Mycothiazole (1) was isolated from Spongia mycofijiensis collected from Vanuatu by Crews et al. and it exhibited anthelmintic activity (in vitro) while high toxicity was observed in mice. The most unique feature of this structure is a thiazole ring which is imbedded between two acyclic polyketide chains. Furthermore the side chains contain nonconjugated and conjugated diene systems. Although 1 has only one stereogenic center on the secondary alcohol, its absolute configuration has been unknown. According to our synthetic strategy, we started to synthesize the left fragment (3) which contains a thiazole ring and a nonconjugated diene system. In order to synthesize a thiazole ring, we attempted the Hantzch method via the thioamide 8. While this procedure was unsuccessful, the thiazole moiety 12 could be obtained by our method, CMD (Chemical Manganese Dioxide) oxidation procedure from the corresponding thiazolidine 11 in one step. In this oxidation, activation of CMD by azeotropic dehydration with benzene was needed to prevent hydrolysis of 11. The skipped diene was constructed by using the Stine cross coupling two times. The coupling of 15 with the tin compound 18 gave 19 in good yield, and the bromination of the primary alcohol in 19 followed by coupling with vinyl tributyltin furnished the nonconjugated diene. As part of synthesis of the right fragment (3), the addition of hydrogen iodine to the alkyne 21 gave the vinyl iodide 22, which was coupled with propargyl alcohol by the Sonogashira coupling to give 23. However, the attempt of semi-hydrogenation of the alkyne which conjugated with exo-olefin failed. Thus we changed the plan that the exo-olefin would be introduced by the Wittig type reaction. The Nozaki-Hiyama-Kishi coupling of the vinyl iodide 29 with the aldehyde 31 gave 32, and the oxidation of the allyl alcohol in 32 afforded the α,β-unsaturated ketone 33. Construction of exo-methylene and further synthetic studies toward total synthesis of mycothiazole (1) are actively in progress.
Tetrodotoxin (TTX, 1) is a well-known marine natural product acting as toxic principle of puffer fish poisoning and has been used as a specific blocker of Na-channel proteins. However, the biosynthesis, the mechanisms of bioaccumulation, the details of bound structure to Na-channel protein, etc, are remained to uncover. In order to solve such problems under molecular level, we have studied syntheses of TTX and its analogs as enantiomerically pure form. Herein we present a highly stereocontrolled synthesis of (-)-5,11-dideoxyTTX (2). The synthesis started from the Diels-Alder reaction between 9 derived from levoglucosenone (11) as a chiral starting material and isoprene (10) to construct cyclohexane ring, which was transformed to exo-allylic alcohol 7 in 5 steps. The amino functionality was introduced by the Overman rearrangement of 7 to give trichloroacetarnide 6 which has all of the carbon atoms of TTX skeleton. Hydroxylation at C-8 position of the key intermediate 6 was achieved using a neighboring group participation of the trichloroacetarnide. Inversion of the resulting hydroxy group, stereoselective epoxidation and ozonolysis of the vinyl group yielded an aldehyde 17. Stereoselective addition of magnesium acetylide to the aldehyde 17, followed by oxidative cleavage of the acetylene group with RuO_4 gave the lactone 5. Construction of guanidine function from trichloroacetamide of 5 by our new procedure resulted in failure because of the labile C-9 acetoxy group. In order to avoid such problems, we considered an urea compound 21 as a candidate of precursor for guanyl formation. The urea 21 was dehydrated to carbodiimide 22, which reacted with benzylamine to afford dibenzylguanidine 23. Finally deprotection of benzyl groups, acetyl groups and acetal furnished the 5,11-dideoxyTTX (2) along with its isomers (25, 26). This studies presents the first asymmetric synthesis of TTX analogs and provides a practical route accessible to the labeled compounds for biochemical studies. Further studies toward naturally occurring TTX (1) and other analogs are in progress in the laboratory.
(-)-Dysiherbaine was isolated from a Micronesian sponge Dysidea herbacea after screening for substances that exhibit potent neurotoxic activity. This amino acid was found to be a selective agonist of non-NMDA type glutamate receptors in CNS. Although the relative structure was determined to be a novel di-amino di-carboxylic acid having a densely substituted pyran ring fused with 2-amino-3-(2'-carboxy-5'-tetrahydrofuranyl)propanoic acid, its absolute structure remains unsolved. (+)-Lycoperdic acid, isolated from the mashroom Lycoperdon perlatum, is the 5'-oxo analogue of 2-amino-3-(2'-carboxy-5'-tetrahydrofuranyl)propanoic acid which is the core structure of dysiherbaine. We describe here a novel enntiocontrolled synthesis of (+)-lycoperdic acid and the first total synthesis of (-)-dysiherbaine based on the strategy involving four major transformations; (i) palladium catalyzed cross-coupling reaction of the organozinc reagent, prepared from a β-iodo alanine derivative, with an alkenyl iodide or triflate, (ii) diastereoselective epoxidation, (iii) acid catalyzed cyclization, (iv) oxidation. The present synthesis of (-)-dysiherbaine allows us to conclude that the absolute configuration of natural dysiherbaine is (2S,4R,6R,7R,8R,9S).
Pinnatoxin A (1) was isolated in 1995 by Uemura and co-workers from the Pinna muricata shellfish. Pinnatoxin A is one of the major toxic principles responsible for outbreaks of Pinna shellfish intoxication in China and Japan. It is also believed to be a calcium ion channel activator. The Uemura group determined pinnatoxin's structure and relative stereochemistry using a variety of 2D NMR techniques; however, the absolute stereochemistry was not assigned. They also proposed a biosynthetic route of 1 (2→1), i. e., an intramolecular Diels-Alder reaction, to construct the G-ring as well as the macrocycle followed by imine formation to establish the 6,7-spiro-ring system or vice versa. Since we were interested in this unique biogenetic pathway, we planned to synthesize 1 based on Uemura's proposal with the intramolecular Diels-Alder reaction as a key step. The requisite diene 18 for the intramolecular Diels-Alder reaction was prepared via a dithiane-based coupling of bis-spiroketal 3 with iodide 4 to form the C.25-C.26 bond and sequential Ni(II)/Cr(II)-mediated couplings between vinyliodides 5 and 6 with suitable advanced C.6 and C.32 aldehydes. During the synthesis of 18, we encountered difficulties of epimerization at the C.19 bis-spiroketal stereocenter during formation of the EF bicyclic ring system with acid treatment (15→16). Fortunately, the C.19 stereocenter could be epimerized back to the desired configuration under silylation conditions (16→17). With the diene 18 in hand, we next attempted the intramolecular Diels-Alder reaction. Heating the diene 18 in toluene at 70℃ for 24h gave a 1:1:1 mixture of three products (19a-19c), including the desired exo adduct 19a, out of the eight possible intramolecular Diels-Alder products, which were separated by HPLC. Changing the solvent from toluene into dodecane gave 19a as the major adduct. After deprotection of the TBS group and Alloc group of 19a, imine formation was employed with heating at 200℃ under high vacuum for 1h to give 21 in 70% yield. Finally, the t-butyl ester was cleaved with TFA to furnish synthetic pinnatoxin A (1), identical in all respects to natural pinnatoxin A except for the sign of optical rotation and biological activities. From this information, we concluded that the absolute stereochemistry of natural pinnatoxin A was the antipode of structure (-)-1. Thus, we employed the synthesis of the natural form of (+)-pinnatoxin A (1) in an almost similar way and, at last, synthetic (+)-1 was completely in accord with natural pinnatoxin A.
Dysidiolide (1), a novel sesterterpenoid isolated from the Caribbean sponge Dysidia etheria de Laubenfels, exhibits inhibition of the protein phosphatase cdc25A. Dysidiolide possesses a unique carbon skeleton with structural features that find no precedent in nature. Thus its unique structure and biological activity prompted us to undertake the total synthesis of dysidiolide. Recently, the total synthesis of dysidiolide has been reported by three groups. Herein, we wish to report a novel total synthesis of dysidiolide using intramolecular Diels-Alder reaction as a key step. Cyclohexenone 2 was converted to α,β,γ,δ-unsaturated ketone 4 via enone 3. Conjugate addition of thiophenol to enone 4 and subsequent DIBAL-H reduction afforded alcohols 8a and 8b (8a:8b=1.6:1). Compound 8a was then converted to sulfoxidoester 10 by mCPBA oxidation and esterification with propiolic acid. Compound 10 was also derived from alcohol 8b by mCPBA oxidation and Mitsunobu reaction with propiolic acid. Sulfoxidoester 10 was refluxed in toluene to afford decalin 6 with elimination of sulfoxide and intramolecular Diels-Alder reaction. Decalin 6 was converted to lactone 13 having requisite four stereocenters at C-6, 7, 11 and 15 via 11 a and 12. Lactone 13 was reduced with DIBAL-H followed by LiBH_4 to give diol 14. Deoxyganation of compound 14 at C-12 and 24 gave alcohol 18 via 15, 16 and 17. Alcohol 18 was converted to iodide 19, followed by cross-coupling with isopropenyllithium in the presence of CuI and removal of THP group delivered alcohol 21. Alcohol 21 was oxidized with Dess-Martin periodinate to give aldehyde. Treatment of the aldehyde with 3-lithiofuran gave a mixture of alcohols 22a and 22b (22a:22b=1:1). After separation of the mixture, photochemical oxidation of 22a furnished (±)-dysidiolide.
