PROTECTED MONOMER AND METHOD OF FINAL DEPROTECTION FOR RNA SYNTHESIS

• US 20100076183A1
• Filed: 05/14/2009
• Published: 03/25/2010
• Est. Priority Date: 09/22/2008
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Dellinger, Geraldine F., Dellinger, Douglas J., Myerson, Joel, Sierzchala, Agnieszka, Timar, Zoltan
• Application Number:
  US12/466,326
• Publication Number:
  US 20100076183A1
• Time in Patent Office: Days
• US Class Current: 536/17.100
• CPC Class Codes: A61K 31/712 Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
• CPC Class Codes: A61K 31/7125 Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 13/12 by acids having the group -X-C(=X)-X-, or halides thereof, in which each X means nitrogen, oxygen, sulfur, selenium or tellurium, e.g. carbonic acid, carbamic acid
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/067 with ribosyl as the saccharide radical
• CPC Class Codes: C07H 19/167 with ribosyl as the saccharide radical
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C08L 85/02 containing phosphorus
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

A nucleoside monomer that is protected by a thionocarbamate protecting group is provided, as well as a method for making a polynucleotide that uses the same. Also provided is a polynucleotide synthesis method that employs a diamine to deprotect a protected polynucleotide.

Claims

1. A compound of the structure:

2. The compound of claim 1 wherein:

one of R¹ and R² is selected from a phosphoramidite group and a H-phosphonate group; and

one of R¹ and R² is a protecting group.

3. The compound of claim 2, wherein said thionocarbamate protecting group (PG) is selected from one of the structures:

4. The compound of claim 2, wherein said thionocarbamate protecting group (PG) is selected from one of the structures:

5. The compound of claim 2, wherein said thionocarbamate protecting group (PG) is of the structure:

6. The compound of claim 5 wherein, R¹ is DMT, R² is beta-cyanoethyl-N,N-diisopropylphosphoramidite and B^P is selected from the group consisting of U, N⁶-benzoyl-A, N⁶-isobutyryl-A, N⁶—

(N,N)-dimethylacetamidine-A, N⁶—

(N,N)-dibutylformamidine-A, N⁶-phenoxyacetyl-A, N⁶-4-tert-butylphenoxyacetyl-A, N⁴-acetyl-C, N⁴-isobutyryl-C, N⁴-phenoxyacetyl-C, N⁴-4-tert-butylphenoxyacetyl-C, N²-isobutyryl-G, N²—

(N,N)-dibutylformamidine-G, N²—

(N,N)-dimethylformamidine-G, N²-phenoxyacetyl-G and N²-4-tert-butylphenoxyacetyl-G.

7. A method of synthesizing a polynucleotide comprising at least one ribonucleotide residue, said method comprising:

contacting a nucleotide residue or a nucleoside monomer having an unprotected hydroxyl group with;

a compound of claim 2 under conditions sufficient to covalently bond said compound to said nucleotide residue or said nucleoside monomer and produce said polynucleotide.

8. The method of claim 7 further comprising:

contacting said polynucleotide with a composition comprising a sulfurization agent to produce an oxidized polynucleotide.

9. The method of claim 7 wherein said nucleotide residue or said nucleoside monomer is bound directly or indirectly to a solid support.

10. The method of claim 9 wherein said solid support is selected from a CPG support and a polystyrene support.

11. The method of claim 9 wherein said solid support is selected from a bead and an array substrate.

12. The method according to claim 7, further comprising:

cleaving said polynucleotide from a solid support to produce a free polynucleotide.

13. The method according to claim 12 wherein said free polynucleotide is retained on said solid support.

14. The method according to claim 12, further comprising:

chemically modifying said free polynucleotide to produce a modified polynucleotide.

15. A polynucleotide product produced by the method of claim 7.

16. A polynucleotide product produced by the method of claim 8.

17. A polynucleotide comprising:

a ribonucleotide residue comprising the structure;

18. The polynucleotide of claim 17 wherein said thionocarbamate protecting group (PG) is selected from one of the structures:

19. The polynucleotide of claim 17, wherein said thionocarbamate protecting group (PG) is selected from one of the structures:

20. The polynucleotide of claim 17, wherein said thionocarbamate protecting group (PG) is of the structure:

21. A method of deprotecting a solid support bound polynucleotide comprising at least one 2′

-protected ribonucleotide residue, wherein said residue is not a 2′

-ester protected ribonucleotide, said method comprising;

contacting said polynucleotide with a composition comprising a diamine under conditions sufficient to deprotect said 2′

-protected ribonucleotide residue.

