Method for manufacturing deoxynucleoside

Abstract

The present invention relates to a method for producing deoxyribonucleoside derivatives, wherein (1) Step of phosphorylating D-GA to D-GAP using the ScDAK1 enzyme (2) A step of converting D-GAP into D-dRib 5P by reacting it with AcH using an EcDeoC enzyme and (3) Provides a method including the step of converting D-dRib 5P to D-dRib 1P with an EcDeoB enzyme. The second aspect of the present invention provides a deoxyribonucleoside derivative produced according to the first aspect.

Description

Technology Field

[0001] The present invention relates to a method for producing deoxynucleoside derivatives. Background Technology

[0002] Nucleoside derivatives are characterized by inhibiting DNA and RNA synthesis, thereby interfering with cell metabolism and viral replication, and have clinical importance as anticancer and antiviral agents. In particular, the U.S. Food and Drug Administration (FDA) has approved several unnatural deoxyribonucleoside derivatives, such as idoxyuridine, ploxyuridine, decitabine, and cladribine, as therapeutic agents.

[0003] However, current chemical synthesis methods for these deoxyribonucleoside derivatives are considered unsustainable due to environmental and economic issues arising from multi-step processes, low yields, high energy consumption, the introduction and removal of protecting groups, intermediate separation, and the extensive use of highly toxic organic solvents. Consequently, there is a growing demand for eco-friendly and economical alternative synthesis pathways.

[0004] Accordingly, enzymatic synthesis methods that align with the principles of green chemistry are attracting attention. Enzymes provide high chemoselectivity and stereoselectivity, and simple one-pot synthesis is possible through a sugar transfer reaction utilizing nucleoside phosphorylase (NP). However, this method also has problems such as the use of expensive nucleoside sugar donor substrates, unfavorable equilibrium conditions, and yield limitations due to the generation of byproducts.

[0005] Recent studies have reported the possibility of enzymatic synthesis of nucleoside derivatives using inexpensive starting materials such as bulk chemicals like D-ribose, 2-ethynylglycerol, and D-glyceraldehyde (D-GA), but methods for the enzymatic production of deoxyribonucleoside derivatives using low-cost substrates have not yet been sufficiently explored. Prior art literature

[0006] (Patent Document 0001) US 2014-0135490 A1 (Patent Document 0002) US 2012-0197010 A1 (Patent Document 0003) US 2011-0046363 A1 The problem to be solved

[0007] The problem that this invention aims to solve includes addressing the issues of existing chemical synthesis methods for deoxyribonucleoside derivatives, such as multi-step processes, low yield, high energy consumption, introduction / removal of protecting groups, intermediate separation, and the use of large amounts of highly toxic organic solvents.

[0008] The above tasks are merely examples, and additional tasks may exist that a person skilled in the art can easily derive by referring to the present invention. means of solving the problem

[0009] The first aspect of the present invention relates to a method for producing deoxyribonucleoside derivatives, wherein

[0010] (1) Step of phosphorylating D-GA to D-GAP using the ScDAK1 enzyme

[0011] (2) A step of converting D-GAP into D-dRib 5P by reacting it with AcH using an EcDeoC enzyme and

[0012] (3) Provides a method including the step of converting D-dRib 5P to D-dRib 1P with an EcDeoB enzyme.

[0013] The second aspect of the present invention provides a deoxyribonucleoside derivative produced according to the first aspect.

[0014] The above means are merely examples, and additional means may exist that a person skilled in the art can easily derive by referring to the present invention. Effects of the invention

[0015] The effect according to the present invention includes providing a technology that can efficiently and sustainably synthesize four major therapeutic deoxyribonucleoside derivatives, such as ploxyuridine, idoxyuridine, decitabine, and cladribine, by designing and constructing a one-pot enzyme cascade that utilizes enzymes of the deoxyribonucleoside recovery pathway in reverse reaction, using glycerol and acetaldehyde (AcH), which are economical starting materials produced in large quantities as byproducts in the biodiesel production process.

[0016] Therefore, the present invention has industrial and clinical value as an economical and eco-friendly enzyme-based deoxyribonucleoside derivative production platform that can replace existing expensive and environmentally burdensome chemical synthesis methods.

[0017] The above effects are merely examples, and additional effects that a person skilled in the art can easily derive by referring to the present invention may exist. Brief explanation of the drawing

