Enzymatic synthesis of 4'-ethyl nucleoside analogs

Abstract

To provide a synthesis method of 4'-ethynyl 2'-deoxy nucleosides and analogs thereof that eliminates the use of protecting groups on intermediates, improves the stereoselectivity of glycosylation, and reduces the number of process steps needed to make the compounds.SOLUTION: A method for synthesizing a 4'-ethynyl 2'-deoxy nucleoside or an analog thereof comprises combining compound 6.5 in the figure [where 2X+ is (a) two protons, (b) one proton and one monovalent cation, (c) two monovalent cations which are the same or different, or (d) one divalent cation] with purine nucleoside phosphorylase and a nucleobase or an analog thereof, in a buffered solution containing a manganese (II) salt.SELECTED DRAWING: None

Description

[Background technology]

[0001] Background of the Invention

[0002] 4'-Ethinyl 2'-deoxynucleoside analogs are used in HIV, AIDS, and related diseases. Its activity against is known. [ka]

[0003] An example of a 4'-ethynyl nucleoside analog is in vitro (Kawamoto, E ., Sarafianos SF et al., Int. J. Bioche m. Cell Biol.; 40(11):2410-20

[2008] ; Ohr ui, H., H. et al., Nucleosides & Nucleic Ac ids, 26, 1543-1546

[2007] ) and in vivo (Hat tori, S., K., Nakata, H. et al, Antimicrobia l. Agents and Chemotherapy, 53, 3887-389 3. Inverse nucleosides that block HIV-1 and SIV virus replication in

[2009] 4'-ethinyl2'-deoxyadenosine, a transcriptase translocation inhibitor. (EFdA, also known as MK-8591). EFdA is a U.S. patent no. 7,339,053 (In Patent No. '053, 2'-deoxy-4'-C-ethynyl 2-fluoroadenosyl It is claimed to be (called "n"). EFdA has the following chemical structure. [ka]

[0004] EFdA is metabolized intracellularly to the active triphosphate anabolite that inhibits HIV reverse transcriptase and becomes a chain terminator by lacking the 3'-OH group that inhibits the binding of incoming nucleotides, contrary to currently available nucleoside reverse transcriptase inhibitors (NsRTIs) and nucleotide reverse transcriptase inhibitors (NtRTIs) for the treatment of HIV infection. EFdA retains the 3'-OH group and prevents the translocation of the primer-template in the reverse transcriptase (RT) active site, preventing the binding of incoming deoxyribonucleotide triphosphates (dNTPs) and acting as a chain terminator. Furthermore, the packing of the modified ribose ring of EFdA places the 3'-OH in a vector where phosphate group transfer from incoming nucleotides is inefficient, contributing to the inhibition of reverse transcriptase. (Michailidis E, et al. , Mechanism of inhibition of HIV-1 rever se 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 (E FdA) inhibits HIV-1 reverse transcriptas e with multiple mechanisms, J Biol Chem 289:24533-24548

[2014] ).

[0005] In in vitro HIV replication assays, EFdA is a potent antiviral drug and exhibit equivalent antiviral activity against clinical isolates across all evaluated subtypes. This is because, in both lymphoid-derived cell lines and peripheral blood mononuclear cells, it is rapidly converted to active triphosphate in vitro and the intracellular half-life of EFdA triphosphate (EFdA-TP) exceeds 72 hours. (Stoddart, C. A., Galkina , et al., Oral Administration of the Nuc leoside EFdA (4’-Ethynyl-2-Fluoro-2’-Deoxya denosine) Provides Rapid Suppression of HIV Viremia in Humanized Mice and Favora ble Pharmacokinetic Properties in Mice a nd the Rhesus Macaque, Antimicrob Agents Chemother, 2015 Jul; 59(7): 4190-4198, Epub 2015 May 4).

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

[0007] Conventional synthesis of 4’-ethynyl nucleoside analogs, including EFdA, involves ethynyldeoxyri Bose sugar and 2-fluoroadenine (also known as 2-fluoro-9H-purine-6-amine) (ru) It suffers from low stereoselectivity in the formation of CN bonds with nucleo bases. In conventional synthesis, protecting groups are also required to carry out glycosylation reactions, which reduce the efficiency of synthesis. ru.

[0008] (Kei Fukuyama, et al., Synthesis of EFd A via a Diastereoselective Aldol Reactio n of a Protected 3-Keto Furanose, Organi c Letters 2015, 17(4), pp. 828-831; DOI: The synthesis described in 10.1021 / ol5036535 is diastereoselective. A 14-step reaction from D-glucose diacetonide to establish three stereocenters. The stereochemistry of the anomalous center is subsequently removed by hydrolysis and deoxygenation. -Controlled by the presence of an acetoxy-directing group. This pathway involves four chromatography steps. Purification using IV and the stoichiometric use of toxic organotin reagents are necessary for late-stage deoxygenation.

[0009] Another route (Mark McLaughlin, et al., Enantiose lective Synthesis of 4′-ethyny.-2-fluoro-2′ -deoxyadenosine (EFdA) via Enzymatic Des ymmetrization, Organic Letters 2017, 19 (4), see pp. 926-929) where desymmetrization by enzymes is stereoselective. A completely substituted 4′ carbinol is generated. The 3'-steric center is established by catalytic asymmetric transfer hydrogenation. The anoma-1' bond was established with appropriate stereoselectivity using substrate control, resulting in a stereochemical The improvement in purity is achieved by crystallizing the intermediate. This process requires 15 steps, and the protecting group It is necessary to use it, and it has low stereoselectivity (1.8:1) for glycosylation between nucleobases and sugar fragments. It generates a ru bond.

[0010] The 12-step synthesis for producing EFdA from R-glyceraldehyde acetonide is K Ageyama, M., et al., Concise Synthesis o f the Anti-HIV Nucleoside EFdA, Biosci. Biotechnol. Biochem, 2012 , 76, pp. 1219 -1225; and Enantioselective Total Synth esis of the Potent Anti-HIV Nucleoside E FdA, Masayuki Kageyama, et al., Organic Letters 2011 13 (19), pp. 5264-5266 [DOI This is described in [10.1021 / ol202116k]. This synthesis is a chiral starting point. Using a material, a 3' stereocenter with moderate diastereoselectivity is established. Stereoisomers After separating them by chromatography, diastereoselective archetypes were used with a new stereocenter. This induces anomalous addition and sets a complete replacement 4'-stereocenter. The anomalous 1'-position has less stereocontrol. In this pathway, two Diastereoisomers need to be separated by chromatography at different stages, which is expensive. Starting from Ral starting material.

[0011] Kohgo, S., et al., Design, Efficient Syn thesis, and Anti-HIV Activity of 4′-C-Cy ano- and 4′-C-ethynyl-2′-deoxy Purine Nucle osides, Nucleosides, Nucleotides and Nuc leic Acids, 2004, 23, pp. 671-690 [ DOI: [10.1081 / NCN-120037508] starts with an existing nucleoside, The synthetic pathway describes modifying both the sugar moiety and the nucleobase moiety. 2-amino- The synthesis involves 18 steps starting with 2'-deoxyadenosine, and the overall yield is low at 2.5%.

[0012] Enzymes such as purine nucleoside phosphorylase (PNP, EC 2.4.2.1) With high stereoselectivity, it can be used in nucleosides and nucleoside analogs without the use of protecting groups. It is known that it can form lycosyl bonds. For example, see the review article New Trends i n Nucleoside Biotechnology, Mikhailopul o, IA, Miroshnikov, AI,. Acta Naturae See 2010, 2, pp. 36-58. However, catalyzed by PNP The current range of sugar fragments that can undergo the reaction includes natural ribose and deoxyribose. The α-1-phosphate derivative and small H, NH2, or F substitutions at the C2' and C3' positions. It is limited to a small number of analogs having substitutions of the group and the C5'OH group. Glycation catalyzed by sugars with substitution groups, or by PNPs that use some substitution at the C4' position. There have been no reports of successful cosylation.

[0013] Ribose and deoxyribose α-1-phosphate substrates for PNP catalytic glycosylation Access is via the enzyme phosphopentumutase (PPM, EC 5.4.2.7) at 5' This is indicated by the movement of the phosphate group from the -hydroxyl position to the 1'-hydroxyl position. Mikhailopulo (see IA above). However, this reaction can be catalyzed. The range of kiltoses includes ribose, arabinose, 2-deoxyribose, and 2,3-dide The reaction is limited to oxyribose. Examples of successful reactions with sugar phosphates containing other substituents have not been reported. It hasn't been announced.

[0014] Deoxyriboside phosphate aldolase (DERA, EC 4.1.2.4) enzyme is another enzyme. It is known to catalyze the aldol addition of acetaldehyde to short-chain aldehydes. Review: Stephen M. Dean, et al., Recent Adva nces in Aldolase-Catal-Cath. Synth. Cata l. 2007, 349, pp. 1308 - 1320; DOI: 10.1 (See 002 / adsc.200700115). However, complete substitution of carbon in aldehydes No cases involving aldehydes containing α have been reported.

[0015] U.S. Patent No. 7,229,797 relates to the use of purine nucleoside phosphorylase (PNP). Furthermore, using enzymes such as sucrose phosphorylase, inorganic phosphate byproducts can be extracted. By removing and driving equilibrium, deoxyribose-1-phosphate from natural unsubstituted deoxyribose-1-phosphate The document describes the formation of xylionucleosides. This document describes the formation of non-natural 4-ethynyl PNP yeast can produce nucleosides from D-2-deoxyribose 1-phosphate. The enzyme engineering for the creation of the raw material is not disclosed, nor is the engineering of PPM and DERA enzymes disclosed. In order to act on non-natural substrates, 4-ethynyl D-2-deoxyribos-1-ri The PNP enzyme capable of producing nic acid is also not disclosed. [Prior art documents] [Patent Documents]

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

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

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

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

[2009] [Non-Patent Document 4] Michailidis E, et al., Mechanism of inhibition of HIV-1 reverse transcriptase by 4'-ethynyl-2-fluoro-2'-deoxyadenosine triphosphate, J Biol Chem 284:35681-35691

[2009] [Non-Patent Document 5] 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] ) [Non-Patent Document 6] 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, online May 4, 2015. [Non-Patent Document 7] 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 [Non-Patent Document 8] Mark McLaughlin, et al., Enantioselective Synthesis of 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) via Enzymatic Desymmetrization, Organic Letters 2017, 19 (4), pp. 926-929

Non-licensed literature 9

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Non-licensed Document 12

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[0018] The development of 4'-ethynyl nucleoside analogs to date has been difficult and time-consuming. Considering the synthesis options, the number of process steps is reduced and the use of protecting groups is minimized. , improving the stereoselectivity of glycosylation and avoiding the use of toxic substances, such as EFdA 4 It is desirable to develop improved enzymatic synthesis for '-ethinyl nucleoside analogs.' This is likely a means to solve the invention.

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

[0020] Furthermore, it was discovered that the PNP enzyme also has activity at the 3-atom ethynyl substituent at the 4-position of deoxyribose, and that introducing mutations into this enzyme improves PNP enzyme activity, enhances the sugar modification reaction catalyzed by PNP, and enables a more efficient method for producing 4'-ethynyl-2'-deoxynucleoside.

[0021] Further improvements to the overall synthesis method involve the DERA enzyme, particularly Shewanella hal DERA derived from ifaxensis has a 2-ethynyl glycerin with a fully substituted α-carbon. This was derived from the finding that it has activity in the aldol reaction with aldehyde 3-phosphate. This discovery relates to, for example, 4'-ethynyl 2'-deoxynucleoside analogs including EFdA. This enables the efficient synthesis of 4-ethynyl D-2-deoxyribose 5-phosphate, a precursor of [the compound]. I did.

[0022] Summary of the Invention

[0023] This invention eliminates the use of protecting groups on intermediates, improves the stereoselectivity of glycosylation, and other Among the process improvements, in particular, the necessary steps to produce the aforementioned compound compared to the previous method. The number of process steps is significantly reduced, and includes EFdA, 4'-ethynyl 2'- This includes the use of engineered enzymes in novel enzymatic synthesis of deoxynucleoside analogs. The invention further relates to a novel intermediate that is an integral part of the enzymatic process.

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

[0025] [ka]

[0026] [ka]

[0027] The acid form or salt of the phosphate intermediate is used in the processes described herein. This is possible, and is not limited to the specific acid or salt forms provided in the examples of process steps herein. It is not the case that all phosphate intermediates described herein are 2X + There are two A proton, one proton and one other monovalent cation, two monovalent cations (identical Represents any combination of (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, in the synthesis steps described herein, aldehydes and Intermediate compounds shown or named herein as hydrates are described herein. In the reactions described, any form of such form or a mixture of such forms They can exist in this way. For example, compounds (4) and (5) are hydrates in reaction formula 1, respectively. And although they are depicted as aldehydes, in the reaction steps in which each exists, hydrates Alternatively, they can exist in aldehyde form or as mixtures thereof. Each of the states is obtained by referring to compound number (4) or (5) in the process of this specification. It is included.

[0030] [ka]

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

[0032] Compound (6) exists as a cyclic or open-chain aldehyde or hydrate, respectively. In the reaction steps, it can exist as an acid or its salt. [ka]

[0033] Detailed description of the invention

[0034] 4-ethynyl 2-deoxynucleic acid and analogs having anoma-CN bond [ka] Its activity against HIV, AIDS, and related diseases is being explored. 4'-Ethyl Nyl 2'-deoxynucleosides and their analogues are purines or pyrimidine nucleos Bases (adenine, guanine, cytosine, thymine, or uracil) or modified purines or 4'-ethynyl 2'- bonded via anoma-CN bond to pyrimidine nucleo base Contains deoxyribose.

[0035] 4-ethynyl 2'-deoxynucleoside analogs, such as EFdA, are 4-ethynyl D- 2-deoxyribose 5-phosphate (6) and two enzymes, phosphopentumutase (PPM) [For example, SEQ ID NO: 8, but not limited to this] and purine nucleoside phosphodinucleotides Combining with lyrase (PNP) [for example, SEQ ID NO: 9, SEQ ID NO: 15] Reaction equation 2 shows that the synthesis can be carried out using a one-pot method in the final step. [ka] [ka]

[0036] As shown in reaction equation 2, the final step of the synthesis involves a two-enzyme reaction (a third enzyme may also be used). Using the reaction, the equilibrium of the reaction is directed toward the desired final product. The final step is compound (6). It begins with the salt, where (6) is the cyclic 4-ethynyl 2-deoxyri It is in the form of bose-5-phosphate or its open-chain aldehyde or hydrate.

