To a solution of the benzyloxycarbonyl nonapeptide methyl ester (XIX) (130 mg.) in methanol (1 ml.) 2 N sodium hydroxide (0.1 ml.) was added. After 1 hr. at room temperature water (2 ml.) was added and after another 15 min. the solution was acidified with N hydrochloric acid and kept in a refrigerator overnight. The supernatant was decanted and the semi-solid precipitate was dried in vacuo over sodium hydroxide. The solid residue thus obtained (88 mg.) was dissolved in a mixture of acetic acid-water (2:1) (12 ml.) and hydrogenated at atmospheric pressure for 48 hr. in the presence of 5% palladium on barium sulfate.14 After removal of the catalyst the solution was freeze-dried. The residue was chromatographed on carboxymethylcelluiose, using a gradually increasing concentration of ammonium acetate for the elution, as described for the purification of XI. The product was freeze-dried several times to remove residual ammonium acetate. The final product (30 mg., [a] mD -91.2°(c, 1.0, ,Y acetic acid) ) was homogeneous on paper chromatograms (butanol-acetic acid-water 4:1:5, Ií¡ 0.30) and by paper electrophoresis (pyridine acetate buffer pH 4.0 and ammonium acetate buffer pH 5.3) when developed with ninhydrin, Ehrlich (pdimethylaminobenzaldehyde) and Sakaguchi reagents.
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