Photolabile protective groups for improved processes to prepare oligonucleotide arrays
View Patent ↗The present invention discloses novel and improved nucleosidic and nucleotidic compounds that are useful in the light-directed synthesis of oligonucleotides, as well as, methods and reagents for their preparation. These compounds are characterized by novel photolabile protective groups that are attached to either the 5′- or the 3′-hydroxyl group of a nucleoside moiety. The photolabile protective group is comprised of a 2-(2-nitrophenyl)-ethyoxycarbonyl skeleton with at least one substituent on the aromatic ring that is either an aryl, an aroyl, a heteroaryl or an alkoxycarbonyl group. The present invention includes the use of the aforementioned compounds in light-directed oligonucleotide synthesis, the respective assembly of nucleic acid microarrays and their application.
1. A compound having the formula (1):
wherein
R 1 is selected from the group consisting of H, NO 2 , CN, OCH 3 , a halogen, an alkyl having up to 4 carbon atoms, and an alkoxyl having up to 4 carbon atoms;
R 2 is selected from the group consisting of an aryl group, a substituted aryl group, a heteroaryl group, substituted heteroaryl group, an aroyl group, and a substituted aroyl group;
R 3 is selected from the group consisting of H, NO 2 and a halogen;
R 4 is selected from the group consisting of H, OCH 3 and an alkyl group having up to 4 carbon atoms;
X is selected from the group consisting of oxygen and sulfur; and
Z is selected from the group consisting of a leaving group, an alcoholate group, —OH, a N-atom of an amine compound, a deoxyribonucleoside and a ribonucleoside as represented by either of the following formulae (2) or (3):
wherein
R 5 is selected from the group consisting of a H, an oligonucleotide, a phosphoramidite group and a protecting group;
R 6 is selected from the group consisting of H, OH , an alkoxyl having up to 4 carbon atoms, an alkenoxyl group having up to 4 carbon atoms, a substituted alkenoxyl group having up to 4 carbon atoms, or R 6 is WR 8 wherein W is selected from oxygen and sulfur and R 8 is a protective group useful in oligonucleotide synthesis;
B is a base selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil, and when B is any one of adenosine, cytosine and guanine the amino functions on the heterocycle may bear a protective group useful in oligonucleotide synthesis.
2. The compound of claim 1 , wherein R 1 is H and R 2 is phenyl or substituted phenyl.
3. The compound of claim 1 , wherein R 1 is H and R 2 is benzoyl or substituted benzoyl.
4. The compound of claim 1 wherein W is O and R 8 is selected from the group consisting of an alkyl, alkenyl, acetal and silylether protective group.
5. The compound of claim 1 , wherein W is S and R 8 is an alkyl protective group.
6. The compound of claim 1 , wherein R 6 is selected from the group consisting of an O-methyl, O-ethyl, O-allyl, O-tetrahydropyranyl- O-methoxytetrahydropyranyl and an O-t-butyldimethylsilyl.
7. The compound of claim 1 , wherein B is selected from the group consisting of adenine, cytosine and guanine and wherein R 8 is selected from the group consisting of phenoxyacetyl, 4-tert-butyl-phenoxyacetyl, 4-isopropyl-phenoxyacetyl and dimethylformamidino.
8. The compound of claim 1 , wherein B is adenine and is selected from the group consisting of benzoyl and p-nitrophenyloxycarbonyl (p-NPEOC).
9. The compound of claim 1 , wherein B is guanine and wherein R 8 is selected from the group consisting of isobutyroyl and p-nitrophenylethyloxycarbonyl (p-NPEOC).
10. The compound of claim 1 , wherein B is cytosine and wherein R 8 is selected from the group consisting of benzoyl, isobutyroyl and p-nitrophenylethyloxycarbonyl (p-NPEOC).
11. The compound of claim 1 , wherein R 5 is a phosphoramidite group.
12. The compound of claim 1 , wherein R 5 is an OH— protective group.
13. The compound of claim 12 , wherein R 5 is a dimethoxytrityl- or a monomethoxytrityl- group.
14. The compound of claim 12 , wherein R 5 is a silyl-group.
15. The compound of claim 1 , wherein Z is a leaving group.
16. The compound of claim 15 , wherein the leaving group is selected from the group consisting of chloride, imidazolyl and nitrophenoxyl.
17. The compound of claim 1 , wherein Z is a deoxyribonucleoside or a ribonucleoside represented by formulae (2) or (3):
wherein R 5 is selected from the group consisting of a H and an oligonucleotide;
R 6 is selected from the group consisting of H, OH , an alkoxyl having up to 4 carbon atoms, an alkenoxyl group having up to 4 carbon atoms, a substituted alkenoxyl group having up to 4 carbon atoms, or R 6 is WR 8 wherein W is selected from oxygen and sulfur and R 8 is a protective group;
B is selected from the group consisting of adenine, cytosine, guanine, thymine and uracil.
18. The compound of claim 1 , wherein Z is selected from the group consisting of a deoxyribonucleoside and a ribonucleoside.
19. The compound of claim 1 , wherein Z is selected from the group consisting of an alcoholate group, —OH and an amine.
20. The compound of claim 1 , wherein
R 2 is a phenyl group;
R 4 is a hydrogen atom or an alkyl group having up to 4 carbon atoms;
X is O;
and Z is a deoxyribonucleoside or a ribonucleoside represented by formulae (2) or (3):
wherein R 5 is H;
R 6 is selected from the group consisting of H, OH , an alkoxyl having up to 4 carbon atoms, and an alkenoxyl group having up to 4 carbon atoms; and
B is selected from the group consisting of adenine, cytosine, guanine, thymine and uracil.
21. The compound of claim 1 , wherein R 1 and R 3 are selected from the group consisting of H and NO 2 , wherein R 1 and R 3 are not both NO 2 ;
R 2 is selected from the group consisting of a phenyl group and a benzoyl group;
R 4 is selected from the group consisting of a methyl group and an ethyl group; and
X is oxygen.
22. The compound of claim 21 , wherein Z is a deoxyribonucleoside;
R 6 =H; and
B is selected from the group consisting of adenine, cytosine, guanidine, thymine and uracil.
23. The compound of claim 1 , wherein Z is a deoxyribonucleoside;
R 6 =H; and
B is selected from the group consisting of adenine, cytosine, guanidine, thymine and uracil.
24. A method for the light-controlled synthesis of oligonucleotides, wherein said method is comprised of the following steps:
a) attaching, as a first building block, a nucleoside or nucleotide of claim 1 comprising the photolabile protective group at its primary hydroxyl group, to a support via its 3′ secondary hydroxyl group;
b) irradiating the support-bound nucleoside or nucleotide resulting from step a), such that the protective group at the primary hydroxyl group is removed, thereby deprotecting the primary hydroxyl group;
c) reacting the support-bound nucleotide resulting from step b) in the presence of an activator with a second nucleotide selected from claim 12 comprising a protective group at its primary hydroxyl group and phosphoramidite functional group at its 3′ secondary hydroxyl group, to form an internucleosidic phosphorous linkage;
d) optionally capping unreacted primary hydroxyl groups with an inert alcohol protecting group;
e) oxidizing the internucicosidic phosphorous linkage to the naturally occurring pentavalent state;
f) iterating steps b) to d) while successively applying the phosphoramidite building blocks in a predetermined order until the desired oligonucleotide strand is completed; and
g) removing of all nucleobase and phosphate protective groups.
25. A method for the light-controlled synthesis of oligonucleotides, wherein said method is comprised of the following steps:
a) attaching, as a first building block, a nucleoside or nucleotide of claim 1 comprising the photolabile protective group at its 3′ secondary hydroxyl group, to a support via its primary hydroxyl group;
b) irradiating the support-bound nucleotide resulting from step a), such that the protective group at the secondary hydroxyl group is removed, thereby deprotecting the 3′ secondary hydroxyl group;
c) reacting the support-bound nucleotide resulting from step b) in the presence of an activator with a second nucleotide selected from claim 12 comprising a protective group at its 3′ secondary hydroxyl group and a phosphoramidite functional group at its primary hydroxyl group, to form an internucleosidic phosphorous linkage;
d) optionally capping unreacted secondary hydroxyl groups with an inert alcohol protecting group;
e) oxidizing the internucleosidic phosphorous linkage to the naturally occurring pentavalent state;
f) iterating steps b) to d) while successively applying the phosporamidite building blocks in a predetermined order until the desired oligonucleotide strand is completed; and
g) removing of all nucleobase and phosphate protective groups.
