A macrocyclic tweezers-shaped receptor for the biomimetic recognition of the Gal(α1-3)Gal disaccharide of the α-Gal antigen.

The Gal(α1-3)Gal is the terminal disaccharide unit of the α-Gal epitope [Gal(α1-3)Gal(ß1-4)GlcNAc], an exogenous antigenic determinant with several clinical implications, found in all non-primate mammals and in several dangerous pathogens, including certain protozoa and mycobacteria. Its absence in humans makes the α-Gal epitope an interesting target for several infectious diseases. Here we present the development of a macrocyclic tweezers-shaped receptor, resulting from the combination of the structural features of two predecessors belonging to the family of diaminocarbazole receptors, which exhibits binding properties in the low millimolar range toward the Gal(α1-3)Gal disaccharide of the α-Gal antigen.

Biomimetic Tweezers for N-Glycans: Selective Recognition of the Core GlcNAc2 Disaccharide of the Sialylglycopeptide SGP.

In recent years, glycomics have shown how pervasive the role of carbohydrates in biological systems is and how chemical tools are essential to investigate glycan function and modulate carbohydrate-mediated processes. Biomimetic receptors for carbohydrates can carry out this task but, although significant affinities and selectivities toward simple saccharides have been achieved, targeting complex glycoconjugates remains a goal yet unattained. In this work we report the unprecedented recognition of a complex biantennary sialylglycopeptide (SGP) by a tweezers-shaped biomimetic receptor, which selectively binds to the core GlcNAc2 disaccharide of the N-glycan with an affinity of 170 µM. Because of the simple structure and the remarkable binding ability, this biomimetic receptor can represent a versatile tool for glycoscience, opening the way to useful applications.

Conformationally Constrained Sialyl Analogues as New Potential Binders of h-CD22.

Here, two conformationally constrained sialyl analogues were synthesized and characterized in their interaction with the inhibitory Siglec, human CD22 (h-CD22). An orthogonal approach, including biophysical assays (SPR and fluorescence), ligand-based NMR techniques, and molecular modelling, was employed to disentangle the interaction mechanisms at a molecular level. The results showed that the Sialyl-TnThr antigen analogue represents a promising scaffold for the design of novel h-CD22 inhibitors. Our findings also suggest that the introduction of a biphenyl moiety at position 9 of the sialic acid hampers canonical accommodation of the ligand in the protein binding pocket, even though the affinity with respect to the natural ligand is increased. Our results address the search for novel modifications of the Neu5Ac-α(2-6)-Gal epitope, outline new insights for the design and synthesis of high-affinity h-CD22 ligands, and offer novel prospects for therapeutic intervention to prevent autoimmune diseases and B-cell malignancies.

A Sulfonated Tweezer-Shaped Receptor Selectively Recognizes Caffeine in Water.

The selective recognition of caffeine in water among structurally related xanthines and purine or pyrimidine bases was achieved by a simple tweezer-shaped receptor featuring sulfonate hydrosolubilizing groups. The remarkable affinity for caffeine, among the highest reported thus far in the literature and larger than that shown by adenosine receptors of all subtypes, stems from a synergistic combination of hydrogen bonding, CH-π, and π-stacking interactions.

Molecular Recognition of Disaccharides in Water: Preorganized Macrocyclic or Adaptive Acyclic?

When facing the dilemma of following a preorganized or adaptive design approach in conceiving the architecture of new biomimetic receptors for carbohydrates, shape-persistent macrocyclic structures were most often chosen to achieve effective recognition of neutral saccharides in water. In contrast, acyclic architectures have seldom been explored, even though potentially simpler and more easily accessible. In this work, comparison of the binding properties of two structurally related diaminocarbazolic receptors, featuring a macrocyclic and an acyclic tweezer-shaped architecture, highlighted the advantages provided by the acyclic receptor in terms of selectivity in the recognition of 1,4-disaccharides of biological interest. Selective recognition of GlcNAc2 , the core fragment of N-glycans exposed on the surface of enveloped viruses, stands as an emblematic example. NMR spectroscopic data and molecular modeling calculations were used to ascertain the differences in binding mode and to shed light on the origin of recognition efficacy and selectivity.

A Simple Biomimetic Receptor Selectively Recognizing the GlcNAc2 Disaccharide in Water.

