Associative chemosensing by fluorescent macrocycle-dye complexes-a versatile enzyme assay platform beyond indicator displacement

Article abstract of DOI:10.1039/c4cc10227d

A label-free in situ method to monitor reactions in real time by using fluorescent supramolecular chemosensors based on cucurbit[8]uril is presented. It allows sensing of enzymatic activity, inhibitor and activator screening, and analyte detection with unprecedented versatility and high sensitivity.

Full text of DOI:10.1039/c4cc10227d

Showcasing research from Werner Nau’s Laboratory/
Department of Life Sciences and Chemistry,
Jacobs University Bremen, Bremen, Germany
As featured in:
Associative chemosensing by fluorescent macrocycle–dye
complexes – a versatile enzyme assay platform beyond indicator
displacement
Enzymatic reactions can be readily followed in the presence of
fluorescent supramolecular receptors, allowing the set-up of
a large range of real-time assays, for example for oxidations or
hydrolyses of aromatic substrates, which have added potential
for high-throughput screening.
Credits to K. Assaf.
See Frank Biedermann,
Werner M. Nau et al.,
Chem. Commun., 2015, 51, 4977.
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Associative chemosensing by fluorescent
macrocycle–dye complexes – a versatile enzyme
assay platform beyond indicator displacement†
Cite this: Chem. Commun., 2015,
51, 4977
Received 22nd December 2014,
Accepted 15th January 2015
Frank Biedermann,* Denisa Hathazi and Werner M. Nau*
DOI: 10.1039/c4cc10227d
www.rsc.org/chemcomm
A label-free in situ method to monitor reactions in real time by using
fluorescent supramolecular chemosensors based on cucurbit[8]uril
is presented. It allows sensing of enzymatic activity, inhibitor and
activator screening, and analyte detection with unprecedented versatility
and high sensitivity.
Methods for monitoring (bio)catalytic activity are indispensable for
the elucidation of reaction mechanisms, the testing of inhibitors and
activators, and for the activity screening of natural and tailor-made
enzymes.1 Emission-based detection is almost always preferred,
especially in pharmaceutical-industrial settings, because of its
sensitivity, economic use, and safety. Only a few substrate–product
pairs are intrinsically fluorescent, and even fewer are spectrally
distinguishable from each other. Therefore, fluorescently labeled
substrate analogues are often resorted to, at the expense of added
costs and frequently non-transferable activity profiles.1a,b Supra-
molecular tandem enzyme assays, using a sensing ensemble
composed of a macrocyclic host and an indicator dye, were previously
introduced by us, allowing for label-free reaction monitoring.1b,2
These operate through an indicator displacement scheme,3 which
requires a subtle balance of the affinities of the substrate, product,
and fluorescent dye.1b,2 To address this shortcoming, we explored a
complementary chemosensor design strategy, which was recently
introduced for the detection and reaction monitoring of chiral
aromatic analytes by induced circular dichroism.4 We now introduce
self-assembled fluorescent chemosensors that associate with aromatic
substrates and, thereby, allow the real-time monitoring of their
Scheme 1 (a and b) Fluorescent chemosensors for aromatic analytes are
self-assembled from the large macrocyclic host cucurbit[8]uril (CB8) and
dicationic dyes. (c) Appearance of the product and the corresponding
depletion of the substrate during the course of an enzymatic reaction are
coupled to a change in the amount free and analyte-bound chemosensor.
enzymatic conversions. This establishes a general enzyme assay dye (e.g., MDAP, MDPP, or PDI) into the large cavity of the macro-
platform responsive to aryl moieties, as they are most abundant in cycle cucurbit[8]uril (CB8),6 see Scheme 1. The residual cavity space
drugs, amino acids, toxins, hormones, antibiotics, and colorants.5 of such CB8-based chemosensors allows for the subsequent binding
The self-assembled fluorescent chemosensors can be obtained of an aryl-functionalized analyte which is typically accompa-
by the spontaneous inclusion of a suitable dicationic fluorescent nied by net fluorescence quenching.4,7 The magnitude of the
intensity change is a function of the concentration, binding
affinity (Ka), and quenching efficiency (QE) of the substrate–
product couple, see also Table S1 (ESI†) for representative Ka
and QE values. Importantly, the non-covalent and reversible
Department of Life Sciences and Chemistry, Jacobs University Bremen, Campus Ring 1,
28759 Bremen, Germany. E-mail: frankbiedermann@daad-alumni.de,
w.nau@jacobs-university.de
chemosensor–analyte complexation is essentially instantaneous:
it responds dynamically to analyte concentration changes.4
† Electronic supplementary information (ESI) available: Materials and methods
and experimental details. See DOI: 10.1039/c4cc10227d
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Consequently, the change in the concentrations of the substrate by our emission-based method. Low mM substrate concentrations
(S) and the product (P) in the course of an enzymatic reaction were sufficient, demonstrating high sensitivity. Additionally,
translates directly into alteration of the ratio of emissive (analyte-free, different chemosensors could be used interchangeably (Fig. 1a).
