Project 1

Modeling the Action of Carbonic Anhydrase.

Carbonic anhydrase (CA) is a zinc-containing enzyme found in every living organism.¹  Its only known physiological role is to catalyze the equilibrium interconversion of CO₂ and bicarbonate.

CO₂ + H₂O ß à HCO₃^- + H⁺

Given the importance of the species in the equilibrium, it is not surprising that CA is actively involved in such diverse processes as photosynthesis, shell formation, respiration, and regulation of extracellular pH. The active site of CA comprises a Zn²⁺-ion bound by three histidine imidazoles in a distorted tetrahedral fashion: the fourth metal site is occupied by a water, or HO^- depending on the pH. The basic, or HO^- -form of the enzyme is responsible for hydrating CO₂. This reaction proceeds with very great efficiency, the k_cat / K_M being 10⁸ M^-1s^-1.¹

There have been numerous attempts to reproduce the spectroscopic properties and catalytic activity of the enzyme using simple small molecule complexes, but none of these has been particularly successful in achieving the rates of the enzyme for hydration of CO₂.² Of the most catalytically effective examples, complex 2³  hydrates CO₂ with a k₂^cat of ~600 M^-1s^-1, and we⁴ have shown that 3 facilitates the equilibration of CO₂ ß à HCO₃^- in 80% EtOH/H₂O with a k₂^cat of ~2700 M^-1s^-1. The major problem to be solved is how to make a ligand:metal bound hydroxide a more effective nucleophile toward CO₂ than hydroxide is, the rate constant for the latter reaction (HO^- + CO₂) being ~8500 M^-1s^-1.⁵

In this project we propose to synthesize a new class of tridentate-binding ligands that will closely approximate the enzymatic site given in 1. Representative examples of such ligands are 4 and 5, which, because of their cyclic structures that orient the N lone pairs inward toward an apex, are expected to bind the metal ion very tightly. We need to develop synthetic proceedures to make these in useable amounts. Ligand 5 is very interesting because the structure of the metal complex only allows the three R groups to be oriented upward (three up, or two up and one down) so that they form a hydrophobic pocket into which the metal fits. After synthesis of the ligands, and physical, structural and spectroscopic study of their metal complexes, we will turn attention to studying how well these promote the hydration of CO₂ or dehydration of HCO₃^-.

1.  For leading references see: Christianson and Fierke, Acc. Chem. Res. 1996, 29, 331.
2.  (a) R. S. Brown in "Enzymatic and Model Carboxylation and Reduction Reactions for
     CO₂ Utilization", NATO ASI Series C., Kluwer Academic Publishers: Dordrecht,
      Netherlands pp 145-180 (1990).
     (b) Kimura E. Prog. Inorg. Chem. 1994, 41, 443-491.
3.  Kimura, E. et al Inorg. Chem. 1993, 32, 5749.
4.  R. S. Brown et al J. Am. Chem. Soc. 1984, 106, 2421.
5.  Sirs, J. A. Trans. Faraday Soc. 1958, 54, 201.