Modifying the Catalytic Activity of Lipopeptide Assemblies with Nucleobases.

Biohybrid catalysts that operate in aqueous media are intriguing for systems chemistry. In this paper, we investigate whether control over the self-assembly of biohybrid catalysts can tune their properties. As a model, we use the catalytic activity of functional hybrid molecules consisting of a catalytic H-dPro-Pro-Glu tripeptide, derivatized with fatty acid and nucleobase moieties. This combination of simple biological components merged the catalytic properties of the peptide with the self-assembly of the lipid, and the structural ordering of the nucleobases. The biomolecule hybrids self-assemble in aqueous media into fibrillar assemblies and catalyze the reaction between butanal and nitrostyrene. The interactions between the nucleobases enhanced the order of the supramolecular structures and affected their catalytic activity and stereoselectivity. The results point to the significant control and ordering that nucleobases can provide in the self-assembly of biologically inspired supramolecular catalysts.

Primitive selection of the fittest emerging through functional synergy in nucleopeptide networks.

Many fundamental cellular and viral functions, including replication and translation, involve complex ensembles hosting synergistic activity between nucleic acids and proteins/peptides. There is ample evidence indicating that the chemical precursors of both nucleic acids and peptides could be efficiently formed in the prebiotic environment. Yet, studies on nonenzymatic replication, a central mechanism driving early chemical evolution, have focused largely on the activity of each class of these molecules separately. We show here that short nucleopeptide chimeras can replicate through autocatalytic and cross-catalytic processes, governed synergistically by the hybridization of the nucleobase motifs and the assembly propensity of the peptide segments. Unequal assembly-dependent replication induces clear selectivity toward the formation of a certain species within small networks of complementary nucleopeptides. The selectivity pattern may be influenced and indeed maximized to the point of almost extinction of the weakest replicator when the system is studied far from equilibrium and manipulated through changes in the physical (flow) and chemical (template and inhibition) conditions. We postulate that similar processes may have led to the emergence of the first functional nucleic-acid-peptide assemblies prior to the origin of life. Furthermore, spontaneous formation of related replicating complexes could potentially mark the initiation point for information transfer and rapid progression in complexity within primitive environments, which would have facilitated the development of a variety of functions found in extant biological assemblies.

The protometabolic nature of prebiotic chemistry.

The field of prebiotic chemistry has been dedicated over decades to finding abiotic routes towards the molecular components of life. There is nowadays a handful of prebiotically plausible scenarios that enable the laboratory synthesis of most amino acids, fatty acids, simple sugars, nucleotides and core metabolites of extant living organisms. The major bottleneck then seems to be the self-organization of those building blocks into systems that can self-sustain. The purpose of this tutorial review is having a close look, guided by experimental research, into the main synthetic pathways of prebiotic chemistry, suggesting how they could be wired through common intermediates and catalytic cycles, as well as how recursively changing conditions could help them engage in self-organized and dissipative networks/assemblies (i.e., systems that consume chemical or physical energy from their environment to maintain their internal organization in a dynamic steady state out of equilibrium). In the article we also pay attention to the implications of this view for the emergence of homochirality. The revealed connectivity between those prebiotic routes should constitute the basis for a robust research program towards the bottom-up implementation of protometabolic systems, taken as a central part of the origins-of-life problem. In addition, this approach should foster further exploration of control mechanisms to tame the combinatorial explosion that typically occurs in mixtures of various reactive precursors, thus regulating the functional integration of their respective chemistries into self-sustaining protocellular assemblies.

Tailored Multivalent Targeting of Siglecs with Photosensitizing Liposome Nanocarriers.