[0018] FIG. 1a is a schematic diagram of an enzyme cascade that converts glycerol into D-glyceraldehyde-3-phosphate (D-GAP) according to one embodiment or one example of the present invention. FIG. 1b is the result of the conversion of glycerol to D-glyceraldehyde (D-GA) using various amounts of GoGdh under NADP+ regeneration conditions under a fixed LpNoxVG171R / L172R positive according to one embodiment or one embodiment of the present invention. FIG. 1c shows the effect of changes in ATP concentration on D-GAP production using GoGdh, LpNoxVG171R / L172R and ScDAK1 according to one embodiment or one example of the present invention. FIG. 1d is the result of D-GAP production according to various AcP concentrations in an ATP regeneration system using BsAckA according to one embodiment or one example of the present invention. FIG. 2 shows the results of producing D-glyceraldehyde-3-phosphate (D-GAP) from D-glyceraldehyde (D-GA) and DL-glyceraldehyde (DL-GA) in an antithioselective manner using ScDAK1 according to one embodiment or one example of the present invention (all data are expressed as mean ± standard deviation; n = 3 independent samples). FIG. 3a is an enzyme cascade that converts D-glyceraldehyde (D-GA) and acetaldehyde (AcH) into D-deoxyribose 5-phosphate (D-dRib 5P) according to one embodiment or one example of the present invention. FIG. 3b shows the relative yield of D-dRib 5P according to various AcH concentrations according to one embodiment or one example of the present invention (all data are expressed as mean ± standard deviation; n = 3 independent samples). Figure 4 shows the HPLC results for the production of deoxyribonucleoside analogs after a 24-hour reaction using EcDeoB and EcUdp or EcDeoD with D-deoxyribose 5-phosphate (D-dRib 5P) and corresponding nucleobases as starting materials: (a) Schematic diagram of the enzyme cascade that converts D-dRib 5P and nucleobases into deoxyribonucleoside analogs (b) Results of ploxyuridine production from 5-fluorouracil (5-FU) (Retention times of 5-FU standard and ploxyuridine standard were 20.4 min and 14.8 min, respectively) (c) Results of idoxyuridine production from 5-iodouracil (5-IU) (Retention times of 5-IU standard and idoxyuridine standard were 38.8 min and 20.9 min, respectively) (d) Results of decitabine production from 5-azacytosine (5-AzaC) (Retention times of 5-AzaC and decitabine standard were 1.2 min and 3.4 min, respectively) (e) Results of cladribin production from 2-chloroadenine (2-CA) (retention time of cladribin is 14.1 min) Figure 5 is the SDS-PAGE analysis result of purified AoSucP and EcDeoD according to one embodiment or one example of the present invention. FIG. 6a is a result of improving the yield of ploxyuridine according to a change in sucrose concentration in the presence of AoSucP according to one embodiment or one example of the present invention. FIG. 6b shows the effect of increasing glycerol concentration on ploxyuridine production under phosphoric acid capture conditions according to one embodiment or one example of the present invention. FIG. 6c shows the yields of four deoxyribonucleoside analogs (floxyuridine, idoxyuridine, decitabine, cladribine) using a phosphate capture system according to one embodiment or one example of the present invention (all data are expressed as mean ± standard deviation; n = 3 independent samples). FIG. 7 shows the progress of a nucleobase glycosylation reaction according to one embodiment or one example of the present invention (all data are expressed as mean ± standard deviation; n = 3 independent samples). Specific details for implementing the invention

[0019] Embodiments of the present invention are described below with reference to the attached drawings so that those skilled in the art can easily implement them. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the explanation have been omitted, and similar parts throughout the specification are denoted by similar reference numerals.

[0020] Throughout this specification, when a part is described as 'comprising' a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0021] Throughout the entire specification, ‘steps’ or ‘steps of’ do not mean ‘steps for’.

[0022] Throughout this specification, the term 'combination(s) of these' included in the Markush-type expression means one or more mixtures or combinations selected from the group consisting of the components described in the Markush-type expression, and means including one or more selected from the group consisting of said components.

[0023] Throughout this specification, the description of 'A and / or B' means 'A or B, or A and B'.

[0024] Throughout this specification, the term "deoxynucleoside derivative" means a nucleoside or a variant thereof containing deoxyribose, wherein one or more chemical or structural modifications are made to the sugar and / or nucleobase portions.

[0025] Specifically, deoxyribonucleoside derivatives include compounds in which the position or substitution of sugars, the type or substitution of nucleobases, etc., of natural deoxyribonucleoside are modified, and these modifications may be intended to improve biological activity, chemical stability, pharmacokinetics, and efficacy.

[0026] Accordingly, in this specification, the term "deoxyribonucleoside derivative" will be understood as a term encompassing various modified nucleoside compounds based thereon that maintain the basic framework of natural deoxyribonucleoside.

[0027] Throughout the entire specification, "ScDAK1" refers to the dihydroxyacetone kinase 1 enzyme derived from Saccharomyces cerevisiae. This enzyme performs the role of phosphorylating D-glyceraldehyde (D-GA) into D-glyceraldehyde-3-phosphate (D-GAP) using ATP as a cofactor and has the function of enabling efficient D-GAP production through enzymatic phosphorylation.

[0028] Throughout this specification, “D-GA” or “D-glyceraldehyde” refers to the simplest aldose monosaccharide having the chemical formula C3H6O3, containing three carbon atoms, one of which contains an aldehyde group (-CHO). D-GA has one chiral center and exists as two optical isomers, D-type and L-type, and the term refers to the D-type.

[0029] D-GA is important as an intermediate in various metabolic pathways in living organisms, such as glycolysis and aldol condensation, and is also produced enantioselectively from glycerol by glycerol dehydrogenase. In the present invention, "D-GA" refers to D-type glyceraldehyde produced through an enzymatic oxidation reaction from glycerol.

[0030] Throughout this specification, "D-GAP" or "D-glyceraldehyde-3-phosphate" refers to a triose phosphate metabolic intermediate in which a phosphate group is ester-bonded to the 3rd carbon of a glyceraldehyde molecule. The chemical formula is H(O)C-CH(OH)-CH2-OPO3 2- It plays an important role in various biochemical metabolic processes, such as glycolysis, photosynthesis, and the pentose phosphate pathway. In the present invention, it is utilized as a starting material for the synthesis of deoxyribonucleoside as an expensive enantioselective intermediate produced through the enzymatic oxidation and phosphorylation of glycerol.

[0031] Throughout this specification, "EcDeoC" refers to deoxyribose-5-phosphate aldolase derived from Escherichia coli. This enzyme catalyzes the aldol condensation reaction between D-glyceraldehyde-3-phosphate (D-GAP) and acetaldehyde (AcH) to produce D-deoxyribose-5-phosphate (D-dRib 5P). It is used as an important enzyme for the synthesis of D-dRib 5P, a key intermediate in the present invention.

[0032] Throughout this specification, "D-dRib 5P" or "D-deoxyribose 5-phosphate" refers to a sugar phosphate metabolic intermediate having a structure in which a phosphate group is ester-bonded to the 5th carbon of 2-deoxy-D-ribose. The chemical formula is C5H 11 It is O7P, an important biochemical molecule involved in glycolysis and the pentose phosphate pathway. In this invention, it is utilized as an intermediate in the reverse synthetic enzyme cascade reaction and serves as a starting material that binds to nucleobases to form deoxyribonucleoside analogs.

