We show here synthetic studies on halicholactone (1), solandelactones (2) and cineromycin B (3) using our chiral building block 6 or 34 as a starting material. Halicholactone (1) was isolated from the marine sponge, Halichondoria okadai, and inhibits 5-lipoxygenase of guinea pig polymorphonuclear leukocytes. Recently, we achieved total synthesis of 1 from 6. The starting material (6) was converted into 5 through an epimerization of 9. A key reaction, NHK reaction of 5 with 15, proceeded in DMSO/DMF solution to afford 16a preferentially. Another key reaction, RCM reaction of 4 which was derived from 16a, gave nine-membered lactone. Finally, methanolysis of two acetates afforded 1. We also tried to synthesize 2, which was isolated from the hydroid, Solanderia secunda, and inhibit farnesyl-protein transferase, using the same key reactions. We have now succeeded in synthesizing 18 by RCM reaction of 9 which was derived from 12. Transformation of 18 to 2 is now in progress. Cineromycin B (3) is a macrolide antibiotic isolated from various strains of Steptomyces sp. and displays biological activity primarily against B. subtilius and S. aureus. Very recently, a new activity that 3 induces apoptosis of tumor cells was taken an interest. These important activity and unique structural features have inspired us to its synthesis. Now we show a total synthesis of 3 using oxidative [2.3]-sigmatropic rearrangement of 40 as a key reaction. Firstly, we synthesized 40 from our chiral building block 34. The key rearrangement of 40 was achieved successfully by treatment with H_2O_2 at 0℃ to give 41. Macrocyclization of 30 derived from 41 was unsuccessful and gave 42. In order to afford the desired ring system, protection of the tertiary hydroxyl group was essential and cyclization of 44 gave 45. Finally, we accomplished the total synthesis of 3 by deprotection of TES group.

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Scytophycin C (1) is a novel class of polyketide-derived 22-membered macrolide isolated from the cultured blue-green alga Scytonema pseudohofmanni, which exhibits potent cytotoxicity against a variety of human carcinoma cell lines as well as broad spectrum antifungal activity. The unique structures and biological activities of the scytophycins have elicited much attention from synthetic organic chemists. Toward the stereoselective total synthesis of Scytophycin C (1), we designed a convergent synthetic strategy. Namely, 1 was divided into three segments, segments A, B and C by disconnecting the lactone O-C21 bond, the C18-C19 and C26-C27 bonds, respectively, and each segment containing polypropionate structure was designed to be stereoselectively synthesized by using stereospecific methylation reactions which have been developed in our laboratory. Thus, the segments A and B containing three and five consecutive asymmetric centers, respectively, were highly stereoselectively synthesized according to the synthetic schemes. Similarly, the segment C was stereoselectively synthesized starting from D-glucal via allylation, stereospecific methylation of the epoxy sulfide 15 with Me_3Al with double inversion of the configuration, and subsequent Mukaiyama aldol reaction. The coupling reaction of the segments A and B was efficiently carried out culminating in the synthesis of the segment AB, which was coupled with the silyl enol ether prepared from the segment C to give 22 as a single stereoisomer. Macrolactonization of the seco acid 24 under Yamaguchi conditions yielded 34% of the desired 22-membered lactone 25 and 51% of the 24-membered lactone 26. Fortunately, the undesired 26 was transformed into 25 with titanium tetraisopropoxide. The remaining task for the synthesis of Scytophycin C was introduction of a terminal trans-enamide moiety. However, it was extremely difficult. Finally, we succeeded in the introduction of the terminal trans-enamide moiety by Takai reaction followed by Buchwald's method and achieved the stereoselective total synthesis of Scytophycin C (1).

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Angiogenesis inhibitors are promising drugs for the treatment of angiogenesis-related diseases including cancer. We have recently reported the isolation and structural determination of unique pentaketide dimers, epoxyquinols A (1) and B (2), which show anti-angiogenic activity, but have different structural properties from the known angiogenesis inhibitors. To facilitate elucidation of the mechanism of action of epoxyquinols A (1) and B (2), the development of a method for their total synthesis and derivatization is highly desirable. Though structurally epoxyquinols A and B have a highly functionalized and complicated heptacyclic ring system containing 12 stereo-centers, biosynthetically it is proposed they are formed via an unusual oxidative dimerization of the much simpler epoxycyclohexenone 3. The synthesis starts from the Diels-Alder reaction between furan and chiral dienophile, planned to establish the correct stereochemistry and introduce all the carbon atoms except those in the side chain. The combination of HfCl_4 and the chiral acrylate ester of Corey's auxiliary enables the highly diastereoselective Diels-Alder reaction of furan, which established the correct stereochemistry. All 12 chiral centers of epoxyquinols A (1) and B (2) are controlled by the highly diastereoselective reactions in the route from the initial Diels-Alder product. Iodolactonization followed by hydrolysis and epoxidation gave the endo epoxide 3 in high yield. The stereoselective epoxidation of the homoallylic alcohol and α-iodination of the cyclohexenone were key steps to form the monomer 3. Practical synthetic route of the monomer 3 was also developed: Column chromatography-free synthesis of iodolactone 6 from furan and acryloyl chloride, and lipase-catalyzed kinetic resolution of racemic cyclohexenol (±)-8 enabled the multi-gram synthesis of both enantiomers of the monomer 3. Epoxyquinols A (1) and B (2) were synthesized from the monomer 3 by the biomimetic oxidative dimerization; MnO_2 oxidation of the primary hydroxy group of the monomer 3 without protection of the secondary hydroxy group affords aldehyde 17. The subsequent 6π-electrocyclization proceeds smoothly, providing dienone derivative 18a and 18b, which dimerize to give epoxyquinols A (1) and B (2) in 40% and 25% yield, respectively.

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Kazusamycin, which was isolated as a metabolite of actinomycete strain 81-484, attracts much attention due to its potent antitumor activity on P388 leukemia and sarcoma 180. It was also effective in completely preventing growth of HeLa cells at a concentration of 3.3ng/ml. On the other hand, from the structural point of view, only its planar structure was elucidated, and both its absolute configuration and complete relative configuration remain unknown. We are making an effort on the total synthesis of Kazusamycin in order to determine its absolute configuration and to establish a practical synthetic route for the biological assay. Retrosynthetic fragmentation of Kazusamycin affords two major segments, which we planned to prepare from medium-sized synthetic units, Segment A-E (Fig. 1). Segment A was prepared by using Pd(0) mediated coupling reaction between a chiral zinc homoenolate 3 and an alkenyl halide 4. On the preparation of Segment B, we initially planned to desymmetrize a dihydroxyketone 1 by enzymatic acetylation, however, the ee of the product never exceeded a moderate level. After several investigations, it was found that enzymatic kinetic resolution of rac-7 was successfully employed in the preparation of almost optically pure hydroxy ketone 7, which was protected by silylation to afford Segment B. The pivotal step in this synthetic study is the construction of four contiguous chiral centers, which might be realized by the stereoselective aldol reaction between Segment A and B. We performed the aldol reaction under the conditions developed by I. Paterson et al., in which high stereoselectivity was obtained with a related substrate by means of chelation control with tin(II) triflate. It was revealed that a comparable level of stereoselection was also observed in our case, and this methodology was applicable to the connection of A and B, affording an intermediate leading to one of the two major segments. We have also succeeded to synthesize another Segment D and E (Segment C is commercially available). Studies on the connection of these segments to construct the whole structure of Kazusamycin are now underway.

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Concerns about magnetic effects on the biological system are growing since the chances for mankind to be exposed to moderate or strong magnetic field, e.g., linear motor cars and MRI, are increasing. Living organisms are functioned by series of chemical reactions, and, therefore, basic knowledge of the magnetic effects on fundamental chemical reactions is required for understanding the effect of a magnetic field on human health. In our studies on the effects of a magnetic field on chemical reactions, superconductive NMR instruments (400MHz) have been used. They can produce a strong and extremely stable magnetic field (9.4T [tesla]). Using these apparatus, we examined the magnetic field effects on several fundamental organic reactions. The results obtained by the present study are summarized as follows: 1) An S_N2 reaction of phenethyl mesylate with sodium iodide to produce phenethyl iodide and an epoxidation of an olefin with mCPBA are unaffected by 9.4T magnetic field. 2) Tributyltin hydride reduction of phenethyl iodide, benzyl bromide, and m-methoxybenzyl bromide is remarkably accelerated by 9.4T magnetic field. 3) A radical oxidation of benzyloxymethylene group with 1-(tert-butylperoxy)-1,2-benziodoxol-3(1H)-one, a hypervalent iodane reagent, is accelerated by both 9.4T and 7.05T magnetic field. 4) Autoxidation of linoleic acid is faster under 9.4T magnetic field than that in the absence of magnetic field.

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Synthesis of two possible diastereomers of (+)-didemniserinolipid B (1), the first natural serinolipid isolated from a tunicate Didemnum sp., were studied. The known butanediacetal (BDA) protected aldehyde 2 was converted to the core coupling component 8. The serinol fragments (S)-13 and (R)-13 were prepared from D-serine and L-serine, respectively. Wittig-Horner reaction of 8 and (S)-13 gave (4"S)-14, which was converted to ester (4"S)-18. Finally, removal of all the protecting group was achieved in acidic EtOH to afford (30S)-1. Similarly, (30R)-1 was synthesized from (R)-13. However, the ^1H and ^<13>C NMR spectra of the synthetic 1 were different from those of natural 1. In view of the fact that related natural products were sulfated on the serinol unit we therefore believed the 31 position of the natural 1 should be sulfated. Based on this assumption, we investigated further derivatization of the synthetic compounds. We studied sulfation of N-Fmoc derivative (30R)-19, however, with the usual reagents and conditions resulted in only recovery of the starting material. Finally we developed new microwave-assisted sulfation conditions which gave the desired monosulfate, followed by the Fmoc deprotection affording 31-monosulfate (30R)-20. As the ^1H and ^<13>C NMR data and the value of the specific optical rotation of (30R)-20 coincided with those of the natural product, we concluded that the real structure of (+)-didemniserinolipid B corresponded to a 31-sulfate and the absolute configuration was 8R,9R,10R,13S,30S. The microwave-assisted sulfation developed in this process are effective for unreactive hydroxyl groups.

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Fostriecin (1, CI-920), produced by Streptomyces pulveraceus, is a promising antitumor antibiotic, which is known to inhibit DNA topoisomerase II by a unique, non-DNA-strand cleavage mechanism, and selectively inhibit protein phosphatase 2A and 4. We describe here synthesis of fostriecin and its analogue, 12,13,14,15,16,17-hexahydrofostriecin (2), via a convergent route involving a three-segment (A-C) coupling procedure (Figure 1). The segment B (4) was synthesized from (R)-malic acid by combination of Wittig reaction and Sharpless asymmetric dihydroxylation (Scheme 1). In the synthesis of hexahydrofostriecin 2 (Scheme 3), the segment C' was, at first, coupled with the segment B by taking account of the reactivity of the unsaturated lactone moiety. After the segment B-C' was coupled with the segment A 3c, the stereochemistry at C-5 position was successfully constructed by asymmetric reduction, and the resultant compound was transformed to hexahydrofostriecin 2. Total synthesis of fostriecin (1) was also achieved as follows (Scheme 4). In the case of fostriecin, the segment B was first coupled with the segment A rather than the segment C due to the labile property of triene moiety, then was transformed to the unsaturated lactone according to a similar procedure. Construction of the triene moiety was eventually achieved by combination of iodomethylenation and Stine coupling reaction. The phosphate moiety was introduced via two routes: (i) direct phosphorylation of the monohydroxyl derivative 35 in which other hydroxyl groups are protected; (ii) cyclic phosphorylation and selective cleavage of the cyclic phosphate derivative 38 (Scheme 5). Although the former is basically the same as those reported by other groups, the latter is novel and more effective than former one. The present total synthesis would serve as a versatile synthetic route to not only fostriecin and hexahydrofostriecin, but also its various analogues including stereoisomers.

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Wortmannin (1) is a potent and specific phosphoinositide 3-kinase inhibitor and widely used for the studies on the signal transduction pathways. In spite of the interesting biological activity, 1 has not yet been applied to medical use due to high toxicity. In addition to the medicinal aspect, the challenging structural features of 1 are very attractive from a synthetic point of view. In 1996, we reported the first chemical synthesis of 1 from hydrocortisone as a starting material. Following this primary achievement, we planned to develop a direct total synthesis of 1, hopefully leading to many more derivatives for new types of drugs. Here, we wish to report the first total synthesis of (±)-1. The highly stereoselective construction of allylic quaternary carbon center was achieved (α:β=1:18) using intramolecular Heck reaction with 27. After several elaborations, we found that diosphenol-Claisen rearrangement of 32 was found to be effective for introducing a C-C bond at the sterically hindered neopentyl position. The rearrangement actually proceeded very smoothly under thermal condition and the obtained 33 could be successfully converted into key intermediate lactone 36. After production of 36, furan ring formation remained. The C1 unit for constructing furan ring was introduced by aminomethylenation with HC(NMe_2)_3 and then diosphenol 44 was synthesized by careful transformations. However, unexpectedly, simple acid treatment of 44 only lead to decomposition. Finally, we found clean consumption of 44 by treatment with Et_2NH and successive treatment with 1N HCl afforded furan 46. Final deprotection of TBS and acetylation proceeded to give (±)-1. We are also trying to develop a catalytic asymmetric synthesis of (+)-1. Currently two strategies are being investigated; kinetic resolution using asymmetric Heck reaction and organocatalyst promoted asymmetric cyclization. These results would be also presented.

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Actinobolin 1 is reported to show potent antibiotic, antitumor and antileukemic activity as well as therapeutic effect on autoimmune encephalomyelitis. Due to its unique structure involving perhydroisochromane skeleton possessing an amino function with five contiguous asymmetric carbon centers, many reports on the total synthesis and chemical modification of 1 have appeared. We report here the chiral and stereoselective total synthesis of actinobolin starting from D-glucose, using Ferrier's carbocyclization reaction and three components coupling reaction as the key transformations. Reaction of a substituted cyclohexenone 8 derived from D-glucose by way of Ferrier's carbocyclization, with vinyl cuprate followed by treatment with C3 aldehyde 9 afforded the carbon framework 10 of actinobolin in high yield and in highly stereoselective manner. Reduction of 10 with Me_4NBH(OAc)_3 stereoselectively gave diol 12, whose reaction with TsCl followed by protection of the remaining hydroxyl group generated 13. The requisite lactone 18 having proper functionalities with correct stereochemistries was obtained by reduction of methyl ketone 15, followed by lactonization and introduction of an azide group. Deprotection of TBS group in 18 and subsequent oxidation afforded perhydroisochromane skeleton 21. Hydrogenation of 21 followed by condensation with Cbz-D-alanine gave protected (-)-actinobolin 22. Finally, removal of the Cbz group successfully furnished (-)-actinobolin, an antipode of the natural product. Based on this methodology, formal synthesis of (+)-actinobolin starting from D-glucose was also achieved.

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In 1979, Ito and Saegusa et al. discovered that the corresponding silyl enol ethers of alkenyl ketones provided the β,γ-unsaturated cyclic ketones in the presence of Pd(II). This methodology has proven to be a powerful tool for the synthesis of complex. polycyclic compounds. However, the process employing stoichiometric amounts of Pd(OAc)_2 suffers from low yields on large scale. To solve this problem, we have developed a novel palladium-catalyzed cycloalkenylation of the olefinic tert-butyldimethylsilyl enol ethers, e.g. 1→2, and the methodology was successfully adapted for the syntheses of polycyclic natural products, such as (-)-methyl atis-16-en-19-oate (3), (-)-methyl trachyloban-19-oate (4), (-)-methyl kaur-16-en-19-oate (5), and C_<20> gibberellins (6, 7, and 8) (Scheme 1). The search for the improvement of the palladium-catalyzed cycloalkenylation has been continued, with the goal of increasing the diversity of possible substrates and reaction products. We have developed a convenient, novel process to construct bicyclo[3.3.0]octane derivatives, potential synthons for the syntheses of linear and angular triquinane sesquiterpenes, such as capnellenes and silphinenes. Although the product yields are moderate, the palladium-catalyzed cycloalkenylations can be adaptable for the construction of bicyclo[4.3.0]nonane compounds (hydrindane systems). Interestingly, the palladium-catalyzed cycloalkenylation of the silyl enol ether (25) gave the tricyclic compound (26). In addition, the catalytic cycloalkenylation turned out to be adaptable to a tandem process. The tricyclic compound 28, the basic framework of cedrene (29), was prepared through the above protocol. Finally, a synthesis of aphidicolin (30) has been demonstrated using the palladium-catalyzed cycloalkenylation.

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(-)-Strychnine, which is the most famous of the Strychnos alkaloids, has seven rings and six asymmetric centers in the molecule and is one of the most complex natural products in its size. Although Woodward succeeded in the total synthesis of (-)-strychnine in 1954, there were no reports on the total synthesis of strychnine for about 40 years. Herein we report the total synthesis of (-)-strychnine. The synthesis of a chiral indoline derivative having the ABC-rings of (-)-strychnine was achieved by palladium-catalyzed asymmetric allylic substitution of a cyclohexenol derivative having substituent at the 2-position followed by palladium-catalyzed cyclization. Construction of the D-ring of (-)-strychine was carried out through palladium-catalyzed allylic oxidation. The G-ring and then the E-ring were constructed by palladium-catalyzed cyclization using Pd(OAc)_2. (+)-Isostrychnine was converted into (-)-strychnine by the known method. The spectral data and [α]_D values agreed with those of (+)-isostrychnine and (-)strychnine reported in the literature. The fact that all cyclizations for synthesis of (+)-isostrychnine were performed using palladium catalysts is indicated the importance of palladium catalyst in modern synthetic organic chemistry.

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Himbacine 1 is a piperidine alkaloid isolated in 1956 from the bark of Galbulimima Baccata in magnolia family. It is reported that 1 behave as a potent antagonist of the muscarinic receptor of M_2 subtype with 10-20 fold selectivity toward the M_1 receptor. Thus, blockage of the presynaptic muscarinic receptor of M_2 subtype leads to an elevation of the synaptic levels of acetylcholine, possibly offsetting some of the losses in the cholinergic system that occurs in Alzheimer's disease. Therefore, much interests have been paid on 1 from the viewpoint of medicinal and/or synthetic organic chemistry. With an aim to disclose novel aspects of the structure-activity relationships of 1, and moreover, to explore the promising himbacine congeners which may show more improved M_2 antagonistic activity and subtype selectivity, we embarked on the synthetic studies on 1. Our novel total synthesis of 1 was completed in 1999 by employing highly stereoselective intermolecular Diels-Alder reaction of the tetrahydrofuran 2 with the chiral furan-2(5H)-one 3 as a key step. Successful application of the explored synthetic route to the synthesis of ent-1 realized its efficiency and directness. Binding affinity assay of ent-1 against the muscarinic receptor of M_2 subtype uncovered that the absolute configuration of 1 was obviously correlated with its activity. In order to explore further novel aspects of the structure-activity relationships of 1, we continued the synthetic studies on various structural types of himbacine congeners. These studies resulted in the successful preparations of 4-epi-himbacine 8, the compounds 10 carrying olefin bioisosteres and aromatic or aliphatic amines in place of an (E)-double bond and a 2,6-disubstituted piperidine, 2',6'-diepi-himbacine 12 and its enantiomer ent-12, 3-norhimbacine 20 and its enantiomer ent-20, 4-epi-3-norhimbacine 21 and its enantiomer ent-21, 11-methylhimbacine 27, and 3-epi-himbacine 29. Synthesis of 10, 12, and ent-12 was achieved starting with the synthetic intermediates 6, 7, 9 and ent-6, 7, 9 for 1 and ent-1, respectively. The 3-norhimbacine derivatives 20, ent-20, 21, and ent-21 were prepared from 2 and achiral furan-2(5H)-one 13 following the same synthetic route as that for 1 and ent-1 and separating four diastereomeric intermediates by CHIRALCEL OD. Preparation of 27 was carried out using (S)-5-ethylfuran-2(5H)-one 23 in place of 3. Direct epimerization of the C_<3α>-methyl group for the synthetic intermediate of 1 afforded 29. All the synthesized congeners were subjected to binding affinity assay against the muscarinic receptor of M_2 subtype. It appeared evident that, while its subtype selectivity is similar to that of 1, 3-norhimbacine 20 shows more potent binding affinity than 1 and that binding affinity of all other congeners is clearly inferior to that of 1. In summary, we have succeeded in preparing various structural types of himbacine congeners by employing the synthetic route previously explored to prepare 1. Binding affinity assay of all the synthesized congeners explored novel aspects of the structure-activity relationships for 1, leading to find 3-norhimbacine 20 which shows more potent binding affinity than 1.

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Pseurotins are a class of secondary microbial metabolites, which were isolated from the cultures of Pseudeurotium ovalis STOLK (Ascomycetes) by Tamm et al. in 1976. Pseurotin F_2 (2) was also isolated from Aspergillus fumigatus DSM 6598. Among them, pseurotin A (1) has a potent neurite formation activity to PC-12 pheochromocytoma cells. Pseurotins possess a highly functionalized 1-oxa-7-azaspiro[4.4]non-2-ene-4,6-dione skeleton with three contiguous stereogenic centers. Very recently, structurally related azaspirene (3) was isolated from a fungus Neosartorya sp. by Osada et al. This antibiotic inhibits the endothelial migration induced by vascular endothelial growth factor. Its relative and absolute stereochemistries remain unclear. We have investigated the syntheses of pseurotins A (1), F_2 (2) and azaspirene (3). Herein we disclose the first total synthesis of these structurally as well as biologically intriguing natural products 1 and 2. For the total syntheses of 1〜3, we chose the known α-D-xylo-hexofuranose derivative 10 as a starting material. The chiral spiro-carbon in 1〜3 was constructed efficiently by the stereoselective vinyl Grignard addition to C3 in the 3-ulose derivative derived from 10. By a synthetic venture including a Cu(I) mediated benzyl Grignard addition to aldehyde 9 and a γ-lactone ring formation via ozonolysis of the vinyl group in 15, the right half synthetic precursor 6 was prepared. Introduction of the left half part was carried out by the aldol reaction of 6 with an aldehyde 7, derived from D-glucose. The 3(2H)-furanone 4 was formed via chemoselective cleavage of the TES group, oxidation, and dehydration of the resultant spiro-ketal 20. The ammonolysis of 21, derived from 4, fowllowed by oxidation provided a γ-lactam ring. The crucial benzylic oxidation was achieved via regioselective oxidation of enamide 24 with m-CPBA. Removal of the protective groups completed the first total sysnthesis of pseurotin F_2 (2). Treatment of 2 with CSA in MeOH gave pseurotin A (1). For the total synthesis of azaspirene (3), we have synthesized a highly functionalized hetero-spiro γ-lactam 31, which possesses the core hetero-spiro ring system with all stereogenic centers required for the total synthesis. Further studies aimed to the total synthesis of 3 are now in progress.

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Nakadomarin A (1) was first isolated from the Okinawan marine sponge Amphimedon sp. by Kobayashi in 1997, and was found to be biogenetically closely related to manzamine alkaloids, such as manzamine A and ircinal A. Its structure was elucidated spectroscopically and consists of an unprecedented hexacyclic ring system (8/5/5/5/15/6) that includes a furan ring. An interesting biogenetic transformation of ircinal A to nakadomarin A has been proposed. Although some biological activities of nakadomarin A, such as cytotoxicity against murine lymphoma L1210 cells, inhibitory activity against Cdk4, and anti-microbial activity, have been reported, its limited availability (1.8×10^<-3>%, wet weight) has prevented a complete survey of its biological activity. In connection with our ongoing project on the total synthesis of manzamine alkaloids, we were interested in nakadomarin A because of its unique structure and began a synthetic study. We report here the first total synthesis of (±)-nakadomarin A (1) and also a different synthetic approach toward chiral nakadomarin A. 1) Total synthesis of (±)-nakadomarin A: 4-Oxopiperidine carboxylate was converted to the spiro-lactam 26 (AD ring intermediate). To this intermediate a furan ring was introduced by Suzuki-Miyaura coupling to give ACD ring intermediate 29. Intramolecular cyclization of the furan ring to acyliminium cation afforded the central core of nakadomarin A, ABCD ring intermediate 35. Both 8-membered ring (E ring) and 15-membered ring (F ring) was constructed using ring closing metathesis to give 20,29-dioxonakadomarin A 40 and its (24E)-isomer 41. Finally, both dioxonakadomarins were reduced to nakadomarin A 1 and its (24E)-isomer 42, respectively. 2) Optically active ABCD ring intermediate 56, which already have suitable substituents for further elaboration of both E and F rings, was synthesized from chiral hydroisoquinolinone 46 derived from Diels-Alder reaction of chiral piperidinone derivative 43 and siloxydiene 44.

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The enantioselective total syntheses of (+)-cylindricine C (2) and (-)-lepadiformine (12), novel tricyclic marine alkaloids isolated from the ascidians Claverina cylindrica and Clavelina lepadiformis respectively, have been achieved by using (S)-N-Boc-5-benzyloxy-2-pyrrolidinone (20) as a common starting material. In addition, direct comparison of synthetic 12 with a natural sample of lepadiformine by chiral HPLC allowed to establish the absolute configuration of the natural alkaloid to be 3S, 5R, 7aS, 11aS. The central feature of these synthetic approaches involves highly stereoselective construction of the 6-substituted azaspiro[4.5]decane systems. The N-Boc-pyrrolidinone 20 was subjected to endocyclic ring opening with the Grignard reagent followed by recyclization of the resulting keto amide 26 to give the (S)-1-pyrroline 28, which underwent allylation with the allyl Grignard reagent in the presence of BF_3・Et_2O to provide the 2,2,5-trisubstituted pyrrolidine 29 with almost complete diastereoselectivity. Azaspirocyclization of the aldehyde 30, derived from 29, was performed via intramolecular alkylation of the enamine 31 to form the (6S)-azaspirododecane 33, which was then converted to the enone 35 and underwent intramolecular Michael addition to yield O-benzylcylindricine C (37) as a single isomer. The synthesis of cylindricine C (2) was completed by subsequent hydrogenolytic debenzylation. On the other hand, lactam ring opening of the (S)-N-Boc-pyrrolidinone 20 with the (5E,7E)-tetradeca-5,7-dienyl Grignard reagent 38 afforded the dienyl keto amide 39. Upon treatment of 39 with formic acid in toluene-THF, intramolecular azaspirocyclization smoothly proceeded via the intermediary formation of an acyliminium ion 40 with complete face selectivity to provide the (6S)-azaspirododecane 41. After transformation of 41 into the enone 44, reduction using (S)-BINAL-H highly stereoselectively (97% de) led to the α-alcohol 43a, which was converted to the amino alcohol 46 and was subjected to dehydrocondensation by treatment with CBr_4-PPh_3 to give the tricyclic amine 47 with complete inversion of the configuration at C3'. Finally, hydrogenolytic removal of the benzyl protecting group afforded (-)-lepadiformine (12). Thus, the highly efficient and stereoselective synthesis of (-)-lepadiformine was accomplished via an extremely short route with overall yield of 31.4%.

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Ecteinascidin 743 (Et 743, 1) is an extremely potent antitumor agent isolated from a marine tunicate, Ecteinascidia turbinata. Based on promising results in phase II clinical trials, Et 743 (1) is likely to become the first anticancer drug among marine natural products. The novelty of its structure, the remarkable biological activities, and its natural scarcity have made it an attractive target for total synthesis. We describe herein an efficient total synthesis of 1 that would potentially lead to the development of a practical synthesis of this important compound. Synthesis of the left segment 23, a highly functionalized (R)-phenylglycinol derivative, involves a Mannich-type reaction of phenol 19 with the chiral template 16 developed recently in our laboratories. The right segment 32, (S)-iodophenylalanine derivative, was synthesized in 13 steps from 3-methylcatechol (24) by employing DuPHOS-mediated asymmetric hydrogenation as the key step. These two segments were efficiently coupled by the Ugi's 4CC reaction, and transformed into the cyclic enamide 38 via diketopiperazine 37. Intramolecular Heck reaction of the enamide 38 proceeded smoothly to give 39, containing the bicyclo[3.3.1] skeleton, in high yield. The stereochemistry at C-3 position was controlled by stereoselective reduction of the acyliminium intermediate to give the key intermediate 42. Construction of B-ring was performed by the phenol-aldehyde cyclization of 44, which was invoked by hydrogenolysis of the benzyl groups, giving the desired pentacycle 45 with requisite oxidation state at C-4 position. An acid-induced intramolecular sulfide formation provided the sulfur-containing ten-membered lactone 48. Finally, construction of tetrahydroisoquinoline moiety by means of the Pictet-Spengler reaction with 4 afforded Et 770 (2), which, upon treatment with AgNO_3, was successfully converted to ecteinascidin 743 (1). The present synthetic route is convergent, high-yielding, and exhibiting high selectivities throughout the process. Furthermore, it would also be applicable to the synthesis of a variety of the analogues.

