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COMMUNICATIONS TO THE EDITOR
Vol. s5
spectral evidence supports structure VI rather than its metacyclophane valence tautomer, the delocalization energy of VI probably is in excess of that of two isolated benzene rings. The synthesis of VI raises the possibility that a variety of such molecules can be prepared to test experimentally the exact nature of an aromatic a-electron cloud with regard to various physical and chemical properties such as steric hindrance, unusual bonding, and unusual interactions with ions or radicals generated within the T-electron cavity. Exploration of these general questions is being ~ n d e r t a k e n . ~
corticotropic activity of three different batches of I by the rat adrenal ascorbic acid depletion methodI6 6.6, 39.2 f 7.2 and gave values of 31.2 f 4.2, 37.8 48.4 f 10.4 IU/mg. Of considerable practical importance is the finding that peptide I appears to exhibit 0.7 times the i.v. potency when administration is by the subcutaneous route; values of 32.4 f 3.0, 29.4 f 3.0, 24.3 A 3 and 21.6 3.3 were obtained in four assays. Preliminary studies have shown peptide (I) to exhibit 30-40ojO the in vitro and approximately 35y0 the in vivo steroidogenic activity of corticotropin AI on a weight basis.16 The melanophoretic activity of (9) The analytical a n d spectral d a t a of all of the compounds described are I is 1.6 X lo7MSH units/g." in accord with t h e assigned structures. These findings exclude the methionine residue as a DEPARTMENT OF CHEMISTRY V. BOEKELHEIDE "functionally active site"'* for adrenocorticotropic UNIVERSITY OF OREGOS JOSEPH B. PHILLIPS activity and suggest that oxidation of the methionine EUGENE,OREGON sulfur lowers markedly the binding affinity of corticoRECEIVEDMARCH16, 1963 tropin and its biologically active fragments for the receptor with a corresponding decrease in biological STUDIES ON POLYPEPTIDES. XXVII. ELIMINATION OF activity. The fact that the sulfoxide or sulfone of a THE METHIONINE RESIDUE AS AN ESSENTIAL biologically active peptide containing methionine may FUNCTIONAL UNIT FOR in vivo ADRENOCORTICOTROPIC possess a significantly lower physiological activity than ACTIVITY1-8 the genuine material appears to provide little informaSir : tion regarding the "functional" importance of the Exposure to hydrogen peroxide lowers markedly methionine residue. the adrenal-stimulating activity of pig corticotropin For the synthesis of I , N-t-butyloxycarbonylseryltyand incubation with thiols brings about essentially rosylseryl-a-amino-n-butyrylglutamine (11) (dihydrate; complete reactivation of oxidized material.4--8 This A n d . Found: C,50.0; H, 6.9; N, 12.3; m.p. 146-148'; phenomenon is also observed with the skin darkening [ O ~ ] ~ ' D-28.6' in methanol; R f l 0.7319) was coupled hormones a- and P-MSH and with parathyroid pepwith histidylphenylalanylarginyltryptophylglycine20 via t i d e ~ . ~ - " All these hormones contain methionine as the azide to give N-t-butyloxycarbonylseryltyrosylserylthe sole sulfur containing residue, and reversible oxidaa - amino - n - butyrylglutaminylhistidylphenylalanylartion of the thioether sulfur to the sulfoxide appears to ginyltryptophylglycine (111) (monoacetate trihydrate; provide the basis for the deactivation-reactivation ,1nd Found: C, 53.4; H, 6.7; N, 15.9; [ a ] " -27.2' ~ behavior. l o ,l 2 in 50% v./v. acetic acid; Rfl 0.53; amino acid ratios in The non-essential nature of the methionine residue acid hydrolysate serz,ogtyrl,oobutl.o~gluo.94hi~~.o~phe~.~ as concerns melanocyte expanding activity follows from a r g I , ~ , ~ g l y OThe . g ~ acetate ). (111)was converted to the hythe observation that a family of peptides related to drochloride which was then treated with the hydrochlothe N-terminus of the a-MSH sequence possesses the ride of Ne-t-butyloxycarbonyllysylprolylvalylglycyl-Nfability to darken frog skin although they do not conf-butyloxycarbonyllysyl-Nf- t - butyloxycarbonyllysylartain methionine.13 ginylarginylprolylvaline amide (triacetate hexahydrate ; In order to clarify the essential nature of the methio24nal.Found: C, 50.0; H, 8.2; N, 16.3; [ a ] 2 -71.8' 7~ nine residue for adrenocorticotropic activity, we synin 10% v./v. acetic acid; Rf1 0.68; amino acid ratios in thesized the eicosapeptide amide seryltyrosylserylacid hydrolysate lys~,C6pr~l.94~a12.02gly~.