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Both enantiomers of (3S)-(-)- and (3R)-(+)-Neodictyoprolenol [(3S,5Z,8Z)-(-)-1,5,8-unde-catrien- 3-ol] were successfully converted to the algal sex pheromone, (1S,2R)-(-)-dictyopterene B and (1R,2S)-(+)-dictyopterene B in high enantiomeric purities (e.e. > 99%), respectively, by the biomimetic reaction involving phosphorylation and elimination under a mild condition

Neodictyoprolenol [(-)-(3 S )-(1,5 Z, 8Z)-undecatrien-3-ol] and dictyoprolenol [ (-) - ( 3 S ) - (1,5 Z , 8Z )-undecadien-3-ol]), which had been proposed as possible biosynthetic intermediates of the sex pheromones of marine brown algae such as dictyopterene B [(-)-trans-1- ((1'E,3' Z )-hexadienyl)-2-vinylcyclopropane], D ' [(+ )-6 -((1' Z )-butenyl)-1,4 -cycloheptadiene] and C' [(+ )-6-butyl-1,4-cycloheptadiene], were again identified in the essential oils from Dictyopteris prolifera, D. latiscula, and in D. undulata, together with the C11-related volatile compounds such as neodictyoprolene, dictyoprolene and dictyopterenes. Incubation of D. prolifela preparation with racemic neodictyoprolenol and dictyoprolenol as substrates showed (S)-enantioselective decreases of the added substrates and increases in dictyopterenes. From these results, a possible pathway to form dictyopterenes is discussed.

Neodictyoprolenol [(-)-(S)-(1,5Z,8Z)-undecatrien-3-ol], dictyoprolenol [(-)-(S )-(1,5Z)- undecadien-3-ol] and their acetates neodictyoprolene [(+)-(5)-3-acetoxy-(1,5Z,8Z)-undecatriene] and dictyoprolene [(+)-(S)-3-acetoxy-(1,5Z)-undecadiene], which are interesting as possible biosynthetic intermediates of the sex pheromones (dictyopterene B, C′ and D′) of brown algae, were synthesized by chemo-enzymatic methods through optical resolution of racemic neodictyoprolenol and dictyoprolenol using two lipases; Amano PS (Pseudom onas sp.) and Novozym 435® (Candida sp.). A combination of acylation of the alcohols and hydrolysis of the acetates by Novozym 435® produced neodictyoprolenol, neodictyoprolene, dictyoprolenol and dictyoprolene with high optical purities over 99% enantiomeric excess (e.e.). This snythetic methods will make it easier to search these compounds in marine algae and to study their biosynthesis.

Chemical structure-odor correlations in the isomers of n-C9-methylene interrupted dienols were explored using synthetic nine isomers of these alcohols. The synthetic dienols were purified by recrystallization or column chromatography of their 3,5-dinitrobenzoate de­ rivatives. Chemical structure-odor correlations in all the isomers of the purified n-nonadien-1-ols were analyzed by treating the data obtained statistically with the principal component analysis method (Sakoda et al., 1995; Cramer et al., 1988) in comparison with those of n-nonen-1-ols. The odor profiles of the n-nonadien-1-ols were attributable largely to the geometries of the isomers, compared with n-nonen -1-ols (Sakoda et al., 1995). With the principal component analysis, the odor profiles of the series of the dienols were successfully integrated into the first and the second principal components. The first component (PC 1) consisted of combined characteristics of fruity, fresh, sweet, herbal and oily-fatty, and the second component (PC 2) leaf or grassy and vegetable-like. Of the methylene interrupted dienol isomers, (2E ,6Z)-and (3Z,6Z)-nonadien-1-ols which are natural products and have (6Z) in the same, deviated markedly from the other isomers as seen in (6Z)-nonen -1-ol of n-nonen-1-ols. That suggests that the double bond of (ω3Z) was an important factor for natural characteristic odor.

