The biosynthetic gene cluster for rebeccamycin, an indolocarbazole antibiotic, from Lechevalieria aerocolonigenes ATCC 39243 has 11 ORFs. To clarify their functions, mutants with rebG, rebD, rebC, rebP, rebM, rebR, rebH, rebT, or orfD2_disrupted were constructed, and the gene products were examined. rebP disruptants produced 11,11'-dichlorochromopyrrolic acid, found to be a biosynthetic intermediate by a bioconversion experiment. Other genes encoded N-glycosyltransferase (rebG), monooxygenase (rebC), methyltransferase (rebM), a transcriptional activator (rebR), and halogenase (rebH). Staurosporine is a representative member of indolocarbazole antibiotics. The entire staurosporine biosynthetic and regulatory gene cluster spanning 20-kb was cloned from Streptomyces sp. TP-A0274 and sequenced. The gene cluster consists of 14 ORFs and the amino acid sequence homology search revealed that it contains three genes, staO, staD, and staP, coding for the enzymes involved in the indolocarbazole aglycone biosynthesis, two genes, staG and staN, for the bond formation between the aglycone and deoxysugar, eight genes, staA, staB, staE, staJ, staI, staK, staMA, and staMB, for the deoxysugar biosynthesis and one gene, staR is a transcriptional regulator. staO, staD, and staP were similar to rehO, rebD, and rebP, respectively, all of which are responsible for indolocarbazole biosynthesis, But a rebC homolog, encoding a putative enzyme oxidizing the C-7 site of pyrrole rings, was not found in the staurosporine biosynthetic gene cluster. These results suggest that indolocarbazole is constructed by oxidative decarboxylation of chromopyrrolic acid (11,11'-dichlorochromopyrrolic acid in rebeccamycin) generated from two molecules of tryptophan by coupling and that the oxidation state at the C-7 position depends on the additional enzyme(s) encoded by the biosynthetic genes. Moreover, heterologous gene expression of a complete set of the biosynthetic genes for staurosporine and rebeccamycin cloned into pTOYAMAcos confirmed their role in staurosporine and rebeccamycin biosynthesis.
Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii HC34, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. We have been interested in the biosynthesis of vicenistatin, because the aglycon is distinct from regular polyketides solely consisting of lower fatty acid units. Isotope tracer experiments of ^<13>C-labeled acetate and propionate showed that C-1 to C-16 of the aglycon was derived from acetate and propionate in a standard polyketide biosynthetic pathway, and incorporation of intact acetate into C-17 and C-18 strongly suggested that a possible starter unit was derived from glutamate via 3-methylaspartate. Feeding experiments of deuterated glutamate, [^<15>N]-glutamate and deuterated (2S,3S)-3-methylaspartate showed that glutamate mutase, which is known to convert glutamate to 3-methylaspartate, was actually involved in the biosynthesis of the aglycon. However, (2S,3R)-3-methylaspartate and DL-3-amino-2-methylpropionate were not incorporated into vicenistatin. Thus, epimerization of the methylated site is involved in the starter biosynthesis, but its timing is yet to be clarified. The vicenistatin gene cluster (vin) spanning ca. 64kbp was successfully cloned and sequenced by using consensus sequences of dTDP-glucose 4,6-dehydratase and dTDP-4-keto-6-deoxyglucose 2,3-dehydratase which were usually involved in the early steps of the 2,6-deoxysugar biosynthesis. The vin cluster contains ORFs encoding proteins homologous to polyketide synthases (vinP1-4), glutamate mutase (vinH, I), acyl CoA ligase (vinN) and decarboxylase (vinO). VinN and VinO appear to be involved in the formation of the starter unit of aglycon from the earlier intermediate 3-methylaspartate generated by VinH and VinI. VinP1-4, consisting of eight extending modules and a terminal thioesterase domain, are responsible to produce the whole aglycon polyketide. Also contained in the cluster are ORFs homologous to 4,6-dehydratase (vinB), 2,3-dehydratase (vinD), aminotransferase (vinF), N-methyltransferase (vinG) and glycosyltransferase (vinC), for the vicenisamine biosynthesis. Furthermore, VinC was heterologously expressed in Escherichia coil and purified. The glycosyl transfer reaction from dTDP-vicenisamine to the aglycon was confirmed with the recombinant VinC. These results proved that the abovementioned gene cluster encodes the vicenistatin biosynthetic enzymes.
Aspergillus fumigatus is an important opportunistic pathogen causing aspergillosis. One of its virulence factors, DHN-melanin, is biosynthesized from pentaketide T4HN. We recently reported that naphthopyrone YWA1, a product of heptaketide synthase Alblp, is converted to pentaketide T4HN by a chain-length shortening enzyme Ayglp. To characterize this novel T4HN biosynthesis system in A. fumigatus, functional studies on Alblp and Ayglp were carried out. Alblp is a typical iterative type I PKS with KS, AT, tandem ACPs, and CYC domains. We first established the functional expression system of this fungal PKS in yeast using pYES-DEST52 expression vector with co-expression of Bacillus subtilis sfp. The yeast transformant produced heptaketide naphthopyrone YWA1 by GAL1 promoter induction. The Alblp C-terminus deletion experiments revealed that the deletion mutant Cd-3 which lacked ACP2 and CYC still produced heptaketide DHCI. Co-expression of Cd-3 with C-terminus region reconstituted the naphthopyrone YWA1 production, indicating that CYC domain can work as a separate enzyme on the heptaketide intermediate anchored on PKS. The Ayglp was purified to homogeneity from the Aspergillus oryzae overexpression transformant. The purified Ayglp converted the heptaketide naphthopyrone YWA1 to the pentaketide T4HN with a release of the diketide acetoacetic acid. Ayglp showed strict substrate specificity to the heptaketide YWA1 and was strongly inhibited by the serine protease inhibitors. Site-directed mutagenesis confirmed that the Ser^<257> residue is the active site of the enzyme. Thus, Ayglp appears to catalyze the hydrolytic cleavage of the C-C bond between the naphthalene ring and the side-chain carbonyl of YWA1 which is attacked by a hydroxyl anion of the Ser^<257> in the catalytic center.
1. A C_<35> polyprene in which a farnesyl C_<15> unit is connected in a head-to-head fashion to a geranylgeranyl C_<20> unit was enzymatically converted to an unnatural hexacyclic polyprenoid by squalene: hopene cyclase from Alicyclobacillus acidocaldarius. The cyclization of the C_<35> polyprene was initiated by a proton attack on the terminal double bond of the C_<15> unit, and proceeded without rearrangement of carbon and hydrogen. The substrate should be folded in chair-chair-chair-chair-boat-boat conformation in order to achieve the stereochemistry of the cyclization product. 2. Substrate specificities of chalcone synthase (CHS) were investigated using analogs of malonyl-CoA, the extension unit of the polyketide chain elongation reactions. When incubated with methylmalonyl-CoA and 4-coumaroyl-CoA, CHS from Scutellaria baicalensis afforded an unnatural C_6-C_5 aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by one-step decarboxylative condensation of the substrates. In contrast, succinyl-CoA was not accepted as a substrate. Next, both the starter and the extension unit were simultaneously replaced with non-physiological substrates. When incubated with benzoyl-CoA and methylmalonyl-CoA, CHS afforded a novel triketide, 4-hydroxy-3,5-dimethyl-6-phenyl-pyran-2-one, along with a tetraketide, 4-hydroxy-3,5-dimethyl-6-(1-methyl-2-oxo-2-phenyl-ethyl)-pyran-2-one. On the other hand, the enzyme also accepted hexanoyl-CoA and methylmalonyl-CoA as substrates to produce an unnatural novel triketide, 4-hydroxy-3,5-dimethyl-6-pentyl-pyran-2-one.
Ansamycins such as rifamycin, ansamitocin and geldanamycin are an important class of polyketide natural products. Their biosynthetic pathways are especially complex since they involve the formation of 3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid nonribosomal peptide synthetase (NRPS) / polyketide synthase (PKS). We have reconstituted the ability to synthesize P8/1-OG. an intermediate in rifamycin biosynthesis, in an extensively manipulated strain of E. coli. The parent strain, BAP1, contains the sfp phosphopantetheinyl transferase gene from Bacillus subtilis, which post-translationally modifies NRPS and PKS modules. AHBA biosynthesis in this host required introduction of seven genes from Amycolatopsis mediterranei (which produces rifamycin) and Actinosynnema pretiosum (which produces ansamitocin). Since the 4-module RifA protein (530kDa) from the rifamycin synthetase could not be efficiently produced in an intact form in E. coli, it was genetically split into two bimodular proteins separated by matched linker pairs to facilitate efficient inter-polypeptide transfer of a biosynthetic intermediate. A derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon, the bicistronic RifA construct and the pccB and accAl genes from Streptomyces coelicolor, which enable methylmalonyl-CoA biosynthesis. Fermentation of this strain of E. coli yielded P8/1-OG and an N-acetyl P8/1-OG analog. In addition to providing a fundamentally new route to shikimate and ansamycin-type compounds, this result enables further genetic manipulation of AHBA-derived polyketide natural products with unprecedented power.
