Minimalist De Novo Design of an Artificial Enzyme

Abstract

We employed a reductionist approach in designing the first heterochiral tripeptide that forms a robust heterogeneous short peptide catalyst similar to the "histidine brace" active site of lytic polysaccharide monooxygenases. The histidine brace is a conserved divalent copper ion-binding motif that comprises two histidine side chains and an amino group to create the T-shaped 3N geometry at the reaction center. The geometry parameters, including a large twist angle (73°) between the two imidazole rings of the model complex, are identical to those of native lytic polysaccharide monooxygenases (72.61°). The complex was synthesized and characterized as a structural and functional mimic of the histidine brace. UV-vis, vis-circular dichroism, Raman, and electron paramagnetic resonance spectroscopic analyses suggest a distorted square-pyramidal geometry with a 3N coordination at pH 7. Solution- and solid-state NMR results further confirm the 3N coordination in the copper center of the complex. The complex is pH-dependent and could catalyze the oxidation of benzyl alcohol in water to benzaldehyde with yields up to 82% in 3 h at pH 7 and above at 40 °C. The catalyst achieved 100% selectivity for benzaldehyde compared to conventional copper catalysis. The design of such a minimalist building block for functional soft materials with a pH switch can be a stepping stone in addressing needs for a cleaner and sustainable future catalyst.

Figure 1

(a) 3D structure of the lytic polysaccharide monooxygenase (PDB: 5FJQ) and active-site residues (cyan). (b) LigPlot showing the ligand interactions with the histidine residues in the LPMO active site. (c) Chemical structure of the designed peptide mimicking the histidine brace. (d,e) Comparison between the structural parameters of the copper-chelated modeled peptide and enzyme active site, respectively (Table S1, Supporting Information).

Figure 2

Analysis of the Cu–HPh complex formation: (a) UV–vis, (b) CD, and (c) Raman spectra at different pH values. Experimental conditions: final concentrations of 5 mM HPh and 4.5 mM CuCl₂ were titrated with 1 μL aliquots of 1 M HCl or NaOH solution, and the reaction was left to equilibrate for 10 min after each addition.

Figure 3

(a) Frozen-solution EPR of the Cu–HPh complex at pH 3 (blue) and pH 7(red) in 30% glycerol (v/v). (b) Solution-state ¹H NMR of the Cu–HPh complex at pH 3 and pH 7. (c) Solid-state ¹H NMR of the Cu–HPh complex at pH 3 and pH 7; * the peak at −5 ppm is due to the spectrometer artifact. (d) Solid-state ¹³C NMR of Cu–HPh complex at pH 3 and 7. The peaks in the ¹³C spectra correspond to the peptide backbone shown in Figure S9, Supporting Information.

Figure 4

Morphology characterization of the complex. (a) FESEM image showing the flake-like morphology (inset) that is clustered to form a flower of around 214 nm in diameter; (b) negatively stained TEM image of the “flower”; and (c) corresponding SEM-EDS line map spectrum of the complex showing carbon (magenta), nitrogen (purple), oxygen (pink), sulfur (blue), phosphorus (cyan), and copper (red).

Scheme 1. Selective Oxidation of Benzyl Alcohol to Benzaldehyde in the Presence of the Cu–HPh Complex at 40 °C

Figure 5

Conversion of benzyl alcohol into benzaldehyde in water: pH = 7 at 40 °C is the standard reaction conditions used except in the pH titration experiment. (a) Conversion comparison between copper alone and the complex at 30% loading as a function of time. (b) Histogram comparing percentage conversion by nascent HPh (red), copper (black), and the Cu–HPh complex (olive) after 3 h. (c) Conversion of benzyl alcohol into benzaldehyde at 40 °C in 6 h under aerobic conditions. (d) Histogram showing the catalytic efficiency of the Cu–HPh complex at different pH (mean ± standard deviation, n = 3).

Figure 6

FESEM characterization of the assemblies formed by the Cu–His complex at different pH. Scale 10 μm.