Multi-omics Investigation into the Mechanism of Action of an Anti-tubercular Fatty Acid Analogue.

The mechanism of action (MoA) of a clickable fatty acid analogue 8-(2-cyclobuten-1-yl)octanoic acid (DA-CB) has been investigated for the first time. Proteomics, metabolomics, and lipidomics were combined with a network analysis to investigate the MoA of DA-CB against Mycobacterium smegmatis (Msm). The metabolomics results showed that DA-CB has a general MoA related to that of ethionamide (ETH), a mycolic acid inhibitor that targets enoyl-ACP reductase (InhA), but DA-CB likely inhibits a step downstream from InhA. Our combined multi-omics approach showed that DA-CB appears to disrupt the pathway leading to the biosynthesis of mycolic acids, an essential mycobacterial fatty acid for both Msm and Mycobacterium tuberculosis (Mtb). DA-CB decreased keto-meromycolic acid biosynthesis. This intermediate is essential in the formation of mature mycolic acid, which is a key component of the mycobacterial cell wall in a process that is catalyzed by the essential polyketide synthase Pks13 and the associated ligase FadD32. The multi-omics analysis revealed further collateral alterations in bacterial metabolism, including the overproduction of shorter carbon chain hydroxy fatty acids and branched chain fatty acids, alterations in pyrimidine metabolism, and a predominate downregulation of proteins involved in fatty acid biosynthesis. Overall, the results with DA-CB suggest the exploration of this and related compounds as a new class of tuberculosis (TB) therapeutics. Furthermore, the clickable nature of DA-CB may be leveraged to trace the cellular fate of the modified fatty acid or any derived metabolite or biosynthetic intermediate.

Synthesis of α-Cyano and α-Sulfonyl Cyclic Ethers via Intramolecular Reactions of Peroxides with Sulfone- and Nitrile-Stabilized Carbanions.

The intramolecular reaction of carbon nucleophiles with oxygen-centered electrophiles has been little explored outside of nucleophilic epoxidation. We now report the synthesis of sulfonyl- and cyano-substituted oxacycles via intramolecular reaction of sulfone- and nitrile-stabilized carbanions with dialkyl peroxides, triethylsilyl/alkyl peroxides, and monoperoxyacetals. The cyclizations are successfully applied to synthesize oxetanes, tetrahydrofurans, and tetrahydropyrans but fail for oxepanes. Cyclizations involving the relatively stabilized anion derived from a benzylic nitrile proceed in high yields for a variety of peroxides, including those in which the electrophilic oxygen is formally isobutyl or neopentyl. Corresponding cyclizations of an alkanenitrile are successful with both dialkyl and alkyl silyl peroxides but demonstrate much greater variability in yields. Reactions of sulfone-containing substrates are successful only with dialkyl peroxides. The success of reactions appears to be strongly influenced by the rate of peroxide decomposition, which appears to be highest for reactions involving poorly stabilized anions. The significant variation in diastereoselectivity observed for different classes of peroxide on a common framework suggests the possibility of substrate-dependent reaction mechanisms.

A Dedicated Type II NADPH Dehydrogenase Performs the Penultimate Step in the Biosynthesis of Vitamin K1 in Synechocystis and Arabidopsis.

Mutation of Arabidopsis thaliana NAD(P)H DEHYDROGENASE C1 (NDC1; At5g08740) results in the accumulation of demethylphylloquinone, a late biosynthetic intermediate of vitamin K1. Gene coexpression and phylogenomics analyses showed that conserved functional associations occur between vitamin K biosynthesis and NDC1 homologs throughout the prokaryotic and eukaryotic lineages. Deletion of Synechocystis ndbB, which encodes for one such homolog, resulted in the same defects as those observed in the cyanobacterial demethylnaphthoquinone methyltransferase knockout. Chemical modeling and assay of purified demethylnaphthoquinone methyltransferase demonstrated that, by virtue of the strong electrophilic nature of S-adenosyl-l-methionine, the transmethylation of the demethylated precursor of vitamin K is strictly dependent on the reduced form of its naphthoquinone ring. NDC1 was shown to catalyze such a prerequisite reduction by using NADPH and demethylphylloquinone as substrates and flavine adenine dinucleotide as a cofactor. NDC1 displayed Michaelis-Menten kinetics and was markedly inhibited by dicumarol, a competitive inhibitor of naphthoquinone oxidoreductases. These data demonstrate that the reduction of the demethylnaphthoquinone ring represents an authentic step in the biosynthetic pathway of vitamin K, that this reaction is enzymatically driven, and that a selection pressure is operating to retain type II NAD(P)H dehydrogenases in this process.

