COMMUNICATIONS TO THE EDITOR
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which forms LTPP from T P P and bound LA. 'The loss of this enzyme is apparently the critical change resulting from mutation of the parent strain. This conclusion is based upon studies of pyruvate oxidatTon systems, resolved atid partially purified by the method of Korkes, e/ d . , ? from the wild a t i d iiiutant strains of this organisni. Pyruvate dismutation requires transacetylase, lactic dehydrogenase, orthophosphate, diphosphopyridine nucleotide, coenzyme A, TPP, and two enzyme fractions from the wild strain (designated - 1 ~ 7 and Bw)' (Table I ) . However, the enzyme fraction Hw and the cofactor T P P cat1 be replaced by a single substance, LTPP, indicating that the basic pyruvate oxidase system, which is activated by the coenzyme LTPP, is present only in fraction -1. The apooxidase fraction iron1 the riiutaiit strain, A l f , can likewise be activated either by LTPP, or by TPP plus Bw, but rteither apooxidase fraction is activated by TPP plus B L ~the , fraction from the mutant corresponding to fraction BIV of the wild strain.
Yol. 74
VERATROBASZNE AND GERALBINE, TWO NEW ALKALOIDS ISOLATED FROM VERATRUM ALBUM'
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.liter separation of the ester alkaloids, protoveratrine and veralbidine,2 and the alkamines jervirie and rubijervine from the mixture of alkaloids coiitained in Veratrum album, the mother liquor was divided into two fractions, one of which contained the inarkedly basic alkaloids, and the other the weakly basic alkaloids. From the fraction containing the first group an unknown alkaloid could be crystallized out of a solution in ethyl acetate.$ 'This was purified as its hydrochloride which is only slightly soluble in water. Xfter cleavage of the ialt with dilute ammonia the pure base was obtained. This we intend to call veratrobasine. 'The new base crystallizes from methanol in large prisrns, which turn yellow from 270" upward, and melt a t 2X5-288", with decomposition. Its optical D and in rotation in pure alcohol is [ c Y ] ~ ~ -76.6") pyridine [ C Y ~ ~ O D - 1%' %'hen veratrobasine was dissolved in ch-L'( sulfuric acid (2 mg. of the base 111 10 cc. acid) an intensely orange fluorescent 'r.4BL.I:. 1 iolutioii w'is obtained. The solution kept this k'TKUV.4TE I ) I S M L T A T I O S \VITII PYRIFIED E S Z Y M E F K A C color for over 24 hours. Anal. Calcd. for C X H ~ ~ IIOSh C, 74 44; H, !).03; N, 3.62. Found: C, p l l ?roducts i n !rO i i i i i i 7 !.-Hi, 74.3h; H,( j . h l , U..5i; N, 3.86, 3 . G . Acetyl Carbon phosIn possessing only 24 carbon atoms, the new Coin pun en t? dioxide phate Lariat? ~tlkCiloid is iigiiificm tly different from the other x \ v + TPP IJ il 02 :iIl\ariiines $( J f,ir obtained from T brcctrum album mv :{ti :;-I : I ; .itit1 17eriifriiiii viritle, which all have 27 carbon 11 0 Ii 9 An TPP + 1,A .itoui\. Veratrobasiiie has one N-CHj group, the 1.; 4.-1 -1.7 Ax + LTPP first to be found in the 1-eratrum alkaloids: N-CH, A W -t Incubated" F i x 3 t- 'TPP 3 , ,-I ::,:I :i 4 calcd. 3.83, found 3.94. 0.1 lJ,2 li2 An; + Control' The base also contains two actil-r hydrogen A M + TPP (1.1 1.1.1 1.1.1 ,itoiiis: calcd. O.Xh, found 0.51. + B\\- w P :$.ti 3.'j :I.,-) 'l'he infrared nbsorptiori spectrurll shows no 1.7 4.4 -1,s Aw + LTPP hand tyI)ical of betones, the ultraviolet absorption A H + i n c u b a t e d ~i R\r- t T P P :j ;, :I 2 :; -1 spectruin, however, shows ;L definite miximum at Ii I/ ri 1 .I\( + Culirrctl' 2;i2 rlly (log € 2,I-l). 0.1 0.1 11:: .-I,!\ -cn\r 7 - TPIJ Froill the fraction contaiiiing the weakly basic A!\- .+ Ii\l T P P f 1,A I),! 0.1 11:: ,&aloitls, ;1 further new alkaloid could be crystalx u -t H\I + 'TPP 0.1 0.1 1J.1 lired iroiii ethyl acetate. This new alkaloid, which Preseiit a t following irvels: exizyiiie fractioiis, 2.11 nig. we hdVe called gevalbine, crystallizes from aqueous protein; T P P , 1UOy; LA, 1Oy; LTPP, 2 i y of crude syn- acetoiie in large prisms, and from a mixture of ethyl thetic preparationlb; final volume, 2 ml. Supplements :tiit1 acetate and ether ( I : I ) in rectangular plates which experimental conditions as previously described.'