Supramolecular Assays for Mapping Enzyme Activity by Displacement-Triggered Change in Hyperpolarized 129Xe Magnetization Transfer NMR Spectroscopy

Article abstract of DOI:10.1002/anie.201507002

Reversibly bound Xe is a sensitive NMR and MRI reporter with its resonance frequency being influenced by the chemical environment of the host. Molecular imaging of enzyme activity presents a promising approach for disease identification, but current Xe biosensing concepts are limited since substrate conversion typically has little impact on the chemical shift of Xe inside tailored cavities. Herein, we exploit the ability of the product of the enzymatic reaction to bind itself to the macrocyclic hosts CB6 and CB7 and thereby displace Xe. We demonstrate the suitability of this method to map areas of enzyme activity through changes in magnetization transfer with hyperpolarized Xe under different saturation scenarios. The supramolecular recognition properties of cucurbiturils are exploited in a modified signal-transfer approach in Xe NMR spectroscopy. Magnetic resonance imaging of enzymatic reactions was possible in this way by displacement of hyperpolarized ¹²⁹Xe.

Full text of DOI:10.1002/anie.201507002

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Angewandte
Communications
International Edition: DOI: 10.1002/anie.201507002
German Edition: DOI: 10.1002/ange.201507002
Xenon MRI
Supramolecular Assays for Mapping Enzyme Activity by
129
Displacement-Triggered Change in Hyperpolarized Xe
Magnetization Transfer NMR Spectroscopy
Matthias Schnurr, Jagoda Sloniec-Myszk, Jçrg Dçpfert, Leif Schrçder,* and Andreas Hennig*
Abstract: Reversibly bound Xe is a sensitive NMR and MRI
reporter with its resonance frequency being influenced by the
chemical environment of the host. Molecular imaging of
enzyme activity presents a promising approach for disease
identification, but current Xe biosensing concepts are limited
since substrate conversion typically has little impact on the
chemical shift of Xe inside tailored cavities. Herein, we exploit
the ability of the product of the enzymatic reaction to bind itself
to the macrocyclic hosts CB6 and CB7 and thereby displace
Xe. We demonstrate the suitability of this method to map areas
of enzyme activity through changes in magnetization transfer
with hyperpolarized Xe under different saturation scenarios.
a remedy, we introduce herein a carefully designed approach
to MRI enzyme activity mapping by hyperpolarized Xe NMR
spectroscopy, in which we 1) use cucurbit[n]uril (CB) hosts
with n = 6 (CB6) and n = 7 (CB7) as contrast agents,
2) rationally exploit the molecular recognition properties of
these hosts, and 3) for the first time apply an optimized
magnetization transfer (MT) experiment for the Xe MRI of
enzyme activity (Figure 1).
M
agnetic resonance imaging (MRI) with hyperpolarized
xenon (129Xe) holds promise as a minimally invasive tech-
nique for diagnosis based on disease-specific markers at low
concentrations,[1] in particular in combination with contrast
agents based on macrocyclic supramolecular hosts[2] and
signal amplification through chemical exchange saturation
transfer (Hyper-CEST).[2c] This led to highly sensitive Xe
MRI applications illustrating intracellular labeling[1d,3] and
the specific detection of cell surface receptors via chemically
modified Xe host molecules.[1c] As a next step, following
reactions with high sensitivity, for example, enzyme activity, is
desirable.[4] This would make it possible to image areas of
altered physiological processes associated with major dis-
eases[5] and includes a pathway for amplifying the sensor
signal.[4b]
Unfortunately, reported approaches based on substrate
labeling with a Xe host are limited, because the chemical shift
difference of the Xe NMR frequency before and after the
enzymatic reaction is comparably small (Dd ꢀ 0.5 ppm).[4b] As
Figure 1. Displacement-based approach for 129Xe biosensors (top),
response in Xe saturation transfer spectra (middle), and the difference
spectrum before and after displacement, which gives the optimum
saturation frequency for MT imaging.
