Plasticity of ether lipids promotes ferroptosis susceptibility and evasion.

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers1. The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions2-5. However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.

Hindered dialkyl ether synthesis with electrogenerated carbocations.

Hindered ethers are of high value for various applications; however, they remain an underexplored area of chemical space because they are difficult to synthesize via conventional reactions1,2. Such motifs are highly coveted in medicinal chemistry, because extensive substitution about the ether bond prevents unwanted metabolic processes that can lead to rapid degradation in vivo. Here we report a simple route towards the synthesis of hindered ethers, in which electrochemical oxidation is used to liberate high-energy carbocations from simple carboxylic acids. These reactive carbocation intermediates, which are generated with low electrochemical potentials, capture an alcohol donor under non-acidic conditions; this enables the formation of a range of ethers (more than 80 have been prepared here) that would otherwise be difficult to access. The carbocations can also be intercepted by simple nucleophiles, leading to the formation of hindered alcohols and even alkyl fluorides. This method was evaluated for its ability to circumvent the synthetic bottlenecks encountered in the preparation of 12 chemical scaffolds, leading to higher yields of the required products, in addition to substantial reductions in the number of steps and the amount of labour required to prepare them. The use of molecular probes and the results of kinetic studies support the proposed mechanism and the role of additives under the conditions examined. The reaction manifold that we report here demonstrates the power of electrochemistry to access highly reactive intermediates under mild conditions and, in turn, the substantial improvements in efficiency that can be achieved with these otherwise-inaccessible intermediates.

Vanillyl alcohol oxidase from Diplodia corticola: Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives.

Vanillyl alcohol oxidases (VAOs) belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg-1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg-1) were observed, which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for É£-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg-1). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts.

Precisely Programmable Degradation and Drug Release Profiles in Triblock Copolyether Hydrogels with Cleavable Acetal Pendants.

Hydrogels hold significant promise as drug delivery systems due to their distinct advantage of sustained localized drug release. However, the challenge of regulating the initial burst release while achieving precise control over degradation and drug-release kinetics persists. Herein, we present an ABA-type triblock copolymer-based hydrogel system with precisely programmable degradation and release kinetics. The resulting hydrogels were designed with a hydrophilic poly(ethylene oxide) midblock and a hydrophobic end-block composed of polyethers with varying ratios of ethoxyethyl glycidyl ether and tetrahydropyranyl glycidyl ether acetal pendant possessing different hydrolysis kinetics. This unique side-chain strategy enabled us to achieve a broad spectrum of precise degradation and drug-release profiles under mildly acidic conditions while maintaining the cross-linking density and viscoelastic modulus, which is unlike the conventional polyester-based backbone degradation system. Furthermore, programmable degradation of the hydrogels and release of active therapeutic agent paclitaxel loaded therein are demonstrated in an in vivo mouse model by suppressing tumor recurrence following surgical resection. Tuning of the fraction of two acetal pendants in the end-block provided delicate tailoring of hydrogel degradation and the drug release capability to achieve the desired therapeutic efficacy. This study not only affords a facile means to design hydrogels with precisely programmable degradation and release profiles but also highlights the critical importance of aligning the drug release profile with the target disease.

Pregnenolone-methyl-ether enhances CLIP170 and microtubule functions improving spine maturation and hippocampal deficits related to CDKL5 deficiency.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a neurodevelopmental disease characterized by severe infantile seizures and intellectual disability. The absence of CDKL5 in mice causes defective spine maturation that can at least partially explain the cognitive impairment in CDKL5 patients and CDD mouse models. The molecular basis for such defect may depend on the capacity of CDKL5 to regulate microtubule (MT) dynamics through its association with the MT-plus end tracking protein CLIP170 (cytoplasmic linker protein 170). Indeed, we here demonstrate that the absence of CDKL5 causes CLIP170 to be mainly in a closed inactive conformation that impedes its binding to MTs. Previously, the synthetic pregnenolone analogue, pregnenolone-methyl-ether (PME), was found to have a positive effect on CDKL5-related cellular and neuronal defects in vitro. Here, we show that PME induces the open active conformation of CLIP170 and promotes the entry of MTs into dendritic spines in vitro. Furthermore, the administration of PME to symptomatic Cdkl5-knock-out mice improved hippocampal-dependent behavior and restored spine maturation and the localization of MT-related proteins in the synaptic compartment. The positive effect on cognitive deficits persisted for 1 week after treatment withdrawal. Altogether, our results suggest that CDKL5 regulates spine maturation and cognitive processes through its control of CLIP170 and MT dynamics, which may represent a novel target for the development of disease-modifying therapies.

