). Then, horseradish peroxidase (PEO-301, 1 mg) and catalase derived from Corynebacterium glutamicum (Roche, lyophilized, #11650645103, 3 mg), resuspended in water (100 μL), were added. The reaction mixture was sealed with a gas-permeable membrane and vigorously shaken at 30°C for 48 hours. After 2 days, the final conversion reached 100%, and (R)-2-ethynylglyceraldehyde 3-phosphate (5) was obtained with >99% ee. The enzyme resin was filtered off, and the solution of (R)-2-ethynylglyceraldehyde 3-phosphate (5) 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): C₅H₃O₆P (MH): Calculated value 193.1; Detected value 193.0.

[0158] "Amino acids" are referred to herein by one of the single-letter symbols recommended by the IUPAC-IUB B Biochemical Nomenclature Commission. For the purposes described herein, the codes used for the genetically coded amino acids used for the enzymes used in the methods herein are conventionally shown in Table 2: [Table 3]

[0159] The sequence numbers of enzymes used or that may be used in the steps for synthesizing EFdA described herein and in the exemplified steps in the experimental procedures described herein are provided, but are not limited to those in Table 3. [Table 4]

[0160] Horseradish peroxidase: A wild-type peroxidase isolated from horseradish root (Amoracia rusticana), derived from commercially available horseradish type I, SIGMA (P8125).

[0161] Catalase: (1) wild-type catalase from commercially available bovine liver from SIGMA(C1345); or (2) CAT-101, biocatalyst; or (3) derived from Corynebacterium glutamicum (Roche, #11650645103).

[0162] Additional embodiments of the present invention, without limitation, include the use of the following enzymes in the synthetic process steps described herein for producing 4'-ethynyl 2'-deoxynucleoside or its analogues, such as EFdA.

[0163] 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 with SEQ ID NO: 9 or SEQ ID NO: 15, wherein the polypeptide sequence of the engineered purine nucleoside phosphorylase comprises at least one amino acid substitution or set of amino acid substitutions compared to SEQ ID NO: 9 or SEQ ID NO: 15. 2A. The manipulated purine nucleoside phosphorylase according to 1A, wherein the manipulated purine nucleoside phosphorylase contains a polypeptide sequence that is identical to SEQ ID NO: 9 or SEQ ID NO: 15 by at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. 3A. A modified purine nucleoside phosphorylase comprising the polypeptide sequence described in SEQ ID NO: 9 or SEQ ID NO: 15. A4. One of the manipulated purine nucleoside phosphorylases from 1A to 3A, which has at least one improved characteristic compared to wild-type E. coli purine nucleoside phosphorylase. 5A. The modified purine nucleoside phosphorylase according to 4A, wherein the improved properties include improved activity toward substrate compound 6.5 (its cyclic or open-chain aldehyde or hydrate, or a salt thereof) compared with wild-type E. coli purine nucleoside phosphorylase. 6A. The modified purine nucleoside phosphorylase described in 4A, having improved properties, including improved production of EFdA (compound 7), compared to wild-type E. coli purine nucleoside phosphorylase. 7A. The manipulated purine nucleoside phosphorylase described in any one of A1 to 6A, wherein the manipulated purine nucleoside phosphorylase is purified. 8A. One of the purine nucleoside phosphorylases from 1A to 7A, wherein at least one amino acid substitution (i.e., one or more amino acid substitutions) is a conservative amino acid substitution.

[0164] B. Phosphopentumutase. 1B. An engineered phosphopentumutase comprising a polypeptide sequence or a functional fragment thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 8, wherein the polypeptide sequence of the engineered phosphopentumutase comprises at least one amino acid substitution or set of amino acid substitutions compared to SEQ ID NO: 8. 2B. The engineered phosphopentumutase according to 1B, wherein the genetically engineered phosphopentumutase contains a polypeptide sequence that is identical to SEQ ID NO: 8 by at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. 3B. An engineered phosphopentumutase comprising the polypeptide sequence described in Sequence ID No. 8. 4B. One of the manipulated phosphopentumutases from 1B to 3B, which exhibits at least one improved characteristic compared to wild-type E. coli phosphopentumutase. 5B. The modified phosphopentumutase according to 4B, wherein the improved properties include improved activity toward substrate compound 6 (its cyclic or open-chain aldehyde or hydrate, or a salt thereof) compared with wild-type E. coli phosphopentumutase. 6B. The modified phosphopentumutase described in 4B, wherein the improved properties include improved production of compound 6.5 or compound 7 (EFdA) compared to wild-type E. coli phosphopentumutase. 7B. One of the manipulated phosphopentumutases from 1B to 6B is purified. 8B. One of the manipulated phosphopentumutases from 1B to 7B, wherein at least one amino acid substitution (i.e., one or more amino acid substitutions) is a conservative amino acid substitution.

