Anticancer effect of altersolanol A,a metabolite produced by the endophytic fungus Stemphylium globuliferum,mediated by its pro-apoptotic and anti-invasive potential via the inhibition of NF-κB activity

Altersolanol A, a natural product from the endophytic fungus Stemphylium globuliferum isolated from the medicinal plant Mentha pulegium (Lamiaceae) growing in Morocco, shows cytotoxic, cytostatic, anti-inflammatory and anti-migrative activity against human chronic myeloid K562 leukemia and A549 lung cancer cells in a dose dependent manner without affecting the viability of non cancerous cells. Altersolanol A induces cell death by apoptosis through the cleavage of caspase-3 and -9 and through the decrease of anti-apoptotic protein expression. Moreover, we report here the importance of the distinct structural features of altersolanol A by testing other related anthracene derivatives in order to identify preliminary structure–activity relationships. Acetylation of altersolanol A did not improve activity where other derivatives such as tetrahydroaltersolanol B and ampelanol that differ from altersolanol A by reduction of one of a carbonyl group and removal of hydroxyl substituents were inactive in comparison. Altogether our results suggest that altersolanol A may be considered as an interesting lead for further development of chemotherapeutic agents.     

Thermostable Enzymes in Organic Synthesis, 4. Tbadh-Catalyzed Preparation of Bifunctional Chirons. Total Synthesis of S-(+)-Z-Tetradec-5-En-13-Olide

Highly enantioselective reduction of various methyl- and ethylketones bearing different functional groups, such as double and triple carbon-carbon bonds, methyl ester, cyano, ethyl ether, phenyl and chloride, employing Thermoanaerobium brockii alcohol dehydrogenase (TBADH) as a catalyst, affords the corresponding optically active, secondary alcohols. As expected on the basis of our previous studies with monofunctional ketones, reduction of most of the substrates yields, uniformly, alcohols with an S configuration, arising from highly selective hydride attack at the re face of the carbonyl. However, with the smaller-sized ketones, there is a clear reversal in stereoselectivity. The synthetic usefulness of these chiral building blocks has been demonstrated by the total synthesis of (S)-(+)-Z-tetradec-5-en-13-olide, one of several synergistic aggregation pheromones produced by male flat grain beetles, Cryptolestes pusillus (Schonherr). The pheromone was prepared from (S)-(+)-methyl-8-hydroxynonanoate with optical purity greater than 99% in a six-step synthesis.     

S‐perindopril assay using a potentiometric,enantioselective membrane electrode

A potentiometric, enantioselective membrane electrode based on graphite paste (graphite powder and paraffin oil) has been constructed. The graphite paste is impregnated with a 10⁻³ mol/L 2‐hydroxy‐3‐trimethylammoniopropyl‐β‐cyclodextrin (as chloride salt) solution. The potentiometric, enantioselective membrane electrode can be used reliably for enantiopurity tests of S‐perindopril using a chronopotentiometric (zero current) technique, in the 10⁻⁵–10⁻² mol/L concentration range (detection limit 5 × 10⁻⁶ mol/L), with an average recovery of 99.58% (RSD = 0.33%). The enantioselectivity was determined over R‐perindopril and d ‐proline. The response characteristics of the enantioselective, potentiometric membrane electrode were also determined for R‐perindopril. It was shown that l ‐proline is the main interfering compound. The surface of the electrode can be regenerated simply by polishing, obtaining a fresh surface ready to be used in a new assay. Chirality 11:631–634, 1999. © 1999 Wiley‐Liss, Inc.     

Systematic Analysis of the Expression of the Mitochondrial ATP Synthase (Complex V) Subunits in Clear Cell Renal Cell Carcinoma

Mitochondrial dysfunction is common in cancer and the mitochondrial electron transport chain is often affected in carcinogenesis. To date, little is known about the expression of the ATP synthase subunits in clear cell renal cell carcinoma (ccRCC). The NextBio database was used to determine an expression profile of the ATP synthase subunits based on published microarray studies. We observed down-regulation of 23 out of 29 subunits of the ATP synthase. Differential expression was validated exemplarily for 12 genes (ATP5A1, ATP5B, ATPAF1, ATP5C1, ATP5D, ATP5O, ATP5F1, ATP5G1, ATP5G2, ATP5G3, ATP5I, ATP5S; screening cohort ccRCC n = 18 and normal renal tissue n = 10) using real-time PCR. Additional eight genes (ATP5A1, ATP5B, ATPAF1, ATP5F1, ATP5G1, ATP5G2, ATP5G3, ATP5S) were internally validated within an enlarged cohort (ccRCC n = 74; normal renal tissue n = 36). Furthermore, down-regulation of ATP5A1, ATPAF1, ATP5G1/G2/G3 was confirmed on the protein level using Western Blot and immunohistochemistry. We observed that altered expression of ATPAF1 and ATP5G1/G2/G3 was correlated with overall survival in patients with ccRCC. In conclusion, down-regulation of many ATP Synthase subunits occurs in ccRCC and is the basis for the reduced activity of the mitochondrial electron chain. Alteration of the expression of ATP5A1, ATPAF1, and ATP5G1/G2/G3 is characteristic for ccRCC and may be prognostic for ccRCC patients' outcome.     

Asymmetric Autocatalysis Induced by Chiral Crystals of Achiral Tetraphenylethylenes

The achiral hydrocarbon tetraphenylethylene crystallizes in enantiomorphous forms (chiral space group: P2₁) to afford right- and left-handed hemihedral crystals, which can be recognized by solid-state circular dichroism spectroscopic analysis. Chiral organic crystals of tetraphenylethylene mediated enantioselective addition of diisopropylzinc to pyrimidine-5-carbaldehyde to give, in conjunction with asymmetric autocatalysis with amplification of chirality, almost enantiomerically pure (S)- and (R)-5-pyrimidyl alkanols whose absolute configurations were controlled efficiently by the crystalline chirality of the tetraphenylethylene substrate. Tetrakis(p-chlorophenyl)ethylene and tetrakis(p-bromophenyl)ethylene also show chirality in the crystalline state, which can also act as a chiral substrate and induce enantioselectivity of diisopropylzinc addition to pyrimidine-5-carbaldehyde in asymmetric autocatalysis to give enantiomerically enriched 5-pyrimidyl alkanols with the absolute configuration correlated with that of the chiral crystals. Highly enantioselective synthesis has been achieved using chiral crystals composed of achiral hydrocarbons, tetraphenylethylenes, as chiral inducers. This chemical system enables significant amplification of the amount of chirality using spontaneously formed chiral crystals of achiral organic compounds as the seed for the chirality of asymmetric autocatalysis.     

Action-mode of Antimicrobial Altersolanol A in Pseudomonas aeruginosa

The antimicrobial mechanism of altersolanol A was studied by using Pseudomonas aeruginosa IFO 3080. This reduced anthraquinone antibiotic inhibited the incorporation of radioactive precursors into macromolecules (DNA, RNA, and Protein) in whole cells nonspecifically. It increased respiration of P. aeruginosa cells, though it had no effect on proton condution. Oxidation of NADH in the membrane fraction isolated from P. aeruginosa was enhanced by altersolanol A. Furthermore, altersolanol A stimulated the oxidation of NADH by the enzyme preparation of cytochrome c reductase in the absence of cytochrome c. Altersolanol A seemed to interfere with the respiratory chain in the bacterial membrane as an electron acceptor.     

Biocatalytic enantioselective approach to 3-aryl-2-nitropropanols: Synthesis of enantioenriched (R)-5-methoxy-3-aminochroman,a key precursor to the antidepressant drug Robalzotan

The results of our studies on the biocatalytic enantioselective synthesis of different 3-aryl-2-nitropropanols are presented. These compounds could be obtained in moderate to good ee both by baker's yeast mediated reduction of (E)-2-nitro-3-arylprop-2-en-1-ol precursors and by lipase kinetic resolution of racemic 3-aryl-2-nitropropanols. The synthesis of enantioenriched (R)-5-methoxy-3-aminochroman is also achieved. This important moiety is present in different molecules active toward the central nervous system and is also the precursor for the synthesis of Robalzotan (NAD299), a potent 5-HT_1A antagonist.     

Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.     

