US 6,642,373 B2 · Activators for oligonucleotide synthesis

Abstract

The present invention relates to improved methods for the preparation of nucleoside phosphoramidites and oligonucleotides. In one aspect, the methods of the invention are used to prepare phosphitylating reagents using pyridinium salts as activators. In a further aspect, the methods of the invention are used to prepare internucleoside linkages using activators which include at least one pyridinium salt and at least one substituted imidazole. In a further aspect, methods are provided preparation of internucleoside linkages between nucleosides having 2′-substituents using imidazolium or benzimidazolium salts as an activator. In a further aspect, methods are provided preparation of internucleoside linkages between nucleosides having bioreversible protecting group that confers enhanced chemical and biophysical properties, without exocyclic amine protection, using imidazolium or benzimidazolium salts as an activator.

Claims71

Dependent claims

A method for the preparation of an oligonucleotide comprising the steps of:

providing a 3′-nucleoside phosphoramidite or 3′-oligonucleotide phosphoramidite;
and
reacting said 3′-nucleoside phosphoramidite or 3′-oligonucleotide phosphoramidite with the 5′-hydroxyl of a nucleoside, or oligonucleotide in the presence of an activating reagent;
said nucleoside or oligonucleotide being optionally bound to a solid support;
said activating reagent comprising at least one pyridinium salt and one substituted imidazole.

2 more elements

A synthetic method comprising:

providing a compound of Formula X:
wherein:
B is a nucleobase;
R8 is H, a hydroxyl protecting group, or a linker connected to a solid support;
W is an optionally protected internucleoside linkage;
q is 0 to about 50;
R4 is H, F, O—R, S—R or N—R(R10);
R is H, a protecting group, or has one of the formulas:
where
each m is independently from 1 to 10;
y is from 0 to 10;
E is H, a hydroxyl protecting group, C1-C10 alkyl, N(R10)(R11) or N═C(R10)(R11);
substituted or unsubstituted C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, wherein the substitutions are selected from one or several halogen, cyano, carboxy, hydroxy, nitro and mercapto residues;
and
each R10 or R11 is, independently, H, substituted or unsubstituted C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, wherein the substitutions are selected from one or several halogen, cyano, carboxy, hydroxy, nitro and mercapto residues;
alkylthioalkyl, a nitrogen protecting group, or R10 and R11, together, are a nitrogen protecting group or wherein R10 and R11 are joined in a ring structure that can include at least one heteroatom selected from N and O;
or R is —CH2—CH2—O—CH2—CH2—N(R10)(R11);
reacting the compound of Formula X in the presence of an activating reagent with a compound of Formula XI:
where r is 0 to about 50;
R5 is a hydroxyl protecting group;
R6 is —N(R7)2 wherein R7 is alkyl having from one to about six carbons;
or R7 is a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
to form a compound of Formula XII:
wherein said activating reagent comprises at least one pyridinium salt and one substituted imidazole.

21 more elements

A synthetic method comprising:

providing a compound of Formula X:
wherein:
B is a nucleobase;
R8 is H, a hydroxyl protecting group, or a linker connected to a solid support;
W is an optionally protected internucleoside linkage;
q is 0 to about 50;
R4 is H, F, O—R, S—R or N—R(R10);
R is H, a protecting group, or has one of the formulas:
where
each m is independently from 1 to 10;
y is from 0 to 10;
E is H, a hydroxyl protecting group, C1-C10 alkyl, N(R10)(R11) or N═C(R10)(R11);
substituted or unsubstituted C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, wherein the substitutions are selected from one or several halogen, cyano, carboxy, hydroxy, nitro and mercapto residues;
and
each R10 or R11 is, independently, H, substituted or unsubstituted C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, wherein the substitutions are selected from one or several halogen, cyano, carboxy, hydroxy, nitro and mercapto residues;
alkylthioalkyl, a nitrogen protecting group, or R and R10, together, are a nitrogen protecting group or wherein R and R2 are joined in a ring structure that can include at least one heteroatom selected from N and O;
or R is —CH2—CH2—O—CH2—CH2—N(R10)(R11);
provided that R14 is not H or OH;
reacting the compound of Formula X in the presence of an activator with a compound of Formula XI:
where r is 0 to about 50;
R6 is a hydroxyl protecting group;
R6 is —N(R7)2 wherein R7 is alkyl having from one to about six carbons;
or R7 is a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
to form a compound of Formula XII:
wherein the activator has the formula G+U−, where
G+ is selected from the group consisting of pyridinium, imidazolium, and benzimidazolium;
and
U− is selected from the group consisting of hexafluorophosphate, tetrafluoroborate, triflate, hydrochloride, trifluoroacetate, dichloroacetate, −O-mesyl, −O-tosyl, −Br, and −O-trifluorosulfonyl.

25 more elements

A synthetic method comprising:

providing a compound of Formula XX:
wherein:
R4 is H, F, O—R, S—R or N—R(R10);
R is H, a protecting group, or has one of the formulas:
where
each m is independently from 1 to 10;
y is from 0 to 10;
E is H, a hydroxyl protecting group, C1-C10 alkyl,
N(R10)(R11) or N═C(R10)(R11);
substituted or unsubstituted C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, wherein the substitutions are selected from one or several halogen, cyano, carboxy, hydroxy, nitro and mercapto residues;
and
each R10 or R11 is, independently, H, substituted or unsubstituted C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, wherein the substitutions are selected from one or several halogen, cyano, carboxy, hydroxy, nitro and mercapto residues;
alkylthioalkyl, a nitrogen protecting group, or R10 and R11, together, are a nitrogen protecting group or wherein R10 and R11 are joined in a ring structure that can include at least one heteroatom selected from N and O;
or R is —CH2—CH2—O—CH2—CH2—N(R10)(R11);
R5 is a hydroxyl protecting group;
Z1 is aryl having 6 to about 14 carbon atoms or alkyl having from one to about six carbon atoms;
Y1 is O or S;
Y2 is O or S;
Y3 is C(═O) or S;
v is 2 to about 4;
B is a nucleobase;
R6 is —N(R7)2 wherein R7 is alkyl having from one to about six carbons;
or R7 is a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
reacting said compound of Formula XX with a compound of Formula XXI:
wherein:
R8 is H, a hydroxyl protecting group, or a linker connected to a solid support;
in the presence of an activator to form a compound of Formula XXII:
wherein said activator has the formula G+U−, where:
G+ is selected from the group consisting of pyridinium, imidazolium, and benzimidazolium;
and
U− is selected from the group consisting of hexafluorophosphate, tetrafluoroborate, triflate, hydrochloride, trifluoroacetate, dichloroacetate, −O-mesyl, −O-tosyl, −Br, and −O-trifluorosulfonyl;
or said activator is a substituted imidazolium triflate.

29 more elements

Dependencies are inferred from the claim text.

Activators for oligonucleotide synthesis

Life of this patent

Activators for oligonucleotide synthesis

Life of this patent

Abstract

Claims71