Cross Metathesis and Ring-Closing Metathesis Reactions of Modified Amino Acids and Peptides.

This thesis investigates the application of cross metathesis and ring-closing metathesis to amino acid and peptide-based substrates that are suitably modified to contain an olefin tether. Chapter One introduces olefin metathesis, describes the mechanism of cross metathesis (CM) and ring-closing metathesis (RCM), and outlines the catalysts that can be used for these transformations. The application of CM and RCM to amino acid and peptide-based systems is reviewed. Chapter Two describes the CM coupling between modified lysine- (2.34 - 2.37, 2.43), serine- (2.45, 2.46), and cysteine-based (2.48, 2.49a, 2.51) amino acids and dipeptides (2.54, 2.57) to a terminal alkene (2.61, 2.65), carbohydrate (1.51b), or fatty acid (2.76) target compound using catalyst 1.17. The amino acid and dipeptide-based CM substrates were prepared by side-chain acylation of the parent amino acid with carboxylic acids containing variable but controllable olefin tether lengths. A CM model study in which these amino acid-based substrates were coupled to terminal alkene 2.61 and 2.65 gave CM products 2.66 - 2.74. CM was then carried out between amino acid-based substrates and a carbohydrate (1.51b) or fatty acid derivative (2.76), that afforded a novel series of glycoamino acids (2.80 - 2.85) and lipoamino acids (2.94 - 2.101). Chapter Three describes the synthesis of amino acid dimers by CM. Two serine-based (3.22 - 3.23) and two cysteine-based (3.24 - 3.25) symmetrical dimers along with two unsymmetrical serine-cysteine dimers (3.26 - 3.27) were prepared from the same side-chain acylated amino acid substrates described in chapter 2. These compounds are examples of novel cross-linked amino acid-based dimers, and further illustrate the versatility of the CM methodology developed in this thesis. Chapter Four describes the synthesis of cyclic amino acids and dipeptides via RCM of acyclic precursors that are suitably modified with acyl olefin tethers of variable length. Cyclic compounds based on lysine (4.6, 4.13), serine (4.31, 4.33), and cysteine (4.40, 4.42) single amino acid residues, and compounds based on lysine (4.16, 4.21, 4.27), serine (4.37), and cysteine (4.45, 4.46) dipeptides were prepared. All these compounds were constructed using the same, versatile general method, which involves acylation of the natural amino acid substrate with a carboxylic acid of controllable olefin tether length followed by RCM with catalyst 1.17 to give cyclic products containing variable ring sizes

Direct visualisation of internalization of the adenosine A3 receptor and localization with arrestin3 using a fluorescent agonist

Fluorescence based probes provide a novel way to study the dynamic internalization process of G protein-coupled receptors (GPCRs). Recent advances in the rational design of fluorescent ligands for GPCRs have been used here to generate new fluorescent agonists containing tripeptide linkers for the adenosine A3 receptor. The fluorescent agonist BY630-X-(D)-A-(D)-A-G-ABEA was found to be a highly potent agonist at the adenosine A3 receptor in both reporter gene (pEC50 = 8.48 ± 0.09) and internalization assays (pEC50 = 7.47 ± 0.11). Confocal imaging studies showed that BY630-X-(D)-A-(D)-A-G-ABEA was internalized with A3 linked to yellow fluorescent protein, which was blocked by the competitive antagonist MRS1220. Internalization of untagged adenosine A3 could also be visualized with BY630-X-(D)-A-(D)-A-G-ABEA treatment. Further, BY630-X-(D)-A-(D)-A-G-ABEA stimulated the formation of receptor–arrestin3 complexes and was found to localize with these intracellular complexes. This highly potent agonist with excellent imaging properties should be a valuable tool to study receptor internalization

Conversion of a non-selective adenosine receptor antagonist into A3-selective high affinity fluorescent probes using peptide-based linkers

