Serendipitous Discovery of α-Hydroxyalkyl Esters as β-Lactamase Substrates

Abstract

O-(1-Carboxy-1-alkyloxycarbonyl) hydroxamates were found to spontaneously decarboxylate in aqueous neutral buffer to form O-(2-hydroxyalkylcarbonyl) hydroxamates. While the former molecules do not react rapidly with serine β-lactamases, the latter are quite good substrates of representative classes A and C, but not D, enzymes, and particularly of a class C enzyme. The enzymes catalyze hydrolysis of these compounds to a mixture of the α-hydroxyacid and hydroxamate. Analogous compounds containing aryloxy leaving groups rather that hydroxamates are also substrates. Structure-activity experiments showed that the α-hydroxyl group was required for any substantial substrate activity. Although both D- and L-α-hydroxy acid derivatives were substrates, the former were preferred. The response of the class C activity to pH and to alternative nucleophiles (methanol and D-phenylalanine) suggested that the same active site functional groups participated in catalysis as for classical substrates. Molecular modeling was employed to explore how the α-hydroxy group might interact with the class C β-lactamase active site. Incorporation of the α-hydroxyalkyl moiety into novel inhibitors will be of considerable interest.

Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-α-hydroxyglycine ester catalyzed by serine β-lactamases and dd-peptidases

“…Interestingly, these same interactions were observed in one of the conformations that may occur on reaction of the P99 β-lactamase with 1. 5 This structure is probably even more likely in this than in the latter case because of the presence in 5 of the amido side chain which is seen in Fig. This journal is © The Royal Society of Chemistry 2012 to hydrogen bond into the active site in just the same way as it is thought to do in natural substrates.…”

“…It may be, therefore, that the α-hydroxydepsipeptides 5 bind and react at the β-lactamase active site in a conformation more closely resembling that of 1 than that of 4 . The occupancy of the active site by 1 5 and by 4 15 has been previously discussed. The interaction of 5 with the β-lactamase active site is explicitly addressed below.…”

“…Given this assignment, the P99 and TEM β-lactamases therefore preferred the l ( S )-enantiomer, although, as in the present case, the P99 enzyme also catalyzed more slowly the hydrolysis of the other [ d ( R )] enantiomer. The structural basis of these preferences has been discussed, 5 where the major point is the presence in dd -peptidases of a small hydrophobic binding pocket for the penultimate d -alanine methyl group, which is not present in β-lactamases. Indeed, the deletion of such a pocket has been proposed to be a major element of β-lactamase evolution.…”

“…Indeed, it has already been established that the presence of the α-hydroxyl group is essential to any substrate activity in 1 . 5 The effect of the amido side chain is particularly striking in the class A and D β-lactamases (TEM-2 and OXA-1) and even more so in the dd -peptidases where no activity is seen at all with 1 .…”

“…Recently, we have shown that α-hydroxyalkyl esters such as 1 can be good substrates of class A and C β-lactamases. 5 The incorporation of substituents α to the scissile amide or ester bond of β-lactamase and DD-peptidase substrates and inhibitors has quite a long history, beginning with the example of the cephamycins, a class of β-lactam natural products containing the 7-α-methoxy group, 2. 6 Synthetic analogues of these, such as cefoxitin, 3, soon followed.…”