The modification of surfaces with multiple ligands allows the formation of platforms for the study of multivalency in diverse processes. Herein we use this approach for the implementation of a photosensitizer (PS)-nanocarrier system that binds efficiently to siglec-10, a member of the CD33 family of siglecs (sialic acid (SA)-binding immunoglobulin-like lectins). In particular, a zinc phthalocyanine derivative bearing three SA moieties (PcSA) has been incorporated in the membrane of small unilamellar vesicles (SUVs), retaining its photophysical properties upon insertion into the SUV's membrane. The interaction of these biohybrid systems with human siglec-10-displaying supported lipid bilayers (SLBs) has shown the occurrence of weakly multivalent, superselective interactions between vesicle and SLB. The SLB therefore acts as an excellent cell membrane mimic, while the binding with PS-loaded SUVs shows the potential for targeting siglec-expressing cells with photosensitizing nanocarriers.

A Janus-Type Phthalocyanine for the Assembly of Photoactive DNA Origami Coatings.

Design and synthesis of novel photosensitizer architectures is a key step toward new multifunctional molecular materials. Photoactive Janus-type molecules provide interesting building blocks for such systems by presenting two well-defined chemical functionalities that can be utilized orthogonally. Herein a multifunctional phthalocyanine is reported, bearing a bulky and positively charged moiety that hinders their aggregation while providing the ability to adhere on DNA origami nanostructures via reversible electrostatic interactions. On the other hand, triethylene glycol moieties render a water-soluble and chemically inert corona that can stabilize the structures. This approach provides insight into the molecular design and synthesis of Janus-type sensitizers that can be combined with biomolecules, rendering optically active biohybrids.

Tuning the Nanoaggregates of Sialylated Biohybrid Photosensitizers for Intracellular Activation of the Photodynamic Response.

In the endeavor of extending the clinical use of photodynamic therapy (PDT) for the treatment of superficial cancers and other neoplastic diseases, deeper knowledge and control of the subcellular processes that determine the response of photosensitizers (PS) are needed. Recent strategies in this direction involve the use of activatable and nanostructured PS. Here, both capacities have been tuned in two dendritic zinc(II) phthalocyanine (ZnPc) derivatives, either asymmetrically or symmetrically substituted with 3 and 12 copies of the carbohydrate sialic acid (SA), respectively. Interestingly, the amphiphilic ZnPc-SA biohybrid (1) self-assembles into well-defined nanoaggregates in aqueous solution, facilitating cellular internalization and transport whereas the PS remains inactive. Within the cells, these nanostructured hybrids localize in the lysosomes, as usually happens for anionic and hydrophilic aggregated PS. Yet, in contrast to most of them (e. g., compound 2), hybrid 1 recovers the capacity for photoinduced ROS generation within the target organelles due to its amphiphilic character; this allows disruption of aggregation when the compound is inserted into the lysosomal membrane, with the concomitant highly efficient PDT response.

Programmed Recognition between Complementary Dinucleolipids To Control the Self-Assembly of Lipidic Amphiphiles.

One of the major goals in systems chemistry is to create molecular assemblies with emergent properties that are characteristic of life. An interesting approach toward this goal is based on merging different biological building blocks into synthetic systems with properties arising from the combination of their molecular components. The covalent linkage of nucleic acids (or their constituents: nucleotides, nucleosides and nucleobases) with lipids in the same hybrid molecule leads, for example, to the so-called nucleolipids. Herein, we describe nucleolipids with a very short sequence of two nucleobases per lipid, which, in combination with hydrophobic effects promoted by the lipophilic chain, allow control of the self-assembly of lipidic amphiphiles to be achieved. The present work describes a spectroscopic and microscopy study of the structural features and dynamic self-assembly of dinucleolipids that contain adenine or thymine moieties, either pure or in mixtures. This approach leads to different self-assembled nanostructures, which include spherical, rectangular and fibrillar assemblies, as a function of the sequence of nucleobases and chiral effects of the nucleolipids involved. We also show evidence that the resulting architectures can encapsulate hydrophobic molecules, revealing their potential as drug delivery vehicles or as compartments to host interesting chemistries in their interior.

Synergy of Electrostatic and π-π Interactions in the Realization of Nanoscale Artificial Photosynthetic Model Systems.

