Enzymatic process for the production of n1-methylpseudouridine 5'-triphosphate

Abstract

The invention relates to a process for the production of N-C1-8 alkyl pseudouridine 5'-triphosphate, such as N1-methylpseudouridine 5'-triphosphate, comprising an enzymatic cascade phosphorylation of N-C1-8 alkyl pseudouridine. The process enables the production of higher concentrations of the triphosphate product than previously known, thereby making it possible to run the cascade on a commercial scale. ATP may be used as the source of phosphate and can be enzymatically regenerated by an acetyl kinase. The process may further comprise the step of an enzyme-catalyzed conversion of pseudouridine to N-C1-8 alkyl pseudouridine, optionally with a simultaneous enzyme-catalyzed recycling of an alkyl-providing cofactor. The overall process provides an efficient method for the preparation of valuable fine chemicals, and is more environmentally friendly than conventional synthetic methods.

Description

[0001] ENZYMATIC PROCESS FOR THE PRODUCTION OF Nl-METHYLPSEUDOURIDINE 5'-TRI PHOSPHATE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to Swedish patent application No. 2430460-2, filed September 10, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] The invention relates to a process for the production of N-Ci-g alkyl pseudouridine 5' -triphosphate, such as Nl-methylpseudouridine 5' -triphosphate, comprising an enzymatic cascade phosphorylation of N-CI-8 alkyl pseudouridine. The process enables the production of higher concentrations of the triphosphate product than previously known, thereby making it possible to run the cascade on a commercial scale. Adenosine triphosphate (ATP) may be used as the source of phosphate and can be enzymatically regenerated by an acetyl kinase. The process may further comprise the step of an enzyme-catalyzed conversion of pseudouridine to N-Ci-g alkyl pseudouridine, optionally with a simultaneous enzyme-catalyzed recycling of an alkyl-providing cofactor. The overall process provides an efficient method for the preparation of valuable fine chemicals, and is more environmentally friendly than conventional synthetic methods.

[0006] BACKGROUND

[0007] Ribonucleoside triphosphates are used as building blocks for the polymerase-mediated synthesis of RNA molecules, and have become of increasing importance with the development of messenger RNA (mRNA) vaccines. The rapid advancement of mRNA-based vaccines has created significant demand for modified nucleosides that enhance mRNA stability, expression, and reduce innate immunogenicity. A particular ribonucleoside that received much attention during recent years is Nl- methylpseudouridine, which was included in the mRNA based COVID-19 vaccines by Pfizer-BioNTech (Comirnaty®) and Moderna Therapeutics (Spikevax®) (Morals et al., Front. Cell Dev. Biol. 2021, vol. 9, art. 789427). As the application of mRNA technology expands, the need for cost-effective, scalable, and regulatory-compliant methods to produce these modified nucleotides has become critical.

[0008] Earl et al. disclose the preparation of Nl-methylpseudouridine by chemical methylation of pseudouridine (J. Heterocycl. Chem. 1977, vol. 14, p. 699-700). A method for the multi-gram preparation of Nl-methylpseudouridine-5'-O-triphosphate by chemical phosphorylation of Nl-methylpseudouridine was disclosed by Shanmugasundaram et al. (Nucleosides Nucleotides Nucleic Acids 2016, vol. 35, p. 356-362; Curr. Protoc. Nucleic Acid Chem 2016, Suppl. 67, 13.15.1-13.15.10). This "one-pot, three-step" method provides the triphosphate product with high purity but only in moderate yields.

[0009] EP 4306646 discloses a process for the enzymatic conversion of an N-nucleoside to the corresponding C-nucleoside or C-nucleotide, in particular for the conversion of uridine to pseudouridine-5'- monophosphate. The process comprises an enzymatic conversion of uridine to pseudouridine-5' - monophosphate, followed by an enzymatic phosphorylation to pseudouridine-5' -triphosphate. The same research group recently described a chemoenzymatic synthesis of Nl-methylpseudouridine-5'- triphosphate from uridine, comprising an enzymatic conversion of uridine to pseudouridine-5' - monophosphate, a chemical transformation to methylpseudouridine-5'-monophosphate, and an enzymatic conversion to methylpseudouridine-5'-triphosphate (Pfeiffer et al., Angew. Chem. Int. Ed. 2025, e202506330).

[0010] WO 2023 / 183921 describes the production of Nl-methylpseudouridine-5' -triphosphate comprising the enzymatic methylation of the pseudouridine-5'-triphosphate residues of an RNA oligonucleotide using a methyltransferase. The residues may then be digested into the corresponding nucleotide monophosphates (i.e., Nl-methylpseudouridine-5'-monophosphate) by a nuclease. The resulting monophosphates may thereafter be enzymatically converted into Nl-methylpseudouridine-5'- triphosphate by a nucleoside diphosphate kinase, such as adenosyl kinase, in the presence of ATP.

[0011] CN117587086 describes a method for the preparation of Nl-methylpseudouridine 5'-triphosphate, comprising a one-pot enzymatic cascade phosphorylation of Nl-methylpseudouridine. Catalyzed by a uridine kinase, an uridylate kinase and an acetate kinase, and using ATP and acetyl phosphate (AcP) as the phosphate donor, the method provides the triphosphate product only in low yields.

[0012] In view of the increasing importance of Nl-methylpseudouridine-5' -triphosphate, an efficient and environmentally friendly process for the preparation of this ribonucleoside is highly desirable. NP0895WQ

[0013] SUMMARY OF THE INVENTION

[0014] The object of the invention is the provision of an efficient process for the conversion of pseudouridine to N-CI-8 alkyl pseudouridine 5'-triphosphates, in particular Nl-methylpseudouridine 5' -triphosphate.

[0015] In a first aspect, the invention provides a process for the enzymatic cascade phosphorylation of an N- Ci-8 alkyl pseudouridine, or a 5'-mono- or diphosphate thereof, to the corresponding N-Ci-g alkyl pseudouridine 5' -triphosphate, wherein the output concentration of the product is at least 15 mM. In this process, the N-Ci-g alkyl pseudouridine (or the 5'-mono- or diphosphate thereof) is reacted with a source of phosphate in the presence of a nucleoside kinase and / or one or more nucleoside-phosphate kinases. In some embodiments, ATP is used as a source of phosphate and wherein enzymatically regenerated by an acetyl kinase using acetyl phosphate (AcP) as a phosphate donor. In some embodiments, acetyl phosphate is added 1 to 5 times over a 48-hour reaction. In some embodiments, the enzymatic cascade phosphorylation is performed as a one-pot reaction. As shown in the experimental section, output concentrations of Nl-methylpseudouridine 5'-triphosphate of up to 75 mM were measured (see Fig. 19D). Such concentrations make it possible to run the cascade on a commercial scale.

[0016] In a second aspect, the invention provides a process for the enzymatic alkylation of pseudouridine, or a 5'-mono-, di- or triphosphate thereof, to the corresponding N-Ci.g alkyl pseudouridine, or a 5'-mono- , di- or triphosphate thereof. This process comprises a methyltransferase-catalyzed reaction between pseudouridine (or a 5'-mono-, di- or triphosphate thereof) and a Ci-s alkyl providing cofactor. The methyltransferase is capable of catalyzing the transfer of the Ci-s alkyl group from the Ci-s alkyl providing cofactor to the pseudouridine (or a 5' -mono-, di- or triphosphate thereof). In some embodiments, the methyltransferase is a xanthosine methyltransferase (XMT). In some embodiments, the xanthosine methyltransferase comprises an amino acid sequence according to any one of SEQ ID NOs: 1 to 8, or has at least 80% sequence identity with any one of SEQ ID NOs: 1 to 8.

[0017] In a third aspect, which is related to the second aspect, the invention provides a process for the regeneration of a Ci-s alkyl providing cofactor. The Ci-s alkyl providing cofactor is enzymatically regenerated with a halide Ci-s alkyl transferase in the presence of a Ci-s alkyl donor. In some embodiments, the halide Ci-s alkyl transferase comprises an amino acid sequence according to any one of SEQ ID NOs: 9 to 11, or has at least 80% sequence identity with any one of SEQ ID NOs: 9 to 11. In some embodiments, the Ci.g alkyl providing cofactor is S-adenosylmethionine (SAM). In some embodiments, the Ci.g alkyl donor is methyl iodide or methyl tosylate.

[0018] In a fourth aspect, the invention provides an engineered xanthosine methyltransferase (XMT) enzyme having activity in catalyzing the transfer of a Ci-g alkyl group from a Ci-g alkyl providing cofactor to pseudouridine or a 5'-mono-, di- or triphosphate thereof to form Nl-Ci.g alkyl pseudouridine or a 5'- mono-, di- or triphosphate thereof. In some embodiments, the enzyme comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ. ID NOs: 5 to 8.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The terms Nl-Me-PSW, lN-Me-PSW and Me-PSW are used interchangeably in the figures.

[0021] FIG. 1 shows the methylation activity formation of Nl-methylpseudouridine (Nl-Me-PSW) of four wildtype xanthosine methyltransferases (XMT_1 to XMT_4).

[0022] FIG. 2 shows the methylation activity (formation of Nl-Me-PSW) and the enzyme melting temperature of XMT_4 and four engineered xanthosine methyltransferase variants (XMT_5 to XMT_8).

[0023] FIG. 3 shows the effect of increasing enzyme concentration on the formation of Nl-Me-PSW under standard reaction conditions.

[0024] FIG. 4 shows the effect of starting concentrations of pseudouridine (PSW) on the formation of Nl-Me- PSW.

[0025] FIG. 5 is a time-course measurement showing the effect of increasing the starting concentrations of S-adenosylmethionine (SAM) on the formation of Nl-Me-PSW.

[0026] FIG. 6 shows the formation of S-adenosylmethionine (SAM) from S-adenosyl homocysteine (SAH), using Mel and MeOTs as methyl donors with different wild-type halide methyltransferases (HMT).

[0027] FIG. 7 shows the compatibility of the methyl donor (Mel and MeOTs) with XMT_6. Formation of Me- PSW and SAH in the absence or presence of methyl donor.

[0028] FIG. 8A shows the influence of pH on SAM stability (reaction without enzyme). FIG. 8B shows the influence of pH on XMT_6 activity and SAM stability.

[0029] FIG. 9 shows the influence of the SAH concentration on the methylation activity of XMT_6.

[0030] FIG. 10 shows a plot of the formation of Nl-Me-PSW (concentrations after 24 and 48 hours) when combining HMT and XMT with SAM and methyl iodide.

[0031] FIG. 11 shows the formation of Nl-Me-PSW (concentration after 48 hours) when combining HMT and XMT with SAM and methyl iodide, and comparison with reaction controls. FIG. 12 shows the effect of adjusting enzyme concentration and pH on the formation of Nl-Me-PSW (concentration after 48 hours) when combining HMT and XMT with SAM and methyl iodide.

[0032] FIG. 13 is a time-course measurement of the multi-enzyme phosphorylation cascade of Nl-Me-PSW without ATP recycling.

[0033] FIG. 14 is a time-course measurement of the selectivity of the multi-enzyme phosphorylation cascade of Nl-Me-PSW with ATP recycling and a 5 mM substrate concentration.

[0034] FIG. 15 is a time-course measurement of the selectivity of the multi-enzyme phosphorylation cascade of Nl-Me-PSW with ATP recycling at 5 mM substrate concentration, in a Radleys reactor, with a reaction volume of 700 ml.

[0035] FIG. 16A shows formation of Nl-MePSW-3P by the multi-enzyme phosphorylation cascade in the presence of varying ATP concentrations.

[0036] FIG. 16B shows Kin 3183 activity in the presence of varying ATP concentrations.

[0037] FIG. 17 is a time-course measurement of the selectivity of the multi-enzyme phosphorylation cascade of Nl-Me-PSW with ATP recycling and an 80 mM substrate concentration.

[0038] FIG. 18 is a time-course measurement of the selectivity of the multi-enzyme phosphorylation cascade of Nl-Me-PSW with ATP recycling with an 80 mM substrate concentration with additions of AcP at 0, 5 and 22 hours.

