June 20, 1963
COMMUNICATIONS TO THE EDITOR
1895
renal adipose tissues of the rabbit. By the in u h hypophysectomized frog assay," the synthetic peptide had only one hundredth of the melanocyte-stimulating TABLEI activity of the native hormones. Recent studiesl3with Total yo various synthetic peptides related to ACTH appeared Ratio Fluorescence conto show that the same amino acid sequeace may be rr1/11 Sensitizer' maximum, wb version important for both lipolytic and melanocyte-stimulating 1:4.5 85 Chlorin e6 670 activities. It is further noted that the decapeptide 1:4.5 80 Hematoporphyrin 630 (a1-10-ACTH) possesses approximately one tenth of 1:1.2 88 Rose Bengal 580 the lipolytic10 and the melanocyte-stimulating12activi1.6:l 71 Erythrosin B 578 ties of the natural hormone whereas the pentapeptide" 3.1:l 82 Eosin Y 565 (a16-19-ACTH) has none. The synthetic pentadeca61 30:1 Riboflavinc 510 peptide described herein is the first instance where a a Photooxygenations were conducted in pyridine for 80 hr. separation of these two activities has been achieved. Measured in pyridine with an Aminco-Bowman SpectrofluoMoreover, as far as we are aware this report represents rometer. Owing to low solubility this run was in pyridinemethanol (4:l)for 110 hr. About the same product ratio was the first synthesis of a biologically active peptide in observed in pyridine alone but the total conversion was only which the natural sequence of the ACTH structure has 35%. been altered. This finding has practical value for synthetic work and Na-~arbobenzoxy-NG-tosyl-~-arginine14 (I) was conraises questions on the precise nature of the intermediates densed with L-proline-t-butyl ester15 by N-ethyl-5phenyl isoxa~olium-3'-sulfonate~~ (11) to give the crysin sensitized oxygenation^.^ Interestingly, there is a talline protected dipeptide (111). I11 was hydrorough parallelism between the trend in product ratio and the trend in fluorescence emission maximum for genated and allowed to react with I, again with the the different sensitizers. aid of 11. The protected tripeptide was converted to the pentapeptide Na-carbobenzoxy-Nf-t-butyloxycar(6) Control experiments showed that both products are stable t o the conditions of photo6xygenation. bonyl-L-lysyl-Nf-t-butyloxycarbonylL - lysyl - N - tosyl(7) An example of sensitizer control of product composition in a photoL-arginyl-NG-tosyl-L-arginyl-L-proline-t-butyl ester (IV) chemical reaction not involving oxygen was reported recently [G. S. Hamby stepwise reaction of the tripeptide base with Nu-carbomond, N. J . Turro and A. Fischer, J . A m . Chcm. SOC.,88,4674 (1961)l. benzoxy-Ne-t-butyloxycarbonyl-L-lysinep-nitrophenyl (8) This work was supported by a U.S. Public Health Service Grant (RG9693),by a Grant-in-Aid from the Hynson, Westcott and Dunning Fund, ester." IV was purified by countercurrent distribution and by the Alfred P. Sloan Foundation. inthetoluenesystem' ( K = 0.25); m.p. 105-110'; [cuI25D (9) Fellow of the Alfred P . Sloan Foundation. -36' (c 1,methanol). Anal. Calcd.: C, 56.4; H, 7.22; DEPARTMENT OF CHEMISTRY~ ALEXNICK ON^ 56.2; H, 7.33; N, 13.0; THEJOHNS HOPKINSUNIVERSITY WILPORD L. MENDELSON N, 13.2; S, 4.64. Found: S, 4.69. 