RESUMO
Cyclopropane and azacyclopropane, also known as aziridine, moieties are found in natural products. These moieties serve as pivotal components that lead to a broad spectrum of biological activities. While diverse strategies involving various classes of enzymes are utilized to catalyze formation of these strained three-membered rings, how non-heme iron and 2-oxoglutarate (Fe/2OG) dependent enzymes enable regio- and stereo-selective C-C and C-N ring closure has only been reported very recently. Herein, we present detailed experimental protocols for mechanistically studying Fe/2OG enzymes that catalyze cyclopropanation and aziridination reactions. These protocols include protein purification, in vitro assays, biophysical spectroscopies, and isotope-tracer experiments. We also report how to use in silico approaches to look for Fe/2OG aziridinases. Furthermore, our current mechanistic understanding of three-membered ring formation is discussed. These results not only shed light on the reaction mechanisms of Fe/2OG enzymes-catalyzed cyclopropanation and aziridination, but also open avenues for expanding the reaction repertoire of the Fe/2OG enzyme superfamily.
Assuntos
Aziridinas , Ciclopropanos , Ácidos Cetoglutáricos , Ciclopropanos/química , Ciclopropanos/metabolismo , Aziridinas/química , Aziridinas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/química , Ferro/química , Ferro/metabolismo , Ferroproteínas não Heme/química , Ferroproteínas não Heme/metabolismo , Biocatálise , Ensaios Enzimáticos/métodos , CatáliseRESUMO
As an important small organic molecule, cyclopropane is widely used in drug design. In this paper, fifty-three amide derivatives containing cyclopropane were designed and synthesized by introducing amide groups and aryl groups into cyclopropane through the active splicing method, and their antibacterial and antifungal activities were evaluated in vitro. Among them, thirty-five compounds were new compounds, and eighteen compounds were known compounds (F14, F15, F18, F20-F26, F36, and F38-F44). Bioassay results disclosed that four, three, and nine of the compounds showed moderate activity against Staphylococcus aureus, Escherichia coli, and Candida albicans, respectively. Three compounds were sensitive to Candida albicans, with excellent antifungal activity (MIC80 = 16 µg/mL). The molecular docking results show that compounds F8, F24, and F42 have good affinity with the potential antifungal drug target CYP51 protein.
Assuntos
Amidas , Antifúngicos , Candida albicans , Ciclopropanos , Desenho de Fármacos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Staphylococcus aureus , Ciclopropanos/farmacologia , Ciclopropanos/química , Ciclopropanos/síntese química , Amidas/química , Amidas/farmacologia , Amidas/síntese química , Candida albicans/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Antifúngicos/farmacologia , Antifúngicos/síntese química , Antifúngicos/química , Escherichia coli/efeitos dos fármacos , Relação Estrutura-Atividade , Anti-Infecciosos/farmacologia , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Antibacterianos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Estrutura MolecularRESUMO
The application of coatings is a strategy for maintaining the freshness of highly perishable fruits. This research aimed to evaluate the quality indices of strawberries (Amaou) coated with new coatings based on the sodium carboxymethyl cellulose (CMC) and cellulose nanofibres (CNF) with incorporated mandarin peel extract (ME) or 1-methylcyclopropene (1-MCP) during storage at 20days at 5 °C and 85% relative humidity (RH). Dissolving the coating solution containing ME in 1-MCP maintained its colour for up to 50 days. Coatings enhanced with ME and/or 1-MCP maintained fresh strawberries more effectively than the control, reducing weight loss and maintaining firmness, total soluble solids (TSS), citric acid, colour, and total phenolic content. The CCM2-2 coating solution showed superior effects on the weight loss and relative percentages of strawberry metabolites compared to the other coatings, as confirmed by the different components.
Assuntos
Citrus , Ciclopropanos , Conservação de Alimentos , Armazenamento de Alimentos , Fragaria , Frutas , Extratos Vegetais , Frutas/química , Fragaria/química , Fragaria/metabolismo , Conservação de Alimentos/métodos , Ciclopropanos/química , Ciclopropanos/farmacologia , Extratos Vegetais/química , Citrus/química , Metaboloma , Temperatura BaixaRESUMO
Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvß3.
