A "Smart" 129Xe NMR biosensor for pH-dependent cell labeling

Article abstract of DOI:10.1021/jacs.5b01938

Here we present a "smart" xenon-129 NMR biosensor that undergoes a peptide conformational change and labels cells in acidic environments. To a cryptophane host molecule with high Xe affinity, we conjugated a 30mer EALA-repeat peptide that is α-helical at pH 5.5 and disordered at pH 7.5. The ¹²⁹Xe NMR chemical shift at room temperature was strongly pH-dependent (Δδ = 3.4 ppm): δ = 64.2 ppm at pH 7.5 vs δ = 67.6 ppm at pH 5.5, where Trp(peptide)-cryptophane interactions were evidenced by Trp fluorescence quenching. Using hyper-CEST NMR, we probed peptidocryptophane detection limits at low-picomolar (10^-11 M) concentration, which compares favorably to other NMR pH reporters at 10^-2-10^-3 M. Finally, in biosensor-HeLa cell solutions, peptide-cell membrane insertion at pH 5.5 generated a 13.4 ppm downfield cryptophane-¹²⁹Xe NMR chemical shift relative to pH 7.5 studies. This highlights new uses for ¹²⁹Xe as an ultrasensitive probe of peptide structure and function, along with potential applications for pH-dependent cell labeling in cancer diagnosis and treatment.

Full text of DOI:10.1021/jacs.5b01938

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Article
A “Smart” 129Xe NMR Biosensor for pH-Dependent Cell Labeling
Brittany A. Riggle, Yanfei Wang, and Ivan J. Dmochowski
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b01938 • Publication Date (Web): 07 Apr 2015
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A “Smart” 129Xe NMR Biosensor forpH-Dependent Cell Labe-
ling
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Brittany A. Riggle, Yanfei Wang, and Ivan J. Dmochowski*
Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United
States
ABSTRACT:Here we present a “smart” xenon-129 NMR biosensor that undergoes a peptide conformational change and labels
cells in acidic environments. To a cryptophane host molecule with high Xe affinity, we conjugated a 30mer EALA-repeat peptide
that is alpha-helical at pH 5.5 and disordered at pH 7.5. The 129Xe NMR chemical shift at rtwas strongly pH-dependent (ꢀδ = 3.4
ppm): δ = 64.2 ppm at pH 7.5 vs. δ = 67.6 ppm at pH 5.5 where Trp(peptide)-cryptophane interactions were evidenced by Trp fluo-
rescence quenching. Using Hyper-CEST NMR, we probed peptido-cryptophane detection limits at low-picomolar (10-11 M) concen-
tration, which compares favorably to other NMR pH reporters at 10-2-10-3 M. Finally, in biosensor-HeLa cell solutions, peptide-cell
membrane insertion at pH 5.5 generated a 13.4 ppm downfield cryptophane-129Xe NMR chemical shift relative to pH 7.5 studies.
This highlights new uses for 129Xe as an ultrasensitive probe of peptide structure and function, along with potential applications for
pH-dependent cell labeling in cancer diagnosis and treatment.
(MPIOs),26ultrasmall dextran-coated iron oxide particles (US-
PIOs),27and superparamagnetic iron oxide (SPIO) glycol chi-
INTRODUCTION
tosan.9,28Pioneering studies by the Tsourkas lab and others
Magnetic resonance imaging (MRI) and spectroscopy
(MRS) are versatile and commonly employed techniques for
the diagnosis and staging of disease.1 The development of
targeted and stimuli-responsive (i.e., “smart”) contrast agents
improves the capabilities of MRI/MRS for molecular imag-
ing.2 Targeted therapeutic and diagnostic imaging techniques
are typically directed to one or more receptors associated with
a disease state. However, in cancer, as a result of large natural
variations between cells and the heterogeneous nature of tissue
within a tumor, there is also need for more general biomark-
ers.3,4For example, hypoxia and acidification occur in 90% of
tumors and are key microenvironmental factors in progression
and treatment resistance in solid tumors.5,6The tumor microen-
vironment is acidified to levels approaching pH 6.0from a
normal pH of 7.4, which increases metastasis, mutation rate,
and cell viability.4,7-9 Therefore, being able to identify cells in
acidic environments has practical importance in the design of
cancer therapies and controlled-release drug delivery mechan-
isms.8 Additionally, acidic environments can mitigate the effi-
cacy of weakly basic chemotherapeutics such as doxorubicin,
necessitating methodologies to probe extracellular pH (pHe).10
Here, we present an ultrasensitive xenon-based MR contrast
agent that can identify and label cell populations based on
their acidic pHe.
