Synthesis of sydnonimines from sydnones and their use for bioorthogonal release of isocyanates in cells.

In this article, we report the synthesis of sydnonimines from sydnones and their use as dipoles for fast click-and-release reactions. The process relies on nucleophilic aromatic substitution of aliphatic and aromatic amines with triflated sydnones. This new methodology allowed the preparation of functionalised sydnonimine probes that are otherwise difficult to prepare. These probes were then used to release a drug and a fluorescent aromatic isocyanate inside living cells.

2'-Modified Thymidines with Bioorthogonal Cyclopropene or Sydnone as Building Blocks for Copper-Free Postsynthetic Functionalization of Chemically Synthesized Oligonucleotides.

The development of facile methods for conjugating relevant probes, ligands, or delivery agents onto oligonucleotides (ONs) is highly desirable both for fundamental studies in chemical biology and for improving the pharmacology of ONs in medicinal chemistry. Numerous efforts have been focused on the introduction of bioorthogonal groups onto phosphoramidite building blocks, allowing the controlled chemical synthesis of reactive ONs for postsynthetic modifications. Among these building blocks, alkyne, cyclooctynes, trans-cyclooctene, and norbornene have been proved to be compatible with automated solid-phase chemistry. Herein, we present the development of novel 2'-functionalized nucleoside phosphoramidite monomers comprising bioorthogonal methylcyclopropene or sydnone moieties and their introduction for the first time to ON solid-phase synthesis. Traceless ON postsynthetic modifications with reactive complementary probes were successfully achieved through either inverse electron-demand Diels-Alder (iEDDA) reactions or strain-promoted sydnone-alkyne cycloaddition (SPSAC). These results expand the set of bioorthogonal phosphoramidite building blocks to generate ONs for postsynthetic labeling.

Investigation of mitochondrial targeting ability of sydnones and sydnonimines and mitochondria-targeted delivery of celecoxib.

Mitochondria are considered to be a promising target in cancer diagnosis and therapeutics. Recently, sydnone and sydnonimine, as mesoionic bioorthogonal reagents, have been used in cell labeling and drug delivery. Here we investigated the mitochondrial targeting ability of sydnones and sydnonimines for the first time. Experimental results show that sydnone and sydnonimine themselves have high mitochondrial distribution. However, the introduction of a phenyl group into the C4 position of sydnone dramatically decreases the mitochondrial affinity. In addition, we took advantage of mitochondrial targeting ability and click-and-release reaction of sydnonimine to evaluate anticancer activities of in-mitochondria delivery of celecoxib against HeLa and HepG2 cells, indicating that celecoxib-induced cancer cell death may not involve mitochondria-related pathway.

Sydnone: Synthesis, Reactivity and Biological Activities.

Sydnones are among the most well-known mesoionic compounds. Since their synthesis in 1935 by Earl and Mecknay, numerous researches have shown that the chemical behavior, physical and biological properties of sydnones make them the most useful compounds in organic chemistry. Sydnones undergo thermal 1,3-dipolar cycloaddition reaction with dipolarophiles (alkynes or alkenes) to give exclusively derivatives containing a pyrazole moiety exhibiting numerous applications, such as pharmaceuticals and agrochemicals. However, the sydnone cycloaddition reaction with alkynes requires harsh conditions, like high temperatures and long reaction times, giving poor regioselectivity to the resulting products. To overcome these constraints, new reactions named CuSAC (Copper- Catalyzed Sydnone-Alkyne Cycloaddition) and SPSAC (Strain-Promoted Sydnone- Alkyne Cycloaddition) have been developed, leading to pyrazoles with interesting constant kinetics.

A mechanochemical approach to the synthesis of sydnones and derivatives.

Sydnones are heterocyclic compounds which display important biological activities, including their abilities to react in 1,3-dipolar additions for applications in the development of new prodrugs. Capitalizing on our preliminary work on the mechanosynthesis of sydnones, an extension of this work to two related families of molecules, diarylsydnones and iminosydnones is reported. A ball-milling approach towards the synthesis of diaryl sydnones was developed, a necessary step for the synthesis of potential sydnone-based ligands of metal complexes. A mechanochemistry-based synthesis of iminosydnones was optimized, including the preparation of active pharmaceutical ingredients (API) related to feprosidnine, linsidomine, mesocarb and molsidomine. This work demonstrated that the ball-milling procedures were efficient and time saving through avoiding purification steps, and reduced the use of organic solvents.

Regioselective synthesis of 3-trifluoromethyl 1,2,4-triazoles via photocycloaddition of sydnone with CF3CN.

