Cryptophane xenon-129 nuclear magnetic resonance biosensors targeting human carbonic anhydrase

Article abstract of DOI:10.1021/ja806092w

¹²⁹Xe NMR biosensors are promising agents for early disease detection, especially when their interactions with target biomolecules can perturb ¹²⁹Xe chemical shifts well beyond the typical field inhomogeneity of clinical MRI. We intr

Full text of DOI:10.1021/ja806092w

Published on Web 12/15/2008
Cryptophane Xenon-129 Nuclear Magnetic Resonance
Biosensors Targeting Human Carbonic Anhydrase
Jennifer M. Chambers,† P. Aru Hill,† Julie A. Aaron,† Zhaohui Han,‡
David W. Christianson,† Nicholas N. Kuzma,‡ and Ivan J. Dmochowski*,‡
Department of Chemistry, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104, and
Department of Biomedical Engineering, UniVersity of Rochester, Rochester, New York 14627
Received August 3, 2008; E-mail: ivandmo@sas.upenn.edu
Abstract: 129Xe NMR biosensors are promising agents for early disease detection, especially when their
interactions with target biomolecules can perturb 129Xe chemical shifts well beyond the typical field
inhomogeneity of clinical MRI. We introduce human carbonic anhydrase (CA) as a single-binding-site enzyme
for studying xenon biosensor-protein interactions. A xenon-binding cryptophane was substituted with linkers
of varying lengths to p-benzenesulfonamide to yield nondiastereomeric biosensors with a single 129Xe NMR
resonance. X-ray crystallography confirmed binding of the eight-bond-linked biosensor containing a single
xenon atom in the CAII active site. Biosensor dissociation constants (Kd ) 20-110 nM) were determined
by isothermal titration calorimetry (ITC) for isozymes CA I and II. The biosensor-CA complexes yielded
“bound” hyperpolarized 129Xe NMR resonances of narrow line width that were shifted by 3.0-7.5 ppm
downfield, signifying much larger shifts than seen previously. Moreover, isozyme-specific chemical shifts
clearly differentiated CA I and II, despite their similar structures. Thus, xenon biosensors may provide a
powerful strategy for diagnosing human diseases characterized by the upregulation of specific CA isozymes
and other protein biomarkers.
frequency-resolved 129Xe resonances associated with medically
relevant biomarkers.7,8 Monatomic 129Xe is spin-1/2 and exhibits
Introduction
Magnetic resonance imaging (MRI) is used widely (∼40
a large chemical shift window (∼300 ppm),9 while laser
polarization enhances 129Xe NMR signals more than 10 000-
fold,10 which helps to compensate for a weaker magnetic
moment and much lower concentration of hyperpolarized 129Xe
million procedures annually) to scan deep tissues in human
1
patients with high spatial resolution. Intrinsic H MRI signals
from water and fat provide low sensitivity, thus contrast is
typically enhanced using gadolinium- or iron-oxide-based
agents.1,2 However, there have been challenges in developing
these contrast agents for in vivo imaging of medically relevant
biomarkers, such as proteins. Moreover, U.S. and European
agencies have recently issued advisories based on studies
showing that gadolinium agents pose risks to patients with
impaired renal function for developing nephrogenic systemic
fibrosis/nephrogenic fibrosing dermopathy (NSF/NFD).3 These
findings motivate the investigation of nonproton-based, hyper-
polarized MRI agents such as 129Xe, 13C, and 3He, which enable
physiological assays, for example of blood flow and lung
function, and provide sensitive methods for studying proteins
and metabolites.4-6
1
compared to H in aqueous solutions. Since the first report of
hyperpolarized 129Xe MRI in excised mouse organs,11 numerous
advances in biological imaging with this agent have been
made.12 In a recent study, the xenon gas exchange kinetics of
frequency-resolved tissue barrier and red blood cell resonances
were measured and used to detect regions of rat lung injury.13
Frequency-resolved MRI of an immobilized xenon biosensor
has also been demonstrated,14 showing the promise of imaging
biosensors bound to a target macromolecule. Most recently, we
showed that peptide-functionalized cryptophanes can be deliv-
(7) Spence, M. M.; Rubin, S. M.; Dimitrov, I. E.; Ruiz, E. J.; Wemmer,
D. E.; Pines, A.; Yao, S. Q.; Tian, F.; Schultz, P. G. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 10654–10657.
Functional xenon biosensors have attracted recent attention
due to their potential for simultaneously detecting multiple
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N.; Schultz, P. G.; Wemmer, D. E.; Pines, A. J. Am. Chem. Soc. 2004,
126, 15287–15294.
† University of Pennsylvania.
‡ University of Rochester.
