Synthesis of obovatol and related neolignan analogues as α-glucosidase and α-amylase inhibitors.

Diabetes mellitus is a metabolic disease characterized by hyperglycemia, which can be counteracted by the inhibition of α-glucosidase (α-Glu) and α-amylase (α-Amy), enzymes responsible for the hydrolysis of carbohydrates. In recent decades, many natural compounds and their bioinspired analogues have been studied as α-Glu and α-Amy inhibitors. However, no studies have been devoted to the evaluation of α-Glu and α-Amy inhibition by the neolignan obovatol (1). In this work, we report the synthesis of 1 and a library of new analogues. The synthesis of these compounds was achieved by implementing methodologies based on: phenol allylation, Claisen/Cope rearrangements, methylation, Ullmann coupling, demethylation, phenol oxidation and Michael-type addition. Obovatol (1) and ten analogues were evaluated for their in vitro inhibitory activity towards α-Glu and α-Amy. Our investigation highlighted that the naturally occurring 1 and four neolignan analogues (11, 22, 26 and 27) were more effective inhibitors than the hypoglycemic drug acarbose (α-Amy: 34.6 µM; α-Glu: 248.3 µM) with IC5O value of 6.2-23.6 µM toward α-Amy and 39.8-124.6 µM toward α-Glu. Docking investigations validated the inhibition outcomes, highlighting optimal compatibility between synthesized neolignans and both the enzymes. Concurrently circular dichroism spectroscopy detected the conformational changes in α-Glu induced by its interaction with the studied neolignans. Detailed studies through fluorescence measurements and kinetics of α-Glu and α-Amy inhibition also indicated that 1, 11, 22, 26 and 27 have the greatest affinity for α-Glu and 1, 11 and 27 for α-Amy. Surface plasmon resonance imaging (SPRI) measurements confirmed that among the compounds studied, the neolignan 27 has the greater affinity for both enzymes, thus corroborating the results obtained by kinetics and fluorescence quenching. Finally, in vitro cytotoxicity of the investigated compounds was tested on human colon cancer cell line (HCT-116). All these results demonstrate that these obovatol-based neolignan analogues constitute promising candidates in the pursuit of developing novel hypoglycemic drugs.

A one pot, two-step synthesis of 5-arylpyrrolo[2,3-d]pyrimidines and screening of their preliminary antibacterial properties.

A one pot two step methodology for the synthesis of ten derivatives of 5-arylpyrrolo[2,3-d]pyrimidine has been reported. The methodology exploits the strong reducing nature of alkaline Na2S2O4 solution coupled with favorability of Michael type addition reaction in alkaline medium. The methodology demands attraction as it is non-catalytic, quite general for wide range of nitrostyrenes and possesses comprehensive advantages over most of the earlier methods in terms of reaction time as well as yield. The methodology enjoys additional advantage of utilizing cheaper and easily available chemicals as reagent for the purpose. Some of the synthesized compounds are found to possess remarkable activity against some of the tested bacterial strains.

Mitsunobu Reaction: A Versatile Tool for PEG End Functionalization.

The Mitsunobu reaction can be efficiently used for the transformation of poly(ethylene glycol) (PEG) terminal OH group(s) into a variety of functions. In comparison to more classical approaches of PEG functionalization, the main advantage of the Mitsunobu reaction attains to the fact that in one step, with no detrimental effect on PEG integrity (e.g., chain cleavage). Here, its quantitative conversion is demonstrated into derivatives that, either directly or after deprotection, are amenable to (bio)conjugation reactions: azides (Huisgen cycloaddition), aldehydes, primary amines (Schiff base formation and reduction), thiols, and N-oxymaleimide (Michael-type addition). Therefore this reaction is proposed as a general tool for the preparation of functionalities for the purpose of PEGylation, and more generally for (bio)conjugation purposes.

Immobilization of Escherichia coli cells expressing 4-oxalocrotonate tautomerase for improved biotransformation of ß-nitrostyrene.

The enzyme 4-oxalocrotonate tautomerase (4-OT) encoded by the xylH gene is a part of the degradation pathway of aromatic compounds in Pseudomonas putida mt-2. 4-OT was described to catalyze Michael-type addition of acetaldehyde to ß-nitrostyrene, and the whole cell system based on recombinantly expressed 4-OT has been developed previously. In this study biocatalytic process based on Escherichia coli whole cells expressing 4-OT was significantly improved using immobilization and ex situ product recovery strategies. Whole cell immobilization in alginate beads was applied in biocatalytic production of 4-nitro-3-phenyl-butanal from ß-nitrostyrene and acetaldehyde. Immobilized biocatalyst showed wider pH activity range and could tolerate twofold higher initial concentrations of substrate in comparison to the free whole cell biocatalyst. Beads retained their initial activity over 10 consecutive biotransformations of the model reaction and remained suitable for the repetitive use with 85% of the initial activity after two months of storage. Bioprocess was further improved by utilizing Amberlite XAD-2 hydrophobic resin for the product recovery. With this modification, the amount of organic solvent was reduced 40-fold in comparison to previously reported method making this biocatalytic process greener.

A Spontaneous In Situ Thiol-Ene Crosslinking Hydrogel with Thermo-Responsive Mechanical Properties.

The thermo-responsive behavior of Poly(N-isopropylacrylamide) makes it an ideal candidate to easily embed cells and allows the polymer mixture to be injected. However, P(NiPAAm) hydrogels possess minor mechanical properties. To increase the mechanical properties, a covalent bond is introduced into the P(NIPAAm) network through a biocompatible thiol-ene click-reaction by mixing two polymer solutions. Co-polymers with variable thiol or acrylate groups to thermo-responsive co-monomer ratios, ranging from 1% to 10%, were synthesized. Precise control of the crosslink density allowed customization of the hydrogel's mechanical properties to match different tissue stiffness levels. Increasing the temperature of the hydrogel above its transition temperature of 31 °C induced the formation of additional physical interactions. These additional interactions both further increased the stiffness of the material and impacted its relaxation behavior. The developed optimized hydrogels reach stiffnesses more than ten times higher compared to the state of the art using similar polymers. Furthermore, when adding cells to the precursor polymer solutions, homogeneous thermo-responsive hydrogels with good cell viability were created upon mixing. In future work, the influence of the mechanical micro-environment on the cell's behavior can be studied in vitro in a continuous manner by changing the incubation temperature.

Liver click dECM hydrogels for engineering hepatic microenvironments.