A general and regioselective synthesis of 3-trifluoromethyl 1,2,4-triazoles has been achieved through photocycloaddition of sydnone with trifluoroacetonitrile. This method employed trifluoroacetaldehyde O-(aryl)oxime as the CF3CN precursor and tolerated various functional groups to furnish 3-trifluoromethyl 1,2,4-triazole products in moderate to good yields. Mechanistic experiments revealed an energy transfer from photocatalyst 4-CzIPN to the sydnone substrates.

Study on Emission Characteristics and Emission Reduction Effect for Construction Machinery under Actual Operating Conditions Using a Portable Emission Measurement System (Pems).

With the acceleration of urban construction, the pollutant emission of non-road mobile machinery such as construction machinery is becoming more and more prominent. In this paper, a portable emissions measurement system (PEMS) tested the emissions of eight different types of construction machinery under actual operating conditions and was used for idling, walking, and working under the different emission reduction techniques. The results showed that the pollutant emission of construction machinery is affected by the pollutant contribution of working conditions. According to different emission reduction techniques, the diesel oxidation catalyst (DOC) can reduce carbon monoxide (CO) by 41.6-94.8% and hydrocarbon (HC) by 92.7-95.1%, catalytic diesel particulate filter (CDPF) can reduce particulate matter (PM) by 87.1-99.5%, and selective catalytic reduction (SCR) using urea as a reducing agent can reduce nitrogen oxides (NOx) by 60.3% to 80.5%. Copper-based SCR is better than vanadium-based SCR in NOx reduction. In addition, the study found that when the enhanced 3DOC + CDPF emission reduction technique is used on forklifts, DOC has a "low-temperature saturation effect", which will reduce the emission reduction effect of CO and THC. The use of Burner + DOC + CDPF emission reduction techniques and fuel injection heating process will increase CO's emission factors by 3.2-3.5 and 4.4-6.7 times compared with the actual operating conditions.

On Demand Flow Platform for the Generation of Anhydrous Dinitrogen Trioxide and Its Further Use in N-Nitrosative Reactions.

Dinitrogen trioxide (N2 O3 ) is a powerful and efficient nitrosating agent that comes with an unprecedented atom economy. However, the synthetic application of N2 O3 is still underdeveloped mostly due to its inherent instability and the lack of reliable protocols for its preparation. This paper presents an open-source setup and procedure for the on-demand generation of anhydrous N2 O3 solution (up to 1 M), which can be further used for reactions under batch and flow conditions. The accuracy and stability of N2 O3 concentration are guaranteed with the absence of head-space in the setup and with the synchronization of the gas flows. The reliability of this protocol is demonstrated by >30 worked examples in the nitrosative synthesis of heterocycles-a library of structurally diverse benzotriazoles and sydnones. Kinetic and mechanistic aspects of the N-nitrosative steps are also explored.

Antiproliferative, apoptosis-inducing activity and molecular docking studies of sydnones compounds.

Objective: To evaluate the antiproliferative and apoptosis inducing activity of different sydnones on cancer cell lines and their interaction with cancer proteins by molecular docking studies. Material and Methods: Antiproliferative activity was carried out by MTT assay and apoptosis inducing activity was performed by DAPI and Annexin V and propidium iodide staining. Molecular docking studies were performed using AutoDock Tools 1.5.6. Pharmacokinetics properties like ADME and toxicity were analysed by pkCSM web server. Result: In this study, four new sydnone compounds 3-(4-nonylbiphenyl-4'-yl) sydnone (MC-182), 3-(4-propylbiphenyl-4'-yl) sydnone (MC-454), 3-(4-hexylbiphenyl-4'-yl) sydnone (MC-433), and 3-(4-methylbiphenyl-4'-yl) sydnone (MC-431) were screened for antiproliferative and apoptotic effect against BT-474 (human breast cancer), HeLa (human cervical cancer) and Jurkat (human myeloid leukemia) Mostly, all the sydnone compounds exhibited decent antiproliferative effectiveness, but compound MC-431, MC-433, and MC-454 showed more antiproliferative activity (IC50 1.71, 10.09 and 2.87 µM against BT-474, Hela and Jurkat cell line, respectively). The changes of morphological characteristics of cancer cells determined by staining techniques indicate the apoptotic cell death. The molecular docking and interaction studies were carried out between sydnones with cancer proteins (epidermal growth factor domain receptor tyrosine kinase [EGF-TK], tumor necrosis factor-alpha [TNF-α] and Caspase3. Among all four sydnone molecules, two compounds MC-454 and MC-431 showed good binding energy with targeted proteins. Drug-like property was predicted by ADME toxicity study. Conclusion: The results indicate sydnone compounds were found to exhibit anticancer activity by inducing apoptosis. The molecular docking study of sydnones with cancer proteins showed a decent interaction affinity. The results of absorption, distribution, metabolism, excretion and toxicity studies by the Insilco approach also proved that MC-454 sydnone showed better In-Vivo administration. Thus, the current research work indicates that these sydnone compounds would be prospective in developing anticancer medicines.