[0033] Throughout this specification, "EcDeoB" refers to phosphopentomutase derived from Escherichia coli. This enzyme catalyzes the isomerization of D-deoxyribose 5-phosphate (D-dRib 5P) to D-deoxyribose 1-phosphate (D-dRib 1P) and is involved in the conversion of a key intermediate in the deoxyribonucleoside retrosynthetic pathway.

[0034] Throughout this specification, "D-dRib 1P" or "D-deoxyribose 1-phosphate" refers to a compound in which a phosphate group is ester-bonded to the 1st carbon of a deoxyribose molecule, and signifies a sugar phosphate intermediate important in the DNA and deoxyribonucleoside biosynthetic pathways. The chemical formula is C5H 11 It is O7P, and this molecule binds to nucleobases and acts as a precursor for nucleosides. It differs from D-dRib 5P in the position where the phosphate group is attached, and D-dRib 5P is isomerized into D-dRib 1P and utilized in nucleoside synthesis.

[0035] Throughout this specification, "LpNoxV" refers to NADH oxidase derived from Lactobacillus plantarum. This enzyme oxidizes NADH to regenerate NAD+, and in this process, it has the function of generating water (H2O) or hydrogen peroxide (H2O2) using molecular oxygen (O2). In particular, the present invention uses a variant (LpNoxVG171R / L172R) in which LpNoxV is modified through site-specific mutation to oxidize NADPH, thereby NADP + By implementing a regeneration system, it is used to promote enzyme reactions along with efficient cofactor regeneration.

[0036] Throughout this specification, "BsAckA" refers to acetate kinase derived from Bacillus subtilis. This enzyme catalyzes the reaction of generating acetyl phosphate (AcP) and ADP from acetate and ATP, and plays an important role in the ATP regeneration system. In the present invention, BsAckA is used as an enzyme that enables an economical and efficient supply of ATP by regenerating ATP from ADP using AcP as a sacrificial substrate.

[0037] Throughout this specification, "AcP (acetyl phosphate) is a high-energy compound in which an acetyl group is linked to a phosphate group and is used as a sacrificial substrate in the ATP regeneration system in enzymatic reactions. In particular, acetate kinase (BsAckA) participates in the reaction of regenerating ATP from ADP using AcP. AcP is synthesized chemically or enzymatically and contributes to increasing the economy and efficiency of enzymatic reactions by efficiently regenerating ATP using high phosphate bond energy.

[0038] Throughout this specification, "EcUdp" refers to a UDP-dependent nucleoside phosphorylase derived from Escherichia coli. This enzyme catalyzes the glycosylation reaction in which a nucleobase and a sugar phosphate bind during nucleoside synthesis, and in particular promotes the binding of a modified nucleobase and D-deoxyribose phosphate to enable the production of deoxyribonucleoside analogs.

[0039] Throughout this specification, "EcDeoD" is a nucleoside phosphorylase enzyme derived from Escherichia coli that catalyzes the N-glycosylation reaction between a modified nucleobase and D-deoxyribose 1-phosphate (D-dRib 1P) to synthesize deoxyribonucleoside analogs. This enzyme recognizes modified nucleosides such as 5-azacytosine (5-AzaC) and 2-chloroadenine (2-CA) as substrates and is used for the synthesis of deoxyribonucleoside containing these nucleosides.

[0040] Throughout the entire specification,

[0041] "5-FU" stands for 5-Fluorouracil, which is a pyrimidine-based modified nucleobase used as an anticancer agent in which fluorine is substituted at the 5th position of the uracil nucleobase.

[0042] "5-IU" stands for 5-iodouracil, a modified nucleus in which iodine is substituted at the 5th position of a uracil nucleus.

[0043] "5-AzaC" stands for 5-Azacytidine, which is a modified nucleus in which the 5th position of the cytosine nucleus is substituted with aza (nitrogen), and is used clinically as an antitumor.

[0044] "2-CA" stands for 2-chloroadenine, which is a modified purine nucleobase in which a chloro group is substituted at the 2nd position of an adenine nucleobase.

[0045] Throughout this specification, "AoSucP" refers to sucrose phosphorylase derived from Alloscardovia omnicolens. This enzyme catalyzes the phosphorylation of sucrose into D-fructose and D-glucose 1-phosphate, in which free phosphorus (HPO4) is produced. 2- It consumes ). Accordingly, it reduces the concentration of free phosphate in the reaction solution, thereby shifting the equilibrium of the nucleoside glycosylation reaction toward product formation and improving the yield.

[0046] The first aspect of the present invention relates to a method for producing deoxyribonucleoside derivatives, wherein

[0047] (1) Step of phosphorylating D-GA to D-GAP using the ScDAK1 enzyme

[0048] (2) A step of converting D-GAP into D-dRib 5P by reacting it with AcH using an EcDeoC enzyme and

[0049] (3) Provides a method including the step of converting D-dRib 5P to D-dRib 1P with an EcDeoB enzyme.

[0050] The second aspect of the present invention provides a deoxyribonucleoside derivative produced according to the first aspect.

[0051] In one embodiment, the method may further include the step of (a) oxidizing glycerol to D-GA using a GoGdh enzyme.

[0052] In one embodiment, step (a) may additionally use an LpNoxV mutation.

[0053] In one embodiment, the LpNoxV mutation may be LpNoxVG171R or LpNoxVG172R.

[0054] In one embodiment, the LpNoxV mutation is NADP + It could be playing.

[0055] In one embodiment, the above step (1) may additionally use BsAckA.

[0056] In one embodiment, the BsAckA may regenerate ATP from ADP using AcP.

[0057] In one embodiment, the above step (3) may additionally use EcUdp or EcDeoD.

[0058] In one embodiment, the above step (3) may be to convert 5-FU to ploxyuridine or convert 5-IU to idoxyuridine by additionally using EcUdp.

[0059] In one embodiment, the above step (3) may be to convert 5-AzC to decitabine or 2-CA to cladribin by additionally using EcDeoD.

[0060] In one embodiment, step (3) may additionally use AoSucP and sucrose.

