picrate (in p. 140-142"),? and reduction to .>,sa,G,7,8,9,10,1Oa-octahydrocyclohept [blindole (1ii.p. 7:; i-f"J . ~ Conversioii of I to TI (calcd. for CIJ1,N. C', S i . 1 2 , 11, . i O ( i I'cwtitl: C, S 7 . X ; 11, $ S i ) clictl by apor-phasc (,'L?O ;MI") palladium- charcoal on magnesiuiii ogenation. The absorption spectra of I1 are: ultraviolet (alcohol solution), A, in nip a t 255, log E 4.47, and 309, log E 4.59 (similar to tliat of benz [alazulene) ; 4 risible (alcohol solution i, liroad peak with maximum a t 500 nip, log E 2.(il ; itifrared in ti to 9 p region (carbon tetracliloritlc 7.+, 7.Sp and 8 . 4 ~ . icilutiori), 6.2p, & 7 p , Further identification of the product as I1 $\as provided by reduction ( 9 i % of the theoretical quantity of hydrogen taken up) to I (identity by 1n.m.p. and infrared spectrum). Acridine has been identified (m.p., m.m.p., infrared spectrum) ;is a by-product of the dehydrogenation of I. Repetition of the reaction of I with iodine and nitrobenzene afforded, in our hands, a low yield of dark red solid which was identical (infrared spectrum) with I1 ohtained by catalytic dehydrogenation, and lesser amounts of other, unidentified substances, but no product correspontlitig t o that tlcscribetl by 'I'reibs. 11, as obtained by us, represents the first exaiiiple of a heteroxulene coinpound of estahlished structure. This work was supported in part by a grant from the Research Corporation.
CIlc CJIj '\/
I1
111
I \'
72-hour cultures. Fifteen hours later, the pcarotene content of treated cultures in a typical run was 21s pg. per gram of dry mycelium, compared with 91.2 pg. per gram for control cultures. In all cases, I, I1 and I11 depressed the rate of culture development. Aerial mycelium was sparse and there were few fruiting bodies, and I and I1 showed no pigment production. The effect of I11 011 @-caroteneproduction relative to the controls is lesseried progressively as the aerial mycelium de velops and the cultures mature. The foregoing was dei1ionstratf.d on media liiriitetl in one or mort. nutrients. \Ye therefore developed a more nearly complete medium in which growth of controls was not limited in the total time of the experiment, GO hours. This medium contained 3 g. of Difco yeast extract and 25 g. of glucose per liter, with thiamine added (4) This is in agreement with the observatioris of G M Badger, 1< S Pearce .ind I(. l'tttit ( f b z d , 3199 (1951)) on similar systems a t the time of inoculation, 6 nig. per liter. Twenty DEPARTMENT OF CHEMISTRY A\ID CHEMICAL ENGINEERINGnil. of medium was added to petri dishes with UNIVERSITYOF WASHIUGTOU A G A N D E R ~ O N , J RRaschig rings or glass beads supporting a tared filter JAIIES TALvv.4 paper, cf. ref. 1. Cultures were inoculated and grown in the light, cu. 23", for 36 hours, when aerial inycelium is thick but short (cu. I/*"), with no fruiting bodies and negligible pigmentation. We then CAROTENOID PRODUCTION IN PHYCOMYCES added cu. 3 pl. of the following to the cultures, subSir : sequently kept in the dark: I, (Eastman Kodak Goodwin and Lijinsky' have reviewed hypotheses Co., redistilled) ; 11, synthesized by coupling concerning carotenoid formation in plants. We I with acetone; I11 (Fritzsche Bros., Inc., Novohave confirmed their findings that leucine with viol, beta, re-distilled) ; IV (Novoviol, alpha, slower growth produces more 0-carotene in the extra). Since IV was demonstrated to contain mold Phycomyces blukesleeunus2 than asparagine some 111, this will not be discussed except to rewith faster growth. We have tested a hypothesis mark that no a-carotene was found. Control that we could influence carotenoid production by cultures grew and developed fruiting bodies in the providing cultures with conipounds theoretically subsequent 12 hours. I- and 11-treated cultures capable of providing terminal groups appropriate showed little development and no pigment producto specific carotenoid molecules. These include tion. Cultures treated with I11 yielded 30 pg. oi citral (I), pseudoionone (11), p-ionone (111) and $-carotene per culture after 7 hours, and 134 pg. a-ionone (IV). Thus I and I1 might be expected after 23 hours. They were bright orange, colito form lycopene if ring closure does not occur; trasted with gray yellow for the controls containing 111 t o form @-carotene; IV to form a-carotene or 4.9 pg. after 23 hours. Similar results were obeven el-carotene synthcsi7ed by Karrer and Eugs- tained in a second run. Carotene formation seemter ingly proceeded in the presence of I11 a t the excrhain (carbon-base) medium in- pense of inetabolites that would otherwise ensure glucose, with 0.2570 tll-leucine, riorrnal culture development. Two possibilities ICS demonstr~teda ~ effect i of $- would explain negative results with I and 11, toxionone, applied L U . 2 pl. per 20 nil. of medium to icity or diversion of metabolites to colorless coinpounds, possibly polyene in nature. (1) T W Goodwin and W Lijinsky, Bzochern J , 50, 268 (1951) ( 2 ) W V t~l u i i k D r Krntirth U Raper Korthrrn Regional Researcli This experiment was repeated a t levels of I, I1 l i n i ( i 1 iiirl ( I +tr 1 IIC I ? t i i r \\err I i i i d arid TI1 one-tenth the previous values, and growth re was more nearly normal. I and I1 under thew ( 3 ) 13 K irrer r i i < l (' I 1 1 t i a ' ( I r l l i h ( h r i n 4 r l I , 3 3 , 1477 IO;.! cv)ntlitions I)rotlitcwl less total pigment, 7.5 a n d 88%, f I 351)) LIII~
lit
July 5, 1‘3.52
COMMUNICATIONS TO TIIE Eurroit
3457
produced maximum growth of the niubant a t a level of 2.5 X 10-6 y per cc. of culture medium. In a similar manner, preparations active in catalytic amounts for the mutant have been obtained by condensing a-lipoic acid or its acid chloride with 2-methyl-5-ethoxymethyl-6-aminopyrimidine, thiamin monophosphate, and thiamin pyrophosphate, respectively. The active principles in chemical preparations of lipothiamide and its monophosphate possessed Rf values identical with the two active principles produced biosynthetically and with two of those present in cellular extracts. Furthermore, treatment of lipothiamide monophosphate or pyrophosphate with intestinal phosphatase degrades these two (4) G. A. Garton. T. W. Goodwm a n d W. Lijinsky, Biochcm. J.’ factors to lipothiamide, as revealed with bioauto49, 154 (1951.). G. MACKINNEYgraphs. I~IVISION OF FOOD TECHNOLOGY T. NAKAYAMA An investigation of possible coenzymatic funcC. D. Buss tions of lipothiamide and its phosphorylated dcOF CALIFORNIA UNIVERSITY BERKELEY, CALIFORNIA C . 0. CIIICHESTER rivatives has revealed : (1) resting cell suspensions RECEIVED M A Y 9, 1032 of the mutant will not oxidize either pyruvate or a-ketoglutarate unless one of these conjugates is added; (2) the growth requirement of the organism LIPOTHIAMIDE AND ITS RELATION TO A THIAMIN for lipothiamide can be completely by-passed by COENZYME REOUIRED FOR OXIDATIVE supplying the products (acetate, citrate and sucDECARBOXYLAfION OF a-KETO ACIDS cinate) of these two blocked reactions. A l l three Sir: products, however, must be present. The induced We wish to report evidence indicating that mutation apparently destroys the capacity of the lipbthiamide, a catalytic agent present in cellular organism to conjugate a-lipoic acid and the pyrimiextracts, is the amide of a-lipoic acid and thiamin, dine moiety of thiamin, resulting in a deficiency of and suggesting that this moiety is part of a thiamin an a-keto acid cooxidase required for the reactions coenzyme required for oxidative decarboxylation of producing the three essential metabolites. a-keto