Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection.
Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure.
Department of Molecular Structure & Characterization, Amgen, Inc., Thousand Oaks, CA 91320, United States. Electronic address: heathere@amgen.com.
Heather
H
Eastwood
ASK-Gene Pharma, Inc., Camarillo, CA 93012, United States.
Fang
F
Xia
Department of Molecular Structure & Characterization, Amgen, Inc., Thousand Oaks, CA 91320, United States.
Mei-Chu
MC
Lo
Department of Molecular Structure & Characterization, Amgen, Inc., Thousand Oaks, CA 91320, United States.
Jing
J
Zhou
Department of Molecular Structure & Characterization, Amgen, Inc., Thousand Oaks, CA 91320, United States.
John B
JB
Jordan
Department of Molecular Structure & Characterization, Amgen, Inc., Thousand Oaks, CA 91320, United States.
John
J
McCarter
Department of Molecular Structure & Characterization, Amgen, Inc., Thousand Oaks, CA 91320, United States.
Wesley W
WW
Barnhart
Department of Molecular Structure & Characterization, Amgen, Inc., Thousand Oaks, CA 91320, United States.
Kyung-Hyun
KH
Gahm
D002021:Buffers; D002241:Carbohydrates / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D005643:Fucose / Q000031:analogs & derivatives / Q000032:analysis; D006600:Hexosephosphates / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D015394:Molecular Structure; D009702:Nucleoside Diphosphate Sugars / Q000032:analysis; D009711:Nucleotides / Q000032:analysis*; D012997:Solvents / Q000737:chemistry; D055672:Static Electricity; D013403:Sugar Phosphates / Q000032:analysis
D002021:Buffers; D002241:Carbohydrates; D006600:Hexosephosphates; D009702:Nucleoside Diphosphate Sugars; D009711:Nucleotides; D012997:Solvents; D013403:Sugar Phosphates; C081214:fucopyranosyl phosphate; D005643:Fucose
High performance liquid chromatography; Mass spectrometry; Mixed-mode stationary phase; Nucleotide-sugar; Nucleotides; Sugar phosphate