GlcNAc2 is the core disaccharide fragment present in N-glycans exposed on the surface of enveloped viruses of high health concern, such as coronaviruses. Because N-glycans are directly involved in the docking of viruses to host cells, recognition of GlcNAc2 by a biomimetic receptor may be a convenient alternative to the use of lectins to interfere with viral entry and infection. Herein, we describe a simple biomimetic receptor recognizing the methyl-ß-glycoside of GlcNAc2 in water with an unprecedented affinity of 160 µM, exceeding that of more structurally complex receptors reported in the literature. The tweezers-shaped acyclic structure exhibits marked selectivity among structurally related disaccharides, and complete discrimination between mono- and disaccharides. Molecular modelling calculations supported by NOE data provided a three-dimensional description of the binding mode, shedding light on the origin of the affinities and selectivities exhibited by the receptor.

A Preorganized Hydrogen-Bonding Motif for the Molecular Recognition of Carbohydrates.

The choice between adaptive and preorganized architectures, or of the most effective hydrogen bonding groups to be selected, are dilemmas that supramolecular chemists must address in designing synthetic receptors for such a challenging guest as carbohydrates. In this paper, structurally related architectures featuring two alternative hydrogen bonding motifs were compared to ascertain the structural and functional origin of their binding differences and the advantages that can be expected in monosaccharide recognition. A set of structurally related macrocyclic receptors were prepared, and their binding properties were measured by NMR and ITC techniques in chloroform vs a common saccharidic target, namely, the ß-octyl glycoside of D-glucose. Results showed that the diaminocarbazolic motif, recently reported as the constituting unit of highly effective receptors for saccharides in water, is a superior hydrogen bonding motif compared to the previously described diaminopyrrolic motif, which was successfully employed in molecular recognition of carbohydrates in polar organic solvents, due to intrinsic structural and functional factors, rather than to hydrophobic contributions. In addition, the occurrence of a rare example of a thermodynamic template effect exerted by the beta-glucoside has been ascertained, enhancing the synthesis outcome of the otherwise low yielding preparation of the described macrocyclic receptors.

Biomimetic Carbohydrate-Binding Agents (CBAs): Binding Affinities and Biological Activities.

Mimicking nature in carbohydrate recognition-that is, by using noncovalent interactions exclusively-is a hot topic that has attracted the interest of many scientists in the last 30 years. Carbohydrates are challenging ligands of high biological relevance, playing central roles in several physiological and pathological processes. Carbohydrate-binding agents (CBAs) of proteic nature, such as lectins, have been extensively used in glycobiology to target carbohydrates, but intrinsic drawbacks conferred on them by their proteic nature limit their therapeutic development. Biomimetic CBAs, artificial small molecules designed for molecular recognition of carbohydrates through noncovalent interactions, have been shown to be effective alternatives in recognising carbohydrates in physiological media, opening the way to biological applications. Herein, we describe the recent achievements in this continually developing field, focusing on those biomimetic CBAs for which biological investigations have been carried out.

A Biomimetic Synthetic Receptor Selectively Recognising Fucose in Water.

Carbohydrate recognition in water by biomimetic receptors is an attractive, but very challenging goal. Despite advances achieved in glucose recognition, little or no success has been obtained in the recognition of other saccharidic epitopes of paramount importance in biological processes. Herein, the unprecedented recognition of fucose in water by an artificial receptor that shows affinities closely comparable to those of several lectins is reported. The receptor has been constructed by assembling a hydrogen-bonding element (carbazole), a hydrophobic aromatic moiety (anthracene), and a water-solubilising function (phosphonate) into a macrocyclic structure to provide the appropriate binding geometry. The described receptor binds fucose with sub-millimolar affinity in water at physiological pH; this shows that enthalpic binding can be ascribed to hydrogen bonding to saccharidic hydroxy groups and to CH-π interactions between the sugar backbone and aromatic moieties. Experimental NOE contacts coupled to conformational search calculations return a picture of a binding site in which fucose assumes a staggered orientation reminiscent of that shown by fucose when bound to the Ralstonia solanacearum lectin (RSL).

Antagonism of Transient Receptor Potential Ankyrin Type-1 Channels as a Potential Target for the Treatment of Trigeminal Neuropathic Pain: Study in an Animal Model.

Transient receptor potential ankyrin type-1 (TRPA1) channels are known to actively participate in different pain conditions, including trigeminal neuropathic pain, whose clinical treatment is still unsatisfactory. The aim of this study was to evaluate the involvement of TRPA1 channels by means of the antagonist ADM_12 in trigeminal neuropathic pain, in order to identify possible therapeutic targets. A single treatment of ADM_12 in rats 4 weeks after the chronic constriction injury of the infraorbital nerve (IoN-CCI) significantly reduced the mechanical allodynia induced in the IoN-CCI rats. Additionally, ADM_12 was able to abolish the increased levels of TRPA1, calcitonin gene-related peptide (CGRP), substance P (SP), and cytokines gene expression in trigeminal ganglia, cervical spinal cord, and medulla induced in the IoN-CCI rats. By contrast, no significant differences between groups were seen as regards CGRP and SP protein expression in the pars caudalis of the spinal nucleus of the trigeminal nerve. ADM_12 also reduced TRP vanilloid type-1 (TRPV1) gene expression in the same areas after IoN-CCI. Our findings show the involvement of both TRPA1 and TRPV1 channels in trigeminal neuropathic pain, and in particular, in trigeminal mechanical allodynia. Furthermore, they provide grounds for the use of ADM_12 in the treatment of trigeminal neuropathic pain.

The role of the transient receptor potential ankyrin type-1 (TRPA1) channel in migraine pain: evaluation in an animal model.

BACKGROUND: Clinical and experimental studies have pointed to the possible involvement of the transient receptor potential ankyrin type-1 (TRPA1) channels in migraine pain. In this study, we aimed to further investigate the role of these channels in an animal model of migraine using a novel TRPA1 antagonist, ADM_12, as a probe. METHODS: The effects of ADM_12 on nitroglycerin-induced hyperalgesia at the trigeminal level were investigated in male rats using the quantification of nocifensive behavior in the orofacial formalin test. The expression levels of the genes coding for c-Fos, TRPA1, calcitonin gene-related peptide (CGRP) and substance P (SP) in peripheral and central areas relevant for migraine pain were analyzed. CGRP and SP protein immunoreactivity was also evaluated in trigeminal nucleus caudalis (TNC). RESULTS: In rats bearing nitroglycerin-induced hyperalgesia, ADM_12 showed an anti-hyperalgesic effect in the second phase of the orofacial formalin test. This effect was associated to a significant inhibition of nitroglycerin-induced increase in c-Fos, TRPA1 and neuropeptides mRNA levels in medulla-pons area, in the cervical spinal cord and in the trigeminal ganglion. No differences between groups were seen as regards CGRP and SP protein expression in the TNC. CONCLUSIONS: These findings support a critical involvement of TRPA1 channels in the pathophysiology of migraine, and show their active role in counteracting hyperalgesia at the trigeminal level.

Antiviral Activity of Synthetic Aminopyrrolic Carbohydrate Binding Agents: Targeting the Glycans of Viral gp120 to Inhibit HIV Entry.

The binding abilities of a set of structurally related aminopyrrolic synthetic receptors for mannosides, endowed with antimycotic activity against yeast and yeast-like pathogens bearing mannoproteins on their cell surface, have been investigated towards the highly mannosylated gp120 and gp41 glycoproteins of the HIV envelope. A pronounced binding interaction with both glycoproteins was observed by SPR for most of the investigated compounds. Comparison of their binding properties towards the glycoproteins with their binding affinities toward mannosides revealed a direct correlation, supporting their role as carbohydrate binding agents (CBAs). Cytostatic activity studies revealed antiproliferative activity dependent on the nature and the structure of compounds. Antiviral activity studies against a broad panel of DNA and RNA viruses showed inhibitory effect against HIV infection of the T-lymphocyte CEM cell line for two compounds, suggesting antiviral activity similar to other CBAs, such as the nonpeptidic pradimicin antibiotics.

Phosphate binding by a novel Zn(II) complex featuring a trans-1,2-diaminocyclohexane ligand. Effective anion recognition in water.

In this work we have investigated the binding properties of a new synthetic receptor for phosphate anions that combines metal ion coordination with electrostatic and H-bonding interactions. The described receptor is obtained by assembling an iminodiacetic (IDA) fragment, as a Zn(II) binding site, with a polyamine macrocyclic portion containing two trans-1,2-diaminocyclohexane (DAC) units and a pyrrole ring, as a cationic binding site, into an adaptive structure appropriately spanning the length of di- and tridentate phosphates. Potentiometric measurements together with (1)H and (31)P NMR investigation showed that, in a wide pH range including values of physiological interest, the Zn(II) complex of the receptor binds di- and triphosphates, such as ADP, ATP, pyrophosphate (PP) and triphosphate (TP), far better than monophosphate (MP), and that TP is poorly bound by methyliminodiacetate (MIDA) as a model for the Zn(II) binding site. Besides the excellent selectivity over other phosphates, the affinity for TP is the largest reported to date for Zn(II) complexes in water.

Systematic dissection of an aminopyrrolic cage receptor for ß-glucopyranosides reveals the essentials for effective recognition.

A set of structures designed for the recognition of glucosides has been obtained by systematically destructuring a tripodal aminopyrrolic cage receptor that selectively recognizes octyl-ß-D-glucopyranoside (OctßGlc). NMR spectroscopy and isothermal titration calorimetry binding measurements showed that cleavage of one pillar of the cage was beneficial to the binding properties of the receptor, as long as two residual amino groups of the cleaved pillar were present. Removal of these two residual amino groups produced a dramatic loss of affinity for OctßGlc of the resulting monocyclic analogue of the parent cage receptor. A significant improvement in the binding ability was achieved by replacing one pillar with two aminopyrrolic hydrogen-bonding arms, despite the loss of a preorganized structure. In contrast to the cage receptor, recognition of OctßGlc was observed, even in a competitive medium (30 % DMF in chloroform). Structural studies in solution, carried out through NMR spectroscopy and molecular modeling calculations, led to the elucidation of the 3D binding modes of the side-armed monocyclic receptors; this highlighted the key role of the amino groups and demonstrated the occurrence of a rotaxane-like complex, which featured the octyl chain of the glucoside threaded through the macrocyclic ring.

Why a diaminopyrrolic tripodal receptor binds mannosides in acetonitrile but not in water?

Intermolecular interactions involving carbohydrates and their natural receptors play important roles in several biological processes. The development of synthetic receptors is very useful to study these recognition processes. Recently, it was synthetized a diaminopyrrolic tripodal receptor that is selective for mannosides, which are obtained from mannose, a sugar with significant relevance in living systems. However, this receptor is significantly more active in acetonitrile than in water. In this work, we performed several molecular dynamics and constant-pH molecular dynamics simulations in acetonitrile and water to evaluate the conformational space of the receptor and to understand the molecular detail of the receptor-mannoside interaction. The protonation states sampled by the receptor show that the positive charges are always as distant as possible in order to avoid large intramolecular repulsions. Moreover, the conformational space of the receptor is very similar in water above pH 4.0 and in acetonitrile. From the simulations with the mannoside, we observe that the interactions are more specific in acetonitrile (mainly hydrogen bonds) than in water (mainly hydrophobic). Our results suggest that the readiness of the receptor to bind mannoside is not significantly affected in water (above pH 4.0). Probably, the hydrogen bond network that is formed in acetonitrile (which is weaker in water) is the main reason for the higher activity in this solvent. This work also presents a new implementation of the stochastic titration constant-pH molecular dynamics method to a synthetic receptor of sugars and attests its ability to describe the protonation/conformation coupling in these molecules.

Towards Biomimetic Recognition of Glycans by Synthetic Receptors.

Carbohydrates are abundant in Nature, where they are mostly assembled within glycans as free polysaccharides or conjugated to a variety of biological molecules such as proteins and lipids. Glycans exert several functions, including protein folding, stability, solubility, resistance to proteolysis, intracellular traffic, antigenicity, and recognition by carbohydrate-binding proteins. Interestingly, misregulation of their biosynthesis that leads to changes in glycan structures is frequently recognized as a mark of a disease state. Because of glycan ubiquity, carbohydrate binding agents (CBAs) targeting glycans can lead to a deeper understanding of their function and to the development of new diagnostic and prognostic strategies. Synthetic receptors selectively recognizing specific carbohydrates of biological interest have been developed over the past three decades. In addition to the success obtained in the effective recognition of monosaccharides, synthetic receptors recognizing more complex guests have also been developed, including di- and oligosaccharide fragments of glycans, shedding light on the structural and functional requirements necessary for an effective receptor. In this review, the most relevant achievements in molecular recognition of glycans and their fragments will be summarized, highlighting potentials and future perspectives of glycan-targeting synthetic receptors.

Pyrrolic tripodal receptors for the molecular recognition of carbohydrates: ditopic receptors for dimannosides.

Synthetic ditopic receptors, designed for the molecular recognition of dimannosides, have been prepared by bridging two monotopic units effectively recognizing mannosides with linkers of the appropriate size and flexibility, endowed with hydrogen-bonding groups. Affinities toward the α and ß glycosides of the biologically relevant Manα(1-2)Man disaccharide were measured by NMR spectroscopy and isothermal titration calorimetry (ITC) in polar organic media (30-40 % DMF in chloroform). Significant selectivities and affinities in the micromolar range were observed in most cases, with two newly designed receptors being the most effective receptors of the set, together with a distinct preference of the dimannosides for the (S) enantiomer of the receptor in all cases. A 3D view of the recognition mode was elucidated by a combined NMR spectroscopic/molecular modeling approach, showing the dimannoside included in the cleft of the receptor. Compared to the monotopic precursors, the ditopic receptors showed markedly improved recognition properties, proving the efficacy of the modular receptor design for the recognition of disaccharides.

Niosomes Functionalized with a Synthetic Carbohydrate Binding Agent for Mannose-Targeted Doxorubicin Delivery.

Niosomes are a potential tool for the development of active targeted drug delivery systems (DDS) for cancer therapy because of their excellent behaviour in encapsulating antitumorals and the possibility to easily functionalise their surface with targeting agents. Recently, some of us developed a synthetic carbohydrate binding agent (CBA) able to target the mannosidic residues of high-mannose-type glycans overexpressed on the surface of several cancer cell lines, promoting their apoptosis. In this article, we modified the structure of this mannose receptor to obtain an amphiphilic analogue suitable for the functionalization of doxorubicin-based niosomes. Several niosomal formulations and preparation methods were investigated deeply to finally obtain functionalized niosomes suitable for parental administration, which were stable for over six months and able to encapsulate up to 85% of doxorubicin (DOXO). In vitro studies, carried out towards triple-negative cancer cells (MDA-MB231), overexpressing high-mannose-type glycans, showed a cytotoxic activity comparable to that of DOXO but with an appreciable increment in apoptosis given by the CBA. Moreover, niosomal formulation was observed to reduce doxorubicin-induced cytotoxicity towards normal cell lines of rat cardiomyocytes (H9C2). This study is propaedeutic to further in vivo investigations that can aim to shed light on the antitumoral activity and pharmacokinetics of the developed active targeted DDS.

Aminopyrrolic synthetic receptors for monosaccharides: a class of carbohydrate-binding agents endowed with antibiotic activity versus pathogenic yeasts.

The biological activity of a set of structurally related aminopyrrolic synthetic receptors for monosaccharides has been tested versus yeast and yeast-like microorganisms and compared to their binding affinity toward mannosides. Antibiotic activity comparable to that of well-known polyene (amphotericin B) or azole (ketoconazole) drugs has been found for some members of the family, along with a general correlation with binding abilities. A systematic structure-activity-affinity investigation shed light on the structural and functional requirements necessary for antibiotic activity and identified the tripodal compound 1 as the most potent compound of the set. Together with toxicity tests and inhibitor localization experiments performed through fluorescence microscopy, these studies led to the characterization of a new class of carbohydrate binding agents possessing antibiotic activity, in which pyrrolic groups precisely structured on a tripodal architecture appear to be responsible for permeability through the cell wall of pathogens, as well as for antibiotic activity inside the cytoplasm.

BC(50): a generalized, unifying affinity descriptor.

Assessing binding affinities is an unavoidable step that we come across any time interactions between binding species are investigated. A quantitative evaluation of binding affinities relies on the determination of binding constants but, whilst the binding constant fully defines the affinity of a reagent for a ligand when only one complex species is formed, the same is not true when the interacting partners form more than one complex of different stoichiometry, because all complexes contribute to the overall binding affinity. Unfortunately, this situation is the rule rather than the exception in chemical systems, but a generally accepted solution for this issue has not yet been settled. In this Personal Account, we describe the evolution, from the initial idea to a fully developed stage, of a binding descriptor that has been developed with the aim of filling this gap, thereby providing scientists in all fields of chemistry with a unifying tool for the assessment of binding affinities based on the knowledge of the binding constants in systems that involve any number of complex species.