[0165] C. Deoxyribose-phosphate aldola-ase. 1C. Deoxyribose-phosphate aldorase consisting of the wild type derived from the Shewanella halifaxensis polypeptide sequence described in Sequence ID No. 5. 2C. A modified deoxyribose-phosphate aldola-ase comprising the polypeptide sequence described in SEQ ID NO: 6 or SEQ ID NO: 14. 3C. An engineered deoxyribose-phosphate aldorase comprising a polypeptide sequence that is identical to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 14. 4C. A polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 14, or a polypeptide sequence comprising at least one amino acid substitution or set of amino acid substitutions with respect to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 14, an engineered deoxyriboside-phosphate aldorase, or a functional fragment thereof. 5C. A 1C to 4C deoxyribose-phosphate aldola-ase that is active on substrate compound 5 ((R)-2-ethynylglyceraldehyde 3-phosphate, its hydrate, or any of the above salts). 6C. A deoxyribose-phosphate aldola-ase of any one of 1C to 5C, comprising the ability to produce compound 6 (4-ethynyl D-2-deoxyribose 5-phosphate, or its open-chain aldehyde or hydrate form, or any of the salts thereof) without requiring a protecting group on substrate compound 5 (((R)-2-ethynylglyceraldehyde 3-phosphate, its hydrate, or any of the salts thereof) during the reaction. 7C. A modified deoxyribose-phosphate aldolase of any one of 2C to 6C having improved properties, including improved production of compound 6 (4-ethynyl D-2-deoxyribose 5-phosphate, or its open-chain aldehyde or hydrate form, or a salt of any of the foregoing), compared to wild-type Shewanella halifaxensis deoxyribose-phosphate aldolase. 8C. Deoxyribose phosphate aldolases are purified from any one of the deoxyribose phosphate aldolases from 1C to 7C. 9C. One of the 2C-7C engineered deoxyribose-phosphate aldolases, in which at least one amino acid substitution (i.e., one or more amino acid substitutions) is a conservative amino acid substitution.

[0166] D. Pantothenate quinase. 1D. An engineered pantothenate quinase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity in SEQ ID NO: 2, SEQ ID NO.: 12, SEQ ID NO.: 13, or SEQ ID NO.: 20, or a functional fragment thereof, wherein the polypeptide sequence of the engineered pantothenate quinase comprises at least one amino acid substitution or set of amino acid substitutions compared to SEQ ID NO: 2, SEQ ID NO.: 12, SEQ ID NO.: 13, or SEQ ID NO.: 20. 2D. The manipulated pantothenate quinase according to 1D, wherein the manipulated pantothenate quinase comprises a polypeptide sequence that is identical to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 20. 3D. Manipulated pantothenate quinase comprising the polypeptide sequence described in SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 20. 4D. One of 1D to 3D manipulated pantothenate quinases, containing at least one improved characteristic compared to wild-type E. coli pantothenate quinase. 5D. The modified pantothenate quinase described in 4D, whose improved properties include improved activity toward substrate compound 4 ((R)-2-ethynylglyceraldehyde or its hydrate form) compared to the wild type e. Pantothenate quinase of Escherichia coli. 6D. The modified pantothenate quinase described in 5D, which has improved properties, including improved production of compound 5((R)-2-ethynylglyceraldehyde 3-phosphate), compared to wild-type pantothenate quinase. 7D. Modified pantothenate quinase as described in 4D, with improved properties including improved activity against substrate compound 3 (2-ethynylpropane-1,2,3-triol) compared to wild-type e. Pantothenate quinase from Escherichia coli. 8D. The modified pantothenate quinase described in 7D, which has improved properties, including improved production of compound 9 ((S)-2-ethynylpropane-1,2,3-triol-1-phosphate), compared to wild-type pantothenate quinase. 9D. One of the manipulated pantothenate quinases from 1D to 8D is purified. 10D. One of the manipulated pantothenate quinases from 1D to 9D, wherein at least one amino acid substitution (i.e., one or more amino acid substitutions) is a conservative amino acid substitution.

