Chiral protein scissors: high enantiomeric selectivity for binding and its effect on protein photocleavage efficiency...

Chiral recognition of protein-binding sites by a simple organic molecule with selectivities >100 is reported here. The l-isomer of 4(1-pyrene)-3-butyroyl-phenylalanine amide (Py-L-Phe) binds to BSA with an affinity constant (K(b)) of 3 x 10(7) M(-1), whereas the corresponding D-isomer (Py-D-Phe) binds 100 times weaker. The enantiomers showed contrasting spectral changes when bound to BSA. Whereas hypochromism was observed with the L-isomer, hyperchromism was observed for the D-isomer, and, whereas the fluorescence of the L-isomer was quenched, the fluorescence of the D-isomer was enhanced. The induced CD spectra of the enantiomers bound to BSA bear a near mirror-image relationship. In contrast, the enantiomers show only moderate binding selectivity with lysozyme. The differences in the enantioselectivities with the two proteins indicate that the binding site of 4(1-pyrene)-3-butyroyl-phenylalanine amide (Py-Phe) in BSA is crowded, whereas that of lysozyme is more accommodative of either isomer. The enantioselective binding of Py-Phe isomers is further examined in protein photocleavage studies. Py-D-Phe cleaves BSA and lysozyme at a single site in a manner similar to Py-L-Phe, but the cleavage yields are lower for the d-isomer. Sequencing of the resulting fragments indicated that the photocleavage sites of Py-D-Phe on BSA and lysozyme are identical to those of Py-L-Phe. Flash photolysis studies indicated only minor differences between the two enantiomers. The large binding selectivities, therefore, do not influence cleavage specificity or cleavage site location. The strong role of the single asymmetric center of Py-Phe in recognition and its minor role in photocleavage chemistry are demonstrated.