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Introduction of bulky silyl protections into 1,2-trans hydroxy groups on a pyranose ring sometimes flips the ring to give a stable chair conformation with more axial substituents (axial-rich form). Such ring-inversion from steric repulsion of the silyl protections has only observed in pyranose rings. We investigated conformational transformation of cyclohexane rings caused by bulky silyl protections. We preliminarily investigated the ring-conformation of 1,2-di-O-trialkylsilyl-cyclohexanediols, 1〜3. X-ray diffraction studies of 1〜3 showed these compounds are all in inverted axial-rich form. The two adjacent bulky silyl protections, therefore, can invert cyclohexane ring to axial-rich form. We then introduced two bulky silyl protections into the adjacent trans hydroxy groups of myo-inositol. When the silyl protections were introduced to C-3 and C-4 hydroxy groups (6p〜8p and 6〜8), the rings maintained the original equatorial-rich chair form. In contrast, among the 4,5-di-O-silylated 11p〜13p and 11〜13, three of them (13p, 12, 13) were flipped to be axial-rich form. Considering the conformation of the 3,4-di-O-silylated compounds, the steric repulsion due to two bulky silyl protections is short to the ring inversion. On the other hand, the rings of some 4,5-di-O-silylated compounds were inverted. In these cases, 1,2-trans-oxgen substituents are in the both sides of the O-silylated positions. If the ring maintained the equatorial-rich conformation, four consecutive equatorial substituents would increase the steric repulsion. To release it, the ring would be flipped. When the bulkiest TBDPS groups were attached to the center of the four 1.2-trans-substituents, the ring was inverted even other hydroxy groups were benzylated.

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Lipid A is an immunostimulating glycoconjugate characteristic to gram-negative bacteria. In order to understand the role of the acyl moieties for the biological activity, the effect of distribution of acyl groups was systematically investigated. A new efficient route for the synthesis of lipid A and its analogs was hence established by using our new strategy, i.e., synthesis based on affinity separation (SAS), where compounds possessing a barbituric acid tag (BA) were selectively and rapidly purified by the aid of its interaction with an artificial receptor. After successful synthesis of E. coli lipid A by the SAS-strategy, a small library of lipid A analogs having a carboxymethyl (CM) group at the 1 position in place of the phosphate was synthesized. CM analogs had proved to exhibit biological activity indistinguishable with that of lipid A, but are chemically stable and are easier to synthesize than the latter. All the target structures possess hexaacyl groups with different acylation pattern: each has two (R)-3-hydroxydecanoyl groups and two (R)-3-(dodecanoyloxy)decanoyl groups. A suitably protected glucosamine α-carboxymethyl glycoside possessing the BA-tag was glycosylated with a 4'-phosphorylated glycosyl trichloroacetimidate to give a disaccharide, which was purified by the affinity separation. Successive removal of protective groups and introduction of acyl groups were then effected and the synthetic intermediate at each step was purified rapidly by the affinity separation. The final deprotection and cleavage of the tag by catalytic hydrogenolysis afforded the desired CM-analogues. Biological tests of them clearly demonstrated that the acylation patterns of the individual compounds determine the biological activity.

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Somamide A (1) is a unique cyclic depsipeptide having the 3-amino-6-hydroxy-2-piperidone (Ahp) unit, 2-amino-2-butenoic acid (Abu), and sulfoxide. It was isolated from assemblages of the marine cyanobacteria Lyngbya majuscula and Schizothrix sp. in Fijian Islands by Gerwick and coworkers. As an extension of our interests on the synthesis of biologically and structurally intriguing aquatic natural products, we succeeded in the total synthesis of somamide A (1). For the synthesis of the Abu-containing depsipeptide fragment of somamide A, we chose the stereospecific dehydrative elimination of the corresponding N-acyl thereonine ester. After several trials utilizing thionyl chloride, Burgess reagent, and DAST, we finally found out that Martin's sulfurane (dephenyl bis(1,1,1,3,3,3-hexafluoro-2-phenyl-2-propyl)sulfurane) was quite effective for the stereospecific dehydration to give the Abu residue. The synthesis of the building blocks, the depsipeptide 4, dipeptide 5, and 5-hydroxynorvaline derivative 6 was efficiently accomplished. After assembling these three fragments, the linear precursor 22 was simultaneously deprotected at the C- and N-terminals and subjected to macrolactamization using pentafluorophenyl diphenylphosphinate (FDPP, Ph_2P(O)OC_6F_5) to give the macrocyclic depsipeptide 23 in 64% yield. Removal of the TBS group from 23 followed by treatment with 1-hydroxy-1,2-benziodoxol-3(1H)-one 1-oxide (IBX) then TBAF afforded somamide A (1) as the major product (50%) together with the minor product 24 (39%). The latter was slowly and spontaneously transformed to 1 in air at room temperature. Oxidation of 24 with aqueous hydrogen peroxide also afforded 1 in 57% yield. Thus we could complete the total synthesis of somamide A (1) and indicate that 24 will be a true natural product whereas 1 will be an artifact. Our strategy for the construction of the Ahp moiety will be useful for the synthesis of the Ahp-containing natural products. Furthermore, we have found that martin's sulfurane is a powerful reagent for the dehydrative elimination of the N-acyl-β-hydroxy-α-amino acid esters to the α,β-dehydroamino esters.

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Aurilide (1), 26-membered cyclic depsipeptide, was isolated from the Japanese sea hare Dolabella auricularia by Yamada and co-workers in 1996. This compound has very strong cytotoxicity against HeLa S_3 cells with an IC_<50> of 0.017μg/mL. To elucidate the mechanism of this cytotoxicity with conformation analysis, we started a library synthesis of aurilide derivatives. Herein we wish to report an approach to synthesize aurilide (1) and their derivatives using solid-support. The peptide moiety of aurilide, 3 was synthesized by Fmoc method using diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) on trityl linked solid-support. Optimization of elongation conditions of the tetrapeptide part including N-methyl amino acids was carried out all at once utilizing Mulipin methods followed by LC/MS analyses. The aliphatic moiety 4 was prepared in solution phase by the esterification of hydroxy acid, unsaturated carboxylic acid, and N-methylalanine. The coupling reaction of the solid-supported 3 and diester 4 was carried out by DIC-HOBt method. After deprotection of Fmoc group and cleavage of seco acid 11 from solid-support, macrocyclization was achieved under high dilution conditions using EDCI and HOAt as coupling reagents. Deprotection of a methylthiomethyl group provided aurilide (1) in 11% overall yield. In the synthesis of a combinatorial library, further optimization of coupling reaction of peptide moiety including N-methyl amino acids was performed. It was found that PyBroP was the best reagent for coupling of sterically hindered amino acids. Synthesis of a combinatorial library of 25 aurilide derivatives 2 was accomplished with a similar protocol using TranSort^<TM> technique. Progress toward the elucidation of the mechanism of cytotoxicity with conformation analysis is underway in our laboratory.

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Antifreeze glycoproteins [AFGPs: {Ala-Thr(Galβ1-3GalNAcα)-Ala}_n, n=4-50] in the serum of polar fishes have been known to depress the freezing point of their blood and help them to survive at the temperature below-1.9℃[1]. Though it is widely accepted that AFGPs bind to the ice surface and prevent its growth, the definitive molecular mechanism of the action has not been solved yet. In this study, structure-activity relationships in AFGPs were studied toward understanding of the mechanism, thorough chemical syntheses, activity evaluations, and conformational analyses of the AFGP and its analogues, in which sugar or peptide moieties were different from those of the wild-type AFGP. The AFGP (1) and its analogues (2-8) were successfully synthesized via simple polymerization of their repeating unit using a DPPA method without any protection of sugar -OH groups [2]. Additionally, sialyl T-bearing glycopolypeptide (9) was successfully prepared through enzymatic elongation of a sialic acid residue to C-3 position of the galactose residue in the AFGP (1), indicating the broad applicability of this methodology for the syntheses of various glycopeptide polymers. In the evaluation assay of the antifreeze activity, artificial AFGP (1) showed similar activity as that of the wild-type AFGP. On the contrary, neither an AFGP repeating unit glycopeptide (19) nor a sugar-lacked polypeptide (poly-ATA, 8) shows the activity. Interestingly, AFGP analogs having GalNAc (2) or LacNAc (3) as sugar moiety showed similar activities as AFGP, while that of Galactose (4), Lactose (5), β-O-linked (6), and ASA (7) did not. These results clearly indicate the importance of i) repeating structure of glycopeptide, ii) NHAc group at C-2 position of the sugar moiety directly attached to the peptide, iii) α-glycosidic linkage between the sugar and the threonyl residue, for the specific interaction with ice. Furthermore, secondary structural analyses of the synthetic compounds were carried out by means of a CD spectroscopic method, in order to clarify the relationships between their conformations and their activities. The CD spectra of active compounds (1-3) were obviously different from those of inactive analogues (4-8), suggesting the presence of an ordered structure, but not α-helix.

