Merging enzymes with chemocatalysis for amide bond synthesis

Abstract

Amides are one of the most fundamental chemical bonds in nature. In addition to proteins and other metabolites, many valuable synthetic products comprise amide bonds. Despite this, there is a need for more sustainable amide synthesis. Herein, we report an integrated next generation multi-catalytic system, merging nitrile hydratase enzymes with a Cu-catalysed N-arylation reaction in a single reaction vessel, for the construction of ubiquitous amide bonds. This synergistic one-pot combination of chemo- and biocatalysis provides an amide bond disconnection to precursors, that are orthogonal to those in classical amide synthesis, obviating the need for protecting groups and delivering amides in a manner unachievable using existing catalytic regimes. Our integrated approach also affords broad scope, very high (molar) substrate loading, and has excellent functional group tolerance, telescoping routes to natural product derivatives, drug molecules, and challenging chiral amides under environmentally friendly conditions at scale.

Fig. 1. Approaches towards the construction of amide bonds.

A Retrosynthetic analysis for classic amide disconnection. B Catalytic approaches for amide bond synthesis. C Amide bond synthesis by integrating nitrile hydratase enzymes with Cu-catalysed Ullmann-type arylation.

Fig. 2. Development of the integrated chemo- and biocatalytic synthesis of amides.

A Optimisation of the integrated reaction. Reaction conditions: 1. Nitrile (1) (50 mM), Equi_NHase (see table) in 0.1 M KPi buffer (pH = 7.8)/10% v/v iPrOH (2 mL) at RT, 24 h; 2. CuBr₂ (10 mol%), L1 (20 mol%), d-glu (20 mol%), 3 (150 mM) at 50 °C, 24 h under N₂ atmosphere (headspace purge). [a] Conversion determined in triplicates by HPLC/UV using benzophenone as external standard. [b] Yield of isolated product after column chromatography. [c] E. coli (Equi_NHase) whole cells from ca. 20 mL cell culture (OD₆₀₀ = ~0.5) were used. B Activity screening of E. coli (NHase) whole cells. Relative conversion was determined in triplicates by HPLC/UV analysis (n = 3). Blue bars represent mean values and error bars represent ± SEM of benzamide (2) conversion. n.d. not detected; w/o without, OD₆₀₀ Optical density measured at 600 nm.

Fig. 3. Scope for the integrated chemo- and biocatalytic synthesis of aromatic amides.

Reaction conditions: 1. Nitrile (50 mM), E. coli (CGA009) whole cells in 0.1 M KPi buffer (pH = 7.8)/10% v/v iPrOH (4 mL) at RT, 24 h; 2. CuBr₂ (10 mol%), L1 (20 mol%), d-glu (20 mol%), iodoarene (150 mM) at 50 °C, 24 h under N₂ atmosphere (Method A). Yields refer to isolated products after column chromatography. [a] Amide 12 was synthesised using PhCN (150 mM) and iodoarene (50 mM). [b] Amide 24 was synthesised using E. coli (CGA009) whole cells for 48 h.

Fig. 4. Integrated chemo- and biocatalytic synthesis of aliphatic amides.

A Optimisation of reaction conditions. B Scope of reaction (Method B). Yields refer to isolated products after column chromatography. [a] Amides 51–53 were synthesised using nitrile (0.2 mmol) and halide (0.6 mmol). n.d. not detected, TPGS-750-M dl-α-Tocopherol methoxypolyethylene glycol succinate, w/o without.

Fig. 5. Integrated chemo- and biocatalytic synthesis of chiral amides.

A Scope of the kinetic resolution of racemic nitriles (Method C). Yields refer to isolated products after column chromatography. TPGS-750-M dl-α-Tocopherol methoxypolyethylene glycol succinate; e.r. enantiomeric ratio. B Docking of nitrile 64 into the active site of AJ270 (pdb code: 2QDY) in a putative active conformation indicates a substrate-binding pose with coordination of the nitrile group to the Fe-centre, with stereoselectivity is controlled by binding of the propyl substituent to a hydrophobic pocket formed by M40, Y72, and Y76. Labelled active site residues (blue) and substrate 64 (green).

Fig. 6. Application of the integrated chemo- and biocatalytic synthesis of amides.

A Integrated synthesis of heterocycles (Method A). B Selective hydration of dinitrile 70 for the synthesis of bioactive product 72 (Method A). C Gram-scale reactions (Method A and B, see Supplementary Information for the details). D Biocatalyst recycling experiment. Experimental procedures can be found in the Supplementary Information. Yields refer to isolated products after column chromatography.