[0061] In one embodiment, the above step (3) further uses AoSucP and sucrose to form HPO4 2- It could be removing.

[0062] The second aspect of the present invention provides a deoxynucleoside derivative produced by the method according to the first aspect.

[0063] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the attached drawings. However, the present invention may not be limited to these embodiments and examples and drawings.

[0064] [Experiment Preparation]

[0065] 1. Reagents and Materials

[0066] The chemical standard substances used in the present invention are glycerol, various sugars, nucleobases, deoxyribonucleoside, ATP, and NAD(P). + It includes NAD(P)H cofactor, D-Glucose-1,6-bisphosphate (G1,6-BP), etc., and was purchased from Sigma-Aldrich (St. Louis, KS, USA). The restriction enzyme and DNA polymerase (Q5) used for the production and analysis of the recombinant enzyme were purchased from New England Biolabs (Beverly, MA, USA). The synthesized DNA primers and DNA sequencing services were provided by Cosmo GeneTech (Daejeon, Korea).

[0067] 2. Construction of Plasmids for Recombinant Enzyme Expression

[0068] The gene encoding the target enzyme was amplified via PCR using genomic DNA as a template. The amplified DNA fragments were cleaved with specific restriction enzymes and then ligated into pET21α(+) (Novagen, Darmstadt, Germany) or pET28α(+) (Novagen, Darmstadt, Germany) vectors.

[0069] 3. Overexpression and Purification of Recombinant Enzyme

[0070] To obtain purified recombinant enzymes, each plasmid containing a specific gene was transformed into chemically inducible E. coli BL21 (DE3) cells (RBC Bioscience, Taipei, Taiwan). Plasmids encoding GoGdh, ScDAK1, and EcUdp were transformed into chemically inducible E. coli Rosetta (DE3) cells purchased from Enzynomics (Daejeon, Korea).

[0071] For the expression of GoGdh, EcDeoC, EcUdp, EcDeoD, and AoSucP, recombinant E. coli strains were cultured in LB medium containing 50 μg / mL kanamycin at 37°C and 200 rpm until the optical density of the culture measured at 600 nm reached 0.4-0.6. For the expression of LpNoxVG171R / L172R, ScDAK1, BsAckA, and EcDeoB, recombinant E. coli strains were cultured in LB medium containing 100 μg / mL ampicillin under the same conditions (37°C and 200 rpm) until the OD600 value reached 0.4-0.6. Overexpression of the recombinant enzyme was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and cultured for 18 hours at 16°C and 200 rpm. Subsequently, the cells were harvested by centrifugation at 9000 rpm (17664 * g) for 20 minutes at 4°C.

[0072] To purify the recombinant enzyme, the harvested cell pellet was resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0). The resuspended cells were pulverized using a sonicator (Branson 450, Marchall Scientific, Hampton, NH) and centrifuged at 13,000 rpm (17,190 * g) at 4°C for 40 minutes. The supernatant (crude protein) of the obtained cell lysate was transferred to a HisTrap column (GE Healthcare, Chicago, IL). Subsequently, the cells were washed with 6-column volumes of wash buffer (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4), and additionally washed with the same wash buffer to remove untagged proteins. The target recombinant protein was eluted using elution buffer (20 mM sodium phosphate, 500 mM NaCl, 200 mM imidazole, pH 7.4). The purity of the eluted enzymes was confirmed by comparing them with protein markers and theoretical molecular weights via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For all enzymes except GoGdh, the enzymes were concentrated using an Amicon ultracentrifuge (MW cutoff 10,000; Millipore, Burlington, MA) by exchanging the solution with a storage buffer (20 mM Tris-HCl, 20 mM NaCl, pH 7.4). The purified proteins were stored at -80°C. The concentration of the purified enzymes was measured by Bradford protein assay at 595 nm using bovine serum albumin (Sigma-Aldrich) as the protein standard.

[0073] To obtain freeze-dried GoGdh powder with cell-derived components removed, the GoGdh fraction eluted from the HisTrap column was desalted using a HisTrap desalting column (GE Healthcare) connected to an AKTA Pure chromatography system (GE Healthcare). The desalted GoGdh fraction was placed in freeze-drying bottles and frozen at -80°C until completely frozen. Subsequently, the GoGdh fraction was freeze-dried for 24 hours at a vacuum of 27 mTorr and a condenser temperature of -80°C using a freeze dryer from IlShinBioBase Co. Ltd. (Yangju-si, Korea).

[0074] 4. Production of D-GAP from Glycerol using GoGdh and ScDAK1 and a Cofactor Regeneration System

[0075] NADP + To determine the optimal GoGdh concentration for converting glycerol to glyceraldehyde (D-GA) with a regeneration system, the reaction was carried out by varying the lyophilized GoGdh concentration from 1.25 g / L to 10 g / L and adding 2 g / L of LpNoxVG171R / L172R. Unless otherwise specified herein, the reaction solution was reacted at 25°C. The reaction mixture contained 50 mM triethylamine (TEA) buffer (pH 7.0), 5 mM glycerol as a substrate, and 10 mM MgCl2 and 1 mM NADPH as cofactors.

[0076] The effect of ATP concentration on the conversion of glycerol to D-GAP was investigated when 10 g / L of freeze-dried GoGdh was used in combination with GoGdh, LpNoxVG171R / L172R, and ScDAK1. The reaction was carried out in the presence of 1 g / L of LpNoxVG171R / L172R and 2 g / L of ScDAK1 while varying the ATP concentration from 1.25 mM to 5 mM.

[0077] In addition, to determine the optimal acetyl phosphate (AcP) concentration for ATP regeneration during the conversion of glycerol to D-GAP, reactions were carried out under conditions of 0.025 mM ATP and various AcP concentrations ranging from 3.125 mM to 25 mM, including 10 g / L GoGdh, 1 g / L LpNoxVG171R / L172R, 2 g / L ScDAK1, and 1 g / L BsAckA.

