ENZYMATIC SYNTHESIS OF NTP AND NQP
The present disclosure provides enzymatic methods for the production of 3′-phosphate-nucleoside-5′-triphosphate (NQP). The present disclosure further provides enzymatic methods for the production of nucleotide-5′-triphosphates.
1 . A method of producing a nucleotide triphosphate with a phosphate group at the 3′ position of the sugar (NQP), the method comprising at least:
contacting a nucleoside diphosphate (NDP) with a nucleoside diphosphate kinase, a 3′-O-kinase, and a phosphate donor under reaction conditions such that a NTP with a phosphate group on the 3′ position of the sugar moiety (NQP) is produced.
2 . The method of claim 1 , wherein the NDP has at the 2′ position of the sugar moiety a H or OH.
3 . The method of claim 2 , wherein the NDP is ADP, GDP, UDP, CDP, or TDP, and wherein the NDP has at the 2′-position of the sugar moiety an OH, thereby resulting in corresponding product rATP, rGTP, rUTP, rCTP, or rTTP, respectively.
4 . The method of claim 2 , wherein the NDP is ADP, GDP, UDP, CDP, or TDP, and wherein the NDP has at the 2′-position of the sugar moiety a H, thereby by resulting in corresponding product dAMP, dGMP, dUMP, dCMP, or dTMP, respectively.
5 . The method of claim 1 , wherein the NDP is a modified NDP comprising a modified 2′-position, wherein the 2′-position of the sugar moiety is —O—CH 3 , —O—CH 2 CH 3 , F, or Br.
6 . The method of claim 1 , wherein the NDP comprises a modified nucleobase, wherein the modified nucleobase is 5-bromo-uracil, 5-iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.
7 . The method of claim 1 , wherein the NDP comprises a 5′-O-(1-thio(diphosphate) (NDPαS).
8 . The method of claim 1 , further comprising contacting a nucleoside monophosphate (NMP) with a nucleoside monophosphate kinase in presence of a phosphate donor NTP under suitable reaction conditions such that a product nucleoside diphosphate (NDP) is produced for reaction with the nucleoside diphosphate kinase and/or 3′-O-kinase.
9 . The method of claim 8 , wherein the NMP has at the 2′ position of the sugar moiety a H or OH.
10 . The method of claim 9 , wherein the NMP is AMP, GMP, UMP, CMP, or TMP, and wherein the NMP has at the 2′-position of the sugar moiety an OH, thereby by resulting in corresponding product rADP, rGDP, rUDP, rCDP, or rTDP, respectively.
11 . The method of claim 9 , wherein the NMP is AMP, GMP, UMP, CMP, or TMP, and wherein the NMP has at the 2′-position of the sugar moiety a H, thereby by resulting in corresponding product dADP, dGDP, dUDP, dCDP, or dTDP, respectively.
12 . The method of claim 8 , wherein the NMP is a modified NMP comprising a modified 2′-position of the sugar moiety, wherein the 2′-position of the sugar moiety is —O—CH 3 , —O—CH 2 CH 3 , F, or Br.
13 . The method of claim 8 , wherein the NMP comprises a modified nucleobase, wherein the modified nucleobase is 5-bromo-uracil, 5-iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.
14 . The method of claim 8 , wherein the NMP comprises a nucleoside 5′-O-thiophosphate (NMPαS).
15 . The method of claim 8 , wherein the phosphate donor NTP has the same nucleotide structure as the substrate NMP.
16 . The method of claim 8 , wherein the nucleoside monophosphate kinase is an adenylate kinase.
17 . The method of claim 16 , wherein the adenylate kinase is an adenylate kinase of an even-numbered SEQ ID NO. of SEQ ID NOs: 1206-2504, with or without amino acid residues 1-12.
18 . The method of claim 8 , further comprising contacting a nucleoside with a nucleoside kinase in presence of a phosphate donor NTP under reaction conditions such that a product nucleoside monophosphate (NMP) is produced.
