Anion-π Enzymes

Abstract

In this report, we introduce artificial enzymes that operate with anion-π interactions, an interaction that is essentially new to nature. The possibility to stabilize anionic intermediates and transition states on an π-acidic surface has been recently demonstrated, using the addition of malonate half thioesters to enolate acceptors as a biologically relevant example. The best chiral anion-π catalysts operate with an addition/decarboxylation ratio of 4:1, but without any stereoselectivity. To catalyze this important but intrinsically disfavored reaction stereoselectively, a series of anion-π catalysts was equipped with biotin and screened against a collection of streptavidin mutants. With the best hit, the S112Y mutant, the reaction occurred with 95% ee and complete suppression of the intrinsically favored side product from decarboxylation. This performance of anion-π enzymes rivals, if not exceeds, that of the best conventional organocatalysts. Inhibition of the S112Y mutant by nitrate but not by bulky anions supports that contributions from anion-π interactions exist and matter, also within proteins. In agreement with docking results, K121 is shown to be essential, presumably to lower the pK a of the tertiary amine catalyst to operate at the optimum pH around 3, that is below the pK a of the substrate. Most importantly, increasing enantioselectivity with different mutants always coincides with increasing rates and conversion, i.e., selective transition-state stabilization.

Figure 1

The concept of anion-π enzymes (top), together with structures of substrates, products (A: addition, D: decarboxylation) and conceivable reactive intermediates (RI1) and transition states (TS1). Highlighted are π surface (blue), base catalyst (blue), fixed Leonard turns (magenta), and top-down approach of 2 (bold) to deprotonated 1 on the π surface (TS1). PMP = p-methoxyphenyl.

Figure 2

Structure of biotin–NDI–base triads. Note, 6 is a mixture of sulfoxide diastereoisomers.

Figure 3

Selected experimental data: (A) GPC chromatograms of S121Y with (solid) and without 8 (dashed, 2 equiv), detected at 220 nm (top) and 570 nm (bottom). (B) Absorption spectrum of 8 (top) and CD spectra of S112Y with increasing concentrations of 8 (0–4 equiv, bottom). (C) Dependence of the ee with WT + 8 on pH (red, pH < 3: Gly buffer, pK_a = 2.4; pH > 3: DMG buffer (3,3-dimethylglutaric acid), pK_a1 = 3.7, pK_a2 = 6.3), compared to the pH dependent deprotonation of MAHT 1 (cyan). (D) Dependence of ee on the conversion into 3 after 24 h at pH 3.0 with 8 and different mutants (Table 1, entries 16-31). (E) Conversion into 3 with time for S112Y + 8 (●, ○) and WT + 8 (□) with (●) and without (□, ○) 200 mM NaNO₃. (F) Dependence of the ee with WT + 8 on the concentration of NaNO₃ (●) and glucose-6-phosphate (○).

Figure 4

Docking simulations of S112Y dimers with 8, zoomed on the active site. Protein surfaces are rendered with their electrostatic potential (red: negative, blue: positive, green: aromatics), β sheets as faint green arrows. Exposed parts of 8 are in wireframe presentation, C green, N, blue, S yellow, O red.