CONXIUKICATIONS TO THE EDITOR
xug. 20, 19.57
78.63; H, 10.71*);IIIe (r1i.p. 192-193", [ a ] D -15". Found for C20H3002: C, 79.63; H, 9.S41). OH
-1.557
nuclear substituents, some previously unknown or unavailable by other methods.
OH
Hydride exchange reactions between cycloheptatrienes and trityl salts occur rapidly and quantitatively only in solvents which effect dissociation of these salts. In consonance with conductivity studies, trityl halides function satisfactorily as hyOlf dride abstractors in liquid sulfur dioxide but trityl perchlorate or fluoborate gives equally good results Y in acetonitrile or sulfur dioxide. Most tropeniuni a. 'k' = €1. R = H b; Y = H: R = Me salts may be prepared by the following general c , Y = H, R = Et procedure (all reactants and conditions anhydrous) : d, Y = H, R = -CH=CHa equivalent quantities of trityl salt and the cycloe, Y = H, R = -C=CH heptatriened in acetonitrile (minimum amount) a t f , Y = Me, R = H 111 room temperature or sulfur dioxide (30-50 ml./g. Similarly, 1a-methyl-19-nortestosterone (If) was triene) a t -20" or lower are allowed to react for a converted to the aiio' compound, 1cr-niethyl-19- few minutes, all solvent then evaporated (aceto1iorandrostan-17P-ol-3-0ne(IIf) (m.p. 186-188", nitrile in vacuo; sulfur dioxide by warming, finally [ a ] D +46". Found for ClYHP002:C , 78.78; H, in vacuo), triphenylmethane removed by trituration and extraction with ether from the tropeniutil 0.94) and to the 3P,17@-diol(IIIf) (n1.p. 204-206", [ a ] +5l". ~ Found for C19H3202: C, 77.55; H , salt product; pure perchlorate and fluoborate salts 10.84'1. obtained by recrystallization from acetonitrile~. This series of nordihydrotestosterone derivatives ethyl acetate, halide salts by sublimation a t SO(11) and the diols (111) are potent anti-estrogens as 100" (1 mm.); yields of crude salts almost quanmeasured by inhibition of the uterotrophic activity titative, of pure salts 60-90%. Tropeniuni salts of estrone in the immature mouse.1° Nordihydro- prepared Omax and cmax in concd. sulfuric acid): testosterone (IIa) exhibits about 8X the activity of (i) X = H, Y = C104,2white, m.p. >300°, 217 19-nortestosterone (Ia) in this assay (subcutaneous mp (41,000) and 273.5 mp (4350)(ii, iii, iv, v show route) while the 17-alkyl compounds (e.g. IIb) are same spectrum), 44.037, C, 3.717, H ; (ii) X = highly active by the oral as well as subcutaneous H, Y = BF4,5cwhite, m.p. dec. slowly above 210°, route. Clinical evaluation of some of these com- 47.1'7% C, 3.59% H ; (iii) X = H, Y = Cl,j white, pounds in certain types of hormone dependent 66.61% C, 5.56% H ; (iv) X = H, Y = Br,2.5yellow, m.p. 203"; (v) X = H, Y = I,5red,m.p. l"', tumors has been initiated. readily transformed into tropenium triiodide, deep (9) H. J. Ringold, G. Rosenkranz and F. Sondheimer, 'THISJ O C R N A I . , red-black, m.p. 1 2 7 O , 292 mM (39,800) and 361 nip 7 8 , 2471 (1950). For assignment of t h e lor-configuration, see C. Djerassi, €2. Riniker and B. Riniker, ibid., 78, 6377 (195G). (22,100) in acetonitrile, 18.06y0 C, 1.5170 H ; (vi) (10) Bioassays carried out a t t h e Worcester Foundation for ExperiX = C1, Y = C104, white, m.p. l f i 4 O , 237 m p mental Biology under the direction of one of t h e authors (R. I. D.). (29,SOO) and 310 nifi (822O), 37.37%, C 2.77% H , ItESEARCH LABORATORIES converted to tropone by water or ethanolsb (analSYXTEX, s.21. A. BOWERS MEXICO,D. F., ASII H. J. RINCOLD