In order to prepare optically active α-monosubstituted cyclohexanones, we developed a new method that was thermodynamically controlled deracemization in basic suspension media. For example, use of (R,R)-(-)-trans-2,3-bis(hydroxydiphenylmethyl)-1,4-dioxaspiro[5.4]decane (TADDOL, 1a) (2.0 equiv.) with alkaline in aqueous MeOH converted racemic 2-(2-methoxyethyl)-cyclohexanone (2a) to the R-isomer of 94% ee in quantitative yield (Scheme 1). In the case of 2-allylcyclohexanone (2b), a modified method that was the deracemization followed by filtration and recomplexation using 1b (1.0 equiv.) increased the optical purity of 2b up to 93% ee (Scheme 2), although (R)-2b of only 76% ee was obtained by the original method (Scheme 1). In order to demonstrate the usefulness of this new method, synthesis of (R)-α-lipoic acid was carried out starting from (R)-2a of 99% ee. Baeyer-Villiger oxidation of (R)-2a afforded 7-membered lactone 3 whose optical purity was determined as 99% ee by GLC analysis using a chiral column. The treatment of the lactone 3 with diiodosilane gave (6S)-8-hydroxy-6-iodooctanoic acid, which was converted to mesylate 6. The reaction of 6 using sodium sulfide nanohydrate and sulfur provided (R)-α-lipoic acid successfully. Thus, the present total synthesis of (R)-α-lipoic acid was accomplished quite satisfactorily in 41% overall yield with 5 steps from the racemic ketone 2a (Scheme 3,4). (S)-(+)-Epilachnene was isolated as a component of defensive droplets from grandular hairs of the pupae of the Mexican been beetle, Epilachna varivestis. The deracemization of racemic ketone 2b was also applied to the synthesis of the antipode of the natural epilachnene. First, (R)-2b obtained by the modified method was converted to lactone 7 by Baeyer-Villiger oxidation. DIBAL reduction of 7 followed by Wittig reaction gave hydroxy nitrite 8, which was subjected to Mitsunobu type reaction with N-[2-(t-butyldimethylsilyloxy)ethyl]tosylamide in the presence of CMMP to yield sulfonamide 9 with complete Walden inversion at the carbinol stereocenter of 8. Hydrolysis of the nitrite 9 provided seco-acid 10, which was successfully cyclized by the reaction of 2-chloro-1-methylpyridinium iodide to afford lactam 11. Deprotection of tosyl group followed by acid treatment gave (R)-(-)-epilachnene in good yield (Scheme 6).
Blasticidin S (1) is a representative peptidyl-nucleoside antibiotic first isolated from Streptomyces griseochromogenes in 1958 by Yonehara and co-workers. This antibiotic was once commercialized as a fungicide against the virulent fungus, Piricularia oryzae, a serious cause of rice blast disease in Asia. Its biological activity results from specific inhibition of protein biosynthesis by interfering with the peptide bond formation in the ribosomal machinery. Yamaguchi found two blasticidin S-resistance genes, bsr from Bacillus cereus and BSD from Aspergillus terreus which code blasticidin S deaminase. Both genes are now widely used for genetic engineering experiments to select both prokaryotic and eukaryotic cells expressing the blasticidin S-resistance gene. The Renaissance of blasticidin S, originally used as a microbial fungicide, is now flourishing in the research area of molecular biology. The structure and absolute configuration of blasticidin S have been elucidated via chemical degradation and spectroscopic studies by Otake and co-workers, who isolated two components, blastidic acid and cytosinine by acid-catalyzed hydrolysis of blasticidin S. Blastidic acidis an unusual b-amino acid counterpart of arginine with a modified N-methyl guanidine group. Such a -amino acid motif is also manifested in cytosinine, and the highly functionalized feature in cytosinine is characterized with a unique hexopyranosyl-nucleoside containing 2,3-unsaturated-4-amino pyranose attached with cytosine. The richness with functional complexity found in blasticidin S poses synthetic challenges. Although a pioneering work of Kondo and Goto reported the first synthesis of cytosinine in 1972 and two reports for the syntheses of blastidic acid appeared in 2001, there has been no report on the total synthesis of blasticidin S. A key step in the synthetic scheme with cytosinine is a sigmatropic rearrangement of an ally cyanate to give rise to an efficient and stereoselective access to 2,3-dideoxy-4-amino-D-hex-2-enopyranose. Further elaboration into methyl hex-2-enopyranouronate, and cytosine N-glycosylation by the Vorbruggen method furnished the differentially protected cytosinine in 11 steps starting from 2-acetoxy-D-glucal (4.7% overall yield). In the synthesis of Boc-protected blastidic acid, the Weinreb protocol for the preparation of benzyl amide and Fukuyama's method for the synthesis of secondary amine effectively worked to result in the 9-step synthesis starting from chiral carboxylic acid (23% overall yield). Assembly of the protected cytosinine and blastidic acid by the Bop method in the presence of HOBT, and final deprotection of the fully protected blasticidin S established the total synthesis route of blasticidin S.
Bongkrekic acid is a toxic antibiotic produced by the microorganism Pseudomonas cocovenenans. The high toxicity of bongkrekic acid has been attributed to its affinity for the ATP / ADP translocator protein residing in the mitochondrial inner membrane, thus preventing oxidative phosphorylation. Recently, bongkrekic acid has been found to be an inhibitor of apoptosis acting on permeability transition pores. Thus, bongkrekic acid is very important and a useful biochemical tool for investigation of apoptosis. Since its availability by fermentation is very difficult and there has been only one report of chemical synthesis, an efficient synthetic method for bongkrekic acid has been required. Herein, we report the total synthesis of bongkrekic acid. Our plan for synthesis of bongkrekic acid is based on a convergent strategy. Bongkrekic acid is devided into the right-hand segment and the left-hand segment. The synthesis of the left-hand segment is as follows: Evans' asymmetric alkylation provided 4 with single isomer. After removal of the chiral auxiliary, Takai's alkenylation of the aldehyde, followed by Suzuki coupling, provided the triene which was converted into the left-hand segment 2. The right-hand segment was synthesized as follows: Me_2CuLi of 1,4-addition to alkynylester 14, prepared from D-mannitol in good yield, provided the (Z)-alkenylester 13 in 98% yield as a single isomer. The ester 13 was converted into the bromide 23, which was combined with 2 via sulfone anion alkylation, and the resulting 29 was successfully converted into bongkrekic acid 1.
Scyphostatin(1), isolated from Dasyscyphus mollisimus SANK-13892 by Ogita et al. in 1997, has been shown to be a potent and specific inhibitor of neutral sphingomyelinase (IC_<50>=1.0μM). This natural product is anticipated to be a promising lead compound for the treatment of ceramide-mediated pathogenic states such as inflammation and immunological and neurological disorders. As well as this remarkable biological activity, its unique structural feature make 1 an exceptionally intriguing and timely target for total synthesis. While a number of synthetic approaches have appeared recently, no total synthesis has been reported to date. We embarked on the project directed toward the total synthesis of 1 in an optically active form, and our earnest endeavors culminated in completing the first total synthesis of (+)-1. Retrosynthetically, scyphostatin can be divided into two segments, the cyclohexene part 2 and the fatty acid part 6. We envisaged that the labile epoxycylohexenone moiety should be elaborated after formation of the amide linkage between 2, readily derived from 17, and 6. Our synthetic strategy for the preparation of 17 and 6 features the following three key steps: i) a coupling reaction of ester 5 with Garner aldehyde (4) to install an amino propanol moiety with the desired C-4 quaternary asymmetric carbon center (5→14); ii) ring closing metathesis reaction of 3 to elaborate the cyclohexene ring (3→16); iii) Negishi coupling reaction between alkyl iodide 9 and vinyl iodide 8 to produce tri-substituted olefin 22 (9+8→22). Our synthesis of the cyclohexene part 17 was commenced with the conversion of the known compound 11 to ester 5. The stereoselective alkylation of 5 with Garner aldehyde (4) was achieved by using NaN(TMS)_2 as a base in THE at -78℃ for 4h. After several steps, crucial ring closing metathesis reaction of the diene 3 was conducted by employing Grubbs catalysis, producing 16, which was then converted to the cyclohexene part 17. On the other hand, the fatty acid side chain 6 was prepared from the known aldehyde 19 and olefin 21. Negishi coupling of vinyl iodide 8 and alkyl iodide 9, which were readily accessible from 19 and 21, respectively, was accomplished by applying Smith's modified condition (t-BuLi, ZnCl_2, Pd(PPh_3)_4) to give rise to tri-substituted olefin 22. After several steps including one carbon elongation and HWE reaction, the acid chloride 6 was obtained. With 17 and 6 in hand, we focused our attention on completion of the synthesis of (+)-1. Towards this end, 17 was transformed to enone 31 in 6 steps involving amide formation between 28 and 6. Finally, 31 was converted to the target (+)-1 by hydrolysis of acetal 31 with CCl_3CO_2H, epoxide formation with base, and the Lipase-catalyzed removal of an acetyl group. The synthesized (+)-1 was identical with a natural sample of 1 in all spectroscopic properties.