binary) and non-emissive (analyte-bound, ternary) chemosensor and, In line with intuition, the more electron-rich substrates were
thus, a change in the measurable emission intensity (Iem). The only oxidized faster, e.g., 4-substituted phenols with R = –OMe 4
condition which needs to be fulfilled is differential affinities, that is, –Me 4 –H 4 –F (see Table S2, ESI† for numerical values and
Ka(S) a Ka(P), see Scheme 1c, which is almost always the case for more examples.) The method can also be extended to the screening
chemical modifications taking place in the vicinity of the aryl anchor. of reaction conditions, e.g., of the pH (Fig. 1b). In fact, the HRP-
Specifically, an increase in Iem is expected for Ka(S) 4 Ka(P), the catalysed oxidation of Trp is known to proceed via a number of
substrate-selective enzyme assay, whereas for the product-selective chemical intermediates,9 whose appearance depends on pH, which
variant, with Ka(S) o Ka(P), a decrease in Iem applies. While we is in agreement with the observed complicated kinetic profiles
successfully tested several fluorescent dyes (Scheme 1b), the dye can (Fig. 1b). The use of different chemosensors with distinct responses
in principle be selected at will to match the demands of a user for a to (some of) the intermediate(s) can in such cases provide additional
particular wavelength or sensitivity, or different dyes can be screened kinetic information: For example, the HRP-catalyzed oxidation of
to achieve a quick optimization of the assay.
aniline with H2O2, known to yield polyaniline in a step-wise
After we had confirmed that the chemosensors are chemically process,10 was successfully monitored with all three receptors,
stable in the reaction mixtures, we demonstrated the versatility of that is, CB8ꢀMDAP, CB8ꢀMDPP, and CB8ꢀPDI (Fig. S3b, ESI†).
this supramolecular approach to enzymatic reaction monitoring. Comparison of the kinetic traces clearly exposed the multi-step
For this purpose a wide range of enzymatic redox and hydrolysis nature of the reaction, for which at least two intermediates need
reactions was tested, see Scheme 2. Representative results are to be invoked.
surveyed below and all are contained in the ESI,† as are the
Chloroperoxidase (CPO) has a higher redox potential than
quantitative aspects related to this supramolecular sensing HRP which allows for the oxidation of chloride ions to produce
concept. Our claim is that there is no other assay known which reactive intermediates, thus enabling the oxidation of organic
covers such a large range of enzymes, not to speak of the ease of molecules that are not subject to HRP-catalyzed reactions.11 For
use and the other advantages named above.
instance, the pharmaceutically important enzymatic production
To benchmark our associative chemosensors, the kinetics of of a representative chiral sulfoxide12 by CPO could be followed in
the horseradish peroxidase-(HRP)-catalysed oxidation of catechol with real time even at low mM substrate concentration with the
H2O2 was compared to the direct UV/vis recording of the resulting chemosensor CB8ꢀMDAP (Fig. 1c), which cannot be achieved
chromophoric 1,2-benzoquinone-catechol product-couple.8 Both by direct optical monitoring. In view of the success with HRP and
methods yielded very similar kinetic traces (Fig. S1, ESI†). CPO, it was not surprising that lactoperoxidase-catalysed reactions
Furthermore, the variation of the enzyme concentration yielded could also be monitored with the fluorescent receptors, see Fig. S4
the expected linear proportionality of the initial rates (Fig. S2, (ESI†) for a representative example.
