Brassinosteroids isolation

Figure 28 Structures of three brassinosteroids first isolated from plants. Numbers of carbon atoms are shown in the structure of castasterone.

Brassinosteroids (BRs) are hydroxylated derivatives of cholestane and their structure variations comprise substitutions pattern on ring A, B and the C-17 side chain (Fig. 6.10). The BRs are classified as C27, C28/ or C29 BRs, depending on the substitutions and the length of the side chain. More than 70 BRs as well as more than 42 BR metabolites have been isolated and identified (Bajguz and Tretyn, 2003). 

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 ... 

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). 

In biological experiments with Raphanus sativus (var. Remo) we could also establish the growth stimulating activity of brassinosteroids on isolated radish cotyledons. Furthermore, we have found that this effect is accompanied by a distinct activation of invertase (Andre-as.W. Vorbrodt, H.M. Adam, G., unpublished). 

By the criteria listed, the evidence is consistent with brassinosteroids being a new family of endogenous plant hormones. However, when Jacobs rules (61) for determining the involvement of a hormone in a process (Presence, Excision, Substitution, Isolation, Generality, Specificity), are considered -... 

Sucrose Absorption by Vascular Bundles. Sucrose uptake into 2-4 mm long pieces of vascular bundles isolated from the petiole of Cyclamen persicum was described in detail in (5). Briefly, the tissue material was preincubated (30 min) with brassinosteroids or related compounds, which were also present during the following incubation with 1 mM 1AC-labelled sucrose (30 min). All solutions contained 1 mM CaSO, 1 mM KC1 and 10 mM morpholinoethanesulfonic acid and were adjusted with NaOH to pH 5.0. After a 5-min-washing procedure and extraction in methanol the uptake rate for sucrose was determined analogously to the experiments with leaves. 

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). 

CF preparation was isolated after dialysis against water the low water solubility of brassinosteroids suggested that it was unlikely that these compounds were present at significant concentration in our aqueous preparation. 

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). 

Effects of brassinosteroids on imaginal discs isolated from two different fly species (Phormia terraenovae, 15 Calliphora vicina, 16, 17) were observed. As expected, the imaginal discs of Phormia and Calliphora larvae evaginated in response to 20-hydroxyecdysone. 

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. 

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. 

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... 

In 1982 Yokota et al. isolated 160 pg of dolicholide (31) and 50 pg of dolichosterone (34) from 34 kg of the immature seeds of a bean, Dolichos lablab.12 So as to confirm the proposed structures, we synthesized these brassinosteroids as shown in Figure 2.24.68,73,74 In this case, too, we used organoaluminum chemistry and epoxide cleavage as the key reactions. 

The amounts of homodolicholide (32)75 and homodolichosterone (35)76 isolated from Dolichos lablab were only 12 pg and 20 pg, respectively. It was therefore necessary to synthesize them in substantial amounts so as to evaluate their plant-growth-promoting activity. We synthesized these two brassinosteroids, as shown in Figure 2.25.68... 

The isolation and structure elucidation of native brassinosteroids is summarized comprehensively by several reviews and books. 4 This section describes recent studies on the occurrence of new brassinosteroids found since 1991. 

From Secale cereale, besides the common brassinosteroids castasterone (3), typhasterol (7), teasterone (8), 6-deoxocastasterone (10), brassinone (16) and 24-ethylbrassinone (20), the hitherto unknown brassinosteroid 32 could be isolated 43 The mass spectrum of this compound obtained by GC/MS after methaneboronation shows a molecular ion at m/z 470 and is very similar to that of the 3,6-diketo compound 3-dehydroteasterone (31) isolated from Distylium racemo-sum44 and Triticum aestivum,45 However, the GC retention data are quite different. Compound 32 displays significant key ions at m/z 454, 399, 316, 286, 245, 155 and 138/137 (Table 1, Scheme 3). The base peak at m/z 155 is the typical ion for a saturated brassinosteroid side chain... 

The structural determination of endogenous brassinosteroids, present only in minute amounts in plant material, requires the availability of corresponding reference standards. Thus, for the final identification of the new brassinosteroid secasterone (32) isolated from Secale cereale (see, Section 2), the four epimeric brassinosteroids with 2,3-epoxy function derived from castasterone (3) and 24-epicastasterone (13), respectively, were synthesized (Scheme 6).69... 

---