Assuntos
Biblioteca de Peptídeos , Peptídeos Cíclicos , Ciclização , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo , Humanos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Cisteína/química , Cisteína/metabolismo , Ciclopropanos/química , Peptídeos/química , Peptídeos/metabolismoRESUMO
Purpose: To investigate the preparation of inclusion complexes of diphenylcyclopropenone (DPCP)/ß-cyclodextrin (ß-CD) derivatives using a three-dimensional (3D) ball mill, and verify the inclusion behavior of the solid dispersion. Additionally, we aimed to investigate the effect of DPCP/ß-CDs complex formation on the spleens of male C57BL/6 mice in terms of anti-inflammatory effects. Methods: The inclusion complexes of DPCP with ß-CD and hydroxypropyl-ß-cyclodextrin (HPßCD) were prepared using a 3D ball mill. Powder X-ray diffraction (PXRD) and Fourier-transform infrared spectroscopy (FT-IR) were used to evaluate the solid-state properties. The solubility of the prepared DPCP/ß-CD and HPßCD complexes and the intermolecular interaction between DPCP and ß-CD derivatives in solution were assessed using 1H nuclear magnetic resonance (NMR). Furthermore, the anti-inflammatory effects of DPCPs in the prepared DPCP/CD complexes were investigated using spleens from male C57BL/6 mice, with measurement of interferon gamma (IFN-γ) secretion as an endpoint. Additionally, the protective effects of each drug on NIH-3T3 cells exposed to ultraviolet (UV) irradiation were examined. Results: Solid-state characterization confirmed the formation of inclusion complexes in the 3D ground mixture (3DGM) (DPCP/ß-CD = 1/1) and 3DGM (DPCP/HPßCD = 1/1) complexes through PXRD and IR analysis. The solubility of 3DGM (DPCP/ß-CD = 1/1) and 3DGM (DPCP/HPßCD = 1/1) was 17.5 µg/mL and 58.4 µg/mL, respectively, indicating higher solubility than that of DPCP alone. NMR analysis of 3DGM samples suggested that DPCP/ß-CD and DPCP/HPßCD form inclusion complexes at a molar ratio of 1/1 but with different inclusion modes. Regarding the anti-inflammatory activity of DPCP, 3DGM (DPCP/HPß-CD) showed anti-inflammatory effects at lower doses compared to 3DGM (DPCP/ß-CD) in terms of IFN-γ and NIH-3T3 cells injured by UV irradiation. Conclusion: We successfully formed inclusion complexes of DPCP/ß-CD and DPCP/HPßCD using the 3D ground mixture method. NMR analysis suggested that DPCP/ß-CD and DPCP/HPßCD form inclusion complexes at a molar ratio of 1/1 but with different inclusion modes. The anti-inflammatory activity of DPCP was more pronounced in 3DGM (DPCP/HPßCD) at lower doses compared to that in 3DGM (DPCP/ß-CD), indicating that the HPßCD derivatives were more effective in enhancing the anti-inflammatory properties of DPCP.
