Fluorogenic reagents for detection of glycoconjugates, .alpha.-ketoacids and diketones
AssigneeIndiana University Foundation
InventorsMilos V. Novotny, Osamu Shirota, Donald Wiesler
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Abstract
Compounds containing .alpha.-discarbonyl groups are rendered capable of fluorescent emission by conjugation with carboxylic acid-terminated ortho-diaminoaryl reagents. The carboxylic acid groups serve as ionizable moieties which facilitate the conjugation reaction, and are retained in the conjugation product to enable, or enhance the ability of, the conjugated compounds to be separated in chromatographic and electromigration separation processes, notably electrophoresis. Ortho-dinitro derivatives of the reagents are also disclosed, these derivatives offering the advantage of stability as well as the ability to be used directly in the reaction in the presence of a reducing agent.
Claims31
A method for detecting the presence of at least one .alpha.-dicarbonyl-containing analyte in a liquid sample, said method comprising:
- (a) treating said sample with a compound having the formula ##STR13## in which:
- Ar is an aryl radical selected from the group consisting of phenyl and fused polycyclic aromatic radicals, with the two H.sub.2 N radicals shown in said formula bonded to Ar at ortho-positions relative to each other,
- R is a radical bonded to Ar through one or two single bonds, and is a member selected from the group consisting of --O--alkyl, ##STR14## m is an integer of 1 to 4, and n is an integer of 1 to 4, wherein m.times.n is at least 2,
- to form fluorescently tagged derivatives of said at least one gem-dicarbonyl-containing analyte, said derivatives each containing at least one carboxylic acid group;
- (b) passing said liquid sample so treated through a chromatographic separation medium to chromatographically separate components of said liquid sample from each other;
- and
- (c) detecting said fluorescently tagged derivatives among said components so separated, by fluorescence emission.
A compound having the formula ##STR19## in which:
- Ar is an aryl radical selected from the group consisting of phenyl and fused polycyclic aromatic radicals, with the two O.sub.2 N radicals shown in said formula bonded to Ar at ortho-positions relative to each other;
- R is a radical bonded to Ar through one or two single bonds, and is a member selected from the group consisting of --O--alkyl, ##STR20## m is an integer of 1 to 4, and n in an integer of 1 to 4, wherein m.times.n is at least 2.
A compound having the formula ##STR23## in which:
- Ar is an aryl radical selected from the group consisting of phenyl and fused polycyclic aromatic radicals, with the two H.sub.2 N radicals shown in said formula bonded to Ar at ortho-positions relative to each other;
- R is a radical bonded to Ar through one or two single bonds, and is a member selected from the group consisting of --O--alkyl, ##STR24## m is an integer of 1 to 4, and n in an integer of 1 to 4,
- wherein m.times.n is at least 2.
Dependencies are inferred from the claim text.