Steric and Electronic Effects on an Antibody‐Catalyzed Diels–Alder Reaction

Abstract

Dedicated to Dieter Seebach on the occasion of his 65th birthday A series of substituted thiophene dioxides was tested as diene substrates for the antibody 1E9, which was elicited with a hexachloronorbornene derivative and normally catalyzes the inverse electron-demand Diels ± Alder reaction between 2,3,4,5-tetrachlorothiophene dioxide (TCTD) and N-ethylmaleimide (NEM). Previous structural and computational studies had suggested that the catalytic efficiency of this system derives in part from a very snug fit between the apolar active site and the transition state of this reaction. Nevertheless, replacing all the Cl-atoms in the hapten with Br-atoms leads to no loss in affinity (K d 0.1 nm), indicating substantial conformational flexibility in the residues that line the binding pocket. Consistent with this observation, the 2,3,4,5-tetrabromothiophene dioxide is a good substrate for the antibody (k cat 1.8 min À1, K NEM 14 mm), despite being considerably larger than TCTD. In contrast, normal electron-demand Diels ± Alder reactions between NEM and unsubstituted thiophene dioxide or 2,3,4,5-tetramethylthiophene dioxide, which are much smaller or nearly isosteric with TCTD, respectively, are not detectably accelerated. These results show that the electronic properties of the 1E9 active site are optimized to a remarkable degree for the inverse electron-demand Diels ± Alder reaction for which it was designed. Indeed, they appear to play a more important role in catalysis than simple proximity effects.

Enzyme Mimics: Advances and Applications

“…The antibody‐catalysed Diels–Alder cycloaddition reaction between tetrachlorothiophene dioxide ( 5 ) and N ‐ethylmaleimide ( 6 ) proceeds via a transition state that looks very much like hexachloronorbornene derivative 7 (Scheme ), which was used as a hapten to generate the catalytic antibodies. The unstable intermediate spontaneously eliminates sulfur dioxide to give a product that rapidly undergoes in situ oxidation to produce N ‐ethylphthalimide . Moreover, site‐specific mutagenesis was utilised to produce a Cu II binding protein motif by introducing coordinating amino acids at geometrically appropriate positions in a thermostable protein and subsequently leading to the construction of a metalloenzyme mimic for Diels–Alder cycloaddition .…”

“…Eur.J.2016, 22,8404 -8430 www.chemeurj.org duce N-ethylphthalimide. [287] Moreover,s ite-specific mutagenesis was utilised to produce aC u II binding protein motif by introducing coordinating amino acids at geometrically appropriate positions in at hermostablep rotein and subsequently leading to the construction of am etalloenzyme mimic for Diels-Alder cycloaddition. [288] Experimental evidence clearly showed that the Cu II is coordinatively bound to the amino acids at the artificial binding site where asymmetric Diels-Alder reactions occur.B yg rafting an ew active site (a Cu II -binding bidentate ligand)t oadimeric protein, Bose tal.…”

“…Scheme7.Diels-Alder cycloaddition reaction between tetrachlorothiophene dioxide ( 5)and N-ethylmaleimide (6;reproduced with permission from ref. [287]).…”

Immunological Optimization of a Generic Hydrophobic Pocket for High Affinity Hapten Binding and Diels–Alder Activity

“…Initial velocities were determined by starch±I 2 bleaching at 606 nm at several concentrations of NEM while holding the concentration of TCTD constant at 150 mm. [26,38] The data were corrected for the uncatalyzed reaction and fitted to the Michaelis±Menten Equation:…”