[en] Aminocitrate (and homolog) derivatives have been prepared by bis-alkylation of glycinate Schiff bases with bromoacetates (and ethyl acrylate), followed by N-acylation and esters (partial or complete) deprotection. Aminoisocitrate was similarly obtained by mono-alkylation with diethyl fumarate. Evaluation against representative beta-lactamases revealed that the free acid derivatives are modest inhibitors of class A enzymes, whilst their benzyl esters showed a good inhibition of OXA-10 (class D enzyme). A docking experiment featured hydrophobic interactions in the active site.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Beck, Joséphine; Université Catholique de Louvain - UCL > Unité de chimie organique et médicinale
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We recently disclosed (bis)phosphonic bioisosters of aminocitrate-related compounds as modest inhibitors of NMCA β-lactamase and R39 DD-peptidase. Beck J., Gharbi S., Herteg-Fernea A., Vercheval L., Bebrone C., Lassaux P., Zervosen A., and Marchand-Brynaert J. Eur. J. Org. Chem. (2009) 85
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Acidic hydrolysis was followed by a basic work-up for recovering the free amines 3. Crude amines 3a-b were used in the acylation step. Amines 3c-e could be purified by column chromatography on silica gel. Thus, pure amines were used for the acylation step. However, the overall yields of amides 4a-e from Schiff bases 1a-c remained in the range of 60% (see Supplementary data).
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Protocol of biochemical evaluation: The enzymes were produced and purified as previously described.10,24-29 The enzymes (1-100 nM) were incubated with the tested compounds (100 μM, otherwise mentioned) and the chromogenic substrate31 (nitrocefin, 100 μM) in phosphate buffer (50 mM, pH 7) or acetate buffer (50 mM, pH 5). In the case of metallo-enzymes (class B), HEPES buffer (10 mM, pH 7) added with ZnCl2 (50 μM) was used. Tested compounds were dissolved in DMSO at 10-100 mM and then diluted with the buffer (final concentration of DMSO in the testing = 2%); DMSO = 2% had no effect on the enzyme activity. The hydrolysis rate of nitrocefin was followed by spectrophotometry at 482 nm with UVIKON 860, 940 and XL apparatus connected to a computer via a RS232 line. The residual activity was obtained by comparison with the variation of the absorbance of the reference (sample without inhibitor) and indicated in Table 1. Results are expressed as % of initial activities. The standard deviation is below 5%. All experiments were performed three times.
The catalytic parameters (kcat, Km, kcat/Km) of tested β-lactamases versus nitrocefin are given as Supplementary data.
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Plot v0/vi (ratios of hydrolysis in the absence and in the presence of inhibitors) versus inhibitor concentration gave the inhibition constant Ki. For 6a,11 Ki = 250 μM (BS3, pH 5) and 150 μM (TEM-1, pH 5); for 9 + 10, Ki = 105 μM (TEM-1, pH 5).
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