In the scientific race to build up photoactive electron donor-acceptor systems with increasing efficiencies, little is known about the interplay of their building blocks when integrated into supramolecular nanoscale arrays, particularly in aqueous environments. Here, we describe an aqueous donor-acceptor ensemble whose emergence as a nanoscale material renders it remarkably stable and efficient. We have focused on a tetracationic zinc phthalocyanine (ZnPc) featuring pyrenes, which shows an unprecedented mode of aggregation, driven by subtle cooperation between electrostatic and π-π interactions. Our studies demonstrate monocrystalline growth in solution and a symmetry-breaking intermolecular charge transfer between adjacent ZnPcs upon photoexcitation. Immobilizing a negatively charged fullerene (C60 ) as electron acceptor onto the monocrystalline ZnPc assemblies was found to enhance the overall stability, and to suppress the energy-wasting charge recombination found in the absence of C60 . Overall, the resulting artificial photosynthetic model system exhibits a high degree of preorganization, which facilitates efficient charge separation and subsequent charge transport.

Porphyrinoid biohybrid materials as an emerging toolbox for biomedical light management.

The development of photoactive and biocompatible nanomaterials is a current major challenge of materials science and nanotechnology, as they will contribute to promoting current and future biomedical applications. A growing strategy in this direction consists of using biologically inspired hybrid materials to maintain or even enhance the optical properties of chromophores and fluorophores in biological media. Within this area, porphyrinoids constitute the most important family of organic photosensitizers. The following extensive review will cover their incorporation into different kinds of photosensitizing biohybrid materials, as a fundamental research effort toward the management of light for biomedical use, including technologies such as photochemical internalization (PCI), photoimmunotherapy (PIT), and theranostic combinations of fluorescence imaging and photodynamic therapy (PDT) or photodynamic inactivation (PDI) of microorganisms.

Photoantimicrobial Biohybrids by Supramolecular Immobilization of Cationic Phthalocyanines onto Cellulose Nanocrystals.

The development of photoactive and biocompatible nanostructures is a highly desirable goal to address the current threat of antibiotic resistance. Here, we describe a novel supramolecular biohybrid nanostructure based on the non-covalent immobilization of cationic zinc phthalocyanine (ZnPc) derivatives onto unmodified cellulose nanocrystals (CNC), following an easy and straightforward protocol, in which binding is driven by electrostatic interactions. These non-covalent biohybrids show strong photodynamic activity against S. aureus and E. coli, representative examples of Gram-positive and Gram-negative bacteria, respectively, and C. albicans, a representative opportunistic fungal pathogen, outperforming the free ZnPc counterparts and related nanosystems in which the photosensitizer is covalently linked to the CNC surface.

Generation-dependent templated self-assembly of biohybrid protein nanoparticles around photosensitizer dendrimers.

In this article, we show the great potential of dendrimers for driving the self-assembly of biohybrid protein nanoparticles. Dendrimers are periodically branched macromolecules with a perfectly defined and monodisperse structure. Moreover, they allow the possibility to incorporate functional units at predetermined sites, either at their core, branches, or surface. On these bases, we have designed and synthesized negatively charged phthalocyanine (Pc) dendrimers that behave as photosensitizers for the activation of molecular oxygen into singlet oxygen, one of the main reactive species in photodynamic therapy (PDT). The number of surface negative charges depends on dendrimer generation, whereas Pc aggregation can be tuned through the appropriate choice of the Pc metal center and its availability for axial substitution. Remarkably, both parameters determine the outcome and efficiency of the templated self-assembly process by which a virus protein forms 18 nm virus-like particles around these dendritic chromophores. Protein-dendrimer biohybrid nanoparticles of potential interest for therapeutic delivery purposes are obtained in this way. Biohybrid assemblies of this kind will have a central role in future nanomedical and nanotechnology applications.

Multifunctional Logic in a Photosensitizer with Triple-Mode Fluorescent and Photodynamic Activity.

