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. [ka]

[0003] One example of a 4'-ethynylnucleoside analog is 4'-ethynyl 2'-deoxyadenosine (EFdA, also known as MK-8591), a nucleoside reverse transcriptase translocation inhibitor that blocks HIV-1 and SIV viral replication in vitro (Kawamoto, E., Sarafianos S. F. 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 in the '053 patent as 2'-deoxy-4'-C-ethynyl 2-fluoroadenosine). EFdA has the following chemical structure: [ka]

[0004] EFdA is metabolized intracellularly to an active triphosphate anabolite that inhibits HIV reverse transcriptase. In contrast to the nucleoside reverse transcriptase inhibitors (NsRTIs) and nucleotide reverse transcriptase inhibitors (NtRTIs) currently available for the treatment of HIV infection, which lack a 3'-OH group that blocks the binding of incoming nucleotides, EFdA retains its 3'-OH group, which prevents primer:template translocation in the reverse transcriptase (RT) active site and acts as a chain terminator by preventing the binding of incoming deoxyribonucleotide triphosphates (dNTPs). Furthermore, the modified ribose ring pucker of EFdA is thought to contribute to reverse transcriptase inhibition by positioning the 3'-OH at a vector where phosphoryl 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 subtypes evaluated. It is rapidly converted to the active triphosphate in both lymphoid-derived cell lines and peripheral blood mononuclear cells in vitro, and the intracellular half-life of EFdA triphosphate (EFdA-TP) exceeds 72 hours. (Stoddart, CA, Galkina, et al., Oral Administration of the Nucleoside EFdA (4'-Ethynyl-2-Fluoro-2'-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, published online May 4, 2015.)

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

[0007] Conventional syntheses of 4'-ethynylnucleoside analogs, including EFdA, suffer from poor stereoselectivity in the formation of the C-N bond between the ethynyldeoxyribose sugar and the 2-fluoroadenine (also known as 2-fluoro-9H-purin-6-amine) nucleobase. Conventional syntheses also require protecting groups for 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 that uses diastereoselective reactions to set up three stereocenters. The stereochemistry of the anomeric center is controlled by the inclusion of a 2'-acetoxy directing group, which is subsequently removed by hydrolysis and deoxygenation. This route requires four chromatographic purifications and the stoichiometric use of toxic organotin reagents for the later deoxygenation.

[0009] Another route (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) stereoselectively generates fully substituted 4'-carbinols by enzymatic desymmetrization. The 3'-stereocenter is set by catalytic asymmetric transfer hydrogenation, and the anomeric 1'-bond is established with moderate stereoselectivity using substrate control. Stereochemical purity is achieved by crystallization of the intermediate. This process requires 15 steps, requires the use of protecting groups, and generates the glycosyl bond between the nucleobase and the sugar fragment with low stereoselectivity (1.8:1).

[0010] A 12-step synthesis for producing EFdA from R-glyceraldehyde acetonide has been 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 a chiral starting material to create a 3'-stereocenter with moderate diastereoselectivity. After chromatographic separation of the stereoisomers, the new stereocenter is used to induce diastereoselective alkyne addition to create a fully substituted 4'-stereocenter. The anomeric 1'-position has less stereocontrol, and chromatography is required to separate the anomers. This route requires chromatographic separation of the diastereoisomers at two different steps and begins 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 pathway that starts from an existing nucleoside and modifies both the sugar and base moieties. The synthesis involves 18 steps starting from 2-amino-2'-deoxyadenosine, with a low overall yield of only 2.5%.

[0012] Enzymes such as purine nucleoside phosphorylase (PNP, EC 2.4.2.1) are known to be able to form glycosyl 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" by Mikhailopulo, IA, and 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 with small H, NH, or F substituents at the C2' and C3' positions and substitutions of the C5' OH group. There have been no reports of successful PNP-catalyzed glycosylation using sugars with carbon substituents on the ring or any substitutions 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 a phosphate group from the 5'-hydroxyl to the 1'-hydroxyl position by the enzyme phosphopentomutase (PPM, EC 5.4.2.7) (Mikhailopulo, IA, supra). However, the range of sugars that can catalyze this reaction is limited to ribose, arabinose, 2-deoxyribose, and 2,3-dideoxyribose. Successful reactions with sugar phosphates containing other substituents have not been reported.

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

[0015] U.S. Patent No. 7,229,797 describes the production of deoxyribonucleosides from natural unsubstituted deoxyribose 1-phosphate by using purine nucleoside phosphorylase (PNP) and, further, by using an enzyme such as sucrose phosphorylase to remove the inorganic phosphate by-product and drive the equilibrium. This document does not disclose enzyme engineering to create a PNP enzyme capable of producing nucleosides 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 unnatural substrates through 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-licensed document 2] Ohrui, H., H. et al., Nucleosides & Nucleic Acids, 26, 1543-1546

[2007] ) [Non-licensed document 3] Hattori, S., K., Nakata, H. et al, Antimicrobial. Agents and Chemotherapy, 53, 3887-3893

[2009]

Non-licensed Document 4

[2009]

Non-licensed Document 5

[2014] )

Non-licensed Document 6

[0018] Given the laborious and lengthy synthetic options developed to date for producing 4'-ethynyl nucleoside analogs, it would be desirable to develop an improved enzymatic synthesis for 4'-ethynyl nucleoside analogs such as EFdA that reduces the number of process steps, minimizes the use of protecting groups, improves the stereoselectivity of glycosylation, and avoids the use of toxic substances.

[0019] Surprisingly, it was found that the PPM enzyme is active at the 3-atom ethynyl substituent at the 4' position of ribose, and by introducing mutations into the enzyme, the PPM enzyme activity could be improved and the PPM-catalyzed isomerization of 4-ethynyl-D-2-deoxyribose 5-phosphate (6) to 4-ethynyl-D-2-deoxyribose 1-phosphate (6.5) could be successfully developed, enabling a more efficient method for producing 4'-ethynyl-2'-deoxynucleosides.

[0020] Furthermore, it has been found that the PNP enzyme also has activity at the 3-atom ethynyl substituent at the 4-position of deoxyribose, and that introducing mutations into the enzyme can improve PNP enzyme activity, improve the sugar modification reaction catalyzed by PNP, and enable a more efficient method for producing 4'-ethynyl-2'-deoxynucleosides.

[0021] Further improvements to the overall synthetic methodology came from the discovery that DERA enzymes, particularly those from Shewanella halifaxensis, are active in the aldol reaction with 2-ethynyl-glyceraldehyde 3-phosphate, which has a fully substituted α-carbon. This discovery enabled the efficient synthesis of 4-ethynyl D-2-deoxyribose 5-phosphate, a precursor to 4'-ethynyl 2'-deoxynucleoside analogs, including EFdA.

[0022] Summary of the Invention

[0023] The present invention involves the use of engineered enzymes in novel enzymatic syntheses of 4'-ethynyl 2'-deoxynucleoside analogs, including EFdA, that eliminate the use of protecting groups on intermediates, improve the stereoselectivity of glycosylation, and, among other process improvements, significantly reduce the number of process steps required to make the compounds compared to previous methods. The present invention further relates to novel intermediates that are an integral part of the enzymatic process.

[0024] The overall process is summarized below in Scheme 1 and Scheme 2. The latter scheme provides an alternative method for making compound 5.

[0025] [ka]

[0026] [ka]

[0027] Acid forms or salts of the phosphate intermediates can be used in the processes described herein, and are not limited to the specific acid or salt forms provided in the process step examples herein. For all phosphate intermediates described herein, 2X + represents any combination of two protons, one proton and one other monovalent cation, two monovalent cations (same 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 synthetic steps herein can exist in either such form or a mixture of such forms in the reactions described herein. For example, compounds (4) and (5), while depicted as a hydrate and an aldehyde, respectively, in Scheme 1, can exist as either the hydrate or the aldehyde form, or a mixture thereof, at the reaction step in which they occur. Each of these forms is encompassed by reference to compound number (4) or (5) in the steps herein.

[0030] [ka]

[0031] Compound (3) is achiral and may be represented herein as either: [ka]

[0032] Compound (6) can be present as the acid or its salt in the reaction step where it is present as its cyclic or open-chain aldehyde or hydrate, respectively. [ka]

[0033] Detailed Description of the Invention

[0034] 4-Ethynyl 2-deoxynucleic acid and its analogs having an anomeric-CN bond [ka] are being explored for activity against HIV, AIDS, and related diseases. 4'-Ethynyl 2'-deoxynucleosides and their analogs contain 4'-ethynyl 2'-deoxyribose linked via an anomeric C-N bond to a purine or pyrimidine nucleobase (adenine, guanine, cytosine, thymine, or uracil) or a modified purine or pyrimidine nucleobase.