Confining Thiolysis of Dinitrophenyl Ether to a Luminescent Metal-Organic Framework with a Large Stokes Shift for Highly Efficient Detection of Hydrogen Sulfide in Rat Brain.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that regulates various physiological and pathological processes in the central nervous system. It is vital to develop an effective method to detect H2S in vivo to elucidate its critical role. However, current fluorescent probes for accurate quantification of H2S still face big challenges due to complicated fabrication, small Stokes shift, unsatisfactory selectivity, and especially delayed response time. Herein, based on simple postsynthetic modification, we present an innovative strategy by confining H2S-triggered thiolysis of dinitrophenyl (DNP) ether within a luminescent metal-organic framework (MOF) to address those issues. Due to the cleavage of the DNP moiety by H2S, the nanoprobe gives rise to a remarkable fluorescence turn-on signal with a large Stokes shift of 190 nm and also provides high selectivity to H2S against various interferents including competing biothiols. In particular, by virtue of the unique structural property of the MOF, it exhibits an ultrafast sensing ability for H2S (only 5 s). Moreover, the fluorescence enhancement efficiency displays a good linear correlation with H2S concentration in the range of 0-160 µM with a detection limit of 0.29 µM. Importantly, these superior sensing performances enable the nanoprobe to measure the basal value and monitor the change of H2S level in the rat brain.

Rational design of novel diaryl ether-linked benzimidazole derivatives as potent and selective BACE1 inhibitors.

Due to the large size and high flexibility of the catalytic active site of BACE1 enzyme, the development of nonpeptide inhibitors with optimal pharmacological properties is still highly demanding. In this work, we have discovered 2-aminobenzimidazole-containg ether scaffolds having potent and selective inhibitory potentials against BACE1 enzyme. We have synthesized novel 29 compounds and optimization of aryl linker region resulted in highly potent BACE1 inhibitory activities with EC50 values of 0.05-2.71 µM. The aryloxy-phenyl analogs 20j showed the EC50 value as low as 0.07 µM in the enzyme assay, whereas, the benzyloxyphenyl dervative 24b was comparatively less effective in the enzyme assay. But interestingly the latter was more effective in the cell assay (EC50 value 1.2 µM). While comparing synthesized derivatives in the cell assay using PC12-APPSW cell, compound 27f appeared as the most potent BACE1 inhibitor having EC50 value 0.7 µM. This scaffold also showed high selectivity over BACE2 enzyme and cathepsin D. Furthermore, the research findings were bolstered through the incorporation of molecular docking, molecular dynamics, and DFT studies. We firmly believe that these discoveries will pave the way for the development of a novel class of small-molecule selective BACE1 inhibitors.

Ether Bond Cleavage of a Phenylcoumaran ß-5 Lignin Model Compound and Polymeric Lignin Catalysed by a LigE-type Etherase from Agrobacterium sp.

A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. has been identified, which assists degradation of polymeric lignins. Testing against lignin dimer model compounds revealed that it does not catalyse the previously reported reaction of Sphingobium SYK-6 LigE, but instead shows activity for a ß-5 phenylcoumaran lignin dimer. The reaction products did not contain glutathione, indicating a catalytic role for reduced glutathione in this enzyme. Three reaction products were identified: the major product was a cis-stilbene arising from C-C fragmentation involving loss of formaldehyde; two minor products were an alkene arising from elimination of glutathione, and an oxidised ketone, proposed to arise from reaction of an intermediate with molecular oxygen. Testing of the recombinant enzyme against a soda lignin revealed the formation of new signals by two-dimensional NMR analysis, whose chemical shifts are consistent with the formation of a stilbene unit in polymeric lignin.

