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fect the acetylation of the a-hydrogen of cyclohex- enol test. The combined alkaline solution was acidified 0' and the &diketone taken up in ether, from which it anone or acetophenone with acetic anhydride to at was recovered by fractionation of the dried solution (Table form the P-diketone; instead some of the ketone I). The ligroin phase was dried and fractionated, yielding underwent self-condensation. .Moreover, neither the monoketone and some high-boiling residue. Experiments with Boron Chloride and Aluminum Chloboron chloride nor aluminium chloride brought mixture of toluene (0.20 mole) and acetic anhyabout acetylation of the a-hydrogen of even $4- ride.-A dride (0.8 mole) was saturated with boron chloride at 10' trophenylacetone, which is relatively reactive. and the reaction mixture worked up as described above for Similar observations were reported recently by boron fluoride. There was obtained a 27% yield of pPerfetti and Levine6 who showed that neither alu- methylacetophenone, b.p. 101-102" a t 13 mm. A mixture of cyclohexanone (0.15 mole) and acetic anminum chloride nor stannic chloride can effect the hydride (0.30 moLe) was saturated with boron chloride in acetylation of acetophenone to form benzoylace- 40 minutes a t 10 . After stirring 30 minutes longer, the tone. However, these workers did realize this ace- reaction mixture was decomposed with excess sodium acetylation with zinc chloride and ferric chloride, al- tate in ice-water. The mixture was extracted with ligroin after drying, the ligroin solution was fractionated to though the temperature employed (110') is much and, give 2-cyclohexylidenecyclohexanon~ (599/0), b.p. 142higher than that (0') known to effect the reaction 143' at 17 mm.6; semicarbazone, m.p. 178-179'.6 with boron fluoride. Mixtures of acetophenone and acetic anhydride and of pExperimental Acetoacetylations by Boron Fluoride.-A mixture of the aromatic compound (0.20 mole) and acetic anhydride (0.80 mole) was saturated with boron trifluoride a t 0-10' in two to three hours and then stirred for an additional period to make a total reaction time of four hours. A solution of 100 g. of sodium acetate in 500 ml. of water was added and the reaction mixture refluxed 15-30 minutes. The mixture was cooled and extracted two or three times with 30-80' ligroin. The combined ligroin solution was washed three times with small portions of water and once with saturated sodium bicarbonate solution. The ligroin solution was then extracted several times with cold 2% sodium hydroxide solution until the ligroin phase no longer gave a positive (6) B, M. Perfetti and R. Levine, THIS JOURNAL, 76, 626 (1963)
nitrophenylacetone and this anhydride were treated similarly with boron chloride. There were obtained some dypnone and tarry material, respectively. A mixture of p-nitrophenylacetone (0.02 mole), acetic anhydride (0.06 mole), aluminum chloride (0.14 mole) and 40 ml. of carbon disulfide was stirred 12 hours at room temperature. After distilling most of the solvent, the mixture was poured onto ice and hydrochloric acid to give the original ketone (colored). In all of these experiments, the products gave negative enol tests with ferric chloride showing the absence of 8diketones. (6) A. D. Petrov, Bvll. soc. chim., [IV]43, 1272 (1928).
DEPARTMENT OF CHEMISTRY DUKEUNIVERSITY DURHAM,N. C.
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C O M M U N I C A T I O N S T O T H E EDITOR A SYNTHESIS OF dl-CORTISONE ACETATE anilinoniethylene group' (m.p. 124-125'. Found: Sir I C, 82.8; 13, 8.3.). The protected ketone was We wish to report a direct synthesis of dl- condensed with P-propiolactone2 in the presence of cortisone acetate from the Woodward tricyclic potassium amide in ether. Removal of the blocking group yielded dZ-l-(P-carboxyethyl)-l,l4-diketone,l dE-1,14-dimethyl-2-keto-A1~11~~6~s-octahydroas a phenanthrene. A distinguishing feature of this methy1-2-ket0-A~~'~-decahydrophenanthrene Found: C, synthesis is that the cortical side chain and the crystalline isomer3 (m.p. 171-173'. eleven oxygen function are introduced without pro- 75.2; H, 8.7). This keto-acid was converted to the enol lactone (m.p. 100-102') and thence by treattecting the a,p-unsaturated ketone in ring A. Selective hydrogenation of the U-oodward tri- ment with methplrnagnesiuin bromide followed by c!-clic ketone with palladium on strontium carbon- cyclization' to the tetracyclic ketone I1 (m.p. 1.17.ate rave the oily dihq-drotricyclic ketone I @Ex,148'. Found: C, S4.9; H, 9.2). I1 was oxidized 250 InM, ~15,300. Found: C, S3.6; H, 9.5). with iodine and silver acetate in wet acetic acid4 I was blocked in the 3 position by the methyl- to give a 0-cis-glycol. Reaction with acetone gave dl-3-keto-16& 17P-dihydro~y-A~,~(~~)-~-homoCHO androstadiene acetonides (m.p. 174-175'). The structure of the acetonide was proved by conver-
0
I
I1
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(1) Cf. R . B . Woodward, F. Sondheimar, D. Trub, R. Hsualer, and W. M. McLamore. THISJOURNAL,I C , 4223 (1062).