[0035] 4-Ethynyl 2'-deoxynucleoside analogs, such as EFdA, can be synthesized using a final one-pot method by combining 4-ethynyl D-2-deoxyribose 5-phosphate (6) with two enzymes, phosphopentomutase (PPM) [e.g., but not limited to, SEQ ID NO:8] and purine nucleoside phosphorylase (PNP) [e.g., SEQ ID NO:9, SEQ ID NO:15], as shown in Scheme 2. [ka] [ka]

[0036] The final step of the synthesis uses a bienzymatic reaction (optionally involving a third enzyme) to drive the equilibrium toward the desired end product, as shown in Scheme 2. The final step begins with compound 6 or a salt thereof, where 6 is the cyclic 4-ethynyl 2-deoxyribose 5-phosphate or its open-chain aldehyde or hydrate form, as described above.

[0037] Compound (6) is combined with phosphopentomutase (PPM), purine nucleoside phosphorylase (PNP), sucrose phosphorylase, sucrose, and a nucleobase such as unsubstituted or substituted adenine in a buffer solution containing a manganese(II) salt and adjusted to a pH of about 6.5 to 8.0 or higher, particularly about 7.0 to 7.5. The molar ratio of sucrose to compound (6) can 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 is stirred within a temperature range that does not denature the enzyme, for example, about 30-45° C., more particularly, about 35-45° C. To some extent, the reaction may proceed at cooler temperatures, but this will slow the reaction rate.

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

[0040] The equilibrium reaction can be advanced to high conversion of the final product by consuming the inorganic phosphate by-product through the phosphorylation 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, any other option for removing the phosphate can be used, such as adding calcium, magnesium, or manganese to the reaction to precipitate the phosphate. This highly efficient and eco-friendly process forms anomeric bonds between sugars and nucleobases with very high stereoselectivity without the use of protecting groups or organic solvents, and has the advantage of being 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 of the final product, or extraction into a suitable solvent followed by crystallization.

[0042] As shown in Scheme 2A, the final step of the synthesis can use a three-enzyme reaction (optionally involving a fourth enzyme) to drive the reaction balance toward the desired end product. The final step begins with compound 5 or a salt thereof, where 5 is (R)-2-ethynylglyceraldehyde 3-phosphate or its hydrate form.

[0043] Compound (5) is combined with deoxyribose phosphate aldolase (DERA), acetaldehyde, phosphopentomutase (PPM), purine nucleoside phosphorylase (PNP), sucrose phosphorylase, sucrose, and their nucleobases or analogs, such as unsubstituted or substituted adenine, in a buffer solution containing a manganese(II) salt and optionally adjusted to a pH in the range of about 4-10, or more particularly about 6.5-8.0 or higher, particularly about 7.0-7.5. The molar ratio of sucrose to compound (5) can 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 is carried out within a temperature range that does not denature the enzyme, for example, about 30-45° C., or particularly about 35-45° C. To some extent, cooler temperatures may work, but will slow the reaction rate.

[0045] Acetaldehyde is added as a solution, particularly 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 in isopropanol, acetaldehyde in ethanol, acetaldehyde in water, and acetaldehyde in THF. The molar ratio of aldehyde to compound (5) can be, but is not limited to, about 0.5:1 to 4:1, more particularly 1.5:1.

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

[0047] The equilibrium reaction can be advanced to high conversion of the final product by consuming the inorganic phosphate by-product through the phosphorylation 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, any other option for removing the phosphate can be used, such as adding calcium, magnesium, or manganese to the reaction to precipitate the phosphate. This highly efficient and eco-friendly process has the advantage of forming anomeric bonds between sugars and nucleobases with very high stereoselectivity without the use of protecting groups or organic solvents and can be performed 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 of the final product, or extraction into a suitable solvent followed by crystallization.

[0049] Several upstream intermediates used in this process for the synthesis of the final product, 4'-ethynyl 2'-deoxynucleoside and its analogs, are also prepared using enzymatic methods, as shown in Scheme 3 (Scheme 3A and Scheme 3B). [ka] [ka] [ka]

[0050] Compound 4: Oxidase reaction

[0051] As shown in Reaction Scheme 3, (R)-2-ethynylglyceraldehyde (4) is prepared by reacting galactose oxidase with 2-ethynylpropane-1,2,3-triol (3) in a buffer solution appropriately adjusted to a pH of about 3 to 10 or higher, particularly about 6 to 8. Any buffer having an appropriate pH range, for example, sodium phosphate; sodium acetate; PIPES, for example, piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, for example, 3-(N-morpholino)propanesulfonic acid or 3-piperazineethanesulfonic acid; HEPES, for example, 4-(2-hydroxyethyl)-1-piperazinesulfonic acid or 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; TRIS, for example, tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol; and TRIS methane, for example, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; boric acid; CAPS, for example, N-cyclohexyl-3-aminopropanesulfonic acid; MES, for example, 2-(N-morpholino)ethanesulfonic acid; CHES, such as N-cyclohexyl-2-aminoethanesulfonic acid, glycine, or bicine (N,N-bis(2-hydroxyethyl)glycine); sodium phosphate are preferred.

[0052] Both copper and peroxidase are used in the reaction to activate galactose oxidase (GOase). Copper is provided 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, or it may be substituted with an oxidizing agent such as ferricyanide, iridatides, manganese(III) salts, persulfates, and other one- or two-electron oxidants, or inorganic or organic oxidants. Preferably, the peroxidase is horseradish peroxidase. Catalase is also added to prevent inactivation of GOase. Catalase may be from mammalian sources (bovine), or from bacterial or fungal sources 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 the use of hydrogen peroxide / catalase, superoxide, or other methods known in the art for this purpose.

[0054] The reaction can be carried out at a substrate concentration of about 10 to 180 g / L, particularly about 20 to 50 g / L, at a temperature of about 0 to 40°C, particularly about 10 to 30°C.

[0055] Compound 8: Aminal formation

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

[0057] Optionally, 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 mixtures thereof, with MTBE being preferred. Crystallization is carried out at a temperature of, for example, about 40° C. but not exceeding 50° C. to avoid decomposition of the aminal.

[0058] The reaction proceeds in the absence of oxygen. One convenient method is to sparge the reaction with N. Alternatively, other systems for excluding 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 the respective aminal by reaction with an organic or inorganic acid in the presence of a water-immiscible organic solvent at a temperature below 50°C, e.g., about 0-15°C, to avoid decomposition of the aminal. Any organic or inorganic acid can be used, including, but not limited to, p-toluenesulfonic acid, methanesulfonic acid, camphorsulfonic acid, acetic acid, hydrochloric acid, phosphoric acid, and sulfuric acid. Due to the low solubility of N,N'-dibenzylethane-1,2-diaminebis-p-toluenesulfonate in water, p-toluenesulfonic acid is preferred for the reaction with aminal 8. Any water-immiscible solvent, e.g., MTBE, 2-MeTHF, CPME, diethyl ether, diisopropyl ether, ethyl acetate, isopropyl acetate, toluene, DCM, or mixtures thereof, is preferred. MTBE and 2-MeTHF are also preferred. The reaction can be carried out at a substrate concentration of about 5 to 100 g / L, particularly 20 to 50 g / L.

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

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

[0063] Compound 5: Kinase reaction [ka] As shown in Scheme 3 and Scheme 3A, (R)-2-ethynylglyceraldehyde 3-phosphate hydrate (5) is prepared by reacting wild-type pantothenate kinase (PanK) derived from Escherichia coli or a variant thereof with compound (4) in a buffer solution adjusted to a pH in the range of about 4 to 10, preferably about 6.5 to 8.5, and more preferably about 5.5 to 8.5. Buffers having an appropriate pH range can be used, including, 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)piperazin-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. The reaction can be carried out in the presence of any suitable divalent metal salt, including, but not limited to, magnesium salts, such as magnesium chloride, and salts of cobalt, manganese, zinc, or calcium.

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

[0065] The reaction can be carried out at a substrate concentration of about 10 to 100 g / L, particularly about 20 to 40 g / L, at a temperature of about 0 to 40°C, particularly about 10 to 25°C.