[0037] Compound (6) contains a manganese(II) salt and has a pH of approximately 6.5 to 8.0 or higher. In particular, in a buffer solution adjusted to a range of approximately 7.0-7.5, phosphopentumutase (PP M), purine nucleoside phosphorylase (PNP), sucrose phosphorylase, sucrose It can be combined with nucleobases such as rose and unsubstituted or substituted adenines. The molar ratio of sugar to compound (6) can range from approximately 1:1 to 4:1, but is not limited to this. The components of this one-pot reaction can be combined in any order.

[0038] The reaction is carried out with stirring at a temperature range that does not denature the enzyme, for example, about 30-45°C, and in more detail... Stir at approximately 35-45°C. While the reaction may proceed to some extent at lower temperatures, It will slow down the reaction rate.

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

[0040] The equilibrium reaction involves converting sucrose to D-fructose and α-D-glucose-1-phosphate. The final product is formed by consuming inorganic phosphates, which are by-products, through chemical decomposition. This can advance the conversion of substances. This is achieved by adding sucrose to the reaction mixture. It is catalyzed by sucrose phosphodiolase (EC 2.4.1.7). However, sucrose phosphodiolase is also involved. Instead of using lilacose and sucrose, any other option for removing phosphates For example, by adding calcium, magnesium, or manganese to the reaction, phosphoric acid Salts can be precipitated. This highly efficient and ecological process allows for the removal of protecting groups and organic solvents. Without using a medium, it forms an anomalous bond between the sugar and the nucleobase with very high stereoselectivity. It has the advantage of being able to be performed as a one-pot reaction.

[0041] Once the reaction is complete, the final product can be isolated by crystallization and collected by filtration, or otherwise... Crystallization after extraction in the appropriate solvent, etc., is not limited to, but is known to those skilled in the art. The final product can be isolated using a standard procedure.

[0042] As shown in reaction equation 2A, the final step of the synthesis involves a three-enzyme reaction (a fourth enzyme may also be used). Using the reaction, the equilibrium of the reaction can be driven toward the desired final product. Final stage The compound begins with compound (5) or a salt thereof, where (5) is (R)-2-ethynylglycerin It is a form of rudehyde 3-phosphate or its hydrate.

[0043] Compound (5) contains a manganese(II) salt and appropriately adjusts the pH to a range of about 4 to 10, or especially about In a buffer solution adjusted to 6.5–8.0 or higher, especially in the range of approximately 7.0–7.5. So, deoxyribo-phosphate aldola-ase (DERA), acetaldehyde, phosphope Phosphate mutase (PPM), purine nucleoside phosphorylase (PNP), sucrose Suphorylase, sucrose, and their nucleobases or analogues, e.g., unsubstituted It can be combined with substituted adenine. The molar ratio of sucrose to compound (5) is approximately 1:1. The ratio can be up to 4:1, but is not limited to this. The components of this one-pot reaction can be in any order. They can be combined in order.

[0044] The reaction takes place within a temperature range that does not denature the enzyme, for example, around 30-45°C, or especially around 35°C. The reaction is carried out at 45°C. While lower temperatures can be used to a certain extent, they slow down the reaction rate. It will probably get worse.

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

[0046] Any buffer solution with an appropriate pH and containing a manganese(II) salt can be used in the reaction. It is possible. Examples of such buffering agents include triethanolamine; PIPES, for example. , piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS, e.g., 3-( N-morpholino)propanesulfonic acid or 3-morpholinopropane-1-sulfonic acid; HEPES, for example, 4-(2-hydroxyethyl)-1-piperazineethanesulfone Acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid ; TRIS, for example, tris(hydroxymethyl)aminomethane or 2-amino-2 -(hydroxymethyl)propane-1,3-diol; and BIS-TRIS methane, For example, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)pro It contains pan-1,3-diol. In particular, the buffer is triethanolamine. Buffer The manganese(II) salts in the agent are, for example, manganese chloride, manganese chloride hydrate, and manganese bromide. Examples include manganese iodide, manganese nitrate, and / or manganese sulfate. Buffer solution The manganese concentration inside ranges from approximately 0.05 mM to approximately 10 mM, with a particularly high concentration of approximately 5 mM.

[0047] The equilibrium reaction involves converting sucrose to D-fructose and α-D-glucose-1-phosphate. The final product is formed by consuming inorganic phosphates, which are by-products, through chemical decomposition. This can advance the conversion of substances. This is achieved by adding sucrose to the reaction mixture. It is catalyzed by sucrose phosphodiolase (EC 2.4.1.7). However, sucrose phosphodiolase is also involved. Instead of using lilacose and sucrose, any other option for removing phosphates Limbs, for example, by adding calcium, magnesium, or manganese to the reaction, Acid salts can be precipitated. This highly efficient and ecological process allows for the precipitation of protecting groups and organic compounds. It forms anomalous bonds between sugars and nucleobases with extremely high stereoselectivity without the use of solvents. It has the advantage of being able to be performed as a one-pot reaction.

[0048] Once the reaction is complete, the final product can be isolated by crystallization and collected by filtration, or otherwise... Crystallization after extraction in the appropriate solvent, etc., is not limited to, but is known to those skilled in the art. The final product can be isolated using a standard procedure.

[0049] For the synthesis of the final product 4'-ethynyl 2'-deoxynucleoside and its analogues Several upstream intermediates used in this process are also related to reaction equation 3 (reaction equations 3A and 3B). It is manufactured using an enzymatic reaction method as shown. [ka] [ka] [ka]

[0050] Compound 4: Oxidase reaction

[0051] As shown in reaction equation 3, (R)-2-ethynylglyceraldehyde (4) is galact- Suoxidase and 2-ethynylpropane-1,2,3-triol (3) are mixed at a pH of approximately In a buffer solution adjusted as appropriate to a range of 3 to 10, or more, especially in the range of approximately 6 to 8. Prepare by reacting. Use any buffer solution with a suitable pH range, such as sodium phosphate. Sodium acetate; PIPES, e.g., piperazine-N,N'-bis(2-ethanes) Morpholic acid); MOPS, e.g., 3-(N-morpholino)propanesulfonic acid or 3- Piperazine ethanesulfonic acid; HEPES, e.g., 4-(2-hydroxyethyl)-1 -Piperazine sulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-I [L]ethanesulfonic acid; TRIS, for example, tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol; and TR IS methane, for example, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl) (Tyl)propane-1,3-diol; boric acid; CAPS, e.g., N-cyclohexyl- 3-aminopropanesulfonic acid; MES, e.g., 2-(N-morpholino)ethanesulfonic acid glycylic acid; CHES, for example, N-cyclohexyl-2-aminoethanesulfonic acid, glycylic acid N, or bis(2-hydroxyethyl)glycine); sodium phosphate M is preferable.

[0052] Both copper and peroxidase activate galactosoxidase (GOase). It is used in reactions. Copper is CuSO4, Cu(OAc)2, CuCl2, Cu(II) or The reaction mixture is supplied by adding other salts of Cu(I). Peroxidase It may be horseradish peroxidase, or peroxidase derived from other organisms. Ishi, felicyanide, iride, manganese(III) salt, persulfate and one other Alternatively, substitution with two electron oxidizing agents, or oxidizing agents such as inorganic or organic oxidizing agents. This can be done. Preferably, the peroxidase is horseradish peroxidase. GOa Catalase is also added to prevent the inactivation of se. Catalase is derived from mammalian sources (bovine). ) from, or Corynebacterium, Aspergillus, or the art for this purpose It may originate from bacterial or fungal sources, such as other organisms known in the field.

[0053] The reaction proceeds in the presence of oxygen. One convenient method is to use a spag (spa) to break down the reaction with air. It is to do (rge). Alternatively, hydrogen peroxide / catalase, superoxide, Other methods for generating oxygen, such as the use of other methods known in the art for this purpose. The system can be used.

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

[0055] Compound 8: Aminal formation

[0056] As illustrated in reaction equation 3A, (R)-2-ethynyl-glyceraldehyde (4) is Then, any amine that forms a stable N,N-acetal or N,O-acetal, Diamines or amino alcohols, for example, N,N'-dibenzylethan-1,2-dia Mine, N,N'-dimethylethane-1,2-diamine, N,N'-diphenylethane-1 By reacting it with 2-diamine and N-benzylethanolamine, It can be isolated in amine form (e.g., compound 8), but N,N'-dibenzyl ethanol n-1,2-diamine is preferred. The reaction is carried out in an organic solvent to avoid the decomposition of the aminal. The process is carried out at a temperature of approximately 50°C or lower, preferably 20-30°C. Any solution that is immiscible with water is used. The medium can be, for example, MTBE, 2-MeTHF, CPME, diethyl ether, or diisopropyl. Ether, ethyl acetate, isopropyl acetate, toluene, DCM, or mixtures thereof. These can be used, but are not limited to them. The reaction can be carried out with a substrate concentration of 10-100 g / L. It can be done to a certain extent, especially around 20-50g / L.

[0057] Optionally, organic solvents, such as MTBE, 2-MeTHF, CPME, diethyl ether, di Isopropyl ether, ethyl acetate, isopropyl acetate, toluene, DCM or these Aminal can be further purified by crystallization from a mixture of these, and MTBE Preferably, crystallization is carried out at a temperature of approximately 40°C or below 50°C to avoid the decomposition of the aminal. It breaks.

[0058] The reaction proceeds in the absence of oxygen. One simple method is to inject N2 into the reaction and stir (spar It is argon, helium, or whatever is known in the art for this purpose. Other systems can be used to eliminate oxygen, such as using other methods. ru.

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

[0060] (R)-2-ethynylglyceraldehyde (4) is used to avoid the decomposition of the aminal. In the presence of an organic solvent that is immiscible with water, at a temperature below 50°C, for example, about 0-15°C, the organic These can be regenerated from their respective aminals by reacting them with inorganic acids. Any organic or inorganic acid, for example, p-toluenesulfonic acid, methanesulfonic acid, kan Phoresulfonic acid, acetic acid, hydrochloric acid, phosphoric acid, and sulfuric acid may be used, but are not limited to these. N,N'-Dibenzyletan-1,2-diaminebis-p-toluenesulfonate to water Due to its low solubility, p-toluenesulfonic acid is preferred in the reaction with aminal 8. Any immiscible solvent, e.g., MTBE, 2-MeTHF, CPME, diethyl ether Toluene, diisopropyl ethyl acetate, isopropyl acetate, toluene, DCM or A mixture of these; MTBE and 2-MeTHF is preferred. The reaction proceeds at approximately 5-100 g / This can be done at substrate concentrations of L, especially 20-50 g / L.

[0061] Optionally, the aldehyde 4 solution may be further treated with resin to remove excess organic or inorganic acids. It is possible. The resin treatment is DOWEX (trademark) MARATHON (trademark) A resin. Basic resins such as (hydroxide type) and AMBERLYST® 15 resin (hydrogen type) Fat, or a mixture thereof, preferably DOWEX® MARATHON® A This can be done using a mixture of resin (hydroxide type) and AMBERLYST® 15 resin. can.

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

[0063] Compound 5: Kinase reaction [ka] As shown in Figures 3 and 3A, (R)-2-ethynylglyceraldehyde 3-phosphorus Acid hydrate (5) is pantothenate quinase (PanK) wild type or variant derived from Escherichia coli. The seeds are divided into a range of approximately 4 to 10, preferably approximately 6.5 to 8.5, more preferably 5.5 to 8. It was prepared by reacting compound (4) in a buffer solution adjusted to a pH of 0.5. A buffer with an appropriate pH range can be used, for example (but not limited to these). (not), sodium phosphate, PIPES, for example, piperazine-N,N'-bis( 2-ethanesulfonic acid; Bis-TRIS methane, for example, 2-[bis(2-hydroxy [Ciethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; borate HEPES, for example, 4-(2-hydroxyethyl)-1-piperazine sulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; trie Tano-amines and TRIS, for example, tris(hydroxymethyl)aminomethane or 2-amino-2-(hydroxymethyl)propane-1,3-diol can be used, and phosphorus Sodium acid is preferred. For example, sodium phosphate is preferred. The reaction is carried out with any suitable Divalent metal salts, for example, magnesium salts, for example, magnesium chloride, and cobalt, man This can be done in the presence of, but is not limited to, cancer, zinc, or calcium salts. .

[0064] This reaction requires adenosine as a phosphate source to be regenerated into 5'-triphosphate (ATP). It utilizes 5'-diphosphate (ADP). ATP is generated on the spot, and then ADP, A Regenerated from denosine 5'-monophosphate (AMP) or adenosine by any known method It is possible. For example, the combination of acetyl phosphate and acetate quinase can regenerate ADP into ATP. It can be used to do this. For example, in the presence of pyruvate, phosphoric acid, and oxygen, The combination of oxidase vinate and catalase produces acetyl phosphate, so acetate quinase It can be used to regenerate ADP into ATP in the presence of ze.

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

[0066] The reaction can be carried out with pantothenate quinase (PanK) immobilized on resin, or with resin immobilized... This can also be done with both PanK and acetate quinase. Any appropriate method known in the art may be used. A suitable enzyme immobilization method, for example, immobilized metal ion affinity chromatography (I Affinity-resin immobilization and covalence immobilization using MAC resin or other biological tags. Immobilization on ionic resin, immobilization by adsorption, encapsulation, and / or use of cross-linking enzymes However, it is not limited to these. For example, metal ion affinity chromatography (IMA) C) Resin can be used, or any suitable combination of IMAC resin and divalent cations. It can be used, where cations are, for example, nickel, cobalt, copper, zinc, It may be, but is not limited to, iron and / or aluminum. In particular, nickel IMAC resins containing quinase acetate and quinase pantothenate can be used. Preferably, quinase acetate and quinase pantothenate. - Both (PanK) are fixed onto the resin.

