Photobiological switches of [alpha]-chymotrypsin
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
This thesis examines the design, synthesis and testing of photobiological switches of α-chymotrypsin. A photobiological switch is a biologically active compound that incorporates a photo active group in order that light-triggered switching of the biological target is afforded. The switches described in this thesis incorporate azobenzene as the photochromic group and α-ketoester or α-ketoamide as the inhibitory moiety. A novel component of these switches is the use of peptidyl groups to enhance the specificity of α-chymotrypsin toward these compounds. Thus, there are three fundamental elements to these photobiological switches; the specificity region, the switch and the inhibitory moiety. The target molecules in this thesis result from permutations of these three elements giving compounds of the type A, Band C. In Chapter One, the concept of the photobiological switch is discussed and existing and potential applications of this technology are outlined. A survey is given of photo chromic materials that are suitable for incorporation into photobiological switches. Examples of enzyme bioswitching methodologies are given and the mechanism, specificity and inhibition of α-chymotrypsin are discussed. Finally, as a distillation of the preceding discussions, the design of target compounds of the type A, Band C is outlined. In Chapter Two, the syntheses of four compounds of the type A (2.1, 2.2, 2.3, 2.4) are detailed. Target compounds 2.1 and 2.2 comprise one amino acid and target compounds 2.3 and 2.4 comprise two amino acids. In addition, the synthesis of 2.52, a compound for the referencing of enzyme kinetic data with reported data, is discussed. The syntheses of five compounds of the type B are discussed in Chapter Three. Compounds 3.1, 3.2 and 3.3 are regioisomeric and allow for structure activity relationship investigations into the substitution of the azobenzene group. In Chapter Four, the syntheses of three compounds of the type C are detailed. In Chapter Five, the hydration, photoisomerisation and racemisation behaviours of the target compounds are examined. Hydration studies of 3.1 (type B) showed that this compound was readily hydrated in solutions containing water. Evidence of hydrate formation was gathered for compounds of the type A (2.4) and compounds of the type C (4.2). The photoisomerisation of the target compounds was studied and trends were observed according the substitution of the azobenzene moiety. For example, the most efficient switching was observed for azobenzenes substituted in the para position with methylene groups and poor results were observed for azobenzenes substituted with a para ketone. Racemisation studies showed that 3.1 was prone to deuteration in a HEPES buffer solution at pH 7.8. In Chapter Six, the bioswitching ability of the target compounds was measured by enzyme assay. The level of a-chymotrypsin inhibition by reference compound 2.52 was measured (Ki = 0.17 μM) to be comparable to the literature value for this compound. The target compounds of the types A and B were in a comparable range (Ki = 0.04-10 μM) of bioactivity to the reference compound and in every case the (Z) isomer was more active against α-chymotrypsin than the (E) isomer. Compounds of the type C, however, did not show activity against α-chymotrypsin at millimolar concentration. As expected, the type of inhibition for all of the active compounds fitted most closely to a competitive model of inhibition. A preliminary study into the viability of an in situ reversible assay for the bioswitches described in this thesis is discussed. From the three buffers tested (HEPES, TRIS, phosphate), the phosphate buffer would be the buffer of choice for an in situ reversible assay. Chapter Seven is a summary chapter, giving an overview of the utility of the target compounds and a critical discussion of their design. Potential future directions for research into photobiological switches of α-chymotrypsin are discussed.