Observation of an Acryloyl−Thiamin Diphosphate Adduct in the First Step of Clavulanic Acid Biosynthesis

Abstract

The first committed biosynthetic step toward clavulanic acid, the clinically-important β-lactamase inhibitor, is catalyzed by the thiamin diphosphate (ThDP)-dependent enzyme N 2 -(2-carboxyethyl)arginine synthase (CEAS). This protein carries out a unique reaction among ThDPdependent processes in which a C-N bond is formed, and an electrophilic acryloyl-thiazolium intermediate of ThDP is proposed to be involved, unlike the nucleophilic enamine species typically generated by this class of enzymes. Here we present evidence for the existence of the putative acryloyl adduct, and report the unexpected observation of a long-wavelength chromophore (λ = 433 nm), which we attribute to this enzyme bound species. Chemical models were synthesized that both confirm its expected absorption (λ = 310-320 nm), and exclude selfcondensation and intramolecular imine formation with the cofactor as its cause. Circular dichroism experiments and others discount charge transfer as a likely explanation for the ~120 nm red shift of the chromophore (~25 kcal). Examples are well-known of charged molecules that exhibit significantly red-shifted UV-visible spectra compared to their neutral forms as, for example, polyene cations and dyes such as indigo and the cyanines. Rhodopsin is the classic biochemical example where the protein (opsin)-bound protonated Schiff base of retinal displays a remarkable range of red-shifted absorptions modulated by the protein environment. Similar tuning of the chromophoric behavior of the enzyme-bound CEAS acryloyl•ThDP species may be occurring.With the rise in resistance to penicillins and cephalosporins, the β-lactamase inhibitor clavulanic acid (1) has grown in importance for the treatment of bacterial infections 1 . Several mechanistically intriguing reactions take place in the biosynthesis of 1, 2 the first of which is mediated by N 2 -(2-carboxyethyl)arginine synthase (CEAS). The primary metabolites D-glyceraldehyde-3-phosphate (G3P) and L-arginine are recruited by this enzyme and reacted in a thiamine diphosphate (ThDP)-dependent internal redox process to yield N 2 -(2-carboxyethyl)arginine (6, CEA; Scheme 1). 3 The mechanism shown in Scheme 1 was proposed in accord with extensive isotopic labeling and stereochemical experiments. 3 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript feature of this mechanism was the suggested intermediacy of an acryloyl-ThDP adduct 4. The observation of overall retention of configuration at the G3P β-carbon is consistent with a β-elimination/addition process proceeding by way of this highly electrophilic 4 partner for reaction with the incoming L-arginine. This is a unique reaction cycle among ThDPdependent enzymes where typically intermediates formed are nucleophilic rather than electrophilic, and N-C bond formation has not been seen previously.In this Communication we demonstrate the formation of the acryloyl species 4 and report the observation of an unexpectedly long-wavelength chromophore, which owes to the presence of the acryloyl inter...

NHC‐Catalyzed Michael Addition to α,β‐Unsaturated Aldehydes by Redox Activation

“…[8][9][10] Based on these results we decided to use oxidatively generated intermediates C as Michael acceptors to conduct 1,4-addition reactions using soft C nucleophiles. There is precedence in biosynthesis for such processes, and very recently Lupton reported NHC-catalyzed 1,4-addition using activated a,b-unsaturated acid derivatives via intermediates of type C. [11][12][13] Starting with an a,b-unsaturated aldehyde our planned process would comprise two consecutive umpolung reactions at the b position of the a,b-unsaturated aldehyde by a redox-type activation. To liberate the NHC catalyst, the C nucleophile should bear an additional nucleophilic site (Nu 2 ) such that subsequent 1,2-addition to the acylazolium ion D releases the NHC catalyst.…”

“…The reaction might also proceed by kinetic O acylation of the enolate with C to form enol esters of type 9 (Scheme 5). Subsequent NHC-mediated fragmentation would regenerate acylazolium ion C and the enolate, [12] which could then undergo 1,4-addition to F (thermodynamic addition product). We currently exclude kinetic O acylation, since the test reaction of preformed 9 under optimized reaction conditions afforded 5 a in only 7 % yield; the reaction starting with cinnamaldehyde gave 91 % yield.…”

A Lewis acid-promoted Pinner reaction

“…Somewhat lower yields were observed with acrylonitrile; nevertheless, reported methods for the preparation of acrylate 5 lead to similar, and in some cases even lower yields [13–14]. A standard protocol for the synthesis of 9 H -fluoren-9-ylmethyl acrylate starting with the moisture-sensitive acryloyl chloride under an inert atmosphere was reported to yield only 33% [15].…”

Convergent biosynthetic pathways to β-lactam antibiotics

“…Although the clavulanic acid and thienamycin biosynthetic pathways have not been completely elucidated, enough is presently known to confidently state that their stepwise progression relies on markedly different chemistries and that they evolved independently. To illustrate these differences, the formation of CEA ( 7 ) captures unprecedented C-N bond formation in thiamin diphosphate (ThDP) catalysis from the primary metabolic building blocks glyceraldehyde-3-phosphate (G3P) and L-arginine [13–15] In contrast, three separately precedented crotonase superfamily enzyme reactions are compressed into the formation of (2 S ,5 R )-carboxyethylproline ( 8 ) to initiate carbapenem biosynthesis [16,17]. Although detailed discussion of these pathways is outside the scope of this review, the principal steps are sketched out in Figure 3A.…”