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Fluorogenic polypropionate fragments for detecting stereoselective aldolases.

R Pérez Carlón1, N Jourdain, J L Reymond

  • 1Departement für Chemie und Biochemie, Universität Bern, Switzerland.

Chemistry (Weinheim an Der Bergstrasse, Germany)
|December 29, 2000
PubMed
Summary

Researchers developed novel fluorogenic substrates to detect stereoselective aldolase enzymes. This assay enables efficient screening for new aldolases, significantly advancing biocatalysis discovery.

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High-throughput screening for biocatalysts.

Current opinion in biotechnology·2002

Area of Science:

  • Biochemistry
  • Organic Chemistry
  • Enzyme Engineering

Background:

  • Aldolases are crucial enzymes catalyzing carbon-carbon bond formation via aldol reactions.
  • Detecting stereoselective aldolases is vital for asymmetric synthesis and biocatalysis.
  • Existing methods for aldolase screening can be laborious and lack sensitivity.

Purpose of the Study:

  • To develop a novel fluorogenic assay for detecting stereoselective aldolases.
  • To create versatile polypropionate substrates for probing different aldolase selectivities.
  • To demonstrate the utility of the assay for high-throughput screening and enzyme discovery.

Main Methods:

  • Synthesis of fluorogenic polypropionate fragments.
  • Utilizing retroaldolization and beta-elimination to release a fluorescent product (umbelliferone).

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  • Designing substrates to probe syn/anti and Cram/anti-Cram selectivity.

    • Main Results:

    • Prepared fluorogenic polypropionate substrates that yield a >20-fold fluorescence increase upon reaction.
    • Demonstrated the assay's ability to detect stereoselective aldolization, including with aldolase antibody 38C2.
    • Validated the assay for high-throughput screening in microtiter plates.

    Conclusions:

    • The developed fluorogenic assay is a convenient and sensitive tool for detecting stereoselective aldolases.
    • The versatile substrates allow for the characterization of various aldolase selectivities.
    • This assay facilitates the isolation of novel stereoselective aldolases from catalyst libraries for biocatalysis applications.