Triplet-Triplet Energy Transfer: A Simple Strategy for an Efficient Visible Light-Induced Photoclick Reaction.

Photoclick reactions combine the advantages offered by light-driven processes and classical click chemistry and have found applications ranging from surface functionalization, polymer conjugation, photo-crosslinking, and protein labeling. Despite these advances, the dependency of most of the photoclick reactions on UV light poses a severe obstacle for their general implementation, as this light can be absorbed by other molecules in the system resulting in their degradation or unwanted reactivity. However, the development of a simple and efficient system to achieve bathochromically shifted photoclick transformations remains challenging. Here, we introduce triplet-triplet energy transfer as a fast and selective way to enable visible light-induced photoclick reactions. Specifically, we show that 9,10-phenanthrenequinones (PQs) can efficiently react with electron-rich alkenes (ERAs) in the presence of a catalytic amount (as little as 5 mol %) of photosensitizers. The photocycloaddition reaction can be achieved under green (530 nm) or orange (590 nm) light irradiation, representing a bathochromic shift of over 100 nm as compared to the classical PQ-ERAs system. Furthermore, by combining appropriate reactants, we establish an orthogonal, blue and green light-induced photoclick reaction system in which the product distribution can be precisely controlled by the choice of the color of light.

Establishing PQ-ERA photoclick reactions with unprecedented efficiency by engineering of the nature of the phenanthraquinone triplet state.

The light-induced photocycloaddition of 9,10-phenanthrenequinone (PQ) with electron-rich alkenes (ERA), known as the PQ-ERA reaction, is a highly attractive photoclick reaction characterized by high selectivity, external non-invasive control with light and biocompatibility. The conventionally used PQ compounds show limited reactivity, which hinders the overall efficiency of the PQ-ERA reaction. To address this issue, we present in this study a simple strategy to boost the reactivity of the PQ triplet state to further enhance the efficiency of the PQ-ERA reaction, enabled by thiophene substitution at the 3-position of the PQ scaffold. Our investigations show that this substitution pattern significantly increases the population of the reactive triplet state (3ππ*) during excitation of 3-thiophene PQs. This results in a superb photoreaction quantum yield (ΦP, up to 98%), high second order rate constants (k2, up to 1974 M-1 s-1), and notable oxygen tolerance for the PQ-ERA reaction system. These results have been supported by both experimental transient absorption data and theoretical calculations, providing further evidence for the effectiveness of this strategy, and offering fine prospects for fast and efficient photoclick transformations.

Modulating Secondary Structure Motifs Through Photo-Labile Peptide Staples.

Peptide-protein interactions (PPIs) are facilitated by the well-defined three-dimensional structure of bioactive peptides, interesting compounds for the development of new therapeutic agents. Their secondary structure and thus their propensity to engage in PPIs can be influenced by the introduction of peptide staples on the side chains. In particular, light-controlled staples based on azobenzene photoswitches and their structural influence on helical peptides have been studied extensively. In contrast, photolabile staples bearing photocages as a structural key motif, have mainly been used to block supramolecular interactions. Their influence on the secondary structure of the target peptide is under-investigated. Thus, in this study we use a combination of spectroscopic techniques and in silico simulations to systematically study a series of helical peptides with varying length of the photo-labile staple to obtain a detailed insight into the structure-property relationship in such photoresponsive biomolecules.

Molecular Engineering To Enhance Reactivity and Selectivity in an Ultrafast Photoclick Reaction.

Light-induced 9,10-phenanthrenequinone-electron-rich alkene (PQ-ERA) photocycloadditions are an attractive new type of photoclick reaction, featuring fast conversions and high biocompatibility. However, the tunability of the reaction was hardly investigated up to now. To this end, we explored the influence of substituents on both reaction partners and the reaction rate between the PQs and ERAs. We identified new handles for functionalization and discovered that using enamines as ERAs leads to drastically enhanced rates (>5400 times faster), high photoreaction quantum yields (ΦP , up to 65 %), and multicolor emission output as well as a high fluorescence quantum yield of the adducts (ΦF , up to 97 %). Further investigation of the photophysical and photochemical properties provided insights to design orthogonal reaction systems both in solution and on nanoparticle surfaces for ultrafast chemoselective functionalization by photoclick reactions.

Artificial peptides to induce membrane denaturation and disruption and modulate membrane composition and fusion.