From chemical investigations of marine microorganisms as new source of bioactive substances, amphidinolide B was isolated from the cultured marine dinofragellate Amphidinium. In addition to the interesting 26-membered macrolide structure, powerful antitumor activities were shown against L1210 and KB cell. Because of low isolation yields from the natural origin, detailed bioassay has not been carried out. Accordingly, total synthesis was investigated to supply large quantity of samples to bioassay, and to understand structure-activity relationship of this molecule. According to the convergent synthetic strategy, amphidinolide B (1) was divided to the bottom-half fragment (C1-C13) 2 and top-half fragment (C14-C26) 3. Synthesis of the bottom-half fragment 2 was started from dimethy L-tartrate. After introduction of the oxazolidinone chiral auxiliary by employing our own reagent, introduction of the methyl group of the C-11 position was achieved by the Evans protocol. Continuously, Johnson Claisen rearrangement of the corresponding allyl alcohol, effected the desired chain homologation, and final transformation toward the bottom-half fragment was succeed by the Wittig reaction. On the other hand, the top-half fragment 3 was obtained by coupling of the aldehyde prepared by the Shioiri method with fragment obtained from L-malic acid. Additionally, the diene moiety ascribed to C13-C15 was efficiently synthesized by coupling of the aldehyde with the corresponding acetylide, followed by methylation and the Wittig reaction. The allylic epoxide was also constructed by employing the Sharpless protocol via a halohydrine In conclusion, the syntheses of the two fragments of amphidinolide B, and their suitable coupling were accomplished, and construction of the allylic epoxide was demonstrated.
Ciguatera is a widespread human scale food poisoning that is caused by fishes dwelling in coral reefs in the tropics and subtropics. Ciguatoxis (CTX1B, 1 and CTX3C, 2), regarded as the principal toxins of ciguatera, were isolated from the moray eel, Gymnothorax javanicus, and the absolute configuration of 1 was determined as shown in Figure 1. Synthesis of ciguatoxins has recieved considarable attention among synthetic chemists due to the striking feature and biological features of these toxins. We developed a new method useful for constructing polycyclic ether systems based on alkylation and ring-closing metathesis reaction, and successfully synthesized the ABCDE ring moieties of ciguatoxin using this technique strating with the iodide 4 and ester 5 corresponding to the AB and E ring, respectively. Alternatively, we synthesized the IJKLM ring fragment via successive esterification of the alcohol 23 and carboxylic acid 24, Tebbe olefination, and ring-closing metathesis sequences, and the chemo- and stereoselective reduction of the methyl acetal. Studies toward total synthesis of ciguatoxin are currently underway.
Tetrahydroisoquinoline alkaloid is a class of biologically potent compounds. The existence of 1,2,3,4-tetrahydroisoquinoline (4: TIQ) and 1-methyl-TIQ (6: 1MeTIQ) in the human brain is now in no doubt. The complete prevention of MPTP (1) induced parkinsonism by pretreatment with 6 and inhibition of monoamine oxidase have renewed interest related to the pathogenesis of Parkinson's disease and have been suggested as a leading compound for development of medicine. 1-Phenyl-TIQ (5: 1PhTIQ), originally developed as a general anesthetic agent, also shows phencyclidine-like stereotyped behavior and ataxia. Since the enantiomers were observed to vary in affinity, the synthesis of chiral 1-substituted TIQ is the current focus in medicinal organic chemistry. We report here a novel asymmetric synthetic methodology involving an asymmetric addition of organolithium to an imine and subsequent cyclization by Moffat oxidation as the key steps. In the chiral ligand mediated asymmetric addition of organolithium to imine, a naphthalene unit was developed as a N-substituent of imine to realize high abilities in activation of imine, enantioselectivity, and oxidative dearylation. In the presence of 13, the reaction of methyllithium with the imine (24) bearing N-naphthyl group gave the corresponding amine (25) in up to 97%ee and 99% yield. Conversion of the resulted chiral amine to 1-substituted TIQ was readily achieved through cyclization by Moffat oxidation as a key step. Addition of methyllithium to imine (29) in the presence of 13 gave 30 in 93%ee and 99% yield. Subsequent hydroboration and oxidative work up gave 31 in 76% yield, and following Moffat oxidation afforded TIQ (32) in 75% yield. Dearylation was done by ammonium cerium(IV) nitrate oxidation to give 33 in 94% yield, and finally deacetylation with KOH-hydrazine gave salsolidine (3) in 69% yield. Combination of enantioselective alkylation of imine with the cyclization reaction realized the asymmetric synthesis of potentially biologically active 1-substituted TIQ (3, 6, 7).
Interleukin 6 (IL-6) is a multifunctional cytokine that act on immune response, hematopoiesis and the nervous system. Thus, IL-6 plays one of the central regulatory roles in host defense mechanisms. However, it has been demonstrated that IL-6 produced by tumor cells causes cancer cachexia, and also stimulates the proliferation of tumor cells in an autocrine/paracrine manner. Therefore, it may be possible that the inhibition of IL-6 activity relieves cancer cachexia and suppress the growth of IL-6 dependent tumor cells. In the course of a search for inhibitors of IL-6 activity obtained from microoragnisms, madindoline A (MDL-A) and B (MDL-B) were discovered from the fermentation broth of Streptomyces nitrosporeus K93-0711. We describe here the determination of the complete absolute stereochemistries of MDL-A and B and the first total syntheses of these materials. As our point of departure, asymmetric aldol reaction of oxazolidinone 5 with acrolein followed by methanolysis afforded β-hydroxyester 7. Stereoselective aldol reaction of 7 with methacrolein furnished diolefin 8. Ring closing metathesis of 8 afforded cyclopentene 10. Selective silylation of 10 with TBSOTf followed by oxidation provided the α,β-unsaturated ketone 12. Michael addition of 12 with n-Bu_2CuLi, phenylselenylation, oxidative elimination, and isomerization of double bond furnished ketone 14. Selective reduction of 14 followed by silylation and DIBAL reduction afforded 15. Dess-Martin oxidation of 15 followed by reductive N-alkylation with indoline 17 furnished 18. Deprotection of 18, selective silylation, and MnO_2 oxidation followed by acid hydrolysis afforded indole 19. Finally asymmetric oxidative ring closing of 19 afforded 1 and 2. The synthetic 1 was identical in all respects with a sample of the natural MDL-A. Moreover fourtunately single-crystal X-ray diffraction has now been employed to elucidate the abusolute stereochemistry of 1. The first total synthesis of MDL-A has thus been achieved via a highly convergent, efficient strategy.
The indole-diterpene tremorgens comprise a rapidly growing family of environmental toxins. The two major structural subgroups contain either unsubstituted benzene rings, as in paspaline (1), paspalicine (2), and paspalinine (3), or complex indole systems, exemplified by the lolitrems (4,5) and the penitrems (7-12). The penitrems are the most complex members of this increasingly important class of natural products. Aside from their novel architecture, our interest in the indole tremorgens stems from their biological activities. Members of this family of toxins have been implicated in a wide range of neurological disorders. Our long-standing interest in these compounds has already led to the first total syntheses of paspaline (1), paspalicine (2), and paspalinine (3). We describe herein the first total synthesis of penitrem D (10), the simplest member of the architecturally complex penitrem subfamily. The first-generation approach to penitrem D (10) featured a one-pot cyclization reaction of key intermediate 24, which was prepared from toluidine 17 and lactone 18. The key cyclization reaction of 24 proceeded to give a single product. Unfortunately, the product was found to have the undesired stereochemistry at the newly formed C28 stereocenter. We then focused on a second-generation strategy, in which coupling of aniline 37 with lactone 38 was planned for the latter stages of the synthesis. The synthesis proceeded via 17 step conversion of lactone 18 to 38. Completion of the synthetic scheme involved a 9 step transformation of 38 to penitrem D (10). The synthetic sample proved identical to the natural sample based on ^1H-NMR and TLC analyses.
The indole nucleus is present in a wide range of natural products, and the synthesis of this important structure has been a steady topic of interest for many years. Herein we report a novel indole synthesis that is carried out under mild radical cyclization conditions. Tin-mediated radical cyclization of 2-alkenylthioanilides at room temperature using triethylborane as radical initiator takes place smoothly to furnish 2,3-disubstituted indoles in good to excellent yields. The indole formation reaction proved to be compatible with both acid and base sensitive substituents including esters, THP ethers, and a β-lactam. We also explored tin-free reaction conditions, and found that the indole formation reaction proceeds smoothly using excess AIBN and hypophosphorous acid in n-PrOH in the presence of triethylamine. In addition, modular synthesis of the necessary thioamide precursors was developed to give a wide range of functionality at the 2- and 3-positions on the indole skeleton, as well as at the 5- and 6-positions. 2-Indolylethanol derivatives, potentially useful building blocks for the construction of many indole-containing natural products, were easily synthesized by this method via a ring-opening reaction of quinolines. The methodology was by no means limited to these structures. Simple 2,3-dialkyl indoles were also synthesized easily by means of Sonogashira-coupling of 2-iodoaniline with terminal acetylenes and subsequent partial reduction by activated zinc. Finally, the reaction was applied to synthesis of an optically active β-carboline derivative and the indole alkaloid, catharanthine.
