Two-stage protective groups for the synthesis of biopolymers
The invention relates to a method for the synthesis of a nucleic acid by gradual breakdown from protected synthesis building blocks carrying two-stage protective groups. The two-stage protective groups are split by means of a first exposure step and a subsequent chemical treatment step
1. A process for the synthesis of a nucleic acid by stepwise assembly of building blocks, wherein the building blocks are building blocks for the synthesis of a nucleic acid, wherein at least one of the building blocks carries a two-stage protective group, wherein the two-stage protective group contains a photoactivatable group selected from the group consisting of nitroveratryloxycarbonyl (NVOC), α-methyl-6-nitropiperonyloxycarbonyl (MeNPOC), 3,5 -dimethoxybenzoincarbonate (DMBOC), 2-(o-nitrophenyl)propyloxycarbonyl (NPPOC), o-nitrobenzyl and 2-(o-nitrophenyl) ethyl, wherein the photoactivable group is removed by an illumination step and the remainder of the two-stage protective group is removed by a subsequent acid treatment step.
2. The process as claimed in claim 1 , characterized in that the building block with the two-stage protective group has the general formula I:
where R 1 and R 2 are each independently selected from hydrogen, OR 3, O(CH 2 ) n COOR 3 and NHZ,
R 3 comprises a C 1 -C 8 alkyl group, a C 2 -C 8 alkenyl group, a C 2 -C 8 alkynyl group or/and C 6 -C 20 aryl group which may optionally have substituents,
X is the building block,
Y is in each case independently the photoactivatable group,
Z is an amino-protective group,
n is an integer from 0 to 4, and
where R 1 or/and R 2 may optionally be replaced by Y.
3. The process as claimed in claim 2 , characterized in that a two-stage protective group which carries at least one fluorescent group is used.
4. The process as claimed in claim 3 , characterized in that Y, R 3 or/and Z carry the fluorescent group.
5. The process as claimed in claim 1 , characterized in that the building blocks are phosphoramidites.
6. The process as claimed in claim 5 , characterized in that phosphoramidite building blocks carry the two-stage protective group on the 5′-O atom.
7. The process as claimed in claim 1 , wherein at least one of the building blocks contains a spacer or linker group.
8. The process as claimed in claim 1 , characterized in that the synthesis is carried out on a solid phase.
9. The process as claimed in claim 8 , characterized in that a site-dependent synthesis of a plurality of nucleic acids, each with a different nucleotide sequence, is carried out on a single support.
10. A compound of the general formula I:
where R 1 and R 2 are each independently selected from hydrogen, OR 3, O(CH 2 ) n COOR 3 and NHZ,
R 3 comprises a C 1 -C 8 alkyl group, a C 2 -C 8 alkenyl group, a C 2 -C 8 alkynyl group or/and C 6 -C 20 aryl group which may optionally have substituents,
X is a building block for the synthesis of a nucleic acid or a leaving group,
Y is in each case independently a photoactivatable group,
Z is an amino-protective group,
n is an integer from 0 to 4,
where R 1 or/and R 2 may optionally be replaced by Y, and
wherein the photoactivatable group is selected from the group consisting of nitroveratryloxycarbonyl (NVOC), α-methyl-6-nitropiperonyloxycarbonyl (MeNPOC), 3,5-dimethoxybenzoincarbonate (DMBOC), 2-(o-nitrophenyl)propyloxycarbonyl (NPPOC), o-nitrobenzyl and 2-(o-nitrophenyl) ethyl and wherein the photoactivatable group is removable by illumination.
11. The compound as claimed in claim 10 , characterized in that it carries at least one fluorescent group.
12. The compound as claimed in claim 11 , characterized in that Y, R 3 or/and Z carry a fluorescent group.
13. A method of synthesizing a nucleic acid from building blocks, wherein at least one of the building blocks is protected by a two-stage protective group, wherein the at least one of the building blocks protected by a two-stage protective group is a compound of Formula (I), said method comprising the steps of:
removing a photoactivatable group of the two-stage protective group by illumination, and
subsequently removing the rest of the two-stage protective group from the building block by acid treatment
wherein formula (I) is
where R 1 and R 2 are each independently selected from hydrogen, OR 3, O(CH 2 ) n COOR 3 and NHZ,
R 3 comprises a C 1 -C 8 alkyl group, a C 2 -C 8 alkenyl group, a C 2 -C 8 alkynyl group or/and C 6 -C 20 aryl group which may optionally have substituents,
X is a building block for the synthesis of a nucleic acid or a leaving group,
Y is in each case independently a photoactivatable group,
Z is an amino-protective group,
n is an integer from 0 to 4,
where R 1 or/and R 2 may optionally be replaced by Y, and
wherein the photoactivatable group is selected from the group consisting of nitroveratryloxycarbonyl (NVOC), α-methyl-6-nitropiperonyloxycarbonyl (MeNPOC), 3,5-dimethoxybenzoincarbonate (DMBOC), 2-(o-nitrophenyl)propyloxycarbonyl (NPPOC), o-nitrobenzyl and 2-(o-nitrophenyl) ethyl and wherein the photoactivatable group is removable by illumination.
14. The process of claim 2 , wherein the substituent is selected from the group consisting of a halogen, —OH, —SH, —O—, —S(O)—, —S(O) 2 —, —NO 2, —CN and —NHZ, wherein Z is an amino-protective group.
15. The compound of claim 10 , wherein the substituent is selected from the group consisting of a halogen, —OH, —SH, —O—, —S(O)—, —S(O) 2 —, NO 2, —CN and —NHZ, wherein Z is an amino-protective group.
16. The method of claim 13 , wherein the substituent is selected from the group consisting of a halogen, —OH, —SH, —O—, —S(O)—, —S(O) 2 —, —NO 2, —CN and —NHZ, wherein Z is an amino-protective group.
17. The method of claim 13 , characterized in that the compound of formula I carries at least one fluorescent group.
18. The method of claim 17 , characterized in that Y, R 3 or/and Z carry a fluorescent group.
19. The compound of claim 10 , characterized in that the building block is a phosphoramidite.
20. The compound of claim 19 , characterized in that the linkage with the phosphoramidite building block is through the 5′-O atom.
21. The process of claim 13 , characterized in that the building blocks are phosphoramidites.
22. The process of claim 21 , characterized in that phosphoramidite building blocks carry the two-stage protective group on the 5′-O atom.