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
We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an <i>N</i>-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-Phe<...
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MDPI AG
2023-03-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/28/7/3150 |
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| author | Shin Ono Masato Koga Yuya Arimura Takahiro Hatakeyama Mai Kobayashi Jun-ichi Sagara Takahiko Nakai Yoshikazu Horino Hirofumi Kuroda Hiroshi Oyama Kazunari Arima |
| author_facet | Shin Ono Masato Koga Yuya Arimura Takahiro Hatakeyama Mai Kobayashi Jun-ichi Sagara Takahiko Nakai Yoshikazu Horino Hirofumi Kuroda Hiroshi Oyama Kazunari Arima |
| author_sort | Shin Ono |
| collection | DOAJ |
| description | We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an <i>N</i>-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-Phe<sup>P</sup>(OPh)<sub>2</sub>]. 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-(<i>R</i>)-Phe<sup>P</sup>(OPh)<sub>2</sub> (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-<i>p</i>NA. 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. |
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| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
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| spelling | doaj.art-3553faefa8914ce1b193d91d3b2181a22023-11-17T17:14:16ZengMDPI AGMolecules1420-30492023-03-01287315010.3390/molecules28073150Site-Selective Incorporation of a Functional Group into Lys175 in the Vicinity of the Active Site of Chymotrypsin by Using Peptidyl α-Aminoalkylphosphonate Diphenyl Ester-DerivativesShin Ono0Masato Koga1Yuya Arimura2Takahiro Hatakeyama3Mai Kobayashi4Jun-ichi Sagara5Takahiko Nakai6Yoshikazu Horino7Hirofumi Kuroda8Hiroshi Oyama9Kazunari Arima10Applied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, JapanApplied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, JapanApplied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, JapanApplied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, JapanApplied Chemistry, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, JapanApplied Bioengineering, Kanazawa Institute of Technology, Hakusan 924-0838, Ishikawa, JapanGraduate School of Science and Engineering, University of Toyama, Toyama 930-8555, Toyama, JapanDepartment of Applied Chemistry and Bioscience, Chitose Institute of Science and Technology, Chitose 066-8655, Hokkaido, JapanDepartment of General Education, National Institute of Technology, Ishikawa College, Tsubata 929-0392, Ishikawa, JapanFaculty of Science and Engineering, Setsunan University, Hirakata 572-8508, Osaka, JapanGraduate School of Science and Engineering, Kagoshima University, Kagoshima 890-0065, Kagoshima, JapanWe previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an <i>N</i>-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-Phe<sup>P</sup>(OPh)<sub>2</sub>]. 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-(<i>R</i>)-Phe<sup>P</sup>(OPh)<sub>2</sub> (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-<i>p</i>NA. 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.https://www.mdpi.com/1420-3049/28/7/3150chymotrypsindansyl groupdiphenyl α-aminoalkylphosphonatesite-selective chemical modification |
| spellingShingle | Shin Ono Masato Koga Yuya Arimura Takahiro Hatakeyama Mai Kobayashi Jun-ichi Sagara Takahiko Nakai Yoshikazu Horino Hirofumi Kuroda Hiroshi Oyama Kazunari Arima 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 Molecules chymotrypsin dansyl group diphenyl α-aminoalkylphosphonate site-selective chemical modification |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | chymotrypsin dansyl group diphenyl α-aminoalkylphosphonate site-selective chemical modification |
| url | https://www.mdpi.com/1420-3049/28/7/3150 |
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