Aug. 20, 1958
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C, 63.53; H, 6.78. Dehydrotosylation with sodium acetate in acetic acid gave 21-acetoxy-17a-h~drdxy - 16a - methyl - 1,4,9(11) - pregnatriene238 mp (e 15,500). 3,20-dione, m.p. 210-213", A",: Anal. Found: C , 72.68; H, 7.65. Addition of hypobromous acid (N-bromoacetamide and perchloric acid) gave a Sa,llp-bromohydrin, which was epoxidized by sodium acetate treatment to 21 - acetoxy - 17a - hydroxy 16a -methyl - 9@,llPDEPARTMENT O F AGRICULTURAL BIOCHEMISTRY m.p. 198-200 O, E. F. NEUFELD epoxy-1,4-pregnadiene-3,20-dione, D. S. FEINGOLD UNIVERSITY OF CALIFORNIA [ a ] +40.1°, ~ AZ::" 249 mp (E 15,600). Anal. W. 2 . HASSID Found: C, 69.55; H, 7.18. Ring opening with BERKELEY 4, CALIFORNIA RECEIVEDJUNE 23, 1958 hydrogen fluoride in chloroform-tetrahydrofuran produced the desired product, 9a-fluoro-160methylprednisolone 21-acetate (I), m.p. 229-231 O , 16-ALKYLATED CORTICOIDS. 11. 9~t-FLUOR0-16a- [a]D t 7 7 . 6 , A,":" 239 mp ( E 14,500). Anal. METHYLPREDNISOLONE 21-ACETATE' Found: C, 66.27; H, 7.18. Sir: Eosinopenic activity in the mouse, dog, and man, A recent report2 on preliminary clinical trials of shows this compound to be a t least four to six Oa-fluoro-1Ga-methylprednisoloneprompts us to times as active as prednisone and prednisolone. describe our synthesis of its 21-acetate (I) from In the granuloma pouch test,5 I is 6.5 times as sapogenin intermediates. The biological activity active as prednisolone acetate, while thymus inof I in animal and human studies is similar to that volution studies in rats and the nitrogen excretion reported for the corresponding alcohol. Addi- in dogs reveals this compound to be about twentytional pertinent animal and clinical data for the five times as active as prednisolone acetate and acetate are recorded below. prednisone, respectively, in these tests. 16a-Methylpregnen~lone~was hydrogenated Metabolic balance studies6 carried out with I with palladium in acetic acid to 3P-hydroxy-16a- in a human subject in doses of 15 mg. and 25 mg. methylallopregnan-20-one, m.p. 203-205 O , [ aID per 24 hours caused an average increase over conf68.2 O (all rotations in dioxane). Anal. Found : trol values in urinary excretion per 24 hours of C, 79.42; H, 11.31. Enol acetylation a t C-20 fol- (1) phosphorus: 53 mg. a t 15 mg. dose and 387 mg. lowed by treatment with peracetic acid, then alka- at 25 mg. dose; (2) nitrogen: 3 g. at 15 mg. dose line hydrolysis, gave 3P-17a-dihydroxy-16a-methyl- and 6.4 g. a t 25 mg. dose; (3) sodium: 13.4 meq. allopregnan-2O-one, m.p. 257-259', [a]D +11.9". at 15 mg. dose and 26.4 meq. at 25 mg. dose; Anal. Found: C, 75.91; H, 10.04. Bromination (4) potassium: 8.9 meq. a t 15 mg. dose and 17.9 and acetoxylation a t C-21 produced 21-acetoxy- meq. a t 25 mg. dose. 3p,17a - dihydroxy - l 6 a - methylallopregnanFasting blood sugar levels were consistently [ a ] ~4-21.0. A n d . elevated above 120 mg. per cent. throughout the 20-one, m.p. 181-185', Found: C, 70.59; H, 8.51. Oxidation with period of administration of I in doses of 15 mg. and chromium trioxide-acetone-sulfuric acid gave 21- 25 mg. per 24 hours. This is in marked contrast acetoxy - 17a - hydroxy - 16a -methylallopregnane- to the results obtained with prednisone a t doses up 3,20-dione, m.p. 205-207', [a!D +46O. Anal. t o 70 mg. per 24 hours. Found: C, 71.15; H, 8.74, Dibromination a t ( 5 ) A. Robert and J. E. Nezamis, A d o Endocrinol., 26, 105 (1957). C-2 and C-4, then dehydrobromination with di(6) E. C. Reifenstein, F. Albright and S. Wells, J . Clin. Endocrind.. methylformamide produced 21-acetoxy-17a-hy- 6 , 367 (1945). droxy-16ru-methyl-l,4-pregnadiene-3,20-dione (16aEUGENE P. OLIVETO RICHARD RAUSSER methyl-1-dehydro-CompoundS 21-acetate) which LABORATORIES LOISWEBER without purification was hydrolyzed with sodium RESEARCH SCHERING CORP. A. L. NUSSBAUM hydroxide to the 21-alcohol, m.p. 209-212', [ a ] ~BLOOMFIELD, N. J. ~YILLIAMGEBERT +45.7", "::A: 244 mp (E 14,900). Anal. Found: C. THOMAS CONIGLIO E. B. HERSHBERG C, 73.98; H, 8.38. 11a-Hydroxylation with S. TOLKSDORF Pestalotia foedans4 gave 11a,17a,21-trihydroxyMILTONEISLER 16a-methyl-1,4-pregnadiene-3,20-dione, m.p. 236P. I,. PERLMAN 238", [a]D +23.9', A",: 247 mp (e 18,200). MASSACHUSETTS GENERALHOSPITAL M. M. PECHET Anal. Found: C, 70.56; H, 8.02. 21-Monoacetate: BOSTON,MASS. m.p. 188-190°, [a]D +45.6, A,":,"" 247 m p ( e RECEIVED J U N E 30, 1938 19,000). Anal. Found: C , 66.96; H, 7.64 (1 mole ethyl acetate). 1la-Tosylate-21-acetate: OF THE PHOSPHOGLYCERIC ACID MUTASE m.p. 182-184' (dec.), [ a ] +87.7", ~ 229.5 STUDIES REACTION WITH RADIOACTIVE SUBSTRATES mp ( e 22,200), shoulder a t 241 mp. Anal. Found:
The mung bean particulate preparation thus contains a 4-epimerase capable of converting UDP glucuronic acid to UDP galacturonic acid, and a decarboxylase which decarboxylates the UDP uronic acid (or acids) t o UDP pentose (or pentoses). Particulate preparations from asparagus shoots, radish roots and leaves, and spinach leaves were also found to catalyze these reactions.
(1) After submission of this manuscript, a Communication appeared
[G Arth, J. Fried, D. Johnston, D. Hoff,L. Sarett, R. Silber, H. Stoerk and C. Winter, THIS JOURNAL, 80, 3161 (1958)l dgscribing the preparation of 9~-fluoro-l6~-methylprednisolone 21-acetate from bile acid intermediates. (2) E. W. Boland, Cal. Mcd., 88, 417 (1968). (3) R. E. Marker and H.Crooks, THISJOURNAL, 64,1280 (1942). (4) Canadian Patent 507,009.
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sir:
I n a previous communication it was confirmed that diphosphoglyceric acid (DPGA) activated phosphoglyceric acid mutase and that during the reaction one of the phosphate groups was transferred to a suitable acceptor.' There was observed, (1) L. I. Pizer and C. E. Ballou, THIS JOURNAL, 79, 3612 (1957).
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however, a small but definite amount of enzymatic activity even when special precautions were taken to eliminate DPGA from the reaction. This activity was ascribed to an activated form of the enzyme, probably a phosphorylated form. Another possibility was that cofactor DPGA was tightly bound to the enzyme and was therefore always present. I n order to differentiate between these two alternatives, use has been made of Ci4and P32-labeledsubstrates. Carboxyl-labeled 3-phosphoglyceric acid (3PGA) was obtained by treating ribulose 1,5-diphosphate with C14-sodium bicarbonate in the presence of purified carboxydismutase as described by Calvin, et a1.' To a solution containing 0.17 Hmole of CI4-3PGA, 0.15 ymole DPGA and 50 pmoles of imidazole-HC1 buffer pH 7.0 was added 0.2 pmole of crystalline enzyme (final volume 1.5 ml.). two hours of incubation a t 30' the solution v a s dialyzed extensively against several changes of distilled water and sodium chloride solution. The final dialysate possessed no radioactivity but the protein solution was radioactive, and the amount of radioactivity in this solution indicated that there was about one mole of phosphoglyceric acid per 100 moles of protein. It appears, therefore, that although substrate is tightly bound to the enzyme, not enough is present to account for all the activity in the reaction mixtures where DPGA is excluded. Phosphate-labeled DPGA was isolated from fluoride-poisoned fermentation mixtures by barium p r e ~ i p i t a t i o nand , ~ was purified by paper chromatography with a 2-propanol/NH40H/water (70.10 20) solvent system. To a solution containing 50 ynioles of DPGX and 200 pmoles of imidazole-HC1 buffer pH 7.0 was added 0.2 ptnole of crystalline enzyme (final volume 5.6 ml,). After incubation and dialysis similar to that described previously, the protein solution was evaporated to dryness I t contained bound P32, which was shown to be linked to the protein: Partial hydrolysis of the protein was carried out by dissolving it in 11 1Y hydrochloric acid and incubating a t 40" for i 2 hours. The hydrolysate, after removal of thc hydrochloric acid, mas passed through a. Domev 50 (H+) resin column '1s described I n I'lnvin ' The eluate was rechromatographed mi piper with the 2-propanol solvent previously described and the region containing the P32-labeledpeptide material was eluted. This material after hydrolysis a t 110' in a sealed tube with 5 7 S hydrochloric acid for 20 hours was chromatographed in the phenolwater system of Block6 Developnit n t of the chromatogram with ninhydrin indicated that the amino x i d s serine, alanine, and glutamic acid were present &long with P32-inorganicphosphate. Prewitiably i n the peptide material resulting from partial hydrolysis the phosphate i q esterified to the Serine hydroxyl. Determination of the Pd2in the phosph(,rylated peptide fraction purified by chroniatographv in0)J Xlayaudon, A A R e n i o n a n d R'i Czl\in Riochr >7 n~ * , I \ ( 4(1n 23, 342 (1957) ( 3 ) l h e c l 4 3PGA was prepared by Ur 2. Pon and tht a u t h o r gratefully acknnnltdge? the gift of tht? yampound (4) C S e u b e r i nd H I uqtig A i r h & z o ~ h ~ m 1 i l l (1442) ( 5 ) M F l a i i n 1 Hid C h r m 210, 771 (1954) ( 6 ) R J Block, Anal Chem 22, 1327 (1950).
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dicated that 94y0 of the protein molecules had beeii phosphorylated. It appears that sufficient phosphate is present on the enzyme to account for the observed activity in the absence of DPGA, and that phosphoglyceric acid mutase has a mechanism of action similar to that of phosphoglu~omutase.~ (7) V. A. S a j j a r and hl. E. Pullman, Science, 119, 631 (195.4). E. P. Kennedy and D. E. Koshland, J . Bioi. Chenz., 228, 419 ( l 9 . 5 i ) .
DEPARTMEST OF BIOCHEMISTRY UNIVERSITY O F CALIFORSIA
BERKELEY 4, CALIFORNIA r