[0078] NADP + To investigate the time course of D-GAP conversion from glycerol using an ATP regeneration system, the reaction was carried out at 25°C with 10 g / L GoGdh, 1 g / L LpNoxVG171R / L172R, 2 g / L ScDAK1, and 1 g / L BsAckA under optimal AcP concentration (15 mM) conditions. The reaction buffer contained 50 mM TEA buffer (pH 7.0), 5 mM glycerol as a substrate, and 10 mM MgCl2, 1 mM NADPH, and 0.025 mM ATP as cofactors.

[0079] For analysis, 20 mL of the reaction sample was collected after 24 hours, and the reaction was terminated by adding a 6 M H2SO4 solution at a 10:1 ratio to stop enzyme activity. The glycerol conversion rate was calculated by comparing the decrease in glycerol concentration in the reaction sample with the concentration of the glycerol control group. Quantitative analysis of the converted glycerol was performed using HPLC with an Aminex HPX-87H column.

[0080] 5. Production of deoxyribonucleoside derivatives via the retrosynthetic enzyme cascade

[0081] To investigate the optimal AcH concentration for the conversion of D-dRib 5P from D-GA and AcH, reactions were carried out at 25°C with 2 g / L ScDAK1, 1 g / L BsAckA, and 0.03 g / L EcDeoC while varying the AcH concentration from 3.125 mM to 50 mM. The reaction solution contained 50 mM TEA buffer (pH 7.0), 5 mM D-GA and 15 mM AcP as substrates, and 10 mM MgCl2, 1 mM MnCl2, 1 mM NADPH, and 0.025 mM ATP as cofactors. The method for the quantitative analysis of D-dRib 5P is described in the experimental methods section of the supplementary information.

[0082] For the enzymatic glycosylation of modified nucleobases and D-dRib 5P, each reaction was performed at 25°C using 5 mM of the target modified nucleobases, 1 g / L EcDeoB, and 1 g / L EcUdp or EcDeoD. The reaction solution contained 50 mM TEA buffer (pH 7.0), 5 mM D-dRib 5P as a substrate, and 1 mM MnCl2 and 0.01 mM G1,6-BP as cofactors. A sample (20 mL) was collected after 24 hours, and enzymatic activity was immediately stopped by adding 6 M H2SO4 solution in a 10:1 ratio. Quantitative analysis of the conversion products was performed by HPLC using an Aminex HPX-87H column.

[0083] 6. Effect of Sucrose Phosphate Reaction on Floxyuridine Phosphate Reaction Equilibrium

[0084] To investigate the equilibrium shift effects induced by AoSucP and sucrose, reactions were carried out at 25°C with 5 mM D-dRib 5P and 5 mM 5-fluorouracil (5-FU) as substrates, along with 0.15 g / L EcDeoB, 1 g / L EcUdp, and 0.5 g / L AoSucP. The reaction solution contained 50 mM TEA buffer (pH 7.0), 10 mM MgCl2, 1 mM MnCl2, 1 mM NADPH, and 0.5 mM ATP. The reaction was performed at 25°C for 1 hour while varying the sucrose concentration from 6.25 mM to 200 mM.

[0085] 7. Effect of Increased Glycerol and AcP Input on Floxyuridine Production from Glycerol and AcH in the Retrosynthetic Enzyme Cascade

[0086] To investigate the effect of increasing the input amounts of glycerol and AcP on ploxyuridine production, the reaction was carried out by varying the glycerol concentration to 6, 12, and 30 mM while maintaining the molar ratio of AcP constant. The reaction mixture used 25 mM AcH, 5 mM 5-fluorouracil (5-FU), and 200 mM sucrose as substrates, and was reacted at 25°C with 10 g / L GoGdh, 2 g / L LpNoxVG171R / L172R, 2 g / L ScDAK1, 1 g / L BsAckA, 0.03 g / L EcDeoC, 0.15 g / L EcDeoB, 1 g / L EcUdp, and 5 g / L AoSucP. The reaction solution contained 50 mM TEA buffer (pH 7.0), 10 mM MgCl2, 1 mM MnCl2, 1 mM NADPH, and 0.5 mM ATP as cofactors. The reaction was carried out at 25°C for 24 hours.

[0087] The time course of ploxyuridine synthesis was monitored by quantitatively analyzing the concentration of ploxyuridine generated at each time point. Quantitative analysis of the converted ploxyuridine and idoxyuridine was performed using HPLC with an Aminex HPX-87H column.

[0088] Example 1. Enzyme design for the production of deoxyribonucleoside derivatives

[0089] The inventors recognized the need to synthesize non-natural deoxyribonucleosides from economical starting materials instead of expensive deoxyribonucleosides, which are common substrates for nucleoside transglycosylation processes, and to this end, synthesized deoxyribonucleosides by using the enzymatic reaction of the deoxyribonucleoside recovery pathway as the reverse reaction.

[0090] This approach can effectively provide the expensive intermediate D-Rib 1P from the more economical precursors D-GAP and AcH. In the deoxyribonucleoside recovery pathway, deoxyribonucleoside is naturally degraded through a sequential catalytic reaction of nucleoside phosphorylase (NP) and phosphopentomutase (PPM) to produce D-dRib 5P, which can be further converted to D-GAP and AcH by deoxyribose-5-phosphate aldolase (DERA). This strategy reverses this pathway to efficiently synthesize non-natural deoxyribonucleoside.