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

[0067] Compound 9: Kinase reaction [ka]

[0068] As shown in Reaction Scheme 3B, (S)-2-ethynylpropane-1,3-triol 1-phosphate (9) can be prepared by reacting wild-type pantothenate kinase (PanK) derived from Escherichia coli or a variant thereof with compound (3) in a buffer solution adjusted to a pH range of about 4 to 10, preferably about 6.5 to 8.5, and more preferably about 5.5 to 8.5. Buffers having an appropriate pH range can be used, including, 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)piperazin-1-yl]ethanesulfonic acid); trimethylolamine and TRIS (e.g., tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol), with sodium phosphate being preferred. The reaction can be carried out in the presence of any suitable divalent metal salt, including, but not limited to, magnesium salts, such as magnesium chloride, and salts of cobalt, manganese, zinc, or calcium.

[0069] This reaction utilizes adenosine 5'-diphosphate (ADP) as the phosphate source, which requires regeneration to 5'-triphosphate (ATP). ATP is generated in situ and can subsequently 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 to ATP. Alternatively, (a) a combination of pyruvate oxidase, catalase, and acetate kinase in the presence of pyruvate, phosphate, and oxygen can be used to regenerate ADP to ATP, or (b) a combination of pyruvate oxidase, catalase, and acetate kinase in the presence of pyruvate, phosphate, and acetate kinase, in combination with acetyl phosphate and acetate kinase, can be used to regenerate ATP from ADP.

[0070] The reaction can be carried out at a substrate concentration of about 10 to 100 g / L, particularly about 20 to 40 g / L, at a temperature of about 0 to 40°C, particularly about 10 to 25°C.

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

[0072] Compound 5: Oxidase reaction [ka]

[0073] As shown in Scheme 3B, (R)-2-ethynylglyceraldehyde hydrate 3-phosphate (5) is prepared by reacting galactose oxidase with (S)-2-ethynylpropane-1,2,3-triol 1-phosphate (9) in a buffer solution adjusted to a pH of about 3 to 10, or more preferably about 6 to 8. Any buffer having a suitable pH range can be used, including, but not limited to, sodium phosphate; sodium acetate; PIPES, such as piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, such as 3-(N-morpholino)propanesulfonic acid or 3-piperazineethanesulfonic acid; HEPES, such as 4-(2-hydroxyethyl)-1-piperazinesulfonic acid or 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; TRIS, such as tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol; Bis-TRIS methane, such as 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borates; CAPS, such as N-cyclohexyl-3-aminopropanesulfonic acid; glycine; or bicine (N,N-bis(2-hydroxyethyl)glycine); sodium phosphate can be used, with sodium phosphate being preferred.

[0074] Both copper and peroxidase are used in the reaction to activate galactose oxidase (GOase). Copper is provided 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 peroxidases derived from other organisms, and can be substituted with oxidizing agents such as ferricyanide, irides, manganese(III) salts, persulfates, and other one- or two-electron oxidants, or inorganic or organic oxidants. Preferably, the peroxidase is horseradish peroxidase. Catalase is also added to prevent inactivation of GOase. Catalase can be from mammalian sources (bovine), or from bacterial or fungal sources 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 the use of hydrogen peroxide / catalase, superoxide, or other methods known in the art for this purpose.

[0076] The reaction can be carried out at a substrate concentration of about 10 to 180 g / L, particularly about 20 to 50 g / L, at a temperature of about 0 to 40°C, particularly about 10 to 30°C.

[0077] Compound 6: Deoxyribose-phosphate aldolase (DERA) reaction

[0078] A key advantage of this new route to producing compound (6) over known processes is that it generates the sugar backbone in the correct oxidation state without the use of protecting groups.

[0079] 4-Ethynyl D-2-deoxyribose 5-phosphate (6) is prepared by reacting deoxyribose phosphate aldolase (DERA) with (R)-2-ethynylglyceraldehyde 3-phosphate (5) as an acid or salt, and acetaldehyde in an aqueous solution, optionally adjusted to a pH of about 5-9 or higher, preferably about 6-8. Examples of salts of (5) include, but are not limited to, calcium, magnesium, zinc, mono- or di-Na salts, mono- or di-K salts, or mono- or di-Li salts; mono- or di-ammonium salts; or monovalent or divalent salts with primary, secondary, or tertiary amines. The reaction is preferably carried out in an open vessel, or, preferably, in a closed vessel to prevent evaporation of acetaldehyde.

[0080] The reaction can be carried out at a substrate concentration of about 10 to 100 g / L, particularly about 30 to 60 g / L, at a temperature of about 0 to 40°C, particularly about 25 to 35°C.

[0081] This reaction can be carried out without a buffer. Alternatively, the following buffers can be used, but are not limited to: triethanolamine; phosphate; MOPS, such as 3-(N-morpholino)propanesulfonic acid or 3-morpholinopropane-1-sulfonic acid; HEPES, such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; BIS-TRIS methane, such as 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borate; PIPES, such as piperazine-N,N'-bis(2-ethanesulfonic acid); MES, such as 2-(N-morpholino)ethanesulfonic acid; and borate; or other buffers with an appropriate pH range that do not have a primary amine group.

[0082] Each of the process steps and methods described herein that involve the use of one or more enzymes is carried out at a temperature that does not denature the enzyme(s). Each of the process steps and methods described herein that involve the use of one or more enzymes can be carried out at a pH ranging from about 3 to 10 or about 4 to 10.

[0083] A "nucleobase" (or "nitrogenous base" or "base") is a pyrimidine or purine heterocycle of nucleic acids such as DNA and RNA. As used herein, nucleobase includes adenine, guanine, cytosine, thymine, or uracil, as well as nucleobases with non-natural modifications, e.g., nucleobases in which the base has one or more non-natural substituents, or modifications affecting a heteroatom in the base except for any change to the anomeric-CN bond.

[0084] 4'-ethynyl 2'-deoxynucleosides include nucleobases. As used herein, an analog of a 4'-ethynyl 2'-deoxynucleoside refers to a non-natural modification to the base of the nucleoside, such as a base having one or more non-natural substituents, or a modification affecting a heteroatom in the base, excluding conversion to an anomeric C-N bond.

[0085] As used herein, a "phosphopentomutase" ("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, a "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 analogs).

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

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

[0089] As used herein, "pantothenate kinase" ("PanK") refers to an enzyme (EC 2.7.1.33) that, in its native form, phosphorylates pantothenate to produce 4'-phosphopantothenate. 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 native function toward pantothenate.

[0090] As used herein, "galactose oxidase" ("GOase"; EC 1.1.3.9) enzymes are copper-dependent enzymes that, in the presence of dimolecular oxygen, catalyze the oxidation of primary alcohols to the corresponding aldehydes. They act regiospecifically and enantiospecifically, enabling a synthetic approach requiring little or no functional group protection to yield the desired stereoisomer. The mode of oxidation is mild, and activity is controlled so as not to lead to overoxidation of alcohols to the corresponding carboxylic acids.

[0091] As used herein, the enzyme "horseradish peroxidase" (HRP, EC 1.11.1.7) 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 the normal GOase catalytic cycle. Type I HRP is used in a catalytic manner in the examples included herein, but is not intended to exclude other electron transfer enzymes in this or other enzyme classes and chemical compounds that perform a similar role.

[0092] As used herein, "catalase" refers to a heme-dependent enzyme (EC 1.11.1.6) that acts on hydrogen peroxide, a by-product of the galactose oxidase or pyruvate oxidase reactions that inactivates these enzymes above a certain level. Catalase is used as a catalytically sustaining enzyme in the examples herein to convert hydrogen peroxide to water and oxygen, although in some embodiments, it can be replaced by other methods, such as the electrochemical decomposition of hydrogen peroxide. While heme-dependent catalase is employed in a catalytic manner in the examples included herein, it is not meant to be limited to this role, as there are other enzymes in this class that can fulfill this role.

[0093] As used herein, "acetate kinase" ("AcK") refers to an enzyme (EC 2.7.2.1) that catalyzes the production of acetyl phosphate from acetate 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 kinase is used in the examples herein to recycle the ATP required by pantothenate kinase (PanK); however, in some embodiments, the acetyl phosphate acetate kinase 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, specifically acting on an aldehyde or oxo group of a donor with oxygen as an acceptor, catalyzing the chemical reaction of pyruvate, phosphate ions, and dimolecular oxygen to produce acetyl phosphate, carbon dioxide, and hydrogen peroxide. In the examples herein, pyruvate oxidase (PO) is used in combination with acetate kinase (AcK) and catalase as a catalytic ATP-regenerating combination, where the enzyme combination catalyzes the production of ATP from ADP in the presence of oxygen, pyruvate, and phosphate ions; however, in some embodiments, it can be substituted by other methods known in the art.

