3202
TO THE EDITOR COMMUNICATIONS
ZYGADENUS ALKALOIDS. II. THE OCCURRENCE OF HYPOTENSIVE GERMINE ESTERS IN ZYGADENUS VENENOSUS
Vol. 74
acid determination, 17.52 mg. of neogermidine was equivalent to 5.77 ml. of 0.009126 N sodium thiosulfate; calcd. for germine monoacetate mono-aSir : methylbutyrate, 6.05 ml. Alkaline hydrolysis of A recent study' of the alkaloidal constituents of neogermidine afforded germine, acetic acid and aZygadenus venenosus revealed the occurrence in the methylbutyric acid. The acids were identified by plant of the esters veratroylzygadenine and vanil- conversion to their p-phenylphenacyl esters which loylzygadenine as well as the alkamines zygadenine were characterized after chromatographic separaand germine. We wish to report now the isolation tion. Methanolysis of neogermidine afforded profrom a batch of this plant collected in June, 1951 of toveratridine. The large change in rotation atthe germine esters neogermitrineJ2ge~midine,~ pro- tending the methanolysis of neogermidine to prot~veratridine,~ and a new diester isomeric with toveratridine suggests that the site of attachment of germidine, for which we propose the name neoger- the acetyl group on the alkamine germine is the midine. same as that of the labile acetyl group in both neoFractionation of the chloroform-extractable al- gerrnitrine and germitrine. kaloids of Zygadenus venenosus (WATS.)5 by 8Acetylation of neogermidine with acetic anhyplate countercurrent distribution using benzene and dride and pyridine yielded acetylneogermitrine, phosphate buffer at pH 7.1 yielded, in addition to 1n.p. 248-249' dec., identical with a sample prethe alkaloids obtained previously, the germine pared by acetylation of germidine.2 Acetylation monoester protoveratridine (from the 0-plate frac- of protoveratridine under the same conditions also tion), Protoveratridine crystallized as rectangular gave acetylneogermitrine. These facts show that plates upon reprecipitation from alcoholic acetic the site of attachment of the a-methylbutyryl acid solution with aqueous ammonia; m.p. 272- group is the same in each of the four germine esters 273' dec.; [a]2 2 -9' ~ (c 0.76, pyr.). Anal. Calcd. isolated from Zygadenus venenosus. C32H510BN: C, 64.73; H, 8.66; N, 2.36, Found: Pharmacological experiments carried out with C, 64.79; H, 8.62; N, 2.62. neogermidine a t the laboratory of Professor 0. The filtrates after removal of the crystalline al- Krayer a t Harvard Medical School indicate that kaloids from the plate-8 fraction were combined the circulatory action in the cat and the veratrinic with the material from plates 4 to 7, and the amor- effect on the frog muscle are similar to those of phous mixture obtained upon lyophilization was germidine. designated the "organophilic" fraction. The filThis work was supported (in part) by grants trates after removal of the crystalline alkaloids from the National Institutes of Health and Eli from the plate-0 fraction were combined with the Lilly and Company. The assistance of Eli Lilly material from plates 1 to 3, and the residue obtained and Company in gathering and extracting Zygadeupon lyophilization was designated the "hydro- nus venenosus'is gratefully 'acknowledged. philic" fraction. OF CHEMISTRY Neogermitrine and germidine were obtained by DEPARTMENT HARVARD UNIVERSITY S. MORRIS KUPCHAN 24-plate countercurrent distribution of the organo- CAMRRIDCE 38, MASSACHUSETTS C. V. DELIWALA philic fraction using benzene and 2M acetate bufRECEIVED MAY22, 1952 fer a t pH 5.5.2 The identity of these substances was confirmed by mixed melting point and infrared spectral comparisons with authentic specimens A NEW DISACCHARIDE PRODUCED BY from Veratrum viride kindly provided by Dr. J. LEUCONOSTOC MESENTEROIDES Fried. Sir : The hydrophilic fraction was subjected to 8-plate In studies on the enzymatic