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Coenzyme Q


DATA No : VCQ0005 INFORMANT : Tetsuya Nakamura

NAME : 2,3-Dimethoxy-5-methyl- farnesylnerolidyl -1,4-benzoquinone

COMMON NAME: Coenzyme Q6
SYMBOL: Co Q6
FORMULA: C39H58O4 MOL.WT (average) : 590.875


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BIOLOGICAL ACTIVITY
A mitochondrial NADH:Q6 oxidoreductase isolated from cells of Saccharomyces cervisiae by a method involving extraction of enzyme from the mitochondrial membrane with TritonX-100, followed by chromatography on DEAE-cellulose and blue Sepharose CL-6B. By this procedure a 2000-fold purification is achieved. The purified NADH dehydrogenase consists of a single subunit with molecular mass 53 kDa as indicated by SDS/polyacrylamide gel electrophoresis. The enzyme contains FAD, non-covalently linked, as the sole prosthetic group with Em,7.6 = -370mV and no iron-sulphur clusters. The enzyme is specific for NADH with apparent Km = 31m M and was found to be inhibited by flavone (I 50 = 95mM), but not by rotenone or piericidin.(Ref. 0015)
PHYSICAL AND CHEMICAL PROPERTIES
MELTING POINT:19-20deg(Ref. 0037)

BOILING POINT:

REFRACTIVE INDEX:

OPTICAL ROTATION:

DENSITY:

SOLUBILITY:
SPECTRAL DATA
UV SPECTRA:lmax 270 mm, E1%1cm = 260 in petroleum ether(Ref. 0037)

IR SPECTRA:

NMR SPECTRA:

MASS SPECTRA:

OTHER SPECTRA:
CHROMATOGRAM DATA

SOURCE
Isolation and separation---One kilogram of dry cells of Escherichia coli 08 was extracted by shaking 15 h with 5 litters of ether-ethanol (3:1). The filtrate was evaporated in vacuo, and the residue was dissolved in 300ml of aqueous methanol (95%). After addition of 300 ml of hexane, the mixture was filtered, and the organic phase was separated. The aqueous phase was further shaken with 150 ml of hexane, and the combined extracts were dried over Na2SO4. The solvent was evaporated, and the the residue (14 g) was dissolved in 80 ml of hexane. The orange-brown solution was chromatographed on a column of silica gel (5.2 x 90 cm). The column was developed by elution with 3 liters of hexane followed by increasing one-percent increments of ether in hexane in 3-liter portions, beginning with 1 % ether in hexane. The ubiquinones were eluted with 5% ether in hexane and were collected in two portions.
The first of these fractions contained practically pure Q8. The residue obtained from the second fraction was dissolved in a small volume of ether and streaked on silica gel G plates (0.5-1 mm). The plates were developed in d chloroform, air dried, and again developed in chloroform. This procedure was repeated three times. The upper and lower halves of the yellow quinone band (1-2 cm wide) were collected separately and eluted with ether. The material obtained from the lower half of the Q-band was subjected to the thin layer chroamtographic procedure described above. Four successive separations were carried out in this manner; each time the upper half of the yellow quinone band was discarded. Finally, a sample was obtained which on chromatography on silicon-coated paper proved to be pure ubiquinone-5. Q8, Q7, Q6, and Q5 were present to different extents in the intervening fractions. (Ref. 0032)
CHEMICAL SYNTHESIS
Six grams of 2,3-dimethoxy-5-methyl-1,4-benzoquinone dissolved in 50 ml of methanol is hydrogenated at room temoperature and atmospheric pressure in the presence of 0.5 g of Lindlar's catalyst until the hydrogen consumptin is terminated (about 30 minutes). The catalyst is removed by filtration, the solution is evapo- rated in vacuo, and the residue is dried under high vacuum for 1 hour. The crystalline hydroquinone is dissolved in 180 ml of absolute ether ; 0.3ml of glacial acetic acid, 3.3 g of anhydrous zinc chloride, and 20 g of all-trans-frarnesylnerolidol are added. The mixtute is shaken overnight at room temperature under nitrogen and then refluxed for 1.5 hours. The solvent is evaporated in vacuo, and the residue is dissolved in 500 ml of petroleum ether (b.p. 30deg-45deg) and 250 ml of methanol-water (7 : 3) .
The petroleum ether layer is extracted with three 250-ml portions of metanol-water (7: 3), and the methanolic solutions are reextracted with 250 ml of petroleum ether in a second separatory funnell. The combined petroleum ether sollutions are washed with water, dried over anhydrous sodium sulfate and evaporated in vacuo. The residual brownish-yellow oil (17.5 g) is dissolved in 50 ml of petroleum ether (b.p. 80deg -110deg) and hydrogenatd in the presence of 1 g of Lindlar's catalyst. The catalyst is removes, the solution is evaporated and the residue is chromatographed on 400 g of aluminum oxide (activity grade I, deactivated with 4% water). Eight grams of by-product are eluted first with 3.5 liters of petroleum ether (b.p. 30deg-45deg) . With ether (1 liter) 8.3 g of condensation product is obtained which is dissolved in 100 ml ether and shaken in the presence of 20 g of silver oxide for 2 hours at room temperature. Continued to Genetic Information
METABOLISM

