COMMUNICATIONS TO THE EDITOR
June 20, 1956 TABLE I"
Compound
Rp in MeOH BuOH, Bz, H i 0 2 : 1 : 1:1 con- UltraEktaining violet man's 1% 1% 15 N duoreAcOH NHlOH rescence DSAb agentb
0.48 0 . 5 0 GB M L-Kynurenine .37 .48 GB M D-Kynurenine 3-Hydroxy-~-kynurenine .38 .25e GY R Y' 3-Hydroxy-D-kynurenine .30 .25e CY R Y ' .60 .60 Vc Kynurenic acid .60 .1S6 LBd R Xanthurenic acid .92 .60 BV M Anthranilic acid .78 .62 GB M NQ-Acetyl-L-kynurenine NU-Acetyl-D-kynurenine .78 .62 GB M "Abbreviations: MeOH, methanol; BuOH, n-butanol; Bz, benzene; AcOH, acetic acid; DSA, diazotized sulfanilic acid; G, green; B, blue; Y , yellow; V, violet; L, light; R, red; M, magenta. * These reagents were prepared and used according to C. E. Dalgliesh, Biochem. J., 52, 3 (1952). Changes to light blue after ultraviolet irradiation. Changes to yellow with ammonia vapor. 'RF is variable in the presence of salts. f Becomes yellow with nitrous acid.
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kynurenine since D-kynurenine, NQ-acetyl-L-kynurenine, Na-acetyl-D-kynurenine, kynurenic acid, and anthranilic acid were not attacked. This is in contrast to the non-enzymatic systems described by Udenfriend, Clark, Axelrod and Brodie4 and by Dalgliesh.6 The enzyme system resembles the one which oxidizes non-aromatic rings in certain Acknowledgments.-We are indebted t o Dr. L. M. Henderson, Division of Biochemistry, Noyes Laboratory of Chemistry, University of Illinois, Urbana, Illinois, for a gift of 3 - h y d r o x y - ~ ~ kynurenine, and to Drs. G. C. Mueller and G. Rumney of the McArdle Memorial Laboratory for gifts of reduced triphosphopyridinenucleotide. This work was supported in part by the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council. (4) S. Udenfriend, C. T . Clark, J. Axelrod and B. B. Brodie. J . B i d . Chem.. 206, 731 (1954). (5) C. E. Dalgliesh, Biochcm. J . , 66, xlv (1954). 77, 5185 (6) M . L. Sweat and M. D . Lipscomb, THISJOURNAL, (1955). (7) A. C. Brownie and J. K . Grant, Biockcm. J . , 62, 29 (1956). (8) Fellow of the National Research Council of Brazil.
incubation mixture (20 pliters) on the paper. In the complete system, much of the L-kynurenine disappeared and spots corresponding to 3CANCER RESEARCH HOSPITAL hydroxy-L-kynurenine, kynurenic acid and xanth- UNIVERSITY F. T. DE CASTRO* HOSPITALS urenic acid appeared. No anthranilic acid nor UNIVERSITY J. M. PRICE OF WISCONSIN R. R. BROWN 6-hydroxykynurenic acid was observed. I n the MADISON,WISCONSIN RECEIVED MARCH22, 1956 absence of either T P N or citrate, 3-hydroxykynurenine was not readily detectable. Reduced T P N (4.0 pmoles per flask) was just as effective as T P N plus citrate in stimulating 3-hydroxykynu- STUDIES ON T H E SYNTHESIS OF LYSINE-VASOrenine formation. Succinate, fumarate or malate PRESSIN was not as effective as citrate in supporting hy- Sir : droxylation. Reduced diphosphopyridinenucleotide du Vigneaud, Lawler and Popenoe' proposed (DPNH) or DPNH plus T P N was not as good as Structure I for lysine-vasopressin, the hormone T P N H . Anaerobic conditions or boiling the mitoisolated from hog posterior pituitary glands. A chondria inhibited the reaction completely. Ion exchange chromatography was used t o isoCsH40H Cab I I late the reaction products in spectroscopically pure NH, 0 CH2 0 CHz form. From an incubation of 7.5 pmoles of LI kynurenine with cat liver mitochondria in the com- CHz-CH-C-h' I H-/I H-C-h-H-CH plete system, 4.26 pmoles of 3-hydroxykynurenine, I I c=o 0.11 prnole of kynurenic acid, and 0.11 pmole of S I xanthurenic acid were produced and 2.95 pmoles SI 0 0 NH of unreacted kynurenine remained. These reac- I I1 I1 I tion products chromatographed on ion exchange CH2-yH-IiH-C-CH-NH-C-CH-( I CH2) n-COPu'H, I resins exactly like the corresponding authentic CHz compounds. From pooled incubation mixtures c=o I containing an estimated 14 mg. of 3-hydroxyCONHz kynurenine, 6.7 mg. of 3-hydroxykynurenine was CH2-L 0 0 obtained as beautiful yellow needles. The ultra\ /I II CH-C-XH-CH-C-NH-CHn-CONHn violet spectrum of this material in 0.1 M phosphate I buffers revealed peaks a t 223, 260 and 369 mp a t CHz-CHz CHzCHzCHzCHzNH2 pH 2.0; a t 228,267 and 368 mp a t PH 7.4; and 248, I 286 and 393 mp