Physalins are 13,14-seco-16,24-cyclosteroidal constituents of Physalis plants. Physalin Q (11) possessing endoperoxy structure was isolated from Physalis alkekengi var. francheti. The reported structures of physalins E, H (13), and K (9) were revised. According to the partial structure around C(27) physalins are classified into three types I-III, and interconversion reaction was found between the physalins of types I and II, e.g., physalins B (1) and C (6). Interrelationships among known physalins were suggested. Physalins F (2) and J (14), which were epoxidation products of physalin B, were actually converted to physalins D (15), H, I (16), etc. Transformation by means of photosensitized oxygenation was also observed, e.g., physalin B to physalin K. Activated charcoal-mediated hydroxylation at C(25) was found affording new physalin analogs. Physalins underwent benzilic acid rearrangement-type skeletal conversion to yield compounds possessing neophysalin skeleton. Some of these physalins and their derivatives exhibited in vitro cytotoxic activity against HeLa cells.
Although much attention has been given to cell differentiation inducers as new types of anti-tumore agent, only a few studies has been reported on differentiation inducers from plant sorces. Therefore, we have searched for naturally occouring substances which induce differnciation of leukemia cells. From Condurango Cortex (Marsdenia condurango) (Asclepidaceae) and Periplocae Cortex (Periploca sepium B.) (Asclepidaceae), many kids of pregnan derivatives and some cardenolides. From withania (Withania somnifera) (Solanaceae) (using Indian market withania and withania cultivated at the medicinal plant garden of this university), more than thirty kinds of withanolides were isolated. From Physalis alkekengi (Solanaceae), physalin and neophysalin derivatives were isolated. Structural elucidation of these 60 kinds of steroid derivatives were carried out by means of spectral methods, especially using NMR spectroscopy including H-H COSY, H-C COSY, HMBC, NOE technics. These steroidal derivatives were tested on cell differentiatio inducing activity against mouse myeloid leukemia (M1) cells. Many kinds steroid derivatives showed the activity. Of these active compounds, some kinds of withanolids, 27, 33, 34 and 35 having 4β-hydroxy-5β,6β-epoxy-2-en-1-one structure of AB ring, showed potent differentiation inducing activity.
In the course of our screening to find biologically active principles contained in Japanese and Chinese traditional medicines, several natural medicines were found to contain inhibitors of ethanol absorption. By monitoring the inhibitory effect on ethanol absorption in rats, many active triterpene oligoglycosides were isolated and their structures were clarified. The inhibitory effects of these triterpene oligoglycosides on ethanol absorption were examined, and it was found that the active triterpene oligoglycosides were classified into the following three types. I. Oleanolic Acid and Hederagenin 3-O-Monodesmosides-Elatosides- Active oleanolic acid 3-O-monodesmosides named elatosides A and B were isolated from the bark of Aralia elata (Araliaceae) together with elatosides C and D. Examination of the inhibitory activity of oleanolic acid and hederagenin glycosides led us to presume the following structure-activity relationships: 1) the 3-O-polar function such as glucuronic acid or disaccharide is essential to the activity; 2) the 28-ester glucoside moiety significantly reduces the activity. II. Acylated Oleanene 3-O-Monodesmosides-Escins and Camelliasaponins- New acylated triterpene 3-O-monodesmosides were isolated as active principles of ethanol absorption bioassay; escins-Ia, Ib, IIa, IIb, and IIIa from the seeds of Aesculus hippocastanum (Hippocastanaceae) and camelliasaponins A1, A2, B1, B2, C1, and C2 from the seeds of Camellia japonica (Theaceae), and it was found that the acyl moieties in these saponins were essential to exerting the activity. III. Oleanene Acylated 3,28-Bisdesmosides-Senegasaponins- New active principles, senegasaponins a and b, were isolated from Polygala senega var. latifolia (Polygalaceae) and their structures were determined. Detailed examination of their structure-activity relationships indicated that the acylated 28-O-oligoglycoside moiety related to the inhibitory effect on ethanol absorption.
On the basis of a literature survey, the effects of methanol extracts of 80 herbs on hair regrowth were investigated. Hair regrowth-promoting activity was measured by Ogawa's method using 9-week-old C3H/He mice, in which the anagen hair regrowth after removal of back telogen hair is evaluated. On the 13th day after topical application, 18 kinds of herb extracts showed apparent hair regrowth promotion on the mice. Bioassay-guided fractionation of two potent herbs, Polygara senega var. latifolia and Polyporus umbellatus, afforded eight active principles, 1〜8, by combination of column chromatography (Amberlite XAD-2, Sephadex LH-20, silica gel and HPLC). The structures of these compounds were determined by physicochemical data, as senegose A, senegin II, III, desmethoxysenegin II, 3,4-dihydroxybenzaldehyde, polyporusterone-A and B, and acetosyringone. The structure of polyporusterone A, which showed an apparent activity at a dose of 0.1μg/mouse, was determined to be (+)-2b,3b,14a,20b,(22R)-pentahydroxy-(24S)-methyl-5b-cholest-7-ene-6-one by X-ray crystallographic analysis. 14 analogs to the active principles promoted hair regrowth on the mice.
During our continuous screening for acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors of microbial origin, a soil-isolated new genus fungus, Albophoma yamanashiensis, was found to produce a new series of ACAT inhibitors named terpendoles A〜L (1〜12). Structures of 1〜12 were elucidated by NMR and other spectroscopic studies. They consist of diterpene and indole moieties in common like indoloditerpene group of paspaline (13) and emindole SB (14). Compounds 5〜7 had the same carbon skeleton as 13. One methyl residue of 13 was removed to form epoxide in 2, 8 and 9 and additional isoprene unit was bonded to them in 4 and 10. The additional isoprene was cyclized to form 1,3-dioxane in 1, 3 and 11 and 12 had one more prenyl residue at indole. The relative configurations of 3, 4 and 5 were confirmed by NOE experiments and X-ray crystallographic analyses. Compounds 3 and 4 showed the most potent ACAT inhibitory activity with an IC_<50> value of 2.1μM and 3.2μM, respectively in an in vitro enzyme assay. Two known compounds, 13 and 14, were also isolated from the culture broth of the producer. But they showed weak activities. Evaluation of their ACAT inhibition in the cell assay using J774 macrophages indicated that 4 inhibited the formation of cholesterylester specifically.
In the course of our screening for endothelin antagonists from microorganisms, we found new compounds stachybocins A(1), B(2) and C(3) from the fermentation broth of Stachybotrys sp. They were extracted with EtOAc and then purified by aluminum oxide and silica gel column chromatographies. The molecular formula of stachybocins were determined to be C_<52>H_<70>N_2O_<10> (1) and C_<52>H_<70>N_2O_<11> (2 and 3) by HR FAB-MS and NMR data. The ^<13>C-NMR spectrum of 1 confirmed the presence of 52 carbons including 46 signals of the pairs or equivalent carbons. All one-bond ^1H-^<13>C connectivities were established by the HSQC spectrum using a pulse-field-gradient (PFG) technique. PFG DQF-COSY and HMBC experiments of 1 generated partial structures 1-a and 1-b in Fig. 3. Further detailed analysis of ^<13>C-^1H long range spin couplings enabled to connect these units to give the total structure of 1 as shown in Fig. 1. 2 and 3, monohydroxy derivatives of 1, showed similar ^1H and ^<13>C NMR spectra each other. Detailed analysis of HMBC spectral data established that 2 and 3 have an additional hydroxy group at C-17 and C-17', respectively. The relative stereochemistry of the sesquiterpene portion of the stachybocins was deduced by the NOESY data. The absolute configuration of C-24 was determined to be S by isolation of L-lysine from 1 by Jones oxidation followed by acid hydrolysis.
