C-Nucleoside Monophosphate Synthesis

Abstract

Provided herein is a one-pot method for synthesizing a C-nucleoside-5'-monophosphate, such as pseudouridine-5'-monophosphate, from an N-nucleoside using a multi-step enzymatic pathway, comprising reacting the N-nucleoside with a phosphate source and a nucleoside phosphorylase to form a pentose-1-phosphate intermediate, reacting the pentose-1-phosphate intermediate with a phosphomutase to form a pentose-5-phosphate intermediate, and reacting the pentose-5-phosphate intermediate with a nucleoside-5'-phosphate C-glycosidase to form the C-nucleoside-5'-monophosphate.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to UK Patent Application No. 2200248.9, filed January 10, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention generally relates to the synthesis of nucleoside derivatives. More specifically, the present invention relates to a method for synthesizing C-nucleoside-5'-monophosphate from an N-nucleoside using a multi-step enzymatic pathway, comprising reacting the N-nucleoside with a phosphate source and a nucleoside phosphorylase to form a pentose-1-phosphate intermediate, reacting the pentose-1-phosphate intermediate with a phosphomutase to form a pentose-5-phosphate intermediate, and reacting the pentose-5-phosphate intermediate with a nucleoside-5'-phosphate C-glycosidase to form a C-nucleoside-5'-monophosphate. [Background technology]

[0003] Nucleosides are the molecular building blocks of DNA and RNA. Nucleosides are glycosylamine compounds that contain a pentose sugar attached to a nitrogenous base. In DNA, the pentose sugar is 2-deoxy-D-ribose, and in RNA, the pentose sugar is D-ribose.

[0004] [ka]

[0005] Nitrogenous bases are heterocyclic organic compounds that can be classified as pyrimidines or purines. Pyrimidine bases have the general structure of a six-membered ring containing four carbon atoms and two nitrogen atoms. Purine bases contain a pyrimidine ring fused with an imidazole ring. There are five main or canonical bases as illustrated below: the purines adenine and guanine; and the pyrimidines cytosine, thymine, and uracil.

[0006] [ka]

[0007] In biological systems, DNA and RNA may contain derivatives of canonical bases that have been modified after the formation of the nucleic acid. As an example, a common non-canonical base found in DNA is 5-methylcytosine. Methylation of cytosine plays a central role in regulating gene expression and subsequent adaptation to environmental changes, differentiation, and development. As a further example, 7-methylguanine is often found at the 5' end of mRNA (7-methylmethyguanosine cap). This modification protects mRNA from degradation and aids in mRNA translation. Altered patterns of nucleic acid modification have been observed in disease states such as cancer. There are also a large number of synthetically modified bases or base analogues, many of which serve as important biochemical tools.

[0008] Nucleosides, both synthetic and derived from natural products, are widely used in clinical applications, particularly as antiviral or anticancer drugs. Nucleosides used in clinical applications are typically N-nucleosides in which the base heterocyclic unit is linked to the pentose moiety via an N-glycosidic (CN) bond. Prominent examples include the antiviral drug vidarabine and the anticancer drug gemcitabine. However, N-nucleosides are susceptible to hydrolysis by N-glycosidases, limiting their therapeutic activity. This has led to increased interest in the development of C-nucleosides in which the canonical N-glycosidic bond between the base and the pentose sugar is replaced by a non-hydrolyzable C-glycosidic (CC) bond.

[0009] In particular, the synthetic C-nucleosides BCX4430 and GS6620 have been shown to be effective against filoviruses and hepatitis C virus, respectively. Although originally developed for use against filoviruses, the synthetic C-nucleoside GS-5734 is currently being evaluated against the SARS-CoV-2 virus. Other C-nucleosides, such as showdomycin, formycin, pseudouridimycin, and minimycin, constitute an emerging class of natural microbial compounds that exhibit diverse biological activity, including antibiotic efficacy.

[0010] Pseudouridine is a C-nucleoside and an isomer of the N-nucleoside uridine. The structures of uridine and pseudouridine are presented below for comparison.

[0011] [ka]

[0012] Pseudouridine is formed post-transcriptionally in RNAs such as tRNA, ribosomal RNA and small RNAs by pseudouridylate synthase (alternative term: pseudouridine-5'-monophophosphate glycosidase). The attachment of uracil to ribose via C5 rather than via N1 allows for the presence of an additional imino proton that can form a hydrogen bond with a water molecule, thus stabilizing the secondary structure of the RNA and strengthening base-stacking interactions.

[0013] In mRNA-based vaccines, pseudouridine and its derivatives have been used to replace uridine to reduce the immunogenicity of mRNA and increase antigen stability and expression. Both the Pfizer-BioNtech vaccine against SARS-CoV-2 (BNT162b2) and the Moderna Therapeutics vaccine against SARS-CoV-2 (mRNA-1273) contain mRNA in which uracil is replaced by N1-methylpseudouridine. The quick adoption of mRNA-based vaccines and their expanding use in various clinical fields has led to high commercial demand for pseudouridine and its derivatives.

[0014] Pseudouridine is not readily available by extraction from natural RNA in mammalian cell lines and tissues due to the relatively low percentage of pseudouridine, which is further reflected in the significantly higher retail price of pseudouridine or its derivatives compared to uridine.

[0015] Attempts have been made to develop methods for synthesizing pseudouridine and its derivatives. Once such a method involves a multi-step semi-enzymatic synthesis of pseudouridine (Riley et al., 2021, Bioorg. Med. Chem. Lett. 44, 128105). In this method, as illustrated below, adenosine monophosphate (AMP) is depurinated under acidic conditions and at an elevated temperature of about 100° C. to form ribose-5-phosphate. Ribose-5-phosphate is extracted and purified from the acidic mixture, where it is formed by isopropanol precipitation and anion exchange chromatography. The purified ribose-5-phosphate is then reacted with uracil in the presence of pseudouridine-5'-monophosphate glycosidase (ψMP glycosidase) to form pseudouridine-5'-monophosphate. Pseudouridine-5'-monophosphate is purified by column chromatography and then reacted with bacterial alkaline phosphatase to produce pseudouridine. Purified pseudouridine is obtained by HPLC, however, the harsh conditions and multiple purification steps required in this method make it time- and cost-inefficient.

[0016] Semienzymatic synthesis of pseudouridine (adapted from Riley et al. 2021)

[0017] [ka]

[0018] China Patent No. 114196715 discloses a similar method for synthesizing pseudouridine. However, in this method, ribose-5-phosphate (obtained by hydrolysis of AMP) is reacted with uracil in the presence of mutant pseudouridine-5'-monophosphate glycosidase to form pseudouridine-5'-monophosphate. Pseudouridine is then obtained after dephosphorylation and separation.

[0019] A further multi-step method for synthesizing pseudouridine-5'-monophosphate is illustrated below. In this method, ribose is reacted with ribokinase in the presence of adenosine triphosphate (ATP) to form ribose-5-phosphate. Ribose-5-phosphate can then be converted to pseudouridine-5'-monophosphate and pseudouridine as in the above method by Riley et al. However, ATP is expensive and this method also requires multiple purification steps that make the method time- and cost-inefficient.

[0020] Further multi-step methods for the synthesis of pseudouridine-5'-monophosphate

[0021] [ka]

[0022] It would therefore be desirable to provide a more efficient method for synthesizing C-nucleosides, such as pseudouridine and its derivatives, that overcomes the shortcomings of existing methods. [Prior art documents] [Patent documents]

[0023] [Patent Document 1] UK Patent Application No. 2200248.9 [Patent Document 2] Chinese Patent No. 114196715 [Patent Document 3] WO2015 / 115993 [Non-patent literature]

[0024] [Non-Patent Document 1] Riley et al., 2021, Bioorg. Med. Chem. Lett. 44, 128105 [Non-Patent Document 2] de Giuseppe et al., 2012, PLoS ONE vol. 7, e44282; Uniprot: O34925

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[0025] Accordingly, there is provided a method for synthesizing a C-nucleoside 5'-monophosphate, comprising the steps of: a) reacting an N-nucleoside with a phosphate source in the presence of a nucleoside phosphorylase to form a first intermediate, wherein the nucleoside comprises a pentose sugar and a first base, and the first intermediate comprises a pentose-1-phosphate; b) reacting the first intermediate comprising a pentose-1-phosphate with a phosphomutase to form a second intermediate, wherein the second intermediate comprises a pentose-5-phosphate; c) reacting the second intermediate comprising a pentose-5-phosphate with a second base in the presence of a nucleoside-5'-phosphate C-glycosidase to form the C-nucleoside-5'-monophosphate; The present invention provides a method comprising:

[0026] Preferred features of the invention are defined in the dependent claims.

[0027] Advantageously, the cost of the starting materials in the process of the present invention is low. Moreover, the inventors have unexpectedly found that the process of the present invention can be carried out as a one-pot reaction without the need for separation or purification of any intermediate products, which further reduces costs and makes the process time-efficient.

[0028] Please refer to the following description and drawings. [Brief description of the drawings]

[0029] [Figure 1] 1 is a thin layer chromatogram (TLC) of samples taken at various time points from a reaction mixture containing reactants for synthesizing pseudouridine-5'-monophosphate. [Diagram 2] 1 is an HPLC-MS chromatogram and peak table of a reaction mixture for synthesizing pseudouridine-5'-monophosphate, which has been treated with alkaline phosphatase. [Diagram 3] 1 is an HPLC-MS chromatogram and peak table of commercially available pseudouridine. [Figure 4] FIG. 2 is an HPLC-MS chromatogram of the reaction mixture for synthesizing pseudouridine-5′-monophosphate that has been treated with alkaline phosphatase, showing the protonated (top) and deprotonated (bottom) forms of pseudouridine. [Figure 5A] FIG. 1 is an HPLC-MS chromatogram of the reaction mixture for synthesizing pseudouridine-5′-monophosphate without alkaline phosphatase treatment illustrating uracil and pseudouridine-5′-monophosphate. [Figure 5B] FIG. 2 is an HPLC-MS chromatogram of the reaction mixture for the synthesis of pseudouridine-5′-monophosphate without alkaline phosphatase treatment, showing the protonated (top) and deprotonated (bottom) forms of pseudouridine-5′-monophosphate. [Figure 6] 1H-NMR spectrum of synthesized pseudouridine-5'-monophosphate in DO. [Figure 7] 13C-NMR spectrum of synthesized pseudouridine-5'-monophosphate in DO. [Figure 8] 31P-NMR spectrum of synthesized pseudouridine-5'-monophosphate in DO. [Figure 9] 1 is a TLC of various reaction mixtures for the synthesis of C-nucleoside 5'-monophosphates. [Figure 10] 1 is a graph of the concentrations of pseudouridine monophosphate, uridine, and uracil over time in a one-pot reaction at 1 L scale. [Figure 11] FIG. 1 is a graph of uridine conversion and pseudouridine monophosphate yield in percent (%) after 24 hours at various potassium phosphate buffer concentrations (mM) at pH 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Methods for synthesizing C-nucleoside 5'-monophosphates The present invention provides a method for synthesizing a C-nucleoside 5'-monophosphate, comprising the steps of: a) reacting an N-nucleoside with a phosphate source in the presence of a nucleoside phosphorylase to form a first intermediate, wherein the nucleoside comprises a pentose sugar and a first base, and the first intermediate comprises a pentose-1-phosphate; b) reacting the first intermediate comprising pentose-1-phosphate with a phosphomutase to form a second intermediate, wherein the second intermediate comprises pentose-5-phosphate; c) reacting the second intermediate comprising a pentose-5-phosphate with a second base in the presence of a nucleoside-5'-phosphate C-glycosidase to form the C-nucleoside-5'-monophosphate; The present invention provides a method comprising:

[0031] The term "comprising" or "comprises" as used herein indicates the inclusion of at least the features that follow the term and does not exclude the inclusion of other features not expressly recited. The term may also indicate an entity that consists of the features that follow the term.

[0032] N-Nucleosides As discussed above, N-nucleosides contain a pentose sugar and a base, which is typically nitrogenous, where the base is attached to the pentose moiety by an N-glycosidic (CN) bond. Typically, in pyrimidine-based nucleosides, the glycosidic bond is between C1' and N-1, and in purine-based nucleosides, the glycosidic bond is between C1' and N-9.

[0033] The pentose sugar is typically, but not exclusively, in the D-isomer form. In a preferred embodiment, the pentose comprises a D-aldopentose. The D-aldopentose may be selected from D-ribose, 2-deoxy-D-ribose, D-arabinose, D-xylose, or D-lyxose and derivatives thereof. In a preferred embodiment, the pentose comprises D-ribose. In other embodiments, the nucleoside may comprise a pentose as depicted in general formula Ia, IIa, IIIa, or IVa:

[0034] [ka]

[0035] wherein R1 and R2 are independently selected from H, OH, OCH3, F, NH2, and N3.

[0036] With particular reference to formula Ia, the following substituents are provided: R1 and R2 are each OH; R1 is OH and R2 is H; R1 and R2 are H; R1 is OH and R2 is OCH3; R1 is N3 and R2 is H; R1 is OH and R2 is N3; R1 is H and R2 is OH; R1 is F and R2 is OH; R1 is OCH3 and R2 is OH; R1 is OH and R2 is F.

[0037] In one preferred embodiment, R1 and R2 are each OH (the pentose comprises D-ribose). In another embodiment, R1 is OH and R2 is H (the pentose comprises 2-deoxy-D-ribose). In yet another embodiment, R1 is OH and R2 is F (the pentose comprises 2-deoxy-2-fluoro-D-ribose).

[0038] Although the above substituent selections are presented specifically with respect to formula Ia, they are also relevant and applicable to formulas IIa, IIIa, and IVa. In preferred embodiments, R1 and R2 are each OH, and thus the pentose of formula IIa comprises D-arabinose, the pentose of formula IIIa comprises D-xylose, and the pentose of formula IVa comprises D-lyxose.

[0039] Although the furanose structure of the pentose sugar has been depicted herein in terms of the Haas projection, in the methods of the present invention the pentose sugar may exist in other structural isomeric forms, such as pyranose or linear forms, or combinations thereof.

[0040] A reaction scheme for the method according to the invention using pentose-containing N-nucleosides according to formulae Ia, IIa, IIIa and IVa, respectively, is illustrated below, where "A", "B" and "C" refer to nucleoside phosphorylase, phosphomutase and nucleoside-5'-monophosphate C-glycosidase, respectively.

[0041] [ka]

[0042] [ka]

[0043] The first base of the N-nucleoside may include, but is not limited to, a standard purine or pyrimidine base selected from adenine, cytosine, guanine, thymine, and uracil. Chemically modified bases are also contemplated within the scope of the method of the present invention. Chemically modified bases for use as the first base may also include 3-methyluracil, 6-aminouracil, 4-thiouracil, 2-thiouracil, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-(3-aminoallyl)uracil.

[0044] The first base referred to herein is illustrated below. For simplicity, the base is illustrated only within the N-nucleoside structure according to formula Ia. Similar structures can be seen in formulas IIa, IIIa, and IVa, respectively.

[0045] [ka]

[0046] [ka]

[0047] [ka]

[0048] In some embodiments, other modified bases or base analogs capable of forming N-glycosidic (CN) bonds with the pentose sugars disclosed herein can also be used in the reactions of the invention.

[0049] One-pot reaction and reaction conditions The method of the present invention can be advantageously carried out as a "one-pot" reaction. By "one-pot" reaction is meant that the synthesis of C-nucleoside-5'-monophosphate is carried out without any separation and purification of intermediate compounds, preferably with all reactants being fed into a single reactor. This advantageously saves time and resources while increasing chemical yields.

[0050] In a preferred embodiment, the N-nucleoside, phosphate source, nucleoside phosphorylase, phosphomutase, second base and nucleoside-5'-phosphate C-glycosidase (reactants) are added simultaneously to a reactor, while in other embodiments, one or more of the reactants are added sequentially to a reactor.

[0051] The reaction is preferably carried out at the optimum temperature and pH of the enzyme involved. Typically, the reaction is carried out at about 37° C., or at the optimum temperature of the enzyme. However, the reaction can also be carried out at a temperature of about 20° C. to about 37° C., or up to any temperature at which the enzyme remains active. Typically, the reaction is carried out at the optimum pH of the enzyme. For example, the reaction can be carried out at a pH of about 6 to about 8 (about 6 to about 7, or about 7 to about 8, etc.), or at any pH at which the enzyme remains active. In some embodiments, the reaction is carried out at a pH of about 7. In some embodiments, the reaction is carried out at a pH of about 8.