(2) Cf.T . L. Gresham, J. S. Jansen. F. W. Shaver, \I. R. Frederick and W. L. Beears, ibid., 73, 2345 (1951),and earlier papers. (3) The mother liquor from the isolation of this material undoubtedly contained the epimeric compound. (4) A reagent described in a private communication from K. B. Woodward; cf. S. Winstein and R. E. Buckles, THISJOURNAL, 64, 2787 (1942). ( 6 ) It is to be noted that oitr acetonide differs f r o a WJodward's' in that it WUI derived from a p-cis-glycol whereas !ais was from an o-cisglycol, where a and 8 duignate configuration corresponding to standard rtereid coaveutiaa.
Xug. 20, 1953
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COMMUNICATIONS TO THE EDTTOR
ATP, CoAI -2E sion to dl-A9(11)~16-21-norprogesterone previously Butyrate BuCoA prepared by Woodward.' (1) (11) The acetonide was converted in excellent yield to Hz0 Crotonyl CoA 4 j3-Hydroxy dl - 3 - keto - l l ~ , 1 6 ~ , 1 7-Ptrihydroxy - A4 - 9a(111) bromo-D-homoandrostene acetonide (m.p. 194196') with N-bromosuccinimide and sulfuric acid DPN CoA in aqueous acetones6 The crude bromohydrin was BuCoA 4 AcAcCoA (11') (V) converted by alkali to dZ-3-keto-9,8,1lp-oxido2Malate, DPN 16p,17p-dihydroxy-A4-~-homoandrosteneacetonide 2AcCoA _____) ?Citrate (VI, VII) (map. 191-193'. Found: C, 74.3; H, 8.7). This crude bromohydrin was also oxidized with pyridine- where (I) represents the fatty acid activating chromium trioxide complex7to give a crude bromo(11) fatty acyl CoA dehydrogenase, ketone (m.p. 195-198' dec.) which without puri- enzyme,zVs (111) unsaturated acyl CoA hydrase, (IV) Pfication was debrominated with zinc and aqueous hydroxyacyl CoA dehydr~genase,~ (V) AcAcCoA acetic acid to give dZ-3,l l-diketo-A4-16P,17/3-dicleavage e n ~ y m e , ~ (VI) * ~ . ~ malic dehydrogenase7 hydroxy-D-homoandrostene acetonide (m.p. 198200'). Treatment with periodic acid followed by and (VII) AcCoA-oxalacetate condensing enzyme.a Enzymes (I-V) have been isolated from beef liver benzene and piperidine acetate' gave dl-ll-keto- mitochondria. Tz is the final electron acceptor with A16-21-norprogesterone I11 (m.p. 207-209'. pyocyanine as intermediary carrier. Diaphoraseg Found: C, 76.5; H, 7.7). Reaction with alkaline (VIII) catalyzes the oxidation of DPNH. The hydrogen peroxidea produced dl-ll-keto-l6ct,17a- over-all balanced reaction is oxido-21-norprogesterone (m.p. 243-245'). Oxidation with silver oxide gave dl-3,ll-diketo-l6a,- BuO- + 4Tz + 2Malate f ATP + 2 Citrate 4Fz AMP PPI (1) 17a-oxido-A4-etiocholenic acid (m.p. 217-220' dec.). Reaction of the dry sodium salt with oxalyl The observed citrate:Fz ratio of 1:2.2 is in good chloride yielded an acid chloride which on treat- agreement with the 1: 2 ratio of equation (1). ment with diazomethaneg gave a crystalline diazoPreparations of (I) a t the highest purity level are ketone (m.p. 193-195') having strong infrared homogeneous in the ultracentrifuge. At ;bH 10 absorption a t 4.75~. Reaction of the diazoketone with heptanoate as substrate, 1 mg. of (I) catalyzes with hot acetic acid gave non-crystalline dl-l6a,- the formation of 3.8 p mole of acyl CoA per min. 17a - oxido - 3,11,20 - triketo - 21 - hydroxy - A4- a t 38'. (I) activates a wide variety of odd or even, pregnene acetate, Opening with hydrogen bro- straight (Cd!,,) , branched chain, or substituted mides produced dl-l6~-bromocortisone acetate fatty acids as well as a,& and B,y-unsaturated (m.p. 238-240' dec.). Debromination with Raney acids. (I) has proved invaluable for preparation