Compositions and methods related to non-templated enzymatic nucleic acid synthesis

Abstract

The invention relates to the use of an amine masked moiety in a method of enzymatic nucleic acid synthesis. The invention also relates to said amine masked moieties per se and a process for preparing nucleotide triphosphates comprising said amine masked moieties.

Claims

1. A method of non-templated enzymatic nucleic acid synthesis comprising: (i) providing a compound of formula (I): ##STR00056## wherein: R.sup.1 represents —ONH.sub.2, —ONC(CH.sub.3).sub.2, —OCH.sub.2N.sub.3, —OCH.sub.2CHCH.sub.2, —OPO.sub.3.sup.2−, —OCH.sub.2SSCH.sub.2CH.sub.3, —OCOCH.sub.2, —OCH.sub.2CH.sub.2CN, —O-methoxyethyl; R.sup.2 represents —H, X represents a triphosphate group; R.sup.3 represents an amine masking group selected from an azide, benzoylamine, isobutyrylamine, dimethylformamidylamine, 9-fluorenylmethyl carbamate, t-butyl carbamate, benzyl carbamate, acetamide, trifluoroacetamide, pthlamide, benzylamine, triphenylmethylamine, benxylideneamine, tosylamide, isothiocyanate, N-allyl or N-anisoyl; and B represents a nitrogenous heterocycle selected from a purine or pyrimidine; (ii) incorporating the compound of formula (I) into a nucleic acid molecule; (iii) repeating steps (i) and (ii) until a desired nucleic acid sequence having the amine masking group is synthesized; and (iv) unmasking the R.sup.3 amine masking group to reveal an amino(—NH.sub.2) group.

2. The method of claim 1, wherein the compound of formula (I) is selected from: ##STR00057## where R.sub.2 represents —H.

3. The method of claim 1, wherein R.sup.1 represents —ONH.sub.2, —ONC(CH.sub.3).sub.2, or —OCH.sub.2N.sub.3.

4. A compound of formula (I).sup.a: ##STR00058## wherein: R.sup.1 represents —ONH.sub.2, —ONC(CH.sub.3).sub.2 or —OCH.sub.2N.sub.3; R.sup.2 represents —H; X represents a triphosphate group; R.sup.3 represents azide, benzoyl amine, isobutyrylamine, dimethylformamidylamine, 9-fluorenylmethyl carbamate, t-butyl carbamate, benzyl carbamate, acetamide, trifluoroacetamide, pthlamide, benzylamine, triphenylmethylamine, benxylideneamine, tosylamide, isothiocyanate, N-allyl or N-anisoyl, and B represents a nitrogenous heterocycle selected from a purine or pyrimidine.

5. The method of claim 1, wherein the unmasking comprises unmasking with a reducing agent.

6. The method of claim 5, wherein the reducing agent is selected from beta-mercaptoethanol, dithiothreitol or a phosphine-based reducing agent.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: A time course showing the extent of deamination of 2′-deoxyadenosine (1 mM) in the presence of sodium nitrite (700 mM), sodium acetate, pH 5.5 (1 M) at room temperature. (A) LC-MS was performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-40% B in 10 minutes was run at 0.5 mL/min. Data is shown as a series of LC chromatograms at 260 nm. The ˜2.6 min peak corresponds to 2′-deoxyadenosine and the peak at ˜1.3 min corresponds to 2′-deoxyinosine (oxidative deamination product). (B) Plot of deamination percent over time. (C) Oxidative deamination reaction shown below converting 2′-deoxyadenosine to 2′deoxyinosine.

(2) FIG. 2: Time course of the extent of deamination of N6-azido 2′-deoxyadenosine (0.5 mM) in the presence of sodium nitrite (700 mM), sodium acetate, pH 5.5 (1 M) at room temperature as analyzed by LC-MS performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-40% B in 10 minutes was run at 0.5 mL/min. Data shown as a series of LC chromatograms at two wavelengths for each time point as labeled above. The peak at ˜4.5 min retention time corresponds with 6-azido 2′-deoxyadenosine. There is a notable absence of peaks at ˜2.6 min (2′-deoxyadenosine) or ˜1.3 min (2′-deoxyinosine; oxidative deamination product).

(3) FIG. 3: Exposure of 6-azido 2′-deoxyadenosine (4.5 min retention time) to TCEP results in quantitative conversion to 2′-deoxyadenosine (2.6 min retention time) as analyzed by LC-MS. Analysis was performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-40% B in 10 minutes was run at 0.5 mL/min.

(4) FIG. 4: A time course showing the extent of deamination of 2′-deoxycytosine (1 mM) in the presence of sodium nitrite (700 mM), sodium acetate, pH 5.5 (1 M) at room temperature. (A) LC-MS was performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-25% B in 10 minutes was run at 0.4 mL/min. Data is shown as a series of LC chromatograms at 260 nm. The ˜1.05 min peak corresponds to 2′-deoxycytidine and the peak at ˜1.15 min corresponds to 2′-deoxyuridine (oxidative deamination product). (B) Plot of deamination percent over time. (C) Oxidative deamination reaction shown below converting 2′-deoxycytidine (dC) to 2′-deoxyuridine (dU).

(5) FIG. 5: Extent of deamination of N4-azido 2′-deoxycytidine (N4-azido dC; 1 mM) in the presence of sodium nitrite (700 mM), sodium acetate, pH 5.5 (1 M) at room temperature as analyzed by LC-MS performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-25% B in 10 minutes was run at 0.4 mL/min. (A) Trace showing initial compound with mass of the primary peak shown, confirming the identity of the compound at ˜2.7 min as N4-azido dC. (B) Time course of N4-azido dC incubated with nitrite solution as described above. Data shown as a series of LC chromatograms at 260 nm. There is a notable absence of peaks corresponding to dC (reduction product) or dU (deamination product), which can be seen in the first trace from a dC standard incubated with nitrite solution. (C) N4-azido dC can be easily converted to dC by treatment with a reducing agent. Here, incubation with TCEP led to quantitative conversion of N4-azido dC to dC.