Species differences in the chirality of the carbonyl reduction of [14C] fenofibrate in laboratory animals and humans

The prochiral carbonyl group of fenofibrate (isopropyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl propionate) is reduced during its metabolism giving rise to a chiral secondary alcohol, "reduced fenofibric acid." Chiral and diastereomeric HPLC methods have been developed for the determination of its enantiomeric composition and these have been applied to the measurement of the "reduced fenofibric acid" enantiomers in urine of rats, guinea pigs, dogs, and human volunteers given [14C]fenofibrate. In the three animal species, the reduction is markedly enantioselective for the (-)-isomer, the enantiomeric ratios (-/+) being 95:5. This was not due to differences in the excretion of the enantiomers, since when racemic "reduced fenofibric acid" was given to rats it was recovered in the urine with the same enantiomeric composition as the dose form. In humans the ratio was 52:48 showing the lack of stereoselectivity of reduction in this species.     

Biotransformation of γ-terpinene using Stemphylium botryosum (Wallroth) yields p-mentha-1,4-dien-9-ol, a novel odorous monoterpenol

A wild strain of Stemphylium botryosum, when grown submerged in the presence of γ-terpinene (1), yielded the novel highly odour active terpene alcohol (2), whose structure was established by spectroscopic means as p-mentha-1,4-dien-9-ol. During cultivation (2) was further oxidized, predominantly to the corresponding aromatic alcohol p-cymene-9-ol (5). The enantioselective enzymatic introduction of the hydroxyl group at the non-activated C9 of (1) resulted in an enantiomeric excess (ee) of 74% for (2) and 70% for (5), respectively.     

Preparation of dicationic palladium catalysts for asymmetric catalytic reactions

The synthesis of Pd(OTf)(2)·2H(2)O is described. This was used to generate two different types of chiral dicationic palladium complexes for highly enantioselective addition of aromatic amines to α, β-unsaturated conjugate alkenes ([(R-BINAP)Pd(OH(2))(2)][OTf](2) and [(R-BINAP)Pd(μ-OH)](2)[OTf](2)). The resulting optically active N-arylated β-amino acid derivatives are valuable synthetic intermediates for the synthesis of biologically active molecules and peptidomimetics. The reaction of (2E)-but-2-enoylcarbamate and aniline is shown as an example of the use of these catalysts for enantioselective aza-Michael addition. For the preparation of palladium(II) triflate, the time scale is 20 h 50 min, plus 5 h 15 min for the monomeric complex and plus 6 h 45 min for the dimeric complex.     

Thermostable Enzymes in Organic Synthesis, 4. Tbadh-Catalyzed Preparation of Bifunctional Chirons. Total Synthesis of S-(+)-Z-Tetradec-5-En-13-Olide

Highly enantioselective reduction of various methyl- and ethylketones bearing different functional groups, such as double and triple carbon-carbon bonds, methyl ester, cyano, ethyl ether, phenyl and chloride, employing Thermoanaerobium brockii alcohol dehydrogenase (TBADH) as a catalyst, affords the corresponding optically active, secondary alcohols. As expected on the basis of our previous studies with monofunctional ketones, reduction of most of the substrates yields, uniformly, alcohols with an S configuration, arising from highly selective hydride attack at the re face of the carbonyl. However, with the smaller-sized ketones, there is a clear reversal in stereoselectivity. The synthetic usefulness of these chiral building blocks has been demonstrated by the total synthesis of (S)-(+)-Z-tetradec-5-en-13-olide, one of several synergistic aggregation pheromones produced by male flat grain beetles, Cryptolestes pusillus (Schonherr). The pheromone was prepared from (S)-(+)-methyl-8-hydroxynonanoate with optical purity greater than 99% in a six-step synthesis.     

Biomimetic Synthesis and Studies Toward Enantioselective Synthesis of Flindersial Alkaloids

A strategy allowing both stereocontrol and control over structural isomer formation has been defined for the antimalarial flindersial alkaloids. The recently reported flinderoles were demonstrated to be derived from the natural product borrerine. The structural isomers of flinderoles, the borreverines, were also produced in vitro along with the flinderoles through the dimerization of borrerine in acidic conditions. This result is thought to replicate the biosynthesis of these compounds. Flinderoles A, B, and C, desmethylflinderole C, isoborreverine, and dimethylisoborreverine can each be synthesized in three steps from tryptamine. Furthermore, progress toward a concise enantioselective synthesis of flinderoles A, B, and C is described. This work includes enantioselective conjugate addition to an unprotected indole‐appended enone. Chirality 27:14–17, 2015. © 2013 Wiley Periodicals, Inc.     

Hypoxia enhances prostaglandin synthesis in renal mesangial cell cultures

In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis. Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner. Production rates of PGE₂ and in specified samples also of 6-keto-PGF_1α, as a measure of PGI₂, and PGF_2α were determined by radioimmunoassay after incubation at either 20% O₂ (normoxic) or 2% O₂ (hypoxic) in gas permeable dishes for 24 hrs. Considerable variation in PGE₂ production was noted among independent cell lines. PGE₂ production appeared to be inversely correlated to the cellular density of the cultures. In addition, PGE₂ production was enhanced in hypoxic cell cultures. The mean increase was 50 to 60%. PGF_2α and 6-keto-PGF_1α increased by about the same rate. These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.     

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Crystal structure determination and stereochemistry of the contact region

The structure of the complex of bovine trypsin and bovine pancreatic trypsin inhibitor has been determined by crystal structure analysis at 2.8 Å resolution. The structure is closely similar to the model predicted from the structures of the components. The complex is a tetrahedral adduct with a covalent bond between the carbonyl carbon of Lys-15I of the inhibitor and the γ-oxygen of Ser-195 of the enzyme. The imidazole of His-57 is hydrogen-bonded to Asp-102 and the bound seryl γ-oxygen in accord with the histidine being charged. The negatively charged carbonyl oxygen of Lys-15I forms two hydrogen bonds with the amide nitrogens of Gly-193 and Ser-195. Protonation of the leaving group N-H of Ala-16I to form an acyl-complex requires a conformational change of the imidazole of His-57. The tetrahedral adduct is further stabilized by hydrogen bonds between groups at the leaving group side and inhibitor and enzyme, which would be weakened in the acyl-enzyme. The kinetic data of inhibitor-enzyme interaction are reconciled with the structural model, and relations between enzyme-inhibitor interaction and productive enzyme-substrate interaction are proposed.     

The Seven Carbohydrate Bioengineering Meeting,Braunschweig, Germany,April, 22–25, 2007

Stereocontrol in bakers' yeast reduction can be achieved by introduction of a sulfur functional group into substrates. α-Methylthio-β-keto esters are reduced to give exclusively (3S)-3-hydroxy esters. α-Substituted β-keto thiol esters and dithioesters afford (2R,3S)-3-hydroxy esters with high diastereo-and enantioselectivity. Ketones possessing 1,3-dithiane, phenylsulfenyl, or phenylsulfonyl groups at the α-position are transformed also into the corresponding (S)-secondary alcohols. Optically pure (S)-(phenylsulfinyl)acetones can be obtained by kinetic resolution of racemic derivatives with the yeast. Diastereo- and enantioselective reduction of 1,2-diketones leading into (1S,2S)-1,2-diol derivatives can be also achieved by introduction of 1,3-dithiane, phenylsulfenyl or phenylsulfonyl groups into the α-position. Reductions of carbon-carbon double bond of sulfur-functionalized prenyl derivatives provide both chiral (R)- and (S)-C₅-building blocks for terpenoid synthesis. The utility of the reduction products as chiral building blocks is demonstrated in the synthesis of biologically active natural products such as pheromones, sugars, antibiotics etc. by functional group transformation and carbon-carbon bond formation reactions with the aid of sulfur functional groups.     

Absolute stereochemistry of altersolanol A and alterporriols

The absolute stereochemistry of altersolanol A (1) was established by observing a positive exciton couplet in the circular dichroism (CD) spectrum of the C3,C4-O-bis(2-naphthoyl) derivative 10 and by chemical correlations with known compound 8. Before the discussion, the relative stereochemistry of 1 was confirmed by X-ray crystallographic analysis. The shielding effect at C7'-OMe group by C1-O-benzoylation established the relative stereochemical relationship between the C8-C8' axial bonding and the C1-C4/C1'-C4' polyol moieties of alterporriols E (3), an atropisomer of the C8-C8' dimer of 1. As 3 could be obtained by dimerization of 1 in vitro, the absolute configuration of its central chirality elements (C1-C4) must be identical to those of 1. Spectral comparison between the experimental and theoretical CD spectra supported the above conclusion. Axial stereochemistry of novel C4-O-deoxy dimeric derivatives, alterporriols F (4) and G (5), were also revealed by comparison of their CD spectra to those of 2 and 3.     

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver

1. 1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis.

2. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enzyme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane.

3. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-P_i exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin.

4. 4. The decrease in ATP synthesis and ATP-P_i exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase.

5. 5. The following electron transport activities in the megamitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc₁-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%.

6. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.