nickel* gave &cortisone acetatelo (m.p. 240-243') of all acyl CoA derivatives required as substrates whose infrared spectrum was identical with natural for (11-IV). The mechanism of activation by ATP cortisone acetate. is the same as for the acetate activation enzyme We thank Dr. R. H. Munch, Mr. G. W. Ash- system. lo worth and Mr. 0. E. Kinast for help with the num(IIg) a green copper flavoprotein1' has been isoerous infrared and ultraviolet spectra needed in lated in a form which is homogeneous in both the this work. In addition, we acknowledge the in- ultracentrifuge and Tiselius apparatus. The ribovaluable advice and assistance of Dr. R B. Wood- flavin content of the homogeneous enzyme is 1.2%. ward. The prosthetic flavin has the same absorption spec(IIg) (6) After the completion of our work, J. Fried and E. F. Sabo [THIS trum and enzymatic activity as FAD.'2q'S JOURNAL, 76,2273 (1953)l reported that they added hypobromous acid can be converted into an apoenzyme at PH 3.7 _____f
__f
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in good yield to a 3-keto-AWW steroid. It now appears that the low yield obtained by Hicks and Wallis [J. Biol. Chcm., 165, 641 (1946)l may be attributed to the fact that in their case rings A and B were cis. (7) A reagent first announced at t h e Gordon Research Conferences, A.A.A.S., New Hampton, N. H., August 4-8, 1952; cf. G. I. Poos, G. E. Arth, R. E. Beyler and L. H. Sarett, THISJOURNAL, 76, 422 (1 953). (8) Cf. P. L. Julian, E. W. Meyer, W. J. Karpel and I. R. Waller, ibid., 72, 5145 (1950). (9) Cf. A. L. Wilds and C. H. Shunk. ibid., 70, 2427 (1948). (IO) L. H. Sarett, G. E. Arth, R. M. Lukes, R. E. Beyler, G. I. Poos, W. F. Johns and J. M. Constantin, ibid., 74, 4974 (1952).
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(1) The following abbreviations will be used: adenosinetriphosphate (ATP); adenoaine-5'-phosphate (AMP); coenzyme A (CoA) ; di- and triphosphopyridine nucleotide (DPN, D P N H and T P N , T P N H ) ; flavin adenine dinucleotide (FAD) ; acetyl (Ac) ; acetoacetyl (AcAc) ; butyryl (Bu); triphenyltetrazolium (Tz); formazan (Fz) ; and inorganic pyrophosphate (PPI). (2) H. R. Mahler, "Phosphorus Metabolism," Vol. 2, 286, Johns Hopkins Press, Baltimore, 1953; H. R. Mahler, S. J. Wakil and R. M. Bock, J. Biol. Chcm., in press. (3) G. Drysdale and H. A. Lardy, "Phosphorus Metabolism," Vol. 2, p. 281, Johns Hopkins Press, Baltimore, Md., 1953. (4) F. Lynen, L. Wessely, 0. Wieland and L. Rueff, Angew. Chem., ORGANIC CHEMICALS DIVISION LLOYDB. BARKLEY 64, 687 (1952). ST.LOUISRESEARCH DEPARTMENT MARTINW. FARRAR (5) J. R. Stern, M. J. Coon and A. del Campillo, Nature, 171, 28 MONSANTO CHEMICAL COMPANY WILLIAMS. KNOWLES (1953). ST. LOUIS,MISSOURI HAROLD RAFFELSON (6) D. E. Green, D. S. Goldman, S. Mii and H. Beinert, J. Biol. Chcm., 409, 137 (1953). RECEIVED JULY7, 1953 (7) F. B. Straub, 2. physiol. Chem., 576, 63 (1942). (8) S. Ochoa, J. R. Stern and M. C. Schneider, J . Bid. Chem., 193, 691 (1951). THE RECONSTRUCTION OF THE FATTY ACID OXI(9) J. G. Dcwan and D. E. Green, Biochcm. J . , 32, 626 (1938). DIZING SYSTEM O F ANIMAL TISSUES (10) H. Beinert, D. E. Green, P. Hele, H. Hift, R. W. Von Korff and C. V. Ramakrishnan, J. B i d . Chcm., 503,35 (1953). (11) H. R. Mahler, THISJOURNAL, 76, 3288 (1953). A system including seven enzymes has been (12) E. Negelein and H. Bromel, Biochem. Z.. 800, 225 (1939). (13) 0. Warburg and W. Christian, Biochcm. Z.,598, 150 (1938). shown to catalyze the following sequence
Sir:
