Please do not adjust margins This document is the Accepted Manuscript version of a Published Work that appeared in final form in Royal Society of Chemistry © peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.rsc.org/en/content/articlelanding/2021/qo/d1qo00517k ARTICLE Supramolecular fluorescence sensing of L-proline and L-pipecolic acid Received 00th January 20xx, Accepted 00th January 20xx Andrés Felipe Sierraa,b, Gemma Aragaya, Guillem Peñuelas-Haroa and Pablo Ballestera,c* DOI: 10.1039/x0xx00000x The current library of synthetic molecular sensors for small polar molecules is limited. In this work, we describe the synthesis of two diastereomeric mono-phosphonate calix[4]pyrrole cavitands, 1in and 1out, acting as fluorescent sensors for amino acids. The two isomeric cavitands differ in the relative orientation, in (1in) and out (1out), of their P=O bridging group and the N-phenyl-naphthalamine fluorophore directly attached to it, with respect to their polar aromatic cavities. Using 1H and 31P NMR spectroscopy and non-fluorescent cavitand analogues (5in and 5out), we demonstrate the formation of 1:1 complexes with L-proline (L-Pro) and the relevance of the inwardly directed P=O group in the binding of the amino acid. Only the L-ProÌ5in complex establishes a charged hydrogen bonding interaction between the receptor P=O group and the protonated amine of the bound zwitterionic amino acid. This interaction is responsible for an increase in the thermodynamic stability of the complex compared to the L-ProÌ5out counterpart. We investigate the binding properties of the fluorescent cavitands, 1in and 1out, with L-Pro and L-pipecolic acid (L-Pip) at micromolar concentration using emission spectroscopy (direct binding-based sensing, BBS). The observed emission changes in the BBS experiments were small but evidenced the role of the cavitands as fluorescent sensors. In agreement with the millimolar concentration results (1H NMR experiments), the fluorescent 1in sensor displays a larger binding affinity for L-Pro than the 1out isomer. Conversely, the 1out isomer experienced larger emission changes upon amino acid binding. We develop FRET-based indicator displacement assays (IDA) owing to the small emission changes observed in the direct BBS experiments. At micromolar concentration, the competitive displacement of the quencher N-oxide 6 from the cavity of the 1:1 supramolecular ensemble (6Ì1in and 6Ì1out), by L-Pro, L-Pip, and L-phenylalanine (L-Phe) produced fluorescence “turn- on”. The results of the BBS and IDA experiments assigned a binding selectivity to the 1in isomer for L-Pro. for the design of synthetic selective molecular sensors, as well Introduction as for limiting the interferences caused by non-specific There is an increasing demand in monitoring small polar binding. Specific binding, a.k.a molecular recognition, builds on molecules, which are relevant for disease diagnosis and shape, size and function complementarity between receptor training status, using portable and even wearable sensing and analyte. In many cases, the transduction mechanism of the devices.1 Ideally, the developed sensing devices should be binding event demands the covalent incorporation of reporter designed for direct manipulation by end-users. This units to the receptor’s scaffolds i.e. the fluorescence, characteristic avoids the intervention of trained medical absorbance or redox properties of the receptor itself are not personnel and the commute to point-of-care facilities or suitable for transduction in practical applications. The hospitals. Moreover, the devices might transmit the results constructs resulting from the covalent connection of a wirelessly to apps installed in mobile phones and share them synthetic receptor to a reporter unit are referred as molecular with the electronic patient’s record or the clinician.2 Biological sensors. Molecular sensors are also key in the design of and synthetic receptors are fundamental components of many selective sensor nanomaterials able to discriminate analytes by small molecule sensing devices related to human health.3,4 The molecular structure (specific binding) rather than by principle at work is supramolecular sensing, which relies on physical/chemical properties i.e. polarity (non-specific transduction mechanisms exclusively activated by molecular