18,MARYLAND BALTIMORE FUKEIVEDAPRIL 1, 1963 IV was hydrogenated to yield the pentapeptide base (V) which was purified by countercurrent distribution in the toluene system' ( K = 0.73); m.p. 112-117'; The Synthesis of a Biologically Active Pentadecapeptide [cY]~~D -33.7' ( G 1, methanol). Anal. Calcd.: Corresponding to an Altered Sequence in the 54.8; H, 7.51; N, 14.6. Found: C, 54.5; H, 7.72; Adrenocorticotropin (ACTH) Structure N, 14.4. Sir: V was treated with crystalline carbobenzoxy-L-serylSince the synthesis' of a nonadecapeptide correspondL- tyrosyl-L-seryl-L-methionyly-benzyl-L-glutamyl-Ling to the NHz-terminal 19-amino acids in the 39histidyl-L- phenylalanyl-NG-tosyl-Larginyl-L-tryptoamino acid chain of ACTH structures, several investiphyl-glycine5in the presence of I1 to give the protected gators2" have reported the synthesis of various chain pentadecapeptide (VI). VI was purified by countercurlengths. We wish to describe herein the synthesis of rent distribution in the carbon tetrachloride system' a pentadecapeptide with a structure consisting of the followed by washing with methanol; m.p. 225-230' dec. ; first ten NHz-terminal residues linked with a sequence of [a]=D -51.5' (c 2.4, dimethylformamide). Anal. positions 15 to 19 in ACTH structures; namely, LCalcd.: C, 57.0; H, 6.48; N, 14.0. Found: C, 56.6; seryl -L-tyrosyl -L-seryl -L-methionyl -L- glutamyl - L- hisH, 6.17; N, 13.8. tidy1- L- phenylalanyl - L - arginyl -L-tryptophyl-glycyl -LVI was treated with trifluoroacetic acid and then with lysyl-L-lysyl-L-arginyl-L-arginyl-L-proline.This synsodium in liquid ammonia1*to remove all the protecting thetic product, designated as a(l-'O) +(15-19)-ACTH, groups. The crude pentadecapeptide was desalted was shown to have an ACTH potency of apand purified by chromatography in a carboxymethyl proximately 1 U.S.P. unit per mg., as estimated by cellulo~e'~ column. The purified at1-l0)+(*5-19)-ACTH in vitro' and in vivos methods in the rat. The product was found to be homogeneous in paper and was found to exhibit the full lipolytic potency of the polyacrylamide gelrn electrophoresis. Amino acid naturalg a.-ACTH, when assayedlo in vitro with perianalysis of an acid hydrolysate of the synthetic penta(1) C. H . Li, J. Meienhofer, E. Schnabel, D. Chung. T. B . Lo and J. decapeptide both by the chromatographic method of
marized in Table I and show that either product can be made to predominate.B
c,
c,
Ramachandran, J . A m . Chcm. Soc.. 81,5760 (1960); 88,4449 (1961). (2) K. Hofmann. H. Yajima, N. Yanaihara. T . Y. Liu and S. Lande, ibid.. 88, 488 (1961). (3) H . Kappeler and R . Schwyzer, tlclo. Chim. A d a , 44, 1136 (1961). (4) K.Hofmann, T.Y . Liu, H. Yajima, N. Yanaihara, C. Yanaihara and J. L. Hume, J . A m . Chcm. Soc., 84, 1054 (1962). (5) C. H . Li, D. Chung. J. Ramachandran and B. Gorup, ibid., 84, 2460
(1962). (6) K . Hofmann, N. Yanaihara, S. Lande and H. Yajima, ibid., 84. 4470
(1962). (7) M.Saffran and A. V. SchaIly, Emdocrimd., 66, 523 (1955). (8) H. S. Lipscomb and D . H. Nelson, ibid., 71, 13 (1962). (9) C. H. Li, 1. I . Geschwind, A. L. Levy, J. I. Harris, J. S. Dixon, N. G . Pon and J. 0. Porath, Nolure. 178, 251 (1954).
(IO) A. Tanaka, 3. T . Pickcring and C. H. Li, Arch. Biochcm. Biophys..
n,294
(1962).
(11) L. T. Hogben and D . Slome, Proc. Roy. Soc. (London), BlO8, 10
(1931). (12) B. T. Pickcring and C. H. Li, Biochcm. Biophyr. A d a , 62, 475 (1962). (13) J. Mcienhpfer and C . H. Li, J . A m . Chcm. Soc.. 84,2434 (1962).
(14) J. Ramachandran and C. H. Li, J . Olg. Chcm., 17,4006 (1962). (15) G . W. Anderson and F. M.Callahan, J . A m . Chem. Soc., 81, 3359 (1960). (16) R. (1961).
