July 20, 1957
COMMUNICATIONS TO THE EDITOR
3933
by Clark and Odell,lb decreases steadily with increasing temperature from -16 to 43’, magnetic measurements have been also made on solutions of the complexes in dibutylphthalate which permits measurements up to 200’. Curves of the magnetic moments of these complexes dissolved in this solvent likewise show a NORTHERN UTILIZATION RESEARCH L. L. MCKINNEY minimum. For the bis-N-methyl- complex this F. B. WEAKLEY minimum falls near 120’. Beyond this point the A N D DEVELOPMEXT DIVISION PEORIA,ILLINOIS A. C. ELDRIDGE magnetic moment rises steadily with increasing VETERISARYMEDICAL R. E. CAKPBELL temperature. This suggests that the mechanism INSTITUTE J. c . COtVAX RESEARCH J . C. PICKEN,J R . of the transition from diamagnetism to paramagIOYASTATECOLLEGE AMES,IOWA H. E. BIESTER netism is the same for solutions as i t is for the molten systems. The results of this investigation also RECEIVED JUNE10, 1957 show that the presence of solvents is not necessary to give rise to paramagnetism in these complexes, THE MAGNETIC SUSCEPTIBILITY OF MOLTEN their diamagnetism being a property peculiar to NICKEL,(II) COMPLEXES the solid state only. Sir: The equilibrium constants K = [paramagnetic The paramagnet.ism exhibited by the bis-N- form]/ [diamagnetic form] have been calculated methylsalicylaldiminenickel(I1) complex, diamag- from values of magnetic susceptibility. The plots netic in the solid state, when dissolved in “non- of log K against 1/T give a minimum which demcoordinating” solvents such as benzene and chloro- onstrates an inversion of sign in the enthalpy of form, has been interpreted as being due to the con- values of the equilibrium. version of a proportion of the molecules of the comThe possibility that the paramagnetism of the plex from a planar to a tetrahedral configuration.’ molten compounds can result from dissociation of A similar behavior is observed for complexes of the the complexes into free Ni++ ions and chelate moleseries from bis-N-ethyl- to bis-N-amylsalicylaldi- cules has been excluded by measurements of elecminenickel(I1) .* trical conductivity made on these complexes in the The electric dipole moment measurements made molten state. on such complexes dissolved in dioxane and benzene LUICISACCONI INSTITUTE OF GENERAL CHEMISTRY have afforded evidence against such a view.2 On THE REXATO CIXI UNIVERS~TY OF PALERMO the other hand, the hypothesis that paramagnetic PALERMO, ITALY FRANCESCO MAGGIO octahedral disolvated complexes are formed, alRECEIVED MAY31, 1987 though improbable in the light of the investigations by Basolo and Matoush3on the coordinating tendencies of the methylbenzenes, cannot be ruled THE STRUCTURE OF ETAMYCIN In fact the existence of a silver perchlorateSir: benzene complex5 shows that benzene and metal This communicatior. reports the complete strucatoms may bind together. ture of the antibiotic Etamycinl (Viridogrisein2), I n order to determine whether or not benzene the isolation of which recently was described indemolecules do coordinate with these nickel(I1) com- pendently and simultaneously by two groups. plexes to yield paramagnetic solutions, magnetic The antibiotic possesses interesting activity against measurements have been made on bis-N-alkyl- Gram-positive organisms, and in addition causes a salicylaldiminenicke1(11) complexes, from bis-N- reversible leucopenia in dogs. Etamycin is a surethyl- t o bis-N-decyl-, in the molten state. The prisingly lipophilic peptide (soluble in benzene and magnetic susceptibilities of the molten compounds carbon tetrachloride) with a molecular weight in were measured between 80 and 200’ by the Gouy the range 800-900. The presence of 3-hydroxymethod. I n order to have samples of a lower melt- picohic acid, L-alanine, allo-hydroxy-D-prche, Ding point, mixtures of two complexes in the mo- leucine and threonine was reported.1J m.e have lecular ratio of l :1 also were used. now shown Etamycin to be a macrocyclic lactone Complexes which are diamagnetic in the solid (22-membered ring) which contains in addition to state are paramagnetic with moments ranging the above-mentioned components sarcosine, afrom 0.8 to 1.15 B.M. in the molten state. Graphs CHB-CH-CH-CH-CO*H of the magnetic moment vs. temperature for all of I l l the complexes examined have very similar shapes CHI CH, NHCH, and sometimes coincide. I n the case of complexes I or mixture of complexes melting below loo’, the curves have a minimum near 120”. Since the paramagnetism of bis-N-methylsalicylaldimine!+N)-CO-Thr-D-Leu---aHyPro4u nickel(I1) in benzene and chloroform, as measured O4C-PheSar-L-Ala-DiMeLeu I I
duces in calves the typical TESOM toxicity syndrome, suggest that S-(dichloroviny1)-L-cysteine may be related in structure to a part of, or may possibly be, the toxic principle of TESOM. Attempts to isolate sufficient toxic material from TESOSI for chemical characterization are under way a t the present time.