synthesis of dextran countercurrent distribution using chloroform and 2M acetate buffer a t pH 5.5. Neogermidine was from sucrose by Leuconostoc mesenteroides we have obtained by chromatography on alumina of the ma- noted that under certain conditions as much as 370 terial recovered from plates 5 to 8. Neogermidine of the sucrose is diverted to the production of a new disaccharide which has the following properties: crystallized from benzene as heavy prisms; m.p. Crystallizes in the form of bars from methanol221-223' dec.; [ C ~ ] ~-60' ~ D (C 2.00, PYr.); [ L Y ] ~ ~ D ethyl acetate; m.p. 161-163'; [ C Y ] * ~-8.8' D after -25' (c 2.00, chf.). Anal. Calcd. CS~HBOION:3 minutes, -6.8' after 24 hours (c, 4; H20); analC, (34.22; H, 8.38. Found: C, 64.15; H, 5.70. yzes for Cl~HzzOll; reducing power by Somogyi Neogermidine thiocyanate crystallized from ace- method 46y0 of that of fructose; low order of reactone as needles, m.p. 247-249' dec. Anal. Calcd. tion with hypoiodite (9% by Willsttitter-Schudel C34H530loN.HNCS: C, 60.50; H, 7.82; S,4.61. method; turanose gave 11%); positive Seliwanoff Found: C, 60.16; H, 7.87; S, 4.65. In a volatile test for fructose; hydrolyzes to glucose and fructose as shown by paper chromatography; phenyl(1) S. M. Kupchan and C. V. Deliwala, THISJ O U R N A L , 74, 2382 iI!l.52). osazone (analyzing for &HsNdOe) as needles from ( 2 ) J. Fried, P. Numerof and N . M. C o y , ibid., 74, 3041 (1952). We wet ethyl acetate (m.p. 186-188') ; yields an amorwish t o thank Dr. J. Fried for private communication of these results phous phenylosotriazole which hydrolyzes to Dprior to publication. glucose ( [ c Y ] ~ ~+52') D and D-glucose phenylosotri( 3 ) J Pried. H. L. Whitcand 0. Wiuterrteiner, ;bid.,?%, 4621 (1950). ( 4 ) G. Salzberger, Arch. Phorm., 298, 462 (1890); W. Poethke. {bid., azole (m.p. 197'; [ a I z 5 D -81'). 275, 571 (1937). From this preliminary work it would appear (5) Plant gathered in northeastern Oregon in Juuc, 1951. We are that the sugar is a D-glUCOSyl-D-frUCtOSe with the grateful to Dr. Reed Kollius, Gray Herbarium. IInrvard University, for glucosyl unit probably in the pyranose form since confirming the identity o f the planl.
COMMUNICATIONS TO THE EDITOR
June 20, 1952
only 1% hydrolysis is observed in 24 hours a t room temperature in 0.2 N HC1. A tentative conclusion as to the point of attachment of the glucosyl group has been based on the following obsewations: Chargaff and Magasanik’ noted that glucose phenylosazone was rapidly cleaved by sodium periodate to give a precipitate of compound I in 85% yield. We have extended this technique to the disaccharides and find it useful in the determination of the point of union of the component parts, since glycosidic linkages in the 3- and 4-positions block the reaction. The method should be applicable to any oligosaccharide giving an osazone. CH=N--NH-C&I,
I I
C=N--T\”-C& CHO
I
CH=N--NH-CsHs
CH=N-NH-CeHs
C=N--NH-CaHb
C=K--NH-CsHs
I
I
HO-C-H
I I H-C--O-Gl I
H-C-OH
CHzOH I1
I
I I
HO-C-H H-C-OH
I I
H-C-OH CHz-0-GI 111
3203 C H -,
CH20H
H-C-OH H-C-0 I
*
H-C-0 I CH~OH
IV
Apparently no 1,5-phenylosazones of the proper configuration are available for comparison. Freudenberg and v. Oertzen2 recently synthesized 5-(& g1ucosido)-glucose but were unable to obtain pure osazones. Our work on the mechanism of dextran formation indicates that the new sugar plays a role in the polymerization process; we believe it advisable, therefore, to assign it the common name of “leucrose” which is suggested by its particular microbial origin. Further structure studies are in progress. We are indebted to Prof. E. L. Hirst and Dr. V. C. Barry for laminaribiose samples, to Dr. Allene Jeanes for the isomaltose, to Drs. N. K. Richtmyer and C. S. Hudson for the turanose, and to Dr. N. Hellman and Mr. H. F. Zobel for the X-ray determinations.
