Covalent Complexation Research Articles

Structural, physicochemical and digestive properties of non-covalent and covalent complexes of ultrasound treated soybean protein isolate with soybean isoflavone

The non-covalent and covalent complexes of ultrasound treated soybean protein isolate (SPI) and soybean isoflavone (SI) were prepared, and the structure, physicochemical properties and in vitro digestion characteristics of SPI-SI complexes were investigated. Ultrasonic treatment increased the non-covalent and covalent binding degree of SPI with SI, and the 240 W ultrasonic covalent complexes had higher binding efficiency. Appropriate ultrasonic treatment caused more uniform particle size distribution, lower average particle size and higher surface charge, which enhanced the free sulfhydryl groups and surface hydrophobicity, thus improving the stability, solubility and emulsifying properties of complexes. Ultrasonic treatment resulted in more disordered secondary structure, tighter tertiary conformation, higher thermal stability and stronger SPI-SI covalent interactions of complexes. These structural modifications of particles had important effects on the chemical stability and gastrointestinal digestion fate of SI. The ultrasonic covalent complexation had a greater resistance to heat-induced chemical degradation of SI and improved its chemical stability. Furthermore, the 240 W ultrasonic covalent complexes showed lower protein digestibility during digestion, and provided stronger protection for SI, which improved the digestion stability and antioxidant activity. Therefore, appropriate ultrasound promoted SPI-SI interactions to improve the stability and functional properties of complexes, which provided a theoretical basis for the development of new complexes and their applications in functional foods.

Dual-ROS-scavenging and dual-lingering nanozyme-based eye drops alleviate dry eye disease

Efficiently removing excess reactive oxygen species (ROS) generated by various factors on the ocular surface is a promising strategy for preventing the development of dry eye disease (DED). The currently available eye drops for DED treatment are palliative, short-lived and frequently administered due to the short precorneal residence time. Here, we developed nanozyme-based eye drops for DED by exploiting borate-mediated dynamic covalent complexation between n-FeZIF-8 nanozymes (n-Z(Fe)) and poly(vinyl alcohol) (PVA) to overcome these problems. The resultant formulation (PBnZ), which has dual-ROS scavenging abilities and prolonged corneal retention can effectively reduce oxidative stress, thereby providing an excellent preventive effect to alleviate DED. In vitro and in vivo experiments revealed that PBnZ could eliminate excess ROS through both its multienzyme-like activity and the ROS-scavenging activity of borate bonds. The positively charged nanozyme-based eye drops displayed a longer precorneal residence time due to physical adhesion and the dynamic borate bonds between phenyboronic acid and PVA or o-diol with mucin. The in vivo results showed that eye drops could effectively alleviate DED. These dual-function PBnZ nanozyme-based eye drops can provide insights into the development of novel treatment strategies for DED and other ROS-mediated inflammatory diseases and a rationale for the application of nanomaterials in clinical settings.Graphical

Sodium metabisulfite mediated self-assembly behavior of silver carp myofibrillar protein and effects on covalent complexation with (−)-epigallocatechin-3-gallate

This study aimed to investigate the influence of different concentrations of sodium metabisulfite on the conformation of the myofibrillar protein (MP) and its covalent complexation with (−)-epigallocatechin-3-gallate (EGCG). Changes in sodium dodecyl SDS‒PAGE and free thiol content indicated that disulfide bonds in MP were disrupted by sodium metabisulfite, resulting in a maximum 3.82-fold increase in free thiol concentration compared to the control group. Fluorescence spectra and circular dichroism analysis revealed that when sodium metabisulfite was added at a concentration of 1 mmol/L, hydrophobic forces drove the self-assembly of MP, leading to a reduction in particle size to 53.21 μm and an increase in β-sheet content to 11.27%. Upon complexation with EGCG, the structure of MP became looser, causing an increase in particle size to 169.01 μm and a significant decrease in α-helix content to a maximum of 39.46%. This study revealed that increasing the concentration of sodium metabisulfite promoted the covalent complexation of MP with EGCG, with the optimal complexation observed at 1 mmol/L sodium metabisulfite, resulting in a notable 97.1% free radical sca0venging activity (compared to 77.8 % in the control group). These findings provide an economical and effective method to alleviate limitations in the food industry regarding MP utilization.

