DOI: 10.1002/chem.201500989

Communication

& Anion Sensor

Ultrasensitive Anion Detection by NMR Spectroscopy: A Supramolecular Strategy Based on Modulation of Chemical Exchange Rate Loı¨se H. Perruchoud,[a, b] Alen Hadzovic,[a] and Xiao-an Zhang*[a, b, c] scale[4]), distinct NMR signals for free and bound species will be detected separately; if the interaction is weak and in the fast exchange regime, a chemical shift change will be observed which corresponds to the average signal between the peaks of the free and bound species.[5] In both scenarios, NMR spectroscopy can typically monitor complex formation with a host concentration in the mm to sub-mm range with regular NMR setup.[6] Sensing anion is generally more challenging than the well-established metal cation detection,[7] in part because the supramolecular anion receptors with high selectivity and affinity are relatively rare and structurally more complicated for practical applications.[8] Herein, we report a fundamentally different and significantly more sensitive approach for anion detection by 1H NMR. This indirect anion sensing technique detects the changes of CE rate of the sensor NMR signals, based on anion-induced conformational flexibility. Trace amounts of anions can be detected by the novel method in a selective manner at very low concentrations (120 nm) due to the transient and catalytic nature of the anion-sensor interaction. Intramolecular HB (HBintra) can provide conformational stability. Conformational fluctuations arising from HBintra formation/ breakage have been well studied by NMR in proteins[9] and a few examples are also known in small compounds.[10] Analyte induced conformational fluctuations can potentially be employed as an NMR readout for sensing purpose, in addition to the well-established approaches aforementioned. To test this unexplored approach, a pre-organized thioureido cryptand, TUC[11] (Figure 1) was chosen for the current study because it displays an exceptional conformational rigidity due to the formation of HBintra between the thioureido moieties. As the smallest bicyclic thiourea cryptand known to date, TUC is composed of three thiourea arms in a C3 symmetric fashion. Widely used in neutral anion receptor design,[6a, 12] the thioureido group consists of two HB donor (NH) and one HB acceptor (C= S) sites, capable of forming bifurcated HBs with an anion intermolecularly or with another thiourea intramolecularly. We hypothesize that exposing TUC to anions induces its conformational flexibility, since HBintra is competitively disrupted by formation of intermolecular hydrogen bonds (HBinter) between the thioureido hydrogens and anions in the solution. The consequent change in NMR spectrum can thus be used as a sensitive readout for anion detection. TUC was prepared from tris-(2-aminoethyl)amine (1) which was converted to a tris-isothiocyanate intermediate (2).[13] Equi-

Abstract: NMR spectroscopy is a powerful tool for monitoring molecular interactions and is widely used to characterize supramolecular systems at the atomic level. NMR is limited for sensing purposes, however, due to low sensitivity. Dynamic processes such as conformational changes or binding events can induce drastic effects on NMR spectra in response to variations in chemical exchange (CE) rate, which can lead to new strategies in the design of supramolecular sensors through the control and monitoring of CE rate. Here, we present an indirect NMR anion sensing technique in which increased CE rate, due to anion-induced conformational flexibility of a relatively rigid structure of a novel sensor, allows ultrasensitive anion detection as low as 120 nm.

Monitoring and quantifying intermolecular interactions, including hydrogen bonding (HB), ion pairing, and dipole interactions is of paramount importance in supramolecular chemistry and can be carried out using different analytical techniques such as UV-visible absorption, fluorescence or NMR spectroscopies.[1] NMR has the unique ability to provide detailed structural and dynamic information at the atomic level. Therefore, it is widely used to characterize supramolecular interactions.[2] This powerful technique, however, is limited for sensing purpose, due to low intrinsic sensitivity for direct signal detection. Conventional approaches to the design of supramolecular sensors typically require a specific receptor for the target analyte.[3] If the non-covalent binding of analyte to the receptor is strong and the exchange rate is slow (slower than the NMR time[a] L. H. Perruchoud, Dr. A. Hadzovic, Dr. X.-a. Zhang Department of Environmental and Physical Sciences University of Toronto Scarborough 1265 Military Trail, Toronto, ON M1C 1A4 (Canada) E-mail: [email protected] [b] L. H. Perruchoud, Dr. X.-a. Zhang Department of Chemistry University of Toronto 80 St George Street, Toronto, ON M5S 3H6 (Canada) [c] Dr. X.-a. Zhang Department of Biological Sciences University of Toronto Scarborough 1265 Military Trail, Toronto, ON M1C 1A4 (Canada) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201500989. Chem. Eur. J. 2015, 21, 1 – 6

