March 5 , 1958
COMMUNICATIONS TO THE EDITOR
of release of free amino acids indicated that the Nterminal sequence was Asp (-NHa)-Ser-Cys-GluGly and this was confirmed by the isolation of a series of labeled peptides from the aminopeptidase digest lacking progressively Asp (-NH2),Asp(-NH2) Ser, Asp(-NH2)Ser CyS03H and Asp(-NHz)Ser CySOsH Glu, as indicated in Fig. 1 (top). I n accordance with the specificity of trypsin, the C-terminal residue of the peptide was found t o be lysine, using carboxypeptidase B.4 PARTIAL ACID HYDROLYSIS OF S-8 PEPTIDE BY 12 N HCI AT 37OC FOR 72 HRS
A-3 1
~
GLY
~
GLY
~
ASP
1%1
GLY
~
PRO
I
1261
Fig. 2.
Detailed sequences are, however, available only for chymotrypsin7 and trypsin (above) and these show complete identity over the sequence Gly-AspDIP
1 CYST VAL
~
I
Ser-Gly. Recently, Westheimers has attempted to explain in detail the orientation of the active site of chymotrypsin by assuming the folding of this hypothetical sequence Fig. 1.-Enzymatic degradation of the peptide 0-Tr-1: the horizontal arrows delineate the peptides obtained by degradation with subtilisin (S) or aminopeptidase (AP). The vertical arrows denote the major points of cleavage by subtilisin. The sequence denoted by “Army” has been established previously by the authors of ref. 5 . Peptides S6 and S7 were present in trace quantitities only and their radioactivities were below the limits of detection.
DIP
-X-S-X-His-X-Gly-Asp-Ser-Gl
I
-
y-Glu Ala,-Val-
in an a-helix, thus bringing the histidine and serine into the favorable orientation previously suggested by C ~ n n i n g h a m . ~ On ~ ~the basis of our evidence for the sequence in trypsin which posse.;cses an essentially identical bond-breaking mechani:sm, the assumption as to the position of the histidine is unjustified. In addition, the presence of two cysteic residues (and proline) close to the: serine would probably preclude the formation of an a-helix in this region. The absence of histidine from the trypsin peptide described above [and in fact even from the largest peptide containing 55 residues) would suggest that maintenance of the histidine and serine in a favorable orientation is a question of the tertiary structure of the protein.1°
The remainder of the sequence was determined by subtilisin digestion which yielded 14 peptides. Of the subtilisin peptides, only S5-B4, SS and S9-B4 were radioactive, and their composition showed in each case the presence of a single serine. Glycine was liberated from both S5-B4 and S8 by aminopeptidase, while neither peptide gave any free amino acid with carboxypeptidase, probably due to the C-terminal cysteic acid (S8) and the penultimate proline (S5-B4). S8 was subjected to (7) N. K. Schaffer, L. Simet, S. Harshman, R. R. lfngle and partial acid hydrolysis and the resulting peptides could be fitted into a unique sequence (Fig. 2 ) . R. W. Drisko, J. Bid. Chem., 226, 197 (1957). (8) F.H. Westheimer, Proc. Not. Acad. Sci., 43, 969 (1957). In Fig. 1, these subtilisin peptides are combined (9) L. W. Cunningham, Science, 126, 1146 (1957). with the sequences found by aminopeptidase and (10) G. H . Dixon and H. Neurath, Biocliim. e l Biophys. Acto, 20, acidic hydrolysis, and provide a unique sequence. 572 (1956). The sequence Asp-Ser-Gly around the DIP-seryl DEPARTMENT OF BIOCHEMISTRY GORDON E[. DIXON DOROTHY L. KAUFFMAN confirms that previously determined by Schaffer, UNIVERSITYOF ~‘ASHINGTON SEATTLE, WASHINGTOS HANSNEURATH et aL6 RECEIVED JANUARY 15, 1955 Much interest has been focused recently upon the similarity in cornposition around the phosphorylated serine in those enzymes inhibited by DFP.6 (4) J. E. Folk, THISJOURNAL, 78, 3541 (1956). (5) N. K. Schaffer, R. E. Engle, L. Simet, R. W. Drisko and S. Harshman, Fed. Proc., 16, 347 (1956). (6) N. K. Schaffer, S. C. M a y a n d W. H. Summerson, J . B i d . Chcm., 202, 69 (1953); R. A. Oosterbaan, H . S. Jansz a n d J. A. Cohen, Biochim. et Biophys. Acta, 20, 402 (1956); G. H . Dixon, S. Go and H . Neurath, i b i d . , 19, 193 (1956); D. E. Icoshland, Jr., and M. J. Erwin, THIS JOURNAL, 79, 2657 (1957); F.T u r b a and G. Gundlach, Biochem. 2.. 837, 186 (1955).
ON THE ABSOLUTE CONFIGURATION O F T H E ANTIBIOTIC ACTIDIONE
Sir: The striking results of optical rotary dispersion measurements among polycyclic ketones,’ have (1) For leading references see C. Djerassi, B I L LSoc. Chim. France, 741 (1957). a n d C. Djerassi, 0. Halpern, V. Halpern, 0. Schindler and C. T a m m , E e l v . Chim. Acta, 41, Xo. 1 (1958), in press.