binding). recognition events.5,6 This approach offers significant benefits Phosphonate calix[4]pyrrole cavitands are synthetic molecular receptors displaying one or more phosphonate bridging groups at the upper rim of “four wall” aryl-extended calix[4]pyrrole aInstitute of Chemical Research of Catalonia (ICIQ), The Barcelona Institute of scaffolds. In previous studies, we described the use of these Science and Technology (BIST), Avgda. Països Catalans 16, 43007 Tarragona, Spain. receptors for the selective recognition of ion-pairs and small E-mail: pballester@iciq.es bUniversitat Rovira i Virgili (URV), Departament de Quıḿica Analıt́ica i Quıḿica polar neutral molecules.7,8 We showed that the mono- Orgànica, c/Marcel·lí Domingo, 1, 43007 Tarragona, Spain phosphonate calix[4]pyrrole cavitand 5in (Figure 1) provided a cICREA, Passeig Lluís Companys 23, 08018 Barcelona, Spain Electronic Supplementary Information (ESI) available: [synthetic procedures, characterization data, UV-Vis and fluorescence titrations, energy minimized structures. CCDC 2053978 and 2053980]. See DOI: 10.1039/x0xx00000x Please do not adjust margins Please do not adjust margins Journal Name ARTICLE scaffolds.13 We also report the binding properties of mono- phosphonate calix[4]pyrrole cavitands and their fluorescent derivatives with a reduced series of amino acids: L-proline (L- Pro), L-pipecolic acid (L-Pip) and L-phenylalanine (L-Phe). Using a direct binding-based sensing (BBS) approach, we determined that the binding constant of the fluorescent 1in isomer for L- Pro was one order of magnitude larger than that of L-Pip. Surprisingly to us, the direct BBS experiments of L-Pro and L- Pip with the 1out isomer produced larger changes in emission intensity in comparison to those of the 1in counterpart. Nevertheless, the binding constant values determined for the 1:1 inclusion complexes of 1out and the amino acids were one order of magnitude smaller than those of the 1in isomer. Due to the small changes observed with the direct BBS approach, we considered that an improved fluorescent sensing of the amino acids required the development of indicator displacement assays (IDA).14 To this end, we used the pyridine- N-oxide 6 as analogue of the well-known DABCYL (4- dimethylaminoazo)benzene-4-carboxylic acid) black-hole quencher (Figure 1). Pyridine N-oxide 6 formed thermodynamically and kinetically highly stable 1:1 non- emissive complexes with 1in and 1out. The displacement of 6 from the 1:1 complexes produced fluorescence “turn-on”. The BBS and IDA experiments produced analogous binding constant values for all complexes. They assigned a superior stability to the L-ProÌ1in complex compared to L-PipÌ1in and Figure 1. Line drawing molecular structures of the monophosphonate calix[4]pyrrole L-PheÌ1in analogues, and the 1out counterparts. The cavitands 1in/1out and 5in/5out, the pyridine N-oxide quencher 6, diethyl 6- obtained binding results are supported by direct inspection of (phenylamino)naphthalene-2-phosphonate 7 and the substrates used in the work (L- Pro, L-Pip and L-Phe). Proton assignment of calix[4]pyrrole cavitands and amino acids is the molecular interactions present in the energy-minimized shown in the structure. structures of the inclusion complexes. three-dimensional polar aromatic cavity suitable for including creatinine and surrounding most of its surface.9 Results and discussion The receptor’s aromatic cavity is functionalized with an Synthesis inwardly directed phosphonate group at its open upper rim The fluorescent phosphonate calix[4]pyrrole cavitands 1in and and by four pyrrole NHs at the opposed and closed end. These 1out were prepared in two synthetic steps starting from the polar groups offer complementary hydrogen bonding donor known α,α,α,α-isomer of tetra-meta-hydroxyphenyl-tetra- and acceptor interactions to those of the included guest. We methyl-calix[4]pyrrole 2 (Scheme 1).7 Firstly, the mono- used the mono-phosphonate calix[4]pyrrole cavitand scaffold methylene bridged calix[4]pyrrole 3 was obtained by reacting for the construction of ion selective electrodes, molecular α,α,α,α-2 tetrol with 1.2 equiv. of bromochloromethane in sensors and indicator displacement assays for creatinine DMSO solution in the presence of potassium carbonate as sensing and quantification.10,11 More