2016
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VOl. 84
cycles) a t 6.85 7 (60 mc., benzene capillary reference, 2.73 used to relate benzene to tetramethylsilane). This is in striking contrast to the n.m.r. spectrum of IIa (CC14solution), which consists of a complex multiplet from 5.9-9.4 7 and a complex multiplet from 9.5-9.8T with areas in the approximate ratio of 13. This reduction of the complex cyclopropyl n.m.r. pattern to a single band is also found in protonated dicyclopropyl ketone, which exhibits a band (with poorly resolved fine structure) a t 7.06 7 in 96% H2SOa, These observations both support the identification of Ia and show the close relation between carbonium ions and protonated ketones. I I a was recovered in 63% yield from solutions of I a in 96% H2S04. The b.p. and both n.m.r. and infrared spectra are identical with those of the original IIa used to form Ia. Addition of a solution of I a in 9Syo DzS04 to ice-lOyo NaOH yielded a CCl4 extract. The n.m.r. spectrum of this ex(9) L. M . Jackman, "Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry," Pergamon Press, London, 1959, tract was virtually identical with that of the original p. 55. IIa demonstrating that no rapid H-D exchange had (10) J. B. Hendrickson, Tetrahedron, 9, 82 (1959). occurred and that IIa was the only water insoluble (11) M. D. Sutherland and 0. J. Waters, A u s f . J . Chem., 1 4 , 596 (1961). product derived from drowning Ia. Even slow (12) National Science Foundation Predoctoral Fellow, 1957-1959 H-D exchanges are absent because the n.m.r. WILLIAMG. DAUBEN spectrum of I a in 96Y0 D2SO4was essentially unDEPARTMENT OF CHEMISTRY WILLIAME. THIESSEN'~ UNIVERSITY OF CALIFORNIA PAULR. RESNICK changed after one hour. This eliminates an olefin BERKELEY 4, CALIFORNIA as a component of the equilibrium and reflects the RECEIVED MARCH16, 1962 high energy of cyclopropylidene derivatives. The initial i-factor of I I a in HzS04 was 4.1 in PHYSICAL PROPERTIES OF THE accord with the equation ROH 2HzS04 = R+ TRICYCLOPROPYLMETHYL CATION' H30+ 2HS04-. The infrared spectrum of Ia Sir: exhibits bands a t 837, 1279, and 1445 ( ~ m . - ' ) . ~I n Recently the physical properties of a simple the ultraviolet, Xmax for Ia (270 mp, t 22,000)5bears aliphatic alkenyl cation were reported. We now a relation to alkenyl cations (310-335 mp) that is report the physical properties of the tricyclopropyl- comparable to the relation between protonated methyl cation, an aliphatic carbonium ion contain- cyclopropyl ketones (dicyclopropyl ketone.H+ ing no formal double bonds. 235 mp, tricyclanone.H+ 250 mp)6 and protonated Hart and Sandri3 showed from solvolysis studies unsaturated ketones (4-methyl-3-penten-2-one." that cyclopropyl groups stabilize carbonium ions. 284 mp)? The p-nitrobenzoates of IIb, IIc, and IId solvolyzed Solutions of Ia in 96% H2S04 show little change with relative rates of 23,500:246:1, indicating that in n.m.r. or ultraviolet spectra after one hour althe effect of cyclopropyl groups was nearly addi- though decomposition is progressing. The ion beti~e.~ comes increasingly unstable (chemically) as the % H&O4 is reduced from 85 to 52%. At 25*, the half-life is 700 seconds in 71y0 acid and 30 seconds in .%yoacid. Extrapolations of the first order rate plots suggest that I I a is completely converted to I a even in 52y0 acid. The mode of decomposition appears to be nucleophilic attack on the -CHzI I1 I11 tached to carbons bearing acetoxyl groups. The downfield shift of 1.08 p.p.m. is characteristic of an acetate of a secondary alcoh01.~ These data agree with structure VI, a structure derived from dihydrocenibrene 11 and cembrene I and would not agree with a similar group of structures derived from IV. Thus, I represents the structure of cembrene. Although no information is available with regard to the stereochemistry of the trisubstituted double bonds, from a study of models the structure written containing one cis bond appears to be more favored than an all trans structure. This diterpene hydrocarbon, the first naturally-occurring fourteen carbon ring compound, is specially significant from the biogenetic standpoint, since i t is the monocyclic diterpene derived from geranylgeraniol in a manner analogous to the formation of the sesquiterpene humulene from farnesol.lol.l1
+
a, R1 = R2 = Rs = cyclopropyl b, R1 = R2 = cyclopropyl
Rs c, R1 R2 d, R1 e , R1
= isopropyl = cyclopropyl = Rs = isopropyl = RZ = Rs = isopropyl = R2 = cyclopropyl RI = -CH2CHzCHtOH2+
Ad( ition of I I a t o 96% produces the tricyclopropylmethyl cation (Ia). This ion has a most remarkable n.m.r. spectrum consisting of a single sharp band (width a t half-height about 3.5 (I) Grateful acknowledgment is made of partial support of this Research b y grants from t h e Petroleum Research Fund of the American Chemical Society, and t h e National Science Foundation. ,2) N. C. Deno, 11. C. Richey, Jr., J D. Hodge, and hl. J. Wisotsky, J . A m . Chem. Soc., 84, 1498 (1462). (3) H . H a r t and J. M. Sandri, ibid., 81, 320 (1959).