Cocrystallization of ubiquitin-deubiquitinase complexes through disulfide linkage.

Structural characterization of the recognition of ubiquitin (Ub) by deubiquitinases (DUBs) has largely relied on covalent complexation of the DUB through its catalytic cysteine with a Ub C-terminal electrophile. The Ub electrophiles are accessed through intein chemistry in conjunction with chemical synthesis. Here, it was asked whether DUB-Ub covalent complexes could instead be accessed by simpler disulfide chemistry using a Ub cysteine mutant in which the last glycine has been replaced with a cysteine. The Ub cysteine mutant displayed a wide variability in disulfide formation across a panel of eukaryotic and prokaryotic DUBs, with some showing no detectable reaction while others robustly produced a disulfide complex. Using this approach, two disulfide-linked ubiquitin-bound complexes were crystallized, one involving the Legionella pneumophila effector SdeA DUB and the other involving the Orientia effector OtDUB. These DUBs had previously been crystallized in Ub-bound forms using the C-terminal electrophile strategy and noncovalent complexation, respectively. While the disulfide-linked SdeA DUB-Ub complex crystallized as expected, in the OtDUB complex the disulfide bond to the Ub mutant involved a cysteine that differed from the catalytic cysteine. Disulfide formation with the SdeA DUB catalytic cysteine was accompanied by local distortion of the helix carrying the active-site cysteine, whereas OtDUB reacted with the Ub mutant using a surface-exposed cysteine.

Computational Investigations on Reaction Mechanisms of the Covalent Inhibitors Ponatinib and Analogs Targeting the Extracellular Signal-Regulated Kinases.

As an important cancer therapeutic target, extracellular signal-regulated kinases (ERK) are involved in triggering various cellular responses in tumors. Regulation of the ERK signaling pathway by the small molecular inhibitors is highly desired for the sake of cancer therapy. In contrast to the routine inhibitors targeting ERKs through long-range non-bonding interactions, Ponatinib, a covalent inhibitor to ERK2 with a macrocyclic structure characterized by the α,β-C=C unsaturated ketone, can form the stable -C(S)-C(H)-type complex via the four-center barrier due to the nucleophilic addition reaction of the thiol group of the Cys166 residue of ERK2 with the C=C double bond of Ponatinib with reaction free-energy barrier of 47.2 kcal/mol. Reaction mechanisms for the covalent binding were calculated using QM/MM methods and molecular dynamics simulations. The interaction modes and the corresponding binding free energies were obtained for the non-covalent and covalent complexation. The binding free energies of the non-covalent and covalent inhibitions are 14.8 kcal/mol and 33.4 kcal/mol, respectively. The mechanistic study stimulated a rational design on the modified Ponatinib structure by substituting the C=C bond with the C=N bond. It was demonstrated that the new compound exhibits better inhibition activity toward ERK2 in term of both thermodynamic and kinetic aspects through the covalent binding with a lower reaction free-energy barrier of 23.1 kcal/mol. The present theoretical work sheds new light on the development of the covalent inhibitors for the regulation of ERKs.

Improving ocular bioavailability of hydrophilic drugs through dynamic covalent complexation.

The clinical benefits of diquafosol tetrasodium (DQS), a hydrophilic P2Y2 receptor agonist for dry eye, have been hindered by a demanding dosing regimen. Nevertheless, it is challenging to achieve sustained release of DQS with conventional drug delivery vehicles which are mainly designed for hydrophobic small molecule drugs. To address this, we developed an affinity hydrogel for DQS by taking advantage of borate-mediated dynamic covalent complexation between DQS and hydroxypropyl guar. The resultant formulation (3% DQS Gel) was characterized by sustained release, low corneal permeation, and extended ocular retention, which were desirable attributes for ocular surface drug delivery. Both in vitro and in vivo studies had been carried out to verify the biocompatibility of 3% DQS Gel. Using corneal fluorescein staining, the Schirmer's test, PAS staining, quantitative PCR and immunohistological analyses as outcome measures, the superior therapeutic effects of 3% DQS Gel over PBS, the hydrogel vehicle and free DQS were demonstrated in a mouse dry eye model. Our DQS delivery strategy reported herein is readily applicable to other hydrophilic small molecule drugs with cis-diol moieties, thus providing a general solution to improve clinical outcomes of numerous diseases.