recently, we base. The mono-methylene bridged compound 3 was isolated demonstrated that the mono-phosphonate calix[4]pyrrole in 48 % yield after column chromatography purification of the cavitand was also an effective synthetic carrier facilitating the reaction crude and crystallization of the isolated fraction in selective diffusion of L-proline across membranes of liposomes acetonitrile. Secondly, the incorporation of the fluorescent and living cells.12 L-Proline is also included in the aromatic unit at the upper rim of 3 involved the room temperature cavity of the mono-phosphonate receptors establishing reaction with freshly prepared 6-(phenylamino)-naphthalen-2- multiple charged hydrogen-bonds (carboxylate-pyrrole NHs, yl phosphonic acid dichloride 4,13 in THF solution during 2h and ammonium-phosphonate) and CH-π interactions. using triethylamine as base. The reaction produced a mixture Herein, we describe the synthesis of two novel fluorescent of the two mono-phosphonate diastereoisomers 1in and 1out. molecular sensors based on a mono-phosphonate The two pure stereoisomers were isolated by separation of an calix[4]pyrrole cavitand scaffold. The introduced approach enriched fraction of the reaction crude (see SI for details) by hinges on the covalent attachment of the fluorescent signalling means of analytical HPLC (Waters Spherisorb®, 5.0 μm Silica, unit directly to the phosphonate bridging group. The design is 4.6 mm × 250 mm) using isocratic elution (DCM:AcOEt 90:10). inspired by the work of Dalcanale and co-workers with mono- phosphonate fluorescent cavitands based on resorcin[4]arene This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 2 Please do not adjust margins Please do not adjust margins Journal Name ARTICLE H HO OH HO O N HO OH HO O Br Cl O P K2CO H 3 H Et3N, THF O O H O O H N H N H N H N N DMSO N H N N O H Cl P H N H N H Cl 2 3 N N N Cl H Ph Et3N 4 P Cl H O THF N 1in + 5in + 5out O O P O O O H H N H N H N N 1out Scheme 1. Synthetic scheme for the preparation of the isomeric mono-phosphonate mono-methylene fluorescent calix[4]pyrrole cavitands 1in and 1out and non- fluorescent analogues 5in and 5out. The 1out isomer eluted first. The 1in isomer, presenting the resorcin[4]arene out isomer directs the fluorescent substituent P=O group inwardly oriented with respect to its aromatic towards the receptor’s cavity experiencing the shielding effect cavity, was more polar and eluted second. This latter exerted by the aromatic rings. arrangement of functional groups should allow that suitable We did not observe significant chemical shift changes for the bound guests can establish simultaneous hydrogen-bonding signals of the hydrogen atoms of the fluorescent substituent when comparing the 1H NMR spectra of the 1in and 1out intermolecular interactions with both the P=O group and the isomers in acetone-d6 or dichloromethane-d2 solutions (Figure NHs of the calix[4]pyrrole core of the fluorescent receptor. The 3). This finding indicated that in agreement with the solid-state isolated 1out isomer will be used as a control to validate this structures, in solution, the two isomers of 1 also featured the hypothesis. fluorescent substituent outwardly directed with respect to The configurational assignment of the two diastereoisomeric their aromatic cavities. However, the signals for H8 and H7 cavitands, 1in and 1out, was achieved by a combination of 1H, (Figure 1), corresponding to the meso-aryl hydrogen atoms 31P NMR spectroscopy and X-ray crystallographic analysis. that are ortho with respect to the bridging phosphonate group, Single crystals of 1in grew from a deuterated acetone solution showed very different chemical shift values in the two isomers. used to acquire its NMR spectra. On the other hand, we used For example, in the 1out isomer H8 resonates significantly acetonitrile to obtain single crystals of 1out. The solid-state structures of the two fluorescent calix[4]pyrrole isomers are depicted in Figure 2. In both of them, the calix[4]pyrrole core adopts the cone conformation and one molecule of the solvent, used to grow the crystals, is included in its polar aromatic cavity. The included solvent forms four simultaneous hydrogen-bonding interactions between its heteroatom (oxygen or nitrogen) and the pyrrole NHs. In the solid state and for the two isomers, the 