Tsuyoshi Watanabe

The Arabidopsis deetiolated2 mutant is blocked early in brassinosteroid biosynthesis.

The Arabidopsis DEETIOLATED2 (DET2) gene has been cloned and shown to encode a protein that shares significant sequence identity with mammalian steroid 5 alpha-reductases. Loss of DET2 function causes many defects in Arabidopsis development that can be rescued by the application of brassinolide; therefore, we propose that DET2 encodes a reductase that acts at the first step of the proposed biosynthetic pathway--in the conversion of campesterol to campestanol. Here, we used biochemical measurements and biological assays to determine the precise biochemical defect in det2 mutants. We show that DET2 actually acts at the second step in brassinolide biosynthesis in the 5 alpha-reduction of (24R)-24-methylcholest-4-en-3-one, which is further modified to form campestanol. In feeding experiments using 2H6-labeled campesterol, no significant level of 2H6-labeled campestanol was detected in det2, whereas the wild type accumulated substantial levels. Using gas chromatography-selected ion monitoring analysis, we show that several presumed null alleles of det2 accumulated only 8 to 15% of the wild-type levels of campestanol. Moreover, in det2 mutants, the endogenous levels of (24R)-24-methylcholest-4-en-3-one increased by threefold, whereas the levels of all other measured brassinosteroids accumulated to < 10% of wild-type levels. Exogenously applied biosynthetic intermediates of brassinolide were found to rescue both the dark- and light-grown defects of det2 mutants. Together, these results refine the original proposed pathway for brassinolide and indicate that mutations in DET2 block the second step in brassinosteroid biosynthesis. These results reinforce the utility of combining genetic and biochemical analyses to studies of biosynthetic pathways and strengthen the argument that brassinosteroids play an essential role in Arabidopsis development.

Biosynthesis of brassinosteroids in cultured cells of Catharanthus roseus

Abstract Precursor administration experiments with 2 H -labeled 6-oxocampestanol, 6-deoxocastasterone and 6α-hydroxycastasterone in cultured cells of Catharanthus roseus were performed and the metabolites were analyzed by GC-MS. [ 2 H 6 ]Cathasterone was identified as a metabolite of [ 2 H 6 ]6-oxocampestanol, whereas [ 2 H 6 ]6α-hydroxycastasterone and [ 2 H 6 ]castasterone were identified as metabolites of [ 2 H 6 ]6-deoxocastasterone, and [ 2 H 6 ]castasterone was identified as a metabolite of [ 2 H 6 ]6α-hydroxycastasterone, indicating that 6-deoxocastasterone is converted to castasterone via 6α-hydroxycastasterone. In addition, 6-deoxocathasterone, a putative biosynthetic intermediate in the late C6-oxidation pathway, was identified as an endogenous brassinosteroid. These studies provide further evidence supporting our proposed biosynthetic pathways for brassinolide.

A general approach to synthesis of labeled brassinosteroids: Preparation of [25,26,27-2H7]brassinolide with 60% isotopic purity from the parent brassinolide

Abstract From brassinolide (BL) 1 , [25,26,27- 2 H n ]BL 10 was synthesized in 5 steps including C-25 hydroxylation, dehydration and catalytic deuteriogenation. In direct oxy-functionalization of tetra- O -acetyl BL 2 with methyl(trifluoromethyl)dioxirane leading to 25-hydroxyl compound 3 , 14-hydroxyl, 25-hydroxy-15-oxo and 14,25-dihydroxyl derivatives, 4 , 5 and 6 , were newly identified; the catalytic deuteriogenation of Δ 25(26) -BL, 8 using 5% palladium-on-charcoal resulted in abundant incorporation of deuterium atoms to give 10 with 60% isotopic purity of [25,26,27- 2 H 7 ]BL.

Synthesis and biological activity of 26-norbrassinolide, 26-norcastasterone and 26-nor-6-deoxocastasterone

26-Norbrassinolide, identified as a metabolite of brassinolide in cultured cells of the liverwort, Marchantia polymorpha, as well as 26-norcastasterone and 26-nor-6-deoxocastasterone were synthesized. Synthesis of these new brassinosteroids was conducted by employing the orthoester Claisen rearrangement and asymmetric dihydroxylation as key reactions. The modified rice lamina inclination test indicated that these three 26-norbrassinosteroids were less active than their corresponding C28 brassinosteroids. Growth-promoting activities were also examined by using the brassinosteroid-deficient, dwarf mutant lkb of garden pea (Pisum sativum L.). In this assay, 26-norbrassinolide was as effective as brassinolide and 26-norcastasterone was more effective than castasterone although 26-nor-6-deoxocastasterone was much less effective than 6-deoxocastasterone. Therefore, removal of C-26 of brassinosteroids does not necessarily reduce the biological activity. The role of C-26 removal in Marchantia cells remains unclear.

2,3,5-Tri-epi-brassinolide : preparation and biological activity in rice lamina inclination test

Abstract With a view to attaining more precise information on the biological activity of 2,3,5-tri- epi -brassinolide analogs, 2,3,5-tri- epi -brassinolide was prepared from 2,3-di- epi -brassinolide by direct epimerization at C-5. Biological activity of 2,3,5-tri- epi -brassinolide in the rice lamina inclination test was nil even at 1xa0μg/plant by the single application technique, while with co-application of indole-3-acetic acid, the activity was ca 1/1000th that of brassinolide, reconfirming that the A/B trans -fused ring junction of brassinosteroids is an essential structural factor for high biological activity.

Synthesis and biological activity of 6a-carbabrassinolide: B-ring homologation of 6-oxo-steroid to 6-oxo-7a-homosteroid with trimethylsilyldiazomethane-boron trifluoride etherate

Abstract From castasterone (10), 6a-carbabrassinolide (2) was synthesized via a highly regioselective B-ring homologation with trimethylsilyldiazomethane and boron trifluoride etherate. A preliminary experiment using a simple 6-oxo-steroid (3) revealed that the actual products of this homologation reaction were α-trimethylsilyl ketones (4) and (5), which were converted to 6 (79%) and 7 (7.2%) by acid treatment. Biological activity of 2 in the rice lamina inclination test was ca. 1 2 compared with 1, suggesting that 6a-oxygen on 1 is an important but not essential factor for the activity.

Epimerization at C-5 of brassinolide with sodium methoxide and the biological activity of 5-epi-brassinolide in the rice lamina inclination test

Brassinolide 1 easily epimerized at C-5 by treatment with sodium methoxide in refluxing methanol via two intermediary methyl esters, and the subsequent re-lactonization with acid led to the formation of 5-epi-brassinolide 2 (42%) and 6(6a→3a)abeo-5-epi-brassinolide 3 (18%), along with recovery of 1 (37%); 3 was quantitatively converted to a 94∶6 equilibrium mixture of 2 and 3 by prolonged treatment with acidic resin at 60xa0°C. The NMR experiments allowed the conformations of the A/B ring moiety of 1 and 2 and the corresponding part of 3 in solution to be elucidated. Biological activity of 2 in the rice lamina inclination test was less than 1/1000 compared with 1, providing clear evidence that the A/B trans fused ring junction of brassinosteroids is an essential structural factor for high biological activity.

Synthesis of New Naturally Occurring 6-Deoxo Brassinosteroids

New natural 6-deoxo brassinosteroids, n6-deoxoteasterone 1, 3-dehydro-6-deoxoteasterone 2 and n6-deoxotyphasterol 3, as well as 6-deoxocastasterone 4, are nsynthesized from n(20S)-20-formyl-6β-methoxy-3α,5-cyclo-5α-pregnane 5.

A Convenient Synthesis of (22S)-22-Hydroxycampesterol and Some Related Steroids

As possible candidates for intermediates in brassinolide biosynthesis, (22S)-22-hydroxycampesterol 1 and its related new steroids 5–7 are conveniently synthesized by employing a Grignard reaction of a known steroidal 22-aldehyde 8 with 2,3-dimethylbutylmagnesium bromide as a key reaction.

Improved Synthesis of Castasterone and Brassinolide

Castasterone 1 is synthesized in 32% overall yield in eight steps from the known (20S)-6,6-ethylenedioxy-20-formyl-3α,5-cyclo-5α-pregnane 4.