Structural and signaling mechanisms of TAAR1 enabled preferential agonist design.

Trace amine-associated receptor 1 (TAAR1) senses a spectrum of endogenous amine-containing metabolites (EAMs) to mediate diverse psychological functions and is useful for schizophrenia treatment without the side effects of catalepsy. Here, we systematically profiled the signaling properties of TAAR1 activation and present nine structures of TAAR1-Gs/Gq in complex with EAMs, clinical drugs, and synthetic compounds. These structures not only revealed the primary amine recognition pocket (PARP) harboring the conserved acidic D3.32 for conserved amine recognition and "twin" toggle switch for receptor activation but also elucidated that targeting specific residues in the second binding pocket (SBP) allowed modulation of signaling preference. In addition to traditional drug-induced Gs signaling, Gq activation by EAM or synthetic compounds is beneficial to schizophrenia treatment. Our results provided a structural and signaling framework for molecular recognition by TAAR1, which afforded structural templates and signal clues for TAAR1-targeted candidate compounds design.

Metabolome profiling and transcriptome analysis filling the early crucial missing steps of piperine biosynthesis in Piper nigrum L.

Black pepper (Piper nigrum L.), the world renown as the King of Spices, is not only a flavorsome spice but also a traditional herb. Piperine, a species-specific piper amide, is responsible for the major bioactivity and pungent flavor of black pepper. However, several key steps for the biosynthesis of piperoyl-CoA (acyl-donor) and piperidine (acyl-acceptor), two direct precursors for piperine, remain unknown. In this study, we used guilt-by-association analysis of the combined metabolome and transcriptome, to identify two feruloyldiketide-CoA synthases responsible for the production of the C5 side chain scaffold feruloyldiketide-CoA intermediate, which is considered the first and important step to branch metabolic fluxes from phenylpropanoid pathway to piperine biosynthesis. In addition, we also identified the first two key enzymes for piperidine biosynthesis derived from lysine in P. nigrum, namely a lysine decarboxylase and a copper amine oxidase. These enzymes catalyze the production of cadaverine and 1-piperideine, the precursors of piperidine. In vivo and in vitro experiments verified the catalytic capability of them. In conclusion, our findings revealed enigmatic key steps of piperine biosynthetic pathway and thus provide a powerful reference for dissecting the biosynthetic logic of other piper amides.

Neuromodulation of Innate Behaviors in Drosophila.

Animals are born with a rich repertoire of robust behaviors that are critical for their survival. However, innate behaviors are also highly adaptable to an animal's internal state and external environment. Neuromodulators, including biogenic amines, neuropeptides, and hormones, are released to signal changes in animals' circumstances and serve to reconfigure neural circuits. This circuit flexibility allows animals to modify their behavioral responses according to environmental cues, metabolic demands, and physiological states. Aided by powerful genetic tools, researchers have made remarkable progress in Drosophila melanogaster to address how a myriad of contextual information influences the input-output relationship of hardwired circuits that support a complex behavioral repertoire. Here we highlight recent advances in understanding neuromodulation of Drosophila innate behaviors, with a special focus on feeding, courtship, aggression, and postmating behaviors.

Ancient and Nonuniform Loss of Olfactory Receptor Expression Renders the Shark Nose a De Facto Vomeronasal Organ.

Cartilaginous fishes are renowned for a keen sense of smell, a reputation based on behavioral observations and supported by the presence of large and morphologically complex olfactory organs. At the molecular level, genes belonging to the four families coding for most olfactory chemosensory receptors in other vertebrates have been identified in a chimera and a shark, but it was unknown whether they actually code for olfactory receptors in these species. Here, we describe the evolutionary dynamics of these gene families in cartilaginous fishes using genomes of a chimera, a skate, a sawfish, and eight sharks. The number of putative OR, TAAR, and V1R/ORA receptors is very low and stable, whereas the number of putative V2R/OlfC receptors is higher and much more dynamic. In the catshark Scyliorhinus canicula, we show that many V2R/OlfC receptors are expressed in the olfactory epithelium in the sparsely distributed pattern characteristic for olfactory receptors. In contrast, the other three vertebrate olfactory receptor families are either not expressed (OR) or only represented with a single receptor (V1R/ORA and TAAR). The complete overlap of markers of microvillous olfactory sensory neurons with pan-neuronal marker HuC in the olfactory organ suggests the same cell-type specificity of V2R/OlfC expression as for bony fishes, that is, in microvillous neurons. The relatively low number of olfactory receptors in cartilaginous fishes compared with bony fishes could be the result of an ancient and constant selection in favor of a high olfactory sensitivity at the expense of a high discrimination capability.

