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 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-ß-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.

Formation of 2,6-dichloro-1,4-benzoquinone from aromatic compounds after chlorination.

Halobenzoquinones are a group of disinfection byproducts formed by chlorination of certain substances in water. However, to date, the identities of halobenzoquinone precursors remain unknown. In this study, the formation of 2,6-dichloro-1,4-benzoquinone (DCBQ), a typical halobenzoquinone, from 31 aromatic compounds was investigated after 60 min of chlorination. DCBQ was formed from 21 compounds at molar formation yields ranging from 0.0008% to 4.9%. Phenol and chlorinated phenols served as DCBQ precursors, as reported previously. Notably, DCBQ was also formed from para-substituted phenolic compounds. Compounds with alkyl and carboxyl groups as para-substituents led to relatively higher molar formation yields of DCBQ. Moreover, p-quinone-4-chloroimide, 2,6-dichloroquinone-4-chloroimide (2,6-DCQC), and para-substituted aromatic amines (e.g., aniline and N-methyl aniline) served as DCBQ precursors upon chlorination. It was deduced that DCBQ was formed from the para-substituted aromatic amines via 3,5-dichloroquinone-4-chloroimide, a structural isomer of 2,6-DCQC. These results suggested that DCBQ was formed by chlorination of natural organic matter containing para-substituted phenolic species and para-substituted aromatic amines, despite the absence of phenol in water.

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

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.

Formaldehyde formation from tertiary amine derivatives during chlorination.

In May 2012, formaldehyde (FA) precursor contamination in the Tone River Basin led to the suspension of water supply to approximately 360,000 homes, which affected approximately 870,000 people in the Tokyo Metropolitan Area. The discharge of industrial effluents containing hexamethylenetetramine (HMT), a tertiary amine and FA precursor, without proper treatment resulted in the formation of FA during chlorination at water purification plants. Tertiary amines are known to be the precursors of aldehydes upon chlorination. In this study, FA formation from 29 separate amine derivatives during chlorination was investigated to determine any other potential causes of this water quality accident. The FA formation yield also included FA formation by the autolysis of the target compounds as well as the chlorination of the autolysis products. The FA molar formation yield of HMT was the highest after 24h of chlorination (440%). Among the various tertiary amine derivatives containing N-methyl groups, tertiary amines and hydrazines were found to be strong FA precursors because the FA molar formation yields per N-methyl group ranged from 25% to 45% (with a mean of 38%) and from 35% to 45% (with a mean of 41%), respectively. Guanidines and sulfamides containing N-methyl groups were also FA precursors but they exhibited lower FA molar formation yields per N-methyl group. The FA molar formation yields of the remaining compounds were <4%. The FA formation yield of HMT was extremely high even on a per weight basis (95 wt.%). The FA weight formation yields of some tertiary amines and hydrazines were greater than 20 wt.%.