Abstract
The adenosine A1 receptor (A1R) is a class A GPCR and is one of four receptor subtypes. It is expressed ubiquitously throughout the body; however, is found in elevated levels in the brain and cardiac tissue. There is growing evidence to suggest that the A1R can signal not only as a receptor monomer, but also as a homo- or heterodimer. Dysregulated signalling of both A1R monomers and dimers has been implicated in neurological diseases such as Alzheimer’s disease. Because of this, there is a strong incentive to develop chemical tools to further investigate the role of A1R in disease.
Bifunctional chemical tools possess dual reactivity; this could take the form of a covalent warhead that enables covalent attachment to the receptor and a bioorthogonal handle that can undergo a bioorthogonal reaction with a complementary reaction partner. There is a growing requirement to develop bioorthogonal reactions that are triggerable and provide a fluorescent read-out of the reaction. The tetrazine ligation is a bioorthogonal inverse electron-demand Diels–Alder (IEDDA) reaction that has been adapted to be photoactivatable or fluorogenic; however, a photoactivable and fluorogenic tetrazine ligation reaction has yet to be reported. Moreover, a photoactivatable fluorogenic bifunctional chemical tool has yet to be developed. These principles could be applied to the detection of A1R homo- or heterodimers. This thesis therefore explores both the design and synthesis of bifunctional chemical tools for the A1R (Chapters 2 and 3) and the development of a photoactivatable and fluorogenic tetrazine ligation reaction (Chapters 4 and 5).
The first-generation bifunctional chemical tool was designed based on the potent and selective A1R antagonist SLV320. Sixteen compounds were designed and evaluated using computational-protein ligand docking to give the sulfonyl fluoride and azide-containing bifunctional lead 2.20 (Chapter 2). Synthesis of the first-generation computational lead 2.20 was unsuccessful due to the inability to install the required trans-cyclohexyl secondary amine moiety onto the pyrrolopyrimidine core despite attempting a range of SNAr and Buchwald–Hartwig amination conditions. Because of this, a second-generation bifunctional chemical tool 3.3 was identified from a second protein-ligand docking screen (Chapter 3). The synthesis of 3.3 was successfully achieved using a Sonogashira coupling in the key derivatisation step. In addition to sulfonyl fluoride and azide-containing bifunctional tool 3.3, several analogues were successfully synthesised with/without key features assess the effect of SLV320 functionalisation. This included the non-covalent sulfonyl methyl 3.13, the acetamide 3.14, the PEG-containing acetamide 3.12, and the sulfonyl fluoride-containing compound without a bioorthogonal PEG handle 3.11.
A photoactivatable fluorogenic tetrazine ligation reaction was investigated using the coumarin scaffold as both a photocaging group and a means to red shift the IEDDA product (Chapter 4). Photocaging a tetrazine using Approaches A, B and C were attempted but unfortunately all were unsuccessful due to the instability and degradation of the coumarin and in some cases also the tetrazine reaction partner. Moving away from a coumarin-based system, the dihydrotetrazine carbamate 4.62 was successfully synthesised using benzyl chloroformate as a non-photoactivatable model system. This enabled the regioisomer and dihydrotetrazine tautomer of 4.62 to be determined using X-ray crystallography. A nitro benzyl photocaged dihydrotetrazine 5.28 was then synthesised (Chapter 5). Dihydrotetrazine carbamate 5.28 quantitively released tetrazine 5.29 upon irradiation with 365 nm light and was stable to ambient light in solution. The IEDDA reaction between 5.29 and 4.14 formed pyridazine 5.30 rapidly. Pyridazine 5.30 had an increase in fluorescence quantum yield and displayed a red-shifted emission spectra compared to tetrazine 5.29, meaning that the reaction was both photoactivatable and fluorogenic.