[0095] As used herein, "wild-type" and "naturally occurring" enzymes refer to the form found in nature. For example, a wild-type polypeptide sequence is a sequence that can be isolated from a natural source and is present in an organism that has not been intentionally modified by human manipulation.

[0096] As used herein, "engineered," "variant," "mutant," and "non-naturally occurring" refer to enzymes, including polypeptides, or substances corresponding to the native or naturally occurring form of a substance, that have been altered in a way that would not occur in nature. 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] The terms "percentage of sequence identity," "percentage of identity," "percentage of identity," and "percentage of identity" with respect to enzymes are used herein to refer to comparisons between polynucleotide or polypeptide sequences and are determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where either the identical nucleobase or amino acid residue occurs in both sequences, or the number of positions where the nucleobase or amino acid residue aligns with a gap and is matched, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. Optimal alignment and percent sequence identity determination are performed using the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402). Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information.

[0098] Briefly, BLAST analysis involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that either match words of the same length in a database sequence or, when matching words of the same length, satisfy some positive threshold score T. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate cumulative scores. Extension of word hits in each direction is stopped when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score becomes zero or less, one or more negative-scoring residue alignments accumulate; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expected euro length of 10, M=5, N=-4, and a comparison of both strands as defaults. For amino acid sequences, the BLASTP program uses a world length (W) of 3, an expected euro length (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915) as defaults.

[0099] Many other algorithms that function similarly to BLAST are available to provide the percent identity of two sequences. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482; the homology algorithm of Needleman and Wunsch, 1970, J. Mol. 2:482; the similarity search algorithm of Pearson and Lipman, 1988, Proc. Chem. Soc. 1999; the similarity algorithm of Pearson and Lipman, 1988, Proc. Chem. Soc. 1999; the similarity algorithm of GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package; computer-generated images; or visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)). Additionally, sequence alignments and determination of percent sequence identity can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelerys, Madison, Wis.), using default parameters.

[0100] "Substantial identity" 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 over a comparison window of at least 20 residue positions, often a window of at least 30-50 residues, where the percentage of sequence identity is calculated by comparing to a sequence that contains deletions or additions totaling no more than 20% of the reference sequence over the comparison window. In a specific embodiment applied to polypeptides, the term "substantial identity" means that two polypeptide sequences share at least 80% sequence identity, preferably at least 89% sequence identity, and more preferably at least 95% sequence identity (e.g., 99% sequence identity), when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

[0101] "Stereoselectivity" refers to the chemical or enzymatic preferential production of one stereoisomer over another. Stereoselectivity may be partial, favoring the production of one stereoisomer over the other, or may result in only one stereoisomer being formed. When the stereoisomers are enantiomers, the stereoselectivity is referred to as enantioselectivity, which is the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is alternatively commonly reported in the art (typically as a percentage) as the enantiomeric 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, which is the proportion (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, and is commonly reported alternatively as diastereomeric excess (de). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.

[0102] The phrase "suitable reaction conditions" refers to conditions in an enzyme conversion reaction solution (e.g., ranges of enzyme load, substrate load, temperature, pH, buffer, cosolvent, etc.) that allow each polypeptide used in the present invention to convert a substrate into a desired product compound. Some exemplary suitable reaction conditions are provided herein.

[0103] As used herein, "substrate" in the context of an enzymatic conversion reaction process refers to the compound or molecule that is acted upon by the engineered enzyme used herein.

[0104] As used herein, "product" in the context of an enzymatic conversion process means a compound or molecule that results from the action of an enzyme polypeptide on a substrate.

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

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

[0107] "Enantiomeric excess" (ee) is a measure of purity used for chiral substances. It reflects the degree to which a sample contains more of one enantiomer than the other. For example, a racemic mixture has an enantiomeric excess of 0%, whereas a completely pure mixture of one enantiomer has an ee of 100%, and a sample containing 70% of one enantiomer and 30% of the other has an ee of 40% (70% - 30%). Diastereomeric excess (de) is calculated as if only two diastereoisomers were present in the mixture.

[0108] "Protein," "enzyme," "polypeptide," and "peptide" are used interchangeably herein to refer to polymers of at least two amino acids covalently joined by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.

[0109] As used herein, "about" refers to an acceptable error for a particular value. In some cases, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% at the low and high ends of a given range of values. With respect to pH, "about" means plus or minus 0.5.

[0110] As used herein, a "substantially pure" polypeptide or "purified" protein refers to a composition in which the polypeptide species is the predominant species present (i.e., greater, on a molar or weight basis, than any other individual macromolecular species in the composition); generally, a composition is substantially purified when the subject species comprises at least about 50% of the macromolecular species present, on a molar or weight percent basis. However, in some embodiments, a composition comprising a polypeptide contains less than 50% pure polypeptide (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%). Generally, a substantially pure polypeptide composition comprises 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 macromolecular species present in the composition, on a molar or weight percent basis. In some embodiments, the polypeptide is purified to essential homogeneity (i.e., contaminating species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered to be macromolecular species. In some embodiments, an isolated polypeptide is a substantially pure polypeptide composition.

[0111] As used herein, an "improved property" of an enzyme refers to at least one improved property of the enzyme. In some embodiments, the invention utilizes recombinant PPM, PNP, DERA, PanK, AcK, SP, and / or GOase polypeptides that exhibit an improvement in any enzymatic property compared to a reference PPM, PNP, DERA, PanK, AcK, SP, or GOase polypeptide, respectively, and / or a wild-type PPM, PNP, DERA, PanK, AcK, SP, or GOase polypeptide, respectively, 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 wild-type and engineered 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 towards the substrate), increased thermal activity, thermostability, increased pH activity, increased stability, increased enzymatic 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 susceptibility to proteolysis), reduced aggregation, increased solubility, and altered temperature profile. In additional embodiments, the term is used in reference to at least one improved property of a PPM, PNP, DERA, PanK, AcK, SP, and / or GOase enzyme. In some embodiments, the invention uses improved PPM, PNP, DERA, PanK, AcK, SP and / or GOase polypeptides, and / or other modified PPM, PNP, DERA, PanK, AcK, SP and / or GOase polypeptides, respectively, that exhibit an improvement in any enzymatic property compared to a reference PPM, PNP, DERA, PanK, AcK, SP and / or GOase polypeptide, respectively. Thus, the level of "improvement" can be determined and compared among various polypeptides, including wild-type and engineered polypeptides.

[0112] As used herein, "conversion" refers to the enzymatic conversion (or biotransformation) of a substrate to a corresponding product, and "percent" conversion refers to the percentage of a substrate that is converted to a product under specified conditions within a given period of time. Thus, the "enzymatic activity" or "activity" of a polypeptide can be expressed as the percentage conversion of a substrate to a product in a specified period of time.

[0113] As used herein, "stereoselectivity" refers to the preferential production of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity may be partial, favoring the production of one stereoisomer over the other, or may result in only one stereoisomer being formed. When the stereoisomers are enantiomers, stereoselectivity is referred to as enantioselectivity, which is the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is alternatively commonly reported in the art (typically as a percentage) as the enantiomeric excess ("ee") calculated therefrom according to the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomers are diastereoisomers, the stereoselectivity is called diastereoselectivity, which is the proportion (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, and is commonly reported alternatively as diastereomeric excess ("de"). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.

[0114] The present invention encompasses the use of engineered 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, referred to as conservatively modified variants of each of SEQ ID NOs: 1-21.

[0115] As used herein, "conservative" amino acid substitutions refer to substitutions of amino acids in a protein with similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) such that they can be frequently changed without altering the biological activity of the protein. This includes one or more substitutions of an amino acid in a polypeptide with a different amino acid 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 ed.)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to destroy biological activity. For example, and not by way of limitation, in some embodiments, an amino acid having an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid having a hydroxyl side chain is substituted with another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain is substituted with another amino acid (e.g., lysine and arginine); an amino acid having an acidic side chain is substituted with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is substituted with another hydrophobic or hydrophilic amino acid, 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 that has an amino-terminal and / or carboxy-terminal deletion and / or an internal deletion, but where the remaining amino acid sequence is identical to corresponding positions in the sequence to which it is compared (e.g., a full-length engineered PPM, PNP, DERA, PanK, AcK, SP or GOase enzyme used in the present invention), and that retains substantially all of the activity of the full-length polypeptide.

[0119] As used herein, a "deletion" refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can 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 comprising the reference enzyme, while retaining enzymatic activity and / or the improved properties of the engineered PPM, PNP, DERA, PanK, AcK, SP, or GOase enzyme. Deletions can be directed to internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include contiguous segments or can be discontinuous. Deletions are typically indicated by a "-" in the amino acid sequence.