14-membered rings delineated by the bridged phosphonate-group, two meso-phenyl groups, their corresponding meso-carbons and one pyrrole ring, present a conformation locating the phenyl-amino-naphthyl substituent of the phosphorous atom in equatorial orientation and pointing away from the aromatic cavity. The outwardly oriented fluorescent unit, which is observed in both solid-state structures of the calix[4]pyrrole diastereoisomers, is in striking contrast to the observation made in structurally related isomers of mono-phosphonate resorcin[4]arene cavitands.13 In the latter case, the 1H NMR spectrum of the out isomer showed the protons assigned to Figure 2. Solid-state structures of the two diastereoisomeric fluorescent receptors, 1in the phenyl-amino-naphthyl substituent upfield shifted (a) and 1out (b). The structures of the receptors are shown in ORTEP view with thermal compared to those of the in counterpart. This difference was ellipsoids set at 50% probability. Hydrogens are shown as fixed-size spheres of 0.3 Å attributed to dissimilar orientations of the substituent with radius. The included solvent molecules (acetone and acetonitrile) are displayed as respect to the receptor’s aromatic cavity. In short, the space-filling models. Please do not adjust margins Please do not adjust margins Journal Name ARTICLE The signals corresponding to the pyrrole NHs were the ones experiencing the most important chemical shift changes. These signals moved downfield compared to those in free 1in (Δδ = 1.8-1.5 and 2.3-1.1 ppm, for L-Pro and L-Pip, respectively), suggesting their involvement in hydrogen bonding interactions with the corresponding included guest. On the other hand, we observed the appearance of a new set of signals in the upfield region of the spectra. These signals were indicative of the inclusion of the guests in the polar aromatic cavity of 1in. Specifically for L-Pro extraction, we observed a broad signal centred at d = 2.7 ppm. We attributed this signal to the proton α to the carboxylate group of the bound L-Pro. This signal appeared upfield shifted compared to that of the free guest in Figure 3. Selected regions of the 1H (400 MHz, 298 K) spectra of 1in (a) and 1out (b) (CD ) SO solution (Δδ = -1.2 ppm). Thus, the proton atoms of isomers in acetone-d6 solution. The pyrrole NH, β-pyrrole, H11 and the two proton 3 2 signals of the meso-aryl groups, H and H , that are ortho with respect to the the included L-Pro experienced the shielding effect exerted by 8 7 phosphonate bridging groups are indicated. See Figure 1 for proton assignment. the four meso-phenyl substituents of 1in. Analogously, the signals of bound L-Pip also appeared upfield shifted compared downfield shifted compared to the chemical shift value for the to those in the free guest in (CD3)2SO solution. The integral values of selected proton signals for the host and the guest analogous proton signal in the 1in counterpart (Figure 3). This indicated the quantitative formation of 1:1 complexes: L- chemical shift difference is due to the positioning of H8 in the Pro⸦1in and L-Pip⸦1in. That is, receptor 1in extracted 1 equiv. deshielding zone of the magnetic anisotropy cone generated of L-Pro and L-Pip in the independent solid-liquid extractions by the out P=O group. For the same token, proton H7 becomes experiments. partially deshielded when the P=O group is inwardly directed. The binding of the guest was also supported by the chemical The chemical shift values of the phosphorous atoms of the 1in shift changes observed in the 31P NMR spectra of the filtered and 1out isomers are in agreement with those observed for solutions. The 31P NMR spectrum of free 1in shows a singlet structurally related mono- and bis-phosphonate calix[4]pyrrole resonating at δ = 17.3 ppm. Remarkably, after the extraction cavitands (Figure S12 and S20).7,9 The phosphorous atom of experiments of L-Pro and L-Pip, the phosphorous signal of the out isomer resonates slightly upfield, possibly due to the bound 1in resonated at d = 17.0 ppm and 17.9 ppm, shielding effect exerted by the aromatic cavity. In acetone-d respectively. (Figure 4 right). 