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. 2014 Jun 20;16(12):3220-3.
doi: 10.1021/ol501241a. Epub 2014 Jun 9.

A general NMR-based strategy for the in situ characterization of sugar-nucleotide-dependent biosynthetic pathways

Shanteri Singh  1 , Pauline Peltier-Pain, Marco Tonelli, Jon S Thorson
Affiliations

A general NMR-based strategy for the in situ characterization of sugar-nucleotide-dependent biosynthetic pathways

Shanteri Singh et al. Org Lett. .

Abstract

A simple method for the study of sugar-nucleotide-dependent multienzyme cascades is highlighted where the use of selectively (13)C-labeled sugar nucleotides and inverse (13)C detection NMR offers fast, direct detection and quantification of reactants and products and circumvents the need for chromatographic separation. The utility of the method has been demonstrated by characterizing four previously uncharacterized sugar nucleotide biosynthetic enzymes involved in calicheamicin biosynthesis.

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Figures

Scheme 1
Scheme 1. Synthesis of 2-Chloro-4-nitrophenyl-β-d-[U-13C]glucose and Proposed Biosynthetic Pathway of TDP-4-Hydroxyamino-6-deoxy-glucose and TDP-3-Methoxy-rhamnose en Route to Calicheamicin
(a) Ac2O pyridine; (b) HBr, AcOH/DCM, 0 °C, 2 h; (c) 2-chloro-4-nitrophenol, TBABr, DCM/NaOH (1:1), rt, 18 h; NaOMe, MeOH, rt, 12 h; d-[U-13C]glucose, 1; penta-O-acetyl-d-[U-13C]glucose, 2; 1-bromo-tetra-O-acetyl-α-d-[U-13C]glucose, 3; 2-chloro-4-nitrophenyl-β-d-[U-13C]glucose, 4; TDP-α-d-[U-13C]glucose, 5; TDP-4-keto-6-deoxy-α-d-[U-13C]glucose, 6; TDP-4-keto-β-l-[U-13C]rhamnose, 7; TDP-β-l-[U-13C]rhamnose, 8; TDP-3-methoxy-β-l-[U-13C]rhamnose, 9; TDP-4-amino-6-deoxy-α-d-[U-13C]glucose, 10; TDP-4-hydroxyamino-6-deoxy-α-d-[U-13C]glucose, 11.
Figure 1
Figure 1
(A) Overlay of 2D-1H–13C-HSQC spectra of TDP16, CalS3 and CalS13 reactions. (B) Overlay of 2D-1H–13C-HSQC spectra of 6 (HSQC with blue signals), CalS1, CalS2, and CalS11 reactions. Colors correlate to the products in Scheme 1.
Figure 2
Figure 2
Progress of TDP16 (square) and CalS3 (circle) reactions. Colors correlate to the reactants/products in Scheme 1.
Figure 3
Figure 3
Overlay of 2D-1H–13C-HSQC spectra of CalS13 (red) and WecE (black) reactions. The C4′–H4′ correlations corresponding to CalS13 and WecE products are circled.
See this image and copyright information in PMC

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