26. A compound having the formula (1):
wherein
R 1 is COOY, wherein Y is selected from the group consisting of an alkyl group of up to 10 carbon atoms,
R 2 is selected from the group consisting of H, NO 2 , CN, OCH 3 , a halogen, an alkyl having up to 4 carbon atoms, an alkoxyl having up to 4 carbon atoms;
R 3 is selected from the group consisting of H, NO 2 and halogen;
R 4 is selected from the group consisting of OCH 3 , an alkyl group having up to 4 carbon atoms and an optionally substituted alkyl group having up to 4 carbon atoms;
X is selected from the group consisting of oxygen or sulfur; and
Z is selected from the group consisting of a leaving group, an alcoholate, —OH, a N— atom of an amine compound, a deoxyribonucleoside and a ribonucleoside as represented by either of the following formulae (2) or (3):
wherein
R 5 is selected from the group consisting of a H, an oligonucleotide, a phosphoramidite group and a protecting group;
R 6 is selected from the group consisting of H, OH, an alkoxyl having up to 4 carbon atoms, an alkenoxyl having up to 4 carbon atoms, or a substituted alkenoxyl having up to 4 carbon atoms, or R 6 is WR 8 wherein W is selected from oxygen and sulfur and R 8 is selected from a protective group useful in oligonucleotide synthesis;
B is base selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil, and in the case of adenosine, cytosine and guanine the amino functions on the heterocycle may bear a protective group useful in oligonucleotide synthesis.
27. The compound of claim 26 , wherein Y is an alkyl group selected from the group consisting of methyl and tertiary-butyl, and R 2 is H.
28. The compound of claim 26 wherein W is O and R 8 is selected from the group consisting of an alkyl, alkenyl, acetal and silylether protective group.
29. The compound of claim 26 wherein W is S and R 8 is selected from the group consisting of an alkyl protective group.
30. The compound of claim 26 , wherein R 6 is selected from the group consisting of an O-methyl, O-ethyl, O-allyl, O-tetrahydropyranyl- O-methoxytetrahydropyranyl and an O-t-butyldimethylsilyl.
31. The compound of claim 26 , wherein B is selected from the group consisting of adenine, cytosine and guanine and said protective group is selected from the group consisting of phenoxyacetyl, 4-tert-butyl-phenoxyacetyl, 4-isopropyl-phenoxyacetyl and dimethylformamidino.
32. The compound of claim 26 , wherein B is adenine and the protective group is selected from the group consisting of benzoyl and p-nitrophenyloxycarbonyl (p-NPEOC).
33. The compound of claim 26 , wherein B is guanine and the protective group is selected from the group consisting of isobutyroyl and p-nitrophenylethyloxycarbonyl (p-NPEOC).
34. The compound of claim 26 , wherein B is cytosine and the protective group is selected from the group consisting of benzoyl, isobutyroyl and p-nitrophenylethyloxycarbonyl (p-NPEOC).
35. The compound of claim 26 , wherein R 5 is a phosphoramidite group.
36. The compound of claim 26 , wherein R 5 is an OH— protective group.
37. The compound of claim 36 , wherein R 5 is selected from a dimethoxytrityl- or a monomethoxytrityl- group.
38. The compound of claim 36 , wherein R 5 is a silyl- group.
39. The compound of claim 26 , wherein Z is a leaving group.
40. The compound of claim 39 , wherein the leaving group is selected from the group consisting of chloride, imidazolyl and nitrophenoxyl.
41. The compound of claim 27 , wherein Z is a leaving group.
42. The compound of claim 41 , wherein the leaving group is selected from the group consisting of chloride, imidazolyl and nitrophenoxyl.
43. The compound of claim 26 , wherein Z is a deoxyribonucleoside or a ribonucleoside represented by formulae (2) or (3):
wherein R 5 is selected from the group consisting of a H and an oligonucleotide;
R 6 is selected from the group consisting of H, OH , an alkoxyl having up to 4 carbon atoms, an alkenoxyl group having up to 4 carbon atoms, a substituted alkenoxyl group having up to 4 carbon atoms, or R 6 is WR 8 wherein W is selected from oxygen and sulfur and R 8 is a protective group;
B is selected from the group consisting of adenine, cytosine, guanine, thymine and uracil.
44. The compound of claim 26 , wherein Z is selected from the group consisting of a deoxyribonucleoside and a ribonucleoside.
45. The compound of claim 26 , wherein Z is selected from the group consisting of an alcoholate group, —OH and an amine.
46. The compound of claim 26 , wherein
R 2 is a phenyl group;
R 4 is a hydrogen atom or an alkyl group having up to 4 carbon atoms;
X is O;
and Z is a deoxyribonucleoside or a ribonucleoside represented by formulae (2) or (3):
wherein R 5 is H;
R 6 is selected from the group consisting of H, OH , an alkoxyl having up to 4 carbon atoms, and an alkenoxyl group having up to 4 carbon atoms; and
B is selected from the group consisting of adenine, cytosine, guanine, thymine and uracil.