Masahiko Nagaki

Artificial substrates for undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26

Abstract Substrate specificity of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26 was investigated with respect to some alkyl- and bromo-group homologs of isopentenyl diphosphate. Among the homologs relating to the 3-methyl group, but-3-enyl diphosphate ( 2b ) and 3-ethylbut-3-enyl diphosphate ( 3b ) were accepted as substrates, with (all- E )-farnesyl diphosphate (FPP) to give 7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate, and a mixture of 3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl- and 3,7-diethyleicosa-2,6,10,14,18-pentaenyl diphosphates, respectively. With respect to the homologs modified at the 4 position of isopentenyl diphosphate, (4 E )-3-methylpent-3-enyl diphosphate ( 2f ) was accepted as a substrate to give (4 S )-(2 Z ,6 E ,10 E ,14 E )-4-methylgeranylgeranyl- and (4 S ,8 S )-(2 Z ,6 Z ,10 E ,14 E ,18 E )-4,8-dimethylgeranylfarnesyl diphosphates. Neither (4 Z )-3-methylpent-3-enyl diphosphate nor 4-bromo-3-methylbut-3-enyl diphosphates was accepted as a substrate at all.

Two sesquiterpene lactones from artemisia species

Abstract Two new sesquiterpene lactones, himeyoshin and montanone, were isolated from Artemisia feddei and A. montana , respectively and identified by chemical and spectral data as1α,2α-epoxy-3-oxo-5,6-dihydroalantolactone and 1,10-dihydro-11,13-dehydromatricarin.

Guaianolides from Artemisia montana

Abstract From the aerial parts of Artemisia montana (‘Ezo-yomogi’ in Japanese), two new sesquiterpene lactones, neozeoguaianin and ezoyomoginin were isolated together with five known guaianolides, yomogiartemin, ezoartemin, yamayomoginin, ezomontanin, and 11,13-dihydroezomontanin. The structures of new compounds were established mainly by high field 1 H NMR spectroscopic method.

Intramolecular exciplex formation and complexing behavior of 1-(2-naphthalenecarboxy)-n-(p-substituted benzenecarboxy)oxaalkanes as fluorescent chemosensors for calcium and barium ions

Abstract 1-(2-Naphthalenecarboxy)- n -( p -substituted benzenecarboxy)oxaalkanes (2NP n X, X=H, Cl, CF 3 , CN; n =5, 6) as fluorescent sensors by exciplex emission for metal ions were synthesized. We investigated the relationship between the substitution effects and the metal ion recognition. Before the metal salts were added, the substitution effect was found. The fluorescence quantum yields ( Φ f ) decrease as the electronegativity of the p -substituent of the acceptor benzoate increases. 2NP n CN ( n =5, 6) with the most negative free energy of electron transfer (Δ G ET ) showed the most efficient quenching and exciplex emission. When Li + , Na + , K + and Mg 2+ were added to the acetonitrile solution of 2NP n X (X=H, Cl, CF 3 , CN; n =5, 6), the shape and intensity of the fluorescence spectra did not change. However, the spectra of all the 2NP n Xs significantly changed with the addition of Ca 2+ and Ba 2+ . Especially, due to the large fluorescent enhancement factor of 2NP n CN ( n =5, 6) for Ca 2+ , they are well-suited for use as a fluorescent chemosensor for Ca 2+ .

Substrate Specificities of Several Prenyl Chain Elongating Enzymes with Respect to 4-Methyl-4-pentenyl Diphosphate

In order to develop synthetic methods for biologically active homoallylic terpene sulfates, we examined the applicability and substrate specificities of several prenyl chain elongating enzymes with respect to 4-methyl-4-pentenyl diphosphate (homoIPP). The reaction of dimethylallyl diphosphate with homoIPP by use of Bacillus stearothermophilus (all-trans)-farnesyl diphosphate synthase resulted in efficient yields of cis-(yield: 45.9%) and trans-4,8-dimethylnona-3,7-dien-1-ol (homoGOH, 25.5%), which has a carbon skeleton of 4,8-dimethylnona-3-en-1-sulfate, an antiproliferative compound from a marine organism (Aiello, A. et al., Tetrahedron, 53, 11489–11492 (1997)). The homoIPP was found to be also active as a homoallylic substrate in place of isopentenyl diphosphate for Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase to give diphosphate of cis- and trans-4,8,12-trimethyltrideca-3,7,11-trien-1-ol, for Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase to give cis- and trans-4,8,12,16-tetramethylheptadeca-3,7,11,15-tetraen-1-ol (homoGGOH), and for Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase to give cis-homoGGOH exclusively.

Substrate specificity of thermostable farnesyl diphosphate synthase with alkyl group homologs of isopentenyl diphosphate

3-Alkyl group homologs of isopentenyl diphosphate were examined for the reactivity as substrates of the thermostable farnesyl diphosphate (FPP) synthase of Bacillus stearothermophilus. Even 3-n-propyl- and 3-n-butyl-but-3-enyl diphosphates, which are hardly acceptable by animal FPP synthases, are accepted by this bacterial enzyme as substrates to react with dimethylallyl- and geranyl diphosphates, yielding 7-methyl-3-n-propylocta-2,6-dienyl- and 7,11-dimethyl-3-n-propyldodeca-2,6,10-trienyl diphosphate, respectively.

Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates.

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH3OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH3OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.

Substrate specificity of thermostable farnesyl diphosphate synthase with respect to 4-alkyl group homologs of isopentenyl diphosphate

In order to investigate substrate spcificity of Bacillus stearothermophilus farnesyl diphosphate synthase (FPS), we examined the reactivity of 4-alkyl group homologs of isopentenyl diphosphate (IPP). The enzymatic reactions of the 4-methyl homologs, (E)-3-methylpent-3-enyl diphophates (1a) and (Z)-3-methylpent-3-enyl diphophates (1b) with geranyl diphosphate (GPP) gave 4-methylfarnesyl diphosphates (2a and 2b), respectively. The stereochemistry of each aldehyde derived from 2a or 2b was determined by CD spectrometry to be (S)-4-methylfarnesal or (R)-4-methylfarnesal, respectively. Similarly, 1a reacted with dimethylallyl diphosphate (DMAPP) to give a mixture of (4S)-4-methylgeranyl diphosphates (3a) and (4S,8S)-4,8-dimethylfarnesyl diphosphates (4a). The (Z)-isomer 1b also reacted with DMAPP to give the corresponding enantiomers with (4R)- and (4R,8R)-configurations. On the other hand, reactions of the 4-ethyl homologs, (E)-3-methylhex-3-enyl diphosphates (1c) and (Z)-3-methylhex-3-enyl diphosphates (1d) with GPP gave two types of 4-ethylfarnesyl diphosphates. Reactions of 1c or 1d with DMAPP also gave two types of 4-ethylgeranyl- and 4,8-diethylfarnesyl diphosphates. Meanwhile, reaction of the 4-propyl homologs, (E)-3-methylhept-3-enyl diphosphates (1e) and (Z)-3-methylhept-3-enyl diphosphates (1f) with GPP gave two types of 4-propylfarnesyl diphosphates. Reactions of 1e or 1f with DMAPP gave only two types of the 4-propyl GPPs. However, neither (E)-3-methyloct-3-enyl diphosphates (1g) or (Z)-3-methyloct-3-enyl diphosphates (1h), nor (E)-4-bromo-3-methylbut-3-enyl diphosphate (1i) or (Z)-4-bromo-3-methylbut-3-enyl diphosphate (1j) was acceptable as a substrate for the thermophilic FPS at all.

Artificial substrates of medium-chain elongating enzymes, hexaprenyl- and heptaprenyl diphosphate synthases

We examined the reactivity of 3-alkyl group homologues of farnesyl diphosphate or isopentenyl diphosphate for medium-chain prenyl diphosphate synthases, hexaprenyl diphosphate- or heptaprenyl diphosphate synthase. But-3-enyl diphosphate, which lacks the methyl group at the 3-position of isopentenyl diphosphate, condensed only once with farnesyl diphosphate to give E-norgeranylgeranyl diphosphate by the action of either enzyme. However, norfarnesyl diphosphate was never accepted as an allylic substrate at all. 3-Ethylbut-3-enyl diphosphate also reacted with farnesyl diphosphate giving a mixture of (all-E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl- and (all-E)-3,7-diethyl-11,15,19-trimethylicosa-2,6,10,14,18-pentaenyl diphosphates by hexaprenyl diphosphate synthase. On the other hand, heptaprenyl diphosphate synthase reaction of 3-ethylbut-3-enyl diphosphate with farnesyl diphosphate gave only (all-E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate.

One-pot syntheses of the sex pheromone homologs of a codling moth, Laspeyresia promonella L.

In order to carry out one-pot syntheses of the sex pheromone homologs of a codling moth, Laspeyresia promonella L. by use of a thermophilic enzyme, we have examined the reactivities of 3-methylhex-2-enyl diphosphates as an allylic substrate and of some 3-alkyl homologs of isopentenyl diphosphate in the reaction catalyzed by farnesyl diphosphate (FPP) synthase of Bacillus stearothermophilus. 3-methylhex-2-enyl diphosphate (1a) was accepted as a substrate with 3-ethylbut-3-enyl diphosphate (2b) to give a mixture of (2E,6E)-3-ethyl-7-methyldeca-2,6-dienyl- (3b) and (2E, 6E, 10E)-3,7-diethyl-11-methyltetradeca-2,6,10-trienyl diphosphate (4b). 1a was also reacted with 3-propylbut-3-enyl diphosphate (2c) to give exclusively (2E,6E)-7-methyl-3 propyldeca-2,6-dienyl diphosphate (3c). On the other hand, Z-3-methylhex-2-enyl diphosphate (1b) was accepted as a substrate with 2b to give a mixture of (2E,6Z)-3-ethyl-7-methyldeca-2,6-dienyl- (3′b) and (2E,6E,10Z)-3,7-diethyl-11-methyltetradeca-2,6,10-trienyl diphosphate (4′b). The Z-isomer (1b) was reacted with 2c to give (2E,6Z)-7-methyl-3 propyldeca-2,6-dienyl diphosphate (3′c). On the basis of these reactivities, one-pot enzymatic syntheses of the sex pheromone homologs of the codling moth, 3c-OH and 3′c-OH, were carried out by the action of the FPP synthase, followed by an alkaline phosphatase, with yields of 9.6% and 0.6%, respectively.