Diphenyl (alpha-aminoalkyl) phosphonates act as mechanism-based inhibitors against serine proteases by forming a covalent bond with the hydroxy group of the active center Ser residue. Because the covalent bond was found to be broken and replaced by 2-pyridinaldoxime methiodide (2PAM), we employed a peptidyl derivative bearing diphenyl 1-amino-2-phenylethylphosphonate moiety (Phe(p)(OPh)(2)) to target the active site of chymotrypsin and to selectively anchor to Lys175 in the vicinity of the active site. Previously, it was reported that the configuration of the a-carbon of phosphorus in diphenyl (alpha-aminoalkyl) phosphonates affects the inactivation reaction of serine proteases, i.e., the (R)-enantiomeric diphenyl phosphonate is comparable to L-amino acids and it effectively reacts with serine proteases, whereas the (S)-enantiomeric form does not. In this study, we evaluated the stereochemical effect of the phosphonate moiety on the selective chemical modification. Epimeric dipeptidyl derivatives, Ala-(R or S)-Phe p (OPh) 2, were prepared by separation with RP-HPLC. A tripeptidyl (R)-epimer (Ala-Ala-(R)-Phe p (OPh) 2) exhibited a more potent inactivation ability against chymotrypsin than the (S)-epimer. The enzyme inactivated by the (R)-epimer was more effectively reactivated with 2PAM than the enzyme inactivated by the (S)-epimer. Finally, N-succinimidyl (NHS) active ester derivatives, NHS-Suc-Ala-Ala-(R or S)-Phe p (OPh) 2, were prepared, and we evaluated their action when modifying Lys175 in chymotrypsin. We demonstrated that the epimeric NHS derivative that possessed the diphenyl phosphonate moiety with the (R)-configuration effectively modified Lys175 in chymotrypsin, whereas that with the (S)-configuration did not. These results demonstrate the utility of peptidyl derivatives that bear an optically active diphenyl phosphonate moiety as affinity labeling probes in protein bioconjugation. (C) 2015 Wiley Periodicals, Inc.