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Communication inequivalency of the geminal hydrogens is consistent with the Xray structure which also reveals that these geminal hydrogen pairs in TUC are found in different chemical environments. The unusual splitting of the geminal -CH2- NMR signals suggests a relatively higher conformational rigidity of TUC in CDCl3 than in DMSO. We postulate that the pair of bifurcated HBintra between the neighboring thioureido groups contributes to this rigidity. Since TUC also exhibits C3 symmetry in CDCl3 solution, the HBintra must be shifting between Figure 1. Molecular structure of TUC. Thermal ellipsoids at 50 % probability. a) Side view. b) Top view along N(2)– N(2)’ axis. Methylene hydrogens omitted for clarity. the three thioureido arms faster than NMR timescale without causing dramatic conformational reorientation of the macrocycle. This dynamic C3 symmetry is molar coupling of 1 and 2 under high dilution condition gave TUC (3) in 97 % yield (see Supporting Information). maintained even at low temperature, as the three arms still exTo confirm the existence of HBintra and to obtain conformahibit identical signals at 40 8C (Figure 2 c), suggesting a low tional information, the structure of TUC was determined by Xenergy barrier for the rapid transfer of the HBintra pair among ray crystallography (Figure 1). Single crystals of TUC can be obthe three arms. This low barrier can be attributed to the headtained from CHCl3 or DMSO/water solutions by solvent evapoto-tail circular orientation of the three thioureido groups as ration or vapor diffusion, respectively. Both procedures gave shown in the X-ray structure (Figure 1), which facilitates the a comparable structure.[11] In the solid state, TUC possesses only one pair of bifurcated HBintra, between the two thioureido hydrogens NH(1) and the neighboring sulfur atom S(2) (Figure 1). The bond angle involved in HB (N1-H1-S2) is 1618 and the N(1)–S(2) distance is 3.473(3) , suggesting a relatively weak hydrogen bond.[14] The other two sulfur atoms point outwards and are involved in HBinter with NH of neighboring TUC molecules instead (see Table S1). Because there is only one pair of HBintra, the three thiourea arms are not identical and TUC does not show the expected C3 symmetry. This is not surprising since the simultaneous formation of more than one pair of HBintra would cause significant increase in conformational strain in TUC. In solution, however, TUC appears to be highly symmetrical, possessing a C3 symmetry axis and a mirror plane, as demonstrated in the NMR spectrum (Figure 2). The 1H NMR spectrum of TUC in [D6]DMSO contains only three peaks at d 7.09, 3.48 and 2.56 ppm with a 1:2:2 ratio corresponding to the NH, the -CH2- close to the thioureido group and the -CH2- directly bonded to the bridgehead nitrogen, respectively (Figure 2 a), consistent with expected high symmetry. Interestingly, the 1 H NMR spectrum of TUC in a less polar solvent such as CDCl3 exhibits a drastically different pattern, containing five peaks instead of three at d 6.66, 4.63, 3.00, 2.80 and 2.42 ppm with equal integrals (Figure 2 b). The significantly broader peaks in CDCl3 indicate possible involvement of CE. All peaks become Figure 2. 1H NMR spectra of TUC at 500 MHz a) in [D6]DMSO at 25 8C; b) in CDCl3 at 25 8C; and c) in CDCl3 at 40 8C. d) Structure of TUC with assignsharper at low temperature and their splitting patterns can be ment of methylene hydrogen atoms. e) Newman projection from the methresolved at 40 8C (Figure 2 c). Based on the chemical shifts, ylene carbons of TUC, used for assigning the methylene hydrogen atoms to coupling constants and 2D NMR data (Figure S1–2), all peaks the corresponding NMR signals. Solvent peaks are labeled * for DMSO, ** for can be reasonably assigned as shown in Figure 2 d–e. The NMR dichloromethane and *** for chloroform. &

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Communication rapid translocation of the single HBintra pair among the three arms without major change in conformation. To maintain a fluctuating HBintra pair, the three thioureido groups must retain the same relative dipole orientation, either clockwise or anti-clockwise (Figure 3). Only switching the relative dipole orientation of the three thioureido groups simultaneously, that is, from clockwise (Figure 3 a) to anti-clockwise (Figure 3 b) or vice versa, would cause the inter-conversion of chemical environments between the geminal hydrogen pairs. This mechanism enables the NMR CE between the geminal H-pairs. In CDCl3, this exchange is slower than NMR time scale.

To further illustrate the high sensitivity of the current method, anion detection was carried out using only trace amounts of Cl . As demonstrated in Figure 5, detectable peak broadening could be reliably observed at [Cl ] as low as 120 nm, corresponding to 1/14 000 equivalents of that of the sensor. Stepwise increase of the anion concentration continuously led to further broadening of the peaks. This catalytic effect of anion decreases the activation barrier of conformational interconversion by weakening of HBintra, and thus increases the kinetics of this reversible process. Similar effects can also be produced with increase in temperature, as followed by the broadening effect on the NMR spectrum (see Figure S3 in the Supplementary Information). Overall, this kinetic-related mechanism is different from the conventional anion sensing methods, which are mainly based on the thermodynamic molecular recognition of anions by the receptors. For practical purposes, all NMR experiments were carried out with regular NMR settings. Higher sensitivity in principle can be obtained by changing acquisition parameters or optimizing the hardware.[15] To compare the sensitivity of the current method Figure 3. Schematic mechanism of conformational chemical exchange between geminal with direct NMR detection, the signal of the counter hydrogens in TUC. Top view of TUC is shown in a dynamic average C3 symmetry, with ion (TBA) was used as a surrogate measure of [Cl ]. the dipole moment of three thioureido groups in a clockwise a) or anti-clockwise, b) orientation. Only selected representative H atoms are shown for clarity. As shown in Figure 4, the N(CH2)4 signal of TBA at 3.43 ppm (black arrow) only appeared at 28 mequiv Cl . As a reference to 1 equiv of [Cl ], this dePolar solvents such as DMSO are HB acceptors, thus can tectable 1H NMR signal corresponds to eight equivalents of H compete with and break HBintra. Therefore, conformational riatoms (~ 400 mm per hydrogen), more than three orders of magnitude higher than the low concentration (120 nm) detectgidity is diminished in DMSO (Figure 2 a) and the otherwise ed by the current method using the same settings. chemically inequivalent geminal H-atoms become dynamically Interestingly, thioureido groups in TUC also display a downequivalent due to rapid CE through conformational reorientafield shift after addition of high concentrations of anions. The tion (Figure 3). The NH signal in [D6]DMSO (7.09 ppm) is signifiNH peak is shifted up to 0.53 ppm after addition of cantly shifted downfield in contrast to that in CDCl3 15 equiv Cl . Because TUC has a relatively low affinity for the (6.66 ppm), supporting the occurrence of extra HBinter involving NH and solvent molecules. Cl , DdNH is rather insensitive for detecting trace amounts of This drastic change in the NMR spectrum in response to alterations of CE rate induced by HB competition can be utilized for sensing purposes. To verify the feasibility of this novel approach, we first exposed TUC to chloride anion, a strong HB acceptor. A solution of TUC (1.7 mm in CDCl3) was titrated with tetrabutylammonium chloride (TBACl) and followed by 1H NMR (Figure 4). Adding a small quantity of Cl (0.0014 equivalents) led the geminal resonances at d 2.42 and 2.80 ppm to coalesce and produced one broad peak at 2.64 ppm. Meanwhile, the other -CH2pair at d 4.63 and 3.00 ppm became significantly broader, but did not coalesce due to larger difference in their chemical shifts. These expected effects confirmed the increase of TUC’s conformational flexibility induced by Cl . Upon increase in [Cl ], the broad single peak at 2.64 ppm became sharper and the separate geminal -CH2- signals at 4.63 and 3.00 ppm became even broader, and eventually merged into a single peak at 3.86 ppm with 0.028 equiv of Cl Figure 4. 1H NMR titration of TUC in CDCl3 at 500 MHz and 25 8C (1.7 mm) with chloride added, consistent with a gradual increase in CE rate. (as a TBA salt). Chem. Eur. J. 2015, 21, 1 – 6

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Communication of chemical shift (fast-exchange scenario), the current approach monitors the increase in CE rate of geminal H signals of TUC. Since anions can catalytically induce the conformational flexibility of the sensor through HB competition, they produce a drastic effect on NMR spectra. To the best of our knowledge TUC is the first NMR sensor based on this unique HB competition strategy.