Covalent and Noncovalent Complexation of Phosvitin and Gallic Acid: Effects on Protein Functionality and In Vitro Digestion Properties.

To investigate the effects of different binding modes on the structure, function, and digestive properties of the phosvitin (Pv) and gallic acid (GA) complex, Pv was covalently and noncovalently combined with different concentrations of GA (0.5, 1.5, and 2.5 mM). The structural characterization of the two Pv-GA complexes was performed by Fourier transform infrared, circular dichroism, and LC-MS/MS to investigate the covalent and noncovalent binding of Pv and GA. In addition, the microstructure of the two Pv-GA complexes was investigated by super-resolution microscopy and transmission electron microscopy. The particle size and zeta potential results showed that the addition of GA increased the particle size and the absolute potential of Pv. The determination of protein digestibility, polyphenol content, SH and S-S group levels, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antioxidant capacity of the digests indicated that noncovalent complexes had greater antioxidant and protective effects on polyphenols. Molecular docking revealed that GA was conjugated with Pv through hydrogen bond interactions.

MALDI-TOF-MS and in-depth dynamic simulations on the molecular forces determining the stability of the 4-hydroxybenzoic acid – β-Casein complex following UHT-like treatment

Previous work has suggested the ability of 4-hydroxybenzoic acid (4HBA) to bind to β -casein following ultra high temperature-like processing (UHT) in model aqueous systems. The present work confirmed directly, using MALDI-TOF-MS, the presence of covalently bound 4HBA following UHT-like treatment. In subsequent molecular dynamics simulations, the 3D structure of the β -casein molecule was modified so that the meta-C of 4HBA ring and the side chain amino group of lys32 were linked covalently. Such simulations further indicated that the covalent addition of the phenolic compound had impacted the protein density and solvent accessibility. Hydrogen bond analysis between lys32 and the remainder of the protein structure revealed that the covalent complexation supported the formation of additional hydrogen bonds. These increased from potentially 9, in the single protein molecule, to 51 in the complex with 4HBA. However, the persistence of hydrogen bonds was reduced, leading overall to decreased stability and increased protein flexibility.

Strategies to Broaden the Applications of Olive Biophenols Oleuropein and Hydroxytyrosol in Food Products.

Oleuropein (OLE) and hydroxytyrosol (HT) are olive-derived phenols recognised as health-promoting agents with antioxidant, anti-inflammatory, cardioprotective, antifungal, antimicrobial, and antitumor activities, providing a wide range of applications as functional food ingredients. HT is Generally Recognised as Safe (GRAS) by the European Food Safety Authority (EFSA) and the Food and Drug Administration (FDA), whereas OLE is included in EFSA daily consumptions recommendations, albeit there is no official GRAS status for its pure form. Their application in food, however, may be hindered by challenges such as degradation caused by processing conditions and undesired sensorial properties (e.g., the astringency of OLE). Among the strategies to overcome such setbacks, the encapsulation in delivery systems and the covalent and non-covalent complexation are highlighted in this review. Additionally, the synthesis of OLE and HT derivatives are studied to improve their applicability. All in all, more research needs however to be carried out to investigate the impact of these approaches on the sensory properties of the final food product and its percussions at the gastrointestinal level, as well as on bioactivity. At last limitations of these approaches at a scale of the food industry must also be considered.