have explored pH-dependent cell labeling with these reagents,
but there remain significant limitations, e.g.,pH-responsive
SPIO typically requiressignificant incubation time (~24 h)
between administration and imaging.9More generally, applica-
tions withconventional MRI contrast agents are limited by low
detection sensitivity on a per-monomer basis (i.e., low
mM).One strategy for improving NMR detection sensitivity
involves the use of exogenously supplied “hyperpolarized”
(hp) nuclei, e.g., 129Xe, 13C, and 3He, with magnetic spin reser-
voirs that exceed the normal Boltzmann distribution by several
orders of magnitude. Xe binds void spaces in materials,29 pro-
teins,30and spores31but shows highest affinity and useful ex-
change kinetics for a class of host molecules known as crypto-
phane.32-39 Perturbation of the large (~42 Å3 volume) 129Xe
electron cloud can produce significant nuclear magnetic chem-
ical shift changes and results in a nearly 300 ppm chemical
shift window when bound to different cryptophanes in
aqueous solution.33,40,41
Based on these principles, we and others have developed
129Xe-cryptophane NMR biosensors42 for the sensitive detec-
tion of protein receptors,42-46 enzymes,47 DNA,48 and metal
ions in solution.49 In one proof-of-concept experiment, Ber-
thaultet al. decorated cryptophane with sixcarboxylic acids to
create a pH reporter: unique chemical shifts were measured
over the pH 3.5-5.5 range with a total ꢀδ of 3.55 ppm.50 How-
ever, solubility issues precluded work near neutral pH.
A variety of pH-responsive MR contrast agents have been
designed previously, including Gd complexes,11,12tunable mi-
celle-encapsulated polymers and 19F compounds,13,14and CEST
agents,15-21 among others.22,23Theseprobes enable measure-
ments of solution pH but do not selectively label cells in acidic
environments, e.g., as needed for identifying small populations
of cancer cells or performing cell tracking experiments. In
parallel efforts over the past two decades,many strategies have
been developedfor labeling cellswith MRI contrast agents such
as membrane-targeting Gd chelates,24monocrystalline iron-
oxide particles (MIONs),25micron-sized iron-oxide particles
Recent studies have moved xenon biosensing from buffer
solutions to lipid membrane suspensions and living cells. The
Pines lab developed ultrasensitive methods for detecting cryp-
tophane in solution using hp129Xe chemical exchange satura-
tion transfer (Hyper-CEST) NMRspectroscopy.51 They also
discovered that cryptophane in association with a dilute sus-
pension of sub-micron Intralipid vesicles yielded a 129Xe NMR
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peak that was shifted ~10 ppm downfield from the aqueous
ically relevant pH range.62As the glutamates are protonated,
129Xe-cryptophane peak;52 similar results were later obtained
with different lipid compositions.53The Schröder labsubse-
quently performed Hyper-CEST NMR spectroscopy and imag-
ing studies in cells loaded with lipophilic cryptophane and
found a similar 9-11 ppm downfield chemical shift change,
likely due to membrane association.54-56These studies highlight
the large 129Xe NMR chemical shift changes that can be
achieved by engineering cryptophane-lipid membrane interac-
tions.
the EALA-repeat peptide becomes more helical and hydro-
phobic, and it inserts into lipophilic membranes.63 This pH-
dependent membrane insertion has been used in living cells to
facilitate endosomal escape of both nanocapsule and gene
payloads.64-66 Thus, by appending cryptophane to a membrane-
inserting EALA peptide, we endeavored to generate a xenon
contrast agent capable of being “activated” in acidic cell envi-
ronments to label cell membranes and give large 129Xe NMR
chemical shift changes. We based the design on our previously
reported tripropargyl cryptophane-A derivative (with two cyc-
lotriveratrylene units tethered by three ethylene link-
ers),67which should allow facile attachment of a pH-responsive
peptide and also two water-solubilizing moieties (Scheme 1)
to mitigate the potential for cryptophane aggregation.55
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Building on these examples, we set out to develop an ultra-
sensitive129Xe NMR contrast agent for labeling cells in acidic
microenvironments.Recent work from our laboratory57 and
elsewhere58,59 has demonstrated nM-to-pM detection of water-
soluble cryptophane using Hyper-CEST NMR spectroscopy.
