Conformational Selection in Anion Recognition: cGMP-Selective

May 22, 2019 - Structure of Macrocycles in Solid and Solution States ..... In summary, chromogenic receptor 3 was successfully synthesized via [2+2] ...
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Cite This: J. Org. Chem. 2019, 84, 9034−9043

Conformational Selection in Anion Recognition: cGMP-Selective Binding by a Naphthalimide-Functionalized Amido-Amine Macrocycle Aleksandr S. Oshchepkov,†,‡ Tatiana A. Shumilova,† Mario Zerson,† Robert Magerle,† Victor N. Khrustalev,‡,§ and Evgeny A. Kataev*,† †

Faculty of Natural Sciences, Technische Universität Chemnitz, Chemnitz 09107, Germany Peoples’ Friendship University of Russia (RUDN University), Moscow 117198, Russia § National Research Center (Kurchatov Institute), Moscow 123098, Russia

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S Supporting Information *

ABSTRACT: Amido-amine macrocycles with two and four naphthalimide dyes were designed to bind nucleoside monophosphates and oligonucleotides in an aqueous buffered solution. Anion-templated synthesis was used to direct the macrocyclization reaction to the [2+2] product, while high dilution conditions favored the formation of the [4+4] macrocycle with an unprecedented geometry, as revealed from the X-ray analysis. The [2+2] product was found to exhibit a remarkable binding strength and fluorescence response for cyclic guanosine monophosphate (cGMP) in an aqueous solution. To our knowledge, this is the first synthetic receptor for cGMP, which also demonstrates a high preference to bind guanine-rich sequences accomplished by a strong fluorescence quenching. The receptor conformation is very sensitive to the guest structure in an aqueous solution, thus modeling the adaptive behavior of proteins. The study of synthetic systems with a detectable conformational equilibrium represents a great potential for understanding highly specific and tightly regulated interactions in biological systems.

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INTRODUCTION Nucleotides are important constituents of a living cell that have a broad range of functions. They work as chemical messengers, energy carriers, and cofactors and participate in phosphorylation reactions that control the activity of enzymes.1 Finding new synthetic hosts for selective recognition of nucleotides in aqueous solution is of great importance for the development of the fluorescent-based assay and quantitative analysis of nucleotide levels in biological samples with confocal microscopy.2 For instance, protein-based cyclic adenosine monophosphate (cAMP)-selective probes have enabled the visualization of a number of biological processes.3 cGMP is another important second messenger that regulates many physiological processes, especially in cardiovascular and nervous systems.4 The receptors with a high binding selectivity for a specific nucleobase can be used as receptors/probes for abasic sites,5 short DNA sequences,6 and mismatched base pairs7,8 and as drugs targeting the specific regions of nucleic acids. Targeting RNA with such systems is a great challenge, since RNA is a single-stranded biopolymer and adopts various conformations such as hairpin loops and bulges with exposed nucleobases.9,10 Synthetic receptors for nucleoside monophosphates and cyclic monophosphates are of particular interest because they © 2019 American Chemical Society

are a part of RNA and DNA structure. As we have recently reported, if a synthetic receptor can bind a nucleoside monophosphate, it is likely that it will also bind the nucleobase residue in the structure of an oligonucleotide.11 There have been only a few selective synthetic receptors/probes reported that demonstrate selectivity for nucleoside monophosphates or cyclic monophosphates.12 For instance, Rebek and co-workers reported an elaborate design of a receptor that can bind cAMP in organic and aqueous solutions.13,14 The group of Kikuchi presented a Cd(II) complex, which has different fluorescence responses for AMP and cAMP in a buffered solution.15 Selective detection of these nucleotides was also demonstrated with the help of a naphthol-rhodamine conjugate16 and an urea-based receptor.17 Feng and co-workers have studied the recognition of GMP in an organic and aqueous solution by using a naphthyridine moiety, which forms complementary hydrogen bonds with guanine.18 One of the most selective receptors for AMP under a physiological pH was reported by Albrecht and co-workers, who synthesized a binuclear europium(III) complex. 19 The distance between two europium(III) centers in the complex perfectly matches the Received: April 5, 2019 Published: May 22, 2019 9034

DOI: 10.1021/acs.joc.9b00947 J. Org. Chem. 2019, 84, 9034−9043

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The Journal of Organic Chemistry

Figure 1. Synthesis of the [1+1] macrocycle (5), [2+2] macrocycle (3), [4+4] macrocycle (4), and reference compound 6.

strategy to achieve a high selectivity for a desired nucleotide. Interestingly, nature follows the same strategy and often separates binding pockets targeting the phosphate residue and the nucleobase. For example, recognition of the phosphate residue is usually achieved by a so-called p-loop motif,31 while the recognition of a nucleobase is realized by different specific interactions or combinations of them: (1) hydrophobic interactions with, e.g., Ala, Val, or Leu residues; (2) stacking interactions with the guanidinium, carboxylate, or phenyl groups; (3) hydrogen bonding. Stacking of a nucleobase with an aromatic ring is one of the most attractive interactions in terms of simplicity of the host design. Stacking interactions are considered as strong interactions in an aqueous solution32−34 and represent a key mechanism of aggregation-induced emission/quenching-based probes for nucleotides.35 With this idea in mind, we have attached naphthalimide dyes to an anion-binding macrocycle and thus combined a recognition motif for phosphate with the aromatic ring that can interact with a nucleobase. The core structure of the macrocycle is similar to that, which demonstrated excellent selectivity for phosphate.36,37 It is well-known that positively charged receptors bearing aromatic rings have a tendency to aggregate in the presence of anions.38 This knowledge has been implemented by a number of research groups in the design of aggregation-induced emission probes for e.g. phosphates and nucleotides.39 Recently, we have also reported a macrocyclic receptor that forms aggregates in the presence of inorganic anions except fluoride.40 The reason for such selectivity was the ability of the receptor to encapsulate two fluoride anions in the cavity of the macrocycle, while larger anions were bound outside of the receptor and thus induced aggregation in aqueous solution. On the basis of these findings, we have proposed that receptor 3, bearing a suitable cavity for binding of the phosphate anion, should form complexes with nucleoside monophosphates in aqueous solution more preferably than with nucleoside triphosphates. The triphosphates are larger than monophosphates and should demonstrate a different binding mode with the receptor.

distance between the phosphate residue and the nucleobase. Duan also used a lanthanide complex with a naphthyridine recognition motif to detect GMP in an aqueous medium and in vivo. However, a little progress has been made in the area of cyclic nucleoside monophosphate recognition. Systems that have a good selectivity for these guest molecules against noncyclic monophosphates and di- and triphosphates remain a great challenge. Herein, we report the synthesis and binding properties of amido-amine macrocycles obtained by [2+2] and [4+4] condensation reactions of a naphthalimide-functionalized diamine with diacid precursors. The [2+2] macrocycle demonstrates selective binding and a fluorescence response for cGMP over other nucleoside mono-, di-, and triphosphates. The macrocyclic core of the receptor is responsible for the recognition of the phosphate residue, while the naphthalimide fragment is able to intercalate with nucleobases and with aromatic rings of the receptor. An interesting feature of both [2+2] and [4+4] receptors is the ability to change their conformations depending on the guest structure. Binding of tetrahedral oxyanions and nucleoside monophosphates to the [2+2] macrocycle results in a rigid receptor conformation with a high complementarity for the guests. The conformation of the receptor is stabilized by intramolecular pyridine− naphthalimide (Py-Naph) stacks. This behavior resembles the conformational selection postulated for proteins to explain the protein−ligand-binding mechanisms.20−22

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RESULTS AND DISCUSSION Design and Synthesis. During the last two decades, the area of the nucleotide recognition has attracted great attention with the emphasis on sensing applications in cells.2,23 The highest selectivity for nucleoside monophosphates under physiological conditions has been achieved by using metalbased receptors.13,15,16,19,24−29 Our recent investigations11 and the reported synthetic receptors for nucleotides30 support the fact that a combination of two binding sites in one receptor (for the phosphate group and for a nucleobase) is a promising 9035

