Design and Synthesis of Visible-Light-Responsive Azobenzene Building Blocks for Chemical Biology.

Tetra-ortho-fluoro-azobenzenes are a class of photoswitches useful for the construction of visible-light-controlled molecular systems. They can be used to achieve spatio-temporal control over the properties of a chosen bioactive molecule. However, the introduction of different substituents to the tetra-fluoro-azobenzene core can significantly affect the photochemical properties of the switch and compromise biocompatibility. Herein, we explored the effect of useful substituents, such as functionalization points, attachment handles, and water-solubilizing groups, on the photochemical properties of this photochromic system. In general, all the tested fluorinated azobenzenes exhibited favorable photochemical properties, such as high photostationary state distribution and long half-lives, both in organic solvents and in water. One of the azobenzene building blocks was functionalized with a trehalose group to enable the uptake of the photoswitch into mycobacteria. Following metabolic uptake and incorporation of the trehalose-based azobenzene in the mycobacterial cell wall, we demonstrated photoswitching of the azobenzene in the isolated total lipid extract.

Strategy for Engineering High Photolysis Efficiency of Photocleavable Protecting Groups through Cation Stabilization.

Photolabile protecting groups (PPGs) enable the precise activation of molecular function with light in many research areas, such as photopharmacology, where remote spatiotemporal control over the release of a molecule is needed. The design and application of PPGs in recent years have particularly focused on the development of molecules with high molar absorptivity at long irradiation wavelengths. However, a crucial parameter, which is pivotal to the efficiency of uncaging and which has until now proven highly challenging to improve, is the photolysis quantum yield (QY). Here, we describe a novel and general approach to greatly increase the photolysis QY of heterolytic PPGs through stabilization of an intermediate chromophore cation. When applied to coumarin PPGs, our strategy resulted in systems possessing an up to a 35-fold increase in QY and a convenient fluorescent readout during their uncaging, all while requiring the same number of synthetic steps for their preparation as the usual coumarin systems. We demonstrate that the same QY engineering strategy applies to different photolysis payloads and even different classes of PPGs. Furthermore, analysis of the DFT-calculated energy barriers in the first singlet excited state reveals valuable insights into the important factors that determine photolysis efficiency. The strategy reported herein will enable the development of efficient PPGs tailored for many applications.

Light-Control over Casein Kinase 1δ Activity with Photopharmacology: A Clear Case for Arylazopyrazole-Based Inhibitors.

Protein kinases are responsible for healthy cellular processes and signalling pathways, and their dysfunction is the basis of many pathologies. There are numerous small molecule inhibitors of protein kinases that systemically regulate dysfunctional signalling processes. However, attaining selectivity in kinase inhibition within the complex human kinome is still a challenge that inspires unconventional approaches. One of those approaches is photopharmacology, which uses light-controlled bioactive molecules to selectively activate drugs only at the intended space and time, thereby avoiding side effects outside of the irradiated area. Still, in the context of kinase inhibition, photopharmacology has thus far been rather unsuccessful in providing light-controlled drugs. Here, we present the discovery and optimisation of a photoswitchable inhibitor of casein kinase 1δ (CK1δ), important for the control of cell differentiation, circadian rhythm, DNA repair, apoptosis, and numerous other signalling processes. Varying the position at which the light-responsive azobenzene moiety has been introduced into a known CK1δ inhibitor, LH846, revealed the preferred regioisomer for efficient photo-modulation of inhibitory activity, but the photoswitchable inhibitor suffered from sub-optimal (photo)chemical properties. Replacement of the bis-phenyl azobenzene group with the arylazopyrazole moiety yielded a superior photoswitch with very high photostationary state distributions, increased solubility and a 10-fold difference in activity between irradiated and thermally adapted samples. The reasons behind those findings are explored with molecular docking and molecular dynamics simulations. Results described here show how the evaluation of privileged molecular architecture, followed by the optimisation of the photoswitchable unit, is a valuable strategy for the challenging design of the photoswitchable kinase inhibitors.

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.

Computational Design, Synthesis, and Photochemistry of Cy7-PPG, an Efficient NIR-Activated Photolabile Protecting Group for Therapeutic Applications.

Photolabile Protecting Groups (PPGs) are molecular tools used, for example, in photopharmacology for the activation of drugs with light, enabling spatiotemporal control over their potency. Yet, red-shifting of PPG activation wavelengths into the NIR range, which penetrates the deepest in tissue, has often yielded inefficient or insoluble molecules, hindering the use of PPGs in the clinic. To solve this problem, we report herein a novel concept in PPG design, by transforming clinically-applied NIR-dyes with suitable molecular orbital configurations into new NIR-PPGs using computational approaches. Using this method, we demonstrate how Cy7, a class of NIR dyes possessing ideal properties (NIR-absorption, high molecular absorptivity, excellent aqueous solubility) can be successfully converted into Cy7-PPG. We report a facile synthesis towards Cy7-PPG from accessible precursors and confirm its excellent properties as the most redshifted oxygen-independent NIR-PPG to date (λmax =746 nm).