Decellularized extracellular matrix (dECM) hydrogels provide tissue-specific microenvironments which accommodate physiological cellular phenotypes in 3D in vitro cell cultures. However, their formation hinges on collagen fibrillogenesis, a complex process which limits regulation of physicochemical properties. Hence, achieving reproducible results with dECM hydrogels poses as a challenge. Here, we demonstrate that thiolation of solubilized liver dECM enables rapid formation of covalently crosslinked hydrogels via Michael-type addition, allowing for precise control over mechanical properties and superior organotypic biological activity. Investigation of various decellularization methodologies revealed that treatment of liver tissue with Triton X-100 and ammonium hydroxide resulted in near complete DNA removal with significant retention of the native liver proteome. Chemical functionalization of pepsin-solubilized liver dECMs via 1-ethyl-3(3-dimethylamino)propyl carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling of l-Cysteine created thiolated liver dECM (dECM-SH), which rapidly reacted with 4-arm polyethylene glycol (PEG)-maleimide to form optically clear hydrogels under controlled conditions. Importantly, Young's moduli could be precisely tuned between 1 - 7 kPa by varying polymer concentrations, enabling close replication of healthy and fibrotic liver conditions in in vitro cell cultures. Click dECM-SH hydrogels were cytocompatible, supported growth of HepG2 and HepaRG liver cells, and promoted liver-specific functional phenotypes as evidenced by increased metabolic activity, as well CYP1A2 and CYP3A4 activity and excretory function when compared to monolayer culture and collagen-based hydrogels. Our findings demonstrate that click-functionalized dECM hydrogels offer a highly controlled, reproducible alternative to conventional tissue-derived hydrogels for in vitro cell culture applications. STATEMENT OF SIGNIFICANCE: Traditional dECM hydrogels face challenges in reproducibility and mechanical property control due to variable crosslinking processes. We introduce a click hydrogel based on porcine liver decellularized extracellular matrix (dECM) that circumnavigates these challenges. After optimizing liver decellularization for ECM retention, we integrated thiol-functionalized liver dECM with polyethylene-glycol derivatives through Michael-type addition click chemistry, enabling rapid, room-temperature gelation. This offers enhanced control over the hydrogel's mechanical and biochemical properties. The resultant click dECM hydrogels mimic the liver's natural ECM and exhibit greater mechanical tunability and handling ease, facilitating their application in high-throughput and industrial settings. Moreover, these hydrogels significantly improve the function of HepaRG-derived hepatocytes in 3D culture, presenting an advancement for liver tissue cell culture models for drug testing applications.

A tough and bioadhesive injectable hydrogel formed with maleimidyl alginate and pristine gelatin.

Injectable hydrogels have wide applications in clinical practice. However, the development of tough and bioadhesive ones based on biopolymers, along with biofriendly and robust crosslinking strategies, still represents a great challenge. Herein, we report an injectable hydrogel composed of maleimidyl alginate and pristine gelatin, for which the precursor solutions could self-crosslink via mild Michael-type addition without any catalyst or external energy upon mixing. This hydrogel is tough and bioadhesive, which can maintain intactness as well as adherence to the defect of porcine skin under fierce bending and twisting, warm water bath, and boiling water shower. Besides, it is biocompatible, bioactive and biodegradable, which could support the growth and remodeling of cells by affording an extracellular matrix-like environment. As a proof of application, we demonstrate that this hydrogel could significantly accelerate diabetic skin wound healing, thereby holding great potential in healthcare.

Biocatalytic Michael-type additions of acetaldehyde to nitroolefins with the proline-based enzyme 4-oxalocrotonate tautomerase yielding enantioenriched γ-nitroaldehydes.

Call me Michaelase: The enzyme 4-oxalocrotonate tautomerase (4-OT) promiscuously catalyzes the Michael-type addition of acetaldehyde to a collection of aromatic and aliphatic nitroolefins with high stereoselectivity producing precursors of γ-aminobutyric acid (GABA) analogues.

Design of Functional Pluronic-Based Precursors for Tailoring Hydrogel Thermoresponsiveness and Cell-Adhesive Properties.

In this study, functional Pluronic F127 precursors were designed and synthesized for the preparation of thermosensitive hydrogels. Using linear Pluronic thioacetate and Pluronic multi-acrylate precursors, F127-based hydrogels were prepared through thioacetate deprotection-mediated Michael-type addition. The properties of these gels were compared to those obtained through free radical crosslinking of F127 diacrylate. Temperature was found to have a clear influence on gel swelling as a result of F127 thermoresponsiveness. The macromolecular architecture and functionality of the precursors were also optimized and characterized in terms of gelation kinetics and drug diffusion. In vitro tests were conducted on fibroblasts and endothelial cells to assess their response to cellular adhesion with Pluronic gels that were functionalized with an RGD peptide or pretreated with serum proteins to promote cell adhesion. This study provides a method for creating tailored hydrogels suitable for various biomedical applications, such as soft-tissue engineering, cell encapsulation, wound healing, and sustained delivery of therapeutic molecules.

"One-step" preparation of thiol-ene clickable PEG-based thermoresponsive hyperbranched copolymer for in situ crosslinking hybrid hydrogel.

A well-defined poly(ethylene glycol) based hyperbranched thermoresponsive copolymer with high content of acrylate vinyl groups was synthesized via a "one-pot and one-step" deactivation enhanced atom transfer radical polymerization approach, which provided an injectable and in situ crosslinkable system via Michael-type thiol-ene reaction with a thiol-modified hyaluronan biopolymer. The hyperbranched structure, molecular weight, and percentage of vinyl content of the copolymer were characterized by gel permeation chromatography and (1)H NMR. The lower critical solution temperature of this copolymer is close to body temperature, which can result in a rapid thermal gelation at 37 °C. The scanning electron microscopy analysis of crosslinked hydrogel showed the network formation with porous structure, and 3D cell culture study demonstrated the good cell viability after the cells were embedded inside the hydrogel. This injectable and in situ crosslinking hybrid hydrogel system offers great promise as a new class of hybrid biomaterials for tissue engineering.

Michael-type addition of azoles of broad-scale acidity to methyl acrylate.

An optimisation of Michael-type addition of azole derivatives of broad-scale acidity - ranging from 5.20 to 15.00 pK(a) units - namely 4-nitropyrazole, 3,5-dimethyl-4-nitropyrazole, 4(5)-nitroimidazole, 4,5-diphenylimidazole, 4,5-dicyanoimidazole, 2-methyl-4(5)-nitroimidazole, 5(4)-bromo-2-methyl-4(5)-nitroimidazole and 3-nitro-1,2,4-triazole to methyl acrylate as an acceptor was carried out. The optimisation process involved the use of an appropriate basic catalyst (DBU, DIPEA, NaOH, NaH, TEDA), a donor/base/acceptor ratio and the reaction temperature. The reactions were performed in DMF as solvent. Target Michael adducts were obtained in medium to excellent yields. Importantly, for imidazole and 1,2,4-triazole derivatives, no corresponding regioisomers were obtained.

Mechanistic evidence for the effect of sulphur-based additive: methionine, on acrylamide reduction.

The unavoidable presence of acrylamide in foods has fuelled the search for a suitable food additive, one that can successfully mitigate dietary acrylamide levels without changing food quality or compromising the health of consumers. The purpose of this study was to investigate the effect of a sulphur-based additive and amino acid, methionine, on acrylamide reduction. Differential scanning calorimetry, supported by chromatographic measurements, has shown that methionine interacts with acrylamide at a possible optimum temperature of 160°C, thereby disfavouring acrylamide polymerisation. Analysis of the methionine-acrylamide interaction via density functional theoretical modelling (DFT/6-31 + G(d)/RCAM-B3LYP) revealed that methionine's reducing effect may be driven by a Michael-type conjugation of the vinyl group of acrylamide at both the sulphur atom (∆Gf = -53 kJ mol-1) and the amino group (∆Gf = -11.84 kJ mol-1) of methionine. The former conjugation pathway results in a product that is more thermodynamically feasible.

QCM-D and XPS study of protein adsorption on plasma polymers with sulfonate and phosphonate surface groups.

As some proteins are known to interact with sulfated and phosphated biomolecules such as specific glycosaminoglycans, this study derives from the hypothesis that sulfonate and phosphonate groups on solid polymer surfaces might cause specific interfacial interactions. Such surfaces were prepared by plasma polymerization of heptylamine (HA) and subsequent grafting of sulfonate or phosphonate groups via Michael-type addition of vinylic compounds. Adsorption of the proteins fibrinogen, albumin (HSA) and lysozyme on these functionalised plasma polymer surfaces was studied by XPS and quartz crystal microbalance with dissipation (QCM-D). It was also studied whether pre-adsorption with HSA would lead to a passivated surface against further adsorption of other proteins. XPS confirmed grafting of vinyl sulfonate and vinyl phosphonate onto the amine surface and showed that the proteins adsorbed to saturation at between 1 and 2 h. QCM-D showed rapid and irreversible adsorption of albumin on all three surfaces, while lysozyme could be desorbed with PBS to substantial extents from the sulfonated and phosphonated surfaces but not from the amine surface. Fibrinogen showed rapid initial adsorption followed by slower additional mass gain over hours. Passivation with albumin led to small and largely reversible subsequent adsorption of lysozyme, whereas with fibrinogen partial displacement yielded a mixed layer, regardless of the surface chemistry. Thus, protein adsorption onto these sulfonated and phosphonated surfaces is complex, and not dominated by electrostatic charge effects.