An atypical two-component system, AtcR/AtcK, simultaneously regulates the biosynthesis of multiple secondary metabolites in Streptomyces bingchenggensis.

Streptomyces bingchenggensis is an industrial producer of milbemycins, which are important anthelmintic and insecticidal agents. Two-component systems (TCSs), which are typically situated in the same operon and are composed of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Here, an atypical TCS, AtcR/AtcK, in which the encoding genes (sbi_06838/sbi_06839) are organized in a head-to-head pair, was demonstrated to be indispensable for the biosynthesis of multiple secondary metabolites in S. bingchenggensis. With the null TCS mutants, the production of milbemycin and yellow compound was abolished but nanchangmycin was overproduced. Transcriptional analysis and electrophoretic mobility shift assays showed that AtcR regulated the biosynthesis of these three secondary metabolites by a MilR3-mediated cascade. First, AtcR was activated by phosphorylation from signal-triggered AtcK. Second, the activated AtcR promoted the transcription of milR3. Third, MilR3 specifically activated the transcription of downstream genes from milbemycin and yellow compound biosynthetic gene clusters (BGCs) and nanR4 from the nanchangmycin BGC. Finally, because NanR4 is a specific repressor in the nanchangmycin BGC, activation of MilR3 downstream genes led to the production of yellow compound and milbemycin but inhibited nanchangmycin production. By rewiring the regulatory cascade, two strains were obtained, the yield of nanchangmycin was improved by 45-fold to 6.08 g/L and the production of milbemycin was increased twofold to 1.34 g/L. This work has broadened our knowledge on atypical TCSs and provided practical strategies to engineer strains for the production of secondary metabolites in Streptomyces.IMPORTANCEStreptomyces bingchenggensis is an important industrial strain that produces milbemycins. Two-component systems (TCSs), which consist of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Coupled encoding genes of TCSs are typically situated in the same operon. Here, TCSs with encoding genes situated in separate head-to-head neighbor operons were labeled atypical TCSs. It was found that the atypical TCS AtcR/AtcK played an indispensable role in the biosynthesis of milbemycin, yellow compound, and nanchangmycin in S. bingchenggensis. This atypical TCS regulated the biosynthesis of specialized metabolites in a cascade mediated via a cluster-situated regulator, MilR3. Through rewiring the regulatory pathways, strains were successfully engineered to overproduce milbemycin and nanchangmycin. To the best of our knowledge, this is the first report on atypical TCS, in which the encoding genes of RR and HK were situated in separate head-to-head neighbor operons, involved in secondary metabolism. In addition, data mining showed that atypical TCSs were widely distributed in actinobacteria.

Properties of Configurationally Stable Atropoenantiomers in Macrocyclic Natural Products and the Chrysophaentin Family.

development of antibiotics, antineoplastics, and therapeutics for other diseases. Natural products are unique among all other small molecules in that they are produced by dedicated enzymatic assembly lines that are the protein products of biosynthetic gene clusters. As the products of chiral macromolecules, natural products have distinct three-dimensional shapes and stereochemistry is often encoded in their structures through the presence of stereocenters, or in the case of molecules that lack a stereocenter, the presence of an axis or plane of chirality. In the latter forms of chirality, if the barrier to rotation about the chiral axis or chiral plane is sufficiently high, stable conformers may exist allowing for isolation of discrete conformers, also known as atropisomers. Importantly, the diverse functions and biological activities of natural products are contingent upon their structures, stereochemistry and molecular shape. With continued innovation in methods for natural products discovery, synthetic chemistry, and analytical and computational tools, new insights into atropisomerism in natural products and related scaffolds are being made. As molecular complexity increases, more than one form of stereoisomerism may exist in a single compound (for example, point chirality, chiral axes, and chiral planes), sometimes creating atypical or noncanonical atropisomers, a term used to distinguish physically noninterconvertable atropisomers from typical atropisomers.Here we provide an account of the discovery and unusual structural and stereochemical features of the chrysophaentins, algal derived inhibitors of the bacterial cytoskeletal protein FtsZ and its associated protein partners. Eleven members of the chrysophaentin family have been discovered to date; seven of these are macrocyclic bis-bibenzyl ethers wherein the site of the ether linkage yields either a symmetrical or asymmetrical macrocyclic ring system. The asymmetrical ring system is highly strained and corresponds to the compounds having the most potent antimicrobial activity among the family. We review the structure elucidation and NMR properties that indicate restricted rotation between axes of two biaryl ethers, and the plane represented by the substituted 2-Z-butene bridge common to all of the macrocycles. Computational studies that corroborate high barriers to rotation about one representative plane, on the order of 20+ kcal/mol are presented. These barriers to rotation fix the conformation of the macrocycle into a bowl-like structure and suggest that an atropisomer should exist. Experimental evidence for atropisomerism is presented, consistent with computational predictions. These properties are discussed in the context of the total synthesis of 9-dechlorochrysophaenin A and its ring C isomers. Last, we discuss the implications for the presence of enantiomers in the biological activity and macrocyclization of the natural product.