GENETIC INFORMATION
The solution is filtered and evaporated to give crude ubiquinone-6 as an orange-yellow oil, exhibiting and ultraviolet absorption maximum at 272 nm. Further purification is achieved by chromatography on 150 g of aluminum oxide (activ ity grade I, deactivated with 7% water). With petroleum ether (b.p. 30deg-45deg), 5.5 g of product is eluted. Of this concentrate [lmax 272 nm ( E1%1cm sim100), in cyclohexane], 116 mg is chromatographed on 10 g of polyethylene powder (Hostalen W) with acetone-water (72 : 28) as mobile phase. One hundred fractions of 5.5 ml each are collected. The yellow fractions are combined (275-380 ml), diluted with water, and excracted with petroleum ether.
The petroleum ether extracts are washed with water and evaporated to give 30 mg of deep orange oil showing an ultraviolet absrption maximum at 272 nm (E1%1cm = 260) and a minimum at 237 nm (E1%1cm = 69).The oil is dissolved in a 10-fold amount of absolute alcohol or acetone and crystallized at 115. The precipitatedcystls melt at 19-290. For paper chromatography Whatman No.1 paper impregnated with Dow-Cornng silicone DC 1107 and isopropanol-glacial acetic acid -water (600:25:375) is recommended. Time 15 hours. Rf 0.54. Alternatively, the conditions described under ubiquinone-9 (Ref. 0002)may be used.(Ref. 0004)
NOTE

REFERENCES
[0002]
AUTHOR:Mayer,H., and Isler, O.
TITLE:Synthesis of ubiquinones
JOURNAL:Methods in Enzymology
VOL:18 PAGE : 182 -213 (1971)
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[0004]
AUTHOR:Gloor, U., Isler, O., Morton, R. A., Rüegg., and Wiss, O.
TITLE:Die Struktur des Ubichinons aus Hefe
JOURNAL:Helv. Chim. Acta
VOL:41 PAGE : 2357 -2362 (1958)
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[0015]
AUTHOR:de Vries, S., and Grivell, L. A.
TITLE:Purification and characterization of a rotenone-insensitive NADH:Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae PubMed ID:3138118
JOURNAL:Eur J Biochem.
VOL:176 PAGE : 377-384 (1988)
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[0032]
AUTHOR:Friis, P., Daves, G. D., Jr., and Folkers, K.
TITLE:Isolation of ubiquinone-5, new member of ubiquinone group PubMed ID:5337718
JOURNAL:Biochem Biophys Res Commun.
VOL:24 PAGE : 252-256 (1966)
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[0037]
AUTHOR:Sommer,P., and Kofler,M.
TITLE:Physicochemical Properties and Methods of Analysis of Phylloquinones, Menaquinones, Ubiquinones, Plastoquinones, Menadione, and Related Compounds. PubMed ID:5340867
JOURNAL:Vitamins and Hormones
VOL:24 PAGE : 349 -399 (1966)
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