a t pH 12.0, corresponding exactly synthesis of biologically active material according to to the spectrum of synthetic 3-hydroxy-~~-kynuthis structure was announced by du Vigneaud, renine. The spectra of the isolated kynurenic acid and xanthurenic acid were also identical with spec- Popenoe and Roeske (footnote, ref. 1).2 Synthesis (1) V. du Vigneaud, H. C.Lawler and E. A. Popenoe, THIS JOURtra of authentic compounds a t each of these pH NAL, 76, 4880 (1953). values. (2) This synthetic approach paralleled the method employed for the The extent to which the added kynurenine was synthesis of oxytocin [V. d u Vigaeaud, C. Ressler, J. M . Swan, C. W. accounted for and the absence of detectable quan- Roberts and P. G. Katsoyannis, i b i d . , 76, 3115 (1954)1, with e-tosyltities of 6-hydroxykynurenic acid suggests that lysine (e-p-toluenesulfonyllysine) replacing leucine and phenylalanine isoleucine in the series of reactions. However, the yield of little if any hydroxylation occurred a t the 5-posi- replacing pressor-antidiuretic material was very low. Confirmation of these tion of kynurenine. results has now been obtained and this method of synthesis has given The hydroxylation system was specific for L- biologically active material in somewhat higher yield.
L
I
I /
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COMMUNICATIONS TO THE EDITOR
Vol. 78
of I was also attempted by the coupling of S-benzylThe N-tosyl and S-benzyl groups were removed N - carbobenzoxy - L - cysteinyl -L- tyrosyl -L- phenyl- from I11 (300 mg.) by reduction with sodium in alanyl-~-glutaminyl-~-asparagine~ with S-benzyl-L- liquid ammonia and the resulting product was cysteinyl-L-prolyl-etosyl-L-lysylglycinamide (11) oxidized to the disulfide form by aeration in aqueous according t o the pyrophosphite p r o c e d ~ r e ,fol~ solution a t pH 6.4. The yield of pressor activity" lowed by removal of the protecting groups from ranged in several experiments from 50,000 to the resulting nonapeptide derivative and subse- 70,000 units (per 300 mg. of 111). After concenquent oxidation to the disulfide form. This ap- tration and lyophilization, the product was puriproach yielded a synthetic product assaying ap- fied by countercurrent distribution between secproximately 100 pressor units/mg. after purification butyl alcohol and 0.08 M p-toluenesulfonic acid by electrophoresis a t pH 5.6 in pyridine acetate and followed by electrophoresis in pyridine acetate countercurrent distribution between sec-butyl al- buffer (pH 5.6) on a cellulose-supporting mecohol and 0.08 M p-toluenesulfonic acid. d i ~ m . ' ~ ,The ' ~ purified material had a pressor In addition to these approaches, another method activity of 250-290 units/mg.'* when assayed was utilized which has led to material which is as as usual against the U.S.P. Standard P ~ w d e r . ' ~ biologically active as the most potent preparation The ratios between pressor, antidiuretic, milkof lysine-vasopressin obtained from natural sources. ejecting and avian depressor activities for the synThe key intermediate in this synthesis is S-benzyl- thetic material are the same as those for natural N - tosyl-L-cysteinyl-L-tyrosyl-L-phenylalanyl-L-glu-lysine-vasopressin.12t14r16 Starch column chromataminyl-L-asparaginyl-S-benzyl -L - cysteinyl - L - protography" of a hydrolysate of this material showed lyl-e-tosyl-L-lysylglycinamide (111), which has solu- the eight amino acids to be present in molar ratios bility properties favorable for its purification. to each other of approximately 1 : 1 and ammonia I11 was synthesized by the coupling of S-benzyl- in a ratio to any one amino acid of approximately N-tosyl-L-cysteinyl-L-tyrosyl-L-phenylalanyl-L-glu-3 :l. The natural hormone and the synthetic prodtaminyl-L-asparagine (IV) with the tetrapeptide uct showed the same infrared spectrum.'* They amide 11. For the synthesis of 11, ethyl L-prolyl- showed the same behavior on countercurrent disc-tosyl-L-lysylglycinate,6 m.p. 81-84', was coupled tribution between sec-butyl alcohol and 0.08 :tlp with S-benzyl-N-carbobenzoxy-L-cysteine6 by the toluenesulfonic acid and also had the same electroN,N'-dicyclohexylcarbodiimide method? and the phoretic mobility on Whatman No. 1 paper in resulting protected tetrapeptide ester was con- pyridine acetate buffer a t pH 5.6 and 4.0. verted t o the corresponding crystalline amide with (11) J. Dekanski, B r i f . J. Pharmacal., 7 , 567 (1952). ammonia in ethanol; m.p. 101-104°, [ c Y ] ~ ' D-29.3' (12) D. N. Ward and V. d u Vigneaud, J . B i d . Chcm., in press. (13) H. G. Kunkel in "Methods of Biochemical Analysis," 1'01. 