ogous preparation of bromide or iodide salts gives mixed halotropenium halides due to halogen interTHE~ ' O R C E S T E RFOUSUATION FOR EXPERIMEXTAL BIOLOGY change); (vii) X = Br, Y = C104, white, 1ll.J). SHREWSBURY, MASS. It. I. DORFMAN 149.5", 247 mp (22,750) and 323 mp (SgOO), 31.27%, RECEIVED J U N E 13, 19.57 C, 1.89% H, converted to tropotie by water or ethanoPh; (viii) X = MeO, Y = Clod, white, m.p. 107", 234 mp (32,100) and 315 mp (10,050), SYNTHESIS OF TROPENIUM (CYCLOHEPTA43.38% C, 4.34% H (similarly prepared bromide or TRIENYLIUM) SALTS BY HYDRIDE EXCHANGE' iodide salts yield tropone and methyl bromide or Sir: iodide on warming); (ix) X = Me, Y = C104,hc The previously reported2 novel method of syn- white, m.p. 10Do, 2% nip (37,100) and 288 nip thesis of tropeniuni3 perchlorate by hydride ab- (3500), 46.89y0 C, 4.73% H (yellow bromide salt straction from cycloheptatriene by trityl per- preparable in sulfur dioxide a t - 70" but deconichlorate in acetonitrile has been found to be ver- poses on warming). Perchlorate and fluoborate satile and adaptable to the preparation of a number salts, due to greater stability and non-hygroscopicof tropenium salts containing various anions and/or ity, are easier to handle than chloride (very hy- I
(1) Supported in part b y t h e Office of Ordnance Research, U. S. Army, Contracts DA-04-200-ORD-235 and 601. (2) H. J. Dauben, Jr.. and D. L. Pearson, Abstracts, 126th Meeting, Sept. 13, 1954, p. 18-0. American Chemical Society, New York, N. Y., ( 3 ) Trfipenium seems preferable t o tropylium as the name for the c7EI7* ion because of its representation of all structural features of the ion and its consistency with benzeuium, azuleniutu, e t c . , names for tel.itcil c.irtjotiiuitt i o n .
(4) Substituted cycloheptatrienes prepared by the method of W.
H. Knox, THISJOURNAL, 76, 297 (1953). ( 5 ) Previously reported by: (a) Doering and Knox, ibid.. 76, 3203 ( 1 9 5 4 ) ; (b) W. v. E . Doering and H. Krauch, Angew. Chem., 68,661 ( 1 1 M i ) : (c) A t . .I. S. Dewar a n d R. Pettit, J . Chem. Soc., 2021, 20% (IKXj); the color. stability and spectrum reported for their methyltro,p?niuni bruniidc monohydrate ure incunsistcnt n i t h (his 5 1 1 urtural aasijiriiucnt. v. E. Doering and L.
4555
COhlbIUNICATIONS
TO THE
EDITOR
1-01, ?!I
groscopic) , bromide (hygroscopic) or iodide (non- possesses a characteristic absorption maximum a t hygroscopic but unstable) salts. The allylically this wave length.4 Recently, a mechanism of reacby tion of &chymotrypsin with NPA has been proposed5 substituted 7-methoxy-l,3,5-~ycloheptatriene,~~ giving with trityl bromide in sulfur dioxide only which accounts for this observation and several tropenium bromide and trityl methyl ether, shows others : (1) The stability of monoacetyl-6-chymotrypsin a t low pH6 (2) a difference of 0.7-0.8 pK methoxide instead of hydride exchange. With more complete spectral data now available, unit for acetylation and deacetylation of the eni t is evident that both predicted bands (4.33ev, zyme6 (3)the reversible sensitivity of the acetylaweak; 6.37 ev, intense)6 for tropenium ion agree tion and deacetylation reactions to denaturation reasonably well with observed values (4.54 ev, by urea,2 (4)the reversible loss of reactivity of the 4350; 5.73 ev, 41,000). Results to date indicate acetyl with hydroxylamine in urea.2 This scheme that substituent effects on both 'ElUand lEsu bands is presented in