acids. INSTITUTE AND Natural materials contain a-lipoic acid, a cataly- BIOCHEMICAL DEPARTMENT OF CHEMISTRY LESTERJ. REEI) tic factor required for pyruvate oxidation,‘ in UNIVERSITY OF TEXAS,A N D BETTYG. DEBUSK bound condition. Acidic or basic hydrolysis de- CLAYTON FOUNDATION FOR RESEARCH grades the bound material predominantly to a- AUSTIN,TEXAS RECEIVED MAY5, 1952 lipoic acid, whereas enzymatic hydrolysis releases “complex” forms of a-lipoic acid.’S2 Our study of a-lipoic acid complexes was directed toward first obtaining an organism which required LYSINE BIOSYNTHESIS IN TORULOPSIS UTILE‘ such a complex for growth. This requirement was Sir : exhibited by a niutant strain of Escherichia coli, In an isotopic study of biosynthetic iiiechanisins developed by irradiating wild-type cells and isolat- in Torulopsis utilis, data from yeast grown on ing the mutant, using the penicillin technique. methyl- and carboxyl-labeled acetate have proWhereas the mutant did not respond to a-lipoic vided a clue to the pathway of lysine synthesis in acid in the presence of all other known vitamins this organism. Cultural details and other experiand growth factors, it did respond to the a-lipoic mental procedures have already been described. acid complexes present in cellular extracts, or to The lysine was isolated from the cell material by substances formed by incubating a-lipoic acid and standard methods, and degraded by the following thiamin with wild-type Escherichia coli or Strepto- steps : decarboxylation with ninhydrin ; oxidation coccus lactis cells. to 6-aminovaleric acid; degradation of the latter Bioautographs of the incubation mixture revealed by the Schmidt reaction3 to carbon dioxide and 1,4the presence of two active principles, possessing Rf diaminobutane ; oxidation of the latter to succinic values identical with those of two a-lipoic acid acid; isolation of succinate carboxyls by the complexes present in cellular extracts. Both Schmidt reaction. Another sample of lysine was biosynthetic “conjugates” gave a negative thio- oxidized to glutaric acid and the glutarate carboxyls chrome test and a positive azo test. These obser- obtained by the Schmidt reaction. These provations suggested that the two conjugates contain cedures yielded individual activities of carbons a-lipoic acid and thiamin conjugated through an 1, 2, 3 and G, and the averages of carbons 4 and 5. amide linkage. ( 1 ) Aided by grants from the U. S Atomic Energy Commission, ConChemical synthesis studies support this conclu- tract No. ATI3O 1)777, and by a n institutional gr.irit f r o m the Amrrision. Reaction mixtures obtained by heating a- can Cancrr Society. ( 2 ) M. Strassmdn a n d s. Weinhouse, T H I S J O ~ J R V A I . , 74, 172G lipoic acid and thiamin in vacuo, and by treating thiamin with the acid chloride of a-lipoic acid, (1952). (3) E. F. Pharcs, Arch. Diochern. Biophys., 33, 173 (1921); w e arc
respectively, of that for tlic control, cf. 450% for 111. However, spectroscopic and chromatographic evidence indicated a small increase in the absolute amount of lycopene for I and 11, compared with I11 and the control. The fluorescence of I- and 11-treated samples was noticeably blue-green, whereas the others were characteristically green. In summary, production of @-carotene can be markedly influenced in a short period (5 to 20 hours) and of lycopene to a minor extent by use of compounds presumably providing terminal groups in the carotenoid molecule. Results are not incompatible with a tentative scheme postulated by Garton, Goodwin and L i j i n ~ k y . ~
(1) I C G u i ~ s ~ l uels al , J Rtol rhein , 194, 82‘3 (1’322) (2) I, J Recrl, cf nl , ibrd , 192, 851 (1051)
greatly in(1cbted to D r . Pliares lor supplying us with this niethod prior to publication.