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9
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2009 American Chemical Society
J. AM. CHEM. SOC. 2009, 131, 563–569 563

A R T I C L E S
Chambers et al.
ered to human cells and achieve intracellular concentrations
(g100 µM) that should allow in vivo hyperpolarized 129Xe MRI
studies.15
Herein, we systematically varied the interaction of a xenon
biosensor with a biomedically relevant protein, human carbonic
anhydrase (CA), in order to investigate whether small changes
in binding are detectable by 129Xe NMR. Cryptophane-A-based
xenon biosensors offer the possibility of tuning the frequency
of the bound 129Xe nucleus, through electronic and mechanical
perturbations of the cage environment. Water-soluble crypto-
phane-A derivatives have been shown to bind xenon with
micromolar dissociation constants under nearly physiological
conditions, such as in blood plasma.16 Functionalizing the
cryptophane with a biological recognition motif creates a
biosensor for the detection of specific proteins, such as matrix
metalloproteinases known to be secreted by various tumor
cells.17 Current strategies for constructing protein-targeted xenon
biosensors are (i) the use of a peptide to solubilize the large
(∼1 kD) nonpolar cryptophane7,8,18 or (ii) the incorporation of
cryptophanes into supramolecular dendrimers.19 While the use
of polyamidoamine (PAMAM) dendrimers affords narrow 129Xe
line widths, it also shields the cryptophane from solution and
renders the bound 129Xe relatively insensitive to protein binding.
In contrast, studies with peptido-biotin cryptophane biosensors
have shown that decreasing the linker length to the biotin ligand
increases the change in chemical shift but also broadens the
line width upon avidin binding.18 Attaching a peptide to the
chiral cryptophane creates diastereomers, which increases the
number of peaks in the 129Xe spectrum, and correspondingly
decreases the signal-to-noise ratio. Computational studies have
helped to assign the various NMR resonances to their respective
diastereomers.20 However, the tetrameric nature of avidin can
further complicate the 129Xe NMR spectrum.18
Figure 1. Crystal structure showing C8B-Xe-CAII interactions.29 The
Xe atom (green) is shown with a van der Waals diameter of 4.3 Å, the
Zn2+ ion is gray, the CAII backbone and surface are tan, and the MoMo
enantiomer of C8B is shown in black (carbon), red (oxygen), and blue
(nitrogen), surrounded by its van der Waals surface (dots). C8B binds in
the active site with the sulfonamidate anion coordinated to Zn2+
.
Numerous crystal structures of CA-inhibitor complexes are
available to guide biosensor development, and CAII in particular
has served as a successful model system for rational drug
design.21,25,26 CA is a monomeric protein, which reduces the
probability of biosensor-biosensor interactions that were
problematic with the tetrameric avidin target.18 Water-soluble
biosensors for CA were developed that avoid the appendage of
additional stereocenters to the cryptophane core. Use of the
arylsulfonamide ligand allows comparison of the biosensor-CA
interaction with other designed CA inhibitors. Solution-based
assays for CA can confirm active-site binding, and identify
protein-biosensor complexes suitable for crystallization.27,28 We
recently reported the crystal structure of CAII bound to a
benzenesulfonamide-linked-cryptophane (Figure 1).29
Experimental Procedures
Chemical Synthesis. Synthetic protocols and characterization
of all new compounds shown in Scheme 1 can be found in the
Supporting Information (Synthetic Methods and Figure S1).
To conjugate the sulfonamide linker to the tripropargyl crypto-
phane-A cage, 45-90 mg of 1 was dissolved in 2.0-4.0 mL dry
DMSO at rt with stirring. Sulfonamide linker 2, 3, or 4 (1.1 equiv)
was added. A 100 mM CuSO4 solution (0.25 equiv) was added,
followed by 2,6-lutidine (0.25 equiv), and 300 mM (+)-sodium-
L-ascorbate (0.75 equiv). The reaction was allowed to stir overnight
and then poured into 50 mL of H2O. This solution was extracted
with ethyl acetate and the combined organic layer was washed with
saturated NaCl solution, dried over Na2SO4, filtered, and evaporated.
The yellow oil recovered was purified by silica gel column
chromatography to give the pure product as a white solid.
In the current study, carbonic anhydrase was chosen as the
target enzyme due to its biomedical relevance and status as a
model system for understanding protein-ligand interactions.21
CA is involved in many physiological processes such as carbon
dioxide transport and pH homeostasis in tissue.22 However,
some CAs also appear to have detrimental effects on human
health: For example, CA I, II, and other isozymes were shown
to be present and probably involved in the formation of certain
tumors and polycystic kidney disease.23,24 Thus, the develop-
ment of isozyme-specific CA biosensors holds considerable
promise for cancer imaging.
Compound 6, 7, or 8 (27.2-54.2 mg) was dissolved in 2.0 mL
dry DMSO at rt with stirring. 5 (10 equiv) was added. A 100 mM
CuSO4 solution (0.25 equiv) was added, followed by 2,6-lutidine
(0.25 equiv), and 300 mM (+)-sodium-L-ascorbate (0.75 equiv).
The reaction was allowed to stir overnight and then poured into 30
mL of H2O. Compound 6, 7, or 8 was dissolved in dry DMSO at
rt with stirring. Solubilizing linker 5 was introduced. A 100 mM
CuSO4 solution (0.25 equiv) was added, followed by 2,6-lutidine
(0.75 equiv), and 300 mM (+)-sodium-L-ascorbate (0.25 equiv).