Quaternized γ-Fe2O3@cellulose ionomer: An efficient recyclable catalyst for Michael-type addition reaction.

Owe to unique advantages of heterogeneous catalytic reactions, there is increasing interest to use this type of chemical transformations in organic synthesis. Among various heterogeneous catalytic systems, magnetic supported ionic liquids are emerging ones in the chemical synthesis. As a result, this research focuses on developing an efficient magnetically recyclable catalytic system for Michael-type addition reaction based on quaternized γ-Fe2O3@cellulose ionomer. Core-shell structured magnetite cellulose nanosphere was synthesized by one step precipitation route and further modified with epichlorohydrin and hexamethylenetetramine. Anion exchange reaction was performed with polytungstophosphate. Synthesized nanocatalyst was characterized with FESEM, FTIR, VSM, EDX and TEM techniques. Vinyl pyridine and three types of functional groups i.e., hydroxyl, thiol, and amine were employed to evaluate the catalyst performance. Results showed that the addition reaction promoted up to 95% within 2h reaction time at moderate temperature (50°C) moreover the nanocatalyst showed good recyclability after three catalytic run as the reaction efficiency was >80% at the end of the third cycle which confirmed high efficiency of the presented system as a green heterogeneous catalyst to synthesis intermediate organic compounds.

Control of thiol-maleimide reaction kinetics in PEG hydrogel networks.

Michael-type addition reactions are widely used to polymerize biocompatible hydrogels. The thiol-maleimide modality achieves the highest macromer coupling efficiency of the reported Michael-type pairs, but the resulting hydrogel networks are heterogeneous because polymerization is faster than the individual components can be manually mixed. The reactivity of the thiol dictates the overall reaction speed, which can be slowed in organic solvents and acidic buffers. Since these modifications also reduce the biocompatibility of resulting hydrogels, we investigated a series of biocompatible buffers and crosslinkers to decelerate gelation while maintaining high cell viability. We found that lowering the polymer weight percentage (wt%), buffer concentration, and pH slowed gelation kinetics, but crosslinking with an electronegative peptide was optimal for both kinetics and cell viability. Including a high glucose medium supplement in the polymer solvent buffer improved the viability of the cells being encapsulated without impacting gelation time. Slowing the speed of polymerization resulted in more uniform hydrogels, both in terms of visual inspection and the diffusion of small molecules through the network. However, reactions that were too slow resulted in non-uniform particle dispersion due to settling, thus there is a trade-off in hydrogel network uniformity versus cell distribution in the hydrogels when using these networks in cell applications. STATEMENT OF SIGNIFICANCE: The polymer network of thiol-maleimide hydrogels assembles faster than individual components can be uniformly mixed due to their fast gelation kinetics. The lack of homogeneity can result in variable cell-based assay results, resulting in batch-to-batch variability and limiting their use in predictive screening assays. Although these hydrogels are incredibly useful in tissue engineering, this network heterogeneity is a known problem in the field. We screened a variety of possible techniques to slow down the reaction speed and improve the homogeneity of these hydrogels, without sacrificing the viability and distribution of encapsulated cells. As others have reported, an electronegative crosslinker was the most effective technique to slow the reaction, but the chemical modification required is technically challenging. Of interest to a broad community, we screened buffer type, strength, and crosslinker electronegativity to find an optimal reaction speed that allows for high cell viability and small molecule diffusion, without allowing cells to settle during gelation, allowing application of these materials to the drug screening industry and tissue engineering community.

