Method for generating nucleotide triphosphates (NTPs)

Abstract

This invention relates to a novel enzymatic method for producing nucleoside triphosphates (NTPs), particularly modified NTPs, using polyphosphate kinase 2. The NTPs produced by the method of this invention can be used in methods for producing polynucleotides, including oligonucleotides, for therapeutic use.

Description

[Technical Field]

[0001] This invention relates to a novel enzymatic method for producing nucleoside triphosphates (NTPs), particularly modified NTPs. The NTPs produced by the method of this invention can be used in methods for producing polynucleotides, including oligonucleotides, for therapeutic use. [Background technology]

[0002] Nucleoside triphosphates (NTPs) are fundamental building blocks of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Naturally occurring NTPs include adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), and uridine triphosphate (UTP). ATP is the primary source of cellular energy, while GTP is frequently used as a cofactor for enzymes and proteins. Commercial supply of naturally occurring NTPs is typically obtained using cell fermentation and purification methods.

[0003] NTPs with modifications to sugars, bases, and / or phosphate groups are invaluable experimental tools and are also therapeutically useful, for example, in the production of nucleotide-based therapeutics such as therapeutic oligonucleotides and mRNAs, because they contribute to enhanced affinity for targets, improved nuclease resistance, altered circulatory properties, and modulation of immunological properties (Kulkarni et al., Nature Nanotechnology, 2021, 16, 630-643).

[0004] Several wild-type polymerases, as well as engineered polymerases, can utilize modified NTPs to generate modified polynucleotides complementary to the template sequence (Milisavljevic et al., Org. Biomol. Chem, 2018, 16, 5800-5807; and Sabat et al., Front Chem., 2023, 11, 1161462). Furthermore, terminal deoxynucleotidyltransferase (TdT) enzymes can utilize modified NTPs to generate modified oligonucleotides in a template-independent manner (International Publication No. 2019 / 121500).

[0005] However, modified NTPs are difficult to produce, especially on a large scale, and are expensive. Synthetic reaction schemes for producing modified NTPs are described in Marcel Hollenstein's 2012 review (Molecules, 2012, 17, 13569-13591), and include the Yoshikawa Protocol, the Ludwig-Eckstein method, the Borch approach, and other strategies (see also Burgess and Cook, Chem. Rev. 2000, 100, 2047-2059).

[0006] More recently, biocatalytic methods for the production of specific NTPs have been studied. MK-1454 is a cyclic dinucleotide-stimulating (STING) agonist of interferon genes and is being developed as an immuno-oncology therapeutic. MK-1454 is diastereoselectively assembled from two non-native monothiophosphate nucleotides in a one-pot cascade, where two thiotriphosphate nucleotides are biocatalyzed and simultaneously produced, followed by coupling and cyclization catalyzed by engineered animal cyclic guanosine-adenosine synthase (cGAS). Three kinase enzymes have been engineered for thiotriphosphate synthesis, and a non-native cofactor recycling system has been developed (McIntosh et al., Nature, 2022, 603, 439-444).

[0007] However, there is a need for simpler and more cost-effective means of producing NTPs, particularly modified NTPs, that can be used on a large scale, especially in the manufacture of polynucleotide therapeutics. [Overview of the project]

[0008] In a first aspect of the present invention, the use of polyphosphate kinase (PPK) is provided for generating glycosylated and / or α-phosphate-modified nucleoside triphosphates from corresponding modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates.

[0009] In a second aspect of the present invention, a method is provided for screening a panel of putative and known PPKs to identify enzymes capable of producing corresponding modified nucleoside triphosphates using sugar-modified and / or α-phosphate-modified nucleoside monophosphate or nucleoside diphosphate as substrates. The method comprises: a) identifying sequences encoding the panel of putative and known PPKs; b) synthesizing the PPKs; c) purifying the PPKs; d) mixing each of the purified PPKs with the sugar-modified and / or α-phosphate-modified nucleoside monophosphate or nucleoside diphosphate and polyphosphate to form a reaction mixture; and e) analyzing each of the reaction mixtures for the presence of the corresponding modified nucleoside triphosphate to determine which enzyme produces the corresponding modified nucleoside triphosphate.

[0010] A third aspect of the present invention provides an enzymatic method for producing glycosylated and / or α-phosphate-modified nucleoside triphosphates, comprising incubating PPK with corresponding modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates under suitable conditions.

[0011] In a fourth aspect of the present invention, the use of a modified nucleoside triphosphate obtained by the method of the present invention in the production of a polynucleotide is provided.

[0012] In a fifth aspect of the present invention, the use of PPK is provided for producing stereospecific α-phosphate-modified nucleoside triphosphates from α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates.

[0013] In a sixth embodiment of the present invention, an manipulated PPK having at least 90% sequence identity with respect to sequence number 22 is provided.

[0014] A seventh aspect of the present invention provides a method for producing a polynucleotide comprising incubating a nucleoside triphosphate, a suitable enzyme, and optionally a template under suitable conditions, wherein the nucleoside triphosphate is produced by incubating the corresponding nucleoside monophosphate and / or nucleoside diphosphate, PPK, and polyphosphate under suitable conditions. [Brief explanation of the drawing]

[0015] [Figure 1] A schematic diagram illustrating preferred catalytic reactions related to ATP production in the PPK2 subfamily is shown. [Figure 2] The chromatogram from Example 5 shows that for the negative control sample, a 2'OMe-pGMP peak is observed, and 2'OMe-pGDP and 2'OMe-pGTP peaks are absent [upper trace]. For the PPK (SEQ ID NO: 31) sample, the 2'OMe-pGMP peak is reduced, and two new peaks corresponding to the formation of 2'OMe-pGTP and 2'OMe-pGTP are observed [lower trace]. [Figure 3] The chromatogram from Example 12 shows the stereoselective preferences of three different PPKs in forming 5'-(α-thio)triphosphate. The PPK of SEQ ID NO: 8 [bottom trace] produced approximately 50:50 Sp:Rp diastereoisomers. The PPKs with SEQ ID NOs: 34 and 47 [middle and top traces] showed reverse stereoselective preferences, with one preferring the Sp diastereoisomer and the other the Rp diastereoisomer. Detailed description of the invention

[0016] definition As used herein, “Ambo” is a prefix indicating that a molecule having two (or more) chiral elements exists in an unspecified proportion as a mixture of two racemic diastereoisomers (Pure and Appl. Chem., 68, 12, 2193-2222, 1996). See also “Nomenclature and Symbolism for Amino Acids and Peptides,” Pure Appl. Chem., 56, 595-624, 1984, and “Nomenclature of Tocopherols and Related Compounds,” Pure Appl. Chem., 54, 1507-1510, 1982.

[0017] In the context of enzymes, as used herein, "derived from" refers to identifying the original enzyme on which the genetic manipulation was based, and / or the gene encoding such an enzyme.

[0018] As used herein in relation to the production of polynucleotides containing oligonucleotides, “enzymatic synthesis” means producing the polynucleotide using enzymes (e.g., polymerases, ligases, transferases, phosphatases, kinases (including polyphosphate kinases)) and nucleases (e.g., endonucleases). These enzymes may be wild-type enzymes, mutant enzymes, or engineered enzymes. Mutant or engineered enzymes capable of acting on modified nucleotide or oligonucleotide substrates are within the scope of this disclosure. “Enzymatic method” is a method that includes enzymatic synthesis.

[0019] As used herein, “manipulated enzyme” refers to an enzyme whose natural or native form, known as the “wild type,” has been modified in a manner not present in nature. Modifications may include, for example, the deletion, insertion, and / or substitution of one or more amino acids in the wild-type sequence of the enzyme.

[0020] As used herein, “modified nucleotide,” “modified nucleoside monophosphate, diphosphate, or triphosphate,” or “modified polynucleotide or oligonucleotide” means a nucleotide residue, nucleoside monophosphate, diphosphate, or triphosphate, or polynucleotide or oligonucleotide that contains at least one aspect of chemical properties different from those of naturally occurring nucleotide residues, nucleoside monophosphates, diphosphates, or triphosphates, or polynucleotides or oligonucleotides. A modified polynucleotide or oligonucleotide contains at least one modified nucleotide. Modifications can occur at any part of a nucleotide residue, such as modification of a sugar moiety (“sugar modification”), modification of a nucleic acid base (“nucleic acid base modification”), and / or modification of a phosphate / backbone (“α-phosphate modification”). One or more identical or different modified nucleotide residues can form part of a modified polynucleotide or oligonucleotide.

[0021] The term “messenger RNA” (mRNA) refers to one specific type of RNA molecule. In vivo, DNA transcription typically produces so-called immature RNA, which must be processed into so-called messenger RNA, usually abbreviated as mRNA. Processing of immature RNA in eukaryotes, for example, involves a variety of different post-transcriptional modifications, such as splicing, 5' capping, polyadenylation, and transport from the nucleus or mitochondria. These processes are collectively referred to as mRNA maturation. Mature mRNA typically provides a nucleotide sequence that can be translated into the amino acid sequence of a particular peptide or protein. Typically, mature mRNA includes a 5' cap, a 5' untranslated region (5'UTR), an open reading frame, a 3' untranslated region (3'UTR), and a homopolymer tail (e.g., a poly-A or poly-C sequence). In the context of this disclosure, mRNA may be an artificial molecule, i.e., a molecule that does not exist in nature. This means that mRNA in the context of this disclosure may include a combination of a 5'UTR, an open reading frame, a 3'UTR, and a poly(A) sequence, which is not naturally occurring in this combination. mRNA in the context of this disclosure includes at least one modified nucleotide residue.

[0022] As used herein, “oligonucleotide,” or simply “oligo,” refers to a polymer of nucleotide residues. The term “oligonucleotide” is typically used for shorter sequences than the term “polynucleotide,” generally ranging from 3 to 30 nucleotides. These may incorporate deoxyribonucleotides (where the resulting oligonucleotide is DNA), ribonucleotides (where the resulting oligonucleotide is RNA), modified nucleotides, or mixtures thereof. Oligos may be single-stranded, such as antisense molecules, or double-stranded, such as siRNA molecules.

[0023] The “identity percentage” or “identity %” is a percentage representing the “identity” value between the query nucleic acid sequence and the target nucleic acid sequence, calculated over the entire length of the query sequence using an appropriate algorithm (e.g., BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle, or EMBOSS infoalign) after pairwise global sequence alignment using an appropriate algorithm (e.g., Needleman-Wunsch or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle, or EMBOSS infoalign). Importantly, the query nucleic acid sequence may be described by the nucleic acid sequences disclosed herein.

[0024] The “identity percentage” or “identity %” between the query amino acid sequence and the target amino acid sequence is an “identity” value expressed as a percentage, calculated over the entire length of the query sequence using an appropriate algorithm (e.g., BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle, or EMBOSS infoalign) after pairwise global sequence alignment using an appropriate algorithm (e.g., Needleman-Wunsch or GenePAST / KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle, or EMBOSS infoalign). Importantly, the query amino acid sequence may be described herein by an amino acid sequence disclosed in particular in one or more claims, especially by Sequence ID No. 22.

[0025] The query sequence may be 100% identical to the target sequence, or it may contain up to a certain integer number of amino acid or nucleotide changes compared to the target sequence, such that the identity percentage is less than 100%. For example, the query sequence may be at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the target sequence. In the case of nucleic acid sequences, such changes may include the deletion, substitution, or insertion of at least one nucleotide residue, and such changes may occur at the 5' or 3' terminal of the query sequence, or at any position between these terminals. These changes may occur individually scattered among nucleotide residues in the query sequence, or in one or more consecutive groups within the query sequence. In the case of amino acid sequences, such alterations include the deletion, substitution (including conserved and non-conservative substitutions), or insertion of at least one amino acid residue, and such alterations may occur at the amino-terminal or carboxy-terminal position of the query sequence, or at any position between these terminal positions, individually scattered among amino acid residues in the query sequence, or in one or more consecutive groups within the query sequence.

[0026] As used herein, “polynucleotide” is a polymer of nucleotide residues. A single polynucleotide molecule may, for example, contain 14 or more nucleotide residues in its chain structure. DNA and RNA are examples of polynucleotides. Polynucleotides include oligonucleotides. Polynucleotides are therapeutically useful and are used, for example, in the production of oligonucleotides that recruit and guide DNA and RNA editing enzymes, such as therapeutic mRNA (which can be used as mRNA vaccines), antisense oligonucleotides, siRNA, miRNA, aptamers, CRISPR-guided RNA, and A-to-I RNA base-editing oligonucleotides (AIMERs).

[0027] As used herein, “polyphosphate kinase,” “PolyP kinase,” or “PPK” are enzymes capable of producing nucleoside triphosphates from polyphosphate and nucleoside monophosphate and / or nucleoside diphosphate. PPK can also produce modified nucleoside triphosphates from polyphosphate and the corresponding modified nucleoside monophosphate and / or diphosphate.

[0028] As used herein, “polymerase” means an enzyme that catalyzes the bonding of a nucleotide to the 3'-OH of another nucleotide, oligonucleotide, or polynucleotide, i.e., covalent bonding, such as by the formation of a phosphodiester bond between the 3' end of one nucleotide or oligonucleotide, or the 5' end of another nucleotide. Therefore, polymerase activity is 5' to 3'. Polymerase may include DNA and / or RNA polymerase. Polymerase may be a wild-type enzyme, a mutant enzyme, or an engineered enzyme.

[0029] As used herein, “primer” means a polynucleotide or oligonucleotide sequence used as a starting point for synthesizing the segment polynucleotides or oligonucleotides of this disclosure. A polymerase may require a primer; for example, DNA polymerase may require a primer. A primer may comprise at least three nucleotides. The primer can bind to the template before the polymerase catalyzes the linking of the nucleotides. The primer does not need to be removed. The primer can form part of the polynucleotide or oligonucleotide template. For example, the template may comprise a hairpin loop containing the primer. The primer can form part of the polynucleotide or oligonucleotide product.

[0030] As used herein, “product polynucleotide or oligonucleotide” means a desired polynucleotide or oligonucleotide having a specific sequence, and is also referred to herein as “target polynucleotide,” “target oligonucleotide,” or “target oligo.”

[0031] As used herein, a “protecting group” is a group attached to another reactive 3'-hydroxyl position of a nucleoside sugar to prevent undesirable side reactions in a given reaction scheme. The protecting group assists the selectivity of the desired reaction and is removed after the desired reaction has occurred.

[0032] "RNA" is a common abbreviation for ribonucleic acid. RNA is a nucleic acid molecule, a polymer composed of nucleotide monomers. These nucleotides are typically adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate monomers or analogs thereof, which are linked to one another along a so-called backbone. The backbone is formed by a phosphodiester bond between a first sugar, i.e., ribose, and the phosphate portion of a second adjacent monomer. The specific order of monomers, i.e., the order of bases linked to the sugar / phosphate backbone, is called the RNA sequence. The term "RNA" generally refers to a molecule or molecular species selected from the group consisting of long RNA, coding RNA, non-coding RNA, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), linear RNA (linRNA), circular RNA (circRNA), messenger RNA (mRNA), RNA oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA (asRNA), CRISPR / Cas9 guide RNA, riboswitches, immunostimulatory RNA (isRNA), ribozymes, aptamers, ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA (vRNA), retroviral RNA or replicon RNA, nuclear small RNA (snRNA), nucleolar small RNA (snoRNA), microRNA (miRNA), and Piwi-binding RNA (piRNA). In the context of this disclosure, this optionally includes all types of therapeutic RNA.

[0033] As used herein, “stopper” is also known as “block” or “3'-adjacent oligonucleotide” and means a polynucleotide or oligonucleotide sequence that stops or prevents polymerase from binding further nucleotides. A stopper can terminate polymerase elongation. A stopper may include 5' phosphate, 5' thiophosphate (which may result in a phosphorothioate bond), 5' amide phosphate (which may result in a phosphoramidate bond), 5' diamide phosphate (which may result in a phosphorodiamidate bond), 5' amide thiophosphate, 5' amide dithiophosphate, 5' diamide thiophosphate, or 5' dithiophosphate (which may result in a phosphorodithioate bond).

[0034] As used herein, “template” means a polynucleotide or oligonucleotide containing a sequence complementary to a single-stranded polynucleotide or oligonucleotide product. A template may contain a sequence 100% complementary to the sequence of the target (or product) polynucleotide or oligonucleotide. A template may consist of a sequence 100% complementary to the sequence of the target (or product) polynucleotide or oligonucleotide. A template may be a longer sequence compared to the product sequence. A template may contain a sequence not used to produce the polynucleotide or oligonucleotide product. If the template is longer than the product sequence, a stopper can be used to control the production and / or length of the polynucleotide or oligonucleotide product. If the template is longer than the product sequence, a primer can be used to control the production and / or length of the polynucleotide or oligonucleotide product. If the template is longer than the product sequence, a hairpin loop may be included. The hairpin loop may act as a primer. The template may contain a segment of the product within the hairpin loop. In such cases, the product can be released from the template via cleavage by a nuclease, nicasse, DNAzyme, or chemical method. Unless otherwise specified, the term “complementary” as used herein means 100% complementary.

[0035] As used herein, “therapeutic oligonucleotide” or “therapeutic oligo” means an oligonucleotide having therapeutic use, for example, in the prevention or treatment of a condition or disease in humans or animals. Such oligos typically contain one or more modified nucleotide residues or linkages. Therapeutic oligos act through one of several different mechanisms, including, but not limited to, antisense, splice switching or exon skipping, immunostimulation and RNA interference (RNAi, e.g., via microRNA (miRNA) or small interfering RNA (siRNA)), and recruitment and induction of DNA and RNA editing enzymes (e.g., A-to-I RNA base editing oligonucleotides (AIMer)). Therapeutic oligos may also be aptamers. Therapeutic oligonucleotides usually have a defined sequence, though not always. Therapeutic oligonucleotides are an example of therapeutic polynucleotides.

[0036] As used herein, “therapeutic polynucleotides” means polynucleotides that have therapeutic applications, for example, in the prevention or treatment of a condition or disease in humans or animals. Such polynucleotides typically contain one or more modified nucleotide residues or linkages. Therapeutic polynucleotides act through one of several different mechanisms, including, but not limited to, antisense, splice switching or exon skipping, immunostimulation, RNA interference (RNAi, e.g., miRNA or siRNA), and recruitment and induction of DNA and RNA editing enzymes (e.g., A-to-I RNA base-editing oligonucleotides (AIMer)). Aptamers are an example of therapeutic polynucleotides. mRNA vaccines are also an example of therapeutic polynucleotides. Therapeutic polynucleotides usually, but not always, have a defined sequence. Therapeutic polynucleotides include therapeutic oligonucleotides and therapeutic RNAs.

[0037] "Therapeutic RNA" is RNA that has therapeutic applications, for example, in the prevention or treatment of a condition or disease in humans or animals. Such RNA is suitable for use in the human or animal body for medical purposes and is clinical grade, for example, with respect to parameters such as purity and integrity, and the underlying manufacturing methods which must comply with current Good Manufacturing Practice (cGMP) requirements.

[0038] As used herein, “transferase” means an enzyme that catalyzes the template-independent binding of one nucleotide to another nucleotide or oligonucleotide. The transferases described herein include terminal nucleotidyl transferases (TdT), also known as DNA nucleotidyl exotransferases (DNTT) or terminal transferases. TdT is a specialized DNA polymerase expressed in immature pre-B, pre-T lymphoid cells, and TdT enables VDJ antibody gene binding diversity. TdT catalyzes the addition of nucleotides to the 3' end of a DNA molecule. The transferases described herein include non-natural or mutant TdT. Transferases capable of binding unmodified nucleotides to unmodified oligonucleotides, transferases capable of binding unmodified nucleotides to modified oligonucleotides, transferases capable of binding modified nucleotides to unmodified oligonucleotides, and transferases capable of binding modified nucleotides to modified oligonucleotides are within the scope of this disclosure.