[0120] As used herein, "insertion" refers to a polypeptide resulting from the addition of one or more amino acids from a reference polypeptide. The insertion may be in an internal portion of the polypeptide, or at the carboxy or amino terminus. As used herein, an insertion includes fusion proteins, which are known in the art. The insertion may be a contiguous segment of amino acids, or may be separated by one or more amino acids in the naturally occurring polypeptide.

[0121] Additional acronyms and abbreviations used herein are as follows: [Table 2]

[0122] Experimental procedure Synthesis of 2-ethynyl-2-hydroxypropane-1,3-diyl diacetate (2) Method A: [ka] To a −35°C solution of diacetoxyacetone (1) (159 g, 914.0 mmol) in THF (1000 mL) was added 1600 mL of a 0.5 M solution of ethynylmagnesium chloride in THF, maintaining the temperature below −20°C. After the reaction was complete, acetic acid (78 mL) in 400 mL of methyl tert-butyl ether (MTBE) was added dropwise, maintaining the temperature below −20°C. MTBE (800 mL) was then added and allowed to warm to room temperature. Saturated NaCl in water (1000 mL) was added, followed by saturated NH₄Cl solution (1050 mL) in water. The organic layer was separated, dried over Na₂SO₄, and evaporated to give compound (2) as an oil (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: [ka] To a solution of 2-ethynyl 2-hydroxypropane-1,3-diyl diacetate (2) (70 g, 350 mmol) in ethanol was added 0.5 M of a methanolic solution of sodium methoxylate (69.9 mL, 35.0 mmol) at room temperature (rt). The reaction was stirred at rt for 2 hours (h) to complete the reaction. 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 sparged with nitrogen to remove residual solvent. Nuclear magnetic resonance (NMR) analysis (maleic acid as internal standard) yielded a 40.9% solution of 2-ethynylpropane-1,2,3-triol (3) (108 g, 100% yield). H NMR (DO, 500 MHz): δ 3.60 (dd, 4 H), 2.85 (s, 1 H).

[0124] Alternative preparation of (R)-2-ethynylglyceraldehyde (4) C1 method: [ka] In a stirred reactor, 2-ethynylpropane-1,2,3-triol (3) (1.1 g, 9.47 mmol) in sodium phosphate buffer (30 mL, 100 mM, pH 7.0) containing antifoam 204 (Sigma A6426, 1 drop ~ 20 μL) was warmed to 30 °C while sparging at 12.5 sccm. Galactose oxidase (GOOase, SEQ ID NO: 1) (250 mg), horseradish peroxidase (type I, 5 mg), and bovine catalase** (5 mg) were dissolved in sodium phosphate buffer (5 mL, 100 mM, pH 7.0), followed by the addition of aqueous CuSO4 (100 mM, 150 μL). The reaction mixture was stirred at 600 rpm with air sparging for 47 h to give (R)-2-ethynylglyceraldehyde (4) in 47% conversion (by NMR) and 72% ee (this product was not isolated). 1H NMR (DO, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). * Horseradish peroxidase: Wild-type peroxidase from horseradish type I, isolated from horseradish root (Amoracia rusticana) and commercially available from SIGMA (P8125). ** Bovine catalase: Heme-dependent catalase derived from bovine sources, commercially available from Sigma (C1345)

[0125] C2 method: [ka] 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 with 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). Galactose oxidase (451.2 g) (SEQ ID NO: 10) was added and stirred for 15 minutes with an air sparge. Horseradish peroxidase* (200.2 g) and catalase** (502.6 g) were added, and the reactor was rinsed with water (2.0 kg). A solution of 2-ethynylpropane-1,2,3-triol (3) in water (9.48%, 30.34 kg, 24.72 mol) was then added, followed by antifoam 204 (A6426, 10 mL). The reaction was sparged with air and stirred overnight to give 94.0 kg of (R)-2-ethynylglyceraldehyde (4) in 66% conversion (by NMR) and 84% ee. Analysis showed 60%: 1H NMR (DO, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). * Horseradish peroxidase: Purified wild-type peroxidase from horseradish, commercially available from Toyobō (PEO-301), 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 galactose oxidase (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 (DO, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H).

[0127] C3 method: [ka]

[0128] A 100 mL Easimax vessel equipped with a sparger and flow controller was charged with water (82 mL) and PIPES potassium buffer (5 mL, 0.5 M). The pH was adjusted to 7.5 with 5 M KOH at 25 °C. Antifoam 204 (200 μL) was added, followed by evolved galactose oxidase (SEQ ID NO: 17, 450 mg enzyme powder) and copper(II) sulfate pentahydrate (100 μL, 100 mM). The reaction mixture was sparged with air at 125 standard cubic centimeters per minute (sccm) for 15 minutes. Bovine catalase (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 was stirred at 30 °C with 125 sccm of aeration and sampled over 20 h using an Easy Sampler. 70% conversion was obtained, producing compound (4) ((R)-2-ethynylglyceraldehyde) in 58% assay yield and 99% ee. 1H NMR (D2O, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). The crude reaction mixture was carried directly to the next phosphorylation step.

[0129] C4 method: Oxidation with immobilized galactose oxidase [ka]

[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 volumes, 160 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution. Lyophilized powder of in-vessel evolved galactose oxidase (SEQ ID NO: 17, 2.00 g) was resuspended in copper(II) sulfate solution (100 μM; 5.00 mL), and binding buffer (50 mL) and resin were added. The solution was mixed on a rotary mixer for 5 hours at 20 °C. The resin was filter-washed with binding buffer (10 column volumes, 160 mL) and potassium PIPES buffer (10 column volumes, 160 mL; 50 mM, pH 7.5) and used directly in the reaction.

[0131] Reaction Procedure: A 100 mL Easymax vessel equipped with a sparger and flow controller was charged with water (82 mL) and PIPES potassium buffer (5 mL, 1 M). The pH was adjusted to 7.5 with 5 M KOH at 25 °C. Antifoam 204 (200 μL) was added, followed by resin-immobilized evolved galactose oxidase (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 air at 125 standard cubic centimeters per minute (sccm) for 15 minutes. Bovine catalase (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 with aeration at 125 sccm. After 22 h, the reaction had reached 91%, yielding a 200 mM (R)-2-ethynylglyceraldehyde (4) solution (100 mL, 68% assay yield, 97% ee). H NMR (DO, 500 MHz): The crude reaction mixture was carried directly to the next phosphorylation step.

[0132] Method C5: Optional isolation of aldehydes via formation of aminal (8) Step 1: Preparation of (S)-2-(1,3-dibenzylimidazolidin-2-yl)but-3-en-1,2-diol [ka] A 100 L jacketed cylindrical vessel equipped with a nitrogen bubbler, mechanical stirrer, and thermocouple was charged with crude oxidase reaction stream containing (R)-2-ethynylglyceraldehyde (4), 26.0 kg, 1.85 wt% aldehyde, 3.64 mol), and inerted with a N2 atmosphere. The aqueous solution was warmed to 20 °C, and N,N-dimethyldodecan-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'-dibenzylethane-1,2-diamine (1.55 kg, 6.43 mol). The brown, biphasic mixture was stirred overnight at 20 °C under a nitrogen atmosphere. After 17 h, stirring was stopped, and the organic phase was removed and discarded. A light brown solution of (S)-2-(1,3-dibenzylimidazolidin-2-yl)diphenyl-3-yn-1,2-diol (56.5 kg, 2.02 wt % aminal, 3.39 mmol, 93% assay yield) in MTBE was obtained.

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

[0134] A 50 L jacketed cylindrical vessel equipped with a mechanical stirrer, distillation head (condenser at -20 °C), and thermocouple was charged with the aminal solution (45 L). A vacuum was applied to the vessel (65-95 torr) and the jacket was set to 40 °C. The solvent was removed by distillation until a volume of 35 L was reached. At this point, the internal temperature was 6.1 °C and an off-white solid had begun to crystallize. The remaining MTBE solution was slowly added, maintaining a constant volume of 35-40 L and an internal temperature of 0-10 °C. Once all the MTBE solution had been added, the volume was reduced to 25 L. The distillation was stopped, the vessel was inerted with nitrogen, and the jacket temperature was reduced to 10 °C. The resulting pale yellow suspension was held at this temperature for 2 h, 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 h. (S)-2-(1,3-Dibenzylimidazolidin-2-yl)-hept-3-yn-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) aminal (M + H+) C21H25N2O2+ 337: calculated 1911; found 337.