Potentiometric determination of the local anesthetic procaine in pharmaceutical samples.

In this study, a new potentiometric sensor was developed for the determination of the local anesthetic drug procaine in pharmaceutical samples. Procaine (Pr)-Tetraphenlyborate (TPB) ion-pair was synthesized and used as a sensor material. Potentiometric sensors using the synthesized ion pair (Pr-TPB), poly(vinyl chloride) (PVC) and o-nitrophenyloctyl ether (o-NPOE) in different proportions were prepared and their performance properties were tested. Among the prepared sensors, the best potentiometric response characteristics were obtained with the sensor composition Pr-TPB:PVC:o-NPOE in the ratio of 6.0:32.0:62.0 (w/w %). The new procaine sensor developed in the present study had a near-Nernstian behavior of 54.1 ± 3.3 mV/per decade and a low detection limit of 3.18 × 10-5 mol L-1 in the concentration range of 1.0 × 10-1-1.0 × 10-4 mol L-1. Additionally, the sensor had a response time of less than 10 s and could work in a wide pH range for two different concentration values without being affected by pH changes. Finally, the new procaine potentiometric sensor was used to detect procaine in injection samples and successfully determined procaine concentrations with high recoveries.

Cooperation in public goods game does not require assortment and depends on population density.

The threshold public goods game is one of the best-known models of non-linear public goods dilemmas. Cooperators and defectors typically coexist in this game when the population is assumed to follow the so-called structured deme model. In this article, we develop a dynamical model of a general N-player game in which there is no deme structure: Individuals interact with randomly chosen neighbours and selection occurs between randomly chosen pairs of individuals. We show that in the deterministic limit, the dynamics in this model leads to the same replicator dynamics as in the structured deme model, i.e., coexistence of cooperators and defectors is typical in threshold public goods game even when the population is completely well mixed. We extend the model to study the effect of density dependence and density fluctuation on the dynamics. We show analytically and numerically that decreasing population density increases the equilibrium frequency of cooperators till the fixation of this strategy, but below a critical density cooperators abruptly disappear from the population. Our numerical investigations show that weak density fluctuations enhance cooperation, while strong fluctuations suppress it.

Bicyclostreptins are radical SAM enzyme-modified peptides with unique cyclization motifs.

Microbial natural products comprise diverse architectures that are generated by equally diverse biosynthetic strategies. In peptide natural products, amino acid sidechains are frequently used as sites of modification to generate macrocyclic motifs. Backbone amide groups, among the most stable of biological moieties, are rarely used for this purpose. Here we report the discovery and biosynthesis of bicyclostreptins-peptide natural products from Streptococcus spp. with an unprecedented structural motif consisting of a macrocyclic ß-ether and a heterocyclic sp3-sp3 linkage between a backbone amide nitrogen and an adjacent α-carbon. Both reactions are installed, in that order, by two radical S-adenosylmethionine (RaS) metalloenzymes. Bicyclostreptins are produced at nM concentrations and are potent growth regulation agents in Streptococcus thermophilus. Our results add a distinct and unusual chemotype to the growing family of ribosomal peptide natural products, expand the already impressive catalytic scope of RaS enzymes, and provide avenues for further biological studies in human-associated streptococci.

