Yasushi Todoroki

Biosynthesis of Abscisic Acid by the Non-mevalonate Pathway in Plants, and by the Mevalonate Pathway in Fungi

The biosynthetic pathways to abscisic acid (ABA) were investigated by feeding [1-13C]-D-glucose to cuttings from young tulip tree shoots and to two ABA-producing phytopathogenic fungi. 13C-NMR spectra of the ABA samples isolated showed that the carbons at 1, 5, 6, 4′, 7′ and 9′ of ABA from the tulip tree were labeled with 13C, while the carbons at 2, 4, 6, 1′, 3′, 5′, 7′, 8′ and 9′ of ABA from the fungi were labeled with 13C. The former corresponds to C-1 and -5 of isopentenyl pyrophosphate, and the latter to C-2, -4 and -5 of isopentenyl pyrophosphate. This finding reveals that ABA was biosynthesized by the non-mevalonate pathway in the plant, and by the mevalonate pathway in the fungi. 13C-Labeled β-carotene from the tulip tree showed that the positions of the labeled carbons were the same as those of ABA, being consistent with the biosynthesis of ABA via carotenoids. Lipiferolide of the tulip tree was also biosynthesized by the non-mevalonate pathway.

8′,8′-Difluoro- and 8′,8′,8′-trifluoroabscisic acids as highly potent, long-lasting analogues of abscisic acid

Abstract Racemic 8′,8′-difluoroabscisic acid (difluoro-ABA) and 8′,8′,8′-trifluoroabscisic acid (trifluoro-ABA) were synthesized as highly potent, long-lasting analogues of abscisic acid (ABA). The individual optical isomers were obtained by optical resolution of the racemic mixture by HPLC with a chiral column. (+)-8′,8′-Difluoro-ABA and (+)-8′,8′,8′-trifluoro-ABA inhibited the elongation of rice seedlings six and 30 times more strongly, respectively, than (+)-ABA. These analogues also showed double the (+)-ABA-induced inhibition of lettuce seed germination. In causing stomatal closure and inhibiting the induction of α-amylase by gibberellin A 3 , these analogues were equally as effective as (+)-ABA. The high activity in the assays over a long period suggested that the metabolism of difluoro- and trifluoro-ABAs was delayed. (−)-Enantiomers were equal to, or weaker than (−)-ABA in the assays.

Synthesis, biological activity and metabolism of (S)-(+)-3′-Fluoroabscisic acid

Abstract (S)-(+)-3′-Fluoroabscisic acid (5a) was synthesized as an analogue of abscisic acid that can resist the metabolic cyclization of 8′-hydroxyabscisic acid to phaseic acid due to increased electron density at C-2′. The inhibitory activity of 5a was slightly higher than that of abscisic acid in lettuce seed germination, and was almost equal to that of abscisic acid in rice seedling elongation. When 5a was applied to bean shoots, 3′-fluoro-8′-hydroxyabscisic acid (8a), 3′α- and 3′β-fluorophaseic acids (9a and 10a) and 3′α- and 3′β-fluorodihydrophaseic acids (11a and 12a) were identified as the free metabolites. The methyl esters 8b, 9b and 10b coexisted at equilibrium in the ratio of 7:6:1. This result indicates that 8a is stable as back-isomerization of 9a and 10a occurs.

RING CONFORMATIONAL REQUIREMENT FOR BIOLOGICAL ACTIVITY OF ABSCISIC ACID PROBED BY THE CYCLOPROPANE ANALOGUES

Abstract For investigating ring conformational requirement for the biological activity of abscisic acid, the cyclopropane analogues 6, 7, 9 , and 10 were synthesized and their biological activities in four bioassays were tested. The activity of the achiral cyclohexadienone analogue 8 also was examined. Analogue 7 in which the 6′ β-substituent is constrained essentially to the axial-like orientation between axial and bisectional showed no activity, while 6 and 8 with no axial-like substituent at C-6′β independent of the conformational preference exhibited the equivalent activity to abscisic acid. This result suggested that the axial substituent at the β-side of the ring is fatal to the activity. The active conformation of abscisic acid would be a conformation where C-9′ is equatorial and the side chain is between pseudo-axial and bisectional, that is, close to the favored half-chair with the side chain pseudo-axial rather than the less favored half-chair with the side chain pseudo-equatorial.