[0039] "Phosphoramidate (PN)" generally refers to a group having the structure -P(O)2NRaRb, where Ra and Rb are either independent substituents or together represent a double bond to an atom, such as carbon. For example, PN is defined as PO2N(R) as defined herein. 3 )2 or PO2R 4 It could refer to that.

[0040] The term "pharmaceutically acceptable" means a compound (including salts), material, composition, and dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, or other problems or complications, within the bounds of sound medical judgment, and that is commensurate with a reasonable benefit / risk ratio.

[0041] Disclosure of the invention The inventors have surprisingly found that polyphosphate kinase (PPK) is a suitable and effective enzyme for producing modified nucleoside triphosphates (NTPs). The NTPs produced by the method of this disclosure provide a simpler and more cost-effective alternative to state-of-the-art methods. In particular, the inventors have used PPK to produce sugar-modified and / or α-phosphate-modified nucleoside triphosphates from corresponding modified nucleoside monophosphates or nucleoside diphosphates, and polyphosphates.

[0042] Inorganic polyphosphates, also known as polyphosphate or PolyP, are typically linear polymers in which tens to hundreds of phosphate residues (Pi) are linked by high-energy phosphate anhydride bonds, such as those found in adenosine triphosphate (ATP). However, unlike other phosphate sources currently used in ATP or NTP production, PolyP is a more stable form of phosphate. It has been proposed that PolyP acted as an energy source in the world before ATP existed and was first utilized by early single-celled organisms. PolyP is found in all organisms from bacteria to higher eukaryotes and has many biological functions, serving not only as a means of energy storage but also as a Pi reservoir, a metal ion chelator, a channel for DNA entry, a stress and survival regulator, and a supporting component in gene regulation and enzyme function (Motomura et al., Applied and Environmental Microbiology, 2014, 80(8), 2602-2608).

[0043] PPKs are traditionally classified into two families, PPK1 and PPK2, based on differences in amino acid sequence and kinetic properties. PPK1 preferentially catalyzes ATP-driven PolyP formation, while PPK2 preferentially catalyzes PolyP-driven nucleotide phosphorylation, which is crucial for the survival of microbial cells under stress or pathogenic conditions. The PPK2 family is further classified into the following subfamilies: single-domain enzymes that catalyze phosphorylation of class I nucleoside diphosphates, mainly two-domain enzymes that catalyze phosphorylation of class II nucleoside monophosphates, and single-domain enzymes that catalyze phosphorylation of both class III nucleoside monophosphates and nucleoside diphosphates (see Figure 1).

[0044] Known PPKs can be found in INTERPRO (EMBL-EBI) entry IPR022488, including those characterized by Nocek et al. (ACS Catal. 2018, 8, 11, 10746-10760).

[0045] The estimated PPK can be identified by searching sequence databases. Examples of sequence databases include Universal Protein Resource (UniProt), UniParc, European Nucleotide Archive, GenBank, Reference Sequence Database (RefSeq), Protein Information Resource (PIR), Protein Data Bank (PDB), Pfam, and InterPro. Suitable bioinformatics tools include Basic Local Alignment Search Tool (BLAST), Clustal Omega, ClustalW, HMMER, InterProScan, UniProt Tools, CD-HIT, MMseqs2, and CLANS.

[0046] Recently, PPK has been used as an ATP recycling system in the industrial implementation of ATP-dependent enzymes, thus avoiding the addition of stoichiometric amounts of cofactors (Tavanti et al., Green Chemistry, 2021, 23, 828-837).

[0047] In this disclosure, PPK may be used to produce sugar-only modified nucleoside triphosphates, where the base or α-phosphate is unmodified, i.e., the base is a naturally occurring base, and the α-phosphate is unmodified. Sugar-only modified nucleoside triphosphates may include 2'-sugar modification, 3'-sugar modification, or both 2' and 3' sugar modification. Alternatively, PPK may be used to produce α-phosphate-only modified nucleoside triphosphates, where the base or sugar is unmodified, i.e., the base is a naturally occurring base, and the sugar is a naturally occurring sugar (e.g., ribose or deoxyribose). PPK may also be used to produce sugar-modified and / or α-phosphate-modified nucleoside triphosphates, which further have a modified base, i.e., a base that does not exist in nature.

[0048] In one embodiment of this disclosure, a compound of formula (I): [ka] or its salt, or a pharmaceutically acceptable salt thereof, By reacting PPK and polyphosphate, Compound of formula (II): [ka] (In the formula, X1 is OH, SH, CH2, NR 3 2, CH3, BH3 - , Se and R 4 Selected from the group consisting of, X2 is either O or S. R 1 H, PO3, PO2S, PO2N(R 3 )2, PO2R 4, POS2, PO2CH3, PO2BH3, PO2Se, N-methyl-anthraniloyl (MANT), acetyl (Ac), benzyl (Bn), benzoyl (Bz), allyl (All), 2-cyanoethyl (CE), azidomethyl (AzM), cyanovinyl (Cv), methoxymethyl (MOM), methoxyethoxymethyl (MEM), methoxymethylphenylmethyl (MPM), diphenylmethyl (DPM), tetrahydropyranyl (THP), dimethoxytrityl (DMTr), methoxycarbonylmethyl (MOMcl), ethoxycarbonyl (EOC), benzylcarbonyl (Cbz), trifluoromethanesulfonate (triflate), methanesulfonate (mesylate), p-toluenesulfonate (tosylate), pivaloyl (Piv), ortho-nitrobenzyl (o-NB), bromomethylcoumarinylmethyl (Bhc), (7-(diethylamino)coumarin-4-yl)methyl (DEACM), 6-nitroveratryloxycarbonyl (NVOC), and 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc-ol), is selected from the group consisting of R 2 is selected from the group consisting of H, OH, O-CH3 (OMe), O-CH2-CH3 (OEt), O-CH2-CH2-CH3, O-CH2-CH2-O-CH3 (MOE), F, O-CH2-CO-NH-CH3, O-CH2-CH2-O-Nme2, an O-methylene bridge having 4' carbon, and an O-constrained ethylene bridge having 4' carbon R 3 is independently H or

Chemical formula

Chemical formula

[0049] As those skilled in the art will understand, in formulas (I) and (II), depending on the conditions, the OH group is O - It may exist as such, and the SH group is S - They may exist as such. Therefore, in formulas (I) and (II), OH and O where applicable. - They are used interchangeably, SH and S - They are used interchangeably.

[0050] In one embodiment, the bases are adenine (A), uracil (U), cytosine (C), guanine (G), thymine (T), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (hm5C), 5-fluorocytosine (5fC), 5-bromouracil (5BrU), 2-aminopurine (2AP), inosine (I), pseudouracil (Ψ), N1-methylpseudracil (m1Ψ), 1-Ethyl pseudouracil, 2-Thio-1-methyl-1-deaza-pseudracil, 2-Thio-1-methyl-pseudracil, 2-Thio-5-aza-uracil, 2-Thio-dihydropseudracil, 2-Thio-dihydrouracil, 2-Thio-pseudracil, 4-Methoxy-2-thio-pseudracil, 4-Methoxy-pseudracil, 4-Thio-1-methyl-pseudracil N6-methyladenine (m6A), N4-acetol, 4-thio-pseudracil, 5-aza-uracil, dihydropseudracil, 5-methyluracil, 5-methoxyuracil (mo5U), 2'-O-methyluracil (2moU), 7-deazaguanine (7-deaza-G), 8-oxoguanine (8-oxo-G), 5-propynyluracil (pU), 5-propynylcytosine (pC), N6-methyladenine (m6A), N4-acetol The following are selected from the group consisting of cytosine (ac4C), O6-methylguanine (O6meG), hypoxanthine (H), xanthine (X), isocytosine (iC), isoguanine (iG), cuin (Q), 2-thiouracil (s2U), 4-thiouracil (s4U), 5-iodouracil (5Iu), 2-thiocytosine (s2C), 5-chlorouracil (5ClU), and isouracil.

[0051] X1 is OH, SH, CH2, NR 3 2, CH3, BH3 - , Se and R 4 Selected from the group consisting of . As will be understood by those skilled in the art, if X1 is OH, then depending on the conditions, OH or O - It may be so, and therefore OH and O - The terms are used interchangeably in this specification. X1 is SH or S depending on the condition. - It may also be the case that SH and S - These terms are used interchangeably in this specification. If X1 is Se, then Se is used interchangeably depending on the condition. - It may be so, and therefore, in this specification, Se is Se - It is interchangeable with it.

[0052] In one embodiment, X1 is selected from the group consisting of OH, SH, and N, and X2 is O or S, and R 1 These are H, PO3, PO2S, and PO2NR. 3 2. PO2R 4 Selected from the group consisting of POS2, MANT, Ac, Bz, Bn, DMTr, and Cbz, R 2 The group is selected from H, OH, OMe, MOE, OEt, and F, and the base is selected from the group A, U, C, G, T, 5mC, I, Ψ, and m1Ψ.

[0053] In one embodiment, X1 is OH, SH, NR 3 2 or R 4 Selected from the group consisting of, X2 is O or S, and R 1 The group is selected from H, PO3, PO2S, and MANT, and R 2 The group is selected from H, OH, OMe, MOE, and F, and the base is selected from the group A, U, C, G, T, 5mC, I, Ψ, and m1Ψ.

[0054] In one embodiment, X1 is OH. In one embodiment, X1 is SH. In one embodiment, X1 is NR 3 2. In one embodiment, X1 is R 4 That is the case.

[0055] In one embodiment, X2 is O. In another embodiment, X2 is S.

[0056] In one embodiment, R 1 is H. In one embodiment, R 1 is PO3. In one embodiment, R 1 It is MANT.

[0057] In one embodiment, R 2 is H. In one embodiment, R 2 is OH. In one embodiment, R 2 is OMe. In one embodiment, R 2 is MOE. In one embodiment, R 2 It is F.

[0058] In one embodiment, the base is A. In one embodiment, the base is U. In one embodiment, the base is C. In one embodiment, the base is G. In one embodiment, the base is T. In one embodiment, the base is 5mC. In one embodiment, the base is I. In one embodiment, the base is Ψ. In one embodiment, the base is m1Ψ.

[0059] In one embodiment, X1 is OH, X2 is O, and R 1 is PO3, R 2 is H, OH, OMe, MOE, or F. In one embodiment, X1 is OH, X2 is O, and R 1 is MANT, R 2 is H, OH, OMe, MOE, or F. In one embodiment, X1 is OH, X2 is O, and R 1 H is R 2is OMe, MOE, or F. In one embodiment, X1 is SH, X2 is O, and R 1 is PO3, R 2 is H, OH, OMe, MOE, or F. In one embodiment, X1 is SH, X2 is O, and R 1 is MANT, R 2 is H, OH, OMe, MOE, or F. In one embodiment, X1 is SH, X2 is O, and R 1 H is R 2 These are H, OH, OMe, MOE, or F.

[0060] In one embodiment, X1 is OH, X2 is S, and R 1 is PO3, R 2 is H, OH, OMe, MOE, or F. In one embodiment, X1 is OH, X2 is S, and R 1 is MANT, R 2 is H, OH, OMe, MOE, or F. In one embodiment, X1 is OH, X2 is S, and R 1 H is R 2 is OMe, MOE, or F. In one embodiment, X1 is SH, X2 is S, and R 1 is PO3, R 2 is H, OH, OMe, MOE, or F. In one embodiment, X1 is SH, X2 is S, and R 1 is MANT, R 2 is H, OH, OMe, MOE, or F. In one embodiment, X1 is SH, X2 is S, and R 1 H is R 2 These are H, OH, OMe, MOE, or F.

[0061] In one embodiment, X1 is OH, X2 is O, and R 1 is PO3, R 2 is OMe. In one embodiment, X1 is OH, X2 is O, and R 1 is PO3, R 2is MOE. In one embodiment, X1 is OH, X2 is O, and R 1 is PO3, and R 2 is F. In one embodiment, X1 is OH, X2 is S, and R 1 is PO3, and R 2 is OMe. In one embodiment, X1 is OH, X2 is S, and R 1 is PO3, and R 2 is MOE. In one embodiment, X1 is OH, X2 is S, and R 1 is PO3, and R 2 is F.

[0062] In one embodiment, X1 is OH, X2 is O, and R 1 is MANT, and R 2 is OMe. In one embodiment, X1 is OH, X2 is O, and R 1 is MANT, and R 2 is MOE. In one embodiment, X1 is OH, X2 is O, and R 1 is MANT, and R 2 is F. In one embodiment, X1 is OH, X2 is S, and R 1 is MANT, and R 2 is OMe. In one embodiment, X1 is OH, X2 is S, and R 1 is MANT, and R 2 is MOE. In one embodiment, X1 is OH, X2 is S, and R 1 is MANT, and R 2 is F.

[0063] In one embodiment, X1 is OH, X2 is O, and R 1 is H, and R 2 is OMe. In one embodiment, X1 is OH, X2 is O, and R 1 is H, and R 2 is MOE. In one embodiment, X1 is OH, X2 is O, and R 1 is H, and R 2is F. In one embodiment, X1 is OH, X2 is S, and R 1 is H, and R 2 is OMe. In one embodiment, X1 is OH, X2 is S, and R 1 is H, and R 2 is MOE. In one embodiment, X1 is OH, X2 is S, and R 1 is H, and R 2 is F.

[0064] In one embodiment, X1 is SH, X2 is O, and R 1 is PO3, and R 2 is H. In one embodiment, X1 is SH, X2 is O, and R 1 is PO3, and R 2 is OH. In one embodiment, X1 is SH, X2 is O, and R 1 is PO3, and R 2 is OMe. In one embodiment, X1 is SH, X2 is O, and R 1 is PO3, and R 2 is MOE. In one embodiment, X1 is SH, X2 is O, and R 1 is PO3, and R 2 is F. In one embodiment, X1 is SH, X2 is S, and R 1 is PO3, and R 2 is H. In one embodiment, X1 is SH, X2 is S, and R 1 is PO3, and R 2 is OH. In one embodiment, X1 is SH, X2 is S, and R 1 is PO3, and R 2 is OMe. In one embodiment, X1 is SH, X2 is S, and R 1 is PO3, and R 2 is MOE. In one embodiment, X1 is SH, X2 is S, and R 1 is PO3, and R 2 is F.

[0065] In one embodiment, X1 is SH, X2 is O, and R 1 is MANT, R 2 is H. In one embodiment, X1 is SH, X2 is O, and R 1 is MANT, R 2 is OH. In one embodiment, X1 is SH, X2 is O, and R 1 is MANT, R 2 is OMe. In one embodiment, X1 is SH, X2 is O, and R 1 is MANT, R 2 is MOE. In one embodiment, X1 is SH, X2 is O, and R 1 is MANT, R 2 is F. In one embodiment, X1 is SH, X2 is S, and R 1 is MANT, R 2 is H. In one embodiment, X1 is SH, X2 is S, and R 1 is MANT, R 2 is OH. In one embodiment, X1 is SH, X2 is S, and R 1 is MANT, R 2 is OMe. In one embodiment, X1 is SH, X2 is S, and R 1 is MANT, R 2 is MOE. In one embodiment, X1 is SH, X2 is S, and R 1 is MANT, R 2 It is F.

[0066] In one embodiment, X1 is SH, X2 is O, and R 1 H is R 2 is H. In one embodiment, X1 is SH, X2 is O, and R 1 H is R 2 is OH. In one embodiment, X1 is SH, X2 is O, and R 1 H is R 2 is OMe. In one embodiment, X1 is SH, X2 is O, and R 1 H is R2 is MOE. In one embodiment, X1 is SH, X2 is O, and R 1 H is R 2 is F. In one embodiment, X1 is SH, X2 is S, and R 1 H is R 2 is H. In one embodiment, X1 is SH, X2 is S, and R 1 H is R 2 is OH. In one embodiment, X1 is SH, X2 is S, and R 1 H is R 2 is OMe. In one embodiment, X1 is SH, X2 is S, and R 1 H is R 2 is MOE. In one embodiment, X1 is SH, X2 is S, and R 1 H is R 2 It is F.

[0067] In one embodiment, R 2 If is OH, the sugar is ribose. In one embodiment, R 2 If the hyphen is OH, the sugar is 2'-arabinose (ANA).

[0068] In one embodiment, R 2 If H is present, the sugar is 2'-deoxyribose.

[0069] In one embodiment, R 2 If is F, the sugar is 2'-deoxy-2'-fluororibose (FNA). In one embodiment, R 2 If the sugar is F, then the sugar is 2'-deoxy-2'-fluoro-β-D-arabinose (FANA).

[0070] In one embodiment of this disclosure, PPK can utilize one or more of the following substrates: locked nucleic acid (LNA), restricted ethyl (cEt), and 1,5-dianehydrohexitol (HNA) nucleoside.

[0071] In one embodiment, the nucleoside triphosphate produced by the PPK catalytic reaction is as follows: [ka] [Table 1] (In the formula, PN-1= [ka] (is) or its salt, or a pharmaceutically acceptable salt thereof It is any one of the following.

[0072] In one aspect of this disclosure, the use of PPK is provided for producing sugar-modified and / or α-phosphate-modified nucleoside triphosphates from sugar-modified and / or α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates.

[0073] In one embodiment, PPK is PPK2. In one embodiment, PPK2 is PPK2-III.

[0074] In one embodiment, PPK contains an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22. In one embodiment, PPK consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22. In one embodiment, PPK contains SEQ ID NO: 22. In one embodiment, PPK is SEQ ID NO: 22.

[0075] In one embodiment, PPK is obtained by a screening method disclosed herein.

[0076] In one aspect of the present disclosure, an enzymatic method is provided for producing glycosylated and / or α-phosphate-modified nucleoside triphosphates, comprising incubating PPK with corresponding modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates under suitable conditions.

[0077] In one embodiment, incubation is performed using a divalent cation. In one embodiment, the divalent cation is Mg 2+ Mn 2+ , Cd 2+ , or Co 2+ In one embodiment, the divalent cation is Mg 2+ In one embodiment, the divalent cation is Mn 2+ In one embodiment, the divalent cation is Cd 2+ In one embodiment, the divalent cation is Co 2+ That is the case.

[0078] In one embodiment, incubation uses magnesium chloride, manganese chloride, cadmium chloride, cobalt chloride, magnesium acetate, manganese acetate, cadmium acetate, or cobalt acetate. In one embodiment, incubation uses magnesium chloride, manganese chloride, magnesium acetate, or manganese acetate. In one embodiment, incubation uses magnesium chloride or magnesium acetate. In one embodiment, incubation uses magnesium chloride. In one embodiment, incubation uses magnesium chloride or magnesium acetate. In one embodiment, incubation uses manganese chloride.

[0079] Sugar modifications include modifications at the 2' and 3' positions. 3'-Sugar modifications can act as protecting groups in certain reactions.

[0080] Examples of 2'-sugar modifications include phosphoric acid (PO), O-methyl (OMe), O-methoxyethyl (MOE), fluoro (FNA), O-ethyl (OEt), O-propyl (OProp), 2'-arabino (ANA), 2'-deoxy-2'-fluoro-β-D-arabino (FANA), O-[2-(methylamino)-2-oxoethyl] (NMA), and 2'-O-[2-{(N,N-dimethylamino)oxy}-ethyl] (DMAOE). In one embodiment, the 2'-sugar modification is selected from the group consisting of OMe, MOE, FNA, OEt, and FANA. In one embodiment, the 2'-sugar modification is selected from the group consisting of OMe, MOE, and FNA. In one embodiment, the 2'-sugar modification is OMe. In one embodiment, the 2'-sugar modification is MOE. In one embodiment, the 2'-sugar modification is FNA. In one embodiment, the 2'-sugar modification is FANA. In one embodiment, the 2'-saccharide modification is OEt.

[0081] In one embodiment, the 2'-sugar modification is a crosslink between the 2' oxygen and the 4' carbon sugar (ribose) position. In one embodiment, the crosslink is a methylene crosslink, i.e., a locked nucleic acid sugar. In one embodiment, the crosslink is a restricted ethyl crosslink.

[0082] Examples of 3'-sugar modifications include H, PO, phosphorothioate (PS), thiophosphate, phosphoramidate (PN), phosphorodithioate, methylphosphonate, boranophosphate, phosphoroselenoate, MANT, Ac, Bn, Bz, All, CE, AzM, Cv, MOM, MEM, MPM, DPM, THP, DMTr, MOMcl, EOC, Cbz, triflate, mesylate, tosylate, Piv, o-NB, Bhc, DEACM, NVOC, and Bhc-ol. In one embodiment, the 3'-sugar modification is selected from the group consisting of H, PO, PS, PN, phosphorodithioate, MANT, Ac, Bn, Bz, and Cbz. In one embodiment, the 3'-sugar modification is selected from the group consisting of H, PO, PS, PN, phosphorodithioate, and MANT. In one embodiment, the 3'-sugar modification is selected from the group consisting of H, PO, PS, and MANT. In one embodiment, the 3'-sugar modification is PO. In one embodiment, the 3'-sugar modification is MANT. In one embodiment, the 3'-sugar modification is H. If the 3'-sugar modification is PO and acts as a protecting group, the PO protecting group can be removed using a phosphatase.

[0083] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) includes a 2'-sugar modification. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) includes 2'-OMe, 2'-MOE, or 2'-FNA. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) does not include a 2'-sugar modification, i.e., the 2' position is H or OH. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) includes 2'-H. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) includes 2'-OH.

[0084] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) includes a 3'-sugar modification. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) includes a 3'-sugar modification and a 2'-H. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) includes a 3'-sugar modification and a 2'-OH. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) includes a 3'-PO, a 3'-PS, a 3'-MANT, or a 3'-hydrogen. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains 3'-PO, 3'-PS, 3'-MANT or 3'-hydrogen, and 2'-H. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains 3'-PO, 3'-PS, 3'-MANT or 3'-hydrogen, and 2'-OH. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) does not contain 3'-sugar modification, i.e., the 3' position is OH.

[0085] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) includes 2'-sugar modification and 3'-sugar modification. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) includes 2'-H, 2'-OH, 2'-OMe, 2'-MOE or 2'-FNA, and 3'-PO, 3'-PS, 3'-MANT or 3'-hydrogen.

[0086] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-OMe and 3'-PO. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-OMe and 3'-PS. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-OMe and 3'-MANT. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-OMe and 3'-hydrogen.

[0087] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-MOE and 3'-PO. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-MOE and 3'-PS. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-MOE and 3'-MANT. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-MOE and 3'-hydrogen.

[0088] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-FNA and 3'-PO. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-FNA and 3'-PS. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-FNA and 3'-MANT. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises 2'-FNA and 3'-hydrogen.

[0089] In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains α-phosphate. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains a naturally occurring base. In one embodiment, the sugar-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains α-phosphate and a naturally occurring base.

[0090] In one embodiment, any one or more of the above-described sugar modifications may be combined with α-phosphate modifications and / or base modifications.

[0091] Examples of α-phosphate modifications include PS, PN, methylphosphonate, boranophosphate, and phosphoroselenoate. In one embodiment, the α-phosphate modification is PS, for example, a compound of the following formula (IIa): [ka] or a salt thereof, or a pharmaceutically acceptable salt thereof In the formula, X2, R 1 , R 2 The bases are as described above.

[0092] In one embodiment, the α-phosphate modification is PN, for example, a compound of the following formula (IIb) or (IIc): [ka] or a salt thereof, or a pharmaceutically acceptable salt thereof In the formula, X2, R 1 , R 2 , R 3 , R 4 The bases are as described above.

[0093] References herein to compounds of formula (I) or (II) or their salts should be understood to include compounds of the formula as free acids or as salts thereof, for example, as pharmaceutically acceptable salts thereof. For their potential use in pharmaceuticals, salts of compounds of formula (I) and (II) will be understood to be preferably pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, in particular, those described in Berge, J. Pharm. Sci., 1977, 66, 1-19, or those listed in P.H. Stahl and C.G. Wermuth, eds., Handbook of Pharmaceutical Salts; Properties, Selection and Use, 2nd edition, Stahl / Wermuth: Wiley-VCH / VHCA, 2011. Examples of pharmaceutically acceptable base addition salts of compounds of formula (I) and (II) include sodium and lithium. Pharmaceutically unacceptable salts are also within the scope of this invention.

[0094] In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains 2'-H. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains 2'-OH. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains ribose or deoxyribose.

[0095] In one embodiment, the α-phosphate-modified nucleoside triphosphate includes a modified sugar.

[0096] In one embodiment, α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) also includes sugar modification. In one embodiment, α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) also includes 2'-sugar modification. In one embodiment, α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) also includes 3'-sugar modification. In one embodiment, α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) also includes 2'-sugar modification and 3'-sugar modification.

[0097] In one embodiment, the α-phosphate-modified nucleoside triphosphate includes α-thiophosphate and also includes sugar modification. In one embodiment, the α-phosphate-modified nucleoside triphosphate includes α-thiophosphate and also includes 2'-sugar modification. In one embodiment, the α-phosphate-modified nucleoside triphosphate includes α-thiophosphate and also includes 3'-sugar modification. In one embodiment, the α-phosphate-modified nucleoside triphosphate includes α-thiophosphate and also includes 2'-sugar modification and 3'-sugar modification.

[0098] In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate and 2'-OMe. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate and 2'-MOE. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate and 2'-FNA.

[0099] In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains α-thiophosphate and 3'-PO. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains α-thiophosphate and 3'-PS. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains α-thiophosphate and 3'-MANT. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) contains α-thiophosphate and 3'-hydrogen.

[0100] In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-OMe, and 3'-PO. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-OMe, and 3'-PS. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-OMe, and 3'-MANT. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-OMe, and 3'-hydrogen.

[0101] In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-MOE, and 3'-PO. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-MOE, and 3'-PS. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-MOE, and 3'-MANT. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-MOE, and 3'-hydrogen.

[0102] In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-FNA, and 3'-PO. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-FNA, and 3'-PS. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-FNA, and 3'-MANT. In one embodiment, the α-phosphate-modified nucleoside triphosphate (and the corresponding modified nucleoside monophosphate or nucleoside diphosphate) comprises α-thiophosphate, 2'-FNA, and 3'-hydrogen.

[0103] NTPs containing α-phosphate modification have a chiral center on phosphorus. For example, the formation of a phosphoanhydride bond to α-thiophosphate creates a chiral center on the phosphorus of α-thiophosphate, resulting in two diastereomers, S. p diastereomers and R p It produces diastereomers.

[0104] As is evident from the examples herein (see, for example, Figure 3), different wild-type PPKs exhibit different control over stereochemistry under the same reaction conditions. The diastereomer ratio produced by PPK may be 1, indicating no stereopreference for phosphorylation, i.e., both diastereomers are a 50:50 mixture (S P :R p ) will be different PPKs, S p It may have a preference for producing diastereomers, but another PPK is R p It may have a preference for generating diastereomers.

[0105] Stereochemical control can sometimes be manipulated by altering the divalent cation.

[0106] In one aspect of this disclosure, the use of PPK is provided for producing stereospecific α-phosphate-modified nucleoside triphosphates from α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates.

[0107] In one embodiment, PPK is S in a ratio of approximately 50:50. p Diastereomer vs. R p Diastereomers are produced. In one embodiment, PPK is R in a ratio of approximately 55:45. p Diastereomer vs. S p Diastereomers are produced. In one embodiment, PPK is S in a ratio of approximately 55:45. p Diastereomer vs. R p It generates diastereomers.

[0108] In one embodiment, PPK is S in a ratio of approximately 55:45, approximately 60:40, approximately 65:35, approximately 70:30, approximately 75:25, or approximately 80:20. p Diastereomer vs. R p It generates diastereomers.

[0109] In one embodiment, PPK is R in a ratio of approximately 55:45, approximately 60:40, approximately 65:35, approximately 70:30, approximately 75:25, or approximately 80:20. p Diastereomer vs. S p It generates diastereomers.

[0110] In one embodiment, PPK is substantially all S p Diastereomers are produced. In one embodiment, S P :R p is greater than 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99:1. In one embodiment, S P :R p is greater than 95:5. In one embodiment, S P :R p It is greater than 99:1.

[0111] In one embodiment, PPK is substantially all R p A diastereomer was produced. In one embodiment, R p :S P is greater than 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99:1. In one embodiment, R p :S P is greater than 95:5. In one embodiment, R p :S P It is greater than 99:1.

[0112] In one embodiment, the stereospecific α-phosphate-modified nucleoside triphosphate is a stereospecific PS-modified nucleoside triphosphate.

[0113] In one embodiment, the stereospecific PS-modified nucleoside triphosphate is nucleoside 5'-(R P It is )-[α-thio]triphosphate. In one embodiment, the stereospecific PS-modified nucleoside triphosphate is nucleoside 5'-(S P It is )-[α-thio]triphosphate.

[0114] In one embodiment, the sugar-modified and / or α-phosphate-modified triphosphate contains a naturally occurring base. In one embodiment, the sugar-modified and / or α-phosphate-modified nucleoside triphosphate also contains a base modification.

[0115] In one embodiment, the bases are A, U, C, G, T, m5C, hm5C, 5fC, 5BrU, 2AP, I, Ψ, m1Ψ, 1-ethylpsuduracil, 2-thio-1-methyl-1-deazapsuduracil, 2-thio-1-methylpsuduracil, 2-thio-5-azapsuduracil, 2-thio-dihydropsuduracil, 2-thio-dihydrouracil, 2-thiopsuduracil, 4-methoxy-2-thiopsuduracil, 4-meth The following are selected from the group consisting of xy-pseudracil, 4-thio-1-methyl-pseudracil, 4-thio-pseudracil, 5-aza-uracil, dihydropseudracil, 5-methyluracil, 5-methoxyuracil (mo5U), 2'-O-methyluracil, 7-deaza-G, 8-oxo-G, pU, pC, m6A, ac4C, O6meG, H, X, iC, iG, Q, s2U, s4U, 5Iu, s2C, 5ClU, and isouracil.

[0116] In one embodiment, the base is selected from the group consisting of A, U, C, G, T, m5C, hm5C, 5fC, 5BrU, 2AP, I, Ψ, m1Ψ, 7-deaza-G, 8-oxo-G, pU, pC, m6A, ac4C, O6meG, H, X, iC, iG, Q, s2U, 5Iu, s2C, and 5ClU.

[0117] In one embodiment, the base is selected from the group consisting of A, U, C, G, T, m5C, and I. In one embodiment, the base is selected from the group consisting of A, U, C, G, and T. In one embodiment, the base is selected from the group consisting of A, U, C, G, and m5C. In one embodiment, the base is selected from the group consisting of A, U, C, and G.

[0118] In one embodiment, the base is A. In one embodiment, the base is U. In one embodiment, the base is C. In one embodiment, the base is G. In one embodiment, the base is T. In one embodiment, the base is 5mC. In one embodiment, the base is I. In one embodiment, the base is Ψ. In one embodiment, the base is m1Ψ.

[0119] In one embodiment, the sugar-modified nucleoside triphosphate includes 2'-sugar modification and base modification. In one embodiment, the sugar-modified nucleoside triphosphate includes 3'-sugar modification and base modification. In one embodiment, the sugar-modified nucleoside triphosphate includes 2'-sugar modification, 3'-sugar modification, and base modification. In one embodiment, the sugar-modified nucleoside triphosphate includes 2'-sugar modification, 3'-sugar modification, base modification, and α-phosphate modification.

[0120] In one embodiment, the α-phosphate-modified nucleoside triphosphate also includes base modification. In one embodiment, the α-phosphate-modified nucleoside triphosphate also includes base modification and 2'-sugar modification. In one embodiment, the α-phosphate-modified nucleoside triphosphate also includes base modification and 3'-sugar modification. In one embodiment, the α-phosphate-modified nucleoside triphosphate also includes base modification, 2'-sugar modification and 3'-sugar modification.

[0121] Nucleoside triphosphates that can be produced by the method disclosed herein include modified deoxyadenosine triphosphate, modified deoxycytidine triphosphate, modified deoxyguanosine triphosphate, modified deoxythymidine triphosphate, modified deoxyuridine triphosphate, modified adenosine triphosphate, modified cytidine triphosphate, modified guanosine triphosphate, modified thymidine triphosphate, modified uridine triphosphate, 2'-deoxyadenosine-5'-(α-thio)-triphosphate, and 2'-deoxycytidine-5' Examples include -(α-thio)-triphosphate, 2'-deoxyguanosine-5'-(α-thio)-triphosphate, 2'-deoxythymidine-(α-thio)-triphosphate, 2'-deoxyuridine-(α-thio)-triphosphate, 2'-adenosine-5'-(α-thio)-triphosphate, 2'-cytidine-5'-(α-thio)-triphosphate, 2'-guanosine-5'-(α-thio)-triphosphate, 2'-thymidine-(α-thio)-triphosphate, and 2'-uridine-(α-thio)-triphosphate.

[0122] In one embodiment, the nucleoside triphosphate produced by the method of the present disclosure is 2'-OMe-,3'-PO3-CTP;2'-OMe-,3'-PO3-CTP-α-PN-1(S P );2'-OMe-,3'-PO3-CTP-α-S(R P );2'-(R)-F-, 3'-PO3-ATP-α-S(R P );2'-(R)-F-, 3'-PO3-GTP-α-S(R P );2'-MOE-,3'-PO3-5mCTP-α-S(R P );2'-MOE-, 3'-PO3-ATP;2'-(R)-F-, 3'-PO3-GTP;2'-(R)-F-, 3'-PO3-CTP-α-S(R P );2'-OMe-,3'-PO3-UTP-α-S(R P );2'-OMe-, 3'-PO3-UTP;2'-OMe-, 3'-PO3-CTP-α-PN-1(R P );2'-(R)-F-, 3'-PO3-ATP;2'-(R)-F-, 3'-PO3-GTP-α-S(R P );2'-(R)-F-, 3'-PO3-UTP-α-PN-1(S P);2’-MOE-、3’-PO3-5mCTP;2’-(R)-F-、3’-PO3-CTP-α-S(R P );2’-OMe-、3’-PO3-CTP-α-S(R P );2’-OMe-、3’-PO3-UTP;2’-OMe-、3’-PO3-UTP-α-PN-1(R P );2’-MOE-、3’-PO3-5mCTP;2’-(R)-F-、3’-PO3-CTP、2’-デオキシ-、3’-PO3-5mCTP-α-S(R P )、2’-デオキシ-、3’-PO3-(N3)-UTP-α-S(R P )、2’-デオキシ-、3’-PO3-ITP-α-S(R P )、2’-OMe-、3’-PO3-UTP-α-PN-1(R P )、2’-(R)-F-、3’-PO3-CTP、2’-OMe-、3’-PO3-GTP-α-S(R P )、2’-OMe-、3’-PO3-ATP-α-S(R P )、2’-OMe-、3’-PO3-UTP-α-PN-1(S P)、2'-OMe-、3'-PO3-CTP-α-S(ambo)、2'-OMe-、3'-PO3-GTP、2'-OMe-、3'-PO3-UTP、2'-OM e-3'-PO3-ATP, 2'-(R)-F-3'-PO3-GTP, 2'-(R)-F-, 3'-PO3-CTP, 2'-OMe-, 3'-PO3-UTP- α-S(ambo), 2'-(R)-F-, 3'-PO3-CTP-α-S(ambo), 2'-OMe-, 3'-PO3-ATP-α-S(ambo), 2'-( R)-F-、3'-PO3-UTP-α-S(ambo)、2'-OMe-、3'-PO3-CTP、2'-(R)-F-、3'-PO3-UTP、2'-(R)-F -、3'-PO3-ATP、2'-OMe-、3'-PO3-GTP-α-S(ambo)、2'-MOE-、3'-PO3-GTP-α-S(ambo)、2'- MOE-、3′-PO3-MeCTP-α-S(ambo), 2′-MOE-、3′-PO3-ATP-α-S(ambo), 2′-dimer-、3′-PO3-GT P-α-S(ambo) and 2'-dimer-3'-PO3-TTP-α-S(ambo) and 2'-dimer-3'-PO3-ATP-α-S(ambo) 3'-PO3-MeCTP-α-S(ambo) and 2'-MOE-, 3'-PO3-MeUTP-α-S(ambo) were smooth.

[0123] In one aspect of the present disclosure, a method is provided for screening a panel of putative and known PPKs to identify enzymes capable of producing corresponding modified nucleoside triphosphates using sugar-modified and / or α-phosphate-modified nucleoside monophosphate or nucleoside diphosphate as substrates. The screening method comprises: a) identifying sequences encoding the panel of putative and known PPKs; b) synthesizing the PPKs; c) purifying the PPKs; d) mixing each of the purified PPKs with the sugar-modified and / or α-phosphate-modified nucleoside monophosphate or nucleoside diphosphate and polyphosphate to form a reaction mixture; and e) analyzing each of the reaction mixtures for the presence of the corresponding modified nucleoside triphosphate to determine which enzyme produces the corresponding modified nucleoside triphosphate.

[0124] In one embodiment, the sequence in step a) is identified using one or more sequence databases and bioinformatics tools. In one embodiment, the sequence database is selected from the group consisting of UniProt, UniParc, European Nucleotide Archive, GenBank, RefSeq, PIR, PDB, Pfam, and InterPro. In one embodiment, the sequence database is UniProt. In one embodiment, the sequence database is InterPro. In one embodiment, the bioinformatics tool is selected from the group consisting of BLAST, Clustal Omega, ClustalW, HMMER, InterProScan, UniProt Tools, CD-HIT, MMseqs2, and CLANS. In one embodiment, the bioinformatics tool is CD-HIT. In one embodiment, the sequence database is UniProt and the bioinformatics tool is CD-HIT.

[0125] In one embodiment, the sequence in step a) encodes a protein with a length of 150 to 800 amino acid residues. In one embodiment, the sequence in step a) encodes a protein with a length of 150 to 600 amino acid residues. In one embodiment, the sequence in step a) encodes a protein with a length of 600 to 700 amino acid residues.

[0126] In one embodiment, PPK is synthesized using an N-terminal hexahistidine tag in step b) and purified using nickel-nitriloacetate in step c).

[0127] In one embodiment, step d) includes incubating the sugar-modified and / or α-phosphate-modified nucleoside monophosphate or nucleoside diphosphate, polyphosphate and PPK at 25°C. In one embodiment, incubation at 25°C is 24 hours. In one embodiment, the reaction is quenched after incubation at 25°C for 24 hours. In one embodiment, the reaction is quenched with EDTA at 80°C for 15 minutes. In one embodiment, the polyphosphate is sodium hexametaphosphate.

[0128] In one embodiment, the reaction mixture is centrifuged and the supernatant is analyzed for the presence of the corresponding modified nucleoside triphosphate. In one embodiment, the reaction mixture is centrifuged at 4000 g for 15 minutes. In one embodiment, the supernatant is analyzed using HPLC or UPLC.