Effects of soybean protein isolate − polyphenol conjugate formation on the protein structure and emulsifying properties: Protein − polyphenol emulsification performance in the presence of chitosan

Herein, we investigated the influence of covalent complexation between (−)-epigallocatechin gallate (EGCG) and soy protein isolate (SPI) on the structural and emulsifying properties of the protein as well as the effect of chitosan (CS) on the stability of emulsions formed by the covalent conjugates. The formation of the covalent complex of SPI and EGCG was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three-dimensional fluorescence spectroscopy and circular dichroism spectroscopy were conducted to analyze the changes in the protein structure at different EGCG concentrations (0.005, 0.015, and 0.025 wt%). The addition of EGCG changed the tertiary conformation and secondary structure of the protein—unfolding of the protein polypeptide chain, decrease in the β-strand content, and increase in α-helix and random coil contents were observed. After covalent bonding of SPI with EGCG (0.015 wt %), the complex exhibited optimal solubility and emulsifying properties. SPI–EGCG–CS emulsions were prepared by layer-by-layer electrostatic assembly at pH 5.0. The effect of environmental stress on the stability of the CS–loaded SPI–EGCG (0.015 wt%) emulsion oil droplets was studied by freezing (−20 ℃, pH 5) and thermal treatment (90 ℃, 30 min, pH 5). The stability of the emulsion was improved when the CS concentration was >0.25 wt%; this improvement was attributed to the increase in electrostatic force and steric hindrance among droplets and the formation of a relatively thick biopolymer interface on the surface of oil droplets. In addition, the rheological properties showed that thicker interface layers and network structures caused deviations from the flow behavior of emulsions. These results indicate that SPI − EGCG conjugates can be used as food additives and for the construction of the lipophilic active ingredient delivery system.

Effect of non-covalent and covalent complexation of (−)-epigallocatechin gallate with soybean protein isolate on protein structure and in vitro digestion characteristics

The present study was aimed to investigate the effects of non-covalent and covalent interactions between soy protein isolate (SPI) and different concentrations (1, 2 and 5 mM) of (−)-epigallocatechin gallate (EGCG) regarding the structural and functional properties of the complex. The combination with EGCG caused changes in the secondary structure of SPI. The covalent complexes formed at low concentrations of EGCG tended to form a network structure. Compared with the SPI-EGCG non-covalent complexes, the covalent complexes exhibited higher thermal stability and oxidation resistance and a polyphenol-protective effect. In addition, the corresponding anti-digestive ability of the covalent complexes was strong and would therefore be more stable in the intestinal tract. The findings of this study provide a theoretical reference and research basis for the use of different SPI-polyphenol complexes as functional food ingredients or as bioactive materials.

Highly U(VI) immobilization on polyvinyl pyrrolidine intercalated molybdenum disulfide: Experimental and computational studies

In this study, polyvinyl pyrrolidine intercalated molybdenum disulfide (PVP/MoS2) with well-interconnected nanosheets were successfully prepared by an effective and versatile one-step hydrothermal strategy. The structural characterization indicated that the PVP molecules were intercalated into MoS2 to produce widened interlayer spacing. The obtained PVP/MoS2 nanomaterials exhibited impressive adsorption performance. Specifically, the maximum adsorption capacity of U(VI) on PVP/MoS2 (∼117.9 mg/g) was apparently superior than that of pristine MoS2 (∼23.7 mg/g) at 298 K and pH = 4.5, which might be related to the enlarged specific surface area and extensive functional groups of PVP/MoS2. The kinetics investigation showed that the uptake of U(VI) with PVP/MoS2 was ultrafast and the adsorption equilibrium could be reached within 10 min. The enrichment of U(VI) on PVP/MoS2 was caused by the existence of UO2-S covalent bonds and surface complexation with PVP, which was jointly verified by systematic investigation of FT-IR, XPS and DFT calculations. These findings were intriguing for both evaluating the adsorption performance of PVP/MoS2 and advancing other polymer interacted layered metal sulfides potential applications in the field of radioactive water treatment.

Sub-picomolar Inhibition of HIV-1 Protease with a Boronic Acid.

Boronic acids have been typecast as moieties for covalent complexation and are employed only rarely as agents for non-covalent recognition. By exploiting the profuse ability of a boronic acid group to form hydrogen bonds, we have developed an inhibitor of HIV-1 protease with extraordinary affinity. Specifically, we find that replacing an aniline moiety in darunavir with a phenylboronic acid leads to 20-fold greater affinity for the protease. X-ray crystallography demonstrates that the boronic acid group participates in three hydrogen bonds, more than the amino group of darunavir or any other analog. Importantly, the boronic acid maintains its hydrogen bonds and its affinity for the drug-resistant D30N variant of HIV-1 protease. The BOH···OC hydrogen bonds between the boronic acid hydroxy group and Asp30 (or Asn30) of the protease are short ( rO···O = 2.2 Å), and density functional theory analysis reveals a high degree of covalency. These data highlight the utility of boronic acids as versatile functional groups in the design of small-molecule ligands.

Nanoporous Anodic Alumina Surface Modification by Electrostatic, Covalent, and Immune Complexation Binding Investigated by Capillary Filling.

The fluid imbibition-coupled laser interferometry (FICLI) technique has been applied to detect and quantify surface changes and pore dimension variations in nanoporous anodic alumina (NAA) structures. FICLI is a noninvasive optical technique that permits the determination of the NAA average pore radius with high accuracy. In this work, the technique is applied after each step of different surface modification paths of the NAA pores: (i) electrostatic immobilization of bovine serum albumin (BSA), (ii) covalent attachment of streptavidin via (3-aminipropyl)-triethoxysilane and glutaraldehyde grafting, and (iii) immune complexation. Results show that BSA attachment can be detected as a reduction in estimated radius from FICLI with high accuracy and reproducibility. In the case of the covalent attachment of streptavidin, FICLI is able to recognize a multilayer formation of the silane and the protein. For immune complexation, the technique is able to detect different antibody-antigen bindings and distinguish different dynamics among different immune species.

Evaluation of structural and functional properties of chitosan⿿chlorogenic acid complexes

The objectives of the present study were to first synthesize chitosan⿿chlorogenic acid (CA) covalent complex and then compare structural and functional properties between chitosan⿿CA covalent complex and physical complex. First, chitosan⿿CA covalent complex was synthesized and its total phenolic content was as high as 276.5±6.2mg/g. Then structural and functional properties of chitosan⿿CA covalent and physical complexes were analyzed. The covalent reaction induced formation of both amide and ester bonds in chitosan. Data of X-ray diffraction (XRD) and scanning electron microscopy (SEM) indicated that the complexations of CA changed crystallinity and morphology of chitosan, and covalent complexation induced a larger change of physical structure than physical complexation. In terms of functional properties, chitosan⿿CA covalent complex exhibited better thermal stability than physical complex in terms of antioxidant activity, and the viscosity of chitosan was significantly increased by covalent modification.

Computational study adsorption of mercaptopurine in generation G3 and G5 PAMAM dendrimers

Due to its unique properties of dendrimers as well as with high-level groups, a special ability to carry drugs. In this study compared the interaction between the two generations of poly (amidoamine) PAMAM (G3and G5) dendrimers and the drug 6-mercaptopurine (6-MP) and the drug entrapment within the structure of dendrimers the electrostatic and covalent complexation of drugs to the dendrimer surface have been studied. The use of drug-dendrimer complex enhanced drug solubility and bioavailability of the drug.

Luminescent hybrid materials based on covalent attachment of Eu(III)-tris(bipyridinedicarboxylate) in the mesoporous silica host MCM-41.

A luminescent inorganic-organic hybrid material was synthesized by covalent immobilization of a europium bipyridine carboxylate complex on the inner pore walls of the mesoporous silica host MCM-41 using the grafting method. Guest-host binding was achieved through double functionalization of the host surface with organosilane reagents (trimethylsilyl, TMS, and aminopropyltriethoxysilane, APTES) followed by reaction of the active amino sites of the APTES residue with the ligand 2,2'-bipyridyl-6,6'-dicarboxylic acid. Addition of EuCl3 solution dissolved in ethanol results in the formation of an immobilized complex having the probable formula Eu(L)x(3 ≥ x ≥ 1)(H2O)y, whose detailed photophysical properties were investigated. In the final step, an additional 2,2'-bipyridine-6 monocarboxylic acid ligand was added in an attempt to complete the coordination sphere of the rare earth ion. Each of the synthesis steps was monitored by (1)H, (13)C, and (29)Si solid state NMR spectroscopies, allowing for a quantitative assessment of the progress of the reaction and the influence of the paramagnetic species on the spectra. Based on these data and additional characterizations by chemical analysis, thermogravimetric analysis (TGA), N2 sorption, X-ray diffraction and FT-IR spectroscopy, a comprehensive quantitative picture of the covalent binding and complexation process was developed.

