Fluorescent reagents for the preparation of 5'-tagged oligonucleotides
First Claim
1. A nonnucleoside-containing chemical reagent having the formula
wherein O is a phosphorus group suitable for attachment to the 5'"'"'-hydroxyl group of an oligonucleotide selected from the group of phosphorus groups consisting of ##STR13## and salts thereof;
wherein R2 and R3 are independently selected from the group consisting of C3 -C10 branched alkyl and C1 -C10 unbranched alkyl;
wherein R4 is selected from the group consisting of --(CRR1)m (A)n (CRR1)m --, ##STR14## wherein A is selected from the group consisting of --O--, --S--, ##STR15## wherein R and R1 are independently selected from the group consisting of H, C3 -C10 branched alkyl, C1 -C10 unbranched alkyl, C6 -C10 aryl, C7 -C12 alkaryl, and C7 -C12 aralkyl;
wherein m=1-6, and n=0-1, provided that 2m+n≦
12;
wherein X is selected from the group consisting of --F, --Cl, --Br, --I, imidazol-l-yl, 1,2,4-triazol-1-yl, and 1-hydroxybenzotriazol-O-yl;
wherein Y is selected from the group consisting of 4-Cl-C6 --H4 --O--, 2-Cl-C6 H4 --O--, 4-NO2 -C6 H4 --O--, 4-NO2 -C6 H4 CH2 CH2 --O--, 2,4-NO2 -C6 H3 CH2 CH2 --O--, 2,4-Cl2 -C6 H3 --O--, 2,3-Cl2 -C6 H3 --O--, NCCH2 CH2 O--, NCCH2 C(CH3)2 --O--, CH3 O--, (Z)3 CCH2 --O--, (Z)3 CC(CH3)2 --O--, R'"'"'S--, R'"'"'SCH2 CH2 --O--, R'"'"'SO2 CH2 CH2 --O--, and R'"'"'NH--;
wherein Z is selected from the group consisting of Cl, Br, and I;
wherein R'"'"' is selected from the group consisting of H, C3 -C10 branched alkyl, C1 -C10 unbranched alkyl, C6 -C10 aryl, C7 -C12 alkaryl, and C7 -C12 aralkyl;
wherein the --O-- in --O--reporter was formerly part of a hydroxyl group and is covalently linked to said Q; and
wherein the --O--reporter is either (i) a fluorescent reporter selected from the group of fluorescent dyes consisting of xanthenes, rhodamines, benzamidizoles, ethidiums, propidiums, anthracyclines, mithramycins, acridines, actinomycins, merocyanines, coumarins, pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes, salicyclic acids, benz-2-oxa-diazoles, and fluorescamine, or (ii) a protected form of one of said fluorescent reporters.
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Accused Products
Abstract
Fluorescent reagents possessing an activated phosphate for the convenient covalent coupling to the 5'"'"'-hydroxyl of oligonucleotides. A class of 5═-fluorescence-tagged oligonucleotides is also disclosed.
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Citations
7 Claims
- 1. A nonnucleoside-containing chemical reagent having the formula
- space="preserve" listing-type="equation">Q-O-reporter
wherein O is a phosphorus group suitable for attachment to the 5'"'"'-hydroxyl group of an oligonucleotide selected from the group of phosphorus groups consisting of ##STR13## and salts thereof;
wherein R2 and R3 are independently selected from the group consisting of C3 -C10 branched alkyl and C1 -C10 unbranched alkyl;wherein R4 is selected from the group consisting of --(CRR1)m (A)n (CRR1)m --, ##STR14## wherein A is selected from the group consisting of --O--, --S--, ##STR15## wherein R and R1 are independently selected from the group consisting of H, C3 -C10 branched alkyl, C1 -C10 unbranched alkyl, C6 -C10 aryl, C7 -C12 alkaryl, and C7 -C12 aralkyl; wherein m=1-6, and n=0-1, provided that 2m+n≦
12;wherein X is selected from the group consisting of --F, --Cl, --Br, --I, imidazol-l-yl, 1,2,4-triazol-1-yl, and 1-hydroxybenzotriazol-O-yl; wherein Y is selected from the group consisting of 4-Cl-C6 --H4 --O--, 2-Cl-C6 H4 --O--, 4-NO2 -C6 H4 --O--, 4-NO2 -C6 H4 CH2 CH2 --O--, 2,4-NO2 -C6 H3 CH2 CH2 --O--, 2,4-Cl2 -C6 H3 --O--, 2,3-Cl2 -C6 H3 --O--, NCCH2 CH2 O--, NCCH2 C(CH3)2 --O--, CH3 O--, (Z)3 CCH2 --O--, (Z)3 CC(CH3)2 --O--, R'"'"'S--, R'"'"'SCH2 CH2 --O--, R'"'"'SO2 CH2 CH2 --O--, and R'"'"'NH--; wherein Z is selected from the group consisting of Cl, Br, and I; wherein R'"'"' is selected from the group consisting of H, C3 -C10 branched alkyl, C1 -C10 unbranched alkyl, C6 -C10 aryl, C7 -C12 alkaryl, and C7 -C12 aralkyl; wherein the --O-- in --O--reporter was formerly part of a hydroxyl group and is covalently linked to said Q; and wherein the --O--reporter is either (i) a fluorescent reporter selected from the group of fluorescent dyes consisting of xanthenes, rhodamines, benzamidizoles, ethidiums, propidiums, anthracyclines, mithramycins, acridines, actinomycins, merocyanines, coumarins, pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes, salicyclic acids, benz-2-oxa-diazoles, and fluorescamine, or (ii) a protected form of one of said fluorescent reporters. - View Dependent Claims (2, 3, 4, 5, 6)
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7. A chemical compound of the structure ##STR18## wherein R5 and R6 are independently selected from the group consisting of --H, C1 -C4 alkyl, --F, --Cl, --Br, --I, C1 -C4 alkoxy, and --CN;
- B is selected from the group consisting of 1-thyminyl, 1-cytosinyl, 1-uracilyl, 9-adeninyl, and 9-guaninyl;
R9 is selected from the group consisting of --H and --OH; and
n=0 to about 100.
- B is selected from the group consisting of 1-thyminyl, 1-cytosinyl, 1-uracilyl, 9-adeninyl, and 9-guaninyl;
Specification
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- Resources
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Current AssigneeNEN Life Science Products Inc (Revvity, Inc.)
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Original AssigneeE. I. du Pont de Nemours and Company (Corteva, Inc.)
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InventorsHobbs, Frank W. Jr.
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Primary Examiner(s)Brown, Johnnie R.
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Assistant Examiner(s)Kunz, Gary L.
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Application NumberUS07/244,982Time in Patent Office901 DaysField of Search435/6, 536/28, 536/27, 536/29, 558/93, 558/95, 549/385, 546/242US Class Current536/24.3CPC Class CodesC07F 9/65586 at least one of the hetero ...C07H 21/00 Compounds containing two or...G01N 33/582 with fluorescent label
