A R T I C L E S
Spence et al.
into high-throughput and combinatorial chemistry applica-
tions.13-18 Utilization of conventional NMR for high-throughput
biosensor applications may be limited by the intrinsically low
sensitivity and the complexity of spectra obtained from bio-
molecules and mixtures. Laser-polarization19 of 129Xe offers an
increase in signal-to-noise by several orders of magnitude
relative to the equilibrium nuclear-spin polarizations measured
in normal NMR experiments.20,21 In addition, 129Xe NMR
1
spectra are less complex than those from H or 13C NMR,
usually showing only a few, easily interpretable lines with no
background signal. Despite its spectral simplicity, xenon exhibits
the important property that it can sensitively report on its local
environment via its chemical shift and relaxation parameters.
Furthermore, xenon NMR and MRI can take advantage of
“remote” detection of signals that can be used to reconstruct
substantially amplified spectra22 and images.23 NMR of 129Xe
has been used to investigate the structure and dynamics of
materials,24-27 molecular cages,28-34 biological systems, and for
biomedical applications.35-37 Recently, extensive reviews re-
garding the history and development of hyperpolarized xenon
NMR have been published.38,39
Figure 1. Structure and schematic representation of biosensor molecule
designed target xenon to avidin with high affinity and specificity. Cryp-
tophane-A (black) binds the xenon atom, and the biotin ligand (red) is
connected to encapsulated xenon via a linker (purple).
themselves.48 While xenon shows appreciable binding to many
proteins, as evidenced by its use in making heavy atom
derivatives of protein crystals for X-ray crystallography,49,50 the
xenon-protein binding constants are relatively low (Ka ≈ 10-
100 M-1). The exchange of bound and free xenon has been
found to be fast on the time-scale of the chemical shift difference
between the protein and solvent environments, leading to NMR
spectra with a single xenon resonance. The chemical shift
reflects a population-weighted average over the xenon chemical
shifts of the available environments: solvent, protein interior,
and protein surface. As a result, the chemical shift value of this
single peak can report on changes in protein interactions.
Experiments with maltose binding protein demonstrated that the
129Xe chemical shift responds to a change in protein conforma-
tion upon ligand binding; the difference in shift results from
distinct xenon-protein interactions between the two conform-
ers.44,45 However, to induce measurable shifts, relatively high
concentrations of the “analyte” are required.
Biological systems studied with 129Xe NMR include globular
proteins such as myoglobin and hemoglobin,40-46 membrane
associated peptides such as gramicidin,47 and lipid membranes
(13) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996,
274, 1531-1534.
(14) Keifer, P. A. Curr. Opin. Biotechnol. 1999, 10, 34-41.
(15) Lacey, M. E.; Subramanian, R.; Olson, D. L.; Webb, A. G.; Sweedler, J.
V. Chem. ReV. 1999, 99, 3133-3152.
(16) Shapiro, M. J.; Gounarides, J. S. Prog. Nucl. Magn. Reson. Spectrosc. 1999,
35, 153-200.
(17) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V.
Science 1995, 270, 1967-1970.
(18) Hou, T.; Smith, J.; MacNamara, E.; Macnaughtan, M.; Raftery, D. Anal.
Chem. 2001, 73, 2541-2546.
(19) Happer, W. ReV. Mod. Phys. 1972, 44, 169-249.
(20) Walker, T. G.; Happer, W. ReV. Mod. Phys. 1997, 69, 629-642.
(21) Zook, A. L.; Adhyaru, B. B.; Bowers, C. R. J. Magn. Reson. 2002, 159,
175-182.
(22) Moule, A. J.; Spence, M. M.; Han, S. I.; Seeley, J. A.; Pierce, K. L.; Saxena,
S.; Pines, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9122-9127.
(23) Seeley, J. A.; Han, S. I.; Pines, A. J. Magn. Reson. 2003, 167, 282-290.
(24) Ripmeester, J. A.; Ratcliffe, C. I.; Tse, J. S. J. Chem. Soc., Faraday Trans.
1 1988, 84, 3731-3745.
Despite the favorable attributes of xenon interacting with
proteins in solution, high-sensitivity molecular sensing is not
compatible with the fast-exchange characteristic of xenon-
protein interactions. This limitation can be overcome by
identifying xenon interactions that report analyte binding via a
unique resonance that is resolved from those averaged into the
single fast-exchange peak. To realize this, we have taken the
approach of “functionalizing” the xenon by providing a physical
coupling between xenon and the ligand that targets a protein.