In the course of searching new substances produced by marine-microorganism, three anti-microalgal substances named halymecins A(1), B(2), C(3) were isolated from the fungus, Fusarium sp. FE-71-1 of an alga isolate, by centrifuged partition chromatography and column chromatography. The molecular formula of 1 was determined to be C_<42>H_<76>O_<14> on the basis of HR FAB-MS. The ^1H and ^<13>C NMR showed the molecule contained one acetyl group, four methyl groups, 16 methylene groups, 8 methine groups bound to oxygen and four carbonyl carbons besides acetyl carbon. The acetyl derivative of 1 simplified ^1H-NMR spectrum, suggested that 1 was the tetramer of single unit and this unit was elucidated to 3,5-dihydroxydecanoate by extensive analyses of COSY and TOCSY spectra. The position of the acetyl group of 1 was determined by HMBC spectra. Acid degradation of 1 afforded an optical active 3-hydroxy-δ-lactone, coincided with the reported 3R,5R stereo-isomers unambiguously by NMR and optical rotation. The structure of 2 was elucidated to a β-D-mannoside of 1 from the spectral analysis of NMR and chemical degradation. 3 was also elucidated to mono acetyl derivative of 3,5-dihydroxydecanoate trimmer as a same manner. We also found the anti-microalgal fraction from another fungus, Acremonium sp. FK-N30 of an alga isolate. NMR spectral features were similar to those observed for 1, 2 and 3 but without having acetyl group. This fraction showed a single peak by HPLC however it was proved to be the mixture of halymecin D (4) and E (5) by negative FAB-MS. 4 had a molecular formular of C_<40>H_<74>O_<15> by HR FAB-MS suggested the existence of trihydroxydecanoate units as well as 3,5-dihydroxydecanoate. Negative FAB CID revealed that 4 was composed of two dihydroxydecanoate and two trihydroxydecanoate and the sequence was explained by fragmentation brought by cleavage of each ester bond. The hydroxy groups of trihydroxydecanoate moieties were elucidated to 3, 5, 9 positions by COSY spectrum. A molecular formular of C_<30>H_<56>O_<11> was established for 5. The structure was elucidated to be composed of two 3,5-dihydroxydecanoate and one 3,5,9-trihydroxydecanoate The sequence was explained by negative FAB CID.
Our continuing interest in marine toxins such as palytoxin focused on the structure determination of the potent shellfish poison, pinnatoxins which was recently reported as a Ca^<2+> channel activator by Chinese inverstigators. We isolated two new toxins, named pinnatoxin A (1), B, C (2) and one congener pinnatoxin D (3) from the 75% MeOH extracts of the Okinawan Pinna muricata. Espesially, the toxicity of pinnatoxin B, C (2) is as significant as that of tetrodotoxin. Molecular fomulas of (1), (2) and (3) were deduced to be C_<41>H_<61>NO_9. C_<42>H_<63>NO_<10> and C_<45>H_<67>NO_<10> from HRFABMS spectra and NMR spectra data respectively. Their planar structures were determined mainly by interpretation of 2D NMR (DQF-COSY, HOHAHA, HSQC-HOHAHA, HMBC) spectra to be a macrocyclic polyether including carboxylate and iminium functionality. The relative stereochemistry was proposed by the detailed analysis of the NOESY, ROESY data.
During our search for new bioactive substances from marine organisms, we have examined extracts of the Floridian bryozoan Amathia convoluta and obtained the six new alkaloids, convolutamides A (1a), B (1b), C (2a), D (2b), E (3a), and F (3b), possessing an N-acyl-γ-lactam moiety with a dibromophenol group. Also, from the same extracts, the five new β-phenylethylamine alkaloids, convolutamines A (4), B (5), C (6), D (7), and E (8) were obtained. The structures have been elucidated on the basis of extensive spectroscopic data such as ^1H-^1H COSY, HMQC, HMBC, NOESY, HMQC-HOHAHA, INADEQUATE experiments. The structures of convolutamides A〜F were shown in Fig. 1. The NMR data of convolutamides A (1a) and B (1b) were shown in Table 1. The partial structure of the γ-lactam moiety was deduced from the COSY correlations as well as the HMBC cross-peaks, in addition to FAB MS/MS spectral data (Table 2). In Fig. 2, the daughter ions observed in the negative FAB MS/MS for the acyl part of 1b and 2b were illustrated. On the other hand, the structures of convolutamines A〜E were shown in Fig. 3. The ^1H and ^<13>C NMR data were indicated in Table 3 and 4. The relative stereochemistry of the morpholine ring in the structure of convolutamine E (8) was illustrated in Fig. 4. To our knowledge, compounds with oxazolidine as convolutamine D (7) are rare in nature. While, convolutamides A〜F (1a〜3b) may belong to an unprecedented class of natural product possessing a unique γ-lactam and dibromophenol ring system. Convolutamides A (1a) and B (1b) exhibited against L1210 and KB cells with IC50 values of 4.8 and 2.8μg/ml, respectively. Also, convolutamines A (1), B (2), and D (4) exhibited against P388 cell with IC50 values of 10.6, 4.8, and 8.6μg/ml, respectively.
A structurally unique and potent cytotoxic macrolide (1), halichomycin, has been isolated from a strain of Streptomyces hygroscopicus which was isolated from the gastrointestinal tract of the marine fish Halichoeres bleekeri. Furthermore, leptosins A (2)-G(8), G_1(9), G_2(10), H(11)-K(14), K_1(15) and K_2(16), belonging to a series of epipolythiodioxopiperazines, have been isolated as cytotoxic metabolites of a strain of Leptosphaeria sp. isolated from the marine alga Sargassum tortile. Their stereo-structures have been elucidated by spectroscopic analyses and some chemical transformations. The X-ray analysis of 14 demonstrated that two conformers are incorporated in a single crystal in a 1:1 ratio. NOE analyses for solution conformation showed 14 and 16 to exist in only one conformer and in a 1:1 mixture of two conformers in CHCl_3 solution, respectively. All the compounds exhibited significant cytotoxicity against cultured P 388 cells. Especially potent cytotoxicities were observed in dimeric epipolythiodioxo-piperazines (2-4, 8-11 and 14-16). Leptosin A (2) and C (4) exhibited significant antitumour activity against Sarcoma 180 ascites.
Three new cytotoxic dimeric steroidal alkaloids, ritterazines A (1), B (2), and C (3) have been isolated fom the tunicate, Ritterella tokioka (family, Polyclinidae) collected off the Izu Peninsula. Ritterazines A, B, and C exhibited cytotoxicity against P388 murine leukemia cells with IC_<50>, values of 3.8, 0.018, 9.6ng/mL, respectively. Their structures have been elucidated by detailed spectroscopic analyses. Ritterazine A had a molecular fomula of C_<54>H_<76>N_2O_<10> as established by HR-FABMS. Partial structures derived from the COSY spectrum were connected by interpretation of HMBC data, which led to two polyoxygenerated steroid skeletons, one with a rearranged skeleton. The presence of a pyrazine ring which linked the two units was deduced from UV, NMR, and HR-FABMS data. Ritterazine B had a molecular formula of C_<54>H_<78>N_2O_9 as determined by HR-FABMS. A gross structure similar to ritterazine A was deduced by interpretation of NMR data. Relative stereochemistry of each steroid nucleus was determined by NOESY data and coupling constants. Ritterazine C was an isomer of ritterazine B. In fact, ritterazine C was readily derived from ritterazine B by acid-catalized isomerization. The structure of ritterazine C including absolute stereochemistry was determined by spectral and chemical methods.
Prymnesium parvum is a notorious red tide organism belonging to Haptophyceae. The flagellate causes serious damages to aquaculture and marine ecology in many parts of the world. In recent years the threat is most significant to salmon culture in Norway. Despite effort of many research groups. the causative ichthyotoxin named prymnesin has never been purified successfully. Hence chemical and toxicological studies of the toxin have been hampered. Recently we have succeeded for the first time to isolate two hemolytic-ichthyotoxic substances, named prymnesin-1 (PRM1) and prymnesin-2(PRM2). Their hemolytic potencies exceeded that of plant saponin more than 1000 times. FAB-MS data on molecular ion species and their isotope distribution pattern containing ^<35>Cl and ^<37>Cl allowed us to deduced a molecular formula C_<107>H_<154>O_<44>NCl_3 for PRM1 and C_M<96>H_<136>O_<35>NCl_3 for PRM2. ^<13>C NMR(BBD, DEPT) measurement on ^<13>C enriched PRM2 N-acetate indicated 96 carbon signals (2-methyls, 24-methylenes, 10-olefinic methines, 53-other methines and 7-quaternary carbons). Further detailed analyses of both ^1H-NMR (COSY, HOHAHA, NOESY) and ^1H observed ^1H-^<13>C correlation NMR (HSQC, HMBC) data disclosed for the first time the unprecedentedly unique structure of PRM2. The molecule is characterized by the 14 ether rings (6/6/6/7/6, 6/6, 6, 6/6, 6/6, 6), polyene-polyine bonds, three chlorine and one nitrogen atoms, and one pentose. Structural confirmation was further made by measuring NMR spectra of PRM2, the N-acetate, and per-acetate in two different solvents. PRM1 probably has additional one pentose and one hexose to the PRM2 skeleton.