Assuntos
Alopecia em Áreas , Anti-Inflamatórios , Ciclopropanos , Camundongos Endogâmicos C57BL , Solubilidade , beta-Ciclodextrinas , Animais , beta-Ciclodextrinas/química , beta-Ciclodextrinas/farmacologia , Masculino , Camundongos , Ciclopropanos/farmacologia , Ciclopropanos/química , Ciclopropanos/administração & dosagem , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Alopecia em Áreas/tratamento farmacológico , Baço/efeitos dos fármacos , 2-Hidroxipropil-beta-Ciclodextrina/química , 2-Hidroxipropil-beta-Ciclodextrina/farmacologiaRESUMO
Hemoproteins have recently emerged as powerful biocatalysts for new-to-nature carbene transfer reactions. Despite this progress, these strategies have remained largely limited to diazo-based carbene precursor reagents. Here, we report the development of a biocatalytic strategy for the stereoselective construction of pyridine-functionalized cyclopropanes via the hemoprotein-mediated activation of pyridotriazoles (PyTz) as stable and readily accessible carbene sources. This method enables the asymmetric cyclopropanation of a variety of olefins, including electron-rich and electrodeficient ones, with high activity, high stereoselectivity, and enantiodivergent selectivity, providing access to mono- and diarylcyclopropanes that incorporate a pyridine moiety and thus two structural motifs of high value in medicinal chemistry. Mechanistic studies reveal a multifaceted role of 7-halogen substitution in the pyridotriazole reagent toward favoring multiple catalytic steps in the transformation. This work provides the first example of asymmetric olefin cyclopropanation with pyridotriazoles, paving the way to the exploitation of these attractive and versatile reagents for enzyme-catalyzed carbene-mediated reactions.
Assuntos
Ciclopropanos , Triazóis , Ciclopropanos/química , Ciclopropanos/síntese química , Triazóis/química , Triazóis/síntese química , Estereoisomerismo , Piridinas/química , Piridinas/síntese química , Estrutura Molecular , BiocatáliseRESUMO
Innovative discovery approaches such as genome-mining and metabolomics-inspired methods have reshaped the natural product research field, complementing traditional bioactivity-based screens and allowing hitherto unseen compounds to be uncovered from previously investigated producers. In line with these trends, we report here imidacins, a novel class of secondary metabolites specific to the myxobacterial genus Stigmatella. A combination of secondary metabolome analysis, genome-mining techniques, spectroscopic analysis, and finally total synthesis was used to allow structure elucidation. Imidacins are urocanate-derived aliphatic acids with an adjacent cyclopropane moiety, structural features unprecedented in natural products to date.
Assuntos
Stigmatella aurantiaca , Estrutura Molecular , Stigmatella aurantiaca/química , Alcaloides/química , Alcaloides/síntese química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/síntese química , Myxococcales/química , Ciclopropanos/química , Ciclopropanos/farmacologia , Ciclopropanos/síntese químicaRESUMO
The evolving use of covalent ligands as chemical probes and therapeutic agents could greatly benefit from an expanded array of cysteine-reactive electrophiles for efficient and versatile proteome profiling. Herein, to expand the current repertoire of cysteine-reactive electrophiles, we developed a new class of strain-enabled electrophiles based on cyclopropanes. Proteome profiling has unveiled that C163 of lactate dehydrogenase A (LDHA) and C88 of adhesion regulating molecule 1 (ADRM1) are ligandable residues to modulate the protein functions. Moreover, fragment-based ligand discovery (FBLD) has revealed that one fragment (Y-35) shows strong reactivity toward C66 of thioredoxin domain-containing protein 12 (TXD12), and its covalent binding has been demonstrated to impact its downstream signal pathways. TXD12 plays a pivotal role in enabling Y-35 to exhibit its antisurvival and antiproliferative effects. Finally, dicarbonitrile-cyclopropane has been demonstrated to be an electrophilic warhead in the development of GSTO1-involved dual covalent inhibitors, which is promising to alleviate drug resistance.
Assuntos
Ciclopropanos , Proteoma , Ciclopropanos/química , Ciclopropanos/farmacologia , Ligantes , Humanos , Proteoma/química , Proteoma/metabolismo , Descoberta de Drogas , Estrutura Molecular , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/químicaRESUMO
We synthesized two new gemini analogues, UG-480 and UG-481, that incorporate a modified longer side chain containing a cyclopropane group. The evaluation of the bioactivities of the two gemini analogues indicated that the 17,20 threo (20S) compound, UG-480, is the most active one and is as active as 1,25(OH)2D3. Docking and molecular dynamics (MD) data showed that the compounds bind efficiently to vitamin D receptor (VDR) with UG-480 to form an energetically more favorable interaction with His397. Structural analysis indicated that whereas the UG-480 compound efficiently stabilizes the active VDR conformation, it induces conformational changes in the H6-H7 VDR region that are greater than those induced by the parental Gemini and that this is due to the occupancy of the secondary channel by its modified side chain.