Herein we describe a photosensitizer (PS) with the capacity to perform multiple logic operations based on a pyrene-containing phthalocyanine (Pc) derivative. The system presents three output signals (fluorescence at 377 and 683 nm, and singlet oxygen ((1)O2) production), which are dependent on three inputs: two chemical (concentration of dithiothreitol (DTT) and acidic pH) and one physical (visible light above 530 nm for (1)O2 sensitization). The multi-input/multioutput nature of this PS leads to single-, double-, and triple-mode activation pathways of its fluorescent and photodynamic functions, through the interplay of various interrelated AND, ID, and INHIBIT gates. Dual fluorescence emissions are potentially useful for orthogonal optical imaging protocols while (1)O2 is the main reactive species in photodynamic therapy (PDT). We thus expect that this kind of PS logic system will be of great interest for multimodal cellular imaging and therapeutic applications.

The systems perspective at the crossroads between chemistry and biology.

During the last century a number of authors pointed to the inherently systemic and dynamic nature of the living, yet their message was largely ignored by the mainstream of the scientific community. Tibor Ganti was one of those early pioneers, proposing a theoretical framework to understand the living principles in terms of chemical transformation cycles and their coupling. The turn of the century then brought with it a novel 'systems' paradigm, which shined light on all that previous work and carried many implications for the way we conceive of chemical and biological complexity today. In this article tribute is paid to some of those seminal contributions, highlighting the importance of adopting a systems view in present chemistry, particularly if plausible mechanisms of chemical evolution toward the first living entities want to be unraveled. We examine and put in perspective recent discoveries in the emerging subfield of 'prebiotic systems chemistry', reaching the conclusion that the functional coupling of protocellular subsystems (i.e., protometabolism, protogenome and membrane compartment) is the most challenging target to make qualitative advances in the problem of the origins of life. For the long-awaited goal of assembling an autonomous protocell from its most basic molecular building blocks, we further suggest that a systems integrative strategy should be considered from the earliest synthetic steps, already at the level of monomer precursors, opening the way to biogenesis.

Selectivity in the chiral self-assembly of nucleobase-arylazopyrazole photoswitches along DNA templates.

The control of supramolecular DNA assembly through external stimuli such as light represents a promising approach to control bioreactions, and modulate hybridization or delivery processes. Here, we report on the design of nucleobase-containing arylazopyrazole photoswitches that undergo chiral organization upon self-assembly along short DNA templates. Chiroptical spectroscopy shows that the specific nucleobases allow selectivity in the resulting supramolecular DNA complexes, and UV light irradiation triggers partial desorption of the arylazopyrazole photoswitches. Molecular modeling studies reveal the differences of binding modes between the two configurations in the templated assembly. Remarkably, our results show that the photoswitching behaviour controls the self-assembly process along DNA, opening the way to potential applications as nano- and biomaterials.

Quantifying Siglec-sialylated ligand interactions: a versatile 19F-T2 CPMG filtered competitive NMR displacement assay.

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are integral cell surface proteins crucial for the regulation of immune responses and the maintenance of immune tolerance through interactions with sialic acids. Siglecs recognize sialic acid moieties, usually found at the end of N-glycan and O-glycan chains. However, the different Siglecs prefer diverse presentations of the recognized sialic acid, depending on the type of glycosidic linkage used to link to the contiguous Gal/GalNAc or sialic acid moieties. This fact, together with possible O- or N-substitutions at the recognized glycan epitope significantly influences their roles in various immune-related processes. Understanding the molecular details of Siglec-sialoglycan interactions is essential for unraveling their specificities and for the development of new molecules targeting these receptors. While traditional biophysical methods like isothermal titration calorimetry (ITC) have been utilized to measure binding between lectins and glycans, contemporary techniques such as surface plasmon resonance (SPR), microscale thermophoresis (MST), and biolayer interferometry (BLI) offer improved throughput. However, these methodologies require chemical modification and immobilization of at least one binding partner, which can interfere the recognition between the lectin and the ligand. Since Siglecs display a large range of dissociation constants, depending on the (bio)chemical nature of the interacting partner, a general and robust method that could monitor and quantify binding would be highly welcomed. Herein, we propose the application of an NMR-based a competitive displacement assay, grounded on 19F T2-relaxation NMR and on the design, synthesis, and use of a strategic spy molecule, to assess and quantify sialoside ligand binding to Siglecs. We show that the use of this specific approach allows the quantification of Siglec binding for natural and modified sialosides, multivalent sialosides, and sialylated glycoproteins in solution, which differ in binding affinities in more than two orders of magnitude, thus providing invaluable insights into sialoglycan-mediated interactions.