B. Woodward, R . A. Olofson and H. Mayer, ibid., W , 1010
(17) R. Schwyzer and W. Rittel, Relo. Chim. A d a , 44, 159 (1961). (18) V. du Vigneaud and 0. K. Behrens, J . B i d . Chcm., 117, 27 (1937). (19) E.A. Peterson and H. A. Sober, 1.A m . Chcm. SOC.,78, 751 (1956). (20) R. A. Reisfeld, U. J. Lewis and D. E. Williams, Nolure, 186, 281 (1982).
1896
COMMUNICATIONS TO THE EDITOR
Vol. 85
Spackman, Stein and Moorez1 and by microbiological trypsin catalyzed reactions, i.e., preference for the Lassayz2showed an amino acid composition consistent antipode. with the theoretically calculated values (chromatoAn example of diminished stereochemical preference graphic: lYs~.~hisl.ooarg2.~serl.76glu1.~prp1.0~gl~0.~7meto.~~ for the L-antipode in compounds of the type R1’tyro.9gphel.oz; microbiological: lysl,~~his~.o~arg~.~~ser~.~~CONHCHRzCOR3, and associated with the nature proo.?zglyl.~meto.Bs tyr1,04phe~,~). The intact pentadecaof RI’, became available when it was found that the peptide was found to contain tyrosine and tryptophan in kinetic constants for the a-chymotrypsin catalyzed a molar ratio of one to one, as determined spectrophotohydrolysis of N-benzoylalanine methyl ester, in aqueous metrically. solutions a t 25.0°, pH 7.90 and 0.20 M in sodium chloride, were KO = 3.3 f 0.2 m M and ko = 0.0107 f (21) D. H . Spackman. W. H . Stein and S. Moore, A n d . Chcm., SO, 1190 (1958). 0.0002 set.-' for the D-antipode, and K O = 9.8 =t0.9 ( 2 2 ) The microbiological assay was carried out by the Shankman Laboram M and ko = 0.26 f 0.01 set.-' for the L-antipode.8 tories, Los Angeles, Calif. For N-acetylalanine methyl ester, K O = 611 f 1 0 (23) T . W. Goodwin and R . A. Morton, Biochcm. J . , 40, 628 (1946). m M and ko = 1.29 f 0.02 sec.-l for the L antipode (in (24) This work was supported in part by a grant (GM-2907) from the National Institutes of Health, United States Public Health Service, and a 0.50 M sodium chloride) with no detectable substrate grant from the American Cancer Society. activity being observable for the D-antipode.’O The sought for example of inversion of antipodal HORMONE RESEARCH LABORATORY CHOHHAOLP4 OF CALIFORNIA JANAKIRAMAN RAMACHANDRANspecificity for substrates of the type R1’CONHCHRzUNIVERSITY BERKELEY, CALIFORNIA DAVIDCHUNG COR3 arising from appropriate selection of the group RECEIVED MARCH9, 1963 RI’, with the nature of Rz and R3 remaining invariant, has now been found. In the a-chymotrypsin catalyzed hydrolysis of N-picolinylalanine methyl ester, in A Further Example of Inversion of the Usual Antipodal aqueous solutions a t 25.0°, pH 7.90 and 0.10 M in Specificity of a-Chymotrypsin‘ sodium chloride, values of KO = 18 f 1 m M and ko Sir : = 0.070 0.003 sec.-I were obtained for the L-antipode -15.3 0.3’ (c 3% in water)) (m.p. 59-60’, [aI25~ In 1948 it was shown that the stereochemical course and K O = 17 f 1 m M and ko = 0.165 =t 0.006 set.-' of the papain catalyzed synthesis of a-N-acylated afor the D-antipode (m.p. 59-60’, [alz5”015.3 f 0.3’ amino acid phenylhydrazides, from certain a-N(c 3% in water)). The experiments were conducted with acylated a-amino acids and phenylhydrazine, could be the aid of a pH-statl1sl2 under conditions where [ E ] determined by the structure of the acyl component.2 = 26 p1W3 and [SI = 2.3-18.4 m M for the L-antipode Subsequent s t u d i e ~ confirmed ~?