Light-driven release of cucurbit[8]uril from a bivalent cage

Article abstract of DOI:10.1039/d1sc01410b

Temporal control over supramolecular systems has great potential for the modulation of binding and assembly events, such as providing orthogonal control over protein activity. Especially light controlled triggering provides unique entries for supramolecular systems to interface in a controlled manner with enzymes. Here we report on the light-induced release of cucurbit[8]uril (CB[8]) from a bivalent cage molecule and its subsequent activation of a proteolytic enzyme, caspase-9, that itself is unresponsive to light. Central to the design is the bivalent binding of the cage with high affinity to CB[8], 100-fold stronger than the UV-inactivated products. The affinity switching occurs in the (sub-)micromolar concentration regime, matching the concentration characteristics required for dimerizing and activating caspase-9 by CB[8]. The light-responsive caged CB[8] concept presented offers a novel platform for tuning and application of switchable cucurbiturils and beyond.

Full text of DOI:10.1039/d1sc01410b

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Light-driven release of cucurbit[8]uril from
a bivalent cage†
Cite this: DOI: 10.1039/d1sc01410b
*
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Pim J. de Vink,
Tim van der Hek
and Luc Brunsveld
Temporal control over supramolecular systems has great potential for the modulation of binding and
assembly events, such as providing orthogonal control over protein activity. Especially light controlled
triggering provides unique entries for supramolecular systems to interface in a controlled manner with
enzymes. Here we report on the light-induced release of cucurbit[8]uril (CB[8]) from a bivalent cage
molecule and its subsequent activation of a proteolytic enzyme, caspase-9, that itself is unresponsive to
light. Central to the design is the bivalent binding of the cage with high affinity to CB[8], 100-fold
stronger than the UV-inactivated products. The affinity switching occurs in the (sub-)micromolar
concentration regime, matching the concentration characteristics required for dimerizing and activating
caspase-9 by CB[8]. The light-responsive caged CB[8] concept presented offers a novel platform for
tuning and application of switchable cucurbiturils and beyond.
Received 10th March 2021
Accepted 10th April 2021
DOI: 10.1039/d1sc01410b
rsc.li/chemical-science
has been used for example for induction of light-mediated self-
sorting.23 Notwithstanding, there is a need for novel switchable
Introduction
CB[8]-based systems for controlling biomolecular assemblies,
including those with affinities matching the concentrations and
assembly characteristics of the biomolecules involved. Here we
Supramolecular systems have shown great potential for the
modulation of biomolecular assemblies, providing orthogonal
and additive control to natural recognition and switching
events.1–4 Several supramolecular host molecules, such as
molecular tweezers, calixarenes, cyclodextrins, and cucurbit[n]
urils, are able to selectively recognize specic peptide and
protein elements through, for example, combinations of side
chain residues.5–10 In particular cucurbit[n]urils have shown to
be successful in protein binding, enabling for example protein
activity modulation, because of their relatively strong affinity to
biomolecular epitopes in water.11–14 Cucurbit[8]uril (CB[8]),
specically, is able to incorporate two N-terminal phenylala-
nine–glycine–glycine (FGG) motifs simultaneously in its
hydrophobic cavity and therewith acts as a supramolecular
glue5 allowing to control the dimerization and assembly of
biomolecules such as caspases15 and larger complexes.16
Light controlled triggering systems offer the possibility to
perturb and modulate supramolecular over biological systems
with spatio or temporal control.17,18 Beautiful examples of
supramolecular light stimulus-responsive systems include:
light induced pH jumps,19 photo redox switches20 and photo-
caged guests to release drug molecules from CB[8].21 Of partic-
ular note are diarylethenes-based switches featuring low to high
nanomolar affinity switches between their two states,22 which
Fig.
1 General concept and design elements for light-triggered
Laboratory of Chemical Biology, Department of Biomedical Engineering, Institute for
Complex Molecular Systems, Eindhoven University of Technology, P. O. Box 513,
5600 MB Eindhoven, The Netherlands. E-mail: l.brunsveld@tue.nl
supramolecular activation of enzymes. CB[8] is temporarily caged by
bivalent FGG-peptide 1 that cleaves upon UV-light radiation, which
substantially reduces the affinity of the individual cage elements for
CB[8]. This liberates the CB[8] to bind and activate two monomers of
caspase-9. Also shown is control cage 2 that cannot be light-
inactivated.
