Method for labeling the 3' terminus of a synthetic oligonucleotide using a unique multifunctional controlled pore glass (MF-CPG) reagent in solid phase oligonucleotide synthesis
First Claim
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1. In an improved automated nucleotide synthesis process yielding an unbound oligonucleotide having a protected primary amine, sulfhydryl, disulfide, or hydroxyl attached to the 3'"'"' terminus, wherein the improvement comprises:
- using a multifunctional solid support reagent having the structure ##STR6## wherein;
C=carbon atom;
W=a solid support which is stable under all conditions of solid phase oligonucleotide synthesis selected from the group consisting ofalkylamine CPG, wherein alkyl is from 1 to 50 carbon atoms, inclusive, and isomeric forms thereof;
chemically modified CPG, with modifications selected from the group consisting of hydroxyl, carboxyl, sulfhydryl, and disulfide; and
copolymers of styrene and divinylbenzene;
X=a cleavable linking arm connecting carbon C to W characterized as a combination of atom groups that covalently connects to the solid phase through a cleavable linkage, is stable to all the conditions of solid phase oligonucleotide synthesis, and which is readily cleaved from the solid phase, selected from the group consisting of --(CH2)n --, where n is an integer from 1 to 50, --OCO--, --CO2, --NHCO--, --CONH--, and --O--CO--CH2 --CH2 --CO--;
Y=a linking arm connecting carbon C to R1 O-- that is at least one carbon atom long characterized as a combination of atom groups that covalently connects to OR1 and is stable to all the conditions of solid phase oligonucleotide synthesis selected from the group consisting of --(CH2)n --, where n is an integer from 1 to 50, --OCO--, --CO2, --NHCO--, --CONH--, and --O--CO--CH2 --CH2 --CO--;
Z=a molecule selected from the group consisting of --(CH2)n --, where n is an integer from 1 to 50, --OCO--, --CO2, --NHCO--, and --CONH--, wherein said molecule is bonded between the variable A-- and carbon C, wherein the ester and amide bonds are directed toward the A or C substituent;
R1 O=a protected hydroxide group wherein R1 is a base-stable/acid-labile hydroxyl protecting group selected from the group consisting of monomethoxytrityl and dimethoxytrityl;
A=a functional group that is capable of attaching a reporter molecule or a detectable complex wherein A is selected from the group consisting of primary amine, sulfhydryl, disulfide, and hydroxyl;
R2 =corresponding protecting group for A that is stable to all the conditions of solid phase oligonucleotide synthesis wherein said protecting group is 9-fluorenylmethyl (Fmoc) or trifluoroacetyl (TFA); and
X'"'"'=a molecule that is inert to solid phase oligonucleotide synthesis selected from the group consisting of H, alkyl from 1 to 50 carbon atoms, inclusive, and isomeric forms thereof, --Z--A--R2, and --Y--OR1.
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Abstract
A method for derivatizing and labeling the 3'"'"'-terminus of an oligonucleotide during solid phase synthesis comprising the use of a multifunctional reagent whose preferred structure is shown below. ##STR1## wherein CPG is controlled pore glass beads, Fmoc is 9-fluorenylmethyoxycarbonyl, and the alkylamine contains 1 to 50 carbon atoms.
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Citations
9 Claims
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1. In an improved automated nucleotide synthesis process yielding an unbound oligonucleotide having a protected primary amine, sulfhydryl, disulfide, or hydroxyl attached to the 3'"'"' terminus, wherein the improvement comprises:
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using a multifunctional solid support reagent having the structure ##STR6## wherein;
C=carbon atom;W=a solid support which is stable under all conditions of solid phase oligonucleotide synthesis selected from the group consisting of alkylamine CPG, wherein alkyl is from 1 to 50 carbon atoms, inclusive, and isomeric forms thereof; chemically modified CPG, with modifications selected from the group consisting of hydroxyl, carboxyl, sulfhydryl, and disulfide; and copolymers of styrene and divinylbenzene; X=a cleavable linking arm connecting carbon C to W characterized as a combination of atom groups that covalently connects to the solid phase through a cleavable linkage, is stable to all the conditions of solid phase oligonucleotide synthesis, and which is readily cleaved from the solid phase, selected from the group consisting of --(CH2)n --, where n is an integer from 1 to 50, --OCO--, --CO2, --NHCO--, --CONH--, and --O--CO--CH2 --CH2 --CO--; Y=a linking arm connecting carbon C to R1 O-- that is at least one carbon atom long characterized as a combination of atom groups that covalently connects to OR1 and is stable to all the conditions of solid phase oligonucleotide synthesis selected from the group consisting of --(CH2)n --, where n is an integer from 1 to 50, --OCO--, --CO2, --NHCO--, --CONH--, and --O--CO--CH2 --CH2 --CO--; Z=a molecule selected from the group consisting of --(CH2)n --, where n is an integer from 1 to 50, --OCO--, --CO2, --NHCO--, and --CONH--, wherein said molecule is bonded between the variable A-- and carbon C, wherein the ester and amide bonds are directed toward the A or C substituent; R1 O=a protected hydroxide group wherein R1 is a base-stable/acid-labile hydroxyl protecting group selected from the group consisting of monomethoxytrityl and dimethoxytrityl; A=a functional group that is capable of attaching a reporter molecule or a detectable complex wherein A is selected from the group consisting of primary amine, sulfhydryl, disulfide, and hydroxyl; R2 =corresponding protecting group for A that is stable to all the conditions of solid phase oligonucleotide synthesis wherein said protecting group is 9-fluorenylmethyl (Fmoc) or trifluoroacetyl (TFA); and X'"'"'=a molecule that is inert to solid phase oligonucleotide synthesis selected from the group consisting of H, alkyl from 1 to 50 carbon atoms, inclusive, and isomeric forms thereof, --Z--A--R2, and --Y--OR1. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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Specification