Manzamine alkaloids constitute a novel family of several marine sponge metabolites that exhibit significant cytotoxic activity against leukemia and antibiotic activity. The unprecedented structures of these highly functionalized heterocyclic ring system and remarkable biological properties have attracted much attention as a challenging synthetic targets. While the simplest congener manzamine C (3) and related compounds have been previously synthesized by us and Langlois' group, the more complex manzamine A (1) has been more challenging target. Quite recently, Winkler, Martin and their coworkers have succeeded in total synthesis of manzamine A and its related compounds. We have also been interested in developing efficient routes to tetraazacyclic intermediate 5 based on the initial construction of tricyclic intermediate 20 by an intermolecular Diels-Alder reaction of functionalized dihydropyridinone 10 as a dienophile with siloxydiene, leading to the construction of a cis relationship in the central AB ring system of this unique structure. Using this strategy, we could synthesize advanced key intermediates 30 and 36. Details of the synthsis and further conversion to ircinal A (4) and manzamine A will be discussed.
FR901464 1, FR901463 2, and FR901465 3 are new antitumor substances which were isolated from a culture broth of a bacterium of Pseudonionas sp. No. 2663. These compounds show transcriptional regulating activity and induce characteristic G1 and G2/M phase arrest in the cell cycle. Related to these activities, they show potent antitumor effect. Especially, FR901464 1 exhibits the most prominent antitumor activity among the three compounds. The unique structure as well as the significant biological activities prompted us to undertake the synthesis of this class of compound. Herein, we describe a stereoselective synthetic approach to 1. The retrosynthetic analysis is presented in Scheme. 1. Our analysis divided 1 into three segments, the carboxylic acid 4, the sulfone 5, and the aldehyde 6. We thought that 4, 5, and 6 would be obtained from commercially available compounds respectively, and the target molecule 1 should be synthesized via convergent route by condensations of these segments. According to this synthetic plan, we carried out our study. The acid 4 was prepared from a lactate 8 using the reported method. N-Boc-L-threonine 11 was used as the starting material to synthesize 5. The Garner aldehyde 14 was prepared from 11. Its side chain was elongated by the Wittig reaction, and was led to a lactone 10 by acid catalyzed ring closure. Treatment with Lawesson's reagent and the following Wittig reaction gave the olefin isomers, 17 and 18. After the base catalyzed deconjugation of olefin, catalytic hydrogenation gave the tetrahydropyran with the desired all-cis stereochemistry as a sole product, and the successive DIBAL reduction afforded the aldehyde 19, which was converted into the sulfone 5 through 4 steps. The aldehyde 6 was synthesized from 2-deoxy-D-glucose 13. First, 4,6-dihydroxyl groups of 13 were protected as benzylidene acetal. Selective oxidation of hemiacetal with bromine, followed by the protection of the remaining free hydroxyl group gave the lactone 12. Methylation and acetalization followed by deprotection of benzylidene group afforded diol 23. It was converted into aldehyde 6 via protection-deprotection process and Dess-Martin oxidation. Having completed the synthesis of the three segments 4, 5 and 6, we next examined the coupling processes. Condensation of 4 with 5 was achieved by using HBTU to give the amide 26. Using Julia olefination method, coupling of 26 and the aldehyde 6 afforded the diene 27. The successive selective desilylation and oxidation afforded the diene 29. The diene 29 possesses most of the carbon framework with the requisite asymmetric carbons involved of 1. Since 29 is considered as a potential key intermediate in our synthetic strategy, conversion of 29 to the target molecule 1 is now under investigation.
Manzacidins isolated from Hymeniacidon sp. have attracted much attention because of their biological activities and their novel structures possessing an ester-linked bromopyrrole carboxylic acid ester, an amino group attached to the quarternary carbon center, and a tetrahydropyrimidine ring. In this report, the first total synthesis of manzacidin A, which accompanied determination of its absolute stereochemsitry, is described. The key feature to the total synthesis relies on stereoselective construction of the aminonitrile intermediate 4a by the asymmetric Strecker synthesis. To this end, three kinds of ketoamide 3a-c prepared by condensation of a chiral amino alcohol 2 with glycine, L-or D-phenylalanine were employed. Treatment of glycyl amide 3c with TMSCN and ZnCl_2 afforded a diastereomeric mixture of aminonitriles 4c and 4d in a ratio of 1:5 in 48% yield. When ketoamide 3a bearing phenylalanine as the asymmetric transferring group was used, the double asymmetric induction occurred to give 4a as a sole product. The addition reaction from 3b was also stereoselective to provide 4b in high yield. Relative stereochemistries of the Strecker adducts 4a and 4b corresponded to those of manzacidin A and C, respectively. Total synthesis of manzacidin A was achieved by the following sequence of reactions: 1) oxidation of aminonitrile 4a to imine 5, 2) hydrolysis of 5 followed by introduction of Boc protective groups to the amino groups, 3) conversion of 6 to the lactone 7, 4) formation of the tetrahydropyrimidine ring, and 5) esterification of 9 with bromotrichloroacetylpyrrole 10. The absolute stereochemistry was determined to be (4S,6R) by comparison of optical rotation of the synthetic manzacidin A with that of natural manzacidin A.
Zoanthamine (1) and norzoanthamine (2) are marine alkaloids with significant biological activities. In particular, norzoanthamine and its hydrochloride suppress decrease in bone weight and strength in ovariectomized mice without increase of the uterine weight, and are considered to be a promising candidate for an ostcoporotic drug. In addition to their potential medicinal interest, zoanthamine and norzoanthamine are challenging target molecules from a synthetic point of view due to their complex heptacyclic ring system including aminal structure. During the course of our studies directed toward total synthesis of norzoanthamine, we examined an enantioselective route to the fully functionalized heterocyclic aminal core 3. Pentacyclic heterocyclic 3 contains four of the five quaternary chiral centers, including three contiguous ones, in zoanthamine and norzoanthamine. Our basic strategy is to construct the pentacyclic 3 from monocyclic precursor 5 by sequential cyclization. As a cyclization precursor we selected 6 in which cyclohexanone is protected as ethyleneketal and aminoalcohol moiety is protected as Cbz- or Boc- substituted N,O-acetal. The synthesis of the key intermediate 6 was accomplished by the coupling of the aldehyde 7 and sulfone 8, which were prepared from (+)-Wieland-Miescher ketone and D-glutamic acid, respectively. It should be noted that the stereochemical control of contiguous quaternary chiral centers in 7 was achieved by the stereoselective cuprate addition to W.-M. ketone. After a number of experiments, N-substituted aminal 19a and 19b were isolated in good yields when 6a and 6b were treated with 2N HCl in THF. N-Cbz aminal 19a was then subjected to a hydrogenolysis, and the pentacyclic aminal 3 was isolated in 71% yield after treatment of the reaction mixture with MS3A. The pentacyclic aminal core 3 was also obtained from Boc derivative 19b by stirring in aqueous acetic acid at 100℃ followed by the Na_2SO_4 treatment. The structure of 3 was supported by ^1H- and ^<13>C-NMR spectra which were in good accordance with those of norzoanthamine. Finally, one-step cyclization of Boc derivative 19b to 3 was achieved in 89% yield. This is the first example of the synthesis of the characteristic moiety of zoanthamine/norzoanthamine family. Concerning the cyclization of monocyclic precursor, we observed that the (R)-methyl isomer 6c gave only spiroketal 21, and 6c did not cyclize to pentacyclic aminal 22 by aqueous acetic acid treatment. We believe that the present methodology developed here could be applicable to the total synthesis of norzoanthamine, and are currently doing effort toward this goal.
The first total syntheses of fasicularin (13) and lepadiformine (9), novel tricyclic marine alkaloids with cytotoxicity against tumor cell lines recently isolated from the marine ascidians Nephteis fasicularis and Clavelina lepadiformis respectively, have been achieved. The key strategic element was the intramolecular hetero Diels-Alder cycloaddition of N-acylnitroso compounds with exocyclic diene moieties, in which anti/syn-face selectivity was controlled. Thus, the B/C trans-fused cycloadduct 22 formed by the acylnitroso-Diels-Alder reaction proceeding with anti-facial selectivity was converted to (±)-fasicularin (13). On the other hand, the B/C cis-fused cycloadduct 35, obtained by the syn face selective acylnitroso-Diels-Alder reaction, was converted to "lepadiformine" having the originally proposed structure 9, which, however, was found to be clearly different from natural lepadiformine. The B/C trans-fused cycloadduct 22 was then converted to the new structrue 10 of lepadiformine identical in all respects with the natural product. These results indicate that the originally proposed structure 9 for lepadiformine is uncorrect and should be revised to the structure 10 we have synthesized in the present investigation.
