Tandem synthetic–biosynthetic procedures were used to prepare two novel fluorescent labelled affinity probes for diadenosine-5′,5‴-P 1 ,P 4 -tetraphosphate (Ap 4 A)-binding studies. These compounds (dial-mant-Ap 4 A and azido-mant-Ap 4 A) are shown to clearly distinguish known Ap 4 A-binding proteins from Escherichia coli (LysU and GroEL) and a variety of other control proteins. Successful labelling of chaperonin GroEL appears to be allosteric with respect to the well-characterized adenosine 5′-triphosphate (ATP)-binding site, suggesting that GroEL possesses a distinct Ap 4 A-binding site.