VOl. 75 Acknowledgments.-The authors wish to thank the Scientific

The authors wish to thank the Scientific Management of Hoffmann-La Roche,. Inc., Nutley, N. J., for the use of their facilities during this study, Dr...
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5428 4.74. Fraction b, m.p. 185-186': 3.93.

C, 50.48; H, 7.94; N,

VOl. 75

tenone should be applied 12 to 24 hours after inoculation. If applied immediately germination 2-(2-Ethylpiperidyl)-cyclohexanol(III).-The reduction of the spores is unduly delayed. of 2-(2-ethylpyridyl)-cyclohexanone was carried out as in The following results are typical of several inthe preparation of IV above, but a t room temperature (50 hours). The reduction mixture was decanted from the dependent runs. Figures for the heptenone-treated catalyst, made basic with aqueous alkali, and extracted with cultures precede values for the controls, in pg. chloroform. The chloroform was dried, evaporated, and carotenoid per g. of dry mycelium, each value the residue distilled, giving 57% of a thick, colorless liquid, representing five plates : (1) cultures treated 24 I3.p. 115-148° (2 mm.). This was crystallized from Skellyso1L-e B , the crystals take11 up in boiling ether, and set at hr. after inoculation, harvested in 60 hr., v s . con0' for 2 days. A small amount of I V was filtered off, and trols; dry weights, 0.400, 0.573 g.; phytofluene the ether filtrate evaporated. The residue was recrystal- 46, 3.7; @-carotene 118, 253; {-carotene 60, not lized from Skellysolve B, giving a 40% yield of white solid, detected. (2) Cultures treated 17.5 hr. after in1n.p. 83-88". Anal. Calcd. for C13HzsNO: C, 73.88; H , 11.92; N, oculation, harvested in 112 hr., vs. controls; dry weights 0.555, 0.582 g.; phytofluene 88.5, 5.8; 6.63. Found: C, 73.83; H , 12.14; N, 6.91. The p-nitrobenzoate (N-p-nitrobenzoyl), prepared in the @-carotene195, 392; [-carotene 43.3, trace. usual manner, crystallized from ethanol in pale yellow In no case was neurosporene detected in the conprisms, m.p. 158-160°. trols, though present in the treated cultures. The Anal. Calcd. for CnH31N307: C, 63.64; H, 6.13; N, lycopene zone was definitely more prominent in the 8.25. Found: C , 63.96; H, 6.32; N, 8.30. treated cultures, but at best was still a minor comAcknowledgments.-The authors wish to thank ponent, not exceeding 5 to 10 pg./g. the Scientific Management of Hoffmann-La Roche, The striking effects of methylheptenone are Inc., Nutley, N. J., for the use of their facilities therefore three: reduction of the @-carotene, a during this study, Dr. A1 Steyermark of the Micro- marked increase in phytofluene and the appearance chemical Department a t Roche, for the micro- of {-carotene as a major constituent. Re-examinaanalyses reported in this paper, and Professor N. J. tion of the absorption curves from extracts of citralLeonard, for his comments. treated cultures makes it apparent that they are DEPARTMENT OF CHEMISTRY intermediate between controls and methylheptenSTEVENS INSTITUTE OF TECHNOLOGY one-treated cultures. Loss in 0-carotene is not so HOBOKEN, NEWJERSEY marked, nor is production of phytofluene so enhanced, under comparable conditions. These qualitative interpretations are supported also by Carotenoids in Phycomyces the observation that the fluorescence of the citralBY G. MACKINNEP, C. 0. CHICHESTER AND PATRICIA S. culture extracts is intermediate in intensity. We WONG cannot as yet comment on possible additional RECEIVED JULY29, 1953 effects ascribable to pseudoionone. We have hitherto been puzzled by failure to A marked effect on @-carotene production in detect numerous minor components observed by Phycomyces may be shown by addition of /3-ionone G ~ o d w i n . ~He obtained phytofluene yields of 70 to cultures.' Recent experiments with purified to 109 pg. per 250 ml. culture solution (Table 9, a-ionone indicate that this isomer also enhances ref. 2 ) after 9 days growth on a 3% glucose-O.2% @-carotene production. By contrast, we reported asparagine in the presence of diphenylthat citral and pseudoionone had a slight effect on amine. Ourmedium, 5 plates, collectively containing 100 lycopene and that methylheptenone had no effect. ml. medium, produced 49 pg, of phytofluene (run This requires revision. Control and ionone-treated 2), from 0.555 g. of dry matter, in a total of 112 cultures, harvested 60 to 100 hours after inoculation when treated, compared with 3.4 pg. for the on a glucose-yeast autolysate medium give extracts hr., It is clear that great variation may be whose carotenoid spectrum is essentially that controls. anticipated in the proportions of the different of @-carotene. Methylheptenone-treated cultures carotenoids comprising the mixture. show weak pigmentation, when compared with (2) The culture response to citral is affected more by the mode of controls, but the carotenoid spectrum is radically application than is the response to ionone different. After chromatography of the crude (3) T W.Goodwin, Biochem J , BO, 550 (1952). petroleum ether extracts on MgO-Si02 columns and spectrophotometric estimation of the components, DEFT.OF FOODTECHNOLOGY OF CALIFORNIA the following effects of methylheptenone may be UNIV. 4, CALIF. BERKELEY shown : production of @-carotene is halved; the phytofluene content is increased 6- to 15-fold; c"-carotene which is not demonstrable in control cultures grown under our conditions is found in The Reaction of Diphosphopyridine Nucleotide with significant amount, neurosporene is detected, and Sodium Borohydride-A Correction and Extension the lycopene content is also increased. BY MARTINB. MATHEWS AND ERICE. CONX Culture conditions and procedures have already RECEIVED JUNE25, 1953 been described. Methylheptenone, 20 pl., (FritzIt was previously observed' that diphosphosche Bros.) was applied to each culture at times varying from 6 to 27 hours after inoculation. To pyridine nucleotide IDPN) was quantitatively minimize adverse growth effects, the methylhep- reduced by sodium borohydride, in agreement with values obtained by reduction with sodium (1) G. Mackinney, T. Nakayama, C. 0.Chichester and C. D.Buss. THISJ O ~ N A L ,74, 3468 (1962); 711, 236 (1963.).

(1) M. B. Mathews, J . Biol. Chtm., 176, 229 (1948).