(6) FIG. 6: Extent of deamination of N4-acetyl 2′-deoxycytidine (N4-acetyl dC) in the presence of sodium nitrite (700 mM), sodium acetate, pH 5.5 (1 M) at room temperature as analyzed by LC-MS performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-25% B in 10 minutes was run at 0.4 mL/min. (A) Trace showing initial compound with mass of the primary peak shown, confirming the identity of the compound at ˜2.1 min as N4-acetyl dC. (B) Time course of N4-acetyl dC incubated with nitrite solution as described above. Data shown as a series of LC chromatograms at 260 nm. There is a notable reduction in the peaks corresponding to dC (reduction product) or dU (deamination product), when compared to a dC standard incubated with nitrite solution (see FIG. 4). (C) N4-acetyl dC can be easily converted to dC. One such method involves treatment with aqueous 40% methylamine, which can be seen to result in loss of the N4-acetyl dC peak at 2.1 min and appearance of the dC peak at 1.05 minutes, as confirmed by mass in panel (D).

(7) FIG. 7: Extent of deamination of N6-acetyl adenosine (N6-acetyl A) in the presence of sodium nitrite (700 mM), sodium acetate, pH 5.5 (1 M) at room temperature as analyzed by LC-MS performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-25% B in 10 minutes was run at 0.4 mL/min. (A) Trace showing initial compound with mass of the primary peak shown, confirming the identity of the compound at ˜1.8 min as N6-acetyl A. (B) Time course of N6-acetyl A incubated with nitrite solution as described above. Data shown as a series of LC chromatograms at 260 nm. While minor deacetylation is seen at 48 hours, no deamination product is detected. (C) N6-acetyl A can be easily converted to dC. One such method involves treatment with aqueous 40% methylamine, which can be seen to result in loss of the N6-acetyl A peak at 1.8 min and appearance of the A peak at 1.9 minutes, as confirmed with the extracted mass traces corresponding to the acetylated (310.50 extraction) and deacetylated (268.00 extraction) compound. (D) Another method to deacetylate N6-acetyl A is with potassium carbonate. Here, treatment with 50 mM aqueous potassium carbonate at room temperature afforded the deacetylated compound, again proven with the disappearance of the 310.50 peak and appearance of the 268.00 peak.

(8) FIG. 8: Extent of deamination of N6-benzyl 2′-deoxyadenosine (N6-benzyl dA; 1 mM) in the presence of sodium nitrite (700 mM), sodium acetate, pH 5.5 (1 M) at room temperature as analyzed by LC-MS performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-25% B in 10 minutes followed by a hold for 5 minutes was run at 0.4 mL/min. (A) Trace showing initial compound with mass of the primary peak shown, confirming the identity of the compound at ˜10.8 min as N6-benzyl dA. (B) Time course of N6-benzyl dC incubated with nitrite solution as described above. Data shown as a series of LC chromatograms at 260 nm. There is a notable absence of peaks corresponding to dA (debenzylated product) or deoxyinosine (dI; deamination product).

(9) FIG. 9: Extent of deamination of N4-anisoyl 2′-deoxycytidine (N4-anisoyl dC) in the presence of sodium nitrite (700 mM), sodium acetate, pH 5.5 (1 M) at room temperature as analyzed by LC-MS performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-25% B in 10 minutes was run at 0.4 mL/min. (A) Trace showing initial compound with mass of the primary peak shown, confirming the identity of the compound at ˜9.8 min as N4-anisoyl dC. (B) Time course of N4-anisoyl dC incubated with nitrite solution as described above. Data shown as a series of LC chromatograms at 260 nm. There is a notable reduction in the peaks corresponding to dC (reduction product) or dU (deamination product), when compared to a dC standard incubated with nitrite solution (see FIG. 4). (C) N4-anisoyl dC can be easily converted to dC. One such method involves treatment with aqueous 40% methylamine, which can be seen to result in loss of the N4-anisoyl dC peak at ˜9.8 min and appearance of the dC peak at 1.05 minutes, as confirmed with the extracted mass traces. (D) Another method to deacylate N4-anisoyl dC to dC involves treatment with potassium carbonate. Here, treatment with 50 mM aqueous potassium carbonate at room temperature afforded the deacylated compound dC.

(10) FIG. 10: Extent of deamination of N6-dimethylallylamino purine in the presence of sodium nitrite (700 mM), sodium acetate, pH 5.5 (1 M) at room temperature as analyzed by LC-MS performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-25% B in 10 minutes was run at 0.4 mL/min. Time course of N6-dimethylallylamino purine incubated with nitrite solution as described above. Data shown as a series of LC chromatograms at 260 nm. There is a notable absence of peaks corresponding to dA or dI.

(11) FIG. 11: Enzymatic incorporation of nucleoside 5′-triphosphates with amine-masking moieties. An engineered terminal deoxynucleotidyl transferase was incubated for 10 minutes at 37° C. in an appropriate buffer containing cobalt chloride and a DNA primer with a 5′-fluorophore as well as the following nucleoside 5′-triphosphates: (A) Lane 1: No extension control; Lane 2: N6-benzyl-dATP; Lane 3: N6-benzyl-rATP; Lane 4: N6-methyl-dATP; Lane 5: N6-methyl-rATP; (B) Lane 1: No extension control; Lane 2: 3′-O-acetyl-N4-benzoyl-dCTP; (C) Lane 1: No extension control; Lane 3: N6-benzoyl-dATP; Lane 4: N4-benzoyl-dCTP. Reactions were analyzed by standard denaturing polyacrylamide gel electrophoresis (TBE buffer) and imaged with a fluorescent scanner.

(12) FIG. 12: Extent of deamination of 3′-azido N4-benzoyl 2′-deoxycytidine (1 mM) in the presence of sodium nitrite (700 mM), sodium acetate, pH 5.5 (1 M) at room temperature as analyzed by LC-MS performed on a Bruker amaZon system, with a Synergi Polar RP column. Solvents were A (20 mM ammonium acetate, pH 4.6) and B (20 mM ammonium acetate, pH 4.6 [5%]/acetonitrile [95%]). A gradient from 5-25% B in 10 minutes followed by a hold for 5 minutes was run at 0.4 mL/min. (A) Trace showing initial compound with mass of the primary peak shown, confirming the identity of the compound at ˜12.5 min as 3′-azido N4-benzoyl 2′-deoxycytidine. (B) Time course of 3′-azido N4-benzoyl 2′-deoxycytidine incubated with nitrite solution as described above. Data shown as a series of LC chromatograms at 260 nm. There is a notable absence of peaks corresponding to dc (deacylatedproduct) or deoxyinosine (deamination product)—which can be seen in the bottom trace showing a dC incubation with nitrite. (C) Treatment of 3′-azido N4-benzoyl 2′-deoxycytidine with potassium carbonate yields 3′-azido dC.

DETAILED DESCRIPTION OF THE INVENTION

(13) According to a first aspect of the invention, there is provided the use of an amine masked derivative of a nitrogenous heterocycle, such as adenine, guanine, cytosine, isoguanine, isocytosine and 2,6-diaminopurine in a method of enzymatic nucleic acid synthesis.

(14) According to a further aspect of the invention which may be mentioned, there is provided the use of an amine masked derivative of a nitrogenous heterocycle, such as adenosine, guanosine, and cytidine, in a method of enzymatic nucleic acid synthesis.

(15) References herein to a derivative of adenosine, guanosine and cytidine refer to deoxy derivatives thereof (i.e. deoxyadenosine, deoxyguanosine and deoxycytidine) and the phosphated derivatives thereof (i.e. adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, guanosine monophosphate, guanosine diphosphate, guanosine triphosphate, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate and all the deoxyribose versions thereof).

(16) According to a further aspect of the invention, there is provided the use of a compound of formula (I):

(17) ##STR00008##
wherein:
R.sup.1 represents a moiety capable of being unmasked to reveal a hydroxyl group, including —H, —OH, —ONH.sub.2, —ONC(CH.sub.3).sub.2, —OCH.sub.2N.sub.3, —OCH.sub.2CHCH.sub.2, —OPO.sub.3.sup.2−, —OCH.sub.2SSCH.sub.2CH.sub.3, —OCOCH.sub.3, —OCH.sub.2CH.sub.2CN, —O-methoxyethyl, —O-alkyl, or —O-alkoxy;
R.sup.2 represents —H, —OH, —ONH.sub.2, —ONC(CH.sub.3).sub.2, —OCH.sub.2N.sub.3, —OCH.sub.2CHCH.sub.2, —OPO.sub.3.sup.2−, —OCH.sub.2SSCH.sub.2CH.sub.3, —OCOCH.sub.3, —OCH.sub.2CH.sub.2CN, —O-methoxyethyl, —O-alkyl, or —O-alkoxy or any other molecular moiety;
X represents an —OH group or one or more phosphate, phosphorothioate, boranophosphate or imidophosphate groups, or any combination thereof, wherein said group is capable of endowing competence for enzymatic addition;
R.sup.3 represents an amine masking group, wherein said amino group would be involved in hydrogen bond base-pairing with a complementary base and deamination of said amino group could result in altered hydrogen bonding with a complementary base; and
B represents a nitrogenous heterocycle;
in a method of enzymatic nucleic acid synthesis.

(18) According to a further aspect of the invention which may be mentioned, there is provided the use of a compound of formula (I):

(19) ##STR00009##
wherein:
R.sup.1 and R.sup.2 independently represent —H, —OH, —ONH.sub.2, —ONC(CH.sub.3).sub.2, —OCH.sub.2N.sub.3, —OCH.sub.2CHCH.sub.2, —O-methoxyethyl, —O-alkyl, —O-alkoxy, cyanoethyl, a thiol or a suitable hydroxy protecting group;
X represents an —OH group or one or more phosphate, phosphorothioate, boranophosphate or imidophosphate groups, or any combination thereof;
R.sup.3 represents an amine masking group, wherein said amino group is involved in hydrogen bond base-pairing with a complementary base; and
B represents a nitrogenous heterocycle;
in a method of enzymatic nucleic acid synthesis.

(20) Enzymatic nucleic acid synthesis is defined as any process in which a nucleotide is added to a nucleic acid strand through enzymatic catalysis in the presence or absence of a template.

(21) For example, a method of enzymatic nucleic acid synthesis could include non-templated de novo nucleic acid synthesis utilizing a PoIX family polymerase, such as terminal deoxynucleotidyl transferase, and reversibly terminated 2′-deoxynucleoside 5′-triphosphates or ribonucleoside 5′-triphosphate. Another method of enzymatic nucleic acid synthesis could include templated nucleic acid synthesis, including sequencing-by-synthesis. Reversibly terminated enzymatic nucleic acid synthesis is defined as any process in which a reversibly terminated nucleotide is added to a nucleic acid strand through enzymatic catalysis in the presence or absence of a template. A reversibly terminated nucleotide is a nucleotide containing a chemical moiety that blocks the addition of a subsequent nucleotide. The deprotection or removal of the reversibly terminating chemical moiety on the nucleotide by chemical, electromagnetic, electric current, and/or heat allows the addition of a subsequent nucleotide via enzymatic catalysis. Thus, in one embodiment, the method of enzymatic nucleic acid synthesis is selected from a method of reversibly terminated enzymatic nucleic acid synthesis and a method of templated and non-templated de novo enzymatic nucleic acid synthesis.

(22) The compound of formula (I) contains three synergistic components which may be summarized as follows: (i)—The R.sup.3 group. R.sup.3 is typically a chemical moiety on the nitrogenous heterocycle that can be unmasked to reveal an amino (—NH.sub.2) group; (ii)—The R.sup.1 group. R.sup.1 is typically a chemical moiety at the 3′-position on the sugar that can be unmasked to reveal a hydroxyl (—OH) group; and (iii)—The X group. X is typically a chemical moiety endowing competence for enzymatic addition (e.g., 5′-triphosphate group).

(23) Without being bound by theory, it is believed that the combination of R.sup.1, R.sup.3 and X result in nucleotide analogs that protect the amino group in component (i) from mutation during the method of enzymatic nucleic acid synthesis described herein. Specifically, a method of enzymatic nucleic acid synthesis would involve nucleotide analogs that have characteristic R.sup.3, X, and R.sup.1, where R.sup.1 is fixed as an —ONH.sub.2 group.

(24) In one embodiment, R.sup.1 and R.sup.2 independently represent —H, —OH, —ONH.sub.2, —ONC(CH.sub.3).sub.2, —OCH.sub.2N.sub.3, —OCH.sub.2CHCH.sub.2, —OPO.sub.3.sup.2−, —OCH.sub.2SSCH.sub.2CH.sub.3, —OCOCH.sub.3, —OCH.sub.2CH.sub.2CN, —O-methoxyethyl, —O-alkyl, or —O-alkoxy or a suitable hydroxyl protecting group.

