Chemoenzymatic synthesis of 3′-O-(carboxyalkyl)fluorescein labels

A general and versatile method is described for the synthesis of fluorescent labels. Coupling of the 3'-phenol of fluorescein methyl ester with hydroxyalkyl benzyl esters, followed by benzyl ester hydrolysis, provided a series of fluorescein carboxyalkyl ethers. Use of the Mitsunobu reaction allowed for the introduction of linkers of different lengths onto the 3'-phenol of fluorescein. Chemoenzymatic benzyl ester hydrolysis was achieved with LPL-80 lipase, providing pH-independent labels useful for the preparation of fluorescent conjugates.