Dioxygenases, key enzymes to determine the aglycon structures of fusicoccin and brassicicene, diterpene compounds produced by fungi

Fusicoccin A and cotylenin A are structurally related diterpene glucosides and show a phytohormone-like activity. However, only cotylenin A induces the differentiation of human myeloid leukemia cells. Since the cotylenin A producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out and a modified fusicoccin A with hydroxyl group at the 3-position showed a similar biological activity with that of cotylenin A. We then searched for an enzyme source that catalyzes the introduction of a hydroxyl group into the 3-position and found that brassicicene C, which is structurally related to fusicoccin A with hydroxyl group at the 3-position, was produced by Alternaria brassicicola ATCC96836. We recently cloned a brassicicene C biosynthetic gene cluster including the genes encoding fusicocca-2,10(14)-diene synthase and two cytochrome P450s, which were responsible for the formation of fusicocca-2,10(14)-diene-8β,16-diol. In this study, we report that a α-ketoglutarate dependent dioxygenase, the gene coding for which was located in the cluster, catalyzed a hydroxylation at the 3-position of fusicocca-2,10(14)-diene-8β,16-diol. On the other hand, a α-ketoglutarate-dependent dioxygenase, which had been identified in a fusicoccin A biosynthetic gene cluster, catalyzed the 16-oxidation of fusicocca-2,10(14)-diene-8β,16-diol to yield an aldehyde (8β-hydroxyfusicocca-1,10(14)-dien-16-al), although both dioxygenases had 51% amino acid sequence identity. These findings suggested that the dioxygenases played critical roles for the formation of the fusicoccin A-type and cotylenin A-/brassicicene C-type aglycons. Moreover, we showed that short-chain dehydrogenase/reductase located in the fusicoccin A biosynthetic gene cluster catalyzed the reduction of the aldehyde to yield fusicocca-1,10(14)-diene-8β,16-diol.     

Synthesis,structure, and dynamic behavior of cyclopentadienyl-lithium, -sodium,and -potassium annelated with bicyclo[2.2.2]octene units: a systematic study on site exchange of alkali metals on a cyclopentadienyl ring in tetrahydrofuran

Novel cyclopentadienyl (Cp)-alkali metal complexes 1-M and 2-M (M = Li, Na, K), in which the Cp ring is annelated with two bicyclo[2.2.2]octene units and substituted with a phenyl group for 1 and a tert-butyl group for 2, were synthesized, and their structures and dynamic behaviors were investigated by means of X-ray crystallography, dynamic (13)C NMR, and DFT calculations. The X-ray crystallography results indicated that 1-Li, 1-Na, and 2-Na form monomeric contact ion pairs (CIP) with three THF molecules coordinated to the metal atom. Also, in THF-d(8), all of the 1-M and 2-M form monomeric CIP in the ground state. However, variable-temperature (13)C NMR measurements of 1-M and 2-M in THF-d(8) demonstrated dynamic behavior in which the metal ion exchanges positions between the upper and lower faces of the Cp ring. From a study of the concentration dependence of the dynamic behavior, the exchange was found to proceed principally as an intramolecular process at concentration ranges lower than 0.2 M. The experimentally observed deltaG values for the intramolecular exchange process for all the 1-M and 2-M (except for 2-Li, whose intramolecular process was too slow to observe) were found to be quite similar in THF-d(8) solution and to fall within the range of 12-14 kcal mol(-)(1). Within this range, a tendency was observed for the deltaG values to increase as the size of the metal decreased. Theoretical calculations (B3LYP/6-31G(d)) afforded considerably large values as the gas-phase dissociation energy for 1-M (162.7 kcal mol(-)(1) for M = Li; 131.6 kcal mol(-)(1) for M = Na; 110.9 kcal mol(-)(1) for M = K) and for 2-M (170.0 kcal mol(-)(1) for M = Li; 137.5 kcal mol(-)(1) for M = Na; 115.4 kcal mol(-)(1) for M = K). These values should be compensated for by a decrease in the solvation energies for the metal ions with increasing size, as exemplified by the calculated solvation energy for M(+)(Me(2)O)(4), which serves as a model for metal ions solvated with four molecules of THF (-122.9 kcal mol(-)(1) for M = Li; -94.7 kcal mol(-)(1) for M = Na; -67.7 kcal mol(-)(1) for M = K). This compensation results in a small difference in the overall energy for dissociation of 1-M or 2-M in ethereal solutions, thus supporting the similar deltaG values observed for the intramolecular metal exchange.     

Studies on taxol biosynthesis. Preparation of 5α-acetoxytaxa-4(20),11-dien-2α,10β-diol derivatives by deoxygenation of a taxadiene tetra-acetate obtained from Japanese yew

The putative metabolite, 5α-acetoxytaxa-4(20),11-dien-2α,10β-diol (7), which is a promising candidate as a biosynthetic pathway triol in taxol biosynthesis, has been prepared by Barton deoxygenation of the C-14-hydroxyl group of a differentially protected derivative of natural 2α,5α,10β-triacetoxy-14β-(2-methyl)-butyryloxytaxa-4(20),11-diene (8), a major taxoid metabolite isolated from Japanese Yew heart wood. The synthetic protocol devised, is amenable for the preparation of isotopically labeled congeners that will be useful to probe further intermediate steps in the biosynthesis of taxol.     