[0052] Nucleoside phosphorylase Nucleoside phosphorylase suitable for use in the present invention is not particularly limited, and includes any enzyme that can catalyze the reaction: N-nucleoside D-pentose-1-phosphate + first base. Nucleoside phosphorylases generally have broad specificity, and typically any given nucleoside phosphorylase can act on multiple substrates. Nucleoside phosphorylases for use in the method of the present invention can be selected based on the starting N-nucleoside, in particular the base portion of the N-nucleoside. Nucleoside phosphorylases for use in the method of the present invention are readily commercially available, and can also be obtained by established methods of recombinant expression.

[0053] In some embodiments, the nucleoside phosphorylases used include, but are not limited to: purine-nucleoside phosphorylase (EC 2.4.2.1), pyrimidine-nucleoside phosphorylase (EC 2.4.2.2), thymidine phosphorylase (EC 2.4.2.4): thymine, uridine phosphorylase (EC 2.4.2.3) and guanosine phosphorylase (EC 2.4.2.15). (The EC numbers provided herein refer to the Enzyme Number, which is an internationally accepted numerical classification scheme for enzymes. The Enzyme Number is based on and specifies the reaction catalyzed by the enzyme, not the enzyme itself).

[0054] In a preferred embodiment, the nucleoside phosphorylase comprises uridine phosphorylase. Other nucleoside phosphorylases having the same EC number as any of those listed above can also be used in the present invention.

[0055] In a preferred embodiment, the N-nucleoside comprises guanine, adenine, hypoxanthine, xanthine or 7-methylguanine as the first base moiety, and the nucleoside phosphorylase used comprises a purine-nucleoside phosphorylase (EC 2.4.2.1). Such purine-nucleoside phosphorylases have been identified, for example, in Bacillus subtilis (de Giuseppe et al., 2012, PLoS ONE vol. 7, e44282; Uniprot: O34925) and Escherichia coli (Jensen et al., 1975, Eur. J. Biochem., vol. 51, pp. 253-265; Uniprot: P0ABP8).

[0056] In another preferred embodiment, the N-nucleoside comprises cytosine, thymine, 5,6-dihydrouracil, 5-methylcytosine, 5-(3-aminoallyl)uracil, uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, or 2-thiouracil as a base moiety, and the nucleoside phosphorylase used comprises a pyrimidine-nucleoside phosphorylase (EC 2.4.2.2). Such pyrimidine-nucleoside phosphorylases have been identified, for example, in Bacillus stearothermophilus (Saunders et al., 1969, J. Biol. Chem., vol. 244, pp. 3691-3697; Pugmire et al., 1998, Structure, vol. 6, pp. 1467-1479; Uniprot: P77836). Pyrimidine / pyrine nucleoside phosphorylases have also been identified in Escherichia coli (Wen et al., 2022, Proteins vol. 90, pp. 1233-1241; Uniprot: P0C037) and Acinetobacter baumannii (Vallenet et al., 2008, PLoS ONE vol. 3, e1805; Uniprot: B0VLV6).

[0057] In a further preferred embodiment, the N-nucleoside comprises uracil as the first base moiety and the nucleoside phosphorylase used comprises uridine phosphorylase (EC 2.4.2.3), which has been identified, for example, in Klebsiella aerogenes (Veiko et al., 1998, Bioorg. Khim., vol. 24, pp. 381-387; UniProt ID: O08444) and Escherichia coli (Walton et al., 1989, Nucleic Acids Res. vol. 17, pp. 6741; Uniprot: P12758).

[0058] In yet further embodiments, the N-nucleoside comprises thymine or uracil as the first base moiety and the nucleoside phosphorylase used comprises thymidine phosphorylase (EC 2.4.2.4); or the N-nucleoside comprises guanine as the first base moiety and the nucleoside phosphorylase used comprises guanosine phosphorylase (EC 2.4.2.4). Thymidine phosphorylase has been identified, for example, in E. coli (Walter et al., 1990, J. Biol. Chem., vol. 265, pp. 14016-14022; Uniprot: P07650).

[0059] However, the above combinations of phosphorylase and base substrates are not intended to be exclusive or limiting, and other combinations are permissible and would be readily determined by one of skill in the art.

[0060] The source of phosphate in step a) of the method of the present invention, which is a substrate required by nucleoside phosphorylase, may comprise a compound selected from soluble alkali metal or alkaline earth metal phosphates. In some embodiments, the source of phosphate comprises a compound selected from sodium dihydrogen phosphate, trisodium phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, tripotassium phosphate, and potassium hydrogen phosphate. In a preferred embodiment, the source of phosphate comprises a potassium phosphate buffer at a pH of about 7. In another preferred embodiment, the source of phosphate comprises a potassium phosphate buffer at a pH of about 8.

[0061] In some embodiments, the concentration of the nucleoside phosphorylase used in step a) is about 0.02 to about 0.6 mg / mL, e.g., about 0.3 to about 0.6 mg / mL, about 0.4 to about 0.6 mg / mL, or about 0.5 mg / mL, or for example, about 0.02 to about 0.5 mg / mL, about 0.02 to about 0.4 mg / mL, about 0.02 to about 0.3 mg / mL, about 0.02 to about 0.2 mg / mL, about 0.02 to about 0.1 mg / mL, about 0.02 to about 0.05 mg / mL, or about 0.025 to about 0.035 mg / mL. In some embodiments, the concentration of nucleoside phosphorylase is about 0.02 mg / mL, about 0.025 mg / mL, about 0.03 mg / mL, about 0.035 mg / mL, about 0.04 mg / mL, about 0.045 mg / mL, or about 0.05 mg / mL. Enzyme concentrations below 0.02 mg / mL may reduce the yield of the final C-nucleoside-5'-monophosphate, and enzyme concentrations above 0.6 mg / mL may have limited economic utility.

[0062] Phosphomutase The phosphomutase used in step b) of the method of the present invention is not particularly limited and includes any enzyme that can catalyze the reaction: D-pentose-1-phosphate D-pentose-5-phosphate. Preferably, said phosphomutase is a phosphopentomutase. Phosphopentomutase also has broad specificity, and a given phosphopentomutase can act on several different D-pentose-1-phosphate intermediates to form D-pentose-5-phosphate intermediates.

[0063] Preferred phosphopentomutases for use in the methods of the invention include enzymes having the EC number 5.4.2.7, such as deoB phosphopentomutase, which can be isolated from several different strains of bacteria, including Escherichia coli (Valentin-Hansen et al., 1984, Nucleic Acids Res., vol. 12, pp. 5211-5224; Uniprot: P0A6K6), Bacillus cereus (Panosian et al., 2011, J. Biol. Chem., vol. 268, pp. 8143-8054; Uniprot: Q818Z9), Streptococcus pneumoniae, and Bacillus anthracis.

[0064] Other phosphopentomutases are commonly known, including, for example, phosphopentomutases isolated from Thermus thermophilus HB8 (GenBank ID: 3169853 or UniProt: Q5SLG9), Streptococcus thermophilus (GenBank ID: 66898918 or UniProt: Q5M482), and Saccharomyces cerevisiae S288C (GenBank ID: 855321 or UniProt: Q03262).

[0065] In a preferred embodiment, step a) of the method of the invention comprises reacting an N-nucleoside containing a D-ribose or 2-deoxy-D-ribose sugar according to formula Ia, where R1 and R2 are each OH, or where R1 is OH and R2 is H, respectively, with a phosphopentose mutase (e.g., a deoB phosphopentose mutase (EC 5.4.2.7) or any other enzyme having the same enzyme classification as a deoB phosphopentose mutase) to form a first intermediate D-ribose-1-phosphate or 2-deoxy-D-ribose-1-phosphate.

[0066] In some embodiments, the concentration of the phosphomutase used in step b) is about 0.2 to about 0.6 mg / mL, e.g., about 0.3 to about 0.6 mg / mL, about 0.4 to about 0.6 mg / mL, or about 0.5 mg / mL, or, e.g., about 0.2 to about 0.5 mg / mL, about 0.2 to about 0.4 mg / mL, or about 0.2 to about 0.3 mg / mL. In some embodiments, the concentration of the phosphomutase is about 0.2 mg / mL, about 0.25 mg / mL, about 0.3 mg / mL, or about 0.35 mg / mL. Concentrations below 0.2 mg / mL may reduce the yield of the final C-nucleoside-5'-monophosphate, and concentrations of the enzyme above 0.6 mg / mL may have limited economic utility.

[0067] Nucleoside-5'-monophosphate C-glycosidase Nucleoside-5'-monophosphate C-glycosidase for use in the method of the present invention is not particularly limited and includes any enzyme that can catalyze the reaction: D-pentose-5-phosphate + second base C-nucleoside-5' monophosphate. C-nucleoside glycosidases generally have broad specificity, and typically a given C-nucleoside glycosidase can act on several different substrates. C-nucleoside glycosidase for use in the method of the present invention can be obtained by established methods of recombinant expression.

[0068] A preferred C-nucleoside glycosidase for use in the methods of the present invention is pseudouridylate synthase (alternatively referred to as pseudouridine-5'-monophosphate glycosidase) (EC 4.2.1.70) or any other enzyme having the same enzyme classification. Pseudouridylate synthase is a metabolic enzyme used in nucleotide catalytic pathways in prokaryotes. Pseudouridylate synthase or related enzymes with activity equivalent to pseudouridylate synthase have been identified and isolated from a number of prokaryotic sources. For example, YeiN(psuG) has been identified in Escherichia coli (Preumont et al., 2008, J. Biol. Chem., vol. 283, No.37, pp. 25238-25246; Pfeiffer and Nidetzky 2020, Nat Commun. 2020 Dec 8;11(1):6270.), AlnA has been identified in Streptomyces species (Oja et al., 2013, PNAS, vol. 100, no.4, pp. 1291-1296), SdmA has been identified in Streptomyces showdoensi (Palmu et al., 2017, ACS Chemical Biology, vol. 12, pp. 1472-1477), and TmYeiN has been identified in Thermotoga maritima (TmYeiN). maritima (Preumont et al., 2008, J. Biol. Chem., vol. 283, No. 37, pp. 25238-25246). In addition, MinB (GenBank: QDX19370.1) has been identified in Streptomyces hygroscopicus (Kong et al., 2019, iScience, vol. 22, pp. 430-440), and IndA (GenBank: AFV27435.1) has been identified in Streptomyces chromofuscus. Eukaryotic homologs of pseudouridylate synthase also exist as multifunctional enzymes. Any of the above-mentioned enzymes would be suitable for the methods of the present invention.

[0069] In some embodiments, pseudouridylate synthase catalyzes the 5-β-C-glycosylation of uracil and its derivatives from pentose-5-phosphate substrates. Pentose may include any one of D-ribose (R1 and R2 are each OH, formula Ic above), 2-deoxy-D-ribose (R1 is OH and R2 is H, formula Ic above), 2-deoxy-2-fluoro-D-ribose (R1 is OH and R2 is F, formula Ic above); D-arabinose (R1 and R2 are each OH, formula Iic above), D-xylose (R1 and R2 are each OH, formula IIIc above), and D-lyxose (R1 and R2 are each OH, formula Ivc above). Uracil derivatives may include, but are not limited to, 3-methyluracil, 6-aminouracil, 4-thiouracil, 2-thiouracil.

[0070] In some embodiments, step c) of the method of the invention comprises reacting a pseudouridylate synthase (e.g., YeiN) with D-ribose-5-phosphate in the presence of a base (second base) selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, or 2-thiouracil.

[0071] In some embodiments, step c) of the method of the invention comprises reacting a pseudouridylate synthase (e.g., YeiN) with 2-deoxy-D-ribose-5-phosphate in the presence of a base (second base) selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, or 2-thiouracil.

[0072] In some embodiments, step c) of the method of the invention comprises reacting a pseudouridylate synthase (e.g., YeiN) with D-xylose-5-phosphate in the presence of a base (second base) selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, or 2-thiouracil.

[0073] In some embodiments, step c) of the method of the invention comprises reacting a pseudouridylate synthase (e.g., YeiN) with D-arabinose-5-phosphate in the presence of a base (second base) selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, or 2-thiouracil.

[0074] In some embodiments, step c) of the method of the invention comprises reacting a pseudouridylate synthase (e.g., YeiN) with D-lyxose-5-phosphate in the presence of a base (second base) selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, or 2-thiouracil.

[0075] In some embodiments, step c) of the method of the present invention comprises reacting a pseudouridylate synthase (e.g., YeiN) with 2-deoxy-2-fluoro-D-ribose in the presence of a base (second base) selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, or 2-thiouracil.

[0076] In the above embodiment, and with respect to Schemes 1-4 presented above, the pentose moiety of the pentose-5-phosphate that is the substrate for pseudouridylate synthase is derived from an N-nucleoside starting compound.

[0077] In embodiments where the first and second bases are different (see discussion of "wasteful" reactions below), the nucleoside-5'-monophosphate C-glycosidase is preferably reactive towards the second base but unreactive or less reactive towards the first base.

[0078] Without wishing to be bound by theory, and with particular reference to uracil or its derivatives as substrate, the reaction catalyzed by pseudouridylate synthase is believed to involve cleavage of the regular N1-C1' glycosidic bond, 180° rotation of the uracil base along the N3-C6 axis, and coupling of the C5 and C1' to form a CC glycosidic bond in a reaction similar to a Mannich addition. Similar chemistry is expected to occur with other combinations of base and pentose substrates, and with other comparable C-nucleoside-5'-monophosphate glycosidase enzymes.

[0079] In some embodiments, the concentration of C-nucleoside-5'-monophosphate glycosidase used in step c) is about 0.2 to about 0.6 mg / mL, e.g., about 0.3 to about 0.6 mg / mL, about 0.4 to about 0.6 mg / mL, or about 0.5 mg / mL, or, e.g., about 0.2 to about 0.5 mg / mL, about 0.2 to about 0.4 mg / mL, or about 0.25 to about 0.35 mg / mL. In some embodiments, the concentration of C-nucleoside-5'-monophosphate glycosidase is about 0.2 mg / mL, about 0.25 mg / mL, about 0.3 mg / mL, about 0.35 mg / mL, or about 0.4 mg / mL. Enzyme concentrations below 0.2 mg / mL may reduce the yield of the final C-nucleoside-5'-monophosphate, while enzyme concentrations above 0.6 mg / mL may have limited economic utility.

[0080] In some embodiments, the nucleoside phosphorylase, phosphomutase and / or nucleoside 5'-phosphate C-glycosidase may comprise a thermophilic enzyme, for example as found in a thermophilic bacterium. Thermophilic enzymes may be advantageous due to their increased robustness and stability (e.g., at high temperatures and in the presence of common protein denaturing agents) compared to their non-thermophilic counterparts. These properties may facilitate storage of the enzyme, allow enzymatic reactions to be carried out over extended periods of time, and facilitate isolation of the enzyme from the reaction mixture by methods such as ultrafiltration for subsequent reuse. For example, thermophilic enzymes may be obtained by recombinant methods including overexpressing the enzyme in E. coli or other suitable host cells, lysing the cells, heat treating the cell lysate to inactivate all native E. coli enzymes (thermophilic enzymes will remain active under these conditions), filtering / centrifuging the heat treated lysate to remove insoluble particles, and using the crude enzyme preparation for the enzymatic synthesis method of the invention.

[0081] Second base The second base is also not particularly limited and may include standard or chemically modified bases as defined herein. Additional bases that may act as second bases include 2-amino-1H-pyrrole-5-carboxylate and 1,4-naphthoquinone. Other modified bases or base analogs that can form C-glycosidic bonds with the pentose-5-phosphate compounds disclosed herein may also be used in the present invention.

[0082] "No waste" reaction In some embodiments, the first base and the second base are the same. In these reactions, the first base generated as a waste product in step a) is reused in step c) to form C-nucleoside-5'-monophosphate. Thus, there is no "waste" of base.

[0083] In these embodiments, the first and second bases are preferably selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil and 2-thiouracil.

[0084] In a specific embodiment, the method for synthesizing pseudouridine-5'-monophosphate comprises the steps of: a) reacting uridine with a phosphate source in the presence of a nucleoside phosphorylase to form ribose-1-phosphate as a first intermediate and uracil; b) reacting the pentose-1-phosphate with a phosphomutase to form ribose-5-phosphate as a second intermediate; c) reacting ribose-5-phosphate with the uracil produced in step a) in the presence of a nucleoside-5'-phosphate C-glycosidase to form pseudouridine-5'-monophosphate; The present invention provides a method comprising:

[0085] The process is illustrated below (Scheme 5), where "A," "B," and "C" refer to nucleoside phosphorylase, phosphomutase, and nucleoside-5'-phosphate C-glycosidase, respectively.

[0086] [ka]

[0087] In the above "no waste" method, the nucleoside phosphorylase, phosphomutase, and nucleoside 5'-phosphate C-glycosidase may be as described above. Preferably, the nucleoside phosphorylase comprises uridine phosphorylase, the phosphomutase comprises phosphopentomutase DeoB, and / or the nucleoside-5'-phosphate C-glycosidase comprises pseudouridylate synthase (psuG or yeiN).

[0088] In another embodiment of the "no waste" reaction, and further with reference to Scheme 5, 2-thiouridine, 4-thiouridine, or 4-thio-2'-deoxyuridine is used as the starting nucleoside instead of uridine to form 2-thio-pseudouridine-5'-phosphate, 4-thio-pseudouridine-5'-phosphate, and 4-thio-2'-deoxy-pseudouridine-5'-phosphate, respectively. The nucleoside phosphorylase, phosphomutase, and nucleoside 5'-phosphate C-glycosidase used in the reaction may be as described above. Preferably, the nucleoside phosphorylase comprises uridine phosphorylase, the phosphomutase comprises phosphopentomutase DeoB, and / or the nucleoside-5'-phosphate C-glycosidase comprises pseudouridylate synthase (psuG or yeiN).

[0089] "There is waste" reaction In some embodiments, the first base is different from the second base. In these embodiments, the first base may be selected from cytosine, guanine, thymine, adenine, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-(3-aminoallyl)uracil. The second base may be selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, 2-thiouracil, 2-amino-1H-pyrrole-5-carboxylate, and 1,4-naphthoquinone. In these embodiments, it is desirable for the nucleoside-5'-phosphate C-glycosidase to be reactive toward the second base but non-reactive or less active toward the first base, to ensure that the reaction proceeds in favor of the formation of N-nucleoside-5'-monophosphate.

[0090] In a specific embodiment, the following method for synthesizing N-3 methylpseudouridine-5'-monophosphate is provided: a) reacting 5-methyluridine with a phosphate source in the presence of a nucleoside phosphorylase to form ribose-1-phosphate as a first intermediate and thymine as a waste product; b) reacting ribose-1-phosphate with a phosphomutase to form ribose-5-phosphate as a second intermediate; c) reacting ribose-5-phosphate with N-3-methyluracil in the presence of nucleoside-5'-phosphate C-glycosidase to form N-3 methylpseudouridine-5'-monophosphate; The present invention provides a method comprising:

[0091] The process is illustrated below (Scheme 6), where "A," "B," and "C" refer to nucleoside phosphorylase, phosphomutase, and nucleoside-5'-phosphate C-glycosidase, respectively.

[0092] [ka]

[0093] In the above "with waste" method, the nucleoside phosphorylase, phosphomutase, and nucleoside 5'-phosphate C-glycosidase may be as described above. Preferably, the nucleoside phosphorylase comprises uridine phosphorylase or thymidine phosphorylase, the phosphomutase comprises phosphopentomutase DeoB, and / or the nucleoside 5'-phosphate C-glycosidase comprises pseudouridine-5'-phosphate glycosidase (e.g., psuG / Yei N).

[0094] Purification of C-nucleoside-5'-monophosphate The C-nucleoside-5'-monophosphate produced by the method of the present invention can be subjected to further separation and / or purification steps. In some embodiments, the C-nucleoside-5'-monophosphate is separated from the reaction mixture by one or more methods selected from high performance liquid chromatography (HPLC), ion exchange chromatography, and reverse phase chromatography. In some embodiments, the C-nucleoside-5'-monophosphate is separated from the reaction mixture and / or purified by crystallization.

[0095] Depending on the desired application, the C-nucleoside-5'-monophosphates produced by the methods described herein can be optionally treated with a phosphatase enzyme prior to any separation or purification steps to produce C-nucleosides (and phosphate waste products). Thus, the present invention further provides a method for producing C-nucleosides. In some embodiments, the phosphatase enzyme is an alkaline phosphatase enzyme. In some embodiments, the phosphatase enzyme is an HAD-like phosphatase, more specifically HAD hydrolase subfamily 1A, e.g., Uniprot ID Q181K6 from Clostridioides difficile. The phosphatases are commercially available or readily produced by standard recombinant methods.

[0096] The C-nucleosides produced by the methods of the invention can be subjected to further separation and / or purification steps. In some embodiments, the C-nucleosides are separated from the reaction mixture by one or more methods selected from high performance liquid chromatography (HPLC), ion exchange chromatography, and reverse phase chromatography. In some embodiments, the C-nucleosides are separated from the reaction mixture and / or purified by crystallization.

[0097] Immobilization of enzymes on solid supports When enzymes are used in organic synthesis (such as in organic solvents), they often tend to aggregate, precipitate, and / or unfold (i.e., denature). Thus, in some embodiments, the enzymes used in the methods disclosed herein are immobilized on solid supports and used as catalysts in the immobilized state. Such immobilization can improve the stability of the enzyme and allow reaction conditions that the enzyme would not normally tolerate. The use of enzymes in the immobilized state also facilitates separation from the reaction mixture and allows recovery of the catalyst. In addition, immobilization allows the use of immobilized enzymes at much higher concentrations than would be possible with the enzymes free in solution. Methods for immobilizing enzymes on solid supports are known in the art.

[0098] In some embodiments, the solid support is a rigid porous material having a pore diameter of about 20 nm to about 100 nm, e.g., about 20 to about 60 nm. In some embodiments, the solid support exhibits limited swelling in solvents and is chemically and dimensionally stable in many organic media and aqueous environments below pH 10. In some embodiments, the solid support material is controlled porosity glass (CPG) or hybrid controlled porosity glass (hybrid CPG) as disclosed in WO2015 / 115993.

[0099] In some embodiments, the enzyme comprises a metal affinity tag, such as a polyhistidine tag (i.e., a His-tag of 2-8 consecutive histidine residues, preferably 6 consecutive histidine residues), to facilitate immobilization of the enzyme via a chelated metal on a solid support.

[0100] In some embodiments, each of the three enzymes used in the methods disclosed herein (i.e., the nucleoside phosphorylase, the phosphomutase, and the nucleoside-5'-monophosphate C-glycosidase) is individually immobilized to a solid support. In some embodiments, two or three of the enzymes are co-immobilized to the same solid support.

[0101] In some embodiments, the immobilized enzyme is used in a batch or continuous stirred tank reactor, hi some embodiments, the immobilized enzyme is loaded into a fixed bed reactor suitable for operation in a continuous flow mode, for example a continuous flow fixed bed reactor.

[0102] As used herein, the term "about" refers to a value or parameter herein, including (and describing) an embodiment that is directed to the value or parameter itself. For example, a statement that refers to "about 20" includes the statement "20." Numerical ranges are inclusive of the numerical values ​​that define the range. Generally speaking, the term "about" refers to the value of the indicated variable and all values ​​of the variable that are within the experimental error of the indicated value (e.g., within a 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater.

[0103] The invention will now be described by the following examples, which are not intended to limit the invention in any way. All cited documents and references described herein are incorporated by reference in their entirety.

[0104] Experimental Method Analytical methods in Examples 3 to 11 For the analysis and separation of compounds uridine, uracil, pseudouridine, and pseudouridine monophosphate, the following setup was used: Instrument: Thermo scientific Vanquish, Agilent 1260 Infinity II, Column: HILICON iHILIC-(P)Classic column (length x inner diameter 100 mm x 2.1), Mobile phase: A: Acetonitrile, B: 100 mM ammonium formate pH 5.8, Gradient: 0 min - 15% B, 2 min - 15% B, 2.5 min - 40% B, 5.5 min - 40% B, Switch back to 5.5 min - 15% B, Hold for 5 min, Flow rate: 0.3 mL / min, Injection volume 2 μL. Sample preparation for analyzing uridine, uracil, pseudouridine, and pseudouridine monophosphate: 10 μL of reaction sample was combined with 50 μL of internal standard stock solution (10 mM caffeine), followed by the addition of 940 μL of HPLC grade water. The solution was vortexed for 15 seconds, then 500 μL of the resulting solution was mixed with 500 μL of acetonitrile (ACN), followed by another vortex for 15 seconds. The solution represented a 200-fold dilution and a final caffeine concentration (standard) of 0.25 mM, which was used for HPLC analysis. If caffeine was present in the reaction mixture as an internal standard, the preparation of the HPLC sample was as follows: 10 μL of reaction sample was combined with 990 μL of HPLC grade water. The solution was mixed by vortexing for 15 seconds, then 500 μL of the resulting mixture was added to 500 μL of ACN, followed by vortexing for 15 seconds. The solution represented a 200-fold dilution and a final caffeine concentration of 0.25 mM (standard) which was used for HPLC analysis.