[0091] For the synthesis of nucleoside derivatives with modified nucleobases, the substrate range for the modified nucleobases of the NPs was a critical factor in the design of the retrosynthetic enzyme cascade. EcUdp, an NP derived from E. coli, possesses broad substrate specificity, enabling the synthesis of nucleosides with modified sugar or base portions, such as 5-FU and 5-IU. For the synthesis of decitabine and cladribine, EcDeoD, another NP derived from E. coli with catalytic activity for 5-AzaC and 2-CA, was used. Additionally, EcDeoB and EcDeoC, which act as E. coli-derived PPM and DERA, were selected because their catalytic activities for D-dRib 1P and D-dRib 5P, respectively, were known. Therefore, deoxyribonucleoside derivatives can be synthesized by using EcDeoC, EcDeoB, EcUdp, and EcDeoD in a retrosynthetic enzyme cascade through aldol condensation between D-GAP and AcH, conversion of D-dRib 5P to D-dRib 1P, and glycosylation of D-dRib 1P using modified nucleobases. However, the overall efficiency of this cascade depends heavily on a sufficient supply of D-GAP. Enantiopure D-GAP is expensive and unstable, so it is not considered a practical starting material.

[0092] To overcome the limitations of using D-GAP as a substrate, synthesizing D-GAP in situ from glycerol, an inexpensive and abundant starting material, is essential for improving the overall economic efficiency of the enzyme cascade. Accordingly, the present invention has constructed an efficient enantioselective enzyme cascade for producing D-GAP from glycerol, which is much more economical than D-GAP and D-GA (Fig. 1a). This cascade consists of two steps: the first is the process of oxidizing glycerol to D-GA, and the second is the process of phosphorylating D-GA to D-GAP. However, since D-GA is an inexpensive material like glycerol, the same effect can be obtained even if the process of oxidizing glycerol to D-GA is omitted and D-GA is purchased and used.

[0093] Example 2. Oxidize glycerol to D-GA

[0094] First, NADP + We selected D-specific glycerol dehydrogenase derived from Gluconobacter oxydans (GoGdh), which enantioselectively oxidizes glycerol to D-GA using [another enzyme] as a cofactor. GoGdh has been reported to exhibit high enantioselectivity, which is only about 4% of L-GA activity. For the second step, the phosphorylation of D-GA, we selected dihydroxyacetone kinase 1 derived from Saccharomyces cerevisiae (ScDAK1), which showed excellent enantioselectivity toward D-GA (Fig. 2). Furthermore, ATP-dependent phosphorylation by ScDAK1 enabled the production of D-GAP from D-GA with a high yield of up to 99.3%.

[0095] In the enantiosectastic oxidation of glycerol to D-GA, GoGdh is equivalent to a molar amount of NADP +It requires [it] as a cofactor. However, previous studies have reported that GoGdh prefers reduction reactions over oxidation reactions, and that its specific activity for D-GA reduction is up to 230 times higher compared to glycerol oxidation. This thermodynamic limit can be overcome by combining oxidation reactions with highly preferred or irreversible reactions using sacrificial substrates, thereby driving the overall reaction toward the desired product. To implement this strategy, NADH is oxidized using molecular oxygen (O2) to produce NAD + NADP using NADH oxidase (NOX) that regenerates and produces water (H2O) or hydrogen peroxide (H2O2) as a byproduct + A regenerative system was developed. While most NOX, such as the wild-type NADH oxidase (LpNoxV) of Lactobacillus plantarum, is specific to NADH, LpNoxVG171R / L172R, which introduces two basic residues—lysine and arginine—through site-specific mutations, has been reported to possess NADPH oxidative activity. Consequently, NADP + The regeneration system supplies necessary oxidation cofactors to promote the conversion of glycerol to D-GA, and external NADP + It alleviates the cost burden associated with supply.

[0096] Example 3. Phosphorylation of D-GA to D-GAP

[0097] Similarly, ScDAK1 uses an equivalent molar amount of ATP for D-GA phosphorylation. While ATP-dependent kinase reactions enable high D-GAP yields, an ATP regeneration system is required to maintain economic viability due to the high cost of ATP. As an ATP regeneration system, B. subtilis-derived acetate kinase (BsAckA) was introduced, which regenerates ATP from ADP using AcP as a sacrificial substrate. AcP can be chemically synthesized from ethyl acetate and phosphate using a simple laboratory-scale method. In this invention, AcP was prepared in-house, and details are described in the supplementary information. Phosphoenolpyruvate (PEP), creatine phosphate, and polyphosphate (polyP) may be considered as alternative sacrificial substrates for ATP regeneration. However, PEP and creatine phosphate are expensive, which limits their use in in vitro ATP regeneration systems. Furthermore, polyP-based ATP regeneration requires a high molar ratio of polyP, which is why Mg 2+ , Mn 2+ There is a concern that it may chelate essential diatomic metal cofactors, etc. In addition, free HPO4 is released upon polyP hydrolysis. 2 It is released and may have a negative effect on phosphorylase yield.

[0098] Overall, this enantioselective enzyme cascade provides a thermodynamically favorable pathway for the production of D-GAP from glycerol through enantioselective conversion and an efficient cofactor regeneration system at each step. By utilizing this enantioselective enzyme cascade to efficiently convert glycerol to D-GAP and incorporating it into a retrosynthetic enzyme cascade for deoxyribonucleoside synthesis, the goal was to produce the target deoxyribonucleoside from glycerol, a stable and inexpensive starting material, without relying on the direct supply of unstable and expensive D-GAP.

[0099] Example 4. Confirmation of the effects of NAD+ and the ATP regeneration system

[0100] As the first step in constructing an en-antiosectonic cascade for producing D-GAP from glycerol, the goal was to convert glycerol to D-GA using GoGdh. However, NADP + In the absence of regeneration, no significant glycerol conversion was observed, which implies that the oxidation reaction is thermodynamically unfavorable, as previously discussed.

[0101] On the other hand, NADP using LpNoxVG171R / L172R + Upon introduction of the regeneration system, the D-GA conversion rate of 5 mM glycerol reached 56.4% under conditions of 10 g / L GoGdh and 1 g / L LpNoxVG171R / L172R (Fig. 1a). This NADP + The regeneration system uses 1 mM NADP + It enabled glycerol conversion with only [amount], and the Total Turns (TTN) of LpNoxVG171R / L172R reached 5. Although it did not achieve complete conversion, NADP, which is favorable for the thermodynamically unfavorable glycerol oxidation reaction, was + It was clearly shown that combining regenerative reactions effectively promotes the conversion of glycerol to D-GA.