[*] Dr. A. Hennig[+]
Department of Life Sciences and Chemistry
Jacobs University Bremen
Campus Ring 1, 28759 Bremen (Germany)
E-mail: a.hennig@jacobs-university.de
Dipl.-Chem. J. Sloniec-Myszk[+]
BAM Bundesanstalt für Materialforschung und -prüfung
Richard-Willstätter-Strasse 11, 12489 Berlin (Germany)
Dr. M. Schnurr,[+] Dr. J. Dçpfert, Dr. L. Schrçder
ERC Project BiosensorImaging
Leibniz-Institut für Molekulare Pharmakologie (FMP)
Campus BerlinBuch
CBs are very stable and nontoxic macrocyclic hosts,[6]
which have been explored in Xe NMR experiments,[2d–h]
although their real potential has been underrated; for
example, the utility of CB6 for Xe Hyper-CEST has been
demonstrated only recently.[7] Their excellent molecular
recognition properties in water have, however, led to
numerous applications,[8] in particular for the detection of
enzyme activity through optical sensing.[9] In this approach,
the substrate and product of an enzymatic reaction bind to
a host with different affinities, such that the enzyme trans-
forms a weakly binding substrate into a strongly binding
product. The concomitant displacement of a fluorescent dye
changes the spectroscopic properties of the dye, which is used
Robert-Rçssle-Strasse 10, 13125 Berlin (Germany)
E-mail: lschroeder@fmp-berlin.de
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201507002.
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to follow the reaction. Our idea was that in a reaction mixture
with Xe, its gradual displacement from the host as the
enzymatic reaction progresses could be followed by overall
changes of Xe saturation transfer spectra from the Xe–host
complex (Figure 1).
To demonstrate the transferability of supramolecular
enzyme assays to MRI, we first chose the strongly Xe-binding
homologue CB6 as the contrast agent and lysine decarbox-
ylase (LDC) as the enzyme. LDC converts the weakly binding
substrate l-lysine (Lys) into the strongly binding product
cadaverine (Cad) and has a key role in tumor growth and
inflammatory processes.[10]
As an initial control and for comparison with the Xe NMR
studies, we developed a fluorescent assay with CB6 and LDC
using a putrescine derivative of 1-aminonaphthalene-5-sul-
fonic acid as fluorescent dye, which responds to CB6 binding
with an increase in fluorescence.[11] The binding constant was
determined by fluorescence titration as (4 Æ 1) ” 104 mÀ1 in the
NH4OAc buffer (Figure SI-1), which lies between 4.3 ” 107 mÀ1
in 1 mm HCl (pH 3.0) and 2.5 ” 103 mÀ1 in 50 mm NaOAc
(pH 5.5).[11] The higher binding constant in 1 mm HCl results
most likely from an increased protonation of the dye in the
more acidic solution and binding in 50 mm NaOAc is lower
because of the competitive binding of Na+ to CB6.
The binding constant of Cad to CB6 (109 to 1010 mÀ1 [9e] is
)
clearly higher, and the binding of Lys is too weak to efficiently
compete with the dye at relevant concentrations (Figure SI-
2). Consequently, dye displacement and fluorescence
decrease were expected upon progress of the enzymatic
reaction, which was in fact observed (Figure SI-3). The
fluorescence traces gave a turnover number of 74 nmol
minÀ1 mgÀ1 at 258C and 300 mm Lys. These fluorescence
results 1) demonstrate the principle feasibility of the enzyme
assay under these conditions, 2) exclude potential undesirable
interactions between CB6 and the enzyme, and 3) ensure that
the enzymatic reaction completes within an acceptable time
to be detected by Xe NMR spectroscopy.
