Reminiscences of research on the chemistry and biology of natural sterols in insects, plants and humans.
Since 1930s biologically active mammalian steroids had been paid attention to many researchers and tremendous studies on steroids had provided numerous brilliant results. On the other hand, plant steroids were known only as final products in the biosynthesis of isoprenoids and thus were not paid attention due to lack of knowledge on their biological activities. In 1957, discovery of cholesterol in algae by Tsuda et al. became a topic in this field. (1) Before 1970s mammalian steroid hormones which have short or no side chains were major targets for the steroid research. (2) Steroids originated from non-mammalian sources such as fungi, insects and plants showed interesting side chains oxygenated at C20, C22, C23, C25 and C26 as found in antheridiol, (3) ecdysteroids (4) and withanolides. (5) These steroids have stigmastane (C29), cholestane (C27) and ergostane (C28) skeletons, respectively.
Chemical and biochemical studies on the steroids having side chains had experienced several difficulties in the isolation of the active steroids due to their faint amount in the biological pool and in the separation of the stereoisomers. Thus, we had established a novel gas-chromatography (GC) technology, which enabled us to cleanly separate natural steroids and also developed the chemical synthetic methods in conjunction with the biochemical methods to solve the problems, which interfered with the research on the steroids having functionalized side chains. These methods were applied to the investigation of plant and insect sterols. (6)
GC technology enabled us the microanalysis of sterol mixtures and the synthetic organic chemistry made it possible to prepare all stereoisomers through the stereoselective reactions at the steroid side chains. Thus, we synthesized numerous kinds of the steroids having oxygenated side chains and revealed the roles of those steroids in their biological activities. (7)
Sterol metabolism in insects
1. Dealkylation mechanism of plant sterols to cholesterol. Insects, unlike most plants and animals, are incapable of de novo sterol biosynthesis and therefore require a dietary or exogenous source of sterol for their normal growth and development. The sterol requirement of insects is, in most cases, satisfied by cholesterol (1), which is the principal sterol in insects and serves as the structural component of cell membranes and as biogenetic precursor of the molting hormone, 20-hydroxyecdysone (2). In phytophagous insects the requirement can be fulfilled also with plant sterols e.g., sitosterol (3), campesterol (4) and stigmasterol (5), since these C-24 alkylated sterols are metabolically converted to cholesterol. Thus, dealkylation of phytosterols is an important metabolic step in many insects.
GC analysis of the sterol fraction of silkworm larvae indicated the presence of cholesterol, together with small amounts of campesterol and sitosterol. (8) The sterol fraction of mulberry leaves contained sitosterol and a smaller amount of campesterol. (9) It was thus suggested that these plant sterols were converted into cholesterol and we had an interest in the conversion mechanism. Indeed we showed that [[sup.3]H]-sitosterol was converted into cholesterol in the silkworm Bombyx mori in 1967. (10) Meanwhile, fucosterol (6), 24-methylenecholesterol (7) and desmosterol (8) were postulated as intermediates of the dealkylative conversion principally by Svoboda's group utilizing tobacco hornworm. (11)
However, the precise C-24-C-28 bond-cleavage mechanism of plant sterols has remained unanswered. We started the study on the dealkylation mechanism around 1968, since we possessed a sizable amount of fucosterol (6), available from brown algae such as Sargassum ringgoldianum through our search for marine resources. (12) It was a lucky break to find out that BF3 treatment of 3-acetate of fucosterol 24,28-epoxide (FE) (9) yielded desmosterol acetate together with usual epoxide-carbonyl rearrangement products during chemical studies of the fucosterol [[DELTA].sup.24(28)]-double bond. (13) The unique rearrangement of FE was accompanied by the formation of acetaldehyde and thus expected to involve hydrogen migration from the C25 to C24 position (See Fig. 2).
The rather unexpected C-C bond cleavage reaction was regarded as a biomimetic version of the phytosterol dealkylation. We proved this hypothesis by several lines of evidence utilizing silkworm larvae. First, [sup.3]H-label of fucosterol administered to larva was trapped in FE and [sup.3]H-label of FE was incorporated into cholesterol. (14) Secondly, hydrogen migration from C-25 to C-24 was verified by feeding [25-[sup.3]H]-24-ethylcholesterol followed by determining chemically
the position of the label of the resulting desmosterol. (15) Thirdly, the 24,28-imine analogue of FE was shown to be a potent inhibitor of the enzyme termed fucosterol epoxide lyase that catalyzes the conversion. (16) The intermediacy of FE in sitosterol dealkylation was supported in three other insects. (17) We subsequently indicated that the same hydrogen migration took place in the dealkylation of stigmasterol and campesterol, now utilizing [[sup.2]H]-labeled substrates coupled with GC-MS analysis. (18) These studies have established the phytosterol dealkylation mechanism as summarized in Fig. 1 (Ikekawa pathway).
We then investigated several stereochemical issues of the dealkylation processes. Incubation with a cell free preparation obtained from midguts of silkworm larvae revealed that (24R,28R)-fucosterol epoxide and (24R,28S)-isofucosterol ([[DELTA].sup.24(28)]-Z isomer of 6) epoxide, out of the four diastereomers at the C-24 and C-28 positions, were converted into desmosterol by the lyase. (19) Similarly, (24R)-24-methylenecholesterol 24,28-epoxide (10), but not the (24S)-isomer, became a substrate for the enzyme. (20) Both (24R,28R)- and (24S,28S)-FEs were, however, characterized from the whole body of silkworm larvae. (21) Yet another stereochemical preference with respect to the epoxide isomers was reported for the insect Tenebrio molitor. (22) These discrepancy needs to be clarified, including the question as to which epoxide diastereomers are formed from the [[DELTA].sup.24(28)]-olefinic substrates.
Metabolic fate of the diastereotopic C-26 and C-27 methyl groups of FE was investigated using a cell free preparation from midguts of silkworm larvae, and [pro-R]-methyl group (designated as open circle in Fig. 2) was turned out to become sterespecifically (E)-methyl of desmosterol. (23),24) The stereochemistry in the desmosterol reduction was explored with the cell-free preparation in the presence of NADPH cofactor by applying deuterium-decoupled [sup.1]H,[sup.13]C shift correlation NMR analysis of the biosynthesized cholesterol. (25) The study established that the hydrogen addition by sterol-[[DELTA].sup.24]-reductase took places in anti fashion from the re-face at C-24 and the si-face at C-25 (Fig. 2). The steric course of the hydrogen addition was also investigated with rat liver cell-free preparation and plant callus, which confirmed the same anti addition. This required revision of syn addition mechanism originally reported by Caspi and co-workers in rat liver system more than 30 years ago. The stereochemistry of the sitosterol dehydrogenation (cis- vs. trans-dehydrogenation) leading to fucosterol, but not isofucosterol, (26) remains an open question. The gene encoding the insect sterol-[[DELTA].sup.24]-reductase of Bombyx mori has been identified recently, (27) although those encoding the enzymes of the preceding three steps remain unidentified. Characterization of these genes may facilitate search for inhibitors of the phytosterol dealkylation, which would be environmentally friendly insecticides for agrochemical use. Also, they can be used for the conversion of plant sterols to useful sterols functionalized in the side-chain by the fermentation of engineered microbes.
2. Biosynthesis of insect molting hormones. At the beginning of the 1940s, Fukuda first proposed that prothoracic ground (PG) secrets a molting hormone. (28) In addition, our group reported a brain hormone-like activity of cholesterol administered to silkworm pupae as early as 1963 when the structure of ecdysone remained unclear. (29) Since then, [alpha]-ecdysone (ecdysone) (11) and [beta]-ecdysone (20-hydroxyecdysone) (2) were isolated from the silkworm and their structures were elucidated. (4),30),31) However, it had not been clear whether PG is a responsible organ that produces a molting hormone, and if so, which substance(s), either ecdysone and/or 20-hydroxyecsysone, is produced by PG.
Chino and co-workers challenged this problem and succeeded in culturing PG organs that produced a hormonally active substance. (32) We had developed a micro-analytical method of ecdysteroids based on GC-MS (mass chromatography mode) analysis of their trimethylsilyl ether derivatives, (33) and successfully characterized the substance released from the cultured PGs to the medium as ecdysone in 1973. (32) Ecdysone thus formed is converted into 20-hydroxyecdysone by 20-hydroxylase distributed in peripheral tissues such as fat body.
GC analysis of a PG extract revealed the occurrence of 7-dehydrocholesterol (12) as well as 7-dehydrositosterol and 7-dehydrocampesterol, the amounts of which changed in proportional to that of ecdysone, (34) supporting a view that 7-dehydrocholesterol is a biosynthetic intermediate next to cholesterol.
Accumulation of highly polar ecdysteroid conjugates in the diapause eggs of the silkworm Bombyx mori was first reported by Ohnishi et al. (35) His group also noted that the ovaries of B. mori exhibited high molting hormone activity. (36) Since then, we worked jointly with his group on the structure analysis of ovarian ecdysteroids, and characterized six ecdysteroids, 2-deoxyecdysone (13), (37) 2,22-dideoxy-20-hydroxyecdysone (14), (39) ecdysone (11), 2-deoxy-20-hydroxyecdysone (15), (38) 20-hydroxyecdysone (2), and a unique ecdysteroid bombycosterol (16) (40) as major ecdysteroids in Bombyx ovaries. Their conjugate forms were elucidated to be all phosphate esters (X = P[O.sub.3.sup.2-] in Fig. 3). (41) Figure 3 also shows the principle biosynthetic route (X = H in Fig. 3) of 20-hydroxyecdysone, although the mechanism of an early stage (so-called black box), leading to the 14-hydroxy-5[beta]-enone (17), has remained unsolved. Our characterization of various free ecdysteroids and their phosphate derivatives in Bombyx ovaries/eggs has contributed to establish the generally accepted view that the ecdysteroid conjugates are a physiologically inactive form that are stored in eggs and serve as a source of an active ecdysteroid like 20-hydroxyecdysone that is indispensable for embryonic developments. It is interesting to note that ecdysteroids work on a membrane receptor in addition to the nuclear ecdysone receptor (EcR). (42)
Synthesis and biochemical research of active forms of vitamin [D.sub.3] metabolites and their analogs
1. Structure determination of vitamin [D.sub.3] metabolites. New era of steroid research was opened by the identification of 25-(OH)-V[D.sub.3] (18, for the structure see Fig. 8) and 1,25-[(OH).sub.2]-V[D.sub.3] (19)as the active metabolites of V[D.sub.3].43) During 1968-71, DeLuca and Tanaka mainly contributed to the discovery of the hydroxylated V[D.sub.3]. The most intriguing findings were the importance of secosteroid derivatives having oxygenated side chains, although 1 ,-hydroxylation is essential to elicit their biological activity. After identification of 1,25-[(OH).sub.2]-V[D.sub.3], 24,25-[(OH).sub.2]-V[D.sub.3] (20) was identified by MS analysis. (44) However, the configuration at C24 of 20 remained unestablished.
At this moment we had a large amount of desmosterol (8) chemically derived from fucosterol (6). Thus, our research work was started from the determination of the C24 configuration. At first, 24-OH cholesterol (21) was prepared from 8 and was separated into two isomers (21[alpha]/21b). Their C-24 configurations were determined with modified Horoeu's method using GC. (45)
Two C24-isomers of 24,25-[(OH).sub.2] cholesterol (22) were prepared from 8 and the resulting mixture was separated by HPLC of the tri-TMS derivatives (Fig. 4). Both 24S and 24R isomers (22a and 22b) were converted into V[D.sub.3] form, and assayed for V[D.sub.3] activity by DeLuca and Tanaka of Univ. of Wisconsin-Madison, showing that the 24R isomer exhibited potent activity (vide infra). This was a start of the long-term joint research between their groups and ours lasting nearly 20 years, and biological activities of our synthetic V[D.sub.3] samples were always evaluated by their group thereafter. HPLC comparison of the synthetic samples with natural 24,25-[(OH).sub.2]-V[D.sub.3] (20) revealed that 24R isomer (20a) was correspondent to the natural metabolite. (46)
Subsequently, 25,26-[(OH).sub.2]-V[D.sub.3] (23) was identified as a metabolite of 25-(OH)-V[D.sub.3].47) Then, two C25-isomers of 25,26-[(OH).sub.2] cholesterol were stereo-selectively synthesized from (24R)- and (24S)-A25-24-hydroxycholesterol derivative (24a/24b) as shown in Fig. 5. Interestingly, 25,26-[(OH).sub.2]-V[D.sub.3] had both 25S and 25R configuration (23a/23b) in an equal amount as revealed by HPLC comparison with the synthetic 25,26-[(OH).sub.2]-V[D.sub.3], (48) while 24R isomer was a sole metabolite for 24,25-[(OH).sub.2]-V[D.sub.3].
A further research on V[D.sub.3] metabolism using [[sup.14]C]-25-(OH)-V[D.sub.3] led to identification of 23,25-[(OH).sub.2]-V[D.sub.3] (25) (49) and 25-(OH)-V[D.sub.3] 26,23-lactone (calcidiol lactone, 26), (50) which are 23-hydroxylated V[D.sub.3] derivatives.
The 23S configuration of 25 was elucidated as follows. (23S)-23,25-[(OH).sub.2]-V[D.sub.3] and its (23R)-isomer were synthesized according to Fig. 6. Coupling of the aldehyde (27) with metallylmagnesium chloride yielded the C-23 hydroxy derivative as an epimeric mixture, which was separated into the epimers (28a and 28b) in the form of 3,23-dibenzoate (R=Bz).
Compounds 28a and 28b were converted to the 3,23S,25-triol (29a,R=H) and its 23R-isomer (29b), respectively. The configuration at C-23 of 29a was determined to be S by X-ray analysis. Compounds 29a and 29b (R=Bz) were further converted into V[D.sub.3] form. HPLC comparison of the stereo-defined synthetic samples with the natural metabolite established the 23S configuration of 25. (51)
The synthesis of four isomers at C23 and C25 of calcidiol lactone was accomplished by the stereo-specific iodolactonization or selenolactonization of the trans-andcis-[[DELTA].sup.22]-26-carboxylic acids (30 and 31). The introduction of the 25-OH group was achieved through the oxidation at the C,-position of the 26-ester or lactone moiety. The stereoselective introduction of the hydroxyl group at C25 was dependent on the configuration at C23 of the lactone and thus, the hydroxyl group of the other configuration was introduced in the ester moiety before the lactonization as shown in Fig. 7. (52) The 23S,25R configuration of calcidiol lactone was also established by HPLC comparison with the natural metabolite. (53)
2. Biological activity for vitamin [D.sub.3] analogs having hydroxylated side chain. Vitamin [D.sub.3] biosynthesized at the skin is transformed at liver and then kidney to the hydroxylated derivative, 1[alpha],25-[(OH).sub.2]-V[D.sub.3] (19). This activated hormone binds vitamin [D.sub.3]-binding protein in blood and is transferred to the target cells. Then, 1[alpha],25-[(OH).sub.2]-V[D.sub.3] binds vitamin [D.sub.3] receptor (VDR) in the cell nucleus. It also forms RXR-VDR-1,,25-[(OH).sub.2]-V[D.sub.3] complex with 9-cis-retinoic acid receptor (RXR), which binds vitamin D-responding element (VDRE) present at the upstream of vitamin [D.sub.3]-dependent gene. Then, translation-coupled co-activator binds this complex to regulate the gene expression. Thus, the complex in the process will become the targets of vitamin [D.sub.3] derivatives.
In relation to the configuration at the 24 position of 24,25-[(OH).sub.2]-V[D.sub.3] (20), we prepared two isomers of 24-(OH)-V[D.sub.3] and found that (24R)-24-(OH)-V[D.sub.3] has the same mobilization of bone [Ca.sup.2+] and calcification activity as 25-(OH)-V[D.sub.3] and a similar activity to 24,25-[(OH).sub.2]-V[D.sub.3], while the 24S isomer showed little activity. (54) Although both isomers were metabolized to the respective 24,25-[(OH).sub.2]-V[D.sub.3], only 24R isomer was further converted to 1,24,25-[(OH).sub.3]-V[D.sub.3] (32). (55) Thus, 1,-hydroxylase recognizes only 24R isomer to give the active metabolite. When we assume that a particular residue in the enzyme requires the interaction with the hydroxyl group at C24 in the substrate-binding site, the conformation of the C25-C27 side chain moiety should affect the binding of the steroid skeleton. This is a unique example that the stereochemistry at the side chain affects the enzymatic reaction at A-ring of steroids. Furthermore, only 25S-isomer of 25,26-[(OH).sub.2]-V[D.sub.3] was converted to 1,25,26-[(OH).sub.3]-V[D.sub.3] (33)by 1[alpha]-hydroxylase. When the 3D-structure of 1[alpha],-hydroxylase is available, it will be revealed whether the same residues are contributed to the highly specific recognition of the stereochemistry of the hydroxylated side chains. According to the V[D.sub.3] metabolites identified so far, the plausible metabolic pathway can be summarized in Fig. 8.
The activity of calcification of these hydroxylated V[D.sub.3] derivatives is as follows: 24,25-[(OH).sub.2]-V[D.sub.3] is almost equal to 25-(OH)-V[D.sub.3] and 25,26-[(OH).sub.2]V[D.sub.3] is weaker than 24,25-[(OH).sub.2]-V[D.sub.3]. Furthermore, 23,25-[(OH).sub.2]-V[D.sub.3] is weaker than 25,26-[(OH).sub.2]-V[D.sub.3]. These results indicate that the hydroxylation at 23, 24 and 26 is a deactivation step of 25-(OH)-V[D.sub.3]. Interestingly, the C22-hydroxylated (R)- and (S)-22,25-[(OH).sub.2]-V[D.sub.3] lost V[D.sub.3] activity, although these derivatives have not been found in the metabolites. This suggests that the hydroxyl group at the C22 position disturbs their binding or affects the binding mode to the V[D.sub.3] receptor.
3. Fluorine derivatives and the enhancing activities of V[D.sub.3]. We prepared 26,27-[F.sub.6]-, 24-[F.sub.2]-, and 23-[F.sub.2]-1,25-[(OH).sub.2]-V[D.sub.3] derivatives (34, 35, 36, respectively) to block the hydroxylation (Fig. 9). As we expected, 24-[F.sub.2]-1,25-[(OH).sub.2]-V[D.sub.3] (35) showed 5-10 times higher activity than 1,25-[(OH).sub.2]-V[D.sub.3] and 26,27-[F.sub.6]-1,25-[(OH).sub.2]-V[D.sub.3] (34) showed 10 times higher activity than 1,25-[(OH).sub.2]-V[D.sub.3], which maintained its activity for a week. (56) This is because compound 34 underwent 23-hydroxylation and the metabolite remained in cells for a longer time. 23-[F.sub.2]-1,25-[(OH).sub.2]V[D.sub.3] derivative (36) did not enhance the activity of 1,25-[(OH).sub.2]-V[D.sub.3].
4. A new rearrangement of the side chain-- Biological activity of side chain carbon homologues. It was reported that 1,25-[(OH).sub.2]-V[D.sub.3] receptor is distributed in not only intestine, kidney, and bone, but also stomach, skin, pituitary gland, parathyloid gland and lung cancer cells. (57) Furthermore, 1,25-[(OH).sub.2]-V[D.sub.3] was reported to strongly facilitate the cell differentiation of human myeloid leukemia cells. (58) In order to develop anticancer and anti-leukemia drugs, it is thus required to seek for derivatives that have potent cell differentiation activity ([10.sup.-7]-[10.sup.-8]M for 1,25-[(OH).sub.2]-V[D.sub.3]) but much more weak calcium regulating activity ([10.sup.-9]-[10.sup.-10]M for 1,25-[(OH).sub.2]-V[D.sub.3]), i.e., separation of the activities.
DeLuca and Tanaka found a new metabolite that strongly bound to the cytosol V[D.sub.3] receptor in addition to the 24- and 1-hydroxylated metabolites when they incubated 24-epi-25-OH-VD2 ((24R)-24-methyl 25-(OH)-V[D.sub.3])(37) with avian kidney. The mass spectrum of the metabolite suggested that it could be either [[DELTA].sup.22]-26-methyl-1,25-[(OH).sub.2]-V[D.sub.3] (38) or 24-homo-[[DELTA].sup.22]-1,25-[(OH).sub.2]-V[D.sub.3] (39). Thus, the two compounds were synthesized and the following comparison with the metabolite established that the metabolite was 38. (59) Although the exact mechanism of this unique reaction is not clear, it might involve the cleavage of the C23-C24 bond in the metabolism as reported by Djerassi et al. in the biosynthesis of the marine sterols (Fig. 10). (60)
In addition to the unique formation of the 26-homo skeleton, the metabolite showed 10 times higher myeloid leukemia cell differentiation activity with little V[D.sub.3] activity than 1,25-[(OH).sub.2]-V[D.sub.3]. This suggested that some homo V[D.sub.3] side-chain skeletons have different activity from the native V[D.sub.3] skeleton. In this course, we have synthesized 26,27-dimethyl-, 26,27-diethyl-, 26,27-dipropyl-1,25-[(OH).sub.2]-V[D.sub.3] (40, 41 and 42).61) The dimethyl and diethyl analogs showed 2.5 and 10 times higher myeloid leukemia cell differentiation activity, respectively, than 1,25-[(OH).sub.2]-V[D.sub.3]. The dipropyl analog did not show the activity. A series of 24-homologated 1,25-[(OH).sub.2]-V[D.sub.3] analogs were also synthesized. Evaluation of the cell differentiation activity revealed that 1- or 2-carbon homologation (39, 43) increased the activity by 10-fold, but 3-carbon homologation (44) reduced the activity by one half (Fig. 11).
5. Development of new drugs. As described in the above two sections, the effects of fluorine substitution and carbon homologation in the side chain have been elucidated. We, therefore, synthesized side chain modified analogues, 24,24-[F.sub.2]-24-homo-1,25-[(OH).sub.2]-V[D.sub.3] (45) and 24,24-[F.sub.2]-26,27-dimethyl-V[D.sub.3] (62) (46) (Fig. 12). The latter showed more potent V[D.sub.3] activity than 1,25-[(OH).sub.2]-V[D.sub.3], whereas the former was much less active. However, the former was at least ten times more active than 1,25-[(OH).sub.2]-V[D.sub.3], for cell growth inhibitor of HL-60 cells. (22S)-24-Homo-26,27-[F.sub.6]-1,22,25-[(OH).sub.2]-V[D.sub.3] (DD-003, 47) was also synthesized as a possible drug candidate. This compound exhibited 10-fold greater inhibiting effect on the growth of HT-29 human colonic adenocarcinoma cells than 1,25-[(OH).sub.2]-V[D.sub.3], without calcium regulating activity. (63)
(24R)-1,24-[(OH).sub.2]-V[D.sub.3], which was synthesized at the early stage of our research in considering the importance of side chain hydroxylation, is an excellent antipsoriasis drug. This compound binds to chick intestinal cytosol receptor for 1,25-[(OH).sub.2]-V[D.sub.3] as equally as 1,25-[(OH).sub.2]-V[D.sub.3], is as active as 1[alpha]-OH-V[D.sub.3], and exhibits reduced toxicity due to rapid clearance. (24R)-1,24-[(OH).sub.2]-V[D.sub.3] is metabolized to 1,24,25-(OH)3-V[D.sub.3] upon the action of 25-hydroxylase. 26,27-Cyclo-[[DELTA].sup.22]-1,24-[(OH).sub.2]-V[D.sub.3] developed by Leo Pharmaceutical Products is a drug also for antipsoriasis. (66) 22-Oxa-1,25-[(OH).sub.2]-V[D.sub.3] has been developed as a drug for antipsoriasis and hyperparathyroidism by Chugai Pharmaceutical Co. Ltd. (64)) 26,27-[F.sub.6]-1,25-[(OH).sub.2]-V[D.sub.3] (34) has been developed as a drug of hyperparathyroidism. (23S,25R)-1,25-[(OH).sub.2]-V[D.sub.3]-26,23-lactone (48) shows only osteogenesis activity without bone resorption. Furthermore, (24R)-24,25-[(OH).sub.2]-V[D.sub.3] (20) was found to have osteogenesis activity. (65) Thus, the compounds synthesized for investigation of the side chain structure of the V[D.sub.3] metabolites have contributed to not only biochemical and biomedical research but also the development of new drugs.
The recent research on hydroxylated steroids and their receptors opens up a new horizon of the research on biological function of steroid and its related compounds. A further investigation of VDR/ NR ligands, in particular, molecular recognition of ligand and receptor would end up with a fruitful result.
Plant hormone brassinosteroids
1. Synthesis of brassinosteroids. In 1970, Mitchell and coworkers of the US Department of Agriculture reported that pollen of rape (Brassica napus) contains a new growth-promoting factor. (67) Their further efforts culminated in the isolation and structure elucidation of brassinolide (49) from 500 lbs. of bee-collected rape pollen in 1979. (68) A recent investigation on its receptor revealed that brassinolide binds a membrane protein which belongs to Toll-like receptor family alike mammal nuclear receptors for steroid hormones. (69)
The synthesis of brassinolide was completed by the use of Sharpless epoxidation as a key reaction to introduce the 23R-hydroxyl group together with 24R-methyl group as outlined in Fig. 13. (70) Starting from 22-aldehyde (50), the reaction with Grignard reagent generated from 3-methyl-1-butyn gave a mixture of 22R and 22S-hydroxy derivatives (51)in a similar ratio. Then, 22R-hydroxy derivative was converted to 23-cis allyalcohol (52). The Sharpless oxidation using vanadyl oxidant specifically provided the desired epoxyalcohol (53). Although direct introduction of the methyl group at C24 by opening the 23,24-epoxide was not successful, a cyano group was introduced specifically at C24 to give 22R,23R-diol (54). The conversion of the cyano group to the methyl group (55) was successfully carried out via the aldehyde.
The AB ring was then derived to the 6,7-secolactone moiety. At first, hydroboration of 5,6-double bond for the acetonide derivative (56) and the following oxidation of the resulting 6[alpha]-hydroxyl group into the oxo group gave 6-oxo derivative (57). Then, dehydration of the hydroxyl group at C3 gave 2,3-olefin(58), which was oxidized to afford the 2[alpha],3[alpha]-diol derivative (59). The Baeyer-Villiger oxidation with m-chloroperbenzoic acid gave the desired lactone (60) as a major product, which was converted to brassinolide through the following deprotection. The selectivity observed in this Baeyer-Villiger reaction is rather unusual, but the electronegative 2,3-diol group would control the selectivity.
The other groups reported similar synthetic routes for the synthesis. (71),72) Furthermore, we have developed the method for synthesizing brassinosteroids such as castasterone (61) and dolicholide (62). (73)
2. Microanalysis of brassinosteroid derivatives. Microanalytical methods for brassinosteroids were developed based on GC-MS and HPLC techniques. For GC analysis, bis-methaneboronate (BMB) ester derivative (63) of brassinosteroids was used. (74) A series of the BMB derivatives were separated with detection limits at nanogram levels (Fig. 15). (75) For HPLC, the bis-naphthaleneboronates (64) were preferably used for monitoring by a UV or a fluorimetric detector which enabled us to measure the amount of the derivatives down to 10-20 pg. (76)
The structures of brassinosteroids were determined by means of MS using several ionization techniques such as FD, CI, EI and FAB methods. The selective ion monitoring in GC-MS revealed the presence of brassinone (28-norcastasterone) in the leaves of green tea and the insect galls of Distylium racemosum, and the presence of castasterone in the leaves and the insect galls. (77) Dolichosterone (65) and castasterone were detected in the rice plant (78) and brassinone, castasterone (61), brassinolide and 24-epibrassinolide (66) were identified in bee pollen of the broad bean. (79) The analysis of various plant sources indicated that brassinosteroids are widely distributed in the plant kingdom as a mixture of several chemical entities (Fig. 14). (80),81)
The microanalytical method also enabled us to quantitatively measure the location and amounts of brassinosteroids in plants. For example, brassinolide and 28-norbrassinolide were detected in leaves of Distylium racemosum in 23 and 156ng/kg, respectively, but not in its insect galls, while castasterone and dolichosterone were detected in the insect galls in 2500 and 5000 ng/kg, respectively, and in the leaves in 133 and 16 ng/kg, respectively.
3. Structure-activity study and practical use of the analogs. The synthetic methods developed by us enabled to prepare more than 50 analogs of brassinosteroids for the study of structure-activity relationship analysis. (82) Since a structure of brassinosteroids/receptor complex is not available yet, it is not clear how the hydroxyl groups in the side chain are recognized at the ligand-binding site of the receptor. However, we found that the 22,23-vicinal diol groups with 22R,23R or 22S,23S configuration are important for the receptor activation. In contrast, the isomers with 22R,23S or 22S,23R configuration were inactive. With respect to the C24 substituent, 28-norbrassinolide was equally active as brassinolide, while 28-homobrassinolide was 10-fold less active than brassinolide. Interestingly, a side chain truncated analog, 26,27-bisnorbrassinolide, was active as brassinolide.
24-Epibrassinolide (66) and 24-epicastasterone are rare natural brassinosteroids that have 24S configuration. The activity of 24-epibrassinolide was comparable to that of brassinolide. Since our group had found a considerable amount of brassicastrol (67) in rape seed oil by GC analysis of sterol content, we developed a production method of 24-epibrassinolide (66) in industrial scale (Fig. 16) as a promising candidate for agricultural application. (83) Field testing on brassinosteroids such as 24-epibrassinolide and homobrassinolide for increasing crop yield was carried out in various countries and promising results were obtained not only for wheat, rice, soybean, and corn, but also for several vegetables. (84)
Collaborative studies between Japan and China on the practical application of 24-epibrassinolide in agriculture were started in 1985. A series of preliminary green house tests using 24-epibrassinolide supplied by our group were carried out at the Shanghai Institute of Plant Physiology. These early studies showed that 24-epibrassinolide accelerated the growth of cereals (wheat and corn), vegetables (watermelon, cucumber and grape), and tobacco. Follow-up field trials have been pursued in Shanghai, Henan and Zhejiang Provinces over a five-years period and the significant effects of 24-epibrassinolide have been observed in this trials. 24-Epibrassinolide as a mixture with its 22S,23S isomer are currently used in China, Belarus and other countries.
Progress in chemistry and biology of the major three steroidal hormones that have oxygenated side chains has been summarized.
In insect, it has been established that fucosterol 24,28-epoxide is a key intermediate in the dealkylative conversion of sitosterol to cholesterol. An ecdysteroid biosynthesized and released in prothoracic gland was unambiguously determined as ecdysone by the application of GC-MS analysis developed by us. Furthermore, detailed analysis of free and conjugated ecdysteroids in silkworm ovaries led to the findings that the phosphate form is an inactive stocked form and converted to the free ecdysteroid during embryonic development.
The stereo-structures of vitamin [D.sub.3] metabolites such as 24,25-[(OH).sub.2]-V[D.sub.3] and calcidiol lactone were unambiguously determined by HPLC comparison of the natural metabolites with synthetic isomers. These basic studies have revealed the details of the metabolism of vitamin [D.sub.3] and clarified the physiological importance of the stereochemistry at the side chain of vitamin [D.sub.3]. These understandings of the roles of the side chains were extended to the design and synthesis of more potent analogs having a specific and enhanced activity, which was eventually linked to the development of drugs for antipsoriasis and hyperparathyroidism.
More than 50 brassinosteroids, including the first synthesis of brassinolide, were synthesized in our laboratory and used as authentic standards in GCMS microanalysis for natural brassinosteroids and in the study of their structure-activity relationships. These studies have ended up with the development of 24-epibrassinolide, which is an active and useful analog in agriculture.
For more than 40 years, we have investigated biologically active steroids focusing on those bearing oxygenated side chains and have clarified the importance of the position and configuration of hydroxyl groups and number of carbons in the oxygenated side chains. It is interesting that simple sterols such as cholesterol and campesterol serve as the substrates of oxidative metabolisms and the resulting oxygenated metabolites, 20-hydroxyecdysone, brassinolide and 1,25-[(OH).sub.2]-V[D.sub.3] commonly play roles of physiologically important hormones in their growth in insects, plants and human, respectively. Thus, the modification and utilization of steroid side chains commonly occur and are important in maintenance of the physiological conditions of the life. It should be finally mentioned that intimate collaborations with prominent biologists enabled us to bring fruitful results mentioned above.
(1) Tsuda, K., Akagi, S. and Kishida, Y. (1957) Discovery of cholesterol in some red algae. Science 126, 927-928.
(2) Gortler, L. and Sturchio, J.L. (1992) The History of Steroid Chemistry Part 1. Steroids 57, 354-418, Gortler, L. and Sturchio, J.L. (1992) The Historyz of Steroid Chemistry Part 2. Steroids 57, 578-648.
(3) McMorris, T.C. and Barksdale, A.W. (1967) Iso lation of a sex hormone from the water mold, Achlya bisexualis. Nature 215, 320-321.
(4) Butenandt, A. and Karlson, P. (1954) Uber die isolierung eines metamorphose-hormons der insekten in kristallisierter form. Z. Naturforsch. 9b, 389-391.
(5) Lavie, D., Glotter, E. and Shvo, Y. (1965) Constit uents of Withania somnifera. Dun. III. The side chain of withaferine A. J. Org. Chem. 30, 1774-1778.
(6) Tsuda, K., Sakai, K. and Ikekawa, N. (1961) Gas chromatography of [C.sub.27],[C.sub.28], and [C.sub.29] sterols. Chem. Pharm. Bull. 9, 835-836.
(7) Ikekawa, N. (1987) Structures and biological activities of vitamin D metabolites and their analogs. Med. Res. Rev. 7, 333-366.
(8) Saito, M., Yamazaki, M., Shimizu, M. and Ikekawa, N. (1963) Studies on the sterol of Bombyx mori L. Agric. Biol. Chem. 27, 572-575.
(9) Miyauchi, K., Saito, M. and Ikekawa, N. (1964) Identification of [beta]-sitosterol and campesterol in mulberry leaf sterol, by gas chromatography. Bull. Sericul. Exp. Sta. 18, 431-439.
(10) Ikekawa, N., Suzuki, M., Kobayashi, M. and Tsuda, K. (1966) Studies on the sterol of Bombyx mori L. IV. Conversion of the sterol in the silkworm. Chem. Pharm. Bull. 14, 834-836.
(11) Thompson, M.J., Kaplanis, J.N., Robbins, W.E. and Svoboda, J.A. (1973) Metabolism of steroids in insects. Adv. Lipid Res. 11, 219-265.
(12) Ikekawa, N., Morisaki, N., Tsuda, K. and Yoshida, T. (1968) Marine algae. II. Sterol compositions in some green algae and brown algae. Steroids 12, 41-48.
(13) Ikekawa, N., Morisaki, M., Ohtaka, H. and Chiyoda, Y. (1971) Reaction of fucosterol 24,28-epoxide with boron trifluoride etherate. J. Chem. Soc. Chem. Commun., 22, 1498.
(14) Morisaki, M., Ohtaka, H., Okubayashi, M., Ikekawa, N., Horie, Y. and Nakasone, S. (1972) Fucosterol 24,28-epoxide as a probable intermediate in the conversion of [beta]-sitosterol to cholesterol in the silkworm. J. Chem. Soc. Chem. Commun., 23, 1275-1276.
(15) Fujimoto, Y., Awata, N., Morisaki, M. and Ikekawa, N. (1974) Migration of C-25 hydrogen of sitosterol to C-24 during conversion to desmosterol in the silkworm Bombyx mori. Tetrahedron Lett. 15, 4335-4338.
(16) Fujimoto, Y., Morisaki, M., Ikekawa, N., Horie, Y. and Nakasone, S. (1974) Synthesis of 24,28 iminofucosterol and its inhibitory effects on growth and steroid metabolism in the silkworm, Bombyx mori. Steroids 24, 367-375.
(17) Allais, J.P., Alcaide, A. and Barbier, M. (1973) Fucosterol-24,28-epoxide and 28-oxo-[beta]-sitosterol as possible intermediates in the conversion of [beta]-sitosterol into cholesterol in the locust Locusta migratoria L. Experientia 29, 944-945.
(18) Maruyama, S., Fujimoto, Y., Morisaki, M. and Ikekawa, N. (1982) Mechanism of campesterol demethylation in insect. Tetrahedron Lett. 23, 1701-1704.
(19) Fujimoto, Y., Morisaki, M. and Ikekawa, N. (1985) Enzymic dealkylation of phytosterols in insects. Methods Enzymol. 111, 346-352.
(20) Nicotra, F., Ronchetti, F., Russo, G. and Toma, L. (1984) Synthesis of (24R)- and (24S)-24,28-epoxyergost-5-en-3[beta]-ols. Substrate stereospecificity in their metabolism in the insect Tenebrio molitor. J. Chem. Soc., Perkin Trans. 1, 2039-2042.
(21) Ikekawa, N., Fujimoto, Y., Takasu, A. and Morisaki, M. (1980) Isolation of fucosterol epoxide from larvae of the silkworm, Bombyx mori. J. Chem. Soc. Chem. Commun., 15, 709-711.
(22) Nicotra, F., Pizzi, P., Ronchetti, F., Russo, G. and Toma, L. (1981) Substrate stereospecificity in the metabolism of fucosterol and isofucosterol 24,28-epoxides in Tenebrio molitor. J. Chem. Soc., Perkin Trans. 1, 480-483.
(23) Fujimoto, Y., Ikuina, Y., Nagakari, M., Kakinuma, K. and Ikekawa, N. (1990) C-25 prochirality in the fragmentation reaction catalyzed by fucosterol epoxide lyase from the silkworm, Bombyx mori. J. Chem. Soc., Perkin Trans. 1, 2041-2046.
(24) Fujimoto, Y., Nagakari, M., Ikuina, Y., Ikekawa, N. and Kakinuma, K. (1991) Stereochemistry of the hydrogen addition to C-25 of desmosterol by sterol-[[DELTA].sup.24]-reductase of the silkworm, Bombyx mori. J. Chem. Soc. Chem. Commun., 10, 688-689.
(25) Takahashi, K., Hashimoto, K., Fujiyama, A., Yamada, J., Kobayashi, N., Morisaki, M., Nakano, S., Hara, N. and Fujimoto, Y. (2003) Stereochemistry of reduction of the C-24, 25 double bond in the conversion of desmosterol into cholesterol. Tetrahedron Lett. 44, 341-344.
(26) Svoboda, J.A., Thompson, M.J. and Robbins, W.E. (1971) Identification of fucosterol as a metabolite and probable intermediate in conversion of [beta]-sitosterol to cholesterol in the tobacco hornworm. Nat. New Biol. 230,57-58.
(27) Ciufo, L.F., Murray, P.A., Thompson, A., Rigden, D.J. and Rees, H.H. (2011) Characterization of a desmosterol reductase involved in phytosterol dealkylation in the silkworm, Bombyx mori. PLoS ONE 6, e21316.
(28) Fukuda, S. (1940) Induction of pupation in the silkworm by transplanting the prothoracic gland. Proc. Imp. Acad. Tokyo 16, 417-419.
(29) Kobayashi, K., Saito, M., Ishitoya, Y. and Ikekawa, N. (1963) Brain hormone activity in Bombyx mori of sterols and physiologically vital active substances. Proc. Soc. Exp. Biol. Med. 114, 316-318.
(30) Huber, R. and Hoppe, W. (1965) Zur chemie des ecdysons, VII: Die kristall- und molekulstrukturanalyse des insektenverpuppungshormons ecdyson mit der automatisierten faltmolekulmethode. Chem. Ber. 98, 2403-2424.
(31) Hocks, P. and Wiechert, R. (1966) 20-Hydroxy ecdysone, isoliert aus insekten. Tetrahedron Lett. 7, 2989-2993.
(32) Chino, H., Sakurai, S., Ohtaki, T., Ikekawa, N., Miyazaki, H., Ishibashi, M. and Abuki, H. (1974) Biosynthesis of ,-ecdysone by prothoracic glands in vitro. Science 183, 529-530.
(33) Ikekawa, N., Hattori, F., Rubio-Lightbourn, J., Miyazaki, H., Ishibashi, M. and Mori, C. (1972) Gas chromatographic separation of phytoecdysones. J. Chromatogr. Sci. 10, 233-242.
(34) Sakurai, S., Yoneyama, N., Fujimoto, Y., Hata, F. and Ikekawa, N. (1986) 7-Dehydrosterols in prothoracic glands of the silkworm, Bombyx mori. Experientia 42, 1034-1036.
(35) Mizuno, T. and Ohnishi, E. (1975) Conjugated ecdysone in the eggs of the silkworm, Bombyx mori. Dev. Growth Differ. 17, 219-225.
(36) Ohnishi, E., Ohtaki, T. and Fukuda, S. (1971) Ecdysone in the eggs of Bombyx silkworm. Proc. Jpn. Acad. 47, 413-415.
(37) Ohnishi, E., Mizuno, T., Chatani, F., Ikekawa, N. and Sakurai, S. (1977) 2-Deoxy-,-ecdysone from ovaries and eggs of the silkworm, Bombyx mori. Science 197,66-67.
(38) Ikekawa, N., Ikeda, T., Mizuno, T., Ohnishi, E. and Sakurai, S. (1980) Isolation of a new ecdysteroid, 2,22-dideoxy-20-hydroxyecdysone, from the ovaries of the silkworm Bombyx mori. J. Chem. Soc. Chem. Commun., 10, 448-449.
(39) Ohnishi, E., Mizuno, T., Ikekawa, N. and Ikeda, T. (1981) Accumulation of 2-deoxyecdysteroids in ovaries of the silkworm, Bombyx mori. Insect Biochem. 11, 155-159.
(40) Fujimoto, Y., Miyasaka, S., Ikeda, T., Ikekawa, N., Ohnishi, E., Mizuno, T. and Watanabe, K. (1985) An unusual ecdysteroid, (20S)-cholesta-7,14-diene-3[beta],5,[alpha],6[alpha],20,25-pentaol (bombycosterol) from the ovaries of the silkworm, Bombyx mori. J. Chem. Soc. Chem. Commun., 1,10-12.
(41) Ohnishi, E., Hiramoto, M., Fujimoto, Y., Kakinuma, K. and Ikekawa, N. (1989) Isolation and identification of major ecdysteroid conjugates from the ovaries of Bombyx mori. Insect Biochem. 19,95-101.
(42) Elmogy, M., Iwami, M. and Sakurai, S. (2004) Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori. Eur. J. Biochem. 271, 3173-3179.
(43) Tanaka, Y., DeLuca, H.F., Ikekawa, N., Morisaki, M. and Koizumi, N. (1975) Determination of stereochemical configuration of the 24-hydroxy group of 24,25-dihydroxy-vitamin [D.sub.3] and its biological importance. Arch. Biochem. Biophys. 170, 620-626.
(44) Holick, M.F., Schnoes, H.K., DeLuca, H.F., Gray, R.W., Boyle, I.T. and Suda, T. (1972) Isolation and identification of 24,25-dihydroxycholecalciferol, a metabolite of vitamin [D.sub.3] made in the kidney. Biochemistry 11, 4251-4255.
(45) Koizumi, N., Morisaki, M., Ikekawa, N., Suzuki, A. and Takeshita, T. (1975) Absolute configurations of 24-hydroxycholesterol and related compounds. Tetrahedron Lett. 16, 2203-2206.
(46) Tanaka, Y., DeLuca, H.F., Ikekawa, N., Morisaki, M. and Koizumi, N. (1975) Determination of stereochemical configuration of the 24-hydroxy group of 24,25-dihydroxy-vitamin [D.sub.3] and its biological importance. Arch. Biochem. Biophys. 170, 620-626.
(47) Suda, T., DeLuca, H.F., Schnoes, H.K., Tanaka, Y. and Holick, M.F. (1970) 25,26-Dihydroxycholecalciferol, a metabolite of vitamin [D.sub.3] with intestinal calcium transport activity. Biochemistry 9, 4776-4780.
(48) Ikekawa, N., Koizumi, N., Ohshima, E., Ishizuka, S., Takeshita, T., Tanaka, Y. and DeLuca, H.F. (1983) Natural 25,26-dihydroxyvitamin [D.sub.3] is an epimeric mixture. Proc. Natl. Acad. Sci. U.S.A. 80, 5286-5288.
(49) Wichmann, J.K., DeLuca, H.F., Schnoes, H.K., Horst, R.L., Shepard, R.M. and Jorgensen, N.A. (1979) 25-Hydroxyvitamin [D.sub.3] 26,23-lactone: a new metabolite of vitamin [D.sub.3]. Biochemistry 18, 4775-4780.
(50) Tanaka, Y., Wichmann, J.K., Schnoes, H.K. and DeLuca, H.F. (1981) Isolation and identification of 23,25-dihydroxyvitamin [D.sub.3], an in vitro metabolite of vitamin [D.sub.3]. Biochemistry 20, 3875-3879.