6 Next, we performed a competitive solid-liquid extraction solution, both isomers showed three highly downfield shifted experiment by adding equimolar amounts of solid L-Pro and L- signals for the pyrrole NHs. This is due to the involvement of Pip (~2 mg of each guest) to a millimolar CD2Cl2 solution of the the NHs in hydrogen bonding interactions with the oxygen fluorescent receptor 1in. The 1H NMR spectra of the filtered atom of one included acetone molecule. The bound acetone solution clearly showed two sets of proton signals for the NHs molecule locks the cavitands in their cone conformation as of bound 1in. Moreover, the integral values of the two sets of observed for 1in in the solid-state (Figure 2a). The 1H-NMR NH signals were significantly different (Figure 4d). By spectra of the two isomers in chlorinated solvents (vide infra) comparison to the 1H NMR spectra of the solid-liquid are significantly different to those registered in acetone. Most extraction experiments performed separately, we easily likely, the receptors adopted alternate conformations of their assigned the two sets of signals to the protons of the bound calix[4]pyrrole core and are involved in conformation receptor in the L-ProÌ1in and L-PipÌ1in complexes. (Figure 4b exchange processes that are fast on the chemical shift and c). Integration of the pyrrole NH signals for the two timescale. complexes assigned a 5:1 molar ratio to the mixture of L- ProÌ1in and L-PipÌ1in complexes.‡ Considering that the NMR binding studies We became interested in investigating the binding properties of these type of cavitands as receptors for L-Pro and the 6- membered ring α-amino acid analogue, L-pipecolic acid (L-Pip). L-Pip has been described as a diagnostic marker of pyridoxine- dependent epilepsy.15 We first performed separate solid-liquid extraction experiments with L-Pro or L-Pip and the fluorescent cavitand 1in in CD2Cl2. We added an excess of the solid amino acid (~ 2mg L-Pro or L-Pip) to a 2 mM CD2Cl2 solution of the cavitand 1in. We hand-shook the suspensions for several minutes and filtered off the remaining solid. The 1H NMR spectra of the filtered solutions showed significant differences compared to that of the free cavitand 1in in the same solvent (Figure 4). In Figure 4. 1H NMR (400 MHz, 298 K) and 31P NMR spectra of a 2 mM CD2Cl2 solution of 1in before (a) and after (b-d) solid-liquid extraction with solid L-Pro (b) and solid L-Pip both cases, only a single set of sharp signals was observed for (c) and with equal amounts of solid L-Pro and L-Pip (d). the hydrogen atoms of the receptor. This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 4 Please do not adjust margins Please do not adjust margins Journal Name ARTICLE solubility of L-Pro is slightly higher than that of L-Pip, the result during the solid-liquid experiment and hampering its use for of the competitive extraction experiment is not conclusive in quantitative purposes. assigning a larger binding affinity of 1in for L-Pro compared to For this reason, we decided to perform a competitive binding L-Pip.§ Nevertheless, it provides an initial hint in this direction. experiment starting from a millimolar CD2Cl2 solution of the L- We were also interested in evaluating the importance of the ProÌ5in complex. The addition of 1 equiv. of the 5out isomer hydrogen bonding interaction established between the to the above solution did not induce significant changes to the inwardly directed P=O group of cavitand 1in and the proton signals of the L-ProÌ5in complex (Figure 5 b). We protonated amino group of the bound amino-acid guests. With detected the appearance of a new set of proton signals that this aim, we performed analogous solid-liquid extraction almost coincided with those of the free 5out receptor. The experiments of L-Pro with non-fluorescent cavitands 5in and broadening and small chemical shift changes observed for the 5out. We used the non-fluorescent receptors as model new set of signals (Figure 5c) suggested the existence of a compounds of the fluorescent counterparts due to their ease binding equilibrium in solution. In short, we propose that the of synthesis (Figure 1). We hypothesized that 5out could bind L-ProÌ5out complex is formed in solution to a reduced extent and extract L-Pro through the formation of four hydrogen and the binding equilibrium between free and bound 5out bonds with the pyrrole NHs and additional CH-π interactions. shows fast/intermediate exchange dynamics on the chemical In contrast, the outwardly directed P=O group