22. The method of claim 21 wherein said 2′

-protected ribonucleotide residue comprises the structure;

23. The method of claim 22 wherein said thionocarbamate protecting group (PG) is selected from one of the structures:

24. The method of claim 22 wherein said thionocarbamate protecting group (PG) is selected from one of the structures:

25. The method of claim 22, wherein said thionocarbamate protecting group (PG) is of the structure:

26. The method of claim 21 wherein said diamine reagent comprises two primary amino groups connected by a linker of about 2 to 12 atoms in length.

27. The method of claim 26 wherein said linker is of about 2 to 6 atoms in length.

28. The method of claim 21 wherein said diamine is selected from 1,2-diaminoethane, 1,2-diaminopropane, 1,3-diaminopropane, 1,4-diaminobutane, 2,2′

-diaminodiethylamine, and substituted versions thereof.

29. The method of claim 21 wherein said diamine is 1,2-diaminoethane.

30. The method of claim 21 wherein said composition comprises at least 50% by volume 1,2-diaminoethane.

31. The method of claim 21 wherein said composition comprises 1,2-diaminoethane and a solvent.

39. A polynucleotide produced by the method of claim 21.

32. A method comprising:

(a) contacting a solid support bound polynucleotide comprising;

a ribonucleotide residue comprising a 2′

-protecting group, a phosphate protecting group, and optionally a nucleobase protecting group;

with a first composition comprising a phosphate deprotection reagent, to remove said phosphate protecting group and produce a first deprotected polynucleotide that remains bound to said solid support;

(b) contacting said first deprotected polynucleotide with a second composition comprising a diamine to remove said 2′

-protecting group and removing the nucleobase protecting group, if present, to produce a second deprotected polynucleotide; and

(c) or (d) wherein;

(c) comprises simultaneously cleaving said second deprotected polynucleotide from said solid support; and

(d) comprises contacting said second deprotected polynucleotide with a third composition comprising a linker cleaving reagent to cleave said second deprotected polynucleotide from said solid support,

to produce a deprotected, cleaved polynucleotide.

33. The method of claim 32 wherein:

said phosphate protecting group is a 2-cyanoethyl group or a methyl group.

34. The method of claim 33 wherein:

said phosphate deprotection reagent is selected from diethylamine, t-butylamine, diaza(1,3)bicyclo[5.4.0]undecane (DBU), thiophenol and disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate; and

wherein said diamine is 1,2-diaminoethane.

35. A method of deprotecting a polynucleotide comprising a nucleobase protecting group;

and a ribonucleotide residue comprising a 2′

-protecting group selected from tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM) and 2′

-O-bis(2-acetoxyethoxy)methyl (ACE);

said method comprising;

(a) contacting said polynucleotide with a first composition comprising a 2′

-deprotection reagent, under conditions sufficient to remove said 2′

-protecting group and produce a first deprotected polynucleotide;

(b) contacting said first deprotected polynucleotide with a second composition comprising a diamine, under conditions sufficient to remove said nucleobase protecting group and produce a fully deprotected polynucleotide.

36. A method of deprotecting a solid support bound polynucleotide comprising a phosphate protecting group, a nucleobase protecting group;

and a ribonucleotide residue comprising 2′

-protecting group;

said method comprising;

(a) contacting said polynucleotide with a first composition comprising a phosphate deprotection reagent, under conditions sufficient to remove said phosphate protecting group and produce a first deprotected polynucleotide;

(b) contacting said first deprotected polynucleotide with a second composition comprising a 2′

-deprotection reagent under conditions sufficient to remove said 2′

-protecting group and produce a second deprotected polynucleotide;

(c) contacting said second deprotected polynucleotide with a third composition comprising a diamine, under conditions sufficient to remove said nucleobase protecting group and produce a fully deprotected polynucleotide.

37. The method of claim 36 wherein said 2′

-protecting group is selected from tert-butyldimethylsilyl (TBDMS), triisopropylsilyloxymethyl (TOM) and 2′

-O-bis(2-acetoxyethoxy)methyl (ACE).

38. A method of deprotecting a solid support bound polynucleotide comprising a nucleobase protecting group and a ribonucleotide residue comprising a thionocarbamate protecting group, said method comprising:

(a) contacting said polynucleotide with a composition comprising a diamine, under conditions sufficient to remove said protecting groups and cleave said polynucleotide from said solid support, and produce a cleaved polynucleotide;

wherein said cleaved polynucleotide is retained on the solid support;

(b) washing the solid support and said cleaved polynucleotide; and

(c) eluting said cleaved polynucleotide from said solid support.

40. A kit for deprotecting a polynucleotide comprising a 2′

protected ribonucleotide residue, said kit comprising;

a composition comprising 1,2-diaminoethane, or a derivative thereof.