Doliculide (1), a cytotoxic 16-membered cyclodepsipeptide has been isolated from the Japanese sea hare Dolabella auricularia. This compound exhibited remarkable cytotoxicity against HeLa-S_3 cells with an IC_<50> of 0.0054μg/mL. The gross structure was elucidated by using primarily 2D NMR technique. The absolute stereostructure was deduced by extensive NOE experiments of doliculide (1) and the chiral HPLC analysis of N-methyltyrosine that was obtained by acidic hydrolysis of doliculide (1). The proposed structure of doliculide (1) was unambiguously confirmed by the efficient enantioselective total synthesis. The polyketide unit (12) was synthesized with almost complete stereocontrol by application of the Evans aldol reaction followed by the Barton deoxygenation reaction. Coupling of the polyketide unit (12) with glycine tert-butyl ester and subsequently with the tyrosine derivative (18) gave the linear depsipeptide (19). Finally, 16-membered ring formation was accomplished successfully by macrolactamization. The yield of the synthesis, based on the longest linear sequence, was 11%. Artificial analogues (21-25) of doliculide (1) were synthesized and the structure-activity relationship was examined. The results revealed the importance of the 3-iodo-N-methyl-D-tyrosine moiety with respect to cytotoxicity.
During the course of our investigations in search of new biologically active substances from marine organisms, we have investigated the chemical constituents of the Okinawan marine sponge Dysidea arenaria by taking advantage of bioassay-guided fractionations and separations. Examinations of cytotoxicity against L1210 and KB cell lines have led us to the isolation of a very potent cytotoxic depsipeptide named arenastatin A (1) [IC_<50> 5pg/ml (KB)]. The plane structure of arenastatin A (1) have been first elucidated on the basis of 2D NMR analysis. Then, the absolute configurations of the 2-hydroxy-4-methylpentanoyl and O-methyltyrosine moieties in 1 have been determined by HPLC identification of the respective phenylethylamide and urethane derivatives. Next, the relative stereostructures of the C-5 C-8 part in 1 has been presumed as 5S, 6S, 7R, and 8R from the ROESY spectrum of 1 together with the accumulated NMR evidence. Arenastatin A (1) is fairly unstable for acidic and basic treatment. Thus, methanolysis of arenastatin A (1) with K_2CO_3-MeOH followed by imidazole treatment furnished a tetrahydrofuran derivatives 4. For the purpose of confirming the absolute stereostructure of 1. we have then synthesized 4 starting from a 2S,3R-diol ester 5. Finally, we have accomplished the total synthesis of 1 starting from 1-O-TBDPS-1,3-propanediol (23). It is very interestingly to mention that arenastatin A (1) corresponds to a β-alanine analog of cryptophycin B. which was recently isolated from a cultured cyanobacteria of Nostoc sp as a significantly potential cytotoxic compound.
Ascidian trypsin inhibitor (ATI) from hemolymph of the solitary ascidian, Halocynthia roretzi, consists of a single polypeptide chain with 55 amino acid residues that shows no extensive homology to other known protease inhibitors. It has four disulfide bridges in a molecule. This is in marked contrast with the better characterized Kunitz-type and Kazal-type inhibitors, most of which contain three disulfide bridges in a molecule. In the present study, we analyzed the solution structure of ATI by means of 2D-NMR and simulated annealing calculations. The resulting structure of ATI is characterized by an α-helical conformation in the sequence, Asn35-His43, and a three-stranded antiparallel β-sheet in the sequence, Leu21-Ile26, Ala29-Arg34 and Glu48-Asn51. The secondary structure and the overall folding of main chain of ATI are similar to those of the third domain of Japanese quail ovomucoid and human pancreatic secretory trypsin inhibitor being a typical Kazal-type inhibitor. These results indicate that ATI is classified as a member of the Kazal-type inhibitor family on the basis of the tertiary structure. On the other hand, ATI has the CSH motif (cystine stabilized α-helical motif), the α-helix structure containing a Cys-X_1-X_2-X_3-Cys sequence stabilized by two disulfide bridges with Cys-X-Cys, which are found in a hormonal peptide (endothelin-1), a honey bee toxin (apamin), a scorpion toxin (charybdotoxin), and so on. It has never been found that Cys at the X_3 position forming the disulfide bridge with other Cys as shown in ATI. These results show that the presence of Cys at the X_3 position does not interfere with the formation of the helix of the CSH motif.
The blue pigments, commelinin, from Commelina communis, and protocyanin, from Centaurea cyanus, are metal-complex anthocyanins, named a metalloanthocyanin. The exact molecular weight of commelinin (ca 8846), of which the structure has already been determined by crystallographic analysis, was succeeded directly to be measured by negative mode ESI-MS. Protocyanin was prepared from its components, succinylcyanin (Sucy), malonylflavone (Mafl), and ferric and magnesium ion. The composition of protocyanin was determined by ICP atomic analysis, ESR, Mossbauer spectra and ESI-MS to be [Sucy_6Mafl_6Fe^<3+>Mg^<3+>]. Protocyanin-like pigment was prepared from Al^<3+> instead of Fe^<3+>, then ^1H NMR was measured. The flavonid components were stacked tightly and the arrangement of their components is very similar to that of commelinin. It was larified that mechanism of blue color development of protocyanin arises from LMCT between Fe^<3+> and anhydrobase anion of Sucy by MCD measurement.
New methods for elucidating the absolute configuration of Type A alcohol and Type B carboxylic acid are reported. Determination of the absolute configurations of such kind of long-chain compounds has been extremely difficult and they have usually remained undetermined. Optically active 2NMA (2-naphthylmethoxyacetic acid; 1) was obtained in gram scale by chromatographic separation of the diastereomers C and D followed by acid hydrolysis. The absolute configurations of the long-chain alcohols 3, 4, 5, and 6 have been determined by analyzing the NMR spectra of their esters of (R) and (S)-1. In all the cases, the protons located on the same plane with the 2-naphthyl groups of 1 greatly shifted upfield, which made the assignment very easy. Magnitude of Δδ values gave hints about the conformation of the respective compounds. PGDA (phenylglycine dimethyl amide; 7) and PGME (phenylglycine methyl ester; 8) have been developed as the chiral anisotropic reagents for elucidation of the carboxylic acid of Type B. The conformation of the amides, B-PGDA and B-PGME, was assumed as H by consideration of the electronic repulsion of the substituents. Eventually the conformation H was verified by X-ray analysis of 9, MM calculation on 11, and NOE study on 10. PGDA and PGME were applied to the carboxylic acids 11-17, the absolute configurations of which are known. Δδ values (Δδ=δ_R-δ_S; see model B) are depicted in each structure. The absolute configurations derived from the present method are in all cases the same as known ones,. which means that the method using PGDA and PGME is applicable to the carboxylic acid whose absolute configuration is unknown.
In 1984, Marfey reported that a mixture of D- and L-amino acid can be separated into each enantiomer by usual reversed phase HPLC after derivatization with FDAA (1-fluoro-2,4-dinitrophenyl-5-L-alanine-amide). The method has been refered to as "Marfey's method" and has an advantage that it can determine sterochemistry of an amino acid by simple operation. Although it has been widely used, it is hard to apply the method to non-proteinogenic amino acid, because it is difficult to obtain authentic sample. In such a case it would be effective to combine Marfey's method with an appropriate mass spectrometry. In this study we tried to establish a total method for determination of constituent amino acids including stereochemistry, even unusual amono acids, in peptides without authentic sample. In order to establish the total system shown in Fig. II, the following four problems have to be resolved: 1. Elucidation of limitation of Marfey's method and its separation mechanism 2. Optimization of various conditions for combination of Marfey's method and mass spectrometry 3. Collection of structural information by LC/MS and optimization of epimerization 4. Application of the advanced method to natural products As a result of extensive experiments Marfey's method proved to have a wide applicability except for a few basic amino acids. A separation mechanism was proposed based on NMR measurement of D,L-Val-FDAA derivative and elution behavior of various amino acids. According to this mechanism both isomers can be resolved due to the difference of their hydrophobicity which is derived from cis or trans configuration of two more hydrophobic substituents at both α-carbons of introduced amino acid and L-Ala-NH2, so that D-isomer interacts more strongly with ODS silica gel and has longer retention time. Although Frit-FAB and ESI were used as interfaces in the present study, derivatized amino acids with FDAA showed poor sensitivities. Since it was considered that this result may be due to poor hydrophobicity of the derivatives, a new derivatization reagent, FDLA (1-fluoro-2,4-dinitrophenyl-5-L-leucine-amide) was prepared. Derivatized amino acids with FDLA showed the almost same retention behavior as that with FDAA and much stronger sensitivity than that with FDAA by both ionization methods. The advanced Marfey's method was successfully applied to analysis of hydrolyzate of a known antibiotic bacitracin A composed of 12 amino acid residues and it was ensured that the combination method is much superior to the original method. The method is being applied to analysis of naturally occurring peptides from cyanobacteria and would contribute to progress of various researches in this field.
In the squid luminescence in S. oualaniensis, the system requires molecular oxygen and monovalent cations (e.g. Na+, K+, etc.) for bioluminescence. We found that only a high molecular fraction (from gel filtration chromatography with Bio-gel P-6) emits light (470nm) by addition of KCl and O_2. When the homogenate was extracted only with MeOH (without acetone), dehydrocoelenterazine (1) was extracted, its amount being estimated about 20% from the total luminescent light. None of 1 nor 5 was, however, detected after luminescence (470nm) of the homogenate of the photogenic organs by addition of KCl, indicating that the bioluminescence of S. oualaniensis consumed dehydrocoelenterazine 1. Although dehydrocoelenterazine (1) itself does not exhibit any chemi-luminescence activity, some derivatives produced by addition (such as 4, 5, 6 etc.) should retain the activity. The dithiothreitol adduct to dehydrocoelenterazine, although existing only as equilibrium, in agreement with higher and longer luminescence activity of this squid as reported by Tsuji et al. Dehydrocoelenterazine absorbs long wavelength light to become reddish color, but the photogenic organ is not red but yellow-brown (identical with the acetone adduct 4), indicating that the existing conjugate system is broken. This phenomenon is interpreted by deconjugative addition of nucleophile in protein (e.g. a functional residue on lysine, cysteine etc.). These facts led us to conclude that dehydrocoelenterazine 1 exists as adduct as 5. In conclusion, the possibility of the dehydrocoelenterazine adduct as the luciferin of S. oualaniensis bioluminescence system becomes apparent through the current studies.
Soles of the genus Pardachirus are characterized by their chemical defense against predation with a copious and ichthyotoxic secretion being discharged upon disturbance. Two chemical classes of bioactive entities, namely, steroid monoglycosides and amphiphilic peptides, have been isolated from each secretion of two different biological species. Based on permeabilization of phospholipid bilayer exhibited by these components at the lower concentrations than their EC's for various bioactivity, their mechanisms of action may well be attributed to nonspecific derangement of animal cell membrane without binding to any particular biomolecule. In this study, an assay method of membrane permeabilization was employed, where calcein was encapsulated in unilamellar liposomes of egg-yolk phosphatidylcholine as the cellular model and its leakage outward was assessed by the increase of fluorescence, in order to evaluate (i) the function of the hydrophobic N-terminal region of pardaxin M-1, a representative peptidic component, as the membrane binding region, and (ii) the significance of the existence in the defense secretion of steroid glycosides, i.e., pavoninins and mosesins, which appear to behave in a similar manner as pardaxins in this membranal action but with lower potencies. As a result of the binding analyses with concentrations of the test sample and liposomes as two independent variants and application of Langmuir's adsorption isotherm with the two-state model to the outcomes, the N-terminal region was revealed not only to function as the strong binding region, but also to enhance when bound the deranging activity putatively of the α-helical region at the middle of the 33 amino acid sequence by the factor of 6 times. In this model assay with varied cholesterol contents in the lipid bilayer, the defensive steroid glycosides were shown to be indifferent to this membranal component in their permeabilizing action, being unlike the conventional hemolytic saponins. In addition, different kinetics exhibited here between the steroid glycosides and pardaxins implied that the former's is rather transient while the latter's is persistent with all-or-none rupture of liposomes. An extent of synergism, possibly with such differently allotted functions in the defensive action, was also shown between the two chemical entities.
3-Isopropylmalate dehydrogenase (IPMDH, EC 126.96.36.199) catalyses dehydrogenation and decarboxylation of threo-Ds-3-isopropylmalate (IPM) to 2-oxoisocaproate in the presence of NAD in the biosynthetic pathway of L-leucine. The features of substrate recognition of IPMDH derived from an extreme thermophile Thermus thermophilus HB8 were studied. Various (2R,3S)-3-alkylmalic acids were synthesized enantioselectively by chirality transcription on a carbohydrate template, and subjected to the kinetic studies. Hydrophobic interactions play an important role in the substrate recognition. The kcat/Km increased according to the order of hydrophobicity of the substrate and reached to maximum with Et and i-Pro, but steric hindrance became counteractive in the bulkier alkyl derivatives. Thr-88 and Leu-90 were proposed to be responsible for these hydrophobic interaction in the active site. Electrostatic interactions between the C-1 carboxylate and Arg-104 are also important for substrate binding, since synthetic C-1-carboxamide of IPM was neither a substrate nor an inhibitor. 2-O-Methyl-IPM was found to be an uncompetitive inhibitor, thereby suggesting little contribution of the OH group. Although site-directed mutagenesis of IPMDH did not alter its substrate specificity to be recognizable for isocutrate, an interesting change of substrate specificity was observed with modified ICDH of T. thermophilus. Modification of the plausible carboxylate recognition site into a hydrophobic group (Asn99Leu) resulted in increase of ethylmalate recognition and decrease of isocitrate recognition. While modification of substrate recognition by site-directed mutagenesis is not simple, this new technology may become important in the future.
Prenyltransferases catalyze the sequential condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates with various chain lengths and stereochemistries. They are extremely interesting from both mechanistic and synthetic view points in that they catalyze the repetition of stereospecific condensation of IPP with prenyl diphosphates to give products with certain chain lengths and stereochemistries fixed by the specificities of the individual enzymes (Scheme 1). In order to extend the comparative study of prenyltransferase structures, and also to elucidate the molecular mechanisms of the catalytic function, we isolated and sequenced the gene for farnesyl diphosphate (FPP) synthase from a thermophilic bacterium, Bacillus stearothermophilus. We also succeeded in the overproduction in Escherichia coli of this thermostable enzyme, its purification to homogeneity, and crystallization. Molecular cloning of heptaprenyl diphosphate (Hep-PP) synthase of the same bacterium was also achieved by application of PCR reactions using some nucleotide probes whose sequences were found to be characteristic of prenyltransferase genes. As a result, this enzyme was proved to consist of two different proteins with molecular masses of 25kDa (Component A) and 36kDa (Component B). The amino acid sequence of Component B showed 31.9% and 31.8% similarities with those of FPP synthase of the same bacterium and hexaprenyl diphosphate (Hex-PP) synthase of Saccharomyces cerevisiae, respectively. In contrast, Compond A had no such similarities.
Astaxanthin is a carotenoid pigment abundant in the animal kingdom, e. g., red color appearing in salmons, porgies and shrimps is attributed to this pigment. But, the genes or enzymes mediating the biosynthesis of astaxanthin are unknown. This is because the late step enzymes in carotenoid biosynthesis are membrane-integrated proteins, which readily lose activity on solubilization, thus hampering their purification and subsequent cloning of the genes coding for them. We cloned the astaxanthin biosynthesis genes from a marine bacterium Agrobacterium aurantiacum by their functional expression in Escherichia coli carrying the Erwinia carotenogenic genes for the biosynthesis of lycopene or β-carotene. The region needed for astaxanthin synthesis is located on a 5.4kb BamHI fragments in the lycopene-producing E. coli. Its nucleotide sequencing showed there were five carotenogenic genes. The biosynthetic pathway from β-carotene to astaxanthin was elucidated for the first time at the level of the biosynthesis genes by chemical analysis of the pigments synthesized in the β-carotene or zeaxanthin-producing E. coli carrying the various combination of the A. aurantiacum carotenogenic genes.
