Journal: Nature chemical biology
Article Title: Using Simple Donors to Drive the Equilibria of Glycosyltransferase-Catalyzed Reactions
Figure Lengend Snippet: Utilizing a colorimetric screen for glycosyl transfer. ( a ) Scheme for colorimetric screen using the single enzyme (TDP-16) coupled format. ( b ) Evaluation of the colorimetric assay with 58 as the final acceptor. The reactions contained 0.5 mM 9 as donor, 0.5 mM 58 as acceptor, 5 μM UDP, and 11μM TDP-16 in a final total volume of 100 μl with Tris-HCl buffer (50 mM, pH 8.5) in a 96-well plate incubated at 25°C for one hour. ( i ) Qualitative color change after one hour for the full reaction (yellow square), a control lacking the final acceptor 58 (white circle), and a control lacking UDP (red triangle). ( ii ) Δ410 nm over one hour for the full reaction (yellow squares), a control lacking the final acceptor 58 (white circles), and a control reaction lacking UDP (red triangles). ( iii ) HPLC chromatograms of full reaction at 1, 5, and 60 min where 1 is desired product, 9 is the donor, 58 is the target aglycon and ⋄ represents 2-chloro-4-nitrophenolate. (c) The absorbance data and HPLC chromatograms of three representative hits [( i ) 62 (genistein), ( ii ) 79 (tyrphostin), or ( iii ) 92 (ciprofloxacin)] from the broad 50 compound panel screen using the single enzyme (TDP-16) coupled format. In HPLC chromatograms 9 indicates donor; 62 , 79 or 92 represent target aglycon; ⋄ indicates 2-chloro-4-nitrophenolate; and ● depicts glucosylated product(s). For the overall results of the 50 compound screen, additional representative absorbance plots and chromatograms, and combined HPLC and LC/MS characterization, see Supplementary Fig. 19–21 and Supplementary Table 6 .
Article Snippet: Following, samples were thawed at 4°C and filtered through a MultiScreen Filter Plate (Millipore, Billerica, MA, USA) according to manufacturer’s instructions and evaluated for formation of UDP- ( 33a ) or TDP-α-D-glucose ( 33b ) by analytical reverse-phase HPLC with a 250 mm × 4.6 mm Gemini-NX 5μ C18 column (Phenomenex, Torrance, CA, USA) using a linear gradient of 0% to 15% CH3 CN (solvent B) over 15 minutes (solvent A = aqueous 50 mM triethylammonium acetate buffer [Sigma-Aldrich, St. Louis, MO, USA], flow rate = 1 ml min−1 , with detection monitored at 254 nm).
Techniques: Colorimetric Assay, Incubation, High Performance Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy