Synthetic Lectin Research Articles

Genetically Encoded Boronolectin as a Specific Red Fluorescent UDP-GlcNAc Biosensor.

There is great interest in developing boronolectins that are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid- and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.

Dynamic Approach to Synthetic Lectin for Glucose with Boosted Binding Affinity through C-H Hydrogen Bonds.

A biomimetic receptor for glucose has been developed with high affinity and selectivity. The receptor was efficiently synthesized in three steps through dynamic imine chemistry followed by imine-to-amide oxidation. The receptor features two parallel durene panels, forming a hydrophobic pocket for [CH⋅⋅⋅π] interactions, and two pyridinium residues directing four amide bonds towards the pocket. These pyridinium residues not only improve solubility but also provide polarized C-H bonds for hydrogen bonding. Experimental data and DFT calculations show that these polarized C-H bonds significantly enhance substrate binding. These findings demonstrate the power of dynamic covalent chemistry for creating molecular receptors and using polarized C-H bonds for boosted carbohydrate recognition in water, providing a foundation for developing glucose-responsive materials and sensors.

Synthesis and Evaluation of Protein-Phenylboronic Acid Conjugates as Lectin Mimetics.

Glycan-binding molecules, such as lectins, are very important tools for characterizing, imaging, or targeting glycans and are often involved in either physiological or pathological processes. However, their availability is far less compared to the diversity of native glycans. Therefore, development of lectin mimetics with desired specificity and affinity is in high demand. Boronic acid reacts with 1,2- and 1,3-diols of saccharides in aqueous media through reversible boronate ester formation and are regarded as synthetic lectin mimetics. In this study, bovine serum albumin (BSA)–phenylboronic acid (PBA) conjugates were synthesized in a density-controlled manner by targeting both aspartic and glutamic acids to afford lectin mimetics with multivalent PBA, as multivalency is a key factor for glycan recognition in both specificity and affinity. The resultant BSA–PBA conjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Their macrophage cell surface glycan-binding capacity was characterized by a competitive lectin-binding assay examined by flow cytometry, and 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed biocompatibility. These novel lectin mimetics will find a broad range of applications as they can be wittingly modified, altering binding specificity and capacity.

Maltodextrin recognition by a macrocyclic synthetic lectin.

A monocyclic "temple" synthetic lectin, with extended electron-rich aromatic surfaces, shows enhanced affinities for carbohydrates in water. The methoxy substituents favour binding to α-linked maltodextrins, against the all-equatorial substrates normally favoured by this family of receptors.

Synthesis and evaluation of a desymmetrised synthetic lectin: an approach to carbohydrate receptors with improved versatility.

A new synthetic lectin features apolar surfaces provided by two different aromatic components (biphenyl and pyrenyl). Affinities up to 260 M(-1) are recorded for carbohydrates in water. The desymmetrised design has potential for variation to give receptors with a broadened range of capabilities.

Molecular recognition of surface-immobilized carbohydrates by a synthetic lectin.

The molecular recognition of carbohydrates and proteins mediates a wide range of physiological processes and the development of synthetic carbohydrate receptors (“synthetic lectins”) constitutes a key advance in biomedical technology. In this article we report a synthetic lectin that selectively binds to carbohydrates immobilized in a molecular monolayer. Inspired by our previous work, we prepared a fluorescently labeled synthetic lectin consisting of a cyclic dimer of the tripeptide Cys-His-Cys, which forms spontaneously by air oxidation of the monomer. Amine-tethered derivatives of N-acetylneuraminic acid (NANA), β-D-galactose, β-D-glucose and α-D-mannose were microcontact printed on epoxide-terminated self-assembled monolayers. Successive prints resulted in simple microarrays of two carbohydrates. The selectivity of the synthetic lectin was investigated by incubation on the immobilized carbohydrates. Selective binding of the synthetic lectin to immobilized NANA and β-D-galactose was observed by fluorescence microscopy. The selectivity and affinity of the synthetic lectin was screened in competition experiments. In addition, the carbohydrate binding of the synthetic lectin was compared with the carbohydrate binding of the lectins concanavalin A and peanut agglutinin. It was found that the printed carbohydrates retain their characteristic selectivity towards the synthetic and natural lectins and that the recognition of synthetic and natural lectins is strictly orthogonal.

Carbohydrate biomarker recognition using synthetic lectin mimics

Carbohydrate biomarkers play very important roles in a wide range of biological and pathological processes. Compounds that can specifically recognize a carbohydrate biomarker are useful for targeted delivery of imaging agents and for development of new diagnostics. Furthermore, such compounds could also be candidates for the development of therapeutic agents. A tremendous amount of active work on synthetic lectin mimics has been reported in recent years. Amongst all the synthetic lectins, boronic-acid-based lectins (boronolectins) have shown great promise. Along this line, four classes of boronolectins including peptide-, nucleic-acid-, polymer-, and small-molecule-based ones are discussed with a focus on the design principles and recent advances. We hope that by presenting the potentials of this field, this review will stimulate more research in this area.

A simple and accessible synthetic lectin for glucose recognition and sensing

Binding carbohydrates from water is a difficult task, even for the natural carbohydrate-binding proteins known as lectins. The design of synthetic lectin mimics is correspondingly challenging, especially if good selectivities are required. In previous work we showed that success is possible, but only for complex polycyclic architectures that require lengthy and low-yielding syntheses; for example, one glucose-selective system was made in 21 steps and only 0.1% overall yield. Here we report the discovery of a simple monocyclic host that matches the earlier designs, but is far more accessible as it is prepared in just five steps and 23% overall yield. The new synthetic lectin binds glucose with excellent selectivity versus other common monosaccharides (for example, ~50:1 versus galactose) and sufficient affinity for glucose sensing at the concentrations found in blood. It also features a built-in signalling system in the form of strong and guest-dependent fluorescence emission. The effectiveness and simplicity of this molecule suggests the potential for development into a new methodology for practical glucose monitoring.

Synthetic lectin arrays for the detection and discrimination of cancer associated glycans and cell lines.

Aberrant glycosylation is a hallmark of various disease states, including cancer, and effective detection and discrimination between healthy and diseased cells is an important challenge for the diagnosis and treatment of many diseases. Here, we describe the use of boronic acid functionalized synthetic lectins (SLs) in an array format for the differentiation of structurally similar cancer associated glycans and cancer cell lines; discrimination is based on subtle variations in glycosylation patterns. We further demonstrate the utility of our SLs in recognizing glycoproteins with up to 50-fold selectivity, even in 95% human serum. Given their robust and selective nature, these SLs were able to effectively distinguish (a) five structurally similar glycans with 94% accuracy; (b) seven normal, cancerous and metastatic colon cancer cell lines, including three isogenic cell lines, with 92% accuracy; and (c) these same seven cell lines using a guided statistical analysis to improve our analysis to 97% accuracy. In total, these data suggest that an SL-based array will be useful for the diagnosis of cancer.

Activity and Safety of Synthetic Lectins Based on Benzoboroxole-Functionalized Polymers for Inhibition of HIV Entry

