Discovery of selective monosaccharide receptors via dynamic combinatorial chemistry.

The molecular recognition of saccharides by synthetic hosts has become an appealing but elusive task in the last decades. Herein, we combine Dynamic Combinatorial Chemistry (DCC) for the rapid self-assembly and screening of virtual libraries of receptors, with the use of ITC and NMR to validate the hits and molecular modelling to understand the binding mechanisms. We discovered a minimalistic receptor, 1F (N-benzyl-L-phenylalanine), with considerable affinity for fructose (Ka = 1762 M-1) and remarkable selectivity (>50-fold) over other common monosaccharides. The approach accelerates the discovery process of receptors for saccharides.

Molecular Recognition of Tyrosine-Containing Polypeptides with Pseudopeptidic Cages Unraveled by Fluorescence and NMR Spectroscopies.

The molecular recognition of Tyr-containing peptide copolymers with pseudopeptidic cages has been studied using a combination of fluorescence and NMR spectroscopies. Fluorescence titrations rendered a reasonable estimation of the affinities, despite the presence of dynamic quenching masking the unambiguous detection of the supramolecular complexes. Regarding NMR, the effect of polypeptide (PP) binding on relaxation and diffusion parameters of the cages is much more reliable than the corresponding chemical shift perturbations. To that, purification of the commercial PPs is mandatory to obtain biopolymers with lower polydispersity. Thus, the relaxation/diffusion-filtered 1H spectra of the cages in the absence vs presence of the PPs represent a suitable setup for the fast detection of the noncovalent interactions. Additional key intermolecular NOE cross-peaks supported by molecular models allow the proposal of a structure of the supramolecular species, stabilized by the Tyr encapsulation within the cage cavity and additional attractive polar interactions between the side chains of cage and PP, thus defining a binding epitope with a potential for implementing sequence selectivity. Accordingly, the cages bearing positive/negative residues prefer to bind the peptides having complementary negative/positive side chains close to the target Tyr, suggesting an electrostatic contribution to the interaction. Overall, our results show that both techniques represent a powerful and complementary combination for studying cage-to-PP molecular recognition processes.

Pseudopeptidic host adaptation in peptide recognition unveiled by ion mobility mass spectrometry.

Complexation of the glutamic-tyrosine-glutamic tripeptide (EYE) with a series of pseudopeptidic cages has been thoroughly investigated using different analytical techniques. The stoichiometry and affinities of the supramolecular host : guest complexes both in aqueous solution and in the gas-phase were obtained from a suitable combination of fluorescence spectroscopy, NMR, and mass spectrometry (MS) methods. The cages bearing basic groups (lysine, ornitine and histidine) display the tightest EYE binding in aqueous media following the order CyHis > CyLys > CyOrn, thus suggesting that Tyr side chain encapsulation is additionally modulated by the identity of the cage side chains and their ability to be engaged in polar interactions with the EYE peptide. Similarly, binding affinities estimated by MS methods clearly point towards a reduced affinity for the Cy cages with acidic pendant groups and a higher affinity of the CyHis cage over CyLys and CyOrn. Ion mobility spectrometry (IMS)-MS, assisted by molecular modelling, has been used to uncover the structural and conformational characteristics of the pseudopeptidic hosts and their supramolecular adducts with the EYE peptide. The cages display a collisional cross-section increase upon EYE inclusion that is associated with the expansion of the binding pocket of the cage cavity, thus constituting a unique example of conformational pseudopeptidic host adaptation to accommodate the inclusion of the guest.

A Degenerate Metal-Templated Catalytic System with Redundant Functional Groups for the Asymmetric Aldol Reaction.

A degenerate zinc-templated catalytic system containing two bipyridine ligands with redundant functional groups for either enamine or hydrogen bond formation was applied to the asymmetric aldol reaction. This concept led to both a higher probability of reaction and rate acceleration. Thus, the catalyst loading could be decreased to a remarkable 2 mol % in what we think is a general approach.

Dynamic Combinatorial Optimization of In Vitro and In Vivo Heparin Antidotes.

Heparin-like macromolecules are widely used in clinics as anticoagulant, antiviral, and anticancer drugs. However, the search of heparin antidotes based on small synthetic molecules to control blood coagulation still remains a challenging task due to the physicochemical properties of this anionic polysaccharide. Here, we use a dynamic combinatorial chemistry approach to optimize heparin binders with submicromolar affinity. The recognition of heparin by the most amplified members of the dynamic library has been studied with different experimental (SPR, fluorescence, NMR) and theoretical approaches, rendering a detailed interaction model. The enzymatic assays with selected library members confirm the correlation between the dynamic covalent screening and the in vitro heparin inhibition. Moreover, both ex vivo and in vivo blood coagulation assays with mice show that the optimized molecules are potent antidotes with potential use as heparin reversal drugs. Overall, these results underscore the power of dynamic combinatorial chemistry targeting complex and elusive biopolymers.

Inhibition of Sema-3A Promotes Cell Migration, Axonal Growth, and Retinal Ganglion Cell Survival.

Purpose: Semaphorin 3A (Sema-3A) is a secreted protein that deflects axons from inappropriate regions and induces neuronal cell death. Intravitreal application of polyclonal antibodies against Sema-3A prevents loss of retinal ganglion cells ensuing from axotomy of optic nerves. This suggested a therapeutic approach for neuroprotection via inhibition of the Sema-3A pathway. Methods: To develop potent and specific Sema-3A antagonists, we isolated monoclonal anti-Sema-3A antibodies from a human antibody phage display library and optimized low-molecular weight Sema-3A signaling inhibitors. The best inhibitors were identified using in vitro scratch assays and semiquantitative repulsion assays. Results: A therapeutic approach for neuroprotection must have a long duration of action. Therefore, antibodies and low-molecular weight inhibitors were formulated in extruded implants to allow controlled and prolonged release. Following release from the implants, Sema-3A inhibitors antagonized Sema-3A effects in scratch and repulsion assays and protected retinal ganglion cells in animal models of optic nerve injury, retinal ischemia, and glaucoma. Conclusions and Translational Relevance: Collectively, our findings indicate that the identified Sema-3A inhibitors should be further evaluated as therapeutic candidates for the treatment of Sema-3A-driven central nervous system degenerative processes.

Molecular cages for biological applications.

Artificial receptors able to recognise biologically relevant molecules or ions have gained interest in the chemical community because they offer a plethora of posibilities. Molecular cage compounds are polycyclic compounds with a cavity designed for the encapsulation of guest species. Once inside the host cavity, the substrate can be transported through membranes and protected from the action of enzymes or other reactive species, thus offering the possibility of interfering with biological systems. Commonly, enzymes have been an inspiration for chemists in the search and design of defined cavities for different purposes. However, the chemical preparation of molecular cages has struggled with many synthetic challenges but this effort is worthwhile as they are a very promising tool for many applications ranging from sensing, delivery, purification or even promotion of/prevention from chemical modifications. Since the early reports at the end of the 60s, this field has experienced a growing interest; this review summarises the progress in the preparation and study of cage-like compounds highlighting their importance in biological applications.

Semaphorin 3A-Glycosaminoglycans Interaction as Therapeutic Target for Axonal Regeneration.

Semaphorin 3A (Sema3A) is a cell-secreted protein that participates in the axonal guidance pathways. Sema3A acts as a canonical repulsive axon guidance molecule, inhibiting CNS regenerative axonal growth and propagation. Therefore, interfering with Sema3A signaling is proposed as a therapeutic target for achieving functional recovery after CNS injuries. It has been shown that Sema3A adheres to the proteoglycan component of the extracellular matrix (ECM) and selectively binds to heparin and chondroitin sulfate-E (CS-E) glycosaminoglycans (GAGs). We hypothesize that the biologically relevant interaction between Sema3A and GAGs takes place at Sema3A C-terminal polybasic region (SCT). The aims of this study were to characterize the interaction of the whole Sema3A C-terminal polybasic region (Sema3A 725-771) with GAGs and to investigate the disruption of this interaction by small molecules. Recombinant Sema3A basic domain was produced and we used a combination of biophysical techniques (NMR, SPR, and heparin affinity chromatography) to gain insight into the interaction of the Sema3A C-terminal domain with GAGs. The results demonstrate that SCT is an intrinsically disordered region, which confirms that SCT binds to GAGs and helps to identify the specific residues involved in the interaction. NMR studies, supported by molecular dynamics simulations, show that a new peptoid molecule (CSIC02) may disrupt the interaction between SCT and heparin. Our structural study paves the way toward the design of new molecules targeting these protein-GAG interactions with potential therapeutic applications.

Modulation of Src Kinase Activity by Selective Substrate Recognition with Pseudopeptidic Cages.

The selective recognition of tyrosine residues in peptides is an appealing approach to inhibiting their tyrosine kinase (TK)-mediated phosphorylation. Herein, we describe pseudopeptidic cages that efficiently protect substrates from the action of the Src TK enzyme, precluding the corresponding Tyr phosphorylation. Fluorescence emission titrations show that the most efficient cage inhibitors strongly bind the peptide substrates with a very good correlation between the binding constant and the inhibitory potency. Structural insights and additional control experiments further support the proposed mechanism of selective supramolecular protection of the substrates. Moreover, the approach also works in a completely different kinase-substrate system. These results illustrate the potential of supramolecular complexes for the efficient and selective modulation of TK signaling.

Spontaneous macrocyclization through multiple dynamic cyclic aminal formation.

The use of aminals in dynamic covalent chemistry is slightly underexplored, probably due to their inherent instability. Here we report the spontaneous [2+2] macrocyclization of tetrakis(aminals). Their unexpected stability and structural modularity, the dynamic nature of the connections and their water tolerance make them appealing systems for future applications as stimulus-responsive materials.

Dynamic Stereoselection of Peptide Helicates and Their Selective Labeling of DNA Replication Foci in Cells*.

Although largely overlooked in peptide engineering, coordination chemistry offers a new set of interactions that opens unexplored design opportunities for developing complex molecular structures. In this context, we report new artificial peptide ligands that fold into chiral helicates in the presence of labile metal ions such as FeII and CoII . Heterochiral ß-turn-promoting sequences encode the stereoselective folding of the peptide ligands and define the physicochemical properties of their corresponding metal complexes. Circular dichroism and NMR spectroscopy in combination with computational methods allowed us to identify and determine the structure of two isochiral ΛΛ-helicates, folded as topological isomers. Finally, in addition to the in-vitro characterization of their selective binding to DNA three-way junctions, cell-microscopy experiments demonstrated that a rhodamine-labeled FeII helicate was internalized and selectively stains DNA replication factories in functional cells.

Exploiting complexity to implement function in chemical systems.

Chemistry deals with complex molecular systems that can be further connected by supramolecular interactions and reaction networks. However, chemists have taken little advantage of the intrinsic complexity of chemical systems, probably due to the lack of appropriate tools to analyse and understand complexity. In the last few decades, the concept of complexity has grown appealing: it allows the design of networks and dynamic systems expressing emerging properties and functions, which would be difficult to achieve from the mere addition of the components of the ensemble. Here we describe a personal overview of the recent state-of-the-art in the field, mainly focused on complex systems providing molecular recognition and catalysis. Far from being a thorough revision of the recent literature, we intend to illustrate the topic to attract the chemical community for considering complexity as an additional parameter in their research.

MCR-ALS analysis of 1H NMR spectra by segments to study the zebrafish exposure to acrylamide.

Metabolomics is currently an important field within bioanalytical science and NMR has become a key technique for drawing the full metabolic picture. However, the analysis of 1H NMR spectra of metabolomics samples is often very challenging, as resonances usually overlap in crowded regions, hindering the steps of metabolite profiling and resonance integration. In this context, a pre-processing method for the analysis of 1D 1H NMR data from metabolomics samples is proposed, consisting of the blind resolution and integration of all resonances of the spectral dataset by multivariate curve resolution-alternating least squares (MCR-ALS). The resulting concentration estimates can then be examined with traditional chemometric methods such as principal component analysis (PCA), ANOVA-simultaneous component analysis (ASCA), and partial least squares-discriminant analysis (PLS-DA). Since MCR-ALS does not require the use of spectral templates, the concentration estimates for all resonances are obtained even before being assigned. Consequently, the metabolomics study can be performed without neglecting any relevant resonance. In this work, the proposed pipeline performance was validated with 1D 1H NMR spectra from a metabolomics study of zebrafish upon acrylamide (ACR) exposure. Remarkably, this method represents a framework for the high-throughput analysis of NMR metabolomics data that opens the way for truly untargeted NMR metabolomics analyses. Graphical abstract.

Live-Cell-Templated Dynamic Combinatorial Chemistry.

Dynamic covalent chemistry combines in a single step the screening and synthesis of ligands for biomolecular recognition. In order to do that, a chemical entity is used as template within a dynamic combinatorial library of interconverting species, so that the stronger binders are amplified due to the efficient interaction with the target. Here we employed whole A549 living cells as template in a dynamic mixture of imines, for which amplification reflects the efficient and selective interaction with the corresponding extracellular matrix. The amplified polyamine showed strong interaction with the A549 extracellular matrix in on-cell NMR experiments, while combination of NMR, SPR, and molecular dynamics simulations in model systems provided insights on the molecular recognition event. Notably, our work pioneers the use of whole living cells in dynamic combinatorial chemistry, which paves the way towards the discovery of new bioactive molecules in a more biorelevant environment.

Molecular Recognition of Zwitterions with Artificial Receptors.

Many biomolecules exist as internal ion pairs or zwitterions within a biologically relevant pH range. Despite their importance, the molecular recognition of this type of systems is specially challenging due to their strong solvation in aqueous media, and their trend to form folded or self-assembled structures by pairing of charges of different sign. In this Minireview, we will discuss the molecular recognition of zwitterions using non-natural, synthetic receptors. This contribution does not intend to make a full in-depth revision of the existing research in the field, but a personal overview with selected representative examples from the recent literature.

Targeting redox metabolism: the perfect storm induced by acrylamide poisoning in the brain.

Exposure to acrylamide may lead to different neurotoxic effects in humans and in experimental animals. To gain insights into this poorly understood type of neurotoxicological damage, we used a multi-omic approach to characterize the molecular changes occurring in the zebrafish brain exposed to acrylamide at metabolite, transcript and protein levels. We detected the formation of acrylamide adducts with thiol groups from both metabolites and protein residues, leading to a quasi-complete depletion of glutathione and to the inactivation of different components of the thioredoxin system. We propose that the combined loss-of-function of both redox metabolism-related systems configure a perfect storm that explains many acrylamide neurotoxic effects, like the dysregulation of genes related to microtubules, presynaptic vesicle alteration, and behavioral alterations. We consider that our mechanistical approach may help developing new treatments against the neurotoxic effects of acrylamide and of other neurotoxicants that may share its toxic mode of action.

Synthesis of second-generation self-assembling Gemini Amphiphilic Pseudopeptides.

HYPOTHESIS: The structural modularity of Gemini Amphiphilic Pseudopeptides (GAPs) allows the tuning of the self-assembling properties by slight modifications in the chemical structures. We hypothesized that the introduction of a flexible linker containing a central nitrogen atom in bipodal and tripodal GAPs would improve their self-assembly properties in aqueous media. EXPERIMENTS: After preparation of the corresponding GAPs, a combination of SEM, TEM and AFM techniques were used to study the morphology of the self-assembled structures in different media. The solution structures in non-aggregated states were also analyzed by combining NMR, UV and CD studies. The transition from the non-aggregated species to the hierarchical self-assembly was monitored by ATR FT-IR spectroscopy, while the critical aggregation concentration in water was determined by fluorescence spectroscopy. FINDINGS: The formation of different morphologies (vesicles or fibers) highly depends on the polarity and the pH of the medium. A reasonable mechanism for the self-assembly has been established in agreement with the experimental techniques used, where the protonation of the nitrogen in the linker must play a key role. In general, the obtained GAPs showed an improved formation of vesicles in aqueous media (different pH or ionic strength) with potential applications in biomedicine and drug delivery.

pH-Dependent Chloride Transport by Pseudopeptidic Cages for the Selective Killing of Cancer Cells in Acidic Microenvironments.

Acidic microenvironments in solid tumors are a hallmark of cancer. Inspired by that, we designed a family of pseudopeptidic cage-like anionophores displaying pH-dependent activity. When protonated, they efficiently bind chloride anions. They also transport chloride through lipid bilayers, with their anionophoric properties improving at acidic pH, suggesting an H+ /Cl- symport mechanism. NMR studies in DPC micelles demonstrate that the cages bind chloride within the lipid phase. The chloride affinity and the chloride-exchange rate with the aqueous bulk solution are improved when the pH is lowered. This increases cytotoxicity towards lung adenocarcinoma cells at the pH of the microenvironment of a solid tumor. These properties depend on the nature of the amino-acid side chains of the cages, which modulate their lipophilicity and interactions with the cell membrane. This paves the way towards using pH as a parameter to control the selectivity of cytotoxic ionophores as anticancer drugs.

An efficient dynamic asymmetric catalytic system within a zinc-templated network.

Enhanced cooperativity leading to high catalytic activity and stereoselectivity has been achieved through a complex network of simple species interacting reversibly. This novel dynamic catalytic system relies on bipyridine-based organocatalytic ligands and zinc(ii) as the template. It demonstrates the effectiveness of dealing with mixtures rather than single species in asymmetric catalysis.

Sensing, Transport and Other Potential Biomedical Applications of Pseudopeptides.

Pseudopeptides are privileged synthetic molecules built from the designed combination of peptide-like and abiotic artificial moieties. Consequently, they are benefited from the advantages of both families of chemical structures: modular synthesis, chemical and functional diversity, tailored three-dimensional structure, usually high stability in biological media and low non-specific toxicity. Accordingly, in the last years, these compounds have been used for different biomedical applications, ranging from bio-sensing, ion transport, the molecular recognition of biologically relevant species, drug delivery or gene transfection. This review highlights a selection of the most remarkable and recent advances in this field.