Assuntos
Ciclopropanos , Desenho de Fármacos , Receptores de Calcitriol , Ciclopropanos/química , Ciclopropanos/síntese química , Ciclopropanos/farmacologia , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/agonistas , Humanos , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Simulação de Acoplamento MolecularRESUMO
The ability to tame high-energy intermediates is important for synthetic chemistry, enabling the construction of complex molecules and propelling advances in the field of synthesis. Along these lines, carbenes and carbenoid intermediates are particularly attractive, but often unknown, high-energy intermediates1,2. Classical methods to access metal carbene intermediates exploit two-electron chemistry to form the carbon-metal bond. However, these methods are usually prohibitive because of reagent safety concerns, limiting their broad implementation in synthesis3-6. Mechanistically, an alternative approach to carbene intermediates that could circumvent these pitfalls would involve two single-electron steps: radical addition to metal to forge the initial carbon-metal bond followed by redox-promoted α-elimination to yield the desired metal carbene intermediate. Here we realize this strategy through a metallaphotoredox platform that exploits iron carbene reactivity using readily available chemical feedstocks as radical sources and α-elimination from six classes of previously underexploited leaving groups. These discoveries permit cyclopropanation and σ-bond insertion into N-H, S-H and P-H bonds from abundant and bench-stable carboxylic acids, amino acids and alcohols, thereby providing a general solution to the challenge of carbene-mediated chemical diversification.
Assuntos
Álcoois , Aminoácidos , Ácidos Carboxílicos , Técnicas de Química Sintética , Ferro , Metano , Fotoquímica , Álcoois/química , Aminoácidos/química , Carbono/química , Ácidos Carboxílicos/química , Catálise , Ciclopropanos/química , Ciclopropanos/síntese química , Ferro/química , Metano/análogos & derivados , Metano/química , Oxirredução , Fotoquímica/métodos , Técnicas de Química Sintética/métodos , ElétronsRESUMO
Cyclopropenone is a valuable electrophilic reagent that can react with electrophilic reagents, nucleophilic reagents, free radicals, organic metals, etc. Furthermore, cyclopropenone derivatives have shown significant biological activity in various diseases, such as triple-negative breast cancer (TNBC), melanoma, and alopecia areata (AA). The cyclopropenone analogue diphenylcyclopropenone (DPCP) has been approved for the treatment of AA. Given the potential therapeutic benefits of cyclopropenone derivatives, this review aims to systematically summarize the structures, synthesis routes, and potential pharmacological functions of cyclopropenone analogues in the hope of offering novel insights for further rational design of more drugs based on the cyclopropenone skeleton for the treatment of human diseases.
Assuntos
Ciclopropanos , Humanos , Ciclopropanos/química , Ciclopropanos/farmacologia , Ciclopropanos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Estrutura Molecular , Alopecia em Áreas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Melanoma/tratamento farmacológico , Melanoma/patologia , Relação Estrutura-AtividadeRESUMO
The C797S mutation of EGFR leads to Osimertinib resistance by blocking the covalent binding of Cys797. To develop new agents that can overcome EGFR mutation resistance, thirty seven new cyclopropane sulfonamide derivatives were synthesized and evaluated as EGFRL858R/T790M/C797S or EGFRDel19/T790M/C797S inhibitors by structure-based screening. Most of the synthesized compounds exhibit good to excellent anti proliferation activity against to BaF3-EGFR L858R/T790M/C797S and BaF3-C797S/Del19/T790M cancer cell lines. Representative compounds 8l showed inhibitory activity against the two cancer cell lines with the IC50 values of 0.0012 and 0.0013 µM, respectively. Another compound 8h, exhibited slightly lower activity (0.0042 and 0.0034 µM of the IC50 values) to both of the two tri-mutation cell lines, but excellent activities against H1975 and PC9 cells with IC50 values of 13 and 19 nM, respectively. Considering the acquired drug resistance of tumors is a gradual process, we chose 8h for further in vivo and mechanism study. 8h was demonstrated significantly inhibited tumor growth with 72.1 % of the TGI in the BaF3/EGFR-TM xenograft tumor model and 83.5 % in the H1975-DM xenograft tumor model. Compound 8h was confirmed to be safe with no significant side effects as showed by the results of in vitro assay of human normal cells and the sections of animals major organs. Mechanism studies showed that in addition to inhibiting EGFR mutations, 8h can also target the tumor microenvironment and induce tumor cell apoptosis. All these results indicate that 8h deserves further investigation as an EGFR inhibitor to overcome C797S-mediated resistance.
Assuntos
Antineoplásicos , Proliferação de Células , Ciclopropanos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB , Mutação , Inibidores de Proteínas Quinases , Sulfonamidas , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Sulfonamidas/farmacologia , Sulfonamidas/química , Sulfonamidas/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Animais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Ciclopropanos/farmacologia , Ciclopropanos/química , Ciclopropanos/síntese química , Camundongos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Estrutura Molecular , Descoberta de Drogas , Linhagem Celular Tumoral , Camundongos NusRESUMO
Design of metal cofactor ligands is essential for controlling the reactivity of metalloenzymes. We investigated a carbene transfer reaction catalyzed by myoglobins containing iron porphyrin cofactors with one and two trifluoromethyl groups at peripheral sites (FePorCF3 and FePor(CF3)2, respectively), native heme and iron porphycene (FePc). These four myoglobins show a wide range of Fe(II)/Fe(III) redox potentials in the protein of +147â mV, +87â mV, +42â mV and -198â mV vs. NHE, respectively. Myoglobin reconstituted with FePor(CF3)2 has a more positive potential, which enhances the reactivity of a carbene intermediate with alkenes, and demonstrates superior cyclopropanation of inert alkenes, such as aliphatic and internal alkenes. In contrast, engineered myoglobin reconstituted with FePc has a more negative redox potential, which accelerates the formation of the intermediate, but has low reactivity for inert alkenes. Mechanistic studies indicate that myoglobin with FePor(CF3)2 generates an undetectable active intermediate with a radical character. In contrast, this reaction catalyzed by myoglobin with FePc includes a detectable iron-carbene species with electrophilic character. This finding highlights the importance of redox-focused design of the iron porphyrinoid cofactor in hemoproteins to tune the reactivity of the carbene transfer reaction.
Assuntos
Mioglobina , Oxirredução , Mioglobina/química , Mioglobina/metabolismo , Engenharia de Proteínas , Ciclopropanos/química , Metaloporfirinas/química , Metano/química , Metano/análogos & derivadosRESUMO
Feline immunodeficiency virus (FIV) is a common infection found in domesticated and wild cats worldwide. Despite the wealth of therapeutic understanding of the disease in humans, considerably less information exists regarding the treatment of the disease in felines. Current treatment relies on drugs developed for the related human immunodeficiency virus (HIV) and includes compounds of the popular non-nucleotide reverse transcriptase (NNRTI) class. This is despite FIV-RT being only 67% similar to HIV-1 RT at the enzyme level, increasing to 88% for the allosteric pocket targeted by NNRTIs. The goal of this project was to try to quantify how well the more extensive pharmacological knowledge available for human disease translates to felines. To this end we screened known NNRTIs and 10 diverse pyrimidine analogs identified virtually. We use this chemo-centric probe approach to (a) assess the similarity between the two related RT targets based on the observed experimental inhibition values, (b) try to identify more potent inhibitors at FIV, and (c) gain a better appreciation of the structure-activity relationships (SAR). We found the correlation between IC50s at the two targets to be strong (r2 = 0.87) and identified compound 1 as the most potent inhibitor of FIV with IC50 of 0.030 µM ± 0.009. This compared to FIV IC50 values of 0.22 ± 0.17 µM, 0.040 ± 0.010 µM and >160 µM for known anti HIV-1 RT drugs Efavirenz, Rilpivirine, and Nevirapine, respectively. This knowledge, along with an understanding of the structural origin that give rise to any differences could improve the way HIV drugs are repurposed for FIV.