le2
(1) (a) J. 8. Willis and D. P. Mellor, THISJOURNAL,69, 1237 (1947); (b) H. C. Clark and A. L. Odell, J . Chem. SOC.,3431 (1955). (2) L. Sacconi, P. Paoletti and G. Del Re, THISJOUBNU, in the press. (3) F.Basolo and W. R . Matoush, ibid., ‘74 5663 (1953). (4) Cf.H . C. Clark and A. I. Odell, J . Chem. soc., 520 (1950). ( 5 ) R . E. Riinrfle and J , H. Goring. TWIS TOTJRNAL.79, ,5337(1960).
I1 (1) B. Heinemann, el ai., Arrtrbiolics A n n r d , 2, 728 (1954-1955). (2) Q. R. Bartz, el ul., ibid., a, 777, 784 (1954-1955): the identity of Vitidogrisein with Etamycin wan established in the laboratories of the authors of refs. 1 and 2 and at M.I.T.
m3.1
phenylsarcosine (PheSar) and p,N-dimethylleucine (DiMeLeu, I), the latter two of which have not been encountered previously in a natural product. The presence of K-methylamino acids was indicated by color tests3 on hydrolysates, and confirmed by an N-methyl determination, which demonstrated the presence of three N-methyl groups per molecule of Etamycin. For the isolation of the three N-methylamino acids, an Etamycin total hydrolysate was deaminated to destroy threonine, alanine and leucine and subjected to preparative paper chromatography. One of the amino acids was identified as sarcosine by conversion to the dinitrophenyl derivative, m.p. 183-186', undepressed upon admixture with an authentic sample. The second amino acid, C ~ H l l N 0 2m.p. , 245-243' (sublimes) (Found: C, 65.30; H, 6.66; N, 8.37) had infrared and ultraviolet spectra indicating the presence of a benzene ring. It was proved to be identical with synthetic D,L-a-phenylsarcosine, m p . 246-247' (sublimes), by infrared and ultraviolet spectra and chromatographic behavior. The optical inactivity is expected since optically active a-phenylglycine is known4 to racemize rapidly with hot 1OyGhydrochloric acid. The third amino acid, C8Hl7SO2,n1.p. 313-:31Go dec., (Found: C, 60.12; H, 10.47; N, 19.77) was optically active, [a]'*D f36.2' ( 6 , 2.4 in 5 N HC1). Hypochlorite degradation5gave niethylamine, identified as the dinitrophenyl derivative, and the isopropylmethylacetaldehyde, isolated as the 2,4ditiitrophenylhydrazone, m.p. 125.2-1 26.0O , [ a ] 2 b ~ -3S.i" (c, 1.3 in CHClai, which was identical (ni.p., mixed m.p., infrared spectra) with a sample of the DNPH of (- )-isopropylmethylacetaldehyde obtained by ozonolysisfiof ergosterol.' Thus, the third amino acid is p,N-dimethylleucine (I). I t may be significant that the new amino acid and ergosterol have a common structural unit. Etamycin contains only one each of the eight components according to quantitative paper chromatography and analyses of Etamycin, C44Hfi2KsOl0 (Found: C, 39.95; H, 7.40; N, 13.07; dried a t 135' (0.01 mm.) and Etamycin hydrate, (241Hs2hTsO1o.H20 (Found: C, 38.96; H, 7.17; N, 12.54; X-CHa, 4.63; eq. wt., 890; dried a t 110' (0.01 111111.).