[0129] In one embodiment, an enzyme capable of producing the corresponding modified nucleoside triphosphate is further manipulated to increase the conversion percentage to the corresponding modified nucleoside triphosphate. Methods of manipulating the enzyme are known to those skilled in the art and include mutagenesis and / or DNA shuffling, as described in Stemmer, 1994, Proc Natl Acad Sci USA 91:10747~10751; International Publication No. 95 / 22625; International Publication No. 97 / 0078; International Publication No. 97 / 35966; International Publication No. 98 / 27230; International Publication No. 00 / 42651; International Publication No. 01 / 75767; and U.S. Patent No. 6,537,746. Other procedures that can be used include, in particular, the staggered extension process (StEP), in vitro recombination (Zhao et al., 1998, Nat. Biotechnol. 16:258~261), mutagenic PCR (Caldwell et al., 1994, PCR Methods Appl. 3:S136~S140), and cassette mutagenesis (Black et al., 1996, Proc Natl Acad Sci USA 93:3525~3529). Techniques for mutation introduction and directed evolution useful for the purposes of this specification are cited in the following literature: Ling et al., 1997, "Approaches to DNA mutagenesis: an overview," Anal. Biochem. 254(2):157~78; Vidal et al., 2023, "A primer to directed evolution: current methodologies and future directions," RSC Chem Biol, 4:271~291; Currin et al., 2021, "The evolving art of creating genetic diversity: From directed evolution to synthetic biology," Biotechnology Advances, 50:107762.Currinら、2015、「Synthetic biology for the directed evolution of protein biocatalysts:navigating sequence space intelligently」、Chem Soc rev、44:1172~1239;Daleら、1996、「Oligonucleotide-directed random mutagenesis using the phosphorothioate method」、Methods Mol.Biol.57:369~74;Smith、1985、「In vitro mutagenesis」、Ann.Rev.Genet.19:423~462;Botsteinら、1985、「Strategies and applications of in vitro mutagenesis」、Science 229:1193~1201;Carter、1986、「Site-directed mutagenesis」、Biochem.J.237:1~7;Kramerら、1984、「Point Mismatch Repair」、Cell 38:879~887;Wellsら、1985、「Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites」、Gene 34:315~323;Minshullら、1999、「Protein evolution by molecular breeding」、Curr Opin Chem Biol 3:284~290;Christiansら、1999、「Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling」、Nature Biotech 17:259~264;Crameriら、1998、「DNA shuffling of a family of genes from diverse species accelerates directed evolution」、Nature 391:288~291;This is also described in Crameri et al., 1997, "Molecular evolution of an arsenate detoxification pathway by DNA shuffling," Nature Biotech 15:436~438; Zhang et al., 1997, "Directed evolution of an effective fructosidase from a galactosidase by DNA shuffling and screening," Proc Natl Acad Sci USA 94:45-4-4509; Crameri et al., 1996, "Improved green fluorescent protein by molecular evolution using DNA shuffling," Nature Biotech 14:315~319; and Stemmer, 1994, "Rapid evolution of a protein in vitro by DNA shuffling," Nature 370:389~391.

[0130] In one aspect of this disclosure, manipulated PPKs are provided that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to SEQ ID NO: 22. A PPK having 100% sequence identity with respect to SEQ ID NO: 22 is not a manipulated PPK.

[0131] The NTPs produced by the method of this disclosure can be used in the production of polynucleotides, including oligonucleotides.

[0132] Polynucleotides may consist solely of naturally occurring nucleotide residues (i.e., all “natural nucleotides” or “naturally occurring nucleotides”), or they may include at least one modified nucleotide residue, or at least one linkage between modified nucleotide residues.

[0133] Examples of naturally occurring nucleotides include deoxyadenosine monophosphate, deoxycytidine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, deoxyuridine monophosphate, adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, thymidine monophosphate, and uridine monophosphate.

[0134] Modified nucleotides are not naturally occurring nucleotides (i.e., they are non-natural nucleotides). Modified nucleotides may be naturally occurring nucleotides that have been modified, for example, chemically modified. Modified nucleotides may include modified phosphate groups, modified sugars, and / or modified nucleic acid bases. Certain modifications are recognized to occur sporadically in nature, i.e., in naturally occurring nucleotides such as 2'-OMe or C5 pyrimidine modifications, but in this disclosure, these are considered modified nucleotides.

[0135] Modifications in the sugar moiety may include modifications at the 2' position of the sugar moiety, modifications at the 3' position of the sugar moiety, bicyclic sugars, or 4'-CH(CH3)-O-2' groups, and combinations thereof. For example, modifications at the 2' position of the sugar moiety may include 2'-F, 2'-OMe, or 2'-MOE.

[0136] Examples of modified nucleic acid bases include modified cytosines, such as 5-methylcytosine, 5-methylpyrimidine, 7-deazaguanosine, and debasalized nucleotides. Further examples of modified nucleic acid bases include m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), mlA (1-methyladenosine); m2A (2-methyladenosine); Am (2'-O-methyladenosine); ms2m6A (2-methylthio-N6-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); io6 A(N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A(2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A(N6-glycylcarbamoyladenosine); t6A(N6-threonylcarbamoyladenosine); ms2t6A(2-methylthio-N6-threonylcarbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyladenosine); ms2h n6A(2-methylthio-N6-hydroxynorvalylcarbamoyladenosine);Ar(p)(2'-O-ribosyladenosine (phosphate));I(inosine);miI(1-methylinosine);m'Im(l,2'-O-dimethylinosine);m3C(3-methylcytidine);Cm(2T-O-methylcytidine);s2C(2-thiocytidine);ac4C(N4-acetylcytidine);f5C(5-formylcytidine);m5Cm(5,2-O-dimethylcytidine);ac4Cm(N4-acetyl2TO-methylcytidine) Lucitidine); k2C (Lycidin); mlG (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywibutosine);OHyW (hydroxywibutosine); OHyW* (unmodified hydroxywibutosine); imG (wyosine); mimG (methylguanosine); Q (queusin); oQ (epoxyqueusin); galQ (galactosylqueusin); manQ (mannosylqueusin); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G (archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine) );s4U(4-thiouridine);m5s2U(5-methyl-2-thiouridine);s2Um(2-thio-2'-O-methyluridine);acp3U(3-(3-amino-3-carboxypropyl)uridine);ho5U(5-hydroxyuridine);mo5U(5-methoxyuridine);cmo5U(uridine 5-oxyacetic acid);mcmo5U(uridine 5-oxyacetic acid methyl ester);chm5U(5-(carboxyhydroxymethyl)uridine));mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester);mcm5 U(5-Methoxycarbonylmethyluridine);mcm5Um(S-Methoxycarbonylmethyl-2-O-methyluridine);mcm5s2U(5-Methoxycarbonylmethyl-2-thiouridine);nm5s2U(5-Aminomethyl-2-thiouridine);mnm5U(5-Methylaminomethyluridine);mnm5s2U(5-Methylaminomethyl-2-thiouridine);mnm5se2U(5-Methylaminomethyl-2-selenouridine);ncm5U(5-Carbamoylmethyluridine);ncm5Um(5-Carbamoylmethyl-2'-O -methyluridine);cmnm5U(5-carboxymethylaminomethyluridine);cnmm5Um(5-carboxymethylaminomethyl-2-LOmethyluridine);cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine);m62A(N6,N6-dimethyladenosine);Tm(2'-O-methylinosine);m4C(N4-methylcytidine);m4Cm(N4,2-O-dimethylcytidine);hm5C(5-hydroxymethylcytidine);m3U(3-methyluridine);cm5U(5-carboxymethyluridine);m6Am(N6,TO-dimethyladenosine);rn62Am(N6,N6-O-2-trimethyladenosine);m2'7G(N2,7-dimethylguanosine);m2'2'7G(N2,N2,7-trimethylguanosine);m3Um(3,2T-O-dimethyluridine);m5D(5-methyldihydrouridine);£5Cm(5-formyl-2'-O-methylcytidine);mlGm(1,2'-O-dimethylguanosine);m'Am(1,2-O-dimethyladeno Syn) Irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine); and ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(Cl-C6)-alkyluracil, 5-methyluracil, 5-(C2-C6)-Alkenyluracil, 5-(C2-C6)-Alkinyluracil, 5-(Hydroxymethyl)uracil, 5-Chlorouracil, 5-Fluorouracil, 5-Bromouracil, 5-Hydroxycytosine, 5-(Cl-C6)-Alkylcytosine, 5-Methylcytosine, 5-(C2-C6)-Alkenylcytosine, 5-(C2-C6)-Alkinylcytosine, 5-Chlorocytosine, 5-Fluorocytosine, 5-Bromocytosine, N2-Dimethylguani Examples include 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, and 7-deaza-8-substituted purine. For example, polynucleotides may contain one or more modified pyrimidine nucleic acid bases such as pseudouridine and / or 5-methylcytosine residues.

[0137] Examples of skeletal modifications include phosphorothioates, phosphoramidates, phosphorodiamidates, phosphorodithioates, and / or methylphosphonates. O, one, more, or all nucleoside-to-nucleoside bonds may be modified nucleoside-to-nucleoside bonds. While the polynucleotides of this disclosure may contain only phosphodiester bonds between nucleosides, other examples may include phosphorothioates, phosphoramidates, phosphorodiamidates, phosphorodithioates, and / or methylphosphonate bonds.

[0138] Polynucleotides, including oligonucleotides, may be single-stranded or double-stranded. The polynucleotides or oligonucleotides of this disclosure may be conjugated to another molecule, for example, N-acetylgalactosamine (GalNAc) or a plurality of them (GalNAc clusters).

[0139] Modified nucleoside triphosphates known in this field include 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate, and 4-thiouridine- 5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodyl De-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-azacitidine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'- Examples include triphosphates, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, and xanthosine-5'-triphosphate.

[0140] Examples of base-modified nucleotides known in the art include 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate, pyridine-4-one-ribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudridine, and 5-propynyl -Uridine, 1-propynyl-pseuduridine, 5-taurinomethyluridine, 1-taurinomethyl-pseuduridine, 5-taurinomethyl-2-thiouridine, 1-taurinomethyl-4-thiouridine, 5-methyluridine, 1-methyl-pseuduridine, 4-thio-1-methyl-pseuduridine, 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine, dihydropseuduridine, 2-thio-dihydrouridine, 2-thio -Dihydropsuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypsuduridine, and 4-methoxy-2-thiopsuduridine, 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methylpsudisocytidine, pyrrolocytidine, pyrrolopsudisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thiopsudisocytidine, 4- O-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methylcytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-Deaza-2-aminopurine, 7-Deaza-8-aza-2-aminopurine, 7-Deaza-2,6-diaminopurine, 7-Deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N 6-dimethyladenosine, 7-methyladenine, 2-methylthioadenine, and 2-methoxyadenine, inosine, 1-methylinosine, uosin, wibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine, 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, 5'-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thiocytidine, α-thiocytidine, pseudoisocytidine, 5-aminoallyl-u Lysine, 5-iodouridine, N1-methylpseudolidine, 5,6-dihydrouridine, α-thiouridine, 4-thiouridine, 6-azauridine, 5-hydroxyuridine, deoxythymidine, 5-methyluridine, pyrrolocytidine, inosine, α-thioguanosine, 6-methylguanosine, 5-methylcytidine, 8-oxoguanosine, 7-deazaguanosine, N1-methyladenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, pseudoisocytidine, 6-chloropurine,N6-methyladenosine, α-thioadenosine, 8-azidoadenosine, 7-deazaadenosine, pseudouridine, N1-methylpseudridine, N1-ethylpseudridine, 2-thiouridine, 4'-thiouridine, 5-methyluridine, 2-thio-1-methyl-1-deazapseudridine, 2-thio-1-methylpseudridine, 2-thio-5-azauridine, 2-thio-dihydropseudridine Examples include 2-thio-dihydrouridine, 2-thio-pseuduridine, 4-methoxy-2-thio-pseuduridine, 4-methoxy-pseuduridine, 4-thio-1-methyl-pseuduridine, 4-thio-pseuduridine, 5-aza-uridine, dihydropseuduridine, 2'-O-methyluridine, pseudouridine(y), N1-methylpseuduridine, 5-methylcytosine, and 5-methoxyuridine.

[0141] In one embodiment, the polynucleotide is a therapeutic polynucleotide. In one embodiment, the therapeutic polynucleotide is therapeutic RNA. In one embodiment, the therapeutic polynucleotide is therapeutic mRNA.

[0142] In one embodiment, the polynucleotide is an oligonucleotide. In one embodiment, the oligonucleotide is selected from the group consisting of antisense oligonucleotides, siRNAs, miRNAs, aptamers, CRISPR guide RNAs, and oligonucleotides used to recruit and guide DNA and RNA editing enzymes. In one embodiment, the oligonucleotide used to recruit and guide DNA and RNA editing enzymes is an A-to-I RNA base-editing oligonucleotide (AIMer). In one embodiment, the oligonucleotide is a therapeutic oligonucleotide.

[0143] In one embodiment, the polynucleotide is produced by an enzymatic method, i.e., by enzymatic synthesis, also referred to herein as enzymatic. In one embodiment, the enzyme is a polymerase. In one embodiment, the polymerase is a template-dependent polymerase. In one embodiment, the polymerase is a template-independent polymerase. In one embodiment, the enzyme is a transferase. In one embodiment, the enzyme is a terminal deoxynucleotidyltransferase (TdT).

[0144] In one aspect of the present disclosure, a method is provided for producing a polynucleotide comprising incubating a nucleoside triphosphate, a suitable enzyme, and optionally a template under suitable conditions, wherein the nucleoside triphosphate is produced by incubating the corresponding nucleoside monophosphate and / or nucleoside diphosphate, PPK, and polyphosphate under suitable conditions.

[0145] In one aspect of the present disclosure, a method is provided for producing a polynucleotide, comprising the steps of: a) providing a nucleoside triphosphate prepared using a corresponding nucleoside monophosphate and / or nucleoside diphosphate, PPK, and polyphosphate under suitable conditions; and b) producing a polynucleotide using the nucleoside triphosphate and a suitable enzyme under suitable conditions.

[0146] In one embodiment, one, two or more, or all of the NTPs are naturally occurring NTPs.

[0147] In one embodiment, one or more or all of the NTPs include sugar, base, and / or α-phosphate modifications. In one embodiment, one or more or all of the NTPs include base modifications. In one embodiment, one or more or all of the NTPs include sugar modifications. In one embodiment, one or more or all of the NTPs include 2'-sugar modifications. In one embodiment, one or more or all of the NTPs include 3'-sugar modifications. In one embodiment, one or more or all of the NTPs include α-phosphate modifications.

[0148] In one embodiment, the polynucleotide is mRNA. In one embodiment, one or more or all of the NTPs include base modifications. In one embodiment, the provided NTPs include or consist of naturally occurring NTPs. In one embodiment, the NTPs are naturally occurring NTPs except that uridine triphosphate is replaced with pseudouridine triphosphate. In one embodiment, the provided NTPs are naturally occurring NTPs except that uridine triphosphate is replaced with N1-methylpseudridine triphosphate.

[0149] In one embodiment, the enzyme used in step b) is a polymerase. In one embodiment, the polymerase is a template-dependent polymerase, and a suitable template is included in step a). In one embodiment, the polymerase is a template-independent polymerase. In one embodiment, the enzyme used in step b) is a transferase. In one embodiment, the enzyme used in step b) is a terminal deoxynucleotidyl transferase (TdT).

[0150] The enzymes disclosed herein, in particular PPK, can be prepared by any of a number of conventional techniques. For example, PPK can be purified from cells that express them naturally or produced in a recombinant expression system.

[0151] Several different expression systems and purification procedures can be used to generate the PPKs of this disclosure. Generally, host cells are transformed with a recombinant expression vector encoding the desired PPK. Depending on the expression system, the expression vector may be maintained by the host as a separate genetic element or may be incorporated into the host chromosome. A wide range of host cells can be used, including prokaryotes (including Gram-negative or Gram-positive bacteria, e.g., Escherichia coli, Bacilli sp., Pseudomonas sp., Corynebacterium sp.), eukaryotes including yeasts (e.g., Saccharomyces cerevisiae, Pichia pastoris), fungi (e.g., Aspergilus sp.), or higher eukaryotes including insect cells and mammalian cell lines (e.g., CHO, NS0, PER.C6, HEK293, and HeLa). In one embodiment, the vector is the pET28a vector.

[0152] The host cell may be an isolated host cell. The host cell is usually not part of a multicellular organism (e.g., a plant or an animal). The host cell may be a non-human host cell. In one embodiment, the host cell is a bacterial cell. In one embodiment, the host cell is a Gram-positive bacterial cell. In one embodiment, the host cell is Escherichia coli. In one embodiment, the host cell is Escherichia coli BL21(DE3).

[0153] Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian host cells are known in the art.

[0154] Cells can be cultured under conditions that promote PPK expression using various devices such as shaking flasks, spinner flasks, and bioreactors. The polypeptide is recovered by conventional protein purification procedures. Protein purification procedures typically consist of a series of unit operations comprising various filtration and chromatographic processes developed to selectively concentrate and isolate PPK. In certain embodiments, hexahistidine is fused to the N-terminus of PPK, and PPK is purified using immobilized metal affinity chromatography. In certain embodiments, hexahistidine is fused to the N-terminus of PPK, and PPK is purified using nickel-nitriloacetate (Ni-NTA). [Examples]

[0155] Example 1: Selection of polyphosphate kinase (PPK) INTERPRO (EMBL-EBI) provides functional analysis of proteins by classifying them into families and predicting their domains and critical regions. (Paysan-Lafosse et al., Nucleic Acids Research, 2023, 51, D418-D427). INTERPRO entry IPR022488 corresponds to a polyphosphate kinase-2 related protein, i.e., a putative PPK2s in contrast to a characterized PPK2s, and was used as a starting point (28,000 proteins).

[0156] Sequences ranging from 150 to 600 amino acids were filtered using a standard Python script, and sequences with at least 90% sequence identity were clustered using CD-HIT V4.6.8 (11,779 clusters).

[0157] Characterized PPK2-III sequences (Nocek et al., ACS Catal. 2018, 8, 11, 10746-10760) and in-house PPK2 sequences were inserted into the dataset without further clustering to preserve sequence diversity.

[0158] Multiple sequence alignment was performed using MAFFT (Multi-Sequence Alignment Program) version 7.520 (March 2023), and phylogenetic tree files were created and visualized using Christian Zmasek's Archaeopteryx software (https: / / sites.google.com / site / cmzmasek / christian-zmasek / software) V0.9928. Clustering of PPK2 belonging to different classes was manually checked by searching for related sequences in the generated tree (i.e., using characterized PPK2-III as well as known PPK2 I and II sequences). 4185 PPK2-III family members were identified for further study.

[0159] PPK2-III family member sequences were searched using UNIPROT. Fragments and poorly annotated sequences were removed by column sorting and filtering, leaving 4002 potentially useful sequences. The sequences were then classified into three groups using UNIPROT: archaea (34 sequences), metagenomics (38 sequences), and bacteria (3930 sequences).

[0160] Further clustering of bacterial sequences was performed using CD-HIT (3-step: 80%, 70%, and 60% sequence identity across sequences), yielding 686 clusters. Representative sequences were aligned using Clustal Omega to create phylogenetic tree files, which were visualized using Archaeopteryx software. Phylogenetic analysis divided the sequences into four major clades, and further phylogenetic trees were created for each of these clades. From each of the four phylogenetic trees, the clade with the fewest sequences was removed, and, where available, sequences from known extremophile organisms within the clade were selected as representative of the clade. After trimming the sequences to ensure maximum sequence diversity, the last 95 genes were selected. Due to gene synthesis failures and sequences not produced in a sufficiently soluble form, the number of PPK sequences was reduced to 66.

[0161] Example 2: 5'-phosphorylation of 2'-hydroxyadenosine 5'-monophosphate (AMP) by PPK to obtain 2'-hydroxyadenosine 5'-triphosphate (ATP) (natural PPK reaction) The wild-type PPKs described in Sequence IDs 1-66 were each fused to a hexahistidine tag (6×His) at the N-terminus. The genes were synthesized, cloned into pET28a, and the gene-coding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using nickel-nitriloacetate (Ni-NTA) and used directly.

[0162] The reaction was set up as shown in Table 1, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding ethylenediaminetetraacetic acid (EDTA) (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a fresh polymerase chain reaction (PCR) plate. The reaction mixture was then analyzed by high-performance liquid chromatography (HPLC) for the presence of adenosine 5'-triphosphate (ATP). The product peaks were assigned using liquid chromatography-mass spectrometry (LCMS).

