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 activI b (or the alkene) is oxidized by 96% as ities are given in Table I. evidenced by liberation of SO2 and rising absorption TABLE I around 300 mp. INHIBITION O F CHYMOTRYPSIN BY I AT PH 5: EFFECT O 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,2 is 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).
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2018
The maximum degree of inactivation so far obtained has been 80 5%, as measured in the standard assay.3 To determine whether this represents a mixture of 20y0of active chymotrypsin with 80% of totally inactive enzyme or an enzyme possessing 20yoactivity, samples of the inactivated enzyme were allowed to react with diisopropylfluorophosphate. Since i t is known that during the complete inactivation of chymotrypsin by diisopropyl fluorophosphate,’ one mole of diisopropyl phosphate per mole of enzyme7 is bound to the serine hydroxyl group a t the active sitejs the phosphorus content of inhibited chymotrypsin, allowed to react with diisopropylfluorophosphate until completely inactive, is a measure of the proportion of enzyme molecules capable of reaction at the active site.g The phosphorylated proteins, which had less than 0.25yo activity, containedJ0 one mole of phosphorus per mole of enzyme (Table I). Amino acid analyses” of chymotrypsin and of the irreversibly inactivated chymotrypsin show that one methionine residue in the latter is destroyed, and that the products (after hydrolysis) from it are the same as those obtained by GundInch, Stein, and Moore (ref. 12, fig. 2c) with ribonuclease inactivated by iodoacetic acid a t pH 2.8. In addition, roughly one mole of a-aminoisobutyric acid is found in the hydrolysate (Table 11). No other change in amino acid composition has been detected.
*
TABLE I1 METHIONINE, ITS DECOMPOSITION PRODUCTS AND a-AMINOISOBUTYRIC ACID IS HYDROLYSATES OF MODIFIEDCHYMOAmino acid
Methionine S-Carboxymethylhomocysteine Homoserine Homoserine lactone a-Aminoisobutyric acid
TRYPSIN Modified C T
CT
DIP-CTa
1.19”
2.08“
2.10
0 .5 0.1 0.3
... ... ...
cn. 0 . 0 1
0.00 0.00
ca. 1 . O C ... trace The DIP (diisopropylphosphory1)-CT, which had 0.25y0 activity, was allowed to react with 25 moles of I for 6 hr. * Average of three determinations. e This amino acid was difficult t o determine with accuracy since it gives a low ninhydrin color yield and falls on one side of the cystine peak in chromatograms.” a
We conclude that the activated bromine atom of the acyl enzyme alkylates a methionine residue in the vicinity of the active site, either while attached (7) E . F. Jansen, h l - D . F. S u t t i n g and A. K. Balls, J . B i d . C h e m . , 179, 201 (1949); A K Balls and E. F. Jansen, Admnces in Enzymol., 13, 321 (19.52). (8) J A. Cohen, R . A Oosterbaan, H S. Jansz and F . Berends, J . Cell. C o m p . P h y s i o i 64, S u p p l . 1, 231 (19591, an0 refs cited therein. (9) This method is analogous t o t h e “all or none” assays of (a) Q‘. J. R a y , J r , J . J Ruscica and D . E Koshland, Jr., J . A m . Chem SOL , 8 2 , 4739 (1960), and (b) %’. J. R a y , J r . and D. E . Koshland, J r . , Abstracts of Papers, American Chemical Society Meeting, Washingt o n , D C , March 21-24, 1962, p. 41-C. (IO) “Official and Tentative LIethods of Analysis of t h e Association of Official Agricultural Chemists,” 6th Ed , A O.A.C., Washington, 11. C , 1943, p. 127. (11) D. H. Spackman, W. H Stein and S . Moore, .4?101.Chrin , 30, 1190 (1958). (12) (a) H G. Gundlach, W. H Stein and S. Moore. J . Bioi. Chein., 984, 1754 (1959); (b) H.G. Gundlach, S. hloore and V d H. Stein, i b i d . , 984, 1761 (1959).
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to the serine or shortly after hydrolysis of the acylserine bond. Since the liberated serine hydroxyl group is capable of reaction with diisopropylfluorophosphate, the active site is still capable of functioning, but much less efficient than i t was before modification of the methionine residue near it. The inefficiency of the modified enzyme is attributable to a considerable increase (about 11-fold) in the Michaelis constant over that of native c h y m o t r y sin.13 Our work corroborates the finding of Kay, et al.,” that destruction of methionine leads to the inactivation of chymotrypsin. I t is unlikely that the sulfide group of the critical methionine behaves as n neighboring nucleophile a t the active site of chymotrypsin (though this is entirely possible i n the case of phosphoglucomutase14), since this activity should be abolished upon alkylation, which gives a ternary sulfonium salt, l Z l 1 jor upon subsequent peptide cleavage.’j3l6 The increase in the Michaelis constant indicates that modification of the critical methionine residue results in decreased affinity for substrate. This general method for the modification of enzymes, as well as the complementary method of Baker, et aZ.,li should prove to be a good tool for the “mapping” of enzyme active sites. Acknowledgments.-VVe are grateful to the National Institutes of Health for grants .4-5299 Bio. and 2E-140 (Cl) which helped to support this work. Procedure of L. W. Cunningham, Jr., ibid., 207, 443 (1934) (14) W. J. R a y , J r , H . G. Latham, Jr., Ll. Katsoulis and D 1C. Koshland, J r . , J A m . Chenz. Soc , 8 2 , 4743 (1960). (15) W, B. Lawson, E Gross, C. iVl. Foltz and R . Witkoi,, i b i d . . 83. 1509 (1961); E . Gross and B. U’itkop, ibid., 83, 1210 (1961). (16) Subsequent peptide cleavage a t t h e carboxyl gloup of t h e methionine is likely t o be slight under t h e conditions used (6.f ref. 1,;) This point, a s well as the possibility of cleavage upon heating, is under investigation. (17) B. R. Baker, W.W. Lee, E. Tong and L. 0. Ross, J . d i n . Chlr’z. Soc., 83, 3713 (1961); cf. also G. Schoellmann and E. Shaw, B Z O . / ~ P J J J Biophys. Res. Cornm.,?, 36 (1962); F e d . Pro,., 21, 2 3 2 t l Q f i ? ) ,
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DIVISIONOF LABORATORIES AND RESEARCH NEW YORKSTATE DEPARTMENT OF HEALTH W. B. LAWSOX ALBANY,SEW YORK H. J. SCHRAMM RECEIVED MARCH 3, 1962
A STEREOSPECIFIC TOTAL SYNTHESIS OF 18-SUBSTITUTED STEROIDS. APPLICATION TO THE SYNTHESIS OF dZ-CONESSINE
Sir: This communication reports a total synthesis of 18-substituted steroids in which the B-C-D systetn is constructed following the lines laid in our previous steroid total synthesis,l but control of the CI,, stereochemistry is achieved by the use of an 8 - h o (cis B/C) structure which finally is inverted to the 8-normal (8)configuration. lVe illustrate the synthesis by building up the steroid alkaloid conessine,? but it will be obvious that a variety of 18-substituted 20-ketosteroids would be of considerably simpler access, e.g., v i a IV. (1) G. Stork, H J E. Loewenthal and P C . htukharji, J . Ain Chriii. J o c , 78, 501 (193i).
( 2 ) D . H R Barton and L . R . Slorgan, J . Chem. S O L . ,C22 ( 1 9 6 2 ) , have accomplished a partial synthesis of natural conessine from A;pregnene-3,9,20B-diol.
