Detailed Mechanism Research Articles

Regio- and Stereoselective Hydroalkynylation of Internal Alkynes with Terminal Alkynes by Half-Sandwich Rare-Earth Catalysts.

The regio- and stereoselective hydroalkynylation of internal alkynes with terminal alkynes is of great interest and importance as a straightforward route for synthesizing multisubstituted 1,3-enynes. However, this transformation often suffers from regio- and stereoselectivity issues when working with unsymmetrical internal alkynes. Herein, we report for the first time the regio- and syn-stereoselective hydroalkynylation of a variety of heteroatom-functionalized unsymmetrical internal alkynes including homopropargyl ethers, thioethers, and tertiary amines with terminal alkynes by half-sandwich rare-earth catalysts. This protocol provides an atom-efficient and straightforward route for the synthesis of a new family of heteroatom (O, S, or N)-functionalized 1,3-enynes, featuring 100% atom-efficiency, broad substrate scope, and high regio- and syn-stereoselectivity (>19:1 r.r. and >19:1 syn/anti). The mechanistic details have been elucidated by deuterium-labeling experiments, control experiments, and isolation and transformations of key reaction intermediates, revealing that the reaction proceeded through the C(sp)-H deprotonation of a terminal alkyne by a half-sandwich scandium alkyl species to form a catalytically active dimeric half-sandwich scandium tetraalkynyl species followed by heteroatom-assisted insertion of internal alkyne into the Sc-alkynyl bond and the subsequent protonolysis of the resulting Sc-alkenyl bond with another terminal alkyne molecule. The coordination of the heteroatom (O, S, or N) of internal alkynes to the catalyst metal center plays a critically important role in achieving a high level of reactivity and regio- and stereoselectivity. Remarkably, the catalytically active dimeric half-sandwich scandium tetraalkynyl species can be recovered and reused, constituting the first example of a recyclable catalyst system for the hydroalkynylation of internal alkynes.

Protective Role of Electroacupuncture Against Cognitive Impairment in Neurological Diseases.

Many neurological diseases can lead to cognitive impairment in patients, which includes dementia and mild cognitive impairment and thus create a heavy burden both to their families and public health. Due to the limited effectiveness of medications in treating cognitive impairment, it is imperative to develop alternative treatments. Electroacupuncture (EA), a required method for Traditional Chinese Medicine, has the potential treatment of cognitive impairment. However, the molecular mechanisms involved have not been fully elucidated. Considering the current research status, preclinical literature published within the ten years until October 2022 was systematically searched through PubMed, Web of Science, MEDLINE, Ovid, and Embase. By reading the titles and abstracts, a total of 56 studies were initially included. It is concluded that EA can effectively ameliorate cognitive impairment in preclinical research of neurological diseases and induce potentially beneficial changes in molecular pathways, including Alzheimer's disease, vascular cognitive impairment, chronic pain, and Parkinson's disease. Moreover, EA exerts beneficial effects through the same or diverse mechanisms for different disease types, including but not limited to neuroinflammation, neuronal apoptosis, neurogenesis, synaptic plasticity, and autophagy. However, these findings raise further questions that need to be elucidated. Overall, EA therapy for cognitive impairment is an area with great promise, even though more research regarding its detailed mechanisms is warranted.

Recent Advance in the Reductive Heck Cyclization for the Formation of Five to Nine Member Rings

Palladium-catalyzed reactions are widely used for creating carbon-carbon and carbon- heteroatom bonds, with the Heck reaction being particularly valuable for forming rings of various sizes, including medium-sized rings. Recent reports have shown the synthetic potential of intramolecular Heck reactions for assembling rings of seven or more members. While the regioselectivity of this cyclization is often unpredictable in the absence of directing groups, the reductive Heck cyclization strategy can help minimize this issue. Nickel catalysts are also valuable due to their abundance and environmentally friendly nature, playing a pivotal role in producing biologically significant carbocycles and heterocycles. The use of both Pd(0) and Ni(0) catalysts, with or without chiral ligands, has been successful in forming five to ninemember ring heterocycles and carbocycles in a simple, cost-effective manner. This review provides a comprehensive survey of the literature from the past decade on the use of reductive Heck cyclization methodology, including mechanistic details as needed.

