Enzymatic synthesis of modified nucleoside triphosphate analogues

A method of enzymatic synthesis of a nucleoside triphosphate having the structure of Formula (I):

• or a salt thereof,
• the method comprising:
• (a) contacting a nucleoside monophosphate having the structure of Formula (II) or a salt thereof with a first phosphate donor in the presence of a nucleoside monophosphate (NMP) kinase to form a nucleoside diphosphate of Formula (III) or a salt thereof:
• and
• (b) reacting the nucleoside diphosphate of Formula (II) or the salt thereof with a second phosphate donor in the presence of a nucleoside diphosphate (NDP) kinase to form the nucleoside triphosphate of Formula (I) or the salt thereof:
• wherein:
• R1 is —ORa, —SRa, —NRbRc, or —BH3;
• each of R2 and R3 is independently H, C1-C6 alkyl, C1-C6 hydroxyalkyl, —C(═O)H, —C(═O)OH, —N3, —ORa, —NRbRc, or
• R4 is H, —CH2N3, —CHRaN3, —CH2—CH═CH2, —CH═CH2, —CH2—O—CH2—CH═CH2, or a 3′ hydroxy blocking group;
• R5 is H, C1-C6 alkyl, vinyl, halo, —ORe or a protected hydroxy group;
• Y is O, S or Se;
• each of Ra, Rb and Rc is independently H, or unsubstituted or substituted C1-C6 alkyl;
• Rd is independently H, halo, C1-C6 alkyl, C1-C6 haloalkyl —CN, —C(═O)H, —C(═O)OH, —SO2 (C1-C6 alkyl), —N3, —OH, or —NRbRc;
• Re is H, unsubstituted or substituted C1-C6 alkyl, —CH2N3, —CHRaN3, —CH2NRbRc, —CH2—CH═CH2, —CH═CH2, or —CH2—O—CH2—CH═CH2;
• and
• Z is a natural nucleobase or a modified natural nucleobase;
• provided that when Y is O, R1 is —OH, R5 is OH or H, then at least one of R2, R3 and R4 is not H.