Application of several types of substrates to ficin-catalyzed peptide synthesis.

The capability of ficin, a cystine protease, to form peptide bonds was investigated using several types of N-Boc-amino acid phenyl and naphthyl esters as acyl donor components. Enzyme-catalyzed peptide synthesis was carried out under optimized reaction conditions of pH, acyl acceptor concentration and selection of the best yield organic solvent. It used a condensation of N-Boc-Ala-OpGu and Ala-pNA as a model reaction. The products were obtained in 72-96% yield using 10 different substrates, within a few minutes of reaction time.

Chum salmon trypsin-catalyzed preferential formation of peptides containing D-amino acid.

Chum salmon trypsin-catalyzed peptide synthesis has been studied by using nine series of "inverse substrates," i.e., p-amidinophenyl, p- and m-guanidinophenyl, p- and m-(guanidinomethyl)phenyl, and four position isomers of guanidinonaphthyl esters derived from Nalpha-(tertbutyloxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of trypsin. All substrates tested in this study undergo less enantioselective coupling reaction, and the coupling product was the favorably obtained D-series rather than L-series (in the present case; Nalpha-Boc-D-Ala and Nalpha-Boc-L-Ala). The optimum condition for the coupling reaction was studied by changing the organic solvent, buffer solution, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible.

X-ray crystallographic analyses of complexes between bovine beta-trypsin and Schiff base copper(II) or iron(III) chelates.

To establish the structural basis underlying the activity of a novel series of metal-chelate trypsin inhibitors, the structures of p-amidinosalicylidene-l-alaninato(aqua)copper(II) (1a), m-amidinosalicylidene-l-alaninato(aqua)copper(II) (1b), bis(p-amidinosalicylidene-l-alaninato)iron(III) (2a), and bis(m-amidinosalicylidene-l-alaninato)iron(III) (2b) bound to bovine beta-trypsin were studied by X-ray crystallography. The amidinium group of the inhibitor donates hydrogen bonds to Asp189, Gly219 and Ser190, as seen before in trypsin-benzamidine complexes. The copper(II) ion of 1a is situated away from trypsin's catalytic triad residues, and is octahedrally coordinated by a Schiff base and three water molecules. In contrast, the copper(II) ion of 1b is situated close to the catalytic triad and adopts a square pyramidal coordination geometry. The iron(III) ion of 2a is octahedrally coordinated by two Schiff base ligands and, like the copper(II) ion of 1a, is situated away from the catalytic triad. The p-amidinophenyl ring of a second Schiff base ligand of 2a is directed toward a hydrophobic groove formed by Trp215 and Leu99. Finally, the iron(III) ion of 2b appears to be replaced by magnesium(II), which is octahedrally coordinated by a Schiff base, Gln192 and two water molecules. One of the Schiff base ligands seen in the trypsin-2a complex or in the unbound form of 2b is replaced by water molecules and Gln192. His57 and Ser195 form water-mediated interactions with the magnesium(II) ion of 2b, and Ser195 also forms a hydrogen bond with the phenolic oxygen atom of the Schiff base ligand. These structures reveal a novel mode of interaction between metal-chelate inhibitors and serine proteases, thus providing a structural basis for the development of more potent inhibitors against a variety of trypsin-like enzymes.

Anionic trypsin from chum salmon: activity with p-amidinophenyl ester and comparison with bovine and Streptomyces griseus trypsins.

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.

Trypsin-catalyzed peptide synthesis with m-guanidinophenyl and m-(guanidinomethyl)phenyl esters as acyl donor component.

Two series of inverse substrates, m-guanidinophenyl and m-(guanidinomethyl)phenyl esters derived from N-(tert-butyloxycarbonyl)-amino acid, were prepared as an acyl donor component for trypsin-catalyzed peptide synthesis. The kinetic behavior of these esters toward tryptic hydrolysis was analyzed. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of trypsin. Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. Within the enzymatic peptide coupling methods, this approach was shown to be advantageous, since the resulting peptides are resistant to the enzymatic hydrolysis.

Application of Schiff base copper(II) and iron(III) chelates to site-specific cleavage of a trypsin.

Amidine-containing Schiff base iron(III) and copper(II) chelates were prepared from alpha-amino acid, metal ion, and salicylaldehyde. These chelates behaved as specific inhibitors of trypsin, with Ki values in the range 10(-5)-10(-6) M. Selective cleavage of the trypsin backbone resulting from specific binding of the chelate to the trypsin active site was investigated. Cleavage was observed when trypsin was incubated with amidine-containing copper(II) or iron(III) chelate, H2O2, and ascorbate. Examination of the three-dimensional structure of trypsin suggests that cleavage occurred at a peptide bond within the Gly195-Ala204 sequence.

Enzymatic peptide synthesis with p-guanidinophenyl and p-(guanidinomethyl)phenyl esters as acyl donors.

Two series of "inverse substrates", N-Boc-amino acid p-guanidinophenyl and p-(guanidinomethyl)phenyl esters, were prepared as acyl donor components for enzymatic peptide synthesis. The kinetic behavior of these esters toward bovine and Streptomyces griseus (SG) trypsin was analyzed. The spatial requirement of the active site of these enzymes for catalytic efficiency is discussed based on the steric characteristics of the substrates. These substrates were found to couple readily with amino acid p-nitroanilides to produce peptides. SG trypsin was the most efficient catalyst among the enzymes tested (bovine, porcine, and SG trypsin).

Synthesis and tryptic hydrolysis of p-guanidinophenyl esters derived from amino acids and peptides.

A facile synthetic method for p-guanidinophenyl esters derived from a variety of amino acids and peptides, including D-amino acids, is presented. The kinetic behavior of trypsin towards these synthetic esters, inverse substrates, was analyzed. The spatial requirement of the enzyme active site for catalytic efficiency is discussed based on the steric characteristics of the substrates.

Trypsin-catalyzed peptide synthesis and various p-guanidinophenyl esters as acyl donors.

Trypsin-catalyzed peptide synthesis has been studied by using p-guanidinophenyl esters of N alpha-(tert-butyloxycarbonyl)amino acid and peptide as acyl donor components. The reaction conditions were optimized for organic solvents, pH, and concentration of acceptor. The method was especially useful for the preparation of various peptides containing D-amino acids. The enzymatic hydrolysis of the resulting products was negligible.

Studies on zoospore attracting activity. II. Synthesis of isoflavones and their attracting activity to Aphanomyces euteiches zoospore.

Nineteen isoflavones were synthesized and their attracting activity to Aphanomyces euteiches zoospore were investigated. Isoflavones (1, 2, 4, 16 and 19) with an unsubstituted B ring were synthesized by the oxidation of corresponding 2'-hydroxychalcones with thallium (III) nitrate trihydrate in methanolic perchloric acid. A hydroxyl group at the C-5 position in isoflavones was necessary for strong attraction to A. euteiches zoospore. The introduction of an additional hydroxyl group at the C-7 or C-4' position strengthened the attracting activity. Moreover, the methylation of the C-7 hydroxyl group strengthened the attracting activity, but the methylation of the C-4' hydroxyl group slightly weakened it. The naturally occurring isoflavones (9 and 10), which were reported to possess estrogen activity, showed moderate attracting activity.