Self-assembled porphyrin-peptide cages for photodynamic therapy.

The development of photodynamic therapy requires access to smart photosensitizers which combine appropriate photophysical and biological properties. Interestingly, supramolecular and dynamic covalent chemistries have recently shown their ability to produce novel architectures and responsive systems through simple self-assembly approaches. Herein, we report the straightforward formation of porphyrin-peptide conjugates and cage compounds which feature on their surface chemical groups promoting cell uptake and specific organelle targeting. We show that they self-assemble, in aqueous media, into positively-charged nanoparticles which generate singlet oxygen upon green light irradiation, while also undergoing a chemically-controlled disassembly due to the presence of reversible covalent linkages. Finally, the biological evaluation in cells revealed that they act as effective photosensitizers and promote synergistic effects in combination with Doxorubicin.

Nucleic-Acid-Templated Synthesis of Smart Polymer Vectors for Gene Delivery.

Nucleic acids are information-rich and readily available biomolecules, which can be used to template the polymerization of synthetic macromolecules. Here, we highlight the control over the size, composition, and sequence one can nowadays obtain by using this methodology. We also highlight how templated processes exploiting dynamic covalent polymerization can, in return, result in therapeutic nucleic acids fabricating their own dynamic delivery vector - a biomimicking concept that can provide original solutions for gene therapies.

Complexation Preferences of Dynamic Constitutional Frameworks as Adaptive Gene Vectors.

The growing applications of therapeutic nucleic acids requires the concomitant development of vectors that are optimized to complex one type of nucleic acid, forming nanoparticles suitable for further trafficking and delivery. While fine-tuning a vector by molecular engineering to obtain a particular nanoscale organization at the nanoparticle level can be a challenging endeavor, we turned the situation around and instead screened the complexation preferences of dynamic constitutional frameworks toward different types of DNAs. Dynamic constitutional frameworks (DCF) are recently-identified vectors by our group that can be prepared in a versatile manner through dynamic covalent chemistry. Herein, we designed and synthesized 40 new DCFs that vary in hydrophilic/hydrophobic balance, number of cationic headgroups. The results of DNA complexation obtained through gel electrophoresis and fluorescent displacement assays reveal binding preferences of different DCFs toward different DNAs. The formation of compact spherical architectures with an optimal diameter of 100-200 nm suggests that condensation into nanoparticles is more effective for longer PEG chains and PEI groups that induce a better binding performance in the presence of DNA targets.

A Triazolium-Anchored Self-Immolative Linker Enables Self-Assembly-Driven siRNA Binding and Esterase-Induced Release.

The increased importance of RNA-based therapeutics comes with a need to develop next-generation stimuli-responsive systems capable of binding, transporting and releasing RNA oligomers. In this work, we describe triazolium-based amphiphiles capable of siRNA binding and enzyme-responsive release of the nucleic acid payload. In aqueous medium, the amphiphile self-assembles into nanocarriers that can disintegrate upon the addition of esterase. Key to the molecular design is a self-immolative linker that is anchored to the triazolium moiety and acts as a positively-charged polar head group. We demonstrate that addition of esterase leads to a degradation cascade of the linker, leaving the neutral triazole compound unable to form complexes and therefore releasing the negatively-charged siRNA. The reported molecular design and overall approach may have broad utility beyond this proof-of-principle study, because the underlying CuAAC "click" chemistry allows bringing together three groups very efficiently as well as cleaving off one of the three groups under the mild action of an esterase enzyme.

Structure-Activity Relationships in Nucleic-Acid-Templated Vectors Based on Peptidic Dynamic Covalent Polymers.

The use of nucleic acids as templates, which can trigger the self-assembly of their own vectors represent an emerging, simple and versatile, approach toward the self-fabrication of tailored nucleic acids delivery vectors. However, the structure-activity relationships governing this complex templated self-assembly process that accompanies the complexation of nucleic acids remains poorly understood. Herein, the class of arginine-rich dynamic covalent polymers (DCPs) composed of different monomers varying the number and position of arginines were studied. The combinations that lead to nucleic acid complexation, in saline buffer, using different templates, from short siRNA to long DNA, are described. Finally, a successful peptidic DCP featuring six-arginine repeating unit that promote the safe and effective delivery of siRNA in live cancer cells was identified.

Bis-Alkylureido Imidazole Artificial Water Channels.