+
+
(4) T h e H2S04 solution was held in plastic films between N a C l prisms. B y taking two spectra, one with high clarity polyethylene and one with high clarity F E P fluorocarbon polymer (kindly supplied by the d u Pout Company), t h e complete 2-15 1.1 reaion can be measured. T h e HBO, has a strong background absorption throughout this region, but t h e intense bands pierce through this background. (5) I I a Solvolyzes a t a moderate r a t e in acetic acid (and probably also methanol and ethanol) so t h a t it was necessary t o introduce IIa directly into 96% H B O , and dilute the resulting solutions. (6) Simple protonated ketones d o not abqorb above 220 mp (H. J. Campbell and J. T. Edward, Can. J . Chem., 38,2100 (1960)). ( 7 ) 4-Methyl-3-penten-2-one. H + has a strong absorption hand a t 1540 cm. -1 in close agreement with t h e value 1533 cm. -1 found in a n alkenyl cation (ref. 2). This is ascribed t o a stretching mode of the C-C-C system and suggests that the hydroxyalkyl cation form makes the dominant contribution t o t h e structuie of this protonated ketone. A second intense band at 1595 cm.-1 m a y be t h e second stretchinp band of t h e allylic system though a band a t 1590-1610 bas been found in several simple protoiiateri kttones including acetone. H
+.
May 20, 1962
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2017
CHI of I a to give ring opening analogous to the reaction I of I I a in dilute acids observed by Hart and SandA3 BrCH2CONH-C-CO,R The decomposition product is a new ion ( h m a x 285 I CHa mp, E > 3000),half-formed above 80% HzS04, and I, R = p-nitrophenyl; 11, R = H tentatively assigned structure Ie. Addition of solutions of Ie to water gives a compound whose b.p. When chymotrypsin (1.4mg./ml.) is treated a t and n.m.r. and infrared spectra are in accord with pH 5 with a tenfold molar excess (dissolved in 10% re- by volume of ethanol) of p-nitrophenyl bromostructure 111. The addition of I11 to generates Ie. acetyl-a-aminoisobutyrate (I), m.p. 149-150' The identification of Ib is less complete. The (Anal. Calcd. for Cl2HI3O5N2Br:C , 41.75; H, ultraviolet spectrum (Amax 293 mp, E > 2600)8 is 3.80; N,8.12. Found: C, 42.02;HI 3.80; N, similar to that of Ia and le. Ib is half formed 7.88), its activity toward tyrosine ethyl ester3 above 80% acid as is Ie. Immediate drowning of slowly decreases: to 40% in 1 hr., to 22% in 3 hr. cold solutions of I b in ice-lO% NaOH gave a 25% After overnight dialysis, the activity remains the yield of polymer and a 20% yield of an olefin which same, which would be expected by analogy with has a b.p. and n.m.r. and infrared spectra in agree- pivalyl-chymotrypsin.' Unlike pivalyl-chymoment with the alkene (1,1-dicyclopropyl-2-methyl-trypsin, however, chymotrypsin inactivated by I propene) derived from IIb by direct dehydration. does not regain activity on treatment with a p H I n addition to decomposition by polymerization, 8 buffer containing glycerol.2 Typical final activas ities are given in Table I. I b (or the alkene) is oxidized by 96% evidenced by liberation of SO2 and rising absorption TABLE I around 300 mp. INHIBITION O F CHYMOTRYPSIN BY I AT PH 5: EFFECTO F A number of C4 and CSalkenes and alkanols form a species (possibly several species) in H2S04 charac- IPA" O N THE INHIBITION; PHOSPHORUS CONTENTOF INHIBITED CHYMOTRYPSINS AFTER REACTION WITH DFP terized by a broad absorption band around 293 % Ratio: Ratio: Ratio: m ~ Its . ~absorption and its thermodynamic staActivity P/CT IPA/CT I/CT ) ~ characterisbility (half-formed in 65% H ~ s 0 4 are 10.0 0 30 ,.. tic of alkenyl cations.2 The slow formation from 10.0 5 35 ... t-butyl alcoho1,'O the same rate of formation from 10.0 25 42 ... t-butyl alcohol and 2-butanoll9and our observation 10.0 100 60 ... that the kinetics of formation are not simply first 0 ... 