TO THE EDITOR COMMUNICATIONS
1262
Lrol.
so
prompted us to extend such studies to optically adipic acid (V) which already had been related3"13 active cyclohexanones. For the proper evaluation to D-glyceraldehyde. Since (+)-2,4-dimethylof the dispersion data i t was important to use cyclohexanone (11) was formed under alkaline cyclohexanones of known absolute configurations conditions the two methyl groups of I1 can be and these are generally best secured from terpenes. assumed to be cis from which it follows that the Thus, ( ) -3-methylcyclohexanone is readily ob- absolute configuration (2R :4R") as depicted in IT tained2Ja from (+)-pulegone and serves as an correctly represents (-+)-cis-2,4-dimethylcyclohesextremely useful standard for many stereochemical anone. correlations3 and for transformations t o substituted H CHI I3 CH? cyclohexanones for conformational studies by rota\ ' '\ . 0 tory dispersion measurements.4 I 4 For these reasons it would be very desirable to CHL , k F € O I I C H have accessible additional alkylated cyclohexanones with known absolute configurations and the present communication deals with (+)-2,4-dimethylcyclohexanone (11). This ketone is formed in one step by alkaline treatment of the antibiotic actidione (I),6 which is prepared commercially in large amounts because of its agricultural applications. The determination of the absolute configuration of I1 affords a reference standard for the eventual elucidation of the absolute configuration of the remaining asymmetric centers of this antibioticG and even more importantly provides a convenient model and starting material for experimental and theoretical rotatory dispersion studies as will become apparent from subsequent papers. (+)-2,4-Dimethylcyclohexanone (aZ5~ +2.4GU, neat) exhibits a single negative Cotton effect cwve7 in methanol solution (c, 0.097) with a trough a t 297.5 mp (-278') and a peak a t 276 mp (-57") and was transformed into its enol acetate 1118 (b.p. 48" (1.5 mm.), [Cr]"D 4-74.3" (octane); (13) K. Freudenberg and W.Hohtnann, e l a l . , Ax?%., 5 8 4 , 54 (1954). Anal. Found for CloHlaO*: C, 71.03; H, 9.96). (14) (a) Postdoctorate research fellow on funds supplied by t h e Ozonolysis provided (+) -4-methyl-6-oxoheptanoic National Science Foundation; (b) Predoctorate research fellow on acid ( I v ) (b.p. 101" (0.02 mm.), [(YIz5D +8.0° funds supplied b y t h e National Cancer Institute (Grant S o . C'i(CHC&), : ' : : :A 5.80 p ; Anal. found for CsH1403: 2919) of t h e U. S. Public Health Service. OF CHEMISTRY E. J. EISEX3RAUN'4" C, 60.10; H, 8.06; neut. equiv., 165) whose single bDEPARTMENT 7 A Y N E STATE UXIVERSITY J. O S I E C K I ' ~ ~ positive Cotton effect cwve7 was opposite in sign DETROIT,MICHIGAN CARL DJERASSI to that of (+)-2-ethyl-4-pentanone (VI). Since RECEIVED FEBRUARY 3, 1958 the latter had been synthesizedg from (-)-2-ethyl1-propanol of known1° absolute configuration (S),llthe 4-methyl group of I1 presurnablyl2 belongs SOME ISOMORPHOUS TERNARY OXIDES CONTAINING TANTALUM to the D-series (R according to the new convention") Sir: Rigorous confirmation was provided by hypoIn attempts to make Bao.6Ta0z.6 and similar bromite oxidation of IV leading to (+)-&methyl- compounds, we have prepared some ternary oxides containing tantalum which have a somewhat (2) 0. Wallach, Ann., 289, 337 (1896). different composition. From powder and single (3) (a) See E. J. Eisenbraun and S. 11. McElvain, THISJOURNAL, 77, 3382 (1958); (b) A. Melera, D. Arigoni, A. Eschenmoser, 0. Jeger crystal X-ray data, these compounds appear to be and L. Ruzicka, Helv. Chim. Acla, 39, 441 (1956). isomorphous. (4) C. Djerassi, L. E . Geller, J. Osiecki and E. J. Eisenbraun, Mixtures were made according to equations (1) paper t o be presented a t "Conformational Analysis" Symposium, and (2) and heated a t 1250' in evacuated, sealed .4CS, San Francisco meeting, April, 1958. ( 5 ) E. C. Kornfeld, R. G. Jones and T. V. Parke, THISJOURNAL. capsules for three 24-hour periods. The sarnples 71, 150 (1949). W e are indebted t o D r . E. C . Kornfeld (Eli Lilly and were reground between heatings. Company) and Dr. D. I. Weisblat (Upjohn Company) for supplies of
+
\
(
1
~
I
actidione. (6) This may also be of help in synthetic studies-see D . D. Phillips, & A. I.Acitelli a n d J. Meinwald, ibid., 79, 3317 (1957). (7) For nomenclature see C. Djerassi and W. Klyne, P Y O C Chem. . Soc., 55 (1957). ( 8 ) T h e presence of some of the double bond isomfr is not excluded (9) L. E , Geller, unpublished observation in these laboratories. ( I O ) L. Crombie and S. H . Harper, J. Chem. Soc., 2685 (1950). (11) K. S. Cahn, C. K. Ingold and V. Prelog, Expeuienlia, XII,81 (1956). (12) T h i s is predicated on t h e assumption t h a t t h e carboxyl groiip of IV r a n be ignored which turned out to be justified.
+
+
O.5OBaO 0.40TazOs 0.20Ta = Ba0.~0Ta'"02.s ( 1 ) 0.50Ba0 f O.20Ta2O6-I- O.1OTa -I0.50NbOz = B a o . ~ ~ ( T a o . s o T V S b o . 5 (2) ~1V)O~.~
Two analyses of the product of reaction (1) for barium and tantalum, plus a determination of weight gain on heating in air: indicated the cotnThe product of position Bao.44(Tao.741VTao.2~v)0*.5,. reaction (2) has not been analyzed. When reaction (1) was run with the reactants wrapped in tantalum foil, platy blue cryst:als
