Fel 20, 1955
COMMUNICATIONS TO THE EDITOR
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of Wilds' method.12 The crude acid chloride reacted with diazomethane, giving the diazo ketone COOR ' c=o IIIc, which, upon decomposition with hydrochloric acid, yielded the chloroketone IIId [m.p. 151', [ C Y ] ~ 48.1' ~D (c 0.890, CHCI3); calcd. for C~H3103Cl: C, 69.73; H, 8.25; C1, 9.36. Found: C, 69.93; H, 8.38; C1, 9.32. Yield from I11 65-70701. The chloride IIId was converted to the iodide IIIe and thence, using a method previously described, l t S a with silver acetate in boiling pyridine, in the pres11, R = R ' = H 1.R = R' = H ence of small amounts of acetic anhydride and IIa, R = H, R' = CHa Ia, R = Br, R' = H IIb, R = Ac, R ' = CHa I b , R = R ' = Br under nitrogen, to the keto1 acetate IIIf [m.p. ~ (c 1.051, CHC13); calcd. R CH~OAC 191.5-192.5', [ a I z 445.9' I I for C24H3405: C, 71.61; H, 8.51. Found: C, c=o c=o 71.67; H, 8.44. Yield from I I I d 65-70%]. Introduction of the A4-double bond, according to Kendall's procedure,13 through the 4-bromide (m.p. 163-164') and the A4-3-semicarbazone (m.p. 210215') of IIIf, gave 11-dehydro-17a-methylcorticosterone acetate (IV) [m.p. 157-158', [ a I z 4 D 170' (c 0.79, CHCI3); 237 mp (log a 4.44); &f;IxClr 1750 and 1720 cm. -l (21-acetoxy-20-ketone 111. R = OH IIIc. R = CHNs IV 111,R = OCHa IIId; R = CHzCi doublet); 1710 cm.-' (11-ketone); 1670 and 1620 IIIe, R = CHd IIIb, R = C1 cm.-l (A4-3-ketone doublet) ; calcd. for C24H3206: IIIf, R = CH~OAC C, 71.97; H, 8.05. Found: C, 7 2 . 2 3 ; H, 7.96. C, 51.85; H, 6.24; Br. 29.821 and the starting Yield from IIIf approximately 60701. material I were isolated. The crude, not easily The adrenal cortical activity exhibited by the separable bromination product was subjected to a new hormone analog, 11-dehydro-17a-methylcorrearrangement of the Aston-Greenburg typelOa,b,c ticosterone acetate, will be the subject of a separate and, after debromination and reacetylation, methyl communication. 3 a-acetoxy- 11-keto- 17a-methyletianate (I Ib) [m.p. I wish to express sincere thanks to the Canadian D (c 0.982, CHCI3); calcd. for Life Insurance Officers Association for financial 184', [ ( Y ] ~ ~63.7' C24H3606: C, 71.25; H, 8.99. Found: C, 71.21; support; to the Ciba Company Limited, Basel, H, 8.801 obtained in approximately 4oY0 yield from Switzerland, for the generous gift of starting mathe neutral fraction of the reaction product. From terial, t o Dr. E. H. Venning, Royal Victoria Hosthe acid fraction the hydroxy acid I1 [m.p. 285- pital, Montreal, for the biological tests; to Dr. ~ D (c 1.099, dioxane); calcd. for: R. N. Jones, National Research Council, Ottawa, 286', [ C ~ ] ~29.5' C21H3204: C, 72.38: H, 9.26. Found: C, 72.34; for the infrared spectral analysis; and to Dean H, 9.041 was isolated. The pure monobromide I a J. B. Collip for his continued encouragement. gave under similar conditions a higher yield of ester The author also wishes to express his appreciation I I b and acid TI. Refluxing of I I b with methanolic Mr. J. F. Alicino, Metuchen, New Jersey, who potassium hydroxide gave the hydroxy ester I I a to carried out the micro analyses. [m.p. 165', [CZ]*~D 41.5' (c 1.012, CHCI,); calcd. (12) A. L. Wilds, U. S. Patent 2,538,611. A. L. Wilds and C. H . for C22H3404: C, 72.80; H, 9.45. Found: C, Shunk, THISJOURNAL, 70, 2427 (1948). Compare also R. Adams 72.78; H, 9.301, also obtained upon methylation and L. H. Ulich, ibid., 42, 599 (1920). of acid I1 and easily reacetylated to the ester IIb. (13) W. F. McGuckin and E. C. Kendall, ibid., 74, 5811 (1952). Prolonged treatment of the latter with methanolic COLLIPMEDICALRESEARCH LABORATORY potassium hydroxide in a sealed tube a t 170' gave a UNIVERSITY O F WESTERN ONTARIO CH. R.ENGEL ONTARIO, CANADA high yield of the free acid 11. Oxidation of acid I1 LONDON, RECEIVED