(3) Paper chromatograms showed three weakly ninhydrin-positive spots, two of which gave red color tests with 9-nitrobenzoyl c h l o r i d t pyridine according t o P. A. Plattner and U. ;r-ager, Hclv. C k i m . A d a . 31, 220 (1948). (4) F. Ehrlich, Biochem. Z.,8 , 446 (1908). ( 5 ) K. Langheld, Bar., 42, 2360 (1909); P. A. Plattnrr and T i . Nagcr, H e l n . China. Acta, 31, 2192 (1948). l e ) (.; Slomp, el ai., THISJ O U R N A L , 7 7 , 1216 (19551 (7) W. Bergmann and H. A. Stansbury, J . Or#. C h r m . , 9 , 281 I I W l i ; t h e I ) S P H prrpared hy these authors had [n]% -37.7 and m.1, i'l-124.50.
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COJIRIVNICATIONS 'ro THE EDITOI..
Evidence that Etamycin contains a lactone structure was obtained as follows. On treatment with 0.1 N sodium hydroxide a t 'room temperature it is converted into an antibiotically inactive acid, which contains the hydroxypicolinic acid and all amino acids. In Etamycin the threonine resists oxidation with chromic acid, whereas under the same conditions, in Etamycin acid the threonine is destroyed t o the extent of 80%. Consequently the lactone link must involve the C-terminal carboxyl group and the hydroxyl group of threonine. The absence of a terminal free amino group and the presence of unusual amino acids presented difficulties in the application of the conventional chemical and enzymatic methods of peptide sequence determination. However, catalytic hydrogenation of the obviously N-terminal hydroxypicolinic acid provided a secondary amino function. and alkali treatment gave a terminal carboxyl group, thus making possible the application of the Edmati method? Seven successive degradations revealed the sequence as hydroxypicolinic acid-Thr-DLeu- D-aHyPro-Sar-DiMeLeu-L-Ala (?)- (PheSar). The course of the degradation was followed by paper chromatography of the phenylthiohydanto in^,^ dinitrophenylation and hydrolysis of each successive peptide, followed by paper chromatography of the resulting DNP-amino acids,'O and by quantitative estimation of the amino acid content of each successive peptide after total hydrolysis and two dimensional paper chromatography. The sequence was completed by degradation from the carboxyl end with hydrazine, l1 which established phenylsarcosine as C-terminal. The structure of Etamycin is represented by 11. NOTE ADDED IS PRooF.-Comparison of the rotation of the DiMeLeu in neutral and acid solution has shown it t o be in the L-series. Another isolation after mild hydrolysis gave PheSar having [ c Y ] ~ ~fD 118" (c, 4.8 in ,V HCl), from which i t can be deduced that it probably is in the 1,series.
\Ye are indebted to Rristol Laboratories of Syracuse, K. U.,for our supply of Etamycin, for microbiological assays and for some financial aid. DEPARTMEST OF CHEMISTRY MASSACHUSETTS INSTITUTE OF TECHNOLOGY
CAXBRrDGE 39,
hIASS.
JOHNC. SHEEHAN HANSGEORCZ A C H A U ' ~ ~ ~ - I L L I A MR . L A L V S O X ~ ~ ~
RECEIVED JKNE19, 1957 ( 8 ) P. Edman, Acta Chein. S c n n d . , 4, 283 (1950). (9) J. Sjijquist, ibid., 7 , 447 (1963). (10) S . Rlackburn and A . G. Lowther, Biochem. J . , 48, 126 (1951). (11) S. Akabori, K. Ohno and K. Narita, AMII.Soc. Chpiit. Jnfi,, 2 6 , 214 (1952). (12) (a) Supported t,y R grant from the Kational InstitttLe.:
