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US 2006/0135759 A1Published application
Reusable universal polymer support for repetitive oligonucleotide synthesis
AssigneeCouncil of Scientific and Industrial Research
InventorsPradeep Kumar, Kailash Gupta
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Abstract
Present invention provides a reusable universal polymer support for repetitive Oligonucleotide synthesis and a method of synthesis thereof.
Claims19
A reusable universal polymer support for repetitive oligonucleotide synthesis having structural formula I:
- where, n is number of alkyl groups selected from 1-6 C chain and R1 is substituted or unsubstituted alkyl groups selected from —CH2CH2—, —CH(CH3)nCH2—, —CH2CH(CH3)n—, —CH(CH3)nCH(CH3)n—, —CH(CH2OCH2)CH—, —CH(CH2CH2CH2)CH and P is an organic or inorganic polymer support.
A process for preparation of reusable universal polymer support of formula I, said process comprises steps of;
- (i) treating the unmodified CPG with 3-mercaptopropyltriethoxysilane to obtain 3-mercaptopropylated CPG;
- (ii) activating the support obtained in step (i) above with a thiol specific reagent, DTNP to obtain an activated polymer support;
- (iii) reacting the polymer support obtained in step (ii) with DMTrO(CH2)6SH followed by TCA in the range of 2-4% in dichloroethane to obtain a disulfide linkage containing hydroxyl-CPG, and
- (iv) coupling universal linker namely 2(3)-O-dimethoxytrityl-anhydroerythritol-3(2)-O-succinate, with the support obtained in step (iii) above in the presence of a condensing reagent to obtain a reusable universal polymer support.
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A method of using universal polymer support of formula I for repetitive oligunucleotide synthesis, said method comprises steps of;
- (i) treating the unmodified CPG with 3-mercaptopropyltriethoxysilane to obtain 3-mercaptopropylated CPG;
- (ii) activating the support obtained in step (i) above with a thiol specific reagent, DTNP to obtain an activated polymer support;
- (iii) reacting;
- the polymer support obtained in step (ii) with DMTrO(CH2)6SH followed by TCA in the range of 2-4% in dichloroethane to obtain a disulfide linkage containing hydroxyl-CPG;
- (iv) coupling universal linker namely 2(3)-O-dimethoxytrityl-anhydroerythritol-3(2)-O-succinate, with the support obtained in step (iii) above in the presence of a condensing reagent to obtain a reusable universal polymer support;
- (v) synthesizing an oligonucleotide sequence on the reusable universal polymer support obtained in step (iv) followed by cleavage of oligomer chains under non-alkaline conditions;
- (vi) refunctionalizing the reusable universal polymer support obtained in step (v) after cleavage reaction repeating the steps (ii) to (iv) followed by synthesizing another cycle of oligomer sequence, and
- (vii) deprotecting the oligonucleotides completely obtained in step (v) with aq. ammonium hydroxide containing spermine in the range of 0.5 to 2M and lithium chloride 0.25 to 0.7M for a time period in the range of 6 to 10 hr. at a temperature in the range of 60-75° C.
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Dependencies are inferred from the claim text.