Epolactaene (1), which was isolated from fungal strain Penicillium sp. BM1689-P by Osada et al., possesses a potent neurite outgrowth activity in a human neuroblastoma cell line SH-SY5Y. The characteristic features of epolactaene include the highly functionalized α,β-epoxy-γ-lactam and the conjugated triene moiety in the side chain. The significant biological activity as well as the structural complexity of epolactaene has stimulated intensive synthetic interests, culminating in the total syntheses and determination of its absolute stereochemistry by two groups. We have accomplished the total synthesis of epolactaene by an application of the bridgehead oxiranyl anion strategy. We first successfully performed an aldol-type condensation of the epoxylactone with a series of aldehydes by a two-step procedure via the trimethylsilyl epoxylactone. Then the methodology was applied to the total synthesis of epolactaene. The present approach provides the first example of the generation and reaction of the highly reactive bridgehead oxiranyl anion derived from the epoxylactone. The most significant feature of the present approach is that the trimethylsilylated epoxylactone serves as a potential intermediate for the synthesis of various substituted epolactaene analogs. Several analogs were also prepared by the coupling of the silylated epoxylactone with aldehydes. Biological studies on the α,β-epoxy-γ-lactam derivatives as well as the synthetic intermediates were investigated. As a result, we observed that the synthetic (+)-epolactaene and α,β-epoxy-γ-lactam derivatives inhibited the activities of mammalian DNA polymerase α,β and human DNA topoisomerase II. We also found that they induced apotosis in BALL-1 cells. In order to investigate further biological studies, we prepared fluorescent and biotinylated derivatives. The biologcal studies using the synthetic probes are now undergoing.
Leustroducsin B (LSN-B, 1b) is a potent colony-stimulating factor inducer isolated from the culture broth of Streptomyces platensis SANK 60191 by Sankyo's groups. LSN-B is known to exhibit a variety of biological activities and is likely to be developed as a new drug candidate. These interesting biological activities coupled with its unique structural features have attracted our attention as a target for total synthesis. We designed the construction of a carbon framework of LSN-B by coupling the aldehyde 2 and the enyne 3. The key point of synthesis of 2 was construction of C8, C9 stereocenters. The stereochemistry of C8 of 2 was controlled by lipase-mediated desymmetrization of 2,2-disubstituted propanediol 10. Chelation-controlled addition of allylmagnesium bromide to the aldehyde 12 in Et_2O at -78℃ afforded the desired sec-alcohol 13 as a single diastereomer. Upon treatment with a catalytic amount of Ti(Oi-Pr)_4 in refluxing benzene, 20 underwent smooth cyclization to give lactone 21. Synthesis of the enyne 3 involves optical resolution of racemic hydroxy ester 23 by lipase, (Z)-selective Horner-Wadsworth-Emmons reaction and Sonogashira coupling reaction. It was found that Zn reagents were very effective for the coupling reaction between the aldehyde 22 and the enyne 25. The alkynylzinc bromide 26, which was prepared from 25, underwent smooth addition to 22 in toluene-Et_2O to give the desired adduct 27 as a single diastereomer. Partial reduction of the conjugated triple bond was best carried out with Zn/LiCuBr_2 according to the method of Brandsma to yield the Z,Z-diene 28. The final stage of our synthesis involves a series of functional group manipulations without disturbing the other delicate functionalities. For the reason mentioned above, we devised this new benzylidene group, which can be deprotected safely under weakly acidic conditions from densely functionalized substrates. The protecting group was easily removed in a 2-step procedure (30 to 31, (HF)_3・Et_3N; AcOH-THE-H_2O). Finally, removal of the phenoxyacetyl group on C11-OH with Er(OTf)_3 in MeOH followed by treatment with Pd(PPh_3)_4, HCO_2H and Et_3N furnished leustroducsin B (1b).
Zaragozic acids, isolated and characterized independently by Merck and Glaxo in 1992, have attracted considerable synthetic attention because of the biological activity of these compounds and their novel structural aspects. In 1997 we achieved a total synthesis of zaragozic acid C; however, this route suffered from poor selectivity (1.6:1) in the aldol fragment coupling reaction to join the C4-C5 bond. Then, we addressed a second-generation synthesis of zaragozic acids, highlighting an alternative construction of the 2,8-dioxabicyclo[3.2.1]octane core system via a tandem carbonyl ylide formation and 1,3-dipolar cycloaddition process. Toward this end, α-diazo ester 7 was prepared from di-tert-butyl D-tartrate (10) (Scheme 2). After considerable experimentation with respect to a dipolarophile, reaction of 7 and 3-butyn-2-one with catalytic Rh_2(OAc)_4 in refluxing benzene was found to give the desired cycloadduct 22 as a single diastereomer out of the four possible diastereomers in 72% yield (Scheme 3). Dihydroxylation of 22 with OsO_4 followed by sequential benzylation, DIBAL-H reduction, oxidative cleavage of diol with Pb(OAc)_4, and reduction with DIBAL-H/ZnCl_2 effected the installation of the C6, C7 trans diol moiety. Upon protecting group interchange and oxidation, alcohol 26 was uneventfully transformed to the fully functionalized core structure 4. As a means for the elaboration of C1 side chain of 2, we were attracted to the possibility of a cross-metathesis between terminal olefin 32 derived from 4 and allyl acetate 33 (Scheme 4). Gratifyingly, the cross-metathesis reaction with Grubbs' second-generation catalyst (34) in refluxing benzene was found to provide the desired cross-product 35 in good yield. As might be expected from the hindered nature of the olefinic functionality in 32, the dimer 36 from self-metathesis of 32 was not detected. Hydrogenation of 35 followed by debenzylation produced a late intermediate 37 in the synthesis of zaragozic acid C by the Carreira^<8b> and our groups.
Benzastatins and (-)-virantmycin (2a) are a family of indoline and tetrahydroquinoline alkaloids isolated from Streptomyces nitrosporeus. Benzastatins show neuronal cell protecting activity that can be used to prevent brain ischemia injury, and (+)-benzastatin E (1) is the most potent inhibitor of glutamate toxicity using neuronal hybridoma N18-RE-105 among the benzastatin family. (-)-Virantmycin (2a) has been found to exhibit antifungal activity as well as inhibitory activities against various RNA and DNA viruses. From the structures of these alkaloids, a biosynthesis involving an aziridine intermediate has been suggested. Based on this hypothesis, we designed the PPh_3-CCl_4 mediated rearrangement from α,α-disubstituted indoline-2-methanol 3 to 2,2,3-trisubstituted tetrahydroquinoline 4 via the aziridine intermediate followed by the ring opening attack of the chloride anion. Chiral α,α-disubstituted indoline-2-methanol 3 were readily synthesized by the diastereoselective addition of Grignard reagents to 2-acylindoline 9. Reaction of 2-acylindoline 9 possessing various alkyl groups with metal reagents in THF at -78℃ gave tert-alcohols 10 as separable diastereoisomers. These additions were highly diastereoselective as demonstrated in Table 1. The stereochemical outcome of this reaction can be explained by the Felkin-Anh model as depicted in Figure 1. The total synthesis of (+)-benzastatin E (1) was accomplished utilizing this method, in three steps from the acetonide 11. The chiral α,α-disubstituted indoline-2-methanol 3, obtained by deprotecting the Boc group of 10, was treated with PPh_3-CCl_4 in CH_2Cl_2 to afford the desired tetrahydroquinoline 4 as a sole stereoisomer. The dechlorinated derivative of 4b was identical with the dechlorinated compound of 4a except for the optical rotation, suggesting that the rearrangement is stereospecific. All the reactions provide single stereoisomers in moderate to good yield as shown in Table 2. The utility of this reaction was clearly demonstrated by the efficient total synthesis of (-)-virantmycin (2a), which was carried out in only nine steps from commercially available starting material. In summary, we have developed a novel synthetic route of chiral trisubstituted tetrahydroquinolines by the combination of the diastereoselective Grignard addition and the stereospecific rearrangement. Using this methodology, we have achieved the efficient total syntheses of (+)-benzastatin E (1) and (-)-virantmycin (2a). We believe that our rearrangement reaction provides some support for the proposed biogenesis of virantmycin and benzastatins involving the aziridine intermediate.
Pseurotin A, isolated in 1976, inhibits chitin synthase and also induces cell differentiation of PC12 cells, while azaspirene, isolated in 2002 by Kakeya and Osada et al., is an angiogenesis inhibitor, and attracted much attention as a lead of a drug for the treatment of cancer. As they both contain a novel, highly-substituted and oxygenated 1-oxa-7-azaspiro[4.4]non-2-ene-4,6-dione skeleton with three continuous chiral centers, their total syntheses are a challenge. We have synthesized pseurotin A and azaspirene from the same key elaborated ketone intermediate. The ketone was stereoselectively synthesized by the following key reactions: a MgBr_2・OEt_2-mediated Mukaiyama aldol reaction affords the desired syn-aldol in good yield with high selectivity. A NaH-promoted, intramolecular cyclization of a non-activated alkynylamide provides (Z)-benzylidene-γ-lactam. The aldol condensation proceeds smoothly between a side chain aldehyde and the ketone containing functionalized γ-lactam moiety without protection of tert-alcohol and amide functionalities. In the synthesis of pseurotin A, introduction of the benzoyl group by the selective oxidation of a benzylidene moiety with dimethyldioxirane (DMD) was successfully performed. In the synthesis of azaspirene, we found that the order of the last two reactions is very important because the reversed reaction sequence afforded a racemic azaspirene. By this total synthesis, the absolute stereochemistry of azaspirene has been determined.