FERMENTATION DIVISION FRANKH. STODOLA NORTHERN REGIONAL RESEARCH LABORATORY Under the conditions used by those workers we HAROLD J. KOEPSELL found that the phenylosazone of the new sugar as PEORIA,ILLINOIS^ EUGENE S. SHARPE well as that of isomaltose (6-(a-~-g~ucopyranosyl)RECEIVED MAY2, 1952
D-glucose) and of gentiobiose (6-(P-~-glucopyrano- (2) K. Preudenberg and K. v. Oertzen, Ann.. 674, 37 (1951). sy1)-D-glucose) gave rapid precipitation of com(3) One of the laboratories of the Bureau of Agricultural and Induspound I in yields of 70-80ojO. On the other hand, trial Chemistry, Agricultural Research Administration, U. S . Departthe phenylosazones of turanose (3-(a-D-glucopyran- ment of Agriculture. osy1)-D-fructose), laminaribiose (3-(P-~-glucopyranosy1)-D-glucose), maltose (4-(a-~-glucopyranoTHE MOLECULAR WEIGHT AND SHAPE OF sy1)-D-glucose), and cellobiose (4-(P-~-glucopyranDESOXYPENTOSE NUCLEIC ACID osy1)-D-glucose) gave no precipitates (other than Sir : colorless inorganic salts) even on standing overThe uncertainty which persists with regard to night. Such behavior is compatible only with the molecular weight and shape of desoxypentose structures I1 and I11 having the glucosidic linkage nucleic acid, DNA, from calf thymus appears to be on positions 5 or 6, respectively. This conclusion is due to the inadequacy of most macromolecular further supported by the fact that the phenylosa- techniques in characterizing very large, charged zone of the new sugar gives an X-ray diffraction polyelectrolytes and to the varying degrees of pattern readily distinguishable from that obtained degradation inherent in the different methods of with the phenylosazones of turanose, laniinaribiose, preparation. In a current paper’ it is shown that maltose, or cellobiose. the measurement of the angular distribution of The possibility of a 1,6 liiikage was also rendered scattered light from DNA solutions in the concenunlikely by the following considerations: The X- tration range of 1 to 10 mg./100 cc. leads to a deray pattern of the phenylosazone of the new sugar termination of the molecular weight as well as to was found to differ from that of gentiobiose phenyl- some definite conclusions about the size and shape osazone; the pattern given by isomaltose phenyl- of several different samples. The sample having osazone, however, was so similar to that of the new the highest molecular weight was that prepared by phenylosazone that conclusive differentiation was Schwander and Signer.2 We wish to report rneasnot possible. Fortunately, identity could be ruled urements on a sample we have prepared by the out by the melting point of isomaltose phenylosa- Signer method which indicate the reproducibility zone (205-207’) and the fact that i t forms a crys- of this preparative method and provide new intalline phenylosotriazole (m.p. 179-180’). formation on the structure of the DNA molecule. Preliminary evidence, then, suggests that the Light scattering measurenients on the new prepnew sugar may be a 5-(D-glucopyranosy1)-D-fructo- aration in 0.2 M NaCl show the molecular weight to pyranose, a possible form of which is shown in be 7,700,000 in comparison with 6,700,000 found’ formula IV. (1) P. Doty and B. H. Bunce, THISJOURNAL, in press. (1) E. Chargaff and B. Magasanik, THIS JOURNAL, 69, 1459 (1847).
(2) H. Schwander and R. Signer, Helu. Chim. Acta, 88, 1521 (1950).