† Electronic supplementary information (ESI) available: Figures and tables,
experimental details of peptide synthesis, protein expression, NMR,
uorometric assays, ITC. See DOI: 10.1039/d1sc01410b
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report on a supramolecular caged CB[8] capable of undergoing
light-triggered release using a photo-cleavable bivalent FGG-
cage (Fig. 1). We illustrate its application for subsequent acti-
vation in downstream processes, such as uorescent dye
aggregation assembly and control over enzyme activity. For the
light triggered CB[8] to work in unison with downstream protein
effectors, the affinities were balanced such that the cage
completely blocks the CB[8] prior to the stimulus, yet releases
the host molecule aer the stimulus for efficient binding to the
proteins. Central to this design is the usage of a bivalent FGG-
cage with a high affinity for CB[8] through an enhanced effec-
tive molarity24 and the usage of FGG-tagged enzymes, whose
affinity for dimerization is enhanced upon two-fold binding to
the CB[8] platform.25 In our design, two FGG-epitopes are
bridged by an amino acid linker including two photo-cleavable
amino-3-(2-nitrophenyl)propanoic acid (Anp) residues (1).26,27
Upon irradiation with 365 nm light, the amino acid backbone
cleaves, resulting in two monovalent FGG-peptide fragments
that have signicant lower overall affinity, releasing the CB[8] to
interact with other guests such as the FGG-tagged caspase-9.
Results and discussion
The peptide cages were synthesized using Fmoc-based solid-
phase peptide synthesis. The two Anp residues are positioned
within the backbone of the supramolecular cage, to ensure
a high photo-cleavage yield. The second FGG-tag was intro-
duced from a branching lysine side chain, temporarily pro-
tected via a 4-methoxytrityl group. Aer assembly of the
complete backbone, this protecting group was selectively
removed, followed by the subsequent installation of the two
FGG-tags. The two N-terminal phenylalanines are 11 amino
acids apart, giving it sufficient exibility to encompass the CB[8]
and bind the host in a bivalent manner without excessive strain
on the peptide backbone (ESI Fig. S1†). As a control, also a non-
cleavable variant (2) was made that has b-alanines instead of the
Anp-residues.
Fig. 2 Cage-cleavage by UV-light. (A) Cleavage followed over time by
LC-MS of 1 and 2 (control) with an internal standard as reference. The
cleavage half-life (s1/2) ꢀ 40 s. The presence of CB[8] has no significant
influence on the cleavage rate. (B) UV-Vis absorption spectra of 1 upon
UV-radiation at different time points. Inset: absorbance at 230 nm over
time. (C) 1H-NMR spectra of peptide cage 1 (500 mM) before (blue) and
after (red) UV-radiation in D2O. Spectrum between 5 and 3.5 ppm is
omitted for clarity (see ESI Fig. S3† for full spectra).
The photo-cleavage of FGG-cage 1 was investigated using LC-
1
MS, UV-Vis and H-NMR. The peptide cage was mixed with an
internal standard as reference for LC-MS quantication and
samples were analyzed at different time points of exposure to
irradiation with UV light (365 nm) (Fig. 2A). Within 5 minutes of
UV light exposure most of cage 1 was cleaved, with a half-life of
around 40 seconds. The control b-alanine-cage 2 remained
intact, also aer prolonged exposure. Gratifyingly, the presence
of CB[8] had no signicant inuence on the cleavage rate.