JANUARY 14, 1955 with chromic acid afforded the keto acid I11 [m.p. 288.5', [ a I z 2 D 45.1' (c 0.941, dioxane); calcd. for C21H3004: C, 72.80; H , 8.73. Found: C, 72.68; FORMATION OF A NEW DINUCLEOTIDE FROM H, 8.75. Yield 85-90y0], further characterized by COZYMASE BY ENZYMIC DESTRUCTION OF its methyl ester IIIa [m.p. 185', [ ( r I z 3 ~ 49.8 (c THE "ONIUM" LINKAGE 1.002, CHC13); calcd. for C22H3204: C, 73.27; Sir : H, 8.95. Found: C, 73.49; H, 8.811 which was Recently it has been shown' that certain enzyalso obtained by chromic acid oxidation of IIa. matically catalyzed syntheses derive their energy Acid I11 was transformed to its chloride IIIb with from the reduction of quaternary ammonium or oxalyl chloride, using Reichstein's modificationll sulfonium salts, rather than from the usual mecha(10) (a) 3. G.Aston and R. B . Greenburg, THISJOURNAL, 6'2,2590 nism of cleavage of energy-rich phosphate esters. (1940). (b) See also Al. Faworsky, J . p 7 a k f . Chem., [2]88,658 (1913). One such "onium" salt, viz., cozymase (DPN) (c) Comparable rearrangements of 17-bromo-20-ketosteroids have been was proposed as a suitable substrate from which to described by R. E. Marker and R. B . Wagner [THISJOURNAL,64, 216, 1273 (19431; P1. A. Plattner, H . Heusser and S. F. Boyce [Helv. derive dinucleotides by this mechanism. We wish Chrm. Acta, 81,603 (1948)j; H . Heusser, Ch. R. Engel, P . T b . Herzig to record the realization of such a reaction. The and PI. A. Plattner [ i b i d . , 33, 2229 (1950)l. substrate was DPN and the acceptor amine was (11) F . Reber, A. Lardon and T . Reichstein, ibid., 37, 45 (1954). CHzR'
I
A. T.ardon and T. Reichstein, ibid., 37, 388,443 (1954).
(1) D. W.Woolley. N a f u r c , 171,323 (1953).
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COMMUh’ICATIONS T O THE E D I T O R
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4-amino-5-carboxamido-imidazole, the known precursor of purines.2 The enzyme was soluble, highly purified, beef-spleen DPNase. The reaction proceeded to completion according to the equation
could be an alternative to the one viewed by Greeiibergs or even the one he has been investigating. The conversion of DPN to this new dinucleotide can be clearly distinguished from the exchangc reactions of DPN with isoniazide and with PThe Imidazole + D P N --+Sicotinamide + H + + acetyl-pyridine which have been described by Zatman, et a1.lO In these exchange re”’ actions, (a) the energy rich quaternary 0 I linkage is not lost; (b) no H + is formed; 132N/c\C/N\ /x\C’c\N and (c) the susceptibility to DPNase /I CH CH II I attack is not lost. On the contrary, in \ x / ” \ ~ / ‘ ~ the new reaction, the quaternary linkH AJC\J HO OH I/O\ I t /O\I age is lost, a H + is formed and the prodCH( CHOH)2CHC€I20POPOCHeCH(CHOH)&H uct is no lonaer attacked by DPNase. It It Similar feat&es which distinguish be0 0 tween base exchange without loss of The dinucleotide was isolated in relatively pure “onium” linkage and destruction of this linkage form, and its structure established. have been discussed fully in the case of thiaminase.’ Although DPNases from animal sources have not FROM THE ROCKEFELLER IXSTITUTE S. G. A . ALIvIsArosll previously been separated from cellular particle^,^,^ FOR MEDICALRESEARCH D. W ,WOOI.LDY it was found possible to do so by treatment of NEWYORK,N. Y. RECEIVED DECEMBER 27, 1954 mashed beef-spleen cell fragments with isoamyl alcohol plus desoxyribonucleic acid. The solu(10) L. J. Zatman, N. 0. Raplan, S. P. Colowick and M . M . Ciotti, bilized enzyme was then purified by conventional J . B ~ O Ithem., . ao9, 453 (1954). (11) Damon Runyon Memorial Fellow. methods (45 U./mg. protein). The dinucleotide was formed by incubation of 33 units of this enzyme with 26.1 micromoles of ISOLATION OF HOMOGENEOUS MELANOCYTE D P N and 3000 micromoles of the imidazole a t p H STIMULATING HORMONE FROM HOG PITUITARY GLAND 7.5 in 10 ml. for 16 hr. a t 37’. I t was then isolated by paper electrophoresis (4O, 0.2 M ammonium Sir: acetate, p H 5.0). Its mobility toward the anode The melanocyte stimulating hormone’ (MSH), was 2.2 times that of DPN, whereas for the free elaborated by the pituitary gland, darkens the skin imidazole it was 0, and for the nicotinamide-free of many chordates,2 including Although hydrolytic product of DPN it was 2.5. Yield was some investigators4v6 suggest that MSH is not a 29 per cent. of the DPN, Rp 0.26 in the ethanol- distinct substance but an adrenocorticotropin, acetic acid system4 and 0.56 in the isoamyl alcohol evidence is good that the major portion of MSH KzHPOb system,6 both a t 4’. It gave the test for activity is distinct from other pituitary hormones.2 diazotizable aminese and, when analyzed quantita- We wish to report the isolation of a fraction homotively, showed a ratio of imidazole to adenine to geneous to electrophoresis and to countercurrent ribose to phosphateof 1.0:1.1:2.0:1.9. distribution possessing the main MSH activity of The structure of the new dinucleotide was further the pituitary gland. established by cleavage with Kornberg’s pyrophosOne hundred g. of acetone dried hog posterior phata~e.~ The products were isolated by electro- pituitary powder6 (4-6 X lo7 MSH u./g.)’ mixed phoresis and shown to be adenosine-5‘-phosphate with 250 ml. acetone and 1 1. acetic acid, was heated and a mononucleotide, the 5’-phosphate of the to 50’ for 10 min. and centrifuged. The residue amino-carboxamidoimidazole riboside. This com- was separated and re-extracted. The combined pound has been postulated by Greenbergs and by extracts (1900 ml.) were mixed with acetone (950 Buchanang as the precursor of inosinic acid in ml.) and 10 ml. saturated NaCl.* The supernate pigeon liver and in bacteria. Mobility relative to obtained by centrifugation was mixed with petroDPN was 1.9, for adenosine-5’-phosphate was 1.6. leum ether (5400 ml.) and allowed to stand overI t had RF 0.69 in the isoamyl alcohol K2HP04 night a t -5’. The precipitate was acetone washed system5 and then dried in vacuo. Sixteen to 18 g. of product These results strongly suggest that inosinic acid (1) Melanocyte stimulating hormone has been referred t o also as and its derivatives could arise in living things melanophore hormone, melanophore dilating principle, intermedin, etc. (2) H. Waring and F. W. Landgrebe in “The Hormones,” edited through the action of DPNase with this new G. Pincus and IC. V. Thimann. Academic Press, Inc., New York, dinucleotide as an intermediate. Such a pathway by N. Y., Vol. 2, 1950,p. 427. (2) W. Shive, W. W. Ackermann, M. Gordon, M. E. Getzendaner and R. E. Eakin, THISJOURNAL, 69, 725 (1947). (3) S. G. A. Alivisatos and 0. F . Denstedt, Science, 114, 281 (1951). (4) L.J. Zatman, N. 0. Raplan and S. P. Colowick, J . Biol. Chem., aoo, 197 (1953). (5) C. E. Carter, THISJOURNAL, 71, 1466 (1950). (6) A. C. Bratton and E. R. Marshall, Jr., J . B i d . Chcm., 118, 537 (1939). (7) A. Romberg and W. E. Pricer, Jr., i b i d . , LEI, 763 (1950). (8) G. R.Greenberg, J. B i d . Chcm., 190,611 (1951). (9) W.J. Williams and J. M. Buchanan, ibid.. 201, 253 (1953).
(3) A. B. Lerner, IC. Shizume and I. Bunding, J . Clin. E n d o c r i d . E- Melab., 14,1463 (1954). 76, 5565 (1954). (4) P. H. Bell, THISJOURNAL, (5) N.G. Brink, G. E. Boxer, V. C. Jelinek, F . A. Kuehl. Jr., J . W. Richter and K. Folkers, i b i d . , 76, 1960 (1953). (6) We are grateful t o the Armour Laboratories and Wilson & Company for supplying us with a total of 3 kg. of hog posterior pituitary powder. (7) An MSH unit is defined by K. Shizume, A. B. Lerner and T. B. Fitzpatrick, Endocrinology. 64, 553 (1954). (8) F.W.Landgrebe and K. A . Munday, Quart. J. E r p . Piiysiol.. 89, 11 (1954).