(51) Ikekawa, N., Eguchi, T., Hirano, Y., Tanaka, Y., DeLuca, H.F., Itai, A. and Iitaka, Y. (1981) Synthesis and determination of the configuration of 23,25-dihydroxyvitamin [D.sub.3], a new metabolite of vitamin [D.sub.3]. X-ray crystal structure of a 3,23,25triol precursor. J. Chem. Soc. Chem. Commun., 1157-1159.
(52) Eguchi, T., Takatsuto, S., Ishiguro, M., Ikekawa, N., Tanaka, Y. and DeLuca, H.F. (1981) Synthesis and determination of configuration of natural 25-hydroxyvitamin [D.sub.3] 26,23-lactone. Proc. Natl. Acad. Sci. U.S.A. 78, 6579-6583.
(53) Ikekawa, N. (1983) Synthesis and biological activity of vitamin [D.sub.3] analogues. J. Steroid Biochem. 19, 907-911.
(54) Eguchi, T., Takatsuto, S., Hirano, Y., Ishiguro, M. and Ikekawa, N. (1982) Syntheses of four isomers of 25-hydroxyvitamin [D.sub.3]-26,23-lactone. Hetero cycles 17, 359-375.
(55) Castillo, L., Tanaka, Y., DeLuca, H.F. and Ikekawa, N. (1978) On the physiological role of 1,24,25 trihydroxyvitamin [D.sub.3]. Miner. Electrolyte Metab. 1, 198-207.
(56) Carradio, R.A., DeLuca, H.F., Tanaka, Y., Ikekawa, N. and Kobayashi, Y. (1980) A fluorinated vitamin [D.sub.3] analog with biopotency greater than 1,25dihydroxyvitamin [D.sub.3]. Biochem. Biophys. Res. Commun. 96, 1800-1803.
(57) Stumpf, W.E., Sar, M., Clark, S.A. and DeLuca, H.F. (1982) Brain target sites for 1,25-dihydroxyvita min [D.sub.3]. Science 215, 1403-1405.
(58) Abe, E., Miyaura, C., Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshiki, S. and Suda, T. (1981) Differentiation of mouse myeloid leukemia cells induced by 1[alpha],25-dihydroxyvitamin [D.sub.3]. Proc. Natl. Acad. Sci. U.S.A. 78, 4990-4994.
(59) Tanaka, Y., Sicinski, R.R., DeLuca, H.F., Sai, H. and Ikekawa, N. (1986) Unique rearrangement of ergocalciferol side chain in vitro: production of a biologically highly active homologue of 1,25dihydroxyvitamin [D.sub.3]. Biochemistry 25, 5512-5518.
(60) Proudfoot, J.R., Catalan, C.A.N. and Djerassi, C. (1986) Biosynthetic studies of marine lipids. 6. Evidence for an unprecedented biomethylation pathway in the biosynthesis of the cyclopropyl-containing marine sterol, petrosterol. Tetrahedron Lett. 27, 423-426.
(61) Honda, A., Mori, Y., Otomo, S., Ishizuka, S. and Ikekawa, N. (1991) Effects of novel 26,27-dialkyl analogs of 1[alpha],25-dihydroxyvitamin [D.sub.3] on differentiation-inducing activity of human promyelocytic leukemia (HL-60) cells in serum-supplemented or serum-free culture. Steroids 56, 142-147.
(62) Harada, M., Miyahara, T., Miyata, M., Komiyama, H., Sugure, A., Ikemoto, Y., Kondo, S., Takemura, T., Higuchi, S., Otomo, S., Kozuka, H., Ikekawa, N. and Eguchi, T. (1993) Calcium regulating activity of 24a-Homo-24,24-difluoro-1[alpha],25-dihydroxyvitamin [D.sub.3] and 26,27-dimethyl-24,24-difluoro-1[alpha],25-dihydroxyvitamin [D.sub.3]. Calcif. Tissue Int. 53, 318-323.
(63) Tanaka, Y., Wu, A.-Y.S., Ikekawa, N., Iseki, K., Kawai, M. and Kobayashi, Y. (1994) Inhibition of HT-29 human colon cancer growth under the renal capsule of severe combined immunodeficient mice by an analogue of 25-hydroxyvitamin [D.sub.3], DD-103. Cancer Res. 54, 5148-5153.
(64) Binderup, L. and Bramm, E. (1988) Effects of a novel vitamin D analog MC 903 on cell proliferation and differentiation in vitro and on calcium metabolism in vivo. Biochem. Pharmacol. 37, 889-895.
(65) Abe, L., Morikawa, M., Miyamoto, K., Kaiho, S., Fukushima, M., Miyaura, C., Abe, E., Suda, T. and Nishii, Y. (1987) Synthetic analogues of vitamin [D.sub.3] with an oxygen atom in the side chain skeleton A trial of the development of vitamin D compounds which exhibit potent differentiation-inducing activity without inducing hypercalcemia. FEBS Lett. 226,58-62.
(66) Endo, H., Kiyoki, M., Kawashima, K., Naruchi, T. and Hashimoto, Y. (1980) Vitamin [D.sub.3] metabolites and PTH synergistically stimulate bone formation of chick embryonic femur in vitro. Nature 286, 262-264.
(67) Mitchell, J.W., Mandava, N., Worley, J.F., Plimmer, J.R. and Smith, M.V. (1970) Brassins-a new family of plant hormones from rape pollen rape. Nature 225, 1065-1066.
(68) Grove, M.D., Spencer, G.F., Rohwedder, W.K., Mandava, N., Worley, J.F., Warthen, J.D. Jr., Steffens, G.L., Flippen-Anderson, J.L. and Cook, J.C. Jr. (1979) Brassinolide, a plant growth-promoting steroid isolated from Brassica napus pollen. Nature 281, 216-217.
(69) Hothom, M., Belkhadir, Y., Dreux, M., Dabi, T., Noel, J.P., Wilson, I.A. and Chory, J. (2011) Structural basis of steroid hormone perception by the receptor kinase BRI1. Nature 474, 467-471.
(70) Ishiguro, M., Takatsuto, S., Morisaki, M. and Ikekawa, N. (1980) Synthesis of brassinolide, a steroidal lactone with plant-growth promoting activity. J. Chem. Soc. Chem. Commun., 20, 962-964.
(71) Fung, S. and Siddall, J.B. (1980) Stereoselective synthesis of brassinolide: a plant growth promoting steroidal lactone. J. Am. Chem. Soc. 102, 6580-6581.
(72) Mori, K., Sakakibara, M. and Okada, K. (1984) Synthesis of naturally occurring brassinosteroids employing cleavage of 23,34-epoxides as key reactions. Synthesis of brassinolide, castasterone, dolicholide, dolichosterone, homodolicholide, homodolichosterone, 6-deoxocastasterone and 6-deoxodolichosterone. Tetrahedron 40, 1767-1781.
(73) Takatsuto, S. and Ikekawa, N. (1983) Stereoselective synthesis of plant growth-promoting steroids, dolicholide and 28-norbrassinolide. Tetrahedron Lett. 24, 773-776.
(74) Takatsuto, S., Ying, B., Morisaki, M. and Ikekawa, N. (1982) Microanalysis of brassinolide and its analogs by gas chromatography and gas chromatography-mass spectrometry. J. Chromatogr. A 239, 233-241.
(75) Ikekawa, N., Takatsuto, S., Kitsuwa, T., Saito, H., Morishita, T. and Abe, H. (1984) Analysis of natural brassinosteroids by gas chromatography and gas chromatography-mass spectrometry. J. Chromatogr. A 290, 289-302.
(76) Gamoh, K., Kitsuwa, T., Takatsuto, S., Fujimoto, Y. and Ikekawa, N. (1988) Determination of trace brassinosteroids by high-performance liquid chromatography. Anal. Sci. 4, 533-535.