19 . The method of claim 18 , wherein the nucleoside has at the 2′ position of the sugar moiety a H or OH.
20 . The method of claim 19 , wherein the nucleoside is adenosine (A), guanosine (G), uridine (U), cytidine (C), or thymidine (T), and wherein the nucleoside has at the 2′-position of the sugar moiety an OH, thereby by resulting in corresponding product rAMP, rGMP, rUMP, rCMP, or rTMP, respectively.
21 . The method of claim 19 , wherein the nucleoside is adenosine (A), guanosine (G), uridine (U), cytidine (C), or thymidine (T), and wherein the nucleoside has at the 2′-position of the sugar moiety a H, thereby by resulting in corresponding product dAMP, dGMP, dUMP, dCMP, or dTMP, respectively.
22 . The method of claim 18 , wherein the nucleoside is a modified nucleoside, wherein the 2′-position of the sugar moiety is —OCH 3 , —OCH 2 CH 3 , F, or Br.
23 . The method of claim 18 , wherein the nucleoside comprises a modified nucleobase, wherein the modified nucleobase is 5-bromo-uracil, 5-iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.
24 . The method of claim 18 , wherein the phosphate donor NTP is a nucleoside-5′-gamma thio-triphosphate (NTPγS) for producing NMP-5′-O-thiophosphate (NMPαS).
25 . The method of claim 18 , wherein the nucleoside kinase is an adenosine kinase.
26 . The method of claim 25 , wherein the adenosine kinase is an adenosine kinase of an even-numbered SEQ ID NO. of SEQ ID NOs: 66-1204, with or without amino acid residues 1-12.
27 . The method of claim 1 , wherein the nucleoside diphosphate kinase is an acetate kinase.
28 . The method of claim 27 , wherein the acetate kinase is an acetate kinase of an even-numbered SEQ ID NO. of SEQ ID NOs: 2506-3058, with or without amino acid residues 1-12.
29 . The method of claim 1 , wherein the 3′-O-kinase is a 3′-O-kinase of an even-numbered SEQ ID NO. of SEQ ID NOs: 3060-3370 and 3376-5126, with or without amino acid residues 202-211.
30 . A method of producing a NTP with a phosphate group at the 3′ position of the sugar (NQP), the method comprising contacting a nucleoside with a nucleoside kinase, a nucleoside monophosphate kinase, nucleoside diphosphate kinase, and a 3′-O-kinase in presence of a phosphate donor under reaction conditions such that a product NTP with a phosphate group at the 3′ position of the sugar (NQP) is produced.
31 . A method of producing a NTP with a phosphate group at the 3′ position of the sugar (NQP), the method comprising (i) contacting a nucleoside with a nucleoside kinase, a nucleotide monophosphate kinase, and a nucleoside diphosphate kinase in presence of a phosphate donor under reaction conditions such that a NTP is produced; and iii) contacting the NTP produced in (i) with a 3′-O-kinase enzyme under suitable reaction conditions, such that a NTP with a phosphate group at the 3′ position of the sugar (NQP) is produced.
32 . A method of producing a NTP with a phosphate group at the 3′ position of the sugar (NQP), the method comprising (i) contacting a nucleoside with a nucleoside kinase, (ii) contacting the product of (i) with a nucleotide monophosphate kinase, (iii) contacting the product of (ii) with a nucleoside diphosphate kinase, and (iv) contacting the product of (iii) with a 3′-O-kinase enzyme, wherein (i), (ii), (iii) and (iv) are carried out in presence of a phosphate donor under reaction conditions such that a product NTP with a phosphate group at the 3′ position of the sugar (NQP) is produced.
33 . The method of claim 30 , wherein the nucleoside has at the 2′-position of the sugar moiety a H or OH.
