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  • br Conclusions In conclusion the

    2019-07-11


    Conclusions In conclusion, the substrates 18c–18e all produced highly fluorescent colonies with the panel of Gram-negative microorganisms. This has been attributed to the combination of two synergistic effects; the wide distribution of l-alanylaminopeptidase in Gram-negative microorganisms and the inhibitory effect of these substrates against Gram-positive microorganisms. Some Gram-positive microorganisms gave fluorescent colonies with substrates that were not inhibitory to their growth (substrates 18a and 22).
    Experimental NMR spectra were recorded on a JEOL ECS400 Delta spectrometer at frequencies of 400MHz for 1H NMR spectra and 101MHz for 13C NMR spectra. All chemical shifts are quoted in ppm relative to TMS as an internal standard. The multiplicity of the signals is expressed as follows; s=singlet, d=doublet, dd=doublet of doublets, t=triplet, q=quartet and m=multiplet, or in combinations (e.g., td=triplet of doublets). High resolution mass spectra (HRMS) were obtained using a Finnigan MAT 900 XLT high resolution double focussing mass spectrometer or a Thermo Scientific LTQ Orbitrap XL mass spectrometer using nanoelectrospray ionisation. Low resolution mass spectrometry (LRMS) was performed via direct injection of dilute methanolic solutions (containing 0.1% formic acid) into a Thermo Finnigan LCQ Advantage MS detector using electrospray ionisation. Infra-red spectra were obtained via a diamond anvil sample cell using a Perkin Elmer 1000FT-IR spectrometer. Melting points are reported uncorrected as determined on a Stanford Research Systems MPA161 melting point apparatus. Thin layer chromatography was performed on Merck plastic foil plates pre-coated with silica gel 60 F254. Merck silica gel 60 was used for column chromatography. The preparation of agar plates followed the procedure described previously.
    Acknowledgements We thank BioMérieux SA for generous financial support and the EPSRC Mass Spectrometry Centre, Swansea, for high resolution mass spectra. We thank Dr E. Fazackerley for the preparation of Benzoquinonium dibromide molecular 16e.
    Introduction Equilibrium binding isotope effects (BIEs) report on differences in the bond vibrational environment of enzyme substrates in the free and bound states preceding catalysis. As such, BIEs inform on substrate structure in the Michaelis complex and provide an experimental approach to probe substrate reactivity prior to the transition state (TS). BIEs have been measured for several enzyme systems, and these data have been leveraged in conjunction with quantum chemical calculations to elucidate the effects of protein binding on substrate conformation and the role of enzyme–substrate (ES) interactions in promoting catalysis (Anderson, 2005, Schramm, 2007, Swiderek and Paneth, 2013). The expression of BIEs on enzyme binding results from perturbing the vibrational state of the substrate at the position of isotopic substitution (Gaylord & Eliel, 1960). For systems in which the free and bound substrates occupy distinct bonding environments, an observable BIE occurs when differential equilibria are established for substrates bearing either “light” or “heavy” isotopes at a fixed position (Swiderek & Paneth, 2013). A normal BIE (>1.0) results from preferential binding of the substrate bearing the light isotope and is generally reported as either a ratio of the equilibrium constants for the isotopically substituted substrates (e.g., KeqLight/KeqHeavy=1.05) or as a positive percentage (e.g., 5%). Normal BIEs occur when the bonding environment of the isotopically substituted position is less constrained in the bound state than in the free state, resulting in a decreased force constant for this bond upon enzyme binding (Fig. 1A). By contrast, inverse BIEs (<1.0) occur when binding of the substrate bearing the heavy isotope is favored and are commonly expressed as a ratio of equilibrium constants (e.g., KeqLight/KeqHeavy=0.95) or as a negative percentage value (e.g., −5%). Inverse BIEs indicate an increased force constant for the isotopically substituted bond in the bound state and illustrate that the enzyme binding site presents a more constrained bonding environment relative to the solution state (Fig. 1B).