Mutualistic Synthesis from Orthogonal Dynamic Covalent Reactions.

Mutualisms are interactions that benefit all species involved. It has been widely investigated in neighbouring subjects, such as biology, ecology, sociology, and economics. However, such a reciprocal relationship in synthetic chemical systems has rarely been studied. Here, we demonstrate a mutualistic synthesis where byproducts from two orthogonal chemical reactions aid each other's production. Disulfide exchange and hydrazone exchange were chosen to generate two dynamic combinatorial libraries. A minor tetrameric macrocycle from the active disulfide library was quantitatively amplified in the presence of the hydrazone library. This incorporation also turned on the previously inert hydrazone reaction, producing a linear species that formed a "handcuffs" catenane with the disulfide tetramer. These findings not only lend robust support to the hypothesis of "RNA-peptide coevolution" for the origin of life but also broaden the scope of synthetic chemistry, highlighting the untapped potential of minor products from different reactions. Additionally, the co-self-assembly of these mutualistic entities to form supramolecular structures opens new avenues for future development of composite nanosystems with synergistic properties.

Self-Adaptive Synthesis of Non-Covalent Crosslinkers while Folding Single-Chain Polymers.

Peptide folding is a dynamic process driven by non-covalent cross-linking leading to functional nanostructures for essential biochemical activities. However, replicating this process in synthetic systems is challenging due to the difficulty in mimicking nature's real-time regulation of non-covalent crosslinking for single-chain polymer folding. Here, we address this by employing anionic dithiol building blocks to create macrocyclic disulfides as non-covalent crosslinkers that adapted to the folding process. Initially, small macrocycles facilitated a low degree folding of a polycation. Then, this preorganized structure catalysed the production of larger macrocycles that enhanced the folding conversely. The self-adaptive synthesis was verified through the encapsulation of an anticancer drug, showing an updated production distribution of non-covalent crosslinkers and maximizing drug-loading efficiency against drug-resistant cancer in vitro. Our research advances the understanding of molecular systems by exploring species evolution via the structural dynamics of polymer folding. Additionally, adaptive synthesis enables controlled, sequential folding of synthetic polymers, with the potential to mimic protein functions.

Base-Filling in Double-Helical Nucleic Acids.

Base-filling, i. e., post-synthetic furnishing of an oligonucleotide scaffold with base moieties or their analogues, is an interesting alternative to the conventional approach of sequential coupling of building blocks (modified or otherwise). Reversible attachment of the base moieties is particularly attractive as it allows the use of dynamic combinatorial chemistry and usually leads to higher fidelity. This concept article summarizes the various backbones and coupling reactions used for base-filling over the past fifteen years, discusses the impact of base stacking and pairing on efficiency and fidelity and highlights potential and realized applications.

Ternary Complex-Templated Dynamic Combinatorial Chemistry for the Selection and Identification of Homo-PROTACs.

Dynamic combinatorial chemistry (DCC) leverages a reversible reaction to generate compound libraries from constituting building blocks under thermodynamic control. The position of this equilibrium can be biased by addition of a target macromolecule towards enrichment of bound ligands. While DCC has been applied to select ligands for a single target protein, its application to identifying chimeric molecules inducing proximity between two proteins is unprecedented. In this proof-of-concept study, we develop a DCC approach to select bifunctional proteolysis targeting chimeras (PROTACs) based on their ability to stabilize the ternary complex. We focus on VHL-targeting Homo-PROTACs as model system, and show that the formation of a VHL2 : Homo-PROTAC ternary complex reversibly assembled using thiol-disulfide exchange chemistry leads to amplification of potent VHL Homo-PROTACs with degradation activities which correlated well with their biophysical ability to dimerize VHL. Ternary complex templated dynamic combinatorial libraries allowed identification of novel Homo-PROTAC degraders. We anticipate future applications of ternary-complex directed DCC to early PROTAC screenings and expansion to other proximity-inducing modalities beyond PROTACs.

Generalist versus Specialist Self-Replicators.

Darwinian evolution, including the selection of the fittest species under given environmental conditions, is a major milestone in the development of synthetic living systems. In this regard, generalist or specialist behavior (the ability to replicate in a broader or narrower, more specific food environment) are of importance. Here we demonstrate generalist and specialist behavior in dynamic combinatorial libraries composed of a peptide-based and an oligo(ethylene glycol) based building block. Three different sets of macrocyclic replicators could be distinguished based on their supramolecular organization: two prepared from a single building block as well as one prepared from an equimolar mixture of them. Peptide-containing hexamer replicators were found to be generalists, i. e. they could replicate in a broad range of food niches, whereas the octamer peptide-based replicator and hexameric ethyleneoxide-based replicator were proven to be specialists, i. e. they only replicate in very specific food niches that correspond to their composition. However, sequence specificity cannot be demonstrated for either of the generalist replicators. The generalist versus specialist nature of these replicators was linked to their supramolecular organization. Assembly modes that accommodate structurally different building blocks lead to generalist replicators, while assembly modes that are more restrictive yield specialist replicators.

Analytical Tools for Dynamic Combinatorial Libraries of Cyclic Peptides.

Target-directed dynamic combinatorial chemistry is a very attractive strategy for the discovery of bioactive peptides. However, its application has not yet been demonstrated, presumably due to analytical challenges that arise from the diversity of a peptide library with combinatorial side-chains. We previously reported an efficient method to generate, under biocompatible conditions, large dynamic libraries of cyclic peptides grafted with amino acid's side-chains, by thiol-to-thioester exchanges. In this work, we present analytical tools to easily characterize such libraries by HPLC and mass spectrometry, and in particular to simplify the isomers' distinction requiring sequencing by MS/MS fragmentations. After structural optimization, the cyclic scaffold exhibits a UV-tag, absorbing at 415 nm, and an ornithine residue which favors the regioselective ring-opening and simultaneous MS/MS fragmentation, in the gas-phase.

Deciphering dynamic combinatorial libraries of glycoclusters with miniaturized weak affinity chromatography coupled with mass spectrometry (nano-FAC-MS).

We report an original methodology based on affinity chromatography coupled with mass spectrometry to decipher the complexity of dynamic combinatorial libraries (DCLs) of glycoclusters. Such libraries are intended to boost the design of potential therapeutic anti-infectious agents targeting Pseudomonas aeruginosa, which is responsible for numerous diseases, mostly found in hospitals as major a cause of nosocomial infections. Dynamic combinatorial chemistry provides a rapid access to an equilibrating mixture of glycocluster candidates through the formation of reversible covalent bonds under thermodynamic control. Identifying each molecule in the complex mixture overcomes challenges due to the dynamic process. Selection of glycoclusters candidates was first realized on a model lectin (Concanavalin A, ConA). Home-made affinity nanocolumns, containing covalently immobilized ConA and have volumes in the microliter range, were used to separate DCLs of glycoclusters with respect to their specific lectin binding properties under buffered aqueous conditions. Miniaturization facilitates the inline coupling with MS detection in such purely aqueous and buffered conditions and reduces target protein consumption. Monolithic lectin-affinity columns prepared by immobilization of ConA were first characterized using a known ligand. The amount of active binding immobilized lectin is 61 ± 5 pmol on 8.5-cm length column. We demonstrated the ability of our approach to evaluate individual dissociation constants of species directly in the complex mixture. The concept was then successfully applied to the screening of DCLs of more complex glycoclusters to identify (by mass spectrometry) and rank the ligands (by relative breakthrough curve delay) according to their affinity for the immobilized lectin in a single experiment.

Development of Urease Inhibitors by Fragment-Based Dynamic Combinatorial Chemistry.

In this study, fragment-based dynamic combinatorial chemistry (DCC) was explored for the development of novel urease inhibitors. Based on a rationally designed fragment, two iteratively evolved dynamic combinatorial libraries (DCLs) were generated and screened in the presence of urease template. The best ligand identified revealed not only strong urease inhibition but also low cytotoxicity. Additionally, a possible inhibitory mechanism was elucidated in the binding kinetics study and docking simulation.

Teaching an Old Compound New Tricks: Reversible Transamidation in Maleamic Acids.

Dynamic combinatorial chemistry is a method widely used for generating responsive libraries of compounds, with applications ranging from chemical biology to materials science. It relies on dynamic covalent bonds that are able to form in a reversible manner in mild conditions, and therefore requires the discovery of new types of these bonds in order to progress. Amides, due to their high stability, have been scarcely used in this field and typically require an external catalyst or harsh conditions for exchange. Compounds able to undergo uncatalysed transamidation at room temperature are still rare exceptions. In this work, we describe reversible amide formation and transamidation in a class of compounds known as maleamic acids. Due to the presence of a carboxylic acid in ß-position, these compounds are in equilibrium with their anhydride and amine precursors in organic solvents at room temperature. First, we show that this equilibrium is responsive to external stimuli: by alternating the additions of a Brønsted acid and a base, we can switch between amide and anhydride several times without side-reactions. Next, we prove that this equilibrium provides a pathway for reversible transamidation without any added catalyst, leading to thermodynamic distributions of amides at room temperature. Lastly, we use different preparation conditions and concentrations of Brønsted acid to access different library distributions, easily controlling the transition between kinetic and thermodynamic regimes. Our results show that maleamic acids can undergo transamidation in mild conditions in a reversible and tunable way, establishing them as a new addition to the toolbox of dynamic combinatorial chemistry.

Dynamic Amino Acid Side-Chains Grafting on Folded Peptide Backbone.

An efficient strategy for the synthesis of large libraries of conformationally defined peptides is reported, using dynamic combinatorial chemistry as a tool to graft amino acid side chains on a well-ordered 3D (3-dimension) peptide backbone. Combining rationally designed scaffolds with combinatorial side chains selection represents an alternative method to access peptide libraries for structures that are not genetically encodable. This method would allow a breakthrough for the discovery of protein mimetic for unconventional targets for which little is known.

Proteomic Tools for the Quantitative Analysis of Artificial Peptide Libraries: Detection and Characterization of Target-Amplified PD-1 Inhibitors.

We report a quantitative proteomics data analysis pipeline, which coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted, amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.

Sulfur in Dynamic Covalent Chemistry.

Sulfur has been important in dynamic covalent chemistry (DCC) since the beginning of the field. Mainly as part of disulfides and thioesters, dynamic sulfur-based bonds (DSBs) have a leading role in several remarkable reactions. Part of this success is due to the almost ideal properties of DSBs for the preparation of dynamic covalent systems, including high reactivity and good reversibility under mild aqueous conditions, the possibility of exploiting supramolecular interactions, access to isolable structures, and easy experimental control to turn the reaction on/off. DCC is currently witnessing an increase in the importance of DSBs. The chemical flexibility offered by DSBs opens the door to multiple applications. This Review presents an overview of all the DSBs used in DCC, their applications, and remarks on the interesting properties that they confer on dynamic chemical systems, especially those containing several DSBs.

Out-of-Equilibrium Self-Replication Allows Selection for Dynamic Kinetic Stability in a System of Competing Replicators.

Among the key characteristics of living systems are their ability to self-replicate and the fact that they exist in an open system away from equilibrium. Herein, we show how the outcome of the competition between two self-replicators, differing in size and building block composition, is different depending on whether the experiments are conducted in a closed vial or in an open and out-of-equilibrium replication-destruction regime. In the closed system, the slower replicator eventually prevails over the faster competitor. In a replication-destruction regime, implemented through a flow system, the outcome of the competition is reversed and the faster replicator dominates. The interpretation of the experimental observations is supported by a mass-action-kinetics model. These results represent one of the few experimental manifestations of selection among competing self-replicators based on dynamic kinetic stability and pave the way towards Darwinian evolution of abiotic systems.

Identification of potent α-amylase inhibitors via dynamic combinatorial chemistry.

In this study, we report for the first time the discovery of potent α-amylase inhibitors using principle of dynamic combinatorial chemistry. The best compound identified exhibited not only high inhibitory efficiency but also low cytotoxicity. The binding mode and possible mechanism are determined in the subsequent kinetic and molecular docking studies.

Identification and synthesis of selective cholesterol esterase inhibitor using dynamic combinatorial chemistry.

In this study, the concept of dynamic combinatorial chemistry (DCC) was applied to explore novel cholesterol esterase (CEase) inhibitors. In the presence of enzyme, two substrates (A1H3 and A2H3) were amplified from the dynamic combinatorial library (DCL), which was generated through reversible acylhydrazone formation reaction. In the in vitro biological evaluation, compound A1H3 exhibited not only potent (IC50 in nanomolar range) but also selective inhibition (>120 folds of selectivity for CEase over AChE). Furthermore, the binding pattern and possible binding mechanism were investigated in the kinetic experiment and molecular docking study, respectively.

Chaotropic and Kosmotropic Anions Regulate the Outcome of Enzyme-Mediated Dynamic Combinatorial Libraries of Cyclodextrins in Two Different Ways.

