Photostable Abscisic Acid Agonists with a Geometrically Rigid Cyclized Side Chain

[14C]ABA fed to avocado fruit is known to be converted into the 1′,4′-trans-diol and [14C]1′,4′-trans-diol has been shown to be converted into ABA by several plant tissues. As a ‘cold trap’of trans-diol becomes labelled with 14C when [14C]mevalonate is converted into ABA, the trans-diol has been suggested to be the immediate precursor of ABA. This proposal has now been tested by feeding [5-14C,5-3H2]mevalonolactone to unripe avocado fruit and measuring the 3H :14C ratio in the ABA and in the 1′,4′-trans-diol isolated from the fruit after 16 h. Little labelled diol was present unless a ‘cold trap’ of unlabelled 1,4-trans-diol was added with the mevalonate. One 3H atom, derived from those at C-5 of mevalonate, would be expected at C-4′ of the diol, adjacent to the hydroxyl group, and another at C-5 of the side chain of the diol if the diol were a precursor of ABA (3H:14C ratio of 2:3). However, if the 4′-hydroxyl group had been oxidised to a ketone to form ABA, then the 3H atom at C-4′ of the diol would have been lost and the 3H:14C ratio would be expected to be 1:3. The normalised 3H : 14C ratios of ABA and 1′,4′-trans-diol biosynthesised from [14C,5-3H2]mevalonate were 0.915:3 and 0.844:3 respectively and after oxidation of the diol to ABA with MnO2 the ratio was 0.869:3 i.e. there was no 3H at C-4′ of the diol. These ratios are as expected for the trans-diol if it had been formed by reduction of ABA. This, and the absence of labelled diol in the fruit unless a ‘cold trap’was added, establishes that the 1′,4′-trans-diol is formed from ABA and it is not a precursor. The formation of the diols from newly synthesised labelled ABA in cell-free systems can be attributed to the addition to the homogenate of compounds with strong reducing potential. NADPH2+ (8.4 nmol) added to a mung bean seedling homogenate caused the reduction of (±)-[14C]ABA (0.37 nmol, 22.5 µCi/mol) to trans-diol (1189 dpm) whereas with NADP+ only 338 dpm were present in trans-diol. Glutathione (46 nmol) caused the formation of 1214 dpm while oxidised glutathione produced 638 dpm. Less 1′,4′-cis-diol was formed.

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The conformation of abscisic acid by n.m.r. and a revision of the proposed mechanism for cyclization during its biosynthesis

Biochemical Journal ◽

10.1042/bj2200325 ◽

1984 ◽

Vol 220 (1) ◽

pp. 325-332 ◽

Cited By ~ 26

Author(s):

B V Milborrow

Keyword(s):

Abscisic Acid ◽

Hydrogen Atom ◽

Pulse Sequence ◽

Chair Conformation ◽

Crystalline Form ◽

Side Chain ◽

Hydrogen Atoms ◽

Methyl Group ◽

Nuclear Overhauser Enhancement ◽

Nuclear Overhauser

The n.m.r. spectrum of abscisic acid (ABA) formed from [1,2-13C2]acetate by the fungus Cercospora rosicola shows 13C-13C coupling between C-6′ (41.7 p.p.m.; 36 Hz) and the downfield 6′-methyl group (6′-Me) (24.3 p.p.m, 36 Hz). This 6′-Me, therefore, is derived from C-3′ of mevalonate [Bennett, Norman & Maier (1981) Phytochemistry 20, 2343-2344]. An i.n.e.p.t. (insensitive nuclei enhanced by polarization transfer) pulse sequence demonstrated that the downfield 13C signal is produced by the 6′-Me that gives rise to the upfield 1H 6′-Me signal (23.1 d). The absolute configuration of this, the equatorial 6′-Me group, was determined as 6′-pro-R by decoupling and n.O.e. (nuclear-Overhauser-enhancement) experiments at 300 MHz using ABA, ABA in which the axial 6′-pro-S 5′-hydrogen atom had been exchanged with 2H in NaO2H and the 1′,4′-cis- and 1′,4′-trans-diols formed from these samples. The configuration at C-1′ and at C-6′ are now compatible with a chair-folded intermediate during cyclization, as proposed for beta- and epsilon-rings of carotenoids. ABA in solution exists, as in the crystalline form, with the ring in a pseudo-chair conformation. The side chain is axial and the C-3 Me and the C-5 hydrogen atoms are predominantly cis(Z).

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