ESI†). Both observations confirm that the chemosensors do not
Copper-containing laccases utilize molecular dioxygen for
interfere with the enzymatic activity and remain stable under the the oxidation of various substrates and have therefore found
experimental conditions. The chemosensors show broad selectivity widespread industrial use, e.g., for lignin degradation, waste-water
to many aromatic substrates, such that they are predestined for cleaning, and in food production.13 Redox mediators such as
rapid substrate-screening studies. To demonstrate, the oxidation of 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)
naphthols and phenols (Fig. 1a and Fig. S3a, ESI†) was monitored and N-hydroxyl-phthalimide (NHA) can significantly speed up
the desired oxidation of a substrate.13c As is demonstrated in
Fig. S5 (ESI†), it was possible to follow the representative
laccase-catalysed oxidation of 4-chlorophenol and to screen
for the activity of the mediators ABTS and NHA.‡
Alcohol dehydrogenases (ADHs) have industrial and phar-
maceutical use for the oxidative conversion of alcohols to
aldehydes and for the (stereoselective) reduction of ketones to
alcohols.14 In contrast to the aforementioned enzymatic reactions,
ADH requires a cofactor, NAD+/NADH,H+, that is commonly
employed in large excess. Fortunately, neither NAD+ nor NADH,H+
interact strongly with the chemosensor CB8ꢀMDAP and their
absorption bands do not interfere with its excitation wavelength.
Hence, the ADH-catalyzed oxidation of 4MeO-benzylalcohol
to 4MeO-benzaldehyde could be readily monitored (Fig. 1d).
A representative reduction reaction is shown in Fig. S6 (ESI†).
Moving from enzymatic redox to hydrolysis reactions, the
exopeptidase L-leucine aminopeptidase (LAP) catalyses the hydrolysis
of peptides and proteins starting from their N-terminus. In addition
Scheme 2 Enzymatic (a) redox and (b) hydrolysis reactions that can be
monitored with the associative chemosensors introduced herein.
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Fig. 1 (a) HRP-catalysed (0.6 mg mLꢁ1) oxidation of 2-naphthol and 4-methoxy phenol (each 46 mM) with H2O2 (100 mM) and NaI (120 mM), reported by
two different chemosensors (each 5 mM). (b) HRP-catalysed (12 mg mLꢁ1) oxidation of Trp with H2O2 (100 mM) and NaI (120 mM) in the presence of CB8ꢀ
MDAP (5 mM). Left: varying Trp concentrations at pH 4. Right: pH variation with 20 mM Trp. (c) CPO-catalysed (25 mg mLꢁ1) sulfoxidation with H2O2
(200 mM) and NaCl (100 mM) in the presence of CB8ꢀMDAP (5 mM). (d) ADH-catalysed (3.8 mg mLꢁ1) alcohol-oxidation (100 mM) with the cofactor NAD+
(1 mM) in the presence of CB8ꢀMDAP (5 mM). (e) LAP-catalysed (50 mg mLꢁ1) hydrolysis of dipeptides (20 mM) in the presence of CB8ꢀMDPP (5 mM).
(f) Pepsin-catalysed (0.0–7.0 mg mLꢁ1) hydrolysis of bovine serum albumin (BSA) protein (80 mg mLꢁ1) in the presence of CB8ꢀMDPP (5 mM) at pH 2.
(g) PLE-catalysed (80 mg mLꢁ1) hydrolysis of phenyl acetate at pH 5 in the presence of CB8ꢀMDAP (5 mM). The plot of the initial rates vs. the substrate
concentration was fitted by a Michaelis–Menten function to yield a KM value of (25 ꢂ 4.) mM. All reactions were carried out in 10 mM sodium phosphate
buffer. See the Materials and methods section in the ESI† for further details.
to its wide natural abundance, LAP found use in peptide industry.18 The applicability and compatibility of the receptor
sequencing.15 Fig. 1e shows the kinetic profile for the LAP-catalyzed was demonstrated for the PLE-catalysed hydrolysis of the model
hydrolysis of AlaPhe into Ala and Phe, as was monitored in the substrates, phenyl acetate and butyl benzoate (Fig. 1g and Fig. S10,
presence of the chemosensor CB8ꢀMDPP. Other examples ESI†), including the determination of its Michaelis–Menten
including the hydrolysis of Trp-containing peptides (Fig. S7) kinetics (Fig. 1g).
and the comparison of the enzymatic activity when incubated
Phosphates catalyze the removal of phosphate groups from
organic molecules, which alters their overall charge and, thus,
with Mg2+ or Zn2+ (Fig. S8) are shown in the ESI.†
Carboxypeptidases are exopeptidases that cleave proteins their affinity for supramolecular hosts. Such charge-altering
and peptides C-terminally16 for which even substrate-labeled reactions are good candidates for dye-displacement assays.1b,2c
assays are scarce.17 The fluorescent chemosensor-based monitoring Nevertheless, a phosphatase reaction can also be monitored
of carboxypeptidase A (CPA) activity on hippuryl-phenylalanine is with our chemosensing ensembles that complex preferentially
shown in Fig. S9 (ESI†), providing a complementary monitoring non-charged over negatively charged analytes4,6,7b (product-
method to our recently reported induced circular dichroism selective assays). For instance, the dephosphorylation of phenyl
detection strategy.4
and naphthyl phosphates by alkaline phosphatase (ALKP) is
The aromatic amino acids of most proteins are located presented in Fig. S11 (ESI†) and shows the expected decrease in
inside the protein hydrophobic core; they are thus inaccessible Iem as a function of substrate conversion.
for direct binding to chemosensors.4 However, digestion of a
Penicillinase belongs to the important enzyme class of
protein with peptidase destroys its tertiary protein structure, b-lactamases, which are largely responsible for the resistance
which makes the aromatic recognition motifs sterically accessible. of certain bacteria to b-lactam antibiotics. It hydrolyses the
The digestion of bovine serum albumin (BSA) with pepsin at pH 2 b-lactam ring of benzylpenicillin, also known as penicillin G,
serves as a model reaction, displaying the expected reduction in followed by spontaneous decarboxylation. We could now monitor
emission intensity in the presence of CB8ꢀMDPP (Fig. 1f). The use this enzymatic reaction by fluorescence in real time (Fig. S12, ESI†),
of the chemosensor-based detection is likely a broadly applicable offering a sensitive detection alternative to direct circular
method to monitor the digestion of proteins since most proteins dichroism.19
contain at least one Trp (or Phe) residue.
While unsubstituted carbohydrates do not bind to the receptors,
Esterases such as porcine liver esterase (PLE) have received their aryl-substituted variants are complexed with mM to mM
interest for synthetic chemical purposes including chiral resolution, affinities.4 We have followed the hydrolysis of phenyl-b-D-
and also in agriculture, as well as the food and pharmaceutical galactopyranoside by b-galactosidase (b-Gal), a commonly studied
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`
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Associative chemosensing ensembles consisting of the large
macrocycle cucurbit[8]uril and suitable fluorescent dyes provide
a versatile platform to monitor in situ enzymatic reactions with
mM sensitivity for a wide range of substrates carrying aromatic
recognition motifs. We demonstrated the large scope of the
method by employing 12 different enzymes catalyzing redox
and hydrolysis reactions over a broad pH range, including
peroxidases, dehydrogenases, laccases, peptidases, esterases,
phosphatases, penicillinase, and b-galactosidase. All enzymatic
conversions were followed in real time and enzyme-kinetic
parameters are directly accessible. The broad selectivity of the
receptors allows for activity screening of substrates and mediators;
the extension to inhibitor screening is logical, albeit limited to
inhibitors that do not (strongly) bind to the receptor. Important
for sensing, the excitation and the emission can be chosen in the
visible region of the spectrum and can be tuned through modifica-
tion of the dye, keeping the macrocycle unchanged. The use of self-
assembled CB8-dye receptors also enables the identification of
reaction intermediates that would escape detection when observing
the fluorescence/absorption of the substrate or product alone. We
believe that the associative chemosensing strategy can find a wide
range of applications in enzymatic reaction screening, in pharma-
ceutical research, white biotechnology, and also as an economic
analytical tool in biochemical research labs.
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This work was supported by the German Academic Exchange
Service (F.B.) and the Deutsche Forschungsgemeinschaft (DFG
Grant NA-686/5, WMN).
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Notes and references
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4980 | Chem. Commun., 2015, 51, 4977--4980
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