Communication pubs.acs.org/JACS
A Bis-calix[4]pyrrole Enzyme Mimic That Constrains Two Oxoanions in Close Proximity Qing He,† Michael Kelliher,† Steffen Baḧ ring,†,‡ Vincent M. Lynch,† and Jonathan L. Sessler*,† †
Department of Chemistry, The University of Texas at Austin, 105 East 24th Street-A5300, Austin, Texas 78712-1224, United States Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
‡
S Supporting Information *
bonds have been noted in recent years.9−12 For instance, Tomisic’s group reported a thermodynamic study of dihydrogen phosphate dimerization based on the use of ureaand thiourea-based receptors.10d In 2015, the Das group published a polyammonium-functionalized tripodal receptor capable of encapsulating a hydrogen sulfate dimer.11 Very recently, Flood12 reported that their macrocyclic hosts could stabilize a bisulfate dimer via borderline-strong hydrogen bonds, albeit in a 2:2 (receptor-bisulfate) binding stoichiometry. These reports notwithstanding, the rational capture of two sulfate anions (as opposed to hydrogen sulfate) within the same synthetic framework remains an unmet challenge. Moreover, to our knowledge, the concurrent trapping of two pyrophosphate anions in a synthetic receptor has not yet been achieved. Here, we report a new bis-calix[4]pyrrole (1) with multiple anion binding sites (up to 12 hydrogen bond donors within each receptor) that is capable of stabilizing cocomplexes with, respectively, two H2PO4−, two SO42−, or two HP2O73− anions. This system thus provides a rudimentary structural mimic of enzymes, such as PPases and ATP sulfurylase, that are capable of binding two tetrahedral oxoanions. To our knowledge, only two examples of bis-calix[4]pyrroles connected by two arms are currently known.13 These systems were both prepared by coupling preformed cis-meso disubstituted calix[4]pyrroles, such as 3, obtained by condensing a functionalized dipyrromethane (e.g., 4) with acetone (Scheme 1a). Unfortunately, the cis form of configurationally locked precursors, such as 3, can be difficult to separate from the corresponding trans form. We thus sought a new approach to preparing doubly linked bis-calix[4]pyrroles (Scheme 1b). Here, a linked bis-dipyrromethane 7 serves as the key intermediate; it was obtained readily via a simple symmetrical coupling of functionalized dipyrromethane 6 with 2,6diaminopyridine 5 in the presence of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI·HCl) and pyridine in DCM for 24 h at room temperature. The desired trimacrocyclic bis-calix[4]pyrrole 1 was obtained in 2% yield via a one-pot, BF3-catalyzed, multiring forming condensation of 7 with acetone. Compound 1 was characterized by NMR spectroscopy and mass spectrometry as well as by X-ray diffraction analyses of single crystals (Figures S2−S4). Given its cavity size (>0.282 nm3, Figure S5),14 we expected that the biscalixpyrrole 1 would act as an effective receptor for relatively large anions, e.g., H2PO4−, HSO4−, SO42−, Cr2O72−, or
ABSTRACT: Herein we describe a large capsule-like biscalix[4]pyrrole 1, which is able to host concurrently two dihydrogen phosphate anions within a relatively large internal cavity. Evidence for the concurrent, dual recognition of the encapsulated anions came from 1H NMR and UV−vis spectroscopies and ITC titrations carried out in CD2Cl2/CD3OD (9/1, v/v) or dichloroethane (DCE), as well as single crystal X-ray diffraction analyses. Receptor 1 was also found to bind two dianionic sulfate anions bridged by two water molecules in the solid state. The resulting sulfate dimer was retained in DCE solution, as evidenced by spectroscopic analyses. Finally, receptor 1 was found capable of accommodating two trianionic pyrophosphate anions in the cavity. The present experimental findings are supported by DFT calculations along with 1H NMR and UV−vis spectroscopies, ITC studies, and single crystal X-ray diffraction analyses.