Disrupted intercellular bridges and spermatogenesis in fatty acyl-CoA reductase 1 knockout mice: A new model of ether lipid deficiency.

Peroxisomal fatty acyl-CoA reductase 1 (FAR1) is a rate-limiting enzyme for ether lipid (EL) synthesis. Gene mutations in FAR1 cause a rare human disease. Furthermore, altered EL homeostasis has also been associated with various prevalent human diseases. Despite their importance in human health, the exact cellular functions of FAR1 and EL are not well-understood. Here, we report the generation and initial characterization of the first Far1 knockout (KO) mouse model. Far1 KO mice were subviable and displayed growth retardation. The adult KO male mice had smaller testes and were infertile. H&E and immunofluorescent staining showed fewer germ cells in seminiferous tubules. Round spermatids were present but no elongated spermatids or spermatozoa were observed, suggesting a spermatogenesis arrest at this stage. Large multi-nucleated giant cells (MGC) were found lining the lumen of seminiferous tubules with many of them undergoing apoptosis. The immunofluorescent signal of TEX14, an essential component of intercellular bridges (ICB) between developing germ cells, was greatly reduced and mislocalized in KO testis, suggesting the disrupted ICBs as an underlying cause of MGC formation. Integrative analysis of our total testis RNA-sequencing results and published single-cell RNA-sequencing data unveiled cell type-specific molecular alterations underlying the spermatogenesis arrest. Many genes essential for late germ cell development showed dramatic downregulation, whereas genes essential for extracellular matrix dynamics and cell-cell interactions were among the most upregulated genes. Together, this work identified the cell type-specific requirement of ELs in spermatogenesis and suggested a critical role of Far1/ELs in the formation/maintenance of ICB during meiosis.

A novel amphiphilic squalene-based compound with open-chain polyethers reduces malignant melanoma metastasis in-vitro and in-vivo.

Squalene (SQ) is a well-known antioxidant and anti-inflammatory agent that provides promising anti-aging and UV-protective roles on human skin. However, its strong hydrophobic nature, accompanied by issues such as poor solubility and limited tissue permeation, has created challenges for scientists to investigate its untapped potential in more complex conditions, including cancer progression. The present study assessed the potent anti-metastatic properties of a newly synthesized amphiphilic ethylene glycol SQ derivative (SQ-diEG) in melanoma, the most fatal skin cancer. In vitro and in vivo experiments have discovered that SQ-diEG may exert its potential on melanoma malignancy through the mitochondria-mediated caspase activation apoptotic signaling pathway. The potent anti-metastatic effect of SQ-diEG was observed in vitro using highly proliferative and aggressive melanoma cells. Administration of SQ-diEG (25 mg/kg) significantly decreased the tumor burden on the lung and inhibited the metastasis-associated proteins and gene markers in B16F10 lung colonization mice model. Furthermore, global gene profiling also revealed a promising role of SQ-diEG in tumor microenvironment. We anticipated that the amphiphilic nature of the SQ compound bearing ethylene glycol oligomers could potentially augment its ability to reach the pathology site, thus enhancing its therapeutic potential in melanoma.

Synthesis of Biobased Poly(butylene Furandicarboxylate) Containing Polysulfone with Excellent Thermal Resistance Properties.

A synthetic biopolymer derived from furandicarboxylic acid monomer and hydroxyethyl-terminated poly(ether sulfone) is presented. The synthesis involves 4,4'-dichlorodiphenyl sulfone and 4,4-dihydroxydiphenyl sulfone, resulting in poly(butylene furandicarboxylate)-poly(ether sulfone) copolyesters (PBFES) through melt polycondensation with titanium-catalyzed polymerization. This facile method yields segmented polyesters incorporating polysulfone, creating a versatile group of high-temperature thermoplastics with adjustable thermomechanical properties. The PBFES copolyesters demonstrate an impressive tensile modulus of 2830 MPa and a tensile strength of 84 MPa for PBFES55. Additionally, the poly(ether sulfone) unit imparts a relatively high glass transition temperature (Tg), ranging from 36.6 °C for poly(butylene 2,5-furandicarboxylate) to 112.3 °C for PBFES62. Moreover, the complete amorphous film of PBFES exhibits excellent transparency and solvent resistance, making it suitable for applications, such as food packaging materials.