Synthesis of boronic acid-functionalized molecularly imprinted silica nanoparticles for glycoprotein recognition and enrichment.

A novel imprinting strategy using reversible covalent complexation of glycoprotein is described for creating glycoprotein-specific recognition cavities on 3-acrylamidophenylboronic acid-immobilized silica nanoparticles (SiO2@AAPBA). Two kinds of organic silanes (3-aminopropyltriethoxysilane (APTES) and n-octyltrimethoxysilane (OTMS)) were then polymerized on the surface of SiO2@AAPBA after the template (horseradish peroxidase (HRP)) was covalently immobilized by forming cyclic boronate complexes and their influence was examined. The results showed that not only the silane composition but also the relative proportions play an important role in glycoprotein imprinting. The template recognition properties were evaluated by single-protein or competitive batch rebinding experiments, and the results showed that the HRP-imprinted silica nanoparticles (HRP-MIP silica NPs) exhibited higher recognition ability and selectivity towards the template than the nonimprinted silica NPs and their corresponding imprinted factor (a) reached 2.71. The as-prepared HRP-MIP silica NPs could not only differentiate the template from another glycoprotein, but also enrich HRP from spiked human serum. The good results demonstrated their potential in glycoproteomic analysis.

Preparation of boronate-functionalized molecularly imprinted monolithic column with polydopamine coating for glycoprotein recognition and enrichment

A novel imprinting strategy using reversible covalent complexation of glycoprotein was described for creating glycoprotein-specific recognition cavities on boronate-functionalized monolithic column. Based on it, a molecularly imprinted monolithic column was prepared by self-polymerization of dopamine (DA) on the surface of 4-vinylphenylboronic acid (VPBA)-based polymeric skeletons after reversible immobilization of horseradish peroxidase (HRP). Due to the combination of boronate affinity and surface imprinting of DA, the stable and accessible recognition sites in the as-prepared imprinted monolith could be obtained after the removal of the template, which facilitated the rebinding of the template and provided good reproducibility and lifetime of use. The recognition behaviors of proteins on the bare VPBA-based, HRP-imprinted and nonimprinted monolithic columns were evaluated in detail and the results showed that the HRP-imprinted monolith exhibited higher recognition ability toward the template than another two monolithic columns. Not only nonglycoproteins but also glycoproteins can be well separated with the HRP-imprinted monolith. In addition, the feasibility of the HRP-imprinted monolith, adopted as an in-tube solid phase microextraction (in-tube SPME), was further assessed by selective extraction and enrichment of HRP from human serum. The good results demonstrated its potential in glycoproteome analysis.

Covalent complexation and functional evaluation of (−)-epigallocatechin gallate and α-lactalbumin

The covalent complexation between (−)-epigallocatechin gallate (EGCG) and α-lactalbumin (α-La), and the effect of EGCG on the antioxidative activity and emulsifying properties of α-La were investigated. The turbidity of a solution of 1% α-La and 0.2% EGCG decreased after 24h reaction at pH 8.0 and 60°C, less than the value of α-La alone. The interaction between α-La and EGCG at pH 8.0 was more pronounced than that at pH 6.0 or 7.0, as evidenced by the reduced amplitude of amide A, and I, and II bands of α-La by FT-IR and the increase in the total phenolic content and denaturation temperature. These results implied that EGCG was covalently linked to α-La at pH 8.0. In addition, compared with the control of α-La, α-La–EGCG complexes had significantly increased antioxidative activity. The emulsions stabilized by α-La–EGCG complexes, compared with that by α-La alone, had much smaller droplet sizes, which indicated the emulsifying property of α-La was improved.