Methods for the synthesis of functionalized nucleic acids

Patent metadata

Field	Value
Publication number	US-9605019-B2
Application number	US-201214233579-A
Country	US
Kind code	B2
Filing date	Jul 13, 2012
Priority date	Jul 19, 2011
Publication date	Mar 28, 2017
Grant date	Mar 28, 2017

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.

First claim

What is claimed is:

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:

wherein:
W is O;
R 3 is —OH, —SH, —NR d R d , —N 3 , halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —P(O)(R e ) 2 , —OR a or —SR c ;
Y 1 is O, NR d , S, or Se;
R a is a blocking group;
R c is a blocking group;
each instance of R d is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, or tertiary silyl, or when R d is attached to a nitrogen atom, —C(O)—O-alkyl, —C(O)—O-alkenyl, —C(O)—O-alkynyl, —C(O)—O-heteroalkyl, —C(O)—O-heteroalkenyl, —C(O)—O-heteroalkynyl, —C(O)—O-aryl, or —C(O)—O-heteroaryl,
each instance of R e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y 2 —, alkenyl-Y 2 —, alkynyl-Y 2 —, aryl-Y 2 —, or heteroaryl-Y 2 —, or a cation which is Na +1 , Li +1 , or K +1 ;
Y 2 is O, NR d , or S;
each instance of R 4 is independently hydrogen, —OH, —SH, —NR d R d , —N 3 , halogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —OR b , or —SR c , and R b is a blocking group;
each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;
R 5 is hydrogen, a blocking group, a linking moiety connected to a solid support or a linking moiety connected to a nucleic acid; and
n is between 1 and about 200; and

the thiosulfonate reagent of structure IIa has the following structure:

wherein:
X is alkyl, cycloalkyl, or heteroaryl;
R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or R 1 —R 2 ;
R 1 is —S-alkenylene-, —S-alkylene-, —S-alkylene-aryl-alkylene-, —S—CO-aryl-alkylene-, or —S—CO-alkylene-aryl-alkylene-;
R 2 is heterocyclo-alkylene-S—, heterocyclo-alkenylene-S—, aminoalkyl-S—, or (alkyl) 4 N-alkylene-S—;

and the oligonucleotide of structure IIIa has the following structure:

wherein:
W is O;
R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or R 1 —R 2 ;
R 1 is —S-alkenylene-, —S-alkylene-, —S-alkylene-aryl-alkylene-, —S—CO-aryl-alkylene-, or —S—CO-alkylene-aryl-alkylene-;
R 2 is heterocyclo-alkylene-S—, heterocyclo-alkenylene-S—, aminoalkyl-S—, or (alkyl) 4 N-alkylene-S—;
R 3 is —OH, —SH, —NR d R d , —N 3 , halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —P(O)(R e ) 2 , —OR a or —SR c ;
Y 1 is O, NR d , S, or Se;
R a is a blocking group;
R c is a blocking group;
each instance of R d is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, or tertiary silyl, or when R d is attached to a nitrogen atom, —C(O)—O-alkyl, —C(O)—O-alkenyl, —C(O)—O-alkynyl, —C(O)—O-heteroalkyl, —C(O)—O-heteroalkenyl, —C(O)—O-heteroalkynyl, —C(O)—O-aryl, or —C(O)—O-heteroaryl;
each instance of R e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y 2 —, alkenyl-Y 2 —, alkynyl-Y 2 —, aryl-Y 2 —, or heteroaryl-Y 2 —, or a cation which is Na +1 , Li +1 , or K +1 ;
Y 2 is O, NR d , or S;
each instance of R 4 is independently hydrogen, —OH, —SH, —NR d R d , —N 3 , halogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —OR b , or —SR c , and R b is a blocking group;
each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;
R 5 is hydrogen, a blocking group, a linking moiety connected to a solid support or a linking moiety connected to a nucleic acid; and
n is between 1 and about 200; and
wherein each heteroaryl group is a mono-, bi-, or poly-cyclic 5-14 membered ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

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:

wherein:
at least one H-phosphonate linkage is non-stereorandom;
W is independently O, NH, or CH 2 ;
R 3 is —OH, —SH, —NR d R d , —N 3 , halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —P(O)(R e ) 2 , —OR a or —SR c ;
Y 1 is O, NR d , S, or Se;
R a is a blocking group;
R c is a blocking group;
each instance of R d is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl, or tertiary silyl, or when R d is attached to a nitrogen atom, —C(O)—O-alkyl, —C(O)—O-alkenyl, —C(O)—O-alkynyl, —C(O)—O-heteroalkyl, —C(O)—O-heteroalkenyl, —C(O)—O-heteroalkynyl, —C(O)—O-aryl, or —C(O)—O-heteroaryl,
each instance of R e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y 2 —, alkenyl-Y 2 —, alkynyl-Y 2 —, aryl-Y 2 —, or heteroaryl-Y 2 —, or a cation which is Na +1 , Li +1 , or K +1 ;
Y 2 is O, NR d , or S;
each instance of R 4 is independently hydrogen, —OH, —SH, —NR d R d , —N 3 , halogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —OR b , or —SR c , and R b is a blocking group;
each instance of Ba is independently a blocked or unblocked adenine, cytosine, guanine, thymine, uracil or modified nucleobase;
R 5 is hydrogen, a blocking group, a linking moiety connected to a solid support or a linking moiety connected to a nucleic acid; and
n is between 1 and about 200; and

the thiosulfonate reagent of structure IIb has the following structure:

wherein:
X is alkyl, cycloalkyl, aryl, or heteroaryl;
R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or R 1 —R 2 ;
R 1 is —S-alkenylene-, —S-alkylene-, —S-alkylene-aryl-alkylene-, —S—CO-aryl-alkylene-, or —S—CO-alkylene-aryl-alkylene-;
R 2 is heterocyclo-alkylene-S—, heterocyclo-alkenylene-S—, aminoalkyl-S—, or (alkyl) 4 N-alkylene-S—;
and the oligonucleotide of structure IIIb has the following structure:

wherein:
at least one phosphorothiotriester linkages is non-stereorandom,
W is O;
R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or R 1 —R 2 ;
R 1 is —S-alkenylene-, —S-alkylene-, —S-alkylene-aryl-alkylene-, —S—CO-aryl-alkylene-, or —S—CO-alkylene-aryl-alkylene-;
R 2 is heterocyclo-alkylene-S—, heterocyclo-alkenylene-S—, aminoalkyl-S—, or (alkyl) 4 N-alkylene-S—;
R 3 is —OH, —SH, —NR d R d , —N 3 , halogen, hydrogen, alkyl, alkenyl, alkynyl, alkyl-Y 1 —, alkenyl-Y 1 —, alkynyl-Y 1 —, aryl-Y 1 —, heteroaryl-Y 1 —, —P(O)(R e ) 2 , —OR a or —SR c ;
Y 1 is O, NR d , S, or Se;
R a

Assignees

• Verdine Gregory L
• Meena
• Iwamoto Naoki
• Butler David Charles Donnell
• Wave Life Sciences Ltd

Inventors

• Verdine Gregory L
• Meena
• Iwamoto Naoki
• Butler David Charles Donnell

Classifications

• C07H21/04 Primary

  with deoxyribosyl as saccharide radical · CPC title

• C07D295/108

  to an acyclic saturated chain · CPC title

• C07H13/04

  having the esterifying carboxyl radicals attached to acyclic carbon atoms · CPC title

• A61K31/70

  Carbohydrates; Sugars; Derivatives thereof (sorbitol A61K31/047) · CPC title

• A61K31/66

  Phosphorus compounds · CPC title