Affiliation:
1. Department of Chemistry and NanoScience Center University of Jyväskylä FI-40014 Jyväskylä Finland
Abstract
AbstractConformationally flexible tertiary amine – thiourea−urea catalysts 1 and 2 for the Mannich reaction between imines and malonate esters are efficiently inhibited by quaternary ammonium halides. NMR titrations, isothermal titration calorimetry (ITC) and NOE experiments showed that the catalysts bind chloride and bromide ions with relatively high affinities (K=103–105 M−1 in acetonitrile). The halide ions not only block the active site of the catalysts, but they also induce refolding into catalytically inactive conformations upon complexation in an allosteric‐like event. At substoichiometric inhibitor:catalyst ratios, the catalysts displayed hypersensitivity to the inhibitors, with overall rates that were lower than those expected from simple 1st order kinetics and 1 : 1 inhibitor:catalyst stoichiometry. To rationalize the observed hypersensitivity, different kinetic scenarios were examined. For catalyst 2 and the Takemoto catalyst (6), the data is consistent with 2nd order dependency on catalyst concentration, suggesting that a mechanism involving only a single catalyst in the catalytic cycle is not operative. For catalyst 1, an alternative scenario involving 1st order in catalyst and catalyst poisoning at low concentrations of 1 could also rationalize the hypersensitivity. Interestingly, inhibition of catalysts 1 and 2 by halide salts led to significant loss of enantioselectivity, in contrast to the Takemoto catalyst 6 which was inhibited but with essentially no change in enantioselectivity.
Reference50 articles.
1. Census of halide-binding sites in protein structures
2. Structural and mechanistic studies of chloride induced activation of human pancreatic -amylase
3. Residues affecting the chloride regulation and substrate selectivity of the angiotensin-converting enzymes (ACE and ACE2) identified by site-directed mutagenesis
4. WNK4 kinase is a physiological intracellular chloride sensor
5. Allosteric effectors bind at a site other than the active site of the catalyst. In our case the chloride ions bind to the urea subunit disrupting the fold of the catalyst and also to one of the thiourea N−Hs at the active site. Since the binding involves a conformational change resulting in the capture of an inactive conformation (i. e. it changes the populations of the conformers) it closely resembles true allosteric response and hence the term “allosteric-like” is used herein. For an example of allosteric capture of inactive conformation in enzymes see: