Development of a novel fluorescent tag O-2-[aminoethyl]fluorescein for the electrophoretic separation of oligosaccharides.
This study describes the development of a novel fluorescent tag, O-2-[aminoethyl]fluorescein, for the separation of sugars by capillary electrophoresis with fluorescence detection using an argon ion laser. The tag was synthesised using three consecutive steps namely: esterification, alkylation and hydrolysis, specifically designed to offer a flexible way in which to make an assortment of fluorescent tags from cheap and readily available starting reagents (typically less than $1 per g of fluorescent tag). Via this flexible synthetic pathway, O-2-[aminoethyl]fluorescein was designed and synthesised with a spacer group to lower steric effects between the fluorescein backbone and the reducing end of the carbohydrate which were anticipated to improve the reactivity of the tag. The newly synthesised tag, O-2-[aminoethyl]fluorescein was evaluated against structurally similar commercial fluorescent motifs namely fluorescent 5-aminomethylfluorescein and non-fluorescent 5-aminofluorescein. Kinetic studies indicated that O-2-[aminoethyl]fluorescein showed similar labeling efficiencies as 5-aminomethylfluorescein, but were achieved in only 30 min, supporting the notion of improved reactivity of the spacer group. The sensitivity of O-2-[aminoethyl]fluorescein was evaluated using maltoheptaose with a detection limit of 1 nM obtained, which was slightly higher than that of 0.3 nM obtained with 5-aminomethylfluorescein, and was due to its lower quantum yield (0.24) when conjugated to the sugar. The separation performance of the tag was also benchmarked with the two commercial reagents using a range of corn syrup oligosaccharides, from 4 to 10 glucose units, typically found in rice starch. Separations were performed using an electrolyte containing 100 mM boric acid, tris at pH 8.65 as background electrolyte, 30 kV applied voltage, 50 microm I.D. x 40 cm (30 cm effective length) capillary. The novel tag showed better resolution of small oligosaccharides, G3 and G4, than the other two reagents, but slightly worse resolution for the longer oligosaccharides, most likely due to the monovalent charge state of the O-2-[aminoethyl]fluorescein compared to the divalent charge of the other two tags.
Kazarian
Artaches A
AA
Australian Centre for Research on Separation Science, School of Chemistry, University of Tasmania, GPO Box 252-75, Hobart, Tasmania 7001, Australia.
Smith
Jason A
JA
Hilder
Emily F
EF
Breadmore
Michael C
MC
Quirino
Joselito P
JP
Suttil
James
J
D004586:Electrophoresis / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D019793:Fluorescein / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D057230:Limit of Detection; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009844:Oligosaccharides; C030864:5-aminofluorescein; D019793:Fluorescein