Sensitive oligonucleotide synthesis using sulfur-based functions as protecting groups and linkers
View Patent ↗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.
1 . 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.
2 . The process of claim 1 wherein said three-valent phosphorus compound is selected from the group consisting (I-V):
Wherein n is an integer selected from 0-10.
3 . The process of claim 1 wherein said phosphoramidite tagging agent is selected from the group consisting (VI-XVII):
Wherein R 1 is a trityl group or monomethoxytrityl group; R 2 are independently H or an alkyl group with less than 10 carbons; and n are independently integers selected from 0-5.
4 . The process of claim 1 wherein said phosphoramidite tagging agent is selected from the group consisting (XVIII-XXI):
Wherein R 1 is a trityl group or monomethoxytrityl group; R 2 are independently H or an alkyl group with less than 10 carbons; R 3 =Me, Et, —(CH 2 ) 2 OMe, Triisopropylsilyloxymethyl (Tom) or tert-Butyldimethylsilyl (TBDMS); and n are independently integers selected from 0-5.
5 . The process of claim 1 wherein said phosphoramidite tagging agent is selected from the group consisting (XXII-XXXII):
Wherein R 1 is a trityl group or monomethoxytrityl group; R 2 are independently H or an alkyl group with less than 10 carbons; and n are independently integers selected from 0-5.
6 . The process of claim 1 wherein said phosphoramidite tagging agent is selected from the group consisting (XXXIII-XLVII):
Wherein R 1 is a trityl group or monomethoxytrityl group; R 2 are independently H or an alkyl group with less than 10 carbons; and n are independently integers selected from 0-5.
7 . Derivatized nucleoside phosphoramidites having the general formula (XLVIII), wherein at least one of the R 4 , R 5 and R 9 groups contain a sulfur-based protecting group as defined in said groups:
Wherein, R 4 is selected from the group consisting (XLIX-LIV):
Wherein R 4a is —SR 4a1 with R 4a1 being independently an alkyl group, derivatized alkyl group, aryl group or derivatized aryl group; and R 4b is independently H, alkyl group, derivatized alkyl group, aryl group, derivatized aryl group, or —SR 4b1 with R 4b1 being independently an alkyl group, derivatized alkyl group, aryl group or derivatized aryl group; or R 4a -R 4b =—S[C(R 4a2 )R 4b3 ] n S— or —S{[C(R 4a2 )R 4a3 ] n O[C(R 4a2 )R 4a3 ] m } p S— wherein independently R 4a2 and R 4b3 are H or alkyl groups, m and n are independently integers larger than 1, and p is a positive integer;
R 4c and R 4d are independently H, alkyl group, derivatized alkyl group, aryl group, or derivatized aryl group including instances wherein R 4c and R 4d are connected to form, cycle;
R 5 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 R 4a , R 4b , R 4c and R 4d are defined for R 4 ;
R 5a are independently alkyl, derivatized alkyl, aryl or derivatized aryl groups;
R 5b is H, alkyl group, or derivatized alkyl group;
R 6 is defined by (LX):
Wherein R 6a , R 6b and R 6c are independently H, alkyl groups, derivatized alkyl groups, alkoxyl groups, aryl groups and derivatized aryl groups;
R 7 and R 8 are independently alkyl groups or derivatized alkyl groups, or R 7 and R 8 taken together form a nitrogen-containing cycle;
R 9 is an alkyl group, derivatized alkyl group, alkoxyl group, derivatized alkoxyl group —O[C(R 9a )R 9b C(H)R 9c ]CN wherein R 9a , R 9b , and R 9c are independently H or alkyl groups, or (LVII).
8 . Derivatized nucleoside phosphoramidites of claim 7 wherein (XLVIII) is selected from the group consisting (XLVIII 1 -XLVIII 7 ):
Wherein R 2 is independently H or alkyl group with less than six carbons; n are independently integers selected from 0-5; and DMTr is 4,4′-dimethoxytrityl.
9 . Derivatized nucleoside phosphoramidites of claim 7 wherein (XLVIII) is selected from the group consisting (XLVIII 8 -XLVIII 14 ):
Wherein R 2 is H or an alkyl group with less than six carbons; R 3 =Me, Et, —(CH 2 ) 2 OMe, Triisopropylsilyloxymethyl (Tom) or tert-Butyldimethylsilyl (TBDMS); n are independently integers selected from 0-5; and DMTr is 4,4′-dimethoxytrityl.
10 . Derivatized nucleoside phosphoramidites of claim 7 wherein (XLVIII) is selected from the group consisting (XLVIII 15 -XLVIII 22 ):
Wherein R 2 are independently H or an alkyl group with less than six carbons; n are independently integers selected from 0-5; and DMTr is 4,4′-dimethoxytrityl.
11 . 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 R 4 is —SR 4a1 with R 4a1 being independently an alkyl group, derivatized alkyl group, aryl group or derivatized aryl group; and R 4b is independently H, alkyl group, derivatized alkyl group, aryl group, derivatized aryl group, or —SR 4b1 with R 4b1 being independently an alkyl group, derivatized alkyl group, aryl group or derivatized aryl group; or R 4 -R 4 =—S[C(R 4a2 )R 4a3 ] n S— or —S{[C(R 4a2 )R 4a3 ] n O[C(R 4a2 )R 4a3 ] m } p S— wherein independently R 4a2 and R 4a3 are H or alkyl groups, m and n are independently integers larger than 1, and p is a positive integer;
R 5 and R 10 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 R 4a and R 4b are defined as for (LXI) above;
R 4c and R 4d are independently H, alkyl group, derivatized alkyl group, aryl group, or derivatized aryl group including instances wherein R 4c and R 4d are connected to form a cycle;
R 5a are independently alkyl, derivatized alkyl, aryl or derivatized aryl groups;
R 5b is H, alkyl group, or derivatized alkyl group;
R 6 is defined by (LX):
Wherein R 6a , R 6b and R 6c 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):
12 . Cleavable linkers of claim 11 wherein formula (LXI) is selected from the group consisting (LXI 1 -LXI 4 ):
Wherein R 2 is H or an alkyl group with less than 10 carbons; R 11 is H, alkyl group, derivatized alkyl group, aryl group, derivatized aryl group, amino group, derivatized amino group, alkoxy group or derivatized alkoxy group; m, n and o are integers 0-18, 0-5, and 0-18, respectively; and DMTr is 4,4′-dimethoxytrityl.
13 . Cleavable linkers of claim 11 wherein formula (LXI) is selected from the group consisting (LXI 9 -LXI 12 ):
Wherein R 2 is H or an alkyl group with less than 10 carbons; R 11 is H, alkyl group, derivatized alkyl group, aryl group, derivatized aryl group, amino group, derivatized amino group, alkoxy group or derivatized alkoxy group; m, n and o are integers 0-18, 0-5, and 0-18, respectively; and DMTr is 4,4′-dimethoxytrityl.
14 . Cleavable linkers of claim 11 wherein formula (LXI) is selected from the group consisting (LXI 13 -LXI 16 ):
Wherein R 3 =Me, Et, —(CH 2 ) 2 OMe, Triisopropylsilyloxymethyl (Tom) or tert-Butyldimethylsilyl (TBDMS); R 11 is H, alkyl group, derivatized alkyl group, aryl group, derivatized aryl group, amino group, derivatized amino group, alkoxy group or derivatized alkoxy group; m, n and o are integers 0-18, 0-5, and 0-18, respectively; and DMTr is 4,4′-dimethoxytrityl.