Aug. 20, 1962
COMMUNICATIONS TO THE
EDITOR
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TABLE I. Noticeable differences in the chemical shifts of the 19-methyl protons for a- and P-epoxides were CALCULATED AND OBSERVED COUPLING CONSTANTS FOR 6 also anticipated? PROTONS OF STEROID ~,~-EPoxIDES~~~~ Eight 5a,6a-epoxides and seven 5P,BP-epoxides, 5a,6aObserved epoxide all having either an ethylene ketal or a 30-acetoxy 60 JK, c i s P,CIS J , CIS substituent a t C-3, were e ~ a r n i n e d . ' ~ , 'I~n no 68H-7aH 94 f 4' 0.28- 0 . 1 0.0-0.27 . .... compound were there C=C or C = O functions 6pH-7BH 2 8 f 4' 5.8 6.8 7.2-8.3 3.3-4.1 which could give rise to long-range shielding of 5888either the 19-protons or the epoxidic proton. epoxide For the a-epoxide-3-ketals the 6P-proton resonance 6cuH-7cuH 75 f 4' 0.03-0.62 0.36-1.06 . . ... always appeared within the range 169.5-171.5 6aH-7pH 49 f 4' 2.8 -4.0 3 . 6 -5.0 2.1-2.7 c./s. from the TMS reference as a doublet, J , a These values of 4 are measured from models. Twelve 3.3-4.1 c./s., while the 6a-proton resonance of the measurements were made in each case and the majority fell 8-epoxide-3-ketals appeared a t 183.0-185.4 c./s. well within the limiting range of f 4 ' . from TMS,15 J , 2.1-2.7 c./s. Replacement of the 2.5 c./s. for 3-ketal by 38-OAc raises the epoxidic proton ab- of 4- 15.0 c./s. for the a-epoxide, 2 c./s. for the 3-ketal of the sorption range by ca. 5 c./s. for the a-epoxides and the @-epoxide,and by ca. 2 c./s. for the &epoxides. A comparison P-epoxide.18 Thus, after epoxidation of a series of of observed and calculated J values is given in A5-steroid 3-ethylene ketals the 19-protons resoTable I. It is of note that these epoxides constitute nated a t 64.4-65.6 c./s. for five a-epoxides and a t very rigid systems, yet the observed J values for 60.2-60.7 c./s. for four @-epoxides. Agreement of the epoxidic protons are lower than either set of calculated and observed 19-proton frequencies is calculated values. The agreement of the ob- a further criterion for the epoxide stereochemistry. served values and those calculated from the Moreover, if the epoxide bears a 38-hydroxy or Karplus equations5 is however acceptable. The 3P-acyloxy function, the 3a-proton is shifted 25range of J values is conveniently narrow in each 30 c./s. away from the TMS frequency in the a-epoxcase and coupled with the value of the chemical ides 0nly.'9 We consider i t important to note that additivity shift completely defines the epoxide stereochemistry. That each proton absorption appears as a doublet values for steroid methyl proton frequency shifts agrees well with the implications5J that for 4 = are applicable only when the introduction of further 70-llOo, J is quite small. In both epoxides only substituents into the steroid nucleus does not cause serious alterations of the relative positions and one 6,7-proton coupling is observed. From the positions of the 19-proton resonances orientations of the methyls and substituents capable of long-range shielding. This is particularly imi t was possible to calculate additivity valuesg~10~17 portant for unsaturated and keto steroids. Thus, (8) The chemical shift of the methyl protons a t C1o or CISin steroids additivity generally holds for steroids containing a is determined by the over.111 shielding experienced, which is a net effect A9(11) double bond or a 58,68-epoxide, but when of t h e position and orientation of all neighboring single and double both groups are present this is no longer true since bonds, plus long-range shielding effects due to magnetic anisotropy uf more distant groups of electrons. Changes i n srcreochcmistry can lead now ring B is forced into a half-boat form and the l o considerable changes i n long-range shielding effects.'-*-lo Failure t o position of the 19-methyl relative to both groups consider fully these effects has led to errors in interpretation in reis changed. For example, 5P,GP-epo~y-A~(~~)cently published work.lo*ll A more detailed discussion of some new pregnene-3,20-dione-l7a,21-diol diacetate 3aspects will appear shortly.', (9) A. D. Cross and P. W.Landis, unpublished results. ethylene ketal has calculated and observed 19(10) R. F. Zurcher, Hclo. C h i n . Acta, 44, 1380 (1961). proton resonances of 70.0 and 73.7 c./s. respectively, (11) J. Jacquesy, J. Lehn a n d J. Levisalles, Bull. SOC.chim. France, a disparity considerably larger than the agreement 2444 (1961). normally observable. g,lO,ll (12) A. D. Cross, forthcoming publication. (13) N.m.r. spectra were obtained for purified chloroform solutions We thank the Universidad Nacional Aut6noma a t 60 Mc. using tetramethylsilane (TMS) as an internal reference. A de Mexico and Prof. A. Sandoval for time on the Varian A-60 spectrometer was employed, but final calibration is against A-60 spectrometer. spectra run on a Varian HR-60 instrument, suitably equipped for cali-
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bration by the standard side-band technique. Accuracy limits are of (18) Positive shifts are away from T M S , for the 19-proton resonance the order of 1 1 c./s. for chemical shifts and 1 0 . 3 c./s. for J values. frequency, due t o the extra deshielding induced by these functional (14) The a-epoxides were: 5a,6a-epoxy-androstan-3B,l78-diol group*, relative t o Sa- or 5p-androstane, according to stereochemistry diacetate, 5a,6a-e~xy-androstan-l7-one-3R-olacetate, Ba,Ba-epoxyat CI. androstan-3-0ne-17@-01-17-acetate 3-ethylene ketal, 5a,6a-epoxy(19) Shifts of the 3 a - H resonance have been noted for the other preman-320-dione 320-diethylene ketal, 5a,6a-epoxy-pregnan3,20- steroids with a highly polar 5a bond., dime-21-01 320-diethylene ketal, 5lr.6a-epoxy-pregnan-3,20-dione3RESEARCH LABORATORIES ethylene ketal, 5a,6a-epoxy-pregnan-3,2O-dione-2l-ol3-ethylene S.A. ALEXANDER D. CROSS ketal 21-acetate, and 5a.6a-epoxy-pregnan-3,20-dione-17a,2l-diol SYNTEX, POSTAL2679 APARTADO 3-ethylene ketal Iln,Zl-diacetate The @-epoxides were: 50,68epoxy-pre~nan-3,20-dione-21-ol3-ethylene ketal 21-acetate, 58,6@- MEXICO,D. F. epoxy-pregnan-3,20-dione 3,20-diethylene ketal, 56,6B-epoxy-pregnanRECEIVED MARCH19, 1962 3,20-dione-17a.21-diol 3.20-diethylene ketal 17a,Zl-diacetate, 58,6& epoxy-androstan-17-one-a5-01 acetate, 5,8,6&epoxy-17a-ethinylandros. tan-3B,17B-diol 38-acetate. 5B,68-epoxy-androstan-3~,17~-diol diTHE ANODIC OXIDATION OF TRIPHENYLMETHANE acetate, and 5~,6~-epoxy-androstan-3-one-l7B-ol 17-acetate 3-ethylene DYES ketal. (15) On t h e r scalen the a-epoxide 6B-proton is ca. 7.2 and the 8epoxide 6a-proton is ca. 6.9. We wish to report an unusual electrochemical (16) G . V. D. Tiers, J . P h y s . Chem., 6 8 , 1151 (1958). (17) J. N. bhoolery and M. T. Rogers, J. Am. Chem. Soc., 80, reaction, which is exhibited in the anodic oxidation 5121 (1958) of crystal violet and related triphenylmethane dyes.
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COMMUNICATIONS TO THE EDITOR
Vol. 84
Crystal Violet (CV) and Malachite Green (RIG), cation radical of TMB. This spectrum recently has as well as 9,p’-methylenebis-(N,N-dimethylaniline)been interpreted in detaiL6 (MBis) all yield N,N,N’,N’-tetramethyl-benzidine Finally, the uncertainty exists that, (a) one (TMB) and its corresponding diquinoid (TRIBOx) molecule of TLSB arises by oxidation of 2 molecules upon oxidation a t platinum and carbon electrodes of dye each losing a K-substituted phenyl, followed in acidic, aqueous buffers. I t can be shown that by chemical coupling reactions, or, (b) that one dye this reaction must take place Yia ejection of an in- molecule loses the central carbon phenyl unit and tegral unit composed of the central carbon atom the remaining two N-substituted phenyl groups attached to a phenyl group. In the case of the couple intra to give TMB. I t a r i be shown that the RfBis, where the central group is merely -CH2--, latter is the case. The peak current due to TMB formaldehyde results. The ethylated dyes Ethyl which arises from AIG, CV or MBis is ca. 1.8 times Violet (EV) and Brilliant Green (BG) which are that obtained from similar oxidation of dimethylanalogous to Crystal Violet and RIalachite Green, aniline. Since it has been shown that 2 moles of respectively, yield the corresponding N,N,N’,N’- dimethylaniline are oxidized to give 1 mole of tetraethylbenzidine. These results are especially TMB, these results can be interpreted to mean that interesting in view of the recent work of Eastman, 1 mole of 1IG, CV or MBis gives 1 mole of T1IB. Engelsma and Calvin, who showed CV was formed =Inother interesting facet of the triphenylmethby oxidation of dimethylaniline with ch1oranil.l ane dye oxidation is that i t occurs in a %step wave. Here the central carbon of the CV cation must I t can be shown that the two waves are due to the originate from a methyl carbon of dimethylaniline. oxidation of the hydrated and non-hydrated forms Our results show the central carbon residue can be of the dye proposed by Cigen.’ I t is only the removed by the relatively mild process of electro- hydrated form which gives rise to the TRSB during chemical oxidation. Thus i t would appear that the anodic oxidation in strong acid medium. il decentral carbon of triphenylmethane dyes (and re- tailed interpretation of the electrochemical results lated compounds) is an unusually facile portion of a will be given soon. rather complex molecule. These results appear to Acknowledgment.-This work was supported be of fundamental interest in complex organic by the Atomic Energy Commission through conoxidation-reduction processes. tract AT( 11-1)-686 and this support is gratefully The cyclic voltammetry and other electrochemi- acknowledged. cal techniques used here were identical with those (6) 2. Galus and R . N. Adams, J . Cltem. Phys., 36, 2814 (19G2). reported in the study of the anodic oxidation of (7) R. Cigen, Acta Chern. Scartd., 12, 1456 (19%). X,N-dimethylaniline. DEPARTMENT OF CHEMISTRY In 1 N sulfuric acid-sodium sulfate medium, CV UNIVERSITY OF KAXSAS Z. GALUS KAXSAS R A L P I I3‘.ADAMS oxidizes a t ca. 0.8 v. vs. s.c.e. Using a 2 v./min. LAWRENCE, triangular wave sweep voltage, no evidence of any RECEIVEI)J U N K 20, 19ti2 oxidation a t less than 0.8 v. is evident on the iirst anodic sweep. However, on the second and all subsequent sweeps, an almost reversible oxidaON THE MECHANISM OF THE ENZYMATIC DECARBOXYLATION OF ACETOACETATE. 11‘ tion-reduction system is found a t the lesser anodic potential of ca. 0.55 v. The anodic and cathodic sir: half-peak potentials of this system correspond The decarboxylatioi~ of acetoacetate by the ‘ioif/iiiz2 millivolts with those of TMB-TRIBOx in decarboxylase2 purified from Cl. acetobutylicuna the same medium. previously had been shown to involve obligatory Chemical oxidatiou of triphenylmethane dyes to exchange of the carboiiyl oxygen atom with the ‘I‘MBOx is fairly well e ~ t a b l i s h e d . ~The , ~ most oxygen of the water used as ~ o l v e n t . ~ These recent work of Hanousek and Matrka 011 1 I G is findings suggest3 that a Schiff base formed between probably most definitive.5 To have further proof the enzyme and its substrate may be an active that the compound formed electrochemically was intermediate in the decarboxylation. We have ’I‘hIBOx, CV, -11G and hIBis were oxidized with therefore tried to trap the postulated Schiff base lead peroxide in sulfuric acid and the corresponding intermediate by reduction with borohydride as oxidation products were isolated as perchlorates. has been done with the Schiff bases present i n Solutions of all these compounds showed cyclic other e n z y ~ i i i c ~and - ~ similar s y s t e ~ n s . ~The suc\roltainmetry in complete agreement with the cessful results of these experiments are reported TlIB-TMB Ox oxidation-reduction system formed below. by electro-chemical means only. (1) This work was supiwrted, in part, by N. I. H. grant number Further, these oxidation products were dissolved RG-6687. iii 30% acetone-50yo aqueous buffer of pH 3.8. ( 2 ) G. Hamilt,m and 1 , €I. Westheimer, .I. A m . Chevi..Sot., 81, 2277 These solutions all showed electron paramagnetic (1959). (3) G. Hamilton and I:. H. Il’estheirner, J . Ant. Chem. Soc.. 81, 6332 resonaiice (e.1l.r.) spectra identical with that of the
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(1959). (4) E. H. Fischer, A. B. Kent, E. R. Snyder and E . G. Krebs, J. Arn. Chem. Soc., SO, 2906 (1968). ( 5 ) E. Grazi, T . Cheni: and R. T,. Horerkrr, Biochem. nitd BioDhyT. Resrrnvch Conmi., 7, 350 (1Sfi2). (6) R. 1.. Horecker. S . P o n t r e m o l i , C. Rirci and T. Cheng, P u n i (1922). .Yuf,Acnd. Si-i..47, 1919 (1961). (j)V Hanousek and 32. l l a t r k a , Coli. Caeclioslor,. Cheiiz. C o f i ~ i i i . , ( 7 ) R. B. Dempsey and H. N. Christensea, J . Bioi. C/icW., 237, 1113 (1962). 24, I 6 (1959).
( I ) J. W. Eastman, G . Engelsma and M. Calvin, J . A m . Cheiii. Sa:.. 84, 1339 (1962). ( 2 ) 2. Galus and R . N. Adams, .I. A m . C h o n .Snr., 84. 2061 (1982). 0 ) J . Knop, Z . n ~ t n i Chrii!., . 8 5 , 2.7:3 (1931). (41 F. Kehrmann, G , R o y 1 1 , and K a m m , H1.12’. ( ‘ I i i i i i . . I , li:, 6, 16.;
