Multistep Cascade Reactions Research Articles

Synthesis of 6/5/3-Fused Tricyclic Scaffolds via Multistep Cascade Cyclization of α-Aryl Vinylsulfoniums with para-Quinamines and para-Quinols.

Herein, we present a straightforward approach to access hydroindoline-5-one-based 6/5/3-fused polycyclic ring structures through multistep cascade reactions involving α-aryl vinylsulfoniums and para-quinamines. The reactions proceed smoothly under mild conditions to deliver the desired products in generally good isolated yields. This protocol is also applicable to the cascade cycloaddition reactions of α-aryl vinylsulfoniums and para-quinols, effectively generating complex tricyclic scaffolds. In addition, the scale-up synthesis and further derivatizations demonstrate the potential synthetic application of the protocol.

Design and one-pot synthesis of new substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine as potential antitumor agents: in vitro and in vivo studies

A new efficient and versatile one-pot three-component synthesis of substituted pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives has been developed. It is based on a multistep cascade reaction from 2-aminothiophenes and 2-hydroxy-4-oxobut-2-enoic acids, and derivatives of cyanoacetic acid catalyzed by diisopropylethylamine. As a result, novel pyrrolo[1,2-a]thieno[3,2-e]pyrimidine derivatives (21 compounds) were synthesized in a mild reaction conditions with a high yield. The structures of the developed compounds were confirmed by NMR and elemental analysis. The influence of electron-withdrawing or electron-donor substituents on the antitumor activity of the developed compounds has been identified. In vitro screening analysis of 21 compounds revealed six lead candidates (12aa, 12dc, 12hc, 12ic, 12lb, and 12mb) that demonstrated the most significant antitumor activity against B16-F10, 4T1 and CT26 cells. Necrosis/apoptosis assay showed that apoptosis was the predominant mechanism of cell death. Molecular docking analysis revealed several potential targets for tested compounds, i.e. phosphatidylinositol 5-phosphate 4-kinase (PI5P4K2C), proto-oncogene serine/threonine-protein kinase (Pim-1), nicotinamide phosphoribosyltransferase (NAMPT) and dihydrofolate reductase (DHFR). The lead compound (12aa) can effectively induce cell apoptosis, possesses a high yield (98 %) and requires low-cost starting chemicals for its synthesis. In vivo experiments with melanoma-bearing mice confirmed that 12aa compound resulted in the significant tumor inhibition on 15 d after the therapy. In particular, tumor volume was ∼0.19 cm3 for 50 mg/kg versus ∼2.39 cm3 in case of untreated mice and tumor weight was ∼71.6 mg for 50 mg/kg versus ∼452.4 mg when considered untreated mice. Thus, our results demonstrated the high potential of the 12aa compound in the treatment of melanoma and can be recommended for further preclinical studies.

“Triple-Hop” nano-bomb combining CDT, PDT and immunetherapy for NIR-triggered cancer therapy

The development of non-interventional combination therapy with immune activation property provides a potential direction for solving the diversity, complexity and heterogeneity of malignant tumors. However, how to induce satisfactory immune activation by non-interventional treatment remains a great challenge. Herein, a “Triple-Hop” nano-bomb UCNPs@SiO2-psoralen-K2FeO4@lauric acid-RGD (USPFLR), was prepared and “detonated” after three steps. For step one, with NIR light as the “ignition” signal, USPFLR promoted the tumor to reach 40℃ and the phase transition of Lauric acid (LA) induced the release of K2FeO4 and psoralen, triggering H2O2/GSH depletion and ·OH /O2 production through multi-step cascade reactions of K2FeO4 and psoralen-involved photodynamic therapy (PDT). For step two, high ROS release and direct DNA damage caused by psoralen further significantly triggered ferroptosis pathway and cGAS-STING pathway, which promoted the maturation of dendritic cells (DCs) and induced immune activation and immune memory in the third step. Therefore, through the first step of O2 supply, GSH consumption and ROS generation, the second step of the activation of the ferroptosis pathway and cGAS-STING pathway and the third step of immune activation and immune memory, USPFLR nano-bombs successfully realized the “Triple-Hop” anti-tumor strategy, providing a referential application paradigm for tumor eradication and prevention of tumor recurrence.

Membrane and Lumen-Compartmentalized Polymersomes for Biocatalysis and Cell Mimics.

Compartmentalization is a crucial feature of a natural cell, manifested in cell membrane and inner lumen. Inspired by the cellular structure, multicompartment polymersomes (MCPs), including membrane-compartmentalized polymersomes and lumen-compartmentalized polymersomes (polymersomes-in-polymersomes), have aroused great expectations for biological applications such as biocatalysis and cell mimics in the past decades. Compared with traditional polymersomes, MCPs have advantages in encapsulating multiple enzymes separately for multistep enzymatic cascade reactions. In this review, first, the design principles and preparation methods of membrane-compartmentalized and lumen-compartmentalized polymersomes are summarized. Next, recent advances of MCPs as nanoreactors and cell mimics to mimic subcellular organelles or artificial cells are discussed. Finally, the future research directions of MCPs are prospected.

Novel co-immobilization of thermostable isomerase and aspartase on NH2-UiO66 for efficient biosynthesis of L-aspartate from maleate

L-aspartate is an important chemical in the food and pharmaceutical industries. Presently, it is meaningful to realize a co-immobilized multi-enzyme biocatalyst that can synthesize L-aspartate in vitro through multi-step cascade reaction. Herein, the immobilization method was first employed to investigate the ability of immobilized double enzymes, isomerase AaMaiA and aspartase AspB, to systhesize L-aspartate. The optimal temperature (50 ℃) of AaMaiA-immobilized NH2-UiO66, a metal organic framework material, was significantly superior to that of its free counterpart. Compared to free enzymes, AaMaiA@NH2-UiO66 retained 57.8% of enzyme activity and exhibited a half-life that was improved 2.59-fold at 50 ℃. The co-immobilized AaMaiA/AspB@NH2-UiO66 (AaMaiA: AspB = 6: 1, v/v) showed high protein loading (27.899 ± 1.945 mg/g) and 84.4% recovery of enzyme activity after 8 consecutive cycles. Compared to free AaMaiA/AspB, the novel AaMaiA/AspB@NH2-UiO66 exhibited far superior tolerance to organic solvents. For the first time, the prepared AaMaiA/AspB@NH2-UiO66 was successfully applied as an efficient biocatalyst and converted 1 M maleate to L-aspartate with a yield of 86.8% within 1 h. Remarkably, AaMaiA/AspB@NH2-UiO66 exhibited great potential for efficient biosynthesis of L-aspartate.

Multistep Cascade Catalytic Reactions Employing Bifunctional Framework Compounds.

Multistep cascade reactions are important to achieve atom as well as step economy over conventional synthesis. This approach, however, is limited due to the incompatibility of the available reactive centers in a catalyst. In the present study, new MOF compounds, [Zn2(SDBA)(3-ATZ)2]·solvent, I and II, with tetrahedral Zn centers as good Lewis acidic sites and the free amino group of the 3-amino triazole ligand as a strong Lewis base center were shown to perform 4-step cascade/tandem reaction in a facile manner. Effective conversion of benzaldehyde dimethyl acetal in the presence of excess nitromethane at 100 °C in water to 1-(1,3-dinitropropan-2-yl) benzene was achieved in 10 h with yields of ∼95% (I) and ∼94% (II). This 4-step cascade reaction proceeds via deacetalization (Lewis acid), Henry (Lewis base), and Michael (Lewis base) reactions. The present work highlights the importance of spatially separated functional groups in multistep tandem catalysis─the examples of which are not common.

Real time optical monitoring of cascade mechanochemical reactions and capture of ultra-unstable intermediates under hydrostatic pressure

The high pressure induced physical and chemical processes of two spiropyran derivatives are optically monitored with diamond anvil cell techniques, in which emission enhancement and multi-step cascade reactions are disclosed.

NADP(H)-dependent biocatalysis without adding NADP(H)

Isocitrate dehydrogenase 1 (IDH1) naturally copurifies and crystallizes in a resting state with a molecule of its exchangeable cofactor, NADP+/NADPH, bound in each monomer of the homodimer. We report electrochemical studies with IDH1 that exploit this property to reveal the massive advantage of nanoconfinement to increase the efficiency of multistep enzyme-catalyzed cascade reactions. When coloaded with ferredoxin NADP+ reductase in a nanoporous conducting indium tin oxide film, IDH1 carries out the complete electrochemical oxidation of 6 mM isocitrate (in 4mL) to 2-oxoglutarate (2OG), using only the NADP(H) that copurified with IDH1 and was carried into the electrode pores as cargo-the system remains active for days. The entrapped cofactor, now quantifiable by cyclic voltammetry, undergoes ~160,000 turnovers during the process. The results from a variety of electrocatalysis experiments imply that the local concentrations of the two nanoconfined enzymes lie around the millimolar range. The combination of crowding and entrapment results in a 102 to 103-fold increase in the efficiency of NADP(H) redox cycling. The ability of the method to drive cascade catalysis in either direction (oxidation or reduction) and remove and replace substrates was exploited to study redox-state dependent differences in cofactor binding between wild-type IDH1 and the cancer-linked R132H variant that catalyzes the "gain of function" reduction of 2OG to 2-hydroxyglutarate instead of isocitrate oxidation. The combined results demonstrate the power of nanoconfinement for facilitating multistep enzyme catalysis (in this case energized and verified electrochemically) and reveal insights into the dynamic role of nicotinamide cofactors as redox (hydride) carriers.

