Brassinolide 26-hydroxylation

From 40 kg of rape pollen, 4 mg of an active compound was eventually isolated and shown to be the novel plant growth substance brassinolide (1) (3). The structure, as determined by X-ray crystallography, was [ (2a, 3a, 22R, 23R)-tetrahydroxy- 24 a-methyl-B-homo-7-oxa-5a-cholestane-6-one]. This structure was unique in possessing a 24a-methyl, a 7-oxalactonic B ring, and vicinal hydroxyls on the A ring (C2a and C3a) plus a side chain (C22R and C23R ). 

Microanalytical techniques, consisting of methaneboronation of the vicinal hydroxyls ( bismethaneboronate or methaneboronate-TMS-ether ) have been developed by Takatsutoet al. for the GC/MS or GC/SIM of brassinosteroids (16). Consequently brassinone (15), 24-ethylbrassinone (12) and 28-norbrassinolide (14) were detected by GC/MS without isolation from Chinese cabbage ( Brassica campestris ), green tea ( Thea sinensis ) and chestnut insect galls ( Distilium racemosum ) (17-19). The presence of brassinolide and castasterone in the tissues of these plants has been also detected. 

The required starting compound for the synthesis of brassinolide by hydroxylation of a A22 double bond is 22-dehydrocampesterol, which is not readily available (7). It can be isolated as a mixture with the C-24 epimer, brassicasterol, from oysters (Crassostrea virginica), but the mixture is difficult to separate. It is more readily prepared by synthesis (6). For the synthesis of 24-epibrassinolide, brassicasterol, which can be obtained from rapeseed oil, is an obvious starting compound (15). Ergosterol, which is readily available, has also been employed (16). 

Recently, by the ligand-accelerated osmium catalyzed asymmetric hydroxylation using a cinchona alkaloid derivative, the 22/ ,23/ -epimer was obtained as the major product of the oxidation (77). Thus, epibrassinolide may be ideal for practical use, and, therefore, is one of the most desirable brassinosteroids because of the ease with which it can be synthesized. This compound showed about one tenth the activity of brassinolide in Raphanus and tomato bioassay (5). In field trials, the activity of epibrassinolide was about the same as of brassinolide. Epibrassinolide is a natural sterol. This was confirmed by GC-MS analysis, which identified epibrassinolide, along with co-existing brassinolide, brassinone, and castasterone, from the bee pollen of the broad bean Vida faba obtained in China (72). 

The spectroscopic information on the crystalline product indicated that it was a steroid molecule that contained four hydroxyl groups, an oxygen function, and a carbonyl function. We also found that the compound contained a side chain (same number of carbons as in cholesterol) and oxygen and carbonyl functions due to a lactone in the steroid skeleton. The exact locations of the hydroxyl groups and the lactone in the steroid molecule were revealed by X-ray crystallography, which showed the structure 1 (Figure 3). This compound was named brassinolide, derived from a combination of words, brassin (after the genus Brassica) and olide for lactone (4). 

The results of the rice lamina inclination test (RLIT) indicated an extraordinary high activity of 25-hydroxy-24-epibrassinolide (90). This compound is about ten times more active than 24-epibrassinolide (12), indicating that the hydroxylation at C-25 is an activating step in the brassinosteroid metabolism. Therefore, 25-hydroxy-24-epibrassinolide (90) is, next to brassinolide (1), one of the most active brassinosteroids known until now. In comparison with 25-hydroxy-24-epibrassinolide (90), the 26-hydroxylated metabolite (91) was clearly less active. As in other groups of steroidal hormones, for instance vitamine D metabolites, hydroxylation at C-25 seems to be essential for high activity. ... 

P450 inhibitors clotrimazole and ketoconazole have been found to suppress 25-hydrox-ylation of 24-epibrassinolide and brassinolide in tomato cell suspension cultures, indicating that the 25-hydroxylation is catalyzed by a P450 enzyme. This 25-hydroxylation activity of 24-epibrassinolide and brassinolide is induced specifically by 24-epibrassinolide and brassinolide but not by 24-epicastasterone, (225,235)-28-homo-brassinolide and some non-specific P450 inducers [73]. 

It is of interest that the structure of brassinolide resembles that of the insect molting hormone, ecdysone, which also is of plant origin. The structure of ecdysone is shown in Figure 1 and differs from the structure of brassinolide in that the orientation of the vicinal hydroxyl group at C-2 and C-3 is beta, the A/B junction is cis rather than trans as in the brassinolide structure and that ecdysone lacks the lactone oxygen in the B-ring. 

Typhasterol. Typhasterol and Teasterone are the first tri-hydroxy 6-ketone sterols (2-deoxy-castasterones and its 3-epimer) isolated that show biological activity in the rice lamina joint bending test despite the lack of a hydroxyl group at C-2. Typhasterol was isolated from cattail (Typha latifolia L) pollen (29), and both teasterone and typhasterol were present at 60 and 15 ng respectively per kg fr. wt. of leaves and were isolated from the less polar active fraction obtained from tea leaves (30). The biological activity of these two ketones as tested in the rice test, is about one tenth that of brassinolide (30). Typhasterol has recently been synthesized (31), but its biological activity was not tested. 

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