(25) In one embodiment, the compound of formula (I) is selected from:

(26) ##STR00010##
where R.sup.2 is as defined herein, such as —OH or —H. In one embodiment, R.sup.2 is H.

(27) In one embodiment, the compound of formula (I) is a compound of formula (I).sup.b:

(28) ##STR00011##

(29) The compound of formula (I).sup.b is known chemically as N6-azido 2′-deoxyadenosine. Upon exposure of the compound of formula (I).sup.b to sodium nitrite, no conversion to 2′-deoxyinosine was observed, as shown in FIG. 2. Conveniently, upon exposure to TCEP or another reducing agent, the compound of formula (I).sup.b is easily converted to 2′-deoxyadenosine, as shown in FIG. 3.

(30) In one embodiment, the compound of formula (I) is a compound of formula (I).sup.b:

(31) ##STR00012##

(32) The compound of formula (I).sup.c is known chemically as N4-azido 2′-deoxycytidine. Upon exposure of the compound of formula (I).sup.c to sodium nitrite, no conversion to 2′-deoxyuracil was observed, as shown in FIG. 4. Conveniently, upon exposure to TCEP or another reducing agent, the compound of formula (I).sup.c is easily converted to 2′-deoxycytidine, as shown in FIG. 5. Thus, the present invention provides the advantage of providing a solution to the problem of oxidative deamination of adenine, guanine and cytosine in the presence of reagents such as sodium nitrite under acidic conditions.

(33) In one embodiment, the compound of formula (I) is a compound of formula (I).sup.d:

(34) ##STR00013##

(35) The compound of formula (I).sup.d is known chemically as N6-acetyl 2′-deoxyadenosine. Upon exposure of the compound of formula (I).sup.d to sodium nitrite, no conversion to 2′-deoxyinosine was observed, as shown in FIG. 7. Conveniently, upon exposure to 40% aqueous methylamine, or potassium carbonate, or ammonium hydroxide, or ammonia (for instance in ethanol) or other appropriate reagents, the compound of formula (I).sup.d is easily converted to 2′-deoxyadenosine, as shown in FIG. 7.

(36) In one embodiment, the compound of formula (I) is a compound of formula (I).sup.e:

(37) ##STR00014##

(38) The compound of formula (I).sup.e is known chemically as N4-acetyl 2′-deoxycytidine. Upon exposure of the compound of formula (I).sup.e to sodium nitrite, no conversion to 2′-deoxyuracil was observed, as shown in FIG. 6. Conveniently, upon exposure to methylamine or other bases, the compound of formula (I).sup.e is easily converted to 2′-deoxycytidine, as shown in FIG. 6. Thus, the present invention provides the advantage of providing a solution to the problem of oxidative deamination of adenine, guanine and cytosine in the presence of reagents such as sodium nitrite under acidic conditions.

(39) In one embodiment, the compound of formula (I) is a compound of formula (I).sup.f:

(40) ##STR00015##

(41) The compound of formula (I).sup.f is known chemically as N6-benzyl 2′-deoxyadenosine. Upon exposure of the compound of formula (I).sup.f to sodium nitrite, no conversion to 2′-deoxyinosine was observed, as shown in FIG. 8. Conveniently, upon treatment with hydrogen in the presence of a suitable catalyst (such as palladium or nickel), the compound of formula (I).sup.f is easily converted to 2′-deoxyadenosine. The triphosphate form of species (I).sup.f can act as a substrate for terminal transferase enzymes in a DNA synthesis process as shown in FIG. 11. Thus, the present invention provides a solution to the problem of oxidative deamination and offers utility in a method of enzymatic DNA synthesis.

(42) In one embodiment, the compound of formula (I) is a compound of formula (I).sup.g:

(43) ##STR00016##

(44) The compound of formula (I).sup.g is known chemically as N4-anisoyl 2′-deoxycytidine. Upon exposure of the compound of formula (I).sup.g to sodium nitrite, no conversion to 2′-deoxyinosine was observed, as shown in FIG. 9. Conveniently, upon treatment with methylamine or potassium carbonate, or another suitable base or reagent, the compound of formula (I).sup.g is easily converted to 2′-deoxycytidine as shown in FIG. 9. The identical chemistry is appropriate for benzoyl moieties, as found in N4-benzoyl 2′-deoxycytidine and N6-benzoyl 2′-deoxyadenosine.

(45) In one embodiment, the compound of formula (I) is a compound of formula (I).sup.h:

(46) ##STR00017##

(47) The compound of formula (I).sup.h is known chemically as N4-methyl 2′-deoxycytidine. As shown here, secondary amines are protected from oxidative deamination induced by nitrite solutions. Thus N-methyl would be an appropriate protecting group. Exocyclic N-methyl can be conveniently removed by treatment with demethylating enzymes such as AlkB (D. Li, et al., Chem. Res. Toxicol. 26 (2013) 1182-1187).

(48) In one embodiment, the compound of formula (I) is a compound of formula (I).sup.i:

(49) ##STR00018##

(50) The compound of formula (I).sup.i is known chemically as N6-methyl 2′-deoxyadenosine. As shown here, secondary amines are protected from oxidative deamination induced by nitrite solutions. Thus N-methyl would be an appropriate protecting group. Exocyclic N-methyl can be conveniently removed by treatment with demethylating enzymes such as AlkB (D. Li, et al., Chem. Res. Toxicol. 26 (2013) 1182-1187). The triphosphate form of species (I).sup.i can act as a substrate for terminal transferase enzymes in a DNA synthesis process as shown in FIG. 11. Thus, the present invention provides a solution to the problem of oxidative deamination and offers utility in a method of enzymatic DNA synthesis.

