Synthesis and structure of clozapine N-oxide hemi(hydro-chloride): an infinite hydrogen-bonded poly[n]catenane.

The structure of the title compound, 2C18H19ClN4O·HCl or (CNO)2·HCl (C36H39Cl3N8O2), at 100 K has tetra-gonal (I4/m) symmetry. The dihedral angle between the benzene rings of the fused ring system of the CNO mol-ecule is 40.08 (6)° and the equivalent angle between the seven-membered ring and its pendant N-oxide ring is 31.14 (7)°. The structure contains a very strong, symmetrical O-H⋯O hydrogen bond [O⋯O = 2.434 (2) Å] between two equivalent R 3N+-O- moieties, which share a proton lying on a crystallographic twofold rotation axis. These units then form a (CNO)4·(HCl)2 ring by way of two equivalent N-H⋯Cl hydrogen bonds (Cl- site symmetry m). These rings are catenated into infinite chains propagating along the c-axis direction by way of shape complementarity and directional C-H⋯N and C-H⋯π inter-actions.

Synthesis of the Alkylsulfonate Metabolites Cysteinolic Acid, 3-Amino-2-hydroxypropanesulfonate, and 2,3-Dihydroxypropanesulfonate.

Chiral hydroxy- and aminohydroxysulfonic acids are widespread in the marine and terrestrial environment. Here we report simple methods for the synthesis of d- and l-cysteinolic acid (from (Boc-d-Cys-OH)2 and (Boc-l-Cys-OH)2, respectively), R- and S-3-amino-2-hydroxypropanesulfonate (from S- and R-epichlorohydrin, respectively), and R- and S-2,3-dihydroxypropanesulfonate (from S- and R-epichlorohydrin, respectively). d-Cysteinolate bile salts were generated by coupling with cholic and chenodeoxycholic acids. A series of single-crystal 3D X-ray structures confirmed the absolute configurations of the aminosulfonates. By comparison of optical rotation, we assign naturally occurring 3-amino-2-hydroxypropanesulfonate from Gateloupia livida as possessing the R-configuration. This simple synthetic approach will support future studies of the occurrence, chemotaxonomic distribution, and metabolism of these alkylsulfonates.

Genome sequences of Arthrobacter spp. that use a modified sulfoglycolytic Embden-Meyerhof-Parnas pathway.

Sulfoglycolysis pathways enable the breakdown of the sulfosugar sulfoquinovose and environmental recycling of its carbon and sulfur content. The prototypical sulfoglycolytic pathway is a variant of the classical Embden-Meyerhof-Parnas (EMP) pathway that results in formation of 2,3-dihydroxypropanesulfonate and was first described in gram-negative Escherichia coli. We used enrichment cultures to discover new sulfoglycolytic bacteria from Australian soil samples. Two gram-positive Arthrobacter spp. were isolated that produced sulfolactate as the metabolic end-product. Genome sequences identified a modified sulfoglycolytic EMP gene cluster, conserved across a range of other Actinobacteria, that retained the core sulfoglycolysis genes encoding metabolic enzymes but featured the replacement of the gene encoding sulfolactaldehyde (SLA) reductase with SLA dehydrogenase, and the absence of sulfoquinovosidase and sulfoquinovose mutarotase genes. Excretion of sulfolactate by these Arthrobacter spp. is consistent with an aerobic saprophytic lifestyle. This work broadens our knowledge of the sulfo-EMP pathway to include soil bacteria.

A Family of Dual-Activity Glycosyltransferase-Phosphorylases Mediates Mannogen Turnover and Virulence in Leishmania Parasites.

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of ß-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.

Synthetic ß-1,2-Mannosyloxymannitol Glycolipid from the Fungus Malassezia pachydermatis Signals through Human Mincle.

Mincle is a C-type lectin receptor of the innate immune system with the ability to sense pathogens and commensals through lipidic metabolites. While a growing number of bacterial glycolipids have been discovered that can signal through human Mincle, no fungal metabolites are known that can signal through the human form of this receptor. We report the total synthesis of a complex ß-1,2-mannosyloxymannitol glycolipid from Malassezia pachydermatis 44-2, which was reported to signal through the murine Mincle receptor. Assembly of 44-2 was achieved through a highly convergent route that exploits symmetry elements inherent within this molecule and delineation of conditions that maintain the delicate l-mannitol triester-triol array. We show that 44-2 is a potent agonist of human Mincle signaling and constitutes the first fungal metabolite identified that can signal through the human Mincle receptor, providing new insights into antifungal immunity.

Comprehensive Synthesis of Substrates, Intermediates, and Products of the Sulfoglycolytic Embden-Meyerhoff-Parnas Pathway.

