VINCA ALKALOIDS. III.1 CHARACTERIZATION OF LEUROSINE AND

CHARACTERIZATION OF LEUROSINE AND VINCALEUKOBLASTINE, NEW ALKALOIDS FROM VINCA ROSEA LINN. N. Neuss, M. Gorman, G. H. Svoboda, ...
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this technique is superior to established assay methods. The carbon-14 form of the amino acid whose activation is being studied is incubated with adenosine triphosphate, hydroxylamine and the enzyme preparation in a total volume of as little as 100 pl. Aliquots are removed a t appropriate time intervals, heated briefly to destroy the enzyme and evaporated onto a line one inch from the end of a 3 / / 4 by 5 inch strip of Aniberlite IR-120 ion exchange paper (sulfonic acid resin Na+ form, courtesy of Rohm and Haas). A sodium phosphate buffer (pH 7.0, 0.05 -14)is allowed to rise by capillarity through the strip which is then dried. Under these conditions all the free unreacted neutral amino acids move with the solvent front and the hydroxamates of the neutral amino acids remain a t the origin. (Other conditions permit the separation of the acidic or basic amino acids from their hydroxamates.) Comparison of the radioactivity a t the two sites indicates the fraction converted and hence the rate of activation. Although the paper absorbs some of the radiation, the use of high activity L-amino acids (20 rncuries/ mmole) and a thin window Geiger counter (Nuclear Chicago D-47, 45% efficiency) permits us to recognize mole of hydroxamate formation in the aliquot. X typical experiment is given:

Vol. 81

VINCA ALKALOIDS. 111.1 CHARACTERIZATION OF LEUROSINE AND VINCALEUKOBLASTINE, NEW ALKALOIDS FROM V I N C A ROSEA LIATAV. Sir :

Leurosine,2 a new alkaloid fror.1 Vinca rosea Linn., was described recently, but no empirical

formula was assigned. Independently Noble, Beer and Cutts have reported the physical and biological properties of another new alkaloid, vincaleukoblastine. In view of the unusual properties of these two alkaloid^,^^^^^ we wish to present the analytical and physical data which led to the establishment of empirical formulas for vincaleukoblastine and leurosine and indicate their close structural relationship. Vincaleukoblastine sulfate5 melted a t 281->85', [CY]% -28' (CHaOH). Calcd. for C46&809h74' HzS04.HzO: C, 59.59; H, 6.74; 0, 24.16; N , 6.04; S,3.46. Found: C,59.68; H,6.72; 0 , 2 4 2 7 ; N, 6.19; S , 3.37. The free base, recrystallized from ether, formed a stable etherate, loss of solvent a t 180-182', m.p. 201-211', [cY]*~D+42' (CHCl,). Calcd. for C46H5809r\r4.(C2H5)20: C , 67.85; H, 7.74; 0, 18.09; N, 6.32; mol. wt., 885. Found: C, 67.89, 67.93; H, 7.63, 7.76; 0, 18.08; N, 6.38, 6.43; mol. wt., 887.8 (X-ray data). Ether of solvation was demonstrated by vapor phase chromatography and a band a t 8.4 p in the inINITIALRATES (M~MOLES/ML./HR.) (2-14 HYDROXAMATE frared disappearing on evaporation of a chloroform FORMATIOX solution of the etherate. The base from methanol +11 m h f . melted a t 211-216', calcd. for C46H5809K4'2CH:3r' no C-12 +11 m M . + I 1 miM. alloisoamino acid valine isoleucine leucine OH.H20: C, 64.55; H, 7.68; N, 6.27; mol. w t . , Valine-C-14, mhl. 894. Found: C, 64.11; H, 7.49; X, 6.36; mol. 0.15 96 46 44 a t . , 889 =k 5 (electrometric titration, HzO; pk", 1 0 94 5.4, 7.4). =Ifter drying a t 180' (1 min.), calcd. for C46H58OgN4: C, 68.12; H, 7.21; 0,17.75; Isoleucine-C-14, mM. N, 6.90; weight loss, 9.19. Found: C, 68.15; 0.15 85 34 30 H, 7.44; 0, 18.05; ?;, 6.65; weight loss, 8.81. 1.0 82 Vincaleukoblastine formed a dihydrochloride dihyAiloisoleucine-C-14, mnif drate, m.p. 244-246' (dec.). Calcd. for C460.15 1 2 no 0 3 H5809N4.2HCl.2H20: C, 60.06; H, 7.01; 0, 1.0 9 0 19.13; N, 6.09; C1, 7.71. Found: C, 60.36, The incubation mixtures contained in addition to 39.95; H , 7.24, 7.18; 0, 19.01; N , 5.94;C1, 7.37. Leurosine? was recrystallized from acetonitrile, the amino acids, 10 mil. adenosine triphosphate, 12 mM. Mg++, 25 mil. KC1, 50 mM. tris-(h>droxy- n1.p. 202-203' (dec.) (loss of solvent a t 172-175'), +72' (CHC13). Calcd. for Cd6HjgOgS4. methyl)-aminomethane, 0.2 Jf sucrose and 2.0 Jf hydroxylamine. The volume was 0.23 ml., the pH 8I-120: mol. wt., 955.09. Found: mol. wt., 955.3 7.4, and the temperature 25'. Eachincubation flask i. lT0 (X-ray data); 032 i 10 (electrometric contained 0.05 ml. of a dilute extract of alumina titration, pK', 5 . 5 and 7.5 in water). ,5fter drying ground E . coli. Aliquots of 23 p1. were removed for a t 130' in vacua, weight loss calcd. : 15.0!1. Found: analysis a t zero, 30, 60 and 120 minutes. The rate 15.60. Calcd. for C46HSSOgN4: C, G8.12; H , of hydroxamate formation was linear except when 7.211; 0, 17.75; N, R.91. Found: C, G8.11. 07.88; H, i.30, 7.45; 0,17.34, lS.0t5; S , 7.10, the substrate was approaching exhaustion. Similar assays can be devised for any system 6.93. The sulfate froni ethanol, [alf611 -X:< in which the starting material and product can (CHaOH), m.p. 238-233' (dec.),was dried a t 1:3(1° C , 6!).77; be caused to differ markedly in charge; e g . , the in Z ~ Q C L I O . Calcd. for C1GH5SOgS,i.H2SOI: (1) Vinca Alkaloids 11, .\I. Gorman e t al , J . . 4 ~ P, h n m as so^ conversion of glucose iiito glucose phosphate, of acetate into acetohydroxamate, or the pyrophos) G. H . Svohoda, J . A m . P h n v n i . Assoc. .Sct. E d . , 47, 834 (1959). phate exchange into adenosine triphosphate (3) R . L. S o b l c , C. T . Beer and J . H. C u t t s , A n x . \- E' A r a d . .S