[0039] FIG. 19 shows the time-course evolution of the concentrations of starting material Nl-Me-PSW (A), intermediate products Nl-Me-PSW-Pi (B) and Nl-Me-PSW-2Pi (C), and final product Nl-Me-PSW-3Pi (D) in the multi-enzyme phosphorylation cascade of Nl-methylpseudouridine.

[0040] FIG. 20 shows the time-course evolution of the concentrations of AMP (A), ADP (B) and ATP (C) in the multi-enzyme phosphorylation cascade of Nl-methylpseudouridine.

[0041] FIG. 21 shows the time-course evolution of the concentrations of phosphate (A) and AcP (B) in the multi-enzyme phosphorylation cascade of Nl-methylpseudouridine.

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] Production ofN-Cl salkyl pseudouridine 5'-triphosphate

[0044] In a first aspect, the present invention relates to a process for the production of N-Ci-g alkyl pseudouridine 5' -triphosphate. In this process, an N-Ci-g alkyl pseudouridine, or a 5'-mono- or diphosphate thereof, is converted to the corresponding N-Ci.g alkyl pseudouridine 5'-triphosphate by reacting the N-Ci.g alkyl pseudouridine or the 5' -mono- or diphosphate thereof with a source of phosphate in the presence of a kinase and / or one or more nucleoside-phosphate kinases, and wherein the output concentration of the N-Ci-g alkyl pseudouridine 5'-triphosphate is at least 15 mM.

[0045] In some embodiments, the process for the production of N-Ci-g alkyl pseudouridine 5' -triphosphate comprises the steps of:

[0046] (a) reacting N-Ci-g alkyl pseudouridine with a source of phosphate in the presence of a nucleoside kinase (NK) to form N-Ci-g alkyl pseudouridine 5'-monophosphate; and

[0047] (b) reacting the N-Ci-g alkyl pseudouridine monophosphate with a source of phosphate in the presence of a nucleoside-phosphate kinase (NPK) to form N-Ci-g alkyl pseudouridine 5'- triphosphate; and wherein the output concentration of the N-Ci-g alkyl pseudouridine 5'-triphosphate is at least 15 mM.

[0048] In some embodiments, the process for the production of N-Ci.g alkyl pseudouridine 5' -triphosphate comprises the step of:

[0049] (a) reacting a N-Ci.g alkyl pseudouridine monophosphate with a source of phosphate in the presence of a nucleoside-phosphate kinase (NPK) to form N-Ci.g alkyl pseudouridine 5'- triphosphate; and wherein the output concentration of the N-Ci.g alkyl pseudouridine 5'-triphosphate is at least 15 mM.

[0050] Nucleoside kinases are enzymes that belong to the transferases family, specifically those transferring phosphorus-containing groups (phosphotransferases). As used herein, a nucleoside kinase (NK) is an enzyme that catalyzes the reversible phosphorylation of nucleosides. As used herein, a nucleoside- phosphate kinase (NPK) is an enzyme that catalyzes the reversible transfer of a phosphate group to a nucleoside mono- or diphosphate.

[0051] Depending on whether the substrate is a nucleoside monophosphate or a diphosphate, a nucleoside- phosphate kinase (NPK) may also be more specifically referred to as a nucleoside-monophosphate kinase (NMPK) or a nucleoside-diphosphate kinase (NDPK), respectively. It should be understood that certain NPKs display both NMPK and NDPK activity (Amiri et al., Int. J. Biochem. Cell Biol. 2013, vol. 45, p. 925-931). In certain embodiments, ATP is used as a source of phosphate, and regeneration of the ATP is catalyzed enzymatically by acetyl kinase using acetyl phosphate as the phosphate donor.

[0052] The high-energy adenosine triphosphate (ATP) molecule usually acts as a phosphate group donor, producing adenosine diphosphate (ADP). ADP can further act as a phosphate group donor, producing adenosine monophosphate (AMP). It is known in the art that recycling of the ATP cofactor can be performed by a variety of enzymes in combination with a variety of phosphate donors (Pfeiffer et al., Nat. Commun. 2020, vol. 11, 6270; Tavanti et al., Green Chem. 2021, vol. 23, p. 828-837). For example, the recycling of the ATP cofactor can be performed by acetyl kinases (AcK) and a phosphate donor group such as acetyl phosphate (AcP). As used herein, an "acetyl kinase" or "acetyl phosphate kinase" (AcPK)) refers to an enzyme that by naming convention is considered to catalyze the reversible phosphorylation of acetate in the presence of ATP and a divalent cation as a cofactor. In the present invention however, an AcPK may be utilized for the reverse reactions ADP+AcP -> ATP+Ac or AMP+AcP -> ADP+Ac in the presence of appropriate relative concentrations of the substrates and products.

[0053] An N-CI-8 alkyl pseudouridine may be reacted with a source of phosphate in the presence of nucleoside kinase (NK) to form the corresponding N-Ci-g alkyl pseudouridine 5'-monophosphate. In some embodiments, the nucleoside kinase belongs to EC 2.7.1.145. In a preferred embodiment, the nucleoside kinase comprises an amino acid sequence according to SEQ ID NO: 12, or has at least 80% sequence identity, such as at least 85%, 90%, 95%, 97%, 98%, 99% or complete sequence identity, with SEQ ID NO: 12. The nucleoside kinase may be present in a concentration of from about 0.6 pM to about 10 pM, such as from about 1 pM to about 10 pM, from about 2 pM to about 8 pM, from about 2 pM to about 6 pM, from about 3 pM to about 5 pM, or in a concentration of from about 1 pM to about 4 pM, such as from about 1 pM to about 3 pM, from about 1 pM to about 2.5 pM, from about 1 pM to about 2 pM, or from about 1 pM to about 1.5 pM. In some embodiments, the concentration of the nucleoside kinase is about 1 pM, about 1.5 pM, about 2 pM, about 3 pM, about 3.2 pM, about 3.5 pM or about 4 pM.

[0054] An N-CI-8 alkyl pseudouridine monophosphate may be reacted with a source of phosphate in the presence of a nucleoside-phosphate kinase (NPK) to form N-Ci-g alkyl pseudouridine 5'-diphosphate. The nucleoside-phosphate kinase may be an NMPK, as defined above. An N-Ci-g alkyl pseudouridine diphosphate may be reacted with a source of phosphate in the presence of nucleoside-phosphate kinase (NPK) to form N-Ci-g alkyl pseudouridine 5'-triphosphate. The nucleoside-phosphate kinase may be an NDPK, as defined above. A single NPK enzyme or a combination of separate NMPK and NDPK NP0895WQ enzymes can be employed for the conversion of N-Ci-g alkyl pseudouridine monophosphate to N-Ci-g alkyl pseudouridine triphosphate. In some embodiments, the nucleoside-phosphate kinase comprises a phosphotransferase (EC 2.7.4.-). In a preferred embodiment, the nucleoside-phosphate kinase comprises an amino acid sequence according to any one of SEQ ID NOs: 13 or 14, or has at least 80% sequence identity, such as at least 85%, 90%, 95%, 97%, 98%, 99% or complete sequence identity, with any one of SEQ ID NOs: 13 or 14, preferably SEQ ID NO: 13. The NPKs may be present in a combined concentration (e.g., either the concentration of NPK alone, or the combined concentrations of NMPK + NMDK) of from about 1 pM to about 200 pM, such as from about 2 pM to about 150 pM , from about 10 pM to about 150 pM, from about 20 pM to about 150 pM, from about 30 pM to about 100 pM, from about 40 pM to about 100 pM, from about 40 pM to 80 pM, from about 40 to about 60 pM, from about 40 to about 50 pM, from about 45 to about 55 pM, from about 50 pM to about 60 pM, from about 40 pM to 45 pM, from about 45 pM to about 50 pM, from about 50 pM to about 55 pM, or in a concentration of from about 50 pM to about 200 pM. In some embodiments, the concentration of the nucleoside-phosphate kinase is about 20 pM, about 30 pM, about 40 pM, about 45 pM, about 50 pM, about 55 pM, about 58 pM, about 60 pM, or about 70 pM.

[0055] In some embodiments, AcPK is used as an enzyme having NPK activity. It is known in the prior art that AcPK can also be used to form triphosphates of certain molecules (Ding et al., Appl Biochem Biotechnol 2020, vol. 190, p. 1271-1288; Winkler et al., Journal of Biological Chemistry 2023, vol. 299, 104746). The inventors have discovered that while AcPK can also be employed to form N-Ci.g alkyl pseudouridine 5'-triphosphate from the corresponding mono- or diphosphate, NPKs SEQ ID Nos: 13 or 14, are more effective for the formation of N-Ci.g alkyl pseudouridine 5' -triphosphate from N-Ci.g alkyl pseudouridine 5' -diphosphate. SEQ ID NO: 13 has been reported to display diphosphate kinase activity for other substrates (Amiri et al., Int. J. Biochem. Cell Biol. 2013, vol. 45, p. 925-931).

[0056] In one embodiment, the phosphorylation cascade is operated under conditions that enable the recycling of ATP and the production of higher concentrations of Nl-methylpseudouridine 5'- triphosphate than previously known. The operation of manufacturing processes at high concentrations affords known economic benefits. For example, production throughput can be higher, commercial reactors with smaller volumes can be deployed, and the energy requirements for the isolation and purification of the product can be reduced.

[0057] The operation of the phosphorylation cascade to enable higher output concentrations of Nl- methylpseudouridine 5'-triphosphate does present certain challenges. For example, it is known that NP0895WQ

[0058] ATP can inhibit enzymes when deployed at high concentrations. (See, for example, Hsu et al., Mol Pharmacol. 2005, vol. 67, p. 806-814, who reported a marked inhibitory effect for certain enzymes at ATP concentrations of 1 mM). For the operation of the phosphorylation cascade to enable higher output concentrations of Nl-methylpseudouridine 5'-triphosphate, the use of ATP concentrations of up to 5 mM is demonstrated herein, without a significant inhibitory effect on the activity of NMPK_1 (SEQ 13, FIG. 16). To further increase output concentrations of Nl-methylpseudouridine 5'- triphosphate, recycling of the ATP cofactor is required. Recycling of ATP removes the need for stoichiometric amounts of ATP (an expensive reagent), and avoids the build-up of high concentrations of ADP and AMP, enabling a simpler isolation and purification of the product, and thereby providing significant economical benefits to the process. Preferably, the recycling entails that on average, each ATP molecule undergoes at least 2 cycles, or in an increasing order of preference at least 3, 4, 5, 8, 10, 15, 20, 25, 30, 40, 50 or 100 cycles of regeneration during the process.

[0059] Recycling of the ATP cofactor can be performed by a variety of enzymes in combination with a variety of phosphate donors. In one embodiment, the preferred enzyme is an AcPK, belonging to EC 2.7.2.1 or EC 2.7.2.12. In a preferred embodiment, the acetyl kinase used in the present invention comprises an amino acid sequence according to any one of SEQ ID NOs: 15 to 17, or has at least 80% sequence identity, such as at least 85%, 90% , 95%, 97%, 98%, 99% or complete sequence identity, with any one of SEQ ID NOs: 15 to 17, preferably SEQ ID NO: 15. The acetyl kinase may be present in a concentration of from about 0.6 pM to about 10 pM, such as from about 1 pM to about 10 pM, from about 1 pM to about 8 pM, from about 1 pM to about 6 pM, from about 1 pM to about 4 pM, from about 1 pM to about 3 pM, such as from about 1 pM to about 2 pM, from about 1.5 pM to about 2.5 pM, from about 2 pM to about 3 pM, or from about 1.5 pM to about 2 pM, from about 2 pM to about 2.5 pM, or from about 2.5 pM to about 3 pM. In some embodiments, the concentration of the acetyl kinase is about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM or about 4 pM.