Lectins derived from plant and microbial sources constitute a vital class of entry inhibitors that target the oligomannose residues on the HIV envelope gp120. Despite their potency and specificity, success of lectin-based entry inhibitors may be impeded by high manufacturing costs, formulation and potential mitogenicity. Therefore, there exists a gap in the HIV microbicides pipeline that underscores the need for mass producible, synthetic, broad-spectrum, and biocomptabile inhibitors of HIV entry. Here, we present the development of a polymeric synthetic lectin, based on benzoboroxole (BzB), which exhibits weak affinity (∼25 M(-1)) for nonreducing sugars, similar to those found on the HIV envelope. High molecular weight BzB-functionalized polymers demonstrated antiviral activity that increased with an increase in ligand density and molecular weight of the polymer construct, revealing that polyvalency improves activity. Polymers showed significant increase in activity from 25 to 75 mol % BzB functionalization with EC(50) of 15 μM and 15 nM, respectively. A further increase in mole functionalization to 90% resulted in an increase of the EC(50) (59 ± 5 nM). An increase in molecular weight of the polymer at 50 mol % BzB functionalization showed a gradual but significant increase in antiviral activity, with the highest activity seen with the 382 kDa polymer (EC(50) of 1.1 ± 0.5 nM in CEM cells and 11 ± 3 nM in TZM-bl cells). Supplementing the polymer backbone with 10 mol % sulfonic acid not only increased the aqueous solubility of the polymers by at least 50-fold but also demonstrated a synergistic increase in anti-HIV activity (4.0 ± 1.5 nM in TZM-bl cells), possibly due to electrostatic interactions between the negatively charged polymer backbone and the positively charged V3-loop in the gp120. The benzoboroxole-sulfonic acid copolymers showed no decrease in activity in the presence of a seminal concentration of fructose (p > 0.05). Additionally, the copolymers exhibit minimal, if any, effect on the cellular viability, barrier properties, or cytokine levels in human reconstructed ectocervical tissue after 3 days of repeated exposure and did not show pronounced activity against a variety of other RNA and DNA viruses.

Boronic Acid Functionalized Peptidyl Synthetic Lectins: Combinatorial Library Design, Peptide Sequencing, and Selective Glycoprotein Recognition

Aberrant glycosylation of cell membrane and secreted glycoproteins is a hallmark of various disease states, including cancer. The natural lectins currently used in the recognition of these glycoproteins are costly, difficult to produce, and unstable toward rigorous use. Herein we describe the design and synthesis of several boronic acid functionalized peptide-based synthetic lectin (SL) libraries, as well as the optimized methodology for obtaining peptide sequences of these SLs. SL libraries were subsequently used to identify SLs with as high as 5-fold selectivity for various glycoproteins. SLs will inevitably find a role in cancer diagnostics, given that they do not suffer from the drawbacks of natural lectins and that the combinatorial nature of these libraries allows for the identification of an SL for nearly any glycosylated biomolecule.

A Synthetic Lectin for β‐Glucosyl

Geschmacksache: Affinität und Selektivität makrotricyclischer Kohlenhdratrezeptoren lassen sich verbessern, indem man Alkoxygruppen (im Bild grün) an den Molekülgerüsten anbringt. So zeigt der Propoxy-substituierte Rezeptor eine starke Selektivität für die β-Glucosylgruppe. Glucose wird sowohl in Wasser als auch in rekonstituiertem Blutplasma gebunden, was zur Überwachung von Blutzuckerspiegeln genutzt werden könnte. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A Synthetic Lectin for β‐Glucosyl

Evolving tastes: Addition of alkoxy groups (green; see picture) to macrotricyclic carbohydrate receptors yields improved affinities and new selectivities. Notably, the propoxy-substituted receptor shows a strong preference for β-glucosyl. Glucose is bound both in water and reconstituted blood plasma, which holds promise for applications in blood glucose monitoring. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A synthetic lectin for O-linked beta-N-acetylglucosamine.

Changing employment: Receptor 1 binds beta-N-acetylglucosaminyl (beta-GlcNAc) up to 100 times more strongly than it does glucose. This synthetic lectin shows affinities similar to wheat germ agglutinin (WGA), a natural lectin used to bind GlcNAc. Remarkably, 1 is more selective than WGA. It favors especially the glycoside unit in glycopeptide 2, a model of the serine-O-GlcNAc posttranslational protein modification.

Design of Lectin Mimetics

A powerful carbohydrate receptor, which can be seen as a “synthetic lectin”, has recently been developed by Davis et al. The meta-terphenyl-based tetracyclic receptor was inspired by carbohydrate-binding proteins, and was designed to provide both polar and apolar contacts to a disaccharide molecule. Image taken from ref. 9 with permission. Copyright 2007, American Association for the Advancement of Science.