Membranes consisting of phospholipid bilayers are an essential constituent of eukaryotic cells and their compartments. The alteration of their composition, structure, and morphology plays an important role in modulating physiological processes, such as transport of molecules, cell migration, or signaling, but it can also lead to lethal effects. The three main classes of membrane-active peptides that are responsible for inducing such alterations are cell-penetrating peptides (CPPs), antimicrobial peptides (AMPs), and fusion peptides (FPs). These peptides are able to interact with lipid bilayers in highly specific and tightly regulated manners. They can either penetrate the membrane, inducing nondestructive, transient alterations, or disrupt, permeabilize, or translocate through it, or induce membrane fusion by generating attractive forces between two bilayers. Because of these properties, membrane-active peptides have attracted the attention of the pharmaceutical industry, and naturally occurring bioactive structures have been used as a platform for synthetic modification and the development of artificial analogs with optimized therapeutic properties to transport biologically active cargos or serve as novel antimicrobial agents. In this review, we focus on synthetic membrane interacting peptides with bioactivity comparable with their natural counterparts and describe their mechanism of action.

Photoswitchable architecture transformation of a DNA-hybrid assembly at the microscopic and macroscopic scale.

Molecular recognition-driven self-assembly employing single-stranded DNA (ssDNA) as a template is a promising approach to access complex architectures from simple building blocks. Oligonucleotide-based nanotechnology and soft-materials benefit from the high information storage density, self-correction, and memory function of DNA. Here we control these beneficial properties with light in a photoresponsive biohybrid hydrogel, adding an extra level of function to the system. An ssDNA template was combined with a complementary photo-responsive unit to reversibly switch between various functional states of the supramolecular assembly using a combination of light and heat. We studied the structural response of the hydrogel at both the microscopic and macroscopic scale using a combination of UV-vis absorption and CD spectroscopy, as well as fluorescence, transmission electron, and atomic force microscopy. The hydrogels grown from these supramolecular self-assembly systems show remarkable shape-memory properties and imprinting shape-behavior while the macroscopic shape of the materials obtained can be further manipulated by irradiation.

Hypothesis-Driven, Structure-Based Design in Photopharmacology: The Case of eDHFR Inhibitors.

Photopharmacology uses light to regulate the biological activity of drugs. This precise control is obtained through the incorporation of molecular photoswitches into bioactive molecules. A major challenge for photopharmacology is the rational design of photoswitchable drugs that show light-induced activation. Computer-aided drug design is an attractive approach toward more effective, targeted design. Herein, we critically evaluated different structure-based approaches for photopharmacology with Escherichia coli dihydrofolate reductase (eDHFR) as a case study. Through the iterative examination of our hypotheses, we progressively tuned the design of azobenzene-based, photoswitchable eDHFR inhibitors in five design-make-switch-test-analyze cycles. Targeting a hydrophobic subpocket of the enzyme and a specific salt bridge only with the thermally metastable cis-isomer emerged as the most promising design strategy. We identified three inhibitors that could be activated upon irradiation and reached potencies in the low-nanomolar range. Above all, this systematic study provided valuable insights for future endeavors toward rational photopharmacology.

A Molecular Pump Facilitates Mechanical Adsorption Away from Equilibrium.

Artificial molecular pumps that perform unidirectional motion on the molecular scale have been used to design systems operating away from equilibrium. Crafting such pumps onto a surface allows their unidirectional movement to be organized in a confined environment as well as the active pumping of adsorbents from the bulk solution to the surface. This study led to the discovery of the phenomenon of mechanisorption and paves the way for the construction of highly complex organized molecular architectures.

Phenylimino Indolinone: A Green-Light-Responsive T-Type Photoswitch Exhibiting Negative Photochromism.

Imines are photoaddressable motifs useful in the development of new generations of molecular switches, but their operation with low-energy photons and control over isomer stability remain challenging. Based on a computational design, we developed phenylimino indolinone (PIO), a green-light-addressable T-type photoswitch showing negative photochromism. The isomerization behavior of this photoactuator of the iminothioindoxyl (ITI) class was studied using time-resolved spectroscopies on time scales from femtoseconds to the steady state and by quantum-chemical analyses. The understanding of the isomerization properties and substituent effects governing these photoswitches opens new avenues for the development of novel T-type visible-light-addressable photoactuators based on C=N bonds.

Directing Coupled Motion with Light: A Key Step Toward Machine-Like Function.

Molecular photoactuators can control shape and chemical or physical properties of the responsive system they are embedded in. These effects are usually mediated by supramolecular interactions and can be amplified to perform work at the micro- and macroscopic scale, for instance, in materials and biomimetic systems. While many studies focus on the observable outcome of these events, photoresponsive structures can also translate their conformational change to molecular components and perform work against random Brownian motion. Stereochemical cascades can amplify light-generated motion to a distant moiety of the same molecule or molecular assembly, via conformationally restricted stereogenic elements. Being able to control the conformation or motion of molecular systems remotely provides prospects for the design of the smallest machines imaginable. This Focus Review emphasizes the emergence of directed, coupled motion of remote functionalities triggered by light-powered switches and motors as a tool to control molecular topology and function.

