A new non-azole inhibitor of ABA 8'-hydroxylase: effect of the hydroxyl group substituted for geminal methyl groups in the six-membered ring

We designed and synthesized AHI4 that has an axial hydroxyl group instead of geminal methyl groups at C-6' of AHI1, previously reported as a lead compound for the development of non-azole inhibitors of ABA 8'-hydroxylase. (+)-AHI4 competitively inhibited 8'-hydroxylation of ABA by recombinant CYP707A3. The K(I) value was found to be 0.14 microM, 10-fold less than that of (+)-AHI1, suggesting that enzyme affinity increased by a factor of 10 due to substitution of the hydroxyl group by the geminal methyls at C-6'. This finding should assist in the design of more effective, non-azole ABA 8'-hydroxylase inhibitors.     

Expression of ABA 8'-hydroxylases in relation to leaf water relations and seed development in bean

In plants, the level of abscisic acid (ABA) is determined by synthesis and catabolism. Hydroxylation of ABA at the 8' position is the key step in ABA catabolism. This reaction is catalyzed by ABA 8'-hydroxylase, a cytochrome P450 (CYP). The cDNAs of PvCYP707A1 and PvCYP707A2 were isolated from bean (Phaseolus vulgaris L.) axes treated with (+)-ABA and that of PvCYP707A3 from dehydrated bean leaves. The recombinant PvCYP707A proteins expressed in yeast were biochemically characterized. Yeast strains over-expressing any of the three PvCYP707As were able to convert ABA to phaseic acid (PA). The microsomal fractions from these yeast strains also exhibited ABA 8'-hydroxylase activity. Expression of PvCYP707A3 in primary leaves was strongly increased by water stress, whereas PvCYP707A1 and PvCYP707A2 mRNA levels were rapidly increased by rehydration of water-stressed leaves. Northern blot analysis of PvCYP707As in bean showed a high level of expression in the mature fruits, senescent leaves, roots, seed coats and axes. All three PvCYP707As were expressed at varying intensities throughout seed development. Imbibed seeds also had high PvCYP707A mRNA levels. Thus, expression of PvCYP707As is both environmentally and developmentally regulated. Transgenic Nicotiana sylvestris plants over-expressing PvCYP707As displayed a wilty phenotype, and had reduced ABA levels and increased PA levels. These results demonstrate that expression of PvCYP707As is the major mechanism by which ABA catabolism is regulated in bean.     

Effect of the minor ABA metabolite 7'-hydroxy-ABA on Arabidopsis ABA 8'-hydroxylase CYP707A3

To examine the effect of the minor abscisic acid (ABA) metabolite 7'-hydroxy-ABA on Arabidopsis ABA 8'-hydroxylase (CYP707A3), we developed a novel and facile, four-step synthesis of 7'-hydroxy-ABA from alpha-ionone. Structural analogues of 7'-hydroxy-ABA, 1'-deoxy-7'-hydroxy-ABA, and 7'-oxo-ABA were also synthesized to evaluate the role of the 7'-hydroxyl group on binding to the enzyme. The result of enzyme inhibition assay suggests that the local polarity at C-7', neither steric bulkiness nor overall molecular hydrophilicity, would be the major reason why (+)-7'-hydroxy-ABA is not a potent inhibitor of CYP707A3.     

Stereochemistry during aflatoxin biosynthesis: cyclase reaction in the conversion of versiconal to versicolorin B and racemization of versiconal hemiacetal acetate.

(1'R,2'S)-(-)-aflatoxins are produced from racemic versiconal hemiacetal acetate (VHA) through complicated pathways, including a metabolic grid involving VHA, versiconol acetate (VOAc), versiconol, and versiconal (VHOH), and a reaction sequence from VHOH to versicolorin A (VA) through (-)-versicolorin B (VB) [or (+/-)-versicolorin C] (K. Yabe, Y. Ando, and Y. Hamasaki, J. Gen. Microbiol. 137:2469-2475, 1991; K. Yabe, Y. Ando, and T. Hamasaki, Agric. Biol. Chem. 55:1907-1911, 1991). In this study, we examined stereochemical changes of substances formed during the conversion of VHA to VA by using chiral high-performance liquid chromatography. In cell-free experiments using the cytosol of Aspergillus parasiticus NIAH-26, both (2'S)- and (2'R)-VOAc enantiomers were formed at about a 1:2 ratio from racemic VHA in the presence of NADPH and dichlorvos (dimethyl 2,2-dichlorovinylphosphate). Also, the esterase activity catalyzing the conversion of VHA to VHOH or of VOAc to versiconol did not show the stereospecificity for the 2' carbon atom of VHA or VOAc. However, when racemic VHA or racemic VHOH was incubated with the cytosol, (1'R,2'S)-(-)-VB was formed exclusively. Furthermore, only (1'R,2'S)-(-)-VB, and not (1'S,2'R)-(+) antipode, served as a substrate for desaturase activity in the microsome fraction catalyzing the conversion of VB to VA. These results demonstrate that the stereoconfiguration of bis-furan moiety in aflatoxin molecules is determined by the cyclase enzyme catalyzing the reaction from VHOH to VB, and the (1'R,2'S)-(-) configuration was further confirmed by the subsequent desaturase reaction. Remarkably, we found nonenzymatic racemization in both the (2'R)- and (2'S)-VHA enantiomers, and it was dependent upon the temperature and alkaline conditions.     

Inhibitors of abscisic acid 8'-hydroxylase

Structural analogues of the phytohormone (+)-abscisic acid (ABA) have been synthesized and tested as inhibitors of the catabolic enzyme (+)-ABA 8'-hydroxylase. Assays employed microsomes from suspension-cultured corn cells. Four of the analogues [(+)-8'-acetylene-ABA, (+)-9'-propargyl-ABA, (-)-9'-propargyl-ABA, and (+)-9'-allyl-ABA] proved to be suicide substrates of ABA 8'-hydroxylase. For each suicide substrate, inactivation required NADPH, increased with time, and was blocked by addition of the natural substrate, (+)-ABA. The most effective suicide substrate was (+)-9'-propargyl-ABA (K(I) = 0.27 microM). Several analogues were competitive inhibitors of ABA 8'-hydroxylase, of which the most effective was (+)-8'-propargyl-ABA (K(i) = 1.1 microM). Enzymes in the microsomal extracts also hydroxylated (-)-ABA at the 7'-position at a low rate. This activity was not inhibited by the suicide substrates, showing that the 7'-hydroxylation of (-)-ABA was catalyzed by a different enzyme from that which catalyzed 8'-hydroxylation of (+)-ABA. Based on the results described, a simple model for the positioning of substrates in the active site of ABA 8'-hydroxylase is proposed. In a representative physiological assay, inhibition of Arabidopsis thaliana seed germination, (+)-9'-propargyl-ABA and (+)-8'-acetylene-ABA exhibited substantially stronger hormonal activity than (+)-ABA itself.     

A lead compound for the development of ABA 8'-hydroxylase inhibitors

(1'S*,2'S*)-(+/-)-6-Nor-2',3'-dihydro-4'-deoxo-ABA (2) was designed and synthesized as a candidate lead compound for developing a potent and specific inhibitor of ABA 8'-hydroxylase. This compound acted as an effective competitive inhibitor of the enzyme, with a K(I) value of 0.40microM, without exhibiting ABA activity. However, compound 2 also functioned as an enzyme substrate, making it a short-lived inhibitor. The 8'-difluorinated derivative of 2 (4) was synthesized as a long-lasting alternative. Compound 4 resisted 8'-hydroxylation, but inhibited ABA 8'-hydroxylation as effectively as 2. These results suggest that compound 2 is a useful lead compound for the future design and development of an ideal ABA 8'-hydroxylase inhibitor.     

A plant growth retardant, uniconazole, is a potent inhibitor of ABA catabolism in Arabidopsis

Plant growth retardants (PGRs) reduce the shoot growth of plants by inhibiting gibberellin biosynthesis. In this study, we performed detailed analyses of the inhibitory effects of PGRs on Arabidopsis abscisic acid (ABA) 8'-hydroxylase, a major ABA catabolic enzyme, recently identified as CYP707As. In an in vitro assay with CYP707A3 microsomes expressed in insect cells, uniconazole-P inhibited CYP707A3 activity more effectively than paclobutrazol or tetcyclacis, whereas the other PGRs tested did not inhibit it significantly. Uniconazole-P was found to be a strong competitive inhibitor (K(i)=8.0 nM) of ABA 8'-hydroxylase. Uniconazole-P-treated Arabidopsis plants showed enhanced drought tolerance. In uniconazole-P-treated plants, endogenous ABA levels increased 2-fold as compared with the control, and co-application of GA(4) did not suppress the effects, indicating that the effects were not due to gibberellin deficiency. Thus uniconazole-P effectively inhibits ABA catabolism in Arabidopsis plants. We also discuss the structure-activity relationship of the azole-type compounds on ABA 8'-hydroxylase inhibitory activity.     

