6402
COMMUNICATIONS TO T H E EDITOR
Vol. 77
135') 171-171.5' or 197-199" (polymorphic forms), Anal. Calcd. for C26H3604: C, 74.96; H , 9.06. [ ~ J D 4- 164' (chl.), "A:= 242 mp (15,125). Anal. Found: C, 75.23; H, 9.17. Oxidation of XVI Calcd. for C24H3406: C, 68.87; H, 8.19. Found: with hydrogen peroxide and osmium tetroxide proc, 68.84; H, 8.15, and llfl,17a,20cy,21-tetrahy- duced 2-ethyl-1 1~,17~~,2I-trihydroxy-4-pregnene-3,droxy-2-methyI-4-pregnene-3-one 21-acetate (VII), 20-dione 21-acetate (XVII), m.p. 160-168", isolated m.p. 215-218.5') [ a ] D +67" (diox.). Anal. Calcd. as a methanol solvate. Anal. Calcd. for C26H36for C H H ~ ~ OC,~ : 68.54; H, 8.63. Found: C, 06.CH30H: C, 67.21; H, 8.67. Found: C, 67.55; 68.82; H, 8.60. The presence of the 17,2O-glycol H, 8.97. These compounds were tested in the Department grouping in VI1 was shown by a negative Tollens test and periodic acid oxidation to 1lo-hydroxy-2- of Endocrinology of the Upjohn Research Division. 2-hfethylhydrocortisone acetate (VI) was found to methyl-4-androstene-3,17-dione,m.p. 206-208', [CYJD +220° (chl.). Anal. Calcd. for C20H2803: be ten times as active as hydrocortisone in the glyC, 75.91; H, 8.92. Found: C, 75.98; H, 9.31. cogen deposition assay, while the corresponding 9 cyHydrolysis of VI with potassium bicarbonate in fluoro derivative XI11 was thirty-eight times as potent. In the salt retention assay VI and XI11 methanol gave 11~,17cy,21-trihydroxy-2-methyl-4pregnene-3,20-dione (VIII), m.p. 237-238", [ f f ] D were found to be more potent than DOCA by fac+185" (95% EtOH), Ag2H 242 mp (15,250). tors of two and six-tenths and ninety, respectively. Anal. Calcd. for C22H3205: C, 70.18; H, 8.57. Xore complete biological data will be p ~ b l i s h e d . ~ The preparation of the 2-alkyl analogs of other Found: C, 70.14; H, 8.61. N-Bromoacetamide in t-butyl alcohol-pyridine oxidized VI to 17a,21- steroid hormones will be reported a t a later date. The authors are indebted t o J. L. Johnson, Mrs. dihydroxy-2-methyl-4-pregnene-3,11 ,ZO-trione 21acetate (IX) in 72% yield, m.p. 205-209", [ f f ] D G. S. Fonken and J. E. Stafford for infrared and ul170" (acetone). Anal. Calcd. for C24H3206: traviolet absorption data, and to W. 4 . Struck and associates for microanalyses. C, 69.25; H, 7.75. Found: C, 68.94; H, 7.69. Dehydration of the 110-hydroxyl group of VI ( 5 ) W. W. Byrnes, L. E. Barnes, B. J. Bowman, W. E. Dulin, E. H. with thionyl chloride in pyridine afforded 17a,21- Morley and R. 0. Stafford, Proc. SOC.E x p . M e d . , in press. dihydroxy-2 - methyl - 4,9 (11)- pregnadiene - 3,20- di- RESEARCH LABORATORIES J. A. HOGG F. H. LINCOLN one 21-acetate (X), m.p. 220-223", [.ID +13S0 THEUPJOHNCOMPANY MICHIGAN R. W. JACKSON (chl.), 240 m p (16,750). Anal. Calcd. for KALAMAZOO, W. P. SCHNEIDER C24H3205: C, 71.97; H, 8.05. Found: C, 72.05; RECEIVED NOVEMBER 15, 1955 H, 8.32. Practically quantitative conversion of X to Sa-bromo-1lo,17cy,21-trihydroxy-2-methyl-4pregnene-3,20-dione 21-acetate (XI), [m.p. 125- AN EFFECT O F PYRIDOXALS-PHOSPHATE IN VITRO 130' dec., [ f f ] D +146' (chl.). Anal. Calcd. for ON HEME SYNTHESIS AND COt PRODUCTION FROM GLYCINE-2-C-14' C24H3306Br:Br, 16.07; Found: Br, 16.27, 16.061 Sir : was accomplished with N-bromoacetamide in t-buVarious species of vitamin &-deficient animals tyl alcohol containing aqueous perchloric acid. XI with potassium acetate in acetone gave 9fl,llp- develop an anemia (dog,2 pig,3 rat,4 ducks). The effect of pyridoxal-5-phosphate on heme synthesis epoxy- 17a,21-dihydroxy-2-methyl-4-pregnene-3,20was studied with duck blood since ducks have nudione 21-acetate (XII), 75% yield, m.p. 185-188') [ a ] D +49" (chl.). Anal. Calcd. for C24H&,: cleated red cells which are able to synthesize laC, 69.20; H, 7.75. Found: C, 69.28; H, 7.90. beled heme in vitro from glycine-2-C-14.6 Day-old Pekin ducklings were made vitamin B6Hydrofluoric acid converted XI1 to 9a-fluoro-110,with a diet described by Hegsted and 17cy,2 l-trihydroxy-2-methyl-4-pregnene - 3,20- dione deficient 21-acetate (XIII) in about 4oyOyield, m.p. 236- Rao.5 After 8 days on the diet, the average 238O, [ a ]4-167" ~ (diox.), 238.5 m p (16,150). weights of the control and deficient ducklings were and 89 g., respectively. Two ml. samples of Anal. Calcd. for C24H3306F: C,66.03; H, 7.62; 271 blood removed from the heart of each animal under F, 4.35. Found: C, 66.12; H, 7.31; F,3.74. The ether anesthesia were incubated in Warburg vescorresponding 21-alcohol XIV, formed from XI11 sels with glycine-2-C-14, in the presence and absence by potassium bicarbonate hydrolysis, melted a t of pyridoxal-5-phosphate. The COz released dur250-253" dec., 239 mp (16,175). Anal. ing the incubation was collected in 0.2 ml.of 1070 Calcd. for C22H310SF: C, 66.98; H, 7.92; F, 4.82. ml. of 10% NaOH contained in the center well. Found: C, 67.14; H, 7.97; F, 4.47. Oxidation of After 2 hours the samples were chilled in ice and 3 XI11 with chromium trioxide in acetic acid pro- i d . of rat blood was added to each vessel to increase duced 9cy-fluoro-17a,21-dihydroxy-2-methyl-4-pregthe yield of heme. The cells were centrifuged and nene-3,11,20-trione 21-acetate (XV), m.p. 22'7- washed twice with 0.9% saline, and hemin was iso229",XEzH235.5 m p (15,500), [ a ]+16'7" ~ (diox.). (1) This work was supported in part b y a research grant (C-1852Anal. Calcd. for C14Ha106F: c, 66.34; H, 7.19; i C 2)) from the Xational Institutes of Health, Public Health Service. ( 2 ) P. J. Fouts, 0 . A I . Helmer, S. Lepkovsky and T. H. Jukes, f. F,4.37. Found: C, 65.79; H, 7.23; F, 3.97. Alkylation of 2-ethoxyoxalyl- 1l@,2l-dihydroxy- S u f u i t i o n , 16,197 (1938). (3) H. Chick, T. F. Macrae, A. J. P. Martin and C. J. Martin, 4,17(20)-pregnadiene-3-one(111) with ethyl iodide, Biochem. J . , 3 2 , 2207 (1938). followed by removal of the ethoxyoxalyl grouping (1) 4.Kornberg, H. Tabor and W. H. Sebrell, A m . f.Physiul., 1 4 3 , and acetylation gave 2-ethyl-11/3,21-dihydroxy- 134 (1945). ( 3 ) D. ?if. Hegsted and M. N. Rao, J . Nulrilion, SO, 307 (1945). 4,17(20)-pregnadiene-3-one 21-acetate (XVT) in 9% ( 6 ) N. S. R n d i n , D. Kittenberg and D. Shemin, f , B i d . C h e m . , 184, yield, m.1). 149-131°," : : A 2-12 mp (15,000). 743 ( l Q j 0 ) .
+
COMMUNICATIONS TO THE EDITOR
Dec. 5, 1955
6403
is not excluded. Assuming complete reaction, the detection of a small amount of glucose by paper chromatography in the hydrolysate of periodateoxidized (with subsequent reduction) amylopectin furnishes analytical evidence' for the presence of a small number of (143) or of both ( 1 4 2 ) and ( 1 4 4 ) linkages in amylopectin. We wish to present definitive evidence herein for the existence of an a - ~ (1+3)-bond in amylopectin. This evidence consists of the isolation of nigerose (8-o-a-D-glUCOpyranosyl-D-glucose) as its crystalline p-D-octaacetate from an amylopectin hydrolysate produced under conditions which are known to minimize its formation by reversion to a negligible quantity.2 A 0.4% solution of amylopectin (130 g., waxy maize starch) in 0.1 N hydrochloric acid, was hydrolyzed by heating a t 97' to 67y0 completion. This hydrolysate, after removal of the acid by ion-exchange resin, was subjected to fractionation on a carbon (Nuchar C, unground) column by the general method of Whistler and D ~ r s o . The ~ fraction known to contain maltose and isomaltose was acetylated to give 40 g. of sirupy material from which most of the maltose was removed as ,&maltose ocTABLE I taacetate by direct crystallization from ethanol, HEMESYNTHESIS AND Cog PRODUCED BY THE INCUBATION yield 18 g., m.p. 155-156", [ O ( ] ~ ~+64" D (c 4.5, chloOF GLYCINE-2-C-14 WITH DUCKBLOOD roform). The material from the mother liquor was Each vessel contained 2 ml. of heparinized blood from subjected to fractionation by silicate column extrueither vitamin BB-deficient or control ducklings and 0.1 ml. of glycine-2-C-14 (23.5 p M . ; 230,000 c.p.m./pM.). In sion chromatography and produced p-isomaltose addition, 1 mg. of crystalline pyridoxal-5-phosphate mono- octaacetate, 1.67 g., m.p. 144-146', [cyI3OD +98" (c hydrate in 0.1 ml. of saline was added to appropriate flasks 4.4, chloroform), and P-nigerose octaacetate, 350 and 0.1 ml. of saline was added to the others. Values given mg., m.p. 140-145', [E]"SD +80° (c 3.0, chloroare the averages f standard errors obtained from 4 deiicient form), X-ray powder diffraction pattern identical and 4 control ducklings. The p-values for 1, 2 and 3 are with that of known P-nigerose (sakCbiose4)octaace0.2 and