[0135] Step 2: Preparation of (R)-2-ethynyl-glyceraldehyde (4) from aminal (8) [ka] A 4 L jacketed cylindrical vessel equipped with a nitrogen bubbler and mechanical stirrer was charged with TsOH·HO (12.0 g, 63.1 mmol), water (60 mL), (S)-2-(1,3-dibenzyl imidazolidin-2-yl)but-3-yn-1,2-diol (110 g, 327 mmol), and MTBE (1700 mL). The biphasic mixture was placed under nitrogen, and the jacket temperature was set to 15 °C. A solution of TsOH·HO (114 g, 599.3 mmol) in water (600 mL) was added dropwise over 1.5 h with stirring (200 rpm). After the addition was complete, the jacket temperature was lowered to 5 °C, and the resulting slurry was allowed to settle for 1 h. The solids were removed by filtration and washed with cold water (270 mL). The biphasic solution was transferred to a separatory funnel, and 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 sparging with N2 at a rate of 200 sccm for 24 hours to remove residual MTBE. The resin was removed by filtration to give a colorless aqueous solution of (R)-2-hydroxy-2-(hydroxymethyl)but-3-ynal (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; Found value 251.0530.

[0136] Alternative preparation of (R)-2-ethynyl-glyceraldehyde 3-phosphate (5): D1 Method: Acetate Kinase: ATP Regeneration System [ka] (R)-2-ethynylglyceraldehyde (4) (1.9 mL, 210 g / L solution in water, 3.51 mmol) was added to a solution of adenosine diphosphate disodium salt (40 mg, 0.087 mmol) and magnesium chloride (38 mg, 0.400 mmol) in HEPES buffer (66 mM, pH 7.5, 30 mL) in a stirred reactor, followed by acetate kinase (SEQ ID NO: 3) (40 mg) and pantothenate kinase (SEQ ID NO: 2) (120 mg). The reaction mixture was warmed to 25 °C, and a solution of lithium potassium acetylphosphate (1.3 g, 7.01 mmol) in HEPES buffer (50 mM, pH 7.5, 10 mL) was added dropwise over 4 h, maintaining the pH at 7.5 with 5 M sodium hydroxide. The reaction was stirred for 18 h to give (R)-2-ethynylglyceraldehyde 3-phosphate (5) in 85% conversion by HPLC (product was not isolated). 1H NMR (DO, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): C5H7O6P (MH): Exp. 193.1; Found 193.0.

[0137] Method D2: Pyruvate oxidase ATP regeneration system [ka] In a stirred reactor, a solution of sodium pyruvate (3.11 g, 28 mmol) and phosphoric acid (0.523 mL, 7.71 mmol) in 76 mL of water, pH 7.5, was charged 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 kinase (SEQ ID NO:3) (80 mg), pyruvate oxidase (SEQ ID NO:4) (80 mg, lyophilized cell-free extract), pantothenate kinase (SEQ ID NO:2) (400 mg), and catalase (800 μL, ammonium sulfate suspension CAT-101, Biocatalytics) were added. The reaction was stirred with an air sparge at 500 rpm and 30°C for 72 h, giving (R)-2-ethynylglyceraldehyde 3-phosphate 5 in 95% conversion by HPLC (product was not isolated). 1H NMR (DO, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): calculated for C5H7O6P (MH): 193.1; found 193.0.

[0138] The above reaction was also carried out using pantothenate kinase (SEQ ID NO: 13), giving product 5 in 66% conversion (this product was not isolated). 1H NMR (DO, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H).

[0139] D3 Method: Acetate Kinase: ATP Regeneration System Using Immobilized Enzyme [ka]

[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 vessel, pantothenate kinase (8.4 g) (SEQ ID NO: 12) and acetate kinase (2.8 g) (SEQ ID NO: 3) were dissolved in binding buffer (500 mL). The washed resin was charged to the vessel, and the solution was stirred at 20°C for 4 hours. The resin was filtered and washed first with binding buffer (1.6 L) and then 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 1 L reactor was charged with a solution of (R)-2-ethynylglyceraldehyde (4) in water (608.7 g, 4.6 wt %, 212 mmol) and cooled to 5 °C. To the cooled solution was added 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), acetyl phosphate diammonium salt (51.8 g, 265 mmol), adenosine diphosphate disodium salt hydrate (1.17 g, 2.12 mmol), and water (192 mL). The solution was stirred and the pH was adjusted to 6.4 using 5 N KOH. The reaction was warmed to 20 °C and 168 mL of resin immobilized with pantothenate kinase (SEQ ID NO: 12) and acetate kinase (SEQ ID NO: 3) was added. The reaction was stirred for 10 h with 5N KOH used to maintain the pH at 6.4, giving (R)-2-ethynylglyceraldehyde 3-phosphate (5) in 92% conversion (by HPLC) and 91% yield (by P NMR with tetraphenylphosphonium chloride as an internal standard; the product was not isolated). H NMR (DO, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): CHOP (MH): calculated 193.1; found 193.0.

[0142] Synthesis of 4-ethynyl D-2-deoxyribose 5-phosphate (6) Method E: [ka] 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 25 mg of deoxyribose phosphate aldolase (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 give 4-ethynyl D-2-deoxyribose 5-phosphate (6) at 99% conversion, i.e., 99% deoxyribose as a 1:1 anomeric mixture (the product was not isolated). α-anomer: 1H NMR (DO, 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 (DO, 151MHz) δ 97.7(s), 81.4(d), 79.4(s), 78.9(s), 71.1(s), 67.7(d), 39.6(s). β-anomer: 1H NMR (DO, 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 (DO, 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): C7H10O7P (MH): 237.0; found 237.0.

[0143] Alternative Preparation of (2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-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 [ka]

[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 MnCl). 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. An enzyme solution was prepared by dissolving phosphopentomutase (SEQ ID NO: 8) (100 mg), purine nucleoside phosphorylase (SEQ ID NO: 9) (50 mg), and sucrose phosphorylase (SEQ ID NO: 7) (10 mg) in 10 mL of pH 7.5 buffer. The enzyme solution was added to the reagent mixture, and the resulting suspension was shaken at 40 °C. After 20 hours, the suspension was cooled to 0°C, filtered and washed with cold water, and the solid was sucked dry to give the title compound (1.12 g, 92%) 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 each derived from Escherichia coli-derived enzymes by mutation. The sucrose phosphorylase (SP) used in this step was derived from Alloscardovia onicolens; SPs from other organisms could also be used.

[0147] F2 method: [ka] Triethanolamine (7.09 g, 47.5 mmol) was added to an aqueous solution of (R)-2-ethynylglyceraldehyde 3-phosphate (5) (950 mL, 157 mmol) in 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 with 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, and the following enzymes were added to the solution: deoxyribose-phosphate aldolase (SEQ ID NO:14) (461 mg), sucrose phosphorylase (SEQ ID NO:7) (494 mg), phosphopentomutase (SEQ ID NO:8) (2.63 g), and purine nucleoside phosphorylase (SEQ ID NO:15) (659 mg). Once the enzymes were 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 2 hours of reaction, the mixture was seeded with EFdA crystalline product (0.96 g, 2 mol%). After 26 hours of reaction at 35°C, the slurry was cooled to 0°C, and the solid was collected by filtration and washed twice with water (40 mL ea.). The solid was dried under a nitrogen sweep. 43.2 g, 92 wt%, 96.2% corrected, was 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). 13C nuclear magnetic resonance (150.92MHz, DMSO-d6, ppm) δ158.5(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; calcd. 316.0818.

[0148] Alternative Preparation of (S)-2-Ethynyl-propane-1,2,3-triol 1-phosphate (9) G1 Method: Acetate Kinase: ATP Regeneration System Using Enzymes SEQ ID NO:2 and SEQ ID NO:3 [ka] A 50 mL reactor was charged with an aqueous solution of 2-ethynylpropane-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), acetyl phosphate diammonium salt (1.851 g, 89 wt%, 9.46 mmol), adenosine diphosphate disodium salt hydrate (ADP, 42 mg, 0.076 mmol, 0.01 eq), and water (28 mL). The pH was adjusted to 6.4 with 5 M KOH, the solution was warmed to 20 °C, and evolved pantothenate kinase PanK SEQ ID NO:2 (264 mg) and acetate kinase AcK SEQ ID NO:3 (88 mg) were added. The reaction was stirred for 16 h with the pH maintained at 6.4 using 5 N KOH. The final reaction yield was (S)-2-ethynylpropane-1,2,3-triol 1-phosphate (9) in >95% ee and 99% conversion ( 31 P-NMR) The product was not isolated. 1H NMR (D2O, 500 MHz) δ 3.89 (m, 2H), 3.72 (d, J = 11.6 Hz, 1H), 3.65 (d, J = 11.6 Hz, 1H), 2.93 (s, 1H). 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; Found 195.0068 [MH] - : 195.0058.

[0149] G2 Method: Acetate kinase: ATP regeneration system using enzymes SEQ ID NO:20 and SEQ ID NO:21. [ka] The jacketed reactor was charged with 2-ethynylpropane-1,2,3-triol (3) (11.47 kg, 8.7 wt%, 8.61 mol) and water (7.5 kg), followed by 1 M BIS-TRIS methanesulfonate buffer, pH 6.5 (1 L) and magnesium chloride (41.4 g). ATP (48 g, 0.086 mol, 0.01 equiv) and diammonium acetyl phosphate (2.021 kg, 89%, 10.33 mmol) were added, warmed to 20 °C, and the pH was readjusted to 6.8 with KOH (270.4 g). Evolved pantothenate kinase SEQ ID NO:20 (20.4 g) and evolved acetate kinase SEQ ID NO:21 (3 g) were charged as solids. The reaction was stirred at 20 °C for 16 h, at which point the pH had dropped to 5.5. Quantitative conversion of 2-ethynylpropane-1,2,3-triol (3) 1 The resulting (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, as determined by H and P NMR. 1H NMR (D2O, 500 MHz) δ 3.89 (m, 2H), 3.72 (d, J = 11.6 Hz, 1H), 3.65 (d, J = 11.6 Hz, 1H), 2.93 (s, 1H).

[0150] G3 Method: Acetate kinase: Enzymes SEQ ID NO:20 and SEQ ID NO:2 and deuterated compound (3) are used to assign absolute stereochemistry and demonstrate desymmetric phosphorylation. [ka]

[0151] The evolved pantothenate kinase SEQ ID NO:20 (100 μL of a 10 g / L solution) and the evolved acetate kinase SEQ ID NO:21 (100 μL of a 2 g / L solution) 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 of a 1 M solution in water), ADP (10 μL of a 100 g / L solution in water), and sodium phosphate buffer (10 μL of a 1 M solution in water) in 800 μL of water. The reaction was incubated at rt for 24 h to give the deuterated 2-ethynylpropane-1,2,3-triol 1-phosphate analogs (S)-9-(3,3-d2) and (S)-9-(1,1-d2) in a 95:5 ratio and 99% overall yield. 31 The ratio of phosphorylated compounds was determined to be approximately 95:5 by P NMR, confirming stereoselective phosphorylation of 2-ethynylpropane-1,2,3-triol (3) at the pro-(S) hydroxyl group (i.e., desymmetric phosphorylation). 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 kinase: ATP regeneration system using immobilized enzymes SEQ ID NO:20 and SEQ ID NO:21. [ka] 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 x 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 powder of pantothenate kinase (SEQ ID NO: 20, 6.0 g) was resuspended in binding buffer (200 mL) in a container and added to the washed resin. Mixing was performed on a rotary mixer at 25°C for 6 hours. The resin was filtered and washed with binding buffer (6 column volumes, 6 x 225 mL) and BIS-TRIS buffer (8 column volumes, 600 mL; 50 mM, pH 6.2). Reaction Procedure: An aqueous solution of 2-ethynylpropane-1,2,3-triol (3) (574 g, 8.7% by weight, 0.430 mol) and water (350 mL) was placed in a jacketed reactor, followed by 1 M BIS-TRIS methanesulfonate buffer, pH 6.5 (50 mL) and magnesium chloride (2.033 g, 0.01 mol). ATP (2.37 g, 0.0043 mol, 0.01 equiv.) and diamonium phosphate (101 g, 89%, 0.530 mmol, 1.2 equiv.) were added, the mixture was warmed to 20 °C, and the pH of the solution was readjusted to 6.8 with 5 M KOH. Resin (25 mL) loaded with immobilized pantothenate kinase SEQ ID NO:20 and evolved acetate kinase SEQ ID NO:21 (0.15 g) was added as a solid. The reaction was stirred at 20 °C for 16 h, during which time the pH decreased to 5.5. Quantitative conversion of 2-ethynylpropane-1,2,3-triol (3) to (S)-2-ethynylpropane-1,2,3-triol 1-phosphate (9) was observed. 1 H and 31P NMR (DO, 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).

[0153] Alternative preparation of (R)-2-ethynyl-glyceraldehyde 3-phosphate (5): H1 Method: Immobilized galactose oxidase SEQ ID NO: 16 [ka] 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 volumes, 100 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). The resin stock solution was removed, yielding 16 g of washed resin. Lyophilized powder of in-vessel evolved galactose oxidase (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. Mixing was performed on a rotary mixer at 20 °C for 5 hours. The resin was filtered, washed with binding buffer (10 column volumes, 100 mL) and BIS-TRIS buffer (10 column volumes, 100 mL; 50 mM, pH 7.5), and used directly in glycosylation reactions.

[0154] Reaction Procedure: Immobilized galactose oxidase resin (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 (35 mM, adjusted to pH 7.2), followed by copper(II) sulfate solution in water (30 μL, 100 mM), and horseradish peroxidase (PEO-301, 18 mg) and bovine catalase (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 to reach a final conversion of 77%, yielding (R)-2-ethynylglyceraldehyde 3-phosphate (5) in 95% ee. The enzyme resin was filtered, and the solution of (R)-2-ethynylglyceraldehyde 3-phosphate (5) was used directly in the glycosylation reaction. H NMR (DO, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): CHOP (MH): Found 193.1; Found 193.0.

[0155] H2 Method: Immobilized galactose oxidase SEQ ID NO: 17 [ka] 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 volumes, 100 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). The resin stock solution was removed, yielding 16 g of washed resin. Lyophilized powder of evolved galactose oxidase (SEQ ID NO: 16, 750 mg) was resuspended in copper(II) sulfate solution (100 μM; 5.00 mL) in a container, and binding buffer (20 mL) and washed resin (3.0 g) were added. The mixture was mixed on a rotary mixer at 20 °C for 5 hours. The resin was then filtered and washed with binding buffer (10 column volumes, 100 mL) and BIS-TRIS methane buffer (10 column volumes, 100 mL; 50 mM, pH 7.5) and used directly in the reaction. Reaction Procedure: Immobilized galactose oxidase 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 (35 mM, adjusted to pH 7.2), followed by a copper(II) sulfate solution in water (30 μL, 100 mM), and horseradish peroxidase (PEO-301, 18 mg) and bovine catalase (C1345, 120 mg) resuspended in water (600 μL). The reaction was sealed with a gas-permeable membrane and vigorously shaken at 22 °C for 4 days to reach a final conversion of 77%, yielding (R)-2-ethynylglyceraldehyde 3-phosphate (5) in 95% ee. The enzyme resin was filtered off, and the solution of (R)-2-ethynylglyceraldehyde 3-phosphate (5) was used directly in the glycosylation reaction. H NMR (DO, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): r CHOP (MH): calculated 193.1; found 193.0.

[0156] H3 Method: Immobilized galactose oxidase SEQ ID NO: 18 [ka] 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 volumes, 30 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). The resin stock solution was removed, yielding 2.4 g of washed resin. Lyophilized powder of vial-evolved galactose oxidase (SEQ ID NO: 18, 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. Mixing was performed on a rotary mixer at 20°C for 5 hours. The resin was then filtered, washed with binding buffer (10 column volumes, 4 mL) and BIS-TRIS methane buffer (10 column volumes, 4 mL; 50 mM, pH 7.5), and used directly in the reaction. Reaction Procedure: Immobilized evolved GOase SEQ ID NO.:18 (400 mg) was added 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 (35 mM, adjusted to pH 7.2), followed by horseradish peroxidase (PEO-301, 1 mg) and catalase from Corynebacterium glutamicum (Roche, lyophilized material, #11650645103, 3 mg) resuspended in water (100 μL). The reaction was sealed with a gas-permeable membrane and vigorously shaken at 30 °C for 48 h. Final conversion after 2 days reached 90%, with (R)-2-ethynylglyceraldehyde 3-phosphate (5) in >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 (DO, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): r C5H7O6P (MH): calculated 193.1; found 193.0.