Fosfomycin is a clinically used antibiotic possessing a unique C-P bond and an epoxide. Through the biosynthetic studies on fosfomycin using Streptomyces wedmorensis, we revealed that its biosynthetic pathway consisted of 4 steps: (1) intramolecular rearrangement of phosphoenolpyruvate to phosphonopyruvic acid (PnPy) catalyzed by phosphoenolpyruvate phosphomutase, (2) decarboxylation of PnPy to generate phosphonoacetaldehyde (PnAA), (3) methylation of PnAA to provide 2-hydroxypropylphosphonic acid (HPP), and (4) epoxide formation to form the final product. It should be emphasized that the epoxide is formed by dehydrogenation of the alcohol function of HPP and that unlike usual epoxidation, the molecular oxygen is not incorporated into fosfomycin. In addition, we have cloned the genes responsible for the biosynthesis of fosfomycin which were clustered in an about 11.0kb fragment on the chromosome. The nucleotide sequence of the fragment revealed the presence of 10 open reading frames including four biosynthetic genes and two resistance genes.
Mugineic acids are a group of compounds having ferric chelating activity. They are secreted from the roots of some gramineous plants under iron deficiency. These compounds play an important role in the acquisition of insoluble iron from soils by the gramineous plants. In the present study, the biosynthetic pathways of five mugineic acids were investigated in different gramineous plants using feeding experiments with ^<13>-and/or ^2H-labeled compounds and by NMR techniques. The results revealed that L-Met serves as a precursor for all mugineic acids tested. All mugineic acids share the pathway from L-Met to 2'-deoxymugineic acid although subsequent steps differ and are dependent on the plant species and cultivars. In oats, avenic acid A is biosynthesized from 2'-deoxymugineic acid by cleavage of the azetidine ring. In barley, hydroxylation at the C-2' position in 2'-deoxymugineic acid yields mugineic acid. Further hydroxylation at the C-3 position in mugineic acid produces 3-epihydroxymugineic acid and hydroxymugineic acid in beer barley and rye, respectively. All mugineic acids were found to have similar effect on iron uptake, suggesting that 2'-deoxymugineic acid is a key phytosiderophore. The biosynthetic pathway between L-methionine and 2'-deoxymugineic acid was investigated by observing the incorporation of label from ^<13>C, ^2H, and ^<15>N-labeled methionine in wheat roots (Triticum aestivum L. cv Minori). As a result, only the deuterium atom from the C-2 position of L-methionine was lost, while other atoms were completely incorporated when three molecules of methionine were converted to 2'-deoxymugineic acid. These observations are consistent with the conversion of L-methionine to azetidine-2-carboxylic acid, suggesting that L-methionine is first converted to azetidine-2-carboxylic acid during biosynthesis leading to 2'-deoxymugineic acid. Based on these results, a hypothetical pathway from L-methionine to 2'-deoxymugineic acid was postulated.
The enzymatic rearrangement in the formation of isoflavone skeleton from flavanone precursor was investigated in elicitor-treated cell suspension cultures of Pueraria lobata Ohwi (Leguminosae). A reaction intermediate formed in the reaction was identified as 2-hydroxyisoflavanone by mass, UV and ^1H NMR spectroscopies. The oxidative aryl migration of flavanone generating 2-hydroxyisoflavanone was catalyzed by a microsomal cytochrome P-450-dependent monooxygenase. The formation of 3-hydroxyflavanone was also observed as a by-product. The dehydration enzyme which catalyzes the conversion of 2-hydroxyisoflavanone into isoflavone was localized in a soluble enzyme fraction. The dehydratase was purified to apparent homogeneity and revealed to be a single polypeptide with a MW of 38 kDa. The treatment of P. lobata cells with yeast extract-elicitor resulted in the induction of the P-450 and the dehydratase, and the treatment of jasmonic acid or methyl jasmonate was also effective. The incorporation experiments using ^<18>O2 gas and ^<18>O-labeled flavanone demonstrated that 2-hydroxyl oxygen of 2-hydroxyisoflavanone was originated from molecular oxygen. Some unnatural flavanones, which lack 4'-hydroxyl function on the side phenyl ring, was also converted into corresponding isoflavones, suggesting that the contribution of spirodienone intermediate involved in the earlier proposals is not essential for the rearrangement reaction. Based on these observations a new mechanism, P-450-mediated hydroxylation associated with migration, was proposed for the oxidative aryl rearrangement.
Mn-salen catalysts (e.g. 3 and 14) were found to be effective catalysts for enantioselective oxygen-transfer reaction. Catalyst 3 showed extremely high enantioselectivity in the epoxidation of cis-olefins conjugated with aryl, alkenyl and alkynyl groups. For example, the epoxidation of 2,2-dimethylchromene and inden proceeded with high enantioselectivity of >99% ee and 98% ee, respectively, to give the corresponding epoxides that were key intermediates for the syntheses of (3S,4R)-4-amino-3-hydroxy-2,2-dimethylchromane derivatives, K-channel opener, and L-735,524, a drug for AIDs. The hypothesis on the mechanism of high asymmetric induction was also presented based on the data about the epoxidation with various types of Mn-salen catalysts. Catalyst 14 catalyzed the oxidation of aryl methyl sulfides with a good level of enantioselectivity up to 90% ee. Mn-salen catalyzed epoxidation could be successfully applied to the enantioselective synthesis of the antipode of the aggregation pheromone(21) of the flat grain beetle, cryprolestes pussilus. The synthesis started with the cross coupling reaction of cis-1-bromo-1-propene and 1-heptyne. The resulting cis-enyne 16a was subjected to the asymmetric epoxidation with Mn-salen catalyst 3 as a catalyst. The reaction gave a mixture of (2R,3S)- and (2R,3R)-epoxides which were treated with LAH without separation to give (R)-2-hydroxy-4-decyne (18a) of 86% ee. The internal acetylenic group was shifted to the terminal position by treatment with KAPA. After hydroxy protection as THP ether, the resulting terminal acetylene was carbon-extended to give 19. Deprotection of MPM ether followed by PDC oxidation and deprotection of THP ether gave hydroxy acid (20) which has been converted into 21 in two steps by Mori et al.
Cobalt(II) porphyrin-catalyzed reduction-oxygenation of α,β,γ,δ-unstaturated carbonyl compounds with molecular oxygen (1 atm) and triethylsilane in CH_2Cl_2-i-PrOH at room temperature proceeded smoothly and regioselectively to afford γ-hydroperoxy-α,β-unsaturated carbonyl compounds in good yields (Scheme 1 and Table 1). The obtained hydroperoxides were converted in one pot into γ-oxo-α,β-unsaturated carbonyl compounds by treating with acetic anhydride and 4-(N,N-dimethylamino)pyridine (DMAP), and into γ-hydroxy-α,β-unsaturated carbonyl compounds by reducing with trimethyl phosphite, respectively. By the application of this reductive oxygenation, we attempted total syntheses of natural products such as antibiotic macrolide (-)-pyrenophorin (1), antioxidant phenol (2) and the related furans (3) isolated from Ginger, 15-HPETE (4), cytotoxic fatty acid (5) isolated from Corn, and furanoid fatty acid (6) (Fig. 1). (-)-Pyrenophorin (1) was synthesized by the Mitsunobu reaction of (2E,7S)-4,4-ethylenedioxy-7-hydroxy-2-octenoic acid (16), which has been prepared via the reductive oxygenation of ethyl (2E,4E,7S)-7-acetoxy-2,4-octadienoate (13) (Scheme 2). Dienone 22 synthesized from vanillin was oxygenated to give γ-hydroxy-α,β-unsaturated ketone 23, which could be converted into 2 and 3. The reductive oxygenation of (2E,4E)-2,4-nonadienal gave (2E)-4-hydroperoxy-2-nonenal (Table1, Entry 3), which can be converted into racemic 4 according to the reported method (Ref. 13). Lipoxygenase-catalyzed oxidation of linoleic acid followed by treatment with CH_2N_2 gave hydroperoxide 26, which was reduced and then acetylated to afford diene 28. The reductive oxygenation of 28 followed by acetylation gave γ-acetoxy-α,β-unsaturated ketone 29, a precursor of 5. On the other hand, dienone 30 obtained from 26 was oxygenated, reduced with (CH_3O)_3P, and then treated with p-TsOH to give 6.
