Kinetic and Mechanistic Evaluation of Biologically Relevant Molecules

This thesis focuses on the hydrolysis of amino acid thioesters and the determination of the pKa values of a series of smoking cessation agent derivatives.

Thioesters play a key role both in nature and synthetic chemistry. To date there exists a gap in literature regarding the hydrolysis of amino acid thioester substrates. This thesis investigates the kinetic evaluation of the hydrolysis of four alanyl and phenylalanyl S-ethyl and S-phenyl thioesters over the pH range 0.46 – 12.9. An HPLC method was developed and applied successfully in all cases. For the S-ethyl thioester substrates, the pH rate profiles show three regions which correspond to acid-catalysed, pH-intermediate and base-catalysed hydrolysis of the thioester moiety. Rate constants were determined for of these forms of catalysis for both N-acetyl-alanine S-ethyl thioester (kH = (5.9 ± 0.6) x 10-7 M-1 s-1, kH2O = (4.8 ± 0.3) x 10-9 s-1 and kHO = 0.19 ± 0.01 M-1 s-1) and N-acetyl-phenylalanine S-ethyl thioester (kH = (3.3 ± 0.2) x 10-7 M-1 s-1, kH2O = (6 ± 2) x 10-9 s-1 and kHO = 0.25 ± 0.02 M-1 s-1).

For the S-phenyl substrates, two regions were seen in the pH-rate profiles corresponding to pH-independent and base-catalysed hydrolysis. No evidence of acid-catalysed hydrolysis was observed for the S-phenyl substrates in the pH range studied. Rate constants for pH-independent and base-catalysed hydrolysis were determined for both N-acetyl-alanine S-phenyl thioester (kH2O = (7.1 ± 0.3) x 10-7 s-1 and kHO = 36 ± 2 M-1 s-1) and N-acetyl-phenylalanine S-phenyl thioester (kH2O = (6.30 ± 0.01) x 10-7 s-1 and kHO = 203 ± 3 M-1 s-1).

Analysis of the pH-rate profiles allowed for the determination and comparison of component rate constants to literature values. Addition-elimination mechanisms (AN + DN) via cationic, neutral and anionic tetrahedral intermediates were proposed for each of the substrates.

Moving on from these we evaluated N-acetyl-histidine S-ethyl thioester to determine whether the high reactivity of histidyl thioesters noted in the literature around Native Chemical Ligation is due to intramolecular catalysis. This substrate was successfully synthesised via two different methods. The hydrolysis of N-acetyl-histidine S-ethyl thioester was monitored across the pH range 0.46 – 11.3 using the same HPLC method used on the other substrates. A pH-rate profile was constructed for this substrate and the rate constants for acid-catalysed, pH-independent and base-catalysed hydrolysis of the substrate were determined (kH = (3.22 ± 0.07) x 10-7 M-1 s-1, kH2O = (1.70 ± 0.04) x 10-8 s-1 and kHO = 2.90 ± 0.03 M-1 s-1) and compared to those of the alanyl and phenylalanyl S-ethyl thioesters. Intramolecular catalysis involving the imidazolyl moiety was observed and determined to proceed via a general-base mechanism from the primary solvent kinetic isotope effect found. The second-order rate constant for the intramolecularly catalysed hydrolysis of N-acetyl-histidine S-ethyl thioester (kHis = (9.6 ± 0.6) x 10-6 s-1) was determined from the global fit of the pH-rate profile and compared to some relevant literature values of intramolecular catalysis. An estimate of 7.0 ± 0.3 for the pKa value of the imidazolyl moiety of the substrate was also determined from this fit.

Finally, the pKa values of a series of smoking cessation agents and their derivatives were determined spectrophotometrically as part of a collaborative project to determine their mechanism of action. The compounds investigated in this study include nicotine (pKa1 = 3.27 ± 0.02 and pKa2 = 8.19 ± 0.03), 4-dimethylamino pyridine (9.67 ± 0.02), cytisine (8.1 ± 0.1), N-Benzyl cytisine (6.28 ± 0.09), varenicline tartrate (8.93 ± 0.09) N-benzyl varenicline (7.68 ± 0.06), isovarenicline hydrochloride (8.5 ± 0.1), N-benzyl isovarenicline (6.20 ± 0.07), N2-varenicline trifluoroacetate (7.31 ± 0.04), N-benzyl N2-varenicline (5.59 ± 0.06), N-benzyl C1-varenicline (8.21 ± 0.06), C2-varenicline hydrochloride (9.61 ± 0.09). It wasn’t possible to determine the pKa value of N-benzyl C2-varenicline due to poor solubility.

Increasing the number of electron-withdrawing nitrogen atoms in the conjugated ring system of the varenicline derivatives was found to increase the acidity (i.e. result in a lower pKa value). This trend was attributed to the destabilising effect of these atoms on the ammonium of the conjugate acid. All N-benzylated derivatives were more acidic than their respective non-benzylated secondary amine derivative counterparts. This was explained by the lower Gibbs free energy of desolvation of the conjugate acid of the N-benzylated derivatives. The trend between the effect of acidity on binding capability of varenicline, C2-varenicline and isovarenicline at the α4β2 nicotinic acetylcholine receptor (nAChR) was rationalised with the aid of molecular dynamics simulations. The speciation of the ammonium group in this series played a key role in binding. The H-bond between the pyrazinyl moiety of varenicline and the hydroxyl group of the residue β2S133 was found to play a key role in its physiological activity.