The first total synthesis of a novel potent antinociceptive monoterpene alkaloid incarvillateine (1) and its congenetic alkaloids incarvine C (2) and incarvilline (3) is presented. The synthetic plan for incarvillateine (1) we conceived relied on [2π+2π] cycloaddition of incarvine C (2). Thus, the synthesis of 2 started with (S)-4-siloxycyclopentenone 12, prepared from L-tartaric acid, upon treatment of 12 with the organozinc reagent generated in situ from (E)-stannylalkene 14, followed by addition of iodomethane, affording the 2,3,4-trisubstituted cyclopentane 15 as a single stereoisomer. The cyclopentenone 20 derived from 15 in five steps was then cyclized to the octahydrocyclopenta[c]pyridine 21 by means of the reductive Heck reaction using palladium(II) catalyst in the presence of formic acid. Compound 21 was stereoselectively converted to 6-epi-incarvilline (4) via reduction of the C-6 carbonyl group and catalytic hydrogenation of exocyclic alkene. Mitsunobu inversion of the C-6 hydroxy configuration of 4 led to the synthesis of (-)-incarvilline (3). On the other hand, Mitsunobu condensation of 4 with ferulic acid resulted in the synthesis of (+)-incarvine C (2). Attempted photodimerization of 2, however, failed to yield the expected [2+2] cycloadduct. The total synthesis of (-)-incarvillateine (1) was successsfully achieved by Mitsunobu condensation of 4 with the α-truxillic acid, prepared by head-to-tail photodimerization of the O- tosyl derivative of ferulic acid.
TMC-95A (1) was isolated as a potent proteasome inhibitor. The structural features of 1 are the axially-chiral biaryl bond in the macrocycle, the highy oxidized tryptophan moiety, the (Z)-propenylamide and the 3-methyl-2-oxopentanoic acid side chain. Here we present the efficient, stereoselective total synthesis of 1. (Z)-selective Mizoroki-Heck reaction of 9, which was derived from D-serine, afforded the oxindole 10. The diastereoselective epoxidation of 10, followed by 6-endo epoxide ring-opening reaction, installed the C6 and C7 stereocenters to give 13. Then, the removal of carbamate group from 15 under Gassman's conditions was accompanied by concomitant loss of the acetonide to produce 16. Finally, 16 was transformed to 19 through regeneration of the oxindole structure. Suzuki coupling reaction between 19 and 31 afforded the biaryl 32. After the introduction of the L-asparagine derivative, the lactamization produced the macrocyclic framework 34 in high yield. Then, 34 was coverted to 39 through 6 steps. On treatment of β-hydroxycarboxylic acid 39 under Mitsunobu conditions, the decarboxylative anti-elimination proceeded smoothly to yield the isomerically pure (Z)-propenylamide 41. Deprotection of 41 and selective oxidation of the C35-alcohol of 42 gave MOM-protected TMC-95A 43. The C7 alcohol was temporarily protected as its chroloacetyl ester 44 to suppress the side reaction that involved the propenylamide. Exposure of 45 to 1N HCl-THF and then saturated aqueous NaHCO_3 in one pot afforeded fully synthetic TMC-95A (1), of which phisical data are identical to those of the natural product.
Serofendic acids A and B, possessing atisane skeleton, isolated from fetal calf serum (FCS) show potent protective action against neurotoxicity induced by glutamete and an No donor. Due to its scarce availability from nature, the development of efficient synthesis is highly demanded. Eisai group synthesized serofendic acids from (-)-isosteviol through methyl atisienoate and established its absolute configuration including the stereochemistry about sulfoxide. Tohoku group studied four approaches for the synthesis of atisane derivatives. The first approach using intramolecular Diels-Alder reaction resulted in a non stereoselective production of atisane skeleton. The second approach utilizing intramolecular double Michael reaction provided the required atisane derivative in a highly stereoselective manner. However, the transformation of the product into natural diterpenes required a number of steps for the removal of ester group and introduction of C_1, unit. In order to prepare a bicyclo[2.2.2]octane ring system carrying exo olefin part, cyclization of vinyl radical was next examined. Although total synthesis of (±)-gumiferolic acid showing potent growth-regulatory activity was achieved by the third approach, the yield of the key step was low. Finally, the bicyclo[3.2.1]octane possessing exo olefin part and oxygen functionality was prepared by cycloalkenylation reaction using catalytic amount of palladium(II) acetate in DMSO under oxygen atmosphere and converted into methyl atisienoate via intramolecular Diels-Alder reaction and homoallyl-homoallyl radical rearrangement. Thus, (-)-enantiomer of atisane diterpene convertible to serofendic acids was effectively synthesized.
Tricycloclavulone (1) was isolated from Okinawan soft coral Clavularia viridis by our group as a novel marine prostanoid. This molecule has a unique tricyclo[5,3,0,0^<1,4>]decane skeleton and 6 chiral centers. Although relative stereochemistries of 1 was determined by spectroscopic analysis, the stereochemistry of a chiral center on the α-chain and the absolute stereochemistry have not been determined yet. The biological activity of the compound 1 has not been examined owing to its small amount obtained as a natural product. For the solution of above mentioned problems, the total synthesis of tricycloclavulone, which might be challenging synthetic target for organic chemists, was performed. We described here the total synthesis of (±)-tricycloclavulone as well as the effort for the synthesis of 1 as an optically active form. We designed the synthetic route as follows (Scheme 1). The tricyclic compound 2, which has a vinyl sulfone moiety on the C-ring, was an important intermediate. Two side chains would be constructed stereoselectively through 1,4-addition of organometallic species to the vinyl sulfone moiety and the following capture of the resulting anion on the sulfone bearing carbon atom with an appropriate electrophile. The compound 2 could be prepared from the compound 4 through [2+2]-cycloaddition reaction and ring closing olefin metathesis as key steps. Copper trifluoromethanesulfonate catalyzed [2+2]-cycloaddition reaction gave bicyclic compound 6 in good yield (Table 1). After introduction of two vinyl groups to 6, the construction of tricyclic skeleton was achieved by ring closing olefin metathesis using Grubbs catalyst (Scheme 2). Stereoselective addition of side chains to the tricyclic skeleton was also achieved on the convex face of the C-ring through 1,4-addition of dibutylcopper lithium to 13 and the following intramolecular ester transfer reaction (Scheme 3). Elongation of the α-chain and reduction of the ketone on the α-chain completed the first total synthesis of 1 as a racemic form (Scheme 4). For the preparation of tricycloclavulone (1) as an optically active form, enantioselective [2+2]-cycloaddition reaction was also examined (Table 2). In the presence of a catalytic amount of ligand 21, CuCl_2, and AgSbF_6, the reaction proceeded to give (1S,5R)-6 as 61% ee in 69% yield (Scheme 5).
We recently reported the intramolecular substitution of an allyl alcohol by a heteroatom using a palladium (II) catalyst without activation of the allylic alcohol. We report here highly stereoselective intramolecular cyclization of 1,11-dihydroxyundece-9-en-5-one derivatives using palladium (II) catalyst via unstable hemiacetal intermediates, in which cyclization occurs without activation of the allylic alcohol to afford spiroketal structures. Stereoselective Tandem Cyclization using Pd (II)-catalyst Treatment of 1,11-dihydroxy-7-phenylundec-9-en-5-one (11) with 10mol% palladium (II) bis(acetonitrile) dichloride in THF afforded 6,6-membered spiroketal 12 in 62% yield as a single stereoisomer. To demonstrate the synthetic potential of this method, we have synthesized a natural product having spiroketal moiety. Synthetic study of Spirofungin B using Pd (II) catalyst Spirofungin B is a polypropionate-type antibiotic isolated from Streptomyces violaceusniger Tu 4113. Now we show a synthesis of spirofungin B using the tandem cyclization of dihydroxy ketone 27 as a key reaction. Retrosynthetic fragmentation of spirofungin B affords three segments. Firstly, we synthesized the key intermediate 17 from (Z)-2-butene-1,4-diol (Scheme 6). The aldehyde segment 19 and sulfone segment 24 was prepared from 17, respectively (Scheme 7,8). The cyclization precursor 27 was prepared by using Julia coupling reaction between aldehyde segment 19 and sulfone segment 24 (Scheme 9). The key tandem cyclization of 27 was achieved successfully by treatment with PdCl_2(PhCN)_2 at room temperature to give segment B (Scheme 10). Segment C was synthesized from alcohol ent-17 by Takai reaction (Scheme 11). Finally, the segment B was introduced to dibromoolefin 32, and 32 was coupled with the segment C by Suzuki-Miyaura coupling to afford the desired(Z,E)-diene 33 (Scheme 12). Studies on the connection of 33 and the segment A to construct the whole structure of spirofungin B are now underway.
(+)-Macquarimicin A (1) was isolated from Micromonospora chalcea in 1995. Later, it was found that 1 is a selective inhibitor of membrane-bound neutral sphingomyelinase (N-SMase) and exhibits an anti-inflammatory activity. The structure of 1 is characterized by a novel tetracyclic framework, which comprises a cis-tetrahydroindanone ring, a β-keto-δ-lactone ring, and a 10-membered carbocycle. In addition, 1 would be biosynthesized via an intramolecular Diels-Alder reaction (IMDA) of a polyketide intermediate. This formidable molecular architecture, combined with the remarkable biological profiles, makes 1 an attracting synthetic target. Herein, we describe the first total synthesis of natural (+)-macquarmicin A (1) and determination of its absolute configuration. (Z)-Stannylalkene 10 and (E)-iodoalkene 11, each synthesized from known 12 and 17 respectively, were converted into 8 via a CuCl promoted Stille coupling reaction. The macroallylation of 8 was successfully carried out to form a 17-membered ring using Pd(PPh_3)_4/dppe (1:1) as a catalyst. Formation of the β-keto-δ-lactone ring under thermal conditions followed by the introduction of the double bond afforded 7 as a mixture of geometrical isomers. Under thermal conditions(130℃), the transannular Diels-Alder reaction of 7 furnished the desired diastereomer 6 as a sole cycloadduct. In this reaction, the geometry of (Z,E)-diene is the origin of endo selectivity, while the lactone ring restricts the conformation of transition state to control the diastereofacial selectivity. Silylation of 6 and deprotection of the MPM group provided 28. Dess-Martin oxidation of 28 and subsequent PPTS catalyzed cleavage of the TES group afforded (+)-1. Spectral properties (^1H and ^<13>C NMR and IR) of synthetic (+)-1 were completely identical with those of a natural sample. Furthermore, optical rotation of synthetic (+)-1 ([α]^<23>_D +285°(c 0.780, MeOH)) established the absolute configuration of natural (+)-1 ([α]^<25>_D +285.6°(c 0.2, MeOH)).
Norzoanthamine (1), a marine alkaloid isolated from the colonial zoanthid Zoanthus sp., has a densely functionalized heptacyclic ring system including two aminal structures and four quaternary asymmetric carbon centers on the C-ring. Since 1 not only inhibits IL-6 production, but also suppresses the decrease in bone weight and strength in ovariectomized mice, norzoanthamine has been regarded as a promising candidate for an osteoporotic drug. One of the critical problems in the total synthesis of zoanthamine alkaloids may be the stereoselective construction of the four quaternary asymmetric carbon centers in the C-ring. In order to overcome this critical problems, we designed a synthetic strategy employing an intramolecular Diels-Alder reacion as the key step. Triene 8, the key precursor for the Diels-Alder reaction, was efficientry and highly stereoselectively synthesized by a tandem conjugate addition-aldol strategy and subsequent photosensitized oxidation of a furan derivative. Stereoselective synthesis of the ABC ring system of the norzoanthamine bearing five asymmetric centers including two quaternary carbon atoms has been successfully accomplished by the intramolecular Diels-Alder reaction of the triene 8. The alkyne 4, a precursor of coupling reaction was synthesized from diketone 7 via stereoselective construction of C9 quaternary carbon atoms by intramolecular acylation and subsequent methylation. By the coupling reaction of the alkyne 4 with the amine segment 3 followed by several synthetic transformations, we have achieved the synthesis of the carboxylic acid 2, the key precursor of succsesive cyclization to the DEFG ring system. Further studies toward total synthesis of the norzoanthamine (1) are in progress.
Angiogenesis, a formation of new blood capillaries from preexisting blood vessels, is critical for tumor growth and metastasis. A growing tumor needs an extensive network of capillaries to provide nutrients and oxygen etc. In addition, the new blood vessels provide a way for tumor cells to enter in the circulation and to metastasize to another organ. Therefore, substances that inhibit the angiogenesis have a considerable potential to be therapeutic agents. In the course of our study on the bioactive substances from marine organisms, we focused on a search for anti-angiogenic substances and isolated bastadins from the Indonesian marine sponge of Ianthella basta. Bastadins were cyclic tetramers of brominated-tyrosine. Among of them, bastadin 6, a major constituent, showed a selective growth inhibition against human umbilical vein endothelial cells (HUVECs) whereas it showed a weak growth inhibition against fibroblast (3Y1) and tumor cells (KB3-1, K562, Neuro2A). The effect was enhanced under the basic fibroblast growth factor (bFGF)- or vascular endothelial growth factor (VEGF)-dependent proliferative conditions. Bastadin 6 also inhibited in vitro VEGF-induced migration and bFGF-induced tube formation of HUVEC. Moreover, bastadin 6 almost completely blocked VEGF and bFGF-induced neovascularization in the in vivo mouse corneal assay. We also studied total synthesis of bastadin 6 in order to establish a practical synthetic method for further biological analysis. According to the reported method by Sih et al., utilizing the enzymatic oxidative coupling of bromophenols as the key reaction, we developed an improved synthetic route by changing starting materials and reaction conditions, and achieved a convergent total synthesis of bastadin 6.
Commelinin is a stoichiometric self-assembled supramolecular pigment from the blue petrals of dayflower, Commelina communis, composed with six molecules of malonylawobanin, six moleclues of flavocommelin and two atoms of magnesium ion. A fine molecular recognition occurs on formation of commelinin. The atomic structure of commelinin was determined by X-ray crystallography. We synthesized its various chiral analogues of flavocommelin and clarified the role of flavocommelin on formation of commelinin and its color development. We succeeded in the first total synthesis of flavocommelin by development of novel method in C-glycosylation to flavan skeleton. At first, naringenin was reducted to the flavan, which was C-glucosylated at C-6 using glucosyl fluoride in presence of Lewis acid followed by oxidation with CAN to give the naringenin 6-C-β-D-glucoside. The glucosylation at OH-4' of the naringenin glucoside according to our method and then oxidation with DDQ achieved total synthesis of flavocomelin. And also flavocommelin-analogues bearing D- and/or L-glucose were prepared. Reproduction experiments of the metalloanthocyanin from the anthocyanin, various chiral flavocomelin or its analogues in the presence of Mg^<2+> was performed. For formation of the metal-complex showing the blue color, D-glucose at 4'-O of apigenin was essential while the solution from the 4'-O-L-glucosides only gave purple color, which was very unstable. The 6-C-D-glucoside played a role for the stability of the color. However, surprisingly, apigenin 6-C-D-glucoside formed the supramolecule to show blue color. Now we are attempting to reveal the reason of the chiral structural recognition for the metal-complex formation by the structural analysis in detail. The metal-complex formation on the basis of the molecular interaction causes the beautiful blue color of dayflower.
Since the discovery of "Enantiomerism" by Louis Pasteur in 18481, chiral discrimination has been one of the major subjects in science. However, the most widely used diastereomer method for chiral discrimination an "intrinsic problem" that it is very difficult or impossible to discriminate the diastereomers having chiral centers separated more than four bonds. In order to discriminate such diastereomers, we have proposed a new concept that the chirality of the chiral deriving reagent in the diastereomer provides the diastereomer with a specific preferred chiral conformation and the other chirality in the diastereomer could be discriminated as the chirality directly attached to the chiral molecules. Based on the concept developed we have developed highly potent chiral deriving reagents; 1-(anthracene-2,3-dicarboximido)-2-propyl-OTf, 2-(anthracene-2,3-dicarboximido)-1-propyl-OTf, and 2-(anthracene-2,3-dicarboximido)cyclohexanol for chiral carboxylic aids, and 2-(anthracene-2,3-dicarboximido)cyclohexane carboxylic acid for chiral alcohols. These reagents gave us a solution of the "intrinsic problem" of diastereomer method [3,4] and opened the door to a new chiral discrimination world. The new concept, design of the reagents, abilities of these reagents, and several examples of the determination of the absolute configuration of natural products by use of them will be presented.
A ^<13>C and ^1H NMR spectral database for organic natural products, CH-NMR-NP, started to build from May 2002 as the development of the SDBS-NMR for general organic compounds. The chemical information, NMR data and the spectral assignments are compiled from the literatures (J. Antibiotics, Bull. Chem. Pharm., Tetrahedron, Tetrahedron Letts., Magn. Reson. Chem., J. Natural Products, J. Org. Chem., and others). The criteria to adopt data are 1) The tables of ^1H and ^<13>C NMR data are given. 2) The chemical structure is clearly drawn and consistent with the molecular formula in the text. 3) The name is described (a trivial name of a chemical name). 4) Clear errors are not found in NMR data. 5) The selection of the compounds is made not to overlap similar compounds with similar NMR spectra. As the results, the database does not include every compounds appeared in the above journals. The input of chemical information is made by a ISIS/Base and the spectral information such as ^<13>C and ^1H shifts and coupling constants are compiled by a Microsoft Excel. The search and display systems have been created on a web system, where a PostgreSQL is used as a DBMS. The searches by NP number, name, molecular formula, molecular weight, ^<13>C shift and ^1H shifts are possible. ^<13>C NMR spectral patterns are shown by a Java applet. The intensities are generated from the number of the carbons for each line. The specification of the carbon property such as C, CH, CH_2 and CH_3 is possible. 1125 data were compiled from the literatures in 2002. Also about 80 data were collected by collaboration with the researchers of natural products. The addition of the data will be continued.
Brasilinolide A (1) is a polyhydroxy macrolide possessing potent immunosuppressive activity isolated from a pathogenic actinomycete Nocardia brasiliensis IFM 0406. The gross structure and partial relative stereochemistry of 1 have been elucidated by 2D NMR data, whereas its absolute stereochemistry remains undefined. Further investigation resulted in isolation of a new related macrolide, brasilinolide C (2), and assignments of the absolute configurations of 1 and 2 on the basis of spectroscopic data and chemical means as follows. Solvolysis of 2 afforded a macrocyclic aglycone (3) and L-2-deoxyfucopyranose (4). Oxidative degradation of the aglycone (3) gave three segments, C1-C15 (7), C17-C27 (8), and C28-C37 (9). On the other hand, oxidative degradation of 2 yielded two segments, C1-C15 (14) and C17-C37 (15). The relative stereochemistry was elucidated mainly by NMR data including NOESY correlations for the acetonide derivatives (10-14 and 15) or the oxidative product (16). Absolute configurations were assigned on the basis of modified Mosher's method applied to C5, C9, C13, C19, and C31. Finally, the absolute configurations at 26 chiral centers in 2 were elucidated. Similarly, the absolute configurations of brasilinolide A (1) were also assigned as the same as 2.
GRP78 acts as a molecular chaperone in endoplasmic reticulum (ER) by associating transiently with incipient proteins as they traverse the ER and aiding in their folding and transport. Furthermore, the GRP78 protein is also induced under various stress condition such as glucose starvation, inhibition of protein glycosylation by tunicamycin, perturbation of ER function and protein movement by brefeldin A, and suppression of ER-calcium-ATPase pump by thapsigargin. The enhancement of ER stress response (also known as the unfolded protein response) takes part in the resistant mechanism against chemotherapy and hypoxic stress in solid tumor. The ER stress response causes an increase in gene expression of a number of ER chaperones such as GRP78 and GRP94. Thus, substances that directly down-regulate grp78 transcription are expected to be useful drugs for the treatment of solid tumor. In the course of our screening for inhibitors of luciferase expression, which is regulated under the control of GRP78 promoter, by the treatment of tunicamycin, we isolated a novel compound designated as versipelostatin from Streptomyces versipellis 4083-SVS6 as a down-regulator of the grp78 gene. The structure of versipelostatin (C_<61>H_<94>O_<17>) was elucidated on the basis of spectral analyses including 2D NMR (HMQC, DQF-COSY, HMBC) to be a 17-membered macrocyclic compound consisting of an α-acyltetronic acid and sugar moieties. Although, α-acyltetronic acid derivatives such as kijanimicin, tetrocarcins, and teronothiodin were reported, the 17-membered macrocyclic skeleton involving the α-acyltetronic acid moiety in versipelostatin was the first example so far. In the evaluation system we employed, versipelostatin reduced this reporter gene expression at the IC_<50> value of 5μM. Versipelostatin exhibited limited cytotoxic effects against various cancer cell lines. Versipelostatin specifically inhibited the induction of endogenous GRP78 elicited by glucose starvation and 2-deoxyglucose (2-DG) treatment. Versipelostatin, glucose starvation and 2-DG treatment alone did not induce cell death in cancer cells. To the contrary, vigorous cell death was induced by the co-treatment of versipelostatin with glucose starvation or 2-DG in accordance to the decreased level of GRP78. Thus, it is expected that versipelostatin would be a promising cancer chemotherapeutic agent against solid tumor.
Treponemes are anaerobic bacteria with a typical helical shape that have been associated with the induction of human chronic diseases. Glycoconjugates distributed on the cell surfaces of some treponemes have been reported to be different from lipopolysaccharide (LPS). However, the chemical and immunobiological properties of the glycoconjugates have not been well characterized. In the present study, we separated a glycoconjugate fraction from Treponema medium ATCC 700293, a medium-sized oral spirochete, to examine the chemical structure and its immunobiological activities. The glycoconjugate was separated by phenol-water extraction, followed by hydrophobic chromatography and gel filtration. The chemical structure was shown by two-dimensional NMR and MALDI-TOF-MS to he a tetrasaccharide with two amino acids, [→4]β-D-GlcpNAc3NAcA(1→4)β-D-ManpNAc3NAOrn(1→3)β-D-GlcpNAc(1→3)α-D-Fucp4NAsp(1→), as a hydrophilic part and a lysophosphatidylglycerol derivative as a hydrophobic part. The glycoconjugate showed no immunostimulating activities, but we demonstrated that the glycoconjugate inhibited the LPS-induced immunostimulation. We also noted that the inhibitory effect was attributable to blocking the binding of LPS with LBP and CD14. Further, a phosphatidylglycerol derivative, which is a treponemal membrane phospholipid, was found to be a principle of the effect. These results suggest that the glycoconjugate and membrane phospholipid may be possible virulent factors of treponemes to evade the host defense system.
Among fungi, the genera Penicillin, Aspergillus, Trichoderma and Fusarium have appeared in a considerable number of reports as producers of not only antimicrobial agents but also a variety of bioactive compounds. We are attempting to develop resources for drug discovery by characterizing previously unknown fungi or even known fungi, which have been rarely reported to produce secondary metabolites. Fungal strain FKA-25 was isolated from a soil sample collected at Yakushima Island, Kagoshima Prefecture. FKA-25 was considered to belong to the genus Pseudobotrytis based on taxonomic characteristics. There have been some reports on this genus, but no reports on the secondary products. Sespendole was purified from EtOAc extracts of the 4 day-old culture broth of the strain by ODS chromatography and HPLC. The molecular formulae was determined to be C_<33>H_<45>O_4N by HR-EI-MS. The structure was elucidated by various NMR experiments. Sespendole possesses the indole-sesquiterpene core with two additional isoprenyl side chains. To our knowledge, sespendole is the first microbial metabolite having the indole-sesquiterpene skeleton. The relative stereochemistries of indole-sesquiterpene moiety are presumed to be 3S^*, 4R^*, 9S^* and 12S^* from the NOE experiments. The biosynthetic origin of sespendole was studied by feeding the kinds of sodium [^<13>C]acetates to the strain, indicating that the indole-sesquiterpene core was derived from a sesquiterpene and an indole ring. Sespendole inhibited the synthesis of cholesteryl ester and triacylglycerol by mouse macrophages with IC_<50> values of 4.0 and 3.2μM, respectively.
The harmful dinoflagellate Heterocapsa circularisquama has caused large-scale red tides in the coastal embayments of central and western Japan. Red tides of H. circularisquama have been responsible for mass mortalities of both natural and cultured bivalves such as pearl oyster, and short-necked clam and oyster. HML (Hydorxy Myristoyl Leucine), a novel compound was found as a promising agent through screening of 5,000 marine bacterial broth extractions. HML(1) showed crucial activity against H. circularisquama and Prorocentrum micans. On the other hand it showed less algicidal activity against the diatom, Skeletonema costaum or Cheatoceros decipiens which are the main foods for shellfishes. In order to enhancement of this selective algicidal effect, more than 40 analogues of HML(1) have been chemically synthesized. Nevertheless the algicidal mechanism of HML have not been clarified, some information about structure-activity correlation was obtained. Stereochemistries of HML(1) were determined to [3(S), 2(R)] by the comparison of chemically synthesized all four possible steroisomers. HML and its enantiomer analogue [3(S), 2'(R)] (2) have algicidal effect for dinofllagellate but not for diatom,. while [3(S), 2'(S)](3), [3(R), 2'(R)] or (4) analogue lacks of selective effect and have some algicidal acictity against diatom. Chain-length of HML is essential for the intense of activity, and longer or shorter analogue on fatty acid moiety, decrease the algicidal activity. Analogues of modified carboxylic acid, such as methyl ester or amide, drastically decreased the acitivity. Enhancement of acitiviy was achieved by the replacement of leucine with aspartic acid and this most potent synthetic analogue HMAsp 6) can terminate H. circularisquam at 0.3mg/L for 4 hours.
Several kinds of shrews produce anesthetic substances which they inject into the bodies of their prey, such as earthworms and insects. The paralyzed prey are then stored in their nest. Due to the interesting ecology of shrews, we sought to isolate these anesthetic substances. We studied the American short-tailed shrew Blarina brevicauda, which produces a potent venom. Despite its classification as an "Insectivore", it consumes not only insects and other invertebrates, but also vertebrates, including prey even larger than itself. Guided by acute toxicity against mice, the extract of its submaxillary gland was separated using column chromatography, and a lethal venom, blarina toxin, and its derivative, blarinasin, were isolated. Mice injected with blarina toxin showed characteristic symptoms, i.e., irregular and quickened respiration, paralysis of the hind limbs, and convulsions before death. The mature form of the primary amino acid sequences of blarina toxin (253 residues) and blarinasin (252 residues) were determined using partial amino acid sequence analysis and cDNA cloning, respectively. Both of these proteins were a type of secretory serine protease. Studies on posttranslational modification and the mode of action of blarina toxin are in progress. We also studied the long-clawed shrew Sorex unguiculatus, which inhabits Hokkaido. Guided by the N-type calcium channel activity, the aqueous extract of its submaxillary gland was subjected to separation using ultrafiltration and column chromatography. Further purification of the active fraction using reversed-phased HPLC is in progress.
The pigmentation of hippopotamus sweat after perspiration is observed. The colorless viscous sweat of the hippopotamus gradually turns red within a few minutes. In spite of the interesting and wonderful phenomenon, the responsible pigment had remained to be elucidated because of it instability. The red solution of the crude extracts of the sweat turns brown with decomposition of the pigment when the solution is subjected to the following conditions: violent acidification, addition of strong bases, standing for a long time at room temperature, addition of abundant organic solvent, and concentration to dryness. Therefore, special precaution to isolate the pigment was required. Gel filtration of the red solution through Sephadex G-15 and G-25 with H_2O indicated the presence of three pigments (orange, red, and brown) in the crude extracts. Then, to each orange and red solution was added an anion exchange resin QAE Sephadex A-25 (0.2M phosphate buffer, pH6.1), and the resin was quickly washed and eluted with NaCl / buffer to afford a comparably concentrated bright red and orange solutions. For NMR analyses, the samples were eluted with deuterated buffer solution. For MS analyses, the samples were eluted with TEA-HCO_2H buffer. In order to obtain further information on the structure, the unstable red pigment 1 was converted to a stable derivative through several steps. X-ray analysis of this derivative suggested that the structure of 1 should be the highly oxidized fluorene. The UV spectra of the red (1) and the orange (2) pigments suggested that they have the same chromophore. Taking these data into consideration, the structures of 1 and 2 were elucidated to be substituted fluorene diquinones. The tautomeric structures of these pigments were also studied by chemical synthesis and by the model studies. These studies will provide a useful information about the role of hippopotamus sweat and the coloring mechanism.
Chemotaxis of sperm toward eggs during fertilization is a crucial event for species conservation, particularly for animals living in aquatic environments. Relevant chemical attractants have been found from a few marine organisms such as sea urchins and corals. We previously showed that sperm of the ascidian Ciona intestinalis is activated and then attracted toward the egg by a factor released from the egg, which led to the purification of sperm-activating and attracting factor (SAAF) from the egg-conditioning medium of Ciona intestinalis. The structure of SAAF was proposed to be a novel polyhydroxysterol sulfate (1) on the basis of 2D-^1H NMR and FAB-MS/MS analysis using no more than approximately 4μg of a specimen. However, there still remained small but serious ambiguity that the biological activity might be attributed to a minor constituent. Synthesis is, therefore, essential for the unequivocal identification of 1 as the active principle, and for the complete structure elucidation including the stereochemistry at C25. SAAF and its C-25 epimer were synthesized from chenodeoxycholic acid (8) in 19 steps via the pivotal intermediate 6 and both the sperm-activating and attracting activity of them were bioassayed. Synthetic SAAF activated the sperms of ascidian Ciona intestinalis at 3.7nM and concurrently exhibited the attracting activity at <10nM, which led to the unambiguous structure determination of SAAF and the verification of its dual activity. It is noteworthy that 25-epi-SAAF turned out to possess the comparative activities as those of SAAF.
(-)-Galanthamine (1), an alkaloid of the Amaryllidaceae family, has been evaluated as a potent agent for the treatment of Alzheimer's disease. Because of the scarcity of natural supply, several syntheses of galanthamine have been reported using biomimetic phenolic oxidative coupling of a norbelladine derivative and recently via an asymmetric Heck reaction. Despite of these efforts, the yield of the pivotal coupling reaction in these syntheses still remains only moderate (〜50%). We accomplished an efficient total synthesis of (±)-galanthamine through the improved phenolic oxidative coupling reaction of norbelladine-type compound 1 having pyrogallol moiety. Namely, the coupling reaction of 1e using phenyliodine(III)bis(trifluoroacetate) (PIFA) gave the spiro-dienone 2e in high yield (85%), which was converted into (±)-galanthamine and (±)-narwedine. On the basis of the above synthetic method, we planned an asymmetric synthesis of (±)-galanthamine using optically active amino acid as a chiral auxiliary. Acidic cyclization of a D-phenylalanine derivative 11a with a benzaldehyde derivative 8a gave the imidazolidinone derivative 12a as a single diastereomer. The oxidative coupling of 12a with PIFA and subsequent debenzylation with boron trichloride gave the cyclic ether 13. In the above reaction, two chiral centers were created by the remote asymmetric induction based on conformational restriction of the chiral imidazolidinone ring. The cyclic ether 13 was effectively converted into (-)-galanthamine. Buflavine, isolated from boophane flava (Amaryllidaceae), has been shown to exhibit strong α-adrenolytic and anti-serotonine activities. We accomplished a total synthesis of buflavine through the above coupling reaction of norbelladine derivative 16 and a dienone-phenol rearrangement of the spiro-dienone 17. We also achieved a total synthesis of (±)-oxocrinine, isolated from Crinum americanum (Amaryllidaceae), through the above coupling reaction of norbelladine derivative 20 and the Michael addition of amino group to the dienone moiety of 21.
Recently, an increasing number of structurally and bioactively unique 3-alkylpyridine alkaloids have been isolated from marine sponges of several genera. In 1999, Kobayashi and co-workers reported the isolation of a structurally novel cis-cyclopent[c]isoxazolidine alkaloid, pyrinodemin A, from the marine sponge Amphimedon sp. Pyrinodemin A shows potent cytotoxicity against murine leukemia L1210 (IC_<50>= 0.058μg/mL) and KB epidermoid carcinoma cells (IC_<50>=0.5μg/mL) in vitro. Although the plane structure and relative configuration of pyrinodemin A were primarily proposed as 1, the absolute configuration has not yet been determined. In this symposium, we report the racemic synthesis of possible structures 2-4 for pyrinodemin A and support the C14'-C15' double-bond positional isomer 3, which Baldwin's group proposed, as the correct structure (Scheme 1). Furthermore, we report the first enantioselective total synthesis of (-)-pyrinodemin A (3) through a highly diastereoselective intramolecular 1,3-dipolar cycloaddition reaction as the key step and the determination of its absolute configuration (Scheme 2). Two novel cytotoxic alkaloids haliclamines A (28) and B (29), isolated from a marine sponge of the genus Haliclona by Fusetani et al. in 1989, are most closely related to the key bisdihydropyridine intermediate 27 of the biogenetically unique manzamine family. We report the first total syntheses of 28 and 29 through a convergent and expeditious assembly of 3-substituted pyridine derivatives with different alkyl chains to the bispyridinium macrocycle as a general approach to the key bisdihydropyridine intermediate 27 (Schemes 3-5). In the course of our synthetic studies on macrocyclic marine alkaloids 28 and 29, it was determined that MsCl resulted in the deoxygenation of pyridine N-oxides 53 and 60 under mesylation conditions (MsCl, Et_3N). The problem is the reaction mechanism of these unusual deoxygenations. After many unsuccessful experiments, products, which are very significant for deducing the reaction mechanism, have been trapped. The treatment of N-oxide 64 with BnSO_2Cl and Et_3N in the presence of alkenes afforded a γ-sultone along with deoxygenated 2,4,6-trimethylpyridine (Scheme 6). Here we suggest the possibility of the generation and/or intermediacy of α-sultones as new reactive intermediates for the first time.
All possible stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin D_3 (4a-d) were designed and convergently synthesized, in view of the important effects of the 2α-methyl substitution on the A-ring of the natural hormone, 1α,25-dihydroxyvitamin D_3 (1). Nine-step conversion of methyl hydroxypivalate (8) provided the desired A-ring enyne synthon (15a,b) in good overall yield. Palladium-catalyzed cross-coupling reaction of the A-ring synthon (15a,b) with the CD-ring portion, followed by deprotection, gave the 2,2-dimethyl analogues (4a-d). The stereochemistry of the C1 and C3 positions was determined by the modified Mosher's method using their bis-MTPA esters. Applying the above procedures, we also synthesized 2,2-ethano-, 2,2-propano- and 2,2-butano-1,25-dihydroxyvitamin D_3 (5-7), as novel spiro-ring analogues having cyclopropane, cyclobutane and cyclopentane fused at the C2 position, respectively. Biological potencies of the synthesized compounds were assessed in terms of vitamin D receptor (VDR) binding affinity, as well as the HL-60 cell differentiation-inducing activity. The 2,2-ethano analogue (5a) showed a comparable activity to the natural hormone (1), while the 2,2-dimethyl analogue (4a) proved to be a weak binder to VDR. These features were explained, in part, by the docking studies based on the X-ray crystal structure of VDR complexed with 1.
Spiroxins A-E (1a-e), isolated from a marine-derived fungal strain LL37H248, have an unique bisnaphthospiroketal octacyclic ring system. Spiroxin A (1a), which is a major component produced from the culture medium, was reported to show not only antibacterial activity against Gram-positive and-negative bacteria, but also antitumor activity against ovarian carcinoma in nude mice, which was caused by its single-stranded DNA cleavage activity. However details of the mechanism are not clear. We report here the first total synthesis of (±)-spiroxin C (1c), in which a TBAF-actvated Suzuki-Miyaura cross-coupling reaction and an intramolecular bromoetherification reaction are included as key reactions. We planned that the basic spiroketal structure was constructed after fomation of a binaphthyl structure, which was expected to be obtained by a Pd(0)-catalyzed cross-coupling reaction (Scheme 1). Organoboronate (4) and enol triflate (5) were prepared from naphthol (6) and tetralone (7), respectively (Scheme 2). After several attempts, a key compound, binaphthyl (3), was found to be successfully obtained from these compounds by a TBAF-activated Suzuki-Miyaura cross-coupling reaction (Scheme 3). As shown in Scheme 4, the basic spiroketal structure of spiroxins was constructed via an intramolecular bromoetheration of 17 employing 2,4,4,6-tetrabromocyclohexa-2,5-dienone (18). Transformations involving a stereospecific epoxidation and a benzilic oxidation afforded spiroxin C.
TAN-1085 (1) is an antibiotic of Streptomyces origin, which has attracted considerable synthetic interest due to the unique structure, a curved tetracyclic core glycosylated with a rhodinose, and important biological activities. These structual features as well as the significant biological activities led us to undertake the synthesis of this class of compounds. We report herein the first total synthesis of 1. Although initial study for the key thermal reaction with 2 occurred only the [1,7]-shift of allylic hydrogen, thereby giving enol ether 4. However, we discovered that compound 18 could be directly converted to naphthodialdehyde 19 under the Swern oxidation conditions, where the ring opening of benzocyclobutane and subsequent 6π-closure occurred at room temperature. Stereoselective pinacol cyclization by using Sml_2, followed by direct quenching of the resulting pinacolization product with benzoyl chloride exclusively gave C(5)-benzoate 21 in good yield. Glycosylation of 22 was conducted by the reaction with glycosyl donor 23 in the presence of BF_3・OEt_2, giving an 1:1 mixture of 24 and 25. Although stereochemistries of aglycon part of these were not determined, each of these was converted to the final product. One of these was identified as the natural form by comparison with authentic specimen 1. In conclusion, the first synthesis of TAN-1085 (1) was achieved. Currently we are studying the asymmetric synthesis of 1 without resort to diastereomer separation.