[0105] To analyze phosphate, ribose-1-phosphate, and ribose-5-phosphate, the following setup was used: Instrument: Dionex Intergion HPIC, Column: Dionex IonPac AS11-HC-4um RFIC & HPIC, Column temperature 35° C., Flow rate: 0.38 mL / min, Detector: Conductivity detector, Detector temperature 35° C., Injection volume 10 μL, Eluent: KOH in water, Gradient: 0 min - 10 mM KOH, 6 min - 35 mM KOH, 7 min - 80 mM KOH, 9 min - 80 mM KOH, 9 min 10 mM KOH, 13 min - 10 mM KOH. Sample preparation for analyzing ribose-phosphate: 10 μL of reaction sample was combined with 200 μL of LC-MS grade ACN, followed by the addition of 100 μL of 10 mM ammonium formate in micropure water. Then, 690 μL of Micropure water was added and the solution was vortexed for 15 seconds. 500 μL of the resulting solution was mixed with 500 μL of Micropure water and vortexed again for 15 seconds. The solution represented a 200-fold dilution and a final ammonium formate concentration of 0.5 mM (as a standard) and was used for HPLC analysis. EXAMPLES

[0106] Example 1 Generation of pseudouridine monophosphate A reaction mixture containing 0.48 mg / mL uridine phosphorylase, 0.4 mg / mL phosphopentomutase, 0.5 mg / mL pseudouridine-5'-monophosphate (ΨMP) glycosidase, 70 mM uridine, 0.5 mM MnCl2, and 125 mM potassium phosphate buffer (pH 7.0) in a total volume of 25 mL was shaken at 37°C. The reaction was monitored by thin layer chromatography (TLC) over a period of 28 hours. As can be seen from Figure 1, as the amount of ΨMP increased, the amount of uracil / uridine decreased over time, confirming effective ΨMP synthesis.

[0107] The identity of the synthesized ΨMP was confirmed by HPLC-MS and NMR spectroscopy. For HPLC-MS analysis, a sample taken from the reaction mixture was treated with alkaline phosphatase to remove phosphate. Figure 2 is an HPLC-MS chromatogram of the alkaline phosphatase treated reaction mixture, where two peaks were observed at 254 nm. The first peak at elution time 1.918 min represents pseudouridine. The second peak at elution time 2.081 min represents uracil. Figure 3 is a comparative HPLC-MS chromatogram of commercially available pseudouridine. Due to the presence of organic solvents (methanol and acetonitrile) used for sample preparation, the elution peak corresponding to pseudouridine is observed at a slightly longer retention time of 2.34 s. Figure 4 is an HPLC-MS mass spectrum of the material in the peak with retention time of 1.918 min from Figure 2. This indicates that the ions detected are protonated pseudouridine (top) with mass MS(ESI+): m / z 244.80 [M+H]+ and MS(ESI+): m / z 242.75 [MH] - (The molecular weight of pseudouridine is 244 g / mol). FIG. 5A is an HPLC-MS chromatogram of the reaction mixture before treatment with alkaline phosphatase. Unconsumed uracil elutes at 2.509 min and ΨMP elutes at 4.193 min. FIG. 5B is an HPLC-MS mass spectrum of the ΨMP peak in FIG. 5A, showing that the detected ions are in mass, MS(ESI+): m / z 324.75 [M + H] + Protonated ΨMP (top) with MS (ESI+): m / z 322.75 [M - H] - (The molecular weight of ΨMP is 324.18 g / mol.)

[0108] For NMR spectroscopy, the synthesized ΨMP was purified from the reaction mixture by ion exchange chromatography (solid-phase DEAE Sephadex A-25, eluent: 0.01–0.1 M NaCl) followed by reverse-phase chromatography (C18 30 g. column, eluent: water). 1 H-NMR, 13 C-NMR and 31 P -NMR spectra were recorded on the purified material to further confirm the identity of ΨMP. 1 7 shows the H-NMR spectrum of the synthesized ΨMP in DO. ΨMP is characterized by two specific signals: a doublet at 4.80 ppm (1'-C hydrogen) and a singlet at 7.77 ppm (6-C hydrogen). The small signals at 5.24 ppm and 5.39 ppm represent ribose-1-phosphate or ribose-5-phosphate. 13 8 shows the C-NMR spectrum of the synthesized ΨMP in DO. The nine different signals represent the nine different carbon atoms of ΨMP. 31 1 shows the P-NMR spectrum. The major signal at 1.45 ppm represents the phosphorus atom of the synthesized pseudouridine-5′-monophosphate, while the smaller signal at 0.49 ppm comes from ribose-1′-phosphate or ribose-5′-phosphate.

[0109] To measure the reaction yield, a sample of the reaction mixture was treated with alkaline phosphatase and analyzed by HPLC-MS. The amount of pseudouridine in the sample was calculated by comparing the area of ​​the pseudouridine peak in the measured sample (1,886,102 area units) with the area of ​​the pseudouridine peak in a standard sample with known concentration as presented in Figure 3 (2,765,690 area units). Approximately 380 mg of ΨMP was formed in a 25 mL reaction, which resulted in a yield of about 70%.

[0110] Example 2 Formation of C-nucleoside-5'-monophosphates from various N-nucleosides A reaction mixture containing 0.48 mg / mL uridine phosphorylase, 0.4 mg / mL phosphopentomutase, 0.5 mg / mL pseudouridine-5'-monophosphate (ΨMP) glycosidase, 0.5 mM MnCl2, and 125 mM potassium phosphate buffer (pH 7.0) was mixed with 10 mM 2-thiouridine or 10 mM 4-thiouridine or 10 mM 4-thio-2'-deoxyuridine in a total volume of 0.1 mL and shaken for 16 hours at 37°C. The resulting products were analyzed by TLC as illustrated by Figure 9. C-nucleoside-5'-monophosphate formation is observed from all three starting nucleosides.

[0111] Example 3 Production of pseudouridine monophosphate on a 1 liter scale A reaction mixture containing 0.03 mg / mL uridine phosphorylase, 0.26 mg / mL phosphopentomutase, 0.29 mg / mL pseudouridine-5'-monophosphate (ΨMP) glycosidase, 200 mM uridine, 0.5 mM MnCl2, and 240 mM potassium phosphate buffer pH 8.0 in a total volume of 1 L was stirred at 300 rpm and 37°C in a jacketed 1 L stirred tank reactor (Radleys, batch reactor, glass). The reaction was monitored over a time course of 24.5 hours by the described high performance liquid chromatography (HPLC) method. Measurements were performed in duplicate and average values ​​were determined.

[0112] As shown in Figure 10, pseudouridine monophosphate concentration increased constantly while uridine concentration decreased. Higher pseudouridine monophosphate formation was achieved while uracil levels steadily decreased over time. After 24.5 hours, the following product distribution was achieved: 0.264 mM uridine, 8.147 mM uracil, 191.58 mM pseudouridine monophosphate. Analysis of ribose-phosphate intermediates using HPLC ion chromatography revealed 1.0 mM ribose-1-phosphate and 6.2 mM ribose-5-phosphate after 24.5 hours. Products, substrates, and intermediates were identified based on HPLC analysis and comparison with commercial standards (Sigma Aldrich, Merck, BOCSCI Inc.).

[0113] After 24.5 h, a uridine conversion rate of 99.7% was achieved along with a pseudouridine monophosphate yield of 95.8%, correlating to approximately 62 g pseudouridine monophosphate, thereby demonstrating an effective and scalable enzymatic cascade reaction.

[0114] Example 4 Conversion of pseudouridine monophosphate to pseudouridine The material obtained from Example 3 was filtered (filtration system: Amicon® Stirred Cell, filter: Ultrafiltration Disk, 10 kDa NMW, 76 mm diameter regenerated cellulose) and used as is as the reaction mixture for the dephosphorylation step. 5 mM MgCl2 and 0.12 g / L phosphatase (Uniprot: Q181K6, origin: Clostridioides difficile (strain 630), stored in 50 mM Tris pH 8.0 at a concentration of 2.38 mg / mL) were added to the reaction mixture, which was stirred at 300 rpm and 37°C in a jacketed 1 L stirred tank reactor (Radleys, batch type reactor, glass). The reaction was monitored by HPLC for 23 hours.

[0115] After 23 hours, the following product distribution was obtained: 0.11 mM uridine, 4.77 mM uracil, 184 mM pseudouridine, 2 mM ribose-5-phosphate, 1.9 mM pseudouridine monophosphate. The reaction mixture was filtered (filtration system: Amicon® Stirred Cell, filter: Ultrafiltration Disk, 10 kDa NMW, 76 mm diameter regenerated cellulose) and frozen. This example demonstrates an efficient dephosphorylation step operation on a liter scale with a pseudouridine monophosphate conversion rate of 98.7% and a pseudouridine yield of 96.2%, corresponding to 44.9 g / L.