Advances in fluorescence-based imaging technologies have helped propel the study of real-time biological readouts and analysis across many different areas. In particular the use of fluorescent ligands as chemical tools to study proteins such as G protein-coupled receptors (GPCRs) has received ongoing interest. Methods to improve the efficient chemical synthesis of fluorescent ligands remain of paramount importance to ensure this area of bioanalysis continues to advance. Here we report conversion of the non-selective GPCR adenosine receptor antagonist Xanthine Amine Congener into higher affinity and more receptor subtype-selective fluorescent antagonists. This was achieved through insertion and optimisation of a dipeptide linker between the adenosine receptor pharmacophore and the fluorophore. Fluorescent probe 27 containing BODIPY 630/650 (pKD = 9.12 ± 0.05 [hA3AR]), and BODIPY FL-containing 28 (pKD = 7.96 ± 0.09 [hA3AR]) demonstrated clear, displaceable membrane binding using fluorescent confocal microscopy. From in silico analysis of the docked ligand-receptor complexes of 27, we suggest regions of molecular interaction that could account for the observed selectivity of these peptide-linker based fluorescent conjugates. This general approach of converting a non-selective ligand to a selective biological tool could be applied to other ligands of interest

Development of novel fluorescent histamine H₁-receptor antagonists to study ligand-binding kinetics in living cells

The histamine H1-receptor (H1R) is an important mediator of allergy and inflammation. H1R antagonists have particular clinical utility in allergic rhinitis and urticaria. Here we have developed six novel fluorescent probes for this receptor that are very effective for high resolution confocal imaging, alongside bioluminescence resonance energy transfer approaches to monitor H1R ligand binding kinetics in living cells. The latter technology exploits the opportunities provided by the recently described bright bioluminescent protein NanoLuc when it is fused to the N-terminus of a receptor. Two different pharmacophores (mepyramine or the fragment VUF13816) were used to generate fluorescent H1R antagonists conjugated via peptide linkers to the fluorophore BODIPY630/650. Kinetic properties of the probes showed wide variation, with the VUF13816 analogues having much longer H1R residence times relative to their mepyramine-based counterparts. The kinetics of these fluorescent ligands could also be monitored in membrane preparations providing new opportunities for future drug discovery applications

Cannabinoid Receptor 2 Signalling Bias Elicited by 2,4,6-Trisubstituted 1,3,5-Triazines

Cannabinoid receptor 2 (CB2) is predominantly distributed in immune tissues and cells and is a promising therapeutic target for modulating inflammation. In this study we designed and synthesised a series of 2,4,6-trisubstituted 1,3,5-triazines with piperazinylalkyl or 1,2-diethoxyethane (PEG2) chains as CB2 agonists, all of which were predicted to be considerably more polar than typical cannabinoid ligands. In this series, we found that triazines containing an adamantanyl group were conducive to CB2 binding whereas those with a cyclopentyl group were not. Although the covalent attachment of a PEG2 linker to the adamantyl triazines resulted in a decrease in binding affinity, some of the ligands produced very interesting hCB2 signalling profiles. Six compounds with notable hCB2 orthosteric binding were functionally characterised in three pathways; internalisation, cyclic adenosine monophosphate (cAMP) and ERK phosphorylation (pERK). These were predominantly confirmed to be hCB2 agonists, and upon comparison to a reference ligand (CP 55,940), four compounds exhibited signalling bias. Triazines 14 (UOSD017) and 15 were biased towards internalisation over cAMP and pERK, and 7 was biased away from pERK activation relative to cAMP and internalisation. Intriguingly, the triazine with an amino-PEG2-piperazinyl linker (13 [UOSD008]) was identified to be a mixed agonist/inverse agonist, exhibiting apparent neutral antagonism in the internalisation pathway, transient inverse agonism in the cAMP pathway and weak partial agonism in the pERK pathway. Both the cAMP and pERK signalling were pertussis toxin (PTX) sensitive, implying that 13 is acting as both a weak agonist and inverse agonist at CB2 via Gαi/o. Compound 10 (UOSD015) acted as a balanced high intrinsic efficacy agonist with the potential to produce greater hCB2-mediated efficacy than reference ligand CP 55,940. As 10 includes a Boc-protected PEG2 moiety it is also a promising candidate for further modification, for example with a secondary reporter or fluorophore. The highest affinity compound in this set of relatively polar hCB2 ligands was compound 16, which acted as a slightly partial balanced agonist in comparison with CP 55,940. The ligands characterised here may therefore exhibit unique functional properties in vivo and have the potential to be valuable in the future development of CB2-directed therapeutics

Cross Metathesis and Ring-Closing Metathesis Reactions of Modified Amino Acids and Peptides.

