Class D beta-lactamases represent a growing and diverse class of penicillin-inactivating enzymes that are usually resistant to commercial beta-lactamase inhibitors. As many such enzymes are found in multidrug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel beta-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2'-substituted penicillanic acid sulfones (1-5) were synthesized and tested against OXA-24, a clinically important beta-lactamase that inactivates carbapenems and is found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 beta-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC50 values against OXA-24 and two OXA-24 beta-lactamase variants ranged from 10 +/- 1 (4 vs WT) to 338 +/- 20 nM (5 vs Tyr112Ala, Met223Ala). Compound 4 possessed the lowest K, (500 +/- 80 nM vs WT), and 1 possessed the highest inactivation efficiency (k(inact)/K-i = 0.21 +/- 0.02 mu M-1 s(-1)). Electrospray ionization mass spectrometry revealed single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 angstrom) reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanion hole. These data provide the first structural evidence that 6-alkylidene-2'-substituted penicillin sulfones are effective mechanism-based inactivators of class D beta-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D beta-lactamases is proposed.