3′-OH unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing
The present invention relates generally to 3′-OH unblocked nucleotides and nucleosides labeled and unlabeled with 5-methoxy-substituted nitrobenzyl-based photocleavable terminating groups for use in methods and systems related to DNA and RNA sequencing and analysis. These compounds may be used as reversible terminators as they exhibit fast nucleotide incorporation kinetics, single-base termination, high nucleotide selectivity, and rapid terminating group cleavage that results in a naturally occurring nucleotide.
1. A compound of the formula:
wherein:
R 1 is hydroxy, triphosphate, or α-thiotriphosphate;
R 2 is hydrogen;
R 3 is tert-butyl;
R 4 is hydrogen;
R 5 is —C≡C—CH 2 —NH 2 or a -linker-reporter; and
R 6 is hydrogen;
or a salt, tautomer, or optical isomer thereof.
2. The compound of claim 1 , further defined as a compound of formula I.
3. The compound of claim 1 , further defined as a compound of formula II.
4. The compound of claim 1 , further defined as a compound of formula III.
5. The compound of claim 1 , further defined as a compound of formula IV.
6. The compound of claim 1 , further defined as a compound of formula V.
7. The compound of claim 1 , further defined as a compound of formula VI.
8. The compound of claim 1 , further defined as a compound of formula VII.
9. The compound of claim 1 , wherein R 1 is hydroxy.
10. The compound of claim 1 , wherein R 1 is a triphosphate.
11. The compound of claim 1 , wherein R 1 is an α-thiotriphosphate.
12. The compound of claim 1 , wherein R 5 is —C≡C—CH 2 —NH 2 .
13. The compound of claim 1 , wherein R 5 is a -linker-reporter.
14. The compound of claim 13 , wherein the linker is —C≡C—CH 2 —NH—.
15. The compound of claim 13 , wherein the reporter is based on a dye, wherein the dye is zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, or a squaraine dye.
16. The compound of claim 13 , wherein the reporter is:
17. The compound of claim 1 , wherein the starred carbon atom is in the S configuration.
18. The compound of claim 1 , wherein the starred carbon atom is in the R configuration.
19. A process for making an amino-propynyl-modified terminating nucleotide of the formula:
wherein R is ═O or ═S, or a salt and/or protonated form of any of these formulas, comprising reacting an iodo-modified terminating nucleoside of the formula:
with N-propargyl trifluoroacetamide in the presence of a palladium catalyst under conditions to form an intermediate of the formula:
and further comprising phosphorylating or thiophosphorylating the intermediate under conditions to form the amino-propynyl-modified terminating nucleotide.
20. The method of claim 19 , wherein the palladium catalyst is Pd(PPh 3 ) 4 .
21. A process for making an iodo-modified terminating nucleoside of the formula:
comprising reacting a starting compound of the formula:
with (S)-1-(4-iodo-5-methoxy-2-nitrophenyl)-2,2-dimethyl-1-propanol under conditions to form an iodo-modified terminating nucleoside.
22. The method of claim 21 , wherein the process further comprises activating the starting compound with mesyl chloride prior to the reaction with (S)-1-(4-iodo-5-methoxy-2-nitrophenyl)-2,2-dimethyl-1-propanol.
23. The method of claim 21 , wherein the process further comprises a deprotection step after the reaction with (S)-1-(4-iodo-5-methoxy-2-nitrophenyl)-2,2-dimethyl-1-propanol.
24. The method of claim 23 , wherein the deprotection step comprises the use of a deprotecting agent, wherein the deprotecting agent is n-Bu 4 NF or NH 4 F.
25. The method of claim 23 , wherein the starting compounds is:
and the process further comprises effecting a chloro for amino replacement after the deprotection step.
26. The method of claim 23 , wherein the starting compounds is:
and the process further comprises effecting a chloro for oxo replacement after the deprotection step.
27. The method of claim 23 , wherein the process further comprises converting the iodo-modified terminating nucleoside of the formula:
into the iodo-modified terminating nucleoside of the formula: