June 20, 1955
COMXIJNICATIONS TO TIIE EDITOR
3419
peptides suggests that this is due to the proximity of aspartic and glutamic acids rather than to abSir: sence of an a-carboxyl terminus. The acid lability and unimolar ratio of amino An earlier communication from these laboratories' reported the methods of purification and essen- acids made partial acid hydrolysis promising for tially all of the structure of &corticotropin. We now study of the structure of PPs(4)Tr(l). Hydrolyses wish to present additional results which are suffi- using 12N HCl a t 25' and O.1N HCl a t 105' for varying times were followed by paper chromatogcient to complete the entire sequence. The previous work established the sequence of raphy. The former conditions were slower and the first twenty-four amino acids from the amino less specific than the latter. Treatment with 0.1N end as well as the first eleven amino acids from the HC1 for 2 hours completely destroyed the parent carboxyl end. The simultaneous failure of the two peptide and gave a maximum amount of intermesequence methods being used left an unknown sec- diate peptides with a minimum of conversion to free tion of four amino acids as shown in the following amino acids. Four peptides were isolated from such an acid digest of PPS(4)Tr(l)using a 5:4: 1 n-butati iagr am. no1 :water :acetic acid paper chroPPS(4) matomaDhic svstem. Amino acid I anal& of t6e hydrolysates of PPs(4)Tr(l) 1 these peptides along with their H,Ser. . . . . .'Val-Tyr-Pro-(Asp, Gly, Ala, G1u)-Asp-Glu-Leu, , . . . . Phe,OH Edman degradations established L that their structures were : HeValTyr-Pro.OH, H.Val-Tyr-Pro-Asp. The trypsin fragment PTr( 1) from 6-corticotropin OH, H.Ala-Glu.OH, and H.Gly-A41a-Glu.0H.Since was too acid-labile in this unknown region to permit valine was N-terminal in the starting material, these sequence determination by the Edman2 method. fragments are sufficient to establish that the seEach of the first three Edman degradations gave r-/ only one amino acid per step: valine, tyrosine and quence in /.3Ps(4)Tr(l)is H.Val-Tyr-Pro-Asp-( -Glyproline, in that order. However, the fourth stage of Ala-Glu.OH. The C-terminal glutamic acid was the Edman study gave a predominance of alanine confirmed by the Dakin reaction for free a-car~ aspartic acid failed to undergo this realong with significant amounts of glutamic and as- b ~ x y l s .The partic acids, glycine, phenylalanine and leucine, action under conditions causing reaction of C-termiwhich clearly indicates extensive side-reactions. nal asparagine in a model peptide. Therefore, we The fifth degradation gave the same amino acids in conclude that the aspartic acid is a-linked to the glycine. This, then, establishes the sequence and nearly equimolar amounts. Although pepsin exposes the unknown sequence the nature of the links in the entire structure of by removing the eleven amino acids from the car- @Ps(4),the smallest active pepsin-digestion product boxyl end, the product pPs(4) is unreactive to car- having clinical activity equal to the native hormone. These results complete the structure of p-corticoboxypeptidase. I t is clear from the diagram that the simplest fragment containing the uncertain tropin, ,8PS(3)land PPs(2),' except for the possible /3 portion would be the heptapeptide cleaved from the linkage of the other aspartic acid and the possible y carboxyl end of FPS(4)Iby trypsin. This PPs(4)Tr- linkage of one of the glutamic acids as illustrated (1) was separated from the trypsin dirXHz gest mixture by a 500-transfer counterSTRUCTURE OF 8-CORTICOTROPIN: QUENCE STUDIES
FINAL SE-
1
rNH2
Its amino acid analysis (1.1 Ala, l.O'Asp, 1.0 Gly, I .O Glu, 0.9 Pro, 0.7 Tyr, 1.1 Val) showed it to be the expected heptapeptide. Edman degradation clearly gave a Val-Tyr-Pro . . . sequence with decomposition in the fourth stage similar to that found in /?Tr(l).3 Carboxypeptidase failed to digest even this simpler peptide. Work on model (1) Paul H . Bell, et at., THISJOURNAL, 76, 5565 (1954). (2) P. Edman, Acta Chem. Scand., 4 , 283 (1950). (3) White and Landman, in a publication [THIS JOURNAL, 77, 1711 (1955) I which appeared after this manuscript was prepared, proposed a sequence based on the Edman method for this region of corticotropinA. This sequence was almost the same as the erroneous one based on our Edman results on a fragment from @-corticotropin.' Our subsequent partial acid degradation conclusively placed aspartic acid and showed that the H.Ala.-Glu.OH, also obtained by the Armour workers, came from t h e -COOH end of @PB(4)Tr(l). White, et al., used a qualitative Edman analysis, but in our work we have found that valid conclusions cannot be drawn even from quantitative Edman data because of concomitant decomposition in this region. This difficulty of interpretation is illustrated by the following Edman results on BPs(4)Tr(l): 4th stage-Ala, Glu, Gly and/or Asp in ratios of 5 : 1 . 5 : 1 ; 5th stage-Ala, Glu, Gly, 1 : l : l ; 6th stage-Ala, Glu, Gly and/or Asp, 4:1:'2.