[0163] [Table 2]

[0164] [Table 3] JPEG2026534216000013.jpg248160JPEG2026534216000014.jpg201168

[0165] conclusion The described experiments and the conversion data obtained showed that many of the tested wild-type PPKs (SEQ ID NOs: 1-5, 7-9, 11-31, 33-41, 43-45, 47-57, and 59-66) synthesized adenosine 5'-triphosphate from adenosine 5'-monophosphate by phosphorylation at 5'-phosphate, and 13 wild-type PPKs (SEQ ID NOs: 11, 12, 15, 21, 23, 26, 38, 41, 49-51, 54, and 65) converted more than three-quarters of the substrate to the product under specific reaction conditions.

[0166] Example 3: 5'-phosphorylation of 2'-methoxy-modified 3'-phosphate-protected adenosine 5'-monophosphate with PPK to obtain 2'-methoxy-adenosine 3'-phosphate-5'-triphosphate The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0167] The reaction was set up as shown in Table 3, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-methoxyadenosine 3'-phosphate-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0168] [Table 4]

[0169] [Table 5] JPEG2026534216000017.jpg248160JPEG2026534216000018.jpg191168

[0170] conclusion The described experiments and the conversion data obtained showed that numerous wild-type PPKs (SEQ ID NOs: 11, 14, 15, 19-23, 25-27, 29-31, 36-38, 40, 49-52, 65, and 66) synthesized 2'-methoxy-modified 3'-phosphate-protected adenosine 5'-triphosphate from 2'-methoxy-modified 3'-phosphate-protected adenosine 5'-monophosphate by phosphorylation at 5'-phosphate, with two wild-type PPKs (SEQ ID NOs: 22, 94.3% conversion; and SEQ ID NOs: 52, 86.5% conversion) showing particularly good results.

[0171] Example 4: 5'-phosphorylation of 2'-fluoro,3'-phosphate-protected adenosine 5'-monophosphate with wild-type PPK to obtain 2'-fluoro,3'-phosphate-protected adenosine 5'-triphosphate The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0172] The reaction was set up as shown in Table 5, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-fluoroadenosine 3'-phosphate-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0173] [Table 6]

[0174] [Table 7] JPEG2026534216000021.jpg249168JPEG2026534216000022.jpg191168

[0175] conclusion The described experiments and the resulting conversion data demonstrated that numerous wild-type PPKs (SEQ ID NOs: 11, 15, 19, 21, 22, 25, 27, 29, 31, 36, 38, 49, 52, 59, and 66) synthesize 2'-fluoro,3'-phosphate-protected adenosine nucleotide 5'-triphosphate by phosphorylating 2'-methoxy-modified 2'-fluoro,3'-phosphate-protected adenosine nucleotide 5'-monophosphate with 5'-phosphate. One wild-type PPK, SEQ ID NO: 22, acted exceptionally well, converting 100% of the substrate to the product under specific reaction conditions.

[0176] Example 5: 5'-phosphorylation of 2'-methoxy-modified 3'-phosphate-protected guanosine 5'-monophosphate with PPK to obtain 2'-methoxy-guanosine 3'-phosphate-5'-triphosphate The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0177] The reaction was set up as shown in Table 7, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-methoxyguanosine 3'-phosphate-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0178] [Table 8]

[0179] Results and Conclusions The described experiments and the resulting conversion data demonstrated that several wild-type PPKs (SEQ ID NOs: 19, 22, 31, and 52) synthesize 2'-methoxy-modified 3'-phosphate-protected guanosine 5'-triphosphate from 2'-methoxy-modified 3'-phosphate-protected guanosine 5'-monophosphate via phosphorylation at 5'-phosphate (substrate-to-product conversion percentages were 5.8%, 99%, 28.6%, and 4.0%, respectively). Representative chromatograms are shown in Figure 2. One wild-type PPK, SEQ ID NO: 22, acted exceptionally well, converting 99% of the substrate to the product under specific reaction conditions.

[0180] Example 6: 5'-phosphorylation of uridine 5'-monophosphate with PPK to obtain 2'-methoxyuridine 3'-phosphate-5'-triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0181] The reaction was set as shown in Table 8 by finally adding PPK. The reaction mixture was incubated at 25°C for 24 hours. Then, the reaction was quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000×g for 15 minutes. Subsequently, 12 μL of the supernatant was transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-methoxy-uridine 3'-phosphate-5'-triphosphate. The product peak was assigned using LCMS.

[0182] [Table 9]

[0183] Results and Conclusions From the described experiments and the obtained conversion data, it was shown that a single wild-type PPK (SEQ ID NO: 22) synthesizes 2'-methoxy-modified 3'-phosphate-protected uridine 5'-triphosphate from 2'-methoxy-modified 3'-phosphate-protected uridine 5'-monophosphate via phosphorylation at the 5' phosphate (39.6% conversion of the substrate to the 5'-triphosphate product, as well as 43.1% conversion to 5'-triphosphate and 5'-tetraphosphate products).

[0184] Example 7: 5'-phosphorylation of 2'-fluoro-modified 3'-phosphate-protected cytidine 5'-monophosphate by PPK to obtain 2'-fluorocytidine 3'-phosphate-5'-triphosphate Each of the wild-type PPKs set forth in SEQ ID NOs: 1 to 66 was fused to 6×His at the N-terminus. The genes were synthesized, cloned into pET28a, and the proteins encoded by the genes were produced in Escherichia coli BL21(DE3) using standard cloning, expression, and extraction methods. PPK was purified using Ni-NTA and used directly.

[0185] The reaction was set up as shown in Table 9, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-fluorocytidine 3'-phosphate-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0186] [Table 10]

[0187] Results and Conclusions The described experiments and the resulting conversion data demonstrate that a single wild-type PPK (SEQ ID NO: 22) synthesizes 2'-fluoromodified 3'-phosphate-protected cytidine 5'-triphosphate from 2'-fluoromodified 3'-phosphate-protected cytidine 5'-monophosphate via phosphorylation at 5'-phosphate (22.6% conversion of substrate to product).

[0188] Example 8: 5'-phosphorylation of 2'-methoxy-ethyl modified methylcytidine 5'-monophosphate by PPK to obtain methylcytidine 2'-methoxy-ethylmethylcytidine 3'-phosphate-5'-triphosphate The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0189] The reaction was set up as shown in Table 10, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-methoxy-ethyl-methylcytidine 3'-phosphate-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0190] [Table 11]

[0191] Results and Conclusions From the described experiments and the conversion data obtained, it was shown that several wild-type PPKs (SEQ ID NOs: 22, 31, and 49) synthesize 2'-methoxy-ethyl-methylcytidine 3'-phosphate-protected 5'-triphosphate from 2'-methoxy-ethyl-methylcytidine 3'-phosphate-protected 5'-triphosphate by phosphorylation at 5'-phosphate, albeit with low substrate-to-product conversion percentages (5.4%, 2.4%, and 0.9%, respectively).

[0192] Example 9: 5'-phosphorylation of 2'-methoxy-modified 3'-phosphate-protected cytidine 5'-monophosphate by PPK to obtain 2'-methoxy-modified 3'-phosphate-protected cytidine 5'-triphosphate The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0193] The reaction was set up as shown in Table 11, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-methoxycytidine 3'-phosphate-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0194] [Table 12]

[0195] Results and Conclusions The described experiments and the conversion data obtained showed that several wild-type PPKs (SEQ ID NOs: 22, 29, 31) synthesize 2'-methoxy-modified 3'-phosphate-protected cytidine 5'-triphosphate from 2'-methoxy-modified 3'-phosphate-protected cytidine 5'-monophosphate via phosphorylation at 5'-phosphate (substrate-to-product conversion percentages were 10.4%, 0.3%, and 38.1%, respectively).

[0196] Example 10: 5'-phosphorylation of 2'-methoxy-modified 3'-phosphate-protected adenosine 5'-monothiophosphate with PPK to obtain 2'-methoxy-adenosine 3'-phosphate-5'α-thio-triphosphate The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0197] The reaction was set as shown in Table 12, with PPK added last. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating to 80°C for 15 minutes, and centrifuging at 4000×g for 15 minutes. Then, 12 μL of the supernatant was transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-methoxy-adenosine 3'-phosphate-5'-triphosphate. The product peak was assigned using LCMS.

[0198] [Table 13]

[0199] [Table 14] JPEG2026534216000030.jpg248160JPEG2026534216000031.jpg210168

[0200] Conclusion From the described experiments and the obtained conversion data, it was shown that a large number of wild-type PPKs (SEQ ID NOs: 1 to 4, 10 to 15, 18 to 23, 25 to 27, 29 to 31, 36 to 42, 47, 49 to 54, 56, 57, 59, 60, 65, and 66) synthesize 2'-methoxy-modified 3'-phosphate-protected adenosine 5'-α-thio-triphosphate from 2'-methoxy-modified 3'-phosphate-protected adenosine 5'-thiophosphate by phosphorylation with 5'-thiophosphate.

[0201] Example 11: 5'-phosphorylation of 2'-methoxy-modified 3'-phosphate-protected cytidine 5'-monothiophosphate by PPK to obtain 2'-methoxycytidine 3'-phosphate-5'α-thio-triphosphate The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0202] The reaction was set up as shown in Table 14, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-methoxycytidine 3'-phosphate-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0203] [Table 15]

[0204] Results and Conclusions The described experiments and the conversion data obtained showed that several wild-type PPKs (SEQ ID NOs: 22, 31, 37, 59) synthesize 2'-methoxy-modified 3'-phosphate-protected cytidine 5'-α-thiotriphosphate from 2'-methoxy-modified 3'-phosphate-protected cytidine 5'-thiophosphate via phosphorylation at 5'-thiophosphate (substrate-to-product conversion percentages were 3.3%, 6.2%, 11.9%, and 17.0%, respectively).

[0205] Example 12: 5'-phosphorylation of adenosine 5'-(α-thio)triphosphate by PPK to obtain adenosine 5'-(α-thio)triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0206] The reaction was set up as shown in Table 15, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of adenosine 5'-(α-thio)triphosphate. The peaks of the product were assigned using LC-MS.

[0207] [Table 16]

[0208] The formation of a phosphoanhydride bond to α-thiophosphate generates a chiral center at the phosphorus of α-thiophosphate, resulting in two diastereomers. The diastereomers of adenosine 5'-(α-thio)diphosphate and adenosine 5'-(α-thio)triphosphate were degradable by ultra-high-performance liquid chromatography (UPLC). The diastereomer ratio (first elution peak / second elution peak) and composition % relative to monophosphate, diphosphate, and triphosphate are shown in Table 16 below.

[0209] [Table 17] JPEG2026534216000035.jpg246168JPEG2026534216000036.jpg245168JPEG2026534216000037.jpg101168

[0210] conclusion The described experiments and the resulting conversion data demonstrated that numerous wild-type PPKs (SEQ ID NOs: 1-9, 11, 12, 15-23, 25-41, 44-54, 56, 57, 59, and 61-66) synthesize adenosine 5'-(α-thio)-triphosphate from adenosine 5'-monothiophosphate via phosphorylation at the terminal 5'-thiophosphate.

[0211] The diastereomer ratio of the generated 5'-(α-thio)-triphosphate to 5'-(α-thio)-diphosphate may be 1 (indicating no stereopreference for phosphorylation), greater than 1 (indicating preference for early-eluting diastereomer formation), or less than 1 (indicating preference for late-eluting diastereomer formation), depending on the PPK used (see Figure 3).

[0212] Example 13: 5'-phosphorylation of 2'-deoxyadenosine 5'-monothiophosphate by PPK to obtain 2'-deoxyadenosine 5'-(α-thio)triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0213] The reaction was set up as shown in Table 17, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-deoxyadenosine 5'-(α-thio)triphosphate. The peaks of the product were assigned using LC-MS.

[0214] [Table 18]

[0215] [Table 19] JPEG2026534216000040.jpg248160JPEG2026534216000041.jpg191168

[0216] conclusion The described experiments and the resulting conversion data demonstrated that numerous wild-type PPKs (SEQ ID NOs: 2-4, 7-9, 11, 12, 14-23, 25-31, 33-40, 45, 47-54, 56, 57, 59, 61, 62, 64-66) synthesize 2'-deoxyadenosine 5'-(α-thio)-triphosphate from 2'-deoxyadenosine 5'-monothiophosphate via phosphorylation at the terminal 5'-thiophosphate.

[0217] Example 14: 5'-phosphorylation of 2'-deoxyguanosine 5'-monothiophosphate by PPK to obtain 2'-deoxyguanosine 5'-(α-thio)triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0218] The reaction was set up as shown in Table 19, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-deoxyguanosine 5'-(α-thio)triphosphate. The peaks of the product were assigned using LC-MS.

[0219] [Table 20]

[0220] [Table 21] JPEG2026534216000044.jpg249162JPEG2026534216000045.jpg163168

[0221] conclusion The described experiments and the resulting conversion data demonstrated that numerous wild-type PPKs (SEQ ID NOs: 8, 9, 11, 15, 19-23, 25, 27, 29, 31, 32, 35, 37, 39, 45, 47, 49, 50, 54, 58, 61, and 64-66) synthesize 2'-deoxyguanosine 5'-(α-thio)-triphosphate from 2'-deoxyguanosine 5'-monothiophosphate via phosphorylation at the terminal 5-thiophosphate.

[0222] Example 15: 5'-phosphorylation of 2'-deoxycytidine 5'-(α-thio)triphosphate by polyphosphate kinase to obtain 2'-deoxycytidine 5'-(α-thio)triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0223] The reaction was set up as shown in Table 21, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-deoxycytidine 5'-(α-thio)triphosphate. The peaks of the product were assigned using LC-MS.

[0224] [Table 22]

[0225] [Table 23] JPEG2026534216000048.jpg249162JPEG2026534216000049.jpg163168

[0226] conclusion The described experiments and the resulting conversion data demonstrated that numerous wild-type PPKs (SEQ ID NOs: 9, 14-16, 19, 20, 22, 23, 25, 27, 29, 31, 37, 39, 44, 47, 59, and 65) synthesize 2'-deoxycytidine 5'-(α-thio)-triphosphate from 2'-deoxycytidine 5'-monothiophosphate via phosphorylation at the terminal 5' thiophosphate.

[0227] Example 16: 5'-phosphorylation of 2'-fluorocytidine-5'-monophosphate by PPK to obtain 2'-fluorocytidine-5'-triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0228] The reaction was set up as shown in Table 23, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-fluorocytidine-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0229] [Table 24]

[0230] [Table 25] JPEG2026534216000052.jpg249162JPEG2026534216000053.jpg163168

[0231] conclusion The described experiments and the resulting conversion data demonstrated that numerous wild-type PPKs (SEQ ID NOs: 10, 11, 15, 19, 21-23, 25-27, 29, 31, 32, 37, 39, 40, 49, 54, 59, and 66) synthesize 2'-fluorocytidine-5'-triphosphate from 2'-fluorocytidine-5'-monophosphate via phosphorylation at the terminal 5' thiophosphate.

[0232] Example 17: 5'-phosphorylation of 2'-methoxy-modified adenosine 5'-monophosphate by PPK to obtain 2'-methoxy-modified adenosine 5'-triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0233] The reaction was set up as shown in Table 25, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 2'-methoxyadenosine-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0234] [Table 26]

[0235] [Table 27] JPEG2026534216000056.jpg249162JPEG2026534216000057.jpg163168

[0236] conclusion The described experiments and the resulting conversion data demonstrated that numerous wild-type PPKs (SEQ ID NOs: 1, 2, 4, 5, 7, 9, 11, 12, 14-23, 25-40, 43-45, 47-54, 56, 57, 59-62, and 64-66) synthesize 2'-methoxy-adenosine 5'-triphosphate from 2'-methoxy-adenosine 5'-monophosphate via phosphorylation at the terminal 5' thiophosphate.

[0237] Example 18: 5'-phosphorylation of 3'-(N'-methyl-anthraniloyl)2'-deoxyadenosine-5'-monophosphate by PPK to obtain 3'-(N'-methyl-anthraniloyl)2'-deoxyadenosine-5'-triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0238] The reaction was set up as shown in Table 27, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 3'-(N'-methyl-anthraniloyl)2'-deoxyadenosine-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0239] [Table 28]

[0240] [Table 29] JPEG2026534216000060.jpg249162JPEG2026534216000061.jpg163168

[0241] conclusion The described experiments and the resulting conversion data demonstrated that numerous wild-type PPKs (SEQ ID NOs: 2, 4, 6-9, 11, 12, 14-17, 19-23, 25-40, 42, 44, 45, 47-54, 56-62, and 64-66) synthesize 3'-(N'-methyl-anthraniloyl)2'-deoxyadenosine-5'-triphosphate from 3'-(N'-methyl-anthraniloyl)2'-deoxyadenosine-5'-monophosphate via phosphorylation at the terminal 5' phosphate.

[0242] Example 19: 5'-phosphorylation of 3'-(N'-methyl-anthraniloyl)2'-deoxyguanosine-5'-monophosphate by PPK to obtain 3'-(N'-methyl-anthraniloyl)2'-deoxyguanosine-5'-triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0243] The reaction was set up as shown in Table 29, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (5 μL, 20 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 12 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 3'-(N'-methyl-anthraniloyl)2'-deoxyguanosine-5'-triphosphate. The peaks of the product were assigned using LC-MS.

[0244] [Table 30]

[0245] [Table 31] JPEG2026534216000064.jpg249162JPEG2026534216000065.jpg163168

[0246] conclusion The described experiments and the resulting conversion data demonstrated that numerous wild-type PPKs (SEQ ID NOs: 2, 4-9, 11, 12, 14-40, 42, 44-47, 49-54, 56, 58, 59, 61, and 63-66) synthesize 3'-(N'-methyl-anthraniloyl)2'-deoxyguanosine-5'-triphosphate from 3'-(N'-methyl-anthraniloyl)2'-deoxyguanosine-5'-monophosphate via phosphorylation at the terminal 5' phosphate.

[0247] Example 21: 5'-phosphorylation of 3'-pivaloyl 2'-methoxyguanosine-5'-monophosphate by PPK to obtain 3'-pivaloyl 2'-methoxyguanosine-5'-triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0248] The reaction was set up as shown in Table 31, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (2 μL, 100 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 20 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 3'-pivaloyl 2'-methoxyguanosine-5'-triphosphate.

[0249] [Table 32]

[0250] [Table 33] JPEG2026534216000068.jpg249162JPEG2026534216000069.jpg163168

[0251] conclusion The described experiments and the conversion data obtained showed that numerous wild-type PPKs (SEQ ID NOs: 19, 20, 24, 32, and 36) synthesized 3'-pivaloyl 2'-methoxyguanosine-5'-triphosphate from 3'-pivaloyl 2'-methoxyguanosine-5'-monophosphate by phosphorylation at 5'-phosphate, with one wild-type PPK (SEQ ID NOs: 22, 72.5% conversion) showing particularly good performance.

[0252] Example 22: 5'-phosphorylation of 3'-pivaloyl 2'-methoxyuridine-5'-monophosphate by PPK to obtain 3'-pivaloyl 2'-methoxyuridine-5'-triphosphate. The wild-type PPKs described in Sequence IDs 1-66 were fused at the N-terminus with a 6×His fusion. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0253] The reaction was set up as shown in Table 33, with the addition of PPK at the end. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (2 μL, 100 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 20 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of 3'-pivaloyl 2'-methoxyuridine-5'-triphosphate.

[0254] [Table 34]

[0255] [Table 35] JPEG2026534216000072.jpg249162JPEG2026534216000073.jpg163168

[0256] conclusion The described experiments and the resulting conversion data showed that only one wild-type PPK (SEQ ID NO: 24) synthesizes 3'-pivaloyl 2'-methoxyuridine-5'-triphosphate from 3'-pivaloyl 2'-methoxyuridine-5'-monophosphate via phosphorylation at the 5' phosphate (SEQ ID NO: 22, 28.5% conversion).

[0257] Example 23: 5'-phosphorylation of deoxyadenosine 5'-(α-thio)triphosphate by PPK to obtain deoxyadenosine 5'-(α-thio)triphosphate. The wild-type PPKs described in SEQ ID NOs. 22, 31, and 34 were fused at the N-terminus with a 6×His molecule. The genes were synthesized, cloned into pET28a, and the gene-encoding protein was produced in E. coli BL21(DE3) using standard cloning, expression, and extraction methods. The PPKs were purified using Ni-NTA and used directly.

[0258] The reaction was set up as shown in Table 35, with PPK added last. The reaction mixture was incubated at 25°C for 24 hours. The reaction was then quenched by adding EDTA (2 μL, 100 mM), heating at 80°C for 15 minutes, and centrifugation at 4000 × g for 15 minutes. 20 μL of the supernatant was then transferred to a new PCR plate. The reaction mixture was then analyzed by HPLC for the presence of deoxyadenosine 5'-(α-thio)triphosphate.

[0259] [Table 36]

[0260] The formation of a phosphoanhydride bond to α-thiophosphate generates a chiral center at the phosphorus of α-thiophosphate, resulting in two diastereomers. The diastereomers of deoxyadenosine 5'-(α-thio)diphosphate and deoxyadenosine 5'-(α-thio)triphosphate were degradable by ultra-high-performance liquid chromatography (UPLC). The diastereomer ratio (first elution peak / second elution peak) and composition percentages relative to monophosphate, diphosphate, and triphosphate are shown in Table 36 below.

[0261] [Table 37]

[0262] conclusion The described experiments and the resulting conversion data demonstrate that wild-type PPK (SEQ ID NOs. 22, 31, and 34) synthesizes deoxyadenosine 5'-(α-thio)-triphosphate from deoxyadenosine 5'-monothiophosphate via phosphorylation at the terminal 5' thiophosphate.

[0263] The diastereomer ratio of the generated 5'-(α-thio)-triphosphate to 5'-(α-thio)-diphosphate may be 1 (indicating no stereopreference for phosphorylation), greater than 1 (indicating preference for early-eluting diastereomer formation), or less than 1 (indicating preference for late-eluting diastereomer formation), depending on the PPK.

[0264] Sequence List [Table 38] JPEG2026534216000077.jpg249162JPEG2026534216000078.jpg169168 Sequence ID 1 MRWIAALAEDDPIGKAVAGLVARYRVTDGEKFRIVDHATDDKAGLDKVTAKRFVKARRHHLAVLQENFAAARTRGIVVVLQGMDTAGKDSLIRHVMSGMNPQGTDVTSFKAPTPEEQAHDFLWRVHAAAPPRGKVGVFNRSHYEDVLVARVHAER LAMDGLPGDPSHPEFWDRRLEDIRHFEQYMTRQGFLFLKIFLHISFDEQRRRILRRLDRPEKRWKFDEGDLVERQFWPRYQVAYEEAIRATATEDAPWLVVPSDHKWLSRLVTMEALVGCLESLNPEPPPATSDLVRKIAEFRRALEKDTIVHPA Sequence ID 2 MGSMDKTVYQYTGDQPVDLTQWATTTDTRIKTAQRNADLETLHDLQRRLYAQKQTGVVIILQGMDTAGKDGLIRHVFSGFNPAGTSVVSFKQPTSVDLQHDFLWRINRQLPARGEIRIFNRSQYEDVLISRVHPEILVGQHLPDV RSVADVTDKFFERRYHDLRHFETYLRHQGIMTYKFFLHISQKEQTARLERRLALPEKNWKFSTSDLKERQFWADYQTAYAQMLEHTATAKNPWYIIPADDKPTARRLVTAILTQELSQLDPQYPQVTADQQAHFQQLLQQLRKGQI Sequence ID 3 MDAGGKDSAIKHLFTGINPQGCEVHSFKRPTFLELDHDFMWRSNIRTPSRGKIGVFNRSYYEEVLVVKVHEQILNSSKLPKELIHEDIWAERFEYIRAFERSMHQNGTNVL KFFLYLSPDEQKKRFISRMKNPEKNWKFDPNDLYERSFWGEYMQAYEEAIQSTACQDSPWYIIPADDKPYSRAVIADAVCQHLASLNLEYPTLSEEVQSNLQSYIEQLQNE Sequence ID 4 MLRPDTTNQIRAGTDGGFSLSDHQPSWEGSGRVDGLTGAGLKKRAKKYVKAQRRLLAKAQEKLYADDRFSVLILLQAMDAAGKDSTIKQVMSGVNPQGCQVFSFKQPSAEELDHNFLWRYWRDVPERGRIGIFNRSYYEEVLVVRVHPELVQRQR LPAAPNGSDFWAARFEDINAFERHMTRNGTVILKFFLNVSKEEQRQRFVRRLTKPKKLWKFSANDLYERAHWDDYQHAYEELITATSTEWAPWYVIPADNKWLMRALVSHVIVEAVEGLGVTYPKVPPGREAEFRKALNALRAEGAASGKKGGIG Sequence ID 5 MPHRHRLDHAPFTVQPGQRVRLKDYDSRETAGFKNKSEAKKALLEDVSLLAEMQQLLWAAKEYSLLIVFQALDAAGKDGAIRHVMSGVNPQGVDVYSFKAPTEEERLHHFLWRPAKVMPARGRIAIFNRSYYEEVLVVRVHPEWLERQW VPPRYRGGHYEELWPERFEEINAFERLMSENGVAILKFFLNVSKAEQRERFLERVTDPEKHWKYSAADLRERGYWDMYQAAYEDMLSHTSTKHAPWHVIPADRKWFTRACVADIIAAKIESLDLSYPTVSEEEHSAIEAARRQLEAESD Sequence ID 6 MELKGPKVKDYIAKFIVEPGKQVRLKDFKTGWAENEQMKKLGEDKSKDLLVEMLEKDRSALAAVQDLLWSSRTHAVLIILQGMDTAGKDGTIRHVMSGVNPQGCSVHGFKVPSTQEHNHNFLWRYSNVLPERGEIGIFNRSYYEDVLVTRVH PNIISGFPKDIGPDADGFWKGRYYDINAFEEHLIRNGTLILKFFLHISKDEQKRRLLDRLEHKEKYWKFSVADLTERQFWDNYIKVYEKMLEATSTDIAPWFVIPGDYKWVARTAVAEAVVTAIKGLNLSYPSVSDDEIKRLMEAKKKLQAE Sequence ID 7 MKINIKDFQVLQGDKVKLNKWPTQVGRVYSSKKNYHKLLKKQIKTLNELQQLQYAANTYSVLLVFQAMDAAGKDGVIRHVMSGINPQGCQVFSFKHPSATELDHDFLWRTNQCLPQRGRIGIFNRSYYEEVLIVRVHPEILHRQGL PDRLVDDETIWNGRYESIVDMENHLFRNGTRVIKFFLHLSKDEQRKRFVDRIDDPEKNWKFSEADIEERQFWDKYMGAYELCLSATSSKRAPWYVVPADDKKNARLIVSQVILDTFKSLNMQYPQTDEGRKQELLSIREQLIAEEE Sequence ID 8 MKIKSGDYRVREGDKVRLKKWPTLVKPVYRSKEQYQEMLAEHVDELSKLQQLQYASDRYAFLLIFQAMDAAGKDGAIKHVMSGINPQGCQVFSFKHPSAQELDHDFLWRTTQALPERGRIGIFNRSYYEEVLIVRVHPEILRAQNIP DELLNTKTIWQERYRSIVDLEQHLHRNGTRIIKFFLHLSKEEQRKRFLARIDDPNKNWKFSPADIEERKYWDQYMLAYEACLSATSTKEAPWYVVPADDKENARLIISRIILDTFRMLKMAYPKSGGEHQHELESIRRHLIKEGNAGK Sequence ID 9 MTKNYLLARNGKVKLKAINPEHTGSFVNKEEVLEDFVILKEKLVDLQDKLIAGKKQSLLIIFQGMDCSGKDGVINKVLSAINPQGFQVTSFKKPTEEETSHDFLWRSHKASPAKGYIAAFNRSYYEDVLVTRVHGV IDDDEADRRFKHIRNFEKLLNDSDVRILKIFLHISKDFQIQKIKDRLENPNKRWKFDPSDLEERKYWDAYQSAYEDVFNHCNSKYAPWYAVPANHRWFRDYIVLKLVVKALEDMDLQYPDPDPELEKKISSLDLTKV Sequence ID 10 MSKDNKQKSNAPKQSKVLSKKDLLQARQFSEQYCVGDGKNFKLKDYKTNADFELGEEGKSLVKKTLKMGVNALSTMQDILYAQDKWSVLLIFQAMDAAGKDGAIKHVMSGINPQGCQVSSFKGPSSEELDHDFLWRCQKHLPERGRIGIFNRSYYEEV LVVRVHKAILEGQKIPKKLISDDIWENRFEDIRNFEKYLNRNGTIVIKFFLNVSKEEQKKRFIERIDNPDKNWKFSATDAKERGYWDDYMHAYEELIKNTSTEKSPWYVIPADNKSYARIAIASAIIHALDEMDLEYPKVSESKIVELNDVKQILLDEKE Sequence ID 11 MPMVAAVEFAKSPAEVLRVGSGFSLAGVDPESTPGYTGVKADGKALLAAQDARLAELQEKLFAEGKFGNPKRLLLILQAMDTAGKGGIVSHVVGAMDPQGVQLTAFKAPTDEEKSHDFLWRIEKQVPAAGMVGVFDRSQYEDV LIHRVHGWADAAELERRYAAINDFESRLTEQGTTIVKVMLNISKDEQKKRLIARLDDPSKHWKYSRGDLAERAYWDDYMDAYSVAFEKTSTEIAPWHVVPANKKWYARIAVQQLLLDALGGLQLDWPKADFDVAAERALVVES Sequence ID 12 MGSMDKTVYQYTGDQPVDLTQWATTTDTRIKTAQRNADLETLHDLQRRLYAQKQTGVVIILQGMDTAGKDGLIRHVFSGFNPAGTSVVSFKQPTSVDLQHDFLWRINRQLPARGEIRIFNRSQYEDVLISRVHPEILVGQHLPDV RSVADVTDKFFERRYHDLRHFETYLRHQGIMTYKFFLHISQKEQTARLERRLALPEKNWKFSTSDLKERQFWADYQTAYAQMLEHTATAKNPWYIIPADDKPTARRLVTAILTQELSQLDPQYPQVTADQQAHFQQLLQQLRKGQI Sequence ID 13 MTINRHDFLASPEKKLSEYATAHPDAASYSTEEVKEALFEEVISTLRDQQTRLFAEAKSGIVVILQAMDAAGKDEAVTTVFSNLSVQGLRESEPGEPSDKELKRDYLWRHHETLPKRGEIAILNRSYYEEVLGSRVHGSYQNEP MPDKWKEGPVWKRRYRHLNEYERYLTENGFVVLKFYLNVNKNVQKKRLLERMNNPNRNWEFSFSDVDDRDKWEDFYHAYDEAIKETSTGYAPWYVIPADDPWFTRLVIAEIFSQTLSELNPKLPVLSGEEATKLEEYKEKLKKQ Sequence ID 14 MQGSQFRLEHGKKVRLANMDPNETGSLHDREEAEPNIERLKERLEALQNMLYAEKKQSVLFVIQGMDCSGKDGVVKRALGGLHPQGFQVHSFRTPTEEEEAHDFLWRAHRIVPAKGMIGAFIRSYYEDVLITRTH KWISEAEVKKRLKHINRFEKLLESSGVKIVKIFLHISKDFQLAKLRNRLTDETKRWKFDKNDLVERESWSAYEEAYEDVFKQCSTESAPWYVVPANHRWYRDLAVLQIAVDALESMRLAYPASDPELDQLLEQLQE Sequence ID 15 MDVSRYRVPPGSGFDPEAWPTREDDDFAGGKKEAKKELARLAVRLGELQARLYAEGRQALLIVLQGMDTAGKDGTIRHVFRAVNPQGVRVTSFKKPTALELAHDYLWRVHRHAPARGEIGIFNRSHYEDVLVVR VHELVPPEVWGRRYDHINAFERLLADEGTRIVKFFLHISKDEQKRRLEARLENPRKHWKFNPADLSERARWGDYAAAYAEALSRTSSDRAPWYAVPADRKWQRNRIVAQVLVDALEAMDPRFPRVDFDPASVRVE Sequence ID 16 MATRTEDRADRIEELLDILRVPPGKKIDLRKDYDPGFTGKWMKEEAEKTLAEGIQILAGMQDKLWAQNQYAVLMILQALDAAGKDGTIKHVMSGVNPQGVDVHSFKGPSSEELDHDYLWRNFKALPSRGRIGIFNRSYYEEVLVVRVHPEFLASQQLPPALKDERIWRRRFE EINNFEKYLVDNGIIVVKIFLYVSKEAQKERFLERAMMPEKNWKFSAADMKERAFWDDYIDAYEDMFNHTSTEWAPWHIVPADHKWFTRLAVAAVLYNTMENLAYPTISEQQKQALLVAKKELENEDGGSKDKSVVKAKAKEAAYPEILDASEVTRDETKKRDRKKGKKNR Sequence ID 17 MNIHWHGNSHAISQSPSRYPIVATPKVPSVAMPRNKGSVCEKNDCSKIRAVRAGGEIRVIDERYHKRLQVRPGIKVRLGEYNTGWAQNDALKEAGKDAVRERAMEILEENRAQLASAQELLWASNTYAVLIILQGMDTAGKDGTIKHVMSGVNPQGCKVHSFRVPTEEELDHTFLWR YWRALPNRGEIGIFNFRSYYEDVLVVKVHPERLVNLPPGKRGKKFWNARYEDINAFERHLIRNGTVILKFYLHISKDEQKKRLLERLDDREKLWKFSLSDIEERKFWDRYQEVYEDALTMTSTDEAPWHIIPADHKWVARTLIADIITTKIQSLDLAFPTVSGEAIAQLKEARRHLEEE Sequence ID 18 MKKTSAVDTSAYRVSNPETFVLSDAAADPPTGFDREQSKRVLKKNTKQLQSLQKLMWAERKQRLLIVLQAIDAGGKDGTIRSVFGPLNPQGVKVASFKRPTKTELAHDYLWRVHPHVPGNGEIVVFNRSHYEDVLV VKVHGYATADAIEKRYSHIRSFEQMLVDEGTTVVKIYLHISKDEQKKRFESRLNTPEKQWKFNKDDLKERAYWNQYQDAFETMLKETSTEDAPWYVVPANNKKYRNELISTLIRETLESLHMTWPKADEGIENVCLE Sequence ID 19 MDKIKVDNYKANKNFKIADNETFEDFGKSDKELKKELSRIRRELGEFQDTIYAHGKYSILVCLQGMDTAGKDSLIREVFKDFNVSGVEVHSFKVPTELELSHNYLWRHYLVLPAKGKFGVFNRTHYENVLVTRVHPEYILGEHIPG IHSVDDIDQKFWDKRFDQINDFERHLAENGTLIFKFFLNLSKEEQRQRLLRRLAIKEKHWKFSPGDLKERKLWNVYQKCYEEAISKTTHDHAPWFAIPADNKKATRIIVASILLQSLKKYKDIKEPMLSDKIKSNLDSYEEQLQGEQ Sequence ID 20 MKTINTDDFKITSPKKLSDLPTLVDLKATKQELKEALNETREALSDLQDKMYAHNRYGVLICLQGMDTSGKDSLIREVFKEFNARGVVVHSFKTPNSTELEHDYLWRHYLALPEKGKFSVFNRTHYENVLVTRVHPEYILNESLPG IEDVKDIPKDFWKKRMEQIVNFEKHIVENGVIVLKFFLHLSKEEQRQRLLRRLEKEKHNWKFSPSDLKERSFWKEYQHHYEEAINATSKEKAPWYVIPADDKNACRFLLAKAILDVLSQYKDIKFPELDEKVQENISFYKKELSNEK Sequence ID 21 MMSAYPANLLEALRAEPGVKLSERATKDADWWPGDAPQDKKAAEKRMVQLAELLSDLQERLFAASVAGGQGPAVLLILQGMDTSGKGGTVRHVLGMLDPQGVKHHAFKAPTEEEAAHDFLWRITRQLPAGGLVGVFDRSHYEDV LVAKVKGFATPDAVESRYGKIVDFEKQLIEQGIHPIKVMLHISREEQFERLSARLERADKHWKYSPGDVDDRLLWDDFQSAYEVAINRTDNQGAPWHIVPADQKWKARLCVAELLLATLERVAPDWPVATFDVEQEKARLAAAK Sequence ID 22 MATDFSKLSKYVETLRVKPKQSIDLKKDFDTDYDHKMLTKEEGEELLNLGISKLSEIQEKLYASGTKSVLIVFQAMDAAGKDGTVKHIMTGLNPQGVKVTSFKVPSKIELSHDYLWRHYVALPATGEIGIFNRSHYENVLVTRVHPEYLLSE QTSGVTAIEQVNQKFWDKRFQQINNFEQHISENGTIVLKFFLHVSKKEQKKRFIERIELDTKNWKFSTGDLKERAHWKDYRNAYEDMLANTSTKQAPWFVIPADDKWFTRLLIAEIICTELEKLNLTFPTVSLEQKAELEKAKAELVAEKSSD Sequence ID 23 MKIKKYFAGDDIKLKYFSTRCEEDLNKGEVKSILMPENLKKMTDYQSKLYAEGKKGLIVVFQAMDTAGKDGVIKHVMTSLNPQGMYVASFKAPSGVEMSHDYLWRIHKHAPSRGCVTVFNRSHYEDVIIARVHDLVKNQKLP DSMKHDGIWNDRYEQIRNYESYLHENGIHIVKFFLHLSKDEQRERLLSRIDEPEKNWKFSSADIHERKYWNDYQDAYEKVLQKTSTEKSPWYIIPADQKWFSRYLVSEILVEKFKDLNPEFPKLPEDELDNLAKWREELLND Sequence ID 24 MSKIILANTTTKTYDRALKEKIKAENVKLVNQIVEFQRRMYSDGRHNLLIIFQGMDASGKDGATRKVFSGVNPLGIKVHAYKKPTDKEFAHDFLWRIHQNVPQQGMIQVFNRSHYEDILVPTIEGYFE ESYIEKRYQHINDFEKLLIEGNNTTILKFYLHTSKEEQLERLTERIEIPEKYWKHNDKDWETRKKWDSYMSVYEQIFEKCNQPEWHIIPSDRNWEKENHIAKIVLKAFEEMDLKYPALDSELFKEKGM Sequence ID 25 MGEAMERYFIKPGEKVRLKDWSPDPPKDFEGDKESTRAAVAELNRKLEVLQERLYAERKHKVLVILQGMDTSGKDGVIRSVFEGVNPQGVKVANFKVPTQEELDHDYLWRVHKVVPGKGEIVIFNRSHYEDVLVVRVHNL VPPEVWKKRYEQINQFERLLHETGTTILKFFLFISREEQKQRLLERLADPAKHWKFNPGDLKERALWEEYEKAYEDVLSRTSTEYAPWILVPADKKWYRDWVISRVLVETLEGLEIQLPPPLADAETYRRQLLEEDAPESR Sequence ID 26 MIEEKYLERLLVKPGKKLKLTDFYTGWAQSNALREAGKEAVKEQAAEILEESRAALAEAQELLWADKQYSILVILQGMDTSGKDGTIKHVLSGVNPQGCRVMSFGVPTNDELHHTFLWRFWKAMPRRGEIGIFNRSYYEDVLVPKVHPV ILEEQQLPPGYGSDAFWEARYEDIRAFERHHHANGTIILKFFLNISKEEQKERLLSRLKTEKKQWKFSLSDLTERQYWSHYQAAYEEAIFQTSTDDAPWYVIPADYKWVARTLVADIIVRAISRLDLRYPEISAEEMKEIEKARIHLESE Sequence ID 27 MGDNGARVLERIRVPAGKKVKLTEYPTSWKKAEEFKLNGLKLTKDNSKEILEKSKGELAAAQELLWAEDSYSMLIILQGMDAAGKDGIIEHVMSGVNPQGCEVTGFKTPSQIELDHDFMWRCYAAMPERGRIGIFNRSYYEEVLVVRVHP EILIGQRLPGKEKGEKLWEERFESINDIERHLTRNGTVILKFFLHESKDEQRSRFLDRLSDTEKQWKFSPGDLAEREFWDQYTKAYEEMLSATSTEHAPWYIIPADQKWLARVMVAFVIVRTIESLDLGYPQLSEAQKKSLGEAKKKLQDE Sequence ID 28 MIKEKNIRKLMVIPGKKVSLNRFETGWAQNDELKKVGKDALKDRAAALLEANRIVLAEAQELLWASNIYSVLIVLQGMDTSGKDGTIKHVMSGVNPQGCRVTSFKVPTSEETDHTFLWRYTRALPAKGEIGIFNRSYYEDVLVVRVHPE ILDNQQVPPGKRGDTFWNARYEDINAFERHLVQNGTLILKFFLNISKEEQRRRLLERLENKDKYWKFSLLDLAERRYWDNYQGAYEIMFLKTSTEMAPWYIIPADYKWVARSLIAEIISQKIQMLDLRFPHVSAEGLKELEKAREELNSE Sequence ID 29 MDIDNYRVKPGKRVKLSDWATNDDAGLSKEEGQAQTAKLAGELAEWQERLYAEGKQSLLLILQARDAAGKDGAVKKVIGAFNPAGVQITSFKQPSAEELSHDFLWRIHQKAPAKGYVGVFNRSQYEDVLVTRV YDMIDDKTAKRRLEHIRHFEELLTDNATRIVKVYLHISPEEQKERLQARLDNPGKHWKFNPGDLKDRSNWDKFNDVYEDALTTSTDDAPWYVVPADRKWYRDLVLSHILLGALKDMNPQFPAIDYDPSKVVIH Sequence ID 30 MLRPDTTNQIRAGTDGGFSLSDHQPSWEGSGRVDGLTGAGLKKRAKKYVKAQRRLLAKAQEKLYADDRFSVLILLQAMDAAGKDSTIKQVMSGVNPQGCQVFSFKQPSAEELDHNFLWRYWRDVPERGRIGIFNRSYYEEVLVVRVHPELVQRQR LPAAPNGSDFWAARFEDINAFERHMTRNGTVILKFFLNVSKEEQRQRFVRRLTKPKKLWKFSANDLYERAHWDDYQHAYEELITATSTEWAPWYVIPADNKWLMRALVSHVIVEAVEGLGVTYPKVPPGREAEFRKALNALRAEGAASGKKGGIG Sequence ID 31 MINIYKIDKLNNFNLNNHKTDDYSLCKDKDTALELTQKNIQKIYDYQQKLYAEKKEGLIIAFQAMDAAGKDGTIREVLKALAPQGVHEKPFKSPSSTELAHDYLWRVHNAVPEKGEITIFNRSHYEDVLIGKVKELYKFQNKADRIDEN TVVDNRYEDIRNFEKYLYNNSVRIIKIFLNVSKKEQAERFLSRIEEPEKNWKFSDSDFEERVYWDKYQQAFEDAINATSTKDCPWYVVPADRKWYMRYVVSEIVVKTLEEMNPKYPTVTKETLERFEGYRTKLLEEYNYDLDTIRPIEK Sequence ID 32 MRLPDAVLQQCHYQHASALNLDPNRDFGLPMRKADARLEDLRKWLDRQQQLLWANNGPAILVWLQGPDCSGKDGAIRNSLRGLNPQGIRVSDFQKPTEQQRSEHFLKRYREQLPTAGMITVFNRTPYEGLVSDLADGD IDSNQAAERLQQLLAFEEQLAEQNIHLLKIYLHISKSEQKKRLQQRFINPEKRWKISAADLIGHQNFEQIQANWNQAFAQSSSLSHPWHILPSHNKALRNLLICSLIAQKLEALDLQWPQPALPFAIEDLDLACSISG Sequence ID 33 MAKTSFLQRFKVLPGKKIHLAEIDTDATPGYKNKQEAKADLADCLQRLRDLQYLLYAENKRSLLIVLQGLDASGKDGTIRHVLGYMNPQGCRVTSFKVPTEEEAAHDFLWRVHKAAPGRRQVAIFNRSHYEDVLVVRVHK LVPKSVWEKRYDQINEFERLLADNGTHILKFFLHISPEEQLRRFRKRLENPKKRWKISEADYSERKYWHDYQQAYEDALSRCSRDHAPWFVIPADKKWFRNLAISQIVVESLESLKMQWPNPQVDLDLIRLKYHEAVENS Sequence ID 34 MTTIPEEMEDRIKAVMDIMRVPPDEKIDLEKDYDPGFTGKWMKKKEAKETLVRGIRLLAEMQNKLYAQDQYALLIVLQALDAAGKDSTIKHVMSGINPQGVDVHSFKTPSGEELDHDYLWRNFKALPARGRIGIFNRSYYEEVLVVRVHPEFLASQKLPPILKDK HIWKRRFEEINNFEKYLVDNGIIVLKFFLYVSKEKQRERFLERALLPEKNWKFSTADMKERARWDEYITAYEDMFNHTSTEWAPWYVVPADHKWFTRLAVAAVIYNTMDKLNLAYPVVSEQQKQALLVAEKELENENGGPDDKAVRKVKAKAVASRKNKKKDKKKK Sequence ID 35 MIKKRYLKKIMVSPGSRVSLERYDTGWAQDDELKKVGKDAIKERAAEILEANRAALAESQELLWASNLYSFLIVLQGMDTSGKDGAIKHVMAGVNPQGCRVTSFKVPTSEETDHNYLWRYTRALPARGEIGIFNRSYYEDVLVVKVHPE ILDHQQMPPGKRGDKFWNARYDDINAFERHLVQNGTFILKFFLHISREEQKKRLMERLEDKEKYWKFSLSDLAERRFWDDYQSAYEMMLSKTSTRIAPWYIIPADYKWIARSLIADIISTKIRALDLKYPEVSAETLKQLEGAREQLARE Sequence ID 36 MKTNLIKDLVVRPNMKVKLNEWDPDYDGGIFKKEVESTLNKDLLDRMSDLQYKLFADKNQSLLIILQGIDTSGKDSTIRHVMGAFNPQSCKVISFKAPNDEELSHDYLWRIHKVTPAKGEIVIFNRSHYEDVIEAR VQRLTPEDMFERYRQINEFERYLYENNIKTVKLLLHISKDEQKKRLEDRIKDPSKHWKFSEADIIKRKYWDQYITAYEEAISRCSTPWAPWYIIPANTKWFRNFAIANIVVKSLEDMKLRFPAPKIDLSKVVIDD Sequence ID 37 MNIKTSEFKVPEGIAVDLSKLPTQVEPVYRSKKEYKELLAEQVAALSAQQQLQYADNSHSLLLIFQAMDAAGKDGVIKHVMSGVNPQGCQVFSFKHPSAQELDHDFLWRTTQCLPERGRIGIFNRSYYEEVLIVRVHPEILKNQK LPDSLLNDDAIWPNRYKSIVDFEQHMHRNGTKVIKFFLHLSKDEQRKRFIDRIDQPEKNWKFTVSDINERKHWTQYMKAYESCLTATSTKEAPWYVVPADDKKNARLIVSQIILDAYKDMDMKYPETSEERRQELLSIREQLIND Sequence ID 38 MKEPILAVLPQGPVDLTAIPTDDTTGDFDGNKADGEKALADLGPELSELQERLFAERTAGSTRRVLVVLQGMDTSGKGGVLRHTVGLVDPQGVRITSFKAPTDEERAHDFLWRIEKALPQPGTIGVFDRSHYEDVLIARVR KFAGDEEIERRYGAINDFEQRLVDDGVTIVKCMLHISAEEQKARLLARLDDPSKFYKFNPGDLDERALWPAYREAYEIALERTNTDAAPWHVIPSDKKWFRNLCVGQLLLEALRGLELQWPAADFDVEAQKARLRDEVSPE Sequence ID 39 MGKSKSSENDKPKKIKSPEKKLKNFQKLSPKERIEHAKKFSEQFCVGDGNSFQLKDYKSDIDFDLGKGTKSLVADTLELGVDALATMQEILYAQDKWSLLLVFQAMDAAGKDGAIKHVLSGVNPQGCQVSSFKAPSSEELDHDFLWRCQKHLPERGRIGIFNRS YYEEVLVVRVHEHILKAQKLAEELITENIWEERFEDIKNFEKYLRRNGTVVVKFFLNVSKDEQKKRFIERIDDADKNWKFSTADAKERQHWDEYMHAYEDLIKNTSTKDSPWYVIPADNKAYARIAIASAVIHTLDEMGLEYPKVSDEKVAELNEIKKALLSEE Sequence ID 40 MKKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALAVLNRRLEKLQELLYAEGQHKVLVVLQAMDAGGKDGTIRVVFDGVNPSGVRVASFGVPTEQELARDYLWRVHQQVPRKGELVIFNRSHYEDVLVVRV KNLVPQQVWQKRYRHIREFERMLADEGTTILKFFLHISKDEQRQRLQERLDNPEKRWKFRMGDLEDRRLWDRYQEAYEAAIRETSTEYAPWYVIPANKNWYRNWLVSHILVETLEGLAMQYPQPETASEKIVIE Sequence ID 41 MPHRHRLDHAPFTVQPGQRVRLKDYDSRETAGFKNKSEAKKALLEDVSLLAEMQQLLWAAKEYSLLIVFQALDAAGKDGAIRHVMSGVNPQGVDVYSFKAPTEEERLHHFLWRPAKVMPARGRIAIFNRSYYEEVLVVRVHPEWLERQW VPPRYRGGHYEELWPERFEEINAFERLMSENGVAILKFFLNVSKAEQRERFLERVTDPEKHWKYSAADLRERGYWDMYQAAYEDMLSHTSTKHAPWHVIPADRKWFTRACVADIIAAKIESLDLSYPTVSEEEHSAIEAARRQLEAESD Sequence ID 42 MKTTTPKPTEGQRLQSTAPSFDGCPDWQALADAARVAPGTHVHLMRDHNPGRHTPGLARDGGEYALEAAKTLLLELQDRFFASSGHALVVVLQAIDAAGKDGTIKHVMSGLNPVGVDVYSFKEPTPIEAAHDYLWRYHRVLPERGRIAVFNRSHYEAV LTTRVHPEFLVPPPVADADLHHLWHRRFQEIKEWERYLHDNGTVIVKLFLNLSKGEQKRRFLQRLENPQKNWKFSVNDLAERTHWDDYQFAFQEMLSHTSTTWAPWYVVPADHKWFSHLTTSAVLVQALRGMDPQYPALTDSARERLTAAKLRLDSEPC Sequence ID 43 MATDFSKLSKYVETLRVKPKQSIDLKKDFDTDYDHKMLTKEEGEELLNLGISKLSEIQEKLYASGTKSVLIVFQAMDAAGKDGTVKHIMTGLNPQGVKVTSFKVPSKIELSHDYLWRHYVALPATGEIGIFNRSHYENVLVT RVHPEYLLSEQTSGVTAIEQVNQKFWDKRFQQINNFEQHISENGTIVLKFFLHVSKKEQKKRFIERIELDTKNWKFSTGDLKERAHWKDYRNAYEDMLANTSTKQAPWFVIPADDKWFTRLLIAEIICTELEKLNLTFPTVSL Sequence ID 44 MASRIEDMETRIEKLLDIMRVPPGKKIDLQKDYDPGFTGKWVTKEEAEETLAKGVQLLAEMQDKLWAQNQYALLMILQALDAAGKDGAIKHVMSGVNPQGVDVHSFKGPSSEELDHDYLWRNFKTLPSRGRIGIFNRSYYEEVLVVRVHPEFLASQQLPPALKDKRIWRRRF EEINNFEKYLVDNGIIVVKIFLYVSKEVQKERFLERAMMPEKNWKFSVADMKERAHWDDYIAAYEDMFNHTSTEWAPWYIVPADHKWFTRLAVAAVLYNTMKNLNLAYPTVSEQQKQALLAAKDELEREDGGLKDKAVVKAETKAATDPQAPDASSAVTKGKAKKGGKKKNK sequence number 45 MANRIEELLDVLRVPSGKKINLKKDYDPDFTGKWVKKEEAKEALTEGIQILAEMQDKLWAQDQYALLIILQALDAAGKDSTIKHVMSGVNPQGVDVHSFKVPSGEELHDYLWRNFKALPARGRIGIFNRSYEEVLAVRVHPEFLAGQKLPPSLKDKNIWNRRFEEIN NFEKYLVDNGIIVVKLFLYVSKETQKERFLERTLEPEKNWKFSAADMKERAFWDDYMAAYEDMFNHTSTEWAPWYIVPADHKWTRLAVAAVLYNTMKSLNLAYPTVSEEQKQALLVAKEELEHEDGGLKDKAVTRAEAKTAADPKTLAARSKVTKEEVKKNSTKGKKNK sequence no. 46 MDVSGPRVRKYLRQFVADPTKKIRLKKYDTARVPRKELSAESDDEAKALLKDLLDKNRQELARAQDLLAASAQYSILIVLQGMDTSGKDGTIRHVMSGINPQGCAVHSFKVPGAEEHAHDFLWRYEKVLPERGMIGIFNRSYYEDVLVVHVH PDRMESLPAAIGPENAGFWEGRYKDINAFEKHLVRNGTVVLKFFLHISRDEQKRRLLDRLSHQEKFWKFSFTDIAERQYWDQYQVAYEEMLSATSTEYAPWFIVPADHKWVARTVVAEVIASAIEGLHLCYPVFPDEKIRQLEKAKAALERE Sequence ID 47 MDTLKDLRSLIVEPGRKFDLADRATALDLPGDPAKEEIEAELATLREQIGDIQDRMFTRHEQALLVVLQGMDSAGKDSTTKAVFTGVNPLGIRAASFSVPTPLERRHDYLWRIHAQVPQLGCIGVFNRSHYEDPVI RRVHGEIKNGAMKQYLQQIAEFERLLTETGTTIVKCFLHIGFDEQRERLQERIDNPAKNWKFDPSDLQERRYWKAYQSAYSEALTSTSTEQAPWYVVPADRKPARNLLVARLVLQALKGLSAPEPAPKPELKGLKVR Sequence ID 48 MARSTAYLKTYRVGRKLRLKDIDPGARPAASSSREADDARLAELAIEIDRLQDLLYANGSAGRPPKLLLVLQGMDTSGKDGTARSVFRQCSPLGVRVAAFKAPTEVERAHDFLWRVHAVAPRAGEVVVFNRSHYEDVL VPFVEGWIDAAERQRRLAHINAFERLLHDSGTTIVKCFLHISKDEQRERLQARLDDPAKRWKFQVGDLETREKWKAYLAAYETALAATSTACAPWHVVPADSKSNRNLMIATLVAQALAGMKLKPPKPDFDPAAVRVV Sequence ID 49 MLFKPERYLWRRQSAPDLRQCATRLPYEDKDSRDQLKREAKDLLEESQKELIESQHRLNVIDTHAVLLILQGMDAAGKDGIIKHVLSGLNPLYCQITGFRAPSSIELEYPFLSRYVAHLPQRGHIGLFNRSWYEETVTVKIFPEFLHKQRI ALEAINDRFWQHRFEDINHFEQHLTRNGIAIIKCFLHVSREEQRQRLLDRVVKPEKFWKFDPFDLETRKRWDEFMQAWNETLTATDSHAPWHVIPADDKPMARALVARLLASTIDALPLKEKSFSETQLERIKEARMILENEKDDSAEHD Sequence ID 50 MNLKDYCFFGNKPFVIEQFDPAQTGKFQLKNEVEELHHTNLLKMQELQDRLYAENKEALLIIFQGMDASGKDSAVKHVMGGVNPQGINVHNFKKPSSEELDHDYMWRSMRVIPERGKIGVFNRSYYEDVLVGKVHKLYQESYLPDR CKTDKIFEQRYQQIKNYERYLYENGIRVIKFFLHISKEEQKKRFLERIEDDQKNWKFSDSDMIERDYWEEYQRAYHDAINATATRIAPWYVVPSDKKWFGHFIISETIIDTLEKINPQYPAVTRERKERLLEFRSKLLREEEQSSF Sequence ID 51 MQLDRYRVPPGQRVRLSNWPTDDDGGLSKAEGEALLPDLQQRLANLQERLYAESQQALLIVLQARDAGGKDGTVKHVIGAFNPSGVQVSNFKVPTEEERAHDFLWRIHRQTPRLGMIGVFNRSQYEDVLVTRV HHLIDDQTAQRRLKHICAFESLLTDSGTRIVKFYLHISPEEQKKRLEARLADPSKHWKFNPGDLQERAHWDAYTAVYEDVLTTSTPAAPWYVVPADRKWFRNLLVSQILVQTLEEMNPQFPAPAFNAADLRIV Sequence ID 52 MDTSQFQVSNPDSYVLADAPTVAPSDFDREESKRRVKQLTKQIQALQEKMWAGRKHRLLVVLQAIDAGGKDGTVRSVFGRTNPQGVKVHSFKRPTEQELAHDYLWRVHPHVPGNGEITVFNRSHYEDVLVVKV HGYAPLDVVEKRYQHIRNFEQMLVDEGTTXIKIFLHISKDEQKKRFEARLNRPEKNWKFKKGDIKERAFWEQYHEVFEQMLKKTTTDDSPWYVVPADNKKYRNEIISSLILETLESFKMQWPQPEEDLSDIVIE Sequence ID 53 MIFNGENSVLAEELIAKFRIDEGQKVKLRDYDTGWKVSDEIAYFQKEEVKNKAKDILEKNREDLARGQSLLWANNTYSLLVIIQGMDTAGKDSLIKHVMSGVNPQGCRVSSFDVPSKEELDHDYFWRCNLKLPKRGEIGIFNRSYYEEVLITK VHPEILEGRHLPQKEFHDEFWKMRYEDINSYERHLSNSGTVVLKFFLHISKEKQKERLLARFENESKYWKISPSDIAERKFWDNYIRAYEDMLNNTSTKSAPWYIIPADYKWVARTIVSDIIASKLISLHDSYPAVSEETLEKIKKAADMLKNE Sequence ID 54 MKNEFKGLIVQGDKKFSLKNHKTDFTGGYNKEKAKDALVNSKIELSHLQEKLYASGKHSVLIIFQAMDAAGKDSAIEHVMSGLNPQGCQVYSFKVPTSEEYQHDFLWRHYKALPERGRIGIHNRSHYENVLVCKVHPEYILSEDIP GITDVKKISKDFWKQRYQSIRNFEQHLTANGTVILKFFLNVSKDEQKQRFLDRIEDPSKNWKFSSGDIKERALWDKYMEAYQDAINETSTAESPWHIIPADKKWFARLISEIIEDRLKNLELKFPVLGAEETLKLSETKNALLAE Sequence ID 55 MIPQDFLDEINPDRYIVPAGGNFHWKDYDPGDTAGLKSKVEAQELLAAGIKKLAAYQDVLYAQNIYGLLIIFQAMDAAGKDSTIKHVMSGLNPQACRVYSFKAPSAEELDHDFLWRANRALPERGCIGIFNRSYYEEVLVVRVHPDLLNR QQLPPETKTKHIWKERFEDINHYERYLTRNGILILKFFLHISKAEQKKRFLERISRPEKNWKFSIEDVRDRAHWDDYQQAYADVFRHTSTKWAPWHIIPANHKWFALMVAHFIYQKLASLNLHYPMLSEAHREQLLEAKALLENEPDED Sequence ID 56 MVGKYRVDGKKSIKLKDFPTADKGIFKNKEEGLLKLGENIEIISELQNKLYAEDTYSLLIIFQAMDAAGKDGTIKHVFSGINPQGFQIFNFKQPSREELDHTYMWRTSKSMPERGRIGVFNRSYYEDVLVVRVHNLLSTQHLP KEKQYKDIWKRRFHNIREQERYLFENGVIPIKFFLNVSKEVQKERFLERIEDPSKNWKFSSADIEERKYWDDYQDAYEDAINHTSTSYAPWYVIPADKKWFAKFAVSEIIKETLESLNLKYPELNKTQKSELKNYKEILLKEK Sequence ID 57 MGFCSIEFLMGAQMKKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALAVLNRRLEKLQELLYAEGQHKVLVVLQAMDAGGKDGTIRVVFDGVNPSGVRVASFGVPTEQELARDYLWRVHQQVPRKGELVIFNRSHYED VLVVRVKNLVPQQVWQKRYRHIREFERMLADEGTTILKFFLHISKDEQRQRLQERLDNPEKRWKFRMGDLEDRRLWDRYQEAYEAAIRETSTEYAPWYVIPANKNWYRNWLVSHILVETLEGLAMQYPQPETASEKIVIE Sequence ID 58 MKINSEDFIVSTAGRKKVNLKHRPTIVKPLYGSKKQYKEILNEHIEELSSLQELLFASNRYSLLVIFEGMDAAGKDSAIKHVMSGVNPQGCQVFSFKRPTPIELQHDFLWRYVVCLPERGRIGIFNRSYYEGVLAARVHPEVDLRSEGFLD QDDGPAADEEKKKEEVWEQRYESIVNLEKHLYHNGTRIIKIFLHLSEEEQRKRFLERIDDPKRNWKFSLADVEERKFWHDYMKAYEDCLSATSTTTYAPWYVVPADNKENARLIISEIILETLNGLEMSYPKLDPKRLDELQSTRKKLLLNK Sequence ID 59 MDTSEFLISNAEDFALSNWQVDPPAEFDEKQSKRTLKQNTKVLQSLQKLMWAERKQRVLVVLQAIDAGGKDGTIRSVFGGLNPQGVKVQSFKRPTKQELAHDYLWRVHKHVPGNGEIMVFNRSHYEDVLVVKV HEYAPLDLVEKRYEHIRNEFEQLLADEGTTIIKIFLHISKDEQKRRFEDRLNEPDKNWKFKKEDLKERAFWDQYQDAFETMIEQTSTKDAPWYVVPSNNKKYRNEIISNLLIETLSGLNMEFPPAADGLDNIVID Sequence ID 60 MSAIRLTDIPTRAPGNMDKKKTRKATHKLKKAIADLQNILYAEAKHSLLVILQGMDASGKDGAIRHVFNRINPQGIQVTSFKVPTPEEAAHDFLWRIHAHAPARGMMHIFNRSHYEDVLVTRVHGWIDDD TAHRRMQHINHFEQLLLDHQTTILKFYLHISREEQAKRLQERLKNPKKRWKYNPSDLEEAQKWHDYLHYYEEVFATCSPEIPWIIVPADQNWYKEFVIAEAVHQALKNMQLRYPRRASLASAGHAHNRKK Sequence ID 61 MSNGTRQPIVYPPHTRVDLRKFDPAATNGMSREEAEAELVGLRLRLNELQNMLYADARYALLVVLQGIDAAGKDGTINSVFEQVGPIGCSVVSFKAPTPEELAHDYLWRYHRVMPERGHITIFNRSYYEAVLVER VKGIVPKATWEQRYEDINRLEEYLLRNGTVVMKFFLAISKEEQRERLQERIDNPKKQWKFRLGDLEERKLWDEYLEAFEDMLERCNTKHAPWHVVPADRKWYRDVVVARTLVEKLASLGLRYPPADPAVLGLKVD Sequence ID 62 MANRTEDMETRIEDVEARIEALMEFMRVPPGRKINLRKDYDPGFTGRWLKKAEAKETLARGIQMLAEMQDKLWAQNQYALLMVLQALDAAGKDGTIKHVMSGVNPQGVDVYSFKAPSGEERDHDYLWRNFRALPARGDIGIFNRSYYEEVLVVRVHPEILAGQQLPPALKDEGIW KRRFEEINNFEKYLVDNGIIVVKFFLYVSKEAQKKRFLKRTMLPEKNWKFSAADIKERAHWDDYLDAYEDMFNHTSTEWAPWYIVPADHKWFTRLAVAAVLYSTMKKLNLAYPTVSEQKKQALLVAKEELENEDGGKKDKAVVKAKAKAAANKKVPASGREGTRNESKKKGKKNR Sequence ID 63 MATPLEKLADIFRVPPGRNIDLRKEYDPGFTSDSVSKENAQKYLETGIQKLAEQQDKLYAQNTLALLINFQAMDAAGKDSTIKQVMSGVNPQGVQVKSFKVPSAEELDHDYLWRTFLELPNRGHIGIFNRSYYEEVLVVRVHPEFLERQQLPPKQ KGEDIWKRRYGEINNFEKYLTDNGIVVLKFFLNVSKEEQKKRFLERIEEPEKNWKFSANDVLERQYWDRYMDAYEEMFNNTSTPWAPWYIIPADHKWFTRFAVVSVINYTIDGMNLAYPSVSAEQKEALQKAKEAMEQEETHPEKTTGTKKKRKR Sequence ID 64 MKIRSKDFRVPPGEKLKLDEWPTLIKPAYKSKKAYAELLQEHVAELSTLQQLHYASGRYALLLIFQAMDAAGKDGAIKHVMSGVNPQGCQVFSFKHPSATELEHDFLWRTNQCLPERGRIGIFNRSYYEEVLIVRVHPEILRSQGLPDG AVDEKTVWKERYRSIVDLENHLYRNGTRIIKFFLHLSAEEQRKRFLERIDEPEKNWKFSLADIQERKFWPQYMQAYEACLGATSTKIAPWHVVPADDKENARLIVSQIILDAFKALKMRYPETDAKRRQELLSIRHGLSEEGRRNFSPQ Sequence ID 65 MIKLIKDDRITITSGKNFKLKNFDTDFSLDFVSKESAGVIMQEHIQRIAALQERLYASDNHAILMIFQARDAAGKDSTIKHVMSGINPSGCQVVSFKVPTSTEIEHDFLWRTAVQLPERGRIGIFNRSYYEEVLVTKVHPEIILNQRL PSVLTEKDLDKKFWEHRYESIRDHEKHLSRNGGFVILKFFLNVSKKEQKKRFLERIDQPDKNWKFSYGDIKERQYWDQYEDAFEQMIEETAEEHAPWFVIPSDKKWFAQLAVARILLETLESLDLKFPELGDEEKAVLEKAKMELESEKD Sequence ID 66 MKAINTEDFKVTGKFDVKGVPTFIDVETSDKKIANSLEEVSIKLGKLQDKMYANNRYSVLICLQGMDTAGKDSLIREVFKNFNARGVNVYSFKKPSSSELEHDYLWRHYIALPEKGKFAVFNRTHYENVLVTRVHPEFLMYENLPG IETVEEIPADLWEQRYEQINNFEKHISANGTIIFKFFLHLGKDEQKKRLLRRLEKQSHNWKFSPGDLTERDLWDKYQQYYEEAINKTSKEHAPWYVIPADKKETARYLVAKTILDTLKKYDDIKEPEVSEDVLANISLYRDQLMNEE