1, (c I, CHC13) (calcd. for C 3 8 H 4 8 0 s N & : C, 58.5; D. Glick, Ed., Interscience Publishers, Inc., New York, p . 141. H, 6.19; N, 10.8; S, 8.21. Found: C, 58.3; (14) A sample of natural lysine-vasopressin with approximately the H, 6.34; N, 10.6; S, 8.10); the carbobenzoxy same pressor activity as the synthetic material was kindly supplied group was then removed with H B ~ - H O A C . ~by Dr. Albert Light of this laboratory. This material was obtained IV was prepared by the coupling of L-phenyl- by purification by countercurrent distribution followed by chromatogalanyl-L-glutaminyl-L-asparagineswith S-benzyl- raphy on Amberlite IRC-50. (15) "The Pharmacopeia of the United States of America," fifteenth N-tosyl-~-cysteinyl-~-tyrosine~ by the isobutyl revision, black Printing Co., Easton, Pa., 1955, p. 776. These assay chlorocarbonate mixed anhydride procedurelo in a values are based on an activity of 0.4 U.S.P. Posterior Pituitary Unit/ mg for the Standard Powder. yield of 55% after recrystallization; m.p. 203-204', (16) For the ratios between these four activities in natural lysine[ a I z 1 D +4.4' (c 2.08, dimethylformamide) (calcd. see H. B. van Dyke, s. L. Engel and K. Adamsons, Jr., C, 57.0; H, 5.65; N, vasopressin, for C44HS1N7011S2-1/2H20: Proc. Soc. Expri. B i d . M c d . , 91, 484 (1956). T h e ratios of antidiuretic 10.6. Found: C, 57.1; H, 5.66; N, 10.6). and milk-ejecting activities to pressor activity for the synthetic maCompounds IV and I1 were coupled by the N,N'- terial were determined by Professor H. B. van Dyke and Mr. s. L. of the College of Physicians and Surgeons. dicyclohexylcarbodiimide method? to give a 39% Engel (17) S. Moore and W. H. Stein, J . B i d . Chem., 178, 53 (1949). yield of analytically pure 111, m.p. 226-230°, [ci]"D (18) The authors wish to thank Dr. Julian R. Rachele of this laboratory for determination of the infrared spectra. -23.0' (c 2.11, dimethylformamide) (calcd. for (274(19) This work was supported in part by grants from the Kational H9i0i6Ni3S4.H20: C, 56.8; H, 5.99; N, 11.6; HzO, Heart Institute, Public Health Service, Grant H-1675, and Lederle 1.15. Found: C,56.7; H,6.02; N, 11.6; HaO, 1.02). Laboratories Division, American Cyanamid Co. (3) V. d u Vigneaud, D. T. Gish and P. G. Katsoyannis, ibid., 76, 4751 (1954). (4) G. W. Anderson, J. Blodinger and A. D. Welcher, ibid., 74, 5309 (1952). (5) This tripeptide ester was prepared from a-carbobenzoxy-r-tosylL-lysine, m.p. 85-88', and ethyl glycinate, removal of the carbobenzoxy group, and coupling of the product with carbobenzoxyL-proline by the o-phenylene chlorophosphite procedure [G. W. Anderson and R. W. Young, THISJOURNAL, 74, 5307 (1952) I followed by removal of the carbobenzoxy group with HBr-HOAc. (6) C. R. Harington and T. H. Mead, Biochem. J . , 8 0 , 1598 (1936). 77, 1067 (1955). (7) J. C. Sheehan and G. P. Hess, THIS JOURNAL, (8) E. A. Popenoe and V. d u Vigneaud, i b i d . , 76, 6202 (1954). (9) This compound was prepared independently b y a method similar to t h a t recently published by J. Honzl and J. Rudinger [Collection Czachoslou. Chem. Communs., 20, 1190 (1955) I They also reported the synthesis of a product of high oxytocic activity from a nonapeptide derivative containing a tosylcysteine residue. (10) J. R . Vaughan, Jr., and J. A. Eichler. THIS JOURNAL, 75, 5556 (1963).
M. FREDERICK BARTLETT ALBERTJOHL ROGER ROESKE ~ E P A R T M R N TOF BIOCHEMISTRY CORNELL UNIVERSITYMEDICALCOLLEGE R. J. STEDMAN F. H. C. STEWART h-EW Y O R K , Tu'. 'ET. DARRELL N. WARD \'INCENT
DU \'IGNEAUD''
RECEIVED MAY18, 1956 THE PREPARATION O F ACID-STABILIZED SUBHALIDES FROM MOLTEN METAL-METAL HALIDE SOLUTIONS' Sir :
The formation of slightly stable subhalides by the solution of certain metals in their fused halides (1) Work was performed in the -4mes Laboratory of the Atomic Energy Commission.
