Molecular Probes, Inc.

Zhijian Huang

Stephen T Yue

Richard P Haugland

Yu-Zhong Zhang

Karen D Larison

John Joseph Naleway

Ewald Alois Terpetschnig

Nels A Olson

Richard P Haughland

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

The invention relates to unsymmetrical cyanine dyes with a cationic side chain, typically benzothiazole or benzoxazole derivatives, that exhibit enhanced fluorescence on binding with DNA or RNA, where such fluorescence can be used for evaluating the presence of nucleic acid polymers. The dyes generally have the formula: ##STR1## where R.sup.1 is an alkyl group having 1-6 carbons; X is 0, S, or NR.sup.2, where R.sup.2 is an alkyl group having 1-6 carbons, or CR.sup.3 R.sup.4, where R.sup.3 and R.sup.4, which may be the same or different, are alkyl groups having 1-6 carbons;

Unsymmetrical cyanine dyes with a cationic side chain

Novel fluorescent precipitating 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 further joined so as to complete a first 5- or 6-membered aromatic ring which may contain at least one of the hetero atoms N, O or S,

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Eugene, OR, United States of America

Zhijian Huang