DOI: 10.1021/acs.joc.9b00947 J. Org. Chem. 2019, 84, 9034−9043

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The Journal of Organic Chemistry Receptor 3 was synthesized by the reaction of activated acyl 1 with fluorescent diamine 2 developed by us recently (Figure 1).41 First, the reaction was carried out with acid chloride 1a in highly diluted conditions. According to the ESI-MS and NMR spectra, three products could be isolated in low yields 3 ([2+2] condensation product, 10%), 4 ([4+4] product, 5%), and 5 ([1+1] product, 8%). In our previous work, we found that slowing down the reaction rate of the amide bond formation positively reflects on the product yields.42 The use of activated acyl 1b, which reacts slower than acid chloride 1a, resulted in a 5% yield of the [2+2] product and in a 10% yield of the [4+4] product. Next, we tested tetrabutylammonium salts TBAH2PO4, TBAHSO4, TBAI, TBABr, and TBACl as templates in the macrocyclization reaction. Sulfate and phosphate salts blocked the reaction completely because of their acidic nature, while chloride showed the strongest effect on the product distribution. With 10 equiv of TBACl, it was possible to obtain almost exclusively the [2+2] product (3) with a 40% yield. As can be seen below, the binding studies confirmed that receptor 3 coordinates chloride with a moderate affinity. This fact explains an excellent templating ability of the chloride anion in the formation of the [2+2] product. Structure of Macrocycles in Solid and Solution States. The macrocycles were isolated from the reaction as free amines and were crystallized from polar solvents. According to the single-crystal X-ray analysis of 3 crystallized from DMSO, the macrocycle has a nearly planar structure with two naphthalimide rings located at different sides of the plane and forming stacking interactions with pyridine rings (Figure 2a). There are also two DMSO (solvent) molecules, which are bound to the pyridine dicarboxamide sites through NH···O hydrogen bonds. It is likely that these NH sites participate in coordination of anions in a solution. Interestingly, two receptor molecules in the crystal lattice interact with each other through naphthalimide−naphthalimide stacking interactions (Figure 3), so that pyridine-naphthalimide−naphthalimide-pyridine (Py-Naph-Naph-Py) stacks are observed. Pyrinde−naphthalimide distances (3.3−3.4 Å) are slightly shorter than those observed for naphthalimide−naphthalimide (3.4−3.5 Å) interactions. To our surprise, the solid structure of the [4+4] condensation product possesses the same Py-Naph−Naph-Py stacks, which can be observed in the middle of the large cycle formed by 4 (Figure 2b). The other pyridine dicarboxamide sites are also involved in the interaction of naphthalimide rings and form hydrogen bonds with DMSO. We found an even more interesting organization of Py-Naph−Naph-Py stacks in the crystal structure of the [4+4] macrocycle crystallized from methanol. In this structure, the stacks are oriented perpendicular to each other (Figure 2c). The macrocycle does not possess any cavity because it is occupied by the aromatic rings. The four pyridine dicarboxamide sites are directed outward and coordinate methanol and water molecules through hydrogen bonds. Crystallization of two macrocycles with different anions as tetrabutylammonium salts or as acids was successful only for the mixture of 3 with an excess of sulfuric acid. The solid structure of 3·H2SO4 featured a perfectly organized cavity for sulfate binding. This structure is stabilized by the Naph-PyNaph stacking interactions. Interestingly, only three oxygen atoms of the sulfate anion participate in hydrogen bonding interactions with the macrocycle. Two oxygen atoms are bound to the pyridine dicarboxamide sites, and the third

Figure 2. Single-crystal structures of macrocycle (a) 3 crystallized from DMSO; macrocycle 4 crystallized from (b) DMSO, (c) MeOH; (d) complex 3·H2SO4 crystallized from a mixture of DMSO−H2O. Most hydrogen atoms and solvent molecules are omitted for clarity. Simplified representation with interaction of π-systems: 2,6pyridinedicarboxamide (blue) and naphthalimide (green) are also shown.