[0067] Compound 9: Kinase reaction [ka]

[0068] As shown in reaction equation 3B, (S)-2-ethynylpropane-1,3-triol-1-phosphorus Acid (9) is derived from pantothenate quinase (PanK) wild type or a variant thereof from E. coli. The pH should be in the range of approximately 4 to 10, preferably approximately 6.5 to 8.5, and more preferably 5.5 to 8. It is prepared by reacting compound (3) with a buffer solution prepared with 0.5. A buffer with an appropriate pH range can be used, for example (but not limited to these). (None), sodium phosphate, PIPES, for example, piperazine-N,N'-bis(2-E Tansulfonic acid; Bis-TRIS methane, e.g., 2-[bis(2-hydroxyethyl) [Lu]amino]-2-(hydroxymethyl)propane-1,3-diol; borate, HE PES, for example, 4-(2-hydroxyethyl)-1-piperazine sulfonic acid or 2-[ 4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; take- Luamines and TRIS, for example, tris(hydroxymethyl)aminomethane or 2- Amino-2-(hydroxymethyl)propane-1,3-diol can be used, and sodium phosphate A sodium phosphate is preferred. For example, sodium phosphate is preferred. The reaction proceeds with any suitable divalent gold. Group salts, for example, magnesium salts, for example, magnesium chloride, and cobalt, manganese, This can be done in the presence of a zinc or calcium salt, but is not limited to these.

[0069] This reaction requires adenosine as a phosphate source to be regenerated into 5'-triphosphate (ATP). It utilizes 5'-diphosphate (ADP). ATP is generated on the spot, and then ADP, A Regenerated from denosine 5'-monophosphate (AMP) or adenosine by any known method It is possible. For example, the combination of acetyl phosphate and acetate quinase can regenerate ADP into ATP. It can be used to do this. Alternatively, (a) in the presence of pyruvate, phosphoric acid, and oxygen Using a combination of pyruvate oxidase, catalase, and acetate quinase, ADP is produced. (b) pyruvate, phosphate and acetate quinase can be regenerated into ATP. The combination of pyruvate oxidase, catalase, and acetate quinase in the presence of acetate To use a combination of phosphate and acetate quinase for ATP regeneration from ADP. It is possible.

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

[0071] The reaction can be carried out with pantothenate quinase (PanK) immobilized on resin, or with resin immobilized... This can also be done with both PanK and acetate quinase. Any appropriate method known in the art may be used. A suitable enzyme immobilization method, for example, immobilized metal ion affinity chromatography (I Affinity-resin immobilization and covalence immobilization using MAC resin or other biological tags. Immobilization on ionic resin, immobilization by adsorption, encapsulation, and / or use of cross-linking enzymes However, it is not limited to these. For example, metal ion affinity chromatography (IMA) C) Resin can be used, or any suitable combination of IMAC resin and divalent cations. It can be used, where cations are, for example, nickel, cobalt, copper, zinc, It may be, but is not limited to, iron and / or aluminum. In particular, nickel IMAC resins containing quinase acetate and quinase pantothenate can be used. Preferably, quinase acetate and quinase pantothenate. - Both (PanK) are fixed onto the resin.

[0072] Compound 5: Oxidase reaction [ka]

[0073] As shown in reaction equation 3B, (R)-2-ethynylglyceraldehyde hydrate 3-phosphate ( 5) is galactosoxidase and (S)-2-ethynylpropane-1,2,3-tri All 1-phosphate (9) and the pH is adjusted to approximately 3-10, or especially to approximately 6-8. Prepare by reacting in a buffer solution. Any buffer solution with a suitable pH range can be used, for example. For example (but not limited to these), sodium phosphate; sodium acetate; PIPES, e.g., piperazine-N,N'-bis(2-ethanesulfonic acid); MOPS For example, 3-(N-morpholino)propanesulfonic acid or 3-piperazine ethanesulfonic acid. HEPES, for example, 4-(2-hydroxyethyl)-1-piperazine sulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl]ethanesulfonic acid; TRIS, for example, tris(hydroxymethyl)aminomethane or 2-amino-2-( Hydroxymethyl)propane-1,3-diol; Bis-TRIS methane, for example, 2 -[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1, 3-Diol; borate; CAPS, e.g., N-cycloexyl-3-aminopropane Sulfonic acid; glycine; or bicine (N,N-bis(2-hydroxyethyl)glycine) Sodium phosphate can be used, and sodium phosphate is preferred.

[0074] Both copper and peroxidase activate galactosoxidase (GOase). It is used in reactions. Copper is known as CuSO4, Cu(OAc)2, CuCl2, or Cu(II) Alternatively, other salts of Cu(I) are added to the reaction mixture. Dase is horseradish peroxidase, or a peroxidase derived from other organisms. Also, felicyanide, irides, manganese(III) salts, persulfates and others Substituted by one or two electronic oxidizing agents, or by oxidizing agents such as inorganic or organic oxidizing agents. This is possible. Preferably, the peroxidase is horseradish peroxidase. Catalase is also added to prevent GOase inactivation. Catalase is derived from mammalian sources. (from cattle), or Corynebacterium, Aspergillus, or for this purpose It may originate from other organisms known in the art, such as bacteria or fungi.

[0075] The reaction proceeds in the presence of oxygen. One convenient method is to use a spag (spa) to break down the reaction with air. It is to do (rge). Alternatively, hydrogen peroxide / catalase, superoxide, Other methods for generating oxygen, such as the use of other methods known in the art for this purpose. The system can be used.

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

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

[0078] A key advantage of this new route for producing compound (6) over known processes is that The goal is to create a sugar skeleton in the correct oxidized state without using protecting groups.

[0079] 4-Ethinyl D-2-deoxyribose 5-phosphate (6) is deoxyribose phosphate Aldra-se (DERA) and (R)-2-ethynylglyceraldehyde 3-phosphate (5) Use an acid or salt, and adjust the pH to approximately 5-9 or higher, especially to a range of approximately 6-8, as needed. Prepare by reacting acetaldehyde in the aqueous solution. (5) Examples of salts include calcium M, magnesium, zinc, mono- or di-Na salts, mono- or di-K salts, or Mono- or di-Li salts; mono- or di-ammonium salts; or primary This includes, but is not limited to, monovalent or divalent salts containing secondary or tertiary amines. It is not specified. The reaction should be carried out in an open container or in a sealed container to prevent evaporation of acetaldehyde. It is preferable to do this in a container.

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

[0081] This reaction can be carried out without a buffer. Alternatively, the following buffers can be used. However, this is not limited to: triethanolamines; phosphates; MOPS, For example, 3-(N-morpholino)propanesulfonic acid or 3-morpholinopropane-1 -Sulfonic acid; HEPES, e.g., 4-(2-hydroxyethyl)-1-piperazine Ethanesulfonic acid or 2-[4-(2-hydroxyethyl)piperazine-1-yl] Tansulfonic acid; BIS-TRIS methane, e.g., 2-[bis(2-hydroxyeth) [Lu]amino]-2-(hydroxymethyl)propane-1,3-diol; borate; P IPES, e.g., piperazine-N,N'-bis(2-ethanesulfonic acid); MES, For example, 2-(N-morpholino)ethanesulfonic acid; and borates; or appropriate pH Other buffering agents that have a range and do not contain primary amine groups.

[0082] Each step and method of the process described herein, which involves the use of one or more enzymes, is this The procedure is carried out at a temperature that does not denature one or more enzymes. This specification includes the use of one or more enzymes. Each step and method of the process described herein is performed at a pH in the range of approximately 3 to 10 or approximately 4 to 10. It is possible.

[0083] A "nucleobase" (or "nitrogen base" or "base") is a base found in DNA and RNA, among others. A pyrimidine or purine heterocycle of a nucleic acid. When used herein, a nucleo salt. The group may be adenine, guanine, cytosine, thymine, or uracil, as well as unnaturally modified compounds. Nucleo bases that have one or more unnatural substituents, Modifications that affect heteroatoms in a base, excluding any changes to the anomalous CN bond. Includes.

[0084] 4'-Ethynyl 2'-deoquinucleoside contains a nucleobase. Used herein In this case, the analogue of 4'-ethynyl 2'-deoxynucleoside is the base of the nucleoside. In contrast, unnatural modifications, such as a base having one or more unnatural substituents, or anoma - This refers to modifications that affect heteroatoms in a base, excluding conversion to a CN bond.

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

[0086] The "purine nucleoside phosphorylase" ("PNP") enzyme used herein (EC 2.4.2.2) are purine ribonucleosides and related compounds (e.g., ribonucleos). Free deoxyribonucleosides and their analogues Catalyst for reversible phosphorylation of purine bases and ribose-1-phosphate (and its analogues). It is an enzyme that does this.

[0087] As used herein, "sucrose phosphorylase" ("SP") enzyme (EC 2. 4.1.7) is reversible to the D-fructose base and glucose-1-phosphate of sucrose. It is an enzyme (and its analogues) that catalyzes sucrose phosphorylation. The combination of P) and sucrose is used to treat purine nucleoside phosphorylase (PNP) and phosphorylase. Used in combination with homutase (PPM) to remove free phosphate ions from the reaction. Here, the combination of enzymes catalyzes the formation of the nucleoside MK-8591 (EFdA), In some embodiments, this can be replaced by other methods known in the art. It is possible.

[0088] As used herein, “deoxyribose-phosphate aldola-ase” (“DERA”) (For example, EC 4.1.2.4) refers to a reversible cleavage or creation of carbon-carbon bonds. This refers to enzymes within the ase family. Deoxyribose-phosphate as used herein. Aldora-se is naturally occurring (wild-type) deoxyribose-phosphate aldora-se. This also includes unnaturally occurring engineered polypeptides produced by human manipulation. Oxyribose-phosphate aldolase is derived from 2-deoxy-D-ribose-5-phosphate. - It catalyzes the reversible reaction between glyceraldehyde 3-phosphate and acetaldehyde.

[0089] As used herein, "pantothenate quinase" ("PanK") is the natural form of pantothenate quinase. An enzyme that phosphorylates pantothenic acid to produce 4'-phosphopantothenic acid (EC 2.7.1 This refers to .33). Such mutant enzymes derived from PanK enzymes are such that such mutants Regardless of whether or not it retains its natural function relative to pantothenic acid, D-ethynylglycerin This may demonstrate improved activity and stereoselectivity of aldehydes towards the 3'OH- group.

[0090] As used herein, "galactosoxidase" ("GOase"; EC 1. 1.3.9) The enzyme is a copper-dependent enzyme, and in the presence of two molecules of oxygen, it is a primary alcohol. It catalyzes the oxidation to the corresponding aldehyde. These processes are both regiospecific and enantiospecific. It acts to enable synthetic approaches that require little to no functional group protection. The desired stereoisomer is produced. The mode of oxidation is mild, and the activity is the corresponding causal relationship of the alcohol. It is controlled to prevent excessive oxidation to rubonic acid.

[0091] As used herein, "horseradish peroxidase" (HRP, EC 1.1 1.1.7) The enzyme is an inactive acid at the active site that occurs during a normal GOase catalytic cycle. Iron-dependent process that activates and maintains GOase catalytic activity by oxidizing the reduction state. It is an enzyme. Type I HRP is used in a catalytic manner in the examples contained herein. , and other electron transfer enzymes belonging to this class or other enzyme classes, and perform similar roles. It is not intended to exclude chemical compounds.

[0092] As used herein, "catalase" refers to galactosoxidase or pyroxidase. Heme-dependent enzyme (EC) that acts on hydrogen peroxide, a byproduct of the vinate oxidase reaction. 1.11.1.6) refers to hydrogen peroxide, which is galactosoxide or pyruvate. It is a byproduct of the acid oxidase reaction and inactivates these enzymes above a certain level. Larze is a catalyst used in the examples herein to convert hydrogen peroxide into water and oxygen. used as a maintenance enzyme, while in some embodiments, the electrochemical decomposition of hydrogen peroxide can be replaced by other methods such as. Hemoglobin-dependent catalase has been employed in a catalytic manner in the examples included herein, but it is not meant to be limited to this role as there are other enzymes belonging to 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 acetic acid and adenosine triphosphate (ATP). It can also catalyze the reverse reaction, phosphorylating adenosine 5'-diphosphate (ADP) to adenosine 5'-triphosphate (ATP) in the presence of acetyl phosphate. Acetate kinase is used in the examples herein to recycle the ATP required by pantothenate kinase (PanK), but 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, and in particular acts on the aldehyde group or oxo group of a donor with oxygen as the acceptor, catalyzing the chemical reaction of pyruvate, phosphate ions and molecular oxygen to produce acetyl phosphate, carbon dioxide and hydrogen peroxide. Pyruvate oxidase (PO) is used in the examples herein ​​​​​​​​​​​Acetate quinase (AcK) and catalytic ATP regeneration combination: It is used in combination with catalase, where the combination of enzymes is oxygen, pyruvic acid. It catalyzes the production of ATP from ADP in the presence of phosphate and phosphate ions, but on the other hand, several In one embodiment, it may be replaced by other methods known in the art. can.

[0095] The terms “wild-type” and “naturally occurring” enzymes used herein refer to enzymes found in nature. It refers to a form that is isolated from a natural source. For example, a wild-type polypeptide sequence is a form that is isolated from a natural source. It is possible to do so, and it is a sequence that exists in organisms that have not been intentionally modified by human manipulation. ru.

[0096] As used herein, the terms "manipulated," "mutant," "mutant," and "non- "Naturally occurring" means that the polypeptide has not been modified in a way that would not occur naturally. It refers to an enzyme, or a substance that corresponds to the natural or natural form of a substance. Several implementation forms In this state, polypeptides are identical to naturally occurring polypeptides, but synthetic materials... They are produced or induced by operations using recombinant technology.

[0097] Regarding enzymes, "percentage of sequence identity", "percentage of identity", "identity" "Percentage of" and "Percentage of Identity" are used herein to mean "polynuclear." This term is used to refer to the comparison between rheotide sequences or polypeptide sequences, and refers to the comparison window of 2 This is determined by comparing two optimally aligned sequences, where the comparison is made by comparing two optimally aligned sequences. The portion of the polynucleotide or polypeptide sequence in the ndou is the optimal combination of two sequences. Compared to the reference sequence for the alignment, it contains additions or deletions (i.e., gaps). It is possible. The percentage is such that either the same nucleobase or amino acid residue is present in both. The number of positions that occur in a sequence, or the alignment of nucleobases or amino acid residues with gaps. Find the number of positions that matched, and divide the number of matching positions by the total number of positions in the comparison window. The result is then multiplied by 100 to obtain the percentage of sequence identity, which is used for calculation. Determining optimal alignment and percent sequence identity is done using BLAST and BLAST This is done using algorithm 2.0 (e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altsc hul et al., 1977, Nucleic Acids Res. 338 (See 9-3402). The software for performing BLAST analysis is Na tional Center for Biotechnology Informat It is available through ion's website.