Rapidly Responsive and Highly Selective Fluorescent Probe for Bisulfite Detection in Food.

The new fluorescent probe 4-hydroxy-3-((2E,4E)-5-phenylpenta-2,4-dienoyl)-2H-chromen-2-one (probe 1) was designed and synthesized for selective detection of sulfite. The fluorescence intensity of the probe was decreased only in the presence of HSO3-; all other anions assessed resulted in an increased fluorescence response. Hence, probe 1 acts as a highly selective sensor for HSO3-. This sulfite sensitivity can also be readily monitored visually, as once treated with sulfite the solution shows a marked color change from yellow to colorless. Moreover, probe 1 can be conveniently used as a signal tool to determine the HSO3- levels in various sugar samples.

Microscale Bioadhesive Hydrogel Arrays for Cell Engineering Applications.

Bioengineered hydrogels have been explored in cell and tissue engineering applications to support cell growth and modulate its behavior. A rationally designed scaffold should allow for encapsulated cells to survive, adhere, proliferate, remodel the niche, and can be used for controlled delivery of biomolecules. Here we report a microarray of composite bioadhesive microgels with modular dimensions, tunable mechanical properties and bulk modified adhesive biomolecule composition. Composite bioadhesive microgels of maleimide functionalized polyethylene glycol (PEG-MAL) with interpenetrating network (IPN) of gelatin ionically cross-linked with silicate nanoparticles were engineered by integrating microfabrication with Michael-type addition chemistry and ionic gelation. By encapsulating clinically relevant anchorage-dependent cervical cancer cells and suspension leukemia cells as cell culture models in these composite microgels, we demonstrate enhanced cell spreading, survival, and metabolic activity compared to control gels. The composite bioadhesive hydrogels represent a platform that could be used to study independent effect of stiffness and adhesive ligand density on cell survival and function. We envision that such microarrays of cell adhesive microenvironments, which do not require harsh chemical and UV crosslinking conditions, will provide a more efficacious cell culture platform that can be used to study cell behavior and survival, function as building blocks to fabricate 3D tissue structures, cell delivery systems, and high throughput drug screening devices.

Synthesis, characterization and properties of a physically and chemically gelling polymer system using poly(NIPAAm-co-HEMA-acrylate) and poly(NIPAAm-co-cysteamine).

The aim of this work was to develop a simultaneous physically and chemically gelling system using NIPAAm co-polymers. The in situ polymer gel was obtained by synthesizing poly(NIPAAm-co-HEMAacrylate) and poly(NIPAAm-co-cysteamine) through free radical polymerization and further nucleophilic substitution. The purpose of the dual gelation is that physical gelation would take place at higher temperatures as the NIPAAm chains associate, while chemical gelation would occur through a Michael-type addition reaction, resulting in a cross-link forming through a nucleophilic attack of the thiolate on the acrylate. The structure of each co-polymer was then verified using (1)H-NMR and FT-IR spectroscopy. The corresponding lower critical solution temperature and phase transition behavior of each co-polymer was analyzed through cloud point and DSC, while mechanical properties were investigated through rheology. Swelling behavior was also monitored at different temperatures. The resulting polymer system demonstrated properties compatible with physiological conditions, forming a gel at pH 7.4 and at temperatures near body temperature. The hydrogel also showed reduced viscoelastic flow at low frequency stress, and increased strength than purely physical or chemical gels. Swelling behavior was determined to be temperature-dependent; however, no difference was observed in swelling percent beyond 48 h. Having the ability to alter these co-polymers through various synthesis parameters and techniques, this hydrogel can potentially be used as an injectable, waterborne gelling material for biomedical applications such as endovascular embolization.