Benzyl Residue Research Articles

Specificity of thrombin: I. Esterolytic properties of thrombin, plasma, trypsin, and chymotrypsin with N β-substituted guanidino derivatives of p-nitrophenyl- p′-guanidinobenzoate

Thrombin exhibits a restricted specificity, relative to plasmin, trypsin, and chymotrypsin, for a series of derivatives of the titrant substrate p-nitrophenyl- p′-guanidinobenzoate (NPGB). Substitution on the beta guanidino nitrogen of NPGB with an n-butyl, n-hexyl, cyclo-hexyl, or benzyl residue does not prevent the esterolytic cleavage of these derivatives but does markedly alter their substrate properties with the four enzymes investigated. All four enzymes cleave NPGB at equivalent concentrations by releasing p-nitrophenol as pre-steady-state burst reactions followed by its steady-state production. Both chymotrypsin and trypsin similarly display burst reactions with the derivatives at corresponding concentrations. The acyl-enzyme intermediates formed with chymotrypsin, however, are more stable for the derivatives than for NPGB, and those formed with trypsin are less stable. In contrast, plasmin and thrombin exhibit incomplete burst reactions with the derivatives at these concentrations. Except for the cyclo-hexyl derivative, with which plasmin does not react, the derivatives relative to NPGB were cleaved faster by plasmin than by thrombin. These cleavages with thrombin, moreover, were competitively inhibited by benzamidine. Kinetic data obtained for thrombin further indicated that the substituent groups of derivatives hindered the initial formation of enzyme-substrate complexes. These results suggest that thrombin and, most likely, plasmin have restricted primary binding-site regions for small molecule substrates which do not readily accommodate bulky substituent groups. In addition, increasing concentrations of glycerol were found to greatly alter the esterolytic properties of thrombin for the compounds studied. This effect was demonstrated by increased deacylation rates with NPGB and by decreased cleavage rates with the n-butyl derivative.

Synthese eines labempfindlichen pentadecapeptids aus kuh- -casein

The solid phase synthesis of the pentadecapeptide H-Lys-His-Pro-Pro-His-Leu-Ser-Phe-Met-Ala-Ile-Pro-Pro-Lys-Lys-OH, 1 1 Alle Aminosäuren besitzen L-Konfiguration a partial peptide sequence from bovine ▪-casein, is described. During the synthesis the N α-amino group of the individual amino acids was blocked by the t-butyloxycarbonyl residue (Boc), the N ε-amino group of Lys by the benzyloxycarbonyl residue (Z). the OH group of Ser by the benzyl residue and the imidazole nitrogen of His by the 2,4-dinitrophenyl group. The stepwise condensation of the protected amino acids on to the peptidyl resin proceeded by means of the carbodiimide method. After the separation of the peptide from the carrier resin with purified HBr/Anisol with simultaneous cleavage of the benzyloxycarbonyl and benzyl protective groups, impure dinitrophenyl peptide hydrobromide was obtained in 59·8% yield calc. on the content of Lys in the Boc-Lys(Z)-polymer. The dinitrophenyl groups could be eliminated quantitatively in aqueous solution at pH 8 with 2-mercaptoethanol within 16 h. After purification of the peptide by gel chromatography on Sephadex G-25 in 0·05 M acetic acid, 13·6% of pure pentadecapeptide acetate were obtained. The purity was controlled by TLC in different solvent systems, by thin-layer electrophoresis and by quantitative amino acid analyses. The following peptides were synthesised by the same method for comparison: the octapeptide H-Lys-His-Pro-Pro-His-Leu-Ser-Phe-OH, the heptapeptide H-Met-Ala-Ile-Pro-Pro-Lys-Lys-OH, the hexapeptide H-His-Leu-Ser-Phe-Met-Ala-OH and the pentapeptides H-Leu-Ser-Phe-Met-Ala-OH and H-Ser-Leu-Phe-Met-Ala-OH. The pentadecapeptide is quantitatively split by rennin between Phe and Met at pH 4·25. The Michaelis-Menten constant for this reaction is 0·45 mmoles/l. The hexapeptide is also split by rennin whereas both pentapeptides were found to be resistant to rennin. The His residues in the peptides thus may be considered to be substrate specific. The pentadecapeptide and the hexapeptide as synthetic substrates are suitable for activity determinations and for a control of rennin preparations.

Azonia-azulene salts. Part III. The thermal decomposition of o-benzylphenyl azides to give azepinoindoles

Thermal decomposition of o-benzylphenyl azides usually gave 10H-azepino[1,2-a]indoles [(2), (3), (19), (26)—(28), (37), and (39)]. In one case steric obstruction led to a 6H-azepino[1,2-a]indole (29) and an 8H-azepino-[1,2-a]indole (30). When a o-methoxy-group was present on the benzyl residue the major products were acridines and acridans; the formation of 1-substituted acridines suggests that an azanorcaradiene is an intermediate. Azepino[1,2-a]indol-6-ones [(38) and (42)] have been isolated; azepino[1,2-a]indol-8-one (21) has been prepared in good yield and converted into hydroxy- and ethoxy-azepino[1,2-a]indolium salts [(23) and (24)]. 1-(o-Azidophenyl)-1-phenylethanol (47) decomposed to give some o-anilinoacetophenone (49), showing the occurrence of another nitrene insertion pathway.

α-arylmethylamino acids

N-substituted amino acids with benzyl, veratryl or skatyl residues on the amino group have been synthesized, and their Chromatographie and electrophoretic behavior has been studied. A method of acetylating N-substituted amino acids has been worked out.

Reductive photoalkylation of flavin nuclei and flavin-catalyzed photodecarboxylation of phenylacetate

AbstractStructure and properties of leucoflavin derivatives obtained through flavinsensitized photodecarboxylation of phenylacetate ions are described. The photoreduction of flavin with phenylacetate as donor is shown to be a quantitative photoalkylation, the benzyl residue entering either position 5 (nitrogen) or the bridge position 4a (carbon) of the flavin nucleus, depending on the conditions. The isomers were isolated, and identified by IR., NMR., and elementary analysis. The benzyl residue migrates from N(5) to C(4a) upon heating.

Aromatische Substitution des Tyrosins bei der sauren Hydrolyse seiner Carbobenzoxyderivate

AbstractThe side reactions observed on acid hydrolysis (6 N HCl; 20 hrs at 115°C) of carbobenzoxy derivatives of tyrosine containing peptides were investigated by hydrolyzing carbobenzoxy‐L‐tyrosine or O‐benzyl‐L‐tyrosine under the same conditions or by reacting L‐tyrosine with benzylchloride in the presence of 6N HCl. In every instance the reaction mixture contained besides tyrosine 3 new products distinguishable by paper chromatography. The fractionation of the mixture yielded a mono‐benzylated tyrosine considered to be 3‐benzyl‐L‐tyrosine (I) and two different products which were shown to contain 2–3 benzyl residues in the aromatic ring. These results demonstrate that the benzyl cation formed in the course of the hydrolysis of carbobenzoxy derivatives of tyrosine containing peptides transforms L‐tyrosine into derivatives substituted in the aromatic nucleus.