Site-specific cleavage of proteins and peptides promoted by redox-active metal chelates

by redox-active metal chelates has recently been reported. Rana and Meares demonstrated that an EDTA–Fe derivative, covalently attached to a cysteine residue of the protein, can mediate cleavage of the protein backbone in a conformationdependent manner. A different EDTA–Fe derivative attached to a protein has been described for the affinity cleavage of proteins. In these cases, cleavage of the peptide bond occurred at the region where the metal attained close contact in its three-dimensional structure. This process is more advantageous than cleavage by proteolytic enzymes or by cyanogen bromide for the investigation of the tertiary structure of a protein. However, the design of metal chelates which are specific and reactive to a particular protein is not a trivial matter. In the previous paper, we reported Schiff base copper(II) chelates (1a—i, 2a—i) carrying an amidinium group that were prepared from a-amino acids, copper(II) acetate, and salicylaldehyde, and which exhibited strong binding affinity for bovine trypsin. These compounds possess a cationic amidinium group which has an electrostatic interaction with the anionic carboxyl group of the Asp177 residue 9) of the trypsin binding cavity. In addition to this primary interaction, these compounds should have hydrophobic interactions with the protein which will promote binding affinity. We were thus interested in site-specific cleavage of trypsin by these amidine-containing Schiff base metal chelates. The binding of the metal chelate to trypsin is expected to be a facile process due to electrostatic interaction by the amidinium group. In addition, this method may be applicable to other trypsin-like enzymes, such as plasmin, thrombin, and urokinase. In the present paper, Schiff base iron(III) chelates carrying an amidinium group (3a—i, 4a—i) have been prepared and the inhibitory activity of these chelates toward bovine trypsin has been studied. In addition, site-specific cleavage reactions of bovine trypsin by Schiff base copper(II) or iron(III) chelates in the presence of hydrogen peroxide and sodium ascorbate have been examined. The amidine-containing iron(III) chelates (3a—i) were obtained by adding iron(III) nitrate to an alcoholic solution of a stoichiometric amount of a-amino acid, 4-formyl-3-hydroxybenzamidine hydrochloride and potassium hydroxide. The reaction mixture was stirred for 3 h at room temperature, and the resulting reddish brown crystalline powder was collected and recrystallized from methanol–acetone. Chelates prepared as above were characterized by infrared and UV absorption spectroscopy and also by elemental analysis. Synthesis of the chelates (4a—i) was carried out in a similar manner using 3formyl-4-hydroxybenzamidine hydrochloride. The absorption spectrum of 3a (prepared from L-alanine, R5CH3) showed marked absorption maxima at 430 nm (e53490) and 475 nm (e52930). The IR spectrum of 3a showed a C5N stretching frequency at 1630 cm. These results indicate that iron(III) coordinates to carboxyl oxygen, imine nitrogen, and the phenolic oxygen of the Schiff base ligand. Elemental analysis indicated a 1 : 2 ratio of metal to Schiff base ligand. The inhibitory activity of the amidine-containing iron(III) chelates towards trypsin was determined according to the reported procedure. Determination of inhibition constants (Ki values, the dissociation constants of enzyme–inhibitor complexes formed from trypsin and inhibitors) was carried out following the method of Dixon. Concentrations of inhibitors used in the kinetic analysis were in the range of 10—10 M, corresponding to their Ki values. A control experiment was carried out with bis(N-salicylidenealaninato)iron(III) chelate (6) lacking an amidinium group. The tryptic activity was entirely unaffected by 6 in this concentration range. At a higher concentration of 6, competitive inhibition with a Ki value of 1.4310 23 M was found which is reasonable for the binding of an aromatic compound to trypsin. All iron(III) chelates behaved as potent competitive inhibitors and the Ki values are listed in Table 1. All were stronger inhibitors than the parent salicylaldehydes. The Ki values indicate that the chelates are involved in a highly specific interaction. No pronounced difference in the inhibitory activity in relation to the a-substituent was seen. Chelates derived from 3formyl-4-hydroxybenzamidine exhibit mostly stronger binding affinity than those derived from 4-formyl-3-hydroxybenzamidine, but the difference was not significant. The binding affinities of iron(III) chelates for bovine trypsin were comparable to those of the copper(II) chelates reported previously (Ki, 1.1310 —1.1310 M). Cleavage of the trypsin backbone resulting from specific binding of the copper(II) or iron(III) chelates was next inves116 Notes Chem. Pharm. Bull. 47(1) 116—119 (1999) Vol. 47, No. 1