[0098] In short, BLAST analysis first identifies words of the same length in a database sequence. A positive threshold score T is satisfied when it matches or matches a word of the same length. By identifying short words of length W in the query sequence, high-scoring sequences can be achieved. This includes identifying Highly Spare Words (HSPs). T is the neighbor word score threshold (Altschul These are called (mentioned above). These first neighboring word hits are longer HSPs that include them. It acts as a seed to initiate the search to find it. Next, cumulative alignment Expand word hits in both directions along each sequence as much as possible to increase the Toscore. Cumulative The score is calculated for nucleotide sequences using 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 cumulative score is calculated using a scoring matrix. The extension of word hits in each direction is stopped in the following cases: the cumulative alignment score drops by amount X from its maximum achievable value; one or more negative-score residue alignments accumulate such that the cumulative score becomes zero or less; or reaches the end of either sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. In the BLASTN program (for nucleotide sequences), word length (W) is 11, expectation value (E) is 10, M = 5, N = -4, and pairwise comparison is used as a gap. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915) as a gap. Many other algorithms that function like BLAST can be used to provide the percent identity of two sequences. Optimal alignment of sequences for comparison can be done, for example, using the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the homology algorithm of Needleman and Wunsch, 1970, J. Mol., the Pears and Waterman, 1981, Adv. Appl. Math. 2:482, the homology algorithm of Needleman and Wunsch, 1970, J. Mol., the Pears and Waterman, 1981, Adv. Appl. Math. 2:482, the homology algorithm of Needleman and Wunsch, 1970, J. Mol., the Pears and Waterman, 1981, Adv. Appl. Math. 2:482, the homology algorithm of Needleman and Wunsch, 1970, J. Mol., the Pears and Waterman, 1981, Adv. Appl. Math. 2:482, the homology algorithm of Needleman and Wunsch, 1970, J. Mol., the Pears and Waterman, 1981, Adv. Appl. Math. 2:482, the homology algorithm of Needleman and Wunsch, 1970, J. Mol., the Pears

[0099] and Waterman, 未见完整引用文献信息 and many other algorithms that function like BLAST can be used to provide the percent identity of two sequences. Optimal alignment of sequences for comparison can be done, for example, using the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the homology algorithm of Needleman and Wunsch, 1970, J. Mol., the Pears and Waterman, 1981, Adv. Appl. Math. 2:482, the homology algorithm of Needleman and Wunsch, 1970, J. Mol., the Pears On and Lipman, 1988, Similarity search of Proc, algorithm (G GAP, BESTFIT, FA in CG Wisconsin Software Package Computer imaging or visual inspection (generally, Curren) of STA and TFASTA t Protocols in Molecular Biology, FM A usubel et al., eds., Current Protocols, Gr. eene Publishing Associates, Inc. and John Wi ley & Sons, Inc.(1995 Supplement) (Ausub This can be done through a joint venture with el). Furthermore, sequence alignment The determination of ment and percent sequence identity is performed by the GCG Wisconsin software package. - BESTFIT or GAP Pants (Accelerys, Madison WI) A program can be used. In this case, default parameters are used.

[0100] "Substantial identity" refers to a comparison window of at least 20 residue positions, often at least Compared to the reference sequence across a 30-50 residue window, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Polynucleotides having 97%, 98%, 99%, or higher sequence identity or Polypeptide sequences, where the total of reference sequences across the comparison window is less than 20 percent. By comparing it with sequences containing deletions or additions, the percentage of sequence identity can be calculated. Refers to a polynucleotide or polypeptide sequence that is calculated. Applies to polypeptides. In specific embodiments, the term "substantial identity" means that the two polypeptide sequences are, Using default gap weights, programs such as GAP or BESTFIT can be used. When optimally aligned, at least 80% sequence identity, preferably at least 89% sequence identity, more preferably at least 95% sequence identity (e.g., 99%) This means sharing sequence identity. Preferably, the positions of non-identical residues are preserved. It varies depending on the amino acid substitution.

[0101] "Stereoselectivity" refers to the chemical or enzymatic preference of one stereoisomer over another. This refers to the selective generation of a particular stereoisomer. Stereoselectivity is partial, meaning that the generation of one stereoisomer is often accompanied by the generation of the other. In some cases, one stereoisomer is more advantageous, while in other cases, only one stereoisomer is formed. When it is a nantiomer, stereoselectivity is called enantioselectivity, and the sum of both is This is the fraction of one enantiomer (typically reported as a percentage). Formula [Major-Enantioma--Minor-Enantioma-] / [Major-Enantioma-+ The technique is calculated as an enantioma excess (e.) according to the minus enantioma []. Stereoisomers are commonly reported (typically as a percentage) as alternatives in the field of science. When two diastereoisomers exist, the stereoselectivity is called diastereoselectivity, and the two di The proportion of one diastereomer in a mixture of astereomers (typically a percentage) (as reported in [publication name]) and is generally reported alternatively as diastereoma excess (de). Enantiomer excess and diastereomer excess are types of stereoisomer excess.

[0102] The phrase "appropriate reaction conditions" refers to the conditions under which each polypeptide used in this invention converts the substrate into the desired product. Conditions in the enzyme conversion reaction solution that can be converted to a compound (e.g., enzyme loading, substrate loading, This refers to the range of temperature, pH, buffer, cosolvent, etc. Several example suitable reaction conditions are Provided herein.

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

[0104] As used herein, the term "product" in relation to an enzymatic conversion process refers to a product obtained by converting a substrate into an enzyme. This refers to a compound or molecule resulting from the action of an enzyme polypeptide.

[0105] As used herein, certain components (e.g., enzymes) present in the reaction are the target Compared to when the component is absent, when more products are produced from the reaction Products (for example, 4'-ethynyl 2'-deoxyriboth phosphate analogs or 4'-ethynyl It increases the yield of 2'-deoxynucleoside analogs.

[0106] As used herein, "equilibrium" or "equilibrium" refers to a chemical or enzymatic reaction in which This refers to the process that brings about the steady-state concentrations of chemical species (for example, the interconversion of two species A and B). This is determined by the forward and reverse rate constants of the chemical or enzymatic reaction. This includes the interconversion of stereoisomers.

[0107] "Enantiomer excess" (ee) is a purity measurement used for chiral substances. This reflects the degree to which one enantiomer is present in the sample in greater quantities than the other enantiomer. For example, a racemic mixture has 0% enantiomer excess, but is completely pure enanth. When there is one thiomer, ee is 100%, while the other enantiomer is 70%, and the other In a sample with 30% enantiomer, the ee is 40% (70% to 30%). Astereomer excess (de) is a condition where only two diastereoisomers are present in a mixture. It is calculated in the same way as the sum.

[0108] "Protein," "enzyme," "polypeptide," and "peptide" are used herein. , length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristylation, 1 Regardless of ubiquitination, etc., at least two covalently bonded by an amide bond Used interchangeably to mean amino acid polymers. This definition includes: These are D- and L-amino acids, as well as mixtures of D- and L-amino acids.

[0109] Here, "approximately" means an acceptable error for a particular value. "Approximately" means that the lower and upper limits of a given value range are 0.05%, 0.5%, and 1.0%. Or it means within 2.0%. Regarding pH, "approximately" means plus or minus 0. It means 0.5.

[0110] The terms "substantially pure" polypeptides or "purified" proteins used herein Quality is the dominant species in which the polypeptide species exists (i.e., on a molar or weight basis). This means a composition that is greater than any other individual polymer species in the composition, and generally refers to the target species If it contains at least about 50% of the polymer species present in moles or by weight, then it is substantially fine The composition is manufactured. However, in some embodiments, the composition includes polypeptides. This includes polypeptides with a purity of less than 50% (e.g., about 10%, about 20%, about 30%). Approximately 40%, or approximately 50%. Generally, substantially pure polypeptide compositions contain All polymer species present are present in molar or weight percent, approximately 60% or more, approximately 70% or more, approximately 8% Includes 0% or more, approximately 90% or more, approximately 95% or more, and approximately 98% or more. Several embodiments In this process, polypeptides are purified to an inherently homogeneous state (i.e., contaminants are removed using conventional methods). (Undetectable in the composition by the method of extraction), where the composition is essentially a single polymer It consists of species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. It is not possible. In some embodiments, the isolated polypeptide is substantially pure poly It is a peptide composition.

[0111] As used herein, “improved properties” of an enzyme means at least one modification of the enzyme. Refers to improved characteristics. In some embodiments, the present invention refers to, respectively, a reference PPM. PNP, DERA, PanK, AcK, SP or GOase polypeptide, and / or These are wild-type PPM, PNP, DERA, PanK, AcK, SP, or GOas, respectively. e polypeptide, and / or other modified PPM, PNP, DERA, respectively. Compared to PanK, AcK, SP, or GOase polypeptides, any of the enzymatic properties Recombinant PPM, PNP, DERA, PanK, AcK, SP and / or G showing improvement Oase polypeptide is used. Therefore, the level of "improvement" is determined, and the wild type is included. Various polypeptides and manipulated polypeptides can be compared. The resulting properties include increased protein expression, increased production of the intended product, substrate specificity, and This includes increased affinity (i.e., increased activity for the substrate), increased thermal activity, increased thermal stability, and pH Increased activity, increased stability, increased enzyme activity, increased specific activity, substrate or end product inhibition Increased resistance to acidity, increased chemical stability, improved chemistry, improved solvent stability, acidity Increased tolerance to pH, increased tolerance to proteolytic activity (i.e., protein (Reduced susceptibility to degradation), decreased aggregation, increased solubility, and temperature profile This includes, but is not limited to, characteristics such as change. Additional embodiments of this use The terms are PPM, PNP, DERA, PanK, AcK, SP and / or GOase yeast. Used with reference to at least one improved characteristic of the basics. In some embodiments, this The inventions refer to PPM, PNP, DERA, PanK, AcK, SP and / or Compared to GOase polypeptide, this is an improved PPM that shows improvements in any enzymatic property. , PNP, DERA, PanK, SP and / or GOase polypeptide; and / or other modified PPM, PNP, DERA, PanK, AcK, SP and / or G Each Oase polypeptide is used. Therefore, the level of "improvement" is determined, and the field It is possible to compare various polypeptides, including the biopolymer form, as well as manipulated polypeptides. ru.

[0112] In this context, "conversion" refers to the enzymatic conversion (or in vivo conversion) of a substrate to its corresponding product. In other words, "percent" conversion refers to the conversion of a substrate into a product within a certain period of time under specific conditions. It means a percentage. Therefore, the "enzyme activity" or "activity" of a polypeptide is specific. This can be expressed as the percentage conversion of the substrate to the product over a given period.

[0113] As used herein, "stereoselectivity" refers to the chemical properties of one stereoisomer compared to another. Alternatively, it means that it is preferentially produced by an enzymatic reaction. Stereoselectivity is partial, on the other hand. In some cases, the formation of one stereoisomer is more favorable than the other, while in other cases, only one stereoisomer is formed. In some cases, the stereoisomer is an enantiomeric character, and the stereoselectivity is enantioselectivity. It is called the fraction of one enantiomer in the sum of both (typically as a percentage and It is reported as [Major Enantio - Minor Enantio]. Calculate from there according to [Major Enantioma + Minor Enantioma] As an enantiomer-excess ("ee"), it is commonly used in the art ( They are reported alternatively (as percentages in terms of type). The stereoisomers are diastereoisomers. In some cases, stereoselectivity is called diastereoselectivity, and it is a mixture of two diastereomers. This is the proportion of one diastereomer (typically reported as a percentage), Generally reported alternatively as diastereomer-excess ("de"). Enantiomers Excess and diastereomer-excess are types of stereoisomer-excess.

[0114] This invention relates to manipulated PPM, PNP, DERA, PanK, AcK, SP and GOa se polypeptides, especially those having sequence numbers 1 to 21, and each of sequence numbers 1 to 21 The use of sequences containing one or more conserved amino acid substitutions, called conservatively modified variants. To include.

[0115] As used herein, the term "conservative" amino acid substitution refers to a substitution that alters the biological activity of a protein. To allow for frequent changes without causing damage, similar properties (e.g., acidity, base) should be used. Sex, positive or negative charge, polar or nonpolar, side chain size, hydrophobic / hydrophilic, skeletal structure This refers to amino acid substitutions in proteins that have properties such as conformation and rigidity. This refers to differences within a defined class of amino acids, whether identical or similar, within a polypeptide. This includes one or more substitutions by amino acids. Those skilled in the art will generally know that polypeptides are It is recognized that single amino acid substitutions in non-essential regions do not substantially alter biological activity. (For example, Watson et al. (1987), Molecular Biology) of the Gene, The Benjamin / Cummings Pub. See Co., p.224 (4th edition). Furthermore, structurally or functionally similar Substitutions of similar amino acids are less likely to disrupt biological activity. I will give examples to limit the possibilities. However, in some embodiments, an amino acid having an aliphatic side chain is another aliphatic amino acid. Substituted with ano acids (e.g., alanine, valine, leucine, and isoleucine); hydr An amino acid having a hydroxyl side chain is another amino acid having a hydroxyl side chain (for example, se Substituted with phosphorus and threonine; amino acids having aromatic side chains have aromatic side chains Other amino acids that do this (e.g., phenylalanine, tyrosine, tryptophan, and histamine) Amino acids with a basic side chain are substituted with another amino acid (e.g., lysine); (and arginine) are substituted; amino acids with acidic side chains are replaced by another amino acid with an acidic side chain. Substituted with an amino acid (e.g., aspartic acid or glutamic acid); and / or Hydrophobic or hydrophilic amino acids are substituted with other hydrophobic or hydrophilic amino acids. 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 set of amino acids that have been replaced with a reference sequence. This refers to a group of amino acid substitutions in a cytoplasmic sequence. The substitution sets are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions It can have.

[0118] A "functional fragment" is a fragment with amino-terminus and / or carboxy-terminus deletions and / or Or it has an internal deletion, but the remaining amino acid sequence is in the corresponding position in the sequence being compared. It is identical to the position (for example, fully length operated PPM, PNP, DE used in the present invention). RA, PanK, AcK, SP or GOase enzymes), the actual activity of full-length polypeptides This refers to a polypeptide that qualitatively retains all its components.

[0119] As used herein, "deletion" refers to the removal of one or more amino acids from a reference polypeptide. This refers to the modification of polypeptides by [deletion]. A deletion is the removal of one or more amino acids, or two or more amino acids. Amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or Removal of 20 or more amino acids, up to 10% of the total number of amino acids, or all of the amino acids that make up the reference enzyme. It can contain up to 20% of the number of amino acids, while retaining and / or manipulating enzyme activity. Modification of the PPM, PNP, DERA, PanK, AcK, SP, or GOase enzymes produced The desired properties can be retained. The deletion occurs in the internal and / or terminal parts of the polypeptide. It can be directed towards the end portion. In various embodiments, the deletion may include a continuous segment. It can be continuous or discontinuous. Deletions are typically indicated by a "-" in the amino acid sequence. .

[0120] As used herein, “insertion” refers to the addition of one or more amino acids from a reference polypeptide. This refers to a polypeptide formed by [a specific mechanism]. Insertion refers to the internal part of the polypeptide, or the carboxyl part. It may be at the end or amino terminus. Inserts used herein may be in the art in which It includes known fusion proteins. Insertions are consecutive segments of amino acids. Alternatively, they may be separated by one or more amino acids in naturally occurring polypeptides. That's fine.

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

[0122] Experimental Procedure Synthesis of 2-ethynyl-2-hydroxypropane-1,3-diyldiacetate (2) Method A: [ka] A solution of diacetoxyacetone (1) in THF (1000 mL) at -35°C (159 g, 914.0 mmol) of ethynyl magnesium chloride in THF maintained at a temperature of -20°C or below. 1600 mL of 0.5 M cium solution was added. After the reaction was complete, 400 mL of methyl te was added. Add 78 mL of acetic acid in rt-butyl ether (MTBE) while maintaining the temperature below -20°C. Added. Then, MTBE (800 mL) was added and heated to room temperature. Saturated Na in water Add Cl (1000 mL), then add saturated NH4Cl solution in water (1050 mL). The organic layer was separated, dried on Na2SO4, and evaporated to obtain compound (2) as an oily substance. (160g, 88%) was obtained. 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] 2-Ethinyl 2-hydroxypropane-1,3-diyl diacetate (2) (70g, 3 A 50 mmol ethanol solution is mixed with a methoxylate sodium methanol solution (69 0.9 mL of 35.0 mmol of 0.5 M solution was added at room temperature (rt). The reaction was carried out at rt for 2 hours. h) Stirring was completed. The solvent was evaporated, and the residue was redissolved in 100 mL of water. Extraction was performed with mL of MTBE. The aqueous layer was sprayed with nitrogen to remove residual solvent, and nuclear magnetic resonance (NM) was performed. When measured with R) (maleic acid as an internal standard), 2-ethynylpropane-1,2 A 40.9% solution of ,3-triol(3) (108g, 100% yield) was obtained. 1H N MR (D2O, 500 MHz): δ 3.60 (dd, 4 H), 2.8 5 (s, 1H).

[0124] Alternative preparation of (R)-2-ethynylglyceraldehyde (4) C1 method: [ka] In a stirred reactor, add the defoaming agent 204 (Sigma A6426, 1 drop to 20 μL). 2-ethynylpro in sodium phosphate buffer (30 mL, 100 mM, pH 7.0) Pan-1,2,3-triol(3) (1.1g, 9.47 mmol) in 12.5 sc The mixture was heated to 30°C while being spagled at cm. Galactosoxidase (GOOase, Sequence ID: 1) (250 mg), Horradish peroxidase (Type I, 5 mg) Yobiushikatara-se** (5 mg) in sodium phosphate buffer (5 mL, 100 mM, p It was dissolved in H 7.0, and then CuSO4 aqueous solution (100 mM, 150 μL) was added. The reaction mixture was stirred at 600 rpm for 47 hours while spraying air, and (R)-2-ethynylg Lysellaldehyde (4) was obtained with a conversion rate of 47% (by NMR) and an ee of 72%. (This drug could not be isolated.) 1H NMR (D2O, 500 MHz): δ 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H) ). * Horseradish peroxidase: horseradish root (Amoracia rusticana) SIGMA (P8125) isolated from wasabi wasabi, and wild-type peroxygen derived from commercially available wasabi type I. Da-ze. ** Bovine Catalase: A heme-dependent catalase derived from bovine sources, sigma (C1345) From commercially available

[0125] C2 method: [ka] In a 100 L jacketed furnace containing deionized water (56.2 kg), sodium phosphate ( 1.212 kg (10 moles) was added. At 25°C, a 10 N sodium hydroxide solution (852 The pH was adjusted to 7.02 using 0.6g of antifoam (A642). Antifoam 204 (A642) was added to the reactor. 6.10 mL was added, followed by CuSO4·5H2O (6.5 g). Galacto- Suoxidase (451.2g) (SEQ ID NO: 10) was added, and while sparging with air... Stirred for 15 minutes. Horseradish peroxidase* (200.2g) and catalase** ( 502.6g) was added, and the reactor was washed with water (2.0kg). Next, water (9.48%, 3 2-ethynylpropane-1,2,3-triol in 0.34 kg, 24.72 mol (3) Add the solution, followed by the addition of the defoaming agent 204 (A6426, 10 mL). The reaction was empty. Spurge with air and stir overnight to obtain (R)-2-ethynylglyceraldehyde(4)94. 0 kg was obtained with 66% conversion (by NMR) and 84% ee. Analysis revealed 60%: 1H NMR (D2O, 500MHz): δ 4.29(s, 1H), 3.6 The values ​​are 5(dd, 2H) and 2.83(s, 1H). * Horseradish peroxidase: Horseradish root (Amoracia rustican) a) A commercially available refined wasabi extract from Toyobo (PEO-301) isolated from The original wild-type peroxidase. ** Bovine catalase: A heme-dependent catalase derived from bovine sources, Sigma (C1 It is commercially available from 345.

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

[0127] C3 method: [ka]

[0128] 100mL EasyMaster with Sparrow and Flow Controller Fill the container with water (82 mL) and PIPES potassium buffer (5 mL, 0.5 M). The pH was adjusted to 7.5 using a 5M KOH solution at 25°C. Antifoaming agent 204 (200 Add μL of evolved galactosoxidase (SEQ ID NO: 17, 450 mg enzyme) and then add the enzyme. Add the powder and copper(II) sulfate pentahydrate (100 μL, 100 mM). Reaction mixture 1 Spagged with 25 standard cubic centimeters / minute (sccm) of air for 15 minutes. Ushikatara-ze ( C1345, Sigma-Aldrich, 150mg, 2000~5000 U / mg Add 0.75 MU of horseradish peroxidase (HRP, Toyobo PEO-301 (100 mg, 130 U / mg, 1.3 kU) and 2-ethinyl pro Aqueous solution of pan-1,2,3-triol(3) (25 wt%, 12 mL, 25.8 mm) (ol) was added. The reaction mixture was stirred at 30°C with 125 sccm of aeration, and 20 After sampling using Easy Sampler over a period of time, a 70% conversion was obtained, and 5 Compound (4)((R)-2-ethynylglycerin) was found in 8% assay yield and 99% ee. A dulaldehyde was generated. ¹H NMR (D2O, 500MHz): δ4.29( s,1H), 3.65(dd,2H), 2.83(s,1H). The crude reaction flow is directly into the next ri It was transported to the oxidation stage.

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

[0130] Enzyme immobilization procedure: Nuvia IMAC Ni electrostatic resin (16 mL based on sedimentation capacity) into a filter-funnel. In addition, binding buffer (10 column volume, 160 mL; 500 mM sodium chloride) Wash with 50 mM sodium phosphate, 15 mM imidazole (pH 8.0), and resin The preservation solution was removed. Galactosoxidase (SEQ ID NO: 17, 2) evolved in the container. Resuspend the freeze-dried powder (00g) in copper(II) sulfate solution (100μM; 5.00mL), 50 mL of binding buffer and resin were added. The solution was mixed using a rotary mixer at 20°C for 5 hours. This was mixed with binding buffer (10 column volume, 160 mL) and potassium P. Filtration and washing with IPES buffer (10 column volume, 160 mL; 50 mM, pH 7.5). And it was used directly during the reaction.

[0131] Reaction procedure: A 100mL EasyMax container equipped with a spurge and flow controller contains water ( 82 mL of PIPES potassium buffer (5 mL, 1 M) was added. KO was performed at 25°C in 5 M. The pH was adjusted to 7.5 using solution H. Defoaming agent 204 (200 μL) was added, followed by resin Lipid-immobilized evolved galactosoxidase (SEQ ID NO: 17, enzyme per 6 mL of resin) (750 mg of powder) and copper(II) sulfate pentahydrate (100 μL, 100 mM) were added. The reaction mixture was spagged with 125 standard cubic centimeters / minute (sccm) of air for 15 minutes. Ushikatara-ze (C1345, Sigma-Aldrich, 210mg, 2000~5 Add 000 U / mg (1.05 MU), followed by horseradish peroxidase (HR P, Toyobo PEO-301, 100mg, 130 U / mg, 1.3 kU) Aqueous solution of 2-ethynylpropane-1,2,3-triol(3) (25 wt%, 13 mL) The 29.4 mmol reaction mixture was stirred at 25°C and aerated at 125 sccm. After 22 hours, the reaction reached 91%, and 200 mM (R)-2-ethynylglyceraldehyde (4) Solution (100 mL, 68% assay yield, 97% ee1H NMR (D2 O,500MHz): δ4.29(s,1H),3.65(dd,2H),2.83( The reaction was s(1H). The crude reaction flow proceeded directly to the next phosphorylation step.

[0132] C5 Method: Arbitrary isolation of aldehydes via the formation of aminal(8) Step 1: (S)-2-(1,3-dibenzylimidazolidine-2-yl)but Preparation of -3-en-1,2-diol [ka] A 100 L jacketed cylindrical container equipped with a nitrogen bubbler, mechanical agitator, and thermocouple. (R)-2-ethynylglyceraldehyde(4), 26.0 kg, 1.85% by weight Add the crude oxidase reaction stream containing dehyde (3.64 mol) and inactivate it in an N2 atmosphere. The aqueous solution was heated to 20°C, and N,N-dimethyldodecane-1-amine oxide (DDA) was added. After adding O) (30% by weight in water, 798g, 0.96mol;), MTBE (55 0.3kg, 76 L) and N,N'-dibenzylethane-1,2-diamine (1.55kg (6.43 mol) was added. The brown two-phase mixture was stirred overnight at 20°C under a nitrogen atmosphere. After 17 hours, stirring was stopped, the organic phase was removed, and the mixture was discarded. (S)-2-(1,3- Dibenzylimidazolidine-2-yl) but-3-in-1,2-diol (56.5 (kg, 2.02 wt% aminal, 3.39 mmol, 93% assay yield) Light brown M A TBE solution was obtained.

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

[0134] A 50 L jacket equipped with a mechanical stirrer, distillation head (-20°C condenser), and thermocouple. Load the amminal solution (45 L) into the cylindrical container with a nozzle, and then fill the container (65-95 torr) Vacuum was applied and the jacket was set to 40°C. The solvent was distilled until it reached a volume of 35 L. It was removed by [method]. At this point, the internal temperature was 6.1°C, and the off-white solid began to crystallize. The remaining MTBE solution was kept in a constant volume of 35-40 L and kept at an internal temperature of 0-10°C. It was added slowly while maintaining the current. Once all of the MTBE solution had been added, the volume was 25 L. It decreased to [value]. Distillation was stopped, nitrogen was deactivated in the vessel, and the jacket temperature was lowered to 10°C. The resulting pale yellow suspension was left at this temperature for 2 hours, and the solid was recovered by filtration. After washing the ruta cake with cold (-2°C) MTBE (12.7 kg), it was left to stand under a nitrogen stream for 7 hours. Dry. (S)-2-(1,3-dibenzylimidazolidine-2-yl)-but-3 -In-1,2-diol was obtained as an off-white crystalline solid (5.75 kg). 1H NMR (500 MHz, DMSO-d6) δ 7.42 - 7.35 (m, 4H), 7.32 (td, J = 7.5, 1.6 Hz, 4H), 7. 27 - 7.21 (m, 2H), 5.10 (t, J = 5.6 Hz, 1H), 5.03 (s, 1H), 4.28 (d, J = 13.3Hz, 1H), 4.16 (d, J = 13.3 Hz, 1H), 3.76 (s, 1H), 3.70 - 3.58 (m, 4H), 3.21 (d, J = 0.9 Hz, 1H), 2.90 - 2.80 (m, 2H), 2.60 - 2.51 (m, 2H).13C NMR (126 MHz, DMSO-d6) δ 140.0, 140.0, 128.5, 128.3, 128.2, 1 28.1, 126.8, 126.8, 88.6, 86.9, 75.0, 74 .0, 66.4, 60.7, 60.5, 50.4, 50.3, 39.5.H R-MS(ESI) Aminal (M + H+) C21H25N2O2+337: Calculated value 1911;337. Detected value 1922.

[0135] Step 2 From aminal (8) to (R)-2-ethynyl-glyceraldehyde (4) preparation [ka] TsOH·H2O in a 4L jacketed cylindrical container equipped with a nitrogen bubbler and mechanical stirrer. (12.0 g, 63.1 mmol), water (60 mL), (S)-2-(1,3-dibenzylimidazolidin-2-yl)but-3-yn-1,2-diol (110 g, 327 mmol) and MTBE (1700 mL) were charged. The biphasic mixture was placed under nitrogen and the jacket temperature was set at 15 °C. An aqueous solution of TsOH·H2O (114 g, 599.3 mmol) in water (600 mL) was added dropwise over 1.5 h with stirring (200 rpm). After addition, the jacket temperature was lowered to 5 °C and the resulting slurry was left for 1 h. The solid was 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), sparged with N2 at a rate of 200 sccm for 24 h to remove residual MTBE. The resins were 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 (126 MHz, D2O) δ 129.4, 125.4, 90.3, 81.0, 76.0, 73.9, 65.3. HRMS (ESI) Aldehyde dimer (2M + Na) C10H12NaO6 calcd 251.0526; found 251.0530. (S)-2-(1,3-dibenzylimidazolidin-2-yl)but-3-yn-1,2-diol (110 g, 327 mmol) was added. The biphasic mixture was placed under nitrogen and the jacket temperature was set at 15 °C. An aqueous solution of TsOH·H2O (114 g, 599.3 mmol) in water (600 mL) was added dropwise over 1.5 h with stirring (200 rpm). After addition, the jacket temperature was lowered to 5 °C and the resulting slurry was left for 1 h. The solid was 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). 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). The aqueous phase was sparged with N2 at a rate of 200 sccm for 24 h to remove residual MTBE. The resins were 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). (R)-2-hydroxy-2-(hydroxymethyl)but-3-ynal (774 g, 4.6 wt% aldehyde, 82% yield) was obtained. 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). 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 (126 MHz, D2O) δ 129.4, 125.4, 90.3, 81.0, 76.0, 73.9, 65.3. 13C NMR (126 MHz, D2O) δ 129.4, 125.4, 90.3, 81.0, 76.0, 73.9, 65.3. HRMS (ESI) Aldehyde dimer (2M + Na) C10H12NaO6 HRMS (ESI) Aldehyde dimer (2M + Na) C10H12NaO6 + Calculated 251.0526; + Calculated 251.0526; Found 251.0530.

[0136] Alternative preparation methods for (R)-2-ethynylglyceraldehyde 3-phosphate (5): D1 Method: Acetate-tokinase: ATP regeneration system [ka] In a stirring reactor, Adenium in HEPES buffer (66 mM, pH 7.5, 30 mL) Nosine disodium diphosphate (40 mg, 0.087 mmol) and magnesium chloride (R)-2-ethynylglyceraldehyde in a solution of (38 mg, 0.400 mmol) (4) Add (1.9 mL, 210 g / L solution in water, 3.51 mmol), followed by acetate Na-ze (SEQ ID NO: 3) (40 mg), and pantothenate quinase (SEQ ID NO: 2) (12 0 mg) was added. The reaction mixture was heated to 25°C and HEPES buffer (50 mM, pH 7.5, 10 mL) Lithium potassium acetyl phosphate solution (1.3 g, 7.01 Add mmol) dropwise over 4 hours, and maintain the pH at 7.5 using 5M sodium hydroxide. The reaction was stirred for 18 hours to obtain (R)-2-ethynylglyceraldehyde 3-phosphate (5). The product was given by 85% conversion (by HPLC) (the product was not isolated). 1H NMR (D2O, 400 MHz): δ 5.02 (s, 1H), 4.00 (d q, 2H), 2.88 (s, 1H).LC-MS: (ES, m / z): C5H7O6P (MH): Measured value 193.1; Detected value 193.0.

[0137] D2 method: Pyruvate oxidase ATP regeneration system [ka] In a stirred reactor, sodium pyruvate (3.11 g) in 76 mL of water at pH 7.5 (R) -2-Ethinylglyceraldehyde (4) (3.8 mL, 210 g / L solution in water, 7.0 1 mmol), adenosine diphosphate disodium salt (80 mg, 0.174 mmol), Thiamine pyrophosphate (40 mg, 0.086 mmol), flavin adenine dinucleotide Disodium chloride hydrate (64 mg, 0.077 mmol), and magnesium chloride ( 400 μL of 1 M solution (0.4 mmol) was added to the solution. The pH was adjusted using 5 M sodium hydroxide. The solution was readjusted to 7.5, and the reaction volume was readjusted to 80 mL with water. Acetate quinase (SEQ ID NO: 3 ) (80 mg), pyruvate oxidase (SEQ ID NO: 4) (80 mg, freeze-dried cell migratory (Separated extract), pantothenate quinase (SEQ ID NO: 2) (400 mg), and catalase (800 μL, ammonium sulfate suspension CAT-101, Biocatalytics) The mixture was stirred at 500 rpm and 30°C for 72 hours while sparging with air. Then, (R)-2-ethynylglyceraldehyde 3 was converted with a 95% conversion rate (by HPLC). -Phosphate 5 was obtained (the product was not isolated). ¹H NMR (D2O, 40 0 MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H).LC-MS: (ES, m / z): calculate d for C5H7O6P (MH): 193.1; Detected value 193.0.

[0138] The above reaction can also be carried out using pantothenate quinase (SEQ ID NO: 13), and product 5 is 66% modified. Obtained by exchange. (This drug was not isolated.) 1H NMR (D2O, 400 MH) z): δ 5.02 (s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H).

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

[0140] Enzyme immobilization procedure: NUVIA® Immobilized Metal Ion Affinity Chromatography (IMAC) Add nickel-charged resin (168 mL based on sedimentation capacity) to the filter-funnel, and the binding buffer solution is added. (1.6L; 500mM sodium chloride, 50mM sodium phosphate, pH 8.0) Washed. In a container, add pantothenate quinase (8.4g) (SEQ ID NO: 12) and vinegar. Acid quinase (2.8g) (SEQ ID NO: 3) was dissolved in binding buffer (500mL). The purified resin was placed in a container, and the solution was stirred at 20°C for 4 hours. The resin was filtered, and the first bond was formed. Wash with buffer (1.6 L), followed by piperazine-N,N′-bis(2-ethanesulfone) The samples were washed with (PIPES) buffer (840 mL; 50 mM, pH 6.5). The cleaned resin was used directly in the next step.

[0141] Reaction procedure: In a 1L reactor, add (R)-2-E in water (608.7g, 4.6% by weight, 212 mmol). A solution of thinylglyceraldehyde (4) was added and cooled to 5°C. Cooling solution 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, 9.3 (3 mmol), diammonium acetyl phosphate (51.8 g, 265 mmol), ade Nosine disodium diphosphate hydrate (1.17 g, 2.12 mmol) and water (192 (mL) was added. The solution was stirred, and the pH was adjusted to 6.4 using 5N KOH. The reaction was then carried out. Heat to 20°C, and then add pantothenate quinase (SEQ ID NO: 12) and acetate quinase (SEQ ID NO: 12). 3) 168 mL of the immobilized resin was added. 5 was used to maintain the reaction at pH 6.4. Stir with N KOH for 10 hours, then (R)-2-ethynylglyceraldehyde 3-phosphate (5 ) converted to 92% (by HPLC) and yielded 91% (tetraphenyl chloride as internal standard). The result was obtained by phosphonium-assisted 31P NMR (the product was not isolated). 1 H NMR (D2O, 400 MHz): δ 5.02 (s, 1H), 4. 00 (dq, 2H), 2.88 (s, 1H).LC-MS: (ES, m / z): C5H7O6P (MH): Calculated value 193.1; Detected value 193.0.

[0142] Synthesis of 4-ethynyl D-2-deoxyribose 5-phosphate (6) Method E: [ka] (R)-2-ethinylglyceraldehyde 3-phosphate (5) (5, 20 mL, 5.3 mL) A 40 wt.% aqueous solution of acetaldehyde (2.02 mL, 15 mol) is added to an aqueous solution of acetaldehyde (40 wt.%, 2.02 mL, 15 After adding 0.9 mmol) at room temperature, triethanolamine hydrochloride buffer (1 mL, 1 M, 25 mg of deoxyribose-phosphate aldorase (DERA) in pH 7 (sequence) Add (number 6). Seal the reactor and stir the mixture overnight at 30°C and 600 rpm. 4-ethynyl D-2-deoxyribose 5-phosphate (6) was obtained in the % convolution. 99 % of the product was not isolated, i.e., 99% of the product was isolated as a 1:1 anoma-mixture. ). α-Anoma: 1H NMR (D2O, 600MHz) δ5.31(t, 1H ), 4.13(t, 1H), 3.81~3.72(m, 2H), 2.89(s, 1H), 2.42~2.34(m, 1H), 1.87~1.79(m, 1H), 13C NMR (D2O, 151MHz) δ97.7(s), 81.4(d), 79.4(s), 78 0.9(s), 71.1(s), 67.7(d), 39.6(s). β-anoma: 1H NMR (D2O, 600MHz) δ 5.40(dd, 1H), 4.28(t, 1 H), 3.88~3.80(m, 2H), 2.87(s, 1H), 2.13~2.06( m, 1H), 2.04~1.97(m, 1H), 13C NMR (D2O, 151MH z) 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): C 7H10O7P (MH): 237.0; Detected value 237.0

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

[0144] F1 method [ka]

[0145] Ammonium hydrogen phosphate ((2R,3S)-2-ethynyl 3,5-dihydroxytetrahydroxyphosphate) (1.00 g, 3.91 mmol) methyl drofuran-2-yl (1.00 g, 3.91 mmol) in pH 7.5 buffer. (100 mM triethanolamine·HCl containing 5 mM MnCl2) Dissolved in 0 mL. The pH of the solution was adjusted to 7.3 with 5N NaOH. Add 2-fluorine to the solution. Roadenine (0.599g, 3.91mmol) and sucrose (2.68g, 7.82mg) (mol) was added. The enzyme solution was phosphopentumutase (SEQ ID NO: 8) (100 mg). , purine nucleoside phosphorylase (SEQ ID NO: 9) (50 mg) and sucrose phosphoryl Prepared by dissolving L-Ze (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... The turbidity was cooled to 0°C, filtered, and washed with cold water. The solid was vacuum-dried, and the labeled compound (1.1 2g, 92%) was considered the 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 H z, 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): 316.0 822; 計算値316.0818。

[0146] The PPM and PNP enzymes used in this step are derived from Escherichia coli, respectively. The mutation originated from an enzyme derived from HIA coli. The sucrose used at this stage Phosphorylase (SP) originated from Alloscardovia onnicolens; derived from other organisms. The incoming SP could also be used.

[0147] F2 method: [ka] Contains piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer. R)-2-ethynylglyceraldehyde 3-phosphate (5) (950 mL, 157 mmol) In an aqueous solution with a pH of approximately 5.5-6.0, add triethanolamine (7.09g, 47.5ml) (mol) was added. The pH of the solution was adjusted from 7.1 using potassium hydroxide (8 mL, 8 M). Adjusted to 7.6. Manganese(II) chloride hydrate (0.592g, 4.70 mmol) Add the following: Then add sucrose (161g, 470 mmol) to obtain a pH of 7.5. The solution contains the following enzyme: deoxyribose-phosphate aldolaze (SEQ ID NO: 14) (46 1 mg), sucrose phosphorylase (SEQ ID NO: 7) (494 mg), phosphopentom Tase (SEQ ID NO: 8) (2.63g), and purine nucleoside phosphorylase ( Add (659 mg) of the enzyme (row number: 15). Once the enzyme is dissolved, add 2-fluoroadenine ( 19.80g (125 mmol) was added. The reaction was heated to 35°C, and acetaldehyde was added. Added (40 wt% in isopropyl alcohol, 29.8 mL, 235 mmol). 2 hours After the reaction, EFdA crystal product (0.96 g, 2 mol%) was seeded onto the mixture. After reacting at 35°C for 26 hours, the slurry is cooled to 0°C, the solid is filtered, and washed twice with water. Collected by (40 mL ea.). The solid was dried under nitrogen sweep. 43 Correction values ​​of 0.2g, 92% by weight, and 96.2% are obtained. 1 1H NMR: (300 M) Hz, 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 = 6.4H z, 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). 13 C nuclear magnetic resonance (150.92MHz, DMSO-d6, ppm) δ158.5(d, JCF = 203.5),157.6(d, JCF = 21.2),150.2(d, J CF = 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+N a): 316.0822; calculated value 316.0818.

[0148] (S)-2-ethinylpropane-1,2,3-triol 1-phosphate (9) Alternative preparation method G1 Method: Acetate-tokinase: ATP regeneration system using enzymes SEQ ID NO: 2 and SEQ ID NO: 3 [ka] A 50 mL reactor containing an aqueous solution of 2-ethynylpropane-1,2,3-triol(3) (9.29g, 9.46wt%, 7.57mmol) Potassium PIPES buffer (1.0 (2 mL, 1 M, pH 6.5, 1.02 mmol), magnesium chloride (292 μL, 1 M, 0.292 mmol), diammonium acetyl phosphate (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) were added. 5M KO The pH was adjusted to 6.4 using H, and the solution was heated to 20°C. Thenate quinase PanK SEQ ID NO: 2 (264 mg) and acetate quinase AcK SEQ ID NO: Adding No. 3 (88 mg). The reaction was carried out using 5N KOH, maintaining the pH at 6.4 for 16 minutes. The mixture was stirred for a specified time. The final reaction content was (S)-2-ethynylpropane-1,2,3-triol 1-phosphate (9) with >95% ee and 99% conversion rate ( 31 (P-NMR) The sample was given. The product was not isolated. 1 1H NMR (D2O, 500 MHz) δ 3.89 (m, 2H), 3.72 (d, J = 11.6 Hz, 1 H), 3.65 (d, J = 11.6 Hz, 1H), 2.93 (s, 1H). 13 C NMR (D2O, 126 MHz) δ 82.9 (s) , 75.1 (s), 71.0 (d, J = 6.9 Hz), 67.0 ( d, J = 4.5 Hz), 64.7 (s).31P NMR (D2O, 202 MHz) δ 3.39.HRMS: (ESI, m / z): [M-1] - C5H8O6P: 195.0058; Detected value 195.0068 [MH] - : 195.0058.

[0149] G2 method: Acetate-tokinase: ATP regeneration using enzymes SEQ ID NO: 20 and SEQ ID NO: 21 Raw system. [ka] 2-ethynylpropane-1,2,3-triol(3)(11) in a jacket reactor aqueous solution Add 0.47 kg (8.7 wt%) and water (7.5 kg), then add 1M BIS-TRIS methane buffer pH 6.5 (1L) and magnesium chloride (41. 4g) was added. ATP (48g, 0.086mol, 0.01 equivalents) and diphosphate were added. Add 2.021 kg of monium acetyl (89%, 10.33 mmol) and allow to rise to 20°C. The solution was heated, and the pH was readjusted to 6.8 using KOH (270.4g). ved) Pantothenate quinase SEQ ID NO: 20 (20.4g) and evolved acetate quinase formulation Row number 21 (3g) was loaded as a solid. This reaction was stirred at 20°C for 16 hours. The pH decreased to 5.5. Determination of 2-ethinylpropane-1,2,3-triol(3) Quantity conversion 1 It was obtained by H and 31P NMR. Such prepared (S)-2-E Thinylpropane-1,2,3-triol-1-phosphate (9) solution (397 mM, 22.5k) The product (g, 98% yield) was used in the subsequent oxidation step without further purification. 1 H NMR (D2O, 500 MHz) δ 3.89 (m, 2H), 3.7 2 (d, J = 11.6 Hz, 1 H), 3.65 (d, J = 11 0.6 Hz, 1H), 2.93 (s, 1H).

[0150] G3 Method: Acetate-toquinase: Enzyme SEQ ID NO: 20 and SEQ ID NO: 2 and deuterated compound ( Using method 3), we assign absolute stereochemistry and demonstrate desymmetric phosphorylation. [ka]

[0151] Advanced pantothenate quinase SEQ ID NO: 20 (10 g / L aqueous solution 100 μL) and evolved 100 μL of quinase acetate SEQ ID NO: 21 (2 g / L aqueous solution) is mixed with diammonium acetate phosphate. (41 mg), 2-ethinylpropane-1,1-d2-1,2,3-triol((R )-3-d2, 20 mg, 170 μmol), magnesium chloride (10 μL of 1 M solution in water) ), ADP (10 μL of 100 g / L solution in water) and sodium phosphate buffer (1 M in water) A 10 μL solution was added to a pH 6.5 solution containing 800 μL of water. This reaction was performed using rt. Incubate for 24 hours to produce deuterated 2-ethynylpropane-1,2,3-triol 1. -The phosphate analogs (S)-9-(3,3-d2) and (S)-9-(1,1-d2) are 9 It was obtained with a 5:5 ratio and a total yield of 99%. 31 The ratio of phosphorylated compounds was determined by P NMR. It was determined to be approximately 95:5, and the pro-(S)hydroxyl group (i.e., desymmetric phosphorylation) is 2 Stereoselective phosphorylation of -ethinylpropane-1,2,3-triol(3) was confirmed. 1 H NMR (D2O, 500MHz) δ3.89(m, 2H), 3.72(d, J = 11.6Hz, 1H), 3.65(d, J = 11.6Hz, 1H), 2.9 3(s,1H). 13 ¹³C NMR (D₂O, 126 MHz) δ 82.9 (s), 75.1 (s), 71.0 (d, J = 6.9 Hz), 67. 0 (d, J = 4.5 Hz), 64.7 (s).

[0152] G4 method: Acetate-tokinase: Using immobilized enzymes SEQ ID NO: 20 and SEQ ID NO: 21 TP regeneration system. [ka] Enzyme immobilization procedure: Nuvia IMAC Ni charged resin (75 mL based on sedimentation capacity) into a filter-funnel In addition, water (9 column volumes, 3 × 225 mL) and binding buffer (1 column volume, 75 mL) are used. 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH Washed in 8.0). Pantothenate quinase (SEQ ID NO: 20, 6.0g) frozen in a container. The dried powder was resuspended in binding buffer (200 mL), and the washed resin was added. At 25°C for 6 The mixture was mixed using a rotational mixer. This resin was filtered and the binding buffer (6 column volume, 6 x 225 mL) and BIS-TRIS buffer (8 column volume, 600 mL; 50 Washed with a solution of mM (pH 6.2). Reaction procedure: 2-Ethinylpropane-1,2,3-triol(3)(574g, 8.7% by weight, 0. A 430 mol solution of (430 mol) and 350 mL of water is placed in a jacketed reactor, followed by a 1 M solution. BIS-TRIS methane buffer pH 6.5 (50 mL) and magnesium chloride (2.0 33g, 0.01mol) was added. ATP (2.37g, 0.0043mol, 0.0 (1 equivalent) and diamonium phosphate (101g, 89%, 0.530 mmol, 1.2 equivalents) The solution was then added, heated to 20°C, and the pH of the liquid was readjusted to 6.8 using 5M KOH. Quinase quinatethenate SEQ ID NO: 20 and evolved quinase acetate SEQ ID NO: 21 (0.15g) A resin (25 mL) containing the immobilized ) was loaded as a solid. The reaction was stirred at 20°C for 16 hours. During that time, the pH decreased to 5.5. 2-Ethinylpropane-1,2,3-triol(3 Quantitative determination of (S)-2-ethynylpropane-1,2,3-triol-1-phosphate (9) Target transformation 1 H and 31 P NMR (D2O, 500MHz) δ3.89(m, 2H ), 3.72(d, J = 11.6Hz, 1H), 3.65(d, J = 11.6H The result was obtained by determining z (1H) and 2.93 (s (1H)).

[0153] Alternative preparation methods for (R)-2-ethynylglyceraldehyde 3-phosphate (5): H1 Method: Immobilized galactosoxidase SEQ ID NO: 16 [ka] Enzyme immobilization procedure: Nuvia IMAC Ni charged resin (10 mL based on sedimentation capacity) into a filter-funnel. In addition, binding buffer (10 column volume, 100 mL; 500 mM sodium chloride) Wash with 50 mM sodium phosphate, 15 mM imidazole (pH 8.0), and resin The preservative solution was removed and 16g of washed resin was obtained. Galactosoxide was evolved in the container. -Ze (SEQ ID NO: 16, 750 mg) freeze-dried powder in copper(II) sulfate solution (100 μM; Resuspend in 5.00 mL, add binding buffer (20 mL) and washed resin (3.0 g) It was added. Mixed using a rotary mixer at 20°C for 5 hours. This resin was used to loosen the binding. Combolus (10 column volume, 100 mL) and BIS-TRIS buffer (10 column volume, Wash by filtration with 100 mL of 50 mM, pH 7.5 solution, and use directly for the glycosylation reaction. Ta.

[0154] Reaction procedure: Immobilized galactosoxidase SEQ ID NO: 16 (3.0g) resin is used in BIS-TRIS (S)-2-ethynylpropane-1,2 in Tan buffer (adjusted to 35 mM, pH 7.2) Add to a solution of 3-triol-1-phosphate (9, 5.4 mmol, 270 mM, 20 mL) Next, a solution of copper(II) sulfate in water (30 μL, 100 mM) and water (600 μL) Horseradish peroxidase (PEO-301, 18 mg) resuspended inside and Ushikata Larze (C1345, 120 mg) was added. This reaction mixture was sealed with a gas-permeable membrane and stored at 22°C. After vigorously shaking for 4 days, a final conversion of 77% is reached, and (R)-2-ethynylglycerin Dehyde 3-phosphate (5) was obtained at 95% ee. The enzyme resin was filtered, and (R)-2-E A solution of thynylglyceraldehyde 3-phosphate (5) was used directly in the glycosylation reaction. H NMR (D2O, 400 MHz): δ 5.02 (s, 1H), 4. 00 (dq, 2H), 2.88 (s, 1H).LC-MS: (ES, m / z): C5H7O6P (MH): Measured value 193.1; Detected value 193.0.

[0155] H2 method: Immobilized galactosoxidase SEQ ID NO: 17 [ka] Enzyme immobilization procedure: Nuvia IMAC Ni charged resin (10 mL based on sedimentation capacity) into a filter-funnel. In addition, binding buffer (10 column volume, 100 mL; 500 mM sodium chloride) Wash with 50 mM sodium phosphate, 15 mM imidazole (pH 8.0), and resin The preservative solution was removed and 16 g of washed resin was obtained. In the container, evolved galactosoxide was observed. -Ze (SEQ ID NO: 16, 750 mg) freeze-dried powder in copper(II) sulfate solution (100 μM; Resuspend in 5.00 mL, then add binding buffer (20 mL) and washed resin (3.0 g). The mixture was then mixed using a rotary mixer at 20°C for 5 hours. This resin was then treated with binding buffer. (10 column volume, 100 mL) and BIS-TRIS methane buffer (10 column volume) The solution was filtered and washed with 100 mL of 50 mM, pH 7.5 solution and used directly in the reaction. Reaction procedure: Immobilized galactosoxidase SEQ ID NO.:17 (3.0g) is used in B (S)-2-ethynyl in IS-TRIS methane buffer (adjusted to 35 mM, pH 7.2) Propane-1,2,3-triol-1-phosphate (9,5.4 mmol, 270 mM, 20 Add the solution (mL), followed by the copper(II) sulfate solution in water (30 μL, 100 mM), and Horseradish peroxidase (PEO-301, 18m) resuspended in water (600 μL) g) and bovine catarase (C1345, 120 mg) were added. This reaction was carried out in a gas-permeable membrane. Sealed, vigorously shaken at 22°C for 4 days until 77% final conversion is reached, (R)-2-ethynyl Glyceraldehyde 3-phosphate (5) was obtained at 95% ee. The enzyme resin was filtered off, ( A solution of R)-2-ethynylglyceraldehyde 3-phosphate (5) was subjected to a glycosylation reaction. It was used directly in response. ¹H NMR (D2O, 400 MHz): δ 5.02 ( s, 1H), 4.00 (dq, 2H), 2.88 (s, 1H).LC- MS: (ES, m / z): r C5H7O6P (MH): Calculated value 193.1 Detected value: 193.0.

[0156] H3 method: Immobilized galactosoxidase SEQ ID NO: 18 [ka] Enzyme immobilization procedure: Add Nuvia IMAC Ni charged resin (3 mL based on sedimentation capacity) to the filter-funnel. Binding buffer (10 column volume, 30 mL; 500 mM sodium chloride) Wash with 50 mM sodium phosphate, 15 mM imidazole (pH 8.0), and store in a resin container. The liquid was removed and 2.4 g of washed resin was obtained. Vial evolution galactosoxidase ( Column number: 18, 75 mg) freeze-dried powder was added to copper(II) sulfate solution (100 μM; 1.00 m The solution was resuspended in (L), and binding buffer (5 mL) and washing resin (400 mg) were added. At 20°C The mixture was mixed using a rotary mixer for 5 hours. This resin was then combined with a binding buffer (10 column volume, 4 m³). L) and BIS-TRIS methane buffer (10 column volume, 4 mL; 50 mM, pH 7.5) The solution was filtered and washed, and then used directly in the reaction. Reaction procedure: The immobilized, evolved GOase SEQ ID NO.:18 (400mg) is used in BIS- (S)-2-ethynyl-pr in TRIS methane buffer (adjusted to 35 mM, pH 7.2) opane-1,2,3-triol 1-phosphate solution ((9), 5.4 mmol, 2 After adding it to 70 mM (1 mL), the horseradish peroxide was resuspended in water (100 μL). Kishidase (PEO-301, 1 mg) and Corynebacterium glu Tamicum-derived catalase (Roche, freeze-drying agent, #11650645103, 3 (mg) was added. This reaction was sealed with a gas-permeable membrane and vigorously shaken at 30°C for 48 hours. After two days, the final conversion reached 90%, resulting in (R)-2-ethynylglyceraldehyde 3- The ethanol (5) was >99% ee. The enzyme resin was filtered off, and (R)-2-ethynylglycerin was found. A solution of ceraldehyde 3-phosphate (5) was used directly without further purification. 1H NMR (D2O, 400 MHz): δ 5.02 (s, 1H), 4 .00 (dq, 2H), 2.88 (s, 1H).LC-MS: (ES, m / z): r C5H7O6P (MH): Calculated value 193.1; Detected value 193. 0.

[0157] H4 method: Immobilized galactosoxidase SEQ ID NO: 19 [ka] Enzyme immobilization procedure: Add Nuvia IMAC Ni charged resin (3 mL based on sedimentation capacity) to the filter-funnel. Binding buffer (10 column volume, 30 mL; 500 mM sodium chloride) Wash with 50 mM sodium phosphate, 15 mM imidazole (pH 8.0), and store in a resin container. The liquid was removed and 2.4 g of washed resin was obtained. The evolved galactosoxidase was placed in the container. Sequence ID: 19, 75 mg) Lyophilized powder in copper(II) sulfate solution (100 μM; 1.00 The solution was resuspended in (mL), and binding buffer (5mL) and washing resin (400mg) were added. 20°C The mixture was then mixed using a rotary mixer for 5 hours. This resin was then combined with a binding buffer (10 column volume, 4 (mL) and BIS-TRIS methane buffer (10 column volume, 4 mL; 50 mM, p The water was filtered and washed with H7.5) and used directly in the reaction. Reaction procedure: Immobilized GOase SEQ ID NO: 18 was added to BIS-TRIS methane buffer (35 mM, pH 7). (S)-2-ethynyl-propane-1,2,3-triol 1- (adjusted to 2) After adding (400 mg) to a phosphate solution (9, 5.4 mmol, 270 mM, 1 mL) Horseradish peroxidase (PEO-301, 1 mg) resuspended in water (100 μL). ) and catalase derived from Corynebacterium glutamicum ( Add 3 mg of freeze-drying agent (#11650645103) to this reaction solution. The sample was sealed with a permeable membrane and vigorously shaken at 30°C for 48 hours. The final conversion after 2 days was 100%. Reaching this point, (R)-2-ethynylglyceraldehyde 3-phosphate (5) has >99% ee The enzyme resin was filtered off, and (R)-2-ethynylglyceraldehyde 3-phosphine was obtained. The solution of phosphate (5) was used directly without further purification. ¹H NMR (D2O, 400MHz): δ 5.02 (s, 1H), 4.00 (dq, 2H ), 2.88 (s, 1H).LC-MS: (ES, m / z): C5H7O6 P (MH): Calculated value 193.1; Detected value 193.0.

