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Phosphonamidate analogues of dipeptides with carboxypeptidase A and beta-lactamase-inhibitory activity: elucidation of the mechanism of beta-lactamase inhibition by electrospray mass spectrometry.

ABSTRACT: A series of phosphonamidate compounds with different P1' amino acid residues have been shown to be irreversible inactivators of the serine beta-lactamase from Enterobacter cloacae P99. The efficiency of inhibition (based on k2/K values) of P99 by these derivatives, ordered in decreasing potency, is: beta-phenyl-beta-Ala > L-Phe > beta-Ala > Gly > D-Phe > D-Pro > D-thiazolidine. The D- and L-Phe compounds also inhibit carboxypeptidase A. The proline and thiazolidine derivatives were phosphonamidate methyl esters, whereas the others were salts of diacids. Electrospray mass spectrometry showed that equimolar mixtures of the P99 enzyme with each of the following derivatives, Gly, D-Phe, L-Phe, beta-Ala and beta-phenyl-beta-Ala, effected efficient adduct formation (70-95% of enzyme modified), illustrating the particularly active nature of some of these compounds. All the primary amino acid derivatives gave a similar mass increment, which suggests the displacement of the variable P1' part of the molecule. This observation provides evidence that the compounds phosphonylate the active-site serine, with the phosphonamidate bond as the scissile bond and the amino acid as the leaving group. The thiazolidine derivative (phosphonamidate methyl ester) also appeared to work by the same mechanism. The comparable proline derivatives caused lower than expected mass shifts of 227-229, and therefore it is proposed that with these compounds both the amino acid and the phosphonamidate ester methoxy group were displaced at the phosphorus atom during the inhibition process. Therefore, electrospray mass spectrometry has provided both a measure of potency and a rationale for the mechanism of inhibition of P99 by these compounds.

SUBMITTER: Payne DJ

PROVIDER: S-EPMC1217072 | biostudies-other | 1996 Mar

REPOSITORIES: biostudies-other

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Project description:The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids, however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication [Thomas, M. C.; Mitchell, T. W.; Blanksby, S. J. J. Am. Chem. Soc. 2006, 128, 58-59], we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including acidic and neutral glycerophospholipids, sphingomyelins, and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges, or the adduct ion (e.g., [M - H](-), [M - 2H](2-), [M + H](+), [M + Na](+), [M + NH(4)](+)). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio (m/z). The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used, in conjunction with conventional tandem mass spectral analysis, for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely, SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1), and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and, when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.

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Phosphonamidate analogues of dipeptides with carboxypeptidase A and beta-lactamase-inhibitory activity: elucidation of the mechanism of beta-lactamase inhibition by electrospray mass spectrometry.

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