[0167] E. galactosoxidase. 1E. An engineered galactosoxidase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NOs.:1, 10, 11, 16, 17, 18, or 19, or a functional fragment thereof, wherein the polypeptide sequence of the engineered galactosoxidase comprises at least one amino acid substitution or set of amino acid substitutions compared to SEQ ID NOs.:1, 10, 11, 16, 17, 18, or 19. 2E. The engineered galactosoxidase according to 1E, wherein the genetically engineered galactosoxidase comprises a polypeptide sequence that is sequence ID NO: 1, 10, 11, 16, 17, 18 or 19 in at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. 3E. A manipulated galactosoxidase comprising the polypeptide sequence shown in SEQ ID NOs: 1, 10, 11, 16, 17, 18, or 19. 4E. One of 1E to 3E engineered galactosoxidases, containing at least one improved characteristic compared to wild-type F. graminearum galactosoxidase. 5E. The modified galactosoxidase described in 4E, having improved properties, including improved activity toward primary alcohol substrates compared to wild-type F. graminearum galactosoxidase. 6E. Modified galactosoxidase of 4E, with improved properties including improved activity toward substrate compound 3 (2-ethynylpropane-1,2,3-triol) compared to wild-type F. graminearum galactosoxidase. 7E. Modified galactosoxidase of 6E, with improved properties compared to wild-type F. graminearum galactosoxidase, including improved production of compound 4 ((R)-2-ethynylglyceraldehyde or its hydrate form). 8E. Modified galactosoxidase of 4E, which exhibits improved properties compared to wild-type F. graminearum galactosoxidase, including improved activity toward substrate compound 9(((S)-2-ethynylpropane-1,2,3-triol-1-phosphate). 9E. The modified galactosoxidase described in 8E, having improved properties compared to wild-type F. graminearum galactosoxidase, including improved production of compound 5 ((R)-2-ethynylglyceraldehyde 3-phosphate or its hydrate form). 10E. One of the manipulated galactosoxidases from 1E to 9E, from which the galactosoxidase is purified. 11E. One of the manipulated galactosoxidases from 1E to 10E, wherein at least one amino acid substitution (i.e., one or more amino acid substitutions) is a conservative amino acid substitution.

[0168] F. Acetate quinase. 1F. Acetate quinase consisting of wild-type derived from the Thermotoga maritima polypeptide sequence described in SEQ ID NO: 3 or SEQ ID NO: 21. 2F. An engineered acetatetokinase comprising a polypeptide sequence which is identical to SEQ ID NO: 3 or SEQ ID NO: 21 by at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. 3F. Manipulated acetate quinase 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 with SEQ ID NO: 3 or SEQ ID NO: 21, wherein the polypeptide sequence of the manipulated acetate quinase comprises at least one amino acid substitution or set of amino acid substitutions compared to SEQ ID NO: 3 or SEQ ID NO: 21. 4F. A 2F or 3F acetate quinase containing at least one improved characteristic compared to wild-type T. maritima acetate quinase. 5F. The acetate quinase described in 4F, whose improved properties include improved activity for ATP-cofactor recycling in the phosphorylation reaction on substrate compound 4((R)-2-ethynylglyceraldehyde or its hydrate form) compared with wild-type Thermotoga maritima acetate quinase. 6F. The acetate quinase according to 5F, wherein the improved properties include improved production of compound 5 ((R)-2-ethynylglyceraldehyde 3-phosphate or its hydrate form, or any salt thereof) compared with wild-type Thermotoga maritima acetate quinase. 7F. The acetate quinase according to 4F, wherein the improved properties include improved activity for ATP-cofactor recycling in the phosphorylation reaction on substrate compound 3 (2-ethynylpropane-1,2,3-triol) compared to wild-type Thermotoga maritima acetate quinase. 8F. The acetate quinase according to 7F, wherein the improved properties include improved production of compound 9(((S)-2ethynyl-propane-1,2,3-triol 1-phosphate) or a salt thereof, compared to wild-type Thermotoga maritima acetate quinase. 9F. The acetate quinase described in any one of items 1F to 8F, wherein the acetate quinase is purified. 10F. One of the manipulated acetate quinases from 2F to 7F, in which at least one amino acid substitution (i.e., one or more amino acid substitutions) is a conservative amino acid substitution.

