Incubation of δ-L-α-aminoadipoyl-L-[3-13Ccysteinyl-D-[3-2H]valine with Isopenicillin N Synthase (IPNS) resulted in the observation of a ‘shunt metabolite’, which we believe is formed from the collapse of an enzyme bound monocyclic β-lactam intermediate. Chemical studies into the origin of the shunt metabolite suggest its formation occurred after initial β-lactam ring closure. Further chemical studies on the decomposition pathway of a free thiol monocyclic β-lactam have indicated it is not the source of the shunt metabolite, as upon decomposition the major product formed retains sulphur in the form of an ene-thiol dehydrocysteine.