Method for preparing 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphate

Abstract

The present invention relates to a method for preparing 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphates with reduced contamination with 3'-hydroxy-2'-deoxyribonucleoside-5'-triphosphate by converting 3'-(N-acetone-oxime)-2'-deoxynucleoside triphosphates into 3'-O-amine-2'-deoxynucleoside triphosphates by treatment with an aryl-hydroxylamine.

Description

[0001] In recent years, interest in enzymatic methods for polynucleotide synthesis has increased, not only because of the increased demand for synthesized polynucleotides in many fields such as synthetic biology, gene modification, high-throughput sequencing, and molecular diagnostics, but also because of the limitations of chemical methods for polynucleotide synthesis, such as the upper limit of product length and the use and required disposal of organic solvents, Jensen et al, Biochemistry, 57: 1821-1832 (2018). Enzymatic synthesis is attractive because it has specificity and efficiency, and because it uses mild aqueous reaction conditions, which eliminates the need to handle hazardous waste.

[0002] Currently, most enzymatic methods use a template-free polymerase to repeatedly add 3'-O-blocked nucleoside triphosphates to a single-stranded initiator or an extended strand attached to a support, followed by deblocking until a polynucleotide of the desired sequence is obtained, such as Jensen et al (cited above). A variety of 3'-O-blocking groups are available, but of particular interest is the use of 3'-amino blocking groups because of their small size and ease of cleavage conditions, such as U.S. Pat. Nos. 7,544,794 and 8,212,020. The synthesis of 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphates is described in Figure 1 In the scheme illustrated in, start from corresponding 2 '-deoxyribonucleoside.After protecting 5 '-hydroxyl, by twice subsequent hydroxyl conversion under Mitsunobu condition, provide O-amino moiety as N-hydroxyphthalimide moiety with "masked" (or "protected") form, it is inserted in 3 ' position (Swamy et al, Chem.Rev.109:2551-2651 (2009)).Then 3 '-O-amino and 5 '-hydroxyl of nucleoside are deprotected.However, the triphosphorylation of 5 '-hydroxyl needs to protect 3 '-O-amino again, and this is realized by converting it into oxime, and oxime is finally deprotected by processing with alkoxyamine after triphosphorylation reaction.However, unfortunately, this conventional deprotection method further causes some cracking of 3 '-O-amine. The presence of 3'-hydroxy-2'-deoxynucleoside triphosphates in the enzymatic coupling reaction in polynucleotide synthesis allows for diaddition to the growing chain to produce difficult-to-remove "n+1" failure sequences in the polynucleotide product.

[0003] In view of the above, enzymatic synthesis of polynucleotides using template-free polymerases would be advanced if methods were available for synthesizing 3'-O-amino-2'-deoxynucleoside triphosphates without the presence of 3'-hydroxy-2'-deoxynucleoside triphosphate contaminants. SUMMARY OF THE INVENTION

[0005] The present invention relates to a method for preparing 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphates with reduced 3'-hydroxy-2'-deoxyribonucleoside-5'-triphosphate contamination. The present invention also includes compositions prepared by the method of the present invention.

[0006] In some embodiments, such methods of the present invention include the following steps: (a) protecting the 5'-hydroxyl group of a 2'-deoxyribonucleoside; (b) converting the (S)-3'-hydroxyl group of the compound obtained in step (a) into the (R)-3'-hydroxyl group; (c) reacting the product of step (b) with N-hydroxyphthalimide to produce a 5'-O-protected-3'-O-phthalimido-2'-deoxynucleoside; (d) reacting the product of step (c) with N-hydroxyphthalimide to produce a 5'-O-protected-3'-O-phthalimido-2'-deoxynucleoside. (e) converting the product of step (d) into 3'-O-(N-acetone-oxime)-2'-deoxynucleoside; (f) triphosphorylating the 5'-hydroxyl group of the product of step (e) to obtain 3'-O-(N-acetone-oxime)-2'-deoxynucleoside triphosphate; and (g) converting the product of step (f) into 3'-O-amine-2'-deoxynucleoside triphosphate by treating the product of step (f) with an aryl-hydroxylamine.