Nature creates aquaporins to effectively transport water, rejecting all ions including protons. Aquaporins (AQPs) has brought inspiration for the development of Artificial Water Channels (AWCs). Imidazole-quartet (I-quartet) was the first AWC that enabled to self-assemble a tubular backbone for rapid water and proton permeation with total ion rejection. Here, we report the discovery of bis-alkylureido imidazole compounds, which outperform the I-quartets by exhibiting ≈3 times higher net and single channel permeabilities (107 H2 O/s/channel) and a ≈2-3 times lower proton conductance. The higher water conductance regime is associated to the high partition of more hydrophobic bis-alkylureido channels in the membrane and to their pore sizes, experiencing larger fluctuations, leading to an increase in the number of water molecules in the channel, with decreasing H-bonding connectivity. This new class of AWCs will open new pathways toward scalable membranes with enhanced water transport performances.

Hierarchical Self-Assembly and Multidynamic Responsiveness of Fluorescent Dynamic Covalent Networks Forming Organogels.

Smart stimuli-responsive fluorescent materials are of interest in the context of sensing and imaging applications. In this project, we elaborated multidynamic fluorescent materials made of a tetraphenylethene fluorophore displaying aggregation-induced emission and short cysteine-rich C-hydrazide peptides. Specifically, we show that a hierarchical dynamic covalent self-assembly process, combining disulfide and acyl-hydrazone bond formation operating simultaneously in a one-pot reaction, yields cage compounds at low concentration (2 mM), while soluble fluorescent dynamic covalent networks and even chemically cross-linked fluorescent organogels are formed at higher concentrations. The number of cysteine residues in the peptide sequence impacts directly the mechanical properties of the resulting organogels, Young's moduli varying 2500-fold across the series. These materials underpinned by a nanofibrillar network display multidynamic responsiveness following concentration changes, chemical triggers, as well as light irradiation, all of which enable their controlled degradation with concomitant changes in spectroscopic outputs─self-assembly enhances fluorescence emission by ca. 100-fold and disassembly quenches fluorescence emission.

Fluorescent Dynamic Covalent Polymers for DNA Complexation and Templated Assembly.

Dynamic covalent polymers (DCPs) offer opportunities as adaptive materials of particular interest for targeting, sensing and delivery of biological molecules. In this view, combining cationic units and fluorescent units along DCP chains is attractive for achieving optical probes for the recognition and delivery of nucleic acids. Here, we report on the design of acylhydrazone-based DCPs combining cationic arginine units with π-conjugated fluorescent moieties based on thiophene-ethynyl-fluorene cores. Two types of fluorescent building blocks bearing neutral or cationic side groups on the fluorene moiety are considered in order to assess the role of the number of cationic units on complexation with DNA. The (chir)optical properties of the building blocks, the DCPs, and their complexes with several types of DNA are explored, providing details on the formation of supramolecular complexes and on their stability in aqueous solutions. The DNA-templated formation of DCPs is demonstrated, which provides new perspectives on the assembly of fluorescent DCP based on the nucleic acid structure.

Cell-Selective siRNA Delivery Using Glycosylated Dynamic Covalent Polymers Self-Assembled In†Situ by RNA Templating.

Dynamic covalent libraries enable exploring complex chemical systems from which bioactive assemblies can adaptively emerge through template effects. In this work, we studied dynamic covalent libraries made of complementary bifunctional cationic peptides, yielding a diversity of species from macrocycles to polymers. Although polymers are typically expressed only at high concentration, we found that siRNA acts as a template in the formation of dynamic covalent polymers at low concentration in a process guided by electrostatic binding. Using a glycosylated building block, we were able to show that this templated polymerization further translates into the multivalent presentation of carbohydrate ligands, which subsequently promotes cell uptake and even cell-selective siRNA delivery.

Growing Prospects of Dynamic Covalent Chemistry in Delivery Applications.