We demonstrate how different anions from across the Hofmeister series can influence the behavior of enzyme-mediated dynamic combinatorial libraries of cyclodextrins (CDs). Using cyclodextrin glucanotransferase to catalyze reversible transglycosylation, dynamic mixtures of interconverting cyclodextrins can be formed wherein the relative concentrations of α-CD, ß-CD and γ-CD is determined by their intrinsic stabilities and any stabilizing influences of added template (guest) molecules. Here, we find that addition of high concentrations of kosmotropic anions can be used to enhance the effects of added hydrophobic templates, while chaotropic anions can themselves act as templates, causing predictable and significant changes in the cyclodextrin composition due to weak, but specific, binding interactions with α-CD.

Heteroleptic Ligation by an endo-Functionalized Cage.

A conceptual approach for the synthesis of quasi-heteroleptic complexes with properly endo-functionalized cages as ligands is presented. The cage ligand reported here is of a covalent organic nature, it has been synthesized via a dynamic combinatorial chemistry approach, making use of a masked amine. Inspired by enzymatic active sites, the described system bears one carboxylate and two imidazole moieties as independent ligating units through which it is able to coordinate to transition metals. Analysis of the iron(II) complex in solution and the solid state validates the structure and shows that no undesired but commonly observed dimerization process takes place. The solid-state structure shows a five-coordinate metal center with the carboxylate bidentately bound to iron, which makes Fe@2 an unprecedentedly detailed structural model complex for this kind of non-heme iron oxygenases. As, as confirmed by the crystal structure, sufficient space for other organic ligands is available, the biologically relevant ligand α-ketoglutarate is implemented. We observe biomimetic reaction behavior towards dioxygen that opens studies investigating Fe@2 as a functional model complex.

Self-Sorting in Dynamic Combinatorial Libraries Leads to the Co-Existence of Foldamers and Self-Replicators.

Nature segregates fundamental tasks such as information storage/transmission and catalysis between two different compound classes (e.g. polynucleotides for replication and folded polyamides for catalysis). This division of labor is likely a product of evolution, raising the question of how simpler systems in which replicators and folded macromolecules co-exist may emerge in the transition from chemistry to biology. In synthetic systems, achieving co-existence of replicators and foldamers in a single molecular network remains an unsolved problem. Previous work on dynamic molecular networks has given rise to either self-replicating fibers or well-defined foldamer structures (or completely un-sorted complex systems). We report a system in which two cross-reactive dithiol (nucleobase- and peptide-based) building blocks self-sort into a replicator fiber and foldamer that both emerge spontaneously and co-exist. The self-sorting behavior remains prevalent across different building block ratios as two phases of emergence occur: replicator growth followed by foldamer formation. This is attributed to the autocatalytic formation of the replicator fiber, followed by enrichment of the system in the remaining building block, which is subsequently incorporated into a foldamer.

Design, Synthetic Strategies, and Therapeutic Applications of Heterofunctional Glycodendrimers.

Glycodendrimers have attracted considerable interest in the field of dendrimer sciences owing to their plethora of implications in biomedical applications. This is primarily due to the fact that cell surfaces expose a wide range of highly diversified glycan architectures varying by the nature of the sugars, their number, and their natural multiantennary structures. This particular situation has led to cancer cell metastasis, pathogen recognition and adhesion, and immune cell communications that are implicated in vaccine development. The diverse nature and complexity of multivalent carbohydrate-protein interactions have been the impetus toward the syntheses of glycodendrimers. Since their inception in 1993, chemical strategies toward glycodendrimers have constantly evolved into highly sophisticated methodologies. This review constitutes the first part of a series of papers dedicated to the design, synthesis, and biological applications of heterofunctional glycodendrimers. Herein, we highlight the most common synthetic approaches toward these complex molecular architectures and present modern applications in nanomolecular therapeutics and synthetic vaccines.

Multivalent butyrylcholinesterase inhibitor discovered by exploiting dynamic combinatorial chemistry.

In this study, we report the generation of a polymer-based dynamic combinatorial library (DCL) incorporating exchangeable side chains using acylhydrazone formation reaction. In combination with tetrameric butyrylcholinesterase (BChE), the most potent binding side chain was identified, and the information obtained was further used for the synthesis of a multivalent BChE inhibitor. In the in vitro biological evaluation, this multivalent inhibitor exhibited not only better inhibitory effect than the commercial reference but also high selectivity on BChE over acetylcholinesterase (AChE).