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COMRTUNICATIONS TO THE
EDITOR
VOl. 78
sure to a solid mass. Fractional crystallization from dry was dissolved in chloroform. The solution was washed with methanol separated clivonine hydrochloride, m.p. 263-275" dilute sodium bicarbonate solution and water. Concentradec., from the sodium chloride. The last traces of clivonine tion of the chloroform solution gave an oil that crystallized were obtained by dissolving all residues containing alkaloidal on trituration with ethyl acetate. Recrystallization from material in water, neutralizing the solution with sodium bi- ethyl acetate gave 87 mg. of colorless needles, m.p. 194carbonate solution and extracting the alkaloid with chloro- 196". The acetate showed basic properties. form-ethanol (4: 1). Concentration of this extract and Anal. Calcd. for CigHziXOe: C, 63.50; H, 5.89; N, chromatography on alumina gave crude clivonine, m.p. 3.90. Found: C, 63.74; H , 5.80; N, 3.81. 190-195". By this procedure, a total of 2.00 g. of clivonine Tetrahydroc1ivonine.-Reduction of 313 mg. of clivonine was obtained. A pure sample of clivonine was obtained by recrystallization from ethyl acetate, m.p. 199-200°, [ C Y ] 23089 in tetrahydrofuran with an excess of lithium aluminum hydride gave 261 mg. of a colorless oil that was chromato+41.24', [ o 1 I z 3 p 3 e +103.44' (c 1.11, chloroform). graphed on 30 g. of aluminum oxide (Merck). Elution with Anal. Calcd. for Cl;H19NOI: C, 64.34; H, 6.04; N, chloroform removed 60 mg. of unreacted starting material. 4.41; NCHa, 4.73. Found: C, 64.39; H , 5.95; X, 4.41; Elution with 5% ethanolic chloroform gave 200 mg. of colorNCH3,2.26. No methoxyl was present. less oil which could not be crystallized. The ultraviolet The ultraviolet absorption spectrum showed maxima a t spectrum of this oil showed maxima at 238 mp (log E 3.58) 226 mp (log e 4.33), 268 mp (log B 3.77) and 308 mp (log e and 292 mp (log e 3.51). A vicinal glycol determinationlo 3.76). on 68.4 mg. of this oil indicated that 8l.6yOof the material Clivonine Hydrochloride.-The crude hydrochloride was had a vicinal glycol structure. recrystallized from methanol-ether (which contained a Niva1ine.-The bulbs of Galanthus nivalis L. were purdrop of water) as colorless prisms, m.p. 282-287" dec. chased from Kipiteijti and Sons, Hillegon, The Xetherlands. Anal. Calcd. for C17HlgN06,HCl: C, 57.81; H , 5.70; By the usual extraction method, 9800 g. of fresh bulbs gave 25.6 g. (0.26%) of crude alkaloids. Ethanolic trituration r\',3.96. Found: C, 57.67; H,5.74; N,3.91. The ultraviolet absorption spectrum showed maxima a t of this material gave 1.76 g. (0.01870) of lycorine, m.p. 226 mp (log e 4.35), 268 mp (log e 3.67) and 308 mM (log e 250-255' dec. The filtrate was concentrated under reduced pressure to remove the ethanol. The benzene-soluble por3.75). tion of the residue was chromatographed on aluminum oxide Clivonine Picrate.-An ethanolic solution of 75 mg. of clivonine was treated with saturated aqueous picric acid un- (Merck) to give 4.01 g. (0.04%) of tazettine, m.p. 200-205'. til the precipitation of picrate ceased. The resulting solid The oils (0.776 9.) eluted prior to the tazettine were rewas recrystallized from aqueous ethanol to give a quantita- chromatographed. Elution with 5% ethyl acetate in benzene gave 92 mg. of crude nivaline, m.p. 121-125', which tive yield of derivative, m.p. 250-254" dec. was recrystallized from ethanol to give 73 mg. (0.000770) of Anal. Calcd. for C17H1gN05.C~H3N30;: C, 50.55; H , pure nivaline, m.p. 131.5-132.5", [aIz4D +268" (C 1.0, 4.06; N, 10.25. Found: C, 50.55; H , 4.07; N, 10.19. ethanol). Clivonine Methopicrate.