[0060] The source of phosphate for the AcPK is preferably acetyl phosphate (AcP). Sources of acetyl phosphate include lithium potassium acetyl phosphate, disodium acetyl phosphate and dilithium acetyl phosphate. Lithium potassium acetyl phosphate is available commercially from Sigma-Aldrich, the synthesis of disodium acetyl phosphate solutions can be performed according to Tasnadi et al. (Adv. Synth. Catal. 2018, vol. 360, p. 2394-2401) and the synthesis of dilithium acetyl phosphate can be performed according to Smith et al. (Org. Process Res. Dev. 2021, vol. 25, p. 1402-1413). Furthermore, AcP can be generated enzymatically in situ using a pyruvate oxidase enzyme (McIntosh et al., ACS Cent. Sci. 2021, vol. 7, p. 1980-1985). A further challenge associated with the operation of the phosphorylation cascade to enable higher output concentrations of Nl-methylpseudouridine 5'-triphosphate is the hydrolysis of AcP, which is known to occur under aqueous conditions such as those used for enzymatic reactions (Whicher et al., Orig Life Evol Biosph. 2018, vol. 48, p. 159-179). Hydrolysis of AcP not only reduces AcP concentration in the reaction, limiting the availability of the phosphate donor, it also leads to the formation of acetic acid which at high concentrations leads to an undesirable reaction pH which can slow or stop the reaction. Hydrolysis of AcP furthermore reduces the reaction selectivity which limits the output product concentration.

[0061] Hydrolysis of AcP can be diminished by reducing the reaction temperature. The reaction may be performed at temperatures from 20-40 °C, or more preferably 25-40 °C. The inventors have also found that the challenges associated with AcP hydrolysis can be further mitigated by sequential additions of AcP throughout the time-course of the reaction (see, for example, Figure 17 vs Figure 18). Controlled dosing of AcP throughout the reaction maintains the required AcP concentration whilst minimising build-up of hydrolysis products. Acetyl phosphate additions of 0.25 - 2 times substrate concentration can be performed 1-5 times over a 48-hour reaction. For example, when operating the phosphorylation cascade with a high substrate concentration of 80 mM, the advantage of sequential additions of AcP can be seen by comparing Figures 17 (AcP addition only at 0 hours) with Figure 18 (3 AcP additions at 0, 5 and 22 hours). In some embodiments, additional acetyl phosphate is added after about 4 hours, after about 8 hours, and after about 12 to 24 hours. In some embodiments, additional acetyl phosphate is added after about 4 hours, after about 8 hours, and after about 12 hours, such as about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours or about 24 hours. In some embodiments, additional acetyl phosphate is added after about 4 hours, after about 8 hours, and after about 24 hours.

[0062] Additionally, careful control of the reaction pH can further improve output product concentration. For example, pH monitoring and adjustment during the reaction can be advantageous. The preferred reaction pH range is from pH 6 to pH 9.5. In some embodiments, the pH of the cascade reaction is between about 6.0 and about 9.5, such as between about 7.0 and about 9.0, or between about 7.5 and 8.5. In some embodiments, the pH of the cascade reaction is about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, or about 8.3. The pH can be controlled during the reaction by dropwise addition of a base such as sodium hydroxide. In some embodiments, the initial pH of the cascade reaction is between about 8.0 and 9.0, such as between about 8.0 and 8.5, or between about 8.5 and 9.0. In some embodiments, the initial pH of the cascade reaction is about 8.5 or about 9.0.

[0063] Using a combination of the preferred conditions described above, it is possible to operate the phosphorylation cascade to enable higher output concentrations of Nl-methylpseudouridine 5'- triphosphate than previously known (FIG. 18).

[0064] In some embodiments, the process of the invention results in output concentrations of the N-Ci-g alkyl pseudouridine triphosphate of at least 15 mM. In some embodiments, the process results in output concentrations of the N-Ci-g alkyl pseudouridine triphosphate of at least 20 mM, or in an increasing order of preference at least 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 300 mM or 400mM.

[0065] In some embodiments, an N-Ci.g alkyl pseudouridine is converted to the corresponding N-Ci.g alkyl pseudouridine 5'-triphosphate comprising the steps of:

[0066] (a) reacting N-Ci.g alkyl pseudouridine with a source of phosphate in the presence of a nucleoside kinase (NK) to form N-Ci.g alkyl pseudouridine 5'-monophosphate;

[0067] (b) reacting the N-Ci.g alkyl pseudouridine monophosphate with a source of phosphate in the presence of a nucleoside-phosphate kinase (NPK) and / or acetyl kinase (AcPK) to form N-Ci. g alkyl pseudouridine 5'-triphosphate.

[0068] In some embodiments, an N-Ci.g alkyl pseudouridine is converted to the corresponding N-Ci.g alkyl pseudouridine 5'-triphosphate comprising the steps of:

[0069] (a) reacting N-Ci.g alkyl pseudouridine with a source of phosphate in the presence of a nucleoside kinase (NK) to form N-Ci.g alkyl pseudouridine 5'-monophosphate;

[0070] (b) reacting the N-Ci.g alkyl pseudouridine monophosphate with a source of phosphate in the presence of a nucleoside-phosphate kinase (NMPK) to form N-Ci.g alkyl pseudouridine 5'- diphosphate; and

[0071] (c) reacting the N-Ci.g alkyl pseudouridine diphosphate with a source of phosphate in the presence of a nucleoside-diphosphate kinase (NDPK) and / or acetyl kinase (AcPK) to form N- Ci-g alkyl pseudouridine 5'-triphosphate.

[0072] In a preferred embodiment, the N-Ci.g alkyl pseudouridine is Nl-methylpseudouridine. In some embodiments, therefore, Nl-methylpseudouridine is converted to Nl-methylpseudouridine 5' -triphosphate comprising the steps of:

[0073] (a) reacting Nl-methylpseudouridine with a source of phosphate in the presence of a nucleoside kinase (NK) to form Nl-methylpseudouridine 5' -monophosphate;

[0074] (b) reacting the Nl-methylpseudouridine monophosphate with a source of phosphate in the presence of a nucleoside-phosphate kinase (NMPK) to form Nl-methylpseudouridine 5'- diphosphate; and

[0075] (c) reacting the Nl-methylpseudouridine diphosphate with a source of phosphate in the presence of a nucleoside-diphosphate kinase (NDPK) and / or acetyl kinase (AcPK) to form Nl-methylpseudouridine 5'-triphosphate.

[0076] As also shown in the example section, the above-mentioned steps (a), (b) and (c) may be performed as a one-pot reaction. In some embodiments, the nucleoside kinase (NK) is present in a concentration of from about 0.6 pM to about 10 pM, such as about 3.2 pM; the nucleoside-phosphate kinase (NPK) is present in a concentration of from about 1 pM to about 200 pM, such as about 58 pM; and the acetyl kinase is present in a concentration of from about 1 pM to about 50 pM, such as about 11 pM. In some embodiments, the nucleoside kinase (NK) is present in a concentration of from about 1 pM to about 2 pM; the nucleoside-phosphate kinase (NPK) is present in a concentration of from about 40 pM to about 60 pM, such as from about 45 to about 50 pM; and the acetyl kinase is preferably present in a concentration of from about 1.5 pM to about 2.5 pM.

[0077] In some embodiments, Nl-methylpseudouridine is converted to Nl-methylpseudouridine 5'- triphosphate in combination with recycling of the ATP cofactor comprising the steps of:

[0078] (a) reacting Nl-methylpseudouridine with a source of phosphate in the presence of a nucleoside kinase (NK) to form Nl-methylpseudouridine 5' -monophosphate;

[0079] (b) reacting the Nl-methylpseudouridine monophosphate with a source of phosphate in the presence of a nucleoside-phosphate kinase (NPK) to form Nl-methylpseudouridine 5'- triphosphate; and

[0080] (c) recycling the ATP cofactor using acetyl kinase and acetyl phosphate as a source of phosphate.

[0081] The above-mentioned embodiment is shown in the scheme below:

[0082] As also shown in the example section, the above-mentioned steps (a), (b) and (c) may be performed as a one-pot reaction. In some embodiments, the nucleoside kinase (NK) is present in a concentration of from about 0.6 pM to about 10 pM, such as about 3.2 pM; the nucleoside-phosphate kinase (NPK) is present in a concentration of from about 1 pM to about 200 pM, such as about 58 pM; and the acetyl kinase is present in a concentration of from about 1 pM to about 50 pM, such as about 11 pM. In some embodiments, the nucleoside kinase (NK) is present in a concentration of from about 1 pM to about 2 pM; the nucleoside-phosphate kinase (NPK) is present in a concentration of from about 40 pM to about 60 pM, such as from about 45 to about 50 pM; and the acetyl kinase is present in a concentration of from about 1.5 pM to about 2.5 pM.

[0083] In some embodiments, Nl-methylpseudouridine 5' -monophosphate is converted to Nl- methylpseudouridine 5' -triphosphate in combination with recycling of the ATP cofactor comprising the steps of:

[0084] (a) reacting Nl-methylpseudouridine 5'-monophosphate with a source of phosphate in the presence of a nucleoside-phosphate kinase (NPK) to form Nl-methylpseudouridine 5'- triphosphate;

[0085] (b) recycling the ATP cofactor using acetyl kinase and acetyl phosphate as a source of phosphate.

[0086] This embodiment is shown in the scheme below: Steps (a) and (b) mentioned above may be performed as a one-pot reaction. In some embodiments, the nucleoside-phosphate kinase (NPK) is present in a concentration of from about 1 pM to about 200 pM, such as about 58 pM and the acetyl kinase is present in a concentration of from about 1 pM to about 50 pM, such as about 11 pM. In some embodiments, the nucleoside-phosphate kinase (NPK) is present in a concentration of from about 40 pM to about 60 pM, such as from about 45 to about 50 pM; and the acetyl kinase is present in a concentration of from about 1.5 pM to about 2.5 pM.

[0087] Production ofN-Cl salkyl pseudouridine

[0088] In a second aspect, the invention relates to a process for the production of N-Ci-g alkyl pseudouridine or a 5'-mono-, di- or triphosphate thereof, comprising catalyzing the reaction between pseudouridine or a 5'-mono-, di- or triphosphate thereof and a Ci.g alkyl providing cofactor with a methyltransferase capable of catalyzing the transfer of a Ci-g alkyl group from the Ci-g alkyl providing cofactor to the pseudouridine or a 5'-mono-, di- or triphosphate thereof.

[0089] Throughout the description, pseudouridine or the 5'-mono-, di- or triphosphate thereof will also be referred to as a compound of formula (I), and the N-Ci.g alkyl pseudouridine or the 5'-mono-, di- or triphosphate thereof will also be referred to as a compound of formula (II).

[0090] In some embodiments, the N-Ci.g alkyl pseudouridine or the 5' -mono-, di- or triphosphate thereof is the N3-Ci.g alkyl pseudouridine or the 5' -mono-, di- or triphosphate thereof. In some embodiments, the N-Ci.g alkyl pseudouridine or the 5'-mono-, di- or triphosphate thereof is the Nl-Ci.g alkyl pseudouridine or the 5'-mono-, di- or triphosphate thereof. The scheme below shows the process for the latter embodiment: NP0895WQ wherein R1is selected from the group consisting of hydroxy, a monophosphate ("O-MP"), a diphosphate ("O-DP") or a triphosphate ("O-TP"); and R2is Ci.g alkyl.

[0091] The process involves the transfer of a Ci.g alkyl group from the cofactor to the compound of formula (I), and is catalyzed by a methyltransferase. In some embodiments, the process involves the transfer of a Ci-4 alkyl group from the cofactor to the compound of formula (I). Preferably, the process involves the transfer of a methyl, ethyl, n-propyl or n-butyl group from the cofactor to the compound of formula (I), more preferably the transfer of a methyl or an ethyl group, and most preferably the transfer of a methyl group. In some embodiments, the N-Ci.g alkyl pseudouridine or the 5'-mono-, di- or triphosphate thereof (i.e., the compound of formula (II)) is Nl-methylpseudouridine or a 5'-mono-, di- or triphosphate thereof.

[0092] Methyltransferase

[0093] The methyltransferase may be any enzyme that is capable of catalyzing the transfer of the Ci-g alkyl group from the cofactor to the compound of formula (I). In some embodiments, the methyltransferase is a xanthosine methyltransferase (EC 2.1.1.158). In nature, xanthosine methyltransferase (XMT) enzymes catalyze the first step in the caffeine biosynthetic pathway, by transferring a methyl group to the nucleoside xanthosine. In other embodiments, the methyltransferase is a theobromine synthase (EC 2.1.1.159) or a toxoflavin synthase (EC 2.1.1.349). In a preferred embodiment, the methyltransferase comprises an amino acid sequence according to any one of SEQ ID NOs: 1 to 8, or has at least 80% sequence identity, such as at least 85%, 90%, 95%, 97%, 98%, 99% or complete sequence identity, with any one of SEQ ID NOs: 1 to 8. In a more preferred embodiment, the methyltransferase comprises an amino acid sequence according to any one of SEQ ID NOs: 5 to 8, or has at least 80% sequence identity, such as at least 85%, 90%, 95%, 97%, 98%, 99% or complete sequence identity, with any one of SEQ ID NOs: 5 to 8. In a more preferred embodiment, the methyltransferase comprises an amino acid sequence according to SEQ ID NO: 6, or has at least 80% sequence identity, such as at least 85%, 90%, 95%, 97%, 98%, 99% or complete sequence identity, with SEQ ID NO: 6. The methyltransferase may be present in a concentration of from about 0.4 pM to about 50 pM, such as from about from about 2 pM to about 45 pM, from about 4 pM to about 35 pM, from about 8 pM to about 35 pM, from about 12 pM to about 35 pM, from about 16 pM to about 35 pM, or from about 20 pM to about 35 pM, or from about 30 to about 50 pM, such as from about 30 pM to about 40 pM, such as from about 35 pM to about 45 pM, or such as from about 40 pM to about 50 pM. In some embodiments, the concentration of the methyltransferase is about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM or about 45 pM.

[0094] Cis alkyl providing cofactor and recycling thereof

[0095] In a third aspect, which is related to the second aspect, the present invention provides a process for regeneration of a Ci-g alkyl providing cofactor, catalyzed enzymatically with a halide Ci-g alkyl transferase (preferably a halide methyltransferase) in the presence of a Ci-g alkyl donor (preferably a methyl donor, most preferably methyl iodide or methyl tosylate). Preferably, the third aspect is incorporated as part of a process according to the second aspect. Needless to say, the process may include intermediate purification / concentrations steps.

[0096] Preferably, the regeneration entails that on average, each Ci-g alkyl providing cofactor molecule undergoes at least 2 cycles, or in an increasing order of preference at least 3, 4, 5, 8, 10, 15, 20, 25, 30, 40, 50 or 100 cycles of regeneration during the process.

[0097] In some embodiments, the Ci-g alkyl providing cofactor is a methyl providing cofactor. In a preferred embodiment, the Ci-g alkyl providing cofactor is S-adenosylmethionine (SAM) or a corresponding alkylated S-adenosyl homocysteine (SAH) analogue (Tang et al., Angew. Chem. Int. Ed. 2021, vol. 60, p. 1524-1527; Schulke et al., ChemBioChem 2022, vol. 23, e202100632).

[0098] When an alkylated SAH analogue (e.g., SAM) transfers its alkyl group (e.g., methyl group) to pseudouridine, it turns into S-adenosyl homocysteine (SAH) or a corresponding non-alkylated S- adenosyl homocysteine (SAH) analogue. The alkylated SAH analogue (e.g., SAM) may however be regenerated by conversion of SAH in the presence of a suitable alkyl donor (e.g., methyl donor) and a halide methyltransferase. This allows the cofactor to be used in only catalytic amounts.

[0099] As used herein, a halide methyltransferase (HMT) is any enzyme that is capable of catalyzing the transfer of a Ci.g alkyl group, more preferably a methyl group, from a donor compound to S-adenosyl- L-homocysteine (SAH). In some embodiments, the conversion of S-adenosyl homocysteine (SAH) to S- adenosylmethionine (SAM) is catalyzed by a halide methyltransferase (EC 2.1.1.165) or a 23S rRNA (uridine2552-2'-O-)-methyltransferase (EC 2.1.1.166). In a preferred embodiment, the halide methyltransferase comprises an amino acid sequence according to any one of SEQ. ID NOs: 9 to 11, or has at least 70% sequence identity, such as at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or complete sequence identity, with any one of SEQ. ID NOs: 9 to 11.

[0100] Examples of suitable alkyl donors include, but are not limited to, methyl iodide, ethyl iodide, n-propyl iodide, n-butyl iodide, methyl tosylate, methyl sulphate, methyl fluorosulphonate, methyl boronic acid and dimethylcarbonate. Preferred methyl donors include methyl iodide and methyl tosylate. In a most preferred embodiment, the methyl donor is methyl iodide.

[0101] A process of the second aspect comprising a catalytic reconversion of a methyl providing cofactor according to the third aspect is shown in the scheme below:

[0102] Experiments have shown that SAM requires an acidic or a neutral pH to avoid degradation, whereas XMT requires a basic pH to ensure activity retention (see Example 6.3 and Figures 8A and 8B). For an effective methylation reaction deploying both SAM and XMT, it is therefore important that the cascade reaction is performed at the optimum pH range. The process of the second aspect may involve the step of selecting a pH for the reaction.

[0103] In some embodiments, the pH of the cascade reaction is between about 7.0 and about 8.0, such as between about 7.0 and about 7.5, or between about 7.5 and 8.0. In some embodiments, the pH of the cascade reaction is between about 7.5 and about 7.7, between about 7.6 and 7.8, between about 7.7 and 7.9, or between about 7.8 and 8.0. In some embodiments, the pH of the cascade reaction is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. It has further been observed that the SAH that is formed during the methylation reaction has an inhibitory effect on XMT activity (see Example 6.4 and Figure 9). For an effective methylation reaction, it is therefore important that the amount of SAH in the reaction mixture is kept as low as possible. The amount of SAH may be controlled by the concentration of halide methyltransferase, as well as by the ratio of the concentrations of HMT and XMT.

[0104] Thus, the process of the second aspect may involve the step of selecting the concentration of the Ci.g alkyl transferase used to catalyze the regeneration of the Ci.g alkyl providing cofactor. The process of the second aspect when combined with the process of the first aspect may additionally involve step of selecting the ratio between (i) the halide Ci-g alkyl transferase (such as halide methyltransferase) used to catalyze the regeneration of the Ci-g alkyl providing cofactor to (ii) the methyltransferase capable of catalyzing the transfer of a Ci-g alkyl group from the Ci-g alkyl providing cofactor to the pseudouridine or a 5'-mono-, di- or triphosphate thereof. The molar ratio between the HMT and the XMT should be in the range of about 1:50, about 1:25, about 1:12, about 1:6 or about 1:2.

[0105] In some embodiments, the halide methyltransferase is present in a concentration of from about 0.8 pM to about 16 pM, such as from about 1.9 pM to about 16 pM, from about 4 pM to about 16 pM , from about 8 pM to about 16 pM, from about 12 pM to about 16 pM, or from about 0.8 pM to about 12 pM, such as from about 1 pM to about 4 pM, from about 1 pM to about 3 pM, from about 1 pM to about 2 pM, or from about 2 pM to about 3 pM, or such as from about 1.9 pM to about 12 pM, from about 4 pM to about 12 pM, or from about 8 pM to about 12 pM, or from about 0.8 pM to about 8 pM, such as from about 1.9 pM to about 8 pM, from about 4 pM to about 8 pM, or from about 0.8 pM to about 4 pM, such as from about 1.9 pM to about 4 pM or from about 0.8 pM to about 1.9 pM.

[0106] The presence of a methyl donor is a necessary feature of the process of both the first and the second aspect. The methyl donor supplies the halide methyltransferase with the necessary methyl group to catalytically regenerate the SAM cofactor. The preferred methyl donor is methyl iodide. The concentration of the methyl iodide is an important feature in the process and must be selected to enable high conversions of pseudouridine with high selectivities for the formation of Nl-Me- pseudouridine. The methyl iodide concentration must not be too low such that conversion remains low, and yet must not be too high such that the enzymatic activity is compromised (see Example 7.2 and Figure 11), or such that a high background of non-selective methylation of pseudouridine may occur. Since the methyl iodide provides the stoichiometric supply of the methyl group, its concentration will need to at least match the concentration of pseudouridine to reach high conversions. It may be beneficial to supply methyl iodide in aliquots during the reaction to keep the concentrations at low enough levels to prevent the suppression of enzyme activity, but high enough to enable the catalytic regeneration of SAM cofactor and high conversion of pseudouridine to Nl-Me- pseudouridine.

[0107] In some embodiments, the methyl iodide is present in concentrations of from about 1 to about 100 mM, such as from about 10 mM to about 80mM , from about 20 mM to about 80 mM, from about 30 mM to about 70 mM, or from about 40 mM to about 70 mM, or from about 40 mM to about 60 mM, from about 45 mM to about 55 mM, or about 50 mM.

[0108] Controlled dosing of methyl iodide aliquots throughout the reaction maintains the required methyl iodide concentration necessary to enable the catalytic regeneration of SAM, whilst minimising the suppression of enzyme activity and background non-selective methylation of pseudouridine. Methyl iodide additions of 0.1 - 2 times substrate concentration can be performed 1-10 times over a 48 hour reaction.

[0109] The recycling of the SAM cofactor is highly advantageous for the methylation reaction for several reasons. SAM is a complex molecule that is unstable under physiological conditions, undergoing e.g. depurination and epimerisation at the sulfur centre. Additionally, the need for stoichiometric amounts of expensive SAM creates significant economic challenges for large-scale industrial applications. Recycling of SAM solves these issues by providing a more stable and continuous supply, thereby reducing overall costs. As already mentioned above, recycling SAM also prevents the build-up of SAH, which inhibits methyltransferases. Recycling of SAM therefore not only optimizes enzyme activity but also enhances the efficiency of methylation processes. Additionally, since the concentration of SAM must be kept low prevent SAH building up, the only way to enable the production of high concentrations of Nl-Me-pseudouridine is to enable SAM recycling

[0110] Preferably, each SAM molecule undergoes at least 2 cycles, or in an increasing order of preference at least 3, 4, 5, 8, 10, 15, 20, 25, 30, 40, 50 or 100 cycles of regeneration during the process.

[0111] In some embodiments, the process of converting pseudouridine to Nl-methylpseudouridine in combination with recycling of the SAM cofactor comprises the steps of: (a) Selecting a reaction pH between about 7.5 and 8.0; and using a buffer concentration or the addition of aliquots of reagents to enable the reaction pH to be maintained at the selected pH between 7.5 and 8.0 during the reaction

[0112] (b) Selecting a reaction temperature < 35°C to prevent the thermal degradation of SAM cofactor during the reaction

[0113] (c) Selecting a SAM cofactor concentration between about 0.5 and 5 mM

[0114] (d) Selecting an initial methyl iodide concentration between about 1.5 and 52 mM and providing aliquots of methyl iodide during the reaction to at least match the concentration of pseudouridine and to enable the production of high concentrations of Nl-Me-pseudouridine.

[0115] As also shown in the example section, the above-mentioned steps (a), (b), (c) and (d) may be performed as a one-pot reaction. Under such conditions, the methyltransferase may be present in a concentration of from about 2 pM to about 45 pM, or from about 30 pM to about 50 pM, such as about 20 pM, about 25 pM, about 30 pM, about 35 pM, about 40 pM or about 50 pM; and the halide methyltransferase may be present in a concentration of from about 0.8 pM to about 16 pM, such as from about 1 pM to about 3 pM, such as about 0.8 pM, about 1.9 pM, about 4 pM or about 8 pM.

[0116] The N-CI-8 alkyl pseudouridine product obtained in the process of the second aspect may be used as the starting material in the enzymatic cascade phosphorylation process of the first aspect. In one embodiment, the N-Ci-g alkyl pseudouridine product is N-methylpseudouridine. An overall process comprising the cascade methylation of pseudouridine and the subsequent phosphorylation of Nl- methylpseudouridine to Nl-methylpseudouridine 5' -triphosphate is shown in the scheme below: and HMT

[0117] The second aspect, and optionally also the third aspect, may be incorporated as part of a process according to the first aspect. Needless to say, the process may include intermediate purification / concentration steps, for instance after the production of N-Ci-g alkyl pseudouridine and before the conversion of the N-Ci-g alkyl pseudouridine to N-Ci-g alkyl pseudouridine 5' -triphosphate.