Most recently, Witte et al. demonstrated effective contrast via
Hyper-CEST by site-specific labeling of cell-surface glycans
with nM concentrations of cryptophane.60Thus, Hyper-CEST
NMR should enable ultrasensitive detection of cryptophane-
labeled cells that reside in acidic environments and differentia-
tion from “normal” cells residing in neutral pH environments,
provided that cryptophane-cell interactions can be modulated
over the pH range 5.5-7.5.
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RESULTS AND DISCUSSION
Synthetic Procedures
Scheme 1 shows the synthesis of the water-soluble EALA-
cryptophane (WEC) pH-responsive biosensor5, the details of
which are provided in the Supporting Information. Briefly, the
synthesis of tripropargyl cryptophane 1 was performed in six
non-linear steps with modifications to previously published
methods,68 with an overall yield of 6.4% (Scheme S1). The
yield for the five linear steps was 9.9%. The azido-EALA-
repeat peptide 2 was prepared with standardFmoc synthetic
methods (Figures S1-S2). The peptide was attached to the
cryptophane via copper(I)-catalyzed [3+2] azide-alkyne cyc-
loaddition (CuAAC) to form 3.69,70The mono-peptide crypto-
phane was achieved in preference by controlling reaction stoi-
chiometry. The resulting triazole-hexyl spacer kept the peptide
in close proximity to the 129Xe nucleus while minimizing steric
clashes with cryptophane during conjugation. Formation of
compound 3 was confirmed by MALDI-MS and the yield
quantified by analytical reverse-phase HPLC to be 60-80%
(Figures S3-S4). A solubilizing linker, 3-azidopropionic acid
4, was synthesized in one step from the commercially availa-
ble β-propiolactone (see Supporting Information)47,71 and
reacted with crude 3 via a second CuAAC. Starting from tri-
propargyl cryptophane 1, WEC5 was isolated in ~40% yield
after sequential CuAAC reactions with 2 and 4 and HPLC
purification to remove unreacted EALA peptide and unreacted
cryptophane (Figures S5-S6).
Scheme 1. Synthesis of water-soluble EALA-cryptophane
(WEC): a. 1 (1 eq), 2 (1 eq), CuSO4 (1 eq), TBTA (5 eq), 2,6-
lutidine (1 eq), NaAsc (10 eq), 12 h; b. 3 (crude), 4 (10 eq),
CuSO4 (1 eq), TBTA (5 eq), 2,6-lutidine (1 eq), NaAsc (10
eq), 12 h.
We hypothesized that modifying cryptophane with an
EALA-repeat
peptide,
WEAALAEALAEALAEHLAEALAEALEALAA,61 should
modulate129Xe NMR chemical shift in response to physiologic
pH changes. By our strategy, 129Xe NMR chemical shift
should vary both from pH-dependent peptide conformational
changes as well as pH-dependent peptido-cryptophane-cell
membrane association. The synthetic EALA-repeat peptide
was inspired originally by hemagglutinin (HA), which mem-
brane inserts in low-pH environments.61The poly-glutamic
acid nature of the EALA-repeat peptide elevates the pKa to
around 6, resulting in a conformational change from random
coil (pH 7.5) to mostly alpha helical (pH 5.5), over thisbiolog-
Figure 1.pH titrations monitored by ECD spectroscopy for a.
azido-EALA peptide and b. water-soluble EALA-cryptophane
(WEC). Samples (30 µM) were in 10 mM sodium phosphate buf-
fer over the pH range 5.5-7.5 at 298 K.
Electronic Circular Dichroism (ECD) Spectroscopy
For ECD studies, all samples of azido-peptide 2 or WEC
were prepared at 30 ꢁM concentration in 10 mM sodium
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phosphate buffer, as confirmed by UV-vis spectroscopy (pep- solvated environment near cryptophane. We note that the fluo-
tide: ε280 = 5,700 M-1cm-1, WEC: ε280 = 17,700 M-1cm-1) and
pH adjusted with 1 M HCl or 1 M NaOH. We used ECD spec-
troscopy to confirm that azido-peptide 2 maintained pH sensi-
tivity (Figure 1a):61,72,73 indeed, percent helicity increased from
25% to 67% as the pH was decreased from 7.5 to 5.5 (Table
S1). The ECD signal at pH 5.5 had pronounced local minima
at 208 and 222 nm, indicative of an alpha-helical secondary
structure. At pH 7.5, the spectrum approached a minimum at
204 nm while subsequently decreasing in negative ellipticity at
222 nm, characteristic of a more disordered state. For the
WEC (Figure 1b, Figure S7), we observed a similar increase in
EALA helicity from 36% (pH 7.5) to 61% (pH 5.5). These
data established that the peptide still undergoes a significant
conformational change when conjugated to the cryptophane.
Samples showed reproducible and reversible secondary struc-
ture changes between pH 5.5 and 7.5 (Figure S8). Interesting-
ly, WEC was more ordered at pH 7.5 than peptide alone, sug-
gesting that the cryptophane elevated the conjugated peptide
pKa. Similar pKa elevation was previously observed for the
analogoustris-propionic acid cryptophane, due to the bulky,
hydrophobic cryptophane disfavoring ionization of the nearby
propionates.35
rescence signal for the amino acid tryptophan is typically not
perturbed by pH changes in the range of 4-8,74whereasTrp
incorporated within peptidescan exhibit emission that is very
sensitive to the peptide folded state. Cryptophanefluoresces
(λmax = 313 nm) with comparable intensity to Trp, which fur-
ther blue-shifts the observed emission spectrum.35,44At all pH
values, cryptophane quenched Trp emission, as compared to
the free peptide (Figure 2c). Plots of F/F0 vs. pH (Figure 2c,
where F is the fluorescence emission at a given pH and F0 is
the maximal fluorescence emission at pH 7.5) confirmed that
cryptophane quenching increased from pH 7.5 down to pH
5.5, where cryptophane-Trp interactions were presumably
more prevalent with the relatively uncharged, alpha-helical
peptide. This analysis is in agreement to an earlier work with a
peptide-cryptophane conjugate where we examined the inte-
raction between Trp-containing peptide and cryptophane with
a temperature-dependent quenching assay and Stern-Volmer
analysis.44 These experiments revealed thatTrp(peptide)-
cryptophane complex formation resulted in loss of Trp fluo-
rescence. Previous studies identified high-affinity interactions
between C60 (an aromatic molecule with similar dimensions
and spherical shape to cryptophane) and Trp-containing pro-
teins, which also resulted in Trp fluorescence quenching and
blue-shifted emission.75-77 These results support a mechanism
by which the EALA peptide can mediate Trp-cryptophane
complex formation in WEC (Figure 2d) and result in pH-
dependent Trp fluorescence quenching. Importantly, Trp-
cryptophane pi-stacking interactions have the potential to de-
shield129Xe within the cryptophane cavity, and produce a
downfield chemical shift.78-80
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Figure 3.HP129Xe NMR spectra (average of 16 scans, line-
broadening = 60 Hz) of WEC (30 µM) in 10 mM sodium phos-
phate buffer at 300 1 K, with peak widths (FWHM) indicated in
Hz: a. pH 5.5, 211 Hz; b. pH 6.5, 317 and 214 Hz; c. pH 7.5, 154
Hz.
129Xe NMR Spectroscopy
We initially performed hp129Xe NMR studies to examine the
sensitivity of the cryptophane-encapsulated 129Xe chemical
shift to the nearby peptide conformational state. NMR samples
were identically prepared at 30 ꢁM concentrations in 10 mM
sodium phosphate buffer. Repeated trials at 300 1 K ([Xe] =
6.2 mM)81 with the WEC at pH 7.5, 6.5, and 5.5 gave repro-
ducible chemical shifts (Figure 3). A single peak was observed
at both pH 5.5 (67.6 0.5 ppm) and pH 7.5 (64.2 0.5 ppm),
with a chemical shift difference of 3.4 ppm. Interestingly, al-
though the cryptophane itself is a racemic mixture of stereoi-
somers and the EALA-repeat peptide is chiral, we did not ob-
serve a pair of diastereomeric peaks at pH 7.5 or pH 5.5 as we
Figure 2. pH titration monitored by Trp fluorescence for a. azido-
EALA peptide; b. water-soluble EALA-cryptophane (WEC); c.
plot of F/F0 for the λmax of peptide only (352 nm) and WEC (336
nm) as a function of pH; and, d. representation of alpha-helicaland
disorderedpeptide(Trp)-cryptophane interaction. Samples (30 µM)
were in 10 mM sodium phosphate buffer over the pH range 5.5-
7.5 at 298 K.
Tryptophan Fluorescence
The EALA-repeat peptide contains a single N-terminal tryp-
tophan that we hypothesized should provide a useful local
probe of peptide conformation, as well as peptide-cryptophane
interaction. Fluorescence studies (λex = 280 nm) with peptide 2
demonstrated blue-shifted and somewhat quenched Trp emis-
sion with decreasing pH: 352 nm (pH 7.5) to 343 nm (pH 5.5),
Figure 2a. Trp maximum emission wavelength for the WEC
decreased from 336 nm to 322 nm over the same pH range
(Figure 2b), which was considerably blue-shifted relative to
peptide 2alone, consistent with the Trp experiencing a less
reported
for
previous
peptide-cryptophane
xenon
biosensors44,45and has been seen for various racemic xenon
biosensors complexed to protein active sites.49 We hypothesize
that the two diastereomers provide a very similar environment
for the bound xenon atom, and produce what appears to be a
single 129Xe NMR peak at both pH values. In this case, the
inclusion of solubilizing propionates promotes open, xenon-
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binding conformations of the cryptophane, regardless of pep-
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tide conformation.Conversely, the equal-intensity peaks ob-
served at pH 6.5 (δ = 67.0 and 64.4 ppm) may result
fromhp129Xe experiencing very different environments within
the two WEC diastereomers.
Hyper-CEST 129Xe NMR
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To improve detection sensitivity ofWEC over direct detec-
tion by nearly six orders of magnitude, we employed Hyper-
CEST NMR spectroscopy. This indirect detection method took
advantage of the exchanging 129Xe population between bulk
aqueous solution and the xenon host molecule (Figure 4) by
selectively saturating the bound signal (Figure 4b). Because of
xenon exchange, the selective depolarization resulted in a con-
comitant signal loss from the 129Xe@water peak, which was
readily monitored (Figure 4a). This signal was compared with
a reference measurement where an “off resonance” saturation
was applied to account for the natural self-relaxation of
hp129Xe@water over time.
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Figure 5. Hyper-CEST signal decay of 34pM WEC at pH 7.5,
310 K. Depolarization rates were measured with radiofrequency
pulses either on-resonance (64.2 ppm) or off-resonance (320.6
ppm) with hp129Xe@WEC.
We also investigated the pH sensitivity of WEC-hp129Xe
NMR chemical shift to look at “normal” (pH 7.5) and acidic
(pH 5.9) buffer solutions, Figure 6. Because depolarization
efficiency is decreased with a narrowersaturation pulse, WEC
was employed at 1 µM concentrations, which is still at least
1
103-fold more dilute than demonstrated for HCEST pH re-
porters.82 Prior to detecting a free xenon signal, a loop of se-
lective Dsnob-shaped saturation pulses was scanned over the
chemical shift range of 40-230 ppm in 5-ppm (700 Hz)
steps,which corresponded to pulse length of 3748.6 µs and
power of 77µT. Two saturation responses centered at 195 ppm
(
129Xe@H2O) and 65 ppm (129Xe@WEC) were observed (Fig-
ure 6, full image). By decreasing the frequency scanning step
size to 1 ppm (138.2Hz), which corresponded to shaped pulse
length of 19014 µs and power of 15 µT, we were able to dis-
tinguish the WEC-encapsulated 129Xe peak for pH 7.5 and pH
5.9 samples at 300 K (Figure 6, inset). The total time to record
the Hyper-CEST NMR spectra was composed of xenon deli-
very time (20s) and data collection time. For the latter, each
data point required timeT:
Figure 4. Hyper-CEST detection scheme for WEC-encapsulated
129Xe. a. Representative spectra are shown for i. the initial spec-
trum and ii. the resulting spectrum from selective “on resonance”
saturation of the WEC-encapsulated 129Xe and commensurate
bulk 129Xe@H2O depolarization; b. selective radio frequency
depolarization of WEC-encapsulated 129Xe.
Using 34pM WEC (pH 7.5, 310 K, [Xe] = 0.15 mM) indi-
rect detection via Hyper-CEST was performed by applying
shaped radiofrequency saturation pulses atthe 129Xe@WEC
resonant frequency and measuring the residual aqueous 129Xe
signal for different saturation duration (Figure 5). WEC was
observed to “catalyze” this depolarization process through on-
resonance (64.2 ppm) saturation rf pulses with 129Xe@WEC in
pH 7.5 buffer. In contrast, saturation pulses applied off-
resonance (320.6 ppm) gave a depolarization time that approx-
imated the natural T1 of hp129Xe in water.
ꢂ
ꢇ
ꢀ ꢁ ꢃꢄ6 ꢅ ꢆ12 ꢈ ꢉ6 ꢅ ꢆ1 ꢅ ꢄ1
In the 5-ppm step scanning experiments, sp6 (saturation
pulse length) =3.748 ms, d12 (delay between saturation
pulses) = 20µs, L6 (number of saturation cycles) = 400, d1
(delay before acquisition pulse) = 0.5s, p1 (acquisition pulse)
= 22 µs. Thus, the total time needed to acquire the whole spec-
trumwas 860s. In the 1-ppm step scanning experiments, sp6 =
19.014ms, L6 = 600, and the total time needed was 478s.The
observed pair ofpeaks at pH 5.9was similar tohp129Xe NMR
data collected for 30 µM WEC by direct detectionat pH 6.5
(Figure 3). As illustrated by these data, the Hyper-CEST 129Xe
NMR spectrum readily distinguished between physiologically
normal and acidic pH values.
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Figure 6. Hyper-CEST scan of WEC (1 µM) at 300 K. Full image
was collected with 5-ppm step and individual peaks with 1-ppm
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Figure 7. Hyper-CEST 129Xe NMR spectra for 5-10 µM WEC in
10 mM sodium phosphate buffer with 0.1% Pluronic L-81 in a
suspension of 1 x 107 cells/mL. Data were collected at pH 7.5, a.
Xe@cells-red trace (196.3 ppm), Xe@aq-blue trace (192.3 ppm);
b. Xe@WECaq(65.0 ppm); and at pH 5.5, c. Xe@cells-red trace
(198.0 ppm), Xe@aq-blue trace (192.0 ppm); d. Xe@WECcells-red
trace (78.4 ppm) and Xe@WECaq-blue trace (68.0 ppm). Expo-
nential Lorentzian fits are shown as colored, solidlines and the
corresponding sums are shown as solid black lines.
step at pH 7.5 and pH 5.9 (inset).
Cellular Hyper-CEST 129Xe NMR
Finally, we investigated the utility of WEC in a biological
setting through 129Xe NMR cell studies. Human cervical carci-
noma (HeLa) cells were grown in a flask to confluency. Cells
were washed and suspended in either pH 7.5 or 5.5 sodium
phosphate buffer containing 5-10 µM WEC to give 1 x
107cells/mL concentrations. Pluronic L-81(0.1% final conc.)
was added to reduce foaming that can result from Xe bub-
bling.54 Cells were incubated in these conditions for 45-60 min
and then transferred to an NMR tube. Spectra were acquired at
both pH values with frequency scanning step size of 1 ppm
(138.2Hz), 400 cycles, which corresponded to shaped pulse
length of 19014 µs and powerof 15 µT. Figure 7a shows xe-
non in cells (196.3 ppm, red trace) and xenon dissolved in
aqueous solution of HeLa cells suspended in pH 7.5 buffer
with WEC (192.3 ppm, blue trace). Figure 7b shows WEC-
encapsulated xenon in the same sample. 129Xe@WECaq, pH
7.5 gave a chemical shift of 65.0 ppm, which corresponds to
free biosensor in buffer at pH 7.5. In pH 5.5 experiments, Fig-
ure 7c shows two peaks, one for Xe@cells (198.0 ppm, red
trace) and one for Xe@aq (192.0 ppm, blue trace). Figure 7d
shows the biosensor region of the same sample and exhibits
two peaks, one at 68.0 ppm corresponding to free alpha-helical
WEC in buffer (blue trace) and one at 78.4 ppm that we assign
to WEC inserted in cell membrane (red trace). Notably, upon
biosensor-membrane insertion at pH 5.5, we observed a 13.4
ppm downfield chemical shift compared to biosensor-cell so-
lutions at pH 7.5. Contrary to previous 129Xe NMR cell studies
performed with a more lipophilic cryptophane, we did not
observe cryptophane-membrane association at pH 7.5.52This
result is also consistent with previous studies with the EALA
peptide that showed no membrane association at pH 7.5.83By
targeting acidic pHe as a general cancer biomarker, along with
membrane association, we increased the chemical shift differ-
ence betweenXe@biosensorcellsandXe@biosensoraq as com-
pared to earlier studies.53-55 We note that in living organisms
there will be additional factors (beyond pHe) that impact the
129Xe@biosensorcells chemical shift, including cell type and
membrane composition. Follow-up studies will be required to
assess this variability.
CONCLUSIONS
In summary, by attaching a pH-responsive, membrane-
inserting peptide and two water-solubilizing moieties to a tri-
propargyl cryptophane host, we were able to generate an ultra-
sensitive 129Xe NMR biosensor capable of labeling cells in
acidic microenvironments. This xenon biosensor is unique for
undergoing a rapid and reversible conformational change
(over a range of physiologic pH values) as well as functional
changes: at pH 5.5, the pendant EALA-repeat peptide was
mostly alpha-helical and gained membrane-insertion capabili-
ties. This represents a “smart” 129Xe MR contrast agent, and
builds on previous examples of xenon biosensors thatbind
specific targets (e.g., protein receptors, DNA, cell-surface
glycans) or undergo a modification event (i.e., enzyme-
mediated peptide cleavage).
Significantly, this study demonstrated that appending the
peptide to the ~1 nm diameter, hydrophobic cryptophane did
not significantly reduce its ability to undergo a conformational
change. Circular dichroism, Trp fluorescence, and hp129Xe
NMR spectroscopies were employed to measurethe change in
helical character of the peptide in the pH range 5.5-7.5. EALA
peptide helix formation resulted in a129Xe NMR downfield
chemical shift change of 3.4 ppm, which was likely enhanced-
by significant cryptophane interactions with thenearby, N-
terminal Trp residue. This suggests a general strategy for en-
gineering larger chemical shift changes withxenon biosensors,
particularly to monitor molecular events occurring nanometers
away from the xenon-cryptophane reporter. These data
represent a significant advance over the previous example of a
peptido-cryptophane biosensor, which monitored MMP-7 ac-
tivity: only a 0.5 ppm chemical shift change was observed
upon enzyme-mediated peptide cleavage, perhaps because the
Trp was positioned much farther from the cryptophane.44For
some in vivo applications, it may be useful to maintain the full
range of pH-dependent conformational changes of the EALA-
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repeat peptide, and it will be interesting to explore different
peptides and conjugation strategies that work to achieve this
goal.
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The design of a cryptophane-EALA peptide conjugate ca-
pable of membrane insertion at acidic pH advances our long-
range goal of developing ultrasensitive 129Xe MR contrast
agents to aid in cancer diagnosis and treatment.63,73Picomolar
(10-11 M) concentrations of WEC were detected by Hyper-
CEST NMR, making this approach 8-9 orders of magnitude
more sensitive than commonly employed MR contrast
agents.We demonstrated a 13.4 ppm downfield chemical shift
change from disordered-peptide biosensor at pH 7.5 to the
helical, membrane-inserted biosensor at pH 5.5. This
represents the largest chemical shift change that has been en-
gineered to date for a 129Xe@cryptophane-biomolecule inte-
raction, the magnitude of which should facilitate multiplexed
detection in many experimental formats. The development and
cellular implementation of this “smart” xenon biosensor are
important steps towards future biomedical applications.
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ASSOCIATED CONTENT
Supporting Information.
Experimental and characterization details, including HPLC analy-
sis and MALDI-MS data for biosensor and precursors. This ma-
terial is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* ivandmo@sas.upenn.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research was supported by NIH R01-GM097478,
CDMRP-LCRP
Concept
Award
#LC130824,
and
GM097478S1 for 795-nm laser purchase. We thank Dr. Ra-
kesh Kohli for assistance with MALDI-MS (supported by NSF
MRI-0820996). We thank Drs. George Furst and Jun Gu for
assistance with NMR spectroscopy. We thank Dr. E. James
Petersson for use of a fluorometer. We also thank Dr. Christo-
pher Lanci of the University of Pennsylvania Biological Che-
mistry Resource Center for automated peptide synthesis sup-
ported by UPenn Laboratory for Research on the Structure of
Matter (NSF MRSEC DMR 0520020, 1120901). Dr. Jacob
Goldberg provided assistance with CD spectroscopy. We
thank Rebecca Wissner for synthetic advice, Judith Currano
and Sean Yeldell for editorial assistance.
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