A
nion binding is ubiquitous in biology.1 Often the recognition events are quite complex. This is true in the case of inorganic pyrophosphatases (PPases), which catalyze the hydrolysis of inorganic pyrophosphate (PPi) to generate two molecules of phosphate (Pi) and are critical for balancing phosphate metabolism.2 Enzymatic protein-bound dimeric anion complexes are not limited to PPase. For instance, sulfate dimers trapped within the same enzymatic pocket can be found in the active sites of Streptococcus mutans pyrophosphatase (Figure S8)3 and in ATP sulfurylase (Figure S9).4 In addition, some so-called transferases are able to bind two pyrophosphate anions within the same pocket (Figure S10).5 In contrast to the cobinding of two oxoanions seen in these natural systems, bringing two polyhedral anions together within the same synthetic receptor system represents a considerable challenge. However, it may hold the key to understanding these and other enzymatic processes. Recently, considerable progress has been made in the area of anion recognition. This is particularly true for relatively simple anions, such as halides, nitrate, etc.6 A number of elegant receptors capable of binding a single polyhedral oxoanion such HSO4−, SO42−, H2PO4−, HPO42−, and PO43− anions have also been reported.7 In addition, the encapsulation of two or three NO3− anions within the pocket of appropriately designed receptors has been achieved.8 Several examples of so-called “dihydrogen phosphate dimers” or mixed phosphate sulfate clusters stabilized by the formation of intermolecular hydrogen © 2017 American Chemical Society
Received: March 8, 2017 Published: May 11, 2017 7140
DOI: 10.1021/jacs.7b02329 J. Am. Chem. Soc. 2017, 139, 7140−7143
Communication
Journal of the American Chemical Society Scheme 1. Synthetic Routes Leading to Bis-calix[4]pyrroles: (a) Method Used by Ballester13b and Lee;13a (b) Strategy Reported Here
Figure 1. (a) Single crystal structure of (H2PO4−)2·H2O⊂1; (b) UV− vis spectroscopic titration. Inset: two isosbestic points at 271 and 296 nm, respectively, were seen. (c) ITC data (normalized heat) obtained by injecting TBAH2PO4 to a DCE solution of 1.
HP2O73−.15 Initial screening studies were carried out in CD2Cl2/CD3OD (9/1, v/v) using 1H NMR spectroscopy (Figure S11). It was found that adding 10 equiv of either HSO4− or Cr2O72− as their tetrabutylammonium (TBA) salts failed to produce any noticeable changes in the proton signals of 1, a finding consistent with a very weak interaction. In contrast, a similar addition of H2PO4− or HP2O73− (as their TBA salts) led to a shift and splitting in the β-pyrrole proton signals (i.e., from 5.94 to 5.57 ppm and 5.77 ppm, respectively). Meanwhile, the addition of only 2 equiv of SO42− as its tetramethylammonium (TMA) salt caused the β-pyrrole CH resonances to shift from 5.94 to 5.62 ppm and the pyrrole NH proton signals to shift from 7.96 to 11.4 ppm. These findings led us to suggest that receptor 1 binds the H2PO4−, SO42−, and HP2O73− anions well in solution. Further studies were thus focused on these three oxoanions. Initial evidence that receptor 1 was able to bind two H2PO4− anions concurrently came from an X-ray diffraction analysis of single crystals obtained by allowing a DCM/CH3OH/CH3CN solution of 1 to undergo slow evaporation in the presence of excess TBAH2PO4. The resulting crystal structure revealed an encapsulated 1:2 complex in the solid state (Figure 1a). The (H2PO4−)2⊂1 complex thus provides a structural mimic for the phosphate dimer complex intermediate seen during the enzymatic function of PPases (Figures S6−S7).16 The two cobound H2PO4− anions in the (H2PO4−)2⊂1 complex were found to be bridged by a water molecule. The total length of the H2PO4− dimer (7.267 Å) is shorter than the length of the free host, leading to a twisting of 1 to accommodate the H2PO4− dimer (Figure S12). The associated induced fit action is reminiscent of the relationship between many protein receptors and their ligand targets. Support for the notion that receptor 1 was able to bind H2PO4− in a 1:2 ratio in solution came from a 1H NMR spectroscopic titration. When 1 in CD2Cl2/CD3OD (9/1, v/v) was subjected to a titration with TBAH2PO4 (Figure S13), the original sharp peaks of the NH pyrrole resonances at δ = 8.07 ppm and the β-pyrrole protons resonances at 5.92 ppm gradually broaden through the addition of 2 equiv. These changes were ascribed to the formation of an initial 1:1 complex, H2PO4−⊂1, with the peak broadening reflecting an intermediate exchange between the complexed and the free
species on the NMR time scale. Upon injection of more than 2 equiv of H2PO4−, new proton resonances at δ = 10.73 ppm (pyrrole NH protons) and 5.57 ppm (β-pyrrolic protons) are seen, indicating the occurrence of the (H2PO4−)2⊂1 complex. Although a water molecule serves to bridge the anions in the solid state, unfortunately, we were unable to assign the H2O signature residues because the signals in question become complex and hard-to-assign at low temperature (Figure S14). In addition, we were unable to see the chemical shift of the amide NHs caused by anion binding under the current conditions, probably due to fast exchange with the deuterated solvent (Figure S15). Further evidence for the formation of a 1:2 complex was obtained from UV−vis absorption spectroscopic titrations carried out in 1,2-dichloroethane (DCE) (Figure 1b). Upon adding up to 2 equiv of TBAH2PO4, two isosbestic points at 272 and 296 nm were observed. This is consistent with the clean conversion between one absorbing species and another. Further titration with more guest led both isosbestic points to disappear, a finding rationalized in terms of formation of a second species as the guest/receptor ratio increases. Plots of absorption intensity at 292 nm for 1 versus [H2PO4−]/[1] were then constructed (Figure S16). It was found that the intensity of the absorption at this wavelength decreased sharply until ∼2 equiv of H2PO4− were added. Thereafter, the absorption increased until saturation was reached at ∼3.3 equiv. The binding events were quantified using isothermal titration calorimetry (ITC). The stepwise injection of a DCE solution of TBAH2PO4 (0.86 mM) into a DCE solution of 1 (0.04 mM) at 298 K resulted in a gradual release of heat as would be expected for an exothermic binding process. The resulting binding isotherm (Figure 1c) was analyzed using the sequential binding sites model (binding sites N = 2). The use of this model as opposed to the two sets of sites binding model reflects the expectation that the two putative binding sites in 1 would not necessarily act independently. The magnitude of the first binding constant (K1 = (1.67 ± 0.15) × 106 M−1) is slightly higher than that of the second one (K2 = (1.16 ± 0.08) × 105 M−1). The interaction parameter (α) for this system was calculated to be 0.4 (α = 4K2/K1).17 The two underlying binding events were both driven by enthalpy (ΔH1 = −7025 ± 7141
DOI: 10.1021/jacs.7b02329 J. Am. Chem. Soc. 2017, 139, 7140−7143
Communication
Journal of the American Chemical Society 97.4 cal·mol−1 and ΔH2 = −5505 ± 180 cal·mol−1) and entropy (ΔS1 = 4.92 cal·K−1·mol−1 and ΔS2 = 4.71 cal·K−1·mol−1). We next sought to explore whether 1 could provide a structural mimic for a cobound, enzymatic sulfate anion dimer complex. As above, initial studies of the solution phase chemistry were carried out using 1H NMR spectroscopy. It was found that the addition of 0.33 equiv of TMA2SO4 to a solution of 1 (1 mM) in CD2Cl2/CD3OD (9/1, v/v) resulted in the signal corresponding to the free pyrrolic NH protons at δ = 8.10 ppm to shift to 11.35 ppm (Figure S18). Concurrently, the β-pyrrole protons, originally resonating at δ = 5.93 ppm, shifted to 5.63 ppm. When 2 equiv of TMA2SO4 were introduced, only one set of proton signals could be observed. These findings lead us to infer that a complex with a 1:2 binding stoichiometry ([1]:[SO42−]) is being formed under the conditions of the titration. Further evidence for receptor 1 recognizing sulfate in a 1:2 ratio came from a UV−vis spectroscopic titration experiment (Figure S18). Upon addition of increasing quantities of TMA2SO4 in DCE/methanol (9/1, v/v) to a solution of 1 in DCE, new bands at 240−272 nm and 291−317 nm were seen. Over the course of the titration these bands increased in intensity, while that of the original absorption features at 272− 291 nm decreased. Three isosbestic points, at 272, 291, and 317 nm, respectively, were observed under these conditions. The absorption intensity at 295 nm reached a maximum value (Figure S19) when the guest/host ratio was ∼2.0, further supporting the inferred 1:2 stoichiometric ratio ([1]/[SO42−]). When the fractional saturation of receptor 1 (y = ΔA/ΔAmax) is plotted as a function of sulfate concentration, a sigmoidal binding isotherm is obtained (Figure S20). The UV−vis data were thus fitted to the Hill equation,18 defined as log(y/1 − y) = n log[G] + log K, where y is the fractional saturation of the host 1 and K and n are the association constant and the Hill coefficient, respectively. This fitting allowed log K and n to be calculated as 15.2 ± 0.7 and 3.3 ± 0.2, respectively. A diffraction-grade single crystal was obtained by slowly evaporating a DCM/CH3OH/CH3CN solution of 1 in the presence of TMA2SO4. The resulting structure confirmed the expectation that in the solid state two sulfate anions are bound concurrently within the large cavity defined by 1 (Figure 2). Each sulfate anion is bound to the NH protons of a single calix[4]pyrrole subunit and the adjacent amide moieties. The two cobound sulfate anions are linked by two water molecules via multiple hydrogen bonding interactions. The net result is the bridged sulfate dimer structure seen in the solid state. The
presence of these bridging water molecules serves to increase the distance between the cobound sulfate anions and, along with the specific hydrogen bonding interactions they provide, serves to offset partially what would otherwise be destabilizing electrostatic repulsions between the individual sulfate anions.12 Four TMA counter cations, which may also serve to enhance the sulfate binding energetics, are found per complex, albeit not within the cavity. They balance the charge and confirm the bound anion is SO42−, rather than HSO4− (Figure S21). Pyrophosphate is inherently more structurally complex than dihydrogen phosphate or sulfate. It is also trianionic in its most common form. Initial evidence that receptor 1 was able to bind the HP2O73− anion came from a 1H NMR spectroscopic titration (Figure S22). Upon addition of increasing quantities of tris(tetrabutylammonium) hydrogen pyrophosphate (up to 30 equiv) to a solution of 1 in CD2Cl2/CD3OD (9/1, v/v), the βpyrrole proton signals gradually shift from 5.97 ppm in the free host to 5.73 ppm. These shifts are consistent with the presence of hydrogen-bonding interactions (N−H···O) between the pyrrole NH protons and the pyrophosphate oxygen atoms. In order to obtain more detailed insights into the proposed binding of HP2O73− by 1, UV−vis spectroscopic titrations were carried out in DCE. The incremental addition of TBA3HP2O7 into a solution containing receptor 1 (34.4 μM) resulted in appreciable changes in the absorption features (Figure 3a).
Figure 3. (a) UV−vis titration of the addition of TBA3HP2O7 to a solution of 1. Insert: Two isosbestic points at 270 and 294 nm were observed in the presence of 0 to ≤1.0 equiv of HP2O73−. (b) Observed binding profile recorded at 313 nm; (c) DFT optimized structure of (HP2O73−)2⊂1 complex.
Two isosbestic points at 270 and 294 nm were observed in the presence of ≤1 equiv of HP2O73−. These two isosbestic points disappeared upon further addition of guest, as would be expected under conditions where a second binding process is operative. The absorption intensity of the band at 313 nm increased until 1 equiv of pyrophosphate was added. Upon further addition, the intensity of this band underwent a decrease until reaching a limiting value following the addition of ∼5 equiv (Figure 3b). These findings are taken as evidence that a 1:2 complex is being formed between receptor 1 and HP2O73− under the conditions of the titration. A possible structure for the (HP2O73−)2⊂1 complex was deduced from DFT calculations carried out at the x3lyp/3-21g level (Figure 3c). On the basis of these modeling studies, we
Figure 2. Single crystal structures of the (SO42−)2·(H2O)2⊂1 complex: (a) Front view; (b) side view. 7142
DOI: 10.1021/jacs.7b02329 J. Am. Chem. Soc. 2017, 139, 7140−7143
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Journal of the American Chemical Society
(6) (a) Sessler, J. L.; Gale, P. A.; Cho, W.-S. In Anion receptor chemistry; Stoddart, J. F., Ed.; Royal Society of Chemistry: Cambridge, U.K., 2006. (b) Busschaert, N.; Caltagirone, C.; Van Rossom, W.; Gale, P. A. Chem. Rev. 2015, 115, 8038. (7) (a) Gale, P. A.; Dehaen, W. Anion Recognition in Supramolecular Chemistry; Springer: Heidelberg, 2010. (b) Ravikumar, I.; Ghosh, P. Chem. Soc. Rev. 2012, 41, 3077. (c) Caltagirone, C.; Hiscock, J. R.; Hursthouse, M. B.; Light, M. E.; Gale, P. A. Chem. - Eur. J. 2008, 14, 10236. (d) Basu, A.; Das, G. Dalton Trans. 2012, 41, 10792. (e) Dey, S. K.; Das, G. Dalton Trans. 2012, 41, 8960. (8) (a) Arunachalam, M.; Ghosh, P. Org. Lett. 2010, 12, 328. (b) Mason, S.; Clifford, T.; Seib, L.; Kuczera, K.; Bowman-James, K. J. Am. Chem. Soc. 1998, 120, 8899. (c) Saeed, M. A.; Fronczek, F. R.; Huang, M. J.; Hossain, M. A. Chem. Commun. 2010, 46, 404. (9) (a) Light, M. E.; Gale, P. A. CCDC 1491437: Experimental Crystal Structure Determination, 2016, DOI: 10.5517/ ccdc.csd.cc1m1ywh. (b) Pandurangan, K.; Kitchen, J. A.; Blasco, S.; Boyle, E. M.; Fitzpatrick, B.; Feeney, M.; Kruger, P. E.; Gunnlaugsson, T. Angew. Chem., Int. Ed. 2015, 54, 4566. (10) (a) Katayev, E. A.; Sessler, J. L.; Khrustalev, V. N.; Ustynyuk, Y. A. J. Org. Chem. 2007, 72, 7244. (b) Rudkevich, D. M.; Verboom, W.; Brzozka, Z.; Palys, M. J.; Stauthamer, W. P. R. V.; Vanhummel, G. J.; Franken, S. M.; Harkema, S.; Engbersen, J. F. J.; Reinhoudt, D. N. J. Am. Chem. Soc. 1994, 116, 4341. (c) Lakshminarayanan, P. S.; Ravikumar, I.; Suresh, E.; Ghosh, P. Chem. Commun. 2007, 5214. (d) Bregovic, N.; Cindro, N.; Frkanec, L.; Uzarevic, K.; Tomisic, V. Chem. - Eur. J. 2014, 20, 15863. (11) Hoque, M. N.; Manna, U.; Das, G. Supramol. Chem. 2016, 28, 284. (12) Fatila, E. M.; Twum, E. B.; Sengupta, A.; Pink, M.; Karty, J. A.; Raghavachari, K.; Flood, A. H. Angew. Chem., Int. Ed. 2016, 55, 14057. (13) (a) Saha, I.; Lee, J. H.; Hwang, H.; Kim, T. S.; Lee, C. H. Chem. Commun. 