(51) In one embodiment, the compound of formula (I) is a compound of formula (I).sup.j:

(52) ##STR00019##

(53) The compound of formula (I).sup.j is known chemically as 3′-azido N4-benzoyl 2′-deoxycytidine. Upon exposure of the compound of formula (I).sup.j to sodium nitrite, no conversion to 3′-azido 2′-deoxyinosine was observed, as shown in FIG. 12. Conveniently, upon treatment with methylamine or potassium carbonate, or another suitable base or reagent, the compound of formula (I).sup.j is easily converted to 3′-azido 2′-deoxycytidine as shown in FIG. 12. The triphosphate species of a closely related compound to (I).sup.j, N4-benzoyl 2′-deoxycytidine triphosphate, is accepted as a substrate by terminal transferase enzyme in a DNA synthesis process as shown in FIG. 11. Thus, the present invention provides a solution to the problem of oxidative deamination and offers utility in a method of enzymatic DNA synthesis.

(54) In one embodiment, X represents an —OH group. In an alternative embodiment, X represents a triphosphate group. The triphosphate group of this embodiment has the advantage of being most commonly utilized with nucleotidyl transferases (e.g., polymerases) or any chemical moieties allowing addition to a nucleic acid molecule through enzymatic or chemical catalysis.

(55) References herein to “amine” refer to a —NH.sub.2 group.

(56) References herein to an “amine masking group” refer to any chemical group which is capable of generating or “unmasking” an amine group which is involved in hydrogen bond base-pairing with a complementary base. Most typically the unmasking will follow a chemical reaction, most suitably a simple, single step chemical reaction. In one embodiment, the hydrogen bond base-pairing is selected from: Watson-Crick, Hoogsteen, or alternative/expanded genetic code base pairing.

(57) Examples of suitable amine masking groups for R.sup.3 include azide (—N.sub.3), benzoylamine (N-benzoyl or —NHCOPh), N-methyl (—NHMe), isobutyrylamine, dimethylformamidylamine, 9-fluorenylmethyl carbamate, t-butyl carbamate, benzyl carbamate, acetamide (N-acetyl or —NHCOMe), trifluoroacetamide, pthlamide, benzylamine (N-benzyl or —NH—CH.sub.2-phenyl), triphenylmethylamine, benxylideneamine, tosylamide, isothiocyanate, N-allyl (such as N-dimethylallyl (—NHCH.sub.2—CH═CH.sub.2)) and N-anisoyl (—NHCOPh-OMe), such as azide (—N.sub.3), N-acetyl (—NHCOMe), N-benzyl (—NH—CH.sub.2-phenyl), N-anisoyl (—NHCOPh-OMe), N-methyl, (—NHMe), N-benzoyl (—NHCOPh), N-dimethylallyl (—NHCH.sub.2—CH═CH.sub.2).

(58) In one embodiment, B represents a nitrogenous heterocycle selected from a purine or pyrimidine, or derivative thereof. In a further embodiment, B and R3 can be combined into the following molecular structures, where the nitrogenous heterocycle is connected to the (deoxy)ribose 1′ position of the compound of formula (I):

(59) ##STR00020##

(60) In a further embodiment, R.sup.3 represents an azide (—N.sub.3) group and B is selected from:

(61) ##STR00021##

(62) The term ‘azide’ or ‘azido’ used herein refers to an —N.sub.3, or more specifically, an —N═N.sup.+═N.sup.− group. It will also be appreciated that azide extends to the presence of a tetrazolyl moiety. The “azide-tetrazole” equilibrium is well known to the skilled person from Lakshman et al (2010) J. Org. Chem. 75, 2461-2473. Thus, references herein to azide extend equally to tetrazole as illustrated below when applied to the R.sup.3 groups defined herein:

(63) ##STR00022##

(64) This embodiment has the advantage of reversibly masking the —NH.sub.2 group. While blocked in the —N.sub.3 state, the base (B) is impervious to deamination (e.g., deamination in the presence of sodium nitrite). The canonical cytosine, adenine, guanine can be respectively recovered from 4-azido cytosine, 6-azido adenine and 2-azido guanine by exposure to a reducing agent (e.g., TCEP). Thus, the —N.sub.3 group serves as an effective protecting group against deamination, especially in the presence of sodium nitrite.

(65) It will be appreciated that the compounds of the invention may be readily applied to methods of enzymatic nucleic acid synthesis which are well known to the person skilled in the art.

(66) Non-limiting methods of nucleic acid synthesis may be found in WO 2016/128731, WO 2016/139477, WO 2017/009663, GB 1613185.6 and GB 1714827.1, the contents of each of which are herein incorporated by reference.

(67) According to a further aspect of the invention, there is provided a compound of formula (I).sup.a:

(68) ##STR00023##
wherein:
R.sup.1 represents a moiety capable of being unmasked to reveal a hydroxyl group, including —H, —OH, —ONH.sub.2, —ONC(CH.sub.3).sub.2, —OCH.sub.2N.sub.3, —OCH.sub.2CHCH.sub.2, —OPO.sub.3.sup.2−, —OCH.sub.2SSCH.sub.2CH.sub.3, —OCOCH.sub.3, —OCH.sub.2CH.sub.2CN, —O-methoxyethyl, —O-alkyl, or —O-alkoxy;
R.sup.2 represents —H, —OH, —ONH.sub.2, —ONC(CH.sub.3).sub.2, —OCH.sub.2N.sub.3, —OCH.sub.2CHCH.sub.2, —OPO.sub.3.sup.2−, —OCH.sub.2SSCH.sub.2CH.sub.3, —OCOCH.sub.3, —OCH.sub.2CH.sub.2CN, —O-methoxyethyl, —O-alkyl, or —O-alkoxy or any other molecular moiety;
X represents one or more phosphate, phosphorothioate, boranophosphate or imidophosphate groups, or any combination thereof, wherein said group is capable of endowing competence for enzymatic addition;
R.sup.3 represents an amine masking group, wherein said amino group would be involved in hydrogen bond base-pairing with a complementary base and deamination of said amino group could result in altered hydrogen bonding with a complementary base; and
B represents a nitrogenous heterocycle.

(69) According to a further aspect of the invention which may be mentioned, there is provided a compound of formula (I).sup.a:

(70) ##STR00024##
wherein:
R.sup.1 and R.sup.2 independently represent —H, —OH, —ONH.sub.2, —ONC(CH.sub.3).sub.2, —OCH.sub.2N.sub.3, —OCH.sub.2CHCH.sub.2, —O-methoxyethyl, —O-alkyl, —O-alkoxy, cyanoethyl, a thiol or a suitable hydroxy protecting group;
X represents one or more phosphate, phosphorothioate, boranophosphate or imidophosphate groups, or any combination thereof;
R.sup.3 represents an amine masking group, wherein said amino group is involved in hydrogen bond base-pairing with a complementary base; and
B represents a nitrogenous heterocycle.