Benzylic quaternary carbon centers are found in various bioactive molecules such as eptazocine (1), morphine (2) and halenaquinol (3), and the construction of such centers in a catalytic, enantioselective manner continues to provide an interesting challenge for organic chemists. We thought that catalytic asymmetric syntheses of such benzylic quaternary carbon centers would be feasible by utilizing an asymmetric Heck reaction (ref. 1). Our strategy for the construction of benzylic quarternary carbon centers in an optically active form is illustrated in Scheme I. It was expected that the prsence of a chiral ligand in the Heck-type arylation of 4 would result in the discrimination of the re and si-faces of the trisubstituted olefin, but the effect of olefin geometry on the asymmetric induction remained ambiguous. The asymmetric Heck reaction of (E)-olefins 10 and 11 was then investigated. Treatment of 10 with Pd_2(dba)_3・CHCl_3 (5 mol%), (R)-BINAP (10 mol %), and K_2CO_3 (3 mol equiv) in THF at 70℃ was found to give the best results, affording (S)-trans-13 of 39% ee as the major product (70% yield) and (S)-cis-13 of 32% ee as the minor product (19% yield). A kinetic resolution in the syn-β-hydrogen elimination step could easily explain the differences observed in the enantiomeric excess of trans- and cis-13. On the oter hand, subjection of 11 to the conditions described above resulted in the formation of (S)-14 of 51% ee as a chromatographically inseparable mixture of olefin isomers in 95% yield (trans: cis= 84:11). Furthermore, treatment of 12 with Pd(OAc)_2 (10 mol%), (R)-BINAP (20 mol%), and K_2CO_3 (3 mol equiv) in THF at 50℃ gave the (R)-isomer 14 of 91% ee in 79% yield (trans: cis=98:2). Apparently, the absolute configuration of the product obtained with the (R)-BINAP system is reversed in going from the (E)- to (Z)-trisubstituted olefin, and the degree of enantioselectivity is influenced significantly by the olefin geometry. With our system, the use of (Z)-trisubstituted olefins appeares essential for the synthesis of benzylic quaternary centers of high ee. Next, we sought to apply this methodology to the synthesis of (-)-eptazocine (1). For therapeutic use, (-)-eptazocine (1), the more biologically active enantiomer, is currently prepared through resolution. The trisubstituted benzene derivative 21 was prepared in three steps (78% yield) from 3-methoxyphenol and then converted to 22 in 69% overall yield by the use of Suzuki cross-coupling reaction as a key step. Exposure of 22 to Pd(OAc)_2 (7 mol%), (R)-BINAP (17 mol%), and K_2CO_3 (3 mol equiv) in THF at 60℃ gave 23 (trans: cis=21:3) of 90% ee in 90% yield. This methoxy-substituted tetralin derivative 23 was converted to (-)-eptazocine in short steps. An approach to a catalytic asymmetric synthesis of 3 is currently under investigation.
Carbohydrates are inexpensive and frequently easy to obtain in the pure form, thus they are widely used as chiral blocks for various natural product synthesis. They are also used as the chiral auxiliaries for asymmetric synthesis . Among the chiral centers on carbohydrates, the anomeric carbon has special characteristics, i. e., it is easily epimerable and its stereochemistry is controlled by the anomeric effect and steric effect of other chiral centers. Here we wish to report new conversion methods of carbohydrates based on carbanion rearrangements at anomeric centers. 1)Asymmetric transmission from the anomeric center of glucose via [2,3]-Wittig rearrangement In the interest of using the anomeric center of carbohydrates as the chiral source and developing a new type of conservative asymmetric synthesis in the [2,3]-Wittig rearrangement, we have undertaken a project to investigate the [2,3]-Wittig rearrangement of a glucose derivative. The rearrangement of glucose derivative 1 was carried out under the standard conditions. The rearrangement was found to proceed with a high degree of diastereoselectivity. 2) [1.2]-Wittig rearrangement of acetal systems and its application to C-glycosydation. Despite the great potential of the [1,2]-Wittig rearrangement as a tool for the conversion of an ether bond to a C-C bond, its synthetic use has been extremely limited so far. To use this rearrangement for practical C-C bond formation, we attempted the Wittig rearrangement of cyclic acetal systems. Upon treatment with BuLi, cyclic acetals which were prepared from lactol and benzylalcohol or (E)-3-trimethylsilyl-2-propene-1-ol underwent highly stereoselective rearrangements affording products in good yield. The application of the present Wittig variants of acetal systems to C-glycosylation will be presented.
Biocatalysis is recognized as a useful tool for organic synthesis, especially for the asymmetric synthesis of natural products. As part of our efforts to synthesize versatile chiral building blocks by adopting biocatalysis, we developed the lipase-catalyzed desymmetrization of the meso-glycol 1 and the meso-diacetate 2 to afford both enantiomers of the ketol acetate 4 as an optically pure state, respectively. The desymmetrization of the meso-glycol 5 was also achieved to give the monoacetate (-)-6, which was led in three steps to the decalin (+)-19. Enantiomerization of (+)-19 was readily accomplished to give (-)-19. On the other hand, baker's yeast reduction of the β-keto ester 7 proceeded in a highly enantioselective manner and furnished the homochiral piperidone (-)-8 in good yield. The enantiomer (+)-8 was obtained by the lipase-mediated optical resolution of (±)-8. Thus, we achieved highly efficient synthesis of both enantiomers of chiral building blocks 4, 6, 8. The versatility of 4, 6 and 8 for natural product synthesis was demonstrated by the first total synthesis of(-)-cassine (11), (+)-spectaline (12), (-)-indolizidine 235B' and the formal synthesis of (-)-dihydropinidine, (-)-indolizidine 207A, 209B, (-)-polygodial, (-)-warburganal, (-)-drimenin, and prosopinie, respectively.
We have used baker's yeast reduction for the synthesis of optically active natural products. We extended this concept to bicyclo[2.2.2]octane ring system, and obtained enantiomerically pure β-hydroxy ketone A (>99%e.e.). We carried out the syntheses of pinthunamide (1), bergamoten-12-oic acids (α-form: 2, β-form: 3), and homogynolide A (4) by employing A as a common chiral starting material. The key reaction in the syntheses of 1,2, and 3, was the intramolecular alkylation to generate the four-membered ring (5a→6a, 5b→6b), which can be a general method to prepare pinane-type ring systems. Oxy-Cope rearrangement (27→26) was employed as an important step in the synthesis of 4, which was the ring transformation from bicyclo[2.2.2]octane to bicyclo[3.3.0]octane system. In summary, we demonstrated the utility of the β-hydroxy ketone A as a chiral building block for the syntheses of several natural products.
Chiral bicyclo[3.3.0]octane derivatives 1 are useful as chiral synthons for efficient synthesis of cyclic natural products. Here we describe the asymmetric transformation using enzymatic method of δ-symmetric bicyclic diketones 2a-c and tetraester 9, and the application of chiral bicyclo[3.3.0]octane derivatives [(+)-3b, (+)- and (-)-11] to natural products syntheses. 1. Baker's yeast-mediated asymmetric reduction of δ-symmetric bicyclic diketones 2a-c afforded the optical active ketols 3a-d in excellent enantioselectivity. Especially, ketol (+)-3b was the chiral intermediate for the total syntheses of (-)-cantabrenonic acids. Furthermore, usefulness of (+)-3b as a chiral bicyclic synthon was revealed by the short transformation of the ketol to the chiral intermediate (+)-4 in (+)-capnellenols syntheses (Scheme 5). 2. PPL-mediated asymmetric demethoxycarbonylation of achiral tetraester 9 obtained by Weiss reaction afforded the chiral triester (+)-10 in 98.3 %ee. The chiral triester was selectively demethoxycarbonylated to prepare chiral C_2-symmetry diester (+)-11. On the other hand, lipase AY-mediated asymmetric demethoxycarbonylation of achiral tetraester 9 directly afforded the chiral C_2-symmetry diester (-)-11 (100 %ee), having opposite chirality compared to the reaction with PPL. The results of lipase-mediated asymmetric transformation of achiral tetraester 9 are shown in Table 1. The chiral diesters (+)- and (-)-11 were very useful as chiral C_2-symmetry synthons for the total syntheses of (+)-carbacyclin, (-)-isoiridomyrmecin, (-)-ajmalicine (Scheme 7, 8, and 9).