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Sphingolipids have been known as lipid second messengers in mammalian cells and cell membranes, and a great deal of attention has been devoted to the studies of the biological process regulated by sphingolipids. Now, it has been well accepted that the sphingolipids play key roles in the cellular signal transmission pathway. Sphingomyelin, which is one of the key sphingolipids, is a ubiquitous constituent in animal tissue and has been known to occur in virtually every cell and in cell membranes. Ceramide and phosphoryl choline, which are the primary catabolites of sphingomyelin, are generated through the action of sphingomyelinase; ceramide is believed to display key roles as a signal transduction factor in cell differentiation and in programmed cell death (apoptosis) derivation (Figure 1). Although the significance of the sphingomyelin pathway, which is initiated by hydrolysis of sphingomyelin by SMase, has been well recognized, none of the tertiary-dimensional structures of these important enzymes have been determined and their hydrolytic mechanism has not been well-defined. It is, therefore, a very attractive challenge to reveal the catalytic mechanism of action of this important enzyme. Strong and selective sphingomyelinase inhibitors would contribute to a better understanding both of the roles of these enzymes and of ceramide in signal transduction. In order to elucidate the detailed catalytic mechanism of SMase, the development of the methods for supplying the sphingomyelin analogues, which competitively act at the catalytic site and strongly inhibit the hydrolytic ability of the enzyme, have strongly been desired. We then designed the substrate analogues 1, 2, 3, 4, 5 and 6 as inhibitor candidates on the basis of our previous results obtained on sphingomyelin analogues. In theses analogues, one of the oxygen atoms of the phosphoester, at which sphingomyelin is hydrolyzed by the enzyme, is replaced by either methylene, ethylene, difluoromethylene, nitrogen, or sulfur group, and the relative configuration of the asymmetric centers is (D)-erythro (3S, 4R) form (Figure 2). We will describe in detail the highly efficient stereocontrolled syntheses of the short chain substrate analogues 1, 2, 4, and 6 and their inhibitory activities toward B. cereus SMase.

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Ptilomycalin A (1) and crambescidines, the unique guanidine alkaloid family, consist of novel pentacyclic guanidine units (so-called vessel part) and a spermidine unit linked by a linear long chain fatty acid (so-called anchor part), and they show significant biological activities including the inhibition of Na^+, K^+, Ca^<2+>-ATPases. In 2000, Braekman group reported the novel guanidine alkaloid crambescidin 359 (3). This newly isolated guanidine alkaloid is the first derivative which lacks the anchor part of crambescidins, so its biological properties as the vessel part alone are intriguing. Firstly, we have developed a facile and stereoselective method for the synthesis of the novel pentacyclic guanidine system. Our procedure features (1) the stereoselective synthesis of 2,5-disubstituted pyrrolidine based on sequential 1,3-dipolar cycloaddition followed by hydrogenation or oxidation-hydrogenation of the resulting isoxazolidines, and (2) efficient synthesis of pentacyclic guanidine based on guanylation followed by double N,O-acetalization. This synthetic method could be successfully applied to the stereoselective total synthesis of (-)-crambescidine 359 (3). This synthesis has established the absolute stereochemsitry of 3 and would suggest the counterion of 3 as chloride. We next have studied the Ca^<2+>-ATPase inhibition activities of 3 and its synthetic analogs 26-29. Although crambescidine 359 (3), 26 and 27 did not show the significant activities, 28 and 29, which have the spermidine contained alkyl long chain, exhibited the inhibition of Ca^<2+>-ATPase with an IC_<50> value of 3μM and 1μM, respectively. We finally designed and synthesized the novel C_2-symmetric chiral pentacyclic guanidine compound 30 as an organocatalyst. The structure of 30 was based on the mother skeleton of 3 and was rationally designed to have a C_2-symmetrical chiral reaction cavity around the substrate recognition/activation site. The catalyst 30 effectively induced the asymmetric induction of alkylation reaction with the glycynate-benzophenone Schiff base 31, and the epoxidation reaction with chalcone and its derivatives 33.

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Tetrodotoxin (TTX, 1), isolated as a toxic principle of puffer fish poisoning, is a highly potent neurotoxin which inhibits electrical excitation of nerve and muscle cell membranes. The mechanism of its action is shown that the specific binding to voltage-gated Na^+ channel blocks depolarization. The structure was elucidated in 1964 independently by three groups (Hirata-Goto, Tsuda and Woodward) and the total synthesis of racemic TTX was accomplished in 1972 by Kishi-Goto and coworkers. The novel structure including contiguous eight stereogenic centers and multifunctional groups such as guanidine, hemilactal and six hydroxyl groups, and the characteristic biological activity still make it challenging target for the total synthesis. Herein, we report the synthesis of protected TTX 26. Carbon skeleton synthesis It was designed that the oxygenated cyclohexane derivative A containing the carbon framework of TTX was constructed by intramolecular aldol reaction as a key step. The aldol precursor 6 was prepared via Sonogashira coupling and Claisen rearrangement from the known diol 3. Hydroxylation at the C-5 and C-11 position of 6 were carried out through oxidation of enol ether to give dihydroxyketone 9. The acetylene moiety was then hydrated to provide methyl ketone 10, which was selectively protected via intramolecular acetal formation at C-6, and then converted to enol ether 11. Aldol condensation with TBAF yielded enone 12. Nitrogen introduction Attempted stereoselective introduction of nitrogen functionality at the C-8a position failed by Overman rearrangement giving an undesired product 27. It turned out that intramolecular Michael addition of carbamate 16→47 was effective for the C-N bond formation at C-8a. Epoxidation and the inversion of configuration at C-5 gave fully functionalized cyclohexane 20. α-Hydroxylactone synthesis Aldehyde 21 was converted to cyclic vinyl ether 22 through enolization with DBU. Three step oxidation-reduction sequence afforded α-hydroxylactone 23 as a single isomer, which was transformed to ortho ether 24. Guanidine synthesis Guanidine group was installed with Boc-protected isothiourea to afford 25, which was subjected to oxidative cleavage of 1,2-diol and subsequent treatment with acid to furnish protected TTX 26. Deprotection of 26 to accomplish the total synthesis of 1 is underway.

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Natural cyclic ethers isolated from marine plants, such as algae and dinoflagellates, have recently attracted much attention to their notable bioactivities and complicated structures. Prelaureatin 1 has been isolated from red alga Laurencia nipponica as an 8-membered cyclic ether which belongs to a laurenan family of C15-acetogenins involving laureatin 3, isolaureatin 4, and laurallene 5. These laurenan compounds show interesting bioactivities, represented by potent insecticidal activity of 3 and 4. Our previous chemoenzymatic studies using bromoperoxidase from Laurencia nipponica or lactoperoxidase have demonstrated that 1 is a key precursor in the biosynthetic route to 3, 4, and 5. These results have also shown a possibility of the chemical syntheses of all these laurenan compounds from 1. On the other hand, ciguatoxin (CTX) 2 has been isolated as a causative toxin of ciguatera seafood poisoning from moray eel Gymnothorax javanicus firstly by Scheuer. Later, Yasumoto et al. have clarified that the structure of 2 is characterized by 13-fused cyclic ethers including 7-, 8-, and 9-membered rings and that the original source of 2 is the dinoflagellate Gambierdiscus toxicus. Strong activation of voltage-sensitive sodium ion channel has been reported as a remarkable bioactivity of 2. As a part of our studies toward total syntheses of 1 and 2, new synthetic routes starting from sugar derivatives for the construction of medium-sized cyclic ether parts included in 1 and 2 have been developed. In order to construct a medium-sized cyclic ether structure by a ring-closing olefin metathesis (RCM) reaction, the stereoselective synthesis of a chiral di-sec-alkyl ether as a precursor for the RCM reaction must be considered. In fact, an asymmetric synthesis of the chiral di-sec-alkyl ether has been difficult and an important challenge in organic chemistry. In this context, we developed a synthetic method for the chiral di-sec-alkyl ether structure adopting the cleavage reaction of a hexose ring in a chiral C-glycoside prepared from a natural monosaccharide. Here, we disclose the detail of the method and the application to the total synthesis of 1 as well as syntheses of the D-, E-, F-, and I-ring parts of 2.

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Gambierol (1) is a marine polycyclic ether isolated as a toxic constituent from cultured cells of the ciguatera causative dinoflagellate, Gambierdiscus toxicus. This compound shows toxicity against mice (LD_<50>, 50μg/kg), and the symptoms resemble those caused by ciguatoxins. The unique structural features have attracted the attention of synthetic chemist. We have already succeeded in the convergent synthesis of the CDEFG ring system (8) via the intramolecular allylation of the α-acetoxy ether 4 and subsequent ring-closing metathesis. Encouraged by this result, we started the total synthetic study of gambierol (1). Treatment of the cyclization precursor 15, prepared from the ABC ring segment 10 and FGH ring fragment 11, with MgBr_2・OEt_2 gave the desired product 9 and its C16 epimer 16 in 22% and 47% yield, respectively. The yield of 9 was improved by using monochloroacetoxy group as a leaving moiety. Thus, the reaction of 19 gave 9 and 16 in 30% and 48% yield, respectively. The diene 9 was subjected to ring-closing metathesis with 7 to give 20 in 84% yield. The octacyclic compound 20 was converted to 28 by several steps. We next examined the construction of the triene side chain. Although the Stille coupling of 29 and 30 gave the cross-coupling product 31 in an allowable yield (63%), the reaction was very slow (4 days). After unfruitful attempts, we succeeded in developping an efficient method for the stereoselective synthesis of Z-iodoolefin 33. Treatment of 32 with Zn-Cu and AcOH gave 33 as a single stereoisomer in 80% yield. As expected, the reaction of 33 and 30 was very fast. The triene 31 was obtained in 95% yield after 1.5h. Similaly, the iodoolefin 35, prepared from 28, was converted to the fully protected gambierol 36 in good yield. The final deprotection of 36 giving 1 is currently under way.

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Gambierol (1) is a marine polycyclic ether toxin isolated from the ciguatera causative dinoflagellate Gambierdiscus toxicus. The toxin exhibits potent toxicity against mice (LD_<50> 50μg/kg, ip), and the symptoms caused in mice resemble those shown by ciguatoxins, implying that gambierol is also responsible for ciguatera fish poisoning. However, extremely limited availability from natural sources hampered further detailed biological studies, and therefore, sample supply by practical chemical synthesis is strongly demanded. The chemical structure is characterized by the octacyclic polyether core with 18 stereogenic centers and labile triene side chain that includes conjugated (Z,Z)-diene. Herein we report the first total synthesis of gambierol, featuring convergent synthesis of the octacyclic polyether core via our B-alkyl Suzuki-Miyaura coupling strategy and stereoselective construction of the sensitive triene side chain through the Pd(PPh_3)_4/CuCl/LiCl promoted Stille coupling. The ABC ring fragment 5 was synthesized from the known olefin 7, representing the B ring. The C6 hydroxy group was installed stereoselectively by Sharpless asymmetric epoxidation of allylic alcohol 8 and subsequent reductive cleavage of the derived hydroxy epoxide. Intramolecular hetero-Michael reaction of 10 was utilized for stereoselective formation of the A ring to give 11 as a single stereoisomer. The C ring was constructed via 6-endo cyclization of vinyl epoxide 14 to afford tricyclic compound 15, which was then converted to 5 without incident. Synthesis of the EFGH ring fragment 6 started from the known ester 16. Construction of the H ring was performed by use of a SmI_2-mediated reductive cyclization methodology developed by Nakata. 6-Endo cyclization of vinyl epoxide 19 cleanly afforded the GH ring fragment 20, which was readily converted to methyl ketone 21. Reductive cyclization of 21 under Nakata conditions gave the FGH ring fragment 22, which was then transformed into the targeted fragment 6 by standard chemistry. The convergent union of the ABC and EFGH ring fragments (5 and 6, respectively) was successfully achieved by using the B-alkyl Suzuki-Miyaura coupling to yield the cross-coupled product 23 in gratifying 86% yield. Subsequent elaboration to the octacyclic polyether core 4 of gambierol was accomplished from 23 in only six steps. Compound 4 was converted to tertiary alcohol 26 in six steps via an introduction of the double bond within the H ring by Ito-Saegusa protocol. An array of careful protective group manipulations and installation of a (Z)-vinyl bromide unit led to triol 29. Cross-coupling of 29 with the known (Z)-vinyl stannane 3 under the Pd(PPh_3)_4/CuCl/LiCl promoted Stille conditions furnished gambierol 1. Spectroscopic data (^1H and ^<13>C NMR, FIRMS, CD) and mouse lethality of synthetic 1 were identical with those of authentic natural sample.

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Brevetoxin-B (BTX-B, 1), a potent neurotoxin produced by the red tide organism Gymnodium breve Davis, is the first and representative member of marine polycyclic ethers. Its unique complex structure and potent biological activity have attracted the attention of synthetic organic chemists and the first total synthesis of 1 was achieved by Nicolaou and co-workers in 1995. We describe herein the stereoselective total synthesis of BTX-B (1). The Michael addition of 5 with MeMgBr-TMSCl in the presence of copper catalyst 6 afforded only α-adduct 7. After construction of the D-ring by SmI_2-induced intramolecular cyclization, treatment of bis(β-alkoxy acrylate) 13 with SmI_2 effected double cyclization with complete stereoselection to give trans-fused ester-lactone 14, corresponding to the CDE-ring. Then, 14 was converted into bis(α-epoxide) 19 via one-pot Wittig reaction of 15 with Ph_3P=C(Me)CO_2Et and Ph_3P=CHCOMe, and consecutive Sharpless AE of bis(allylic alcohol) 17, and double olefination. The ABCDEFG-ring was then constructed via consecutive 6-endo-cyclization of 19 and 20, ring-closing olefin metathesis of 22, and 6-endo-cyclization of 26, to give 27, which was successfully converted into the Wittig reagent 2. The UK-ring 3 was stereoselectively constructed via SmI_2-induced cyclization of 30, 6-endo-cyclization of 33, SmI_2-induced intramolecular Reformatsky-type reaction of 35, and direct introduction of the side chain. Both segments 2 and 3 were coupled by Wittig reaction to give 41, which was converted into 43 by intramolecular cyclization to monothioacetal followed by Ph_3SnH-AIBN reduction. Double oxidation of the left and right sides with PCC followed by deprotection of TBS group furnished BTX-B (1).

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Bioluminescence is one of the most attractive natural phenomena, displayed by various different types of organisms from bacteria to fishes. Recently, considerable attention has been directed to bioluminescence reactions due to potential as highly sensitive, nondestructive analytical tools, particularly for monitoring gene expressions and studying protein-protein interactions (luminescence energy transfer). Luciferases catalyze the bioluminescence reactions that involve the oxidations of the luciferins resulting in light emission. Despite their wide applications, little is known about their molecular mechanisms in bioluminescence reactions. We are interested in applying catalytic antibody technology to explore the molecular mechanisms of bioluminescence reactions in proteins. In this work, we have generated catalytic antibodies that catalyze the oxidation reaction of MCLA (1). Hapten 6 was designed as a transition-state analog for the oxidation. The diol moiety in hapten 6 possessed two distinct features. 1) The hydroxylmethyl group at the C-2 position of indanone with a sp^3 carbon mimics a transition-state for the addition of molecular oxygen to 1. 2) The hydroxy group at the C-3 position mimics a transition-state for the formation of the dioxetanone(4) and is also introduced to avoid the decomposition of the hapten via retro-aldol reaction. Of the 70 monoclonal antibodies specific to 6-BSA, 14 accelerated the bioluminescent oxidation of 1. The rate of the oxidation of 2 by antibody 7D10 followed Michaelis-Menten kinetics; apparent values of Micahaelis constant K_m, and rate constant k_<cat> were 52μM and 0.01min^<-1>, respectively. Remarkably, in the presence of 7D10, the luminescent intensity increased 30 fold over the background level of 1. The kinetic characterization and the catalytic mechanism will be discussed.

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More than 20,000 people suffer annually from ciguatera, a disease caused by consumption of reef fish. A problem in avoiding the disease is that there is currently no rapid and reliable method of detecting the causative toxins, namely the ciguatoxins, at fisheries. Furthermore, the content of ciguatoxin is extremely low, which has hampered the preparation of anti-ciguatoxin antibodies. Herein, we have demonstrated a rational approach for the preparation of specific antibodies to targeted sites of the ciguatoxins. Monoclonal antibodies (mAbs) elicited by using tricyclic ABC rings as haptens, however, were not bound to CTX3C itself. After considerable experimentations, specific mAbs with strong affinities to CTX3C itself were prepared by using pentacyclic haptens (ABCDE and IJKLM). The requisite surface area of the hydrophobic polycyclic ether parts was estimated to be more than 400A. We then developed a direct sandwich immunoassay employing the mAbs to both ends of CTX3C. This specific and sensitive sandwich protocol with detection limits of CTX3C [〜1ng/mL] will provide a reliable method for the screening of toxins for public health protection. This study therefore paves the way for the development of sandwich immunoassays using specific antibodies to targeted sites of non-biopolymer organic molecules.

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In the early stage of atherosclerogenesis, macrophages which have penetrated into the intima efficiently take up modified low density lipoprotein store cholesterol and fatty acids as a form of neutral lipids such as cholesteryl ester and triacylglycerol in the cytosolic lipid droplets, and are converted into foam cells, leading to the development of atherosclerosis in the arterial wall. During the course of our screening program for anti-atherosclerotic agents of microbial origin, a culture broth of Beauveria sp. FO-6979 was found to inhibit lipid droplet formation in mouse peritoneal macrophages. Eight structurally related cyclodepsipeptides, beauveriolide I and III to IX, were isolated by solvent extraction, column chromatographies and HPLC from the mycelium fraction cultured in the tryptone-supplemented medium. Beauveriolide I was previously reported as an insectcidal agent and beauveriolide IX was identical to beauverolide Fa, but the others were found to be new. Beauveriolide III was white powder, and its molecular formula was established as C_<27>H_<41>N_3O_5 by high resolution FAB-MS. The structure of beauveriolide III was elucidated to be cyclo-[(3S,4S)-3-hydroxy-4-methyloctanoyl-L-phenylalanyl-L-alanyl-D-alloisoleucyl] by NMR spectral analyses and chemical degradation. Addition of L-Ile to the culture medium yielded a high and selective production of beauveriolide III. Beauveriolides I and III caused a reduction in the number and size of cytosolic lipid droplets in macrophages at 10μM without any cytotoxic effect. Studies on the mode of action revealed that they inhibit ACAT activity specifically. Their in vivo efficiency was shown in LDL receptor knockout mice, reducing the atherogenic lesion in aortas and hearts after 2-month oral administration of beauveriolides. Beauveriolides are expected to develop as a lead for a new type of anti-atherosclerotic drugs.

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Telomeres are guanine-rich, simple repeat sequence of TTAGGG that constitute the physical termini of eukaryotic chromosomes. Maintenance of telomeres, in which specialized ribonucleoprotein complex known as telomerase mediates, is significant for immortalization in cancer cells. Since the correlation between telomerase activity and tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents, we attempted to screen telomerase inhibitors from the metabolites of microorganism. Wide-range of screening resulted in the isolation of a potent specific telomerase inhibitor designated as telomestatin from Streptomyces anulatus 3533-SV4. The structure of telomestatin (C_<26>H_<14>N_8O_7S) was elucidated on the basis of spectral analyses including 2D NMR (HMQC, DQF-COSY, HMBC) to be a cyclic compound consisting of a thiazoline, two methyloxazole, five oxazole rings. To the best of our knowledge, telomestatin is the first example with a macrocyclic system containing a sequential pentaoxazole moiety Telomestain specifically inhibited telomerase activity with an IC_<50> value of 5nM, whereas it did not showed activities against DNA polymerases such as Taq polymerase. Since telomerase is a multisubunit ribonucleoprotein complex which includes an RNA component and a reverse transcriptase (RT) catalytic subunit, the inhibitory effects of telomestatin against RT were investigated. It showed weak activities against RTs such as HIV- and MMLV (Moloney murine leukemia virus)-RTs with IC_<50> values of 19.4 and 13.4μM, respectively. Thus, telomestatin was proved to be a specific telomerase inhibitor. Further experiments of the mode of action mechanism proved that telomestatin acts to stabilize G-quadruplex structure, which follows telomerase inhibition.

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Amphotericin B (AmB, 1) has been a choice for treatment of systemic fungal infections for over 30 years. Its selective toxicity is generally attributed to the higher affinity for ergosterol as a principal fungal sterol over cholesterol occurring mammalian plasma membranes upon forming an ion-permeable channel, in which AmB together with sterol is thought to self-assemble to shape a barrel-stave complex. Although the structure of this assemblage in biomembranes has been a target of extensive investigations for a long time, the precise picture has not been obtained. To reducie serious side effects of the drug, the mechanism of molecular recognition has to be clarified. As the first step toward this goal, we prepared several AmB dimers and AmB-sterol conjugates, and examined for their channel-forming activity. AmB-sterol conjugates were tested for their K^+ flux activity using phosphatidylcholine (PC) liposome systems. The ergosterol conjugate 2 exhibited the prominent activity, which substantially exceeded that of its cholesterol homologue 3. The results indicate that specific intermolecular recognition between AmB and ergosterol could be reproduced to a certain extent by conjugate 2. AmB-sterol conjugates with a longer linker with C_6-diaminocarbamate showed much weaker K^+-flux activity, which suggests that the amino group of AmB mycosamine resides close to 3-OH of ergosterol in a channel assemblage. Molecular recognition between AmB and PC is known to be essential for ion-channel formation. To examine the effect of the length of fatty acyl chains on ion-channel formation, we prepared AmB-PC conjugates 6 and 7, dimyristoylphosphatidyl-AmB and dilauroyl-phosphatidyl-AmB. Conjugate 7 evidently revealed more potent activity, thus disclosing that a direct interaction between AmB and phospholipids is of great importance in ion-channel formation. AmB dimers with an aminoalkyl-dicarboxylate linker between amino groups of AmB have showed potent ion-permeabilizing activity as reported in the previous Symposium. For feasibility in synthesis, another series of AmB dimers linked via carboxylic acid were prepared and examined for their activity. A dimer with a 1,6-hexanediamine linkage (9) bearing potent hemolytic activity gave rise to large K^+ flux in phospholipid liposomes regardless of the presence or absence of sterols. Some of these conjugates may possibly serve as a molecular probe to investigate the precise mechanism of action for the selective toxicity by AmB. Currently, we attempt to prepare isotope-labeled conjugates for measuring solid state NMR. Interatomic distance estimated from the NMR data would help elucidating the structure of a complicated AmB-ergosterol assemblage formed in membrane. Preliminary experiments of ^<13>C-labeled AmB methyl ester will be related.

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A great many host-parasite and host-pathogen interactions depend upon specific oligosaccharide-protein interactions, and the techniques which we have developed are ideally suited for detailed studies on the molecular basis of such interactions. Systems in which we have a particular interest are the carbohydrate-binding A-B class bacterial toxins such as cholera toxin and the toxin derived from E. coli O157. A novel development concerns the use of oligosaccharide ligands which have been enriched with stable isotopes, in order to delineate their bound-state conformations in the absence of detailed structural information on the protein, using isotope edited NMR. This approach involves the chemical synthesis of oligosaccharides which are variously enriched at certain atomic positions, using commercially available isotopically enriched monosaccharides as starting materials. In fact, this synthesis stage plays the most important role. An added bonus is that additional NMR parameters become available as a result of isotopic enrichment, which can be utilised to define more accurately the solution behaviour of the oligosaccharide in the 'free' state, which greatly assists our understanding of the thermodynamics of the oligosaccharide-protein interaction. In our research of VTB (B Subunit of Verotoxin-1) with a ligand, Gb_3 trisaccharide, we successfully obtained several TRNOEs which indicated Gb_3 trisaccharide occupied onto Site2 although Sitel was thought as the primary binding site. Measurement of dipolar couplings by liquid crystal NMR confirmed strongly our result. We have, now, applied these methods for other host-guest systems (it may weaker than VTB-Gb_3 system) and applied liquid crystalline phase NMR for assay of libraries.

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Angiogenesis is a complex process involving several distinct and sequential steps such as membrane degradation, migration (chemotaxis), proliferation, and formation of capillary tubes in endothelial cells. Abnormal angiogenesis often occurs in pathological conditions such as cancer, rheumatoid arthritis, diabetic retinopathy, and other chronic inflammatory diseases. An important step in the development of pathological angiogenesis is thought to involve the production of vascular endothelial growth factor (VEGF) by normal and tumor cells, and the subsequent hyperactivation of downstream signaling pathways in endothelial cells. Inhibition of angiogenesis is emerging as a promising strategy for the treatment of angiogenesis-related diseases including cancer. In our screening program to aim at identifying angiogenesis inhibitors of microbial origin, we found novel pentaketide dimers designated as epoxyquinols A (1) and B (2) produced by a filamentous fungal strain isolated from a soil sample. The structures of 1 and 2 were elucidated mainly by NMR experiments including several 2D-NMR methods, X-ray crystallographic analysis, and other spectroscopic analysis. Epoxyquinol A (1) possesses an attractive structure consisting of a highly functionalized cyclohexenone moiety. Moreover, epoxyquinol B (2) is a unique diastereomer of 1 in respect of the structure, biosynthesis, and biological properties. In addition, a related monomer 3 was also identified, which would be a key intermediate of epoxyquinols biosynthesis. The intermolecular Diels-Alder reaction of a corresponding 2H-pyran monomer resulted from 3 via oxidative 6π-electrocyclization is proposed as possible biosynthetic pathways of 1 and 2. Epoxyquinols A (1) and B (2) inhibited VEGF-induced migration in human vein endothelial cells (HUVECs) with the IC_<50> values of 2.8 and 0.8μM, respectively. Further studies on the structure-activity relationships and the biological activities of epoxyquinols are in progress.

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Corals are important sources of revenue for the fishing and tourism industries, but they are rapidly disappearing. Their decline has been attributed to several causes, including pollution, global warming, overgrowth of hard-coral communities by other organisms and coral predators, such as the sea star Acanthastar planci and the sea shell Drupella cornus. Recently, we were interested in how the Okinawan sponge Terpios hoshinota overgrows and kills corals. We report here the isolation, guided by cytotoxicity to P388 cells, and structural determination of two new cytotoxic compounds, nakiterpiosin (1) and terpiodiene (2). The molecular formulas of 1 and 2 were deduced to be C_<27>H_<31>O_7BrCl_2 and C_<11>H_<14>O_3, respectively, based on ESI-MS and NMR spectral data. Their planar structures were determined based on 2D NMR (COSY, HOHAHA, HMQC, HMBC) spectra. Their relative stereochemistries were proposed based on a detailed analysis of NOESY data. We previously reported feeding attractants for the sea star A. planci at the 42nd Symposium. Using the same bioassay system as before, we also investigated feeding attractant(s) for the coral predator Drupella cornus. The latest result of this study will be described here.

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Microtubules play a pivotal role in mitotic spindle assembly and cell division. These cytoskeletal elements are formed by the self-association of the αβ tubulin heterodimers. There are a number of natural compounds which inhibit the microtuble formation and the mitotic arrest of eucaryotic cells. The antimitotic agents have potential applications in drug development. During our search for bioactive compounds from medicinal plants, we have found that moroidin (10) obtained from the seeds of C. argentea (Amaranthaceae), which is a unique bicyclic peptide originally isolated from Laportea moroides (Labiatae), remarkably inhibits the tubulin polymerization. Further investigation of the extract of C. argentea resulted in the isolation of nine new moroidin-type bicyclic peptides, celogentins A-H and J (1-9). The structures and absolute stereochemistry were determined by using extensive NMR methods and chemical means. Celogentins A-H, and J (1-9) inhibited the polymerization of tubulin. and celogentin C (3) was 4 times more potent than moroidin (10) in the inhibitory activity. Celogentin C with the Pro residue in the right hand ring showed 30 times more potent activity than celogentin A (1), while moroidin (10), celogentins E (5), and F (6) with the Gly residue in the right hand ring showed 10 times more than celogentin A (1). Potency of celogentins G (7), H (8), and J (9) with Ile^4 residue was comparable to that of celogentins E (5) and F (6) with Val^4 residue. The presence of additional Lys^9 residue in celogentin B (2) seems not to influence severely the inhibitory activity. Structure-activity relationship study using moroidin derivatives (14-16) and analog (17) as well as celogentins A-H, and J (1-9) and moroidin (10) indicates that the ring size and conformations of the bicyclic peptides including unusual non-peptide connections among β^5-Leu, Trp, and His residues would be important for their biological activity.

