Chemoenzymatic Cascade Synthesis of Metal-Chelating α-Amino Acids

Abstract

Metal-chelating noncanonical amino acids (ncAAs) are uniquely functional building blocks for proteins, peptide catalysts, and small molecule sensors. However, catalytic asymmetric approaches to synthesizing these molecules are hindered by their functional group variability and intrinsic propensity to ligate metals. In particular, bipyridyl-L-alanine (BpyAla) is a highly sought ncAA, but its complex, inefficient syntheses have limited utility. Here, we develop a chemoenzymatic approach to efficiently construct BpyAla. Three enzymes that can be produced in high titer together react to convert Gly and an aldehyde into the corresponding β-hydroxy ncAA, which is subsequently deoxygenated. We explore approaches to synthesizing biaryl aldehydes and show how the three-enzymatic cascade can access a range of α-amino acids with bulky side chains, including a variety of metal-chelating amino acids. We show that newly accessible BpyAla analogues are compatible with existing amber suppression technology, which will enable future merging of traditional synthetic and biosynthetic approaches to tuning metal reactivity.

Keywords: Biocatalysis; Genetic code expansion; Noncanonical amino acid; Pyridoxal phosphate.

Figure 1.

Overview of bipyridyl alanine and previous synthetic approaches. (A) Structure of l-BpyAla and proteins containing this unique metal-chelating moiety. PDB ID right: 2PXH and left: 5EIL. (B) Previous synthetic route deployed by Xie et al.^[9] (C) Synthetic biology route to in vivo production of amino acids by Song et al.^[24] (D) Strategy proposed here.

Figure 2.

Selection of C─C bond-forming pyridoxyal-dependent aldolase. Both TmLTA and ObiH catalyzed the formation of β-hydroxy BpyAla with an appropriate amino acid donor. Addition of a reducing system (RS) to remove acetaldehyde from the ObiH reaction improved yields. The potential for formation of diverse shunt products from ObiH catalysis (purple) directed us to use TmLTA for subsequent work.

Figure 3.

Dehydratase screening for formation of Bpy α-keto acid. Two dehydratases were tested in a cascade with TmLTA. TtPSD was screened at 65 °C, where threonine dehydration was maximized, while RpicPSD was screened at 37 °C. TtPSD was found to stall at Bpy β-hydroxy amino acid, while RpicPSD carried the reaction to near completion and high yield, 85%.

Figure 4.

Time course analysis of complete three-enzyme cascade. Interconversion of intermediates was observed for the full cascade across 24 h. Bpy aldehyde (1) was observed to rapidly deplete with no significant buildup of β-hydroxy amino acid. α-ketoacid (3) conversion to BpyAla (4) is the rate-limiting step of the cascade.

Figure 5.

Preparative-scale cascade synthesis of noncanonical amino acids.

Figure 6.

Amber suppression with new ncAAs bearing metal-chelating motifs. Fluorescence of superfolder GFP (sfGFP) with the ncAA BpyA incorporated at position 190. In the absence of amino acids, no fluorescence is observed. In the presence of BpyAla (1), fluorescence is restored. Weaker, but observable amber suppression was also observed in the presence of analogues 2 and 3. Incorporation was confirmed through top-down mass spectrometry. See Supporting Information for further details.