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

Rydzik AM, Leung IK, Kochan GT, McDonough MA, Claridge TD, Schofield CJ - Angew. Chem. Int. Ed. Engl. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA (UK).

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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.
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fig01: 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.

Mentions: To explore the biocatalytic potential of BBOX, a set of cyclic GBB analogues was screened as potential substrates (Figure S1 in the Supporting Information) by using a mass spectrometry (MS)-based assay. The screen led to the identification of N,N-dimethyl-piperidine-4-carboxylic acid acid (1) as a potential human BBOX substrate (Figure S2). NMR analysis of the reaction product resulting from the incubation of N,N-dimethyl-piperidine-4-carboxylic acid (1) with BBOX revealed the formation of a single alcohol product: 3-hydroxy-N,N-dimethyl-piperidine-4-carboxylic acid (2; Scheme 1 B, Figure 1, and Figure S3). The product stereochemistry was assigned as (3R)-hydroxy-(4S)-N,N-dimethyl-piperidine-4-carboxylic acid based on coupling constant analysis, which revealed that the C3=H and C4=H bonds are both axial (H4: Jaa=12.4 Hz, Jaa=10.5 Hz, Jae=4.7 Hz; H3: Jaa=10.8 Hz×2, Jae=4.7 Hz), and protein crystallographic analysis (see below). Kinetic analysis revealed a KM value of 40 μm and a kcat value of 0.14 μm s−1 when using 1 as the BBOX substrate, compared to values of 4 μm and 0.83 μm s−1 for GBB (Table S1). Interestingly, substrate inhibition was observed only above 0.2 mm with 1, whereas substrate inhibition occurs at >20 μm of GBB[8] (Figure S14).


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

Rydzik AM, Leung IK, Kochan GT, McDonough MA, Claridge TD, Schofield CJ - Angew. Chem. Int. Ed. Engl. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4497603&req=5

fig01: 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.
Mentions: To explore the biocatalytic potential of BBOX, a set of cyclic GBB analogues was screened as potential substrates (Figure S1 in the Supporting Information) by using a mass spectrometry (MS)-based assay. The screen led to the identification of N,N-dimethyl-piperidine-4-carboxylic acid acid (1) as a potential human BBOX substrate (Figure S2). NMR analysis of the reaction product resulting from the incubation of N,N-dimethyl-piperidine-4-carboxylic acid (1) with BBOX revealed the formation of a single alcohol product: 3-hydroxy-N,N-dimethyl-piperidine-4-carboxylic acid (2; Scheme 1 B, Figure 1, and Figure S3). The product stereochemistry was assigned as (3R)-hydroxy-(4S)-N,N-dimethyl-piperidine-4-carboxylic acid based on coupling constant analysis, which revealed that the C3=H and C4=H bonds are both axial (H4: Jaa=12.4 Hz, Jaa=10.5 Hz, Jae=4.7 Hz; H3: Jaa=10.8 Hz×2, Jae=4.7 Hz), and protein crystallographic analysis (see below). Kinetic analysis revealed a KM value of 40 μm and a kcat value of 0.14 μm s−1 when using 1 as the BBOX substrate, compared to values of 4 μm and 0.83 μm s−1 for GBB (Table S1). Interestingly, substrate inhibition was observed only above 0.2 mm with 1, whereas substrate inhibition occurs at >20 μm of GBB[8] (Figure S14).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA (UK).

Show MeSH