(77) Ikeda, M., Takatsuto, S., S., assa, T., Ikekawa, N. and Nukina, M. (1983) Identification of brassinolide and its analogs in chestnut gall tissue. Agric. Biol. Chem. 47, 655-657.
(78) Abe, H., Nakamura, K., Morishita, T., Uchiyama, M., Takatsuto, S. and Ikekawa, N. (1984) Endogenous brassinosteroids of the rice plant: castasterone and dolichosterone. Agric. Biol. Chem. 48, 1103-1104.
(79) Yokota, T., Koba, S., Kim, S.K., Takatsuto, S., Ikekawa, N., Sakakibara, M., Okada, K., Mori, K. and Takahashi, N. (1987) Brassinosteroids in Phaseolus vulgaris. Part II. Diverse structural variations of the brassino-steroids in Phaseolus vulgaris seed. Agric. Biol. Chem. 51, 1625-1631.
(80) Ikekawa, N. (1990) The analysis of brassinoste roids--plant growth-promoting substances. Trends Analyt. Chem. 9, 337-342.
(81) Ikekawa, N., Nishiyama, F. and Fujimoto, Y. (1988) Identification of 24-epibrassinolide in bee pollen of the broad been, Vicia faba. L. Chem. Pharm. Bull. 36, 405-407.
(82) Takatsuto, S., Yazawa, N., Ikekawa, N., Takematsu, T., Takeuchi, Y. and Koguchi, M. (1983) Structure-activity relationship of brassinosteroids. Phy tochemistry 22, 2437-2441.
(83) Takatsuto, S. and Ikekawa, N. (1984) Short-step synthesis of plant growth-promoting brassinoste roids. Chem. Pharm. Bull. 32, 2001-2004.
(84) Ikekawa, N. and Zhao, Y.-J. (1991) Application of 24-epibrassinolide in agriculture. ACS Symposium Series 474, 280-291.
(Received July 3, 2013; accepted Aug. 21, 2013)
Nobuo IKEKAWA [*1], [[dagger]], Yoshinori FUJIMOTO [*2] and Masaji ISHIGURO [*3]
[*1] Emeritus Professor of Tokyo Institute of Technology, Tokyo, Japan.
[*2] Department of Chemistry and Materials Science, Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo, Japan.
[*3] Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan.
[[dagger]] Correspondence should be addressed: N. Ikekawa, 2-21-5 Kichijoujihigashimachi, Musashino, Tokyo 180-0002, Japan (email: email@example.com).
Nobuo Ikekawa was born on October 12, 1926, in Iiyama, Nagano, Japan. He received his B.S. degree from the University of Tokyo, faculty of pharmaceutical sciences. He continued his studies at Kyushu University, faculty of medicine and then, moved to the Institute of Applied Microbiology of the University of Tokyo in 1955 as a research associate. He received his Ph.D. degree from the University of Tokyo in 1958. After two-year stay at national Institute of Health, NIAMD, in the United States as a visiting scientist, he was appointed a senior chemist at the Institute of Physical and Chemical Research (RIKEN) in 1961. In 1966 he spent four months as an associate professor at the Institute of Lipid Research of Baylor College of Medicine in Houston. In 1969, he joined Tokyo Institute of Technology as an associate professor and then was appointed a full professor of department of chemistry in 1981. He moved to Iwaki-Meisei University in 1987 and then he was appointed as the president of Niigata University of Pharmacy and Applied Life Sciences in 1995 through 2002.
He is the author and co-author of more than 400 original papers and close 100 review articles, dealing mainly with investigation of naturally occurring biologically important substances. He is a pioneer in the field of the separation and isolation of a number of steroids and also contributed to the synthesis of natural and designed steroids.
He edited four-volume book on Modern Gas Chromatography (Hirokawa Pub.) and served on the editorial board of journals of the Pharmaceutical Society of Japan.
He received Sato Memorial Award (1979), Society Award (1981) and Member of Merit Award (1998) from Pharmaceutical society of Japan, and Order of the Rising Sun, Gold Rays with Neck ribbon (2002) from the Japanese Government. He also received M. S. Tswett Chromatography Award (1982), Shimadzu Award (1992) and Career Recognition Award of Vitamin D (1994).
He was a board of directors of Pharmaceutical Society of Japan (1984-1991) and also a director of Niigata Bio-Research Center (2004-2012).
He is a member of merit of Pharmaceutical Society of Japan, Royal Society of Chemistry and a Fifty-Year member of American Chemical Society.
He is a professor emeritus of Tokyo Institute of Technology, Niigata University of Pharmacy and Applied Life Sciences, and Changchun University of Chinese Medicine.
Yoshinori Fujimoto was born in Yamaguchi prefecture in 1949. He completed his Ph.D. for a thesis entitled "Studies on the mechanism of the conversion of phytosterol to cholesterol in insects" under the supervision of Prof. Nobuo Ikekawa in Faculty of Science and Engineering at Tokyo Institute of Technology in 1979. In the same year, he joined Prof. Charles J. Sih's laboratory, School of Pharmacy, University of Wisconsin-Madison as a research fellow and spent three years there, where he worked on the mechanism of microbial degradation of sitosterol side chain and quantitative analyses of biochemical kinetic resolutions of enantiomers. He was appointed to an assistant professor in Department of Chemistry of Tokyo Institute of Technology in 1984 and then promoted to an associate professor in 1990. In 1996, he was promoted to professor, and joined a newly organized Department of Chemistry and Materials Science, Graduate School of Science and Engineering of the same university in 1997. He has been carried out researches in the field of natural product chemistry including biosynthesis of plant and insect sterols, isolation and structure elucidation of new secondary metabolites from plants, insects and marine invertebrates, and synthesis of steroidal compounds having unique side chains. His current research is focused on the identification of genes encoding enzymes that catalyze sterol metabolism in insects as well as plants.
Masaji Ishiguro was born in 1950 and received his Ph.D. in 1977 from Tokyo Institute of Technology. He worked at the laboratory of Prof. E. J. Corey in Harvard University as postdoctoral fellow in 1977 through 1979. While he worked on the synthesis of natural products in Harvard, he was greatly influenced on the computer-aided chemistry developed in the laboratory and thus applied the technology to designing biologically active molecules based on the structures of target biomolecules. Since he joined Suntory Institute for Biomedical Research in 1980, he directed the computer-aided drug design and succeeded in development of new O-lactam antibiotics. A penem Olactam named Farom (Faropenem) was launched in the market in 1995. For this work, he was the first recipient of The Pharmaceutical Society of Japan Award for Drug Research and Development (2001). In 1995 he moved to Suntory Institute for Bioorganic Research (SUNBOR) and started the investigation of receptor-ligand interactions using computational methods focusing on G protein-coupled receptors (GPCR). In 2006, he was appointed to a professor in chemical biology laboratory of Niigata University of Pharmacy and Applied Life Sciences (NUPALS), continuing his research in SUNBOR as senior research fellow until his retirement in 2010. His major subject in chemical biology field has been molecular recognition in vision, odor, and taste. The crystal structure of squid rhodopsin, a member of GPCR, was solved in collaboration with Dr. S. Miyano's group of RIKEN in Harima and the molecular base of recognition of sweet taste molecules by sweet taste receptor (T1R2/T1R3) was established with collaboration with Prof. K. Abe's group of the university of Tokyo. Currently, he is a professor and dean of the faculty of Applied Life Sciences of NUPALS.
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|Author:||Ikekawa, Nobuo; Fujimoto, Yoshinori; Ishiguro, Masaji|
|Publication:||Japan Academy Proceedings Series B: Physical and Biological Sciences|
|Date:||Aug 1, 2013|
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