Synthesis of unsymmetrical carboxyphthalocyanines by palladium-catalyzed hydroxycarbonylation of iodo-substituted precursors.

Phthalocyanines (Pcs) containing a carboxylic acid functionality directly bound to the macrocycle have been synthesized, in one step and in good yields, from iodo-substituted Pc precursors. This methodology represents a convenient alternative to the commonly used method based on two consecutive oxidations of hydroxymethyl Pcs.

Collective adaptability in a replication network of minimal nucleobase sequences.

A major challenge for understanding the origins of life is to explore how replication networks can engage in an evolutionary process. Herein, we shed light on this problem by implementing a network constituted by two different types of extremely simple biological components: the amino acid cysteine and the canonical nucleobases adenine and thymine, connected through amide bonds to the cysteine amino group and oxidation of its thiol into three possible disulfides. Supramolecular and kinetic analyses revealed that both self- and mutual interactions between such dinucleobase compounds drive their assembly and replication pathways. Those pathways involving sequence complementarity led to enhanced replication rates, suggesting a potential bias for selection. The interplay of synergistic dynamics and competition between replicators was then simulated, under conditions that are not easily accessible with experiments, in an open reactor parametrized and constrained with the unprecedentedly complete experimental kinetic data obtained for our replicative network. Interestingly, the simulations show bistability, as a selective amplification of different species depending on the initial mixture composition. Overall, this network configuration can favor a collective adaptability to changes in the availability of feedstock molecules, with disulfide exchange reactions serving as 'wires' that connect the different individual auto- and cross-catalytic pathways.

Encapsulation of phthalocyanine supramolecular stacks into virus-like particles.

We report herein the encapsulation of a water-soluble phthalocyanine (Pc) into virus-like particles (VLPs) of two different sizes, depending on the conditions. At neutral pH, the cooperative encapsulation/templated assembly of the particles induces the formation of Pc stacks instead of Pc dimers, due to an increased confinement concentration. The Pc-containing VLPs may potentially be used as photosensitizer/vehicle systems for biomedical applications such as photodynamic therapy.

Peripherally Crowded Cationic Phthalocyanines as Efficient Photosensitizers for Photodynamic Therapy.

Photodynamic therapy is a treatment modality of cancer based on the production of cytotoxic species upon the light activation of photosensitizers. Zinc phthalocyanine photosensitizers bearing four or eight bulky 2,6-di(pyridin-3-yl)phenoxy substituents were synthesized, and pyridyl moieties were methylated. The quaternized derivatives did not aggregate at all in water and retained their good photophysical properties. High photodynamic activity of these phthalocyanines was demonstrated on HeLa, MCF-7, and EA.hy926 cells with a very low EC50 of 50 nM (for the MCF-7 cell line) upon light activation while maintaining low toxicity in the dark (TC50 ≈ 600 µM), giving thus good phototherapeutic indexes (TC50/EC50) above 1400. The compounds localized primarily in the lysosomes, leading to their rupture after light activation. This induced an apoptotic cell death pathway with secondary necrosis because of extensive and swift damage to the cells. This work demonstrates the importance of a bulky and rigid arrangement of peripheral substituents in the development of photosensitizers.