[0118] Engineered XMT enzymes

[0119] In a fourth aspect, the invention relates to a xanthosine methyltransferase (XMT) enzyme having activity in catalyzing the transfer of a Ci-g alkyl group from a Ci-g alkyl providing cofactor to pseudouridine or a 5'-mono-, di- or triphosphate thereof to form Nl-Ci.g alkyl pseudouridine or a 5'- mono-, di- or triphosphate thereof, wherein the enzyme comprises an amino acid sequence having at least 95% sequence identity, preferably at least 96% sequence identity, more preferably at least 97% sequence identity, more preferably at least 98% sequence identity, even more preferably least 99%, most preferably complete sequence identity according to any one of SEQ ID NOs: 5 to 8. In a more preferred embodiment, the enzyme comprises an amino acid sequence having at least 95% sequence identity, preferably at least 96% sequence identity, more preferably at least 97% sequence identity, more preferably at least 98% sequence identity, even more preferably least 99%, most preferably complete sequence identity to SEQ ID NO: 6.

[0120] The Ci-g alkyl group may be methyl. The Ci-g alkyl providing cofactor may be SAM. By "catalyzing" in this context is meant that the enzyme is at least as catalytically active as XMT_4 (SEQ ID NO: 4) in the conditions used in Example 3.

[0121] Production of pseudouridine

[0122] If pseudouridine or pseudouridine-5'-monophosphate is used as the starting material for the production of N-Ci.g alkyl pseudouridine (i.e., the process of the second aspect), these starting materials may conveniently be produced from uridine in an enzyme-catalyzed process, such as disclosed in WO 2023 / 131727. In one embodiment, this process comprises the steps of:

[0123] (a) reacting uridine with a source of phosphate in the presence of a nucleoside phosphorylase to form ribose-l-phosphate and uracil;

[0124] (b) reacting ribose-l-phosphate in the presence of a phosphomutase to form ribose-5-phosphate;

[0125] (c) reacting ribose-5-phosphate with uracil in the presence of nucleoside-5'-phosphate C-glycosidase to form pseudouridine-5' -monophosphate; and

[0126] (d) optionally, reacting pseudouridine-5'-monophosphate with an alkaline phosphatase to form pseudouridine. The reaction according to steps (a)-(d) above is shown in the following scheme:

[0127] In some embodiments, the nucleoside phosphorylase of step (a) comprises a uridine phosphorylase (EC 2.4.2.3). Uridine phosphorylases have e.g. been identified in Klebsiella aerogenes (Veiko et al., Bioorg. Khim. 1998, vol. 24, pp. 381-387; UniProt ID: 008444) and Escherichia coll (Walton et al., Nucleic Acids Res. 1989, vol. 17, p. 6741; Uniprot: P12758). The nucleoside phosphorylase may be present in a concentration of from about 0.7 pM to about 21 pM, such as from about 1 pM to about 21 pM, from about 1.4 pM to about 21 pM, or about 17.5 pM, or in a concentration of from about 0.7 pM to about 17.5 pM, such as from about 0.7 pM to about 14 pM, from about 0.7 pM to about 10.5 pM, from about 0.7 pM to about 7 pM, from about 0.7 pM to about 3.5 pM, from about 0.7 pM to about 1.7 pM, or from about 0.9 pM to about 1.2 pM. In some embodiments, the concentration of nucleoside phosphorylase is about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1.1 pM, about 1.2 pM, about 1.3 pM, about 1 .4 pM, about 1.5 pM, about 1.6 pM , or about 1.7 pM. Concentrations of enzyme below 0.7 pM may reduce the yield of the final C-nucleoside-5'- monophosphate, and concentrations of enzyme above 21 pM may have limited economical utility.

[0128] In some embodiments, the phosphomutase of step (b) comprises a phosphopentomutase (EC 5.4.2.7). In a preferred embodiment, the phosphomutase is deoB phosphopentomutase, which may be isolated from several different bacterial strains including Escherichia coli, (Valentin-Hansen et al., Nucleic Acids Res., 1984, vol. 12, p. 5211-5224; Uniprot: P0A6K6), Bacillus cereus (Panosian et al., J. Biol. Chem. 2011, vol. 268, pp. 8043-8054; Uniprot: Q818Z9), Streptococcus pneumoniae and Bacillus anthracis. Other commonly known phosphopentomutases include, 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). The phosphomutase may be present in a concentration of from about 4 pM to about 13 pM , such as from about 6.5 pM to about 13 pM, from about 9 pM to about 13 pM, or about 11 pM, or in a concentration of from about 4 pM to about 11 pM, such as from about 4 pM to about 9 pM, or from about 4 pM to about 6.5 pM. In some embodiments, the concentration of phosphomutase is about 4.5 pM, about 5.5 pM, about 6.5 pM, or about 7.5 pM. Concentrations of enzyme below 4 pM may reduce the yield of the final C-nucleoside-5'- monophosphate, and concentrations of enzyme above 13 pM may have limited economical utility.

[0129] In some embodiments, the nucleoside-5'-phosphate C-glycosidase of step (c) comprises a pseudouridylate synthase (EC 4.2.1.70) or any other enzyme having the same enzyme classification. Pseudouridylate synthase or related enzymes with comparable activity to pseudouridylate synthase have been identified and isolated from many prokaryotic sources. For example, YeiN (psuG) has been identified in Escherischia coli (Preumont et al., J. Biol. Chem. 2008, vol. 283, p. 25238-25246; Pfeiffer et al., Nat. Commun. 2020, vol. 11, 6270), AlnA has been identified in Streptomyces species (Oja et al., PNAS 2013, vol. 100, p. 1291-1296), SdmA has been identified in Streptomyces showdoensi (Palmu et al., ACS Chem. Biol. 2017, vol. 12, p. 1472-1477), and TmYeiN has been identified in Thermotoga maritima (Preumont et al., J. Biol. Chem. 2008, vol. 283, p. 25238-25246). Additionally, MinB (GenBank: QDX19370.1) has been identified in Streptomyces hygroscopicus (Kong et al., iScience 2019, vol. 22, p. 430-440) and IndA (GenBank: AFV27435.1) has been identified in Streptomyces chromofuscus. Eukaryotic homologues of pseudouridylate synthase also exist as multifunctional enzymes. In a preferred embodiment, the pseudouridylate synthase is YeiN, SdmA or AlnA. The C- nucleoside-5'-monophosphate glycosidase may be present in a concentration of from about 5.5 pM to about 17.5 pM, such as from about 9 pM to about 17.5 pM, from about 11.5 pM to about 17.5 pM, or about 14.5 pM , or in a concentration from about 5.5 to about 14.5 pM , such as from about 5.5 pM to about 11.5 pM, or from about 7 to about 10 pM. In some embodiments, the concentration of C- nucleoside-5'-monophosphate glycosidase is about 5.5 pM, about 7 pM, about 8.5 pM, about 10 pM, or about 11.5 pM. Concentrations of enzyme below 5.5 pM may reduce the yield of the final C- nucleoside-5'-monophosphate, and concentrations of enzyme above 17.5 pM may have limited economical utility.

[0130] In some embodiments, uracil is added in step (c). In some embodiments, the uracil used in step c) is the uracil produced in step a). In such an embodiment, there is no "waste" of base.

[0131] In some embodiments, steps (a), (b) and (c) are performed as a one-pot reaction.

[0132] In some embodiments, the phosphate source is a phosphate anion, such as a compound selected from soluble alkaline 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 of about pH 7. In another preferred embodiment, the source of phosphate comprises a potassium phosphate buffer of about pH 8.

[0133] If desired, the pseudouridine-5'-monophosphate formed in step (c) may further be treated with a phosphatase enzyme, optionally prior to any separation or purification steps, to generate pseudouridine. In some embodiments, the phosphatase enzyme is an alkaline phosphatase enzyme. In some embodiments, the phosphatase enzyme is a HAD-like phosphatase, more specifically a HAD hydrolase subfamily 1A, e.g. Uniprot ID Q181K6 from Clostridioides difficile. The phosphatase is commercially available or readily produced by standard recombinant methods.

[0134] The pseudouridine or pseudouridine-5' -monophosphate produced by the steps described above may be subject to further separation and / or purification steps prior to the conversion to N-Ci-g alkyl pseudouridine or a 5'-mono-, di- or triphosphate thereof. In some embodiments, pseudouridine or pseudouridine-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, pseudouridine or pseudouridine-5' - monophosphate is separated from the reaction mixture and / or purified by crystallization.

[0135] Immobilization of enzymes on solid support

[0136] When enzymes are used in organic synthesis (such as in organic solvents), they often tend to aggregate, precipitate and / or unfold (i.e., denature). In some embodiments, therefore, the enzymes used in the process disclosed herein are immobilized on a solid support material and used as catalysts in an immobilized state. Such immobilization may improve the stability of the enzymes and allow for reaction conditions which the enzymes normally would not tolerate. The use of enzymes in an immobilized state also facilitates the separation from the reaction mixture and allows for recovery of the catalyst. Additionally, immobilization makes it possible to use immobilized enzymes in much higher concentrations than would have been possible with the free enzymes in solution. Methods for the immobilization of enzymes on a solid support material are known in the art.

[0137] The solid support material is preferably a rigid porous material having a pore diameter of from about

[0138] 20 nm to about 100 nm, such as from about 20 to about 60 nm. In some embodiments, the solid support displays limited swelling in solvents and is chemically and dimensionally stable in most organic media and aqueous environments at pH below 10.

[0139] One useful solid support material is controlled porosity glass (CPG) or hybrid controlled porosity glass (hybrid CPG), as disclosed in WO 2015 / 115993. The support material contains a chelated metal ion such as Fe2+, Fe3+, Co2+, N i2+, Cu2+or Zn2+, allowing enzymes to be immobilized through affinity binding. 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 the chelated metal ion on the solid support.

[0140] Another useful solid support material is controlled porosity silica (CPS) with a pore diameter from about 20 to about 100 nm and comprising an amino-functionalized surface, as disclosed in WO 2023 / 227795. In some embodiments, enzymes are immobilized to the amino-functionalized surface of the support material by a covalent linker comprising a bond selected from amino, amide and imidoamide. In other embodiments, enzymes are immobilized to the amino-functionalized surface of the support material via non-covalent interactions. Such interactions may be mediated by a chelated metal ion, such as Ni2+, Cu2+, Mg2+, Fe3+or Zn2+.

[0141] In some embodiments, a single enzyme is immobilized on a solid support material. In some embodiments, two or more different enzymes are co-immobilized on the same solid support material. For instance, the enzymes used for catalyzing the conversion of Nl-methylpseudouridine to Nl- methylpseudouridine 5'-triphosphate (e.g., a nucleoside kinase, a nucleoside-phosphate kinase, a nucleoside-monosphosphate kinase (NMPK) and a nucleoside-diphosphate kinase (NDPK) or acetyl kinase) may be individually immobilized on a solid support material, or two, three or all of the enzymes may be co-immobilized on the same solid support material. Similarly, the enzymes used for catalyzing the conversion of pseudouridine to Nl-methylpseudouridine with concomitant recycling of the SAM cofactor (e.g., a methyltransferase and a halide methyltransferase) may be individually immobilized on a solid support material, or all the enzymes may be co-immobilized on the same solid support material. Similarly, the enzymes used for catalyzing the conversion of uridine to pseudouridine (e.g., a nucleoside phosphorylase, a phosphomutase and a nucleoside-5'-monophosphate C-glycosidase) may be individually immobilized on a solid support material, or two or three of the enzymes may be co-immobilized on the same solid support material. In some embodiments, the immobilized enzymes are used in batch or continuous stirred tank reactors. In some embodiments, the immobilized enzymes are packed within a fixed-bed reactor suitable for operation in a continuous-flow mode, such as a continuous flow fixed bed reactor.

[0142] DEFINITIONS

[0143] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0144] As used herein, the term "Ci.g alkyl" refers to a straight or branched alkyl group having from 1 to 8 carbon atoms. Examples of Ci.g alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secbutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl.

[0145] Throughout this disclosure, the term "SAM cofactor" is used to refer to a Ci-g alkyl providing cofactor in general. The term encompasses both SAM and alkylated SAH analogues suitable for acting as a Ci-g alkyl providing cofactors.

[0146] As used herein, "EC" number refers to the Enzyme Commission number (EC number), a numerical of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). The enzyme nomenclature is based on the chemical reactions they catalyze.