Annulation by multifold Michael reaction initiated by intermolecular Michael reaction has been a useful tool to construct carbocyclic molecules. We delineate our new synthetic route toward total synthesis of (+)-compactin 1, employing double Michael reaction as a key reaction. (+)-Compactin 1 efficiently inhibits HMG-CoA reductase, a rate limiting enzyme in cholesterol biosynthesis, to lower cholesterol level in blood. Alkoxyacetylcyclohexene 6 was prepared by lipase catalysed kinetic resolution. Double Michael reaction of kinetic enolate of 6 with methyl crotonate, provided a mixture of decalones 11 and 12 which settled by base treatment into trans-decalone 12 having correct stereochemistry at C-9, 11a, and 15a (compactin numbering) for compactin synthesis. Carbonyl group at C-11 of 12 was reduced to give axial alcohol which was protected as xanthate ester. After inversion of configuration of the alkoxygroup at C-15 of 13 by deprotection-oxidation-reduction sequence, double bond at C-10 was introduced by the Chugaev reaction to give lactone 17. Reduction of lactonic portion provided diol 18 which was transformed into aldehyde 22 after a series of protection-deprotection sequence and Swern oxidation. Stereochemistry at C-8 was controlled by base catalyzed isomerization to give aldehyde 23 having desired stereochemistry. Chain elongation from C-8 of 23 was achieved by Horner-Emmons reaction. Diene part was introduced by bromination to olefin 26 and dehydrobromination of 27. After esterification with 2-methylbutyric anhydride to give 29, β-hydroxy-δ-lactonic moiety was installed by aldol condensation of dianion of methyl acetoacetate followed by reduction and finally lactonization. Thus, total syntheses of (+)-compactin 1 and (+)-dihydrocompactin 38 have been accomplished.
Members of taxane family (e.g. 1 and 2) with their unique tricyclo[188.8.131.52^<3,8>]-pentadecane skeleton and significant anticancer activities of a congener, taxol (2), are important targets in synthetic organic chemistry. Aiming the total synthesis of taxol (2) as the final goal, we have examined synthetic studies on taxusin (1). Cyclization precursor 13 possessing the desired 1,4-relative stereochemistry was prepared. The 1,4-relative stereochemistry was controlled by Michael addition of lithium enolate of isobutyrate ester to enone 8 under high 1,4-diastereoinduction and the Mitsunobu inversion. The tricyclic skeleton of taxusin 14 was constructed by means of the TMSOTf-mediated intramolecular eight-membered ring cyclization reaction of 13 between the dienol silyl ether and the acetal. Though the cyclization yielded undesired spirocyclization product 15 as the major product, TiCl_4-induced isomerization of 15 afforded the desired 14 in good yield. In addition that 14 has the correct stereochemistry at C9 and C10 it appeared to possess the endo conformation which is suitable for the subsequent stereoselective transformations on the ring system. Chemo- and stereoselective functional group transformations of 14 gave allyl alcohol 17, setting up the stage for investigation of the 19β-methyl group installation. The 4β-hydroxy group directed cyclopropanation of Δ^<3,8>-double bond smoothly afforded β-methylenation product, PDC oxidation of which led to cyclopropyl ketone 18. Though attempted reductive opening of the cyclopropane ring of 18 failed, resulting in formation of presumed hydroperoxide 19, reduction of 13α-hydroxy derivative 20 accomplished cyclopropane ring opening and at the same time stereoselective protonation at C3 to afford 21. Thus, we succeeded in introduction of the 19β-methyl group on the ring system in high yield. The obtained intermediate is suitably functionalized for the synthesis of taxusin (1). We are currently pursuing the accomplishment of the total synthesis.
(+)-Eremantholide A (1) was isolated by Le Quesne and co-workers from the bark of eremanthus elaeagnus. This natural product exhibits a significant antitumor activity. The relative stereochemistry of 1 was determined by a single crystal X-ray analysis. The first total synthesis of (+)-1 completed by Boeckman and co-workers in 1991 finally confirmed its absolute stereochemistry. The structural characteristics of 1 are a highly oxygenated 3,7-dioxabicyclo[3.3.0]octane skeleton (the A/B ring system) and a strained nine membered ring. The A/B ring system includes five consecutive stereogenic centers, one of them is an asymmetric quaternary carbon. The structural novelty of 1 is also interesting from a synthetic point of view. We wish to report here the enantiospecific total synthesis of (+)-eremantholide A from D-glucose. Furthermore, the synthesis of a novel compound, 10-epi-eremantholide A (36), is also presented. As the first task, we undertook an efficient construction of the A/B ring system. As an enantiomerically pure starting material, we selected a chiron 2 including a stereochemically defined quaternary carbon. This compound 2 was prepared from D-glucose by employing the ortho ester Claisen rearrangement strategy. The substrate 16 for the key five membered ring formation was prepared from 2 in a 14 step reaction. The intramolecular radical cyclization of 16 proceeded regio- and stereoselectively to provide 18. Compound 18 was converted into 21 featured by homologation with an organo copper reagent. Luche reduction of 21 provided the allylic alcohol 22 exclusively. The A/B ring equivalent 25 was prepared from 22. Therefore, we have established an efficient synthetic route to the A/B ring system. Next, we investigated the coupling of the A/B ring and D ring. This reaction was realized by using the triflate 27 and the furanone 28 to give C-alkylation products 29 and 30, and O-alkylation product 31. The nine membered ring formation from the intermediate 29 was achieved by the intramolecular vinylogous aldol reaction providing 33, which was converted to (+)-eremantholide A (1) by a 3 step-reaction. Analogously, 10-epi-eremantholide A (36) was synthesized from 30.
Aphidicolin (1), isolated from the fungus Cepharosporium aphididicola, is an antibiotics that shows marked activity against Herpes simplex Type I virus. In addition to its antifeedant property, aphidicolin (1) exhibits a considerable antitumor activity. Herein we report conceptually distinct two access to 1 featuring (i) successive intramolecular Diels-Alder reactions and (ii) intramolecular Heck reaction and intramolecular Diels-Alder reaction as the key steps. (i) First Generation Synthesis of 1 via the Successive Intramolecular Diels-Alder Reactions Formal synthesis of aphidicolin (1) has been achieved by means of the successive Diels-Alder reactions (5→6 and 13→14) as the key steps of the first generation synthesis of aphidicolin (1). (II) Second generation Synthesis of 1 by Intramolecular Heck Reaction and Intramolecular Diels-Alder Reaction Intramolecular Heck reaction of the bromide (19) and intramolecular Diels-Alder reaction of the triene (13) have been utilized as the key steps for 13-step formal total synthesis of (±)-aphidicolin (1).
Hydroxyl groups play a major role in binding to a receptor protein. To clarify the side chain conformation of 1,25(OH)_2D_3 (1) to bind to VDR, we analyzed the mobility of the side chain of 1 and its 20-epimer (2) in terms of the spatial region accessible by the 25-hydroxyl group and the results were displayed as a dot map. The dot map indicates that each vitamin D has two major densely populated regions: A and G for 1 and EA and EG for 2. We designed six 22-substituted analogs of active vitamin D, 3-8, whose side chain hydroxyl is restricted to occupy one of the four spatial regions defined above and the analogs were classified into group A, G, EA, and EG accordingly. Syntheses of analogs, 3-8, were performed via diastereoselective conjugate addition of alkyl cuprate to steroidal (E)-and (Z)-22-en-24-ones (12 and 13) and -22-en-24-oates (16, 17, 20 and 21) as the key step. The activity of the analogs, 3-8, to bind to VDR was examined in compared with 1. It is apparent that the members of A ( 4 and 6) and EG (7) group analogs show much higher activity than the others, G(3, 5) and EA (8). The contrasting activities of the two 22-methyl analogs (3 and 4) are especially outstanding: VDR binding (3:4:1=1/50:1/3:1); differentiation of HL-60 cells (3:4:1=1/70:1:1). The results suggest that the conformation of 1,25(OH)_2D_3 (1) involved in binding to VDR and responsible for activity is the C(17,20,22,23)-anti form and the region where the 25-hydroxyl group occupies is the A.
(-)-Ircinianin (1) and (+)-wistarin (2) are a rare cyclic furanosesterterpenetetronic acid as a secondary metabolite in marine sponge of Ircinia sp. Hofheinz reported the isolation of 1 from genus ircinia and determined the relative structure in 1977. The structurally related 2 was also found in sponge, ircinia wistarii at by Gregson in 1982. However the absolute structures have not been revealed. Herein, we like to report the first asymmetric total synthesis of 1 and 2, and the reported structure of (+)-wistarin is revised. Our synthesis started methyl R-(-)-3-hydroxy-2-methylpropionate to reach tetronic acid intermediate 5 in 14 steps. On the other hand, iodo triene intermediate 6 was derived from 3-furfural in 12 steps involving the key stereospecific trisubstituted iodo olefin formation. A key coupling of 5 with 6 was performed by NiCl2-CrCl2 mediated reaction. The coupling products were readily cyclized by heating in xylene to give the tricyclic carbon skeleton. Deoxygenation and deprotection completed the total synthesis of 1 in good yields. All the physical and spectroscopic data including specific rotation of the synthetic 1 were identical with the reported data in literature. Iodo ether formation of 1 with iodine in the presence of K_2CO_3 followed by radical reduction with tributyltin hydride afforded 2 which was identified by a comparison with the reported data of natural wistarin. However, the reported structure conflicts with our result concerning the quaternary center at C-8. The structure of the iodo ether suggests the furanopropyl group exists at an equatorial position, which means the quaternary carbon should possesses R-configuration.