Loss of temporal coherence in the circadian metabolome across multiple tissues during ageing in mice.

Circadian clock function declines with ageing, which can aggravate ageing-related diseases such as type 2 diabetes and neurodegenerative disorders. Understanding age-related changes in the circadian system at a systemic level can contribute to the development of strategies to promote healthy ageing. The goal of this study was to investigate the impact of ageing on 24-h rhythms in amine metabolites across four tissues in young (2 months of age) and old (22-25 months of age) mice using a targeted metabolomics approach. Liver, plasma, the suprachiasmatic nucleus (SCN; the location of the central circadian clock in the hypothalamus) and the paraventricular nucleus (PVN; a downstream target of the SCN) were collected from young and old mice every 4 h during a 24-h period (n = 6-7 mice per group). Differential rhythmicity analysis revealed that ageing impacts 24-h rhythms in the amine metabolome in a tissue-specific manner. Most profound changes were observed in the liver, in which rhythmicity was lost in 60% of the metabolites in aged mice. Furthermore, we found strong correlations in metabolite levels between the liver and plasma and between the SCN and the PVN in young mice. These correlations were almost completely abolished in old mice. These results indicate that ageing is accompanied by a severe loss of the circadian coordination between tissues and by disturbed rhythmicity of metabolic processes. The tissue-specific impact of ageing may help to differentiate mechanisms of ageing-related disorders in the brain versus peripheral tissues and thereby contribute to the development of potential therapies for these disorders.

Modulating the Distribution of Formamidinium Iodide by Ultrahigh Humidity Treatment Strategy for High-Quality Sequential Vapor Deposited Perovskite.

The quality of two-step processed perovskites is significantly influenced by the distribution of organic amine salts. Especially, modulating the distribution of organic amine salts remains a grand challenge for sequential vapor-deposited perovskites due to the blocking effect of bottom compact PbI2. Herein, an ultrahigh humidity treatment strategy is developed to facilitate the diffusion of formamidinium iodide (FAI) from the top surface to the buried bottom interface on the sequential vapor-deposited bilayer structure. Both experimental and theoretical investigations elucidate the mechanism that moisture helps to i) create FAI diffusion channels by inducing a phase transition from α- to δ-phase in the perovskite, and ii) enhance the diffusivity of FAI by forming hydrogen bonds. This ultrahigh humidity treatment strategy enables the formation of a desired homogeneous and high-quality α-phase after annealing. As a result, a champion efficiency of 22.0% is achieved and 97.5% of its initial performance is maintained after aging for 1050 h under ambient air with a relative humidity of up to 80%. This FAI diffusion strategy provides new insights into the reproducible, scalable, and high-performance sequential vapor-deposited perovskite solar cells.

Dual-Stimuli-Responsive Modulation Organic Afterglow Based on N─H Proton Migration Mechanism.

Organic afterglow materials have significant applications in information security and flexible electronic devices with unique optical properties. It is vital but challenging to develop organic afterglow materials possessing controlled output with multi-stimuli-responsive capacity. Herein, dimethyl terephthalate (DTT) is introduced as a strong proton acceptor. The migration direction of N─H protons on two compounds Hs can be regulated by altering the excitation wavelength (Ex) or amine stimulation, thereby achieving dual-stimuli-responsive afterglow emission. When the Ex is below 300 nm, protons migrate to S1-2 DTT, where strong interactions induce phosphorescent emission of Hs, resulting in afterglow behavior. Conversely, when the Ex is above 300 nm, protons interact with the S0 DTT weakly and the afterglow disappears. In view of amine-based compounds with higher proton accepting capabilities, it can snatch proton from S1-2 DTT and redirect the proton flow toward amine, effectively suppressing the afterglow but obtaining a new redshifted fluorescence emission with Δλ over 200 nm due to the high polarity of amine. Moreover, it is successfully demonstrated that the applications of dual-stimuli-responsive organic afterglow materials in information encryption based on the systematic excitation-wavelength-dependent (Ex-De) behavior and amine selectivity detection.

Achieving High Operating-Temperature Self-powered X-Ray Detection in Multilayered Hybrid Perovskites through Arylamine Intercalation.

Polar metal halide hybrid perovskites (PHPs) that exhibit outstanding bulk photovoltaic effect (BPVE), excellent semiconductor features, and strong radiation absorption ability, have shown prominent advantages in highly sensitive direct X-ray detection. However, it is still a challenge to explore PHPs with high BPVE temperature ranges, answering the demand of developing thermally stable passive X-ray detection. Herein, by intercalating arylamine into lead tribromide and inducing order-disorder phase transition, a 2D multilayered PHPs (BZA)2(MA)Pb2Br7 (BZPB, BZA = benzylamine, MA = methylamine) is synthesized. BZPB crystallizes in a polar space group Aea2 at a low-temperature phase and demonstrates a significant open-circuit of 0.3 V deriving from BPVE under X-ray irradiation. Meanwhile, the strong X-ray absorption coefficient and outstanding carrier transport capability of the bilayered lead halide framework associated with the polar BPVE give BZPB excellent X-ray detection abilities. At 0 V bias, the impressive sensitivity of BZPB is 98 µC Gy-1 cm-2. Importantly, the introduction of the rigid BZA ring increases the energy barrier of phase transition and thus dramatically enhances the X-ray detection operating temperature of BZPB up to 409 K without significant performance degradation. This work strongly reveals the great potential of rational design of metal halide hybrid perovskites for X-ray detection applications.

Aminic Organoselenium Compounds as Glutathione Peroxidase Mimics and Inhibitors of Ferroptosis.

The synthesis of diarylamine-based organoselenium compounds via the nucleophilic substitution reactions has been described. Symmetrical monoselenides and diselenides were conveniently synthesized by the reduction of their corresponding selenocyanates using sodium borohydride. Selenocyanates were obtained from 2-chloro acetamides by the nucleophilic displacement with potassium selenocyanate. Selenides were synthesized by treating the 2-chloro acetamides with in situ generated sodium butyl selenolate as nucleophile. Further, the newly synthesized organoselenium compounds were evaluated for their glutathione peroxidase (GPx)-like activity in thiophenol assay. This study revealed that the methoxy-substituted organoselenium compounds showed significant effect on the GPx-like activity. The catalytic parameters for the most efficient catalysts were also determined. The anti-ferroptotic activity for all GPx-mimics evaluated in a 4-OH-tamoxifen (TAM) inducible GPx4 knockout cell line using liproxstatin as standard.

Crystallization Assisted Dynamic Kinetic Resolution for the Synthesis of (R)-ß-Methylphenethylamine.

This study explores a combination of the concept of enantioselective enzymatic synthesis of ß-chiral amines through transamination with in situ product crystallization (ISPC) to overcome product inhibition. Using 2-phenylpropanal as a readily available and easily racemizing substrate of choice, (R)-ß-methylphenethylamine ((R)-2-phenylpropan-1-amine) concentrations of up to 250 mM and enantiomeric excesses of up to 99 % are achieved when using a commercially available transaminase from Ruegeria pomeroyi in a fed-batch based dynamic kinetic resolution reaction on preparative scale. The source of substrate decomposition during the reaction is also investigated and the resulting unwanted byproduct formation is successfully reduced to insignificant levels.

High Cell Density Cultivation Combined with high Specific Enzyme Activity: Cultivation Protocol for the Production of an Amine Transaminase from Bacillus megaterium in E. coli.

High cell density cultivation is an established method for the production of various industrially important products such as recombinant proteins. However, these protocols are not always suitable for biocatalytic processes as the focus often lies on biomass production rather than high specific activities of the enzyme inside the cells. In contrast, a range of shake flask protocols are well known with high specific activities but rather low cell densities. To overcome this gap, we established a tailor-made fed-batch protocol combining both aspects: high cell density and high specific activities of heterologously produced enzyme. Using the example of an industrially relevant amine transaminase from Bacillus megaterium, we describe a strategy to optimize the cultivation yield based on the feed rate, IPTG concentration, and post-induction temperature. By adjusting these key parameters, we were able to increase the specific activity by 2.6-fold and the wet cell weight by even 17-fold compared to shake flasks. Finally, we were able to verify our established protocol by transferring it to another experimenter. With that, our optimization strategy can serve as a template for the production of high titers of heterologously produced, active enzymes and might enable the availability of these catalysts for upscaling biocatalytic processes.

Sequence-Guided Redesign of an Omega-Transaminase from Bacillus megaterium for the Asymmetric Synthesis of Chiral Amines.

ω-Transaminases (ω-TAs) are attractive biocatalysts asymmetrically catalyzing ketones to chiral amines. However, poor non-native catalytic activity and substrate promiscuity severely hamper its wide application in industrial production. Protein engineering efforts have generally focused on reshaping the substrate-binding pockets of ω-TAs. However, hotspots around the substrate tunnel as well as distant sites outside the pockets may also affect its activity. In this study, the ω-TA from Bacillus megaterium (BmeTA) was selected for engineering. The tunnel mutation Y164F synergy with distant mutation A245T which was acquired through a multiple sequence alignment showed improved soluble expression, a 3.7-fold higher specific activity and a 19.9-fold longer half-life at 45 °C. Molecule Dynamics simulation explains the mechanism of improved catalytic activity, enhanced thermostability and improved soluble expression of BmeTAY164F/A245T(2 M). Finally, the resting cells of 2 M were used for biocatalytic processes. 450 mM of S-methoxyisopropylamine (S-MOIPA) was obtained with an ee value of 97.3 % and a conversion rate of 90 %, laying the foundation for its industrial production. Mutant 2 M was also found to be more advantageous in catalyzing the transamination of various ketones. These results demonstrated that sites that are far away from the active center also play an important role in the redesign of ω-TAs.

Identification, characterization and application of M16AT, a new organic solvent-tolerant (R)-enantio-selective type IV amine transaminase from Mycobacterium sp. ACS1612.

Biocatalysis has emerged as a powerful alternative to traditional chemical methods, especially for asymmetric synthesis. As biocatalysts usually exhibit excellent chemical, regio- and enantioselectivity, they facilitate and simplify many chemical processes for the production of a broad range of products. Here, a new biocatalyst called, R-selective amine transaminases (R-ATAs), was obtained from Mycobacterium sp. ACS1612 (M16AT) using in-silico prediction combined with a genome and protein database. A two-step simple purification process could yield a high concentration of pure enzyme, suggesting that industrial application would be inexpensive. Additionally, the newly identified and characterized R-ATAs displayed a broad substrate spectrum and strong tolerance to organic solvents. Moreover, the synthetic applicability of M16AT has been demonstrated by the asymmetric synthesis of (R)-fendiline from of (R)-1-phenylethan-1-amine.

Evaluation of the molecular origin of amide proton transfer-weighted imaging.

PURPOSE: To evaluate the assumption in amide proton transfer weighted (APTw) imaging that the APT dominates over the relayed nuclear Overhauser enhancement (rNOE) and other CEST effects such as those from amines/guanidines, thereby providing imaging of mobile proteins/peptides. METHODS: We introduced two auxiliary asymmetric analysis metrics that can vary the relative contributions from amine/guanidinium CEST and other effects. By comparing these metrics with the conventional asymmetric analysis metric on healthy rat brains, we can approximately assess the contribution from amines/guanidines to APTw and determine whether the APT dominates over the rNOE effect. To further investigate the molecular origin of APTw, we used samples of dialyzed tissue homogenates to eliminate small metabolites and supernatants of homogenates to separate lipids from other components. RESULTS: When the APTw signal is positive using high saturation amplitudes (e.g., 2-3 µT), the contributions from amines/guanidines are significant and cannot be ignored. The APTw signal from the dialyzed homogenates and the controls has negligible changes, indicating that it primarily originates from macromolecules rather than small metabolites. Additionally, the APTw signals with low saturation amplitudes (e.g., 1 µT) were negative in tissue homogenates but positive in their supernatants, suggesting that proteins contribute positively to APTw signals, whereas lipids contribute negatively to it. CONCLUSION: The positive APTw signal using high saturation amplitudes could have significant contributions from soluble proteins through CEST, including amide/amine/guanidine proton transfer effects. In contrast, the negative APTw signal using low saturation amplitudes has significant contribution from lipids through rNOE.

Evolving ω-amine transaminase AtATA guided by substrate-enzyme binding free energy for enhancing activity and stability against non-natural substrates.

In the field of chiral amine synthesis, ω-amine transaminase (ω-ATA) is one of the most established enzymes capable of asymmetric amination under optimal conditions. However, the applicability of ω-ATA toward more non-natural complex molecules remains limited due to its low transamination activity, thermostability, and narrow substrate scope. Here, by employing a combined approach of computational virtual screening strategy and combinatorial active-site saturation test/iterative saturation mutagenesis strategy, we have constructed the best variant M14C3-V5 (M14C3-V62A-V116S-E117I-L118I-V147F) with improved ω-ATA from Aspergillus terreus (AtATA) activity and thermostability toward non-natural substrate 1-acetylnaphthalene, which is the ketone precursor for producing the intermediate (R)-(+)-1-(1-naphthyl)ethylamine [(R)-NEA] of cinacalcet hydrochloride, showing activity enhancement of up to 3.4-fold compared to parent enzyme M14C3 (AtATA-F115L-M150C-H210N-M280C-V149A-L182F-L187F). The computational tools YASARA, Discovery Studio, Amber, and FoldX were applied for predicting mutation hotspots based on substrate-enzyme binding free energies and to show the possible mechanism with features related to AtATA structure, catalytic activity, and stability in silico analyses. M14C3-V5 achieved 71.8% conversion toward 50 mM 1-acetylnaphthalene in a 50 mL preparative-scale reaction for preparing (R)-NEA. Moreover, M14C3-V5 expanded the substrate scope toward aromatic ketone compounds. The generated virtual screening strategy based on the changes in binding free energies has successfully predicted the AtATA activity toward 1-acetylnaphthalene and related substrates. Together with experimental data, these approaches can serve as a gateway to explore desirable performances, expand enzyme-substrate scope, and accelerate biocatalysis.IMPORTANCEChiral amine is a crucial compound with many valuable applications. Their asymmetric synthesis employing ω-amine transaminases (ω-ATAs) is considered an attractive method. However, most ω-ATAs exhibit low activity and stability toward various non-natural substrates, which limits their industrial application. In this work, protein engineering strategy and computer-aided design are performed to evolve the activity and stability of ω-ATA from Aspergillus terreus toward non-natural substrates. After five rounds of mutations, the best variant, M14C3-V5, is obtained, showing better catalytic efficiency toward 1-acetylnaphthalene and higher thermostability than the original enzyme, M14C3. The robust combinational variant acquired displayed significant application value for pushing the asymmetric synthesis of aromatic chiral amines to a higher level.

Calcium imaging of adult olfactory epithelium reveals amines as important odor class in fish.

The odor space of aquatic organisms is by necessity quite different from that of air-breathing animals. The recognized odor classes in teleost fish include amino acids, bile acids, reproductive hormones, nucleotides, and a limited number of polyamines. Conversely, a significant portion of the fish olfactory receptor repertoire is composed of trace amine-associated receptors, generally assumed to be responsible for detecting amines. Zebrafish possess over one hundred of these receptors, but the responses of olfactory sensory neurons to amines have not been known so far. Here we examined odor responses of zebrafish olfactory epithelial explants at the cellular level, employing calcium imaging. We report that amines elicit strong responses in olfactory sensory neurons, with a time course characteristically different from that of ATP-responsive (basal) cells. A quantitative analysis of the laminar height distribution shows amine-responsive cells undistinguishable from ciliated neurons positive for olfactory marker protein. This distribution is significantly different from those measured for microvillous neurons positive for transient receptor potential channel 2 and basal cells positive for proliferating cell nuclear antigen. Our results suggest amines as an important odor class for teleost fish.

General Construction of Asymmetric Amine Ethers via Efficient Transesterification.

A novel method to prepare asymmetric amine ethers is reported. Tertiary amine alcohol hydrogen sulfate intermediates are prepared through a reactive distillation process, followed by the transesterification process to afford eventually asymmetric amine ethers. Experiments and DFT calculations revealed the essential roles the sulfate group plays in the highly selective monoesterification process. This clean method is tolerant towards various functional groups with good yields under mild condition, which is obviously superior compared to the conventional processes.

Catalyst-Free Transfer Hydrogenation from Amine-Borane Small Oligomers.

Amine-borane dimers and oligomers with varied steric and electronic profiles were prepared via capping agent-controlled AA/BB polycondensations. They were used for transfer hydrogenations to aldehydes, ketones, imines as well as electron-poor alkene/alkyne moieties. The amine-borane Lewis-paired oligomers and the congested bis(amine-borane)s provided the highest yields. This was likely helped by facilitated dissociation (oligomers) or H-bond assistance. In the case of the oligomers, the second equivalent of H2 present was also engaged in the reaction. Solid-state NMR characterization provides evidence that the boron-containing materials obtained after transfer dehydrogenation are highly similar to those obtained from thermal dehydrogenation. The oligomers bridge the gap between simple amine-borane molecular reductants and the poly-amine-boranes and provide a full picture of the reactivity changes at the different scales.

Synthesis and Performance of Bio-Based Amphoteric Surfactants.

As the global surfactant market continues to expand, there is an increasing need to develop bio-based alternatives in the shift towards a circular economy. This study focuses on the synthesis of polar, amphoteric, amine-oxide surfactants starting from biomass-derived monosaccharides and demonstrating their potential in various applications. The synthesis involved a reductive amination of the sugars with an alkylamine and formaldehyde followed by oxidation to produce N-oxide surfactants. These bio-based surfactants exhibited promising properties, including high solubility, foamability, surface tension reduction, and critical micelle concentration. In particular, N-GalA1.10 and N-GalA1.12 showed comparable performance to commercial surfactants. Furthermore, these bio-based surfactants demonstrated significantly lower skin irritation potential when compared to petrochemical-derived counterparts like sodium laureth sulfate (SLES), making them potentially suitable for personal care products. The biodegradability assessment revealed that N-GalA1.12 exhibited good biodegradation, indicating its potential environmental compatibility. In conclusion, this study highlights the potential of bio-based N-oxide surfactants derived from monosaccharides as sustainable and skin-friendly alternatives to traditional amphoteric surfactants, like cocamidopropyl betaine (CAPB).

A Phosphoramidate Prodrug Platform: One-Pot Amine Functionalization of Kinase Inhibitors with Oligoethylene Glycol for Improved Water-Solubility.

Small molecular kinase inhibitors play a key role in modern cancer therapy. Protein kinases are essential mediators in the growth and progression of cancerous tumors, rendering involved kinases an increasingly important target for therapy. However, kinase inhibitors are almost insoluble in water because of their hydrophobic aromatic nature, often lowering their availability and pharmacological efficacy. Direct drug functionalization with polar groups represents a simple strategy to improve the drug solubility, availability, and performance. Here, we present a strategy to functionalize secondary amines with oligoethylene glycol (OEG) phosphate using a one-step synthesis in three exemplary kinase inhibiting drugs Ceritinib, Crizotinib, and Palbociclib. These OEG-prodrug conjugates demonstrate superior solubility in water compared to the native drugs, with the solubility increasing up to 190-fold. The kinase inhibition potential is only slightly decreased for the conjugates compared to the native drugs. We further show pH dependent hydrolysis of the OEG-prodrugs which releases the native drug. We observe a slow release at pH 3, while the conjugates remain stable over 96 h under physiological conditions (pH 7.4). Using confocal microscopy, we verify improved cell uptake of the drug-OEG conjugates into the cytoplasm of HeLa cells, further supporting our universal solubility approach.