Experimental Section See Supporting Information for experimental procedures, including syntheses, characterization and NMR titrations.

Figure 5. Partial 1H NMR spectra of 1.7 mm TUC in CDCl3 at 500 MHz and 25 8C in the presence of trace amount of Cl .

Acknowledgements This work was mainly supported by NSERC through a Discovery Grant (# 489075) and Connaught New Researcher Award. We are grateful to the University of Toronto Scarborough, Canada Foundation for Innovation, and Ontario Research Fund.

anions. This signal, however, expands the range of anion concentrations that can be quantified with TUC. Overall the combination of three sets of NMR signals, including two pairs of -CH2- signals and the NH signal, offers a large dynamic range for anion detection, covering concentrations from nm to mm, uniquely advantageous for analytical purpose. We next examined the selectivity of this new method for other anion species which is expected to correlate to the HB acceptor ability of anions. Three representative anion species, Br , I and BF4 were chosen, and similar NMR titration studies with TUC were conducted (Figure S4–S7). As expected, Br is slightly less effective than Cl to generate coalescence of the geminal H signals. The difference between these two anions could be detected using peak widths (Figure S8): while 2.8 mequiv Cl generated a coalescent peak at 2.64 ppm with peak width at half height of 100 Hz, same amount of Br produced a broader peak (123 Hz). This is in agreement with Cl being a stronger HB acceptor and therefore generating faster CE. The different selectivity of TUC for I is even more evident: 2.8 mequiv of I was not enough to cause peak coalescence, but induced significant peak broadening (~ 100 Hz) of all four methylene signals. As expected, the effects of BF4 , a relatively inert anion, were the most modest among all anions tested, causing minimal peak broadening (~ 50 Hz). Polar solvents such as DMSO, are neutral and thus form even weaker HB with TUC, in contrast to anions. Small amount of DMSO (up to 14 mequiv) did not cause observable peak broadening in NMR of TUC. Therefore, impact of solvent impurity on anion sensing is rather small. These observations were consistent with the prediction that the magnitude of anion-induced CE rate increase correlates with HB acceptor ability. We have established a new NMR strategy to detect anions with unprecedented high sensitivity, predictable selectivity and a broad dynamic range of analyte concentrations. For the first time, nanomolar concentrations of anions were detected by NMR with regular instrumental setup, using a synthetic anion sensor, TUC. Unlike the conventional NMR sensing methods, which either detect the appearance of new anion-receptor complex peaks (slow-exchange scenario) or follow the change &