Phosphorylated phoshoinositides(PI Pn) and inositol phosphates(IPn) play pivotal roles as second messengers in intracellular signal transduction. D- and L-myo-inositol 3,4,5,6-tetrakisphosphate (D-IP4, and L-IP4) and phosphatidylinositol 3,5-bisphosphate(PI 3,5-P2) were recently identified as novel second messengers. 1. Total synthesis of D- and L-IP4 from D-glucose The 1,2-addition of vinyl copper reagent to the chiral aldehyde derivative from D-glucose gave the desired allyl alcohol as the sole diastereomer. Protection of hydroxyl group, acid hydrolysis, Wittig methylenation and ring-closing metathesis(RCM) gave the conduritol B derivatives(3). The catalytic OsO_4 dihydroxylation of C_2 symmetric conduritol B derivative(4) gave the single diastereomer which was successfully converted to D-IP4 by established amidite procedure. On the other hand, L-IP4 was stereoselectively synthesized by OsO_4 catalyzed diastereoselective dihydroxylation of non-C_2 symmetric conduritol B derivatives(6j). Oxidation of allylic benzoate gave the desired cis-diol with high diastereofacial selectivity, and 7 was converted to L-IP4. 2. Synthesis of PI 3,5-P2 By catalytic OsO_4 oxidation of non-C2 symmetric conduritol B derivatives(6c), we synthesized desired key intermediate of PI 3,5-P2. Finally, this diol was successfully converted to PI 3,5-P2 by established amidite procedure. 3. Enantioselective desymmetrization of meso-1,2,3-Triol Recently, Matsumura and co-workers reported the CuCl_2-Ph_2box catalyzed asymmetric acylation of racemic and meso-1,2-diols. We could succeed the highly enatioselective one-pot desymmetrization of meso-1,2,3-triol derivatives of myo-inositol by asymmetric acylation followed by successive kinetic resolution catalyzed by CuCl_2-chiral diamine complexes. When 18c was used as the chiral diamine, benzoylation of 14 gave D-15 in 81% and 100%ee, respectively. Kinetic resolution of DL-15 under the same reaction condition gave the less reactive D-enantiomer in 45.3% (90.6% theoretical) and 99.4%ee, respectively. 4. Improved synthesis of PI 3,5-P2 by enantioselective desymmetization Enantioselective desymmetrization of meso-myo-inositol derivative(19) under the same condition provided the benzoate (20) in 81% 100%ee. The synthetic intermediate of our previous total synthesis of PI 3,5-P2.
Macrosphelide A and B, isolated from the culture medium of macrospaeropsis sp. FO-5050 by the Omura group, have been shown to strongly inhibit the adhesion of human leukemia HL-60 cells to human-umbilical-vein endothelial cells (HUVEC) in a dose-dependent fashion. Consequently, macrosphelides have received much attention as a lead compound for the development of new anti-cancer drugs and hence, a synthetic method for producing those molecules and their analogues is urgently required. We wish to report a highly convergent synthesis of a library of macrosphelide analogues on a solid-support utilizing a palladium-catalyzed chemoselective carbonylation of vinyl halides. In considering an efficient strategy for the combinatorial synthesis of macrosphelide analogues, we chose a solid-phase synthesis utilizing the three synthetic building blocks A, B, and C as illustrated in Scheme 1. The process involves: (i) attachment of the secondary alcohol in building block A to solid-support, (ii) esterification with building block B, (iii) chemoselective carbonylation of the vinyl iodide in unit A with alcohol C (iv) carbonylative macrolactonization utilizing the rather less reactive vinyl bromide on solid-support, (v) cleavage from the polymer-support. The synthesis of a macrosphelide library was accomplished by split & pool method by means of radiofrequency encoded combinatorial chemistry. The 128 IRORI MicroKans, each containing ca. 30mg of PS-DHP resin were split into individual vessels, according to radiofrequency signals. After attachment of building block A with PPTS, MicroKans were pooled together for deprotection of the TBS group. These microreactors were decoded and split, followed by esterification with building block B to afford the polymer-bound ester. Further sorting, followed by carbonylative esterification of vinyl iodides with building block C to provide the polymer-bound diester. The microreactors were again pooled for the deprotection of the MPM group with DDQ and the carbonylative macrolactonization utilizing Pd_2(dba)_3/dppf as a catalyst to provide 16-membered lactone on solid-support. Finally, the microreactors were divided and treated with 4N-HCl in parallel for removal of the MEM group and cleavage for the polymer-support. Purification by automated preparative HPLC provided 122-member macrosphelide analogues among 128 trials.
Intensive efforts have been recently aimed at the synthesis of oligosaccharides because of their numerous important biological functions. Because oligosaccharides consist of several monosaccharides by means of anomeric C-O bond linkage, the synthesis would necessarily require iterative glycosylation. While various new glycosylation methods have been developed recently to increase the efficiency for the iterative sequences, only a few methods are available to assemble oligosaccharides under one set of glycosylation conditions using a single anomeric substituent for both glycosyl donors and acceptors. We report here two examples of the iterative glycosylation using seleno- or thioglycosides as both glycosyl donors and acceptors. These methods relied on the generation of glycosyl cations or their equivalents from seleno- or thioglycosides and subsequent coupling with seleno- or thioglycosides possessing free hydroxyl groups, respectively. In both case, the choice of counter anion of the "glycosyl cation" is critical for effective glycosyl coupling reaction. We found that selenoglycosides could be selectively converted to the corresponding β-bromoglycosides, which undergo the coupling reaction with selenoglycosides bearing hydroxyl group. Since the products also possess the arylselenyl group as the anomeric substituent, they can be used for the next glycosylation reaction. We also found that thioglycosides could be converted to the corresponding glycosyl triflate and trifluimide intermediates, and that the coupling reaction of the intermediates with thioglycosides bearing hydroxyl group afforded desired thioglycosides. We also initiated the study on construction of oligoglucosamines library based on the iterative glycosylation.
Bacterial cell wall peptidoglycan (PGN) has been well-known as a strong immunopotentiator. We previously demonstrated that the minimum structure required for the immunostimulation is N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide: MDP) but the activity of MDP recently proved to be not identical with that of PGN. Partial structures of PGN were hence synthesized for precise biological study. A key disaccharide glucosaminyl-β(1→4)-muramic acid was prepared by stereoselective glycosylation of an N-Troc muramic acid acceptor with N-Troc-glucosaminyl trichloroacetimidate. The disaccharide was converted to both disaccharide acceptor and donor, which were then coupled together by the same glycosylation method to give a tetrasaccharide. Octasaccharide was also obtained in a similar manner. Introduction of the dipeptide moiety to the 3-O-lactyl groups followed by deprotection afforded the peptidoglycan tetrasaccharide and octasaccahride fragments. Toll like receptor 2 (TLR2) was described as the receptor for PGN but recent studies have shown that MDP acts via a TLR2 independent pathway. The above synthetic fragments also showed TNF-α inducing activity via TLR2-independent pathway. Recently, we found that intracellular protein NOD2 is a cellular receptor for MDP. The above partial structures containing tetra- and octasaccharide also showed NOD2 dependent activity, indicating NOD2 is an intracellular receptor for PGN. We also showed that NOD1 recognized a dipeptide γ-D-glutamyl-meso-diaminopimelic acid, which is a partial structure typical of Gram-negative bacterial PGN.
The nuclear receptors liver X receptor (LXR) α and LXRβ serve as oxysterol receptors and regulate the expression of genes involved in lipid metabolism. LXR activation induces the expression of ATP-binding cassette (ABC) transporters, such as ABCG5 and ABCG8, which inhibit intestinal absorption of cholesterol and phytosterols. Although several synthetic LXR agonists have been generated, these compounds have limited clinical application because they cause hypertriglycemia by inducing the expression of lipogenic genes in the liver. We synthesized derivatives of phytosterols and found some of them to act as LXR agonists. Among them, YT-32 [(22E)-ergost-22-ene-1α,3β-diol], which is related to ergosterol and brassicasterol, is the most potent LXR agonist. YT-32 directly bound to LXRα and LXRβ and induced the interaction of LXRα with cofactors, such as SRC-1, as effectively as the natural ligands, 22(R)-hydroxycholesterol and 24(S),25-epoxycholesterol. YT-32 is a sterol compound with a saturated cholesterol structure and the same side chain as ergosterol and brassicasterol. YT-33, which differs from YT-32 only in the absence of the 1α-hydroxyl group, completely lacks LXRα agonist activity. To further examine the structure-function relationship between YT-32 and LXRα, we synthesized several YT-32 derivatives containing the 1α-hydroxyl group. The data indicate that the 1α-hydroxyl group and saturated ring structure are important for LXRα and LXRβ activation. These structures are also important for induction of the LXR target gene in intestinal cells. YT-32 selectively activated intestinal ABC transporters in mice and inhibited intestinal cholesterol absorption without increasing plasma triglyceride levels. The phytosterol-derived LXR agonist YT-32 might selectively modulate intestinal cholesterol metabolism.