Title: Fluorogenic substrates for BETA-lactamase gene expression

Abstract

Fluorescent substrates for beta-lactamases having the general formula shown above are provided. Z includes a fluorophore or chromophore and includes a group that may link to the lactam-containing group; R1 and R2 are independently selected from H, aliphatic, aromatic, alkyl, and acyl; R4 may be H and lower alkyl; B may be H, physiologically acceptable salts or metal, ester groups, ammonium cations, —CHR5OCO(CH2)nCH3, —CHR5OCOC(CH3)3, acylthiomethyl, acyloxy-alpha-benz, deltabutyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, beta-morpholinoethyl, dialkylaminoethyl, and dialkylaminocarbonyloxymethyl, in which R5 is selected from the group consisting of H and lower alkyl; n is an integer between 0 and 10, inclusive; and A may be S, O, SO, SO2 or CH2. In embodiments, the beta-lactam ring may be cleaved by a beta-lactamase enzyme effective to free a fluorophore. Methods of assaying beta-lactamase activity and monitoring expression in systems using beta-lactamase as a reporter gene are also disclosed.

Inventors:

Tsien, Roger Y.; Rao, Jianghong

Issue Date:

2008-09-23

Research Org.:

Univ. of California, Oakland, CA (United States)

Sponsoring Org.:

USDOE

OSTI Identifier:

1531564

Patent Number(s):

7427680

Application Number:

10/884,019

Assignee:

The Regents of the University of California (Oakland, CA)

Patent Classifications (CPCs):

C - CHEMISTRY C07 - ORGANIC CHEMISTRY C07D - HETEROCYCLIC COMPOUNDS

C - CHEMISTRY C07 - ORGANIC CHEMISTRY C07F - ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM

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C - CHEMISTRY C07 - ORGANIC CHEMISTRY C07D - HETEROCYCLIC COMPOUNDS

C07D463/18 - further acylated by radicals derived from carboxylic acids or by nitrogen or sulfur analogues thereof
C07D463/20 - with the acylating radicals further substituted by hetero atoms or by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
C07D501/00 - Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula
C07D505/00 - Heterocyclic compounds containing 5-oxa-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula

C - CHEMISTRY C07 - ORGANIC CHEMISTRY C07F - ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM

C07F9/65613 - {containing the ring system [IMAGE cpc-sch-C07F-1004.gif]

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

C12Q1/6897 - involving reporter genes operably linked to promoters

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DOE Contract Number:  

FG03-01ER63276

Resource Type:

Patent

Resource Relation:

Patent File Date: 2004-07-02

Country of Publication:

United States

Language:

English