Assuntos
Transcriptase Reversa do HIV , Vírus da Imunodeficiência Felina , Inibidores da Transcriptase Reversa , Animais , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/química , Gatos , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , Humanos , Relação Estrutura-Atividade , Pirimidinas/química , Pirimidinas/farmacologia , Alcinos/química , Alcinos/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Ciclopropanos/farmacologia , Ciclopropanos/química , Simulação de Acoplamento Molecular , Benzoxazinas/química , Benzoxazinas/farmacologiaRESUMO
BACKGROUND: Ulcerative colitis (UC) is one chronic and relapsing inflammatory bowel disease. Macrophage has been reputed as one trigger for UC. Recently, phosphodiesterase 4 (PDE4) inhibitors, for instance roflumilast, have been regarded as one latent approach to modulating macrophage in UC treatment. Roflumilast can decelerate cyclic adenosine monophosphate (cAMP) degradation, which impedes TNF-α synthesis in macrophage. However, roflumilast is devoid of macrophage-target and consequently causes some unavoidable adverse reactions, which restrict the utilization in UC. RESULTS: Membrane vesicles (MVs) from probiotic Escherichia coli Nissle 1917 (EcN 1917) served as a drug delivery platform for targeting macrophage. As model drugs, roflumilast and MnO2 were encapsulated in MVs (Rof&MnO2@MVs). Roflumilast inhibited cAMP degradation via PDE4 deactivation and MnO2 boosted cAMP generation by activating adenylate cyclase (AC). Compared with roflumilast, co-delivery of roflumilast and MnO2 apparently produced more cAMP and less TNF-α in macrophage. Besides, Rof&MnO2@MVs could ameliorate colitis in mouse model and regulate gut microbe such as mitigating pathogenic Escherichia-Shigella and elevating probiotic Akkermansia. CONCLUSIONS: A probiotic-based nanoparticle was prepared for precise codelivery of roflumilast and MnO2 into macrophage. This biomimetic nanoparticle could synergistically modulate cAMP in macrophage and ameliorate experimental colitis.
Assuntos
Aminopiridinas , Benzamidas , AMP Cíclico , Ciclopropanos , Macrófagos , Compostos de Manganês , Óxidos , Probióticos , Animais , Aminopiridinas/farmacologia , Camundongos , AMP Cíclico/metabolismo , Probióticos/farmacologia , Ciclopropanos/farmacologia , Ciclopropanos/química , Compostos de Manganês/química , Compostos de Manganês/farmacologia , Benzamidas/farmacologia , Benzamidas/química , Óxidos/farmacologia , Óxidos/química , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , Inibidores da Fosfodiesterase 4/química , Colite/tratamento farmacológico , Colite/induzido quimicamente , Células RAW 264.7 , Escherichia coli/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Modelos Animais de DoençasRESUMO
The introduction of an abiological catalytic group into the binding pocket of a protein host allows for the expansion of enzyme chemistries. Here, we report the generation of an artificial enzyme by genetic encoding of a non-canonical amino acid that contains a secondary amine side chain. The non-canonical amino acid and the binding pocket function synergistically to catalyze the asymmetric nitrocyclopropanation of α,ß-unsaturated aldehydes by the iminium activation mechanism. The designer enzyme was evolved to an optimal variant that catalyzes the reaction at high conversions with high diastereo- and enantioselectivity. This work demonstrates the application of genetic code expansion in enzyme design and expands the scope of enzyme-catalyzed abiological reactions.
