Lethal metabolism of Candida albicans respiratory mutants.

The destructive impact of fungi in agriculture and animal and human health, coincident with increases in antifungal resistance, underscores the need for new and alternative drug targets to counteract these trends. Cellular metabolism relies on many intermediates with intrinsic toxicity and promiscuous enzymatic activity generates others. Fuller knowledge of these toxic entities and their generation may offer opportunities of antifungal development. From this perspective our observation of media-conditional lethal metabolism in respiratory mutants of the opportunistic fungal pathogen Candida albicans was of interest. C. albicans mutants defective in NADH:ubiquinone oxidoreductase (Complex I of the electron transport chain) exhibit normal growth in synthetic complete medium. In YPD medium, however, the mutants grow normally until early stationary phase whereupon a dramatic loss of viability occurs. Upwards of 90% of cells die over the subsequent four to six hours with a loss of membrane integrity. The extent of cell death was proportional to the amount of BactoPeptone, and to a lesser extent, the amount of yeast extract. YPD medium conditioned by growth of the mutant was toxic to wild-type cells indicating mutant metabolism established a toxic milieu in the media. Conditioned media contained a volatile component that contributed to toxicity, but only in the presence of a component of BactoPeptone. Fractionation experiments revealed purine nucleosides or bases as the synergistic component. GC-mass spectrometry analysis revealed acetal (1,1-diethoxyethane) as the active volatile. This previously unreported and lethal synergistic interaction of acetal and purines suggests a hitherto unrecognized toxic metabolism potentially exploitable in the search for antifungal targets.

Chemistry and biology of spiroacetals from myxobacteria.

This review details the isolation, biosynthesis, biological activity and synthesis of spiroacetals from the myxobacterium Sorangium cellulosum. The strategies utilised to access the challenging structures and stereochemistry of these natural products are highlighted.

Synthesis of new N,S-acetal analogs derived from juglone with cytotoxic activity against Trypanossoma cruzi.

A series of 11 new N,S-acetal juglone derivatives were synthesized and evaluated against T. cruzi epimastigote forms. These compounds were obtained in good to moderate yields using a microwave irradiation protocol. Among all compounds, two N,S-acetal analogs, showed significant trypanocidal activity. Notably, one compound 11g exhibited selectivity index 10-fold higher than the reference drug benznidazole for epimastigote. The compound 11h was more effective for amastigote forms. Both prototypes exhibited S.I. higher than the benznidazole description. Thus, both compounds proving to be useful candidate molecules to further studies in infected animals.

Fabrication of zwitterionic and pH-responsive polyacetal dendrimers for anticancer drug delivery.

Dendrimer micelles are considered to be the most promising synthetic macromolecules for biomedical applications, but their instability in the blood circulation and undesired release at a non-targeted location have become the major concerns for recent research. Herein, zwitterionic sulfobetaine (SB) functionalized polyacetal dendrimers were fabricated through the alternant aza-Michael addition and thiol-ene click reactions. On account of the acid-labile characteristics of acetal segments and charge reversal effects of sulfobetaine moieties, these uniform dendrimers could form biocompatible and biodegradable micelles in aqueous solutions, which present excellent structural stability, good resistivity to protein absorption and high internalization efficiency for a long period. More importantly, effected by the charge shielding of zwitterions on the surface of polyacetal dendrimers, these DOX-loaded nanoparticles exhibited extremely significant pH-responsive drug release behaviors and remarkable anticancer activity. Thus, we believe that this work not only sheds new light on the application of polyacetal dendrimers but also paves a better way for the development of robust on-demand anti-cancer nanosystems with significant potential for clinical translation.

Candidate genes involved in spiroacetal biosynthesis in the oriental fruit fly, Bactrocera dorsalis.

Spiroacetals are widespread in nature as components of volatile semiochemical secretions from many insect species. The general pathway for spiroacetal biosynthesis in Bactrocera sp. is preliminarily established, but many genes involved in this pathway remain to be characterized. By analyzing transcriptomes of the rectal glands (RGs) from immature and mature females of the oriental fruit fly, Bactrocera dorsalis, a set of genes encoding two acetyl-CoA carboxylases (ACCs), two fatty acid synthases (FASs), eight desaturases (DESs), twelve fatty acyl-CoA reductases (FARs), seventy-two cytochrome P450 enzymes (CYPs), and twenty-three odorant binding proteins (OBPs) were identified. We investigated the expression of candidate genes in immature and mature stages based on the RNA-seq data and Real-time quantitative PCR. Expression profiling revealed that some of these genes were primarily expressed in female rectal glands among different tissues, and were up-regulated in mature females. Semi-quantitative RT-PCR assays were also adapted to examine tissue-specific expression of selected candidate genes. Additionally, their putative functions in spiroacetal synthesis and transportation are proposed. Our study provided large-scale sequence information for further functional studies on spiroacetal biosynthetic pathways.

