Phenyl Boronate Research Articles

DFT Study in an Asymmetric Organocatalytic Homologation: Mechanism, Origin of Stereoselectivity

AbstractIn the present study, the mechanism, origin of stereoselectivity, and role of catalyst of the organocatalytic homologation reaction between phenyl boronate and trifluoromethyl diazomethane have been theoretically investigated using the density functional theory (DFT) method. Based on the calculations, the in situ generated ethanol plays significant role in triggering the reaction. Moreover, the [1,2]‐boron migration process is the stereoselectivity‐determining step, with the S‐configured isomer being generated predominantly. In addition, NCI analysis is performed to disclose the origin of stereoselectivity.

Chitosan nanomedicine containing RGD peptide and PAD4 inhibitor based on phenyl boronate coupling inhibition of primary tumor growth and lung metastasis

Stimulus-responsive nanodrugs have been extensively studied and their structural changes in the cells are important for controlled intracellular drug release. Histone citrullination of peptidylarginine deiminase 4 (PAD4) regulates the expression of tumor suppressor genes. In our previous study, compounds such as YW3–56 (356) were developed as potent PAD4 inhibitors with excellent anti-tumor activity in vitro and in vivo. To enhance the antitumor activity and improve the bioavailability, we further optimized the structure by modifying the phenylboronic acid moiety to the PAD4 inhibitor (4B). Taking advantage of the oxidative stress responsiveness of the phenylboronic acid moiety, in this study, we covalently attached 4B to RGD sequence peptide modified chitosan (K-CRGDV) to construct this new oxidative stress responsive nanodrug (K-CRGDV-4B). The modification of RGD sequence peptide conferred the nanodrug the ability to actively target tumors. The release mechanism was verified by UV-Vis spectroscopy, NMR. The anti-tumor and anti-metastatic properties of K-CRGDV-4B were demonstrated by in vitro cytotoxicity assay and in vivo mouse Lewis lung cancer metastasis model. In addition, K-CRGDV-4B modulates the ratio of immune cells in LLC tumor-bearing mice. Immunosuppressive proteins such as PD1 were inhibited, while IFN-γ and IFN-β, which are stimulators of tumor immune responses, were upregulated. Overall, K-CRGDV-4B is a stimulus-responsive nanodrug that responds to the tumor microenvironment by inhibiting PAD4 activity, blocking the formation of neutrophil extracellular traps (NETs), and improving the tumor immune microenvironment.

Microenvironment responsive nanocomposite hydrogel with NIR photothermal therapy, vascularization and anti-inflammation for diabetic infected wound healing.

Bacterial infection, excessive inflammation and damaging blood vessels network are the major factors to delay the healing of diabetic ulcer. At present, most of wound repair materials are passive and can't response to the wound microenvironment, resulting in a low utilization of bioactive substances and hence a poor therapeutic effect. Therefore, it's essential to design an intelligent wound dressing responsive to the wound microenvironment to achieve the release of drugs on-demand on the basis of multifunctionality. In this work, metformin-laden CuPDA NPs composite hydrogel (Met@ CuPDA NPs/HG) was fabricated by dynamic phenylborate bonding of gelatin modified by dopamine (Gel-DA), Cu-loaded polydopamine nanoparticles (CuPDA NPs) with hyaluronic acid modified by phenyl boronate acid (HA-PBA), which possessed good injectability, self-healing, adhesive and DPPH scavenging performance. The slow release of metformin was achieved by the interaction with CuPDA NPs, boric groups (B-N coordination) and the constraint of hydrogel network. Metformin had a pH and glucose responsive release behavior to treat different wound microenvironment intelligently. Moreover, CuPDA NPs endowed the hydrogel excellent photothermal responsiveness to kill bacteria of >95% within 10min and also the slow release of Cu2+ to protect wound from infection for a long time. Met@ CuPDA NPs/HG also recruited cells to a certain direction and promoted vascularization by releasing Cu2+. More importantly, Met@CuPDA NPs/HG effectively decreased the inflammation by eliminating ROS and inhibiting the activation of NF-κB pathway. Animal experiments demonstrated that Met@CuPDA NPs/HG significantly promoted wound healing of diabetic SD rats by killing bacteria, inhibiting inflammation, improving angiogenesis and accelerating the deposition of ECM and collagen. Therefore, Met@CuPDA NPs/HG had a great application potential for diabetic wound healing.

Photoactivation of Boronic Acid Prodrugs via a Phenyl Radical Mechanism: Iridium(III) Anticancer Complex as an Example.