The photo-cleavage of 1 was also followed by UV-Vis spectra
at various time points during irradiation (Fig. 2B). Upon radi-
ation, the characteristic peak at 260 nm of the ortho-nitro group
rapidly disappears, while two new peaks at 230 and 300 nm
appeared, indicating the successful photo-conversion. The
presence of CB[8] had no signicant inuence on the optical
To verify complexation of the bivalent cage 1 with CB[8], 1H-
NMR complex formation studies were performed. The aromatic
signals belonging to the (N-terminal) phenylalanines, shied
up-eld upon addition of CB[8] from 7.2 to 6.4 ppm, indicative
of the de-shielding inside the CB[8] cavity for FGG-peptides (ESI
Fig. S6†).5 Binding studies were performed to explore whether
photo-cleavage of cage 1 would result in differential binding
properties between both chemical states. Initial experiments
were performed using a uorometric assay via the displacement
of acridine orange (AO) from the cavity of CB[8]. The AO dye is
1
properties of 1 (ESI Fig. S4†). H-NMR spectra of 1 before and
aer UV radiation (Fig. 2C and ESI Fig. S5†) revealed that aer
10 minutes of irradiation the signals characteristic of the Anp
groups had all disappeared.
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auto-quenched inside the cavity of CB[8], and becomes uo- displace AO from the CB[8] cavity (IC50 62 mM). In comparison,
rescent again when another guest displaces it from the cavity the bivalent FGG-cages 1 and 2 had a much stronger affinity and
(Fig. 3A).28 The FGG-cages 1 and 2 were titrated to a solution of displayed AO with IC50's of 400 and 500 nM respectively. Upon
pre-complexed CB[8] and AO. In previous studies,28 a relative UV irradiation of the titration plate, the affinity of the light-
high concentration of the FGG-mono peptide was required to sensitive 1 weakened towards the value of the monovalent
Fig. 3 (A) Schematic representation of fluorometric assay. (B) Titration of bivalent cage 1 into a solution of acridine orange (1 mM) and CB[8] (1 mM)
and titration with bivalent control 2 in HBS (10 mM HEPES, pH 7.4, 150 mM NaCl) at various time points after UV-radiation, performed in triplicate;
error bars represent the standard deviation. (C) Apparent KD determined by isothermal titration calorimetry before and after photo cleavage of
cage 1 and control 2; error bars represent the confidence interval of the fit. (D) ITC trace of 1 before and after UV-radiation; (see ESI Table S1† for
an overview of the isothermal titration calorimetry characteristics).
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peptide (Fig. 3B on the le, blue to red). In contrast, the IC50 of
non-cleavable 2 was not affected (Fig. 3B on the right).
Isothermal titration calorimetry (ITC) studies were per-
formed to provide insights into the thermodynamic parame-
ters of the supramolecular caging and light induced uncaging
(Fig. 3C, D; ESI Table S1; Fig. S7 and S8†). Titration of FGG-
cage 1 to CB[8] resulted in a dissociation constant (KD) of
22 nM. Irradiation of 1 resulted in a 100-fold weakening of the
apparent KD to 2.4 mM. Interestingly, the DH of the binding
interactions before and aer UV-light induced cleavage
remained similar and the lowering of the DG results from the
change of the DS. These results strongly support a binding
mode that is molecularly very similar in both states (same
molecular interactions), but that has changed from bivalent to
monovalent.29 In line with previous results, the affinity of the
non-cleavable cage 2 was not affected signicantly upon UV
irradiation.
With the light-controllable CB[8] in hand, we investigated its
utility as downstream control element on enzyme activity.
Protein dimerization is a ubiquitous mechanism to regulate
protein activity in a broad range of biological processes
including apoptosis. Molecular control over these processes is
critical to elucidate and perturb the molecular mechanisms of
the proteins involved.30 Caspase-9 (casp-9) is a critical cysteine
protease and initiator of the apoptosis pathway and is respon-
sible for cleaving proteins at specic aspartate residues. In the
cell, caspase-9 exists primarily in its inactive monomeric form,31
becoming activated only upon induced dimerization by regu-
latory factors as part of controlled cell death.32 For example
extensive UV-damage to the DNA33 leads to mitochondrial
release of cytochrome-C which triggers the assembly of the
apoptosome that acts a scaffolding platform that recruits
multiple copies of caspase-9, initiating apoptosis.34 This scaf-
folding can be mimicked by bringing two caspase-9 proteins in
close proximity by virtue of dual binding of CB[8] to N-terminal
FGG-tags on the caspase-9.15 The intrinsic affinity between two
caspase monomers (KD ꢀ 100 mM)35 causes the caspases to act as
a supramolecular bidentate.28 As a consequence, the caspases
primarily form active dimers, even when CB[8] is in 10-fold
excess over the caspase monomers. We employed a proteolytic
activity assay for caspase-9 using the synthetic substrate Ac-
LEHD-AFC (where AFC is 7-amino-4-(triuoromethyl)
coumarin), that becomes uorescent aer cleavage by
caspase-9 (Fig. 4A). Kinetic traces were recorded by following
the uorescence over time. The optimal concentration of CB[8]
for caspase-9 activation, independent of the bivalent cage, was
determined to be 10 mM.