[0147] As used herein, the term "methylation" refers to a reaction in which a methyl group is added onto a substrate, or the substitution of an atom (e.g., a hydrogen atom) by a methyl group.

[0148] As used herein, the term "phosphorylation" refers to a reaction in which a phosphate group is added to a substrate molecule or an ion. The terms "isolated" and "purified" are used to refer to a molecule, such as an enzyme, or other component that is removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, rather it is intended as a relative definition.

[0149] As used herein, the term "wild-type" refers to the form found in nature. For example, a wild-type enzyme is an amino acid sequence present in an organism that can be isolated from a source in nature, and which has not been intentionally modified by human manipulation.

[0150] As used herein, the terms "parent" and "reference sequence" refer to a defined sequence used as a basis for a sequence and / or activity comparison, as well as the starting sequence used for an engineering cycle.

[0151] The term "sequence identity" as used herein is understood as the relatedness between two amino acid sequences. It is a way to measure the occurrence of exactly the same amino acid in the same position in optimally aligned sequences. The term "Percent sequence identity" as used herein may be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions (protein length) and multiplying the result by 100. Unless indicated otherwise, the comparison window is the entire length of the sequence being referred to. In this context, optimal alignment is the alignment produced by the BLASTP algorithm as implemented online by the US National Center for Biotechnology Information (see The NCBI Handbook, 2ndedition [https: / / www.ncbi.nlm.nih.gov / books / NBK143764 / ]), with the following input parameters: Word length=3, Matrix=BLOSUM62, Gap cost=ll, Gap extension cost=l.

[0152] As used herein, the terms "mutant", "engineered" and "variant" when used with reference to nucleic acid or polypeptide, refer to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature.

[0153] As used herein, the term "activity" refers to the measurement of active enzyme capable of producing a target product in a given amount of time, under certain conditions. Enzymatic activity can be affected by factors that disrupt protein structure (temperature and pH), as well as by factors that affect catalysts in general, such as substrate and / or product concentration, as well as enzyme concentration. 1 As used herein, the terms "increased enzymatic activity" and "enhanced catalytic activity" refer to an improved property of the engineered polypeptide which can be represented by an increase of specific activity (e.g., produced product / time / protein weight) compared to the reference enzyme.

[0154] As used herein, the term "conversion" refers to the amount of substrate that has been consumed in the reaction towards product formation, at a certain time under specified conditions.

[0155] As used herein, the terms "regioselectivity" and "regioselective reaction" refer to a reaction in which one direction of bond making or breaking occurs preferentially over all other possible directions. Reactions can be completely (100%) regioselective if the discrimination is complete, substantially regioselective (at least 75%) or partially regioselective (e.g., 60%, wherein the percentage is set dependent upon the reaction interest), if the product of the reaction at one site predominates over the products of reaction at other sites.

[0156] As used herein, the term "mass balance" refers to an application of conservation of mass to the analysis of physical systems. The mass of reactants must be equal to the total mass of products in any chemical reaction, mass cannot disappear or be created spontaneously.

[0157] As used herein, the term "melting temperature" (Tm) refers to the temperature along a thermal denaturation gradient at which 50% of the protein is folded, and 50% is unfolded.

[0158] As used herein, the term "one-pot" reaction refers to the synthesis strategy to produce a target product in which a substrate is subjected to one or more successive chemical reaction(s) in one single reactor or vessel, without any separation nor purification of the intermediate compound(s). This strategy is used to improve reaction efficiency, increase recovery yields, and save time and resources.

[0159] As used herein, the term "telescoped reaction" refers to a sequential one-pot synthesis with reagents added to the reaction vessel one at a time and without isolation nor purification.

[0160] As used herein, the terms "multi-enzyme cascade" and "multi-step enzyme pathway" refer to the combination of several enzymatic transformations in concurrent one-pot processes or telescoped processes. They offer considerable advantages such as the demand of time, costs and chemicals for product recovery may be reduced, reversible reactions can be driven to completion and the concentration of harmful or unstable compounds can be kept to a minimum. As used herein, the term "comprising" is to be interpreted as including, but not being limited to.

[0161] As used herein, the term "about" refers to a value or parameter herein that includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about 20" includes description of "20." Numeric ranges are inclusive of the numbers defining the range. Generally speaking, the term "about" refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater.

[0162] The invention will now be described by the following examples which do not limit the invention in any respect. All cited documents and references mentioned herein are incorporated by reference in their entireties.

[0163] EXPERIMENTAL SECTION

[0164] Abbreviations

[0165] Nl-Me-PSW Nl-methylpseudouridine

[0166] Nl-Me-PSW-Pi Nl-methylpseudouridine-monophosphate

[0167] Nl-Me-PSW-2Pi Nl-methylpseudouridine-diphosphate

[0168] Nl-Me-PSW-3Pi Nl-methylpseudouridine-triphosphate

[0169] AcP acetyl phosphate

[0170] AcPK acetyl phosphate kinase

[0171] AMP adenosine monophosphate

[0172] ADP adenosine diphosphate

[0173] ATP adenosine triphosphate

[0174] EC Enzyme Class

[0175] HMT halide methyltransferase

[0176] Me-R methyl donor

[0177] NDPK nucleoside-diphosphate kinase

[0178] NMPK nucleoside-phosphate kinase

[0179] NK nucleoside kinase NPK nucleoside-phosphate kinase

[0180] PSW pseudouridine

[0181] SAH S-adenosyl- / .-homocysteine

[0182] SAM S-adenosyl- / .-methionine XMT xanthosine methyltransferase

[0183] The enzymes described here are summarized in the following Table 1.

[0184] Table 1. Analytical Methods

[0185] A. Analytical reference materials and instruments

[0186] The pseudouridine starting material was obtained by the multi-step enzymatic synthesis disclosed in WO 2023 / 13172. Reference materials were purchased from BOC Sciences. All evaluated reactions were monitored using a Thermo Scientific Vanquish UHPLC system equipped with a Variable Wavelength UV Detector. The identity of the target reaction products was confirmed using a triple quadrupole Mass Spectrometer system TSQ Fortis with Electrospray Ionisation (ESI-MS / MS).

[0187] B. Methylation reactions

[0188] Chromatographic separation was performed using a tandem of three columns in the following order: 1) HILICON iHILIC-Fusion(P), 20 x 2.1 mm, 5 pm, 200 A (guard); 2) HILICON iHILIC-Fusion(P), 100 x 2.1 mm, 5 pm, 200 A; 3) HILICON iHILIC-(P) 20 x 2.1 mm, 5 pm, 200 A (guard). The mobile phase consisted of (A) acetonitrile and (B) 100 mM ammonium formate pH ~5.8. The mobile phase gradient programme was: 0 min - 3% B, 2 min - 3% B, 7.5 min - 35% B, 10.5 min - 35% B, 10.5 min - switch back to 3% B in A, hold for 4.5 minutes. The flow rate was constant at 0.3 mL / min, injection volume was 2 pL and the detection wavelength was 260 nm. The column temperature was 40 °C.

[0189] MS detection

[0190] Product identification was conducted via a search of the characteristic product fragments from a Selective Reaction Monitoring (SRM) scan in positive ion mode with electrospray ionization. Argon was used as a collision gas. The capillary voltage was maintained at +3670 V. The following fragments were scanned: 259 -> 139, 259 -> 193, 259 -> 223, 259 -> 169, 259 -> 205.

[0191] Confirmation of methylated position

[0192] The methylation position was confirmed by using chromatographic separation with SRM mode on Mass Spectrometer.

[0193] Chromatographic separation was performed using a tandem of two columns in the following order: 1) SeQuant® ZIC®-cH I LIC 3pm 20 x 2.1 mm (guard); 2) SeQuant® ZIC®-cH I LIC 3 pm, 100 A, 150 x 2.1 mm. The mobile phase consisted of (A) acetonitrile and (B) 100 mM ammonium formate pH ~5.8. The mobile phase gradient programme was: 0 min - 8% B, 4 min - 8% B, 11 min - 40% B, 14 min - 40% B, 14 min - switch back to 8% B, hold for 7 min. The flow rate was maintained at 0.2 mL / min. The injection volume was 2 pL. Detection wavelength was fixed to 260 nm. Column temperature was 40 °C.

[0194] MS identification was conducted via a search for the characteristic product fragments from a SRM scan in positive ion mode with electrospray ionization. Argon was used as a collision gas. The following fragments were scanned: 259 -> 139, 259 -> 193, 259 -> 223 for both Nl- and N3-Me-PSW.

[0195] C. SAM recycling reactions

[0196] Chromatographic separation of the SAM recycling reaction compounds was performed using the same method as for the methylation reaction.

[0197] D. Phosphorylation reactions

[0198] Chromatographic separation of the phosphorylation reaction compounds was based on ion-pairing mechanism and was performed using a C18 column (Phenomenex Gemini C18, 5 pm, 110 A, 250 x 4.6 mm) paired with the guard column (Phenomenex Gemini C18, 4 x 3.0 mm). The mobile phase consisted of (A) 50 mM triethylammonium acetate (TEAA) in HPLC water and (B) 50 mM TEAA in watenacetonitrile 80:20, v / v (HPLC grade). The mobile phase gradient programme was as follows: 0 min - 3% B, 8 min - 25% B, 11.5 min - 25% B, 11.6 min - switch back to 3% B, hold for 4 min before the next injection. The flow rate was 1 mL / min. The injection volume was 2 pL. Detection wavelength was fixed to 260 nm. Column temperature was 40 °C.

[0199] MS detection

[0200] Product identification was conducted via a search for the characteristic product fragments from a selective reaction monitoring (SRM) scan in negative mode, in addition to a full scan in negative ion mode with electrospray ionization. Argon was used as a collision gas. Sample was introduced via direct infusion with a pump flow rate set to 10 pL / min. The capillary voltage was maintained at 3027 V (negative ion).

[0201] EXAMPLES

[0202] Example 1

[0203] Enzymatic methylation of pseudouridine by XMTs Reactions (1 mL and / or 5 mL) were performed in 50 mM Tris-HCI buffer pH adjusted to 8.0, with a starting concentration of 10 M for PSW, 1 mM SAM and 1 mM MgCL. Additionally, 10% (v / v) of either purified or cell-free extract (CFE) of wild-type xanthosine methyltransferases (XMT_1, XMT_2, XMT_3 or XMT_4) was added to the reaction mixture and incubated at 35 °C. Product formation was measured by UV detection after 4 hours of incubation. As shown in Figure 1, different levels of methylation activity were detected. XMT_4 had the highest detected activity, with a 50-fold increase in methylation activity when compared with XMT_1.

[0204] Example 2

[0205] Regioselectivity of enzymatic synthesis of methylpseudouridine by XMTs

[0206] Identity of the methylation position was verified by the combination of chromatographic separation with Selected Reaction Monitoring (SRM) mode on mass spectrometry (see analytical methods B). Commercial standards of Nl- and N3-Me-PSW were separated with a retention time of 9.35 min and 10.44 min, respectively. Reactions mixtures containing starting concentrations of 10 mM for PSW, 1 mM SAM and 1 mM MgCL and 10% (v / v) of purified xanthosine methyltransferase (XMT_1 or XMT_4) in 50 mM Tris-HCI pH 8.0 were incubated at 35 °C. After 24 hours of incubation, the peak observed in the reaction mixture had a retention time of 9.431 min, which agrees with Nl-Me-PSW commercial standard. Enzymatic synthesis Nl-Me-PSW using xanthosine methyltransferase was confirmed to be >99% regioselective.