(71) In one embodiment, X represents a triphosphate group.

(72) In one embodiment, R.sup.1 and R.sup.2 independently represent —H, —OH, —ONH.sub.2, —ONC(CH.sub.3).sub.2, —OCH.sub.2N.sub.3, —OCH.sub.2CHCH.sub.2, —OPO.sub.3.sup.2−, —OCH.sub.2SSCH.sub.2CH.sub.3, —OCOCH.sub.3, —OCH.sub.2CH.sub.2CN, —O-methoxyethyl, —O-alkyl, or —O-alkoxy.

(73) In an alternative embodiment, R.sup.1 and R.sup.2 independently represent —H, —OH, —ONH.sub.2, —N.sub.3, —OCH.sub.2N.sub.3, —ONC(CH.sub.3).sub.2, —OCH.sub.2CHCH.sub.2, —O-methoxyethyl, —O-alkyl, —O-alkoxy, cyanoethyl, a thiol or a suitable hydroxy protecting group.

(74) Examples of suitable amine masking groups for R.sup.3 include azide (—N.sub.3), benzoylamine (N-benzoyl or —NHCOPh), N-methyl, (—NHMe), isobutyrylamine, dimethylformamidylamine, 9-fluorenylmethyl carbamate, t-butyl carbamate, benzyl carbamate, acetamide (N-acetyl or —NHCOMe), trifluoroacetamide, pthlamide, benzylamine (N-benzyl or —NH—CH.sub.2-phenyl), triphenylmethylamine, benxylideneamine, tosylamide, isothiocyanate, N-allyl (such as N-dimethylallyl (—NHCH.sub.2—CH═CH.sub.2)) and N-anisoyl (—NHCOPh-OMe), such as azide (—N.sub.3), N-acetyl (—NHCOMe), N-benzyl (—NH—CH.sub.2-phenyl), N-anisoyl (—NHCOPh-OMe), N-methyl, (—NHMe), N-benzoyl (—NHCOPh), N-dimethylallyl (—NHCH.sub.2—CH═CH.sub.2).

(75) In one embodiment, B represents a nitrogenous heterocycle selected from a purine or pyrimidine. In a further embodiment, B and R3 can be combined into the following molecular structures, where the nitrogenous heterocycle is connected to the (deoxyribose) 1′ position of the compound of formula (I):

(76) ##STR00025##

(77) In one embodiment, R.sup.3 represents an azide (—N.sub.3) group and B is selected from:

(78) ##STR00026##

(79) One particular compound of formula (I).sup.a which may be mentioned (1) is one wherein R.sup.1 represents —ONH.sub.2, R.sup.2 represents H, X represents a triphosphate group, B represents:

(80) ##STR00027##
and R.sup.3 represents N.sub.3, thus a compound of formula (1):

(81) ##STR00028##

(82) The compound of formula (1) may be prepared in accordance with the following synthetic scheme:

(83) ##STR00029## ##STR00030##

(84) One further particular compound of formula (I).sup.a which may be mentioned (2) is one wherein R.sup.1 represents —ONH.sub.2, R.sup.2 represents H, X represents a triphosphate group, B represents:

(85) ##STR00031##
and R.sup.3 represents N.sub.3, thus a compound of formula (2):

(86) ##STR00032##

(87) The compound of formula (2) may be prepared in accordance with the following synthetic scheme:

(88) ##STR00033## ##STR00034##

(89) One further particular compound of formula (I).sup.a which may be mentioned (3) is one wherein R.sup.1 represents —ONH.sub.2, R.sup.2 represents H, X represents a triphosphate group, B represents:

(90) ##STR00035##
and R.sup.3 represents N.sub.3, thus a compound of formula (3):

(91) ##STR00036##

(92) The compound of formula (3) may be prepared in accordance with the following synthetic scheme:

(93) ##STR00037## ##STR00038##

(94) In another embodiment, R.sup.3 represents an acetyl (—Ac) group and B is selected from:

(95) ##STR00039##

(96) In another embodiment, R.sup.3 represents an anisoyl group and B is selected from:

(97) ##STR00040##

(98) In another embodiment, R.sup.3 represents a benzyl group and B is selected from:

(99) ##STR00041##

(100) In another embodiment, R.sup.3 represents a benzyl group and B is selected from:

(101) ##STR00042##

(102) In another embodiment R.sup.3 represents a methyl group and R is selected from:

(103) ##STR00043##

(104) In another embodiment, R.sup.3 represents an allyl group and B is selected from:

(105) ##STR00044##

(106) Particular compounds of formula (I).sup.a which may be mentioned (4-27) are those wherein R.sup.1 represents —ONH.sub.2, R.sup.2 represents H, X represents a triphosphate group and B represents the bases described above, resulting in compounds:

(107) ##STR00045## ##STR00046## ##STR00047## ##STR00048## ##STR00049##

(108) According to a further aspect of the invention, there is provided a process of preparing a compound of formula (V):

(109) ##STR00050##
wherein X, R.sup.1, R.sup.2 and B are as defined herein, which comprises reacting a compound of formula (I):

(110) ##STR00051##
wherein X, R.sup.1, R.sup.2, R.sup.3 and B are as defined herein, with a chemical, with electromagnetic radiation, with heat and/or with an electric current.

(111) According to a further aspect of the invention, there is provided a process of preparing a compound of formula (II), (III) or (IV):

(112) ##STR00052##
wherein X, R.sup.1 and R.sup.2 are as defined herein, which comprises reacting a compound of formula (II).sup.a, (III).sup.a or (IV).sup.a, respectively:

(113) ##STR00053##
wherein X, R.sup.1 and R.sup.2 are as defined herein, with a chemical, with electromagnetic radiation and/or with an electric current.

(114) According to a further aspect of the invention, there is provided a process of preparing a compound of formula (II), (III) or (IV) as defined herein, which comprises reacting a compound of formula (VI):

(115) ##STR00054##
wherein X, R.sup.1, R.sup.2 and B are as defined herein, with a reducing agent.

(116) In one embodiment, the reducing agent is selected from beta-mercaptoethanol, dithiothreitol or a phosphine-based reducing agent such as tris(hydroxymethyl)phosphine (THP). tris(hydroxypropyl)phosphine (THPP) and tris(2-carboxylethyl)phosphine (TCEP).

(117) According to a further aspect of the invention, there is provided a compound of formula (VII):

(118) ##STR00055##
wherein R.sup.2 represents —H, —OH, —ONH.sub.2, —ONC(CH.sub.3).sub.2, —OCH.sub.2N.sub.3, —OCH.sub.2CHCH.sub.2, —OPO.sub.3.sup.2−, —OCH.sub.2SSCH.sub.2CH.sub.3, —OCOCH.sub.3, —OCH.sub.2CH.sub.2CN, —O-methoxyethyl, —O-alkyl, or —O-alkoxy or a suitable hydroxy protecting group;
X represents one or more phosphate, phosphorothioate, boranophosphate or imidophosphate groups, or any combination thereof; and
R.sup.4 represents C.sub.2-6 alkyl, —F, —Cl, —Br, —I, alkoxy, biotin, alkylamine or azide.

(119) According to a further aspect of the invention, there is provided the use of a compound of formula (VII) in a method of enzymatic nucleic acid synthesis.

(120) In one embodiment, the method of enzymatic nucleic acid synthesis is selected from a method of reversibly terminated enzymatic nucleic acid synthesis and a method of templated and non-templated de novo enzymatic nucleic acid synthesis.

(121) The following studies illustrate the invention:

Example 1: Enzymatic DNA Synthesis Using Azide-Masked Nitrogenous Heterocycles

(122) In the following method of DNA synthesis, engineered terminal deoxynucleotidyl transferase is used to add 3′-O-aminoxy reversibly terminated 2′-deoxynucleoside 5′-triphosphates to the 3′-end of DNA strands. This addition process is repeated until a desired sequence is synthesized. The 3′-O-aminoxy moiety must be deaminated (e.g., with acidic sodium nitrite) after each addition cycle to effect reversible termination. The process of deamination after each addition cycle also results in the mutagenic deamination of nitrogenous heterocycles containing amines (e.g., adenine, cytosine and guanine).

(123) Thus, in this example, amino moieties on the nitrogenous heterocycles are masked with an azido group to prevent oxidative deamination (FIG. 1-4). For example, one or a combination of 2′-deoxy-3′-O-aminoxy-N4-azidocytidine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N6-azidoadenine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N2-azidoguanosine 5′-triphosphate and 2′-deoxy-3′-O-aminoxy-5-ethyluridine 5′-triphosphate are used as nucleotide building blocks during each addition cycle in the presence of engineered TdT and required buffer components.

(124) A DNA polymer with amine-masked nitrogenous heterocycles (e.g., N4-azidocytosine, N6-azidoadenine, N2-azidoguanine) is thus synthesized. All amine-masked nitrogenous heterocycles are unmasked to reveal an amino group through exposure to a reducing agent (e.g., TCEP). The DNA polymer is now composed of nitrogenous heterocycles with unmasked amino groups (e.g., N4-azidocytosine is unmasked to cytosine, N6-azidoadenine is unmasked to adenine and N2-azidoguanine is unmasked to guanine). The DNA polymer can now be used for downstream molecular biology applications.

Example 2: Enzymatic DNA Synthesis Using N-Acetyl-Masked Nitrogenous Heterocycles

(125) In the following method of DNA synthesis, engineered terminal deoxynucleotidyl transferase is used to add 3′-O-aminoxy reversibly terminated 2′-deoxynucleoside 5′-triphosphates to the 3′-end of DNA strands. This addition process is repeated until a desired sequence is synthesized. The 3′-O-aminoxy moiety must be deaminated (e.g., with acidic sodium nitrite) after each addition cycle to effect reversible termination. The process of deamination after each addition cycle also results in the mutagenic deamination of nitrogenous heterocycles containing amines (e.g., adenine, cytosine and guanine).

(126) Thus, in this example, amino moieties on the nitrogenous heterocycles are masked with an acetyl group to protect from oxidative deamination (FIGS. 6 and 7). For example, one or a combination of 2′-deoxy-3′-O-aminoxy-N4-acetylcytidine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N6-acetyladenine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N2-acetylguanosine 5′-triphosphate and 2′-deoxy-3′-O-aminoxy-5-ethyluridine 5′-triphosphate are used as nucleotide building blocks during each addition cycle in the presence of engineered TdT and required buffer components.

(127) A DNA polymer with amine-masked nitrogenous heterocycles (e.g., N4-acetylcytosine, N6-acetyladenine, N2-acetylguanine) is thus synthesized. All amine-masked nitrogenous heterocycles are deacetylated and thus unmasked to reveal an amino group through exposure to a base (e.g., potassium carbonate) as shown in FIGS. 6 and 7. The DNA polymer is now composed of nitrogenous heterocycles with unmasked amino groups (e.g., N4-acetlycytosine is unmasked to cytosine, N6-acetyladenine is unmasked to adenine and N2-acetylguanine is unmasked to guanine). The DNA polymer can now be used for downstream molecular biology applications.

Example 3: Enzymatic DNA Synthesis Using N-Benzoyl- and N-Anisoyl-Masked Nitrogenous Heterocycles

(128) In the following method of DNA synthesis, engineered terminal deoxynucleotidyl transferase is used to add 3′-O-aminoxy reversibly terminated 2′-deoxynucleoside 5′-triphosphates to the 3′-end of DNA strands. This addition process is repeated until a desired sequence is synthesized. The 3′-O-aminoxy moiety must be deaminated (e.g., with acidic sodium nitrite) after each addition cycle to effect reversible termination. The process of deamination after each addition cycle also results in the mutagenic deamination of nitrogenous heterocycles containing amines (e.g., adenine, cytosine and guanine).

(129) Thus, in this example, amino moieties on the nitrogenous heterocycles are masked with a benzoyl group to protect from oxidative deamination (FIGS. 9 and 12). For example, one or a combination of 2′-deoxy-3′-O-aminoxy-N4-benzoylcytidine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N6-benzoyladenine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N2-benzoylguanosine 5′-triphosphate and 2′-deoxy-3′-O-aminoxy-5-ethyluridine 5′-triphosphate are used as nucleotide building blocks during each addition cycle in the presence of engineered TdT and required buffer components.