100 1.04 order identify the 293 mp species as an alkenyl cat2.5 ... 77 ... ion rather than the t-butyl c a t i ~ n . ~ 5.0 ... 39 1.05 (8) The t values for I h and Ie are for 96% HlSO, solutions. The ions are not completely formed from their alkenes a t these acidities. (9) J. Rosenbaum and M. C. R. Symons. Mol. Phys., 3, 205 (1960). (10) The 293 mp species is reported t o require two hours t o half form from a -10-4 molar solution of t-butyl alcohol in 80 and 98% HIS04 (ref. 9). In our experience with over a hundred carbonium ions including the aliphatic carbonium ions in this communication and ref. 2 # the alcohol-carbonium ion equilibria were established within seconds,
10.0 ... 23 1.04 20.0 ... 22 1.03 0 Abbreviations: IPA = 3-indolepropionic acid; DFP diisopropylfluorophosphate; CT = chymotrypsin.
(1) C. E. McDonald and A. K. Balls, J. Bioi. Chem., 337, 727 (1957). (2) R. A. Oosterbaan and M. E. Van Adrichem, Biochim. Bio9hys.
(5) H. Neurath, J. A. Gladner and 0. De Maria, J. Bioi. Chcm., 188,407 (1951). (6) E. Abderhalden and E. Haase, Fcrmenlforschung. 12, 313
A C ~ O ,57,423
(1931).
5-
At pH 6, the decrease in activity takes place more rapidly to give about the same (30%) end activity, NORMAN C. DENO while a t pH 7, after a quick drop in activity (to DEPARTMENT OF CHEMISTRY HERMAN G. RICHEY,JR. 40% in 1 min.), recovery occurs slowly (3 hr.) to PENNSYLVANIA STATEUNIVERSITY JANES. LIU UNIVERSITY PARK,PENNSYLVANIA JAMES D. HODCE give a product having about 75% a ~ t i v i t y . ~ The irreversible inactivation occurs subsequent JOHN J. HOUSER MAXJ . WISOTSKY to a specific reaction a t the active site since (a) the RECEIVED MARCH23, 1962 rate of the "burst" of nitrophenol during the formation of the acyl enzyme is retarded by the presence MODIFICATION OF A METHIONINE RESIDUE NEAR of the chymotrypsin inhibitor,6 3-indolepropionic acid, which inhibits the inactivation of the enzyme THE ACTIVE SITE OF CHYMOTRYPSIN (Table I) ; (b) bromoacetyl-a-aminoisobutyric Sir: We wish to describe a new method for the selec- acid (11)6in a 100-fold molar excess does not cause tive chemical modification of enzymes, and its any inactivation under the usual reaction condiapplication to the pancreatic protease, chymotryp- tions (4hrs. a t pH 5 , overnight dialysis); and (c) sin. The basic principle is to combine an enzyme methionine (see below) in diisopropylphosphorylwith a bifunctional reagent, so designed that i t be- chymotrypsin is not attacked by I (Table 11). A comes covalently bound to an amino acid side random alkylation reaction is thus excluded. Reincubation of inhibited chymotrypsin with chain a t the active site, and then, fixed in position, reacts with another amino acid residue in the vicin- the inhibitor I failed to reduce its activity further. (3) G. W.Schwert and Y. Takenaka. ibid , 16, 570 (1955). T h e ity. The reaction of chymotrypsin with nitro- final percentage activity is roughly the same toward N-acetyl-Lphenyl esters,' during which the acyl group of the tyrosine ethyl ester. ester becomes attached to the hydroxyl group of (4) The activities measured during the course of t h e reaction probthe serine residue a t the active site,2is a good one ably represent the combined activities of reversibly and irreversibly inhibited enzyme, while those measured after long standing (dialysis) to demonstrate the utility of the method. are due t o irreversibly inhibited chymotrypsin.
(195s).
