title: Site-selective chemical modification of chymotrypsin using peptidyl derivatives bearing optically active diphenyl 1-amino-2-phenylethylphosphonate: Stereochemical effect of the diphenyl phosphonate moiety.
abstract: Diphenyl (α-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 α-carbon of phosphorus in diphenyl (α-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. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 521-530, 2016.
authors.affiliation: Genome Biotechnology Laboratory, Kanazawa Institute of Technology, Hakusan, 924-0838, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Ono
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, Toyama, 930-8555, Japan.
authors.forename: Takahiko
authors.initials: T
authors.lastname: Nakai
authors.affiliation: Department of General Education, Ishikawa National College of Technology, Ishikawa, 929-0392, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Kuroda
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, Toyama, 930-8555, Japan.
authors.forename: Ryuta
authors.initials: R
authors.lastname: Miyatake
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, Toyama, 930-8555, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Horino
authors.affiliation: Graduate School of Science and Engineering, University of Toyama, Toyama, 930-8555, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Abe
authors.affiliation: Institute of Natural Medicine, University of Toyama, Toyama, 930-0194, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Umezaki
authors.affiliation: Faculty of Science and Engineering, Setsunan University, Osaka, 572-8508, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Oyama
mesh_terms: D000818:Animals; D002918:Chymotrypsin / Q000737:chemistry*; D004151:Dipeptides / Q000737:chemistry*; D063065:Organophosphonates / Q000737:chemistry
chemical_list: D004151:Dipeptides; D063065:Organophosphonates; D002918:Chymotrypsin
keywords: 2-pyridinaldoxime methiodide; optically active diphenyl (1-aminoalkyl)phosphonate; serine protease; site-selective chemical modification; stereoselective reactivation