[0116] Example 5 Purification and crystallization of pseudouridine from the crude reaction mixture An 8 mL aliquot of the material from Example 4 was evaporated to dryness in vacuo using a rotary evaporator, followed by further azeotropic drying with i-PrOH (1 mL). The resulting solid was redissolved in 2 mL of warm (60° C.) distilled water to give a total volume of about 2.25 mL. A 1.8 mL aliquot of this mixture was cooled to room temperature, whereupon small white crystals appeared. The mixture was further cooled to 4° C. and left at this temperature overnight. The resulting white crystals were filtered, washed with cold ethanol (0.5 mL), and subsequently dried. 104 mg of white crystalline pseudouridine was obtained (38% yield). The purity of the crystals was determined by HPLC as described. Results: pseudouridine >99%, uracil 0.3%, pseudouridine monophosphate about 0.1%, uridine <0.1%.

[0117] Example 6 Conversion of uridine to pseudouridine-5'-monophosphate at elevated uridine concentrations (200 mM) in the presence of various potassium phosphate buffer concentrations A set of 1 mL reaction mixtures was prepared in a 96-deep-well plate (2 mL, round well) containing 0.14 mg / mL phosphorylase, 0.26 mg / mL phosphopentomutase, 0.29 mg / mL pseudouridine-5'-monophosphate (ΨMP) glycosidase, 200 mM uridine, 0.5 mM MnCl2, 50 mM caffeine (as an internal standard for the analysis), and varying concentrations of potassium phosphate buffer at pH 8.0 (250, 245, 240, 235, 230, 220, 210 mM). The reaction mixtures were incubated at 37°C and 800 rpm in a plate shaker (MB100-4A Thermoshaker, Allsheng) for 24 h. Measurements were performed in triplicate (n=3). A decrease in uridine conversion and pseudouridine monophosphate yield was observed when the potassium phosphate buffer (pH 8.0) concentration was below 235 mM (see FIG. 11; the average of the measurements is shown with the indicated error bars). At a potassium phosphate buffer concentration of 240 mM at pH 8.0, the following product distribution was obtained: 187 mM pseudouridine monophosphate, 5 mM uridine, and 8 mM uracil. This corresponds to a uridine conversion of 97.5% and a pseudouridine monophosphate yield of 93.5%. Thus, an increase in substrate loading is possible if the buffer capacity is increased as well.

[0118] Example 7 Conversion of uridine to pseudouridine-5'-monophosphate in the presence of reduced enzyme concentrations A 50 mL reaction mixture containing 0.03 mg / mL phosphorylase, 0.26 mg / mL phosphopentomutase, 0.29 mg / mL pseudouridine-5'-monophosphate (ΨMP) glycosidase, 240 mM potassium phosphate buffer pH 8.0, 0.5 mM MnCl2 was prepared in an Erlenmeyer shake flask. The reaction mixture was incubated at 37°C and 150 rpm (25 mm orbital shaker, New Brunswick Innova 42) for 24 hours. After 24 hours, the following product composition was obtained: 0.3 mM uridine, 4.2 mM uracil, 0.4 mM pseudouridine, 195.2 mM pseudouridine monophosphate. This procedure resulted in a uridine conversion rate of 99.9% and a pseudouridine monophosphate yield of 97.6%. Additionally, the phosphate and ribose-phosphate contents were determined to be 0.2 mM ribose-1-phosphate, 2.1 mM ribose-5-phosphate, and 30.8 mM phosphate. Thus, high uridine conversion rates and pseudouridine monophosphate yields can be achieved at enzyme concentrations of less than 0.3 mg / mL per enzyme.

[0119] Example 8 Conversion of uridine to pseudouridine-5'-monophosphate in potassium phosphate buffers with various pH values A set of 1 mL reaction mixtures was prepared in a 96 deep-well plate (2 mL, round well) containing 0.14 mg / mL phosphorylase, 0.26 mg / mL phosphopentomutase, 0.29 mg / mL pseudouridine-5'-monophosphate (ΨMP) glycosidase, 70 mM uridine, 0.5 mM MnCl2, 50 mM caffeine (internal standard) with 200 mM potassium phosphate at various pHs (6, 7, 8). The reaction mixtures were incubated at 37°C and 800 rpm in a plate shaker (MB100-4A Thermoshaker, Allsheng) for 24 h. After 24 h, for the reaction carried out at pH 6, the product composition was 21.82 mM uridine, 24.96 mM uracil, 23.21 mM pseudouridine monophosphate. This corresponds to a uridine conversion of 68.8% and a pseudouridine monophosphate yield of 33.1%. The reaction carried out at pH 7 contained 2.0 mM uridine, 11.74 mM uracil, and 56.24 mM pseudouridine monophosphate after 24 hours of reaction time, corresponding to a uridine conversion of 97.1% and a pseudouridine monophosphate yield of 79.9%. The reaction carried out at pH 8 contained 0.25 mM uridine, 6.32 mM uracil, and 63.42 mM pseudouridine monophosphate after 24 hours of reaction time, corresponding to a uridine conversion of 99.6% and a pseudouridine monophosphate yield of 90.9%. The results demonstrate that reactions at pH 8 in potassium phosphate buffer are preferred compared to pH 6 or pH 7.

[0120] Example 9 Effect of varying enzyme concentration on the conversion of uridine to pseudouridine-5'-monophosphate A set of 1 mL reaction mixtures were prepared in 96 deep-well plates (2 mL, round wells) containing 50 mM potassium phosphate buffer pH 8.0, 200 mM uridine, 0.5 mM MnCl2, 50 mM caffeine (internal standard) and the concentrations of phosphorylase (udp), phosphopentomutase (deoB) and pseudouridine-5'-monophosphate (ΨMP) glycosidase (psuG) were varied ±10% or ±15% for one, two or all three enzymes. As shown below, this resulted in an evaluated concentration range of 0.0255-0.0345 mg / mL for phosphorylase, 0.221-0.299 mg / mL for phosphopentomutase, and 0.2465-0.3335 mg / mL for pseudouridine-5'-monophosphate (ΨMP) glycosidase.

[0121] [Table 1]

[0122] Product formation was monitored for 24 hours. It was observed that the enzyme ratios tested had the same reaction profile and analyte distribution over the course of the 24 hour reaction. The reaction appeared to proceed at the same rate even when all three enzymes were reduced by 15%. Thus, the results demonstrate a robust cascade reaction that tolerates ±15% deviation in enzyme amount.

[0123] Example 10 The right enzyme ratio allows for pseudouridine-5'-monophosphate formation A 1 mL reaction mixture was prepared in a 96 deep-well plate (round well) containing 0.03 mg / mL phosphorylase, 0.26 mg / mL phosphopentomutase, 0.29 mg / mL pseudouridine-5'-monophosphate (ΨMP) glycosidase, 70 mM uridine, 0.5 mM MnCl2, 50 mM caffeine (internal standard for analysis) and 200 mM potassium phosphate buffer pH 7.0. The mixture was incubated at 37°C for 24 hours. After 24 hours, the following product formation was observed: 51 mM pseudouridine monophosphate, 15.4 mM uracil, 3.6 mM uridine. This corresponds to a pseudouridine monophosphate yield of 72% and a uridine conversion rate of 95%. The yield is comparable to that of Example 1 (about 70%), although lower individual and total enzyme concentrations were used in this example. The results demonstrate that enzyme loading is not the only factor to consider in achieving high yields of pseudouridine-5'-monophosphate. Together with Example 9, the results demonstrate that the enzyme ratio of phosphorylase, phosphopentomutase, and pseudouridine-5'-monophosphate (ΨMP) glycosidase is also important.

[0124] Example 11 Dephosphorylation of pseudouridine-5'-monophosphate A reaction mixture was prepared containing 5.1 mM uracil, 0.17 mM uridine, 50 mM caffeine (internal standard), 184.1 mM pseudouridine monophosphate, 5 mM MgCl2, and 0.09 mg / mL phosphatase (Uniprot: Q181K6, origin: Clostridioides difficile (strain 630), stored in 50 mM Tris pH 8.0 at a concentration of 2.07 mg / mL). 785 mL of the resulting reaction mixture was stirred in a jacketed 1 L stirred tank reactor (Radleys, batch reactor, glass) at 300 rpm and 37 °C for 23.5 hours. The reaction was monitored by HPLC over a period of 23.5 hours.