Assuntos
Aldeídos , Ciclopropanos , Aldeídos/química , Aldeídos/metabolismo , Ciclopropanos/química , Ciclopropanos/metabolismo , Estereoisomerismo , Biocatálise , Nitrocompostos/química , Nitrocompostos/metabolismo , Estrutura MolecularRESUMO
Cyclopropane fatty acid synthases (CFAS) are a class of S-adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene-based approaches. This work describes the characterisation of E.â coli CFAS (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with up to 58 % conversion and 73 % ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ecCFAS was found to be correlated with the electronic properties of phospholipid headgroups and for the first time ecCFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ecCFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH3I and CD3I respectively.
Assuntos
Biocatálise , Ciclopropanos , Escherichia coli , Ciclopropanos/química , Ciclopropanos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Estereoisomerismo , Metiltransferases/metabolismo , Metiltransferases/química , Ácido Graxo Sintases/metabolismo , Ácido Graxo Sintases/química , Metano/análogos & derivados , Metano/química , Metano/metabolismo , Ácidos GraxosRESUMO
Our gut microbiota directly influences human physiology in health and disease. The myriad of surface glycoconjugates in both the bacterial cell envelope and our gut cells dominate the microbiota-host interface and play a critical role in host response and microbiota homeostasis. Among these, peptidoglycan is the basic glycan polymer offering the cell rigidity and a basis on which many other glycoconjugates are anchored. To directly study peptidoglycan in gut commensals and obtain the molecular insight required to understand their functional activities we need effective techniques like chemical probes to label peptidoglycan in live bacteria. Here we report a chemically guided approach to study peptidoglycan in a key mucin-degrading gut microbiota member of the Verrucomicrobia phylum, Akkermansia muciniphila. Two novel non-toxic tetrazine click-compatible peptidoglycan probes with either a cyclopropene or isonitrile handle allowed for the detection and imaging of peptidoglycan synthesis in this intestinal species.
Assuntos
Akkermansia , Microbioma Gastrointestinal , Peptidoglicano , Peptidoglicano/metabolismo , Peptidoglicano/química , Peptidoglicano/biossíntese , Akkermansia/metabolismo , Humanos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Química Click , Ciclopropanos/química , Ciclopropanos/metabolismoRESUMO
Dual bioorthogonal labeling enables the investigation and understanding of interactions in the biological environment that are not accessible by a single label. However, applying two bioorthogonal reactions in the same environment remains challenging due to cross-reactivity. We developed a pair of differently modified 2'-deoxynucleosides that solved this issue for dual and orthogonal labeling of DNA. Inverse-electron demand Diels-Alder and photoclick reactions were combined to attach two different fluorogenic labels to genomic DNA in cells. Using a small synthetic library of 1- and 3-methylcyclopropenyl-modified 2'-deoxynucleosides, two 2'-deoxyuridines were identified to be the fastest-reacting ones for each of the two bioorthogonal reactions. Their orthogonal reactivity could be evidenced in vitro. Primer extension experiments were performed with both 2'-deoxyuridines investigating their replication properties as substitutes for thymidine and evaluating subsequent labeling reactions on the DNA level. Finally, dual, orthogonal and metabolic fluorescent labeling of genomic DNA was demonstrated in HeLa cells. An experimental procedure was developed combining intracellular transport and metabolic DNA incorporation of the two 2'-deoxyuridines with the subsequent dual bioorthogonal labeling using a fluorogenic cyanine-styryl tetrazine and a fluorogenic pyrene-tetrazole. These results are fundamental for advanced metabolic labeling strategies for nucleic acids in the future, especially for live cell experiments.
Assuntos
Ciclopropanos , DNA , Corantes Fluorescentes , Humanos , DNA/química , DNA/metabolismo , Células HeLa , Ciclopropanos/química , Ciclopropanos/metabolismo , Corantes Fluorescentes/química , Reação de Cicloadição , Estrutura MolecularRESUMO
Amino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5'-phosphate-dependent enzyme, PazB, via an SN2-like attack at C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.