Selective Colorimetric "Turn-On" Probe for Efficient Engineering of Iminium Biocatalysis

Abstract

The efficient engineering of iminium biocatalysis has drawn considerable attention, with many applications in pharmaceutical synthesis. Here, we report a tailor-made iminium-activated colorimetric "turn-on" probe, specifically designed as a prescreening tool to facilitate engineering of iminium biocatalysis. Upon complexation of the probe with the catalytic Pro-1 residue of the model enzyme 4-oxalocrotonate tautomerase (4-OT), a brightly colored merocyanine-dye-type structure is formed. 4-OT mutants that formed this brightly colored species upon incubation with the probe proved to have a substantial activity for the iminium-based Michael-type addition of nitromethane to cinnamaldehyde, whereas mutants that showed no staining by the probe exhibited no or very low-level "Michaelase" activity. This system was exploited in a solid-phase prescreening assay termed as activated iminium colony staining (AICS) to enrich libraries for active mutants. AICS prescreening reduced the screening effort up to 20-fold. After two rounds of directed evolution, two artificial Michaelases were identified with up to 39-fold improvement in the activity for the addition of nitromethane to cinnamaldehyde, yielding the target γ-nitroaldehyde product with excellent isolated yield (up to 95%) and enantiopurity (up to >99% ee). The colorimetric activation of the turn-on probe could be extended to the class I aldolase 2-deoxy-d-ribose 5-phosphate aldolase, implicating a broader application of AICS in engineering iminium biocatalysis.

Figure 1

4-OT catalyzed Michael-type addition, Brooker’s merocyanine, and the reaction between Pro-1 of 4-OT and cinnamaldehydes. (a) 4-OT catalyzed Michael-type addition of 2 to 1a. Product R-3 is a precursor for Phenibut. (b) Resonance forms of Brooker’s merocyanine. (c) Turn-on probes 1b and 1c reacting with Pro-1 of 4-OT, forming a zwitterionic resonating species. (d) Compounds 1a, 1d, and 1e reacting with Pro-1 of 4-OT, forming a species without zwitterionic resonance.

Figure 2

Absorption spectra of 1b–1e in the presence and absence of 4-OT M45T/F50A. (a) Absorption spectrum of 1b with and without 4-OT M45T/F50A. (b) Absorption spectrum of 1c with and without 4-OT M45T/F50A. (c) Absorption spectrum of 1d with and without 4-OT M45T/F50A. (d) Absorption spectra of 1e with and without 4-OT M45T/F50A. Conditions: 2.5% dimethyl sulfoxide (DMSO), 20 mM sodium phosphate (pH 7.3), 1 mM 1b–1e, 50 μM 4-OT M45T/F50A, 1 cm quartz cuvettes.

Figure 3

Activity for the Michael-type addition of 2 to 1a catalyzed by different 4-OT mutants. (a) Progress curve of the Michael-type addition of 2 to 1a catalyzed by different 4-OT mutants. (b) Initial rate of the Michael-type addition of 2 to 1a catalyzed by different 4-OT mutants. The data are presented as fold increase compared to the initial rate of starting mutant 4-OT F50A. Assay conditions: 5% ethanol, 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) (pH 6.5), 1 mM 1a, 100 mM 2, 50 mM sodium formate.

Figure 4

Agar plates 10 s (left) and 4 min (right) after performing the AICS assay. Transformants of library 21–40_IA were stained with the AICS assay as described in the Experimental Section.

Figure 5

Activity for the Michael-type addition of 2 to 1a catalyzed by CFEs of 92 stained and 92 unstained colonies. Colonies from cells that were freshly transformed with library 21–40_IA were stained using the AICS procedure. In total, 92 stained colonies and 92 unstained colonies were picked, grown overnight, and the CFE was assayed for the Michaelase activity using substrates 2 and 1a. The Michaelase activity was measured in a plate reader by following the depletion of 1a at 290 nm. Assay conditions: 5% ethanol, 30% CFE, 0.5 mM 1a, 100 mM 2, 50 mM sodium formate. Analysis of CFEs of unstained colonies by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that eight out of 16 show significant 4-OT expression, indicating that the AICS procedure can efficiently be used to eliminate colonies not producing enzyme or producing enzyme with low-level activity.