Optimization of α-amido boronic acids via cryo-electron microscopy analysis: Discovery of a novel highly selective immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual inhibitor.

The immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual blockade has been reported to suppress B cell differentiation and activation, suggesting that the dual inhibition of LMP7/LMP2 is a promising approach for treating autoimmune diseases. In contrast, the inhibition of the constitutive proteasome subunit ß5c correlates with cytotoxicity against non-immune cells. Therefore, LMP7/LMP2 dual inhibitors with high selectivity over ß5c may be desirable for treating autoimmune diseases. In this study, we present the optimization and discovery of α-amido boronic acids using cryo-electron microscopy (cryo-EM). The exploitation of structural differences between the proteasome subunits led to the identification of a highly selective LMP7/LMP2 dual inhibitor 19. Molecular dynamics simulation based on cryo-EM structures of the proteasome subunits complexed with 19 explained the inhibitory activity profile. In mice immunized with 4-hydroxy-3-nitrophenylacetyl conjugated to ovalbumin, results indicate that 19 is orally bioavailable and shows promise as potential treatment for autoimmune diseases.

Phenylboronic Acid Modification Augments the Lysosome Escape and Antitumor Efficacy of a Cylindrical Polymer Brush-Based Prodrug.

Timely lysosome escape is of paramount importance for endocytosed nanomedicines to avoid premature degradation under the acidic and hydrolytic conditions in lysosomes. Herein, we report an exciting finding that phenylboronic acid (PBA) modification can greatly facilitate the lysosome escape of cylindrical polymer brushes (CPBs). On the basis of our experimental results, we speculate that the mechanism is associated with the specific interactions of the PBA groups with lysosomal membrane proteins and hot shock proteins. The featured advantage of the PBA modification over the known lysosome escape strategies is that it does not cause significant adverse effects on the properties of the CPBs; on the contrary, it enhances remarkably their tumor accumulation and penetration. Furthermore, doxorubicin was conjugated to the PBA-modified CPBs with a drug loading content larger than 20%. This CPBs-based prodrug could eradicate the tumors established in mice by multiple intravenous administrations. This work provides a novel strategy for facilitating the lysosome escape of nanomaterials and demonstrates that PBA modification is an effective way to improve the overall properties of nanomedicines including the tumor therapeutic efficacy.

Merging the Versatile Functionalities of Boronic Acid with Peptides.

Peptides inherently feature the favorable properties of being easily synthesized, water-soluble, biocompatible, and typically non-toxic. Thus, boronic acid has been widely integrated with peptides with the goal of discovering peptide ligands with novel biological activities, and this effort has led to broad applications. Taking the integration between boronic acid and peptide as a starting point, we provide an overview of the latest research advances and highlight the versatile and robust functionalities of boronic acid. In this review, we summarize the diverse applications of peptide boronic acids in medicinal chemistry and chemical biology, including the identification of covalent reversible enzyme inhibitors, recognition, and detection of glycans on proteins or cancer cell surface, delivery of siRNAs, development of pH responsive devices, and recognition of RNA or bacterial surfaces. Additionally, we discuss boronic acid-mediated peptide cyclization and peptide modifications, as well as the facile chemical synthesis of peptide boronic acids, which paved the way for developing a growing number of peptide boronic acids.

An intramolecular coupling approach to alkyl bioisosteres for the synthesis of multisubstituted bicycloalkyl boronates.

Bicyclic hydrocarbons, and bicyclo[1.1.1]pentanes (BCPs) in particular, are playing an emerging role as saturated bioisosteres in pharmaceutical, agrochemical and materials chemistry. Taking advantage of strain-release strategies, prior synthetic studies have featured the synthesis of bridgehead-substituted (C1, C3) BCPs from [1.1.1]propellane. Here, we describe an approach to access multisubstituted BCPs via intramolecular cyclization. In addition to C1,C3-disubstituted BCPs, this method also enables the construction of underexplored multisubstituted (C1, C2 and C3) BCPs from readily accessible cyclobutanones. The broad generality of this method has also been examined through the synthesis of a variety of other caged bicyclic molecules, ranging from [2.1.1] to [3.2.1] scaffolds. The modularity afforded by the pendant bridgehead boron pinacol esters generated during the cyclization reaction has been demonstrated through several downstream functionalizations, highlighting the ability of this approach to enable the programmed and divergent synthesis of multisubstituted bicyclic hydrocarbons.

Aza-Matteson Reactions via Controlled Mono- and Double-Methylene Insertions into Nitrogen-Boron Bonds.

Boron-homologation reactions represent an efficient and programmable approach to prepare alkylboronates, which are valuable and versatile synthetic intermediates. The typical boron-homologation reaction, also known as the Matteson reaction, involves formal carbenoid insertions into C-B bonds. Here we report the development of aza-Matteson reactions via carbenoid insertions into the N-B bonds of aminoboranes. By changing the leaving groups of the carbenoids and altering Lewis acid activators, selective mono- and double-methylene insertions can be realized to access various α- and ß-boron-substituted tertiary amines, respectively, from common secondary amines. The derivatization of complex amine-containing bioactive molecules, diverse functionalization of the boronate products, and sequential insertions of different carbenoids have also been achieved.

Dual-Functional Surfaces Based on an Antifouling Polymer and a Natural Antibiofilm Molecule: Prevention of Biofilm Formation without Using Biocides.

Pathogenic biofilms formed on the surfaces of implantable medical devices and materials pose an urgent global healthcare problem. Although conventional antibacterial surfaces based on bacteria-repelling or bacteria-killing strategies can delay biofilm formation to some extent, they usually fail in long-term applications, and it remains challenging to eradicate recalcitrant biofilms once they are established and mature. From the viewpoint of microbiology, a promising strategy may be to target the middle stage of biofilm formation including the main biological processes involved in biofilm development. In this work, a dual-functional antibiofilm surface is developed based on copolymer brushes of 2-hydroxyethyl methacrylate (HEMA) and 3-(acrylamido)phenylboronic acid (APBA), with quercetin (Qe, a natural antibiofilm molecule) incorporated via acid-responsive boronate ester bonds. Due to the antifouling properties of the hydrophilic poly(HEMA) component, the resulting surface is able to suppress bacterial adhesion and aggregation in the early stages of contact. A few bacteria are eventually able to break through the protection of the anti-adhesion layer leading to bacterial colonization. In response to the resulting decrease in the pH of the microenvironment, the surface could then release Qe to interfere with the microbiological processes related to biofilm formation. Compared to bactericidal and anti-adhesive surfaces, this dual-functional surface showed significantly improved antibiofilm performance to prevent biofilm formation involving both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus for up to 3 days. In addition, both the copolymer and Qe are negligibly cytotoxic, thereby avoiding possible harmful effects on adjacent normal cells and the risk of bacterial resistance. This dual-functional design approach addresses the different stages of biofilm formation, and (in accordance with the growth process of the biofilm) allows sequential activation of the functions without compromising the viability of adjacent normal cells. A simple and reliable solution may thus be provided to the problems associated with biofilms on surfaces in various biomedical applications.

The synthesis and efficiency investigation of a boronic acid-modified magnetic chitosan quantum dot nanocomposite in the detection of Cu2+ ions.

We prepared the magnetic chitosan carbon quantum dot nanoparticles (Fe3O4@CQD NPs) via the hydrothermal treatment of chitosan biopolymer and then its magnetization with Fe3O4 nanoparticles. (4-Acetylphenyl)boronic acid compound was utilized for the modification of surface of Fe3O4@CQD nanoparticles via the covalent imine bond formation between NH2 groups of chitosan quantum dot with carbonyl functional of acetyl-substituted arylboronic acid. The synthesized Fe3O4@CQD@AP-B(OH)2 was characterized by FE-SEM, EDS, XRD, VSM and ICP-OES analysis and its fluorescence property was studied. This magnetic multifunctional nanoplatform sensor has shown high potential sensitivity for Cu2+ ions (in the range of 1.0-30.0 µM with limit of detection 0.3 µM) through interaction of cupric ions with the boronic-acid moiety.

Recent Developments and Strategies for Mutually Orthogonal Bioorthogonal Reactions.

Over the past decade, several different metal-free bioorthogonal reactions have been developed to enable simultaneous double-click labeling with minimal-to-no competing cross-reactivities; such transformations are termed 'mutually orthogonal'. More recently, several examples of successful triple ligation strategies have also been described. In this minireview, we discuss selected aspects of the development of orthogonal bioorthogonal reactions over the past decade, including general strategies to drive future innovations to achieve simultaneous, mutually orthogonal click reactions in one pot.

Caffeic Acid Phenethyl Ester-Incorporated Radio-Sensitive Nanoparticles of Phenylboronic Acid Pinacol Ester-Conjugated Hyaluronic Acid for Application in Radioprotection.

We synthesized phenylboronic acid pinacol ester (PBPE)-conjugated hyaluronic acid (HA) via thiobis(ethylamine) (TbEA) linkage (abbreviated as HAsPBPE conjugates) to fabricate the radiosensitive delivery of caffeic acid phenetyl ester (CAPE) and for application in radioprotection. PBPE was primarily conjugated with TbEA and then PBPE-TbEA conjugates were conjugated again with hyaluronic acid using carbodiimide chemistry. CAPE-incorporated nanoparticles of HAsPBPE were fabricated by the nanoprecipitation method and then the organic solvent was removed by dialysis. CAPE-incorporated HAsPBPE nanoparticles have a small particle size of about 80 or 100 nm and they have a spherical shape. When CAPE-incorporated HAsPBPE nanoparticles were irradiated, nanoparticles became swelled or disintegrated and their morphologies were changed. Furthermore, the CAPE release rate from HAsPBPE nanoparticles were increased according to the radiation dose, indicating that CAPE-incorporated HAsPBPE nanoparticles have radio-sensitivity. CAPE and CAPE-incorporated HAsPBPE nanoparticles appropriately prevented radiation-induced cell death and suppressed intracellular accumulation of reactive oxygen species (ROS). CAPE and CAPE-incorporated HAsPBPE nanoparticles efficiently improved survivability of mice from radiation-induced death and reduced apoptotic cell death. We suggest that HAsPBPE nanoparticles are promising candidates for the radio-sensitive delivery of CAPE.

Boronic acid complexes with amino phenolic N,O-ligands and their use for non-covalent protein fluorescence labeling.

Phenylboronic acid (PBA) forms neutral tetrahedral N,O-coordinated 6-membered cyclic complexes with stability constants reaching the values as large as 1.3 × 104 M-1 at pH 7.4 in water with amino phenolic compounds including 2-(2'-hydroxyphenyl)-1H-benzimidazole (HPBI) often used for protein probing and labeling. The crystal structures of isolated complexes demonstrate unusually high for boronate adducts degree of the tetrahedral character of the boron atom with short B-N bonds in agreement with their high solution stability. The complexation of PBA with HPBI, causes a strong enhancement of the fluorescence of the "enol" form of the ligand, increases the affinity of the dye to a protein (bovine serum albumin) and makes more pronounced the shift in emission maximum induced by the protein binding. Similar, but larger effects are observed with an amino HPBI derivative and with a stronger boronic acid benzoxaborole. Thus, the binding constant to the protein about 2 × 104 M-1 for free HPBI increases to 1.2 × 106 M-1 for the complex of 5-amino-HPBI with benzoxaborole making it suitable for an efficient non-covalent protein labeling or bioconjugation.

Peptidic boronic acids are potent cell-permeable inhibitors of the malaria parasite egress serine protease SUB1.

Malaria is a devastating infectious disease, which causes over 400,000 deaths per annum and impacts the lives of nearly half the world's population. The causative agent, a protozoan parasite, replicates within red blood cells (RBCs), eventually destroying the cells in a lytic process called egress to release a new generation of parasites. These invade fresh RBCs to repeat the cycle. Egress is regulated by an essential parasite subtilisin-like serine protease called SUB1. Here, we describe the development and optimization of substrate-based peptidic boronic acids that inhibit Plasmodium falciparum SUB1 with low nanomolar potency. Structural optimization generated membrane-permeable, slow off-rate inhibitors that prevent Pfalciparum egress through direct inhibition of SUB1 activity and block parasite replication in vitro at submicromolar concentrations. Our results validate SUB1 as a potential target for a new class of antimalarial drugs designed to prevent parasite replication and disease progression.

Bioadhesive glycosylated nanoformulations for extended trans-corneal drug delivery to suppress corneal neovascularization.

Eye-drop formulations as conventional regimens to tackle ocular diseases are far from efficient due to the rapid clearance by eye tears and the blockage of the corneal epithelium barrier. Here, we describe a bioadhesive glycosylated nanoplatform with boric acid pendants as a drug carrier for noninvasive trans-corneal delivery of drugs to treat corneal neovascularization (CNV), a serious corneal disease resulting in significant vision impairment. This biocompatible nanoplatform is formulated from a synthetic amphiphilic boric acid-based copolymer self-assembling to form highly stable micelles with a high loading capacity for dexamethasone (DEX). The nanoplatform is demonstrated to be in contact with the corneal epithelium for a long period under the bioadhesive function of boric acid modules and releases the drug over 96 h in a controlled manner. Our results also suggest that the nanoplatform can be efficiently internalized by corneal epithelial cells in vitro and realize transcytosis in vivo to greatly enhance the transcorneal penetration of the loaded drugs into the pathological corneal stroma. On topical application against rat corneal alkali burn, the nanoformulation presents more robust efficacy on neovascularization suppression and inflammation elimination than free DEX with a negligible effect on normal tissues. This bioadhesive strategy which focuses on extending ocular drug retention and improving trans-corneal drug delivery not only highlights an approach for alternative noninvasive therapy of CNV but also provides a versatile paradigm for other biomedical applications by overcoming protective barriers.

Towards Enhanced MRI Performance of Tumor-Specific Dimeric Phenylboronic Contrast Agents.

It is known that phenylboronic acid (PBA) can target tumor tissues by binding to sialic acid, a substrate overexpressed by cancer cells. This capability has previously been explored in the design of targeting diagnostic probes such as Gd- and 68Ga-DOTA-EN-PBA, two contrast agents for magnetic resonance imaging (MRI) and positron emission tomography (PET), respectively, whose potential has already been demonstrated through in vivo experiments. In addition to its high resolution, the intrinsic low sensitivity of MRI stimulates the search for more effective contrast agents, which, in the case of small-molecular probes, basically narrows down to either increased tumbling time of the entire molecule or elevated local concentration of the paramagnetic ions, both strategies resulting in enhanced relaxivity, and consequently, a higher MRI contrast. The latter strategy can be achieved by the design of multimeric GdIII complexes. Based on the monomeric PBA-containing probes described recently, herein, we report the synthesis and characterization of the dimeric analogues (GdIII-DOTA-EN)2-PBA and (GdIII-DOTA-EN)2F2PBA. The presence of two Gd ions in one molecule clearly contributes to the improved biological performance, as demonstrated by the relaxometric study and cell-binding investigations.

Installation of an aryl boronic acid function into the external section of N-aryl-oxazolidinones: Synthesis and antimicrobial evaluation.

N-aryl-oxazolidinones is a prominent family of antimicrobials used for treating infections caused by clinically prevalent Gram-positive bacteria. Recently, boron-containing compounds have displayed intriguing potential in the antibiotic discovery setting. Herein, we report the unprecedented introduction of a boron-containing moiety such as an aryl boronic acid in the external region of the oxazolidinone structure via a chemoselective acyl coupling reaction. As a result, we accessed a series of analogues with a distal aryl boronic pharmacophore on the oxazolidinone scaffold. We identified that a peripheric linear conformation coupled with freedom of rotation and no further substitution on the external aryl boronic ring, an amido linkage with hydrogen bonding character, in addition to a para-relative disposition between boronic group and linker, are the optimal combination of structural features in this series for antimicrobial activity. In comparison to linezolid, the analogue comprising all those features, compound 20b, displayed levels of antimicrobial activity augmented by an eight-fold to a thirty-two-fold against a panel of Gram-positive strains, and a near one hundred-fold against Escherichia coli JW5503, a Gram-negative mutant strain with a defective efflux capability.

Boronic Acids and Their Derivatives in Medicinal Chemistry: Synthesis and Biological Applications.

Boron containing compounds have not been widely studied in Medicinal Chemistry, mainly due to the idea that this group could confer some toxicity. Nowadays, this concept has been demystified and, especially after the discovery of the drug bortezomib, the interest for these compounds, mainly boronic acids, has been growing. In this review, several activities of boronic acids, such as anticancer, antibacterial, antiviral activity, and even their application as sensors and delivery systems are addressed. The synthetic processes used to obtain these active compounds are also referred. Noteworthy, the molecular modification by the introduction of boronic acid group to bioactive molecules has shown to modify selectivity, physicochemical, and pharmacokinetic characteristics, with the improvement of the already existing activities. Besides, the preparation of compounds with this chemical group is relatively simple and well known. Taking into consideration these findings, this review reinforces the relevance of extending the studies with boronic acids in Medicinal Chemistry, in order to obtain new promising drugs shortly.

In Vivo Targeting Using Arylboronate/Nopoldiol Click Conjugation.

Bioorthogonal click reactions yielding stable and irreversible adducts are in high demand for in vivo applications, including in biomolecular labeling, diagnostic imaging, and drug delivery. Previously, we reported a novel bioorthogonal "click" reaction based on the coupling of ortho-acetyl arylboronates and thiosemicarbazide-functionalized nopoldiol. We now report that a detailed structural analysis of the arylboronate/nopoldiol adduct by X-ray crystallography and 11B NMR reveals that the bioorthogonal reactants form, unexpectedly, a tetracyclic adduct through the cyclization of the distal nitrogen into the semithiocarbazone leading to a strong B-N dative bond and two new 5-membered rings. The cyclization adduct, which protects the boronate unit against hydrolytic breakdown, sheds light on the irreversible nature of this polycondensation. The potential of this reaction to work in a live animal setting was studied through in vivo capture of fluorescently labeled molecules in vivo. Arylboronates were introduced into tissues through intradermal injection of their activated NHS esters, which react with amines in the extracellular matrix. Fluorescently labeled nopoldiol molecules were administered systemically and were efficiently captured by the arylboronic acids in a location-specific manner. Taken together, these in vivo proof-of-concept studies establish arylboronate/nopoldiol bioorthogonal chemistry as a candidate for wide array of applications in chemical biology and drug delivery.

Designed Parasite-Selective Rhomboid Inhibitors Block Invasion and Clear Blood-Stage Malaria.

Rhomboid intramembrane proteases regulate pathophysiological processes, but their targeting in a disease context has never been achieved. We decoded the atypical substrate specificity of malaria rhomboid PfROM4, but found, unexpectedly, that it results from "steric exclusion": PfROM4 and canonical rhomboid proteases cannot cleave each other's substrates due to reciprocal juxtamembrane steric clashes. Instead, we engineered an optimal sequence that enhanced proteolysis >10-fold, and solved high-resolution structures to discover that boronates enhance inhibition >100-fold. A peptide boronate modeled on our "super-substrate" carrying one "steric-excluding" residue inhibited PfROM4 but not human rhomboid proteolysis. We further screened a library to discover an orthogonal alpha-ketoamide that potently inhibited PfROM4 but not human rhomboid proteolysis. Despite the membrane-immersed target and rapid invasion, ultrastructural analysis revealed that single-dosing blood-stage malaria cultures blocked host-cell invasion and cleared parasitemia. These observations establish a strategy for designing parasite-selective rhomboid inhibitors and expose a druggable dependence on rhomboid proteolysis in non-motile parasites.

α-Triazolylboronic Acids: A Promising Scaffold for Effective Inhibitors of KPCs.

Boronic acids are known reversible covalent inhibitors of serine ß-lactamases. The selectivity and high potency of specific boronates bearing an amide side chain that mimics the ß-lactam's amide side chain have been advanced in several studies. Herein, we describe a new class of boronic acids in which the amide group is replaced by a bioisostere triazole. The boronic acids were obtained in a two-step synthesis that relies on the solid and versatile copper-catalyzed azide-alkyne cycloaddition (CuAAC) followed by boronate deprotection. All of the compounds show very good inhibition of the Klebsiella pneumoniae carbapenemase KPC-2, with Ki values ranging from 1 nM to 1 µM, and most of them are able to restore cefepime activity against K. pneumoniae harboring blaKPC-2 . In particular, compound 1 e, bearing a sulfonamide substituted by a thiophene ring, proved to be an excellent KPC-2 inhibitor (Ki =30 nM); it restored cefepime susceptibility in KPC-Kpn cells (MIC=0.5 µg/mL) with values similar to that of vaborbactam (Ki =20 nM, MIC in KPC-Kpn 0.5 µg/mL). Our findings suggest that α-triazolylboronates might represent an effective scaffold for the treatment of KPC-mediated infections.

Design, synthesis and structure of a frustrated benzoxaborole and its applications in the complexation of amines, amino acids, and protein modification.

This study describes the design and synthesis of arylboronic acid 2, the first example of a permanently open "frustrated" benzoxaborole, along with an exploration of its application in bioconjugation. An efficient and high yielding seven-step synthesis was optimized. NMR experiments confirmed that compound 2 exists in the open ortho-hydroxyalkyl arylboronic acid structure 2-I, a form that is effectively prevented to undergo a dehydrative cyclization as a result of unfavorable geometry. Compound 2-I conjugates effectively with amines to form stable hemiaminal ether structures, including a highly effective reaction with lysozyme. Complexation with cysteine induces an open structure containing a free hydroxymethyl arm, with the amino and thiol groups reacting preferentially with the formyl group to form a N,S-acetal.

Dynamic monitoring and quantitative characterization of intracellular H2O2 content by using SERS based boric acid nanoprobe.

Quantitative characterization of intracellular H2O2 content, which is still difficult by the conventional biochemical methods due to the lack of real-time and non-invasive technique of single cell measurement, is a useful solution for cell state assessment. Based on the surface enhanced Raman scattering (SERS), we construct a novel boric acid (BA) nanoprobe to perform quantitative characterization of H2O2 content, in which the p-thiol benzene boric acid (4-MPBA) reporter molecule modified with gold nanorods (AuNRs) is employed for Raman signal enhancement. The achieved result demonstrates obvious advantages of the synthesized AuNRs/4-MPBA/BA nanoprobe in measurement sensitivity of H2O2 content. Importantly, this AuNRs/4-MPBA/BA nanoprobe will provide a powerful tool for dynamic monitoring and quantitative characterization of intracellular H2O2 content during cell apoptosis or other cell growth processes, and then achieve important reference data for studying the corresponding molecular mechanism.