(130) A DNA polymer with amine-masked nitrogenous heterocycles (e.g., N4-benzoylcytosine, N6-benzoyladenine, N2-benzoylguanine) is thus synthesized. All amine-masked nitrogenous heterocycles are debenzoylated and thus unmasked to reveal an amino group through exposure to a base (e.g., methylamine) as shown in FIGS. 9 and 12. The DNA polymer is now composed of nitrogenous heterocycles with unmasked amino groups (e.g., N4-acetlycytosine is unmasked to cytosine, N6-benzoyladenine is unmasked to adenine and N2-benzoylguanine is unmasked to guanine). The DNA polymer can now be used for downstream molecular biology applications.

Example 4: Enzymatic DNA Synthesis Using N-Benzyl-Masked Nitrogenous Heterocycles

(131) In the following method of DNA synthesis, engineered terminal deoxynucleotidyl transferase is used to add 3′-O-aminoxy reversibly terminated 2′-deoxynucleoside 5′-triphosphates to the 3′-end of DNA strands. This addition process is repeated until a desired sequence is synthesized. The 3′-O-aminoxy moiety must be deaminated (e.g., with acidic sodium nitrite) after each addition cycle to effect reversible termination. The process of deamination after each addition cycle also results in the mutagenic deamination of nitrogenous heterocycles containing amines (e.g., adenine, cytosine and guanine).

(132) Thus, in this example, amino moieties on the nitrogenous heterocycles are masked with a benzyl group to protect from oxidative deamination (FIG. 8). For example, one or a combination of 2′-deoxy-3′-O-aminoxy-N4-benzylcytidine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N6-benzyladenine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N2-benzylguanosine 5′-triphosphate and 2′-deoxy-3′-O-aminoxy-5-ethyluridine 5′-triphosphate are used as nucleotide building blocks during each addition cycle in the presence of engineered TdT and required buffer components.

(133) A DNA polymer with amine-masked nitrogenous heterocycles (e.g., N4-benzylcytosine, N6-benzyladenine, N2-benzylguanine) is thus synthesized. All amine-masked nitrogenous heterocycles are debenzylated and thus unmasked to reveal an amino group through hydrogenolysis (e.g., Pd-C) or tert-butoxide and O.sub.2 in DMSO. The DNA polymer is now composed of nitrogenous heterocycles with unmasked amino groups (e.g., N4-acetlycytosine is unmasked to cytosine, N6-benzyladenine is unmasked to adenine and N2-benzylguanine is unmasked to guanine). The DNA polymer can now be used for downstream molecular biology applications.

Example 5: Enzymatic DNA Synthesis Using N-Methyl-Masked Nitrogenous Heterocycles

(134) In the following method of DNA synthesis, engineered terminal deoxynucleotidyl transferase is used to add 3′-O-aminoxy reversibly terminated 2′-deoxynucleoside 5′-triphosphates to the 3′-end of DNA strands. This addition process is repeated until a desired sequence is synthesized. The 3′-O-aminoxy moiety must be deaminated (e.g., with acidic sodium nitrite) after each addition cycle to effect reversible termination. The process of deamination after each addition cycle also results in the mutagenic deamination of nitrogenous heterocycles containing amines (e.g., adenine, cytosine and guanine).

(135) Thus, in this example, amino moieties on the nitrogenous heterocycles are masked with a methyl group. For example, one or a combination of 2′-deoxy-3′-O-aminoxy-N4-methylcytidine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N6-methyladenine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N2-methylguanosine 5′-triphosphate and 2′-deoxy-3′-O-aminoxy-5-ethyluridine 5′-triphosphate are used as nucleotide building blocks during each addition cycle in the presence of engineered TdT and required buffer components.

(136) A DNA polymer with amine-masked nitrogenous heterocycles (e.g., N4-methylcytosine, N6-methyladenine, N2-methylguanine) is thus synthesized. All amine-masked nitrogenous heterocycles are demethylated and thus unmasked to reveal an amino group through exposure to demethylases. For example, the amine-masked DNA polymer can be exposed to a cocktail of known demethylases or one single demethylase such as the DNA repair enzyme AlkB. The DNA polymer is now composed of nitrogenous heterocycles with unmasked amino groups (e.g., N4-acetlycytosine is unmasked to cytosine, N6-methyladenine is unmasked to adenine and N2-methylguanine is unmasked to guanine). The DNA polymer can now be used for downstream molecular biology applications.

Example 6: Enzymatic DNA Synthesis Using N-Allyl-Masked Nitrogenous Heterocycles

(137) In the following method of DNA synthesis, engineered terminal deoxynucleotidyl transferase is used to add 3′-O-aminoxy reversibly terminated 2′-deoxynucleoside 5′-triphosphates to the 3′-end of DNA strands. This addition process is repeated until a desired sequence is synthesized. The 3′-O-aminoxy moiety must be deaminated (e.g., with acidic sodium nitrite) after each addition cycle to effect reversible termination. The process of deamination after each addition cycle also results in the mutagenic deamination of nitrogenous heterocycles containing amines (e.g., adenine, cytosine and guanine).

(138) Thus, in this example, amino moieties on the nitrogenous heterocycles are masked with a allyl group to protect from oxidative deamination (FIG. 10). For example, one or a combination of 2′-deoxy-3′-O-aminoxy-N4-allylcytidine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N6-allyladenine 5′-triphosphate, 2′-deoxy-3′-O-aminoxy-N2-allylguanosine 5′-triphosphate and 2′-deoxy-3′-O-aminoxy-5-ethyluridine 5′-triphosphate are used as nucleotide building blocks during each addition cycle in the presence of engineered TdT and required buffer components.

(139) A DNA polymer with amine-masked nitrogenous heterocycles (e.g., N4-allylcytosine, N6-allyladenine, N2-allylguanine) is thus synthesized. All amine-masked nitrogenous heterocycles are deallylated and thus unmasked to reveal an amino group through exposure to tetrakis(triphenylphosphine) palladium. The DNA polymer is now composed of nitrogenous heterocycles with unmasked amino groups (e.g., N4-acetlycytosine is unmasked to cytosine, N6-allyladenine is unmasked to adenine and N2-allylguanine is unmasked to guanine). The DNA polymer can now be used for downstream molecular biology applications.