Fast and Efficient Postsynthetic DNA Labeling in Cells by Means of Strain-Promoted Sydnone-Alkyne Cycloadditions.

Sydnones are highly stable mesoionic 1,3-dipoles that react with cyclooctynes through strain-promoted sydnone-alkyne cycloaddition (SPSAC). Although sydnones have been shown to be valuable bioorthogonal chemical reporters for the labeling of proteins and complex glycans, nucleic acids have not yet been tagged by SPSAC. Evaluation of SPSAC kinetics with model substrates showed fast reactions with cyclooctyne probes (up to k=0.59 M-1 s-1 ), and two different sydnones were effectively incorporated into both 2'-deoxyuridines at position 5, and 7-deaza-2'-deoxyadenosines at position 7. These modified nucleosides were synthetically incorporated into single-stranded DNAs, which were successfully postsynthetically labeled with cyclooctyne probes both in vitro and in cells. These results show that sydnones are versatile bioorthogonal tags and have the premise to become essential tools for tracking DNA and potentially RNA in living cells.

Novel trifluoromethyl sydnone derivatives: Design, synthesis and fungicidal activity.

Crop pathogens reduce the yield and quality of agricultural production. The development of new fungicides will help to sustain this protection and overcome fungicide resistance. Sydnone is a kind of mesoionic, which has a wide range of biological activities. The application of sydnones in agriculture is less, and the study of these compounds will lead to the discovery of new active compounds. In this study, we designed and synthesized a series of noval sydnone mesoionic derivatives by active substructure splicing. All compounds were characterized using 1H and 13C NMR spectroscopy. Among them, trifluoromethyl compound D17 showed good bioactivity against Pseudoperonospora cubensis (EC50 = 49 mg L-1) in vivo, the activity was similar to that of the control Kresoxim-methyl (EC50 = 44 mg L-1). However, the target of these compounds should not only be tyrosinase, and the mode of action needs to be further studied. In addition, the structure-activity relationship indicated that the trifluoromethyl group was more beneficial for antifungal activity. This is the first report that fluorine-containing N(3)-benzyl sydnone compounds have good fungicidal activity. These results will provide a basis for the development of sydnone mesoionic as new lead fungicidal agents.

Discovery of novel iminosydnone compounds with insecticidal activities based on the binding mode of triflumezopyrim.

Triflumezopyrim (TFM) is a new mesoionic insecticide developed by DuPont. Like other neonicotinoid insecticides, it binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR), but the binding mode has not been reported. Nicotinic acetylcholine binding proteins (nAChBPs) are ideal alternative structure for nAChRs. In this study, molecular docking, molecular dynamics (MD) simulations, binding free energy calculation, and per-residue binding free energy decomposition were used to study the binding modes of TFM and other 12 mesoionic insecticides. By comparing the binding free energy and the insecticidal activity, it was found that the sub-pocket around the benzyl group of the mesoionic insecticide is the key area for maintaining its activity, which is composed of A: Val116, A: Met124, A: Ile126, B: Trp155 and B: Val156. In order to verify the druggability of the sub-pocket, a series of iminosydnone compounds were designed and synthesized based on the structure of the sub-pocket. The lethality rate of compound 1 against Mythimna separata were 100% at 500 mg/L. Our research provides a basis for designing new mesoionic insecticides based on structure.

Fluorogenic iminosydnones: bioorthogonal tools for double turn-on click-and-release reactions.

In this article, we report the synthesis and use of iminosydnone-based profluorophores as bioorthogonal cleavable linkers for imaging applications. These linkers react with cycloalkynes via subsequent [3+2] cycloaddition and retro Diels-Alder reactions, allowing simultaneous release of two dyes in biological media.

Bioorthogonal Phosphorogenic Rhenium(I) Polypyridine Sydnone Complexes for Specific Lysosome Labeling.