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The EtOH extract of a marine sponge of the genus Theonella collected off Shimokoshiki-jima Island was subjected to solvent partitioning, ODS flash chromatography, and gel-filtration followed by ODS HPLC to afford koshikamide B (1). The planar structure of 1 was elucidated by interpretation of 2D NMR data to be composed of fifteen common and two novel amino acid residues. Assignment of the pyrrolidone moiety was accomplished by interpretation of the COSY, HMBC, and ^<15>N HMQC spectra, while the structure of N^ω-carbamoyl Asn was determined by NMR data together with comparing the NMR data with a model compound. The sequence of the amino acid residues was unambiguously determined by interpretation of HMBC data. The absolute stereochemistries of amino acid residues in 1 were determined by Marfey and GC methods. Ala and N-MeaIle were shown to be 1:1 mixture of D- and L-forms. Their positions in the peptide were determined by analysis of two fragments obtained by partial acid hydrolysis. The absolute stereochemistry of the pyrrolidone moiety was established by chemical degradation and NMR data. Koshikamide B (1) exhibited cytotoxicity against P388 murine leukemia cells with an IC_<50> value of 0.45μg/mL.

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Clavulones were isolated from the Okinawan soft coral, Clavularia viridis, and have been recognized as marine prostanoids having unique structures and significant biological activities. Our continuous studies to find new prostanoids from C. viridis have resulted in the isolation of about 50 congeners of clavulones. The present study deals mainly with the true producer for clavulones, the host soft coral or its symbiotic alga, and the biosynthetic pathway for clavulones. The cultured symbiotic alga, isolated from C. viridis and identified as a dinoflagellate of the genus Symbiodinium, was extracted with MeOH. The AcOEt soluble portion of the MeOH extract was examined to find clavulones, however clavulones as well as their congeners were not detected. Arachidonic acid, the biosynthetic precursor for clavulones, was also not detected in the AcOEt soluble portion by gas chromatographic analysis. The quantitative analysis of clavulones in the fractions obtained by the centrifugal separations of C. viridis indicated the majority of clavulones in the coral fraction rather than the symbiotic alga fraction. These findings strongly suggested that clavulones are produced by the host soft coral itself. In order to obtain biosynthetic intermediates in the biosynthesis for clavulones, the hexane extract of freeze-dried C. viridis was searched, to give preclavulone-A methyl ester (1) and its stereoisomer 2 as the minor prostanoids. Preclavulone-A is an important intermediate in the biosynthetic pathway for clavulones mediated by lipoxygenase, but has not been found as a natural product so far. The biosynthetic experiment using a crude enzyme system (acetone powder) prepared from C. viridis was also carried out. Arachidonic acid in a Tris-HCl buffer solution was treated with the acetone powder at room temperature to give clavulones, whose spectral data were identical with those for the natural products. These findings provided the evidences for the pathway shown in Scheme 1 for the biosynthesis of clavulones proposed by Corey et al. The molecular phylogenetic analysis for the soft corals of the genus Clavularia was also performed.

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A class of Streptomyces aromatic polyketide antibiotics, the benzoisochromanequinone (BIQs) antibiotics all show trans stereochemistry at C-3 and C-15 in the pyran ring. The opposite stereochemical control is found in actinorhodin (3S, 15R, ACT) from S. coelicolor A3(2) and dihydrogranaticin (3R, 15S, DHGRA) from S. violaceoruber Tu22. A common bicyclic intermediate, which is produced by the early biosynthetic genes encoding a type II minimal polyketide synthase, C-9 ketoreductase (KR), aromatase, and cyclase, was postulated to undergo stereospecific reduction to provide either (S)-DNPA or (R)-DNPA. In the ACT biosynthesis, RED1 encoded by act VI-ORF 1 was proved to reduce C-3 of bicyclic intermediate to determine the 3-(S)-configuration of DNPA. Although the homolog of act VI-ORF 1 was not found in the gra cluster, RED2 was suggested to reduce bicyclic intermediate. An explored RED-2 coding gene, gra-6, was subjected to updated BLAST analysis. The gra-6 product, a putative short-chain alcohol dehydrogenase, has virtually no sequence similarity with RED1. Functional analysis of RED1/2 was made from the following points. 1) Introduction of gra-ORF 6 and gra-ORF 5 under translational coupling (gra-5+6) into the act VI-ORF 1 mutant, S. coelicolor B22, led to ACT-like pigmentation, demonstrating gra-ORF 6 to complement the function of act VI-ORF 1 possibly under unnatural stereochemical control. 2) Combinations of the ketoreductase genes were co-expressed with the early biosynthetic genes required for the bicyclic intermediate formation. gra-ORF6 was essential to produce (R)-DNPA in DHGRA biosynthesis. gra-5+6 led to the most efficient production of (R)-DNPA, implying a possible unique cooperative function as RED2. 3) A series of synthetic analogues was applied to the biotransformations based on ketosynthase-deficient recombinants of S. coelicolor carrying either RED1 or RED2. In all cases for RED1, the β-keto ester substrates were reduced with good to excellent enantioselectivity. However, the simpler substrates were not accepted by RED2, indicating the significant difference in substrate specificity between the two reductases. 4) 3D structures of RED1 and RED2 were predicted based on homology modeling (FAMS) using the templates, L-3-hydroxyacyl-CoA dehydrogenase from human heart (for RED1) and tropinone reductase II (for RED2). Catalytically key amino acid residues were revealed for the both enzymes. 5) RED1 and RED2 were overexpressed in E. coli, and in vitro assay system were successfully established. Optimization of the system, purification of both enzymes and site directed mutagenesis to the suggested key residues are in progress.

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Macrophomate synthase catalyzes unusual multistep reaction from oxalacetate and 2-pyrone to fungal metabolite, macrophomate involving Diels-Alder reaction. To elucidate its reaction mechanism, requirement of metal ion, kinetic parameter and stereochemical course were investigated. In addition, the structure of the active site of macrophomate synthase was determined by X-ray crystallography.

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2-Deoxy-scyllo-inosose (DOI) synthase is the key enzyme in the biosynthesis of aminocyclitol antibiotics and catalyzes cyclization of D-glucose-6-phosphate (G-6-P) into the 6-membered carbocycle DOI. DOI synthase from Bacillus circulans was isolated as a heterodimeric protein comprising from a 20kDa and a 40kDa subunits. While the 40kDa subunit (BtrC) is known to be responsible for catalysis, the function of the 20kDa subunit (BtrC2) has yet to be revealed. In this study, we first carried out identification of the gene encoding BtrC2 from B. circulans by reverse genetics. The amino acid sequence of BtrC2 deduced from btrC2 closely resembled to that of YaaE (function unknwon) of Bacillus subtilis. Instead, BtrC2 appeared to have sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Supplementation of NH_4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. These observations suggest that, in addition to the secondary metabolism, BtrC2 is mainly involved in the primary metabolism in B. circulans. In order to study the reaction mechanism and substrate-enzyme interaction of BtrC, a carbacyclic analog (C-6-P) was designed as a inhibitor. The synthesized C-6-P was found to have strong inhibitory activity with inhibition constant K_1, of 12μM. Furthermore, the C-6-P analog showed time- and concentration-dependent inactivation of BtrC (K_1=224μM, k_<inact>=3.22×10^<-3>s^<-1>). Subsequent LC/ESI-MS analysis of the enzyme after reaction with C-6-P showed the molecular mass of 40768, which was ca. 160mass unit larger than that of native BtrC (40608). These results clearly indicated that C-6-P was a mechanism-based irreversible inhibitor by a covalent bond formation. In order to precisely determine the amino acid residue binding to C-6-P, BtrC-C-6-P complex was digested by chymotrypsin, and the resulting peptide mixture was analyzed by LC/MS. From comparison of the peptides derived from native BtrC, C-6-P was found to bind to a 139-146 peptide fragment, SIKQAVNL, and further, Lys-141 was determined as a binding site of C-6-P by LC/MS/MS analysis. Lys-141 appears to recognize the phosphate group of the substrate G-6-P and play an important role in the DOI synthase reaction.

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Eubacterial diterpene cyclase genes had previously been cloned from a diterpenoid-antibiotic, terpentecin producer. Their products, Cyc1 and Cyc2, were essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same basic skeleton as terpentecin. In this study, functional analyses of these two enzymes were performed by using purified recombinant enzymes. The Cyc1 product converted GGDP into a cyclized intermediate (terpentedienol diphosphate, TDP), which was then transformed into TTE by the Cyc2 product. Interestingly, the Cyc2 product directly catalyzed conversion of GGDP into three olefinic compounds. Moreover, the Cyc2 product utilized farnesyl diphosphate (FDP) as a substrate to give three olefinic compounds, which had the same structures as those formed from GGDP except for the chain-lengths. These results suggested that the Cyc1 product with a DXDD motif converted GGDP into TDP by a protonation-initiated cyclization and that the Cyc2 product with a DDXXD motif completed the transformation of TDP to the olefin, terpentetriene by an ionization-initiated reaction followed by deprotonation. Kinetics of the Cyc2 product indicated that the affinity for TDP and GGDP were higher than that of FDP and that the relative activity of the reaction converting TDP into TTE was highest among the reactions using TDP, GGDP, or FDP as the substrate. These results suggested that an actual reaction catalyzed by the Cyc2 was the conversion of TDP into TTE in vivo.

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