Azadirachtin is a C-seco limonoid, which was isolated as an insect antifeedant from the seeds of Azadirachta indica A. Juss in 1968. The highly functionalized structure, along with its biological activities, attracts many synthetic chemists; however, the total synthesis has not been achieved yet. Our strategy involves the coupling of the two fragments by Claisen rearrangement. We describe the syntheses of the decalin compound, the left fragment of azadirachtin, and the tricyclic compound, the right fragment. Furthermore, we investigated the Claisen rearrangement for construction of the C-8 and C-14 bond using the model compounds. At first, we elucidated the intramolecular Diels-Alder (IMDA) reaction to construct the decalin part of azadirachtin. The IMDA reaction of the hydroxyl-triene substrate proceeded under thermal conditions, which afforded a 2:1:1 mixture of decalin compounds. On the basis of these experiments, we developed the chiral synthesis of the decalin compound via asymmetric reduction, and obtained the chiral decalin product, which was converted into the desired four-cyclic compound in 16 steps. However, the attempted introduction of α-OH to C-1 of the product was unsuccessful. Therefore, we investigated an alternative route, and the carbonate of 1,3-dihydroxy triene compound was prepared in 17 steps from ethyl malonate. Its thermal IMDA reaction proceeded smoothly to afford two desired 1,3-oxidized decalin compounds, exclusively. On the other hand, the tricyclic acetal moiety could efficiently be obtained as an enantiomerically pure form from cyclopentadiene and the known acryloyl derivative in 21 steps in 25% overall yield. Furthermore, we also studied the coupling of the right and the left segments of azadirachitin. The Ireland-Claisen rearrangement using two model compounds proceeded selectively to afford the coupling products in good yields, respectively.
The sandfly Lutzomyia longipalpis is the only vector of the protozoan parasite Leishmania chagasi, the causative agent of visceral leishmaniasis in South and Central America. In 1994 Hamilton et al. isolated the sex pheromone of male Lutzomyia longipalpis from Brazil, and confirmed its activity by bioassay. Subsequently in 1996, 9-methylgermacrene-B (1) and 3-methyl-α-himachalene (2) were proposed as the structures of the pheromone components on the basis of their MS and ^1H NMR studies. We synthesized all of their stereoisomers, and established the relative and absolute configuration of the natural pheromone components on the basis of their NMR and GC analysis. 9-Methylgermacrene-B [(±)-1] was synthesized by employing a cyclization reaction [(±)-12→(±)-13] as the key step. The product (±)-1 was shown to be the racemate of the male-produced sex pheromone of the sandfly Lutzomyia longipalpis from Lapinha, Brazil. Both the enantiomers of 9-methylgermacrene-B (1) were synthesized by starting from the enantiomers of methyl 3-hydroxy-2-methylpropanoate (2). The male-produced sex pheromone of the sandfly Lutzomyia longipalpis from Lapinha, Brazil, was identified as (S)-1 by GC comparison. Four stereoisomers (2a-d) of (±)-3-methyl-a-himachalene were synthesized by employing the intramolecular Diels-Alder reaction [(±)-45 to (±)-46] as the key-step. The male-produced sex pheromone of the sandfly Lutzomyia longipalpis from Jacobina, Brazil, was shown to possess the structure and possible absolute configuration as depicted in 2c.
Since the discovery of anticancer activity of taxol, much attention has been paid for the isolation of new taxane diterpenoids from various yew trees and the structure-activity relationships and the total syntheses of taxoids. In our continuing chemical derivatization from taxinine, one of major taxoids obtained from the Japanese yew Taxus cuspidata Sieb. et Zucc., we found unusual reactions of taxinine derivatives. Here we describe the formation of unexpected products and propose plausible mechanism of these reactions. 1. Stereoselective Epoxidation of 4(20)-Exomethylene in Taxinine Derivatives Epoxidation of taxinine (1), taxinine A (2), and taxinine derivative 7 with m-chloroperbenzoic acid afforded the α-4(20)-epoxides selectively (α:β=99:1), while epoxidation of taxinine derivatives 7 and 8 with dimethyldioxirane gave the β-4(20)-epoxides predominantly (α:β=1:4〜5). 2. Unusual Boron Trifluoride-catalyzed Reactions of Taxinine Derivatives with α- and β-4(20)-Epoxides Boron trifluoride-catalyzed reaction of β-4(20)-epoxy-5-O-triethylsilyltaxinine A (8b) gave the 3,5-diene (9, 42%), the 3,8-cyclopropane (10, 16%), the cyclobutane (11, 4%), and the dioxane (12, 3%) derivatives, while the similar reaction of the corresponding α-4(20)-epoxide (8a) afforded the ring contracted derivative (13, 41%), its hemiacetal dimer (14, 16%), and the orthoester (15, 9%) derivatives. The structures of 9〜15 were elucidated by spectral data including MS and 2D NMR data. Plausible mechanisms of these reactions containing 1,2-hydride shift (9 and 10 from 8b) or pinacol-type rearrangement (13 and 14 from 8a) are proposed. 3. Occurrence of a New Dimeric Compound of 5-Oxotaxinine A through Diels-Alder Cycloaddition Oxidation of taxinine A (2) with tetrapropylanunonium perruthenate afforded 5-oxotaxinine A (16) which subsequently gave a new dimeric compound (17) through regio- and stereo-specific Diels-Alder cycloaddition. The relative stereostructure of 17 was established by spectral data and X-ray analysis.
Archaea (archaebacteria) grow under the extreme environments such as hot springs, highly saline lakes, and deep-sea volcanoes. These microorganisms possess unique membrane lipids composed of isoprenoid chains which bind to glycerol by ether linkage. More interestingly, some archaea have macrocyclic ether lipids as large as 36- or 72-membered ring sizes. Our interest focuses on the biochemical significance of the macrocyclic molecular structures. We were the first in synthesizing the archaeal 36- and 72-membered macrocyclic lipids (2, 3a and 3b) using McMurry coupling. In addition, a new and efficient approach to the macrocyclic lipids using olefin metathesis has been accomplished recently. In the presence of a Grubbs' ruthenium-alkylidene complex, RuCl_2(=CHPh)(PCy_3)_2, a ring closing metathesis (RCM) of α,ω-diene 17 efficiently proceeded in 79% yield under high dilution conditions to give 36-membered 18. By changing the reaction conditions, an acyclic diene metathesis (ADM) product 19 was predominantly formed from the same substrate 17. The acyclic product 19 was subsequently subjected to the RCM reaction under high dilution conditions to provide 72-membered compound 20 in 46%. Thus, both 18 and 20 can be obtained at will from the same starting material only by changing the order and conditions of the metathesis reaction. The physicochemical features of the 36-membered macrocyclic phospholipids 21 were investigated in comparison with the corresponding acyclic counterpart 22 in terms of fluidity, thermostability and permeability of the liposomes. Thermal analyses and monolayer studies indicated that 21 can aggregate to more tightly packed structures than 22 due to its less motional freedom at the alkyl chain region. Furthermore, we found that the macrocyclic structure contributed to the formation of thermally stable membrane. These tendencies appear to be related to the tolerance of thermophilic archaea Methanococcus jannaschii against high temperature.
The zaragozic acids and squalestatins are a family of naturally occurring fungal metabolites isolated and characterized independently by researchers at Merck and Glaxo. These natural products are potent inhibitors of squalene synthase and have potential as therapeutic agents for the treatment of hypercholesterolemia. All the zaragozic acids and squalestatins show a common 2,8-dioxabicyclo[3.2.1]octane core with an array of six stereogenic centers including contiguous quaternary carbons, and are different only at the C1 alkyl and C6 acyl side chains. Zaragozic acid A (squalestatin S1) (1) is a representative example of this novel class of compounds. Herein, we wish to report the efficient, convergent synthesis of 1 highlighting acetal [1,2]-Wittig rearrangement to join the C4-C5 bond with a simultaneous creation of the contiguous quaternary carbons as the key steps. Core part synthesis Our synthesis begins from the O-glycoside 4, which can be prepared from in four steps from protected L-arabino-γ-lactone 3 and bis(ethynyl)carbinol. The [1,2]-Wittig rearrangement of O-glycoside 4 to afford the (S, β)-C-glycoside 5 (87% dr) in 55% yield. The major isomer, separated by column chromatography was then converted to the key precursor 7 as follows. Hydroalumination of the TBDPS-substituted ethynyl group of 5 followed by ozonolysis gave the aldehyde which was then subjected to the Grignard reaction (vinyl magnesium bromide) to afford the desired alcohol 7 as a single diastereomer. Further oxidations of ethynyl, vinyl and α-oxy carbon (C1) gave the desired lactone 11, successfully. C1 side chain synthesis C1 side chain segment 15 has been synthesized from (S)-methyl lactate via [1,2]-Wittig rearrangement as a key step. C6 side chain synthesis C6 side chain segment 20 has been synthesized from the chiral α,β-unsaturated imide 16 via the sequential allylation/Cope rearrangement as a key step. Union of segments Generation of the alkyllithium C1 side chain derived from iodide 15 followed by addition of lactone 11 provided hemiketal 21 as a mixture of anomers. Oxidative cleavage of MPM ether followed by acetylation of the C4' hydroxyl completed the assemblage of hemiketal 22, precursor to the bicyclic core and associated C1 side chain. Hydrolysis/ketalization of hemikatal 22 with TFA, followed by the esterification and TBAF treatment to provide the desired bicyclic ketal 23. Protection of C7 hydroxyl with Boc group followed by the acylation with C6 side chain 20, acid treatment afforded the zaragozic acid A (1), whose spectral and chromatographic properties are identical with those of a comparison sample of the natural product.
Reveromycins are novel polyketide-type antibiotics isolated from Streptmyces sp. and inhibit the signal transduction pathway associated with EGF receptors. The characteristic structural features involve spiroketal core having a hemi-succinate, two unsaturated side chains ending in carboxilic acids, and two alkyl groups. The potent biological activity and the unique structure attracted the attention of synthetic organic chemists, and the first total synthesis of 2 was recently achieved by Theodorakis et al. We have already reported the stereoselective synthesis of 6,6-spiroketal system in 1, and elucidated the absolute configuration through the synthesis. Here, we report the stereoselective total synthesis of reveromycin B (2). Our synthetic strategy involved the Wittig reaction, Horner-Wadsworth-Emmons reaction and one-pot Julia coupling reaction for construction of the unsaturated side chains. The spiroketal core was provided by acetylide coupling reaction between Weinreb amide 3 and alkyne 4, which were prepared from epoxides 8 and 15, respectively. Exo-cyclization of the epoxide 8 followed by regioselective oxidation gave the sterically hindered lactone 12, which was efficiently converted into the Weinreb amide 3 via aminolysis using Me_2AlCl-MeNHOMe・HCl. (Z)-Selective Homer-Wadsworth-Emmons reaction of aldehyde 15 using Still's reagent afforded alkenyl oxirane 16. Pd-HCO_2H reduction of 16 proceeded regio- and stereoselectively to give the desired anti-alcohol 17 which was converted into the alkyne 4 by silylation, oxidative cleavage of olefin, and Colvin rearrangement. The coupling reaction of 3 and 4 using nBuLi following by hydrogenation afforded ketone 22, which was treated with TsOH to give the thermodynamically stable spiroketal 23. After succination of 25, deprotection of TES group, and TPAP oxidation, the resulting aldehyde 28 was subjected to the Horner-Wadsworth-Emmons reaction with phosphonate 21 to give the (E, E)-diene 29 exclusively. The Wittig reaction of 37 gave the α,β- unsaturated aldehyde 38 which was reduced with ZnBH_4 to afford allyl alcohol 39. The subsequent Mitsunobu reaction using 2-mercaptobenzothiazole and Mo(VI) catalyzed oxidation gave sulfone 41, which was coupled with aldehyde 36 in the presence of LiHMDS to afford the (E)-olefin 42. Deprotection of TES group in 42 followed by Dess-Martin oxidation provided aldehyde which was subjected to the Wittig reaction with a neutral phosphorane to give the protected reveromycin B (44). Finally, successive desilylation and deallylation gave reveromycin B (2), which was identical (^1H-NMR, ^<13>C-NMR, [α]_D, IR, HRMS) with the natural product.
To elucidate the mechanism of the biological events caused by bacterial endotoxin (lipopolysac-charide, LPS), isotope and fluorescence labelings were effected on lipid A, which is the partial structure of LPS essential for its bioactivity. The labelings were performed by means of chemical synthesis, and physicochemical as well as biological functions of the labeled derivatives were evaluated. 6-^<13>C-Labeled derivatives were synthesized of a biosynthetic precursor of lipid A (6-^<13>C-2) and its analogue with shorter (C10) acyl chains (6-^<13>C-3). NMR study of the biosynthetic precursor 2 using ^<13>C-labeled and unlabeled specimens enabled us to figure out its supramolecule structure formed by self-assembly. By contrast, the short-acyl analogue 3 was found to form no aggregate. Molecular modeling revealed the origin of this striking behavioral and conformational difference: unnatural C10 length for the acyl moieties is not sufficient to obtain stabilization by hydrophobic interaction. Because it has already shown that the short-acyl analogue 3 no longer retains the biological activity of the biosynthetic precursor 2, it is strongly suggested such a supramolecule-forming ability relates with the biological activity of lipid A analogues. The fluorescence-labeled lipid A analogue 5 was next synthesized to obtain a deeper insight on the self-assembly of lipid A. BODIPY^[○!R] was employed as a fluorescence group, and the labeling was effected on the phosphonooxyethyl (PE) analogue 4 wherein the chemically labile glycosyl phosphate of lipid A is replaced with the stable 2-(phosphonooxy)ethyl group. The synthesis was achieved using allyl-type groups for the persistent protection of all active functionalities to afford the highly pure final product. The labeled analogue 5 was found to retain definite activities of lipid A1. Fluorescence spectra indicated the critical micelle concentration of 5 being below 0.46nM. From these experiments it can be clearly concluded that the labeled lipid A analogue 5 forms aggregate around the concentration range where it exhibits the biological activities. To identify the possible receptor(s) on the competent animal cells, tritium-labeling was effected on the ethylene glycol moiety of the PE analogue 4 of lipid A. Careful two-step oxidation of the ally group and partition chromatographic purification after final deprotection led to complete removal of over-oxidized by-products and eventually enabled us to obtain a highly pure (>98% radiochemical purity) preparation of ^3H-4 with high specific radio activity (62GBq/mmol). Using the ^3H-labeled and unlabeled PE analogues, four binding proteins were detected in macrophages from both LPS-responder C3H/HeN mice and LPS-hyporesponder C3H/HeJ mice.
It is now widely recognized that attachment of carbohydrate is one of the most important posttranslational modifications which affects their biological activities by way of controlling higher order structure, stability, immunogenicity, and carbohydrate-protein interaction. In most cases, protein glycosylation can be classified into two major subtypes: O-glycosylation, where an N-acetylgalactosamine residue is linked to the hydroxyl group of either serine or threonine, and N-glycosylation, where a glycan chain is linked via a glycosylamido linkage to an asparagine residue. However, in 1994, a new class of glycoprotein structural motif was identified in human RNase, where a mannose residue is connected to tryptophan via a C-glycosidic linkage. More recently, the same structural motif was found from recombinant human IL12. The total synthesis of this novel type of glyco-amino acid, C^2-α-L-C-mannosylpyranosyl-L-tryptophan has been achieved in a stereocontrolled manner. Mannose moiety and (L)-tryptophanol derivative were connected via epoxide opening reaction. After several functional group transformation, the target molecule was synthesized in a concise manner. With rigorously defined synthetic molecule in hand, ^1H NMR analysis cleanly revealed that mannosylated tryptophan itself adopts the ^1C_4 conformation with the equatorially oriented tryptophan moiety. Peptide elongation reaction was also accomplished by using intermediate azide acid in solution phase. By use of tetramethylfluoroformidium hexafluorophosphate, coupling with tripeptide was achieved in high yield. After selective reduction of azide at N-termini, further peptide elongation was successfully performed to afford the protected hexapeptide sequence, which corresponds to the partial structure of human RNase.
Carbohydrates and peptides play key roles in a variety of disease-related processes; however, use of naturally occurring carbohydrates and peptides as chemotherapeutic agents has often been hampered by their susceptibility to digestion by glycosidases and proteases. There has been a considerable interest in designing and constructing mimetics of the oligosaccharide and peptide molecules with the expectation that these molecules may provide stable alternatives to the biologically important natural carbohydrates and peptides. Glycamino acids, are a unique class of carbohydrate-based molecules that possess both a carboxylate group at the anomeric position and an amino group replacing one of the OH groups of the carbohydrate. As a part of our ongoing program to develop a new class of molecules that possess the structural and functional features of both carbohydrates and peptides, we have synthesized the first homooligomers of glycamino acids in which the glycamino acid residues are connected via β(1→2)-, 13(1→3)-,β(1→4)-, and β(1→6)-linkages, and hybrid molecules composed of a repeating unit of glycamino acid and aspartic acid. We found that, upon O-sulfation, the amido-linked oligosaccharide analogs were able to inhibit the replication of HIV-1, sialyl Lewis x-dependent cell adhesion and heparanase activity.
