Brassinolides function

The Arabidopsis mutant dwf7/ste 1 is defective in C5-desaturation of episterol (Fig. (2)) [18], thus impaired in an enzyme function involved in a very early step of brassinosteroid precursor biosynthesis. The enzymatic block of dwf7/stel was determined by feeding experiments using l3C-labelled mevalonic acid and a subsequent analysis of endogenous sterol and brassinosteroid precursors. The mutant accumulates episterol with a simultaneous decrease of downstream intermediates (24-methylenecholesterol, campesterol, castasterone, brassinolide). 

Brassinosteroids are grouped into C, Cg, and steroids like the typical phytosterols. These classifications result basically from different alkyl substitutions in the side chain, that is no substituent in steroids (14, 15 ), a methyl (1, 3,5, 7-9, 16-22,29) or an exomethylene ( 2,4, 6) at C24 in steroids, and an ethyl at C24 (12 )or an ethylidene at C24 ( 10,11, 13) or an exomethylene at C24 with a methyl at C25 ( 23-28 ) in steroids. The structure-activity relationship reveals that alkylation in the side chain is necessary for biological activity. More especially, the presence of a saturated alkyl ( a methyl or an ethyl ) at C24 and a methyl at C25 makes brassinosteroids biologically more active. Thus, 25-methylbrassinolide bearing both 24-methyl and 25-methyl functions has been chemically synthesized and it is more potent than brassinolide which, until now, had been considered to be the most potent brassinosteroid (44). 

Before discussing our synthetic studies, mention should be made of different approaches employed in the synthesis of brassinolide by other investigators. The most practical method would be to start with a readily available steroid having the same carbon skeleton as brassinolide, and then introduce the required functional groups in rings A and B and the sidechain. In the case of 28-homobrassinolide (5), tiie ideal starting compound would be the abundant sterol stigmasterol (4). 

A more widely used approach to brassinolide is reaction of a C-22 aldehyde, derived from stigmasterol, with a carbanion containing a double bond (or potential double bond). In nearly all cases, epoxidadon of die double bond was employed to introduce one or both oxygen functions (17-24). A synthesis of the brassinolide sidechain starting from pregnenolone has also been reported (25). 

Molecular modelling of ecdysteroids (7) and brassinosteroids is still in its infancy. A comparison of the three dimensional (3D-) structures of the two biologically most active representatives of brassinosteroids and ecdysteroids, i.e. of brassinolide and 20-hydroxyecdysone, suggests that functional groups relevant for bioactivity are found at similar positions in both molecules in spite of the structural differences mentioned above. More detailed comparative studies of the 3D-structures of ecdysteroids and brassinosteroids are eagerly awaited. 

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). 

There is further evidence to support the claim that brassinosteroids are hormonal in their action (6). These are the effects of brassinolide on gravitropism (7), effects in conjunction with light quality (8), effects on photosynthate partitioning (9), probable effects on phytochrome (10), substitution for indole-3-acetic acid in soybean epicotyls (11), enhancement of xylem differentiation (11), stimulation of membrane permeability in cucumber hypocotyls (12), and stimulation of ATPase activity (12). Taken objectively, many of these specific physiological and biochemical functions which are attributed to brassinolide, and by inference to the brassinosteroids in general, have been attributed to the other plant hormones, especially indole-3-acetic acid and the gibberellins (6, 13). 

The stereocontrolled syntheses of steroid side chains for ecdysone, crustecdysone, brassinolide, withanolide, and vitamin D3 have been reviewed (185). Also, other manuscripts, including reviews on the partial synthesis of steroids (186), steroid drugs (187—189), biologically active steroids (190), heterocyclic steroids (191), vitamin D (192), novel oxidations of steroids (193), and template-directed functionalization of steroids (194), have been published. 

A 6-0X0 function is required for high activity, with the seven-membered lactone ring being preferred. Brassinolides and castasterone have comparable activities, but castasterone analogues are generally less active. Alterations to the oxo function drastically reduce activity. 

Dolichosterone. Dolichosterone is the methane derivative of caststerone and differs from the brassinolide structure by the absence of the lactone function in the B-ring and by having a methylene rather than a methyl function at C-24. The brassinosteroid was isolated from immature seeds of Dolichos lablab (hyacinth-bean)... 

Norbrassinolide. 28-Norbrassinolide was isolated from Chinese cabbage (about 1 ng/kg) (16). It differs in structure from brassinolide by the absence of an alkyl function at C-24 and is biologically as active as its 6-ketone analogue (brassinone). It has 1% of the activity of brassinolide as assayed in the bean test (12) but was as active as brassinolide in the radish test and had 10% of the activity of brassinolide in the tomato test (18),... 

In several studies concerning the synthesis of brassinolides such as compound 343 described by Takatsuto [77, 78], the Claisen-Johnson rearrangement afforded stereoselectively functionalized side chains. As previously, formation of the center in of ester group was stereoselective. A mixture of epimers at the a-stereocenter was formed during the process, but since this carbon atom was no longer stereo-genic in compound 343, this lack of stereoselectivity was of no consequence (Scheme 6.56). 

The role of steroids as mammalian hormones has been known since 1930, and steroidal hormones have also been found in insects and fungi. Plants can biosynthesize a large variety of steroids, but it was not known until 1979 that steroids with plant growth-promoting activity were discovered. In that year. Grove et al. [1] reported the discovery of a new steroidal lactone called brassinolide from the pollen of Brassica napus L. To date, more than 70 structurally and functionally related steroids have been isolated from plant materials [2]. These compounds have been identified as members of a new group of plant hormones -the brassinosteroids (BRs). 

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