Figure 3. Intermolecular interactions between receptors found in the solid state for (a) 3·H2SO4 and (b) 4.

oxygen atom forms hydrogen bonds with the NH group at the fourth position of the naphthalimide moiety and with the protonated tertiary nitrogen. The fourth oxygen atom forms a hydrogen bond with the water molecule (Figure 2d). We 9036

DOI: 10.1021/acs.joc.9b00947 J. Org. Chem. 2019, 84, 9034−9043

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The Journal of Organic Chemistry consider this three-oxygen coordination mode as a very promising one in terms of recognition of phosphate monoesters and hence nucleoside monophosphates. A detailed analysis of all obtained crystal structures reveals that naphthalimide−pyridine interactions are energetically favorable in the solid state. They drive the packing of molecules on intra- and intermolecular levels. The intermolecular π−π interactions are present in all structures. As can be seen in Figure 3, the receptors pack either through naphthalimide−naphthalimide or pyridine−pyridine interactions. Since the sulfate complex of 3 is a poorly soluble compound in water and even in organic solvents, we expected that our receptors and complexes might be prone to aggregation in an aqueous solution. The crystallographic data suggest that this aggregation is caused by favorable interactions between aromatic rings. We also suggested that these interactions could be the reason for the formation of the [4+4] macrocycle in a relatively good yield under slow reaction rates. The crystallographic data of the receptor 3 obtained with and without sulfate suggest that the conformation of macrocycles in a solution can be different from that observed in the solid state. According to the 1H−1H ROESY spectrum of 3, there are no interactions between the naphthalimide and the pyridine ring in both DMSO-d6 and in DMSO-d6−D2O mixtures or the interactions are very weak (Supporting Information, Figures S1−S3). The addition of excess sulfuric acid to a DMSO-d6−D2O solution of 3 resulted in broad signals; therefore, NMR experiments of 3 with and without sulfuric acid did not allow us to identify the exact conformation of the receptor in a solution. On the contrary, we found interactions between the pyridine and naphthalimide moieties for receptor 4 in DMSO-d6 and in DMSO-d6−D2O mixtures. Thus, the conformation of 4 in a solution could be similar to those observed in the solid state (Figure 3). The addition of different acids to a solution of 4 also resulted in strong broadening of the signals and precipitation of the salts. To understand the protonation behavior in a solution, we measured fluorescence and UV−vis spectra at different pH values. Because of the low solubility of our receptors in water, the pKa values of tertiary amines were determined with the help of UV−vis measurements in aqueous solutions containing 2% of DMSO (Figure 4). For receptor 3, two protonation constants could be extracted: pKa1 = 3.4, pKa2 = 5.0. For receptor 4, the changes in the UV−vis spectra by changing the pH were much smaller, so we could only determine the average value for all protonation events (pKa = 4.3). The fluorescence−pH relationship in the presence of anions suggests that 5 has a small cavity and does not bind anions with a detectable affinity (Figure S6). Interestingly, the protonation constants of receptors have much lower values than those expected for tertiary amines. The reason for this could be the influence of the amide function, which is located in a close proximity to the tertiary amine. To clarify this fact, we prepared control compound 6 by acylation of diamine 2 with acetic anhydride. According to the UV−vis measurements at different pH values, 6 has a pKa value of 5.9. Thus, it is likely that the pyridine dicarboxamide site incorporated in the macrocyclic structure has a strong influence on the protonation equilibria of the tertiary amines. As can be seen in Figure 4, the protonation of macrocycles induces hypsochromic shifts of the absorption maximum.

Figure 4. UV−vis spectra of (a) 3, (b) 4, and (c) 6 depending on the pH of the solution. Conditions: 0.01 mM receptor, 2% of DMSO in Britton−Robinson buffers, I = 0.1 M.43

Reference compound 6 and receptor 3 demonstrate a 20 nm shift after full protonation, while receptor 4 shows only a 6 nm shift. These data indicate that a hypsochromic shift originates mainly from the protonation and not from the changes in the conformation of the receptors. The smaller shift of 4 can be explained in terms of more possible conformations of the tetraprotonated macrocycle in a solution. Binding Studies with Anions. To understand the binding properties of macrocycles 3 and 4 toward anions, we measured UV−vis and fluorescence spectra at different pH values in the presence and in the absence of sulfate, oxalate, and phosphate. The solutions with fixed pH values were generated from NaOH−acetic acid mixtures because receptors weakly interact with the acetate anion (log K < 2). As can be inferred from Figure 5a, receptor 3 has an unusual fluorescence−pH dependence with the inflection point at about pH 4.5. As expected from the pKa values obtained by UV−vis spectroscopy, the protonation of the macrocycle leads to an increase of the emission intensity because the protonation process blocks the photoinduced electron transfer (PET) from the tertiary amine to the naphthalimide dye. As we have shown recently, the coordination of anions induces the protonation of the 9037

DOI: 10.1021/acs.joc.9b00947 J. Org. Chem. 2019, 84, 9034−9043

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The Journal of Organic Chemistry

Figure 5. (a) Fluorescence intensity of receptor 3 at 540 nm in the presence of an excess of different anions depending on the pH of the solution. Content of the solution with different pH values: 0.01 mM receptor in 50 mM NaOH−acetic acid mixtures containing 2% DMSO and 1 mM anions as sodium salts. (b) Fluorescence titration of 3 with Na2SO4 and (c) with NaCl (added until 200 equiv, ex. 450 nm). Proposed conformational changes of 3 induced by coordination of chloride and sulfate.

amine due to the pKa shift, which results in a fluorescence enhancement.44,45 This is the behavior that we observe now for 3 between pH 4.5 and 5.5. However, test titrations of receptors 3 and 4 at pH 4.5 with anions and nucleotides led to aggregation and precipitation of the complexes. Therefore, we have decided to use a 50 mM acetate buffer with a pH of 3.6. At this pH value, both macrocycles are fully protonated (approximately 98%) and less prone to aggregation. An interesting feature of the [2+2] macrocycle is the fact that the addition of sulfate results in an increase of the Stokes shift of the naphthalimide fluorescence (Figure S7). The overall change in the Stokes shift is generated from the hypsochromic shift in the UV−vis spectrum and the bathochromic shift in the fluorescence spectrum. The hypsochromic shift in the UV−vis titration is similar to that, which was observed for naphthalimide probes;46 it is caused by the completion of protonation of both tertiary amines, as it is seen in Figure 5a. Interestingly, the addition of sulfate resulted in a 6 nm bathochromic shift of the emission spectrum (Figure 5b), while chloride (Figure 5c) and bromide induced 4 and 8

nm hypsochromic shifts, respectively (ESI). The bathochromic shift appears usually only if an anion forms a strong hydrogen bond with the NH group in the fourth position of the naphthalimide moiety or when the naphthalimide moiety is involved in π−π interactions.47 Indeed, these interactions were observed in the solid state structure of 3·H2SO4. Thus, it is likely that the structure of the complex in a solution is similar to that in the solid state (Figure 5). Further evidence of these specific interactions came from the 1H NMR titration of 3 with TBAHSO4, which was used as a source of sulfate to increase the solubility of the complex. The addition of 10 equiv of sulfate resulted in a downfield shift (0.2 ppm) of the NHproton signal (formation of the hydrogen bond with sulfate), while the pyridine CH proton signals shifted to a higher field (0.2 ppm) due to the π−π interactions with the naphthalimide moiety (Figure S5). On the basis of this data, we proposed that the hypsochromic shift of the emission maximum of receptor 3 indicates a loss of stacking interactions during chloride and bromide recognition. Receptor 3 may exist in an aqueous 9038

DOI: 10.1021/acs.joc.9b00947 J. Org. Chem. 2019, 84, 9034−9043

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Figure 6. Tapping-mode AFM images of receptor 3 aggregates prepared in an aqueous solution (pH 3.6, 50 mM acetate buffered, 2% DMSO) and deposited on a Si substrate. (a, b) 3 + Na2SO4 and (c, d) 3 + ATP. The zoomed-in phase images (b, d) are of the areas indicated in the corresponding height images (a, c).

Figure 7. Changes in fluorescence intensity of 3 at 540 nm observed during the addition of (a) nucleoside monophosphates and (d) tetranucleotides. Conditions: 50 mM acetate buffer (2% DMSO, pH 3.6, ex. 450 nm). (b) 1H NMR spectra of 3in the presence of an increasing amount of cGMP. Conditions: 1 mM receptor in a 1:1 mixture of DMSO-d6−D2O in the presence of 5 equiv of HClO4(pH 3); nucleotide additions 0−10 equiv. (c) DFT optimized structure of 3H22+ with cGMP− shown as a ball-and-stick model and as a 2D drawing representation with observed NOEs.

form complexes with structures similar to that observed in the crystal structure of 3·H2SO4. Binding Studies with Nucleotides and Oligonucleotides. Naphthylimides and naphthyldiimide are known to interact with nucleic acids through intercalation.49−51 Measurements of binding constants with di- and triphosphates (ADP, ATP, GTP, CTP, TTP, dTTP) were not possible due to the strong aggregation and precipitation during the titrations experiments. We compared the structures of the formed aggregates of 3 with sulfate and ATP by atomic force microscopy (AFM). AFM images show that 3 forms fibrillar and globular aggregates (Figure 6) with sulfate and ATP, respectively. In both cases, the fibrils are between 1−5 nm high and ≥20 nm wide; the globules are between 5−10 nm high and ≥40 nm wide. The height values are good estimates of the objects diameter since height differences are not affected by the convolution with the tip shape. In a control experiment without receptor 3 added, we observe no fibril formation. Therefore, we suggest that the fibril formation of receptor 3 is caused by π−π interactions between neighboring receptor units similar to those in the solid state. Presumably, ATP helps to connect receptor molecules to form larger aggregates, as compared to those observed with the sulfate anion.

solution in a form where at least one naphthalimide moiety interacts with the pyridine ring and binding of chloride or bromide favors the conformation of 3 that has no intramolecular contacts. On the contrary, the binding of selected oxyanions induces a conformational switching of the receptor favoring intramolecular π−π interactions. The binding mode discovered for 3 with the sulfate anion can also explain the spectroscopic changes of receptor 4 upon the addition of sulfate, where an even stronger, 10 nm bathochromic shift is detected in fluorescence titrations with sulfate. Since receptor 4 possesses pyridine−naphthalimide π−π interactions in a solution (according to NMR studies), two sulfate anions seem to bind with the receptor. The sulfate anion forms hydrogen bonds including the one with the NH group of the naphthalimide moiety and shifts the conformational equilibrium of the receptor to that with more intramolecular π−π contacts. This was evidenced from the bathochromic shift observed for 4 in the presence of sulfate. Binding properties of receptors 3 and 4 toward anions were determined by NMR, UV−vis, and fluorescence titrations by fitting the whole spectrum changes with the HypSpec program.48 Receptor 3 binds sulfate, phosphate, and oxalate with high binding constants (log K values are 4.41, 4.32, and 4.25, Table S2). Thus, we suggested that sulfate and phosphate 9039