Calonyctin A isolated from Calonyction aculeatum L. House (evening-glory) has a potent promoting activity on the plant growth. One of the homologue 1 of calonyctin A has a unique 22-memberd macrolidic structure, which consists of 11-hydroxytetradecanoic acid and four deoxygenated monosaccharides. In order to investigate the mechanism of its physiological activity and assign the absolute configuration of fatty acid, we planned to synthesize 1 and its analogues. We found that an interglucosidic acetal of phenyl 1-thio-β-laminaribioside 6 was a good starting material for the synthesis of 1. After allylation, the fully protected disaccharide 7 was treated with PPTS in MeOH-CHCl_3 and subsequently with BH_3・Me_2NH-BF_3・OEt_2 in CH_2Cl_2, giving the 2,6,2',6'-tetraol 9. A key disaccharide intermediate 5 was prepared by tosylation of 9 and subsequent benzoylation and NaBH_4-reduction of the tosyl groups in 48% overall yield. Coupling of 5 and monosaccharide acceptor 13 with NIS-TfOH gave the trisaccharide derivative 14. After saponification of three ester functions in 14, the resulting 15 was subjected to intramolecular esterification by a mixed anhydride procedure under high-dilution conditions in toluene to give a macrolide. Although two hydroxyl groups are presented, 15 afforded desired 22-membered macrolide 16 as a single product in the yield of 60%. Final L-rhmnosylation of 16 with 4 furnished the tetrasaccharide17, of which de-O-allylation followed by estification with (2R,3R)-3-O-benzyl-2-methylbutyric acid and by hydrogenolysis gave 1.
Sulfoxides are sometimes found in natural compounds and many important pharmaceutically active compounds such as omeprazole are composed of sulfoxide group. A sulfoxide can exist as an enantiomer because racemization between two enantiomers is generally very slow or impossible under the usual conditions. There are few method for determining the absolute configuration of chiral sulfoxide except for X-ray crystallography or synthetic approach. As NMR methods, aromatic solvent-induced shift (ASIS) and some chiral shift reagents such as 9-anthryl-1,1,1-trifluoroethanol, methoxyphenylacetic acid, and N-(3,5-dinitro-benzoyl)-α-phenylethylamine (Kagan's reagent) have been used for this purpose. However, the difference of the chemical shifts between two diastereomeric pairs is generally very small and sometimes nonsystematic. In this paper, we report the reliable method for elucidating the absolute stereochemistry of chiral sulfoxides by means of NMR spectroscopy. The new method consists of two major processes: (i) conversion of a chiral sulfoxide to a chiral sulfoximide with retention of the stereochemistry using mesitylsulfonylhydroxylamine (MSH), and (ii) coupling reaction of the chiral sulfoximide with (S)- and (R)-methoxyphenylacetic acid (MPA). MPA was selected as the chiral anisotropic reagent because the molecular model analysis revealed that the N-MPA-sulfoximide would exist in only one stable conformation. Several racemic and chiral sulfoxide were prepared by the known synthetic method such as the diacetone-D-glucose (DAG) method, and the sulfoxides were transferred by the two-step reactions into N-MPA-sulfoximides. The Δδ values for the protons were calculated according to the equation, Δδ=δ_<S-MPA>-δ_<R-MPA>. In all the examples, the systematic distribution of Δδ values were observed without exceptions, and the results obtained by the present method agreed with the known absolute configurations.
The ciguatoxins are the causative agents responsible for a peculiar form of fish poisoning termed "ciguatera". Precursor toxins produced by the dinoflagellate Gambierdiscus toxicus undergo structural modification in fish to produce various metabolite toxins including ciguatoxin (CTX), the principal toxin in ciguatera. Because of the extreme difficulty to obtain these congeners in sufficient amounts for NMR studies, we carried out FAB MS/MS experiments to elucidate their structures. A total of 53 toxic fractions were prepared from the flesh and viscera of moray eels Gymnothorax javanicus, the flesh of red snappers Lujanus bohar, the flesh of parrotfish Scarus gibbus, and G. toxicus collected from three different places (Tahiti, Tubuai, and Rangiroa). Many of these samples were mixtures and in amounts less than 10μg. Collision induced ions generated from Na-adduct precursor ions were characteristic for the structural modifications. Supportive data were also obtained by measuring spectra of a 2-sulfobenzoate of a CTX congener. Out of 20 congeners detected by LC/MS, the following 17 congeners were identified: CTX4A, M-seco-CTX4A, 52-epi-54-deoxy-CTX, CTX, 52-epi-CTX, 54-deoxy-50-hydroxy-CTX, 7-oxo-CTX, 7-hydroxy-CTX, 3-hydroxy-7-oxo-CTX, CTX3C, 49-epi-CTX3C, 51-hydroxy-CTX3C, 3-hydroxy-CTX3C, 51-hydroxy-3-oxo-CTX3C, 2,3-dihydroxy-CTX3C, 2,3,51-trihydroxy-CTX3C, and A-seco-51-hydroxy-CTX3C.
Many plants close their leaves in evening as if to sleep and open them early in the morning. Such a circadian rhythm has been known to be controlled by their internal clock. As already reported, recently, we succeeded in isolating a true active stimulant (activity: ca. 10^<->8〜10^<-9>M) from the same plant, which consists of three different components (potassium L-malate; magnesium trans-acconitate; dimethyl ammonium salt). All of these components are required for causing the rapid leaf-movement of Mimosa pudica. In addition, the leaf-closing and -opening substances were also isolated from Mimosa pudica. They concern with nyctinastic movement controlled by an internal clock. Therefore, the rapid movement is different from the slow nyctinastic one in mechanism. Further extensive study on other nyctinastic plants led to the isolation of a variety of leaf-closing and -opening substances. As represented by Lespedeza cuneata G. Don. and Phyllanthus urinaria L., their leaf movement was proved to be controlled by the balance of concentration between these two leaf-opening and -closing substances, which was controlled by the activation of β-glucosidase.
Mutations in the Streptomyces peucetius dnrD gene block the ring cyclization leading from aklanonic acid methyl ester to aklaviketone, an intermediate in the biosynthetic pathway to daunorubicin and doxorubicin. To investigate the role of DnrD in this transformation its gene was overexpressed in Escherichia coli and the DnrD protein was purified to homogeneity and characterized. The enzyme was shown to catalyse the conversion of aklanonic acid methyl ester to aklaviketone presumably via an intramolecular aldol condensation mechanism. In contrast to the analogous intramolecular aldol cyclization catalyzed by the TcmI protein from the tetracenomycin C pathway in Streptomyces glaucescens, where a tricyclic anthraquinol carboxylic acid is converted to its fully aromatic tetracyclic form, the conversion catalyzed by DnrD occurs after anthraquinone formation and requires activation of a carboxylic acid group by esterification of aklanonic acid, the aklanonic acid methyl ester precursor. Also, the cyclization is not coupled with a subsequent dehydration step that would result in an aromatic ring. As the substrates for the DnrD and TcmI enzymes are among the earliest isolable intermediates of aromatic polyketide biosynthesis, an understanding of the mechanism and active site topology of these proteins will allow one to determine the substrate and mechanistic parameters important for aromatic ring formation. In the future, these parameters may be able to be applied to some of the earlier polyketide cyclization processes that currently are difficult to study in vitro.
Macrophomate synthase which catalyzes unusual multiple reactions from 2-pyrone (3) to macrophomic acid (1) has been purified as homogenous state. The macrophomate synthase is a dimeric enzyme which requires Mg^<2+> as a co-factor and whose molecular mass is 40kDa. Study on substrate specificity showed that the enzyme is capable of converting various 2-pyrones to the corresponding benzoates. The substrate specificity of this enzyme was also examined with various substrate analogs. Formation of an aberrant product 13 in the enzymatic reaction with 2-pyrone 11 strongly indicated involvement of a bicyclic intermediate which is readily degraded into aromatic product by a concerted dehydration-decarboxylation.
Several eubacteria including Escherichia coli utilize an alternative mevalonate-independent pathway (nonmevalonate pathway) for the biosynthesis of isopentenyl diphosphate. In the nonmevalonate pathway, 2-C-methyl-D-erythritol or its 4-phosphate which was proposed to be formed from 1-deoxy-D-xylulose 5-phosphate (DXP) via intramolecular rearrangement followed by a reduction process is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for the synthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP), we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments which complemented the defect in synthesizing MEP of these mutants contained the yaeM gene which located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in several eubacteria. The yaeM gene product overexpressed in E. coli was found to catalyze the formation of MEP from DXP in the presence of NADPH. These data clearly show that the yaeM gene encodes a novel enzyme, designated DXP reductoisomerase, which synthesizes MEP from DXP in a single step by intramolecular rearrangement and reduction. Since the nonmevalonate pathway is absent in mammals, all the enzymes involved in this pathway are considered to be new targets for screening of antibacterial substances. Therefore, we attempted by database search to find antibiotics active against E. coli and Bacillus subtilis with the nonmevalonate pathway, but inactive against Staphylococcus aureus possessing the mevalonate pathway with an expectation that such inhibitors might have already been reported as antibacterial substances. As a result, fosmidomycin showing the expected antibacterial spectrum came up as a candidate for an inhibitor of the nonmevalonate pathway. In this presentation we describe inhibitory effects of fosmidomycin on DXP reductoisomerase.
Triterpenoids are one of the most abundant natural products commonly occuring in plants and exhibit a wide range of structural diversity. These triterpene frameworks are believed to be biosynthesized from a common precursor 2,3-oxidosqualene by distinct triterpene synthases. In order to identify the origin of the product specificities exhibited by triterpene synthases, we have chosen β-amyrin synthase (PNY) and lupeol synthases (LUP1, OEW) for mechanistic studies. The cyclization mechanisms leading to β-amyrin and lupeol are identical up to lupenyl cation stage where proton abstraction from the methyl group results in lupeol while ring expansion and hydride shift will generate β-amyrin. To determine the polypeptide region important for the product specificity, several chimeric enzymes were constructed. Chimera 1, in which N-terminal half is PNY and C-terminal half is LUP1, produced both β-amyrin and lupeol in 3:1 ratio. In addition, minor amount of butyrospermol (4) was produced. The results from other chimeric enzymes indicated that the 80 amino acid sequence located in the second quarter from N-terminus was important for β-amyrin formation. [1,2-^<13>C_2]Acetate feeding experiment was conducted to identify from which methyl group is proton abstracted during lupeol formation. The result from LUP1 showed that the proton is abstracted from both methyl groups in non-specific manner. On the other hand, OEW exhibited specific proton abstraction from (7)-methyl group of 2,3-oxidosqualene. These results suggested the occurence of two types of lupeol synthases in nature. Furthermore, site-directed mutagenesis was carried out in order to define the amino acid residue responsible for product specificity. PNY W259L mutant gave significant amount of lupeol together with β-amyrin, while OEW L258W mutant gave exclusively β-amyrin. PNY Y261H mutant gave neither β-amyrin nor lupeol and instead it produced mixture of 5 and 6. These results suggested that Trp 259 of PNY stabilizes the oleanyl cation during β-amyrin formation, while Tyr 261 is responsible for the formation of pentacyclic triterpenes.
Squalene cyclization mechanism and the active sites of the cyclase are discussed on the basis of the site-directed mutagenesis experiments. 1. DXDDTA motif: the initiation of the polycyclization and stabilization of carobocation intermediate of the initialy formed A-ring through the carboxylate anion of D377. 2. Phe365 stabilizes the C-8 carbocation intermediate of A/B-fused ring system through cation/π interaction. This interaction was confirmed by constructing the mutants F365Y and F365W; these mutants accelerated the reaction velocity and the significant lowering of activation energy for the polycyclization reaction. Tyr420 is also responsible for the formation of B-ring. 3. Phe 601is crucial for the construction of the 6-membered C/D-ring system. The mutant F601A produced the partially cyclized tricyclic 6/6/5-fused13,14 and tetracyclic 6/6/6/5-fused 18. These products could be formed by a Markovnikov closure and indicated the involvements of ring-expansion processes from the 5-membered into the 6-membered ring system for the construction of the C/D-ring system. Compound 18 was also isolated from the mutant W169F and W169H. The presumed carbocationic intermediates 12 and 17 was trapped by using the substrate analogues 27 and 32(34), respectively, with a high nucleophilic hydroxyl group, resulting in the formation of 31 and 35. Thus, we propose that the polycyclization mechanism proceeds via two ring-expansion steps for the formation of C- and D-rings, leading to the formation of anti-Markovnikov adduct 2. 4. The mutant I261A produced a series of carbocationic intermeidates 6/6/5-, 6/6/6/5-, and 6/6/6/6-fused ring systems, giving further insight into the polycycliation mechansim. In the enzymatic products, unnatural natural products are included. 5. Phe 605 would stabilize the cations of 17, 19 and 20 through cation-π interaction, as verified by the isolations of 6/6/6/5- and 6/6/6/6/5-ring skeletons. 6. The methyl at C10-posisiton of 1 is crucial to the correct folding in the active center, this being demonstrated by the substrate analouge 40. The proposed cyclization mechanism has been verified both by the kinetic measurments and by the isolation of the differently cyclized products resulting from each stage of the carbocationic intermediates. Alteration of the cyclase active sites afforded multiple triterpenes in addtion to the understanding of the fundamental issues of squalene cyclization mechanism, suggesting the possibility that we will be able to generate novel triterpenes (unnatural natural products) by rational genetic engineering of squalene cyclase.
To investigate the defense mechanism of plant cells against chemical stress, we have examined the defense reaction of plant cells against exogenous monoterpenoids. We have screened various isoprenoids to search for inducers of apoptosis in the cultured cells of several plants such as tabacco, chamomile, celery, soybean and liverwort. These studies have revealed that geraniol has the most potent apoptosis-inducing activity, as judged from DNA fragmentation in the cells. The apoptosis-inducing activity of geraniol is concentration- and time-dependent. Morphologically, the nuclei of the cells which have been treated with 5mM of geraniol were condensed and fragmented. On the other hand, additoion of 1ppm of N^6-(Δ^2-isopentenyl) adenosine and kinetin prevented the geraniol-induced DNA fragmentation in the cells. Furthermore, cycloheximide and actinomycin D, inhibitors of protein and RNA syntheses, respectively, did not suppress the apoptosis of the plant cells. This result suggests that the apoptotic process induced by geraniol does not require the synthesis of new proteins. The cytokinin inhibition mechanism of the apoptosis induced by geraniol will be discussed.
Ferula ferulioides (STEUD.) KOROVIN (Umbelliferae) grows in Bulgan Somon of Hovd City, Mongolia, and has been used as a traditional medicine for the treatment of spasm. The present investigation deals with the isolation and the structural elucidation of twenty four novel sesquiterpenoid derivatives (5-28), together with four known compounds (1-4) from F. ferulioides. The dried root powder was extracted with methanol, and removal of the solvent gave waxy solid which was successively extracted with ethyl acetate and water. From the ethyl acetate extract, twenty four novel compounds (5-28) were isolated, together with four known compounds, myristicin (1), guaiol (2), nerolidol (3) and dshamirone (4). Myristicin is a phenylpropanoid derivative; nerolidol and guaiol are sesquiterpenes. Compounds 5-28 were identified as farnesyl-acetophenone type (5-8), fanesyl-benzofranone type (9), prenyl-benzoylfuranone type (10-12), 3-prenyl-furocoumarin type (13-16), 2-prenyl-furocoumarin type (17-25), and 2-prenyl-furochromone type (26-28) new sesquiterpenoid derivatives by comprehensive spectral analysis. A series of NOESY experiments was carried out with 10-28 to identify the relative stereochemistry. The biosynthetic pathway leading to these compounds is proposed based on their structures. Compounds 4-28 were divided into groups A-F by their biosynthetic routes. In addition, the presence of the hypothetical precursors classified into three groups (I-III) was suggested.
In Chinese traditional medicine, Zedoariae Rhizoma (the rhizome of Curcuma zedoaria), Saussureae Radix (the roots of Saussurea lappa), Nupharis Rhizoma (the rhizome of Nuphar pumilum), and Rhei Rhizoma (the rhizome of Rheum undulatum) has been prescribed for the treatment of "Oketsu" syndrome caused by blood stagnation and inflammation. Inducible NO synthase (iNOS) is involved in pathological processes mediated with overproduction of NO and is expressed in response to pro-inflammatory agents such as interleukin-1β, tumor necrosis factor-α and lipopolysaccharide (LPS) in various cell types including macrophages. Inhibition of iNOS enzyme activity or its induction may have therapeutic effects in various types of inflammation. In the course of our studies on bioactive principles from Chinese herbal medicines, we have found that sesquiterpenes (furanodinene, costunolide, dehydrocostus lactone, santamarine, saussureamines A and B) from Zedoariae Rhizoma and Saussureae Radix, dimeric sesquiterpene thioalkaloids (6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythio-nuphlutine B) from Nupharis Rhizoma, and stilbenes (rhapontigenin, piceatannol, resveratrol) from Rhei Rhizoma showed NO production inhibitory activity in LPS-activated macrophages. And some structural requirements for the activity was elucidated. Several sesquiterpenes (curcumenone, furanodiene, germacrone, etc.) from Zedoariae Rhizoma and the stilbenes and stilbene glycosides (rhaponticin, piceatannol 3'-O-β-D-glucopyranoside, etc.) from Rhei Rhizoma showed hepato-protective activity in D-galactosamine/LPS-induced liver injury in mice. Many sesquiterpenes from Zedoariae Rhizoma and Saussureae Radix showed vasorelaxant activity in vitro. These results may be related to the treatment effects of those Chinese herbal medicines for "Oketsu" syndrome.
With age, fluorescent granules called lipofuscin or age pigments accumulate in the retinal pigment epithelium. These granules may be involved in age related macular degeneration (AMD), a leading cause of blindness in elderly people for which no remedy exists. The major hydrophobic human fluorescent pigment of lipofuscin (less than 100μg) was isolated from 250 human eyes and was assigned as structure 1 in 1993 by Eldred et al. In these studies, we revised the structure of this compound as A2E (2), containing two molecules of vitamin A and one molecule of ethanolamine. This amphiphilic structure contains a pyridinium head group and two retinoid side chains. The identity of this fluorophore was confirmed by total chemical synthesis that involved coupling of a pyridine framework with the aliphatic side chains. Further, an investigation aimed at optimizing the yield of a biomimetic synthesis from two mols of all-trans-retinal and ethanolamine provided A2E in 49% yield in only one step. These results have allowed for the development of HPLC conditions that allow nanogram quantities of A2E to be detected from the extracts of tissue samples. By using 5% of individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of iso-A2E, a new double bond isomer, and the identification of retinols in these HPLC chromatograms. Exposure of either A2E or isa-A2E to light gave rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. The biological properties of A2E in retinal pigment epithelium cells will also be discussed.
During the course of our investigation searching for new bioactive substances from marine organisms, we isolated an extremely potent cytotoxic polyketide designated callystatin A (1) from the marine sponge Callyspongia truncata and determined its absolute stereostructure. In order to create a new anti-tumor lead compound, the structure-activity relationships of 1 using several synthetic analogues have been studied. As a result of evaluating their cytotoxicities, the following crucial factors have been disclosed; 1) α, β-unsaturated δ-lactone moiety is a conclusive pharmacophore; 2) 5-R configuration, asymmetric center at C-10, and β-hydroxy ketone moiety contribute to affinity to receptor molecule. From the experiment utilizing the fission yeast expressing an NES-GFP-NLS fusion protein, callystatin A (1) was found to inhibit nuclear export signals (NES) dependent transport of proteins from the nucleus to the cytoplasm as well as leptomycin B (2). In addition, 1 was shown to inhibit direct binding between NES and CRM1 through the competitive experiment by use of the biotinylated leptomycin B.
In the course of screening for apoptosis-inducing agents, three novel compounds named polyoxypeptins A (1) and B (2), and chloptosin (3) were isolated from the cultured broth of Streptomyces strain MK498-98F14 by solvent extraction and column chromatography. Structural elucidation of 1 (C_<45>H_<76>N_8O_<15>) by MS and NMR analyses revealed that it is a cyclic hexadepsipeptide having a novel amino acid and a previously unrecognized N-acyl side chain. The depsipeptide consisted of 3-hydroxyleucine, N-hydroxyvaline, N-hydroxyalanine, piperazic acid, 5-hydroxyhexahydropiperazine-3-carboxylic acid, and an unusual and hitherto unreported amino acid, 3-hydroxy-3-methylproline. Chiralities of L-alanine and L-valine were determined by chiral TLC analysis. Then, the absolute structure of 1 was determined with the relative structure obtained from X-ray crystallographic analysis, and the new amino acid was confirmed to be (2S,3R)-3-hydroxy-3-methylproline. MS and NMR of 2 (C_<45>H_<76>N_8O_<14>) exhibited that it is a monodeoxy compound of 1. Stereochemistry of each amino acid was determined by hydrolysis of 2. By the spectroscopic and chemical degradation studies, the structure of 3 (C_<68>H_<94>N_<18>O_<18>Cl_2) was elucidated to be a dimeric cyclohexapeptide consisting of D-valine, (3S)-and (3R)-piperazic acids, O-methyl-L-serine, D-threonine, and (2S,3aR,8aR)-6-chloro-3a-hydroxy-hexahydropyrrolo[2,3b]indole-2-carboxylic acid. Both 1 and 2 at a concentration of about 0.1μg/ml induced early cell death, nuclear fragmentation, and internucleosomal DNA scission, all of which are characteristic of apoptosis, in the apoptosis-resistant human pancreatic adenocarcinoma cell line AsPC-1. Compound 3 also showed apoptosis-inducing activity and a strong antimicrobial activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus.
In the course of a screening program for specific inhibitors of human topoisomerase I using a recombinant yeast, we have discovered two new active compounds in the culture broth of a fungus, Phoma sp. BAUA2861. We designated these compounds as topopyrone A (1) and B (2). The physico-chemical properties of 1 and 2 are summarized in Table 1. The molecular weight of 1 and 2 were deduced to be 372 from m/z 371 [M-H^-] in ESI-Neg. and the presence of one chlorine atom was indicated by a peak intensity ratio of isotope peaks. The molecular formulae of 1 and 2 were finally established as C_<18>H_9O_7Cl by HRFAB-MS. Treatment of 1 with an excess of diazomethane afforded the methyl derivative (3) whose HREI-MS gave the molecular formula C_<22>H_<17>O_7Cl. The ^1H and ^<13>C NMR data of 3 are summarized in Table 2. As a result of 2D correlation NMR experiment, these signals were assigned as shown in Fig. 2. Acetylation of 1 gave triacetate (5), which had no epoxide ring. These results indicated that epoxide ring of 3 was formed by addition of diazomethane with loss of N_2 to the ketone of 1. Methylation of 2 with diazomethane gave compound 4. From the result of 2D correlation NMR experiment, the ^1H and ^<13>C NMR data of 4 (Table 2) was assigned as shown in Fig. 3. The structures of 1 and 2 were thus elucidated as shown. Topopyrone A (1) and B (2) inhibited selectively recombinant yeast growth dependent on expression of human topoisomerase I with IC_<50> value of 1.22ng/ml and 0.15ng/ml, respectively. The activity and selectivity of 2 were comparable to those of camptothecin. The relaxation of supercoiled pBR322 DNA by human DNA topoisomerase I was inhibited by these compounds as potently as by camptothecin. 1 and 2 were cytotoxic to all the tumor cell line when tested in vitro, but slightly weaker than camptothecin. We also found that 2 has a potent inhibitory activity against herpes virus, especially varicella zoster virus (VZV). It inhibited VZV growth with EC_<50> value of 0.038μg/ml, which is 24-fold stronger than that of acyclovir (0.9μg/ml). 1 and 2 were inhibitory to Gram-positive bacteria including quinolone-resistant MRSA.
Aflastatin A (1) is a specific inhibitor of aflatoxin production by Aspergillus parasiticus. It is produced by Streptomyces sp. MRI142 and has a novel structure of a tetramic acid derivative with a long alkyl side chain. The side chain is polyhydroxylated and acyclic except for a tetrahydropyran ring moiety. Determination of the absolute configuration of 1 is very important for further studies, but there has been a little imformation about the stereochemistry of 1. Untill now, we have determined the absolute configuration of chromophore moiety (fragment A) and the relative stereochemistry around the tetrahydropyran ring. In this experiment, absolute configurations of remaining 26 chiral centers in 1 were chemically elucidated. First, two small fragment molecules were prepared from 1 or its methyl ether (5) by degradation experiments mainly using NaIO_4, and thier absolute structures were assigned as (R)-3-hydroxydodecanoic acid (2) and (R)-1,2,4-butanetriol tribenzoate (3). Next, an acyclic fragment molecule (fragment B) with 13 chiral centers was obtained from 1 by NaIO_4 oxidation, and its relative stereochemistry was elucidated by J-based configuration analysis. By analyzing coupling constants of ^3J_<H,H> and ^<2,3>J_<C,H> obtained from E. COSY, HETELOC and phase-sensitive HMBC spectra and partly supplemented ROE data, the relative configuration of fragment B was established. Finally, by futher J-based configuration analysis using a fragment molecule prepared from 5 with 28 chiral carbons, all relative configurations in the alkyl side chain of 1 were clarified. By connecting these relative configurations with the absolute configurations of the first four fragment molecules, absolute stereochemistry of 1 was fully determined. Aflastatin B (6), a minor component of 1, was purified and its structure was identified as a N-demethylated derivative of 1. Blasticidin A (7) was found in 1955 in the culture broth of S. griseochromogenes, and its isolation and detailed physicochemical properties were reported in 1968. However, the structure of 7 was unknown. Since close homology was noticed between the physicochemical properties of 1 and 7, the biological activity of 7 was examined, and 7 was found to inhibit aflatoxin production. This finding prompted us to investigate the structure of 7, and we determined the structure of 7 as a structurally related compound of 1. The biological activities of aflastatin B (6) and blasticidin A (7) were assessed, and it was shown that these compounds inhibited the aflatoxin production as strongly as 1.
Absolute stereochemistry of mycalolides, actin-depolymerizing macrolides, has been determined on the basis of spectral and chemical synthesis methods. Relative stereochemistry was disclosed by interpretation of NMR data of the natural products, fragment 8 obtained by oxidative degradation, and synthetic compounds 9 and 10. Absolute stereochemistry was determined by a combination of Mosher analysis of the natural products and degradation products and chiral HPLC analysis of degradation products.
CrTX-A and -B which have molecular masses of 43 and 46kDa (caliculated from SDS-PAGE result), respectively, are potent hemolytic proteinous toxins isolated from the tentacle of the box jellyfish (sea wasp) Carybdea rastoni. From the immunoblotting experiment results, it was shown that CrTX-A and CrTX-B have quite closely related molecular structure with each other. CrTX-A was mainly localized in the namatocyst and CrTX-B was detected from the tentacle and not from the nematocyst. It is proposed that there is the passway which CrTX-B was produced at first in the tentacle, then processed to CrTX-A and migrated into nematocyst. CrTX-A caused inflammation on mice skin when subcutaneously injected. Furthermore, CrTX-A showed fatal toxicity to mice with 20mg/kg (i.v.) potency. The full-length cDNA (1600 bp) which encodes CrTXs was sequenced. The deduced amino acid sequence, composed of 450 amino acids, of CrTXs shows no significant homology with any known protein. This is the first complete sequence of a jellyfish proteinous toxin to be reported.