Carbocyclic 5'-noruridine

A convenient preparation of (1'R,2'S,3'R,4'S)-1-(2',3',4'-trihydroxycyclopent-1'-yl)-1H-uracil (carbocyclic 5'-noruridine, 1) is described in 2 steps from the palladium complex of (+)-(1R,4S)-4-hydroxy-2-cyclopenten-1-yl acetate (3) and the sodium salt of uracil (2). Compound 1 was sought as a previously unknown member of the series of carbocyclic 5'-nor nucleosides needed as moieties for new oligomers. With 1 available, its antiviral properties and those of its enantiomer (5) are reported with 5 showing promising activity towards Epstein-Barr virus.     

Carotenoids with a 5,6-dihydro-5,6-dihydroxy-beta-end group, from yellow sweet potato "Benimasari", Ipomoea batatas Lam

A series of carotenoids with a 5,6-dihydro-5,6-dihydroxy-beta-end group, named ipomoeaxanthins A (1), B (2), C1 (3) and C2 (4) were isolated from the flesh of yellow sweet potato "Benimasari", Ipomoea batatas Lam. Their structures were determined to be (5R,6S,3'R)-5,6-dihydro-beta,beta-carotene-5,6,3'-triol (1), (5R,6S,5'R,6'S)-5,6,5',6'-tetrahydro-beta,beta-carotene-5,6,5'6'-tetrol (2), (5R,6S,5'R,8'R)-5',8'-epoxy-5,6,5',8'-tetrahydro-beta,beta-carotene-5,6-diol (3), and (5R,6S,5'R,8'S)-5',8'-epoxy-5,6,5',8'-tetrahydro-beta,beta-carotene-5,6-diol (4) by UV-Vis, NMR, MS and CD data.     

Changes in ABA turnover and sensitivity that accompany dormancy termination of yellow-cedar (Chamaecyparis nootkatensis) seeds.

Yellow-cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds exhibit prolonged coat-imposed dormancy following their dispersal from the parent plant. Analyses were undertaken using S-(+)-[(3)H] abscisic acid (ABA) to monitor the capacity of embryos to metabolize ABA following their isolation from seeds subjected to various dormancy-breaking and control treatments. Radiolabelled phaseic acid (PA) and dihydrophaseic acid (DPA) were detected in embryos and, to a greater extent in the surrounding media, by 48 h regardless of whether the embryos had been excised from seed previously subjected to only a 3 d soak or to a full dormancy-breaking treatment. Of the two enantiomers of ABA, only the natural S-(+)-ABA effectively inhibited germination of isolated embryos. A metabolism-resistant synthetic ABA analogue S-[8',8',8',9',9',9']-hexadeuteroabscisic acid, S-(+)-d6-ABA, consistently slowed the germination rate of excised embryos to a greater extent than that caused by natural S-(+)-ABA. The deuterium-labelled ring methyl groups of the analogue made it more resistant to oxidation by yellow-cedar embryos and thus rendered the analogue more persistent and possessing greater activity. With increasing time of exposure to moist chilling, yellow-cedar embryos became increasingly insensitive to both ABA and to the analogue. Subjecting seed to chemical treatments (GA(3) in combination with 1-propanol) prior to moist chilling strongly enhanced the germinability of whole seeds. This treatment also had a relatively greater impact on ABA metabolism than did moist chilling alone, as indicated by a greater capacity of S-(+)-d6-ABA to inhibit the germination of embryos as compared to S-(+)-ABA. Moist chilling was most critical for reduced ABA sensitivity of embryos. A change in the embryo's ability to metabolize ABA and reduced embryo sensitivity to ABA are two factors associated with dormancy termination of whole seeds of yellow cedar; a change in only one of these factors is insufficient to elicit high germinability.     

