Brassinolide biological activity

The biosynthesis of brassinosteroids has been investigated by in vivo feeding of plant cell cultures, in addition to the analysis of native brassinosteroid-pattems in diverse plant species. The proposed pathway (Fig. (1)) leads from unpolar sterol precursors to the polyhydroxylated phytohormone brassinolide, that is known to exhibit the highest biological activity. 

T. Yokota K. Mori, Molecular Structure and Biological Activity of Brassinolide and Related Brassinosteroids. In Molecular Structure and Biological Activity of Steroids M. Bohl, W. L. Duax, Eds. CRC Press Boca Raton, FL, 1992 pp 317-340. 

The structure of the unique biologically active plant growth promoter, brassinolide, from pollen of the rape plant (Brassica napus L.) was published in 1979 (1). That... 

Among all naturally-occurring brassinosteroids, brassinolide and castasterone are considered to be the most important brassinosteroids because of their wide distribution as well as their potent biological activity. 

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). 

This brief review covers the period of the last ten years in the development of brassinosteroid chemistry in our laboratory. Our interest has been focused on the synthesis of natural brassinosteroids (brassinolide, homobrassinoli-de, epibrassinolide, norbrassinolide, etc.), their analogues and intermediates starting from stigmasterol, ergosterol, or pregnenolone. Some aspects of biological activity are discussed. 

Figure 3 shows hypothetical biosynthetic pathway of brassinolide. Namely campesterol or its analogs will be converted to teasterone via several steps of oxidation, then successively oxidized to typhasterol, castasterone and brassinolide. In parentheses, biological activities in the rice lamina inclination assay are shown. The... 

This chapter deals with metabolism of castasterone and brassinolide and discusses the question as to whether brassinolide is synthesized from castasterone. Furthermore the question as to whether the biological activity of castasterone is mediated through conversion to brassinolide is also discussed. 

Biological activity was determined by the rice lamina inclination assay. HPLC support and mobile phase A, Cjg silica and 45% acetonitrile B, Cjg silica and 45% acetonitrile C, silica (Aquasil) and chloroform-methanol (95 5, 1% water). Abbreviations CS, castasterone BL, brassinolide. 

It is now evident that castasterone is not converted to brassinolide, indicating, in mung bean, that the biological activity of castasterone is exerted by castasterone itself instead of a conversion to brassinolide. The major polar metabolite derived from brassinolide is its 23-0-glucoside while, in contrast, that derived from castasterone is non-glycosidic. 

Thus castasterone is not converted to brassinolide in either rice seedlings or etiolated leaf explants, but it is metabolized to seemingly non-glycosidic compounds. Therefore it might be postulated that castasterone seems to be biologically active by itself in rice. 

Following the discovery of brassin in 1970 (1 ), the isolation and chemical characterization of the active ingredient brassinolide (BR) in 1979 (2) and the subsequent identification of a number of other naturally-occurring brassinosteroids (BS) (see 2) considerable research effort has been directed towards determining the taxonomic and morphological distribution and biological activity of these compounds in plants. 

Discovery of Brassinolide. Being thoroughly convinced that the pollen growth factors were different from the known hormones, Mitchell collaborated with Bhushan Mandava to determine the factors responsible for biological activity. 

Early in our synthetic program for brassinolide and its analogs, we learned that there were certain structural requirements in the steroid molecule for eliciting the biological activity. These requirements include the following (l, 5-7) ... 

Our synthetic work on brassinosteroids showed that the stereochemistry in the steroid molecule is very important to retain the biological activity. We found that, of the 13 asymmetric centers in the molecule, only 3 asymmetric centers (at C-22, C-23, and C-24) can be altered. On the basis of this information, we prepared brassinolide and all the analogs (23) by using the general synthetic scheme (Figure 3). Additionally, we prepared compounds having different substituents (H, CH3, and C2H5) at C-24 (2, 7). 

This is a relatively new group of naturally occurring steroids with remarkable biological activity (see Section XV.2). The first member isolated was brassinolide... 

Distribution of biological activity determined by the rice-lamina inclination bioassay after reversed-phase HPLC of the purified extract of germinated seeds of Raphcmus sativus (BR = brassinolide, 1)... 

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. 

Deoxocastasterone, an important intermediate in this pathway, is usually contained in plant tissues at the highest level [28,50]. However, this BR and some other 6-deoxoBRs have been considered to be end-pathway BRs because these show very low biological activity when examined by the rice lamina inclination assay [28]. In this bioassay, brassinolide promotes the bending of rice leaf joints at as low as 0.1 nM. Nonetheless... 

Brassinolide (BR), (2a,3a,22a,23a-tetrahydroxy-24a-methyl-B-homo-7-oxa-5a-cholestan-6-one), a biologically active steroidal lactone first isolated from rape Brassica napus L.), pollen, stimulates growth of green plant tissues. Although the mechanism responsible for observed BR effects remains to be determined, the action of BR on growth is oligodynamic, and rapid. 

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