Claims

1. Use of polyphosphate kinase (PPK) to produce glycosylated and / or α-phosphate-modified nucleoside triphosphates from corresponding modified nucleoside monophosphates or nucleoside diphosphates and polyphosphates.

2. The use according to claim 1, wherein the PPK is PPK2.

3. The use according to claim 1 or 2, wherein the PPK is PPK2-III.

4. The use according to any one of claims 1 to 3, wherein the PPK has a sequence that is at least 90% identical to sequence number 22.

5. The use according to any one of claims 1 to 4, wherein the PPK has the sequence of sequence number 22.

6. A method for screening a panel of putative and known PPKs to identify enzymes capable of producing corresponding modified nucleoside triphosphates using glycosylated and / or α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates as substrates, a) The step of identifying sequences encoding the panels of the estimated and known PPKs, b) The step of synthesizing the PPK, c) A step of purifying the PPK, d) A step of mixing each of the purified PPKs with the sugar-modified and / or α-phosphate-modified nucleoside monophosphate or nucleoside diphosphate and polyphosphate to form a reaction mixture, e) A step of analyzing each of the reaction mixture for the presence of the corresponding modified nucleoside triphosphate to determine which enzyme produces the corresponding modified nucleoside triphosphate. Methods that include...

7. The method according to claim 6, wherein the sequence in step a) is identified using a sequence database and bioinformatics tools.

8. The use according to claim 6 or 7, wherein the sequence in step a) encodes a protein having a length of 150 to 600 amino acid residues.

9. The method according to any one of claims 6 to 8, wherein the PPK is synthesized using an N-terminal hexahistidine tag in step b) and purified using nickel-nitriloacetate in step c).

10. The method according to any one of claims 6 to 9, wherein step d) comprises incubating the sugar-modified and / or α-phosphate-modified nucleoside monophosphate or nucleoside diphosphate, polyphosphate and PPK at 25°C for 24 hours to quench the reaction.

11. The method according to claim 10, wherein the reaction is quenched with EDTA at 80°C for 15 minutes.

12. The method according to any one of claims 6 to 11, wherein the reaction mixture is centrifuged and the supernatant is analyzed for the presence of the corresponding modified nucleoside triphosphate.

13. The method according to claim 12, wherein the reaction mixture is centrifuged at 4000 g for 15 minutes.

14. The method according to claim 12 or 13, wherein the supernatant is analyzed using HPLC or UPLC.

15. The method according to any one of claims 6 to 14, wherein the enzyme capable of producing the corresponding modified nucleoside triphosphate is further manipulated to increase the percentage of conversion to the corresponding modified nucleoside triphosphate.

16. An enzymatic method for producing glycosylated and / or α-phosphate-modified nucleoside triphosphates, The step of incubating PPK with the corresponding modified nucleoside monophosphate or nucleoside diphosphate and polyphosphate under appropriate conditions. Methods that include...

17. The method according to claim 16, further comprising incubation with a divalent cation.

18. The aforementioned divalent cation is Mg ２＋ Mn ２＋ Or Co ２＋ The method according to claim 17.

19. The method according to claim 16, further comprising incubation with magnesium chloride.

20. The method according to any one of claims 16 to 19, wherein the PPK is selected using the method according to any one of claims 6 to 15.

21. Use of a modified nucleoside triphosphate obtained by the method of any one of claims 16 to 20 in the production of a polynucleotide.

22. The use according to claim 21, wherein the polynucleotide is a therapeutic polynucleotide.

23. The use according to claim 22, wherein the polynucleotide is therapeutic mRNA.

24. The use according to claim 21 or 22, wherein the polynucleotide is an oligonucleotide.

25. The use according to claim 24, wherein the oligonucleotide is selected from the group consisting of antisense oligonucleotides, siRNA, miRNA, aptamers, CRISPR guide RNA, and oligonucleotides used to recruit and guide DNA and RNA editing enzymes.

26. The use according to claim 25, wherein the oligonucleotide used to recruit and guide DNA and RNA editing enzymes is an A-to-I RNA base editing oligonucleotide (AIMer).

27. The use according to any one of claims 21 to 26, wherein the production of the polynucleotide is carried out by an enzymatic method.

28. The use according to claim 27, wherein the enzyme is a polymerase.

29. The use according to claim 28, wherein the polymerase is a template-dependent polymerase.

30. The use according to claim 28, wherein the polymerase is a template-independent polymerase.

31. The use according to claim 27, wherein the enzyme is terminal deoxynucleotidyltransferase (TdT).

32. The use or method according to any one of claims 1 to 31, wherein the sugar modification is a 2'-sugar modification.

33. The 2'-sugar modification is H, OH, O-CH ３ (OMe), O-CH ２ -CH ３ (OEt), O-CH ２ -CH ２ -CH ３ , O-CH ２ -CH ２ -O-CH ３ (MOE), F, O-CH ２ -CO-NH-CH ３ , O-CH ２ -CH ２ -O-Nme2, an O-methylene bridge with 4' carbon, and an O-constrained ethylene bridge with 4' carbon, selected from the group consisting of, the use or method according to claim 32.

34. The use or method according to claim 32, wherein the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate comprise locked nucleic acid (LNA).

35. The use or method according to claim 32, wherein the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate contain restricted ethyl (cEt).

36. The use or method according to claim 33, wherein the 2'-glycosylation is selected from the group consisting of 2'-OMe, 2'-MOE, and 2'-FNA.

37. The use or method according to claim 36, wherein the 2'-sugar modification is 2'-OMe.

38. The use or method according to claim 36, wherein the 2'-sugar modification is 2'-MOE.

39. The use or method according to claim 36, wherein the 2'-glycosylation is 2'-FNA.

40. The use or method according to any one of claims 1 to 39, wherein the sugar modification is a 3'-sugar modification.

41. The aforementioned 3'-sugar modification is phosphate (PO), phosphorothioate (PS), phosphoramidate (PN), phosphorodithioate, methylphosphonate, boranophosphate, phosphoroselenoate, N-methyl-anthraniloyl (MANT), acetyl (Ac), benzyl (Bn), benzoyl (Bz), allyl (All), 2-cyanoethyl (CE), azidomethyl (AzM), cyanovinyl (Cv), methoxymethyl (MOM), methoxyethoxymethyl (MEM), methoxymethylphenylmethyl (MPM), diphenylmethyl (DPM), tetrahydropyranyl (THP), dimethoxytrityl (DMTr), methyl The use or method according to claim 40, selected from the group consisting of toxiccarbonylmethyl (MOMcl), ethoxycarbonyl (EOC), benzylcarbonyl (Cbz), trifluoromethanesulfonate (Triflate), methanesulfonate (Mesylate), p-toluenesulfonate (Tosylate), pivaloyl (Piv), ortho-nitrobenzyl (o-NB), bromomethylcoumarinylmethyl (Bhc), (7-(diethylamino)coumarin-4-yl)methyl (DEACM), 6-nitroberatryloxycarbonyl (NVOC), and 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc-ol).

42. The use or method according to claim 41, wherein the 3'-sugar modification is PO or MANT.

43. The use or method according to any one of claims 1 to 42, wherein the α-phosphate modification is selected from the group consisting of PS, PN, methylphosphonate, boranophosphate, and phosphoroselenoate.

44. The use or method according to any one of claims 1 to 43, wherein the α-phosphate modification is stereospecific.

45. The use or method according to claim 43 or 44, wherein the α-phosphate modification is PS.

46. The use or method according to claim 43 or 44, wherein the α-phosphate modification is PN.

47. The bases in the modified nucleoside triphosphate and modified nucleoside monophosphate / diphosphate are adenine (A), uracil (U), cytosine (C), guanine (G), thymine (T), 5-methylcytosine (m5C), 5-hydroxymethylcytosine (hm5C), 5-fluorocytosine (5fC), 5-bromouracil (5BrU), 2-aminopurine (2AP), inosine (I), pseudouracil (Ψ), and N1-methyl Pseudouracil (m1Ψ), 1-ethylpseudracil, 2-thio-1-methyl-1-deazapseudracil, 2-thio-1-methylpseudracil, 2-thio-5-azapseudracil, 2-thio-dihydropseudracil, 2-thio-dihydrouracil, 2-thiopseudracil, 4-methoxy-2-thiopseudracil, 4-methoxypseudracil, 4-thio-1-methylpseudracil Cyl, 4-thio-pseudracil, 5-aza-uracil, dihydropseudracil, 5-methyluracil, 5-methoxyuracil (mo5U), 2'-O-methyluracil (2moU), 7-deazaguanine (7-deaza-G), 8-oxoguanine (8-oxo-G), 5-propynyluracil (pU), 5-propynylcytosine (pC), N6-methyladenine (m6A), N4-acetylcytosine (ac4C) The use or method according to any one of claims 1 to 46, selected from the group consisting of O6-methylguanine (O6meG), hypoxanthine (H), xanthine (X), isocytosine (iC), isoguanine (iG), cuin (Q), 2-thiouracil (s2U), 4-thiouracil (s4U), 5-iodouracil (5Iu), 2-thiocytosine (s2C), 5-chlorouracil (5ClU), and isouracil.

48. The use or method according to claim 47, wherein the base in the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate is selected from the group consisting of A, U, C, G, T, m5C, I, Ψ, and m1Ψ.

49. The use or method according to claim 47 or 48, wherein the base in the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate is selected from the group consisting of A, U, C, G, and T.

50. The use or method according to claim 47 or 48, wherein the base in the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate is m5C.

51. The use or method according to claim 47 or 48, wherein the base in the modified nucleoside triphosphate and the modified nucleoside monophosphate / diphosphate is m1Ψ.

52. Use of PPK to produce stereospecific α-phosphate-modified nucleoside triphosphates from α-phosphate-modified nucleoside monophosphates or nucleoside diphosphates, and polyphosphates.

53. The use according to claim 52, wherein the stereospecific α-phosphate-modified nucleoside triphosphate is stereospecific PS-modified nucleoside triphosphate.

54. S ｐ Diastereomer vs. R ｐ The diastereomer ratio is approximately 50:50, S ｐ Diastereomer vs. R ｐ The diastereomer ratio is approximately 55:45, or R ｐ Diastereomer vs S ｐ The use according to claim 53, wherein the diastereomer ratio is approximately 55:

45.

55. S Ｐ : R ｐ The use according to claim 53, wherein the ratio is greater than 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99:

1.

56. R ｐ : S Ｐ The use according to claim 53, wherein the ratio is greater than 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, or 99:

1.

57. An engineered PPK having at least 90% sequence identity with respect to sequence number 22.

58. A method for producing a polynucleotide comprising nucleoside triphosphate, a suitable enzyme, and optionally incubating a template under suitable conditions, wherein the nucleoside triphosphate is produced by incubating the corresponding nucleoside monophosphate and / or nucleoside diphosphate, PPK, and polyphosphate under suitable conditions.

59. The method according to claim 58, wherein the nucleoside triphosphate contains or consists of a naturally occurring nucleoside triphosphate.

60. The method according to claim 58, wherein the nucleoside triphosphate includes or consists of a modified nucleoside triphosphate.

61. The method according to claim 59, wherein the nucleoside triphosphate consists of naturally occurring nucleoside triphosphates, except that the uridine triphosphate is substituted with N1-methylpseudridine triphosphate.

62. The method according to claim 60, wherein one or more or all of the nucleoside triphosphates are modified nucleoside triphosphates comprising sugars, α-phosphates and / or base modifications.

63. The method according to claim 62, wherein the modified nucleoside triphosphate is a base-modified nucleoside triphosphate.

64. The method according to any one of claims 58 to 63, wherein the enzyme is a polymerase.

65. The method according to claim 64, wherein the polymerase is a template-dependent polymerase.

66. The method according to claim 64, wherein the polymerase is a template-independent polymerase.

67. The method according to any one of claims 58 to 63, wherein the enzyme is a terminal deoxynucleotidyltransferase (TdT).

Images