Molecular photoswitches in aqueous environments.

Molecular photoswitches enable dynamic control of processes with high spatiotemporal precision, using light as external stimulus, and hence are ideal tools for different research areas spanning from chemical biology to smart materials. Photoswitches are typically organic molecules that feature extended aromatic systems to make them responsive to (visible) light. However, this renders them inherently lipophilic, while water-solubility is of crucial importance to apply photoswitchable organic molecules in biological systems, like in the rapidly emerging field of photopharmacology. Several strategies for solubilizing organic molecules in water are known, but there are not yet clear rules for applying them to photoswitchable molecules. Importantly, rendering photoswitches water-soluble has a serious impact on both their photophysical and biological properties, which must be taken into consideration when designing new systems. Altogether, these aspects pose considerable challenges for successfully applying molecular photoswitches in aqueous systems, and in particular in biologically relevant media. In this review, we focus on fully water-soluble photoswitches, such as those used in biological environments, in both in vitro and in vivo studies. We discuss the design principles and prospects for water-soluble photoswitches to inspire and enable their future applications.

Predicting the substituent effects in the optical and electrochemical properties of N,N'-substituted isoindigos.

Isoindigo, the structural isomer of the well-known dye indigo, has seen a major revival recently because of the increasing interest of its use as a potential drug core structure and for the development of organic photovoltaic materials. Highly beneficial for diverse applications are its facile synthesis, straightforward functionalisation and the broad absorption band in the visible range. Moreover, its intrinsic electron deficiency renders isoindigo a promising acceptor structure in bulk heterojunction architectures. Here we present new insights into the substituent effects of N-functionalised isoindigos, developing a reliable and fast in silico screening approach of a library of compounds. Using experimental UV-Vis and electrochemical data increased the accuracy of the TD-DFT method employed. This procedure allowed us to accurately predict the optical and electrochemical properties of N-functionalised isoindigos and the elucidation of the relationship between substituent effects and electronic properties.

Rational design of a photoswitchable DNA glue enabling high regulatory function and supramolecular chirality transfer.

Short, complementary DNA single strands with mismatched base pairs cannot undergo spontaneous formation of duplex DNA (dsDNA). Mismatch binding ligands (MBLs) can compensate this effect, inducing the formation of the double helix and thereby acting as a molecular glue. Here, we present the rational design of photoswitchable MBLs that allow for reversible dsDNA assembly by light. Careful choice of the azobenzene core structure results in excellent band separation of the E and Z isomers of the involved chromophores. This effect allows for efficient use of light as an external control element for duplex DNA formation and for an in-depth study of the DNA-ligand interaction by UV-Vis, SPR, and CD spectroscopy, revealing a tight mutual interaction and complementarity between the photoswitchable ligand and the mismatched DNA. We also show that the configuration of the switch reversibly dictates the conformation of the DNA strands, while the dsDNA serves as a chiral clamp and translates its chiral information onto the ligand inducing a preference in helical chirality of the Z isomer of the MBLs.

Ultrafast Photoclick Reaction for Selective 18F-Positron Emission Tomography Tracer Synthesis in Flow.

The development of very fast, clean, and selective methods for indirect labeling in PET tracer synthesis is an ongoing challenge. Here we present the development of an ultrafast photoclick method for the synthesis of short-lived 18F-PET tracers based on the photocycloaddition reaction of 9,10-phenanthrenequinones with electron-rich alkenes. The respective precursors are synthetically easily accessible and can be functionalized with various target groups. Using a flow photo-microreactor, the photoclick reaction can be performed in 60 s, and clinically relevant tracers for prostate cancer and bacterial infection imaging were prepared to demonstrate practicality of the method.

Towards Photochromic Azobenzene-Based Inhibitors for Tryptophan Synthase.

Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene-based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto-unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light.

Significance of the Protein Interface Configuration for Allostery in Imidazole Glycerol Phosphate Synthase.

Imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima is a model enzyme for studying allostery. The ImGPS complex consists of the cyclase subunit HisF and the glutaminase subunit HisH whose activity is stimulated by substrate binding to HisF in a V-type manner. To investigate the significance of a putative closing hinge motion at the cyclase:glutaminase interface for HisH activity, we replaced residue W123 in HisH with the light-switchable unnatural amino acid phenylalanine-4'-azobenzene (AzoF). Crystal structure analysis employing angle, buried surface area, and distance measurements showed that incorporation of AzoF at this position causes a closing of the interface by ∼18 ± 3%. This slightly different interface configuration results in a much higher catalytic efficiency in unstimulated HisH due to an elevated turnover number. Moreover, the catalytic efficiency of HisH when stimulated by binding of a substrate to HisF was also significantly increased by AzoF incorporation. This was caused by a K-type stimulation that led to a decrease in the apparent dissociation constant for its substrate, glutamine. In addition, AzoF improved the apparent binding of a substrate analogue at the HisF active site. Remarkably, light-induced isomerization of AzoF considerably enhanced these effects. In conclusion, our findings confirm that signal transduction from HisF to HisH in ImGPS involves the closing of the cyclase:glutaminase subunit interface and that incorporation of AzoF at a hinge position reinforces this catalytically relevant conformational change.

General Principles for the Design of Visible-Light-Responsive Photoswitches: Tetra-ortho-Chloro-Azobenzenes.

Molecular photoswitches enable reversible external control of biological systems, nanomachines, and smart materials. Their development is driven by the need for low energy (green-red-NIR) light switching, to allow non-invasive operation with deep tissue penetration. The lack of clear design principles for the adaptation and optimization of such systems limits further applications. Here we provide a design rulebook for tetra-ortho-chloroazobenzenes, an emerging class of visible-light-responsive photochromes, by elucidating the role that substituents play in defining their key characteristics: absorption spectra, band overlap, photoswitching efficiencies, and half-lives of the unstable cis isomers. This is achieved through joint photochemical and theoretical analyses of a representative library of molecules featuring substituents of varying electronic nature. A set of guidelines is presented that enables tuning of properties to the desired application through informed photochrome engineering.

Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids.

Imidazole glycerol phosphate synthase (ImGPS) is an allosteric bienzyme complex in which substrate binding to the synthase subunit HisF stimulates the glutaminase subunit HisH. To control this stimulation with light, we have incorporated the photo-responsive unnatural amino acids phenylalanine-4'-azobenzene (AzoF), o-nitropiperonyl-O-tyrosine (NPY), and methyl-o-nitropiperonyllysine (mNPK) at strategic positions of HisF. The light-mediated isomerization of AzoF at position 55 (fS55AzoFE ↔ fS55AzoFZ) resulted in a reversible 10-fold regulation of HisH activity. The light-mediated decaging of NPY at position 39 (fY39NPY → fY39) and of mNPK at position 99 (fK99mNPK → fK99) led to a 4- to 6-fold increase of HisH activity. Molecular dynamics simulations explained how the unnatural amino acids interfere with the allosteric machinery of ImGPS and revealed additional aspects of HisH stimulation in wild-type ImGPS. Our findings show that unnatural amino acids can be used as a powerful tool for the spatiotemporal control of a central metabolic enzyme complex by light.

Unraveling the Thermal Isomerization Mechanisms of Heteroaryl Azoswitches: Phenylazoindoles as Case Study.

The research on heteroaromatic azoswitches has been blossoming in recent years due to their astonishingly broad range of properties. Minimal chemical modifications can drastically change the demeanor of these switches, regarding photophysical and (photo)chemical properties, promoting them as ideal scaffolds for a vast variety of applications based on bistable light-addressable systems. However, most of the characteristics exhibited by heteroaryl azoswitches were found empirically, and only a few works focus on their rationalization. Herein we report on a mechanistic study employing phenylazoindoles as a model reference, combining spectroscopic experiments with comprehensive computational analysis. This approach will elucidate the intrinsic correlations between the molecular structure of the switch and its thermal behavior, allowing a more rational design transferable to various heteroaryl azoswitches.

Comparative Study of Photoswitchable Zinc-Finger Domain and AT-Hook Motif for Light-Controlled Peptide-DNA Binding.

DNA-peptide interactions are involved in key life processes, including DNA recognition, replication, transcription, repair, organization, and modification. Development of tools that can influence DNA-peptide binding non-invasively with high spatiotemporal precision could aid in determining its role in cells and tissues. Here, the design, synthesis, and study of photocontrolled tools for sequence-specific small peptide-DNA major and minor groove interactions are reported, shedding light on DNA binding by transcriptionally active peptides. In particular, photoswitchable moieties were implemented in the peptide backbone or turn region. In each case, DNA binding was affected by photochemical isomerization, as determined in fluorescent displacement assays on model DNA strands, which provides promising tools for DNA modulation.