~ and extended these and [E]= 74 p M and [SI = 1.5-12 m M for the Dresults but attempts to observe the same phenomenon antipode. The primary data were evaluated using a with the comparable a-chymotrypsin catalyzed reacDatatron 220 digital computer programmed as detion were U ~ S U C C ~ S S ~ ~ ~ . ~ scribed previously. l 4 In 1960 an inversion of the usual antipodal specificity With three examples of inversion of the usual antiof a-chymotrypsin was demonstrated when it was podal specificity of a-chymotrypsin, involving both found that the rate of the a-chymotrypsin catalyzed hydrolysis of D-3-carbomethoxydihydroisocarbostyril, conformationally constrained and unconstrained substrates, two of which are a-N-acylated a-amino acid to the corresponding acid, was markedly greater than derivatives, it is evident that substantial support has that of the L-antipode.6 In providing an explanation for the preceding observations a theory was d e v e l ~ p e d ~ . ~now been provided for the explanation of this phenomenon given earlier.7-9 It also follows that the more which not only accounted for the above results but also general t h e ~ r ywhich ~ , ~ envisions non-productive comforecast in general terms the existence of other examples bination of substrate that is fully competitive with its of inversion of antipodal specificity as well as those productive combination with the active site of the involving diminished stereochemical preference in enzyme has acquired added significance. favor of the L-antipode for compounds of the type (10) J . P . Wolf, 111, and C . Niemann, Biochemislry, 2 , 493 (1963). R1’CONHCHR&ORa and cognate structures. (11) T . H . Applewhite, R . B . Martin and C. Niemann, 1.A m . Chcm. Soc., In a recent communication Cohen, et al.,9describe an 80, 1457 (1958). inversion of the usual antipodal specificity in the a(12) T . H . Applewhite, H . Waite and C . Niemann, ibid., 80, 1465 (1958). chymotrypsin catalyzed hydrolysis of ethyl a-acetoxy(13) Based upon a molecular weight of 25,000 and a nitrogen content of 16.5%. propionate. This behavior was explained in terms of (14) H . I . Abrash, A. N . Kurtz and C. Niemann, Biochim. B i o p h y s . A d o , a t h e ~ r ywhich , ~ was similar to that developed earlier,’J 46, 378 (1960). and provided a needed example of inversion of antipodal JAMES R. RAPP CONTRIBUTION No. 2948 specificity in a case where the structures were not LABORATORIES OF CHEMISTRY GATESA N D CRELLIN conformationally constrained. However, there reCARLNIEMANN INSTITUTEOF TECHNOLOGY CALIFORNIA mained a need for a demonstration that the nature of PASADENA, CALIFORNIA the group R1’ in compounds of the type RI’CONHCHRECEIVED MARCH18, 1963 RzCOR,, with the nature of R2 and R3 remaining invariant, could determine the degree of stereochemical preference for a given antipode or cause an inversion of Chemistry of Cephalosporin Antibiotics. 111. the antipodal specificity usually observed for a-chymoChemical Correlation of Penicillin and Cephalosporin ( 1 ) Supported in part by a grant from the National Institutes of Health, Antibiotics
*
U S. Public Health Service. ,2) E. L . Bennett and C. Niemann. J . A m . Chcm. Sac., 70, 2610 (1948). (3) (a) H . B. Milne and C. M. Stevens, ibid., 72, 1742 (1950); (b) E. L. Bennett and C. Niemann, ibid., 73, 1798 (1950). (4) W. H . Schuller and C . Niemann, ibid., 79, 1644 (1951). (5) W. H . Schuller and C. Niemann, ibid., 74, 4630 (1952). (6) G. E. Hein, R. B. McGriff and C. Niemann, i b i d . , 82, 1830 (1960). (7) G. Hein and C . Niemann, Proc. Nail. Acnd. Sci., 47, 1341 (1961). ( 8 ) G. E. Hein and C. Niemann, J. A m . Chcm. SOL.,94, 4487, 4495
Sir : Recent reports have shown that a series of new, potent, p-lactam-containing antibiotics can be synthesized from the naturally occurring substance, cephalosporin (2.1 These substances have the same carbon skeleton as penicillins but differ by the state of oxida-
(1962). (9) S. G. Cohen. J. Crossley, E. Khedouri and (1962).
(1) (a) B . Loder, G . G . F. Newton and E . P. Abraham, Biochcm. J.. 79, 408 (1961); (b) R. R . Chauvette, e f d ,J . A m . Chcm. SOL.,84, 3401 (1962).
K. Zand, ibid., 84, 4163