Boronic acid (or ester) is a well-known temporary masking group for developing anticancer prodrugs responsive to tumoral reactive oxygen species (ROS), but their clinic application is largely hampered by the low activation efficiency. Herein, we report a robust photoactivation approach that can spatiotemporally convert boronic acid-caged iridium(III) complex IrBA into bioactive IrNH2 under hypoxic tumor microenvironments. Mechanistic studies show that the phenyl boronic acid moiety in IrBA is in equilibrium with phenyl boronate anion that can be photo-oxidized to generate phenyl radical, a highly reactive species that is capable of rapidly capturing O2 at extremely low concentrations (down to 0.02%). As a result, while IrBA could hardly be activated by intrinsic ROS in cancer cells, upon light irradiation, the prodrug is efficiently converted into IrNH2 even in limited O2 supply, along with direct damage to mitochondrial DNA and potent antitumor activities in hypoxic 2D monolayer cells, 3D tumor spheroids, and mice bearing tumor xenografts. Of note, the photoactivation approach could be extended to intermolecular photocatalytic activation by external photosensitizers with red absorption and to activate prodrugs of clinic compounds, thus offering a general approach for activation of anticancer organoboron prodrugs.

Engineered multifunctional nanocomposite hydrogel dressing to promote vascularization and anti-inflammation by sustained releasing of Mg2+ for diabetic wounds

Diabetic ulcers are common and severe chronic wound. For the treatment of diabetic ulcers, due to the existence of sinus and fistula in the wound sites, the prefabricated hydrogel could not cover the deep wounds well and result in further presence and proliferation of bacteria at the deep wound site, which increases the risk of secondary infection. In addition, most current hydrogel dressings do not rapidly repair wounds due to the lack of bioactivity. Therefore, it's essential to develop a bioactive hydrogel dressing with self-adaption to solve the above clinical problems. In this work, HQ hydrogel was fabricated by the self-crosslinking of hyaluronic acid modified with phenyl boronate acid (HA-PBA) by boronate ester dynamic bonds and electronic interaction between quaternized chitosan (QCS) and HA-PBA, which possessed good injectability, self-healing, adhesion and antibacterial properties. Especially, HQ hydrogel with good self-adaption can be shaped as needed and automatically adapt to the wounds with all kinds of shapes. In addition, Magnesium-containing bioactive glasses (BGs) with a uniform spherical morphology and amorphous structure were incorporated into HQ hydrogel to achieve nanocomposite hydrogel (HQB), which possessed good bioactivity and could slowly release magnesium to recruit cells, promote vascularization and anti-inflammation. More importantly, animal experiments showed that HQB hydrogel can strikingly accelerate wound healing of diabetic SD rats by improving granulation tissues formation, collagen deposition, the formation of blood vessels and decreasing inflammation in the wound sites, indicating a good clinical application prospect as wound dressing for diabetic wound healing.

Expeditious base-free solid-state reaction between phenyl boronates and hydrogen peroxide on silica gel

The expeditious base-free reaction between a phenyl boronate film and H2O2 vapor can be realized on a silica gel surface, playing an important role in sensor manufacturing applications and chemical production.

AIE-based fluorescent boronate probe and its application in peroxynitrite imaging

Fluorescent probes have contributed greatly to our understanding of the biological role of peroxynitrite (ONOO–). The ONOO– fluorescence probe characterized by the arlyboronate received a moderate opening fluorescence response, and the borate-masked probe significantly increased the sensitivity of ONOO–. Thus, two simple fluorescent probes (ADB and ANB) with the recognition receptor of phenyl boronate moiety were constructed for the detection of ONOO–. The change of emission spectrum was affected differently by the electron donating (or withdrawing) of the substituents. ANB was shown to have a low sensitivity and quantum yield towards ONOO– in aqueous solution, whereas ADB with aggregation-induced emission (AIE) process exhibited not only good sensitivity for ONOO– with a detection limit of 75 nM, but also ADB could be used to quantitative detecting ONOO– in response to concentrations of ONOO– within 20 s. Importantly, ADB had good performance for the detection of exogenous ONOO– in the RAW 264.7 cells.

A Facile Route to Melaminophenyl Boronates using 2, 4-Diazido-6- Chloro-1, 3, 5-Triazine as an Electrophilic Reagent

We have synthesised a novel class of melaminyl derivative, containing a phenyl boronate or boronate ester group. The synthetic methodology employed was designed to be facile involving the use of 2, 4-diazido-6-chloro-1, 3, 5-triazine as an electrophilic reagent for nucleophilic substitution of chlorine with a weakly reactive amine. The reagent was chosen with a view to improve the organic solubility of the product and aid isolation by allowing the reaction to be followed by fluorescence quenching (Tlc, F234) and IR (the presence of two N3 peaks).

ROS-responsive and multifunctional anti-Alzheimer prodrugs: Tacrine-ibuprofen hybrids via a phenyl boronate linker.

Current drugs available in clinic for Alzheimer's disease (AD) treatment can only alleviate disease symptoms without clearly curing or delaying the process of AD. And some AD drugs failed in Phase III clinical trials are only focused on targeting amyloid-β (Aβ). Therefore, an alternative strategy in AD drug design is meaningful to be involved in the multiple pathogenic factors which can affect each other at multiple levels. Herein, we report a series of ROS-responsive prodrugs based on multi-target-directed ligands (MTDLs) approach, which can specifically release tacrine derivatives and ibuprofen under oxidation of ROS and show acetylcholinesterase (AChE)-inhibiting, neuron-protective and anti-inflammatory effects in extracellular or intracellular assays. Related biological study illustrated that compound 22 was able to permeate blood-brain-barrier (BBB) showing little hepatotoxicity in comparison to tacrine. Besides, 22 hinted a therapeutic clue in AD-treatment by regulating proinflammatory factors (IL-1β and TNF-α) and apoptosis related proteins (Bax, Bcl-2 and cleaved caspase-3). Further spatial memory assays in Aβ-induced AD model showed that 22 enhanced the ability of learning and memory. Our study proves that the strategy of ROS-responsive prodrugs has promise for AD treatments in future and offers a way for AD drug development.

Boron Effect on Sugar-Based Organogelators.

The reaction of several alkylglucosides with phenyl boronic acid permitted easy access to a series of alkylglucoside phenyl boronate derivatives. This type of compound has structures similar to those of known benzylidene glucoside organogelators except for the presence of a boronate function in place of the acetal one. Low to very low concentrations of these amphiphilic molecules produced gelation of several organic solvents. The rheological properties of the corresponding soft materials characterized them as elastic solids. They were further characterized by SEM to obtain more information on their morphologies and by SAXS to determine the type of self-assembly involved within the gels. The sensitivity of the boronate function towards hydrolysis was also investigated. We demonstrated that a small amount of water (5 % v/v) was sufficient to disrupt the organogels leading to the original alkylglucoside and phenyl boronic acid; an important difference with the stable benzylidene-based organogelators. Such water-sensitive boronated organogelators could be suitable substances for the preparation of smart soft material for topical drug delivery.

An Optimised Di-Boronate-ChemMatrix Affinity Chromatography to Trap Deoxyfructosylated Peptides as Biomarkers of Glycation.

We report herein a novel ChemMatrix® Rink resin functionalised with two phenylboronate (PhB) moieties linked on the N-α and N-ε amino functions of a lysine residue to specifically capture deoxyfructosylated peptides, compared to differently glycosylated peptides in complex mixtures. The new PhB-Lys(PhB)-ChemMatrix® Rink resin allows for exploitation of the previously demonstrated ability of cis diols to form phenylboronic esters. The optimised capturing and cleavage procedure from the novel functionalised resin showed that only the peptides containing deoxyfructosyl-lysine moieties can be efficiently and specifically detected by HR-MS and MS/MS experiments. We also investigated the high-selective affinity to deoxyfructosylated peptides in an ad hoc mixture containing unique synthetic non-modified peptides and in the hydrolysates of human and bovine serum albumin as complex peptide mixtures. We demonstrated that the deoxyfructopyranosyl moiety on lysine residues is crucial in the capturing reaction. Therefore, the novel specifically-designed PhB-Lys(PhB)-ChemMatrix® Rink resin, which has the highest affinity to deoxyfructosylated peptides, is a candidate to quantitatively separate early glycation peptides from complex mixtures to investigate their role in diabetes complications in the clinics.

Preparation and Characterization of pH Responsive Complex Micelles with Dynamic Boronate Cross-Linking

Two kinds of diblock copolymers containing glucose and phenylboronic acid moieties, respectively, poly (ethylene glycol)-b-poly (gluconamidoethyl methacrylate) (PEG-b-PGAMA) and poly (ethylene glycol)-b-poly (2-aminoethyl methacrylate-co-3-nitrophenyboronic acid methacrylate) (PEG-b-P(AMA-co-NPBMA)) were synthesized via atom transfer radical polymerization (ATRP) and post polymerization modification (PPM). Well-defined structure and narrow molecular weight distribution of the polymers were confirmed by proton Nuclear Magnetic Resonance (1H NMR) and Gel Permeation Chromatography (GPC). Based on the cross-linking between the diol groups of the glycopolymer and phenylboronic acid under physiological pH (7.4), complex micelles composed of PEG outer shell and boronate ester cross-linking core with a hydrodynamic diameter around 20nm were formed. Morphology, size and assembly behavior of the complex micelles were investigated by 1H NMR, transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results showed the formation/cleavage of boronate ester linkage is reversible upon the variation of solution pH, the complex micelles displayed pH sentiveties of assembling/disassembling behavior. Above pH 7.4, stable spherical micelles can be formed, whereas pH less than 5.5, the micelles dissociated into unimers. Therefore, such pH-responsive micelles based on dynamic complexation of phenyl boronate bonds are expected to be applied to pH-responsive nanodrug carriers

Studies on the purification of antibody fragments

Abstract Antigen-binding fragments (Fabs) are becoming increasingly prevalent as an alternative to full-length monoclonal antibodies. In this work, the digestion of a mixture of different human antibodies isotypes was optimized in terms of different parameters, including the concentration of papain and cysteine, and the digestion time. The recovery of the Fab fragments was subsequently evaluated by designing four different downstream purification schemes, where the use of affinity chromatography (protein A and L) was the most efficient to isolate the Fab fragments after IgG digestion. A rapid screening of optimal conditions for the binding of pure Fab fragments to four non-affinity chromatography resins was then performed using micro-columns fabricated in a PDMS microfluidic device. The goal of these studies was to screen and evaluate the performance of two cation exchange (carboxymethyl and heparin) and two multimodal (Capto MMC and phenyl boronate) ligands for the capture of Fabs under a wide range of pH (5–9) and conductivity (up to 8 mS/cm) conditions. Multimodal resins showed the best results in binding Fab fragments, particularly at pH 5, well below the range of isoelectric points of the target Fab molecules. In addition, these resins demonstrated to have a salt-tolerant behaviour, meaning that the binding of Fab fragments was not significantly impacted when the conductivity of the adsorption buffer was increased to near-physiological conditions (8 mS/cm).

A Fluorescent Probe for Hydrogen Peroxide in Vivo Based on the Modulation of Intramolecular Charge Transfer.

Endogenous hydrogen peroxide in vivo is related to many diseases, including cancer, diabetes, cardiovascular disease, and neurodegenerative disorders. Although many probes for detection of H2O2 have been explored, rapid response probes are still expected for in vivo application. Here, a new probe (PAM-BN-PB) was designed based on an intramolecular charge transfer (ICT) process with three parts: phenanthroimidazole, benzonitrile, and phenyl boronate. By modulation ICT process of PAM-BN-PB, H2O2 in solution systems can be detected with good selectivity. The exogenous and endogenous H2O2 in normal living cells, ischemia-reperfusion injury cells, and animals all can be imaged by PAM-BN-PB.

Synthesis, characterization and antimicrobial activity of novel Schiff base tethered boronate esters of 1,2-O-isopropylidene-α-d-xylofuranose

A series of twenty one Schiff bases based on boronate ester of 1,2-O-isopropylidene-α-d-xylofuranose scaffold were designed and synthesized by condensation of formyl or amino phenyl boronate esters with substituted anilines or 2-hydroxybenzaldehydes, respectively. All the imines are remarkably stable crystalline solids and were obtained in good yields. All the products were fully characterized by FT-IR, multinuclear NMR (1H, 13C and 11B) spectroscopy, and elemental analysis. Furthermore, the molecular structures of two of the Schiff bases were established by single crystal X-ray diffraction analysis. All the compounds have been screened for in vitro antimicrobial activity against various Gram-positive and Gram-negative bacterial and fungal strains. They exhibited moderate to good inhibitory activity against most of the tested organisms in comparison with standard drugs.

Phenylboronate chromatography selectively separates glycoproteins through the manipulation of electrostatic, charge transfer, and cis-diol interactions.

Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis-diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation-exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge-transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins.

Binding of mucin to water-soluble and surface-grafted boronate-containing polymers

The binding of mucin to water-soluble copolymers of N,N-dimethylacrylamide and N-acryloyl-m-aminophenylboronic acid grafted on the surface of glass is studied. Atomic force microscopy studies show that many graft copolymer islands 20-200 nm in diameter and 50 nm in height occur on the modified surface of flat glass. Owing to the presence of phenyl boronate groups, the copolymer behaves as a weak polyelectrolyte (pK(a) = 9.0) and, in the grafted state in an aqueous solution, experiences reversible transitions between states with higher and lower degrees of ionization. As evidenced by spectral correlation interferometry, this phenomenon brings about a change in the thickness of the grafted layer by approximately 0.5 nm. The ability of phenyl boronate groups to form cyclic esters with diol and polyol groups results in complexation of the soluble copolymer with mucin oligosaccharides and entails the appearance of slowly growing submicron particles formed by similarly charged polymers. The specificity of complexation is confirmed by dissolution of particles in the presence of fructose: a saccharide with a strong affinity for phenyl boronate groups. The binding of mucin to glass, which is chemically modified with the above copolymer, leads to formation of an adsorption layer with a thickness of 1.2-1.8 nm. Thus, boronate-containing copolymers are suitable for preparing carriers with controllable adsorption properties with respect to polyols, including mucinlike proteins of cellular glycocalyxes. (Less)

Studies on the adsorption of cell impurities from plasmid-containing lysates to phenyl boronic acid chromatographic beads

Plasmid DNA (pDNA) is purified directly from alkaline lysis-derived Escherichia coli (E. coli) lysates by phenyl boronate (PB) chromatography. The method explores the ability of PB ligands to bind covalently, but reversibly, to cis-diol-containing impurities like RNA and lipopolysaccharides (LPS), leaving pDNA in solution. In spite of this specificity, cis-diol free species like proteins and genomic DNA (gDNA) are also removed. This is a major advantage since the process is designed to keep the target pDNA from binding. The focus of this paper is on the study of the secondary interactions between the impurities (RNA, gDNA, proteins, LPS) in a pDNA-containing lysate and 3-amino PB controlled pore glass (CPG) matrices. Runs were designed to evaluate the role of adsorption buffer composition, feed type (pH, salt content), CPG matrix and sample pretreatment (RNase A, isopropanol precipitation). Water was chosen as the adsorption buffer over MgCl(2) solutions since it maximised pDNA yield (96.2±4.9%) and protein removal (61.3±3.0%), while providing for a substantial removal of RNA (65.5±3.5%) and gDNA (44.7±14.1%). Although the use of pH 3.5 maximised removal of impurities (~75%), the best compromise between plasmid yield (~96%) and RNA clearance (~60-70%) was obtained for a pH of 5.2. Plasmid yield was maximal (>96%) when the concentration of acetate and potassium ions in the incoming lysate feed were 1.7 M and 1.0 M, respectively. The pre-treatment of lysates with RNase A deteriorated the performance since the resulting oligoribonucleotides lack the cis-diol group at their 3' termini. Overall, the results support the idea that charge transfer interactions between the boron atom at acidic pH and electron donor groups in the aromatic bases of nucleic acids and side residues of proteins are responsible for the non-specific removal of gDNA, RNA and proteins.

Isolation and characterization of tetrahydrofuran-degrading Rhodococcus aetherivorans strain M8

We isolated tetrahydrofuran (THF)-degrading bacteria from waste sludge obtained from a chemical factory in Japan. The isolate designated as strain M8 was identified as Rhodococcus aetherivorans by sequence analysis of its 16S rRNA gene. It grew in a medium containing THF as the sole source of carbon and energy, and its optimal growth pH range and temperature were 6–9 and 37°C, respectively. Strain M8 grew even in the presence of 35mM THF. For its growth, this bacterium used 1,4-butanediol and γ-butyrolactone, which are supposed to be metabolites of THF. To elucidate the pathway involved in THF metabolism in strain M8, the resting cell reaction was performed, and the metabolites of THF were analyzed. In the resting cell reaction, 5mM THF was completely degraded within 5h. Cells were harvested at 2, 3, and 4h after the initiation of the reaction; the intermediates accumulated in the cells were extracted using methanol and were derivatized using phenyl boronate. Gas chromatography–mass spectrometry (GC–MS) analysis of the derivatized products showed 4-hydroxybutyrate accumulating in the resting cells. This result suggests that R. aetherivorans strain M8 degrades THF via the oxidation pathway.

Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5-8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris-HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved.