Site-Selective Incorporation of a Functional Group into Lys175 in the Vicinity of the Active Site of Chymotrypsin by Using Peptidyl α-Aminoalkylphosphonate Diphenyl Ester-Derivatives-Reference-Cited by-同舟云学术

Site-Selective Incorporation of a Functional Group into Lys175 in the Vicinity of the Active Site of Chymotrypsin by Using Peptidyl α-Aminoalkylphosphonate Diphenyl Ester-Derivatives

Published:2023-03-31 Issue:7 Volume:28 Page:3150
ISSN:1420-3049
Container-title:Molecules
language:en
Short-container-title:Molecules

Author:

Ono Shin¹,Koga Masato¹,Arimura Yuya¹,Hatakeyama Takahiro¹,Kobayashi Mai¹,Sagara Jun-ichi²,Nakai Takahiko³,Horino Yoshikazu⁴^ORCID,Kuroda Hirofumi⁵,Oyama Hiroshi⁶,Arima Kazunari⁷

Affiliation:

1. Applied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, Japan

2. Applied Bioengineering, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, Japan

3. Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Toyama, Japan

4. Department of Applied Chemistry and Bioscience, Chitose Institute of Science and Technology, Chitose 066-8655, Hokkaido, Japan

5. Department of General Education, National Institute of Technology, Ishikawa College, Tsubata 929-0392, Ishikawa, Japan

6. Faculty of Science and Engineering, Setsunan University, Hirakata 572-8508, Osaka, Japan

7. Graduate School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Kagoshima, Japan

Abstract

We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an N-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-PheP(OPh)2]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-β-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-(R)-PheP(OPh)2 (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin. Ion-exchange chromatography (IEC), fluorescence spectroscopy, and LC-MS/MS were used to analyze the products from the reaction of Csin with DanD or DanE. By IEC and LC-MS/MS, the results showed that DanE reacted with Csin more effectively than DanD to produce the modified Csin (DanMCsin) bearing Dan at Lys175. DanMCsin exhibited an enzymatic activity corresponding to 1/120 of Csin against Suc-Ala-Ala-Phe-pNA. In addition, an effect of Lys175 modification on the access of the proteinaceous Bowman–Birk inhibitor to the active site of DanMCsin was investigated. In conclusion, by using a peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester, we demonstrated that a functional group could be incorporated into Lys175 in Csin.

Publisher

MDPI AG

Subject

Chemistry (miscellaneous),Analytical Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Molecular Medicine,Drug Discovery,Pharmaceutical Science

Link

https://www.mdpi.com/1420-3049/28/7/3150/pdf

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