The second article by Yu et al.** describes the synthesis of a new chromogenic substrate for β-lactamase (bla). The bla enzyme is a commonly used reporter in cell-based assays in which a fluorescence-resonance-energy-transfer (FRET) substrate is used to measure activity.
The FRET substrate CCF2, consisting of a cephalosporin core linking 7-hydroxycoumarin to a fluorescein moiety, results in blue fluorescence when the substrate is cleaved by bla and green fluorescence when uncleaved, therefore a ratiometric assay is enabled using the blue/green fluorescence value. However, CCF2 is expensive as is Nitrocefin (the most commonly used absorbance-based substrate) and therefore the authors sought to synthesize improved absorbance-based substrates.
The authors describe a two-step synthesis to synthesize a chromogenic cephalosporin-[3-(4-nitrostyryl)-7-(2-phenylacetamido)-ceph-3-em-4-carboxylicacid (Chromacef). Cleavage of the β-lactam ring by bla results in a bathochromic shift of 64 nm (λmax from 378 nm to 442 nm; see Figure 2). The KM value for Chromacef using VIM-2 metallo-β-lactamase was 30 ± 7 μM and for TEM-1 it was 51 ± 15 μM, which are values similar to that obtained for Nitrocefin. The kcat values were improved compared to Nitrocefin.
Chromacef was shown to be cleaved by a variety of β-lactamases and should be a useful substrate to develop bla assays as well as serve as an orthogonal substrate.