POM-mediated MOFs-based probes facilitate multistep cascade reaction for ultrasensitive detection of tumor marker

Metal-organic frameworks (MOFs) are often used as carriers for catalytically active substances and play the role of signal amplifiers in electrochemical immunosensors. However, catalytically active species often form aggregates in MOFs, resulting in negative effects such as reduced specific surface area and reduced catalytic activity. Here, we use the spatial confinement of zeolitic imidazolate framework-8 (ZIF-8) to anchor polyoxometalate (POM), and cooperate with xanthine oxidase (XOD) to achieve multistep cascade amplification of electrochemical signals. In the presence of the substrate hypoxanthine, XOD catalyzes the production of H2O2, which is mediated by POM to amplify the electrochemical signal of thionine. The proposed immunosensor of cytokeratin 19 fragments 21–1 exhibited a wide detection range (100 fg mL-1 to 1 μg mL-1) and an ultralow detection limit of 44.92 fg mL-1. Our work provides the pathway for the gentle preparation of probes with high catalytic site dispersion.

Cascades of Evolved Enzymes for the Synthesis of Complex Molecules

Thanks to advances in enzyme discovery and protein engineering combined with the development of enzymatic multistep reaction cascades, new efficient routes for drug synthesis have been created that are superior to chemical syntheses. This supports the goal of the chemical and pharmaceutical industries to move to more sustainable and environmentally friendly processes. Recently described outstanding examples include the biocatalytic cascade syntheses of the cyclic dinucleotide MK-1454, molnupiravir, and islatravir, as well as the efficient fixation of CO2 to make starch using an artificial enzyme cascade.

Solid acid-catalyzed one-pot multi-step cascade reaction: Multicomponent synthesis of indol-3-yl acetates and indol-3-yl acetamides in water

A solid acid-catalyzed cascade reaction of condensation/intermolecular nucleophilic addition/intramolecular cyclization, esterification and amination has been developed to rapidly construct complex polysubstituted indole compounds. This one-pot, three-step five-component cascade reaction affords a series of 2-(4-oxo-1,2-diphenyl-4,5,6,7-tetrahydro-1H-indol-3-yl) acetamide derivatives with good yield under mild conditions. The present procedure exhibited some attractive features, including inexpensive and easy available reagents, large substrate scope, separable intermediate products, recyclable solid acid catalyst, and more importantly, it can be conveniently performed by simple one-pot three-step procedure, involving the sequential mixture of 3-(arylamino)cyclohex-2-en-1-ones, 2,2-dihydroxy-1-arylethan-1-ones, meldrum acid, and alcohols, anilines in the of solid acid at 80 °C. Significantly, this strategy not only expands the application range of solid acid, but also provides a series of 2-(4-oxo-1,2-diphenyl-4,5,6,7-tetrahydro-1H-indol-3-yl)acetate/acetamide derivatives.

Catalytic asymmetric transformation of nitrones and allenes to dihydropyridoindoles via chiral N,N′-dioxide/cobalt(ii) catalysis

A dia- and enantioselective multistep cascade reaction of nitrones with allenes to chiral dihydropyridoindoles promoted by a chiral N,N′-dioxide/Co(ii) complex was described.

A Four-enzyme Nanoassembly Consisting of Hydrolases and Oxidoreductases for Multi-step Cascade Reactions.

Cascade reactions catalyzed by multi-enzymatic systems have attracted enormous scientific interest over the last decade. They are an emerging technology that significantly expands the applicability of biocatalysts in several biotechnological processes, such as the synthesis of high value-added products. Immobilization of enzymes on a solid carrier is a commonly used strategy to improve the stability and reuse of multiple enzyme systems. Magnetic nanoparticles have been applied as promising nanocarriers for either the immobilization of one enzyme or the co-immobilization of multiple enzymes. In this chapter, we describe the preparation of magnetic iron oxide nanoparticles γ-Fe2O3 modified with 3-(aminopropyl)-triethoxysilane (APTES), for the simultaneous covalent co-immobilization of oxidoreductases and hydrolytic enzymes, such as cellulase, β-glucosidase (bgl), glucose oxidase (GOx), and horseradish peroxidase (HRP). Several spectroscopic techniques that are used to characterize the structure and the catalytic performance of such systems are also described.

Chemo-, Regio-, and Stereoselective Assembly of Polysubstituted Furan-2(5H)-ones Enabled by Rh(III)-Catalyzed Domino C–H Alkenylation/Directing Group Migration/Lactonization: A Combined Experimental and Computational Study

Exploring multistep cascade reactions triggered by C–H activation are recognized as appealing, yet challenging. Herein, we disclose a Rh(III)-catalyzed domino C–H coupling of N-carbamoyl indoles an...

Capillary-Bound Dense Micelle Brush Supports for Continuous Flow Catalysis.

Flow reactors are appealing alternatives to conventional batch reactors for heterogeneous catalysis. However, it remains a key challenge to firmly immobilize the catalysts in a facile and flexible manner and to simultaneously maintain a high catalytic efficiency and throughput. Herein, we introduce a dense cylindrical micelle brush support in glass capillary flow reactors through a living crystallization-driven self-assembly process initiated by pre-immobilized short micelle seeds. The active hairy corona of these micellar brushes allows the flexible decoration of a diverse array of nanocatalysts, either through a direct capture process or an in situ growth method. The resulting flow reactors reveal excellent catalytic efficiency for a broad range of frequently utilized transformations, including organic reductions, Suzuki couplings, photolytic degradations, and multistep cascade reactions, and the system was both recyclable and durable. Significantly, this approach is readily applicable to long capillaries, which enables the construction of flow reactors with remarkably higher throughput.

Capillary‐Bound Dense Micelle Brush Supports for Continuous Flow Catalysis

AbstractFlow reactors are appealing alternatives to conventional batch reactors for heterogeneous catalysis. However, it remains a key challenge to firmly immobilize the catalysts in a facile and flexible manner and to simultaneously maintain a high catalytic efficiency and throughput. Herein, we introduce a dense cylindrical micelle brush support in glass capillary flow reactors through a living crystallization‐driven self‐assembly process initiated by pre‐immobilized short micelle seeds. The active hairy corona of these micellar brushes allows the flexible decoration of a diverse array of nanocatalysts, either through a direct capture process or an in situ growth method. The resulting flow reactors reveal excellent catalytic efficiency for a broad range of frequently utilized transformations, including organic reductions, Suzuki couplings, photolytic degradations, and multistep cascade reactions, and the system was both recyclable and durable. Significantly, this approach is readily applicable to long capillaries, which enables the construction of flow reactors with remarkably higher throughput.

Two (Chemo)-Enzymatic Cascades for the Production of Opposite Enantiomers of Chiral Azidoalcohols

Multi-step cascade reactions have gained increasing attention in the biocatalysis field in recent years. In particular, multi-enzymatic cascades can achieve high molecular complexity without workup of reaction intermediates thanks to the enzymes’ intrinsic selectivity; and where enzymes fall short, organo- or metal catalysts can further expand the range of possible synthetic routes. Here, we present two enantiocomplementary (chemo)-enzymatic cascades composed of either a styrene monooxygenase (StyAB) or the Shi epoxidation catalyst for enantioselective alkene epoxidation in the first step, coupled with a halohydrin dehalogenase (HHDH)-catalysed regioselective epoxide ring opening in the second step for the synthesis of chiral aliphatic non-terminal azidoalcohols. Through the controlled formation of two new stereocenters, corresponding azidoalcohol products could be obtained with high regioselectivity and excellent enantioselectivity (99% ee) in the StyAB-HHDH cascade, while product enantiomeric excesses in the Shi-HHDH cascade ranged between 56 and 61%.

The power of electrified nanoconfinement for energising, controlling and observing long enzyme cascades

Multistep enzyme-catalyzed cascade reactions are highly efficient in nature due to the confinement and concentration of the enzymes within nanocompartments. In this way, rates are exceptionally high, and loss of intermediates minimised. Similarly, extended enzyme cascades trapped and crowded within the nanoconfined environment of a porous conducting metal oxide electrode material form the basis of a powerful way to study and exploit myriad complex biocatalytic reactions and pathways. One of the confined enzymes, ferredoxin-NADP+ reductase, serves as a transducer, rapidly and reversibly recycling nicotinamide cofactors electrochemically for immediate delivery to the next enzyme along the chain, thereby making it possible to energize, control and observe extended cascade reactions driven in either direction depending on the electrode potential that is applied. Here we show as proof of concept the synthesis of aspartic acid from pyruvic acid or its reverse oxidative decarboxylation/deamination, involving five nanoconfined enzymes.

Cascade communication in disordered networks of enzyme-loaded microdroplets.

A network of aqueous emulsion droplets that exhibits programmed and directional chemical inter-droplet communication is described. A non-reciprocal transfer of substrates between enzyme-containing aqueous emulsion droplets is realized by (biochemically) induced osmolarity gradients and concomitant concentration gradients are used to direct a multistep enzymatic cascade reaction across multiple droplets.

Microfluidic platform enables tailored translocation and reaction cascades in nanoliter droplet networks

In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our platform opens doors for creating complex artificial systems for bottom-up synthetic biology.