Site-selective Chemical Modification of Chymotrypsin Using a Peptidyl Diphenyl 1-Amino-2-phenylethylphosphonate Derivative

Ono, Satoshi; Murai, Junya; Nakai, Takahiko; Kawakubo, Hirofumi; Horino, Yoshikazu; Yoshimura, Toshiaki; Oyama, Hiroshi; Umezaki, Masahito

doi:10.1246/cl.130244

Cited by 4 publications

(18 citation statements)

References 24 publications

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“…For the modification using ( R )‐ 1 , a major product was first eluted at 19.8 min. The elution profile was similar to that observed for the chymotrypsin modified using the previously prepared racemic derivative ( RS )‐ 1 and the major product (19.8 min) exhibited a hydrolytic activity against Suc‐Ala‐Ala‐Phe‐pNA (data not shown); therefore, the major product was the desired chymotrypsin modified at Lys175. The second large peak at 24.3 min was identical to that with native chymotrypsin.…”

Section: Resultssupporting

confidence: 71%

“…The comparison of these changes in the enzymatic activities suggests a stereochemical effect of the configuration of the phosphonate moiety, but the progress of the selective modification at Lys175 could not be clearly evaluated simply by monitoring changes in the enzymatic activity after the addition of 2PAM, particularly for the modification by ( S )‐ 1 . This was because of the lower enzymatic activity (about 1/100) of the modified chymotrypsin at Lys175 than that of the native enzyme …”

Section: Resultsmentioning

confidence: 99%

“…To identify the site modified by ( R )‐ 1 , the modified chymotrypsin was subjected to reduction, alkylation, tryptic digestion, and LC‐MS/MS analysis, as described previously . Figure shows the RP‐HPLC analysis profile for the digested fragments.…”

Section: Resultsmentioning

confidence: 99%

“…A hydrobromide salt of racemic diphenyl (1‐amino‐2‐phenylethyl)phosphonate (( RS )‐Phe p (OPh) 2 ) was synthesized by the procedure of Oleksyszyn et al The epimeric dipeptidyl derivative mixture, Ala‐(RS)‐Phe p (OPh) 2 , was prepared by coupling with Boc‐L‐Ala‐OH and deblocking Boc group, and each epimeric derivative was separated on an RP‐HPLC column using a TFA‐acetonitrile solvent system. Dipetidyl phosphonate epimers were used for further synthesis of the final products [( R )‐ 1 and ( S )‐ 1 ] by a classical conventional method with EDC‐HOBt coupling reagents and Boc‐strategy as described previouly . The purity of each compound synthesized was checked by 1H NMR, TLC, RP‐HPLC, and ESI‐TOFMS, and each analytical result was consistent with the proposed structure.…”

Section: Methodsmentioning

confidence: 97%

“…Recently, we designed and synthesized an N ‐succinimidyl (NHS) ester of Suc‐Ala‐Ala‐Phe p (OPh) 2 , and we demonstrated that this derivative can be selectively introduced onto the side chain amino group of Lys175 in bovine α‐chymotrypsin . The Lys175 residue was chosen as a target for modification because it is the nearest to the active site and is located in a direction similar to that in which the pentapeptide TPGVY (PDB ID: 1ab9) is bound to chymotrypsin.…”

Section: Introductionmentioning

confidence: 99%

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Site‐selective chemical modification of chymotrypsin using peptidyl derivatives bearing optically active diphenyl 1‐amino‐2‐phenylethylphosphonate: Stereochemical effect of the diphenyl phosphonate moiety

et al. 2016

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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.

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Section: Resultssupporting

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Site‐selective chemical modification of chymotrypsin using peptidyl derivatives bearing optically active diphenyl 1‐amino‐2‐phenylethylphosphonate: Stereochemical effect of the diphenyl phosphonate moiety

et al. 2016

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A Molecular Probe with Both Chromogenic and Fluorescent Units for Detecting Serine Proteases

et al. 2021

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A molecular probe with l-phenylalanine p-nitroanilide and l-lysin 4-methylcoumaryl-7-amide, in which these amino acid derivatives are connected through a succinic-acid spacer, was prepared. Trypsin and papain were detected by blue-fluorescence emission of generated 7-amino-4-methylcoumarin (AMC). α-Chymotrypsin and nattokinase were detected from both the blue-fluorescence emission of AMC and the UV absorbance of p-nitroaniline. In addition, different time courses of p-nitroaniline and AMC were observed between the reaction of P1 with α-chymotrypsin and that with nattokinase. In the case of nattokinase, both the fluorescence emission and UV absorbance slowly increased. In contrast, the increasing UV absorbance was saturated at the early stage of the reaction of the present probe with chymotrypsin, whereas the fluorescence emission continuously increased in the following stages.

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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

et al. 2023

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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.

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Site-selective Chemical Modification of Chymotrypsin Using a Peptidyl Diphenyl 1-Amino-2-phenylethylphosphonate Derivative

Cited by 4 publications

References 24 publications

Site‐selective chemical modification of chymotrypsin using peptidyl derivatives bearing optically active diphenyl 1‐amino‐2‐phenylethylphosphonate: Stereochemical effect of the diphenyl phosphonate moiety

Site‐selective chemical modification of chymotrypsin using peptidyl derivatives bearing optically active diphenyl 1‐amino‐2‐phenylethylphosphonate: Stereochemical effect of the diphenyl phosphonate moiety

A Molecular Probe with Both Chromogenic and Fluorescent Units for Detecting Serine Proteases

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

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