[0007] In some embodiments, such compositions comprise an aqueous solution comprising 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphate and an aryl-hydroxylamine at a concentration of less than 2% by volume.

[0008] These above-characterized aspects and other aspects of the present invention are exemplified in many illustrated embodiments and applications, some of which are shown in the accompanying drawings and characterized in the claims section that follows. However, the above summary is not intended to describe each illustrated embodiment or every implementation of the present invention.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 A general synthetic scheme for preparing 3'-O-amine-2'-deoxynucleoside triphosphate monomers is illustrated.

[0011] Figure 2 An embodiment of the invention is illustrated in which benzylhydroxylamine is used to convert or deprotect 3'-acetone-oxime-nucleoside-triphosphate to 3'-ONH2-nucleoside-triphosphate.

[0012] Figure 3A is a chromatogram of the crude reaction product described in Example 1.

[0013] Figure 3B is a chromatogram of the desired 3'-O-amino-2'-deoxyguanosine-5'-triphosphate product of Example 1.

[0014] Figure 4 Examples of bases of 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphate according to the present invention are illustrated, wherein Z is the 1' carbon of 2'-deoxyribose-5'-triphosphate, R is H, CH3 or a linking group, and X is N or CR.

[0015] Detailed description of the invention

[0016] The general principles of the invention are disclosed in more detail herein, particularly by way of example, as shown in the accompanying drawings and described in detail. However, it should be understood that it is not intended to limit the invention to the specific embodiments described. The invention is applicable to various modifications and alternative forms, details of which are shown for several embodiments. It is intended to cover all modifications, equivalents and substitutes that fall within the principles and scope of the invention. .

[0017] The present invention relates to a process for preparing 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphates using the following general steps:

[0018] (a) protecting the 5'-hydroxyl group of 2'-deoxyribonucleoside;

[0019] (b) converting the (S)-3'-hydroxyl group of the compound obtained in step (a) into the (R)-3'-hydroxyl group;

[0020] (c) reacting the product of step (b) with N-hydroxyphthalimide to produce a 5'-O-protected-3'-O-phthalimido-2'-deoxynucleoside;

[0021] (d) deprotecting the 5'-hydroxyl group of the product of step (c);

[0022] (e) converting the product of step (d) into 3'-O-(N-acetone-oxime)-2'-deoxynucleoside;

[0023] (f) triphosphorylating the 5'-hydroxyl group of the product of step (e) to obtain 3'-O-(N-acetone-oxime)-2'-deoxynucleoside triphosphate; and

[0024] (g) converting the product of step (f) into 3'-O-amine-2'-deoxynucleoside triphosphates.

[0025] The term "(S)" or "(R)" preceding a group or moiety, such as the (R) in "(R)-3'-hydroxy", refers to the absolute configuration of the carbon substituted by the group. The position of the carbon may also follow the term "(S)" or "(R)". In "(R)-3'-hydroxy", the carbon at the 3' position substituted by the hydroxy group has the R absolute configuration.

[0026] The 2'-deoxynucleosides of adenine, guanine, cytosine, thymine and uracil are 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, 2'-deoxythymidine and 2'-deoxyuridine, respectively.

[0027] The above general steps and many specific embodiments are disclosed in the following references: DeClercq et al, European Patent Application EP0381335; Kondo et al, Symp. Nucleic Acids Chem., 16:93-96 (1985); Burgess et al, J. Chem. Soc. Chem. Comm., 8:915-916 (1994); Hutter et al, Nucleosides, Nucleotides & Nucleic Acids, 29 (11): 879-895 (2010); Sarac et al, International Patent Publication WO2020 / 043846; and US Patents 7544794; 8034923; 8212020; and 10472383.