Fig. 4 (A) Light-triggered dimerization of caspase-9 monomers by
CB[8] induces caspase-9 activity, resulting in the conversion of
Ac-LEHD-AFC into a fluorescent product. (B) Time-activity traces of
increasing molarity corresponding to cleavage of synthetic substrate
Ac-LEHD-AFC (200 mM) by FGG-caspase-9 (1 mM) in 100 mL activity
assay buffer (20 mM Na2HPO4, 150 mM NaCl, 1 mM EDTA, 2 mM TCEP,
pH 7.0, RT). 10 mM 1 or 2 and 10 mM CB[8] was used; the ‘minimum’
contains only casp-9 the ‘maximum’ only casp-9 and CB[8].
Measurements were performed in triplicate. (C) Initial rate (v0) of
substrate cleavage in mM minÀ1
.
The UV-light activated caspase-9 reaches a comparable level
activity as the CB[8]-only positive control (13 mM minÀ1). As
expected, reference inhibitor 2 effectively blocked caspase-9
activity, also aer UV-light treatment. Even though the
caspase-9 monomers feature a similar FGG-tag as the mono-
valent fragments of the inhibitor, the intrinsic interaction
between two caspase monomers results in self-sorting and
hetero complexation in the CB[8] only starts to play a more
dominate role when concentrations exceed the effective
molarity.36
We screened for the optimal concentration of cage 1 that had
the highest on/off ratio before and aer UV cleavage (ESI
Fig. S10†). We then investigated the effect of photo-cleavage of
cage 1 in comparison with caspase with and without CB[8] as
maximum and minimum effect (Fig. 4B). Without UV radiation
the caspase-9 activity in the presence of CB[8] and cage 1 is
similar to the characteristic caspase-9 background activity,
around 1 mM minÀ1 (Fig. 4C). Aer irradiation the activity
increases dramatically to an initial slope of 11 mM minÀ1 (the
rate decreases over time due to depletion of the AFC substrate).
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In conclusion, a versatile photo-triggered release system for CB
[8] is presented, as well as its application for subsequent
downstream modulation of uorescent guest binding and
binding and activation of caspase-9 enzymes. The UV-sensitive
cage exhibits a 100-fold higher affinity in its intact bivalent,
inhibitory mode as compared to its cleaved state. The UV-
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highly favourable for supramolecular chemical biology appli-
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concentration regime.37 Light-activatable supramolecular
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system at hand, for example by varying the concentration of
cage and CB[8] or by tuning of the affinities by utilizing stronger
or weaker binding FGG analogues.39 Similarly, the conceptual
approach of a cleavable bivalent inhibitor, as presented here,
can be extended to other types of triggers, which are currently
being explored.
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Author contributions
P. J. d. V. and T. v. d. H. performed experiments, P. J. d. V. and L.
B. conceptualized the studies, all authors contributed to writing
the manuscript.
Conflicts of interest
´
21 M. A. Romero, N. Basılio, A. J. Moro, M. Domingues,
´
J. A. Gonzalez-Delgado, J. F. Arteaga and U. Pischel, Chem.–
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This research was funded by the Netherlands Organization for
Scientic Research (NWO) through Gravity program
024.001.035 and VICI grant 016.150.366. We thank Bas Rosier
for the discussions about caspases.
´
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