Fluorescent N-methylanthranilyl (Mantyl) tag for peptides: its application in subpicomole determination of kinins.
Highly fluorescent N-methylanthranilyl (Mantyl) peptide derivatives were prepared by a one-step reaction with N-methylisatoic anhydride (MIA) for quantitative detection in HPLC. Reactions were carried out in an organic medium of acetonitrile-triethylamine, in aqueous alkaline sodium carbonate and sodium phosphate buffers. 4-Dimethylaminopyridine (DMAP) catalyzed specific mantylation of -NH2 groups of peptides in the organic reaction medium. The DMAP had no effect in the aqueous buffered reaction systems. Proline amino-terminal peptides reacted equally well with MIA. Mantyl-bradykinin had excitation and fluorescence maxima at 350 nm and 426 nm in water and water/acetonitrile (ACN)/trifluoroacetic acid (TFA) solvent mixtures, respectively. Fluorescence intensity increased with an increase in ACN concentration and decreased with an increase in acid content. Mantyl kinins were completely resolved on a C18 reversed phase HPLC column using an ACN-0.1% TFA gradient and their behavior on the column was similar to having an extra amino acid. Di-Mantyl derivatives obtained with Lys-BK and Met-Lys-BK did not exhibit fluorescence appreciably higher than Mantyl-BK. Fluorescence detection of Mantyl kinins was about 50-100 times more sensitive (lower limits of 0.1 to 0.5 picomole) than UV detection of the phenylisothiocyanate-derivatized kinins under typical HPLC conditions.
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Department of Biochemical Pharmacology, State University of New York, Buffalo 14260.
Schulz
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D015761:4-Aminopyridine / Q000031:analogs & derivatives*; D000812:Anhydrides; D001920:Bradykinin / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry*; D007705:Kinins / Q000032:analysis / Q000737:chemistry*; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014867:Water; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D000812:Anhydrides; D005456:Fluorescent Dyes; D007705:Kinins; D010455:Peptides; D012997:Solvents; D062367:ortho-Aminobenzoates; D014867:Water; C077537:N-methylisatoic anhydride; C038892:methyl anthranilate; D015761:4-Aminopyridine; C003885:4-dimethylaminopyridine; D001920:Bradykinin