Background: Galactose oxidase (GOase) catalyses the highly selective oxidation of
terminal galactosides on a wide range of natural glycoconjugates and has found wide applications
in biotechnology – particularly in biocatalysis. GOase is copper dependent and uses oxygen to oxidise
the C6-primary alcohol of galactose and produces hydrogen peroxide. The enzyme activity can
be conveniently assessed by a colorimetric assay.
Objectives: The objective of the present study was to develop an assay system, which is independent
of the hydrogen peroxide formation to identify possible fluorinated GOase inhibitors. In case
that the inhibitor bears a primary or secondary alcohol, it could also be oxidised by the enzyme. In
such case, the colorimetric assay is not able to distinguish between substrate and inhibitor, since
oxidation of both molecules would result in the formation of hydrogen peroxide.
Methods: D-galactose (D-Gal) was immobilised onto a gold surface functionalised by selfassembled
monolayers (SAMs,). A GOase solution was then added to the surface in a droplet for a
certain period of time and thereafter washed away. The activity of GOase on the immobilised D-Gal
can then be quantified by MALDI-ToF MS.
Results: For inhibition studies, GOase was incubated together with 62.5 mM of deoxy-fluorinated
monosaccharides on the D-Gal displaying platform. Five deoxy-fluorinated D-Gal showed a >50%
inhibition of its activity. The array system has been moreover utilised to determine the apparent
IC50 value of 3-F-Gal 15 as a proof of principle.
Conclusion: The developed array platform allows the fast identification of GOase substrates and
inhibitors from a library of deoxy-fluorinated sugars using MALDI-ToF MS as a label–free readout
method. In addition, the enzymatic reaction enables for the in situ activation of sugar-coated
surfaces to bioorthogonal aldehydes, which can be utilised for subsequent chemical modifications.