250
COMRIUNICATIONS TO THE
STEROIDS. XCIII.1 INTRODUCTION OF T H E CORTICAL HORMONE SIDE-CHAIN Sir :
EDITOR
1’01.
so
sium acetate and potassium bicarbonate in aqueous dimethylformamide solution. (11) Department of Chemistry, Columbia LJuiver3lty, Kew I’urk 27,
Introduction of the important 21-hydroxyl func- N. Y. tion into A4-3-keto steroids with the pregnane RESEARCH LABORATORTFS HOWARD J. RINCOLD S. A. (C-20 ketone) side chain has been accomplished SYNTEX, GILBERT STORK” microbiologically2 or by a chemical reaction se- MEXICO,D. F. RECEIVED SOVEMBER 18, 1937 quence3 involving selective oxalate condensation ___ a t C-21 followed by iodination, hydrolysis and iodide displacement. STRUCTURE OF GLYCOPEPTIDES FROM A HUMAN *,-GLOBULIN We wish t o report a simple procedure for the direct preparation of 21-iodo steroids and thus of Sir: the cortical hormone side-chain. Iodination of The presence of carbohydrate in many proteins 1ia-hydroxyprogesterone in a mixture of tetra- has long been recognized. Neubergerl was able to hydrofuran-methanol with an excess of iodine in isolate a polysaccharide which accounted for the the presence of a base such as solid calcium oxide carbohydrate content of ovalbumin. We wish to or aqueous sodium hydroxide gave mainly the 21- report the isolation and characterization of three iodo steroid which without purification was con- glycopeptides from human ?-globulin, in which the i-erted directly to Reichstein’s substance “SI’ace- carbohydrate is covalently linked to an aspartyl tate4 by treatment with potassium acetate in ace- residue of the peptide. This represents the first tone followed by brief reaction with aqueous- case in which the nature of protein binding to niethanolic bisulfite to remove residual iodine. carbohydrate has been ascertained. Over-all yields of 60% have been consistently The glycopeptides were obtained from human realized in this reaction. This thus constitutes a 7-globulin, Fraction 11-1, Y by digestion with facile three stage5 conversion of 1Ga,l7a-oxido- papain a t pH 6.5, removal of amino acids and small pregnenolone, readily accessible from diosgenin, peptides on Dowex 50 x 8 (hydrogen cycle, 20-50 to substance “S” acetate (A4-pregnene-l7a,21- mesh), ethanol precipitation of the glycopeptides, diol-3,20-dione acetate) and one additional micro- and zone electrophoresis on a starch column at fiH biological step to hydrocortisone. Similarly, pro- 8.5. The isolation was followed by an orcinolgesterone has been converted to desoxycorticos- sulfuric acid method3 and a ninhydrin m e t h ~ d . ~ terone acetate in 40% to 45% yield making this Analysis of effluent fractions from the starch by far the simplest method for the large scale syn- column permitted separation of three glycopeptides. thesis of this hormone. Furthermore, 1lp-hydroxy- The hexose in the glycopeptides before electroprogesterone’ was converted to corticosterone phoresis accounted for about (30% of the hexoses in acetate by the same reaction sequence. the 7-globulin. Versatility of this reaction was further demonThe similarity of the three glycopeptides (Table strated by the preparation of A1-dehydro substance I) suggests that they are derived from the same “S” acetate* from A1*4-pregnadiene-17a-ol-3,20structure. The largest, Glycopeptide 1, probably diene* and by the conversion of ll-ketoproges- represents the prosthetic group of the intact 7terone and pregnan-3cr-ol-l1,20-dione to the corre- globulin. Glycopeptides 2 and 3 appear t o be sponding 21-acetoxy compounds, A4-pregnen-21- partially degraded; they contain less than 1 01-3,11,20-trione acetateg (dehydrocorticosterone residue of sialic acid and Glycopeptide 3 has only acetate) and pregnane-3a,21-diol-11,20-dione1°ace- half the glucosamine of the others. The peptide tate. portions differ only in the number of glutamyl resiThe 21-acetoxy introduction could be carried out dues. Some variation in peptide chain length can in one step but with inferior yield by treatment of be expected since enzymic hydrolysis a t one site the corresponding steroid with excess iodine, potas- would inhibit splitting a t adjacent sites. Traces of other amino acids were present in less than (1) Paper XCII, A. Bowers and H. J. Ringold, T e t r a h e d m n , in stoichiometric amount as judged by the finding press. ( 2 ) For pertinent references see A. Wettstein, Experientia, 11, 465 that the approximate molecular weight was less ( 1 9 3 3 , and G. M. Shull. Trans. N. Y . Acad. Sci., 19, 147 (1956). than 5,000 for a mixture of the three glycopeptide^.^ ( 3 ) J. A. Hogg, P. F. Beal, A. H. Nathan, F. H. Lincoln, W. P. Analyses for amino acids and glucosamine were Schneider, B. J. Magerlein, A. R. Hanze and R. W. Jackson, THIS performed on ion-exchange columns and for carboJOURNAL, 77, 4436 (1955). (4) Compounds were characterized by mixture melting point, rotahydrate components by suitable colorimetric tion, ultraviolet and infrared spectral determination. methods. All components were also identified by ( 5 ) A. Ercoli and P. de Ruggeri, Gazr. chim. i f a l . , 84. 479 (19.54), paper chromatography. Galactose and mannose recorded the conversion of 16~,17u-oxidc-progesterone, derived from were present in a ratio of about 3 to 5. Other Oppenauer oxidation of 16ar,l7u-oxido-pregnenolone,t o 17u-hydroxyprogesterone through Raney nickel reduction of the 16.17-iodohydrin. hexoses, pentoses, hexonic acids, hexuronic acids ( 6 ) P. L. Julian, E. W. Meyer, W. J. Karpel and I. R. Waller, THIS and hexosamines could not be detected. JOURXAL, 72, 5145 (1950). 17) T. Reichstein and H. G. Fuchs. Helu. Chim. Acta, 23, 684 (1940): G, Rosenkranz, J. Pataki and C. Djerassi, J . Org. Chem., 17, 290 (1952). (8) C. Rozenkranz, J. Pataki, St. Kaufmann, J. Berlin and C. Djerassi THISJOURNAL, 72, 4081 (1950). (9) T. Reichytein, Helu. Chim. A d a , 20, 953 (1937). (10) J v. Euw, A. Lardon and T. Reichstein, ibid., 27, 1287
(1944).
(1) A. Neuberger, Btochem. J., 32, 1435 (1938) (2) J. L. Oncley, M. Rlelin, D. A Richert, J. W. Cameron and P. M. Gross, THISJOURNAL, 71, 541 (1949). (3) R. J. Winder, “Methods of Biochemical Analysis.” edited by D. Glick, Vol. 2, 1955, p. 279. (4) S. Moore and W. H.Stein, J . Bid. Chem., 211, 907 (1954). (5) We are indebted t o Dr. H. K. Schachman for a sedimentation determination on this material.