Figure 2. a) Details and overview (inset) of Hyper-CEST spectra (Xe
gas as d=0 ppm reference). Details: only CB6 (dark red), with
15 mgmLÀ1 LDC (light blue), with LDC and 100 mm Lys (dark blue),
with 50 mm Cad (pink), and with 100 mm Lys (yellow). Saturation
parameters: B1 =16 mT, tsat =4 s. b)–e) Hyper-CEST MRI (on-resonant
(b) and off-resonant saturation (c), Hyper-CEST effect (d), and two-
compartment setup (e)). In addition to Lys, 15 mgmLÀ1 LDC was
present only in the inner compartment, which reduced its Hyper-CEST
effect (d) significantly. Conditions: 20 mm CB6, 10 mm NH4OAc, 258C,
[Xe]ꢀ975 mm. No. of averages: 6. Total acquisition time: ca. 4.6 min.
Next, we explored the reporter system with hyperpolar-
ized Xe NMR spectroscopy and MRI. The Hyper-CEST
spectrum of CB6 (Figure 2) displayed resonances at d =
193 ppm ascribed to direct saturation of free dissolved Xe,
and at d = 105 ppm originating from saturation transfer via
Xe encapsulated in CB6,[12] demonstrating that the exchange
kinetics of Xe are compatible with Hyper-CEST.[7] After
addition of LDC to the mixture containing only CB6, the
Xe@CB6 resonance remained unchanged, indicating no
undesirable interaction of CB6 or Xe with the enzyme in
line with the fluorescence results. The Xe@CB6 resonance
vanished completely after addition of 100 mm Lys and
incubation for 30 min. This is due to decarboxylation of Lys
by LDC producing strongly binding Cad, which efficiently
suppresses the saturation transfer by occupying the CB6
cavity. This was also observed when Cad was added to
a solution containing CB6 only. Another control, in which Lys
was added to a CB6-containing solution, showed a small
response, in which the CEST signal became slightly smaller
and shifted to lower ppm values. This is likely due to weak
binding of Lys as also observed by fluorescence (Figure SI-2).
These results clearly show the applicability of Hyper-CEST to
detect enzyme activity through competitive macrocycle
occupation and allow Hyper-CEST MRI in solution, where
only the area not containing LDC displayed a high Hyper-
CEST effect (Figures 2d and SI-4).
When we attempted to transfer this approach to cell
lysates, no clearly resolved Xe@CB6 resonance was observed
at 20 mm CB6, but the CEST response at d = 193 ppm was
significantly broadened as previously observed in blood
plasma (Figure SI-6).[7a] This is attributed to two factors:
1) CB6 is partially occupied by more competitive guests and
2) Xe now additionally interacts with other potential binding
partners, for example, proteins and lipids, with a mutual
influence on the exchange kinetics causing line broadening
per se,[4i] such that it becomes impractical to detect the
presence of CB6 by saturating at d = 105 ppm (Figure SI-6).
As a modified strategy, application of saturation pulses
slightly off-resonant from an exchange-broadened bulk signal
1
is, however, a well-established method in H NMR spectros-
copy called magnetization transfer (MT)[13] and has been
used, for example, to follow myelination of neuronal cells in
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vivo.[14] We therefore reasoned that a similar approach may be
applied to Xe NMR spectroscopy. To further reinforce the
exchange-mediated line broadening, we considered the yet
unexplored CB7 with its larger cavity, which should facilitate
fast exchange. Surprisingly, CB7 gave clear Hyper-CEST
spectra in pure H2O and a well-defined Xe@CB7 resonance at
d = 100 ppm (Figure SI-7), which can be used for Hyper-
CEST MRI (Figure SI-8) and vanished completely upon
addition of Cad. Most importantly, the dissolved Xe reso-
nance at d ꢀ 190 ppm was further broadened compared to the
CB6 spectra confirming the anticipated faster Xe exchange
with CB7.
Fluorescence-based LDC assays were originally estab-
lished with CB7,[9a] such that no control was required and
imaging of LDC activity using Hyper-CEST MRI was directly
pursued. As for CB6, Hyper-CEST spectral resolution with
CB7 is expected to decrease in cell lysates rendering the
contrast of Figure SI-8 unachievable under such conditions.
To identify the ideal frequency of the saturation pulse for MT,
CB7 in a lysate of macrophage cells before and after addition
of LDC was measured (Figure 3a). Therein, the original
CEST response of CB7 at d ꢀ 108 ppm is indeed hard to
identify due to the fast exchange, but the change in the direct
saturation response of the bulk Xe is well pronounced. This is
also reflected in the difference spectrum, in which the curve
has a maximum at d ꢀ 178 ppm. This frequency was thus used
for imaging with optimized MT conditions, which gave
a strong MT ratio (MTR) in the area not containing LDC
and weak signal changes in the other compartment (Fig-
ure 3b–d). The MTR image (Figure 3d) clearly demonstrates
that the enzyme-active compartment can be easily identified
through a pronounced decrease in the MTR signal. The
superior performance of the MT experiment without a con-
ventional CEST pool resonance frequency becomes most
apparent in direct comparison with MR imaging by conven-
tional Hyper-CEST, which gave much less image contrast
(Figure SI-10). Moreover, the typical effort to avoid spillover
effects and preserve specificity for the selected frequency by
avoiding strong saturation pulses for fast exchanging systems
is not an issue for MT experiments.
Figure 3. a) Hyper-CEST spectra of CB7 in the presence of lysed
macrophage cells and 700 mm Lys before (yellow) and 45 min after
adding 50 mgmLÀ1 LDC (dark blue), and the difference spectrum (light
blue). b)–e) Transversal Xe MT MRI of two nested NMR tubes with
50 mgmLÀ1 LDC in the inner tube only (incubation time 45 min).
Saturation at b) d=278 ppm and c) d=178 ppm, and d) resulting
MTR image. e) Schematic representation of the setup. Hyper-CEST
images were recorded using a RARE pulse sequence with an in-plane
resolution of 0.14 mm2 and 20 mm slice thickness. Saturation param-
eters: B1 =20 mT, tsat =4 s. Conditions: 500 mm CB7, 10 mm NH4OAc
(pH 6.0), 228C, 25”106 lysed macrophage cells/mL (ca. 9 mgmLÀ1
protein), [Xe]ꢀ975 mm. No. of averages: 10. Total acquisition time: ca.
7.5 min.
These results show that the concept of a dynamic equi-
librium, in which Xe is gradually displaced from its host as an
enzymatic reaction progresses, can be used to map enzyme
activity by Xe MRI. The decreased spectral resolution in
a complex cellular lysate with CB6 inspired the application of
MT in combination with the first-time use of CB7 as an even
faster exchanging host for Xe NMR experiments. This led to
improved signal contrast compared to that from conventional
Hyper-CEST acquisition and shows that current challenges in
molecular imaging as well as biological applications of
supramolecular systems can be accounted for by a thoughtful
experimental design. Future developments include the exten-
sion to other enzyme classes as previously demonstrated for
optical sensing,[9] as well as improvements inspired by
ratiometric probes in which a reference signal provides
direct information about the local biosensor concentration,[15]
and by developments in supramolecular chemistry, such as the
first demonstration of fluorescent supramolecular displace-
ment assays in living cells.[16]
Acknowledgements
This work was supported by the BAM Federal Institute for
Materials Research and Testing, the DFG (DFG Grant HE-
5967/2-1), and the European Research Council under the
European Communityꢀs Seventh Framework Program (FP7/
2007–2013)/ERC grant agreement no. 242710. We thank
Prof. Dr. W. M. Nau for a gift of the dye used in the
fluorescence studies and Dr. U. Resch-Genger for access to
the spectroscopic facilities of the BAM.
Keywords: cucurbituril · enzymes · NMR spectroscopy ·
supramolecular assays · xenon MRI
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Received: July 28, 2015
Published online: October 1, 2015
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