University of Groningen Controlling Biological Function with Light Hansen, Mickel Jens

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University of Groningen

Controlling Biological Function with Light

Hansen, Mickel Jens

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Publication date: 2018

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Hansen, M. J. (2018). Controlling Biological Function with Light. Rijksuniversiteit Groningen.

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526510-L-sub01-bw-Hansen 526510-L-sub01-bw-Hansen 526510-L-sub01-bw-Hansen 526510-L-sub01-bw-Hansen Processed on: 26-11-2018 Processed on: 26-11-2018 Processed on: 26-11-2018

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Summary, Prospect and Conclusion

In this thesis, the control of biological function with light has been described. To this end, both the field of photopharmacology and photoactivated therapy have been introduced, reviewed and exploited. Close collaboration between synthetic organic chemistry and biology has been essential for the development of these emergent research areas. Chapter 2 gave an introduction to photopharmacology, which potentially allows for unprecedented spatiotemporal resolution together with the possibility to reduce systemic side effects and environmental toxicity. With this chapter, a critical overview and classification of pharmacological targets was given supported by descriptive examples from recent literature.

Chapter 3 focused on the development of a straightforward synthetic protocol to convert known, clinical drugs into their photoswitchable derivatives by a single synthetic step. This modular synthetic approach allows the synthesis of photoswitchable ciprofloxacin derivatives utilizing both azobenzene and spiropyran as the photoswitchable motif. Interestingly, one of the azobenzene-derived antibiotics showed a 50-fold increase in potency compared to the original ciprofloxacin. Moreover, spatiotemporal control of antibacterial activity was showcased by bacterial patterning studies. The application of similar systems with photoswitches that can be controlled with visible light might offer a simple, future way of derivatizing known drugs into clinically relevant photopharmaceuticals. To bring the concept of photopharmacology closer to clinical application, photoswitches that can be controlled with visible light are essential. Despite the design of such derivatives, their synthesis remained challenging. In chapter 4, synthetic methodology for the synthesis of tetra-ortho-substituted azobenzenes has been described. The developed synthetic method employs an ortho-lithiation of 1,3-disubstituted aromatic substrates followed by reaction with aryldiazonium salts. The products were obtained in good to excellent yields after short reaction time and simple purification. Utilizing this method, a diverse set of (unsymmetrically substituted) azobenzenes was synthesized, paving the way for the application of red-shifted azobenzenes in photopharmacology.

Next we aimed at the application of this method to control antibacterial activity with visible light (Chapter 5). First, a UV-light switchable diaminopyrimidine derivative with suitable antibacterial properties and satisfying difference in activity between the trans and cis isomer has been identified. Subsequent replacement with

tetra-ortho-substituted azobenzenes led to photoswitchable antibiotics that could be

controlled in both directions with visible (green and red) light. Moreover, the bidirectional visible light control was, for the first time, attained in situ in the presence of bacteria. Finally, an at least eight-fold difference in antimicrobial activity was obtained for the tetra-ortho-chloro derivative which could be activated with 652

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nm red light within the therapeutic window. The described system holds great promise for in vivo application. Future research should focus on a rational structure-based design, potentially allowing for a significantly larger difference in activity between the two photoisomers.

Chapter 6 described the methodology development, synthesis, characterization and biological evaluation of a privileged series of photomodulators of bacterial communication (quorum sensing). With the developed synthetic methodology, a library of 16 photoswitchable quorum sensing modulators were synthesized via a rapid 4-step synthetic sequence. The lead compounds allowed up to 70% quorum sensing induction and 59% quorum sensing inhibition. Remarkably, for the lead agonist, a >700 times difference in activity between the irradiated and non-irradiated forms was obtained showing unparalleled control in photopharmacology. A similar approach could be envisioned, especially utilizing the methodology described in chapter 4, in which medically relevant quorum sensing inhibitors are designed and synthesized. However, to this end, specific future optimization of the in this chapter described methodology is crucial allowing its application with tetra-substituted azobenzene derivatives.

Photo-activated therapy, more specific, the wavelength-selective application of photo-activated therapy has been described in chapter 7. An introduction and overview of photocleavable groups was given, together with illustrative examples of the application of multiple photocleavable groups in a single system to control diverse functions/properties with different wavelengths of light.

Chapter 8 showed the application of photo-activated therapy to control protein-protein interactions (MDM2-p53). The design, synthesis and biological evaluation of a photoactivatable MDM2 inhibitor, allowing the selective, non-invasive activation of anti-tumor properties with visible light (in collaboration with dr. F.M. Feringa, NKI) was described. Western blot analysis revealed a significant difference in p53 stabilizing activity between the masked and photodeprotected idasanutlin, allowing the application of the synthesized derivative in four different cell lines to photocontrol cellular growth. Moreover, the spatiotemporal precision of this approach was showcased by microsecond laser activation with micrometer resolution. Future studies will aim at the evaluation of the pharmacokinetic properties of the described system and confirming this proof of concept in vivo. Finally, chapter 9 described the design and synthesis of a photocleavable siderophore-antibiotic conjugate as a potential Trojan horse strategy. The pyochelin siderophore, natively utilized by P. Aeruginosa, for the active uptake of iron, was conjugated to a model quinolone antibiotic through a photocleavable linker allowing the photocontrolled release of antibiotic with >400 nm visible light. A 22-steps total synthesis towards the desired conjugate was achieved. Unfortunately, biological activity remains elusive due to insufficient solubility and aggregation of the conjugate in assay buffers at relevant concentrations. However, we believe that a

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Summary, Prospect and Conclusion

Trojan horse strategy, using photocleavable linkers, still holds great promise for fighting resistant bacteria. Towards this end, generic, water-soluble siderophores should be combined with a diverse set of potent antibiotics to overcome penetration problems of the cell-membrane of especially Gram-negative bacteria.

In conclusion, this thesis describes synthetic methodologies and their application to attain visible light control of biological functions. The reported applications range from antibiotic development to anti-tumor drug modification. We believe that the application of visible light to control biological processes is essential for the (future) development of photopharmacology and photo-activated therapy to reach the ultimate goal of clinical application. Especially, the future application and development of multiple light activatable moieties within a single biological system potentially allows for unparalleled light control of complex biological networks. Moreover, rational, structure-based design of photopharmacological systems will be a formidable challenge which, if successful, might allow an even faster academic expansion of this emergent research field and its clinical applications. To do so, the development of light-controllable systems should start in the hospital, searching for biomedical targets with the potential to have a large positive impact on patient life and healthcare. Towards this end, synthetic simplicity and availability of target photoactive compounds is crucial to make these technologies available for the broad chemical biology community. Light control of biological function remains a diverse and challenging research area with the exciting potential to change future healthcare.

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