Methods for the synthesis of functionalized nucleic acids

• US 9,605,019 B2
• Filed: 07/13/2012
• Issued: 03/28/2017
• Est. Priority Date: 07/19/2011
• Status: Active Grant

• Current Assignee: WAVE Life Sciences Ltd.
• Original Assignee: WAVE Life Sciences Ltd.
• Inventors: Verdine, Gregory L., Meena, Iwamoto, Naoki, Butler, David Charles Donnell
• Primary Examiner(s): Crane, Lawrence E
• Application Number:
  US14/233,579
• Publication Number:
  US 20140194610A1
• Time in Patent Office: 1,719 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 31/66 Phosphorus compounds
• CPC Class Codes: A61K 31/70 Carbohydrates; Sugars; Derivatives thereof sorbitol A61K31/047
• CPC Class Codes: C07C 309/66 Methanesulfonates
• CPC Class Codes: C07D 295/108 to an acyclic saturated chain
• CPC Class Codes: C07H 13/04 having the esterifying carboxyl radicals attached to acyclic carbon atoms
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

The present application, among other things, provides technologies, e.g., reagents, methods, etc. for preparing oligonucleotides comprising phosphorothiotriesters linkages, e.g., oligonucleotides having the structure of IIIa, IIIb or IIIc. In some embodiments, provided methods comprise reacting an H-phosphonate of structure Ia or Ib with a silylating reagent to provide a silyloxyphosphonate, and reacting the silyloxyphosphonate with a thiosulfonate reagent of structure IIa or IIb to provide an oligonucleotide of structure IIIa or IIIb. In some embodiments, provided methods comprise reacting an H-phosphonate of structure Ic with a silylating reagent to provide a silyloxyphosphonate, reacting the silyloxyphosphonate with a bis(thiosulfonate) reagent of structure IVc to provide a phosphorothiotriester comprising a thiosulfonate group of structure Vc, and then reacting the phosphorothiotriester comprising a thiosulfonate group of structure Vc with a nucleophile of structure VIc to provide an oligonucleotide of structure IIIc.

Claims

1. A process for the preparation of an oligonucleotide of structure IIIa comprising the steps of:

i) reacting an H-phosphonate of structure Ia with a silylating reagent to provide a silyloxyphosphonate; and

ii) reacting the silyloxyphosphonate with a thiosulfonate reagent of structure IIa to provide an oligonucleotide of structure IIIa;

wherein;

the H-phosphonate of structure Ia has the following structure;

2. A process for the preparation of an oligonucleotide of structure IIIb, comprising the steps of:

i) reacting an H-phosphonate of structure Ib with a silylating reagent to provide a silyloxyphosphonate; and

ii) reacting the silyloxyphosphonate with a thiosulfonate reagent of structure IIb to provide an oligonucleotide of structure IIIb;

wherein;

the H-phosphonate of structure Ib has the following structure;

3. The process of claim 2, wherein each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or 5-methylcytosine.

4. The process of claim 2, wherein R¹ is:

5. The process of claim 2, wherein the silylating reagent is

1,1,3,3-tetramethyl-1,3-diphenyldisilazane;

1,3-dimethyl-1,1,3,3-tetraphenyldisilazane;

1-(trimethylsilyl)imidazole;

N-trimethylsilyl-N-methyl trifluoroacetamide;

bis(dimethylamino)dimethylsilane;

bromotrimethylsilane;

chlorodimethyl(pentafluorophenyl)silane;

chlorotriethylsilane;

chlorotriisopropylsilane;

chlorotrimethylsilane;

dichlorodimethylsilane;

hexamethyldisilazane;

N,N′

-bis(trimethylsilyl)urea;

N,N-bis(trimethylsilyl)methylamine;

N,N-dimethyltrimethylsilylamine;

N,O-bis(trimethylsilyl)acetamide;

N,O-bis(trimethylsilyl)carbamate;

N,O-bis(trimethylsilyl)trifluoroacetamide;

N-methyl-N-(trimethylsilyl)trifluoroacetamide;

N-methyl-N-trimethylsilylacetamide;

N-methyl-N-trimethylsilylheptafluorobutyramide;

N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide;

N-methyl-N-trimethylsilylheptafluorobutyramide;

trimethylsilyltriflate;

triethylsilyltriflate;

triisopropylsilyltriflate;

or

tert-butyldimethylsilyltriflate.

6. The process of claim 2, wherein the silylating reagent is N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyltriflate, chlorotrimethylsilane, or 1-(trimethylsilyl)imidazole.

7. The process of claim 2, wherein the silylating reagent is N,O-bis(trimethylsilyl)trifluoroacetamide.

8. The process of claim 2, wherein the H-phosphonate is covalently linked to a solid phase.

9. A process for the preparation of an oligonucleotide of structure IIIc comprising the steps of:

i) reacting an H-phosphonate of structure Ic with a silylating reagent to provide a silyloxyphosphonate;

ii) reacting the silyloxyphosphonate with a bis(thiosulfonate) reagent of structure IVc to provide a phosphorothiotriester comprising a thiosulfonate group of structure Vc;

iii) reacting the phosphorothiotriester comprising a thiosulfonate group of structure Vc with a nucleophile of structure VIc to provide the oligonucleotide of structure IIIc;

wherein;

the H-phosphonate of structure Ic has the following structure;

10. The process of claim 9, wherein the oligonucleotide of structure IIIc comprises non-stereorandom phosphorous linkages and the H-phosphonate of structure Ic comprises non-stereorandom phosphorous linkages.

11. The process of claim 10, wherein each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or 5-methylcytosine.

12. The process of claim 9, wherein R⁶ is methyl.

13. The process of claim 9, wherein the bis(thiosulfonate) reagent of structure IVc is:

14. The process of claim 9, wherein the nucleophile of structure VIc has the following structure:

15. The process of claim 9, wherein the silylating reagent is

1,1,3,3-tetramethyl-1,3-diphenyldisilazane;

1,3-dimethyl-1,1,3,3-tetraphenyldisilazane;

1-(trimethylsilyl)imidazole;

N-trimethylsilyl-N-methyl trifluoroacetamide;

bis(dimethylamino)dimethylsilane;

bromotrimethylsilane;

chlorodimethyl(pentafluorophenyl)silane;

chlorotriethylsilane;

chlorotriisopropylsilane;

chlorotrimethylsilane;

dichlorodimethylsilane;

hexamethyldisilazane;

N,N′

-bis(trimethylsilyl)urea;

N,N-bis(trimethylsilyl)methylamine;

N,N-dimethyltrimethylsilylamine;

N,O-bis(trimethylsilyl)acetamide;

N,O-bis(trimethylsilyl)carbamate;

N,O-bis(trimethylsilyl)trifluoroacetamide;

N-methyl-N-(trimethylsilyl)trifluoroacetamide;

N-methyl-N-trimethylsilylacetamide;

N-methyl-N-trimethylsilylheptafluorobutyramide;

N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide;

N-methyl-N-trimethylsilylheptafluorobutyramide;

trimethylsilyltriflate;

triethylsilyltriflate;

triisopropylsilyltriflate;

or

tert-butyldimethylsilyltriflate.

16. The process of claim 9, wherein the silylating reagent is N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyltriflate, chlorotrimethylsilane, or 1-(trimethylsilyl)imidazole.

17. The process of claim 9, wherein the silylating reagent is N,O-bis(trimethylsilyl)trifluoroacetamide.

18. The process of claim 9, wherein the H-phosphonate is covalently linked to a solid phase.