Recently, as a proof-of-principle case, we reported a molecule
designed to be an NMR biosensor that targeted the biotin-
binding protein avidin.51 The sensor consists of a modified
cryptophane-A cage, to which xenon binds as the NMR reporter,
(25) Chmelka, B. F.; Raftery, D.; Mccormick, A. V.; Demenorval, L. C.; Levine,
R. D.; Pines, A. Phys. ReV. Lett. 1991, 66, 580-583.
(26) Springuel-Huet, M. A.; Bonardet, J. L.; Gedeon, A.; Fraissard, J. Magn.
Reson. Chem. 1999, 37, S1-S13.
(27) Sozzani, P.; Comotti, A.; Simonutti, R.; Meersmann, T.; Logan, J. W.;
Pines, A. Angew. Chem., Int. Ed. 2000, 39, 2695-2698.
(28) Cram, D. J.; Tanner, M. E.; Knobler, C. B. J. Am. Chem. Soc. 1991, 113,
7717-7727.
(29) Bartik, K.; Luhmer, M.; Heyes, S. J.; Ottinger, R.; Reisse, J. J. Magn.
Reson., Ser. B 1995, 109, 164-168.
(30) Branda, N.; Grotzfeld, R. M.; Valdes, C.; Rebek, J. J. Am. Chem. Soc.
1995, 117, 85-88.
(31) Bartik, K.; Luhmer, M.; Dutasta, J. P.; Collet, A.; Reisse, J. J. Am. Chem.
Soc. 1998, 120, 784-791.
(32) Syamala, M. S.; Cross, R. J.; Saunders, M. J. Am. Chem. Soc. 2002, 124,
6216-6219.
(33) El Haouaj, M.; Luhmer, M.; Ko, Y. H.; Kim, K.; Bartik, K. J. Chem. Soc.,
Perkin Trans. 2 2001, 804-807.
(43) Locci, E.; Dehouck, Y.; Casu, M.; Saba, G.; Lai, A.; Luhmer, M.; Reisse,
J.; Bartik, K. J. Magn. Reson. 2001, 150, 167-174.
(34) Song, Y. Q.; Goodson, B. M.; Taylor, R. E.; Laws, D. D.; Navon, G.;
Pines, A. Angew. Chem., Int. Ed. Engl. 1997, 36, 2368-2370.
(35) Albert, M. S.; Cates, G. D.; Driehuys, B.; Happer, W.; Saam, B.; Springer,
C. S.; Wishnia, A. Nature 1994, 370, 199-201.
(44) Rubin, S. M.; Spence, M. M.; Dimitrov, I. E.; Ruiz, E. J.; Pines, A.;
Wemmer, D. E. J. Am. Chem. Soc. 2001, 123, 8616-8617.
(45) Rubin, S. M.; Lee, S. Y.; Ruiz, E. J.; Pines, A.; Wemmer, D. E. J. Mol.
Biol. 2002, 322, 425-440.
(36) Albert, M. S.; Schepkin, V. D.; Budinger, T. F. J. Comput. Assist. Tomo.
1995, 19, 975-978.
(46) Bowers, C. R.; Storhaug, V.; Webster, C. E.; Bharatam, J.; Cottone, A.;
Gianna, R.; Betsey, K.; Gaffney, B. J. J. Am. Chem. Soc. 1999, 121, 9370-
9377.
(37) Albert, M. S.; Balamore, D. Nucl. Instrum. Methods A 1998, 402, 441-
453.
(38) Cherubini, A.; Bifone, A. Prog. Nucl. Magn. Reson. Spectrosc. 2003, 42,
1-30.
(47) Mckim, S.; Hinton, J. F. Bba-Biomembranes 1994, 1193, 186-198.
(48) Xu, Y.; Tang, P. Bba-Biomembranes 1997, 1323, 154-162.
(49) Schiltz, M.; Prange, T.; Fourme, R. J. Appl. Crystallogr. 1994, 27, 950-
960.
(50) Soltis, S. M.; Stowell, M. H. B.; Wiener, M. C.; Phillips, G. N.; Rees, D.
C. J. Appl. Crystallogr. 1997, 30, 190-194.
(51) 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.
(39) Goodson, B. M. J. Magn. Reson. 2002, 155, 157-216.
(40) Miller, K. W.; Reo, N. V.; Uiterkamp, A. J. M. S.; Stengle, D. P.; Stengle,
T. R.; Williamson, K. L. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4946-
4949.
(41) Tilton, R. F.; Kuntz, I. D. Biochemistry 1982, 21, 6850-6857.
(42) Rubin, S. M.; Spence, M. M.; Goodson, B. M.; Wemmer, D. E.; Pines, A.
Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9472-9475.
9
15288 J. AM. CHEM. SOC. VOL. 126, NO. 46, 2004