Molecular Probes, Inc.

Richard P Haughland

Hee Chol Kang

Yu-Zhong Zhang

Zhijian Huang

Steven L Young

Michael H Melner

Karen D Larison

Activity of enzymes and enzyme conjugates is detected using novel substrates made from a class of fluorophores, generally including quinazolinones (quinazolones), benzimidazoles, benzothiazoles, benzoxazoles, quinolines, indolines, and phenanthridines, having the general formula: ##STR1## where carbon atoms of --C.sup.1 .dbd.C.sup.2 -- are joined to complete a first 5- or 6-membered aromatic ring which optionally contains one of the hetero atoms N, O or S, and carbon atoms of --C.sup.4 --N.dbd.C.sup.3 -- are joined to complete a second 5- or 6-membered aromatic ring that contains a nitrogen between C.sup.3 and C.sup.4 and optionally contains an additional hetero atom N, O or S, where the first and second aromatic rings may be joined by a 5- or 6-membered bridging ring that contains at least the C.sup.2 from the first aromatic ring and the C.sup.3 from the second aromatic ring, where each of the first and second aromatic rings may be fused to at least one additional aromatic ring that may contain at least one of the hetero atoms N, O or S, and where each of said aromatic rings may be further modified by substitution of any hydrogens on an aromatic carbon by substituents that are halogen, nitro, cyano, aryl, lower alkyl (1-4 carbons), perfluoroalkyl (1-4 carbons), or alkoxy (1-4 carbons), or any combination thereof; and the fluorophore is covalently linked to a blocking group through an oxygen --O-- at C.sub.1 ; such that removal of the blocking group by enzyme activity yields a precipitate.

Enzymatic analysis using substrates that yield fluorescent precipitates

Fluorescent compounds useful in the determination of chloramphenicol acetyltransferase (CAT) enzyme activity are described. The compounds are fluorescent derivatives related in structure to chloramphenicol comprising a base, ##STR1## substituted at one to five aromatic ring positions by substituents, which may be the same or different, that are alkyl-, hydroxy-, alkoxy-, aryl-, halo-, nitro-, amino-, alkylamido-, or arylamido-; and a fluorescent moiety linked to the base at --NH-- by an aliphatic chain of 1-12 atoms other than hydrogen, where such atoms are carbon, nitrogen, oxygen, or sulfur, or combinations thereof. The substrate compounds are acylated in the presence of CAT to produce fluorescent mono- and diacylated products, which are then physically separated from the reaction mixture and quantitated by means of their fluorescence and/or absorbance. Fluorescent molecules conjugated to chloramphenicol include derivatives of fluorescein, rhodamine, coumarin, dimethylaminonaphthalene sulfonic acid (dansyl), pyrene, anthracene, nitrobenzoxadiazole (NBD), acridine and dipyrrometheneboron difluoride.

Fluorescent chloramphenicol derivatives for determination of

Growing community of inventors

Eugene, OR, United States of America

Richard P Haughland