[0028] According to the present invention, the 3'-O-(N-acetone-oxime)-2'-deoxynucleoside triphosphate of step (f) is converted into 3'-O-amine-2'-deoxynucleoside triphosphate by treating with an aryl-hydroxylamine, such as Figure 2As illustrated. In some embodiments, the aryl-hydroxylamine is unsubstituted or substituted O-benzylhydroxylamine or substituted or unsubstituted phenylhydroxylamine. Exemplary substituents include methoxy, nitro, halogen, wherein particularly, halogen is fluorine, chlorine or bromine. Exemplary substituted O-benzylhydroxylamines include but are not limited to O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine and O-(4-nitrobenzyl)hydroxylamine. In one embodiment, the aryl-hydroxylamine used in the method of the present invention is O-benzylhydroxylamine. In another embodiment, the aryl-hydroxylamine used in the method of the present invention is substituted or unsubstituted phenylhydroxylamine. As described above, exemplary substituents include methoxy, nitro, halogen, wherein particularly halogen is fluorine, chlorine or bromine. The conversion step (g) can be carried out using an aqueous solution of aryl-hydroxylamine. In some embodiments, pyrimidine 3'-O-(N-acetone-oxime)-2'-deoxynucleoside triphosphate is converted or deprotected to form 3'-O-amine-2'-deoxynucleoside triphosphate at pH 6.8-pH 7.0. In some embodiments, purine 3'-O-(N-acetone-oxime)-2'-deoxynucleoside triphosphate is converted or deprotected to form 3'-O-amine-2'-deoxynucleoside triphosphate at pH 6.0-pH 6.8. In some embodiments, for such conversion, a certain concentration of aryl-hydroxylamine is used, and its concentration is high enough to cause conversion but the concentration is low enough to not need to purify 3'-O-amine-2'-deoxynucleoside triphosphate before being used for polynucleotide synthesis. In some embodiments, this concentration of aryl-hydroxylamine is 1% to 100% (v / v). In some embodiments, this concentration of aryl-hydroxylamine is 1% to 2% (v / v). In other embodiments, such concentration of the aryl-hydroxylamine is less than 2% (v / v). In other embodiments, such concentration of the aryl-hydroxylamine is less than 1% (v / v).

[0029] In some embodiments, the aryl-hydroxylamines used in the methods of the present invention include substituted or unsubstituted O-benzylhydroxylamine, substituted or unsubstituted O-phenylhydroxylamine, substituted or unsubstituted O-phenylethylhydroxylamine, substituted or unsubstituted O-benzyloxyethylhydroxylamine, and substituted or unsubstituted O-phenoxyethylhydroxylamine, wherein the substituent is selected from halogen, methoxy, and nitro. In some embodiments, the halogen is fluorine, chlorine, or bromine. In some embodiments, the aryl-hydroxylamines used in the methods of the present invention include but are not limited to O-benzylhydroxylamine, O-phenylhydroxylamine, O-[(2-methoxyphenyl)methyl]hydroxylamine, O-[(3,5-dichlorophenyl)methyl]hydroxylamine, O-(3-chlorobenzyl)hydroxylamine, O-(4-nitrobenzyl)hydroxylamine, O-(4-methoxybenzyl)hydroxylamine, O-[(3,4-dichlorophenyl)methyl]hydroxylamine, O-(2-chlorobenzyl )hydroxylamine, O-[4-(trifluoromethyl)benzyl]hydroxylamine, O-[(2,5-dichlorophenyl)methyl]hydroxylamine, O-(2-methylbenzyl)hydroxylamine, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, O-(4-nitrobenzyl)hydroxylamine, O-(2-(benzyloxy)ethyl)hydroxylamine, O-(4-bromophenyl)hydroxylamine, O-[2-(trifluoromethyl)phenyl]hydroxylamine or O-(2-phenoxyethyl)hydroxylamine.

