Nucleoside triphosphates with stable aminoxy groups
View Patent ↗This invention claims aqueous compositions that comprise triphosphates of 2′-deoxynucleoside derivatives that have, instead of a 3′-OH moiety, a 3′-ONH 2 moiety; wherein said compositions contain less than 0.5 mole percent contaminating triphosphate having a 3′-OH moiety, as well as processes for providing such compositions. The compositions further must contain insubstantial amounts of hydroxylamine.
1. A composition comprising an aqueous solution containing an alkoxylamine, hydroxylamine at a concentration of less than 1 micromolar and a molecule having the structure:
or one of its ionized forms, wherein B is a heterocycle selected from the group consisting of
wherein Su indicates the point of attachment of the heterocycle to the sugar, R is either H, CH 3 , or a functionalized side chain, and X is either N or C—R.
2. The composition of claim 1 , wherein said alkoxylamine is methoxylamine.
3. The composition of claim 1 , wherein said heterocycle B is selected from the group consisting of thymine, uracil, cytosine, guanine, adenine, 7-deazaadenine, 7-deazaguanine, and isocytosine and the alkoxylamine is methoxylamine.
4. The composition of claim 1 , wherein said heterocycle B is selected from the group consisting of thymine and isocytosine and the alkoxylamine is methoxylamine.
5. The composition of claim 1 , wherein appended to heterocycle B is a linker that comprises either a disulfide moiety or a 1,2-diol moiety and the alkoxylamine is methoxylamine.
6. A process for preparing a compound of having the structure:
or one of its ionized forms, wherein B is a heterocycle selected from the group consisting of
wherein Su indicates the point of attachment of the heterocycle to the sugar, R is H, CH 3 , or a functionalized side chain, and X is either N or C—R, wherein said process comprises:
(a) contacting a solid phase having an immobilized aldehyde or ketone moiety with compound having the following structure
wherein Q is either H or a dimethoxytrityl group, and B is a heterocycle selected from the group consisting of
wherein Su indicates the point of attachment of the heterocycle to the sugar, R is H, CH 3 , or a functionalized side chain, and X is either N or C—R, followed by
(b) contacting the composition from step (a) with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, followed by
(c) contacting the composition from step (b) with an ammonium salt of a pyrophosphate, followed by
(d) oxidizing the composition from step (c), followed by
(e) washing the composition from step (d) with an aqueous solution comprising an alkoxylamine.
7. The process of claim 6 , wherein appended to heterocycle B is a linker that comprises either a disulfide moiety or a 1,2-diol moiety.
8. The process of claim 6 , wherein said heterocycle B is selected from the group consisting of thymine, uracil, cytosine, guanine, adenine, 7-deazaadenine, 7-deazaguanine, and isocytosine.
9. The process of claim 6 , wherein said heterocycle B is selected from the group consisting of thymine and isocytosine.
10. The process of claim 6 , wherein the alkoxylamine is methoxylamine.
11. The composition of claim 1 , wherein said solution contains less than 1 nanomolar hydroxylamine.
12. The composition of claim 1 , wherein the composition contains less than 0.5 mole percent of the molecule having the structure with a 3′-OH group relative to the molecule having the structure with a 3′-O—NH 2 group.
13. The composition of claim 1 , wherein the composition contains less than 0.05 mole percent of the molecule having the structure with a 3′-OH group relative to the molecule having the structure with a 3′-O—NH 2 group.
14. The process of claim 6 , comprising in step e) washing the composition from step (d) with the aqueous solution comprising the alkoxylamine, wherein the aqueous solution does not contain hydroxylamine.
15. The process of claim 6 , wherein the process does not comprise using hydroxylamine in any step after step d).