34 . The method of claim 33 , wherein the nucleoside is adenosine (A), guanosine (G), uridine (U), cytidine (C), or thymidine (T), and wherein the nucleoside has at the 2′-position of the sugar moiety an OH, thereby by resulting in corresponding product rAQP, rGQP, rUQP, rCQP, or rTQP, respectively.
35 . The method of claim 33 , wherein the nucleoside is adenosine (A), guanosine (G), uridine (U), cytidine (C), or thymidine (T), and wherein the nucleoside has at the 2′-position of the sugar moiety a H, thereby by resulting in corresponding product dAQP, dGQP, dUQP, dCQP, or dTQP, respectively.
36 . The method of claim 30 , wherein the nucleoside comprises a modified 2′-position of the sugar moiety, wherein the 2′-position of the sugar moiety is —OCH 3 , —OCH 2 CH 3 , F, or Br.
37 . The method of claim 30 , wherein the nucleoside comprises a modified nucleobase, wherein the modified nucleobase is 5-bromo-uracil, 5-iodo-uracil, 6-mCEPh-purine, 6-phenylpyrrolocytidine, N2-alkyl 8-oxoguanosine, difluorotoluene, difluorobenzene, dichlorobenzene, imidazole, or benzimidazole.
38 . The method of claim 30 , wherein the phosphate donor comprises phosphate donor NTP.
39 . The method of claim 38 , wherein the phosphate donor NTP is a nucleoside-5′-gamma thiotriphosphate (NTPγS).
40 . The method of claim 30 , wherein the nucleoside kinase is an adenosine kinase.
41 . The method of claim 40 , wherein the adenosine kinase is an adenosine kinase of an even-numbered SEQ ID NO. of SEQ ID NOs: 66-1204, with or without amino acid residues 1-12.
42 . The method of claim 30 , wherein the nucleoside monophosphate kinase is an adenylate kinase.
43 . The method of claim 42 , wherein the adenylate kinase is an adenylate kinase of an even-numbered SEQ ID NO. of SEQ ID NOs: 1206-2504, with or without amino acid residues 1-12.
44 . The method of claim 30 , wherein the nucleoside diphosphate kinase is an acetate kinase, and the phosphate donor includes acetyl phosphate.
45 . The method of claim 44 , wherein the acetate kinase is an acetate kinase of an even-numbered SEQ ID NO. of SEQ ID NOs: 2506-3058, with or without amino acid residues 1-12.
46 . The method of claim 30 , wherein the 3′-O′-kinase comprises a 3′-O′-kinase of an even-numbered SEQ ID NO. of SEQ ID NOs: 3060-3370 and 3376-5126, with or without amino acid residues 202-211.
47 - 70 . (canceled)
71 . A method of producing a nucleoside triphosphate (NTP), comprising contacting a nucleoside with a nucleoside kinase, a nucleoside monophosphate kinase, and a nucleoside diphosphate kinase in presence of a phosphate donor under reaction conditions such that NTP is produced.
72 - 86 . (canceled)
87 . The method of claim 1 , further comprising a NTP regenerating system.
88 . The method of claim 87 , wherein the NTP regenerating system comprises creatine kinase, pyruvate kinase, polyphosphate kinase, and/or R-acetate-kinase.
89 . The method of claim 88 , wherein the NTP regenerating system is pyruvate kinase and substrate phosphoenolpyruvate.
90 . The method of claim 88 , wherein the NTP regenerating system is creatine kinase and substrate creatine phosphate.
91 . The method of claim 88 , wherein the NTP regenerating system is polyphosphate kinase and substrate polyphosphate.
92 . The method of claim 88 , wherein the NTP regenerating system is R-acetate kinase and the substrate acetyl phosphate.
93 . The method of claim 92 , wherein the R-acetate kinase is same or different than the acetate kinase of any one of claims 27 , 28 , 44 , 45 , 69 , 70 , 85 , and 86 .
94 . The method of claim 92 , further comprising pyruvate oxidase and substrate pyruvate and inorganic phosphate.
95 . The method of claim 94 , further comprising a catalase.