Biological catalysis regulated by cucurbit[7]uril molecular containers.

We report the synthesis of two-faced inhibitors 1-5 that contain both enzyme inhibitor and cucurbit[n]uril binding domains. The enzyme binding domains of 1-5 bind to the active sites of bovine carbonic anhydrase (BCA) or acetylcholinesterase (AChE) and inhibit their catalytic activities. Addition of CB[7] to BCA1 and BCA2 results in the transient formation of the BCA1CB[7] and BCA2CB[7] ternary complexes that undergo rapid dissociation to form free catalytically active BCA along with CB[7]1 and CB[7]2. The on-off cycle can be performed repetitively by the sequential addition of competitive guest 8 and CB[7]. The detailed origins of this on-off switching of the catalytic activity of BCA is delineated by the combined inference of UV/vis catalytic assays, fluorescence displacement assays, (1)H NMR, along with measurement of the fundamental values of K(a), k(on), and k(off) for the various complexes involved. In contrast, addition of CB[7] to AChE4(4) and AChE5(4) results in the formation of thermodynamically stable ternary complexes AChE*4(4)CB7 and AChE5(4)*CB7 that are catalytically inactive. We highlight some of the advantages and disadvantages of the strategy, based on the direct competition between two receptors (e.g., enzyme and CB[7]) for a common inhibitor, used in this paper to control enzyme catalytic activity compared to the strategy employed by Nature involving the binding of an allosteric small molecule remote from the enzyme active site.