Multistep Cascade Research Articles

Confinement and Diffusion of Small Molecules in a Molecular-Scale Tunnel

Multi-step reaction cascades can be designed to include channeling mechanisms, which provide electrostatic or steric control over intermediate transport such that intermediates do not escape to the bulk between active sites. Physical confinement of the intermediate pathway between sites retains intermediate from bulk access and thus provides high transport efficiency. In this work, we use molecular dynamics to study the transport of intermediates (charged oxalate and neutral ethanol) inside a nanochannel represented by a single-walled carbon nanotube (SWCNT). This approach reveals that solvent orientation highly impacts intermediate transport. At small nanochannel diameter near 1 nm, highly structured solvent water and Knudsen diffusion decreases effective intermediate diffusivity. Finally, modified SWCNT termini with electrostatically-charged carboxylate groups are shown to increase intermediate retention for both charged and uncharged intermediates by up to five-fold. When catalyst sites are located within the nanochannel, decreased diffusion rate and increased retention time will enhance cascade efficiency.

Hollow zeolite microspheres as a nest for enzymes: a new route to hybrid heterogeneous catalysts.

In the field of heterogeneous catalysis, the successful integration of enzymes and inorganic catalysts could pave the way to multifunctional materials which are able to perform advanced cascade reactions. However, such combination is not straightforward, for example in the case of zeolite catalysts for which enzyme immobilization is restricted to the external surface. Herein, this challenge is overcome by developing a new kind of hybrid catalyst based on hollow zeolite microspheres obtained by the aerosol-assisted assembly of zeolite nanocrystals. The latter spheres possess open entry-ways for enzymes, which are then loaded and cross-linked to form cross-linked enzyme aggregates (CLEAs), securing their entrapment. This controlled design allows the combination of all the decisive features of the zeolite with a high enzyme loading. A chemo-enzymatic reaction is demonstrated, where the structured zeolite material is used both as a nest for the enzyme and as an efficient inorganic catalyst. Glucose oxidase (GOx) ensures the in situ production of H2O2 subsequently utilized by the TS-1 zeolite to catalyze the epoxidation of allylic alcohol toward glycidol. The strategy can also be used to entrap other enzymes or combination of enzymes, as demonstrated here with combi-CLEAs of horseradish peroxidase (HRP) and glucose oxidase. We anticipate that this strategy will open up new perspectives, leveraging on the spray-drying (aerosol) technique to shape microparticles from various nano-building blocks and on the entrapment of biological macromolecules to obtain new multifunctional hybrid microstructures.

Design of an in vitro biocatalytic cascade for the manufacture of islatravir.

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

Maximal efficiency from enzyme cascades

Biocatalysis Enzymes are highly selective catalysts that can be useful for specific transformations in organic synthesis. Huffman et al. combined designer enzymes in a multistep cascade reaction (see the Perspective by O'Reilly and Ryan). The approach eliminates purification steps, recycles expensive cofactors, and couples favorable and unfavorable reactions. With the target molecule islatravir, an experimental HIV drug, they optimized five enzymes by directed evolution to be compatible with unnatural substrates and stable in the reaction conditions. Stereochemical purity was amplified at every enzymatic step, and the final synthesis was both atom economical and efficient. Science , this issue p. [1255][1]; see also p. [1199][2] [1]: /lookup/doi/10.1126/science.aay8484 [2]: /lookup/doi/10.1126/science.aaz7376

Development of a multistep reaction cascade for the synthesis of a sacubitril precursor in continuous flow

The active pharmaceutical ingredient sacubitril acts as a neprilysin inhibitor in the body and is administered to patients suffering from high blood pressure and chronic heart failure. In this paper, we report the development of a three-step setup for the synthesis of an advanced sacubitril precursor in continuous flow. The key transformation of our cascade is a Suzuki-Miyaura coupling facilitated by a heterogeneous palladium catalyst. Its implementation in a packed-bed reactor and the application of continuous flow methodologies allow intensification of the cross-coupling reaction compared to batch processing. The subsequent steps for the synthesis of the target molecule involve Boc-deprotection as well as N-succinylation, which have been optimized using the statistical “Design of Experiments” (DoE) approach. In this way, the individual as well as interactive effects of selected parameters on the output of the reactions could be investigated very efficiently. The consecutive performance of the three reaction steps using an integrated setup enabled the synthesis of a late-stage sacubitril precursor in continuous flow with 81% overall yield.Graphical .