Comparison of Mannose, Ethylene Glycol, and Methoxy-Terminated Diluents on Specificity and Selectivity of Electrochemical Peptide-Based Sensors.

We report the synthesis and application of three new antifouling diluents for the fabrication of an E-PB HIV sensor. Among the three thiolated antifouling diluents used in this study, the methoxy-terminated diluent (C6-MEG) is the most effective in alleviating both nonspecific binding and adsorption of matrix contaminants onto the sensor surface, especially when compared to the mannose- (C6-MAN) and ethylene-glycol-terminated (C6-EG) diluents. The sensor fabricated with C6-MEG has a specificity factor (∼13.5) substantially higher than the sensor passivated with only 6-mercapto-1-hexanol (∼1.5). It is functional even when employed directly in 25% serum, an achievement that has not been observed with this class of E-PB sensors. More importantly, incorporation of these antifouling diluents has negligible impact on other important sensor properties such as sensitivity and binding kinetics. This sensor passivation strategy is versatile and can potentially be used with other E-PB sensors, as well as surface-based sensors that utilize thiol-gold self-assembled monolayer chemistry.

Dietary Plant Sterol Esters Must Be Hydrolyzed to Reduce Intestinal Cholesterol Absorption in Hamsters.

BACKGROUND: Elevated concentrations of LDL cholesterol are associated with the development of atherosclerosis and therefore are considered an important target for intervention to prevent cardiovascular diseases. The inhibition of cholesterol absorption in the small intestine is an attractive approach to lowering plasma cholesterol, one that is addressed by drug therapy as well as dietary supplementation with plant sterols and plant sterol esters (PSEs). OBJECTIVE: This study was conducted to test the hypothesis that the cholesterol-lowering effects of PSE require hydrolysis to free sterols (FSs). METHODS: Male Syrian hamsters were fed atherogenic diets (AIN-93M purified diet containing 0.12% cholesterol and 8% coconut oil) to which one of the following was added: no PSEs or ethers (control), 5% sterol stearate esters, 5% sterol palmitate esters (PEs), 5% sterol oleate esters (OEs), 5% sterol stearate ethers (STs; to mimic nonhydrolyzable PSE), or 3% FSs plus 2% sunflower oil. The treatments effectively created a spectrum of PSE hydrolysis across which cholesterol metabolism could be compared. Metabolic measurements included cholesterol absorption, plasma and liver lipid concentration, and fecal neutral sterol and bile acid excretion. RESULTS: The STs and the PEs and SEs were poorly hydrolyzed (1.69-4.12%). In contrast, OEs were 88.3% hydrolyzed. The percent hydrolysis was negatively correlated with cholesterol absorption (r = -0.85; P < 0.0001) and positively correlated with fecal cholesterol excretion (r = 0.92; P < 0.0001), suggesting that PSE hydrolysis plays a central role in the cholesterol-lowering properties of PSE. CONCLUSIONS: Our data on hamsters suggest that PSE hydrolysis and the presence of FSs is necessary to induce an optimum cholesterol-lowering effect and that poorly hydrolyzed PSEs may lower cholesterol through an alternative mechanism than that of competition with cholesterol for micelle incorporation.

Synthesis of Ethers via Reaction of Carbanions and Monoperoxyacetals.

Although transfer of electrophilic alkoxyl ("RO+") from organic peroxides to organometallics offers a complement to traditional methods for etherification, application has been limited by constraints associated with peroxide reactivity and stability. We now demonstrate that readily prepared tetrahydropyranyl monoperoxyacetals react with sp(3) and sp(2) organolithium and organomagnesium reagents to furnish moderate to high yields of ethers. The method is successfully applied to the synthesis of alkyl, alkenyl, aryl, heteroaryl, and cyclopropyl ethers, mixed O,O-acetals, and S,S,O-orthoesters. In contrast to reactions of dialkyl and alkyl/silyl peroxides, the displacements of monoperoxyacetals provide no evidence for alkoxy radical intermediates. At the same time, the high yields observed for transfer of primary, secondary, or tertiary alkoxides, the latter involving attack on neopentyl oxygen, are inconsistent with an SN2 mechanism. Theoretical studies suggest a mechanism involving Lewis acid promoted insertion of organometallics into the O-O bond.

Self-healing gels based on constitutional dynamic chemistry and their potential applications.

As representative soft materials with widespread applications, gels with various functions have been developed. However, traditional gels are vulnerable to stress-induced formation of cracks. The propagation of these cracks may affect the integrity of network structures of gels, resulting in the loss of functionality and limiting the service life of the gels. To address this challenge, self-healing gels that can restore their functionalities and structures after damage have been developed as "smart" soft materials. In this paper, we present an overview of the current strategies for synthesizing self-healing gels based on the concept of constitutional dynamic chemistry, which involves molecular structures capable of establishing dynamic networks based upon physical interactions or chemical reactions. The characterization methods of self-healing gels and the key factors that affect self-healing properties are analyzed. We also illustrate the emerging applications of self-healing gels, with emphasis on their usage in industry (coatings, sealants) and biomedicine (tissue adhesives, agents for drug or cell delivery). We conclude with a perspective on challenges facing the field, along with prospects for future development.

Oxacycle synthesis via intramolecular reaction of carbanions and peroxides.

The intramolecular reaction of dialkyl peroxides with carbanions, generated via chemoselective metal-heteroatom exchange or deprotonation, provides a new approach to cyclic ethers. Applied in tandem with C-C bond formation, the strategy enables a one-step annelation to form oxaospirocycles.

ß-Keto and ß-hydroxyphosphonate analogs of biotin-5'-AMP are inhibitors of holocarboxylase synthetase.

Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin to cytoplasmic and mitochondrial carboxylases, nuclear histones, and over a hundred human proteins. Nonhydrolyzable ketophosphonate (ß-ketoP) and hydroxyphosphonate (ß-hydroxyP) analogs of biotin-5'-AMP inhibit holocarboxylase synthetase (HLCS) with IC50 values of 39.7 µM and 203.7 µM. By comparison, an IC50 value of 7 µM was observed with the previously reported biotinol-5'-AMP. The Ki values, 3.4 µM and 17.3 µM, respectively, are consistent with the IC50 results, and close to the Ki obtained for biotinol-5'-AMP (7 µM). The ß-ketoP and ß-hydroxyP molecules are competitive inhibitors of HLCS while biotinol-5'-AMP inhibited HLCS by a mixed mechanism.

Tandem application of C-C bond-forming reactions with reductive ozonolysis.

Several variants of reductive ozonolysis, defined here as the in situ generation of aldehydes or ketones during ozonolytic cleavage of alkenes, are demonstrated to work effectively in tandem with a number of C-C bond-forming reactions. For reactions involving basic nucleophiles (1,2-addition of Grignard reagents, Wittig or Horner-Emmons olefinations, and directed aldol reactions of lithium enolates), the one-pot process offers a rapid and high-yielding alternative to traditional two-step protocols.

Synthesis of alkyl hydroperoxides via alkylation of gem-dihydroperoxides.

2-Fold alkylation of 1,1-dihydroperoxides, followed by hydrolysis of the resulting bisperoxyacetals, provides a convenient method for synthesis of primary and secondary alkyl hydroperoxides.

Design and Synthesis of a New Class of Twin-Chain Amphiphiles for Self-Assembled Monolayer-based Electrochemical Biosensor Applications.

A new class of twin-chain hydroxyalkylthiols (mercaptoalkanols) featuring a nearly constant cross-section and the potential for modification of one or both termini are available with complete regioselectivity through Pd-mediated couplings of benzene diiododitriflate, including an example of a previously unreported coupling to generate an ortho-substituted arene bis acetic acid. Self-assembled monolayers (SAMs) prepared from the new amphiphiles demonstrate improved stability in an electrochemical sensor system compared with monolayers prepared from analogous single chain thiols.

Re2O7-catalyzed reaction of hemiacetals and aldehydes with O-, S-, and C-nucleophiles.

Re(VII) oxides catalyze the acetalization, monoperoxyacetalization, monothioacetalization and allylation of hemiacetals. The reactions, which take place under mild conditions and at low catalyst loadings, can be conducted using hemiacetals, the corresponding O-silyl ethers, and, in some cases, the acetal dimers. Aldehydes react under similar conditions to furnish good yields of dithioacetals. Reactions of hemiacetals with nitrogen nucleophiles are unsuccessful. 1,2-Dioxolan-3-ols (peroxyhemiacetals) undergo Re(VII)-promoted etherification but not allylation. Hydroperoxyacetals (1-alkoxyhydroperoxides) undergo selective exchange of the alkoxide group in the presence of either Re2O7 or a Brønsted acid.

In vitro and in vivo activity of 3-alkoxy-1,2-dioxolanes against Schistosoma mansoni.

OBJECTIVES: Compounds characterized by a peroxidic skeleton are an interesting starting point for antischistosomal drug discovery. Previously a series of 3-alkoxy-1,2-dioxolanes, which are chemically stable cyclic peroxides, demonstrated significant in vitro activity against Plasmodium falciparum. We aimed to evaluate the potential of these compounds against Schistosoma mansoni and elucidate the roles of iron and peroxidic groups in activity. METHODS: Drugs were tested against juvenile and adult stages of S. mansoni in vitro and in vivo. Selected structures were assessed in vitro against schistosomes in the presence of additional iron sources. In addition, drugs were tested in vitro and in vivo against Echinostoma caproni, a non-blood-feeding intestinal fluke. Finally, the activity of non-peroxidic analogues was evaluated. RESULTS: Three dioxolanes displayed IC50s ≤ 20.1 µM against adult schistosomes and values as low as 4.2 µM against newly transformed schistosomula. Nonetheless, only moderate, non-significant worm burden reductions were observed after treatment of mice harbouring adult infections. Drugs lacked activity against juvenile schistosomes in vivo. Two selected dioxolanes showed in vitro activity against E. caproni down to concentrations of 5 mg/L, but none of the compounds revealed in vivo activity. All tested non-peroxidic analogues lacked activity in vitro against both parasites. CONCLUSIONS: Selected dioxolanes presented interesting in vitro activity, but low in vivo activities have to be overcome to identify a lead candidate. Although the inactivity of non-peroxidic analogues underlines the necessity of a peroxide functional group, incubation of adult schistosomes with additional iron sources did not alter activity, supporting an iron-independent mode of activation.

Generation of singlet oxygen from fragmentation of monoactivated 1,1-dihydroperoxides.