Hydrogel Based on Riclin Cross-Linked with Polyethylene Glycol Diglycidyl Ether as a Soft Filler for Tissue Engineering.

Hydrogels composed of natural polysaccharides have been widely used as filling materials, with a growing interest in medical cosmetology and skin care. However, conventional commercial dermal fillers still have limitations, particularly in terms of mechanical performance and durability in vivo. In this study, a novel injectable and implantable hydrogel with adjustable characteristics was prepared from succinoglycan riclin by introducing PEG diglycidyl ether as a cross-linker. FTIR spectra confirmed the cross-linking reaction. The riclin hydrogels exhibited shear-thinning behavior, excellent mechanical properties, and cytocompatibility through in vitro experiments. Furthermore, when compared with subcutaneous injection of a commercial hyaluronic acid hydrogel, the riclin hydrogels showed enhanced persistence and biocompatibility in Balb/c mice after 16 weeks. These results demonstrate the great potential of the riclin-based hydrogel as an alternative to conventional commercial soft tissue fillers.

Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system.

BACKGROUND: Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. RESULTS: Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. CONCLUSION: The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Novel orexin receptor agonists based on arene- or pyridine-fused 1,3-dihydro-2H-imidazole-2-imines.

A structurally novel class of benzo- or pyrido-fused 1,3-dihydro-2H-imidazole-2-imines was designed and evaluated in an inositol phosphate accumulation assay for Gq signaling to measure agonistic activation of the orexin receptor type 2 (OX2R). These compounds were synthesized in 4-9 steps overall from readily available starting materials. Analogs that contain a stereogenic methyl or cyclopropyl substituent at the benzylic center, and a correctly configured alkyl ether, alkoxyalkyl ether, cyanoalkyl ether, or α-hydroxyacetamido substituted homobenzylic sidechain were identified as the most potent activators of OX2R coupled Gq signaling. Our results also indicate that agonistic activity was stereospecific at both the benzylic and homobenzylic stereogenic centra. We identified methoxyethoxy-substituted pyrido-fused dihydroimidazolimine analog 63c containing a stereogenic benzylic methyl group was the most potent agonist, registering a respectable EC50 of 339 nM and a maximal response (Emax) of 96 % in this assay. In vivo pharmacokinetic analysis indicated good brain exposure for several analogs. Our combined results provide important information towards a structurally novel class of orexin receptor agonists distinct from current chemotypes.

Synthesis, fungicidal activity and molecular docking of novel pyrazole-carboxamides bearing a branched alkyl ether moiety.

Succinate dehydrogenase inhibitors are essential fungicides used in agriculture. To explore new pyrazole-carboxamides with high fungicidal activity, a series of N-substitutedphenyl-3-di/trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamides bearing a branched alkyl ether moiety were designed and synthesized. The in vitro bioassay indicated that some target compounds displayed appreciable fungicidal activity. For example, compounds 5d and 5e showed high efficacy against S. sclerotiorum with EC50 values of 3.26 and 1.52 µg/mL respectively, and also exhibited excellent efficacy against R. solani with EC50 values of 0.27 and 0.06 µg/mL respectively, which were comparable or superior to penflufen. The further in vivo bioassay on cucumber leaves demonstrated that 5e provided strong protective activity of 94.3 % against S. sclerotiorum at 100 µg/mL, comparable to penflufen (99.1 %). Cytotoxicity assessment against human renal cell lines (239A cell) revealed that 5e had low cytotoxicity within the median effective concentrations. Docking study of 5e with succinate dehydrogenase illustrated that R-5e formed one hydrogen bond and two π-π stacking interactions with amino acid residues of target enzyme, while S-5e formed only one π-π stacking interaction with amino acid residue. This study provides a valuable reference for the design of new succinate dehydrogenase inhibitor.