[0125] After 23.5 hours the following product distribution was obtained: 0.15 mM uridine, 5.6 mM uracil, 181.7 mM pseudouridine, and 2 mM pseudouridine monophosphate. The reaction mixture was filtered (filtration system: Amicon® Stirred Cell, filter: Ultrafiltration Disk, 10 kDa NMW, 76 mm diameter regenerated cellulose) and frozen.

Claims

1. 1. A method for synthesizing C-nucleoside-5'-monophosphate, comprising: a) reacting an N-nucleoside with a phosphate source in the presence of a nucleoside phosphorylase to form a first intermediate, wherein the nucleoside comprises a pentose sugar and a first base, and the first intermediate comprises a pentose-1-phosphate; b) reacting the first intermediate comprising a pentose-1-phosphate with a phosphomutase to form a second intermediate, wherein the second intermediate comprises a pentose-5-phosphate; c) reacting the second intermediate comprising a pentose-5-phosphate with a second base in the presence of a nucleoside-5'-monophosphate C-glycosidase to form the C-nucleoside-5'-monophosphate; The method includes:

2. 2. The method of claim 1, wherein steps a), b) and c) are carried out as a one-pot reaction.

3. the N-nucleoside comprises a compound according to formula Ia; the first intermediate comprises a compound according to formula Ib; the second intermediate comprises a compound according to formula Ic; and the C-nucleoside-5'-monophosphate comprises a compound according to formula 1d. 【Chemistry 1】 [In the formula, R 1 and R 2 are H, OH, and OCH. 3 , F, N.H. 2 , and N 3 are independently selected from The method according to claim 1 or claim 2.

4. R 1 and R 2 The method of claim 3, wherein each is OH.

5. R 1 is OH and R 2 is H, or R 1 and R 2 is H, or R 1 is OH and R 2 OCH 3 or R 1 N 3 and R 2 is H, or R 1 is OH and R 2 N 3 or R 1 is H and R 2 is OH, or R 1 is F and R 2 is OH, or R 1 OCH 3 and R 2 is OH, or R 1 is OH and R 2 The method of claim 3, wherein is F.

6. 6. The method of claim 1, wherein the first base is selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, and 2-thiouracil.

7. 7. The method of claim 1, wherein the second base is selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, and 2-thiouracil.

8. 8. The method of claim 1, wherein the first base is the same as the second base.

9. 9. The method of claim 8, wherein the second base in step c) is the first base produced as a by-product of the reaction in step a).

10. 10. The method of claim 8 or claim 9, wherein the first and second bases comprise uracil.

11. 11. The method of any one of claims 1 to 4 and 6 to 10, wherein the N-nucleoside in step a) comprises uridine and the C-nucleoside-5'-monophosphate comprises pseudouridine-5'-monophosphate.

12. 10. The method of claim 8 or claim 9, wherein the first and second bases comprise 4-thiouracil or 2-thiouracil.

13. 6. The method of claim 1, wherein the first base and the second base are different.

14. 14. The method of claim 13, wherein the nucleoside-5' phosphate C-glycosidase is reactive toward the second base but unreactive toward the first base.

15. 15. The method of claim 13 or 14, wherein the first base is selected from cytosine, guanine, thymine, adenine, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-(3-aminoallyl)uracil, and / or the second base is selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, 2-thiouracil, 2-amino-1H-pyrrole-5-carboxylate, and 1,4-naphthoquinone.

16. 16. The method of any one of claims 1 to 4 and 13 to 15, wherein the first base comprises thymine, the second base comprises N3-methyluracil, the N-nucleoside in step a) comprises 5-methyluridine, and the C-nucleoside-5'-monophosphate comprises N3-methylpseudouridine-5'-monophosphate.

17. the N-nucleoside comprises a compound according to formula IIa, the first intermediate comprises a compound according to formula IIb, the second intermediate comprises a compound according to formula IIc, and the C-nucleoside 5' monophosphate comprises a compound according to formula IId. 【Chemistry 2】 [In the formula, R 1 and R 2 are H, OH, and OCH. 3 , F, N.H. 2 , and N 3 are independently selected from The method according to claim 1 or claim 2.

18. R 1 and R 2 The method of claim 17, wherein each is OH.

19. In step a), the N-nucleoside comprises a compound according to formula IIIa, the first intermediate comprises a compound according to formula IIIb, the second intermediate comprises a compound according to formula IIIc, and the C-nucleoside 5' monophosphate comprises a compound according to formula IIId. 【Chemistry 3】 [In the formula, R 1 and R 2 are H, OH, and OCH. 3 , F, N.H. 2 , and N 3 are independently selected from The method according to claim 1 or claim 2.

20. R 1 and R 2 20. The method of claim 19, wherein each is OH.

21. the N-nucleoside comprises a compound according to formula IVa, the first intermediate comprises a compound according to formula IVb, the second intermediate comprises a compound according to formula IVc, and the C-nucleoside 5' monophosphate comprises a compound according to formula IVd. 【Chemistry 4】 [In the formula, R 1 and R 2 are H, OH, and OCH. 3 , F, N.H. 2 , and N 3 are independently selected from The method according to claim 1 or claim 2.

22. R 1 and R 2 22. The method of claim 21 , wherein each is OH.

23. 23. The method of any one of claims 17 to 22, wherein the first base and / or the second base is selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, and 2-thiouracil.

24. 24. The method of any one of claims 17 to 23, wherein the first base is the same as the second base.

25. 25. The method of claim 24, wherein the second base of step c) is the first base produced as a by-product of the reaction of step a).

26. 23. The method of any one of claims 17 to 22, wherein the first base and the second base are different.

27. 27. The method of claim 26, wherein the nucleoside 5'-phosphate C-glycosidase is active toward the second base but inactive toward the first base.

28. 28. The method of claim 26 or 27, wherein the first base is selected from cytosine, guanine, thymine, adenine, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-(3-aminoallyl)uracil, and / or the second base is selected from uracil, 3-methyluracil, 6-aminouracil, 4-thiouracil, 2-thiouracil, 2-amino-1H-pyrrole-5-carboxylate, and 1,4-naphthoquinone.

29. 29. The method according to any one of claims 1 to 28, wherein the nucleoside phosphorylase comprises an enzyme selected from purine-nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, thymidine phosphorylase, uridine phosphorylase and guanosine phosphorylase, preferably, the nucleoside phosphorylase comprises an enzyme selected from pyrimidine-nucleoside phosphorylase, thymidine phosphorylase and uridine phosphorylase.

30. 30. The method of claim 29, wherein the nucleoside phosphorylase comprises a uridine phosphorylase.

31. 31. The method of any one of claims 1 to 30, wherein the nucleoside 5'-phosphate C-glycosidase comprises a pseudouridylate synthase, and optionally the pseudouridylate synthase is selected from YeiN, SdmA, and AlnA.

32. 32. The method of any one of claims 1 to 31, wherein the phosphomutase comprises a phosphopentomutase, and optionally, the phosphopentomutase comprises a deoB phosphopentomutase.

33. 33. The method according to any one of claims 1 to 32, wherein the C-nucleoside-5'-monophosphate obtained in step c) is further treated with alkaline phosphatase.

34. 34. The method of any one of claims 1 to 33, wherein the source of phosphate comprises a compound selected from soluble alkali metal or alkaline earth metal phosphates, and optionally wherein the source of phosphate comprises a compound selected from sodium dihydrogen phosphate, sodium trisodium phosphate, sodium hydrogen phosphate, potassium dihydrogen phosphate, potassium tripotassium phosphate, potassium hydrogen phosphate, potassium phosphate buffer at about pH 7, and potassium phosphate buffer at about pH 8.

35. 35. The method of any one of claims 1 to 34, wherein steps a), b) and c) are carried out at about 37°C.

36. 36. The method according to any one of claims 1 to 35, wherein the C-nucleoside-5'-monophosphate obtained in step c) is purified by one or more methods selected from HPLC, ion exchange chromatography and reverse phase chromatography.

37. 37. The method according to any one of claims 1 to 36, wherein the concentration of the phosphorylase used in step a) is between 0.02 and 0.6 mg / mL, and / or the concentration of the phosphomutase used in step b) is between 0.2 and 0.6 mg / mL, and / or the concentration of the nucleoside 5'-phosphate C-glycosidase used in step c) is between 0.2 and 0.6 mg / mL.

38. 38. The method of any one of claims 1 to 37, wherein the enzyme is immobilized on a solid support.

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