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3Z-Nonenal and 3Z, 6Z-nonadienal, potential biosynthetic precursors of 2E-nonenal and 2E, 6Z-nonadienal, were for the first time synthesized stereoseleclively.

Z-3-Hexenal, a precursor in the biosynthesis of leaf alcohol and leaf aldehyde, was first isolated from fresh tea leaves, Thea sinensis and its structure was confirmed by unequivocal synthetic evidence.

Optically active dictyopterenes A and B and their geometrical isomers were stereoselectively synthesized by condensation of acrolein with carboethoxymethyl dimethylsulfonium bromide and by the Wittig reaction between (+)-2-vinylcyclopropylcarbaldehyde, which was derived from partially resolved (+)-(1,S, 2R)-2-vinylcyclopropanecarboxylic acid, and phosphonium salts in liquid-solid two-phase systems using crown ethers.

A droplet gel-entrapping method used for enzyme immobilization was improved to simplify the procedure and to increase the enzyme stability. This immobilization technique is suitable for coupled enzyme reactions requiring cofactors. Leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) were freeze-dried with bovine serum albumin, dextrin and stabilizers. The freeze-dried enzyme powder was suspended in a methylcellosolve solution containing polyethyleneglycol(#4000)diacrylate, N, N'-methylenebisacrylamide and 2-hydroxyethylacrylate, and the suspension was gelled with initiators. The gel was cut up and the pieces were washed in a buffer to remove the methylcellosolve and the dextrin inside. The maximum conversion ratio for a LeuDH-FDH gel column was determined to be 99.8% by means of the recycling reaction. On longterm operation at 30°C for leucine production, the initial conversion ratio (7.2%) gradually decreased to 6.6% over the first 10 days. However, the conversion ratio remained almost constant after the 10th day. The effects of flow rate, temperature, pH, and the concentrations of formate, α-ketoisocaproate, ammonium and NAD on the leucine productivity with the gel column were also investigated.

3Z-Hexenal, 2E-hexenal (leaf aldehyde) and 1-undecene were first found in Farfugium japonicum Kitamura (Japanese silver) leaves. On blending the leaves in the presence of oxygen, 2E-hexenal was enzymatically generated via a similar biosynthetic pathway to that in Thea sinensis leaves: via 3Z-hexenal from linolenic acid. However, unlike Thea sinensis leaves neither 3Z-hexenol (leaf alcohol) nor 2E-hexenol was found in F. japonicum leaves.

3 Z, 6 Z-Dienoic acids (C8-C12 and C18) were for the first time synthesized by coupling 2-acetylenic bromides and 2-(3'-butynyloxy)-tetrahydropyrane followed by stereoselective hydrogenation and oxidation.

Alcohol dehydrogenase (alcohol: NAD oxidoreductase, E. C. 1. 1. 1. 1.) from Thea sinensis seeds (variety: Zairai) was isolated and purified about 1, 500-fold using preparative disc electrophoresis. The specific activity was about 3.4units/mg protein against ethyl alcohol. Its s020. w value was 6.96S and its molecular weight was approximately 150, 000 using gel filtration on Sephadex G-200. The physical, chemical and catalytic properties of the enzyme are described. The oxidoreduction products formed by the enzyme were identified by gas chromatography, and for the unsaturated compounds the conversion of double bond and geometrical isomerization was observed. The substrate specificity of tea enzyme is discussed in comparison with the enzymes from Leuconostoc mesenteroides, and horse and human livers. Paticularly a tendency for reactivity in the oxidoreduction of unsaturated alcohols and aldehydes were discribed by comparing the effects of geometry, the position of the double bond and the length of chain in substrates.

(—)-Epicatechin-3-gallate (ECG) and (— )-epigallocatechin-3-gallate (EGCG), major tea catechins, formed precipitates with soybean lipoxygenase (LOX) in the pH range of 4~7, although with accompanying 10 ~30% loss of the LOX activity. Yeast alcohol dehydrogenase also was precipitated by EGCG. Polyvinylpyrrolidone, Tween 20 and Triton X-100 dissociated the LOX activity from the EGCG-precipitated LOX. However, the MW of the dissociated LOX (114,000) differed from that of the native LOX (100,000). Enzyme activities of the EGCG-precipitated LOX and the dissociated LOX from the precipitate were less stable than the activity of the native LOX. These findings suggest the altered natures of proteins in the presence of tea catechins, ECG and EGCG.

Lipoxygenase (linoleate: oxygen oxidoreductase, EC 1. 13. 1. 13) from tubers of potato (Solanum tuberosum) was purified 210-fold with a specific activity of 25 units/mg protein, using DEAE-Sephadex column chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme was electrophoretically pure. Some properties of this enzyme were the same as those of potato lipoxygenase previously reported.The enzyme was inhibited by saturated alcohols and SH-reducing reagents as well as soybean lipoxygenase was. The trapped radical of linoleic acid in potato lipoxygenase reaction was detected by ESR spectroscopy, using 2-methyl-2-nitrosopropane as a radical scavenger. Those suggest that the reaction of potato lipoxygenase proceeds by a very similar mechanism to soybean lipoxygenase.

The synthetic activity for 3Z-hexenal, an important precursor of 3Z-hexenol (leaf alcohol), was localized in chloroplasts of Thea sinensis leaves. 3Z-Hexenal, which is easily isomerized to 2E-hexenal (leaf aldehyde), was formed from linolenic acid in the presence of oxygen. 13-l-Hydroperoxy-linolenic acid also served as a precursor, but the triglyceride and methyl ester of linolenic acid did not. This enzyme system appeared to be tightly bound to the lamellae membranes of chloroplasts.

3Z,6Z-Dienoic acids (C8-C12 and C18) were for the first time synthesized by coupling 2-acetylenic bromides and 2-(3′-butynyloxy)-tetrahydropyrane followed by stereoselective hydrogenation and oxidation.

Tyrosine residues in ricin D were modified with N-acetylimidazole and the saccharide binding properties of the resulting derivatives were examined. The cytoagglutinating activity of ricin D was not altered by acetylation of one tyrosine residue/mol, but decreased greatly upon modification of two tyrosine residues/mol. In the cytoagglutination test, only 6% residual activity was found in the derivative, 2-Ac-ricin D, in which 2.4 tyrosine residues/mol were acetylated. In the presence of lactose, however, one tyrosine residue/mol was protected from acetylation by N-acetylimidazole with a retention of the saccharide binding ability, suggesting the involvement of one tyrosine residue in the saccharide binding. Fluorescence and UV-difference spectroscopic data indicate that the binding ability of the low affinity saccharide-binding site (LA-site) of ricin D remained unchanged after modification of 2 tyrosine residues/mol. The affinity chromatography of the acetylated derivatives of ricin D on the lactamyl- and...

Biological asymmetric hydrolysis of ethyl (±)-cycloheptadienecarboxylate with Rhodotorula minuta var. texensis IFO 1102 and chemical resolution of the corresponding carboxylic acid with (−)-quinine provided (R)-(+)-ethyl 2,5-cycloheptadienecarboxylate (78% e.e.) and (S)-(+)-2,5-cycloheptadienecarboxylic acid (95% e.e.), respectively. The (R)-(+)-carboxylate was converted to (R)-(−)-2,5-cycloheptadienylcarbaldehyde and the (S)-(+)-carboxylic acid to (S)-(+)-2,5-cycloheptadienylcarbaldehyde. Ectocarpene (78% e.e.), male-gamete attractant of marine brown alga, and its antipode (95% e.e.) were synthesized by stereoselective Wittig reaction between the (R)-(−)- and (S)-(+)-aldehydes and propyltriphenylphosphonium bromide in a liquid-solid two phase system using 18-crown ether-t-BuOK, respectively.

Leaf aldehyde, (E)-2-hexenal, in diethyl ether, was converted to an ether adduct, 3-(1-ethoxyethyl)-hexanal (diastereomeric mixture) regioselectively, by irradiation with a 100 W high-pressure lamp.

