Oxygenase-catalyzed desymmetrization of N,N-dialkyl-piperidine-4-carboxylic acids

Abstract

γ-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate dependent oxygenase that catalyzes the final hydroxylation step in the biosynthesis of carnitine. BBOX was shown to catalyze the oxidative desymmetrization of achiral N,N-dialkyl piperidine-4-carboxylates to give products with two or three stereogenic centers.

Keywords: C-H activation; biocatalysis; desymmetrisation; oxygenases; γ-butyrobetaine hydroxylase.

Scheme 1

BBOX catalyzes oxidative desymmetrization. A) BBOX catalyzes the stereoselective hydroxylation of γ-butyrobetaine (GBB) to (3R)-carnitine during carnitine biosynthesis. B) The BBOX-catalyzed oxidation of achiral N,N-dimethyl piperidine-4-carboxylic acid (1) gives (3R)-hydroxy-(4S)-N,N-dimethyl-piperidine-4-carboxylic acid (2). In the BBOX-catalyzed oxidation of a mixture of cis-N,N-ethylmethyl-piperidine-4-carboxylic acid (3 a) and trans-N,N-ethylmethyl-piperidine-4-carboxylic acid (3 b), only the cis isomer undergoes effective hydroxylation to give (3R)-hydroxy-(1R)-N,N-dimethyl-piperidine-(4S)-carboxylic acid (4 a).

Figure 1

¹H NMR assignments of the BBOX-catalyzed hydroxylation of achiral N,N-dimethyl piperidine-4-carboxylic acid (1). An overlay of the ¹H NMR spectrum of a reaction mixture containing 1 prior to (red) and after (blue) BBOX incubation reveals the formation of a new species (2). The pattern of the coupling constants for H3 and H4 of 2 demonstrate that the C3=H and C4=H bonds are both axial. The signal for C4=H partly overlaps with other signals of the reaction mixture.

Figure 2

A crystal structure of BBOX complexed with N,N-dimethyl piperidine (1), Ni^II (as a substitute for Fe^II), and NOG (N-oxyalyl glycine, a non-reactive 2OG mimetic). A) The view from the active site of the BBOX 1 Ni^II NOG complex (PDB ID: 4CWD) reveals that 1 adopts a chair conformation, with its pro(R) hydrogen atom, which is hydroxylated, pointing towards the metal. B) An overlay of the active sites of BBOX complexed with GBB, Zn^II, and NOG (blue, PDB ID: 3O2G) or with 1, Ni^II, and NOG (orange, PDB ID: 4CWD) reveal similar positions of the active-site residues and NOG. The carboxylate groups of GBB and 1 are similarly positioned, both interacting with the side chains of Asn191 and Asn292. The quaternary ammonium groups of both GBB and 1 occupy an identical aromatic cage formed by Tyr177, Tyr194, Tyr205, and Trp181. M=metal. Note: the carbons that undergo hydroxylation (C3 in both GBB and 1) are near identically positioned relative to the metal ion in both structures.