The first singlet excited state of molecular oxygen ((1)O(2)) is an important oxidant in chemistry, biology, and medicine. (1)O(2) is most often generated through photosensitized excitation of ground-state oxygen. (1)O(2) can also be generated chemically through the decomposition of hydrogen peroxide and other peroxides. However, most of these "dark oxygenations" require water-rich media associated with short (1)O(2) lifetimes, and there is a need for oxygenations able to be conducted in organic solvents. We now report that monoactivated derivatives of 1,1-dihydroperoxides undergo a previously unobserved fragmentation to generate high yields of singlet molecular oxygen ((1)O(2)). The fragmentations, which can be conducted in a variety of organic solvents, require a geminal relationship between a peroxyanion and a peroxide activated toward heterolytic cleavage. The reaction is general for a range of skeletal frameworks and activating groups and, via in situ activation, can be applied directly to 1,1-dihydroperoxides. Our investigation suggests the fragmentation involves rate-limiting formation of a peroxyanion that decomposes via a Grob-like process.

Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes.

HSAF was isolated from Lysobacter enzymogenes , a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here we describe the HSAF biosynthetic gene cluster, which contains only a single-module polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/NRPS are six genes that encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activates L-ornithine and that the four-domain NRPS is able to catalyze the formation of a tetramic acid-containing product from acyl-S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms.

Deciphering the mechanism of action of antitubercular compounds with metabolomics.

Tuberculosis (TB), one of the oldest and deadliest bacterial diseases, continues to cause serious global economic, health, and social problems. Current TB treatments are lengthy, expensive, and routinely ineffective against emerging drug resistant strains. Thus, there is an urgent need for the identification and development of novel TB drugs possessing comprehensive and specific mechanisms of action (MoAs). Metabolomics is a valuable approach to elucidating the MoA, toxicity, and potency of promising chemical leads, which is a critical step of the drug discovery process. Recent advances in metabolomics methodologies for deciphering MoAs include high-throughput screening techniques, the integration of multiple omics methods, mass spectrometry imaging, and software for automated analysis. This review describes recently introduced metabolomics methodologies and techniques for drug discovery, highlighting specific applications to the discovery of new antitubercular drugs and the elucidation of their MoAs.

Fragmentation of chloroperoxides: hypochlorite-mediated dehydration of hydroperoxyacetals to esters.

Hypochlorites efficently dehydrate hydroperoxyacetals to furnish the corresponding esters. The reaction, which can be accomplished with stoichometric Ca(OCl)(2) or with catalytic amounts of t-BuOCl, appears to involve formation and heterolytic fragmentation of secondary chloroperoxides, species not previously described in solution chemistry.

A click-based modular approach to introduction of peroxides onto molecules and nanostructures.

Copper-promoted azide/alkyne cycloadditions (CuAAC) are explored as a tool for modular introduction of peroxides onto molecules and nanomaterials. Dialkyl peroxide-substituted alkynes undergo Cu(i)-promoted reaction with azides in either organic or biphasic media to furnish peroxide-substituted 1,2,3-triazoles. Heterolytic fragmentation of the peroxide to an aldehyde, a side reaction that appears to be related to the formation of the triazole, can be suppressed by use of excess alkyne, the presence of triethylsilane, or by use of iodoalkyne substrates. Complementary reactions of simple alkynes with azido-substituted peroxides are much less efficient. Click reactions of alkynyl peroxyacetals are also reported; reductive fragmentation can be minimized by increasing the distance between the peroxyacetal and the alkyne. The strategy enables modular introduction of dialkyl peroxides and peroxyacetals onto gold nanoparticles, the first such process to be reported.

Spiroadamantyl 1,2,4-trioxolane, 1,2,4-trioxane, and 1,2,4-trioxepane pairs: relationship between peroxide bond iron(II) reactivity, heme alkylation efficiency, and antimalarial activity.

These data suggest that iron(II) reactivity for a set of homologous spiroadamantyl 1,2,4-trioxolane, 1,2,4-trioxane, and 1,2,4-trioxepane peroxide heterocycles is a necessary, but insufficient, property of animalarial peroxides. Heme alkylation efficiency appears to give a more accurate prediction of antimalarial activity than FeSO(4)-mediated reaction rates, suggesting that antimalarial activity is not merely dependent on peroxide bond cleavage, but also on the ability of reactive intermediates to alkylate heme or other proximal targets.