This thesis investigates the application of cross metathesis and ring-closing metathesis to amino acid and peptide-based substrates that are suitably modified to contain an olefin tether. Chapter One introduces olefin metathesis, describes the mechanism of cross metathesis (CM) and ring-closing metathesis (RCM), and outlines the catalysts that can be used for these transformations. The application of CM and RCM to amino acid and peptide-based systems is reviewed. Chapter Two describes the CM coupling between modified lysine- (2.34 - 2.37, 2.43), serine- (2.45, 2.46), and cysteine-based (2.48, 2.49a, 2.51) amino acids and dipeptides (2.54, 2.57) to a terminal alkene (2.61, 2.65), carbohydrate (1.51b), or fatty acid (2.76) target compound using catalyst 1.17. The amino acid and dipeptide-based CM substrates were prepared by side-chain acylation of the parent amino acid with carboxylic acids containing variable but controllable olefin tether lengths. A CM model study in which these amino acid-based substrates were coupled to terminal alkene 2.61 and 2.65 gave CM products 2.66 - 2.74. CM was then carried out between amino acid-based substrates and a carbohydrate (1.51b) or fatty acid derivative (2.76), that afforded a novel series of glycoamino acids (2.80 - 2.85) and lipoamino acids (2.94 - 2.101). Chapter Three describes the synthesis of amino acid dimers by CM. Two serine-based (3.22 - 3.23) and two cysteine-based (3.24 - 3.25) symmetrical dimers along with two unsymmetrical serine-cysteine dimers (3.26 - 3.27) were prepared from the same side-chain acylated amino acid substrates described in chapter 2. These compounds are examples of novel cross-linked amino acid-based dimers, and further illustrate the versatility of the CM methodology developed in this thesis. Chapter Four describes the synthesis of cyclic amino acids and dipeptides via RCM of acyclic precursors that are suitably modified with acyl olefin tethers of variable length. Cyclic compounds based on lysine (4.6, 4.13), serine (4.31, 4.33), and cysteine (4.40, 4.42) single amino acid residues, and compounds based on lysine (4.16, 4.21, 4.27), serine (4.37), and cysteine (4.45, 4.46) dipeptides were prepared. All these compounds were constructed using the same, versatile general method, which involves acylation of the natural amino acid substrate with a carboxylic acid of controllable olefin tether length followed by RCM with catalyst 1.17 to give cyclic products containing variable ring sizes

Novel Cannabinoid Receptor 2 (CB2) Low Lipophilicity Agonists Produce Distinct cAMP and Arrestin Signalling Kinetics without Bias

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and β-arrestin-2 translocation with temporal resolution (BRET-based biosensors). For the 3-carbamoyl-2-pyridone derivatives, we found that modifying the previously reported compound UOSS77 (also known as S-777469) by appending a PEG2-alcohol via a 3-carbomylcyclohexyl carboxamide (UOSS75) lowered lipophilicity, and preserved binding affinity and signalling profile. The 1,8-naphthyridin-2(1H)-one-3-carboxamide UOMM18, containing a cis configuration at the 3-carboxamide cyclohexyl and with an alcohol on the 4-position of the cyclohexyl, had lower lipophilicity but similar CB2 affinity and biological activity to previously reported compounds of this class. Relative to CP55,940, the new compounds acted as partial agonists and did not exhibit signalling bias. Interestingly, while all compounds shared similar temporal trajectories for maximal efficacy, differing temporal trajectories for potency were observed. Consequently, when applied at sub-maximal concentrations, CP55,940 tended to elicit sustained (cAMP) or increasing (arrestin) responses, whereas responses to the new compounds tended to be transient (cAMP) or sustained (arrestin). In future studies, the compounds characterised here may be useful in elucidating the consequences of differential temporal signalling profiles on CB2-mediated physiological responses