No abstract

-Lactamases are a family of bacterial enzymes that cleave penicillins and cephalosporins with high catalytic efficiency and render these bacteria resistant to -lactam antibiotics. 1 Rapid and sensitive detection of -lactamase activity in biological samples is thus of large clinical importance. While -lactamases are the biochemical markers for identification of -lactam antibioticsresistant bacterial pathogens, the -lactamase activity can also serve as a "reporter" or "sensor" for monitoring biological processes and interactions of interest. TEM-1 -lactamase (Bla), the 29 kDa isoform product of the ampicillin resistance gene (amp r ), is especially useful because it is relatively small and monomeric, does not exist in eukaryotes, but can be easily expressed in eukaryotic cells without any noticeable toxicity. 2,3 TEM-1 Bla has been developed as a reporter for examining the promoter/regulatory elements activity in living mammalian tissue culture cells. [4][5][6] Protein fragment complementation assays based on Bla have been successfully applied to monitor constitutive and inducible protein interactions both in vitro and in vivo. 7-9 Incorporation of Bla into HIV-1 virions allows convenient detection of HIV-1 virion fusion in primary T lymphocytes in complex cell populations. 10 Substrates that detect Bla activity with high sensitivity are critical for the successful implementations of Bla assays. Fluorogenic substrates are superior to chromogenic substrates (such as wellknown nitrocefin and PADAC) [11][12][13] in detecting enzyme activity because of the high sensitivity of fluorescence detection. The first reported fluorogenic Bla substrate shown to work with cells was CCF2, which consists of a donor 7-hydroxycoumarin linked via a cephalosporin to an acceptor fluorescein. 4 CCF2 fluoresces green because of fluorescence resonance energy transfer from the coumarin donor to the fluorescein acceptor. Hydrolysis of the cephalosporin by Bla splits off the fluorescein, disrupts energy transfer, and shifts the emission to blue. 4 As the only fluorogenic Bla substrate currently available, CCF2 has many successful applications in tissue culture, 4-10 but its high molecular weight and low aqueous solubility have prevented applications in intact mammalian tissues or in cells with thick walls such as in yeast or plants. More fluorogenic substrates would expand the usefulness of Bla as a biosensor. Here, we report a new class of small fluorogenic substrates that work by releasing a phenolic dye from a vinylogous cephalosporin.Cleavage of the -lactam ring of a cephalosporin creates a free amino group, which triggers spontaneous elimination of any leaving group previously attached to the 3′ position. 14 Umbelliferone is a widely used fluorophore with maximal excitation at 360 nm and emission at 460 nm. When its 7-hydroxy group is alkylated, the compound becomes essentially nonfluorescent. We thus designed and synthesized a -lactam called CC1 in which the 3′ position of the cephalosporin is linked to the 7-hydroxy group of umbellif...