Benzoxazinone derivatives: new fluorescent probes for two-color flow cytometry analysis using one excitation wavelength.
A new class of fluorescent dye which upon excitation at 488 nm turns red is shown to be probe-suitable for using in flow cytometry alone or in conjunction with fluorescein derivatives. 7-dimethylamino 3-(p-formylstyryl) 1,4 benzoxazin 2-one is suitable for rendering microorganisms, such as Plasmodium merozoites and cells detectable by flow cytometry, allowing a dual fluorescence analysis when the cells are labelled with suitable fluoresceinylated ligands such as fluorescein labeled neoglycoproteins or antibodies. The synthesis of the new benzoxazinone derivatives is described: p-[beta-(7-dimethylamino 1,4 benzoxazin 2-one 3-yl)-vinyl]-phenylpropenoic acid can be easily activated as a hydroxysuccinimide derivative and linked to amino groups of polypeptides. Hydrophilic polypeptides such as poly-L-lysine or glycosylated polymers combined with this new fluorescent dye are shown to be helpful in analyzing cell surface receptors, in dual fluorescence flow cytometry analysis, using a single excitation wavelength and two sets of compounds labeled with the new benzoxazinone derivative and with fluorescein isothiocyanate, respectively. The new benzoxazinone derivative has a high molar absorbance, a good quantum yield fluorescence when it is bound to hydrophilic polypeptides and its fluorescence intensity is not dependent on pH in the physiological pH range.
Monsigny
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Département de Biochimie des Glycoconjugués et Lectines Endogènes, CNRS, INSERM et Université, Orléans, France.
Midoux
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P
Le Bris
M T
MT
Roche
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AC
Valeur
B
B
D000818:Animals; D000911:Antibodies, Monoclonal; D048588:Benzoxazines; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D008264:Macrophages; D051379:Mice; D010078:Oxazines / Q000138:chemical synthesis*; D010962:Plasmodium berghei / Q000166:cytology; D011107:Polylysine; D011134:Polysaccharides; D013057:Spectrum Analysis; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D048588:Benzoxazines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010078:Oxazines; D011134:Polysaccharides; C063622:4-(beta-(7-dimethylamino-1,4-benzoxazin-2-one-3-yl)vinyl)phenylpropenoic acid; D011107:Polylysine