THE DETERMINATION OF HYDROXYLATED NATURAL PRODUCTS

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7-[(Chlorocarbonyl)methoxy]-4-methylcoumarin 1 has been under development in LPC for the fluorescent labeling of primary and secondary hydroxyl groups. We have developed synthetic procedures which allow the gram-scale production of 1 in high purity, and we have found this reagent to react smoothly with a variety of primary and secondary hydroxyl compounds to give the corresponding esters. These esters readily elute from reversed- phase HPLC columns, thereby allowing detection by standard methods such as UV, fluorescence, or mass spectrometry. However, a small, but highly fluorescent impurity was discovered, isolated, and subsequently identified. The origin of this impurity was traced to a side-reaction of 7-(carboxymethoxy)-4-methylcoumarin with thionyl chloride, leading us to a new, coumarin-based, acylation reagent, 3-chloro-7- [(chlorocarbonyl)methoxy]-4-methyl-coumarin. We have developed synthetic procedures which now allow the gram-scale production of this new reagent. This new reagent provides a fluorophore which is many times more fluorescent than the corresponding non-chlorinated analog, thus lowering the limit of detection in potential applications. Upon derivatizing compounds which possess a hydroxyl moiety, we find the reactivity and selectivity of this new reagent similar to that of 7- [(chlorocarbonyl)methoxy]-4-methylcoumarin; the resulting derivatives also readily elute from reversed-phase HPLC columns, thereby allowing for facile analysis.