J d y 20, 19G1
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3161
Prolonged (72 hr.) catalytic hydrogenation of 1- group coplanar with the aromatic nucleus (carbonyl (a-carbethoxy-/3-indolyl)-2-nitrobutane (I) in gla- infrared absorption of the ester function appears cial acetic acid over 3oy0 Pd/C a t about 3 atm. near 1695 cm.-l), is such that the nitro group can and a t room temperature led to I1 (48'%), m.p. occupy only a restricted number of positions. 122.5-124.3', A%?. 288, 227 (E, 16,240,6,380), Addition of the methyl group to the 3-carbon n.ni.r., no aromatic protons. Anal. Calcd. for atom (to give I) blocks the only remaining apCI6Hz2N2O4: C, 61.20;H, 7.33; N,9.52;mol. wt., proach to the close proximity of the nitro group. 294.3. Found: C, 61.30;H, 7.40; N,9.48; mol. The observation that hydrogenation of the benzene wt. (Rast, camphor), 291. When the reaction was ring in I occurs when the nitro function is isolated repeated in a different hydrogenator with catalyst from the catalyst surface further suggests that from a different lot, I1 was isolated in 50% yield electron-attraction by the nitro group facilitates after 50 hr. Some of the Bz-tetrahydroamme hydrogenation of the benzene ring. Several known (111, about 22%) was isolated in each ex eriment examples of hydrogenation of the benzene ring of an as the perchlorate, m.p. 232-234" dec., X i i z indole n ~ c l e u s ,especially ~~~ in the field of P287, 245 (E, 18,420, 4,240). Anal. Calcd. for carboline alkaloids,415support the hypothesis that C1ST-125N208Cl:C, 40.39;H, 6.91;N,7.68. Found: a more or less remote electron-attracting group C, 49.52; H, 7.17; N,7.69. Compound I could be may produce such an effect. regenerated from I1 by chloranil dehydrogenation. (2) V. Boekelheide and Chu-tsin Liu, J. A m Chcm. Soc., 74, 4920 Hydrogenation of 1-(a-carbethoxy-P-indolyl) -2- (1952). (3) H. M. Kissman and B. Witkop. ibid., 76, 1967 (1053). nitropropane (IV) under essentially the same (4) H . Schwarz and E. Schlittler, He2o. Chim. A c f a , 34, 629 (1951), conditions (except for a shorter time employed and references therein. because of the more rapid consumption of hydro(5) A. LeHir, R. Goutarel and M. hl. Janot, Bull. m c . chim. France, gen) occurred in normal fashion, producing a mix- 19, 1091 (1952). THEWILLIAM ALBERTKOYESLABORATORY ture of 1-(a-carbethoxy-~-indolyl)-2-aminopropane OF ILLINOIS DAVIDV. YOUNG (V, 46%) and the corresponding lactam, 1-oxo& UNIVERSITY ILLISOIS H. R. SSYDER methyl-l,2,3,4-tetrahydropyrido[3,4-b]indole (VI, URBANA, 13Y0).The amine (V) was isolated as the perDEHYDROGENASE-A PROTEIN 296, GLUTAMICOFACID chlorate, m.p 240.5-241.5' dec. ; ?.A:! UNUSUAL CONFORMATION1 228 (E, 21,240, 24,900) Anal., Calcd. for C14- Sir: HISNzOeCl: C, 48.47; H , 5.52; N,8.08. Found: A systematic study of the optical rotatory disC , 48.57;H, 5.60;N,7.77. persion of globular proteins showed that about 40% Compounds I and I V also were reduced by a chemical method to form the respective lzctams of them have relatively high rotatory dispersion (VII) and (VI). Treatment of I with zinc in constants (Ac) of 250-290 mp, and that all proteins ~ ~ ~ ~ the enzymes, the aqueous acetic acid for 5 hr. a t 40' gave VI1 (41%), are l e v ~ r o t a t o r y . ?Among slightly levorotatory dehydrogenases exhibit high m.p. 190-191.5° subl., infrared, lactam carbonyl a t 1660 cm.-'. Anal. Calcd. for Cl3Hl4N20: C, Xc values indicating a high a-helix content in these It was, however, most sur72.88; H , 6.59; N , 13.08. Found: C,73.12; HI macromolecules.5.6 6.31; N, 12.92. Similar treatment of I V gave VI prising that the glutamic acid dehydrogenase (5970), m.p. 226-227' subl., infrared, lactam car- (GAD)g was dextrorotatory. Upon a mild denaturabonyl 1660 ern.-'. A n d . Calcd. for C12H12N20: tion with alkali of p H 9.5 a negative shift of the C, 71.97; H, 6.04; N, 13.09. Found: C, 71.92; rotation was observed, and a X, of 316 (&15) mp was obtained a t the beginning of the alkali treatH, 5 9-1; N, 13.82. ment. The negative shift could be followed about x0 2 4-5 hours after the addition of alkali. ilt the end 1 21 3 of this period the Xc dropped to 240 nip, and further CH?-C-CH:-CHj observation was impossible because of increasing turbidity of the solution. The data, which were NO, obtained with a Rudolph model 80 photoelectric H1 COOCZH, I spectropolarimeter, are shown in Table I. ,'v8
a H ;BH~-~-cH~ I H H
"H
1
COOCAH,
11
The slow hydrogenation of the nitro function in I, in contrast to the relatively rapid hydrogenation of that in IV, suggests that the methyl group attached to the %carbon atom of I , in conjunction with the steric requirements of the remainder of the molecule I, hinders contact between the nitro function and the catalyst surface. Indeed, zn examination of models (Stuart and Briegleb) reveals that the nitro function in both T and I V is in a sterically crowded environment The fundamental structure of IV, having the carbethoxy
(1) This study is supported in part by grants from the h'ational Cancer Institute, National Institutes of Health, U. S Public Healtll Service, grant (2-1785, and the Robert A. Welch Foundation, Houston, Texas, grant G-051. (2) J. T . Yang and P. Doty, J . A m . Chcm. SOC.,79, 761 (1957). (3) B. Jirgensons, Arch. Biochcm. Bicph>s., 74, 57, 70 (1958); 78, 235 (1958). (4) J. A. Schellman and Ch. G. Schellman, J . Polymcr Sci , 49, 129
(1961).
( 5 ) B. Jirgensons. Arch. Biochem. Biophys., 86, 532 (1959); 92, 216 (1961). (6j H. Sund, Biochenr. Z.,333, 205 (1960). (7) B. K. Joyce and S. Grisolia, J. Biol. Chcm., 236, 725 (1961). (8) D. D. Ulmer and B. L. Vallee, ibid.. 236, 730 (1961). (9) The GAD preparations were obtained from California Corporation for Biochemical Research, Log Angeles; the crystallized enzyme was isolated from bovine liver by the C. P. Boehringer & Soehne G.m.b.H., Mannheim, Germany. The enzyme was obtained as a suspension of the crystals in either sodium sulfate or ammonium sulfate solutions. The crystals were dissolved bv dialysis against dilute solutions of sodium phosphate or sulfate.
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3162 T.4BLE
I
0.02-0.10 111 SGDIUM SULFATE OR PHOSPHATE (fXaOH), T 26'
SPECIFIC
ROTATION OH 0.80$70 G.4D pH
A , mp
Native GAD, P H 6.9-8.6
IN
9,s'
30 minutes after adding S a O H "
OH 9.5, 4 hours later
546.1 -t13" & 5 -22" 1 3 -49" & 4 492 -28 - 72 435.8 4-21 -eo - 97 404.7 -k20 - 63 - 122 366 +9 - 121 - 164 ba -170 -f. 40 -357 - 70 These values are corrected for the cliange occurring during observation.
Although the accuracy, because of the weak rotatory power and turbidity, was low, the dextrorotation was ascertained using three different preparations, e.g., the [011435.8 of one of them (in 0.02-4fsodium sulfate, fiH 7.1) was +2il0, while another preparation (in 0.1 114 phosphate, p H 6.9) yielded a t the same wave length 18", and a third (in 0.01 1l.I sulfate, pH 7.4) +19". Treatment of the native enzyme with sodium decyl sulfate (0.05 M , pH 7.8) a t 25" resulted in a negative shift of the specific rotation ([a14z6. -87"), and upon this treatment the turbidity disappeared. The Xc of the detergent treated GAD was 247 mp. The rotatory data of the native GA.D did not fit the one-term Drude rule. The data then were evaluated according to the hloffitt-Yang method,lO i.e., the [ a ]X~ (Az - Ao*) values were plotted against X4/(X2 - Xo2), where XO is 212 mp, and the bo values of the Moffitt-Yang equationlo were calculated (see Table I). The bo for the detergent treated CAD was found to be - 98. (The refractive index and residual molecular weight factors were disregarded, since they do not affect bo greatly.") Recently the bo of another preparation of native GAD was rechecked with an improved Rudolph model 80-AQ6 instrument, and the bo was found to be -2208 (.t2o). The [ a l ~= f(X) curve had a fiat minimum a t 290-300 mp. The XioffittYang equation was applicable only within the near ultraviolet and visible spectral range. The enzymic activity 'was tested according to Strecker,'? and the neutral solutions were found as active as the most active preparations rlescribed in the l i t e r a t ~ r e . ' . ~At , ~ ~@H , ~ 8.6, ~ however, the activity was considerably diminished, whereas no significant change could be seen in the rotatory power. Denaturation with alkali a t PH 9.n (4.5 hours a t 23") or with sodium decy! sulfate resulted in complete inactivation. Since the molecular weight of native G-4.D is known to be a.bout IO6, and since the enzyme dissociates and aggregates readily,13 it seems likely that the -11 = IO6 particle may be composed of several polvpeptide chains or subunits. Terminal
+
Vol. 83
amino acid analysis of three specimens after Sanger16 showed that 17-23 moles of N-terminal alanine and 1-2 moles of N-terminal aspartic and/or glutamic acids are present per mole of enzyme. Thus the Af = 106particle is composed a t least of 17-23 peptide chains. After adding the decyl sulfate, a complete dissociation occurred, since a low sZooof 3.7 S and a low intrinsic viscosity of 0.025 dl./g. was observed. Application of the ScheragaMandelkern treatment17 by taking a B factor of 2.16, yielded for the subunits an 251 of 43,000. This is in reasonable agreement with the N-terminal group analysis, ;.e., each subunit possibly represents one polypeptide chain. The results indicate that GAD is a protein of unusual conformation. The negative bo and the dextrorotation of the native enzyme suggest the presence of a-helical conformation. (15) A. Ramel, E. Stellwagen and H. K. Schachman, Ped. Proc.. 20, 387 (1961). (16) F. Sanger, Biochem. J., 39, 507 (1945). (17) H. A. Scheraga and L. Atandelkern, J . A m . Chem. S O L . ,76, 179 (1953); see also H. Schachman in "Methods in Enzymology" (S. P. Colowick and N. 0. Kaplan, eds.). Vol. IV, Academic Press, Inc., Xew York, N. Y., 1957. pp. 32-103.
THEUNIVERSITS OF TEXAS M. D. ANDERSON HOSPITAL AND TUMOR INSTITUTE DEPARTMEST OF BIOCHEMISTRY BRUNOJIRGENSOSS HOUSTON, TEXAS RECEIVED MARCH20, 1961 T H E COPPER SALT CATALYZED PEROXIDE REACTIONS
Sir: The versatility of the copper salt catalyzed reaction of peroxides with various substrates has been well d e s ~ r i b e d . ~ In~ ~particular, ~ ~ ~ ~ , ~the reaction between peroxides and olefins has been postulated to proceed via a complex containing copper, peroxide and olefin. 1 s 4 * 5 This termolecular mechanism is based in part on the non-rearrangement of allylic We have examined this reaction between tei tbutyl peracetate and perbenzoate with the three isomeric butenes in the presence of cuprous bromide a t 75-83'. The stoichiometry is described by the equation RCOaBu-t
+ C4Hr
-+
RCO?C&i $- t-BuOH
The over-all yields of butenyl acetates and benzoates are in the range of 70-S37,. The compositions of the butenyl ester mixtures were examined by gas-liquid chromatography and found to be imariaat (within 5yo) with the reactant butene. Thus, butene- 1, cis-butene-? and trarzs-butene-2 all yield a mixture of butenyl esters consisting of 89-94% 3-acyloxybutene-1 and 1l-G% crotyl esters. Under the conditions of these experiments there is no allylic isomerization of either the reactant butenes6 or product esters.
(10) 11'. 1,loffitt a n d J. T Yang, Proc. %'all. A r a d . S c i , 42, 5DR (1) M. Kharasch, el ol., J . A m . Chem. Soc., 80, 756 (1958); 81, (1956). !* (11) I
