Optimization of Prodrug Activation by Enzymatic Cleavage of the β‐lactam Ring of Carbapenems

β-lactam antibiotics remain the best treatment to fight bacterial infections. Among various antibiotics used today, β-lactams belonging to the carbapenem class are last resort drugs active against many multiresistant Gram-negative bacteria. Carbapenem antibiotics are prone to hydrolysis by certain β-lactamases, leading to the opening of the β-lactam ring and drug inactivation. This mechanism can be repurposed for targeted molecular fragmentation. Here, we report the synthesis of model prodrugs consisting of two carbapenems connected by original self-immolative linkers. A mass spectrometry assay was developed to simultaneously monitor acylation of a β-lactam target by the prodrugs and by their sacrificial and warhead moieties. The linker connecting the two carbapenems was optimized with respect to prodrug stability and effective release of the activated warhead upon carbapenem ring opening.