Biosynthetic investigation of γ-lactones in Sextonia rubra wood using in situ TOF-SIMS MS/MS imaging to localize and characterize biosynthetic intermediates.

Molecular analysis by parallel tandem mass spectrometry (MS/MS) imaging contributes to the in situ characterization of biosynthetic intermediates which is crucial for deciphering the metabolic pathways in living organisms. We report the first use of TOF-SIMS MS/MS imaging for the cellular localization and characterization of biosynthetic intermediates of bioactive γ-lactones rubrynolide and rubrenolide in the Amazonian tree Sextonia rubra (Lauraceae). Five γ-lactones, including previously reported rubrynolide and rubrenolide, were isolated using a conventional approach and their structural characterization and localization at a lateral resolution of ~400 nm was later achieved using TOF-SIMS MS/MS imaging analysis. 2D/3D MS imaging at subcellular level reveals that putative biosynthetic γ-lactones intermediates are localized in the same cell types (ray parenchyma cells and oil cells) as rubrynolide and rubrenolide. Consequently, a revised metabolic pathway of rubrynolide was proposed, which involves the reaction between 2-hydroxysuccinic acid and 3-oxotetradecanoic acid, contrary to previous studies suggesting a single polyketide precursor. Our results provide insights into plant metabolite production in wood tissues and, overall, demonstrate that combining high spatial resolution TOF-SIMS imaging and MS/MS structural characterization offers new opportunities for studying molecular and cellular biochemistry in plants.

Tandem Mass Spectrometry Imaging and in Situ Characterization of Bioactive Wood Metabolites in Amazonian Tree Species Sextonia rubra.

Driven by a necessity for confident molecular identification at high spatial resolution, a new time-of-flight secondary ion mass spectrometry (TOF-SIMS) tandem mass spectrometry (tandem MS) imaging instrument has been recently developed. In this paper, the superior MS/MS spectrometry and imaging capability of this new tool is shown for natural product study. For the first time, via in situ analysis of the bioactive metabolites rubrynolide and rubrenolide in Amazonian tree species Sextonia rubra (Lauraceae), we were able both to analyze and to image by tandem MS the molecular products of natural biosynthesis. Despite the low abundance of the metabolites in the wood sample(s), efficient MS/MS analysis of these γ-lactone compounds was achieved, providing high confidence in the identification and localization. In addition, tandem MS imaging minimized the mass interferences and revealed specific localization of these metabolites primarily in the ray parenchyma cells but also in certain oil cells and, further, revealed the presence of previously unidentified γ-lactone, paving the way for future studies in biosynthesis.

Synthesis and fungicidal activity of novel imidazole-based ketene dithioacetals.

Novel imidazole-based ketene dithioacetals show impressive in planta activity against the economically important plant pathogens Alternaria solani, Botryotinia fuckeliana, Erysiphe necator and Zymoseptoria tritici. Especially derivatives of the topical antifungal lanoconazole, which bear an alkynyloxy or a heteroaryl group in the para-position of the phenyl ring, exhibit excellent control of the mentioned phytopathogens. These compounds inhibit 14α -demethylase in the sterol biosynthesis pathway of the fungi. Synthesis routes starting from either benzaldehydes or acetophenones as well as structure-activity relationships are discussed in detail.

Evidence that Cerambycid Beetles Mimic Vespid Wasps in Odor as well as Appearance.

We present evidence that cerambycid species that are supposed mimics of vespid wasps also mimic their model's odor by producing spiroacetals, common constituents of vespid alarm pheromones. Adults of the North American cerambycids Megacyllene caryae (Gahan) and Megacyllene robiniae (Forster) are conspicuously patterned yellow and black, and are believed to be mimics of aculeate Hymenoptera, such as species of Vespula and Polistes. Adult males of M. caryae produce an aggregation-sex pheromone, but both sexes produce a pungent odor when handled, which has been assumed to be a defensive response. Headspace aerations of agitated females of M. caryae contained 16 compounds with mass spectra characteristic of spiroacetals of eight distinct chemical structures, with the dominant compound being (7E,2E)-7-ethyl-2-methyl-1,6-dioxaspiro[4.5]decane. Headspace samples of agitated males of M. caryae contained five of the same components, with the same dominant compound. Females of M. robiniae produced six different spiroacetals, one of which was not produced by M. caryae, (2E,7E)-2-ethyl-7-methyl-1,6-dioxaspiro[4.5]decane, and five that were shared with M. caryae, including the dominant (2E,8E)-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane. The latter compound is the sole spiroacetal produced by both males and females of a South American cerambycid species, Callisphyris apicicornis (Fairmaire & Germain), which is also thought to be a wasp mimic. Preliminary work also identified spiroacetals of similar or identical structure released by vespid wasps that co-occur with the Megacyllene species.

Enzyme classification using complex dynamic hemithioacetal systems.

A complex dynamic hemithioacetal system was generated for the evaluation of lipase reactivities in organic media. In combination with pattern recognition methodology, twelve different lipases were successfully classified into four distinct groups following their reaction selectivities and reactivities. A probe lipase was further categorized using the training matrix with predicted reactivity.

3CAPS - a structural AP-site analogue as a tool to investigate DNA base excision repair.

Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP-sites. With its 3'-phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase ß but repaired only by strand displacement as the 5'-deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

Site-specific modification of the anticancer and antituberculosis polyether salinomycin by biosynthetic engineering.

The complex bis-spiroacetal polyether ionophore salinomycin has been identified as a uniquely selective agent against cancer stem cells and is also strikingly effective in an animal model of latent tuberculosis. The basis for these important activities is unknown. We show here that deletion of the salE gene abolishes salinomycin production and yields two new analogues, in both of which the C18C19 cis double bond is replaced by a hydroxy group stereospecifically located at C19, but which differ from each other in the configuration of the bis-spiroacetal. These results identify SalE as a novel dehydratase and demonstrate that biosynthetic engineering can be used to redirect the reaction cascade of oxidative cyclization to yield new salinomycin analogues for use in mechanism-of-action studies.

Double parallel dynamic resolution through lipase-catalyzed asymmetric transformation.

Dynamic systems based on double parallel reactions have been generated and resolved in situ by secondary lipase-catalyzed asymmetric transformation, resulting in high chemo- and enantioselectivities.

Definition of metabolism-dependent xenobiotic toxicity with co-cultures of human hepatocytes and mouse 3T3 fibroblasts in the novel integrated discrete multiple organ co-culture (IdMOC) experimental system: results with model toxicants aflatoxin B1, cyclophosphamide and tamoxifen.

The integrated discrete multiple organ co-culture system (IdMOC) allows the co-culturing of multiple cell types as physically separated cells interconnected by a common overlying medium. We report here the application of IdMOC with two cell types: the metabolically competent primary human hepatocytes, and a metabolically incompetent cell line, mouse 3T3 fibroblasts, in the definition of the role of hepatic metabolism on the cytotoxicity of three model toxicants: cyclophosphamide (CPA), aflatoxin B1 (AFB) and tamoxifen (TMX). The presence of hepatic metabolism in IdMOC with human hepatocytes was demonstrated by the metabolism of the P450 isoform 3A4 substrate, luciferin-IPA. The three model toxicants showed three distinct patterns of cytotoxic profile: TMX was cytotoxic to 3T3 cells in the absence of hepatocytes, with slightly lower cytotoxicity towards both 3T3 cells and hepatocytes in the IdMOC. AFB was selective toxic towards the human hepatocytes and relatively noncytotoxic towards 3T3 cells both in the presence and absence of the hepatocytes. CPA cytotoxicity to the 3T3 cells was found to be significantly enhanced by the presence of the hepatocytes, with the cytotoxicity dependent of the number of hepatocytes, and with the cytotoxicity attenuated by the presence of a non-specific P450 inhibitor, 1-aminobenzotriazole. We propose here the following classification of toxicants based on the role of hepatic metabolism as defined by the human hepatocyte-3T3 cell IdMOC assay: type I: direct-acting cytotoxicants represented by TMX as indicated by cytotoxicity in 3T3 cells in the absence of hepatocytes; type II: metabolism-dependent cytotoxicity represented by AFB1 with effects localized within the site of metabolic activation (i. e. hepatocytes); and type III: metabolism-dependent cytotoxicity with metabolites that can diffuse out of the hepatocytes to cause toxicity in cells distal from the site of metabolism, as exemplified by CPA.

Molecular imaging of cytochrome P450 activity in mice.

Detailed knowledge of drug metabolism is relevant information provided by preclinical drug development research. Oxidative enzymes such as those belonging to P450 family of cytochromes (CYP) play a prominent role in drug metabolism. Here, we propose an innovative method based on bioluminescence in vivo imaging which has the potential to simplify the in vivo measurement of CYP activity also providing a dynamic measure of the effects of a drug on a specific P450 enzyme complex in a living mouse. The method is based on a pro-luciferin which can be converted into the active luciferase substrate by a specific P450 activity. The pro-luciferin is administered to a luciferase reporter mouse which produces luminescent signals in relation to the cytochrome activity present in each tissue. The photon emission generated can be easily localized and quantified by optical imaging. To demonstrate the validity of the system, we pharmacologically induced hepatic Cyp3a in the reporter mouse and proved that pro-luciferin administration generates a Cyp3a selective signal in the chest area that can be efficiently detected by optical imaging. The kind of tool generated has the potential to be exploited for the study of additional CYPs.

Mechanism of a chemical glycosylation reaction.

Glycosylation is arguably the most important reaction in the field of glycochemistry, yet it involves one of the most empirically interpreted mechanisms in the science of organic chemistry. The beta-mannopyranosides, long considered one of the more difficult classes of glycosidic bond to prepare, were no exception to this rule. A number of logical but circuitous routes for their preparation were described in the literature, but they were accompanied by an even greater number of mostly ineffective recipes with which to access them directly. This situation changed in 1996 with the discovery of the 4,6-O-benzylidene acetal as a control element permitting direct entry into the beta-mannopyranosides, typically with high yield and selectivity. The unexpected nature of this phenomenon demanded study of the mechanism, leading first to the demonstration of the alpha-mannopyranosyl triflates as reaction intermediates and then to the development of alpha-deuterium kinetic isotope effect methods to probe their transformation into the product glycosides. In this Account, we assemble our observations into a comprehensive assessment consistent with a single mechanistic scheme. The realization that in the glucopyranose series the 4,6-O-benzylidene acetal is alpha- rather than beta-directing led to further investigations of substituent effects on the stereoselectivity of these glycosylation reactions, culminating in their explanation in terms of the covalent alpha-glycosyl triflates acting as a reservoir for a series of transient contact and solvent-separated ion pairs. The function of the benzylidene acetal, as explained by Bols and co-workers, is to lock the C6-O6 bond antiperiplanar to the C5-O5 bond, thereby maximizing its electron-withdrawing effect, destabilizing the glycosyl oxocarbenium ion, and shifting the equilibria as far as possible toward the covalent triflate. beta-Selective reactions result from attack of the nucleophile on the transient contact ion pair in which the alpha-face of the oxocarbenium ion is shielded by the triflate counterion. The alpha-products arise from attack either on the solvent-separated ion pair or on a free oxocarbenium ion, according to the dictates of the anomeric effect. Changes in selectivity from varying stereochemistry (glucose versus mannose) or from using different protecting groups can be explained by the shifting position of the key equilibria and, in particular, by the energy differences between the covalent triflate and the ion pairs. Of particular note is the importance of substitutents at the 3-position of the donor; an explanation is proposed that invokes their evolving torsional interaction with the substituent at C2 as the chair form of the covalent triflate moves toward the half-chair of the oxocarbenium ion.

Spiroacetal biosynthesis in fruit flies is complex: distinguishable origins of the same major spiroacetal released by different Bactrocera spp.

The major spiroacetal ((E,E)-1) of the pestiferous fruit flies, Bactrocera tryoni and Bactrocera cucumis, is biosynthesised from fatty acids by distinguishable pathways which utilise modified beta-oxidation and C-H hydroxylation, generating a putative ketodiol which cyclises.

Characterization of cyclic acetal hydroxyapatite nanocomposites for craniofacial tissue engineering.