Characterization of genes encoding ABA 8'-hydroxylase in ethylene-induced stem growth of deepwater rice (Oryza sativa L.)

Ethylene and submergence enhance stem elongation of deepwater rice, at least in part, by reducing in the internode the endogenous abscisic acid (ABA) content and increasing the level of gibberellin A1 (GA1). We cloned and characterized the CYP707A5 and CYP707A6 genes, which encode putative ABA 8'-hydroxylase, the enzyme that catalyzes the oxidation of ABA. Expression of CYP707A5 was upregulated significantly by ethylene treatment, whereas that of CYP707A6 was not altered. Recombinant proteins from both genes expressed in yeast cells showed activity of ABA 8'-hydroxylase. This finding indicates that CYP707A5 may play a role in ABA catabolism during submergence- or ethylene-induced stem elongation in deepwater rice. Taken together, these results provide links between the molecular mechanisms and physiological phenomena of submergence- and ethylene-induced stem elongation in deepwater rice.     

Differences between the structural requirements for ABA 8'-hydroxylase inhibition and for ABA activity

A major catabolic enzyme of the plant hormone abscisic acid (ABA) is the cytochrome P450 monooxygenase ABA 8'-hydroxylase. For designing a specific inhibitor of this enzyme, the substrate specificity and inhibition of CYP707A3, an ABA 8'-hydroxylase from Arabidopsis thaliana, was investigated using 45 structural analogues of ABA and compared to the structural requirements for ABA activity. Substrate recognition by the enzyme strictly required the 6'-methyl groups (C-8' and C-9'), which were unnecessary for ABA activity, whereas elimination of the 3-methyl (C-6) and 1'-hydroxyl groups, which significantly affected ABA activity, had little effect on the ability of analogues to competitively inhibit the enzyme. Fluorination at C-8' and C-9' resulted in resistance to 8'-hydroxylation and competitive inhibition of the enzyme. In particular, 8',8'-difluoro-ABA and 9',9'-difluoro-ABA yielded no enzyme reaction products and strongly inhibited the enzyme (K(I) = 0.16 and 0.25 microM, respectively).     

Reversible cleavage of the cobalt-carbon bond to coenzyme B12 catalysed by methylmalonyl-CoA mutase from Propionibacterium shermanii. The use of coenzyme B12 stereospecifically deuterated in position 5'

1. (5'R)-(5'-2H1)Adenosine [(5'R):(5'S) = 85:15] was prepared by a procedure which involved inter alia the reduction of 6-N-benzoyl-2',3'-O-isopropylidene-5'-oxoadenosine with a reagent obtained from LiAl2H4 and (-)-isoborneol. 2. (5'S)-(5'-2H1)AdoCbl [(5'S):(5'R) = 74:26] (AdoCbl = 5'-deoxyadenosylcobalamin) was synthesized by reacting cobal(I)amin with (5'R)-2'-3'-O-isopropylidene-5'-tosyl-(5'-2H1) adenosine followed by acid hydrolysis to remove the isopropylidene protective group. 3. (5'R)-(5'-2H1)AdoCbl [(5'R):(5'S) = 77:23] was prepared by reacting cobalt(I)amin with (5'S)-5'-chloro-5'-(5'-2H1)deoxyadenosine [(5'S):(5'R) = 80:20] obtained in turn from (5'R)-(5'-2H1)adenosine. The reaction sequence involved two consecutive inversions at the C-5' atom of adenosine 4. Comparison of the 500-MHz 1H-NMR spectra of unlabelled, (5'S)- and (5'R)-(5'-2H1)AdoCbl allowed assignment of the triplet at 0.58 ppm and the doublet at 1.525 ppm to the diastereotopic 5'-HRe and 5'-HSi atoms, respectively. On acidification, these two protons gave rise to two triplets at 0.11 ppm and 1.78 ppm indicating that torsion had occurred around the C-4'--C-5' bond. 5. Samples of (5'R)- and (5'S)-(5'-2H1)AdoCbl were incubated with methylmalonyl-CoA mutase from Propionibacterium shermanii. Examination by 1H-NMR spectroscopy at 500 MHz revealed partial loss and stereochemical scrambling of the deuterium at the 5' position. This indicates transient conversion of the C-5' atom into a torsiosymmetric group and hence cleavage of the cobalt-carbon bond during interaction with the enzyme. The mechanism by which deuterium is lost remains to be elucidated.     