DNA Release from a Modified Electrode Triggered by a Bioelectrocatalytic Process.

DNA release from an electrode surface was stimulated by application of a mild electrical potential (0 V vs Ag/AgCl). The release process was activated by interfacial pH increase originating from H+ consumption during O2 reduction bio-electrocatalyzed by bilirubin oxidase immobilized at the electrode surface. The pH increase resulted in a change of the electrical charge from positive to negative at the surface of SiO2 nanoparticles (200 nm) associated with the electrode surface and functionalized with trigonelline and boronic acid. While the negatively charged DNA molecules were electrostatically bound to the positively charged surface, the negative charge produced upon O2 reduction resulted in the DNA repulsion and release from the modified interface. The small electrical potential for O2 reduction resulting in the interface recharge was allowed due to the bio-electrocatalysis using bilirubin oxidase enzyme. While, in the first set of experiments, the potential was applied on the modified electrode from an electrochemical instrument, later it was generated in situ by biocatalytic or photo-biocatalytic processes at a connected electrode. A multistep biocatalytic cascade generating NADH or photosynthetic process in thylakoid membranes was used to produce in situ a small potential to stimulate the DNA release catalyzed by bilirubin oxidase. The designed system can be used for different release processes triggered by various signals (electrical, biomolecular, and light signals, etc.), thus representing a general interfacial platform for the controlled release of different biomolecules and nanosize species.

Development of a Four-Enzyme Magnetic Nanobiocatalyst for Multi-Step Cascade Reactions

We report the preparation, characterization and application of a novel magnetic four-enzyme nanobiocatalyst prepared by the simultaneous covalent co-immobilization of cellulase (CelDZ1), β-glucosidase (bgl), glucose oxidase (GOx) and horseradish peroxidase (HRP) onto the surface of amino-functionalized magnetic nanoparticles (MNPs). This nanobiocatalyst was characterized by various spectroscopic techniques. The co-immobilization process yielded maximum recovered enzymatic activity (CelDZ1: 42%, bgl: 66%, GOx: 94% and HRP: 78%) at a 10% v/v cross-linker concentration, after 2 h incubation time and at 1:1 mass ratio of MNPs to total enzyme content. The immobilization process leads to an increase of Km and a decrease of Vmax values of co-immobilized enzymes. The thermal stability studies of the co-immobilized enzymes indicated up to 2-fold increase in half-life time constants and up to 1.5-fold increase in their deactivation energies compared to the native enzymes. The enhanced thermodynamic parameters of the four-enzyme co-immobilized MNPs also suggested increment in their thermal stability. Furthermore, the co-immobilized enzymes retained a significant part of their activity (up to 50%) after 5 reaction cycles at 50 °C and remained active even after 24 d of incubation at 5 °C. The nanobiocatalyst was successfully applied in a four-step cascade reaction involving the hydrolysis of cellulose.

Microbial synthesis of the type I polyketide 6-methylsalicylate with Corynebacterium glutamicum.

Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4'-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered Corynebacterium glutamicum strain as a novel microbial cell factory for type I PKS-derived products. Suitability of C. glutamicum for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from Streptomyces antibioticus. Challenges related to protein folding could be overcome by translation fusion of ChlB1Sa to the C-terminus of the maltose-binding protein MalE from Escherichia coli. Surprisingly, ChlB1Sa was also active in the absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptACg of C. glutamicum can also activate ChlB1Sa. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41mg/L (0.27mM) 6-methylsalicylic acid within 48h of cultivation. Further experiments showed that PptACg of C. glutamicum can also activate nonribosomal peptide synthetases (NRPSs), rendering C. glutamicum a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.

CeO2 -Encapsulated Hollow Ag-Au Nanocage Hybrid Nanostructures as High-Performance Catalysts for Cascade Reactions.

Inspired by bio-enzymes, multistep cascade reactions are highly attractive in catalysis. Despite extensive research in recent years, it remains a challenge to promote the stability and activity of catalysts. Here, well-defined core-shell structured Ag-Au nanocage@CeO2 (Ag-Au NC@CeO2 ) are designed by a simple and facile self-assembly method. The results indicate that the Ag-Au NC@CeO2 has glucose oxidase-like activity and intrinsic peroxidase-like activity at the same time. As expected, Ag-Au NC@CeO2 hybrid nanomaterials exhibit cascade reactions activity. Moreover, the hybrid materials are promising to detect glucose without bio-enzymes. This research has potential applications in biomedicine and biomimetic catalysis.

Nano-Scale Confinement for Efficient Multi-Step Reaction Cascades

Strategic operation of multi-step reaction cascades for energy conversion/storage applications such as biofuel cells is a necessary step to increase process efficiency [1]. Beyond maximizing catalytic activity, efficient transport of reaction intermediates between active sites with minimal diffusion loss into the bulk is critical. Therefore, implementation of intermediate channeling mechanisms, many of which exist in nature, to multi-step energy cascades is a critical aspect of an overall design strategy. Molecular tunneling is one mechanism of substrate channeling that minimizes intermediate diffusional losses into the bulk by confining intermediate pathway [2].In this work, we studied intermediate transport between two active sites confined by single-walled carbon nanotube (SWCNT) using continuum and molecular dynamics (MD) approaches [3]. MD was performed using model intermediates (oxalate and ethanol) inside SWCNTs with diameter 1 nm to 4 nm. Due to water molecule orientation inside the SWCNT, the intermediates experienced a constrained environment that resulted in Knudsen diffusion and decreased diffusion coefficient [4]. In order to increase intermediate residence time inside the nanotube, SWCNT termini were modified with varying numbers of negatively-charged carboxylate groups. Due to attractive-repulsive electrostatic interactions, ethanol, which is uncharged but polar, was retained for a longer time inside the SWCNT. Like-charged intermediates such as oxalate were less affected. This approach represents one possible technique to enhance confinement and therefore increase reaction efficiency.

Cascade Reactions in Nanozymes: Spatially Separated Active Sites inside Ag-Core-Porous-Cu-Shell Nanoparticles for Multistep Carbon Dioxide Reduction to Higher Organic Molecules.

Enzymes can perform complex multistep cascade reactions by linking multiple distinct catalytic sites via substrate channeling. We mimic this feature in a generalized approach with an electrocatalytic nanoparticle for the carbon dioxide reduction reaction comprising a Ag core surrounded by a porous Cu shell, providing different active sites in nanoconfined volumes. The architecture of the nanozyme provides the basis for a cascade reaction, which promotes C–C coupling reactions. The first step occurs on the Ag core, and the subsequent steps on the porous copper shell, where a sufficiently high CO concentration due to the nanoconfinement facilitates C–C bond formation. The architecture yields the formation of n-propanol and propionaldehyde at potentials as low as −0.6 V vs RHE.

Activation of the extracytoplasmic function σ factor σV by lysozyme.

σV is an extracytoplasmic function (ECF) σ factor that is found exclusively in Firmicutes including Bacillus subtilis and the opportunistic pathogens Clostridioides difficile and Enterococcus faecalis. σV is activated by lysozyme and is required for lysozyme resistance. The activity of σV is normally inhibited by the anti-σ factor RsiV, a transmembrane protein. RsiV acts as a receptor for lysozyme. The binding of lysozyme to RsiV triggers a signal transduction cascade which results in degradation of RsiV and activation of σV . Like the anti-σ factors for several other ECF σ factors, RsiV is degraded by a multistep proteolytic cascade that is regulated at the step of site-1 cleavage. Unlike other anti-σ factors, site-1 cleavage of RsiV is not dependent upon a site-1 protease whose activity is regulated. Instead constitutively active signal peptidase cleaves RsiV at site-1 in a lysozyme-dependent manner. The activation of σV leads to the transcription of genes, which encode proteins required for lysozyme resistance.