Synthesis and biological activity of 3′-chloro, -bromo, and -iodoabscisic acids, and biological activity of 3′-fluoro-8′-hydroxyabscisic acid

Abstract (+)-3′-Chloro, -bromo and -iodoabscisic acids were synthesized, and their biological activities and metabolism were examined, as well as that of (+)-3′-fluoroabscisic acid. This was in order to estimate the effects of altered electron density at the C-2′ position of abscisic acid on both biological activity and metabolic stability. The biological activities of (+)-3′-chloro and -bromoabscisic acids were similar to, or higher than, those of (+)-abscisic acid in four bioassays, while (+)-3′-iodoabscisic acid was more potent in two bioassays and less potent than (+)-abscisic acid in the other two bioassays. The biological activities of 3′-haloabscisic acids seem to be dependent on the 3′-halogens, but not on the electron density at C-2′. The metabolism of (+)-3′-haloabscisic acids to ca. 50% of their original levels in the media of rice cell suspension culture was similar to that of (+)-abscisic acid. Metabolites from (+)-3′-chloro, -bromo and -iodoabscisic acids were not found in the culture media and cells, while (+)-3′-fluoroabscisic acid gave (+)-3′-fluoro-8′-hydroxyabscisic acid as a stable metabolite. (+)-3′-fluoro-8′-hydroxyabscisic acid showed biological activities lower than (+)-3′-fluoroabscisic acid, but higher than (−)-3′α-Fluorophaseic acid. These findings suggested that the biological activity of (+)-8′-hydroxyabscisic acid is intermediate between those of (+)-abscisic acid and (−)-phaseic acid. Abscisic acid may be inactivated in a stepwise manner by metabolism in plants.

Analysis of Isomerization Process of 8′-Hydroxyabscisic Acid and its 3′-Fluorinated Analog in Aqueous Solutions

Abstract 8′-Hydroxyabscisic acid (8′-HOABA), the first metabolite of abscisic acid (ABA) in plants, is spontaneously isomerized to low bioactive phaseic acid (PA). We investigated thermodynamic and kinetic properties of the isomerization process in aqueous solution buffered at the various pHs, along with the effects of 3′-fluorine. The 8′-HOABA/PA ratio at equilibrium was 2:98 at 25°C, while the 3′-fluoro-8′HOABA/3′α-fluoro-PA ratio was 16:84, indicating that introduction of a fluorine at C-3′ thermodynamically reduced isomerization of 8′-HOABA to PA. The isomerization became more rapid as pH increased; the rate constant at pH 10 was higher than that at pH 3 by a factor of 2000. Introduction of a fluorine at C-3′ reduced the reaction rate by raising the activation enthalpy. This indicated that 8′-HOABA was also kinetically stabilized by the 3′-fluorine. These findings suggested that the control of pH and modification of the enone moiety of the ring would be useful to manipulate the catabolic inactivation rate of ABA.

Biological Activities of Abscisic Acid Analogs in the Morphological Change of the Green Alga Haematococcus pluvialis

Abstract The plant hormone abscisic acid (ABA) induces a morphological change from green vegetative cells to red cyst cells, of Haematococcus pluvialis containing carotenoids, in plate culture. Long-lasting analogs of ABA, (+)-8′,8′,8′-trifluoro and (+)-8′,8′-difluoro-ABAs, which can resist metabolic inactivation, induce carotenoid production at 100-fold lower concentration than (+)-ABA. These analogs can be used as effective regulators to produce carotenoids in H. pluvialis cells.