* lnmelt a t 221-223" with slight yellowish discoloracubated 90 miri. a t 35O, boiled 10 inin., atid superiiatant :added to A N or A w R \ r incubated :rnd hoilerl prior t o tion. In contrast to the other alkaloids isolated (mnt:tc't with T P P from T'erntrum, geralbine exhibited no measurable iricubation of fractiori Hu- aloiie with 'TPP rotation in pure alcohol, chloroform or pyridine. I'roduces a heat stable product, presumably I,TPP, M'hen dissolved in 84% or pure sulfuric acid 'i which can subsequently activate the apooxidase of light yellow solutiori was obtained which had not either strain ; however, heating fraction Bly prior lost its color after twelve hours. In crystalline to its contact with T P P results in an incubation form geralbine is fairly stable but when dissolved mixture havirig no cooxidase activity. Fractioii in alcohol or chloroform, the solution turns yellow I3Liv must furnish lipoic. acid rorijugase :is well ;IS withiii J iew hours. A m l Calcd. for CZZHuOdY lipoic acid, presumably bound to the coijugase, or C, 70.91, ti, 5.68, N , 4.07 Found: C, 7 6 . i ; ; , less likely t o a contaminating protein, by a union 76.78; H, 9.79, 9.78; N, 3.96, 3.98. Geralbine hydrochloride crystallizes from methnot dissociable by dialysis. anol-ether in fine needles, and melts a t 2iO'. BIOCHEMICAL INSTITUTE AND Anal. Calcd. for CzzHa402NCl: C, 69.55; H, 9.02: LESTERJ. REED D E P A R ~ E NOF T CHEMISRY BETTY G. DEBUSK CI, 9.3.5. Fourld C, 69.96; H, 9.23; CI, 9.35, 9.3; IJN+VERSrfY O F TEXAS, AND ' j
+ +
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CLAYTOX FOCSDATION FOR RESEARCII AL'STIN, TEXAS
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i c c o o r l D-glucosamine. This (111) described above. structure follows from transformation of the diThe methyl ester hydrochloride (II), on oxidasaccharide to D-ghcopyranosido~-arabi- tion with yellow mercuric oxide, followed by sodium nose, a new compound whose structure follows in borohydride reduction, gives glucosido-glucosturn from its preparation from laminaribiose aminic acid (20% yield from I), needles, [aI3'D (glucopyranosido< p-1,3>glucose5) by an applica- -34" (water; c, 0.9). Found: neut. equiv. tion of the ZemplCn4degradation. (formol), 353. Degradation of this aniino acid The crystalline glucuronido-glucosamine is pro- with ninhydrin gives a glucosido-arabinose, isolated duced from uiiibilical cord hyaluronic acid in as the heptaacetate (IV), needles m.p. 198-199' yields as high as 6l(,& by the combined enzymatic (micro-block), [ a ] j 3-47" ~ (chloroform). Found: and acid hydrolysis earlier described, in somewhat C, 49.92; H, 6.00. This acetate gives a melting lower yield by direct acid hydrolysis. The picture2 point depression with ZeniplCn's4 heptaacetylof the polysaccharide as a chain of alternating glu- glucosidoarabinose, [a]= - li", and gives cosamine and glucuronic acid residues must there- no depression with heptaacetylglucosido< p-1,2>fore be essentially correct. Also, the @-1,3- arabinose (IV) from laminaribiose. linkage now found in the disaccharide is apparently Synthetic laminaribiose3n7is treated with hythe predominating if not sole glucuronidic linkage droxylamine. The resulting glass with acetic in the polysaccharide. anhydride and sodium acetate at 110' gives octaI n earlier structural investigations, a methylated acetyllaniinaribionitrile, m.p. 140-141", [ p 1 I 3 O ~ +3" glucopyruronoside derivative has been isolated in (chloroform). Found: N, 2.10. Reaction of the trace quantity on methanolysis of the methylated nitrile with sodium inethoxide and acetylation of polysaccharide,: and various workers have inferred the product gives heptaacetyl-glucopyranosido< pfrom the periodic acid consumption of the poly- 1,z>D-arabinose (Iv), i n . ~ . 199.5-200" (microsaccharide and its derivatives the presence of 1,3-,6a block), [ ( Y ] ~ O D -46" (chloroform). Found: c, (1) hl. SI. Kagport, li. Weissmann, F. Linker and K. Meyer, Salui.e, -29.10; H, 5.62; CHJCO, 47.8. Like veratrobasine, geralbine has one N-CH; group: calcd. 4.38%) found 3.Y6Yc. In the infrared spectrum geralbine shows a band typical of ketones a t 171.5 cm.-l.
+
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168, 996 (1951). (2) K. Meyer, Physiol. Keuiews, 27, 336 (1047). (3) P. Bachli and E. G. Percival, J . Chem. Soc., 1243 (1952). (4) G. Zemplkn, Be?., 69, 1254 (1926). (5) M. A. G. Kaye and M. Stacey, Biochem. J . , 48, 249 (1951) (6) (a) R . W. Jeanloz and E. Forchielli, J . Biol. Chcm., 190, 537 (1951); (b) K. H. Meyer, J . Fellig and E. H. Fischer, Helv. Chim. Acla, 34, 939 (1951); H. Masamune. Z. Yosjzawa and T. Tsikawa, TohokacJ. E x p . Mmf.. 66, 166 ( l a n a i : id C.: R l i u . A r l n ( ' h e m . . S , - f l ? ? d , I,Q8l (1P.511.
DEPARTMEST OF MEDICIYE C O L U M B I A LTSIVERSITI' COLLEGEOF PHYSICIANS AND
BERNARDWEISSMANN SURGEONS AND THE EDWARD DANIELS FAULKNER ARTHRITIS CLINIC PRESBYTERIAS HOSPITAI K A R L MEYER SEW YORK32, N Y. RECEIVEI) J r r r \ I T , 19.72 171
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