Many novel bioorthogonal reactions have been developed for labeling, such as the strain-promoted sydnone-alkyne cycloaddition (SPSAC), but sydnone-based probes with phosphorogenicity (i. e., phosphorescence turn-on upon reaction) have not been investigated to date. Herein, we report the synthesis, characterization, and photophysical properties of rhenium(I) polypyridine complexes containing a sydnone moiety as bioorthogonal phosphorogenic probes. Their reactions with strained alkyne derivatives and the associated photophysical changes were examined. Upon SPSAC with bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN-OH), the complexes exhibited emission enhancement in the range of 8.8 to 17.3. Importantly, conjugation of the complexes with BCN-modified bovine serum albumin (BCN-BSA) led to the increase in emission enhancement to as high as 38.9 and extended lifetimes in the range of 1.80 to 4.71 µs. Additionally, the bioorthogonal ligation of one of the complexes with a morpholine derivative was shown to induce specific lysosomal labeling in live cells; colocalization studies with LysoTracker Deep Red indicated a Pearson's coefficient of 0.83.

Sydnone-based turn-on fluorogenic probes for no-wash protein labeling and in-cell imaging.

We report the synthesis and use of sydnone-based profluorophores as tools for imaging applications. These new probes display exquisite reactivity towards strain promoted cycloaddition reactions with cycloalkynes allowing fast, efficient and selective labeling in biological media. Styryl-pyridinium sydnone probes were found particularly interesting for click reactions to proceed selectively inside cells.

Selective Engineering of Linkage-Specific α2,6-N-Linked Sialoproteins Using Sydnone-Modified Sialic Acid Bioorthogonal Reporters.

The metabolic oligosaccharide engineering (MOE) strategy using unnatural sialic acids has recently enabled the visualization of the sialome in living systems. However, MOE only reports on global sialylation and dissected information regarding subsets of sialosides is missing. Described here is the synthesis and utilization of sialic acids modified with a sydnone reporter for the metabolic labeling of sialoconjugates. The positioning of the reporter on the sugar significantly altered its metabolic fate. Further in vitro enzymatic assays revealed that the 9-modified neuraminic acid is preferentially accepted by the sialyltransferase ST6Gal-I over ST3Gal-IV, leading to the favored incorporation of the reporter into linkage-specific α2,6-N-linked sialoproteins. This sydnone sugar presents the possibility of investigating the roles of specific sialosides.

Sydnone-Based Approach to Heterohelicenes through 1,3-Dipolar-Cycloadditions.

The first approach to pyrazole-containing helicenes via sydnone-aryne [3 + 2]-cycloaddition is described. An unprecedented regioselectivity in the cycloaddition step toward the more sterically constrained product was observed in the presence of extended aromatic scaffolds. DFT calculations enabled understanding the origin of this unexpected selectivity.

Bioorthogonal release of sulfonamides and mutually orthogonal liberation of two drugs.

Sulfonamide derivatives have been used in pharmaceutics for decades. Here we report a new approach to release sulfonamides efficiently using a bioorthogonal reaction of sulfonyl sydnonimines and dibenzoazacyclooctyne (DIBAC). The second-order rate constant of the cycloaddition reaction can be up to 0.62 M-1 s-1, and the reactants are highly stable under physiological conditions. Most significantly, we also discovered the mutual orthogonality between the sydnonimine-DIBAC and benzonorbornadiene-tetrazine cycloaddition pairs, which can be used for selective and simultaneous liberation of sulfonamide and primary amine drugs.

Evaluation of Sydnone-Based Analogues of Combretastatin A-4 Phosphate (CA4P) as Vascular Disrupting Agents for Use in Cancer Therapy.

The combretastatins have attracted significant interest as small-molecule therapies for cancer due to their ability to function as vascular disrupting agents. We have successfully prepared a range of combretastatin analogues that are based on a novel sydnone heterocycle core, and their potential as tubulin binders has been assessed in vitro and in vivo. The most potent candidate was found to disrupt microtubules and affect cellular morphology at sub-micromolar levels. Moreover, it was found to bind reversibly to tubulin and significantly increase endothelial cell monolayer permeability, in a similar manner to combretastatin A4. Surprisingly, the compound did not exhibit efficacy in vivo, possibly due to rapid metabolism.

Sydnone-alkyne cycloaddition: applications in synthesis and bioconjugation.

Sydnones are among the most popular mesoionic compounds studied so far for cycloaddition reactions. However, despite their good chemical stability and versatility, only a limited number of research groups have worked on their chemistry and use in organic synthesis. This feature article aims at providing an overview of the most recent developments in sydnone-alkyne cycloadditions, with particular attention on the strategies that allow us to achieving high regiocontrol and milder reaction conditions. The recent discovery that this dipole is able to undergo click and biorthogonal reactions with cycloalkynes may stimulate renewed interest from the scientific community. Given the high potential and flexibility of this family of mesoionics, we believe that major developments are to be expected both in terms of organic synthetic methodologies and biorthogonal chemistry applications in the field of chemical biology.