[0030] The object of the present invention is also to provide a composition comprising an aqueous solution containing 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphate and an aryl-hydroxylamine in a concentration of less than 2% by volume. In a specific embodiment, the aryl-hydroxylamine is O-benzylhydroxylamine or O-phenylhydroxylamine. In particular, the 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphate can have a base selected from adenine, thymine, guanine, cytosine, uracil, inosine, xanthine or hypoxanthine. In one embodiment, the 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphate has a base selected from adenine, thymine, guanine, cytosine, uracil, inosine, xanthine or hypoxanthine. Figure 4 Advantageously, the linking group is an alkyl linker, an alkenyl linker, a thioether linker, an aminoalkyl, an aminoallyl, an azidoalkyl, an azidoallyl propargylamine, a bispropargyl or a bispropargyl-PEG.

[0031] Example 1: Deprotection of purine nucleobase (A, G) 3'-O-amino-2'-deoxyguanosine-5'-triphosphate

[0032] To a solution of 3'-O-(N-acetone-oxime)-2'-deoxyguanosine-5'-triphosphate (5.0 mL, 5.0 mmol, 100 mM stock solution) was added water 5.0 mL and O-benzylhydroxylamine (6.2 mL, 50 mmol) and the pH was adjusted to 6.5 by adding 10% w / w aqueous acetic acid. The reaction was stirred for 6 hours. The aqueous phase was extracted with ether (3×50 mL). The aqueous phase was frozen and freeze-dried for 1 hour. Figure 3Ais a chromatogram of the crude reaction mixture after 2 hours. Excess O-benzylhydroxylamine was removed by liquid-liquid extraction with an organic solvent. The aqueous composition of 3'-ONH2-nucleoside triphosphate may contain small amounts of O-benzylhydroxylamine, sodium acetate and sodium chloride. Figure 3B The HPLC chromatogram of the 3'-O-amino-2'-deoxyguanosine-5'-triphosphate product is shown in Figure 1. The liquid-liquid extraction solvent in the examples is ether, but it can also be any organic solvent, such as methyl tert-butyl ether, dichloromethane, toluene, pentane, etc. and mixtures thereof.

[0033] Example 2: Deprotection of pyrimidine nucleobase (T,C) 3'-O-amino-2'-deoxycytidine-5'-triphosphate

[0034] To a solution of 3'-O-(N-acetone-oxime)-2'-deoxycytidine-5'-triphosphate (5.0 mL, 5.0 mmol, 100 mM stock solution) was added water 5.0 mL and O-benzylhydroxylamine (6.2 mL, 50 mmol) and the pH was adjusted to 6.8 by adding 10% w / w aqueous acetic acid. The reaction was stirred for 6 hours. The aqueous phase was extracted with ether (3×50 mL). The aqueous phase was frozen and freeze-dried for 1 hour.

Claims

1. A method for preparing 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphate, comprising the following steps: (a) protecting the 5'-hydroxyl group of 2'-deoxyribonucleoside; (b) converting the (S)-3'-hydroxyl group of the compound obtained in step (a) into the (R)-3'-hydroxyl group; (c) reacting the product of step (b) with N-hydroxyphthalimide to produce a 5'-O-protected-3'-O-phthalimido-2'-deoxynucleoside; (d) deprotecting the 5'-hydroxyl group of the product of step (c); (e) converting the product of step (d) into 3'-O-(N-acetone-oxime)-2'-deoxynucleoside; (f) triphosphorylating the 5'-hydroxyl group of the product of step (e) to obtain 3'-O-(N-acetone-oxime)-2'-deoxynucleoside triphosphate; and (g) converting the product of step (f) into a 3'-O-amine-2'-deoxynucleoside triphosphate by treating the product of step (f) with an aryl-hydroxylamine, wherein the aryl-hydroxylamine is substituted or unsubstituted O-benzylhydroxylamine, substituted or unsubstituted O-phenylhydroxylamine, substituted or unsubstituted O-phenylethylhydroxylamine, substituted or unsubstituted O-benzyloxyethylhydroxylamine and substituted or unsubstituted O-phenoxyethylhydroxylamine, wherein the aryl-hydroxylamine is substituted by halogen, methoxy or nitro, and wherein the 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphate has a base selected from: wherein Z is the 1' carbon of 2'-deoxyribose-5'-triphosphate, R is H, CH3 or a linking group, and X is N or CR.