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Keywords: 1H NMR spectroscopy · anion sensor · chemical exchange · hydrogen bonds · non-covalent interactions [1] a) J. M. Lehn, Supramolecular Chemistry: Concepts and Perspectives, Wiley-VCH, Weinheim, 1995; b) C. A. Schalley, Analytical Methods in Supramolecular Chemistry, Wiley-VCH, Weinheim, Germany, 2012; c) P. D. Beer, P. A. Gale, D. K. Smith, Supramolecular Chemistry, Oxford University Press, Oxford, New York, 2003. [2] a) M. Pons, NMR in Supramolecular Chemistry, Kluwer Academic Publishers, Dordrecht; London, 1999; b) Y. Cohen, L. Avram, L. Frish, Angew. Chem. Int. Ed. 2005, 44, 520 – 554; Angew. Chem. 2005, 117, 524 – 560; c) A. Pastor, E. Martinez-Viviente, Coord. Chem. Rev. 2008, 252, 2314 – 2345. [3] a) J. L. Atwood, J. E. D. Davies, D. D. MacNicol, F. Vçgtle, J. M. Lehn, Comprehensive Supramolecular Chemistry, 1st ed., Pergamon/Elsevier, Oxford, 1996; b) J. P. Desvergne, A. W. Czarnick, in Proceedings of the NATO Advanced Research Workshops on Chemosensors of Ion and Molecule Recognition, Vol. 492, Bonas, France, 1997; c) L. Fabbrizzi, A. Poggi, Chem. Soc. Rev. 1995, 24, 197 – 202; d) P. D. Beer, P. A. Gale, Angew. Chem. Int. Ed. 2001, 40, 486 – 516; Angew. Chem. 2001, 113, 502 – 532. [4] R. G. Bryant, J. Chem. Educ. 1983, 60, 933 – 935. [5] a) K. Hirose, J. Inclusion Phenom. Macrocyclic Chem. 2001, 39, 193 – 209; b) P. Thordarson, Chem. Soc. Rev. 2011, 40, 1305. [6] a) A. F. Li, J. H. Wang, F. Wang, Y. B. Jiang, Chem. Soc. Rev. 2010, 39, 3729 – 3745; b) P. A. Gale, N. Busschaert, C. J. E. Haynes, L. E. Karagiannidis, I. L. Kirby, Chem. Soc. Rev. 2014, 43, 205 – 241. [7] B. Valeur, I. Leray, Coord. Chem. Rev. 2000, 205, 3 – 40. [8] N. H. Evans, P. D. Beer, Angew. Chem. Int. Ed. 2014, 53, 11716 – 11754; Angew. Chem. 2014, 126, 11908 – 11948. [9] a) K. Henzler-Wildman, D. Kern, Nature 2007, 450, 964 – 972; b) A. G. Palmer, Chem. Rev. 2004, 104, 3623 – 3640; c) V. L. Schramm, Annu. Rev. Biochem. 2011, 80, 703 – 732; d) A. Mittermaier, L. E. Kay, Science 2006, 312, 224 – 228; e) A. K. Mittermaier, L. E. Kay, Trends Biochem. Sci. 2009, 34, 601 – 611. [10] a) C. J. Haynes, N. Busschaert, I. L. Kirby, J. Herniman, M. E. Light, N. J. Wells, I. Marques, V. Flix, P. A. Gale, Org. Biomol. Chem. 2014, 12, 62 – 72; b) A. Jansma, Q. Zhang, B. Li, Q. Ding, T. Uno, B. Bursulaya, Y. Liu, P. Furet, N. S. Gray, B. H. Geierstanger, J. Med. Chem. 2007, 50, 5875 – 5877; c) I. M. Jones, A. D. Hamilton, Angew. Chem. Int. Ed. 2011, 50, 4597 – 4600; Angew. Chem. 2011, 123, 4693 – 4696; d) E. Fan, S. A. Vanarman, S. Kincaid, A. D. Hamilton, J. Am. Chem. Soc. 1993, 115, 369 – 370. [11] X.-a. Zhang, University of Basel (Basel), 2005.

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Communication [12] a) M. M. G. Antonisse, D. N. Reinhoudt, Chem. Commun. 1998, 443 – 448; b) D. E. Gmez, L. Fabbrizzi, M. Licchelli, E. Monzani, Org. Biomol. Chem. 2005, 3, 1495 – 1500. [13] X.-a. Zhang, W.-D. Woggon, J. Am. Chem. Soc. 2005, 127, 14138 – 14139. [14] G. A. Jeffrey, An Introduction to Hydrogen Bonding, Oxford University Press, New York, 1997.

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[15] D. L. Olson, T. L. Peck, A. G. Webb, R. L. Magin, J. V. Sweedler, Science 1995, 270, 1967 – 1970.

Received: March 12, 2015 Published online on && &&, 0000

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COMMUNICATION & Anion Sensor L. H. Perruchoud, A. Hadzovic, X.-a. Zhang* && – && Ultrasensitive Anion Detection by NMR Spectroscopy: A Supramolecular Strategy Based on Modulation of Chemical Exchange Rate

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Tuc tuc: The thiourea-based anion sensor, TUC, exhibits a conformational rigidity due to intramolecular H-bonding (HBintra), which can be competitively disrupted by catalytic amount of anion. The weakening of HBintra induces a con-

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formational flexibility, and consequently a more rapid chemical exchange between geminal -CH2- protons, which can be used as an ultrasensitive NMR readout for anion detection.

 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Ultrasensitive anion detection by NMR spectroscopy: a supramolecular strategy based on modulation of chemical exchange rate.

NMR spectroscopy is a powerful tool for monitoring molecular interactions and is widely used to characterize supramolecular systems at the atomic leve...
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