2015, 51, 5679. (b) Valderrey, V.; Escudero-Adan, E. C.; Ballester, P. J. Am. Chem. Soc. 2012, 134, 10733. (14) Voss, N. R.; Gerstein, M. Nucleic Acids Res. 2010, 38, W555. (15) (a) Light, M. E.; Camiolo, S.; Gale, P. A.; Hursthouse, M. B. Acta Crystallogr., Sect. E: Struct. Rep. Online 2001, 57, o727. (b) Light, M. E.; Gale, P. A.; Hursthouse, M. B. Acta Crystallogr., Sect. E: Struct. Rep. Online 2001, 57, o705. (16) (a) Oksanen, E.; Ahonen, A. K.; Tuominen, H.; Tuominen, V.; Lahti, R.; Goldman, A.; Heikinheimo, P. Biochemistry 2007, 46, 1228. (b) Heikinheimo, P.; Tuominen, V.; Ahonen, A. K.; Teplyakov, A.; Cooperman, B. S.; Baykov, A. A.; Lahti, R.; Goldman, A. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 3121. (17) Thordarson, P. Chem. Soc. Rev. 2011, 40, 1305. (18) Hill, A. V. J. Physiol. (Oxford, U. K.) 1910, 40, 190.
conclude that the cavity of 1 is large enough to accommodate two HP2O73− concurrently, with the resulting 1:2 complex being stabilized by multiple (N−H···O−P) hydrogen bonding between the NHs of 1 and two HP2O73−. This conclusion is supported by a preliminary single crystal structure (Figures S23−S24). ITC analyses of the interactions between 1 and (TBA)3HP2O7 were carried out in DCE (Figure S25). The ITC data were fitted to the theoretical isotherm derived from the sequential sites binding model. This allowed the binding constants to be estimated as K1 = (6.52 ± 0.56) × 104 M−1 and K2 = 49.0 ± 0.56 M−1. In conclusion, we detail here a new synthetic strategy for the construction of bis-calix[4]pyrroles. Receptor 1 was found to concurrently trap two monovalent H2PO4−, two dianionic SO42−, and two trianionic HP2O73− anions. The ubiquity of related complexation events in the context of enzymatic transformations leads us to propose that systems such as the one described here could serve as useful structural models that may help increase our understanding of natural oxoanion recognition processes.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b02329. Synthetic details, NMR, UV−vis and ITC titration, DFT calculation, and X-ray structural data for 1and its complexes (PDF) Crystallographic data for free 1 (CIF) Crystallographic data for 7 (CIF) Crystallographic data for (H2PO4−)·H2O⊂1 (CIF) Crystallographic data for (SO42−)2·(H2O)2⊂1 (CIF) Crystallographic data for (H2P2O72−)2⊂1 (CIF)
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AUTHOR INFORMATION
Corresponding Author
*
[email protected] ORCID
Steffen Bähring: 0000-0001-5113-2458 Jonathan L. Sessler: 0000-0002-9576-1325 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The work in Austin was supported by the National Institutes of Health (Grant No. GM103790 to J.L.S.), the Robert Welch Foundation (chair funds to J.L.S.), and The Danish Council for Independent Research, Technology, and Production Sciences (FTP, Project 5054-00052 to S.B.).
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REFERENCES
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DOI: 10.1021/jacs.7b02329 J. Am. Chem. Soc. 2017, 139, 7140−7143