Delivery remains a major obstacle restricting the potential action of small molecular drugs as well as novel biologics which cannot readily enter cells without the help of a vector. A successful active delivery process involves three steps: (a) tagging the drug with a vector, (b) effective trafficking of this [drug-vector] conjugate through biological barriers, and finally (c) controlled drug release. While covalent bond formation and/or supramolecular association is involved in the making of the [drug-vector] conjugate, the final step requires precisely a controlled dissociation in order to trigger drug release. Therefore, in pursuit of smart, effective, and nontoxic delivery systems, it has become widely recognized that control over dynamic self-assembly could unleash the efficacy of artificial vectors. In this Account, I discuss our endeavors, and those of colleagues, in the recent implementation of Dynamic Covalent Chemistry (DCvC) in delivery applications. DCvC exploits reversible covalent reactions to generate covalent systems that can self-fabricate, adapt, respond, and fall apart in a controlled fashion. A privileged set of reversible covalent reactions has emerged in the community working on delivery applications and is based on condensation reactions (imine, acylhydrazone, oxime), and disulfide and boronate ester formations. The latest developments making this chemistry particularly attractive for such a DCvC approach are discussed. The rational justifying the potential of DCvC in delivery is based on the principle that using such reversible covalent reactions afford transient [drug-vector] conjugates which form spontaneously and chemoselectively, then adapt and self-correct their structure during self-assembly and trafficking thanks to the dynamic nature of the reversible covalent bonds, and finally respond to physicochemical stimuli such as pH and redox changes, thereby enabling controlled dissociation and concomitant drug release. For these reasons, DCvC has recently emerged as a leverage tool with growing prospects for advancing toward smarter delivery systems. The implementation of DCvC can follow three approaches that are discussed herein: (1) dynamic covalent bioconjugates, involving the transient covalent conjugation with a vector, (2) dynamic covalent vectors, involving the controlled dynamic and adaptive assembly and disassembly of vectors that complex drugs through supramolecular association, and (3) dynamic covalent targeting, involving the transient chemoselective formation of covalent bonds with the constituents of cell membranes. While DCvC has already attracted interest in material sciences, the recent results described in this Account showcase the vast potential of DCvC in biological sciences, and in particular in delivery applications where self-fabricated, adaptive, and responsive devices are of utmost importance.

Switching Multivalent DNA Complexation using Metal-Controlled Cationic Supramolecular Self-Assemblies.

We provide a proof-of-principle that coordination chemistry drives the in situ self-assembly of an inactive ligand into a multivalent cluster capable of effectively complexing DNA. We show that metal coordination and scavenging can be used to switch the multivalency of the system. Thus, controlled DNA complexation and decomplexation could be achieved.

Multivalent Metallosupramolecular Assemblies as Effective DNA Binding Agents.

We report the implementation of coordination chemistry onto the generation of new types of metallosupramolecular complexes with laterally appended cationic moieties for DNA binding in buffered aqueous media. Utilization of an N,N,O-type coordination pocket along with an octahedral zinc(II) metal ion allowed us to obtain mono- and tetranuclear complexes in both solution and solid state, as confirmed by NMR spectroscopy and single-crystal X-ray diffraction, respectively. By using isothermal titration calorimetry and gel electrophoresis, multiply charged cationic assemblies were observed to effectively bind to DNA through multivalent electrostatic interactions. Furthermore, we observed a correlation between the multivalency of the compounds employed and the effectiveness of DNA binding.

Photomodulation of DNA-Templated Supramolecular Assemblies.

A new type of DNA ligand that contains a phosphate-binding group and a photoresponsive azobenzene moiety is reported. When the azobenzene is in trans configuration, the ligand binds to the minor groove of a double-stranded DNA, whereas it partially desorbs upon trans-cis isomerisation with light. The ability to photoswitch the ligand upon interaction with DNA is evidenced by (chir)optical signatures, and deciphered by the differences of binding geometry, stability, and dynamics of the DNA/ligand complexes for the two isomers. We exploit these properties to photomodulate DNA-templated self-assembly, through the incorporation of another π-stacking DNA ligand, which together with the photoresponsive ligand form mixed supramolecular complexes along DNA. Our study demonstrates that well-designed photoresponsive DNA binders can be used to modulate multicomponent supramolecular DNA assemblies.

Self-assembly and chiroptical properties in supramolecular complexes of adenosine phosphates and guanidinium-bispyrene.

Supramolecular systems that respond to the hydrolysis of adenosine phosphates (APs) are attractive for biosensing and to fabricate bioinspired self-assembled materials. Here, we report on the formation of supramolecular complexes between an achiral guanidinium derivative bearing two pyrene moieties, with each of the three adenosine phosphates: AMP, ADP, and ATP. By combining results from circular dichroism spectroscopy and molecular modeling simulations, we explore the induced chirality, the dynamics of the complexes, and the interactions at play, which altogether provide insights into the supramolecular self-assembly between APs and the guanidinium-bispyrene. Finally, we identify the chiroptical signals of interest in mixtures of the guanidinium derivative with the three APs in different proportions. This study constitutes a basis to evolve toward a chiroptical detection of the hydrolysis of APs based on organic supramolecular probes.

Functional interplay between NTP leaving group and base pair recognition during RNA polymerase II nucleotide incorporation revealed by methylene substitution.