The reaction was allowed to stir overnight and then poured into
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Gudiksen, K. L.; Weibel, D. B.; Whitesides, G. M. Chem. ReV. 2008,
108, 946–1051.
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Cryptophane Xenon-129 NMR Biosensors
A R T I C L E S
Scheme 1. Synthesis of Trifunctionalized Cryptophane Biosensors C6B-C8B, MoMo Enantiomers Showna
a Reagents and conditions for two functionalization steps: (a) Sulfonamide linker (1.1 equiv), 100 mM CuSO4 (0.25 equiv), 2,6-lutidine (0.25 equiv), 300
mM sodium ascorbate (0.75 equiv) 10:1 DMSO/H2O, 4 h, 41-57% yield; (b) 5 (10 equiv), 100 mM CuSO4 (0.25 equiv), 2,6-lutidine (0.25 equiv), 300 mM
sodium ascorbate (0.75 equiv), 10:1 DMSO/H2O, 12 h, 41-63% yield (17-36% overall yield, as shown, from tripropargyl cryptophane, and 0.8-1.8%
overall yield from starting materials).16
Table 1. Dissociation Constants for CA-Linker and CA-Biosensor
H2O. This solution was extracted with ethyl acetate, and the
combined organic layer was washed with saturated NaCl solution,
Complexes at 298 K
enzyme
ligand
Kd (nM)
dried over Na2SO4, filtered, and evaporated. The yellow oil
recovered was dissolved in minimal 1 M NaOH, and a white solid
was precipitated with the addition of 1 M HCl. The liquid was
decanted after a 5-min centrifugation at 13.2 krpm. The solid was
then redissolved in minimal 1 M NaOH, precipitated with 1 M
HCl, and isolated. The solid was then sonicated for 10 min in DI
H2O and dried under vacuum. If impurities remained, solid was
dissolved in minimal CH2Cl2 and precipitated with diethyl ether
until desired purity was achieved. Pure product was recovered as
a white solid.
CAI
4
30 ( 10
20 ( 10
80 ( 10
30 ( 20
100 ( 10
100 ( 20
110 ( 30
60 ( 20
C6B
C7B
C8B
4
C6B
C7B
C8B
CAII
Isothermal Titration Calorimetry (ITC). All calorimetry
experiments were conducted at 298 K on a VP-ITC titration
microcalorimeter from MicroCal, Inc. (Northhampton MA), fol-
lowing standard protocols and data analysis.30,31
titrations subtracted from the experimental data. The amount of
heat produced per injection was calculated by integration of the
area under each peak. Data were fit to the equation q ) V∆H[E]tK[L]/
(1 + K[L]), where q is the heat evolved during the course of the
reaction, V is the cell volume, ∆H is the binding enthalpy per mole
of ligand, [E]t is the total enzyme concentration, K is the binding
constant, and [L] is inhibitor concentration. Nonlinear regression
fitting to the binding isotherm (ORIGIN 5.0 software, MicroCal)
using a one-site model gave the equilibrium dissociation constant
of the ligand, Kd, and estimates of the standard error, as provided
in Table 1. The error is σi ) ꢀ(Ciiꢀ2), where Cii is the diagonal
element of the variance-covariance matrix.
CA I and II were exhaustively dialyzed against 50 mM Tris-
SO4 (pH 8.0). Compounds (∼10 mM stock solution in DMSO)
were dissolved at a concentration of 135-300 µM in an aliquot of
the same buffer, and an equivalent volume of DMSO was added
to the enzyme solution. Prior to the titration experiment, samples
were degassed under vacuum for 5 min. The sample cell (effective
volume ) 1.4 mL) was overfilled with 1.8 mL of CA at a
concentration of 14-26 µM, and the reference cell was filled with
water. The contents of the sample cell were titrated with 30 aliquots
(10 µL each) of inhibitor (two initial 2 µL injections were made,
but not used in data analysis). After each injection, the heat change
was measured and converted to the corresponding enthalpy value.
The reaction mixture was continuously stirred at 300 rpm during
titration. Control experiments were carried out by titrating the
inhibitor into the buffer solution under identical experimental
conditions. The calorimetric data are presented with the background
Hyperpolarized 129Xe NMR Spectroscopy. Isotopically en-
riched xenon (86% 129Xe and 0.13% 131Xe, Spectra Gases) was
polarized via spin-exchange optical pumping with rubidium at 175
°C32 and cryogenically separated33 from the buffer-gas mixture (1%
Xe, 10% N2, 89% He by volume) in a Nycomed-Amersham (now
GE) IGI.Xe.2000 gas-flow polarizer (output 129Xe polarization
10-20% for 300-mL batches of pure xenon gas). Immediately after
(32) Walter, D. K.; Griffith, W. M.; Happer, W. Phys. ReV. Lett. 2001, 86,
(30) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Anal. Biochem.
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Chambers et al.
thawing, xenon was transferred to a special aluminum container
inside a home-built 129Xe probe in the bore of a 9.4 T supercon-
ducting magnet (Oxford Instruments). Spin relaxation time, T1, of
129Xe gas in the container ranged from 70 to 120 min.
see Table S1) at which these data were collected, using the measured
temperature dependence of -0.28 ppm/°C.18
Results
Hyperpolarized 129Xe NMR measurements12 with water-soluble
cryptophane biosensors were performed similarly to our previous
studies,17 with the exception of frequency-shift referencing de-
scribed below. For experiments not involving CA, 650 µL solutions
of C6B (96 µM), C7B (186 µM), or C8B (121 µM) in 50 mM
Tris-SO4 water-based buffer (pH 8.0) were added to a 5-mm NMR
tube fitted vertically onto a removable probe. A 1-mm outer
diameter glass capillary was inserted into the NMR tube to allow
bubbling of hyperpolarized Xe gas through the sample. NMR
samples were interchanged without removing the hyperpolarized
Xe container from the vertical magnet bore.
Biosensor Design and Synthesis. Unlike prior biotin-func-
tionalized Xe biosensors, which gained water solubility through
the attachment of a chiral peptide,7,8,18 biosensors for CA were
designed to avoid the incorporation of tetrahedral stereocenters.
Parent cryptophane-A is chiral, with D3 symmetry,35 as the top
and bottom cyclotriveratrylene moieties are oriented in an anti
configuration. With the original biotin-labeled Xe biosensor, the
use of chiral peptide and maleimide-functionalized biotin-
generated four diastereomers,8 which was later reduced to two
diastereomers through direct coupling of the biotin to an
orthogonally protected lysine residue.18 In each case, avidin
binding typically produced numerous overlapping, and cor-
respondingly broadened resonances. In targeting CA, biosensors
were modeled into CAII and structures were identified that
allowed coordination of the sulfonamidate anion to the active-
site Zn2+. We designed relatively low molecular weight bio-
sensors (1372-1423 g/mol, not including Xe), in order to
maximize rotational mobility, and achieve narrow hyperpolar-
ized 129Xe NMR resonances. Scheme 1 shows cryptophanes
attached via triazole linkers with 6-, 7-, and 8-bonds to
benzenesulfonamide (C6B, C7B, C8B). The shortest biosensor,
C6B fit inside the protein cleft and positioned the cryptophane
edge at the protein surface, ∼15 Å from the Zn2+ active site.
Longer biosensors C7B and C8B were conceived as a way to
modulate the interaction between the protein and cryptophane
and to observe the resulting changes in 129Xe NMR chemical
shift. Modeling studies were validated by the X-ray crystal
structure determination of C8B-CAII (Protein Data Bank
accession code 3CYU, Figure 1), which used anomalous
scattering information to confirm the encapsulation of Xe within
the cryptophane.29
Benzenesulfonamide was para-substituted with azido-func-
tionalized linkers 2-4 that were readily synthesized from the
amine analogues (see Supporting Information, Synthetic Meth-
ods), and provided a variable 6-8 bond spacer between the
cryptophane and benzenesulfonamide recognition unit. As seen
in Scheme 1, linkers 2-4 were reacted with tripropargyl
cryptophane (1) by a copper (I)-catalyzed [3 + 2] cycloaddition
process previously developed by others36,37 and applied by our
laboratory.16,17 It was determined that 2 was degraded with light
when dissolved in DMSO; thus, conjugate 6 was successfully
synthesized in the absence of light. Conjugates 7 and 8 did not
require this additional precaution.
Compounds 6-8 exhibited poor solubility in aqueous media.
To enable subsequent solution NMR and crystallography
experiments, two carboxylic acid groups were attached to the
cryptophane. 3-Azidopropionic acid 5 was readily prepared
following literature protocols38 and reacted in 5-fold excess with
the two remaining alkynes by the same copper (I)-catalyzed [3
+ 2] cycloaddition process. In neutral aqueous solutions, the
dianionic biosensors C6B-C8B exhibited favorable solubility
for X-ray crystallography studies with CAII.
After thermal equilibrium inside the magnet had been achieved
(temperature range for all experiments was 20.0-20.5 °C, with
thermal fluctuation of only 0.1 °C), hyperpolarized Xe gas was
bubbled through the biosensor solution for ∼1 min, and the
129Xe@cryptophane signal was monitored via a 1-5 µs pulse every
10 s. Once the 129Xe@cryptophane signal indicated saturation, Xe
bubbling was stopped, and four pulses at 7-11 µs were used to
obtain data (n ) 4, 129Xe frequency f0 ) 110.74668 MHz, 40-70°
tipping pulse) and then averaged. Raw free induction decay (FID)
signals were recorded in quadrature. Where useful, multiple
processed spectra were averaged to increase signal-to-noise ratio.
The biosensor-CA samples were prepared in 50 mM Tris-SO4
buffer (pH 8.0) using the following concentrations: C6B (188 µM)
+ CAI (141 µM), C6B (148 µM) + CAII (123 µM), C7B (136
µM) + CAI (100 µM), C7B (132 µM) + CAII (105 µM), C8B
(189 µM) + CAI (141 µM), and C8B (189 µM) + CAII (153 µM).
The biosensor-CA samples were then subjected to the same data
collection procedure as the unbound biosensor samples. NMR data
were processed using standard fast Fourier transform combined with
baseline and phase corrections. Spectra were fitted to linear
combinations of Lorentzians using IGOR Pro 5.0 (Wavemetrics,
Inc., Oregon). Gaussian broadening of 20 Hz was applied to obtain
the shown spectra, however the fits were done without any
broadening.
A new procedure, detailed in the Supporting Information (129Xe
NMR Methods) was used to reference NMR frequencies to the 129Xe
gas line. In the low-frequency region, two narrow 129Xe peaks (from
the gas in the vertical capillary and in the spherical bubbles) were
observed, superimposed on a broad peak from the gas above the
liquid surface. As xenon bubbling was stopped, the down-frequency
peak from the bubbles disappeared, and all other peaks shifted by
-31 Hz (-0.3 ppm). The narrow gas peaks were split diamag-
netically by 343 ( 20 Hz (3.1 ( 0.2 ppm), consistent with -(scyl
- ssph)ꢀw ) 3.01 ppm, where scyl ) 1 and ssph ) 2/3 are
magnetization shape factors for a vertical cylinder and a sphere,
and ꢀw is diamagnetic susceptibility of the surrounding solution
based on water (-9.04 ppm in SI units). Because the chemical shift
of 129Xe gas at 20.2 °C and 1 atm is 0.509 ppm relative to 129Xe
gas at 0 atm,34 we used the following expression to convert peak
frequencies f to ppm: σ [ppm] ) 106(f - fc + 343)/f0 + 0.509,
where fc is the measured peak frequency of 129Xe gas in the capillary
and f0 is the spectrometer frequency, both in Hz. The uncertainties
in 129Xe chemical shifts from peak fits were small (∼4 Hz, 0.04
ppm), with additional sources of error (such as the fit to the gas
reference peak) being comparable or smaller. These contributions
resulted in peak uncertainties of ∼ (0.07 ppm. 129Xe chemical shifts
are reported to a precision of (0.1 ppm. The calculated 129Xe NMR
chemical shifts for biosensor C7B were decreased by 0.1 ppm to
compensate for the slightly higher temperatures (20.3-20.46 °C,
Biosensor Binding Studies. Initial binding studies were
conducted using a dansylamide displacement assay,27,28 which
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A R T I C L E S
The spectrum of C8B with CAII (Figure 3i) gave an unbound
resonance at 63.2 ppm and also showed two resonances for the
bound cryptophane enantiomers, with the first at 68.2 ppm (∆δ
) 5.0 ppm) and the second at 66.9 ppm (∆δ ) 3.7 ppm). The
difference between the peaks observed for C8B when bound to
CAI (1.5 ppm) and CAII (1.3 ppm) are at the upper bound of
the values observed by Lowery et al. for the diastereomeric
biotin-labeled cryptophanes bound to avidin.18 The paired
resonances observed for C8B are also likely diastereomeric in
nature, based on differences in the chiral potential of 129Xe in
the two enantiomers bound to the chiral protein. The protein
crystal structure identifies one possible site of interaction
between C8B and CAII, with the MoMo and PoPo enantiomers
occupying very similar positions around a central xenon atom.29
Interestingly, the laser-polarized 129Xe NMR spectrum of C6B
with CAI (Figure 3b) showed only a single peak for bound
cryptophane biosensor at 69.2 ppm with the unbound peak at
63.7 ppm (∆δ ) 5.6 ppm). The spectrum of C6B with CAII
(Figure 3c) also showed a single peak for bound cryptophane,
at 68.2 ppm, with the unbound peak at 63.1 ppm (∆δ ) 5.1
ppm). On the basis of previous results with biotin-labeled
biosensors, it was expected that the short linker of C6B would
cause a strong binding-induced shift on the encapsulated 129Xe
resonance due to the proximity of the biosensor to the protein.18
It was not expected, however, that C6B bound to CA I or II
would exhibit only a single bound 129Xe resonance. Diastereo-
merically resolved resonances were previously identified in
Lowery’s biotin-linked cryptophanes, and were also observed
in C8B-CA. It is not possible that only a single enantiomer of
C6B is binding CA, based on the stoichiometries obtained from
ITC measurements (see Supporting Information, Figure S2).
These data and the relatively narrow (40 ( 10 Hz)
129Xe@C6B-CA line width, indicate that the two enantiomers
of C6B must induce the same change in the potential of the
129Xe nucleus when bound to the chiral protein pocket. In
addition to the single “bound” peak, the short linker of C6B
provides significantly larger chemical shift changes compared
to the previous biosensors that targeted avidin,18 without
sacrificing line width.
The laser-polarized 129Xe NMR spectrum of C7B with CAI
(Figure 3e) also showed only a single peak for bound crypto-
phane biosensor at 66.9 ppm with the unbound peak at 63.7
ppm (∆δ ) 3.2 ppm). Most interestingly, when complexed with
CAII (Figure 3f), C7B showed a bound resonance at 67.0 ppm,
and a second bound resonance at 71.2 ppm (∆δ ) 7.5 ppm,
relative to the unbound resonance at 63.7 ppm). This shift is
the largest ever reported for a xenon biosensor. Moreover, the
peak at 71.2 ppm for C7B-CAII clearly provides isozyme
discrimination between CA I and II, which creates opportunities
for 129Xe NMR multiplexing experiments.
The observation of a single “bound” peak for C6B-CAI,
C6B-CAII, and C7B-CAI (Table 2) suggests that this is a
common feature of xenon biosensors when the cryptophane is
confined by a rigid linker within the CA environment. The
cryptophane in close proximity to CA will be partially desol-
vated and experience a lower dielectric environment29 but is
not expected to bind to the protein surface. The net environ-
mental changes felt by the xenon in this case appear to dominate
the observed 129Xe NMR chemical shift and outweigh dia-
stereomeric considerations.
Figure 2. Representative ITC experiment with enthalpograms for
CA-biosensor complexation at 298 K in 50 mM Tris buffer, pH 8.0. (left)
CAI (14.2 µM) titrated with C8B (135.7 µM); (right) CAII (14.9 µM) titrated
with C8B (200.0 µM).
confirmed biosensor binding to the active sites of CA I and II
in solution (data not shown). Isothermal titration calorimetry
was used to determine the affinity of binding of 4, C6B, C7B,
and C8B to CA I and II (Figure 2 and Table 1). Biosensor C8B
exhibited higher affinity for CAII than the other compounds.
However, all four compounds bound slightly better to CAI, with
C6B exhibiting the highest affinity for CAI and 5-fold dis-
crimination over CAII. Further discrimination may be possible
by varying the linker joining benzenesulfonamide and crypto-
phane, based on small differences between the isozymes in
active-site architecture.39,40 Data in Table 1 indicate that the
cryptophane and tether length had little effect on the CA I and
II dissociation constants (Kd ) 20-110 nM) for this range of
linkers. Larger refined thermal (B) factors for C8B (<B> )
42 Å2) compared with the overall CAII model (<B>main chain
)
31 Å2; <B>side chain ) 35 Å2) reflect the mobility and confor-
mational heterogeneity of the bound biosensor.29 Indeed, the
cryptophane in C8B sits a few angstroms above the active-site
cleft, and there are few hydrogen bonds or other specific
interactions with the protein. These structural data are consistent
with the relative insensitivity of affinity to tether length. A more
detailed investigation into the thermodynamics of the biosensor-
protein interaction will be reported in due course.
Hyperpolarized 129Xe NMR Spectroscopy. As expected, the
racemic mixture of unbound MoMo and PoPo enantiomers
produced a single isotropic peak for C6B (63.5 ppm), C7B (63.9
ppm), and C8B (62.9 ppm) in their hyperpolarized 129Xe NMR
spectra (Figure 3a, d, g and Table 2).20 In the presence of
substoichiometric CA, the 129Xe NMR peak corresponding to
unbound biosensor was typically shifted 0.2-0.4 ppm down-
field, which reflects differences in solution properties.
Trials with the three biosensors demonstrated repeatable and
significant changes in 129Xe NMR chemical shift upon addition
of CA I or II. Addition of CAI to C8B gave an unbound
resonance at 63.3 ppm and two peaks at 67.9 and 66.3 ppm for
the bound enantiomers (Figure 3h), with ∆δ ) 4.5 and 3.0 ppm.
(39) Turkmen, H.; Durgun, M.; Yilmaztekin, S.; Emul, M.; Innocenti, A.;
Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2005, 15, 367–372.
In an attempt to explain the spectral differences between
C7B-CAI (one “bound” peak) and C7B-CAII (two “bound”
peaks), we inspected the two isozyme sequences. Using
(40) Srivastava, D. K.; Jude, K. M.; Banerjee, A. L.; Haldar, M.;
Manokaran, S.; Kooren, J.; Mallik, S.; Christianson, D. W. J. Am.
Chem. Soc. 2007, 129, 5528–5537.
9
J. AM. CHEM. SOC. VOL. 131, NO. 2, 2009 567

A R T I C L E S
Chambers et al.
Figure 3. Laser-polarized 129Xe NMR spectra with biosensors in 50 mM Tris, pH 8.0, buffer solution. (a,d,g) 129Xe NMR spectra showing individual
biosensors free in solution, (b,e,h) 129Xe NMR spectra showing each biosensor bound to CAI. (c,f,i) 129Xe NMR spectra showing each biosensor bound to
CAII. (a) C6B alone (96 µM); (b) C6B (188 µM) and CAI (141 µM); (c) C6B (148 µM) and CAII (123 µM); (d) C7B alone (186 µM); (e) C7B (136 µM)
and CAI (100 µM); (f) C7B (132 µM) and CAII (105 µM); (g) C8B alone (121 µM); (h) C8B (189 µM) and CAI (141 µM); (i) C8B (189 µM) and CAII
(153 µM).
Table 2. 129Xe NMR Chemical Shifts for C6B, C7B, and C8B Free
in Solution, Bound to CA I or II
) 1.3 ppm) may be due to conformational differences between
the two biosensors. In C7B, the benzenesulfonamide is kinked
unbound CAI [ppm] unbound;
[ppm]
∆ CAI
[ppm]
CAII [ppm] unbound;
bound
∆ CAII
[ppm]
by 109° relative to the linker, which may enforce interactions
of one or both enantiomers with the protein surface.
biosensor
bound
C6B
C7B
C8B
63.5
63.9
62.9
63.7; 69.2
63.7; 66.9
5.6
3.2
63.1; 68.2
63.7; 71.2; 67.0
5.1
7.5, 3.3
5.0, 3.7
Importantly for biosensing applications, the line widths of
“bound” xenon biosensors (23-50 Hz) were only somewhat
broadened compared to free xenon in solution (12 ( 1 Hz) and
were relatively unchanged from the “free” xenon biosensors
C7B (27 ( 1 Hz) and C8B (25 ( 3 Hz). The apparently broader
line width of C6B (79 ( 20 Hz) was likely the result of poor
mixing and low cryptophane-bound hyperpolarized 129Xe NMR
signal. Nonetheless, all three biosensors exhibited excellent
solubility in aqueous solutions. In the crystal structure of
C8B-CAII,29 the enantiomers appear to interact very little with
the CAII surface, which should allow rotational and translational
motions of the biosensor, and promote narrow NMR line widths.
Furthermore, because the cryptophane in C8B-CAII appears
to be unperturbed by the protein,29 the xenon within the
cryptophane can continue to behave isotropically. This should
make the noncovalently bound 129Xe nucleus fairly insensitive
to the larger rotational correlation time of the biosensor-CA
complex. By comparison, the previous use of polypeptide
solubilizing moieties and linkers in biotin-functionalized bio-
sensors may have promoted binding interactions with avidin
that contributed to line-broadening.18 The CA data support the
design of minimally chiral, low-molecular-weight 129Xe bio-
sensors that target proteins with single binding sites while
minimizing binding interactions between the cryptophane-linker
and the protein surface.
63.3; 67.9, 66.3 4.5, 3.0 63.2; 68.2, 66.9
a C7B data were temperature corrected by subtracting 0.1 ppm in
order to match 129Xe NMR data for C6B and C8B.
EMBOSS-Align:Needle, CA I and II were found to have high
sequence identity (60%) and sequence similarity (72%).41
Structural homology (rmsd ) 0.92 Å) was calculated using the
TOP3D program of the CCP4 package,42 with ligand-free crystal
structures for CAI (Protein Data Bank accession code 2CAB),43
and CAII (Protein Data Bank accession code 2CBA).44 CA I
and II possess active-site clefts of virtually identical width (15
Å) and depth (15 Å).40 Thus, the 10-Å diameter cryptophane
must experience similar steric environments in the two CA
isozymes.
Despite these structural similarities, the cavities of CA I and
II vary considerably in the distribution of polar and nonpolar
residues. For example, two hydrophobic patches that are unique
to the CAII active-site channel previously guided the synthesis
of high-affinity enzyme inhibitors.45 These patches, as well as
other sites rich in aromatic residues, are accessible to the Zn2+
-
bound cryptophane biosensor and may serve to discriminate
between the enantiomers of C7B. We hypothesize that in
C7B-CAI, the bound enantiomers produce equivalent 129Xe
NMR resonances by being in close proximity to the protein
environment, whereas in C7B-CAII, cryptophane interactions
with the protein surface lead to diastereomerism. The variation
in 129Xe NMR chemical shift observed for the two enantiomers
of C7B-CAII (∆δ ) 4.2 ppm) compared to C8B-CAII (∆δ
Discussion
In cancer patients, tumors can exist for long periods in a
dormant, asymptomatic state before development and diagnosis
of disease.46 Furthermore, human cancers are characterized by
the overexpression of many different proteins.47 Thus, the ability
to detect the overexpression of multiple protein biomarkers that
co-localize and identify a population of abnormal cells has the
potential to vastly improve the accuracy and efficacy of early
cancer detection.
(41) Rice, P.; Longden, I.; Bleasby, A. Trends Genet. 2000, 16, 276–277.
(42) , Acta Crystallogr. 1994, D50, 760–763, CCP4 Collaborative Project.
(43) Kannan, K. K.; Ramanadham, M.; Jones, T. A. Ann. N.Y. Acad. Sci.
1984, 429, 49–60.
(44) Hakansson, K.; Carlsson, M.; Svensson, L. A.; Liljas, A. J. Mol. Biol.
1992, 227, 1192–1204.
(46) Almog, N.; Henke, V.; Flores, L.; Hlatky, L.; Kung, A. L.; Wright,
R. D.; Berger, R.; Hutchinson, L.; Naumov, G. N.; Bender, E.; Akslen,
L. A.; Achilles, E.-G.; Folkman, J. FASEB J. 2006, 20, 947–949.
(45) Jain, A.; Whitesides, G. M.; Alexander, R. S.; Christianson, D. W.
J. Med. Chem. 1994, 37, 2100–2105.
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568 J. AM. CHEM. SOC. VOL. 131, NO. 2, 2009

Cryptophane Xenon-129 NMR Biosensors
A R T I C L E S
Toward this goal, we have shown that water-soluble 129Xe
NMR biosensors can be targeted with nanomolar affinity to
biomedically relevant proteins, human carbonic anhydrases I
and II. A xenon-binding tripropargyl cryptophane was func-
tionalized in two facile copper(I)-catalyzed [3 + 2] cycloaddition
steps, where the linker to benzenesulfonamide was varied
between 6 and 8 bonds. X-ray crystal structure analysis of CAII
complexed with C8B confirmed ligation of the sulfonamide
moiety to Zn2+ in the CA active site, with the cryptophane at
the upper rim of the active-site cleft. CA is the first monomeric,
single-binding-site protein that has been studied with a xenon
biosensor. ITC measurements revealed that C6B, C7B, and C8B
exhibit similar dissociation constants for CA as the bare linker
4, indicating that the cryptophane has little effect on CA binding
affinity for this range of linkers.
CA serves as a useful model system for studying xenon
biosensor-protein interactions. In order to improve detection
sensitivity, HyperCEST technique48,49 was proposed to irradiate
and selectively quench the hyperpolarized 129Xe NMR signal(s)
corresponding to the bound species. The presence of a biomarker
would be detected as a decrease in the bulk 129Xe NMR signal,
based on rapid exchange between xenon populations. 129Xe
NMR peaks with narrow line widths and large changes in
chemical shift will facilitate selective irradiation of one or more
“bound” xenon species in solution, as required for indirect
detection. Additionally, well-resolved resonances will facilitate
direct detection. 129Xe NMR studies with C6B, C7B, and C8B
indicated significant and reproducible downfield shifts of 5.6,
7.5, and 5.0 ppm upon binding to CA, which are considerably
larger than those observed previously for 129Xe biosensors
targeting avidin.18 The magnitude of the shifts, relative to typical
MRI field inhomogeneity of ∼1 ppm,50 holds considerable
promise for in vivo imaging studies.
of ∼0.5 ppm between C6B-CAI and C6B-CAII and between
C8B-CAI and C8B-CAII. Significantly, a peak was observed
exclusively for C7B-CAII that was 4.3 ppm downfield from
the single bound resonance for C7B-CAI. Isozyme-specific
chemical shifts clearly differentiate between CA I and II, despite
their homologous structures.
Conclusion
In summary, we synthesized 129Xe NMR biosensors for CA
I and II by modifying a cryptophane-A cage with two water-
solubilizing groups and attaching an arylsulfonamide recognition
moiety via a triazole linker. The linker was varied between 6
and 8 bonds, in order to tether the biosensors to active-site Zn2+
and modulate the interaction between cryptophane and CA. ITC
confirmed nanomolar affinity for CA I and II. When bound to
CA, biosensors C6B, C7B, and C8B demonstrated repeatable
changes in 129Xe chemical shifts of 5.6, 7.5, and 5.0 ppm, with
7.5 ppm being the largest chemical shift change ever reported
for a xenon biosensor. CA I and II were distinguished by C6B
and C8B by reproducible 0.5 ppm chemical shift differences.
Remarkably, C7B produced a unique resonance when bound
to CAII that was shifted 4.3 ppm downfield from the resonance
assigned to CAI. On the basis of the design principles that have
been advanced through this work, xenon biosensors now provide
avenues for detecting diseases associated with CA and other
proteins by in vivo hyperpolarized 129Xe NMR spectroscopy.
Acknowledgment. This work was supported by an NIH
Chemical Biology Interface training grant to J.A.A., NIH Grant
No. GM49758 to D.W.C., and DOD (W81XWH-04-1-0657), NIH
(1R21CA110104), and Camille and Henry Dreyfus Teacher-Scholar
awards to I.J.D. We thank Ronen Marmorstein and Jeffery Saven
for access to instrumentation, and Luigi Di Costanzo, George Furst,
and M.G. Finn for valuable discussions. Thanks go to Kevin Jude
for providing CAII, and Garry Seward and Jan-Oliver Janda for
experimental assistance.
Finally, isozyme discrimination was observed for all three
129Xe biosensors, with a 129Xe NMR chemical shift difference
(47) Welsh, J. B.; Sapinoso, L. M.; Kern, S. G.; Brown, D. A.; Liu, T.;
Bauskin, A. R.; Ward, R. L.; Hawkins, N. J.; Quinn, D. I.; Russell,
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Frierson, J.; Hampton, G. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
3410–3415.
Supporting Information Available: Complete ref 4, detailed
synthetic methods and compound characterization, 129Xe NMR
methods, 129Xe NMR data set parameters and full spectrum,
ITC data for 4, C6B, and C7B. This material is available free
of charge via the Internet at http://pubs.acs.org.
(48) Schroder, L.; Lowery, T. J.; Hilty, C.; Wemmer, D. E.; Pines, A.
Science 2006, 314, 446–449.
(49) Garcia, S.; Chavez, L.; Lowery, T. J.; Han, S.-I.; Wemmer, D. E.;
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