[0102] ScDAK1 was introduced for the next step, the conversion of glycerol to D-GAP. When the ATP concentration was increased from 0 to 2.5 mM, glycerol conversion increased twofold compared to the GoGdh and LpNoxVG171R / L172R combinations without ScDAK1 (Fig. 1b). In particular, complete conversion of glycerol occurred when the ATP concentration was 2.5 mM or higher. However, since the absence of an ATP regeneration system requires an equal molar amount of ATP as glycerol, this concentration was insufficient to phosphorylate all of the intermediate D-GA to D-GAP. To overcome this limitation and reduce the cost burden associated with ATP, an enzymatic ATP regeneration system using BsAckA and AcP as sacrificial substrates was introduced.

[0103] The introduction of an ATP regeneration system significantly improved reaction efficiency and economic feasibility. The effect of ATP regeneration was investigated to optimize D-GAP production at very low ATP inputs (Fig. 1c). As the AcP concentration increased from 3.125 to 25 mM, the conversion rate of glycerol to D-GAP also continuously increased from 59.2% to 100%. Notably, complete conversion of glycerol was achieved even under conditions where the ATP concentration was only 25 μM, which is 1 / 200th the level of the 5 mM ATP concentration required without ATP regeneration. Time-course analysis under the AcP 12.5 mM condition showed that 5 mM glycerol was completely converted within 12 hours, and no accumulation of the intermediate D-GA was detected (Fig. 1d).

[0104] Overall, the integration of the four enzymes GoGdh, LpNoxVG171R / L172R, ScDAK1, and BsAckA is efficient NADP + Supported by an ATP regeneration system, high-yield en-antioselective D-GAP conversion from glycerol was enabled, and the designed enzyme cascade demonstrated the ability to produce effective D-GAP from glycerol. The main objective of the present invention was to establish an en-antioselective and atomic-efficient catalytic system to maximize D-GAP yield from glycerol, but economic feasibility must also be considered. In this context, given that glycerol oxidation has an unfavorable equilibrium compared to D-GA reduction, it may be more advantageous to increase the initial glycerol concentration rather than increase the GoGdh input. Accepting the trade-off of a lower glycerol conversion rate but increased D-GA accumulation can help lower overall production costs, as the cost of purified enzymes is much higher than the cost of glycerol.

[0105] Example 5. Convert D-GAP to D-dRib 5P

[0106] The initial reaction step of the retrosynthetic enzyme cascade using D-GAP produced from glycerol via an anti-antiselective enzyme cascade is to generate D-dRib 5P through an aldol condensation reaction between D-GAP and AcH catalyzed by EcDeoC. In the present invention, instead of using D-GAP directly as a substrate, D-GA was used as a more stable and economical alternative. D-GAP was generated in situ from D-GA by ScDAK1 in conjunction with an ATP regeneration system (Fig. 3a). Since D-GA is much more stable than D-GAP, this in situ generation method enabled an economical and stable process for the synthesis of D-dRib 5P. In the aldol condensation reaction between D-GAP and AcH by EcDeoC, the yield of D-dRib 5P increased with increasing AcH concentration, depending on the equilibrium characteristics of the aldol reaction. However, high AcH concentrations are known to have a negative effect on the activity and stability of the enzyme, so optimization of the AcH input was necessary. As the AcH concentration increased from 3.125 mM to 25 mM, the relative yield of D-dRib 5P increased by 54.7% (Fig. 3b). At 25 mM AcH, D-dRib 5P production reached saturation. However, due to a mass balance mismatch during the derivatization process, the accurate quantification of D-dRib 5P produced from D-GA and AcH was limited. Despite this analytical limitation, the complete conversion of D-GA and the observed saturation of D-dRib 5P yield strongly suggest that the optimal AcH concentration for the synthesis of D-dRib 5P via aldol condensation between in situ-generated D-GAP and AcH in this enzyme cascade is 25 mM.

[0107] Example 6. Switch D-dRib 5P to D-dRib 1P

[0108] After the production of D-dRib 5P by EcDeoC, D-dRib 5P was converted into deoxyribonucleoside derivatives through a series of enzymatic steps involving EcDeoB and either EcUdp or EcDeoD, depending on the nucleobase. Specifically, D-dRib 5P was first converted to D-dRib 1P by EcDeoB, followed by an N-glycosylation reaction between the target nucleobase and D-dRib 1P via EcUdp or EcDeoD. To verify this enzymatic cascade, glycosylation reactions were performed using D-dRib 5P as the initial substrate with four modified nucleosides.

[0109] For 5-FU and 5-IU, EcUdp was used as the NP, while for 5-AzaC and 2-CA, EcDeoD was used to produce ploxyuridine, idoxyuridine, decitabine, and cladribine, respectively (Fig. 4a). HPLC analysis confirmed the production of each target deoxyribonucleoside derivative. After a 24-hour reaction, distinct peaks corresponding to the retention times of the genuine standard and the synthesized nucleosides (Fig. 4b to e) verified the successful enzymatic glycosylation of D-dRib 5P and the modified nucleobase. These results demonstrate the competence of the retrosynthetic enzyme cascade for the synthesis of commercially valuable therapeutic deoxyribonucleoside derivatives.

[0110] Example 7. HPO 4 2- Minimize accumulation

[0111] To confirm the yield of the method for producing deoxynucleoside derivatives according to the present invention, the process of producing ploxyuridine was verified.

