Proceedings of the 33rd European Peptide Symposium

Emilia Naydenova, Tamara Pajpanova (Editors)

European Peptide Society, 2014
Active Site Titration of Lipase Using New Fluorogenic
Coumarine Inhibitors
Daniela Petkova1, Radostin Dimitrov1, Teodora Tsvetkova2 and Ivanka
Stoineva1
1
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, 1113
Sofia, Bulgaria
2
Faculty of Chemistry, University of Sofia, 1164 Sofia, Bulgaria

Introduction
Lipases are very prominent biocatalysts because of their ability to catalyze a wide variety of
reactions in aqueous and non-aqueous media [1]. The natural substrates of lipases are
triacylglycerols, but a wide range of structurally diverse esters, are accepted as substrates by
lipases. The chemo-, regio- and enantio-specific behaviour of these enzymes has caused
tremendous interest among scientists and industry.
For determination of values for the kinetic parameters and ligand-binding stoichiometries
associated with an enzyme-catalysed reaction, it is necessary to know the concentration of
functional active sites of the enzyme. The latter value cannot be calculated simply from the total
concentration of protein and molecular weight of the enzyme; even when the enzyme
preparation has been shown to be homogeneous by a variety of techniques, it is possible that
inactive enzyme is present. Therefore, before undertaking detailed kinetic investigations on any
enzyme, it is important to determine the concentrations of the active sites that can both bind the
ligands and are active. One of the most promising inhibitors used for active-site titration of
lipases are organophosphonate esters. They react irreversibly with the hydroxyl group of the
active-site serine.
The aim of this study is to design and synthesize new fluorogenic inhibitors for active site
titration of lipases .

Results and Discussion

Organophosphorus ester of 3-(benzothiazol-2-yl)-7-hydroxy-2H-chromen-2-on was
synthesized and evaluated as active-site titrant of lipases. It has been shown that the presence in
the third position of the coumarin ring of a heterocyclic substituent and the prolongation of the
conjugated system have a pronounced influence on their photophysical properties. [2]
The synthesis of the organophosphorus ester of benzothiazolyl chromenon (BTC) was
implemented by three steps reaction (Fig. 1). On the first stage a diethyl-n-heptyl phosphonate
was synthesized by Arbuzov reaction. The phosphonate was purified by vacuum distillation and
then analysed by 31P NMR. On the second stage an ethyl heptyl phosphorus chloride was
obtained and purified by fraction distillation and analysed by 31P NMR. On the next stage the
coumarin derivative was complexed with the purified phosphorous chloride to obtain the
fluorophore.
 Fig.1. Synthetic schemes for the synthesis of ethyl-heptyl-benzothiazolyl phosphonate

Normal phase HPLC was performed for purification of the crude compound on “Waters-
Millipore”, UV detector “Waters”-model 441 column Thermo Quest Hypersil Silica 250x10mm
5µ, isocratic elution, mobile phase: AcCN:H2O (85:15) , flow 0,8 ml/min at 254 nm. The
structure and properties of the novel inhibitors were determined by different chromatography
methods, NMR analyses, and spectroscopy methods. Typical absorption and fluorescence
spectrums of the inhibitor are presented on Fig.2.

Fig. 2. Absorbance and fluorescence spectrums of ethyl-heptyl-benzothiazolyl phosphonate

Pancreatic lipase from Candida rugosa AY “Amano” was used for determination of the
properties of the organophosphorus ester of benzothiazolyl chromenon. Qualitative
spectrophotometric measurements were performed (Fig.3). The solution of organophosphorus
ester of benzothiazolyl chromenon is colourless and it turns immediately into yellow when it
reacts with the lipase.

=372nm

=460nm

Fig.3. Absorbance spectrums of organophosphorus ester of benzothiazolyl chromenon, A -

inhibitor without lipase and B in the presence of lipase.

The preliminary results show that the newly synthesized organophosphorus ester of
benzothiazolyl chromenon has the advantage of high sensitivity and rapidity. It can be used as
tool for qualitative and quantitative determination of active sites of lipases in different
heterogeneous systems or of immobilized enzymes which are hard to follow by others spectral
methods.

Acknowledgments

We gratefully acknowledge the WFS for providing WFS National Scholarship to D.P.

References
[1] A.Intra, A.Bava, G.Nasini, S. Riva, J. Mol. Catal. B: Enzym 29 95-98 (2004).

[2] T. Deligeorgiev, T. Tsvetkova, D. Ivanova, I. Timtcheva, J. Color. Technol. 124, 195–203 (2008).