[0158] "Amino acids" are defined in IUPAC-IUB B Biochemical Nomencla. This book is made possible by one of the single-letter symbols recommended by the Nature Commission. As mentioned in the specification. Used for the purposes described herein and in the manner described herein. The codes used for the genetically coded amino acids for the enzymes are shown in Table 2. It is idiomatic: [Table 3]

[0159] The process for synthesizing EFdA as described herein and the experimental procedure described herein The sequence numbers of the enzymes used or that may be used in the exemplified steps in order are: The items provided are not limited to those listed in Table 3, but are available. [Table 4] TIFF0007860034000043.tif214162TIFF0007860034000044.tif207163TIFF0007860034000045.tif165162TIFF00078600340 00046.tif220162TIFF0007860034000047.tif228161TIFF0007860034000048.tif227162TIFF0007860034000049.tif220162

[0160] Horseradish peroxidase: Derived from horseradish root (Amoracia rusticana) Isolated wild-type peroxida from commercially available wasabi type I, derived from SIGMA (P8125). -Ze.

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

[0162] Additional embodiments of the present invention, without limitation, include 4'-ethynyl 2'-deoxy Nucleosides or their analogues, such as EFdA, are described herein. This includes the use of the following enzymes in the synthesis process steps.

[0163] A. Purine nucleoside phosphorylase. 1A. Sequence ID: 9 or Sequence ID: 15 and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Manipulated polypeptide sequences having 98%, 99%, or more sequence identity A purine nucleoside phosphorylase, wherein the manipulated purine nucleoside phosphorylase The polypeptide sequence of lylase is less than that of SEQ ID NO: 9 or SEQ ID NO: 15. Manipulated purine nucleosycetes containing either one amino acid substitution or a set of amino acid substitutions. Dophosphorylase. 2A. The manipulated purine nucleoside phosphorylase is sequence number 9 or sequence number Number 15 and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% Polypeptides that are identical at 93%, 94%, 95%, 96%, 97%, 98%, and 99% or more. A modified purine nucleoside phosphorylase described in 1A, containing the D sequence. 3A. Manipulated purine nucleoside phosphorylase, wherein sequence number 9 or sequence number Contains the polypeptide sequence described in item 15. A4. At least one modified E. coli purine nucleoside phosphorylase compared to wild-type E. coli purine nucleoside phosphorylase One of the manipulated purine nucleoside phosphorylases from 1A to 3A, including those with good properties. Ze. 5A. Improved properties compared to wild-type E. coli purine nucleoside phosphorylase. In comparison, substrate compound 6.5 (its cyclic or open-chain aldehyde or hydrate, or pre The modified prune nuclei described in 4A, containing improved activity against any of the salts listed below. Oside phosphorylase. 6A. Improved properties compared to wild-type E. coli purine nucleoside phosphorylase. In comparison, the modified pre-production described in 4A, including the improved production of EFdA (compound 7), Nucleoside phosphorylase. 7A. The manipulated purine nucleoside phosphorylase is purified in A1-6A. A modified purine nucleoside phosphorylase as described in any one of the items. 8A. At least one amino acid substitution (i.e., one or more amino acid substitutions) is conservative. Amino acid substitution, one of the operations 1A to 7A, purine nucleoside phosphorylation Ze.

[0164] B. Phosphopentumutase. 1B. Sequence ID 8 and at least 85%, 86%, 87%, 88%, 89%, 90%, 9 Sequences with 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or higher are the same. Manipulated phosphopentomes comprising a monogenetic polypeptide sequence or a functional fragment thereof A tase, wherein the polypeptide sequence of the manipulated phosphopentumtase is the sequence number Compared to 8, the manipulated form includes at least one amino acid substitution or set of amino acid substitutions. Phosphopentummutase. 2B. Genetically modified phosphopentumtase is at least 85% of SEQ ID NO: 8. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 1B containing 96%, 97%, 98%, 99%, or more identical polypeptide sequences. The manipulated phosphopentumutase described. 3B. Manipulated phosphopentumta consisting of the polypeptide sequence described in Sequence ID No. 8. -Ze. 4B. At least one improved characteristic compared to wild-type E. coli phosphopentummutase A modified phosphopentumutase containing one of the 1B-3B types. 5B. Improved properties compared to wild-type E. coli phosphopentumutase, Compound 6 (its cyclic or open-chain aldehyde or hydrate, or any of the above salts) The modified phosphopentumutase described in 4B, comprising improved activity against ). 6B. Improved properties compared to wild-type E. coli phosphopentumutase, The modified method described in 4B, including the improved production of compound 6.5 or compound 7 (EFdA) Phosphopentummutase. 7B. The manipulated phosphopentumutase is purified in one of the operations 1B-6B. The phosphopentumutase produced. 8B. At least one amino acid substitution (i.e., one or more amino acid substitutions) is conservative. A modified phosphopentumutase with one amino acid substitution, from 1B to 7B.

[0165] C. Deoxyribose-phosphate aldola-ase. 1C. Shewanella halifaxensis polypeptide as described in Sequence ID: 5 Deoxyribo-phosphate aldora-ase derived from the wild type of the ptido sequence. 2C. Modified deoxyribose-phosphate aldolase, wherein SEQ ID NO: 6 or Contains the polypeptide sequence described in Sequence ID No. 14. 3C. The manipulated deoxyribose-phosphate aldolaase is sequence number 5, sequence number Number 6 or Sequence ID 14 and at least 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more are the same. A modified deoxyribo-phosphate aldola-ase containing the polypeptide sequence. 4C. At least 85% for SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 14 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% polypeptide sequences having 96%, 97%, 98%, 99% or more sequence identity, or For SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 14, at least one amino acid A modified deoxygenated polypeptide sequence containing a substitution or amino acid substitution set. Ribose phosphate aldola-ase, or its functional fragment 5C. Substrate compound 5((R)-2-ethynylglyceraldehyde 3-phosphate, its hydration A deoxyriboside of any of the 1C to 4C compounds that is active in the substance or any of the salts mentioned above. Aldra-ase phosphate. 6C. During the reaction, the substrate compound 5(((R)-2-ethynylglyceraldehyde 3-phosphate Compound 6 (4-ethyl) does not require a protecting group on its hydrate or any of the aforementioned salts. Nyl D-2-deoxyribose 5-phosphate, or its open-chain aldehyde or hydrate form. A deoxygenator with the ability to produce any one of the above salts (1C to 5C) Siribose-phosphate aldola-ase. 7C. Deoxyribo-phosphate aldora-ase is found in wild-type Shewanella ha Compared to lifaxensis deoxyribo-phosphate aldolase, compound 6(4 -Ethinyl D-2-deoxyribose 5-phosphate, or its open-chain aldehyde or water Having improved properties, including improved production of the hydrate form or any of the aforementioned salts. , one of the 2C to 6C modified deoxyribose-phosphate aldola-ase molecules. 8C. Deoxyribose-phosphate aldorase is purified in one of the 1C to 7C cells. Two deoxyribose-phosphate aldola-ase. 9C. At least one amino acid substitution (i.e., one or more amino acid substitutions) is conserved. Modified deoxyribose-phosphate is an amino acid substitution, involving one of the 2C to 7C amino acids. Aldra-ze.

[0166] D. Pantothenate quinase. 1D. At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% SEQ indicates sequence identity of 93%, 94%, 95%, 96%, 97%, 98%, and 99% or higher. ID NO: 2, SEQ ID NO.: 12, SEQ ID NO.: 13 or SEQ ID NO.:20, or the polypeptide contained in their functional fragments The sequence includes, where the polypeptide sequence of the engineering pantothenate quinase is SEQ ID NO: 2, SEQ ID NO.: 12, SEQ ID NO.: 13 or S Compared to EQ ID NO.:20, at least one amino acid substitution or amino acid placement Engineering pantothenate quinase, including replacement sets. 2D. The manipulated pantothenate quinase is used in SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 13 or Array No. 20 and at least 85%, 86%, 87%, 88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more are the same. The manipulated pantothenate quinase described in 1D, comprising the polypeptide sequence. 3D. Manipulated pantothenate quinase, with SEQ ID NO: 2, SEQ ID NO: 12, Contains the polypeptide sequence described in column number 13 or sequence number 20. 4D. Wild-type E. coli pantothenate quinase has at least one improved characteristic. One of the 1D to 3D manipulated pantothenate quinases, including sex. 5D. Improved properties compared to wild-type e, substrate compound 4((R)-2-ethynylg The operation described in 4D, including improved activity against cellulose or its hydrate form. Pantothenate quinase produced. Pantothenate quinase from E. coli. 6D. The improved properties of compound 5((R)) compared to wild-type pantothenate quinase The procedure described in 5D, including the improved production of -2-ethynylglyceraldehyde 3-phosphate. The pantothenate quinase produced. 7D. Improved properties compared to wild-type e, substrate compound 3(2-ethynylpropane- The modified panto(s) described in 4D, including improved activity against 1,2,3-triol. Thenic acid quinase. Pantothenic acid quinase from E. coli. 8D. The improved properties of compound 9((S)) compared to wild-type pantothenate quinase Includes improved production of -2-ethinylpropane-1,2,3-triol-1-phosphate. , the manipulated pantothenate quinase described in 7D. 9D. Pantothenate quinase is purified in one of the manipulated processes from 1D to 8D. Calcium quinase. 10D. At least one amino acid substitution (i.e., one or more amino acid substitutions) is conserved. A modified pantothenate quinase with one of the amino acid substitutions 1D to 9D.

[0167] E. galactosoxidase. 1E. At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% SEQ indicates sequence identity of 93%, 94%, 95%, 96%, 97%, 98%, and 99% or higher. Polypeptides with ID NOs.: 1, 10, 11, 16, 17, 18, or 19 An engineered galactosoxidase containing the sequence or functional fragments thereof The polypeptide sequence of the aforementioned engineered galactosoxidase is SEQ ID NOs .: Compared to 1, 10, 11, 16, 17, 18 or 19, at least one amino acid Engineered galactosoxidases, including acid substitution or amino acid substitution sets. 2E. Genetically modified galactosoxidases, sequence ID NO: 1, 10, 1 1, 16, 17, 18 or 19 and at least 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% The manipulated galactosoxidil described in 1E comprises a polypeptide sequence that is or greater than or equal to a polypeptide sequence. Sidaze. 3E. Manipulated galactosoxidases, such as SEQ ID NOs: 1, 10, 11, 16 , containing the polypeptide sequence shown in 17, 18, or 19. 4E. Wild type F. graminearum galactosoxidase is less compared to 4E. One of the 1E to 3E manipulated galactosoxidil containing at least one improved characteristic Sidaze. 5E. Improved characteristics of the wild type F. graminearum galactosoxid - Compared to -ze, it includes improved activity toward primary alcohol substrates, as described in 4E. Manipulated galactosoxidase. 6E. Improved characteristics of the wild type F. graminearum galactosoxid -Compared to -ze, for substrate compound 3 (2-ethynylpropane-1,2,3-triol) 4E engineered galactosoxidase, including improved activity. 7E. Improved characteristics of the wild type F. graminearum galactosoxid -Compared to -ze, compound 4((R)-2-ethynylglyceraldehyde or its hydrate 6E engineered galactosoxidase, including improved production of the morphology. 8E. Improved characteristics of the wild type F. graminearum galactosoxid -Compared to -ze, the substrate compound 9(((S)-2-ethynylpropane-1,2,3-trio 4E engineered galactosoxy (including improved activity against -1-phosphate) Da-ze. 9E. Improved characteristics of the wild type F. graminearum galactosoxid -Compared to -ze, compound 5((R)-2-ethynylglyceraldehyde 3-phosphate or The modified production of the hydrate form of the manipulated galactosoxidant described in 8E, including improved production of the hydrate form. -Ze. 10E. One of the operations from 1E to 9E in which the galactosoxidase is purified. Galactosoxidase produced. 11E. At least one amino acid substitution (i.e., one or more amino acid substitutions) is conserved. The target amino acid substitution is one of the 1E to 10E modified galactosoxid -Ze.

[0168] F. Acetate quinase. 1F. The Thermomotoga marit described in Sequence ID: 3 or Sequence ID: 21 Acetate quinase derived from the wild-type ima polypeptide sequence. 2F. Manipulated acetate-tokinase, wherein the manipulated acetate-tokinase is , with at least 85%, 86%, 87%, 88%, 89% of sequence number 3 or sequence number 21 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Manipulated acetate-tokinase containing the same polypeptide sequence as described above. 3F. Sequence ID: 3 or Sequence ID: 21 and at least 85%, 86%, 87%, 8 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 Manipulated polypeptide sequences containing 8%, 99%, or more sequence identity Acetate quinase, the polypeptide sequence of the acetate quinase manipulated here is sequence number 3, also This is at least one amino acid substitution or amino acid substitution set compared to Sequence ID: 21. A modified acetate quinase containing. 4F. Compared to wild-type T. maritima acetate quinase, at least one improved characteristic Acetate quinase of 2F or 3F containing the properties of hydroxylase. 5F. Improved properties of the wild-type Thermotoga maritima acet Compared to ate kinase, the substrate compound 4((R)-2-ethynylglyceraldehyde or for ATP-cofactor recycling in phosphorylation reactions on its hydrate form Acetate quinase as described in 4F, including improved activity. 6F. Improved properties of the wild-type Thermotoga maritima acet Compared to ate quinase, compound 5((R)-2-ethynylglyceraldehyde 3-ly 5F The acetate quinase described. 7F. The improved characteristics described above are found in the wild-type Thermotoga maritima ac Compared to etate kinase, substrate compound 3(2-ethynylpropane-1,2,3-) Improved ATP-cofactor recycling in phosphorylation reactions on (Li-L) Acetate quinase, as described in 4F, containing activity. 8F. The improved characteristics described above are found in the wild-type Thermotoga maritima ac Compared to etate kinase, compound 9(((S)-2ethynyl-propane-1 This includes improved production of 2,3-triol 1-phosphate or a salt of either of these. Acetate quinase as described on page 7F. 9F. The acetate quinase is purified according to any one of the items 1F to 8F. Ze. 10F. At least one amino acid substitution (i.e., one or more amino acid substitutions) is conserved. A modified acetate quinase with one of the 2F-7F amino acid substitutions.

Claims

1. The compound shown in the following formula. 【Chemistry 1】

2. The compound shown in the following formula. 【Chemistry 2】 [In the formula, 2X ＋ is (a) two protons, (b) one proton and one monovalent cation, (c) two identical or different monovalent cations, or (d) one divalent cation.

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