Efficient conversion of primary azides to aldehydes catalyzed by active site variants of myoglobin

Abstract

The oxidation of primary azides to aldehydes constitutes a convenient but underdeveloped transformation for which no efficient methods are available. Here, we demonstrate that engineered variants of the hemoprotein myoglobin can catalyze this transformation with high efficiency (up to 8,500 turnovers) and selectivity across a range of structurally diverse aryl-substituted primary azides. Mutagenesis of the 'distal' histidine residue was particularly effective in enhancing the azide oxidation reactivity of myoglobin, enabling these reactions to proceed in good to excellent yields (37-89%) and to be carried out at a synthetically useful scale. Kinetic isotope effect, isotope labeling, and substrate binding experiments support a mechanism involving heme-catalyzed decomposition of the organic azide followed by alpha hydrogen deprotonation to generate an aldimine which, upon hydrolysis, releases the aldehyde product. This work provides the first example of a biocatalytic azide-to-aldehyde conversion and expands the range of non-native chemical transformations accessible through hemoprotein-mediated catalysis.

Fig. 1. (a) Distal pocket of sperm whale myoglobin (pdb 1A6K 12). Active site residues (yellow), heme (wheat), ‘proximal His’ (green), and heme-bound water molecule (grey sphere) are highlighted. (b) Electronic absorption spectra for wild-type Mb and Mb variants Mb(H64V) and Mb(H64V,V68A) in phosphate buffer at pH 7.0. The Soret bands have maxima at 408, 398, and 403 nm, respectively. (c) Ligand-induced decrease of Soret band intensity upon incubation of Mb in phosphate buffer at pH 7 with 20 mM benzyl amine (BnNH₂) and para-substituted BnNH₂ derivatives (λ_max = 408 nm (WT); 409 nm (WT + BnNH₂); 410 nm (WT + 4-(CF₃)–BnNH₂); 409 nm (WT + 4-(NO₂)–BnNH₂); 412 nm (WT + 4-(OMe)–BnNH₂).

Scheme 1. Substrate scope for Mb(H64V,V68A)-catalyzed azide oxidation. Reaction conditions: 10 mM azide, 10 mM Na₂S₂O₄, 1 μM Mb(H64V,V68A).

Scheme 2. Proposed mechanism and catalytic steps for the myoglobin-catalyzed oxidation of alkyl azides to aldehydes (black). The pathway leading to the minor alkyl amine byproduct is also shown (brown).

Fig. 2. (a) Measurement of kinetic isotope effect (KIE) for H/D substitution of αC–H bond on Mb(H64V,V68A)-catalyzed oxidation of benzyl azide. See also ESI Fig. S4. (b) Isotope labelling experiment demonstrating ¹⁸O incorporation in the aldehyde product in the presence of H₂¹⁸O-containing buffer. See also ESI Fig. S5.

Scheme 3. Mb(H64V,V68A)-catalyzed oxidation of secondary alkyl azides.