~oarg~.~~) using a - amino - n - butyrylglutamylhistidylphenylalanylN, N'-carbonyldiimidazole?l as the condensing reagent. arginyltryptophylglycyllysylprolylvalylglycyllysylThe ensuing protected eicosapeptide amide t-butyloxylysylargjnylarginylprolylvaline amide (I) ar.d evaluated carbon ylseryltyrosylseryl- a-amino - n - bu tyrylglutaminits in vivo adrenocorticotropic activity in the rat. ylhistidylphenylalanylarginyltryptophylglycy1-N'-t-buPeptide I is the a-amino-n-butyric acid analog of the tyloxycarbonyllysylprolylvalylglycyl - Nf -t-butyloxycaradrenocorticotropically fully active eicosapeptide amide bonyllysyl - Nf- t - butyloxycarbonyllysylarginylarginylwhich corresponds to the N-terminal half of the pig prolylvaline amide (IV) (triacetate hydrate; [ a l Z 6 D corticotropin m ~ l e c u l e . ' ~Evaluation of the adreno-54.7Oin 10% v./v. acetic acid; single ninhydrin nega(1) T h e authors wish t o express their appreciation t o t h e U. S. Puhlic Health Service and the National Science Foundation for generous support of this investigation. (2) T h e peptides and peptide derivatives mentioned are of t h e L-configuration. I n the interest of space conservation t h e customary L-designation for individual amino residues is omitted. (3) See J . A m . Chem. Soc., 86. 833 (19fi3). for paper XXVI in this series. (4) M. L. Dedman, T. H. Farmer a n d C. J. 0. R. Morris, Biochem. J . , 69, xii (1955). (5) H . B. F. IXxon, Biochim. Biofihys. Acta, 18, 599 (1955). (0) H. R. F. Dixon and M. P. Stack-Dunne, Biochem. J., 6 1 , 4 8 3 il9.5.5). (7) 1'.H . Farmer a n d C. J. 0. R . Morris, Naluue, 1'78, 1405 (1950). (8) M . L. Dedman, T. H. Farmer and C. J. 0. R. Morris, Biorhem. J . , 66, 10G (1957). (9) H. B. F. Dixon, Biochim. Biofihy.!. Acla, 19, 392 (1950). (10) K . Hofmann and H. Yajima, in "Recent Progress in Hormone Research," G. Pincus, Ed., Vol. 18, Academic Press I n c . , New York, N. Y., 19w, p. 41. (11) H. Rasniussen and L. C. Craig, ibid., p. 20H. (12) M. L. Dedman, T. H. Farmer and C. J , 0. R. Morris, Biochem. J . , 7 8 , 3.18 (1901). ( 1 8 ) See K . Hofmann, A n n . Rev. Biochem., 31, 213 (1962), for tabulation of a series of melanophoretically active peptides. (1.1) ( a ) K. Hofmann, T. Liu, H. Yajima, N. Yanaihara, C. Yanaihara and J. I.. Humes, J . A m . Chem. Sos., 64, 1054 (1962); (b) K. Hofmann,
H . Yajima, T. Liu, Ti. Yanaihara, C. Yanaihara and J. I,. Humes, ibid.,
84, 4481 (1962) (15) Ascorbic acid depleting activity was determined in 24-hr. hypophysectomized rats according t o the method of "U. S. Pharmacopeia," VOl. xv. against t h e U.S.P. reference standard, We are indebted t o Dr. Joseph D. Fisher of Armour Pharmaceutical Company, Kankakee, Illinois, f o r these biological determinations. (16) We wish t o express our gratitude t o Drs. F. L. Engel and H. E. Lebovitz of Duke University Medical School for these assays. (17) We wish t o express our thanks t o Dr. A. B. Lerner of Yale University School of Medicine, for t h e M S H assays which were performed according t o the method of K . Shizume, A. B. 1,erner a n d T. B. Fitzpatrick, EndOtYi?ZO!., 64, 5.53 (19.54). (18) See ref. 13 for definition of this term. (19) Rr' values refer t o t h e Partridge system (S. M. Partridge, Bicchcnr. J . , 42, 238 (1948); Rr2 values refer t o the system 1-butanol, pyridine, acetic acid, water 3 0 : 2 0 : 6 : 2 1 (S. G.Waley and J. Watson, ibid., 66, 328 (1953)). With t h e latter system Ry values are expressed as multiples of t h e distance traveled by a histidine marker. (20) (a) K . Hofmann, M. E . Woolner, G. Spiihler and E, T. Schwartz, J . A m . Chem. S O L . 80, , 1486 (1958); (h) K . Hofmann and S. Lande, ibid.. 63, 2286 (1961). (21) (a) H. A. Staab, A n n . Chem., 609, 7 5 (1957); (b) G. W . Anderson and R. Paul, J . A m . Chem. Soc., 80, 4-123 (1958); (c) R . Paul and G. Anderson, i b i d . , 82, +.ill(i (19fj0).
w.
May 20, 1963
COMMUNICATIONS TO THE EDITOR
tive Pauly, Sakaguchi and Ehrlich positive spot with
1547
identical with paromomycin and paromomycin 11,
respectively. Rfl 0.61; single component on paper electrophoresis a t pH 3.5; amino acid ratios in acid hydrolysateser~.~~tyr~.g~-
R’”NH7NHR-I‘
but1.~3glu~.~0hisl.~4phe1.0garg3.04glyl.sllys~.80~r0~.0~vall.s1) was exposed to the action of anhydrous trifluoroaceticacid for ‘‘OR!! RO‘ 20 min. a t room temperature to give crude I which was OR‘ purified by chromatography on carboxymethylcelluIa-m losez2; ( [CX]’~D-61.7’ in 10% v./v. acetic acid; single ninhydrin, Pauly, Sakaguchi and Ehrlich positive spot Ia (Deoxystreptamine): R = R’ = R ” = R”’ = H Ib: R = R’ = H ; R ” = CHI; R”’ = COCHa with Rf20.7 X his; single component on disk electroIC: R = CH3; R’ = R ” = H ; R”’ COCH, phoresis a t pH 4.6; amino acid ratios in acid hydrolysate I d : R = H ; R’ = R ” = CHI; R”’ = COCH3 serl.97tyr0.9lbut0.94glu1.0~his1.~~p he~.98argz.91gly1.891YS3.31- Ie (Neamine): R = Neosamine C; R’ = R” = R”’ = H pr02.~~vall.8~). The observation that leucine aminopeptiR = Neosamine C; R’ = Neobiosamine C ; If (Neomycin C ) : RO = R”’ = H dase digests of I contain glutamic acid and not glutaminez3demonstrates that the glutamine amide group is R = Neosamine C ; R’ = Neobiosamine B; Ig (Neomycin B): R ” = R”’ = H susceptible to hydrolysis by trifluoroacetic acid. Schwyzer, et al.,2 4 exposed N-t-butyloxycarbonylseryltyrosylIh (Paromomycin): R = n-Glucosamine; R’ = Neobiosamine B; R” = R//’ = H serylmethionylglutaminylhistidylphenylalanylarginylIi(Paromomycin 11): R = D-Glucosamine; R’ = Xeobiotryptophylglycyl - NE- t - butyloxycarbonyllysylprolylsamine C; R” = R”‘ = H (suggested) valylglycyl - Ne- t - butyloxycarbon yllysyl-Nf-t-butyloxyR = D-Glucosamine; R = R” = R”’ = H I j (Paromamine): carbonyllysylarginylarginylproline-t-butylester to the Ik (Kanamycin C): R = D-Glucosamine; R’ = R”’ = H ; action of trifluoroacetic acid and stated, without experiR ” = Kanosamine mental support, that this treatment did not remove the I1 (Kanamycin A ) : R = 6-Aminoglucose; R‘ = R”’ = H ; amide group from the glutamine residue. Our own reR” = Kanosamine sults indicate that the glutamine amide group in seIm (Kanamycin B ) : R = Diaminohexose; R’ = R”’ = H ; quences related to the N-terminus of the corticotropins R ” = Kanosamine undergoes significant hydrolysis on exposure to trifluoroThe work of Reeves5 provides a tool for the determiacetic acid. nation of the stereochemical relationship between the Acknowledgment.