Although natural systems demonstrate that simple organic features and metal ion cofactors can combine extremely effectively to lead to efficient catalysis of RNA and DNA cleavage, achieving similar activity with artificial systems has yet to be achieved. One motivation for attempting this would be to create artificial nucleases, which could offer a generic therapeutic approach to controlling gene expression, but require more reactive catalytic sites than have been created to date. This thesis describes studies to develop more effective catalysts for phosphate diester cleavage through the design of small Zn(II) complexes.
Initially, a series of mononuclear Zn(II) catalysts designed to hydrolyse simple phosphate diesters were studied. A comparison between the effect of positioning a sulfonamide or a primary amine in the second coordination sphere was carried out, but the stronger hydrogen bond donor did not lead to an effective catalyst. This was rationalised in terms of the unfavourable ionisation state of the complex at neutral pH.
The amine functional group was then combined with a range of nucleophilic chains to explore how effectively these two activating strategies can be combined. Detailed mechanistic studies were carried out, revealing the mechanistic pathways and the response to inhibition by phosphate diesters and monoesters. The accelerating effects were significant, with the cleavage of bis-4-nitrophenyl phosphate (BNPP) 625 fold faster when an oxime was present as a nucleophile instead of an alcohol group. The effect of exchanging a methyl group for a secondary amine in the secondary sphere was smaller, with a 19 fold increase in activity. Compared to the methyl substituted analogue, the sensitivity to the leaving group of the substrate is reduced. This increase was accompanied by a strong tendency for the complex to dimerise, reducing the observed activity, but provided the most reactive mononuclear complex for BNPP reported to date.
Based on the observation that strongly acidic groups do not enhance the activity of these complexes, the properties of the amino groups were modified by substitution with aromatic rings. The hypothesis was that this would increase the hydrogen bonding capacity, without creating an acidic site. This strategy was effective, and anisidine lead to a rate enhancement of 10 fold over the parent amino substituted complex for cleaving 2-hydroxypropyl 4-nitrophenyl phosphate (HPNPP). Examining the effect of different substituents on activity revealed that electron donating substituents led to greater activity, in contrast to the expected increase with electron withdrawing groups as observed in related complexes.
This substitution pattern was then incorporated into a dinucleating ligand, and the Zn(II) complex exhibited remarkable activity towards HPNPP cleavage, reducing its half life to a few seconds at neutral pH. This represents an increase in activity of about 1000 fold over the parent amino substituted complex at 0.6 mM. The activity towards a nucleotide dimer was also enhanced, but only about 3 fold. Despite the absence of a nucleophilic functionality in the ligand, this complex also showed high activity towards BNPP. Detailed studies on each of these activities revealed complex behaviour when pH and concentration were varied.
Overall, the modifications introduced have led to enhanced activities for three different types of Zn(II) complexes, with remarkable activity for HPNPP cleavage; however, the combination of these modifications has also revealed complex behaviour which has been partly but not completely resolved.