A
An attempt to resolve this uncertainty by means of the Dakin reaction on the nonapeptide r (
r
[
rNH2
H.Val-Tyr-Pro-Asp-Gly-Ala-Glu\ -Asp- -Glu.OH
gave equivocal results. The cleavage of the aspartyl-glutamine bond by carboxypeptidase and of the glutamyl-aspartic bond by pepsin suggests a-peptide linkages. (4) G. H. Cleland and C. Niemann, T H I J~O U R N A L , 71, 841 (1949). ( 5 ) The amide-free corticotropin-A of White and Landman (ref. 3)
could have been derived from an amide-containing precursor, since we observed in our earlier work that alkaline conditions similar to theirs caused decomposition of the glutamine amide without loss of activity. Recently Li, Geschwind, Dixon, Levy and Harris [ J . Biol. Chem., 418, 171 (1955) ] reported a similar observation on sheep corticotropin, T h e identity of the peptide chains of 8-corticotropin and of corticotropin-A is to be expected if the latter is simply a deamidation product of this mild alkaline degradation. In addition, the homogeneity of corticotropin-A in terms of our Q-, 8- and y-corticotropins' needs to be established before comparisons are made. Corticotropin-A comprises approximately 3 / r of the activity of oxycellulose ACTH, which we have shown contains eight active components, the most abundant of which (@-corticotropin)amounts to only 33%.
Lack of time and material does not permit US to obtain additional evidence regarding these alternate linkages. Logical approaches would be by the use of the Edman method on the aspartyl-glutaminyl-leucine peptide' and by partial acid degradation of the nonapeptide mentioned above. Details of this and the earlier work will be reported shortly. EXPERIMENTAL THERAPEUTICS A Y D KATHERINE S. HOWARD MEDICINAL CHEMICAL SECTIONS ROBERT G. SHEPHERD RESEARCH DIVISION E. ANNEIGNER AMERICASCYANAMID COMPAXY DAVIDS. DAVIES STAMFORD, CONNECTICUT PAVLH. BELL RECEIVED MAY11, 1963
agents such as hydrogen over platinum to the corresponding hydroquinone. I can be converted to a variety of stable substitution products (11-VII) which have some chemical properties analogous to those of substituted benzoand naphthoquinones. X
11,
x = c1
111, X = Br
V, X = -0CH3 VI, X = -NHu YII, x = -OH
0
I reacted with chlorine or bromine in acetic acid to yield the yellow halogen derivatives I1 and 111, respectively. I1 had m.p. 114-115'. Anal. Calcd. PHENYLCY CLOBUTADIENOQUINONE for CloHsOzCl: C, 62.36; H, 2.62. Found: C, Sir: H , 2.54. I11 had m.p. 128-129'. Anal. No authentic cylobutadiene derivatives appear to 62.20; Calcd. for C10H602Br:C, 50.66; H , 2.13. Found: have yet been prepared except for biphenyleneIa and related substanceslb)?.Bwhere the four-niem- C, 50.64; H , 2.13. Reduction of I11 with amalgamated zinc and hydrochloric acid gave phenylcybered rings are part of fused-ring system^.^ The clobutane. 111 reacted rapidly with alcoholic silfailure to detect cyclobutadiene or its non-fused ring ver nitrate and, with sodium iodide in acetone, derivatives among the products of appropriate yielded IV, m.p. 163-165' (dec.). Anal. Calcd. synthetic reactions can be ascribed either to excesCloH50zI: C, 42.26; H, 1.77. Found: c, sive ring strain or unfavorable electronic configura- for 42.38; H , 1.87. Methanolysis of 111 afforded pale t i o n ~ . ~Strong ,~ evidence that ring strain is not the yellow V, m.p. 151-152' (Anal. Calcd. for most important factor is now provided by a syntheCI'Ha03: C, 70.20; H, 4.28. Found: C, 69.51; sis of the quite stable phenylcyclobutadienoquinH , 4.16) while ammonia in dry benzene gave the one (phenylcyclobutendione, I ) which to a first ap- colorless essentially neutral amino derivative (VI), proximation is expected to have the same degree of m.p. 279-281' (dec.). Anal. Calcd. for C U I H ~ O ~ N : ring strain as phenylcyclobutadiene. C, 69.36; H, 4.07; N, 8.09. Found: C, 69.37; H, 4.04; N, 8.08. With dilute acetic acid, 111 CBHb- / =O was hydrolyzed to colorless VII, m.p. 205-208' I1 (dec.). VI1 is soluble in water, is an exceptional I O strong acid for a C, H, 0 compound ( ~ K A 1) and gives a magenta color with ferric chloride soluPhenylacetylene with trifluorochloroethylene a t tion. Anal. Calcd. for CloHaOs: C, 68.96; H, 120' for 24 hours gave 1,1,2-trifluoro-2-chloro-3-3.48; neut. equiv., 176. Found: C, 68.87; H, phenylcyclobutene6; b.p. 52-53' (0.4 mm.), n% 3.61; neut. equiv., 174. VI1 is converted to V 1.,5117. Anal. Calcd. for Cl~H6F3C1:C, 54.92; with diazomethane. H, 2.77. Found: C, 54.83; H, 2.89. Hydrolysis6 The stable existence of I-VI1 suggests the possiof the adduct with 92% sulfuric acid a t 100' af- bility that quinone-type derivatives of unknown forded bright-yellow crystalline I in 73% yield; cyclic polyolefins such as pentalene and heptalene m p . 152-1 53' (dec.) after recrystallization from might be more stable than the parent hydrocarbons. acetone. Anal. Calcd. for C10H602: c, 75.94; Investigation of the chemical and physical propH , 3.52; mol. wt., 155. Found: C, 76.00; H , erties of I-VI1 is being continued. 3.85; mol. wt. (Rast), 151. I had Amax = 286 mp, CONTRIBUTION No. 2000 E 2.3 X lo5and strong infrared absorption a t S.6 p . GATESAND CRELLINLABORATORIES EDGAR J. SMUTNY -1chloroform solution of I with 30%. hydrogen per- CALIFORNIA INSTITUTE OF TECHNOI,OGY J O H N D. ROBERTS oxide yielded phenylmaleic anhydride, m.p. 119- PASADENA 4. CALIF. 120', which did not depress the m.p. of an authenRECEIVED MAY20, 1955 tic sample.' Reduction of I with amalgamated zinc and hydrochloric acid yielded phenylcyclobuNEW REAGENT FOR RESOLUTION BY COMPLEX tane which was identified by comparison of its in- A FORMATION ; THE RESOLUTION OF PHENANTHROfrared spectrum with material previously prepared.6 13.4-cl PHENANTHRENE' I is much more stable to water and oxygen than o- Sir: benzoquinone. It is apparently not reduced by We wish to report the synthesis of 2-(2,4,5,7-tet(1) (a) W. C. Lothrop, THISJ O U R N A L , 63, 1187 (1941); (h) 64, ranitro-9-fluorenylideneaminooxy)-propionic acid, 1098 (1942). 111, a useful reagent for the optical resolution of cer(2) R . F. Curtis and G. Viswanath, Chem. ond Ind., 1174 (1954). tain aromatic compounds which do not have the (3) M. P. Cava and J. F. Stucker, ibid., 440 (1955). functional groups usually needed to effect resolution (4) An excellent survey of attempts t o prepare non-fused ring cyclobutadiene derivatives was recently presented by E. R . Buchman, Ahst. by conventional reagents. of A.C.S. meeting, Sept. 13. 1954, p. 9-0. The dextro and levo forms of 111 were made by ( 5 ) For references and discussion see J . D. Roberts, A. Streitwieser, condensing 2,4,5,7-tetranitrofluorenone,I, with Jr.. and Clare hl. Regan, THISJ O U R N A L , 7 4 , 4579 (1952).
-
.
(6) Cf.J . D. Roberts, 0 . R . Kline and H. E. Simmons, J r . , ibid., 76, 4765 (1953). ( 7 ) I , . E. Ililler, H. R . Staler and n. J . hlann, Ibid., 71, 374 (1949).
I
I
(1) A part of this material was presented before the Division of Organic Chemistry a t the 127th meeting of the American Chemical Society, Cinclnnatl, Ohio, >larch, 19n5.