[0207] Example 3

[0208] Improved xanthosine synthase stability variants for pseudouridine methylation

[0209] Mutant libraries designed to increase enzyme stability were generated using well established engineering techniques, including physics-based atomistic modeling and bioinformatic scoring. Sequence ID No. 4 (XMT_4) was used as parent. Samples containing 11 pM of purified xanthosine methyltransferase (XMT_4, XMT_5, XMT_6, XMT_7 or XMT_8) in 50 mM MOPS pH 7.0 were measured in a temperature range of 25 - 95 °C with a ramp of 1 °C / min. Methylation activity was determined by mixing 10 mM PSW, 1 mM SAM, 1 mM MgCL and 4.5 pM of purified enzyme in 50 mM Tris-HCI pH adjusted to 8.0 in a total volume of 5 mL. Reaction mixtures were incubated at 35 °C for 24 hours and monitored by HPLC (UV detection). Figure 2 shows melting temperature and activity results. XMT_8 showed a melting temperature increase of 26 °C whilst maintaining a similar methylation activity compared to the parent. With a sequence identity of 79% in comparison with XMT_4, XMT_8 was the most thermostable variant. Interestingly, both XMT_5 and XMT_7 revealed to have higher Tm than XMT_4, however the methylation activity was decreased. These variants have a sequence identity of 78% and 91% respectively, in comparison with XMT_4. Variant XMT_6 showed the best activity improvement, as a ~3-fold methylation activity increase was observed when compared to the parent. Additionally, thermal stability was also improved, which was confirmed by a ~10 °C increase of the melting temperature. XMT_6 has a sequence identity of 89% compared to XMT_4.

[0210] Example 4

[0211] Enzyme concentration effect on Nl-Me-PSW formation

[0212] A set of 1 mL reactions was prepared with 10 mM PSW, 1 mM SAM, 1 mM MgCL, 40 mM NaCI, 27% (v / v) glycerol in 50 mM Tris-HCI pH 8, and variable concentrations of xanthosine methyltransferase (XMT_6) between 2.2 and 33 pM. Reaction mixtures were incubated at 37 °C and 800 rpm for 24 hours and monitored by HPLC (UV detection). As shown in Figure 3, Nl-Me-PSW product formation increased non-linearly with a protein concentration up to 27.5 pM, at which concentration a plateau is reached at ~42% SAM conversion.

[0213] Example 5

[0214] Pseudouridine methylation response to substrate concentration variation

[0215] A set of 1 mL reactions was prepared with 1 mM SAM, 1 mM MgCL, 40 mM NaCI, 27% (v / v) glycerol and 22 pM of xanthosine methyltransferase (XMT_6) in 50 mM Tris-HCI pH 8, with variable concentrations of PSW (1 to 50 mM). Reaction mixtures were incubated at 37 °C and 800 rpm for 24 hours. Higher substrate concentrations resulted in increased catalytic activity (see Figure 4), under a fixed SAM concentration.

[0216] Another set of 1 mL reaction mixtures was prepared with 40 mM PSW, 1 mM MgCL, 40 mM NaCI, 27% (v / v) glycerol and 22 pM of xanthosine methyltransferase (XMT_6) in 50 mM Tris-HCI pH 8, with variable concentrations of SAM (1 to 10 mM). Reaction mixtures were incubated at 37 °C and 800 rpm over the course of 48 hours. As shown in Figure 5, under the stated conditions, initial product formation rate was similar when using 1 or 2.5 mM of SAM, both faster than 5 mM of SAM. However, after 23 hours of incubation, 1 and 5 mM of SAM reached similar product formation. Concentrations higher than 5 mM had a detrimental effect on enzyme activity. Under these conditions, the optimal SAM concentration therefore lies between 1 and 5 mM. Example 6

[0217] Regeneration of the methyl providing cofactor (SAM)

[0218] 6.1 SAM formation from SAH

[0219] Reactions were performed in 50 mM Tris-HCI pH 8.0 containing 1 mM SAH and 1.5 mM Mel or methyl p-toluenesulfonate (MeOTs) in a total volume of 0.5 mL. Reactions were started by the addition of about 2 pM of halide methyltransferase (HMT_1, HMT_2 or HMT_3), and the reaction mixtures were incubated at 30°C and 400 rpm. After 20 min incubation, SAM formation was measured by HPLC (Figure 6). With Mel as the methyl donor, SAM conversion varied between about 55 and 70% for the different HMTs, and with MeOTs as the methyl donor, SAM conversion varied between about 35 and 65% for the different HMTs. The experiments confirmed that both Mel and MeOTs are effective as methyl donors.

[0220] 6.2 Methyl donor (Mel and MeOTs) compatibility with XMT_6

[0221] A set of 1 mL reactions was prepared with 1 mM SAM, 1 mM MgCL, 10 mM PSW, 4.4 pM XMT_6 and 1.5 mM methyl donor (Mel or MeOTs) in 50 mM Tris-HCI at pH 8. Reaction mixtures were incubated at 30 °C and 800 rpm for 24 hours. In parallel, control reactions (without enzyme) were run under the same conditions. As shown in Figure 7, product formation was similar when either Mel or MeOTs was added to the reactions, compared to no donor addition. This indicates that both methyl donors, necessary for SAM recycling using HMT, are compatible with XMT_6. Background chemical methylation was observed with MeOTs, resulting in the formation of both N1 and N3-Me-PSW (see analytical methods B).

[0222] 6.3 Influence of pH on XMT_6 activity and SAM stability

[0223] Since SAM stability is known to be pH dependent, the effect of the pH for the methylation reactions was investigated over a pH range of 6.5 to 8.5. Reactions (1 mL) were performed in 50 mM buffer (MES pH 6.5-7 or Tris-HCI pH 7.5-8.5) containing 1 mM SAM, 10 mM PSW, and either without or with enzyme (4.4 pM XMT_6). Reaction mixtures were incubated at 35°C and 800 rpm for 24 hours. As shown in Figures 8A and 8B, acidic or neutral pH had a positive effect on SAM stability, but a negative effect on XMT_6 activity.

[0224] 6.4 Influence of SAH concentration on XMT_6 activity Reactions (1 mL) were performed in 100 mM Tris-HCI pH 8.0 buffer containing 2.5 mM SAM, 40 mM PSW, 22 pM XMT_6, 75 M NaCI, 25% glycerol, and with 0.5 or 1 mM SAH or without SAH. Reaction mixtures were incubated at 35°C and 800 rpm for 24 hours. As shown in Figure 9, SAH had a detrimental effect on XMT_6 activity, leading to a 40 and 65% activity drop when 0.5 and 1 mM SAH were added to the reaction, respectively.

[0225] Example 7

[0226] SAM recycling in a methylation cascade for the production of Nl-methylpseudouridine

[0227] 7.1 Combining HMT and XMT with SAM and methyl iodide as a methyl donor

[0228] A set of 1 mL reactions was prepared with 0.5-0.75 mM SAM, 52 mM Mel, 40 mM PSW, 2 pM HMT_2, 22 pM XMT_6, 25% glycerol in 100 mM Tris-HCI pH 7.5-8.0. Reaction mixtures were incubated at 30 or 35 °C and 800 rpm for 24 and 48 hours (Figure 10). After 48 hours of reaction, an improved product concentration was observed, although the reaction rate decreased between the 24 and 48-hour marks. Under certain conditions, the maximum product formation reached 1.74 mM Me-PSW. Since SAM was deployed at concentrations of 0.5-0.75 mM, these results illustrate that SAM was regenerated by HMT / Mel during the course of the reaction and thus can be used catalytically as a cofactor for the XMT methylation.

[0229] 7.2 Comparison with reaction controls

[0230] To further confirm the efficacy of the combination of HMT / XMT with SAM and a methyl donor, a set of control reactions was performed:

[0231] • Condition set 1, to assess chemical background methylation in the presence of the methyl donor. Conditions: 52 mM Mel, 40 mM PSW, 25% glycerol in 100 mM Tris-HCI pH 8.

[0232] • Condition set 2, to assess XMT_6 methylation with SAM, but without any external addition of recycling components (Mel or HMT). Conditions: 0.75 mM SAM, 40 mM PSW, 22 pM XMT_6, 25% glycerol, in 100 mM Tris-HCI pH 8.

[0233] • Condition set 3 to assess XMT_6 methylation activity in the presence of SAM and Mel but without HMT. Conditions: 0.75 mM SAM, 52 mM Mel, 40 mM PSW, 22 pM XMT_6, 25% glycerol, in 100 mM Tris-HCI pH 8.

[0234] The results shown in Figure 11 indicate that the chemical background methylation reaction (condition set 1) occurs at a low level, producing approximately 0.2 mM of Nl-Me-PSW. The fact that the product formed in Condition set 3 is not equal to the sum of Condition sets 1 and 2 indicates a slight decrease in enzyme activity, likely due to the addition of 52 mM Mel. To evaluate the efficacy of the combination of HMT / XMT with SAM and a methyl donor, the results obtained from the cascade combining XMT_6 and HMT_2 with SAM and methyl iodide (Condition set 4 in Figure 11) were compared to those of Condition set 3. Under the same conditions, Nl-Me-PSW product formation using Condition set 4 was 2.5 times higher than those obtained in Condition set 3, indicating that SAM recycling is effective.

[0235] 7.3 Adjusting enzyme concentration and pH

[0236] A set of 1 mL reactions was prepared with 0.75 mM SAM, 52 mM Mel, 40 mM PSW, 2 pM HMT_2, 22.0- 26.4 pM XMT_6, 25% glycerol in 100 mM Tris-HCI pH 7.8-8.0. Reaction mixtures were incubated at 30 and 400 rpm for 48 hours. The results in Figure 12 show the formation of up to 2 mM Nl-Me- PSW after 48 hours, again illustrating that SAM can be recycled and used catalytically as a cofactor for the XMT methylation.

[0237] 7.4 Addition of Mel aliquots during the reaction for the production of Nl-methylpseudouridine

[0238] A set of reactions were prepared in 5 mL reaction vessels with 2.5 mM SAM, 152 mM Mel (a 52 mM aliquot added at the beginning of the reaction and a 100 mM aliquot added after 16 hours), 40 mM PSW, 2 pM HMT_2, 44 pM XMT_6, 25% glycerol in 100 mM Tris-HCI pH 8.5. Reaction mixtures were incubated at 30 °C and 700 rpm for 40 hours. The results illustrated that after 16 hours of reaction, 5.79 mM of Nl-Me-PSW was formed. At this point, a further 100 mM aliquot of Mel was added and the reaction was allowed to proceed to 40 hours. After 40 hours, 7.64 mM Nl-Me-PSW was formed. Since SAM was deployed at concentration of 2.5 mM, these results illustrate that SAM was regenerated by HMT / Mel during the course of the reaction and thus can be used catalytically as a cofactor for the methylation reaction.

[0239] Example 8

[0240] One pot multi-enzyme cascade phosphorylation of Nl-methylpseudouridine with ATP recycling

[0241] 8.1 Screening conditions

[0242] 1 mL reactions mixtures were prepared to determine key factors in NlMePSW3Pi formation, including optimal enzyme ratio. The reaction mixture which produced the highest product concentration contained 2.5 mM Nl-Me-PSW, 2 mM MgCL, 2 mM MnCL, 5 mM DTT, 0.25 mM ATP, 10 mM AcP in 50 mM Tris-HCI pH 7.5, with 3.2 pM nucleoside kinase (NK), 20.5 pM nucleosidephosphate kinase (NMPK_1) and 2.1 pM acetyl phosphate kinase (AcPK_l) (See Table 1). The highest product concentration was 2 mM Nl-Me-PSW-3Pi (80% conversion) after 4 h incubation at 37°C and 300 rpm (see Figure 13).

[0243] 8.2 Reaction with ATP recycling at 5 mM substrate

[0244] A reaction mixture containing 5 M Nl-Me-PSW, 4 mM MgCL, 4 mM MnCL, 10 mM DTT, 0.75 mM ATP, 20 mM AcP in 50 mM Tris-HCI pH 8, was prepared with 3.2 pM nucleoside kinase, 20.5 pM nucleoside-phosphate kinase (NMPK_1) and 8.5 pM acetyl phosphate kinase (AcPK_l). The highest product concentration was 3.33 mM Nl-Me-PSW-3Pi (67% conversion) after 24 h incubation at 37°C and 300 rpm (see Figure 14).

[0245] 8.3 Reaction scale up to 700 mL at 5 mM substrate

[0246] A 700 mL reaction mixture containing 5 mM Nl-Me-PSW, 4 mM MgCL, 4 mM MnCL, 10 mM DTT, 0.75 mM ATP, 20 mM AcP in 50 mM Tris-HCI pH 8 was prepared and the pH was adjusted with 10 M NaOH. 3.2 pM nucleoside kinase, 20.5 pM nucleoside-phosphate kinase (NMPK_1) and 8.5 pM acetyl phosphate kinase (AcPK_l) were added. The reaction was incubated at 37°C in a Radleys jacketed glass reactor and stirred with a tip speed of 0.4 m / s. The highest product concentration was 4.4 mM Nl-Me-PSW-3Pi (87% conversion) after 6 hours (see Figure 15).

[0247] 8.4 Influence of ATP on Kin 3183 activity

[0248] 1 mL reaction mixtures were prepared containing 80 mM Nl-Me-PSW, 320 mM LijAcP, 1.5-10 mM ATP, 50 mM MgCL, 10 mM DTT, 50 mM Tris-HCI pH 8. Enzymes were added in the following concentration: 3.2 pM nucleoside kinase, 37 pM nucleoside-phosphate kinase (NMPK_1) and 10.6 pM acetyl phosphate kinase (AcPK_l). The reaction was heated to 37 °C, 800 rpm for 6.5 hours. The highest final MePSW-3P concentration was obtained using 5 mM ATP. (Figure 16A)

[0249] A 30 mL reaction mixture containing 5 mM Nl-Me-PSW, 2 mM MgCL, 2 mM MnCL, 5 mM DTT, 10 mM ATP in 50 mM Tris-HCI pH 8 with 3.2 pM nucleoside kinase was prepared and incubated at 37°C, 150 rpm for 24 hours. The crude reaction mixture was filtered through a 10 kDa centrifugal filter and used to set up a second reaction step containing 2.5 mM Nl-Me-PSW-P, 3 mM MgCL, 3 mM MnCL, 7.5 mM DTT, 10 mM AcP, 5.5- 6.25 mM combined concentration of ATP, ADP and AMP in 100 mM Tris-HCI pH 8 .5 pM nucleoside-phosphate kinase (NMPK_1) and 8.5 pM acetyl phosphate kinase (AcPK_l) The reaction was incubated at 37°C, 300 rpm for 17 hours. The results showed reduced kinase activity above 5.5 mM combined ATP, ADP and AMP concentration. (Figure 16B) 8.5 High concentration one pot multi-enzyme cascade phosphorylation of Nl-methylpseudouridine with ATP recycling and AcP dosing

[0250] 1 mL reaction mixtures were prepared containing 80 mM Nl-Me-PSW, 240 mM LijAcP, 5 mM ATP, 50 mM MgCL, 30 M DTT and 100 mM Tris-HCI pH 9. The pH was adjusted to 9 with 10 M NaOH. Enzymes were added in the following concentrations: 3.2 pM nucleoside kinase, 57.4 pM nucleoside-phosphate kinase (NMPK_1) and 10.6 pM acetyl phosphate kinase (AcPK_l). The reaction was heated to 30 °C and stirred at 800 rpm. 30 pL samples were taken for analysis at 0, 5, 6, 22, 24 and 28 hours (see Figure 17). At 5 hours and 22 hours, 100 pL of IM LijAcP pH 9 was added to some of the reactions. The highest product concentration was 47.3 mM Nl-Me-PSW-3Pi (72% conversion) (see Figure 18).

[0251] Example 9

[0252] Effect of AcP dosing on one pot multi-enzyme cascade phosphorylation of Nl-methylpseudouridine

[0253] Two similar cascade reactions were performed on a 60 and a 150 mL scale, respectively. The reaction mixtures had the following starting concentrations: 100 mM Nl-Me-PSW, 50 mM MgCL, 30 mM DTT, 5 mM ATP, 100 mM Tris-base pH 9, and 24.3 g / L LijAcP. Enzymes used were 0.05 g / L (1.6 pM) nucleoside kinase, 1.1 g / L (45 pM) nucleoside-phosphate kinase (NMPK_1) and 0.1 g / L (2.1 pM) acetyl phosphate kinase (AcPK_l).

[0254] The reactor vessels were filled with deionized water to 80% of the total desired reaction volume. The weighed amounts of Nl-MePSW, Tris-base, MgCL, DTT and ATP were added, and stirring was turned on to a sufficient speed to dissolve the reagents in the water. Once all reagents had dissolved, the weighed amount of initial LijAcP was added to the reaction mixtures. While stirring, the pH of the reaction mixtures was adjusted to pH 9, using 10 M NaOH at room temperature. The reaction volume was then topped up to the total desired reaction volume with deionized water, and the reaction mixtures were heated up to 30°C. The reactions were then started by adding the individually weighed enzyme powders in the following order: 1 - acetyl phosphate kinase (AcPK_l); 2 - nucleoside-phosphate kinase (NMPK_1); 3 - nucleoside kinase. The reaction temperature was kept constant at 30°C for the rest of the process. Subsequent doses of 15.2 g / L LijAcP were added to the reaction mixture after 4, 8 and 24 hours.

[0255] Two samples (technical replicates) were taken from the reaction mixture at 0 hours and after 1, 2, 3,

[0256] 4, 5, 6, 7, 8, 9, 24, 25, 26 and 27 hours. At 0 hours, samples were taken before and directly after addition of the enzymes. At 4, 8 and 24 hours, samples were taken before and directly after addition of the subsequent dose of LijAcP. Samples were quenched immediately after withdrawal.

[0257] The time-course evolutions of the concentrations of the starting material (Nl-Me-PSW), the two intermediate products (Nl-Me-PSW-Pi and Nl-Me-PSW-2Pi) and the final product (Nl-Me-PSW-3Pi) are shown in Figures 19A-D, respectively. Product concentrations up to 75 mM were measured.

[0258] The time-course evolution of the concentrations of AMP, ADP and ATP are shown in Figures 20A-C, respectively, and the time-course evolutions of the concentrations of phosphate and AcP are shown in Figures 21A-B, respectively.

Claims

1. CLAIMS1. A process for the production of N-Ci-g alkyl pseudouridine 5'-triphosphate, comprising converting an N-Ci-g alkyl pseudouridine, or a 5' -mono- or diphosphate thereof, to the corresponding N-Ci-g alkyl pseudouridine 5' -triphosphate by reacting the N-Ci-g alkyl pseudouridine or the 5' -mono- or diphosphate thereof with a source of phosphate in the presence of a nucleoside kinase and / or one or more nucleoside-phosphate kinases, and wherein the output concentration of the N-Ci-g alkyl pseudouridine 5' -triphosphate is at least 15 mM.

2. The process according to claim 1, comprising the steps of:(a) reacting N-Ci-g alkyl pseudouridine with a source of phosphate in the presence of a nucleoside kinase (NK) to form N-Ci-g alkyl pseudouridine 5'-monophosphate; and(b) reacting the N-Ci-g alkyl pseudouridine monophosphate with a source of phosphate in the presence of a nucleoside-phosphate kinase (NPK) to form N-Ci-g alkyl pseudouridine 5'- triphosphate; and wherein the output concentration of the N-Ci-g alkyl pseudouridine 5' -triphosphate is at least 15 mM.

3. The process according to claim 1, comprising the step of:(a) reacting the N-Ci.g alkyl pseudouridine monophosphate with a source of phosphate in the presence of a nucleoside-phosphate kinase (NPK) to form N-Ci.g alkyl pseudouridine 5'- triphosphate; and wherein the output concentration of the N-Ci.g alkyl pseudouridine 5' -triphosphate is at least 15 mM.

4. The process according to any one of claims 1 to 3, wherein ATP is used as a source of phosphate and wherein regeneration of the ATP is catalyzed enzymatically by an acetyl kinase using acetyl phosphate (AcP) as a phosphate donor.

5. The process according to any one of claims 1 to 4, wherein acetyl phosphate is added sequentially throughout the time-course of the reaction.NP0895WQ6. The process according to claim 5, wherein acetyl phosphate is added 1-5 times over a 48-hour reaction.

7. The process according to claim 5, wherein additional acetyl phosphate is added after about 4 hours, after about 8 hours and after about 24 hours.

8. The process according to any one of claims 4 to 7 , wherein the source of acetyl phosphate is dilithium acetyl phosphate salt (LijAcP).

9. The process according to claim 2, wherein the N-Ci-g alkyl pseudouridine 5'-triphosphate is N- methylpseudouridine 5' -triphosphate and wherein the process comprises the steps of:(a) reaction N-methylpseudouridine with a source of phosphate in the presence of a nucleoside kinase (NK) to form N-methylpseudouridine 5'-monophosphate; and(b) reacting the N-methylpseudouridine monophosphate with a source of phosphate in the presence of a nucleoside-phosphate kinase (NPK) to form N-methylpseudouridine 5'- triphosphate.

10. The process according to claim 2, wherein the nucleoside kinase (NK) comprises an amino acid sequence according to SEQ ID NO: 12, or has at least 80% sequence identity with SEQ ID NO: 12.

11. The process according to claim 2 or 10, wherein the nucleoside kinase (NK) is present in a concentration of from about 1 to about 2 pM.

12. The process according to any one of claims 2 to 11, wherein the nucleoside-phosphate kinase (NPK) comprises an amino acid sequence according to SEQ ID NOs: 13 or 14, or has at least 80% sequence identity with SEQ ID NOs: 13 or 14.

13. The process according to claim 10, wherein the nucleoside-phosphate kinase (NPK) comprises an amino acid sequence according to SEQ ID NO: 13.

14. The process according to any one of claims 2 to 13, wherein the nucleoside-phosphate kinase (NPK) is present in a concentration of from about 40 to about 60 pM, preferably from about 45 to 50 pM.NP0895WQ15. The process according to any one of claims 2 to 14, wherein the acetyl kinase comprises an amino acid sequence according to any one of SEQ ID NOs: 15 to 17, or has at least 80% sequence identity with any one of SEQ ID NOs: 15 to 17.

16. The process according to claim 15, wherein the acetyl kinase comprises an amino acid sequence according to SEQ ID NO: 15.

17. The process according to any one of claims 4 to 14, wherein the acetyl kinase is present in a concentration of from about 1.5 pM to about 2.5 pM.

18. The process according to any one of claims 1-17, wherein the N-Ci-g alkyl pseudouridine is prepared from pseudouridine in a process comprising catalyzing the reaction between pseudouridine and a Ci.g alkyl providing cofactor with a methyltransferase capable of catalyzing the transfer of a Ci-g alkyl group from the Ci-g alkyl providing cofactor to the pseudouridine.

19. The process according to claim 18, wherein the N-Ci.g alkyl pseudouridine is Nl- methylpseudouridine.

20. The process according to claim 18 or 19, wherein the methyltransferase is a xanthosine methyltransferase (XMT).

21. The process according to claim 20, wherein the xanthosine methyltransferase comprises an amino acid sequence according to any one of SEQ ID NOs: 1 to 8, or has at least 80% sequence identity with any one of SEQ ID NOs: 1 to 8.

22. The process according to claim 21, wherein the xanthosine methyltransferase comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 5 to 8.

23. The process according to claim 22, wherein the xanthosine methyltransferase comprises an amino acid sequence according to SEQ ID NO: 6.

24. The process according to any one of claims 18 to 23, wherein the Ci-g alkyl providing cofactor is S-adenosylmethionine (SAM).

25. The process according to any one of claims 18 to 24, wherein regeneration of the Ci.g alkyl providing cofactor is catalyzed enzymatically with a halide Ci.g alkyl transferase in the presence of a Ci-g alkyl donor.

26. The process according to claim 25, wherein the halide Ci-g alkyl transferase comprises an amino acid sequence according to any one of SEQ ID NOs: 9 to 11, or has at least 80% sequence identity with any one of SEQ. ID NOs: 9 to 11.

27. The process according to any one of claims 25 or 26, wherein the Ci-g alkyl donor is methyl iodide or methyl tosylate.

28. The process according to any one of claims 18 to 27, wherein the pseudouridine is produced in a process comprising the steps of:(a) reacting uridine with a source of phosphate in the presence of a nucleoside phosphorylase to form ribose-l-phosphate and uracil;(b) reacting ribose-l-phosphate in the presence of a phosphomutase to form ribose-5- phosphate;(c) reacting ribose-5-phosphate with uracil in the presence of nucleoside-5'-phosphate C- glycosidase to form pseudouridine-5'-monophosphate; and(d) optionally, reacting pseudouridine-5' -monophosphate with an alkaline phosphatase to form pseudouridine.

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