Developing deprotectase biocatalysts for synthesis

Abstract

Organic synthesis often requires multiple steps where a functional group (FG) is concealed from reaction by a protecting group (PG). Common PGs include N-carbobenzyloxy (Cbz or Z) of amines and tert-butyloxycarbonyl (O^tBu) of acids. An essential step is the removal of the PG, but this often requires excess reagents, extensive time and can have low % yield. An overarching goal of biocatalysis is to use "green" or "enzymatic" methods to catalyse chemical transformations. One under-utilised approach is the use of "deprotectase" biocatalysts to selectively remove PGs from various organic substrates. The advantage of this methodology is the exquisite selectivity of the biocatalyst to only act on its target, leaving other FGs and PGs untouched. A number of deprotectase biocatalysts have been reported but they are not commonly used in mainstream synthetic routes. This study describes the construction of a cascade to deprotect doubly-protected amino acids. The well known Bacillus BS2 esterase was used to remove the O^tBu PG from various amino acid substrates. The more obscure Sphingomonas Cbz-ase (amidohydrolase) was screened with a range of N-Cbz-modified amino acid substrates. We then combined both the BS2 and Cbz-ase together for a 1 pot, 2 step deprotection of the model substrate CBz-L-Phe O^tBu to produce the free L-Phe. We also provide some insight into the residues involved in substrate recognition and catalysis using docked ligands in the crystal structure of BS2. Similarly, a structural model of the Cbz-ase identifies a potential di-metal binding site and reveals conserved active site residues. This new biocatalytic cascade should be further explored for its application in chemical synthesis.

Fig. 1. Reaction schemes for the chemical and biocatalytic deprotection of a doubly protected amino acid (Cbz-l-AA-O^tBu). Top route (blue): treatment with trifluoroacetic acid (% TFA) removes the O^tBu ester protecting group from the acid that yields the Cbz-protected amino acid. The Cbz group is removed by catalytic hydrogenation over palladium. This results in the free l-amino acid. Bottom route (green): the same sequential reactions are catalysed by the combination of BS2 esterase and Cbz-ase biocatalysts.

Fig. 2. Analysis of the Cbz-ase catalysed reaction. (A) Reaction scheme for the Cbz-ase catalysed deprotection of Z-l-Phe. (B) Reaction monitoring using HPLC analysis showing the formation of l-Phe (9.6 min, blue circle) and benzyl alcohol (14.2 min, orange square) and the disappearance of the Z-l-Phe substrate (20.1 min, green triangle). (C) Plot of concentration (mM) vs. reaction time (min) displaying the direct formation of by-product benzyl alcohol (orange square) in relation to the depletion of Z-l-Phe (green triangle). Reaction conditions: Z-l-Phe (10 mM), Cbz-ase (0.075 mg mL⁻¹) in sodium phosphate buffer (50 mM, pH 7.5), 1 mL reaction volume, 37 °C, 250 rpm. Samples (100 μL) taken at specified time intervals, quenched with TFA (2 μL, 10% in water) and diluted to 1 mL with water.

Fig. 3. Analysis of the BS2 catalysed reaction. (A) Reaction scheme for the BS2-catalysed deprotection of l-Phe-O^tBu. (B) Reaction monitoring by HPLC analysis showing the formation of l-Phe (9.6 min, blue) from the l-Phe-O^tBu substrate (16.6 min, peach). Reaction conditions: l-Phe-O^tBu (10 mM), BS2 (0.9 mg mL⁻¹) in sodium phosphate buffer (50 mM, pH 7.5), 1 mL reaction volume, 37 °C, 250 rpm. Samples (200 μL) taken and quenched with acetonitrile (200 μL) and diluted to 1 mL with water.

Fig. 4. HPLC chromatograms showing product formation after each step of the sequential biocatalytic deprotection cascade. (A) BS2 step in which Z-l-Phe-O^tBu is deprotected to form Z-l-Phe (green, 20.1 min). (B) Cbz-ase step in which the Z-Phe formed in step A is converted to l-Phe (blue, 9.6 min) and benzyl alcohol (orange, 14.2 min).

Fig. 5. Structural models of the BS2 and Cbz-ase biocatalysts. (A) Model of the BS2 based on the X-ray crystal structure (PDB: 1QE3). The inset shows the putative active with the catalytic triad (Ser₁₈₉, Glu₃₁₀, His₃₉₉) with the key Ser₁₈₉ nucleophile highlighted. The docked l-Phe-O^tBu ligand is shown to H-bond with the side chains of the potential oxyanion hole (Gly₁₀₆ and Ala₁₀₇) and the amine of the substrate engages in an ionic interaction with Glu₁₈₈. The Phe side chain binds in a hydrophobic pocket. (B) Model of the Cbz-ase structure with two Zn²⁺ ions docked in putative metal-binding pockets. Putative binding residues are shown. A highly conserved Cys₁₁₈ is highlighted and Zn²⁺ ions are shown as grey-blue spheres.