Base‐Mediated Four‐Component Intramolecular Cyclization Reaction: One‐Pot Access to Imidazole‐4‐(2H)‐Ones

AbstractA base‐mediated one‐pot, two‐step, four‐component reaction has been developed to synthesize imidazole‐4(2H)‐ones, utilizing commercially available amino acid esters, aldehydes, alkynes, and amino alcohols. Control experiments and isolation of the intermediate revealed the mechanistic details. This four‐component reaction proceeds via imine formation, followed by the nucleophilic addition of alkyne to form a propargylamine precursor. Subsequently, the propargylamine precursor under undergoes base‐mediated conversion into 1‐azadiene, followed by in situ ketene formation to generate (allylideneamino)prop‐1‐en‐1‐one. The nucleophilic addition of amino alcohol and subsequent intramolecular cyclization provides imidazole‐4 (2H)‐ones exclusively.

Lymphatic-derived oxysterols promote anti-tumor immunity and response to immunotherapy in melanoma.

In melanoma, lymphangiogenesis correlates with metastasis and poor prognosis and promotes immunosuppression. However, it also potentiates immunotherapy by supporting immune cell trafficking. We show in a lymphangiogenic murine melanoma that lymphatic endothelial cells (LECs) upregulate the enzyme Ch25h, which catalyzes the formation of 25-hydroxycholesterol (25-HC) from cholesterol and plays important roles in lipid metabolism, gene regulation, and immune activation. We identify a role for LECs as a source of extracellular 25-HC in tumors inhibiting PPAR-γ in intra-tumoral macrophages and monocytes, preventing their immunosuppressive function and instead promoting their conversion into proinflammatory myeloid cells that support effector T cell functions. In human melanoma, LECs also upregulate Ch25h, and its expression correlates with the lymphatic vessel signature, infiltration of pro-inflammatory macrophages, better patient survival, and better response to immunotherapy. We identify here in mechanistic detail an important LEC function that supports anti-tumor immunity, which can be therapeutically exploited in combination with immunotherapy.

Replication across O6-methylguanine activates futile cycling of DNA mismatch repair attempts assisted by the chromatin remodeling enzyme Smarcad1.

SN1-type alkylating reagents generate O6-methylguanine (meG) lesions that activate the mismatch repair (MMR) response. Since post-replicative MMR specifically targets the nascent strand, meG on the template strand is refractory to rectification by MMR and, therefore, can induce non-productive MMR reactions. The cycling of futile MMR attempts is proposed to cause DNA double-strand breaks in the subsequent S phase, leading to ATR-checkpoint-mediated G2 arrest and apoptosis. However, the mechanistic details of futile MMR cycling, especially how this reaction is maintained in chromatin, remain unclear. Using replication-competent Xenopus egg extracts, we herein establish an in vitro system that recapitulates futile MMR cycling in the chromatin context. The meG-T mispair, but not the meG-C pair, is efficiently targeted by MMR in our system. MMR attempts on the meG-strand result in the meG-to-A correction, while those on the T-strand induce iterative cycles of strand excision and resynthesis. Likewise, replication across meG generates persistent single-strand breaks on the daughter DNA containing meG. Moreover, the depletion of Smarcad1, a chromatin remodeler previously reported to facilitate MMR, impairs the retention of single-strand breaks. Our study thus provides experimental evidence that chromatin replication across meG induces futile MMR cycling that is assisted by Smarcad1.

Cell-free assays reveal that the HIV-1 capsid protects reverse transcripts from cGAS immune sensing.

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect viral genomes from cGAS even after completing reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, the capsid inhibitor lenacapavir also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling.

The intestinal microbiota in inflammatory bowel diseases

The intestinal microbiota comprises all living microorganisms in the gastrointestinal tract and is crucial for its function. Clinical observations and laboratory findings confirm acentral role of the microbiota in chronic inflammatory bowel diseases (IBD). However, many mechanistic details remain unclear. Changes in the microbiota and the causal relationship with the pathogenesis of IBD are described and current and future diagnostic and therapeutic options are discussed. Narrative review. The intestinal microbiota is altered in composition, diversity, and function in IBD patients, but specific (universal) IBD-defining bacteria have not been identified. The healthy microbiota has numerous anti-inflammatory functions such as the production of short-chain fatty acids or competition with pathogens. In contrast, the IBD microbiota promotes inflammation through the destruction of the intestinal barrier and direct interaction with the immune system. The balance between pro- and anti-inflammatory effects of the microbiota appears to be crucial for the development of intestinal inflammation. Microbiota-based IBD diagnostics show promise but are not yet ready for clinical use. Probiotics and fecal microbiota transplantation have clinical effects, especially in ulcerative colitis, but the potential of microbiota-based therapies is far from being fully realized. IBD dysbiosis remains undefined so far. It is unclear how the many parallel pro- and anti-inflammatory mechanisms contribute to IBD pathogenesis. An inadequate mechanistic understanding hinders the development of microbiota-based diagnostics and therapies.

PCET-Driven Reactivity of Neptunyl(VI) Yields Oxo-Bridged Np(V) and Np(IV) Species.

Two unconventional polynuclear complexes of neptunium (Np) featuring mono- -oxo motifs have been accessed by proton-coupled electron transfer (PCET) reactivity involving the dissolution of neptunyl(VI) diacetate dihydrate ( HOAc) in methanol followed by addition of a pentadentate Schiff-base ligand. One complex is a mixed-valent [Np ,Np ,Np ] trimer with two bridging -oxos and the other is a [Np ,Np ] dimer featuring a single -oxo. In both complexes the outer Np centers are capped with terminal oxo ligands. Spectroscopic and spectrokinetic studies aimed at elucidating mechanistic details of complex formation in this system show that intermediate multinuclear [Np (OAc)] species form prior to metal chelation by the ligand; electrolysis experiments demonstrate that production of Np(V) gives rise to asynchronous proton transfer that does not occur otherwise (in the Np(VI) state) as well as condensation with loss of and formation of the polynuclear complexes. We attribute the oxo-deficient nature of these products, with respect to conventional actinyl ([AnO ] ) species, to the reduction/condensation reaction sequence of PCET.

Molecular insights into the modulation of the 5HT2A receptor by serotonin, psilocin, and the G protein subunit Gqα.

5HT2AR is a G-protein-coupled receptor that drives many neuronal functions and is a target for psychedelic drugs. Understanding ligand interactions and conformational transitions is essential for developing effective pharmaceuticals, but mechanistic details of 5HT2AR activation remain poorly understood. We utilized all-atom molecular dynamics simulations and free-energy calculations to investigate 5HT2AR's conformational dynamics upon binding to serotonin and psilocin. We show that the active state of 5HT2AR collapses to a closed state in the absence of Gqα, underscoring the importance of G-protein coupling. We discover an intermediate "partially-open" receptor conformation. Both ligands have higher binding affinities for the orthosteric than the extended binding pocket. These findings enhance our understanding of 5HT2AR's activation and may aid in developing novel therapeutics. Impact statement This study sheds light on 5HT2AR activation, revealing intermediate conformations and ligand dynamics. These insights could enhance drug development for neurological and psychiatric disorders, benefiting researchers and clinicians in pharmacology and neuroscience.