In order to elucidate chemical structure-odor correlation in the all isomers of n-nonen-1- ols, an entire series of these alcohols were synthesized stereo-selectively in high purity. For unequivocal syntheses of them, geometrically selective hydrogenation of the respective acetylenic compound was adopted. The synthesized alcohols were converted to their 3,5-dinitrobenzoate derivatives with 3,5-dinitrobenzoyl chloride, and then purified by repeated recrystallization. Chemical structure-odor correlations in all the isomers of n-nonen-1-ols were elucidated by introducing a novel method to evaluate odor characteristics and by treating the obtained data statistically with the principal component analysis method (Cramer et al., 1988). The odor profiles of the tested compounds were attributable largely to the positions of the carbon- double bond. The geometries of compounds had only a little effect. With the principal component analysis, the odor profiles of the series of compounds were successfully integrated into the first and the second principal components. The first component (PC-1) consisted of combined characteristics of fruity, fresh, sweet, herbal and oily-fatty, in which herbal and oily-fatty were conversely correlated each other to the position of double-bond of the tested compounds. Of these, only (6Z)-nonen-1-ol deviated markedly from the correlation, indicative of some special interaction between the spatial structure of this compound and the sensory machinery of human.

Arginine residues of soybean lipoxygenase-1 was modified with an arginine-directed chemical modifier, 2,3-butanedione. Although inactivation was not visible if the enzyme reaction was monitored under the standard assay condition (83.3 μᴍ linoleic acid dispersed in 200 mᴍ sodium borate, pH 9.0), rapid inactivation was observed with 5 mᴍ sodium borate, pH 8.0. The inactivation was protected by the addition of a substrate, linoleic acid, in the modification mixture. Kinetic analyses indicated that one arginine residue accounted for the inactivation. Enzymological analyses showed that the modification narrowed the pH-activity profile of L-1 and made L-1 sensitive to salt concentration of the assay solution. Strong inactivation by modification was found at low salt concentration and low pH. This was not due to a physical change of the linoleic acid. On the other hand, product specificity of L-1 was not altered after modification. Taken together, the modified arginine residue(s) was thought to be not essential to the catalysis but have an important role in supporting an ideal electrostatic interaction within L-1 and/or between L-1 and a substrate even in sub-optimal reaction conditions.

The distribution (or locarization) of lipoxygenase (LOX) and hydroperoxide lyase (HPO lyase) activities in ripening and ripe tomato fruits was investigated. The highest LOX activity existed between skin and outer flesh of tomato fruits. HPO lyase showed no tissue specificity. LOX specifically formed linoleic acid 9-E, Z-hydroperoxide (9-E, Z-HPO) from linoleic acid (LA), whereas HPO lyase specifically cleaved 13-Z, E-HPO. Although a low level (0.36 ± 0.069 nmol/g fr. wt.) of hexanal was detected in the intact tomato fruit, HPOs were not detected. When a tomato fruit was injured by cutting it into 8 fragments and incubated at 25 °C, hexanal increased to 1.642 nmol/g fr.wt. by 30 min. By homogenizing at pH 6.3, hexanal increased to 21.1 nmol/g fr.wt. during a 30 min incubation. UV irradiation of tomato fruits also increased the formation of hexanal. From these results, LOX and HPO lyase are considered to exist as latent forms and to begin the expression of the activity upon injury.

Lipoxygenases were highly purified from soybean seed, wheat seed and cucumber cotyledons. Substrate specificities of these lipoxygenases were studied by using an entire series of (ω6Z ,ω9Z )-C13~C24-dienoic acids as synthetic substrate analogues. Soybean lipoxygenase-1 and cucumber lipoxygenase showed broad specificities for these substrates while wheat lipoxygenase showed narrow specificities. Position of dioxygenation to each substrate was analyzed by high performance liquid chromatography. With soybean lipoxygenase-1 elongation of the distance between the terminal carboxyl group and the site of hydrogen removal in a substrate decreased the positional specificity of dioxygenation, while, with cucumber lipoxygenase, shortening the distance decreased the specificity. It was suggested that cucumber lipoxygenase and soybean lipoxygenase-1 recognized the terminal carboxyl group of a substrate to arrange it only in one orientation at the reaction center. In case of wheat lipoxygenase, recognition of the carboxyl group was thought to have crucial and essential role to secure the activity.

Substrate recognition mechanism of soybean lipoxygenase-1 (L -1) was studied by using an entire series of (ω3Z,ω6Z,ω9Z)-C12~C22-trienoic acids and -trienols. The maximum activity of L-1 was obtained with C20-trienoic acid of all the substrates used. Among trienols, the maximum activity was obtained with C15-trienol. Analysis of the products obtained by the reaction of these substrates with L-1 revealed that trienoic acids were oxygenated mostly at ω6 position independently on the chain length. The hydroperoxy group at ω6 was introduced at (S)-configuration with over 98% enantioselectivity. Although the products formed from most trienols were also selectively oxygenated at ω6 position, only C15-trienol was oxygenated at both ω6 and ω10 positions in the ratio of ω6/ω10 = 48/52.

Substrate specificity of tea leaf fatty acid hydroperoxide lyase was systematically investigated using an entire series of ω 6-(5)-hydroperoxy-C14-C24 dienoic and trienoic acids as substrates. Unexpectedly, the hydroperoxides of C22 but not natural substrates, i.e., those of C18, showed the highest reactivities for the lyase. The reactivities of the hydroperoxides of trienoic acids were always four to ten times higher than those of the dienoic acids.

Incubation of synthetic 12-hydroxy-(3 Z)-dodecenal (3 ZHD) with tea chloroplasts revealed that 12-hydroxy-(2 E)-dodecenal (2 EHD) was the product of isomerization reaction of 3 ZHD during the incubation. The isomerization was caused also by incubation of 3 ZHD with the heat-inactivated chloroplasts. The isomerization activity was extracted with hot ethanol. These results indicate that the isomerization was caused by heat-stable factor(s) but not by enzyme(s) in tea chloroplasts.

Substrate specificity of soybean lipoxygenase-1 (EC 1.13.11.12) was studied using as synthetic substrate analogs an entire series of (ω-6 Z,ω-9 Z)-C13 ~ C20 (rdienoic acids and (ω-6 Z,ω-9 Z)- C12 ~ C20 (i-dienols. The relative activity of lipoxygenase-1 against linoleic acid (C18:2COOH) was the highest among the dienoic acids and that of (6Z.9Z)-pentadecadienol (C15:2OH) was the highest among the dienols. Soybean lipoxygenase-1 oxygenated almost at to- 6 position of the dienoic acids independently of the chain length. With the dienols as substrate, the position of oxygenation was much influenced by the chain length. Above all, C15:2OH was selectively oxygenated at ω-10 rather than at ω-6 . Kinetic analyses revealed that longer and more hydrophobic dienols had higher affinity but lower velocity of oxygenation reaction. But higher velocity was obtained when smaller Km value was observed for the dienoic acids. To clucidate stereospecificity of the oxygenation to C15:2OH, optically active authentic samples were prepared via biological asymmetric reduction with baker’s yeast. The ω- 6 oxygenation to C15:2OH was found to be (S)-specific (R/S = 26/74) as is that to C18:2COOH. whereas the ω-10 oxygenation showed low enantioselectivity (R/S - 60/40).

A study on distribution of hydroperoxide lyase activities in cucumber seedlings was performed. Hydroperoxide lyase activities were at elevated level in root, and in the upper and greener sections the lower activities were observed.

In cucumber cotyledons, both C6 - and C9- aldehyde were formed via hydroperoxide (HPO) lyase activity. Because it has not been elucidated whether these activities are attributed to one enzyme which can cleave both 13-and 9-HPO or to two or more enzymes each of which specifically cleaves 13-or 9-HPO , an attempt to separate HPO lyase activity was done. Ion exchange chromatography separated this activity into two fractions, one of which specifically cleaved 13-hydroperoxylinoleic acid and the other specifically cleaved the 9-isomer. 13-HPO-specific activity was most active at pH 8.0 and 9-HPO-specific one was at pH 6.5. SH -reagents inhibited both the lyases but to different extents.