Synthesis and evaluation of a series of 6-chloro-4-methylumbelliferyl glycosides as fluorogenic reagents for screening metagenomic libraries for glycosidase activity

• Sensitive fluorogenic substrates are required for high-throughput screening of enzyme libraries.
• The synthesis of a series of nine different glycosides bearing a 6-chloro-4-methylumbelliferyl chromogen is described.
• These substrates are used in a pooled fashion to screen a small expressed metagenomic library.
• Simple deconvolution identified the individual glycosidase activities.

Screening of large enzyme libraries such as those derived from metagenomic sources requires sensitive substrates. Fluorogenic glycosides typically offer the best sensitivity but typically must be used in a stopped format to generate good signal. Use of fluorescent phenols of pKa < 7, such as halogenated coumarins, allows direct screening at neutral pH. The synthesis and characterisation of a set of nine different glycosides of 6-chloro-4-methylumbelliferone are described. The use of these substrates in a pooled format for screening of expressed metagenomic libraries yielded a “hit rate” of 1 in 60. Hits were then readily deconvoluted with the individual substrates in a single plate to identify specific activities within each clone. The use of such a collection of substrates greatly accelerates the screening process.

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