RNA polymerase II (pol II) utilizes a complex interaction network to select and incorporate correct nucleoside triphosphate (NTP) substrates with high efficiency and fidelity. Our previous 'synthetic nucleic acid substitution' strategy has been successfully applied in dissecting the function of nucleic acid moieties in pol II transcription. However, how the triphosphate moiety of substrate influences the rate of P-O bond cleavage and formation during nucleotide incorporation is still unclear. Here, by employing ß,γ-bridging atom-'substituted' NTPs, we elucidate how the methylene substitution in the pyrophosphate leaving group affects cognate and non-cognate nucleotide incorporation. Intriguingly, the effect of the ß,γ-methylene substitution on the non-cognate UTP/dT scaffold (∼3-fold decrease in kpol) is significantly different from that of the cognate ATP/dT scaffold (∼130-fold decrease in kpol). Removal of the wobble hydrogen bonds in U:dT recovers a strong response to methylene substitution of UTP. Our kinetic and modeling studies are consistent with a unique altered transition state for bond formation and cleavage for UTP/dT incorporation compared with ATP/dT incorporation. Collectively, our data reveals the functional interplay between NTP triphosphate moiety and base pair hydrogen bonding recognition during nucleotide incorporation.

Polyhedral Oligomeric Silsesquioxane (POSS) Bearing Glyoxylic Aldehyde as Clickable Platform Towards Multivalent Conjugates.

The straightforward access to octafunctional "cubic" silsesquioxane platform grafter with pendant glyoxylic aldehydes is described. This clickable hybrid platform readily reacts with oxyamine or hydrazide compounds to provide, respectively, oxime and acylhydrazone conjugates, thereby offering a new and effective access from which one can elaborate multivalent systems for the targeting of biomolecules of interest.

Effective Access to Multivalent Inhibitors of Carbonic Anhydrases Promoted by Peptide Bioconjugation.

Multivalency has impressive effects on (bio)molecular recognition, through the simultaneous presentation of multiple copies of a ligand, which can change a weak millimolar binder into a potent nanomolar one. The implementation of multivalency in enzyme inhibition is rather recent, being exemplified by few serendipitous discoveries, and hitherto relying on the random exploration of new multivalent structures as potential enzyme inhibitors. Here, a straightforward and versatile method is reported that enables the construction of multivalent systems for the inhibition of carbonic anhydrases (CA), widespread enzymes that catalyze a fundamental biochemical reaction. Oxime and hydrazone click-type bioconjugation techniques were successfully used for the preparation of tetravalent peptide conjugates tethered with sulfonamide CA inhibitors. The enzyme inhibition assays show that multivalent effects were present with these novel compounds, but also reveal various structural effects provided by the scaffolds. The versatility of this approach may facilitate the exploration of structure-activity relationships for other types of enzyme inhibitors.

One-Pot Self-Assembly of Peptide-Based Cage-Type Nanostructures Using Orthogonal Ligations.

The designed arrangement of biomolecular entities within monodisperse nanostructures is an important challenge toward functional biomaterials. We report herein a method for the formation of water-soluble peptide-based cages using orthogonal ligation reactions-acylhydrazone condensation and thiol-maleimide addition. The results show that using preorganized cyclic peptides and heterobifunctional spacers as building blocks and a set of orthogonal and chemoselective ligation reactions enable cage formation in one pot from six components and through eight reactions. Molecular modelling simulations reveal the structural dynamics of these structures. Finally, we exploited the reactional dynamics of the acylhydrazone by demonstrating the controlled dissociation of the cage through directed component exchange.

Generation of Multicomponent Molecular Cages using Simultaneous Dynamic Covalent Reactions.

Cage compounds are very attractive structures for a wide range of applications and there is ongoing interest in finding effective ways to access such kinds of complex structures, particularly those possessing dynamic adaptive features. Here we report the accessible synthesis of new type of organic cage architectures, possessing two different dynamic bonds within one structure: hydrazones and disulfides. Implementation of three distinct functional groups (thiols, aldehydes and hydrazides) in the structure of two simple building blocks resulted in their spontaneous and selective self-assembly into aromatic cage-type architectures. These organic cages contain up to ten components linked together by twelve reversible covalent bonds. The advantage provided by the presented approach is that these cage structures can adaptively self-sort from a complex virtual mixture of polymers or macrocycles and that dynamic covalent chemistry enables their deliberate disassembly through controlled component exchange.

Fluorescent Silica Nanoparticles with Multivalent Inhibitory Effects towards Carbonic Anhydrases.

Invited for the cover of this issue are Jean-Yves Winum and co-workers at University of Montpellier (France) and University of Florence (Italy). The image depicts the multivalency approach applied to zinc metalloenzyme carbonic anhydrases. Read the full text of the article at 10.1002/chem.201501037.