Sulfoglycolysis is a metabolic pathway dedicated to the catabolism of the sulfosugar sulfoquinovose (SQ) into smaller organosulfur fragments. An estimated 10 billion tonnes of SQ fluxes through sulfoglycolysis pathways each year, making it a significant aspect of the biogeochemical sulfur cycle. Delineating the molecular details of sulfoglycolysis requires authentic samples of the various metabolites in these pathways. To this end, we have established chemical and chemoenzymatic methods for the synthesis of the key organosulfur metabolites sulfoquinovosylglycerol, SQ (also in 13C6-labeled form), sulfofructose, sulfofructose-1-phosphate, sulfolactaldehyde, and 2,3-dihydroxypropanesulfonate, as well as an improved route to the chromogenic sulfoquinovosidase substrate 4-nitrophenyl α-sulfoquinovoside.

Discovery of N-Aryloxypropylbenzylamines as Voltage-Gated Sodium Channel NaV 1.2-Subtype-Selective Inhibitors.

We previously reported that a lipophilic N-(4'-hydroxy-3',5'-di-tert-butylbenzyl) derivative (1) of the voltage-gated sodium channel blocker mexiletine, was a more potent sodium channel blocker in vitro and in vivo. We demonstrate that replacing the chiral methylethylene linker between the amine and di-tert-butylphenol with an achiral 1,3-propylene linker (to give (2)) maintains potency in vitro. We synthesized 25 analogues bearing the 1,3-propylene linker and found that minor structural changes resulted in pronounced changes in state dependence of blocking human NaV 1.2 and 1.6 channels by high-throughput patch-clamp analysis. Compared to mexiletine, compounds 1 and 2 are highly selective NaV 1.2 inhibitors and >500 times less potent in inhibiting NaV 1.6 channels. On the other hand, a derivative (compound 4) bearing 2,6-dimethoxy groups in place of the 2,6-dimethyl groups found in mexiletine was found to be the most potent inhibitor, but is nonselective against both channels in the tonic, frequency-dependent and inactivated states. In a kindled mouse model of refractory epilepsy, compound 2 inhibited seizures induced by 6 Hz 44 mA electrical stimulation with an IC50 value of 49.9±1.6 mg kg-1 . As established sodium channel blockers do not suppress seizures in this mouse model, this indicates that 2 could be a promising candidate for treating pharmaco-resistant epilepsy.

Gram scale preparation of clozapine N-oxide (CNO), a synthetic small molecule actuator for muscarinic acetylcholine DREADDs.

Chemogenetics uses engineered proteins that are controlled by small molecule actuators, allowing in vivo functional studies of proteins with temporal and dose control, and include Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). One major class of DREADDs are mutated muscarinic receptors that are unresponsive to acetylcholine, and are activated by administration of clozapine N-oxide (CNO). However, CNO is available in only small amounts and large scale studies involving animals and multiple cohorts are prohibitively expensive for many investigators. The precursor, clozapine, is also expensive when purchased from specialist suppliers. Here we report: •A simple extraction method of clozapine from commercial tablets;•A simple preparation of CNO from clozapine, and for the first time its single-crystal X-ray structure; and•That the CNO prepared by this method specifically activates the DREADD receptor hM3Dq in vivo. This method provides large quantities of CNO suitable for large-scale DREADD applications that is identical to commercial material.

Lipid structure influences the ability of glucose monocorynomycolate to signal through Mincle.

Mincle (macrophage-inducible C-type lectin) is a C-type lectin receptor that provides the capacity for immune sensing of a range of pathogen- and commensal-derived glycolipids. Mincle can recognize mycolic and/or corynomycolic acid esters of trehalose, glycerol and glucose from mycobacteria and corynebacteria. While simple straight-chain long fatty acids (e.g. behenic acid) can substitute for mycolic acid on trehalose and glycerol and maintain robust signalling through Mincle, glucose monobehenate has been reported to be much less active than glucose monocorynomycolate (GMCM). We report the preparation of a range of analogues of GMCM to explore structural requirements in the lipid chain for signalling through Mincle. GMCM analogues bearing simple straight chain or branched fatty acid esters provided only weak signalling through human and mouse Mincle. A GMCM variant with a truncated (pentyl) α-chain provided attenuated signalling, whereas an analogue with an extended (tricosyl; C23) α-chain signalled as potently as GMCM. This work suggests that Mincle has the ability to survey mycolate-derived glycolipids from actinomycetes, distinguishing non-pathogenic (e.g. Rhodococcus spp.) and pathogenic (e.g. Mycobacterium tuberculosis) species on the basis of α-chain length. Finally, an α-phenyldodecyl analogue of GMCM possessed similar potency to GMCM and was only slightly less potent than trehalose dimycolate (cord factor), showing that large functional groups may be tolerated in the α-chain.

Investigation of benzoyloximes as benzoylating reagents: benzoyl-Oxyma as a selective benzoylating reagent.

Hydroxybenzotriazole (HOBt) and HOBt-derived reagents have been classified as Class I explosives, with restrictions on their transportation and storage. We explored a range of benzoylated oxime-based reagents as alternatives to benzoyloxybenzotriazole (BBTZ) for the selective benzoylation of carbohydrate polyols. Benzoylated oximes derived from 2-hydroximino-malononitrile, ethyl 2-hydroximino-2-cyanoacetate (Oxyma), and tert-butyl 2-hydroximino-2-cyanoacetate were most effective for benzoylation of a simple primary alcohol, with yields approaching that obtained for BBTZ. When applied to carbohydrate diols, the most effective reagent was identified as benzoyl-Oxyma. Benzoyl-Oxyma is a highly crystalline, readily prepared alternative to BBTZ, useful in the selective benzoylation of carbohydrate polyols.

Corynomycolic acid-containing glycolipids signal through the pattern recognition receptor Mincle.

An enantioselective synthesis of (+)-corynomycolic acid, and its elaboration to esters of trehalose, glucose and glycerol, is described. Trehalose dicorynomycolate and trehalose monocorynomycolate activate human and mouse Mincle as effectively as trehalose dicorynomycolate (cord factor). Glucose monomycolate is revealed to be a potent activator of both mouse and human Mincle. Glycerol monocorynomycolate signals through human Mincle, with the activity predominantly residing in the 2'S-isomer.

Acetylation of trehalose mycolates is required for efficient MmpL-mediated membrane transport in Corynebacterineae.

Pathogenic species of Mycobacteria and Corynebacteria, including Mycobacterium tuberculosis and Corynebacterium diphtheriae, synthesize complex cell walls that are rich in very long-chain mycolic acids. These fatty acids are synthesized on the inner leaflet of the cell membrane and are subsequently transported to the periplasmic space as trehalose monomycolates (TMM), where they are conjugated to other cell wall components and to TMM to form trehalose dimycolates (TDM). Mycobacterial TMM, and the equivalent Corynebacterium glutamicum trehalose corynomycolates (TMCM), are transported across the inner membrane by MmpL3, or NCgl0228 and NCgl2769, respectively, although little is known about how this process is regulated. Here, we show that transient acetylation of the mycolyl moiety of TMCM is required for periplasmic export. A bioinformatic search identified a gene in a cell wall biosynthesis locus encoding a putative acetyltransferase (M. tuberculosis Rv0228/C. glutamicum NCgl2759) that was highly conserved in all sequenced Corynebacterineae. Deletion of C. glutamicum NCgl2759 resulted in the accumulation of TMCM, with a concomitant reduction in surface transport of this glycolipid and syntheses of cell wall trehalose dicorynomycolates. Strikingly, loss of NCgl2759 was associated with a defect in the synthesis of a minor, and previously uncharacterized, glycolipid species. This lipid was identified as trehalose monoacetylcorynomycolate (AcTMCM) by mass spectrometry and chemical synthesis of the authentic standard. The in vitro synthesis of AcTMCM was dependent on acetyl-CoA, whereas in vivo [(14)C]-acetate pulse-chase labeling showed that this lipid was rapidly synthesized and turned over in wild-type and genetically complemented bacterial strains. Significantly, the biochemical and TMCM/TDCM transport phenotype observed in the ΔNCgl2759 mutant was phenocopied by inhibition of the activities of the two C. glutamicum MmpL3 homologues. Collectively, these data suggest that NCgl2759 is a novel TMCM mycolyl acetyltransferase (TmaT) that regulates transport of TMCM and is a potential drug target in pathogenic species.

A click chemistry approach to 5,5'-disubstituted-3,3'-bisisoxazoles from dichloroglyoxime and alkynes: luminescent organometallic iridium and rhenium bisisoxazole complexes.

5,5'-Disubstituted-3,3'-bisisoxazoles are prepared in one step by the dropwise addition of aqueous potassium hydrogen carbonate to a mixture of dichloroglyoxime and terminal alkynes. The reaction exhibits a striking preference for the 5,5'-disubstituted 3,3'-bisisoxazole over the 4,5'-regioisomer. Organometallic iridium and rhenium bisisoxazole complexes are luminescent with emission wavelengths varying depending upon the identity of the 5,5'-substituent (phenyl, butyl).