Broad substrate scope C-C oxidation in cyclodipeptides catalysed by a flavin-dependent filament

Cyclic dipeptides are produced by organisms across all domains of life, with many exhibiting anticancer and antimicrobial properties. Oxidations are often key to their biological activities, particularly C-C bond oxidation catalysed by tailoring enzymes including cyclodipeptide oxidases. These flavin-dependent enzymes are underexplored due to their intricate three-dimensional arrangement involving multiple copies of two distinct small subunits, and mechanistic details underlying substrate selection and catalysis are lacking. Here, we determined the structure and mechanism of the cyclodipeptide oxidase from the halophile Nocardiopsis dassonvillei (NdasCDO), a component of the biosynthetic pathway for nocazine natural products. We demonstrated that NdasCDO forms filaments in solution, with a covalently bound flavin mononucleotide (FMN) cofactor at the interface between three distinct subunits. The enzyme exhibits promiscuity, processing various cyclic dipeptides as substrates in a distributive manner. The reaction is optimal at high pH and involves the formation of a radical intermediate. Pre-steady-state kinetics, a significant solvent kinetic isotope effect, and the absence of viscosity effects suggested that a step linked to FMN regeneration controlled the reaction rate. Our work elucidates the complex mechanistic and structural characteristics of this dehydrogenation reaction, positioning NdasCDO as a promising biocatalyst and expanding the FMN-dependent oxidase family to include enzyme filaments.

On the Mechanism of Light-Driven O2 Evolution by the Mn(III) Complex [Mn(salpd)(OH2)]+ and Quinone.

In this study, we apply TD-DFT and DFT calculations to explore the mechanistic details of O2 evolution in an artificial system that closely resembles Photosystem II (PSII). The reaction involves mononuclear Mn(III) complex [Mn(salpd)(OH2)]+ and p-benzoquinone under light-driven conditions. Our calculations reveal that the Schiff-base ligand salpd plays a crucial role in several key steps of the reaction, including the light-mediated oxidation of [Mn(salpd)(OH2)]+ to [Mn(salpd)(OH)]+ by p-benzoquinone, the subsequent oxidation of [Mn(salpd)(OH)]+ to the key Mn(V) intermediate [Mn(salpd)(O)]+, and the critical O-O bond formation step. This role is primarily due to the high propensity of the salpd ligand to undergo oxidation by one unit. This characteristic allows the salpd ligand to reduce Mn(IV) in the intermediate [Mn(salpd)(OH)]+ to Mn(III), triggering a Jahn-Teller effect that increases the ionic character of the hydroxide ligand. This transformation makes the resulting complex a strong nucleophile, facilitating O-O bond formation through a reaction between [Mn(salpd)(OH)]+ and [Mn(salpd)(O)]+ with a moderate overall activation free energy of 18.6 kcal/mol. The mechanistic insights presented in this study may provide a useful foundation for developing novel systems that catalyze water oxidation under light-driven conditions, mimicking Photosystem II, and could potentially contribute to advancements in sustainable energy generation.

Protein prenylation in mechanotransduction: implications for disease and therapy.

The process by which cells translate external mechanical cues into intracellular biochemical signals involves intricate mechanisms that remain unclear. In recent years, research into post-translational modifications (PTMs) has offered valuable insights into this field, spotlighting protein prenylation as a crucial mechanism in cellular mechanotransduction and various human diseases. Protein prenylation, which involves the covalent attachment of isoprenoid groups to specific substrate proteins, profoundly affects the functions of key mechanotransduction proteins such as Rho, Ras, and lamins. This review provides the first comprehensive examination of the connections between prenylation and mechanotransduction, exploring both the mechanistic details and its impact on mechanosensitive cellular behaviors. We further highlight recent evidence linking protein prenylation to diseases associated with disrupted mechanical homeostasis, and outline emerging targeted therapeutic strategies.

The mechanism of amyloid fibril growth from Φ-value analysis.