Fig. 1 with certain modifications, of substituted tropenium ions are in accord with the evidence for which will be presented below. A solution of monoacetyl-6-chymotropysin (1.95 those on 'Elu' and I B l u , but not 'Bzu, bands of monosubstituted benzenes and are interpretable in mgm./ml.) a t $H 3.5 was allowed to deacetylate a t $H 9.0 in a Beckman DK-1 spectrophotometer terms of highly polar excited states for the ions. Additional hydride exchange studies on tropen- which was set a t 245 mp. The resulting record of ium and other systems for preparative and theo- AE2h6 with time is seen in Fig. 2, curve 1; a control retical purposes are in progress. I 0.21
(6) J . K. Murre11 and H. C. Longuet-Higgins, J . Chem. Phys., 23, 2347 (1955). ( 7 ) H. B. Klevens and J. R. Platt, Technical Report, P a r t 1, 19j3-4, Laboratory of Molecular Structure and Spectra, University of Chicago, p. 145. (8) (a) National Science Foundation Predoctoral Fellow, 1936-7; (b) Allied Chemical and Dye Co. Fellow, 1953-4. HYPJ. DAUBEN, JR. FILON A. GADECKI DEPARTMENT OF CHEMISTRY M. HARMON& KENNETH UXIVERSITY OF WASHINGTON
DAVIDL. PEAR SON^^ SEATTLE 5 , WASHINGTON RECEIVED JULY 3, 1957
230 240 250 260 270 Wovelength in Millimicrons
AN INTERMEDIATE IN THE DEACETYLATION OF MONO-ACETYL-&CHYMOTRYPSIN HAVING T H E PROPERTIES OF ACETYL-IMIDAZOLYL1
Sir: In a previous communication,2evidence was presented which indicated that upon acetylation of 6chymotrypsin by p-nitrophenylacetate, there was no detectable change in the spectral characteristics of the enzyme in the region of 245 mp. This region was studied carefully since a postulated intermediate in the reaction, acetyl-imida~olyl-,~~~
sloble ocyl-enzyme
I
0-
Fig. 1.-Proposed
mechanism cf enzymatic hydrolysis.
(1) The authors wish to acknowledge the financial support of grant S o . RG-4617 from the National Institutes of Health, U. S. Public Health Service. (2) G. H. Dixon, W. J. Dreyer and H. Neurath, TEISJ O U R N A L , 7 8 , 4810 (1956). (3) H. Gutfreund. Tvaiis. F U Y ~ ~ QSor.. . V 81, 4 4 1 (10.55); R. J. Jandorf, H. 0 Michel, N. K. Schafftr, K. Egan and W , H. Summerson, F a v a d a y Soc. Discussions, 20, 134 (1955). (4) E. Stadtman in "Mechanism of Enzyme Action," Johns Hopkins Press, Baltimore, Md., 1954, p. 581.
I ----20 30 Time (Minutes) Fig. 2.-Both sample and reference cuvettes in a Beckman DK-1 spectrophotometer contained 3.0 ml. of the same solution of acetyl-6-chymotrypsin7 a t pH 3.5; the temperature was 10.0 & 0.2". The pH of the sample was raised t o 8.9 & 0.1 by adding a small aliquot of a triethylanimoniumammonium acetate buffer on a plunger type rapid mixer.
10
of non-acetylated &chymotrypsin is seen in curve 2. Curve 2 was also followed when a portion of the acetyl-&chymotrypsin was allowed to deacetylate a t @H 7.2 for 15 minutes a t room temperature and then adjusted to pH 3.5 with acid before the reaction. I t is clear that only in the case of acetyl-dchymotrypsin is there the rapid formation of a species absorbing a t 245 mp which decreases slowly in concentration with time. The control curve indicates that part of the initial increase a t 245 mp is due to a non-specific, pH dependent, spectral change in the enzyme. (5) L. W. Cunningham, Science,in press. (6) G. H. Dixon and H. Neurath, Fed. Proc., 16, 173 (1957); G. H. Dixon and H. Neurath, J . Biol. C h c n . , 223, 1049 (1957). (7) A. K. Balls and H . N. Wood, J . Biol. Chein. 219, 245(19BU).
