Programming A Molecular Relay for Ultrasensitive Biodetection through 129XeNMR

Article abstract of DOI:10.1002/anie.201508990

A supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized ¹²⁹Xe NMR is reported. A cucurbit[6]uril (CB[6])-based molecular relay was programmed for three sequential equilibrium conditions by designing a two-faced guest (TFG) that initially binds CB[6] and blocks the CB[6]-Xe interaction. The protein analyte recruits the TFG and frees CB[6] for Xe binding. TFGs containing CB[6]- and carbonic anhydrase II (CAII)-binding domains were synthesized in one or two steps. X-ray crystallography confirmed TFG binding to Zn²⁺ in the deep CAII active-site cleft, which precludes simultaneous CB[6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB[6] was detected in buffer and in E.coli cultures expressing CAII through ultrasensitive ¹²⁹XeNMR spectroscopy.

Full text of DOI:10.1002/anie.201508990

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Communications
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International Edition: DOI: 10.1002/anie.201508990
German Edition: DOI: 10.1002/ange.201508990
Biosensors
Programming A Molecular Relay for Ultrasensitive Biodetection
through 129Xe NMR
Yanfei Wang, Benjamin W. Roose, John P. Philbin, Jordan L. Doman, and Ivan J. Dmochowski*
Abstract: A supramolecular strategy for detecting specific
proteins in complex media by using hyperpolarized 129Xe
NMR is reported. A cucurbit[6]uril (CB[6])-based molecular
relay was programmed for three sequential equilibrium con-
ditions by designing a two-faced guest (TFG) that initially
binds CB[6] and blocks the CB[6]–Xe interaction. The protein
analyte recruits the TFG and frees CB[6] for Xe binding. TFGs
containing CB[6]- and carbonic anhydrase II (CAII)-binding
domains were synthesized in one or two steps. X-ray crystal-
lography confirmed TFG binding to Zn2+ in the deep CAII
active-site cleft, which precludes simultaneous CB[6] binding.
The molecular relay was reprogrammed to detect avidin by
using a different TFG. Finally, Xe binding by CB[6] was
detected in buffer and in E. coli cultures expressing CAII
Scheme 1. Molecular relay produces an 129Xe NMR signal upon analyte
detection.
through ultrasensitive 129Xe NMR spectroscopy.
M
ethods for controlling the sequential interaction of
molecules in solution can yield new functionality and com-
plexity, as evidenced by many enzyme- and small-molecule-
mediated tandem reactions, drug delivery strategies, and
nanoscale and mesoscale architectures and working
“machines”.[1] Less investigated are supramolecular strategies
for improving in situ sample analysis, e.g., for generating high
contrast in bioassays and molecular imaging. Our group and
Schrçderꢀs recently showed that commercially available
cucurbit[6]uril (CB[6], Scheme 1) provides exceptional con-
trast at pm concentrations through hyperpolarized (hp) 129Xe
chemical exchange saturation transfer (Hyper-CEST)
NMR.[2] In Hyper-CEST, encapsulated hp 129Xe is selectively
depolarized by radiofrequency pulses, and through rapid
exchange, the depolarized 129Xe accumulates in the solvent
pool where the decrease in signal can be readily monitored
(Scheme S1 in the Supporting Information).[3] CB[6] is not
readily functionalized for biosensing applications, however.
We thus exploited the versatile host–guest chemistry of CB[6]
to develop a de novo “molecular relay” that reports on
specific proteins in solution.
encapsulating agent is required since Xe has low affinity for
endogenous biomolecules. Cryptophane-A and its derivatives
are the most studied Xe-binding cages and they exhibit the
largest association constants.[7] However, cryptophane bio-
sensors require multistep synthesis that yields only milligram
quantities.[8] This has motivated the search for new 129Xe-
binding scaffolds.[9]
The rigid structure and unique molecular recognition
properties of cucurbit[n]uril (CB[n]) compounds make these
useful as drug delivery vehicles, chemical reaction chambers,
components of enzyme assays, and building blocks in stimuli-
responsive supramolecular architectures.[10,11] Importantly,
CB[6] has a suitable cavity size to bind Xe (D ꢀ 4.4 Š), and
CB[6] and a water-soluble derivative were reported to bind
Xe with useful affinities (KA = 500–3000mÀ1 at RT).[12,2a]
Herein, we describe CB[6]–129Xe NMR biosensors pro-
grammed for a molecular relay with three sequential recog-
nition events (Scheme 1). A two-faced guest (TFG) is
engineered to control this relay, such that the CB[6]–129Xe
NMR signal is absent until addition of analyte.
CB[6] guest-binding affinities first measured by Mock and
co-workers[13] guided our design of TFGs that bind CB[6] with
intermediate affinity (displacing Xe) and are readily seques-
tered upon binding of the opposite TFG face to a higher-
affinity protein target. Butylamine was measured by ITC to
exhibit intermediate affinity for CB[6] (KA = 2.85 ” 105 mÀ1 in
pH 7.2, PBS solution at 300 K; Figure S1a in the Supporting
Information). We chose human carbonic anhydrase II (CAII,
EC 4.2.1.1) as the initial target because this is a model single-
site enzyme with biomedical significance[14] and its inhibitors
have been well studied.[15] The active site of CAII resides at
the base of an approximately 15 Š deep conical pocket that
can differentiate between TFGs even if they share the same
129Xe NMR/MRI has enabled the investigation of many
complex porous systems, from inorganic and organic materi-
als[4] to bacterial spores[5] and mammalian lungs.[6] For the
detection of specific proteins in solution, however, a specific
[*] Y. Wang, B. W. Roose, J. P. Philbin, J. L. Doman,
Prof. I. J. Dmochowski
Department of Chemistry, University of Pennsylvania
231 South 34th Street, Philadelphia, PA 19104-6323 (USA)
E-mail: ivandmo@sas.upenn.edu
Supporting information and ORCID(s) from the author(s) for this
article are available on the WWW under http://dx.doi.org/10.1002/
anie.201508990.
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Table 1: The two-faced guests (TFGs) used in this study and thermody-
namic data for binding to CB[6], CAII, or avidin.
a hydrophobic pocket lined by residues L198, P202, L204,
F131, V135, and L141. Other sulfonamide inhibitors with
flexible hydrophobic tails have also been observed to occupy
this pocket.[18] The amine nitrogen in the tail of 1 is positioned
near residues L198 and P202 but does not form hydrogen
bonds with the protein or solvent. This limits enthalpic
contributions to TFG binding, whereas solvent displacement
contributes significant entropy to DG.
A crystal structure of CAII bound to the longest TFG (3;
Figure 1) shows that the amine nitrogen is only 4.8, 5.1, and
5.4 Š from the three neighboring residues, respectively. Based
TFG Host
KD
[mm]
DH
TDS
DG
[kcalmolÀ1
]
[kcalmolÀ1
]
[kcalmolÀ1
]
CB[6]
CAII
CB[6]
CAII
CB[6]
CAII
CB[6]
CAII
4.07Æ0.55 À1.17Æ0.02 6.24
À7.41
À8.16
À7.12
À7.74
À7.22
À8.24
À7.48
À8.67
À4.99
ꢀÀ20
1
1.11Æ0.29
1.23Æ0.03 9.39
6.58Æ0.89 À0.91Æ0.03 6.21
2.29Æ0.46 À2.19Æ0.08 5.55
5.55Æ0.72 À2.33Æ0.05 4.89
1.00Æ0.14 À3.86Æ0.06 4.38
3.61Æ0.90 À2.08Æ0.10 5.40
0.48Æ0.07 À2.22Æ0.02 6.45
2
3
4
CB[6]
236Æ30
À4.79Æ0.66 0.20
N.D. N.D.
5
Avidin [a] ꢀ10À19
Figure 1. X-ray crystal structure of TFG 3 bound to CAII. a) A
simulated annealing omit map (contoured at 3s) shows 3 bound to
Zn2+ (green sphere) in the active site of CAII. Interatomic distances
(solid purple lines) are given in Š. Water molecules are omitted for
clarity. b) The CAII active-site channel is represented as a surface
model (hydrophobic residues in red; hydrophilic residues in white).
[a] Estimated values shown are based on measurements from Ref. [21].
Zn2+-targeting moiety, as previously demonstrated for many
CA inhibitors.[16] We therefore designed TFGs 1–4 (Table 1),
which all have a CAII-binding p-benzenesulfonamide moiety
and CB[6]-binding butylamine tail, but with varying length
and chemical structure of the linker.
Synthetic details for TFGs 1–4 are provided in Scheme S2.
Briefly, by following procedures modified from Salvatore
et al.,[17] 1 and 2 were synthesized from the primary amine and
alkyl bromide in a single step with cesium hydroxide
monohydrate as the base in 30% and 43% yield, respectively.
TFGs 3 and 4 were synthesized in two steps. In the first step, 2-
chloroacetyl chloride was reacted with 4-(2-aminoethyl)ben-
zenesulfonamide or 4-aminobezenesulfonamide in 68% or
70% yield and subsequently reacted with 1-butylamine to
deliver 3 or 4 in 56% or 50% yield, respectively.
To test whether the TFGs function as desired in the
prototypal CB[6] molecular relay, binding affinities were
measured by ITC separately for CB[6] and CAII (Table 1 and
Figure S1). The association constants for CB[6] titrated with
1, 2, 3, or 4 were all in the range of 1–3 ” 105 mÀ1 at 300 K in
pH 7.2, PBS buffer with 1% DMSO. We then performed ITC
to test the binding of 1–4 to CAII in the same buffer, and 4
displayed the highest affinity. The likely origins of the
enthalpic and entropic contributions towards the binding of
1 to CAII are observed in the crystal structure of the enzyme–
TFG complex (Figure S2). The benzenesulfonamide moiety
of 1 forms the interactions typically observed for arylsulfo-
namide inhibitors: the sulfonamidate nitrogen atom (N)
coordinates to the catalytic Zn2+ and donates a hydrogen
bond to the hydroxy oxygen of T199, and a sulfonamide
oxygen atom accepts a hydrogen bond from the backbone
amide nitrogen of T199. The n-butyl tail of 1 occupies
on these distances, TFG 3 is not available to bind simulta-
neously to CB[6], which has an outer diameter of 6.9 Š at its
portal and 14.4 Š at its widest point.[19] Molecular modeling
(Figure S3) helps to illustrate that a stable ternary structure
cannot form between CAII, TFG 3, and CB[6] owing to steric
clashes. Finally, a crystal structure of CAII with TFG 4 (which
lacks an ethyl linker), confirms that the butyl tail is even less
solvent accessible in this complex (Figure S4).
The CB[6]–TFG complexes were incubated with CAII for
20 min, and the hydrolysis rates of p-nitrophenyl acetate
(pNPA) enzymatically catalyzed by CAII were determined by
monitoring an increase in absorbance at l = 400 nm
(Figure 2). Compounds 3 and 4 displayed stronger CAII
inhibition compared to the other TFGs, which is consistent
with their higher CAII affinity. CB[6] alone did not show any
CAII inhibition, which suggests a lack of direct interaction
between CAII and CB[6]. When complexed with CB[6], all of
the TFGs inhibited CAII activity only slightly less than TFG
alone, which provides further evidence that the TFGs shuttle
from CB[6] to CAII, as designed. Furthermore, titration of
CB[6] led to recovery of CAII activity (Figure S5), thus
demonstrating that the shuttling of TFG is under thermody-
namic control. This is also consistent with butylamine under-
going fast exchange (texch < 10 ms) with CB[6].[20]
Based on its highest affinity for CAII and intermediate
affinity for CB[6] (Table 1), TFG 4 was selected for testing
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mated to be much higher (KA ꢀ 1014 mÀ1).[21] In order to block
xenon access initially, we added 50 mm TFG 5 to 1 mm CB[6]
solution, and as expected, the 129Xe–CB[6] Hyper-CEST
signal was completely turned off. Subsequently, administra-
tion of 50 mm avidin fully restored the 129Xe–CB[6] signal
(Figure 4). These data highlight the ease of reprogramming
the CB[6]–TFG relay for assaying a wide range of biomol-
ecules in solution.
Figure 2. Comparison of CAII esterase activity with different TFGs and
CB[6]–TFG complexes monitored by using pNPA as a substrate. All
assays were performed after 20 min incubation. Standard errors were
determined from three or more replicates for each condition.
our “turn-on” 129Xe NMR biosensing strategy. First, 1 mm
CB[6] was dissolved in pH 7.2 PBS solution, and multiple
selective Dsnob-shaped saturation pulses were scanned over
the chemical shift range of d = 90–230 ppm in 5 ppm steps.
Two saturation responses were observed (Figure 3), centered
Figure 4. Frequency-dependent 129Xe NMR saturation spectra in pH 7.2
PBS at 300 K show avidin detection through a CB[6]–pAB molecular
relay.
Finally, we investigated our molecular relay in cellular
environments. BL21(DE3) E. coli expressing recombinant
CAII was cultured in LB medium and induced with 1 mm
isopropyl b-D-1-thiogalactopyranoside (IPTG) and 1 mm
ZnCl2. Cells were then pelleted, washed, resuspended in
PBS buffer, and lysed with two freeze–thaw cycles. Quanti-
tative SDS gel analysis indicated expression levels of 12 nmol
CAII (3 mL of 4 mm sample solution) from cell lysate
equivalent to OD600 nm = 2 (Figure S6). Non-transformed
E. coli were grown and lysed in the same manner to serve
as a negative control. Screening experiments revealed that
16 mm CB[6] gave useful residual 129Xe Hyper-CEST NMR
signal, despite CB[6] binding to many components of the
bacterial lysate. Addition of 4 mm TFG 4 to control sample
with 16 mm CB[6] greatly reduced the Hyper-CEST signal,
whereas in lysate from CAII-overexpressing E. coli, the
original NMR signal intensity was observed (Figure 5).
These experiments highlight the ability of the CB[6] detection
approach to identify a specific protein target within a complex
mixture. The molecular relay thus extends the utility of CB[6]
for ultrasensitive 129Xe NMR biosensing.
Figure 3. Frequency-dependent 129Xe NMR saturation spectra in pH 7.2
PBS at 300 K show CAII detection through a CB[6]–4 molecular relay.
at d = 193 ppm (129Xe-aq) and 122 ppm (129Xe-CB[6]). Upon
addition of 4 mm TFG 4, the 129Xe–CB[6] Hyper-CEST signal
at d = 122 ppm was greatly reduced as a result of less free
CB[6] (ca. 0.5 mm) in the sample solution. Finally, when 4 mm
CAII was added to the solution, the 129Xe–CB[6] Hyper-
CEST signal was mostly restored, thus confirming that TFG 4
was sequestered by CAII.
To apply the molecular relay to a system with different
thermodynamic properties, we employed a commercially
available pentylamine biotin (pAB) TFG 5 that binds CB[6]
much less tightly than its target protein avidin. TFG 5 was
determined by ITC (Table 1) to bind to CB[6] with modest
affinity (KA = 4240mÀ1), whereas avidin affinity was esti-
In summary, by exploiting versatile CB[6] host–guest
chemistry and facile attachment of a CB[6]-binding domain to
a protein-binding moiety, we were able to generate novel
129Xe NMR biosensors. This work was guided by X-ray crystal
structure determination of three CAII–TFG complexes.[22]
The TFG programs the molecular relay for exquisite control
over chemical equilibria between several interacting compo-
nents: CB[6], TFG, the target molecule, and Xe. This strategy
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This work was supported by NIH R01-GM097478 and
CDMRP-LCRP Concept Award no. LC130824. The ITC
was purchased via NIH S10-DO016260. We thank Drs.
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Keywords: bioinorganic chemistry · biosensors · host–
guest systems · molecular recognition · NMR spectroscopy
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Received: September 25, 2015
Published online: December 21, 2015
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