Amyloid fibrils are highly stable misfolded protein assemblies that play an important role in several neurodegenerative and systemic diseases. Although structural information of the amyloid state is now abundant, mechanistic details about the misfolding process remain elusive. Inspired by the Φ-value analysis of protein folding, we combined experiments and molecular simulations to resolve amino-acid contacts and determine the structure of the transition-state ensemble-the rate-limiting step-for fibril elongation of PI3K-SH3 amyloid fibrils. The ensemble was validated experimentally by Tanford β analysis and computationally by free energy calculations. Although protein folding proceeds on funnel-shaped landscapes, here we find that the energy landscape for the misfolding reaction consists of a large 'golf course' region, defined by a single energy barrier and transition state, accessing a sharply funnelled region. Thus, misfolding occurs by rare, successful monomer-fibril end collisions interspersed by numerous unsuccessful binding attempts. Taken together, these insights provide a quantitative and highly resolved description of a protein misfolding reaction.

Combining CRISPR activation and interference capabilities using dCas9 and G-quadruplex structures.

We demonstrate that both Clustered regularly interspaced short palindromic repeats (CRISPR) interference and CRISPR activation can be achieved at RNA and protein levels by targeting the vicinity of a putative G-quadruplex (GQ)-forming sequence (PQS) in the c-Myc promoter with nuclease-dead Cas9 (dCas9). The achieved suppression and activation in Burkitt's Lymphoma cell line and in in vitro studies are at or beyond those reported with alternative approaches. When the template strand (contains the PQS) was targeted with CRISPR-dCas9, the GQ was destabilized and c-Myc mRNA and protein levels increased by 2.1- and 1.6-fold, respectively, compared to controls in the absence of CRISPR-dCas9. Targeting individual sites in the nontemplate strand (NTS) with CRISPR-dCas9 reduced both the c-Myc mRNA and protein levels (by 1.8- and 2.5-fold, respectively), while targeting two sites simultaneously further suppressed both the mRNA (by 3.6-fold) and protein (by 9.8-fold) levels. These were consistent with cell viability assays when single or dual sites in the NTS were targeted (1.7- and 4.7-fold reduction in viability, respectively). We also report extensive in vitro biophysical studies which are in quantitative agreement with these cellular studies and provide important mechanistic details about how the transcription is modulated via the interactions of RNA polymerase, CRISPR-dCas9, and the GQ.

Fluorescence Detection of DNA/RNA G-Quadruplexes (G4s) by Twice-as-Smart Ligands.

Fluorescence detection of DNA and RNA G-quadruplexes (G4s) is a very efficient strategy to assess not only the existence and prevalence of cellular G4s but also their relevance as targets for therapeutic interventions. Among the fluorophores used to this end, turn-on probes are the most interesting since their fluorescence is triggered only upon interaction with their G4 targets, which ensures a high sensitivity and selectivity of detection. We reported on a series of twice-as-smart G4 probes, which are both smart G4 ligands (whose structure is reorganized upon interaction with G4s) and smart fluorescent probes (whose fluorescence is turned on upon interaction with G4s). The fine mechanistic details behind the excellent properties of the best prototype N-TASQ remain to be deciphered: to investigate this, we report here on the synthesis and studies of two analogues, TzN-TASQ and AlkN-TASQ, and on a careful analysis of their G4-interacting properties, investigated both in vitro and in silico. Our results show that fine-tuning their constitutive structural elements allows for increasing the efficiency of both their 'off' (i. e., a conformation with a low fluorescence) and 'on' states (i. e., a conformation with a high fluorescence), which opens interesting ways for the design of more efficient fluorogenic G4 probes.

Light-Dependent Amide or Thioamide Formation of Acylsilanes with Amines using Elemental Sulfur.

Due to the diverse chemical and physical properties of functional groups, mild and controllable ligation methods are often required to construct complex drugs and functional materials. To make diverse sets of products with tunable physicochemical properties, it is also useful to employ complimentary ligation methods that adopt the same starting materials. Here, we disclose the efficient and modular synthesis of amides or thioamides through the chemical ligation of acylsilanes with amines, simply by turning a light on or off. This method is fast, mild, high-yielding and displays excellent functional-group tolerance. The versatility of these reactions is highlighted by their ability to perform post-synthetic modifications on a variety of marketed medications, peptides, natural substances, and compounds with biological activity. In-depth computational and experimental studies clarified the photo-dependent umpolung of reactivity of acylsilanes, namely: photoexcitation leads to nucleophilic O-silyl carbenes that react with S8 to form O-silyl thionoesters and eventually amides. In contrast, acylsilanes react as electrophiles with amines thermally in the dark, with C→O silyl transfer, prior to reacting with S8 to form thioamides. These mechanistic details are expected to guide the development of similar coupling reactions.

Time-resolved and theoretical analysis of Mo-carbene transformations in metathesis of ethylene with 2-butene.

Although supported Mo-containing catalysts have been extensively investigated in the metathesis of ethylene with 2-butene to propene, the mechanisms of the formation and transformation of catalytically active Mo-carbenes in the course of the reaction are still not fully understood. The difficulties arise because only a tiny fraction of MoO x species can form Mo-carbenes in situ, making the detection of the latter by spectroscopic means very unlikely. Herein, purposefully designed steady-state and transient experiments including their kinetic evaluation and density functional theory calculations enabled us to elucidate mechanistic and kinetic details of the above reaction-induced processes in the metathesis reaction over a Mo/P/SiO2 catalyst at 50 °C. We established that, in parallel with the desired reaction cycle, molybdacyclobutanes also undergo reversible structural transformations which might be one of the reasons for low steady-state catalyst activity. Based on the results obtained, strategies for controlling the concentration of the inactive species and accordingly catalyst activity have been suggested and experimentally validated.

Transmission of unfolded protein response-a regulator of disease progression, severity, and spread in virus infections.

The unfolded protein response (UPR) is a cell-autonomous stress response aimed at restoring homeostasis due to the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses often hijack the host cell machinery, leading to an accumulation of misfolded proteins in the ER. The cell-autonomous UPR is the immediate response of an infected cell to this stress, aiming to restore normal function by halting protein translation, degrading misfolded proteins, and activating signaling pathways that increase the production of molecular chaperones. The cell-non-autonomous UPR involves the spreading of UPR signals from initially stressed cells to neighboring unstressed cells that lack the stressor. Though viruses are known modulators of cell-autonomous UPR, recent advancements have highlighted that cell-non-autonomous UPR plays a critical role in elucidating how local infections cause systemic effects, thereby contributing to disease symptoms and progression. Additionally, by utilizing cell-non-autonomous UPR, viruses have devised novel strategies to establish a pro-viral state, promoting virus spread. This review discusses examples that have broadened the understanding of the role of UPR in virus infections and disease progression by looking beyond cell-autonomous to non-autonomous processes and mechanistic details of the inducers, spreaders, and receivers of UPR signals.

Metabolic and physiological functions of Patatin-like phospholipase-A in plants.

Patatin-like phospholipase-A (pPLA) is a class of lipid acyl hydrolase enzymes found in both, the animal and plant kingdoms. Plant pPLAs are related to the potato tuber storage protein patatin in solanaceous plants. Despite extensive investigation of pPLA functions in the animal system, the mechanistic functional details and regulatory roles of pPLA are poorly understood in plants. In recent years, research pertaining to pPLAs has gain some momentum as some of the key members of pPLA family have been characterized functionally. These findings have provided key insights into the structural features, biochemical activities, and functional roles of plant pPLAs. In this review, we are presenting a holistic overview of pPLAs in plants and providing the latest updates on pPLA research. We have highlighted the genomic diversity and structural features of pPLAs in plants. Importantly, we have discussed the role of pPLAs in lipid metabolism, including sphingolipid metabolism, lignin and cellulose accumulation, lipid breakdown and seed oil content enhancement. Moreover, regulatory roles of pPLAs in physiological processes, such as plant stress response, plant-pathogen interactions and plant development have been discussed. This information will be critical in the biotechnological programs for crop improvement.