Tumor cell detection method using complement-mediated cytolytic reaction and imaging sensor system

A novel tumor-detection system consisting of complementmediated cytolytic reaction and an image processing system was developed for the simple and rapid determination of tumor cells. The present system consists of a CCD image sensor, image memory board, personal computer, and microscope. When monoclonal antibody 3C4, which is specific to the guinea pig hepatoma L-10, was added to cell suspension, only L-10 cytolysis occurred. Cytolysis caused a decrease in brightness of the cells observed by phase-contrast microscopy. The phase contrast image of the cells before cytolysis was converted to a digitalized signal and stored in computer memory. After cytolysis, a brightness threshold above that of lysed cells was subtracted from the digitalized signal and compared to the signal stored before reaction. L-10 cells in mixed cell suspension were determined specifically by the system. Measurement time was only 2 sec and overall time, including reaction time, was approximately 30 min. Since this method does not require a cell washing process, automation of the whole system is possible.     

Hyrtimomines,indole alkaloids from Okinawan marine sponges Hyrtios spp.

Six new indole alkaloids, hyrtimomines F–K (1–6), were isolated from Okinawan marine sponges Hyrtios spp. The structures of 1–6 were elucidated on the basis of spectroscopic analysis. Hyrtimomine F (1) is a structurally unique bisindole alkaloid possessing an α-keto-?-caprolactam ring, while hyrtimomine G (2) is a symmetrical bisindole alkaloid. Hyrtimomines H–K (3–6) are indole alkaloids possessing β-carboline skeleton with an imidazolium unit. Antimicrobial activities of hyrtimomines F–K (1–6) were evaluated.     

A Bioinspired Synthesis of (±)‐Rubrobramide, (±)‐Flavipucine,and (±)‐Isoflavipucine

A biomimetic synthesis of naturally occurring lactams rubrobramide, flavipucine, and isoflavipucine is described. The key step is a regioselective Darzens reaction between isobutyl glyoxal and an α‐bromo‐β‐ketoamide. The construction of the core tricyclic ring system of rubrobramide was achieved by a cascade reaction in a single step from an α,β‐epoxy‐γ‐lactam. Furthermore, the absolute configuration of naturally occurring (+)‐rubrobramide was determined by vibrational circular dichroism. (±)‐Flavipucine and (±)‐isoflavipucine were synthesized from an epoxyimide, which was prepared by reaction of isobutyl glyoxal with a protected α‐bromo‐β‐ketoamide. Deprotection of the epoxyimide and formation of the pyridone ring gave (±)‐flavipucine, which was converted into (±)‐isoflavipucine by thermal isomerization.     

Amphotericin B covalent dimers forming sterol-dependent ion-permeable membrane channels

Polyenemacrolides such as amphotericin B (AmB) were thought to assemble together and form an ion channel across plasma membranes. Their antimicrobial activity has been accounted for by this assemblage, whose stability and activity are dependent on sterol constituents of lipid bilayer membranes. The structure of this channel-like assemblage formed in biomembranes has been a target of extensive investigations for a long time. For the first step to this goal, we prepared several AmB dimers with various linkers and tested for their channel-forming activity. Among these, AmB dimers that bore an aminoalkyl-dicarboxylate tether covalently linked between amino groups of AmB showed potent hemolytic activity. Furthermore, K+ influx actions monitored by measuring the pH of the liposome lumen by 31P NMR revealed that the dimers formed the molecular assemblage similar to that of AmB in phospholipid membrane. Judging from changes in 31P NMR spectra, the dimers appeared to induce "all-or-none"-type ion flux across the liposome membrane in the presence of ergosterol, which suggested that the ion channel formed by ergosterol/dimer is similar to that of AmB. With these data in hand, we are now trying to elucidate the structure of the ion-channel complex by making the labeled conjugates of AmB for NMR measurements.     

Preconcentration of trace silver with yeast for river water analysis

A new preconcentration method with yeast is presented. The method was evaluated for the determination of trace silver in river waters by graphite furnace atomic absorption spectrometry (GFAAS). A suitable cultivation bed for preconcentration of silver was 1.75 mg ml-1 2-ammonium hydrogen phosphate. The optimal cultivation time and temperature were 2 h and 25 degrees C. Under optimal conditions, silver in aqueous sample was concentrated to 6.9-fold by yeast. The detection limit was 4.6 pg ml-1 (3S/N) for silver in river water. The yeast preconcentration method was applied to the determination of silver in river waters. The recovery of spiked silver was in the range of 89 to 110%. By the preconcentration, it was found that ultra trace silver in river waters could be determined without interferences of matrix elements, after only the cultivation and with no chemical treatment.