-The skillful technical assistance remaining adjacent hydroxyl groups of Ib, by measuring of Mrs. Chizuko Yanaihara, Mrs. Maria Gunther, the change in optical rotation, A [ M l c u p r a B, when Miss Priscilla Holland and Mr. John Humes is gratecuprammonium hydroxide solution (“Cupra B is fully acknowledged. substituted for water as the solvent. The above compound (Ib) has +9.8’ (c 0.6, (22) E. A. Peterson and H. A. Sober, J. A m . Chem. Soc., 1 8 , 751 (1956). H20) and [a]% +620° (c 0.47, Cupra B), giving (23) The enzyme preparation used in these studies fails t o show glutaminase activity. A [ M ] ~ u p r aB = +1590. The high positive increment (24) R. Schwyzer, W.Rittel, H . Kappeler and B. Iselin, A n g c v . Chem., is similar to that observed with methyl 2-O-methyl-P72, 915 (1960). D-glucoside, I1 ( A [ M ] ~ u p r a B = +2190) but of OPBIOCHEMISTRY DEPARTMENT KLAUSHOFMANN posite sign to that of methyl 4-O-methyl-P-~-g~ucoside, UNIVERSITY OF PITTSBURGH ROBERTD. WELLS I11 (A[hl]cupraB = -2020).6 Thus, the adjacent SCHOOL OP MEDICINE HARUAKI YAJIMA hydroxyl groups in 6-0-methyldeoxystreptamine are PITTSBURGH, PENNSYLVANIA JOACHIM ROSENTHALER related as those a t C-3 and C-4 of the glucopyranose RECEIVED MARCH7 , 1963 ring,7 i.e., 6-0-methyldeoxystreptamine has structure Ib, rather than that of its mirror image IC. CHEMISTRY OF THE NEOMYCINS. XII’ THE H ’I)
ABSOLUTE CONFIGURATION OF DEOXYSTREPTAMINE IN THE NEOMYCINS, PAROMOMYCINS AND KANAMYCINS
NHAc
H N O H -
Sir:
Ho
The final uncertainty in the stereochemistry‘ of neomycin C, the absolute configuration of the unsymmetrically substituted deoxystreptamine portion of the antibiotic, now has been resolved. N,N’-Diacetyl-6-O-methyldeoxystreptamine (Ib),2-4 m.p. 280-284’ dec., [ a I z 3+5.0° ~ (c 0.64, water), was prepared from poly-0-methyl-hexa-N-acetyl-neomycin B. This compound has been isolated previously from neomycin B (reported m.p. 282-284’dec., [cY]’~D+12°),2 frOm paromomycin (reported m.p. 280-282’, [aIz7o +15°),3a and from zygomycins A1 and Az (reported map. 280-283’, [ r u I z 4 ~ +4°),3b which from their properties and degradation products are probably (1) Paper XI in this series: K . L. Rinehart, Jr., W. S. Chilton, M. Hichens and W. v. Phillipsborn, J. A m . Chem. Soc., 84,3216 (1962). (2) K. L. Rinehart, Jr., M. Hichens, A. D. Argoudelis, W. S. Chilton, H. E. Carter, M. Georgiadis, C. P. Schaffner and R . T. Schillings, ibid., 84, 3218
(1962). (3) (a) T. H. Haskell, J. C. French and (2. R . Bartz, ibid., 81,3483 (1959); (b) S.Horii, J . Anfibiofics(Tokyo), Ser. A , 15, 187 (1962). (4) The numbering shown is that defined recently2 for deoxystreptamine in neamine in order t o place neosamine C on the lower numbered carbon. I t is seen in the present report t o have the additional advantage that it gives the lower number t o the hydroxyl-bearing carbon (C-4) of absolute stereochemistry R.
Ac HO
CHsO Ib
H
k
I1
H
H
I11
(5) R. E. Reeves, Advan. Carbohydrafe Chem., 6 , 107 (1951). (6)
A[M1cupraB
=
([alia6
Cupra B
- [ a I 4 s s water)
mol. wt. X
-.
100 (7) The possibility of unexpected interference by the acetamido groups in the cuprammonium complex is eliminated by the observation that crystalline N,N,-diacetyl-5,6-di-O-methyldeoxystreptamine (Id, sublimes above 260’ without melting; A n a l . Found: C, 52.70; H, 8.24; N, 10.68; OCHa, 22.6), prepared from poly-0-methyl-tetra-N-acetylneamine, exhibited no enhanced rotation in Cupra B solution.