1. Etiolated seedlings of alfalfa and cucumber evolved n-hexanal from linoleic acid and cis-3-hexenal and trans-2-hexenal from linolenic acid when they were homogenized.2. The activities for n-hexanal formation from linoleic acid, lipoxygenase and hydro-peroxide lyase were maximum in dry seeds and 1~2 day-old etiolated seedlings of alfalfa, and in 6~7 day-old etiolated seedlings of cucumber.3. n-Hexanal was produced from linoleic acid and 13-hydroperoxylinoleic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. cis-3-Hexenal and trans-2-hexenal were produced from linolenic acid and 13-hydroperoxylinolenic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. But these extracts, particulariy cucumber one, showed a high isomerizing activity from cis-3-hexenal to trans-2-hexenal.4. When the C8-aldehydes were produced from linoleic acid and linolenic acid by the crude extracts, formation of hydroperoxides of these C18-fatty acids was observed.5. When 9-hydroperoxylinolei...

The decomposition of lipid hydroperoxide by soybean lipoxygenase-1 (EC 1.13.11.12) under aerobic conditions was studied by high performance liquid chromatography and the spin trapping method. The reaction followed first order kinetics for lipid hydroperoxide. Its rate was proportional to the enzyme concentration and was independent of pH. A decrease in reactivity due to thermal denaturation and H202-modification of the enzyme indicated that native lipoxygenase was involved in the decomposition of the lipid hydroperoxide. The free radical, possibly the alkyl radical, was trapped by a radical scavenger, 2-methyl-2-nitrosopropane, in the mixture of soybean lipoxygenase-1 and the lipid hydroperoxide. These results indicated a free-radical chain reaction mechanism for decompositon of the lipid hydroperoxide initiated by soybean lipoxygenase-1.

Alcohol dehydrogenase (alcohol; NADP oxidoreductase, EC 1.1.1.2) was purified from Leuconostoc mesenteroides IFO 3426.The native enzyme in sonic extract was dissociated to NAD-dependent subunit S1 and to NAD- and NADP-dependent subunit S2 by DEAE-Sephadex A-50, DEAE-cellulose column chromatography and disc electrophoresis. The molecular weights of the native enzyme, subunit S1 and S2 were approximately 240,000, 80,000, and 160,000, respectively, as determined from gel filtration on Sephadex G-200. Subunit S1 was very unstable. The physical, chemical and catalytic properties of native enzyme were compared with those of subunit S1 and S2.

2-Methyl-4, 6-cyclohexadienaldehyde and n-butyraldehyde were treated with sodium in p-xylene to yield the aromatized “leaf alcohol reaction” product, 2-methyl-benzylalcohol, in a better yield than that with the cyclohexadienaldehyde alone. n-Butyric acid isolated from the reaction mixture unequivocally showed the operation of the “crossed Cannizzaro disproportionation” in this reaction, aliphatic aldehyde serving as the hydride donor. 2-Propyl-5-ethyl-4, 6-cyclohexadienaldehyde was obtained by the NaOH/H2O-EtOH Michael-Aldol condensation of leaf aldehyde, gave 2-propyl-5-ethyl-benzylalcohol along with caproic acid.On the basis of “leaf alcohol-reaction” mechanism, it was obtained following benzyl-alkohols; 2-methyl-, 2-propyl-, 2-methyl-5-ethyl-, 2-propyl-5-ethyl-benzylalcohol, from leaf alcohol and crotylalcohol.

The substrate specificity of enzyme system producing C6-aldehyde in Thea chloroplasts was clarified with an entire series of synthesized positional isomers, in which the position of cis-1, cis-4-pentadiene system varies from C-3 to C-13 in C18 fatty acid and geometrical isomers of linoleic acid. The structural requirement for the substrate of enzyme system producing C6-aldehyde is the presence of cis-1, cis-4-pentadiene system between ω-6 and ω-10.

2-Propyl-5-ethyl-benzylalcohol (IX) was synthesized by an unequivocal route from propylbenzene, thereby establishing the previous deduction tentatively assigned to the leaf alcohol reaction product.1) This benzyl alcohol surmises one of a lemon-like flavor characteristic of manufactured black tea and an attempted search for this compound in the essential oil obtained by steamdistillation of manufactured black tea was made, but its existence has not so far been confirmed with a neutral fraction examined.