Cyclic acetal hydrogels are a novel group of biomaterials which may facilitate osteogenic differentiation of encapsulated bone marrow stromal cells (BMSCs) because of their neutral degradation products. Here, we have incorporated hydroxyapatite nanoparticles within cyclic acetal hydrogels to create cyclic acetal nanocomposites for craniofacial tissue engineering applications. We hypothesized that inclusion of nanosized hydroxyapatite particles within cyclic acetal hydrogels would upregulate osteogenic signal expression of encapsulated BMSCs, due to enhanced cell adhesion, and therefore promote osteodifferentiation. Experimental nanocomposite groups consisted of lower (5 ng/mL) and higher (50 ng/mL) concentrations of nanoparticles. The nanocomposites were characterized by scanning electron microscopy, transmission electron microscopy, and energy dispersive spectroscopy. Swelling parameters of hydrogels in the presence of nanoparticles was studied. Osteoblastic differentiation was characterized by alkaline phosphatase (ALP) and osteocalcin (OC) expression, whereas endogenous osteogenic signal expression was characterized by morphogenetic protein-2 (BMP-2) expression. Finally, immunohistochemistry was performed to detect the presence of OC at the protein level. Results indicated that hydroxyapatite nanoparticles were uniformly distributed throughout the hydrogels and did not affect material properties of the gels. Viability of cells was not affected by nanoparticle concentration, and BMP-2 and OC mRNA expression was enhanced in the presence of nanoparticles. However, a difference in BMP-2, ALP, and OC mRNA expression was not noted between the lower and higher concentrations of nanoparticles. This work demonstrates that inclusion of hydroxyapatite nanoparticles within a cyclic acetal nanocomposite hydrogel may enhance BMSC differentiation by promoting endogenous osteogenic signal expression.

Effects of dietary docosahexaenoic acid connecting phospholipids on the learning ability and fatty acid composition of the brain.

The effects of dietary docosahexaenoic acid (DHA, C22:6n-3) connecting phospholipids on the learning ability and fatty acid composition of the brain were investigated in hypercholesterolemic mice. ICR mice were subjected to a very low level of n-3 fatty acids through two generations. At 4 wk of age, the F(1) generation, n-3 fatty acid deficient male mice were provided with an experimental diet containing four kinds of lipids (safflower oil: Saf, DHA connecting triacylglycerols: DHA-TG, DHA connecting phospholipids: DHA-PL, soybean phospholipids: Soy-PL) for 5 wk. Another group of ICR mice were obtained and fed a commercial diet (CE-2, CLEA Japan, Inc.) as a control. The learning and memory abilities of the mice were evaluated by the modified avoidance procedure. The learning and memory ability level was significantly higher in mice fed the DHA-PL diet than in those fed the Saf and Soy-PL diets, and was the same level as the control. The DHA levels of phosphatidylethanolamine in the brain were significantly higher in the mice fed the two types of DHA-containing diets than in those fed the Saf and Soy-PL diets and was not significantly different between DHA-TG and DHA-PL. The dimethylacetal levels in the brain were significantly higher in the mice fed the DHA-PL diet than in those fed the Saf and DHA-TG diets. These results suggest that the dietary DHA connecting phospholipids have the effect of improving memory learning, and may be related to the both the DHA and plasmalogen levels in the brain.

Evaluation of luciferin-isopropyl acetal as a CYP3A4 substrate for human hepatocytes: effects of organic solvents, cytochrome P450 (P450) inhibitors, and P450 inducers.

This study represents the first report on the characterization of luciferin-isopropyl acetal (LIPA) as a CYP3A4 substrate in human hepatocytes. LIPA metabolism by human hepatocytes was found to be linear with time up to 120 min and followed Michaelis-Menten kinetics, with apparent K(m) value of 15 muM and V(max) of 41 pmol/min/million hepatocytes for the hepatocytes used in the study. The nonspecific cytochrome P450 (P450) inhibitor 1-aminobenzotriazole (ABT) and the CYP3A4-selective inhibitor ketoconazole (KTZ) caused concentration-dependent inhibition of LIPA metabolism, with more than 50% inhibition observed at the lowest concentrations evaluated of 7.8 microM (ABT) and 0.78 microM (KTZ), and near 100% inhibition observed at higher concentrations. Substantially lower inhibitory effects were observed for the non-CYP3A4 inhibitors diethyldithiocarbamate, furafylline, omeprazole, orphenadrine, sulfaphenazole, and quinidine. The commonly used organic solvents-acetonitrile, dimethyl sulfoxide (DMSO), and methanol-were found to inhibit LIPA metabolism, with approximately 50% inhibition at concentrations of 5, 1.25, and 5% (by volume), respectively. The comparatively higher inhibitory effects of DMSO relative to that for acetonitrile and methanol on LIPA metabolism were consistent with its known CYP3A4 inhibitory effects reported by others. LIPA metabolism in human hepatocytes was found to be induced by the treatment of human hepatocytes with the prototypical CYP3A4 inducers rifampin, carbamazepine, omeprazole, phenobarbital, and phenytoin but not by the CYP1A2 inducer 3-methylcholanthrene. Although the selectivity toward CYP3A4 needs to be definitively evaluated using cDNA-expressed P450 isoforms, the results suggest that LIPA is a suitable substrate to be used with human hepatocytes for the evaluation of CYP3A4 activities.