[0112] Despite the innovativeness of this enzyme cascade for floxyuridine production, the final floxyuridine yield is directly inorganic HPO4 upon AcP hydrolysis. 2- HPO4 is released as a byproduct 2-It can be affected by accumulation. To overcome this, free HPO4 in the reaction solution 2- HPO4 that removes 2- A capture strategy was introduced. Specifically, free HPO4 by phosphorylating sucrose into D-fructose and D-glucose 1-phosphate (D-Glc 1P) using sucrose phosphorylase. 2- It was made to consume. This reaction is thermodynamically favorable in the direction of phosphate decomposition, and free HPO4 in the reaction solution 2- It effectively reduces the concentration. This contributes to shifting the equilibrium of the nucleobase glycosylation reaction catalyzed by EcUdp toward nucleoside production. To implement this strategy, sucrose phosphorylase (AoSucP) derived from Alloscardovia omnicolens was overexpressed and purified (Fig. 5).

[0113] HPO4 2- To evaluate the efficacy of the capture system, a retrosynthetic enzyme cascade containing EcDeoB and EcUdp was tested at various sucrose and AoSucP concentrations using D-dRib 5P and 5-FU as substrates (Fig. 6a). The addition of 200 mM sucrose increased the ploxyuridine yield 1.83-fold, from 30% to 55%, compared to the reaction without sucrose. According to these results, AoSucP pharmacologically shifted the reaction equilibrium toward nucleoside glycosylation when provided with an excess of sucrose. However, the yield reached saturation when the sucrose input was 50 mM or higher. This yield saturation is attributed to the fact that sucrose phosphatization is a reversible reaction and free HPO4 in the reaction system 2- This may be because the concentration is lowered sufficiently to further induce the reaction toward the product. The reported equilibrium constant for sucrose phosphate breakdown (KeqK_{eq}Keq) is 44, which shows that it is favorable but not irreversible.

[0114] To further improve the yield, a time-course study was conducted to supply additional sugar phosphate intermediates to the integrated retrosynthetic enzyme cascade by over-injecting glycerol at concentrations of 6, 12, and 30 mM while maintaining the same molar ratio with AcP, and converting to floxyuridine in the presence of 200 mM sucrose and AoSucP (Fig. 6b). Under the 6 mM glycerol condition, the maximum floxyuridine yield was 13.0% at 6 hours, while at 12 mM and 30 mM, significantly higher yields of 28.4% and 51.1% were observed within 3 hours, respectively. However, under all conditions, the floxyuridine yield decreased over time, dropping to 9.0%, 13.6%, and 16.1% at the 9-hour mark for the 6, 12, and 30 mM conditions, respectively. This decrease is due to HPO4 2- It appears to be due to the hydrolysis of phosphate-containing intermediates despite the presence of a capture system.

[0115] Overall, the introduction of AoSucP and Sucrose is free HPO4 2- It was demonstrated that capturing [the substance] could shift the reaction equilibrium toward product formation, and contributed to an increase in yield by expanding the supply of sugar phosphate intermediates with increased substrate input.

[0116] As discussed in the cascade design, this integrated retrosynthetic enzyme cascade has a modular structure and can be applied by introducing other nucleobases for the synthesis of various other therapeutic deoxyribonucleoside. Specifically, idoxyuridine, decitabine, and cladribine were produced by utilizing the broad substrate specificity of EcUdp and EcDeoD using 5-IU, 5-AzaC, and 2-CA as substrates, respectively. To verify the suitability of this cascade for producing three commercial therapeutic deoxyribonucleoside derivatives, the progression of phosphorylase reactions by EcUdp or EcDeoD for four nucleosides was investigated (Fig. 7). The progression of reactions for each nucleoside was similar, and both enzymes exhibited approximately the same magnitude of catalytic activity for the corresponding substrates. A total of four deoxyribonucleosides (floxyuridine, idoxyuridine, decitabine, and cladribine) were synthesized by the conversion of EcUdp and EcDeoD in the presence of 30 mM glycerol and corresponding nucleobases (including 200 mM sucrose and AoSucP). To minimize yield loss due to reaction reversal, the reaction time was limited to 3 hours. Cladribine production was carried out as a heterogeneous reaction due to the low water solubility of 2-CA (0.3 mg per 1 mL).

[0117] This integrated retrosynthetic enzyme cascade successfully produced idoxyuridine, decitabine, and cladribine using glycerol and AcH (Fig. 6c). After 3 hours, the yields were 51.1% for ploxyuridine, 37.3% for idoxyuridine, 4.19% for decitabine, and 48.0% for cladribine. These results demonstrate the flexibility and applicability of the enzyme cascade, which allows for the control of product specificity by switching nucleoside phosphorylases.

Claims

Claim 1 A method for producing a deoxyribonucleoside derivative comprising: (1) a step of phosphorylating D-GA to D-GAP using an ScDAK1 enzyme; (2) a step of converting D-GAP to D-dRib 5P by reacting it with AcH using an EcDeoC enzyme; and (3) a step of converting D-dRib 5P to D-dRib 1P using an EcDeoB enzyme. Claim 2 The method of claim 1, wherein the method further comprises the step of (a) oxidizing glycerol to D-GA using a GoGdh enzyme. Claim 3 In paragraph 2, the method wherein step (a) further uses an LpNoxV mutant. Claim 4 In paragraph 3, the method wherein the LpNoxV mutation is LpNoxVG171R or LpNoxVG172R. Claim 5 In paragraph 3, the above LpNoxV mutation is NADP + A method of playing. Claim 6 In claim 1, the above (1) step is a method of additionally using BsAckA. Claim 7 In paragraph 6, the method wherein the above BsAckA regenerates ATP from ADP using AcP. Claim 8 In claim 1, the above (3) step is a method of additionally using EcUdp or EcDeoD. Claim 9 In claim 8, the above step (3) is a method of converting 5-FU to pliuridine or 5-IU to idoxyuridine by additionally using EcUdp. Claim 10 In claim 8, the above step (3) is a method of converting 5-AzC to decitabine or 2-CA to cladribine by additionally using EcDeoD. Claim 11 In claim 1, the above (3) step is a method of additionally using AoSucP and sucrose. Claim 12 In paragraph 11, the above step (3) further uses AoSucP and sucrose to produce HPO4 2- A method of removing. Claim 13 delete

Images