Recently, stannyl anion has become a useful synthetic tool for organic synthesis. We have developed various cyclization using stannyl anion, generated from Me_3SiSnBu_3 and F^-. Cephalotaxine (28), the major alkaloid of C.harringtonia, has a unique skeleton, which has potential pharmacological activity, such as antileukemia activity. In order to synthesize (-)-cephalotaxine (28), we planned on the basis of the following points. The unique skeleton, 1-azaspiro[4.4]nonane moiety fused benzazepine system, would be prepared by the reaction of 44 with stannyl anion generated from Me_3SiSnBu_3 and F^-. For the synthesis of optically active cephalotaxine, the optically pure starting material 44 would be obtained from D-(+)-proline via 36 by Seebach's procedure. The methylenedioxy group on the aromatic ring is not favorable for the cyclization of the seven membered ring. From (D)-proline, we could obtain compound 44 as an optically active form. To a DMF solution of 44 and CsF was added Me_3SiSnBu_3 (2.0 eq.) at 0℃ and the solution was stirred at room temperature for 8 h. After usual work up, desired cyclized products 45 and 46, were obtained in 85% and 3% yields, respectively. Compound 46 was easily converted into 45 by treatment with Bu_4NF in good yield. Reaction of 45 with PPA followed by treatment with BBr_3 and then CH_2Br_2 gave 47. Treatment of 47 with OsO_4 in the presence of trimethylamine N-oxide in AcOH afforded the diol, which was oxidized with DMSO and trifluoroacetic anhydride to give diketone 48. A dioxane solution of 48 and dimethozypropane in the presence of TsOH was refluxed for 8 h to give 49. The spectral data of cephalotaxinone (49) was fully identical with those of the product reported^<6a>, but it was a racemic product [α]_D-8.2°(c, 0.4, EtOH), lit. [α]_D-135° (c 0.95, EtOH)]. Thus, 48 was treated with methyl orthoformate in the presence of TsOH in CH_2Cl_2 to give 49 in 47% yield with 88% ee. Finally, treatment of 49 with NaBH_4 in MeOH provided (-)-cephalotaxine.
Dynemicin A is a potent antibacterial and antitumor antibiotic having a striking hybrid structure combining the characteristics of both the anthraquinone as a DNA intercalator and diynene as a DNA strand breaker. We investigated the DNA-binding properties of five non-diynene dynemicins and of several related tri-and pentacyclic aza-anthraquinones (1-4). Pentacyclic quinones 1 and 2 were synthesized as shown in Scheme 4. The key step was photochemical cyclization of 11 that proceeded smoothly to afford 17 and 18 with no diastereoselectivity (Scheme 2). Tricyclicquinone 3 was prepared according to the reported procedure (Scheme 5). Since O-methylation of 3 was unsuccessful, tricyclicquinone 4 was synthesized as shown in Scheme 6. Addition of excess DNA to a drug solution induced a pronounced red shifts in UV absorption spectrum of the drug. All dynemicins examined showed similar binding ability with DNA (Figure 3). As expected, a binding of the synthetic anthraquinones with DNA varied depend on their structure. Since supercoiled DNA incubated with these non-diynene dynemicins was retarded on electrophoresis (Figure 4), intercalative binding of these drugs to DNA is suggested. On the other hand, the synthetic anthraquinones 1-4 had no effect on the electrophoretic mobility, suggesting that the gross structure of supercoiled DNA is not changed by the binding of these drugs. This is surprising since the non-diynene dynemicins are presumed to bind intercalatively to DNA through the aza-anthraquinone moiety. In order to investigate the function of this partial structure in dynemicins in their intercalation with DNA, synthesis of anthraquinones more closely related to the dynemicin substructure is currently in progress.
In the course of synthetic studies on benzo[c]phenanthridine alkaloids for their structure-activity relationship macarpine (1), a fully aromatized one carrying six oxygen functions, was synthesized as shown in Scheme 1. In the synthesis we succeeded in regioselective introduction of a nitrogen function into the para position of a hydroxyl group in a naphthol (9) by newly developed SE reaction under basic condition (basic nitrosation). Bischler-Napieralski (B. N.) reaction has been used for the construction of isoquinoline skeleton in our synthetic strategy. Treatment of a naphthylformamide (12) with POCl_3 followed by NaBH_4 reduction afforded dihydromacarpine (13), which could be easily converted into 1, along with an undefined product (14). The corresponded product (17) to 14 was also obtained in the reactions using a naphthylformamide (15). (Scheme 2) The unexpected benz[g]indole structure fused to oxabicyclooctene system for 17 was unambiguously established by X-ray crystallographic analysis. (Fig.1) Thus, it could be reasonable to deduce a benz[g]indole structure for 14 . Fractional recrystallization of the B. N. products of 15 from methanol allowed us to isolate an anomalous cyclized product of the azoniaazulene derivative (19), easily reducible to a benz[g]indole (17), in addition to a normal cyclized product (18). The structure of 19 was determined by its spectral data including NOE (Fig. 2) and COLOC (Fig. 3) experiments. Formation of an azoniaazulene (20) was also found in the B. N. reaction of 12 (Scheme 3) Interestingly, normal cyclization was observed in the B. N. reactions of 21 (R=Me or ^iPr) in which a methylenedioxy group in the 2-aryl substituent was replaced by methoxy groups. On the other hand, a corresponding phenolic 21 (R=H) caused abnormal B. N. reaction. (Scheme 4)
Nephilatoxins (NPTX-1〜12) isolated from the venom of Joro spider (Nephila clavata) have been characterized as structurally new type of neurotoxins consisted of an aromatic acid, amino acids, and some polyamines. These toxins induce histamine release from rat peritoneal mast cell and exhibit potent blocking activities toward glutaminergic neurotransmission in the lobster legs. We have reported the first and efficient syntheses of NPTX-9,10,11, and 12 by the use of azide intermediates (1 and 2) which played key roles in the construction of the characteristic polyamine components such as cadaverine and putreanine. By designing a novel azide compound 3 as 9-aminopropylputreanine equivalent, we were successful in the first synthesis of NPTX-8 (Scheme 2). We also synthesized NPTX-7, the only spider toxin containing an acidic amino acid as a component, according to Scheme 3. In addition enantiomers of NPTX-8 and NPTX-12, i.e., D-NPTX-8 and D-NPTX-12 containing D-asparagine, have been synthesized to investigate the structure-toxicity relationship. From the biological tests of the seven synthetic toxins (paralyzing activity against Blattela germanica L.), it was revealed that the reported order of Nephilatoxins should be revised as NPTX-12>NPTX-10>NPTX-8> NPTX-11>NPTX-9. Interestingly, D-NPTX-8 and D-NPTX-12 have been shown to exhibit considerably potent glutaminergic blocking activities. Further synthetic studies on other spider toxins and analogues are in progress.
The cytotoxic metabolites of pulmonate of the family of Onchidacea, a shelless marine mollusc phylum, were extensively investigated by some research groups. Their biosynthetic origin and stereochemical relationships have attracted some interest. The γ-pyrones are one of major class of those metabolites. Ilikonapyrone (1), peroniatriols (2, 3), and onchitriols (4, 5), are new members of the class; polyhydroxylated linear polypropionates with two fully substituted γ-pyrone rings. The structures of this family were in general, proposed by comparison with that of ilikonapyrone (1), which had been determined by an X-ray crystallographic analysis, but ambiguities on relative stereochemistries of asymmetric centers existing across γ-pyrone or trisubstituted olefin, could not be avoided. In order to confirm these points, we describe herein the effective cyclization of triketides under DMSO-(COCl)_2 or Ph_3P - CCl_4 conditions to the corresponding γ-pyrones without any serious epimerization and/or elimination of adjacent stereogenic centers, which must establish the absolute stereochemistries of peroniatriols and ilikonapyrone by synthesis of their degradation products. We also report the first total synthesis of onchitriol II (5) and some of its diastereoisomers, using the new synthetic methodology for γ-pyrone developed by us. This synthesis allowed the complete assignment of the stereochemistry for onchitriol II (5), and the structural revision of onchitriol I (1).