2. The method of claim 1, wherein the aryl-hydroxylamine is selected from the group consisting of O-benzylhydroxylamine, O-phenylhydroxylamine, O-[(2-methoxyphenyl)methyl]hydroxylamine, O-[(3,5-dichlorophenyl)methyl]hydroxylamine, O-(3-chlorobenzyl)hydroxylamine, O-(4-nitrobenzyl)hydroxylamine, O-(4-methoxybenzyl)hydroxylamine, O-[(3,4-dichlorophenyl)methyl]hydroxylamine, O-(2-chlorobenzyl) Hydroxylamine, O-[4-(trifluoromethyl)benzyl]hydroxylamine, O-[(2,5-dichlorophenyl)methyl]hydroxylamine, O-(2-methylbenzyl)hydroxylamine, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, O-(4-nitrobenzyl)hydroxylamine, O-(2-(benzyloxy)ethyl)hydroxylamine, O-(4-bromophenyl)hydroxylamine, O-[2-(trifluoromethyl)phenyl]hydroxylamine, and O-(2-phenoxyethyl)hydroxylamine.

3. The method of claim 2, wherein the aryl-hydroxylamine is O-benzylhydroxylamine or O-phenylhydroxylamine.

4. The method of claim 1, wherein the linking group is an alkyl linker, an alkenyl linker, a thioether linker, an aminoalkyl, an aminoallyl, an azidoalkyl, an azidoallyl propargylamine, a bispropargyl, or a bispropargyl-PEG.

5. A composition comprising an aqueous solution containing 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphate and an aryl-hydroxylamine at a concentration of less than 2% by volume, wherein the aryl-hydroxylamine is substituted or unsubstituted O-benzylhydroxylamine, substituted or unsubstituted O-phenylhydroxylamine, substituted or unsubstituted O-phenylethylhydroxylamine, substituted or unsubstituted O-benzyloxyethylhydroxylamine, and substituted or unsubstituted O-phenoxyethylhydroxylamine, wherein the aryl-hydroxylamine is substituted with halogen, methoxy or nitro, and wherein the 3'-O-amino-2'-deoxyribonucleoside-5'-triphosphate has a base selected from the group consisting of: wherein Z is the 1' carbon of 2'-deoxyribose-5'-triphosphate, R is H, CH3 or a linking group, and X is N or CR.

6. The composition of claim 5, wherein the aryl-hydroxylamine is selected from the group consisting of O-benzylhydroxylamine, O-phenylhydroxylamine, O-[(2-methoxyphenyl)methyl]hydroxylamine, O-[(3,5-dichlorophenyl)methyl]hydroxylamine, O-(3-chlorobenzyl)hydroxylamine, O-(4-nitrobenzyl)hydroxylamine, O-(4-methoxybenzyl)hydroxylamine, O-[(3,4-dichlorophenyl)methyl]hydroxylamine, O-(2-chlorobenzyl) Hydroxylamine, O-[4-(trifluoromethyl)benzyl]hydroxylamine, O-[(2,5-dichlorophenyl)methyl]hydroxylamine, O-(2-methylbenzyl)hydroxylamine, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, O-(4-nitrobenzyl)hydroxylamine, O-(2-(benzyloxy)ethyl)hydroxylamine, O-(4-bromophenyl)hydroxylamine, O-[2-(trifluoromethyl)phenyl]hydroxylamine, and O-(2-phenoxyethyl)hydroxylamine.

7. The composition of claim 5, wherein the aryl-hydroxylamine is O-benzylhydroxylamine or O-phenylhydroxylamine.

8. The composition of claim 5, wherein the linking group is an alkyl linker, an alkenyl linker, a thioether linker, an aminoalkyl, an aminoallyl, an azidoalkyl, an azidoallyl propargylamine, a bispropargyl, or a bispropargyl-PEG.

Images