[0157] H4 Method: Immobilized galactose oxidase SEQ ID NO: 19 [ka] 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 volumes, 30 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). The resin stock solution was removed, yielding 2.4 g of washed resin. Lyophilized powder of evolved galactose oxidase (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 on a rotary mixer at 20 °C for 5 hours. The resin was then filtered, washed with binding buffer (10 column volumes, 4 mL) and BIS-TRIS methanesulfonate buffer (10 column volumes, 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 (35 mM, adjusted to pH 7.2), followed by the addition of horseradish peroxidase (PEO-301, 1 mg) and catalase from Corynebacterium glutamicum (Roche, lyophilized material, #11650645103, 3 mg) resuspended in water (100 μL). The reaction mixture was sealed with a gas-permeable membrane and vigorously shaken at 30°C for 48 h. After 2 days, the final conversion reached 100%, and (R)-2-ethynylglyceraldehyde 3-phosphate (5) was obtained in >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 (DO, 400 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H). LC-MS: (ES, m / z): C5H7O6P (MH): calculated 193.1; found 193.0.

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

[0159] SEQ ID NOs for enzymes that are or can 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] TIFF0007773906000043.tif214162TIFF0007773906000044.tif207163TIFF0007773906000045.tif165162TIFF00077739060 00046.tif220162TIFF0007773906000047.tif228161TIFF0007773906000048.tif227162TIFF0007773906000049.tif220162

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

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

[0162] Additional embodiments of the present invention include, but are not limited to, the use of the following enzymes in the synthetic process steps described herein to produce 4'-ethynyl 2'-deoxynucleosides or analogs thereof, 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 to 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 engineered purine nucleoside phosphorylase of 1A, wherein the engineered purine nucleoside phosphorylase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:9 or SEQ ID NO:15. 3A. An engineered purine nucleoside phosphorylase comprising the polypeptide sequence set forth in SEQ ID NO:9 or SEQ ID NO:15. A4. An engineered purine nucleoside phosphorylase of any one of 1A to 3A, comprising at least one improved property compared to wild-type E. coli purine nucleoside phosphorylase. 5A. The engineered purine nucleoside phosphorylase described in 4A, wherein the improved properties include improved activity toward substrate compound 6.5 (its cyclic or open-chain aldehyde or hydrate, or a salt of either of the foregoing) compared to wild-type E. coli purine nucleoside phosphorylase. 6A. An engineered purine nucleoside phosphorylase as described in 4A, wherein the improved properties include improved production of EFdA (compound 7) compared to wild-type E. coli purine nucleoside phosphorylase. 7A. The engineered purine nucleoside phosphorylase of any one of A1 to 6A, wherein the engineered purine nucleoside phosphorylase is purified. 8A. The engineered purine nucleoside phosphorylase of any one of 1A to 7A, wherein at least one amino acid substitution (ie, one or more amino acid substitutions) is a conservative amino acid substitution.

[0164] B. Phosphopentomutase. 1B. An engineered phosphopentomutase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the polypeptide sequence of the engineered phosphopentomutase comprises at least one amino acid substitution or set of amino acid substitutions compared to SEQ ID NO:8. 2B. The engineered phosphopentomutase described in 1B, wherein the engineered phosphopentomutase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:8. 3B. An engineered phosphopentomutase consisting of the polypeptide sequence set forth in SEQ ID NO:8. 4B. The engineered phosphopentomutase of any one of 1B-3B, comprising at least one improved property compared to wild-type E. coli phosphopentomutase. 5B. The engineered phosphopentomutase described in 4B, wherein the improved properties include improved activity toward substrate compound 6 (its cyclic or open-chain aldehyde or hydrate, or a salt of either of the foregoing) compared to wild-type E. coli phosphopentomutase. 6B. The engineered phosphopentomutase of 4B, wherein the improved properties include improved production of compound 6.5 or compound 7 (EFdA) compared to wild-type E. coli phosphopentomutase. 7B. The engineered phosphopentomutase of any one of 1B to 6B, wherein the engineered phosphopentomutase is purified. 8B. The engineered phosphopentomutase of any one of 1B to 7B, wherein at least one amino acid substitution (ie, one or more amino acid substitutions) is a conservative amino acid substitution.

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

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

[0167] E. Galactose oxidase. 1E. An engineered galactose oxidase comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs.: 1, 10, 11, 16, 17, 18 or 19, or a functional fragment thereof, wherein the polypeptide sequence of the engineered galactose oxidase 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 galactose oxidase of 1E, wherein the engineered galactose oxidase comprises a polypeptide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1, 10, 11, 16, 17, 18 or 19. 3E. An engineered galactose oxidase comprising the polypeptide sequence set forth in SEQ ID NO: 1, 10, 11, 16, 17, 18 or 19. 4E. An engineered galactose oxidase of any one of 1E-3E, comprising at least one improved property compared to wild-type F. graminearum galactose oxidase. 5E. The engineered galactose oxidase of 4E, wherein the improved properties include improved activity toward a substrate that is a primary alcohol, compared to wild-type F. graminearum galactose oxidase. 6E. An engineered galactose oxidase of 4E having improved properties, including improved activity toward the substrate compound 3 (2-ethynylpropane-1,2,3-triol), compared to wild-type F. graminearum galactose oxidase. 7E. An engineered galactose oxidase of 6E, wherein the improved properties include improved production of compound 4 ((R)-2-ethynylglyceraldehyde or its hydrate form) compared to wild-type F. graminearum galactose oxidase. 8E. An engineered galactose oxidase of 4E, having improved properties including improved activity toward the substrate compound 9 (((S)-2-ethynylpropane-1,2,3-triol 1-phosphate) compared to wild-type F. graminearum galactose oxidase. 9E. The engineered galactose oxidase of 8E, wherein the improved properties include improved production of compound 5 ((R)-2-ethynylglyceraldehyde 3-phosphate or its hydrate form) compared to wild-type F. graminearum galactose oxidase. 10E. The engineered galactose oxidase of any one of 1E-9E, wherein said galactose oxidase is purified. 11E. The engineered galactose oxidase of any one of 1E-10E, wherein at least one amino acid substitution (ie, one or more amino acid substitutions) is a conservative amino acid substitution.

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

Claims

1. The following formula 【change】 'A method for synthesizing compound 6.5 in a buffer containing a manganese(II) salt. 【Chemistry 1】 [In the formula, 2X ＋ is (a) two protons, (b) one proton and one monovalent cation, (c) two monovalent cations that may be the same or different, or (d) one divalent cation. with purine nucleoside phosphorylase and 2-fluoroadenine; The method, 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.

2. The following formula 【Transformation 3】 Compounds of 10. The method of claim 1, further comprising isolating

3. Compound 6 in a buffer containing manganese(II) salt 【Chemistry 4】 and further comprising combining phosphopentomutase with purine nucleoside phosphorylase and 2-fluoroadenine; 2. The method of claim 1, wherein the phosphopentomutase comprises a polypeptide sequence having at least 90% identity to SEQ ID NO: 8 and has phosphopentomutase activity.

4. 4. The method of claim 3, further comprising removing inorganic phosphate by-products from the reaction mixture.

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

6. The following formula 6. The method of any one of claims 3 to 5, further comprising isolating the compound of formula (I).

7. 4. The method of claim 3, further comprising the step of synthesizing compound 6 by dissolving compound 5 in an aqueous solution. 【Transformation 7】 [In the formula, 2X ＋ is (a) two protons, (b) one proton and one monovalent cation, (c) two monovalent cations that may be the same or different, or (d) one divalent cation. with acetaldehyde and deoxyribose-phosphate aldolase to produce compound 6; 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 method.

8. 8. The method of claim 7, wherein the reaction is carried out in a closed vessel.

9. 9. The method of claim 7 or 8, further comprising the step of synthesizing compound 5, wherein said synthesis is carried out by synthesizing compound 4. 【Transformation 8】 with pantothenate kinase in a buffer in the presence of a divalent metal salt and in situ regenerated ATP as a phosphate source to produce compound 5.

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

12. 10. The method of claim 9, further comprising the step of synthesizing compound 4, said synthesis comprising the step of reacting compound 3 in a buffer solution in the presence of oxygen. 【Chemistry 9】 with (a) galactose oxidase, copper, catalase, and (b) peroxidase or an oxidizing agent to produce compound 4.

13. 13. The method of claim 12, wherein the galactose oxidase is immobilized.

14. The following formula 【change】 The method for synthesizing the compound of formula (I) comprises reacting compound 5 in a buffer solution containing a manganese (II) salt. 【Chemistry 10】 [In the formula, 2X ＋ is (a) two protons, (b) one proton and one monovalent cation, (c) two monovalent cations, which may be the same or different, or (d) one divalent cation. combining acetaldehyde and 2-fluoroadenine 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 method, 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.

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

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

17. The compound of the following formula:

17. The method of any one of claims 14 to 16, further comprising isolating the compound of formula (I).

Images