DOI: 10.1021/acs.joc.9b00947 J. Org. Chem. 2019, 84, 9034−9043

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The Journal of Organic Chemistry According to the UV−vis and fluorescence titration data (Table S1), receptor 3 has the highest affinity for cGMP (log K = 4.91) among other nucleoside monophosphates (AMP, 4.40; cAMP, 4.40; GMP, 4.31; TMP, 4.43; CMP, 4.35; UMP, 4.38). cGMP demonstrates the strongest fluorescence quenching (Figure 7a). It is known that, in the complexes with dyes, guanine possesses the strongest quenching ability among other nucleobases.52 Thus, we suggest that in complex of 3H22+ with cGMP the guanine base is ideally located to form stacking interactions with naphthalimide. These interactions, in turn, lead to the strongest quenching effect. The receptor binds nucleoside monophosphates in a 1:1 stoichiometry, as revealed from the fitting analysis and Job plots (Figure S8).53,54 We also observed the second binding event in some cases, which was very weak and could not be accurately assessed. In both UV− vis and fluorescence titrations, a small bathochromic shift with a maximum value of 10 nm for cGMP was observed. This shift serves as additional evidence for the interaction of the naphthalimide dye with nucleobases. The support for this fact was obtained from the 1H NMR titration of 3 with cGMP (Figure 7b), as well as from the 1H−1H ROESY experiment. The addition of nucleotides induced downfield shifts of the guanine proton signals, indicating the existence of a stacking interaction of nucleobases with naphthalimide. The evidence for the naphthalimide π−π interactions was obtained from the 1 H−1H ROESY NMR experiments of 3 in the presence of 5 equiv of cGMP (Figure S4). Proton Hd of the naphthalimide moiety showed cross signals with the purine ring proton HG1 and the sugar protons. Thus, it is suggested that the imidazole ring is oriented in space parallel to the naphthalimide directed to the amine substituent. A possible structure of the cGMP complex with receptor 3 was deduced from DFT calculations carried out at the BP86/ def2-TZVP level with the D3 dispersion correction (Figure 7c).55,56 The obtained structure corroborates our spectroscopic measurements. The NH group of the naphthalimide moiety is involved in hydrogen bonding with the phosphate group. This interaction is responsible for the bathochromic shift observed in the fluorescence titration of 3 with both sulfate and cGMP. A close proximity in space of protons HG1 (guanine) and Hd (naphthalimide) is in a good agreement with the results of the ROESY experiment. The ability of receptor 3 to differentiate between nucleobases in the oligonucleotide chain was also tested with tetranucleotides A4 (5′-AAAA-3′), G4, C4, and T4. These nucleotides contain three phosphate residues similar to nucleoside triphosphates. However, as inferred from the test measurements, they do not form precipitates upon interaction with the receptor. According to Job plots, a 1:1 binding stoichiometry was observed. Fluorescence measurements revealed that receptor 3 has a preference to bind the tetranucleotide with guanine, demonstrating an exceptionally high binding constant of log K > 8 (Figure 7d). Coordination of G4 to the receptor is approximately 10 times stronger than that for other nucleotides. The binding is accomplished with a strong quenching (3-fold) and a 10 nm bathochromic shift. This shift again suggests a strong naphthalimide−guanine interaction. Thus, it is likely that the interaction of our systems with nucleotides is due to the intercalation and electrostatic interactions. While A4 (log K = 7.3) and T4 (log K = 6.1) demonstrated a lower affinity, the coordination of C4 was significantly weaker (log K = 4.0). Analysis of the binding data obtained for nucleoside monophosphates and tetranucleotides

leads to the conclusion that the selectivity for guanine was retained. The larger macrocycle (4) demonstrated small changes in the emission spectra (Figure S8) upon increasing amounts of tetranucleotides. The titration data could not have sufficient precision by using a simple binding model, suggesting that receptor 4 retains its packed conformation, which results in nonselective binding.

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CONCLUSIONS In summary, chromogenic receptor 3 was successfully synthesized via [2+2] macrocyclization reactions. The larger [4+4] macrocycle was found to be a major product when the reaction was carried out at high dilution conditions. Binding studies revealed that 3 possesses a moderate selectivity for cGMP over inorganic anions and other nucleoside monophosphates. Strong quenching by cGMP, a bathochromic shift in UV−vis titrations, ROESY NMR experiments, and DFT calculations suggest the optimal orientation of the guanine and naphthalimide rings to form a stacking interaction in the complex and be responsible for the observed selectivity. Recognition of cGMP by 3 is accomplished by similar changes in UV−vis and fluorescence spectra as those detected for sulfate. Thus, we suggested that 3 adopts a similar conformation to that of the sulfate complex involving pyridine−naphthalimide−pyridine−guanine stacking interactions. These conformational changes upon anion recognition are similar to the conformational selection postulated for proteins to explain the protein−ligand-binding mechanisms. Conformational selection postulates that the proteins exist in different conformations, and the ligand selects the most favorable one to achieve a tight binding. The study of the synthetic systems with a detectable conformational equilibrium could shed light on the mechanisms of highly specific and tightly regulated interactions in biological systems. The design of new systems containing different dye molecules in the macrocyclic structures is underway. The results of this work represent the first step toward the design of selective receptors for nucleoside monophosphates, which have a great potential in the construction of sequence-specific binders.

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EXPERIMENTAL SECTION

All solvents were dried according to standard procedures. The reactions were performed in oven-dried round-bottom flasks. Crude products were purified by column chromatography on silica gel (100−200 mesh). TLC plates were visualized by exposure to ultraviolet light and/or by exposure to acidic ethanolic solution of ninhydrin followed by heating (