-A methanolic solution of 129 Anal. Calcd. for ClsHlgNOa: C, 65.64; H , 5.82; N, mg. of clivonine was refluxed overnight with excess methyl iodide. The neutral gum obtained on concentration was 4.25; OCH3, 9.42; NCHa, 4.56. Found: C, 65.88; H , triturated with ethanol-ether to give a very hygroscopic 5.84; N, 4.36; OCHa, 9.34; NCHa, 3.30. solid. The material was converted to the methopicrate in The ultraviolet absorption spectrum showed maxima at the usual manner and recrystallized from aqueous ethanol, 230 mp (log 4.43), 269 mp (log e 3.75) and 308 mp (log e 111 mg., m.p. 285' dec. 3.77). N ~ O ~ : A similar processing of 26,900 g. of Hymenocallis occidenAnal. Calcd. for C I ~ H ~ ~ ~ T O ~ . C H B . C BC,H ~51.43; H,4.32; N, 10.00. Found: C, 51.66; H,4.88; N,9.89. lalis (le Conte) Kunth gave lycorine (0.007%), tazettine Attempted Hydrogenation of C1ivonine.-Clivonine ab- (0.0370) and nivaline (0.00027,). Dihydroniva1ine.-A solution of 48 mg. of nivaline in 20 sorbed no hydrogen and was recovered unchanged from atml. of ethanol absorbed 3.05 ml. of hydrogen when reduced mospheric hydrogenation conditions employing either palladium-on-charcoal (10%) or platinum oxide catalysts. a t atmospheric pressure and room temperature with 40 mg. 0-Acetylc1ivonine.-A solution of 110 mg. of clivonine in of l o YO palladium-on-charcoal catalyst. The dihydro de2 ml. of acetic anhydride containing twenty drops of pyridine rivative was an oil. was allowed to stand one day a t room temperature and then BETHESDA, MARYLAND concentrated under reduced pressure to a colorless gum that
COMMUNICATIONS T O T H E EDITOK REDUCED TRIPHOSPHOPYRIDINENUCLEOTIDE REQUIREMENT FOR THE ENZYMATIC FORMATION OF 3-HYDROXYKYNURENINE FROM L-KYNURENINE Sir :
Two recent reviews on the metabolism of tryptophan's2 have stressed the lack of information concerning the formation of 3-hydroxykynurenine from kynurenine. I n an effort to account for the absence of the usual tryptophan metabolites from cat urine3 certain in vitro studies were carried out. This letter describes a cell-free system occurring in (1) C. E. Dalgliesh in Advances in Protein Chemistry, 10, 86 (1955). (2) A. H. Mehler in "Amino Acid Metabolism," edited by W. D. McElroy and H. B. Glass, T h e Johns Hopkins Press, Baltimore, hfd , 1955, p. 898. (3) R. R . Brown and J , M. Price, J . B i d . Chcm., P19, 985 (1956).
liver mitochondria of cats and rats which forms 3hydroxykynurenine from L-kynurenine. The disappearance of substrates and the appearance of the metabolites were followed by ascending paper chromatography on Whatman No. 1 filter paper. The spots were identified by comparison with authentic compounds according to the criteria in Table I. Washed mitochondria equivalent to 200 to 400 mg. wet weight of liver was incubated aerobically a t 37' for two hours in 0.05 M phosphate buffer a t pH 7.4 containing 5.0 pmoles of citrate, 0.67 pmole of triphosphopyridinenucleotide (TPN), 33.0 p moles of nicotinamide and 2.5 pmoles of L-kynurenine. The final volume was 1.5 i d . I t was not necessary to deproteinize before spotting the
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 THE 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
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