Mobility of Intermediates in Highly Channeled Reaction Cascades

Novel and energy systems and synthetic processes can benefit from multistep chemical cascades, which allow combination of multiple reaction in a single overall process. Such cascades are ubiquitous in nature, and have been applied to of biosensors and biofuel cell designs as well. But, the efficiency of cascade reactions is significantly limited by the transport of intermediates. Intermediate channeling phenomenon is found to be one of the solutions to this problem in nature, and can include confinement, electrostatic interactions, and other mechanisms. Here we describe the quantitative tools we have developed to describe these channelling systems quantitatively, and to interrogate them experimentally. We specifically apply analysis techniques for infrequent events such as occur with tightly bonded intermediates to probe the tradeoff between retention of intermediates near or within catalytic complexes, and the reduction of mobility such retention entails. We show that because of the very short time scales of transport as compared to reaction, strong retention of intermediates is advantageous even if such retention dramatically reduces intermediate mobility.

Bioinspired Architecture of a Hybrid Bifunctional Enzymatic/Organic Electrocatalyst for Complete Ethanol Oxidation

Enzymatic biofuel cells utilize enzymes to electrochemically catalyze the anodic oxidation of a fuel or the cathodic reduction of an oxidant due the high volumetric catalytic activity towards oxidation of biofuels, providing clean and renewable energy, besides presenting great potential as alternative power sources for low-power electronic devices. One way to amplify the electrocatalytic activity of the biosystem and also extend its service life is mix the proper enzyme with organic oxidation catalysts. This hybrid system enables improvement at the rate of electrocatalytic oxidation of several biofuels[1-3].Herein, we describe the development of a hybrid catalytic architecture combining OxOx with a recently developed TEMPO-modified linear poly(ethylenimine) (TEMPO-LPEI) coupled with carboxylated multi-walled carbon nanotubes (MWCNT-COOH) to transform ethanol into CO2, extracting 12 electrons per ethanol molecule at a single bioanode. We identify and quantify the products from electrochemical ethanol oxidation by high performance liquid chromatography (HPLC). The electrochemical experiments were carried out using an AUTOLAB potentiostat / galvanostat (Software NOVA 1.11) with a three-electrode standard cell. The cascade oxidation of ethanol was performed by the chronoamperometric technique (electrolysis) with applied potential of 0.600 V and analysis time of 43,200 s (12 hours). The products obtained after 12 hours of electrolysis were analyzed by HPLC-UV / RID in Full-Scan mode. On the basis of chromatographic results, the catalytic hybrid electrode system completely oxidized ethanol to CO2 after 12 h of electrolysis. The fact that the developed system can catalyze ethanol electrooxidation at a carbon electrode confirms that organic oxidation catalysts combined with enzymatic catalysts allow up to 12 electrons to be collected per fuel molecule. The Faradaic efficiency of the hybrid system investigated herein lies above 85%. Complete ethanol electrooxidation was achieved by using a hybrid bifunctional enzymatic/organic electrocatalyst, MWCNT-COOH/TEMPO-LPEI/OxOx, This new hybrid system represents a good alternative as compared to previous systems [1], not to mention its excellent selectivity, the high yields of CO2 as the final product, and the high electrochemical oxidation rates. This system also represents an example of a hybrid multifunctional catalyst that may be valuable and open up a number of exciting opportunities to fabricate new hybrid bioanode architectures as energy conversion/storage devices and also as part of multistep reaction cascade systems.

Preparation and evaluation of porous starch/chitosan composite cross-linking hemostatic

Composite porous starch (PS) with chitosan (CS) was easily cross-linked by sodium trimetaphosphate (STMP) to obtain a hemostat called STMP/PS/CS (SPC) with ideal hemostatic effect. The water absorption ratio and swelling ratio of SPC reach up to 150.8%, 355.0% respectively and it was observed by scanning electron microscopy (SEM) that a large amount of CS was combined with PS, which indicated the composite effect was ideal. Synergistic hemostatic effect of SPC was indicated in blood cells evaluation experiment and the activation of platelets triggers a multistep cascade that produces a clot by adsorbing more blood cells on the surface of SPC. The average hemostasis time of SPC in tail amputation 1 cm, 2 cm and liver laceration was shortened by 29%, 42%, 37% respectively compared with blank control, and had better hemostatic effect than CS, PS in the hemostatic performance on the rat tail amputation and liver laceration. Besides, we found SPC displayed a superior adhesiveness in rat injuries.

Porous Organic Polymer-Derived Nanopalladium Catalysts for Chemoselective Synthesis of Antitumor Benzofuro[2,3- b]pyrazine from 2-Bromophenol and Isonitriles.

An efficient strategy for the synthesis of benzofuro[2,3- b]pyrazines was developed. These tricyclic scaffolds were formed through a multistep cascade sequence, which includes double insertion of isonitriles and chemoselective bicyclization. In this reaction, a nanopalladium was used as a recyclable catalyst. Product 3w exhibited excellent anticancer activity toward T-24 (IC50 = 12.5 ± 0.9 μM) and HeLa (IC50 = 14.7 ± 1.6 μM) cells. We also explored the action mechanism of 3w on T-24 cells.

Endophytic Consortium With Diverse Gene-Regulating Capabilities of Benzylisoquinoline Alkaloids Biosynthetic Pathway Can Enhance Endogenous Morphine Biosynthesis in Papaver somniferum

Secondary metabolite biosynthesis in medicinal plants is multi-step cascade known to be modulated by associated endophytes. While a single endophyte is not able to upregulate all biosynthetic steps, limiting maximum yield achievement. Therefore to compliment the deficient characteristics in an endophyte we tried consortium of endophytes to achieve maximum yield. Here, efforts were made to maximize the in planta morphine yield, using consortium of two endophytes; SM1B (Acinetobacter sp.) upregulating most of the genes of morphine biosynthesis except T6ODM and CODM, and SM3B (Marmoricola sp.) upregulating T6ODM and CODM in alkaloid-less Papaver somniferum cv. Sujata. Consortium-inoculation significantly increased morphine and thebaine content, and also increased the photosynthetic efficiency of poppy plants resulted in increased biomass, capsule weight, and seed yields compared to single-inoculation. The increment in morphine content was due to the modulation of metabolic-flow of key intermediates including reticuline and thebaine, via upregulating pertinent biosynthetic genes and enhanced expression of COR, key gene for morphine biosynthesis. This is the first report demonstrating the endophytic-consortium complimenting the functional deficiency of one endophyte by another for upregulating multiple genes of a metabolic pathway similar to transgenics (overexpressing multiple genes) for obtaining enhanced yield of pharmaceutically important metabolites.

Constraints on the dipole photon strength functions from experimental multistep cascade spectra

Constraints on the dipole photon strength functions from experimental multistep cascade spectra

Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC

Ovarian cancer (OvCa) exhibits a specific predilection for metastasis to the omentum. Our earlier studies highlighted the tumour-suppressor effect of secreted protein acidic and rich in cysteine (SPARC) in OvCa through multi-faceted roles inhibiting cancer cell interactions within the peritoneal milieu. The goal of this study is to investigate the role of SPARC in OvCa interactions with omental adipocytes and its role in OvCa colonization in the omentum. We employed multi-pronged approach using primary omental adipocytes from Sparc knockout mice, genetically engineered human omental adipocytes in 3D co-cultures with OvCa cells, as well as treatment with recombinant SPARC protein. We show that SPARC suppresses multistep cascade in OvCa omental metastasis. SPARC inhibited in vivo and adipocyte-induced homing, proliferation, and invasion of OvCa cells. SPARC suppressed metabolic programming of both adipocytes and OvCa cells and exerted an inhibitory effect of adipocyte differentiation and their phenotypic switch to cancer-associated phenotype. Mechanistic studies revealed that this effect is mediated through inhibition of cEBPβ-NFkB-AP-1 transcription machinery. These findings define a novel and functionally important role of SPARC in OvCa and not only bridge the knowledge gap but highlight the need to consider SPARC protein expression in therapeutic development.

Cascade reaction including a formal [5 + 2] cycloaddition by use of alkyne-Co2(CO)6 complex

A new cascade reaction including formal [5 + 2] cycloaddition was developed. Treatment of homocinnamyl alcohol and Co2(CO)6-complexed arylpropynal with BF3·OEt2 resulted in the generation of hydrobenzocycloheptafuran having an alkyne-Co2(CO)6 complex. The reaction consists of 5-membered ring selective Prins cyclization and subsequent Friedel-Crafts cyclization. The cascade reaction was applied to a further multi-step cascade cyclization, which resulted in the formation of more complex polycyclic hydrofurans.