Claims

1. A method for synthesizing a compound of the following formula 【Chem.】 comprising combining compound 6.5 in a buffer containing a manganese (II) salt with purine nucleoside phosphorylase and 2-fluoroadenine, 【Chemical Formula 1】 [wherein, 2X ＋ is (a) two protons, (b) one proton and one monovalent cation, (c) two identical or different monovalent cations, or (d) one divalent cation.] wherein the purine nucleoside phosphorylase comprises a polypeptide sequence having at least 90% identity to SEQ ID NO: 9 or SEQ ID NO: 15 and has purine nucleoside phosphorylase activity, said method.

2. The method according to claim 1, further comprising isolating a compound of the following formula

3. 【Chemical Formula 3】 further comprising combining compound 6 and phosphopentomutase with purine nucleoside phosphorylase and 2-fluoroadenine in a buffer containing a manganese (II) salt, wherein the phosphopentomutase comprises a polypeptide sequence having at least 90% identity to SEQ ID NO: 8 and has phosphopentomutase activity, the method according to claim 1.

4. 【Chemical Formula 4】 The method according to claim 3, further comprising removing inorganic phosphate by-products from the reaction mixture.

5. The method according to claim 4, comprising removing inorganic phosphate by-products from the reaction solution by (a) adding sucrose phosphorylase and sucrose to the reaction mixture, or (b) adding calcium, magnesium or manganese to the reaction mixture.

6. The method according to any one of claims 3 to 5, further comprising isolating a compound of the following formula

7. The method according to claim 3, further comprising a step of synthesizing compound 6, comprising combining compound 5 with acetaldehyde and deoxyribose-phosphate aldolase in an aqueous solution to produce compound 6, wherein the deoxyribose-phosphate aldolase comprises a polypeptide sequence having at least 90% identity to SEQ ID NO: 6 or SEQ ID NO: 14 and has deoxyribose-phosphate aldolase activity, said method.

8. The method according to claim 7, wherein the reaction is carried out in a sealed container. 【Chemical Formula 7】 [wherein, 2X ＋ is (a) two protons, (b) one proton and one monovalent cation, (c) two identical or different monovalent cations, or (d) one divalent cation.]

9. The method according to claim 7 or 8, further comprising a step of synthesizing compound 5, wherein the synthesis comprises combining compound 4 with pantothenate kinase in a buffer in the presence of a divalent metal salt and combining the in-situ regenerated ATP as a phosphate source to produce compound 5, said method.

10. ​ ​ ​ 【Chemical 8】 ​ ​ The method according to claim 9, wherein 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.

11. The method according to claim 10, wherein (a) pantothenate kinase is immobilized, or (b) pantothenate kinase and acetate kinase are immobilized.

12. The method according to claim 9, further comprising the step of synthesizing compound 4, wherein the synthesis is carried out in a buffer in the presence of oxygen, and compound 3 【Chemical Formula 9】 is combined with (a) galactose oxidase, copper, catalase and (b) peroxidase or an oxidizing agent to produce compound 4.

13. The method according to claim 12, wherein galactose oxidase is immobilized.

14. A method for synthesizing a compound of the following formula 【Chem.】 in a buffer containing a manganese (II) salt, wherein compound 5 【Chemical Formula 10】 [wherein, 2X ＋ is (a) two protons, (b) one proton and one monovalent cation, (c) two identical or different monovalent cations, or (d) one divalent cation.], is combined with acetaldehyde and 2-fluoroadenine in combination with deoxyribose-phosphate aldolase, phosphopentomutase and purine nucleoside phosphorylase, the deoxyribose-phosphate aldolase comprises a polypeptide sequence having at least 90% identity to SEQ ID NO: 6 or SEQ ID NO: 14 and has deoxyribose-phosphate aldolase activity, the phosphopentomutase comprises a polypeptide sequence having at least 90% identity to SEQ ID NO: 8 and has phosphopentomutase activity, the purine nucleoside phosphorylase comprises a polypeptide sequence having at least 90% identity to SEQ ID NO: 9 or SEQ ID NO: 15 and has purine nucleoside phosphorylase activity.

15. The method according to claim 14, further comprising removing inorganic phosphate by-products from the reaction mixture.

16. The method according to claim 15, comprising removing inorganic phosphate by-products from the reaction mixture by (a) adding sucrose phosphorylase and sucrose to the reaction mixture, or (b) adding calcium, magnesium or manganese to the reaction mixture.

17. The method according to any one of claims 14 to 16, further comprising isolating a compound of the following formula ​ ​

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