Brassinolide isolation

Among the compounds that have been isolated from the brassins were a new group of glucosyl esters of fatty acids, the structures of which were established by spectral methods (254, 255) as well as by chemical and biochemical synthesis (256, 257, 258). Although these esters were much less active than brassinolide in the bean second internode bioassay, their presence may be essential for seed germination since the enzymatic synthesis of these esters was correlated to germination (257, 258). 

In 1979, a novel plant growth regulating steroidal substance called brassinolide was isolated from rape (Brassica napus) pollen. Brassinosteroids are thought by some to be a new class of plant hormones. The evidences are ... 

The isolation and structural determination of brassinolide [BR] was reported in Nature by Dr. N. B. Mandava and co-workers at the USDA in 1979(5), and was subsequently announced in Chem, Eng, News 6). The steroidal skeleton of the chemical structure of BR prompted us to examine the effect on the LJT. (225,235)-homobrassinolide was synthesized by Mori in 1980( 7) at our request just after the structural determination of BR. BR itself was also produced by Ikekawa and his co-workers the same year(3). Wada, a co-worker in our group, tested the activity of BR and homobrassinolide on rice lamina inclination and found that both compounds dramatically induced the inclination of laminae (see Table I) as expected(5). These findings strongly suggested... 

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 occurrence of brassinosteroids in gymnosperms has been reported from conifers. Yokota ef al. isolated typhasterol ( 2-deoxycastasterone ) from pollen of Japanese black pine ( Pinus thunbergii ) (38) and identified castasterone and typhasterol from shoots of sitka spruce (Picea sitchensis ) (39). Kim ef al. also identified castasterone and brassinolide from cambial scrapings of Scots pine (Pinus silverstris ), using a modified dwarf rice lamina inclination assay which showed a synergistic response of brassinosteroids with indole-3-acetic acid (40). 

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). 

Actually, the data from Table II indicates a slight, but distinctive stimulation of sucrose uptake into isolated vascular bundles of Cyclamen by brassinolide, 24-E... 

In 1970, Mitchell et al. (1) isolated from pollen of rape a lipoidal complex termed "brassins" that has growth-promoting effects on plants. The active compound was eventually identified as a steroid derivative, which was given the name brassinolide (2). In the same year, two highly physiologically active brassinosteroids (a generic term for all compounds structurally related to brassinolide), 225,235,245 (tri-epi)-brassinolide (further referred to as BR-1) and 22/ ,231 ,245 (epi)-brassinohde (BR-2) were synthesized (3, 4). Ever since various synthetic routes have been developed to synthesize BR and its isomers (see elsewhere in this volume). 

Since the discovery of brassinolide, ten years ago, research on brassinolide and related steroids has attained a remarkable degree of development. So far, twenty-three brassinosteroids [BS] have been isolated and identified from twenty-two plant species. Furthermore, many unknown brassinolide-like active substances have been detected, so that the number of identified ones is likely to increase in the future. The background to successful isolation of new BS from various plant sources is due to the use of a rice-lamina inclination test for screening that is sensitive and specific for brassinosteroids. 

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. 

Since the first isolation and identification of brassinolide (BR) from rape pollen by Grove et al in 1978 (7), a wide occurrence in the plant kingdom of related steroidal compounds which are now collectively called brassinosteroids, as well as BR itself, has been confirmed (2). 

The discovery of brassinolide by scientists at USDA led to the development of a new group of plant growth substances, termed brassinosteroids, which have the potential for increasing crop yields and biomass. The chapter summarizes the USDA s contributions to brassinosteroids, which include isolation and identification of brassinolide, synthesis of brassinolide and its analogs, and limited biological and field studies on brassinosteroids. An overview of the current data requirements for the registration of brassinosteroids as biochemical pesticides under the Federal Insecticide, Fungicide, and Rodenticide Act is presented. 

Until the discovery of brassinolide by USDA scientists in 1979, it was thought that only five groups (indole auxins, gibberellins, cytokinins, abscisic acid, and ethylene) of hormones were responsible for regulating plant growth and development. Following this discovery, a number of compounds similar to brassinolide both in structure and physiological activity were isolated from different parts of plants. On the basis of published... 

Brassinosteroids are ubiquitous in nature. Brassinolide was first isolated from the pollen of rape (Brassica napus L.) plant and has plant-growth-promoting activity on various plants. Following the brassinolide discovery, other (about 20) brassinosteroids including brassinolide were found mostly from various higher plants. 

Because brassinolide, and probably other brassinosteroids, occurs in pollen, other aspects which may affect plant, insect, and animal physiology will have to be considered in the future. For example, what about the effects of brassinosteroid laced nectar and pollen to foraging birds and insects It will be recalled that the collection of sufficient rape pollen to isolate brassinolide came not from manual collection, but from pollen collected by honeybees (4). That fact leads to at least three thoughts. 

In 1979, Grove and coworkers isolated 4 mg of crystalline brassinolide from 40 kg of the pollen of rape plant Brassica napus, and determined its structure as shown in 30 (Figure 2.21) by X-ray analysis.6... 

The first synthesis of brassinolide (30) was achieved by Fung and Siddall in 1980,62 and also by Ikekawa and coworkers in the same year.63 Since then, many plant-growth promoters related to 30 with steroidal structures have been isolated from plants (Figure 2.21). They are called brassinosteroids. The early phase of our synthetic works on brassinosteroids has been reviewed.64... 

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