Studies on polypeptides XLI₁H NMR studies of non‐covalent, semi‐synthetic ribonuclease A analogues in which histidine‐119 has been replaced by L‐homohistidine, N^τ‐methyl‐L‐histidine, N^π‐methyl‐L‐histidine and 3‐(3‐pyrazolyl)‐L‐alanine, respectively

Abstract

A solution of 400 mg (0.92 mmol) I2 in a mixture of 50 ml ethyl acetate and 3 ml methanol was stirred in a hydrogen atmosphere with 250 mg Lindlar's catalyst6 and 0.1 ml freshly distilled quinoline at 37°C (bath). After about 20 min the hydrogen uptake almost stopped, 200 mg catalyst and 0.1 ml quinoline were added and stirring was continued at 37°C. After another 20 rnin the hydrogen uptake (23 ml, calcd. 22.0) stopped. The reaction mixture was cooled and the catalyst was filtered off (G4 filter). The filtrate was washed twice with aqueous H,SO, (1 rnol/l). After washing with water until neutral, drying and evaporation 397 mg 16, m.p. 73-74.9"C, was obtained. IR: 3500-2500, 1695, 945 and 1430 cm-' ( -COOH) 3600-3200 and 1065 cm-' (prim. (Z)-28-Hydroxy-l9-octacosenoic acid ester of linoleic acid (18) 329 mg 16 was esterified in the way described for 14. Reaction time 5 h. Purification by reversed-phase chromatography, followed by chromatography over silica H with CH,CI, as eluent, yielded I35 mg 18 (25.7%), m.p. 47.3-48.3"C. IR: 3200-2500, 1695, 1425 and 950 cm-I (-COOH) 1727, 1215 and 1178 crn-' (ester) 3008 and 720 c m -' ( Z double bond). MS: Parent peak at m/e 700 (calcd. 700). - Abstract'H NMR titration studies carried out at 300 or 360 MHz are described for RNase 1-120, RNase 1-1 18 and five non-covalent complexes of RNase 1-1 18IRNase 11 1-124, having L-histidine, L-homohistidine, L-N"-methylhistidine, LN-methyl-histidine and 3-(3-pyrazolyl-~-alanine, respectively, as the activesite amino acid in position 119. The pH titrations were performed both in the absence and in the presence of a RNase A inhibitor (3'-CMP). From least-squares analysis of the titration data, the most probable pK, values and intrinsic chemical shifts of the histidine side-chain in position 12 and 105 and of the amino acid residue in position 119 were obtained. On the basis of these results, the effects of replacing the amino acid residue in position 119 and of inhibitor binding are discussed.