Application of antibody-mediated extraction for the stereoselective determination of the active metabolite of loxoprofen in human and rat plasma.

Antibody-mediated extraction followed by chiral high-performance liquid chromatography (HPLC) was applied to stereoselective determination in human and rat plasma of the active metabolite [(2S,1'R,2'S)-trans-alcohol] with three chiral centers of Loxoprofen, a 2-arylpropionic acid antiinflammatory agent after oral administration. Antiserum against the (1'R,2'S)-cyclopentanol moiety was obtained from a rabbit immunized with bovine serum albumin conjugate linked to the propionic acid moiety, in which another chiral center is located. Then, the immunoglobulin G purified by a protein A column was coupled to BrCN-activated Sepharose 4B. Plasma samples were applied to the immobilized antibody column. After washing the column to remove unrequired stereoisomers, a mixture of two diastereomers whose configurations were 1'R,2'S in the cyclopentanol moiety was extracted with 95% methanol. The solvent was evaporated and the residue was derivatized with (+)-(R)-1-(1-naphthyl)ethylamine as a chiral reagent to separate the diastereomers by HPLC. This combined analytical method showed the stereoselective metabolism of Loxoprofen in human, that is, 64% of the total amount of four trans-alcohol stereoisomers was in the 2S,1'R,2'S form, which is the active metabolite. This phenomenon was also observed in rats given Loxoprofen and its (2S)- and (2R)-isomers, and is explained by stereoselective ketone reduction of Loxoprofen to the (1'R,2'S)-trans-alcohol and inversion from 2R to 2S in the propionic acid moiety. Antibody-mediated extraction should be a selective and simple clean-up method for determining haptens with complicated structures, combined with an appropriate analytical method.     

Synthesis of epimers of L-cyclopentenylglycine using enzymatic resolution

Both epimers of the naturally occurring nonproteinogenic amino acid L-cyclopentenylglycine, (2S,1'S)- and (2S, 1'R)-2-(cyclopent-2'-enyl)glycine, were obtained via a procedure involving condensation of 3-chlorocyclopentene with diethyl acetylaminomalonate, deethoxycarbonylation, chromatographic separation of the resulting two pairs of enantiomers, and enzymatic resolution of the racemates employing enantioselective hydrolysis of the ethyl ester group with alpha-chymotrypsin. The method was used for preparation of (13)C-labeled compounds of interest for biosynthetic tracer experiments. Enantiomeric purity of the products was determined by chiral HPLC on a Crownpak CR(+) column. The biologically active (2S,1'R) isomer was obtained as a pure compound and characterized for the first time. The (2R,1'R) and (2R,1'S) isomers were obtained as N-acetyl ethyl ester derivatives.     

The Arabidopsis cytochrome P450 CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism

The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8'-hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8'-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1-CYP707A4, encode ABA 8'-hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.     

Partial synthesis of serum carotenoids and their metabolites

Human serum and tissues contain in excess of 12 dietary carotenoids and several metabolites that originate from consumption of fruits and vegetables. Among these are hydroxycarotenoids: (3R,3'R,6'R)-lutein (1), (3R,3'R)-zeaxanthin (2), (3R,6'R)-α-cryptoxanthin (3), and (3R)-β-cryptoxanthin (4). In addition, several dehydration products of 1 have also been identified in human serum, these are: (3R,6'R)-3-hydroxy-3',4'-didehydro-β,γ-carotene (5), (3R,6'R)-3-hydroxy-2',3'-didehydro-β,ε-carotene (6), and (3R)-3-hydroxy-3',4'-didehydro-β,β-carotene (7). Several metabolites of 1 and/or 2, namely, (3R,3'S,6'R)-lutein (3'-epilutein, 8) and (3R,3'S;meso)-zeaxanthin (9) have also been characterized in human serum and ocular tissues. Semi-synthetic processes have been developed that separately transform commercially available 1 into 4 via 7 as well as 1 into 8. While 8 is converted into 2 by base-catalyzed isomerization, 7 is transformed into 2 and its (3R,3'S;meso)-stereoisomer (9) by regioselective hydroboration.