US 12,552,828 B2 · Sensitive oligonucleotide synthesis using sulfur-based functions as protecting groups and linkers

Abstract

Embodiments for the synthesis of sensitive oligonucleotides as well as insensitive oligonucleotides are provided. Sulfur-based groups are used for the protection of exo-amino groups of nucleobases, phosphate groups and 2′-OH groups, and as cleavable linker for linking oligonucleotides to a support. Oligonucleotide syntheses are achieved under typical conditions using phosphoramidite chemistry with important modifications. To prevent replacing sulfur-based protecting groups by acyl groups via cap-exchange, special capping agents are used. To retain hydrophobic tag to assist RP HPLC purification, special phosphoramidites are used in the last synthetic cycle. With the sulfur-based groups for protection and linking, oligonucleotide deprotection and cleavage are achieved via oxidation followed by beta-elimination under mild conditions. Therefore, besides for insensitive oligonucleotide synthesis, the embodiments of the invention are capable for the synthesis of oligonucleotide analogs containing sensitive functional groups that cannot survive the harsh conditions used in prior art oligonucleotide synthesis technologies.

Claims14

Dependent claims

A process for the synthesis of oligonucleotides, wherein the oligonucleotides comprise modified oligonucleotides, and the process comprises the use of at least one sulfur-based protecting group, wherein the sulfur-based protecting group is capable of being deprotected via oxidation followed by beta-elimination, wherein the process further comprise:

capping failure sequences with a three-valent phosphorus compound containing at least one phosphorus-nitrogen bond,
tagging full-length sequences of the oligonucleotides with a phosphoramidite tagging agent comprising a hydrophobic group based on the triphenylmethyl (trityl) structure comprising more than one electron-donating alkoxy or dialkylamino group,
or a combination of the capping and the tagging step,
whereby said process makes easier the separation of the full-length sequences from the failure sequences using reversed phase chromatography.

1 more element

Derivatized nucleoside phosphoramidites having the general formula (XLVIII), wherein at least one of the R4, R5 and R9 groups contain a sulfur-based protecting group as defined in said groups:

Wherein, R4 is selected from the group consisting (XLIX-LIV):
Wherein R4a is —SR4a1 with R4a1 being independently an alkyl group, derivatized alkyl group, aryl group or derivatized aryl group;
and R4b is independently H, alkyl group, derivatized alkyl group, aryl group, derivatized aryl group, or —SR4b1 with R4b1 being independently an alkyl group, derivatized alkyl group, aryl group or derivatized aryl group;
or R4a-R4b=—S[C(R4a2)R4b3]nS— or —S{[C(R4a2)R4a3]nO[C(R4a2)R4a3]m}pS— wherein independently R4a2 and R4b3 are H or alkyl groups, m and n are independently integers larger than 1, and p is a positive integer;
R4c and R4d are independently H, alkyl group, derivatized alkyl group, aryl group, or derivatized aryl group including instances wherein R4c and R4d are connected to form, cycle;
R5 is selected from the group consisting —H, —F, alkyl group, derivatize alkyl group, alkoxyl group, derivatized alkoxyl group, amino group, derivatized amino group, and (LV-LIX):
Wherein R4a, R4b, R4c and R4d are defined for R4;
R5a are independently alkyl, derivatized alkyl, aryl or derivatized aryl groups;
R5b is H, alkyl group, or derivatized alkyl group;
R6 is defined by (LX):
Wherein R6a, R6b and R6c are independently H, alkyl groups, derivatized alkyl groups, alkoxyl groups, aryl groups and derivatized aryl groups;
R7 and R8 are independently alkyl groups or derivatized alkyl groups, or R7 and R8 taken together form a nitrogen-containing cycle;
R9 is an alkyl group, derivatized alkyl group, alkoxyl group, derivatized alkoxyl group —O[C(R9a)R9bC(H)R9c]CN wherein R9a, R9b, and R9c are independently H or alkyl groups, or (LVII).

10 more elements

Cleavable linkers comprising a sulfur-based group between a support and nucleoside derivative of the general formula (LXI), whereby cleavage can be achieved by oxidation of the sulfur-based group followed by beta elimination:

Wherein R4 is —SR4a1 with R4a1 being independently an alkyl group, derivatized alkyl group, aryl group or derivatized aryl group;
and R4b is independently H, alkyl group, derivatized alkyl group, aryl group, derivatized aryl group, or —SR4b1 with R4b1 being independently an alkyl group, derivatized alkyl group, aryl group or derivatized aryl group;
or R4-R4=—S[C(R4a2)R4a3]nS— or —S{[C(R4a2)R4a3]nO[C(R4a2)R4a3]m}pS— wherein independently R4a2 and R4a3 are H or alkyl groups, m and n are independently integers larger than 1, and p is a positive integer;
R5 and R10 are independently selected from the group consisting —H, —F, alkyl group, derivatize alkyl group, alkoxyl group, derivatized alkoxyl group, amino group, derivatized amino group, carboxylate group, carbonate ester group, carbamate group and (LV-LIX):
Wherein R4a and R4b are defined as for (LXI) above;
R4c and R4d are independently H, alkyl group, derivatized alkyl group, aryl group, or derivatized aryl group including instances wherein R4c and R4d are connected to form a cycle;
R5a are independently alkyl, derivatized alkyl, aryl or derivatized aryl groups;
R5b is H, alkyl group, or derivatized alkyl group;
R6 is defined by (LX):
Wherein R6a, R6b and R6c are independently H, alkyl groups, derivatized alkyl groups, alkoxyl groups, aryl groups and derivatized aryl groups;
L is a chain of atoms that links the molecule to a support;
and Base is defined by (LXII-LXV) with the nitrogen atom shown in the formula (LXI) connected to the carbon atom in (LXII-LXV):

9 more elements

Dependencies are inferred from the claim text.

Sensitive oligonucleotide synthesis using sulfur-based functions as protecting groups and linkers

Life of this patent

Sensitive oligonucleotide synthesis using sulfur-based functions as protecting groups and linkers

Life of this patent

Abstract

Claims14