of 5out should shift timescale. The 31P NMR spectrum of the mixture also not participate in hydrogen bonding interactions with the supports this hypothesis. It displayed two singlets centred at d protonated amino group of bound L-Pro. Thus, we expected a = 15.8 and 13.4 ppm. The one appearing downfield is quite decrease in the binding constant of the L-Pro⸦5out complex sharp and corresponds to the L-ProÌ5in complex. In contrast, compared to that of the 5in isomer. the upfield-shifted singlet is slightly broadened due to the The solid-liquid extraction experiments of L-Pro using the non- chemical exchange between free and bound 5out. The lack of fluorescent model receptor 5in produced similar changes in separate signals for the phosphorous atoms of free 5in results the 1H and 31P NMR spectra to those described above for the from its low concentration in solution (Figure 5b, right). Taken fluorescent cavitand 1in (Figure 4). To our surprise, the 1H together, these results indicate that the binding affinity of 5in NMR spectrum of the filtered solution obtained after for L-Pro is significantly larger (approximately 7-10-fold, vide extraction of L-Pro with 5out showed broad proton signals. infra) than that of the 5out isomer. We attributed the The corresponding 31P NMR spectrum did not produce any increased binding affinity to the additional hydrogen bond observable signal. We detected a significant increase in the interaction provided by the inwardly directed P=O group in the signal-to-noise ratio of the spectra. We considered this L-ProÌ5in complex. observation as indicative of a diminution of the concentration of 5out in solution after the extraction experiment. To clarify UV-Vis and emission spectroscopy binding studies this issue, we evaporated the CD2Cl2 and re-dissolved the solid Direct binding-based sensing (BBS) residue in (CD3)2SO. The obtained (CD3)2SO solution was After having demonstrated the important contribution of the analysed using 1H NMR spectroscopy. We observed the inwardly directed P=O group for the binding of L-Pro with the diagnostic signals of the protons of free 5out and free L-Pro. 5in model cavitand, we carried out direct binding-based The L-Pro signals displayed a significantly reduced intensity. sensing (BBS) studies with the analogous fluorescent 1in This result suggested that 5out was not able to extract 1 equiv. cavitand. Some years ago, Dalcanale and co-workers reported of L-Pro. To investigate the solubility of the putatively formed the use of somewhat structurally related fluorescent L-ProÌ5out complex in CD2Cl2, we acquired a 1H NMR resorcin[4]arene receptors for the optical sensing of alkyl- spectrum of the filtered solid by dissolving it in (CD3)2SO. The chain C1-C4 alcohols.13 The authors claimed that the hydrogen 1H NMR spectrum of the solution displayed the diagnostic bonding interaction established between the alcohol OH signals of free 5out and free L-Pro. The observation of the function and the P=O group of the receptor could decrease the signals of free 5out indicates that the L-ProÌ5out complex electronic density on the phosphorus atom and modify the features a reduced solubility in CD2Cl2, falling out of solution energy of the excited state of the naphthalene unit directly attached to it. The electronic modification was expected to alter the maximum of the emission band. The UV-Vis absorption spectra of 1in and 1out showed very similar features (Figure S21). Concretely, two intense absorption bands with maxima at 275 (Ɛ = 34000 M-1cm-1) and 328 nm (Ɛ = 24000 M-1cm-1) and a shoulder at 370 nm. These bands are characteristic of π - π* and n - π*-transitions of the phenyl-amino-naphthyl moiety, as demonstrated by simple comparison with the UV-Vis spectrum of diethyl 6- Figure 5. 1H NMR (400 MHz, 298 K) and 31P NMR spectra of a CD2Cl2 solution of L- (phenylamino)naphthalene-2-phosphonate 7 (Figure 1) used ProÌ5in complex before (a) and after (b) the addition of 1 equiv. of 5out. c) 1H and 31P as model compound (Figure S22). NMR of a CD2Cl2 solution of 5out. This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5 Please do not adjust margins Please do not adjust margins Journal Name ARTICLE The incremental addition of L-Pro to separate micromolar constant values for the complexes of 1in with L-Pro and L-Pip dichloromethane solutions of 1in and 1out did not produce as, K(L-Pro⸦1in)BBS = 3.2 ´ 105 M-1 and K(L-Pip⸦1in)BBS = 6.3 ´ significant changes in their corresponding UV-Vis spectra 104 M-1, respectively. Surprisingly to us, the titrations of the (Figure S24). Spectral changes were more evident using 1out isomer with L-Pro and L-Pip produced greater emission emission spectroscopy to monitor the titration experiment. intensity changes (Figure 6b and S27).§§ Conversely, the The excitation at 335 nm of a 5 ´ 10-7 M dichloromethane bathochromic shift experienced by the emission band of 1out solution of 1in resulted in an intense and broad emission band was reduced compared to that of 1in (Δλmax < 1 nm). The with a maximum at 422 nm. The incremental addition of L-Pro mathematical analysis of the titration data of 1out, using a 1:1 provoked a concomitant red-shift of the maximum (Δλmax = 3-4 binding model considering two emissive species, returned very nm) and a decrease in emission intensity (Figure 6a). Similar similar binding constant values for the two complexes of the changes were observed during the incremental addition of L- amino-acids, K(L-ProÌ1out)BBS = 3.9 ´ 104 M-1 and K(L- Pip to 1in using analogous experimental conditions (Figure PipÌ1out)BBS = 2.3 ´ 104 M-1. The magnitudes of the binding S26). constants for the complexes with 1out are close to one order We also performed emission titration experiments with the of magnitude lower than those of the 1in isomer. fluorescent compound 7. This compound was used as The observation of larger emission changes in the titrations of reference of the fluorescent signalling unit incorporated in the the 1out isomer was completely unexpected. The spatial 1in and 1out receptor cavitands. In this case, the incremental orientation of the P=O group in the free isomer and its addition of L-Pro did not produce noticeable changes in the complexes is not geometrically suitable for the involvement in emission of 7 (Figure S23). This result evidenced the relevance charged hydrogen bonding interactions with the bound guests. of the aryl-extended calix[4]pyrrole scaffold for the efficient Dalcanale et al. hypothesized that hydrogen bonding binding of L-Pro by 1in and the transduction of the binding interactions with the inwardly directed P=O group were event in the modification of the emission properties of the responsible for the observed emission changes in the gas- signalling unit. phase sensing of short alkyl chain alcohols using structurally The changes in emission intensity observed during the related phosphonate resorcin[4]arene cavitands. The authors titrations of 1in with L-Pro and L-Pip were rather small. Even did not observe emission changes in the sensing experiments so, we fit the obtained experimental titration data to a using the out isomer. Based on our findings, we suggest that theoretical 1:1 binding model considering two emissive the cavitand-amino acid binding, especially for the 1out species: free and bound 1in. We determined the binding isomer, induces changes in the conformation (and possibly steric effects) of the receptor, which directly affects the properties of the signalling unit. These changes are transduced into non-radiative decay processes of the excited states of the 1:1 complexes. Accordingly, the binding event is responsible for the observed decrease in fluorescence intensity of the signaling unit.V Development of an Indicator Displacement Assay (IDA): a study of the interaction of the fluorescent receptors 1in and 1out with N- oxide 6. The reduced changes observed in the direct BBS titrations of 1in with the two amino acids prompted us to explore an alternative sensing approach using Indicator Displacement Assays (IDA). Recently, we described the synthesis and characterization of the pyridyl-N-oxide derivative 6 (Figure 1). The molecular structure of 6 is based on the popular and efficient acceptor 4-(dimethylaminoazo)benzene-4-carboxylic acid (DABCYL) (λabs,max = 480 nm, Ɛ = 39 500 M-1·cm-1). DABCYL is used in the development of Förster resonance energy transfer (FRET)-based nucleic acid probes. We already applied N-oxide 6 for the optical sensing of creatinine using an IDA. We used a calix[4]pyrrole phosphonate cavitand equipped with a Figure 6. Normalized emission spectra of 1in (a) (0.5 µM) and 1out (1 µM) (b) dansyl group as fluorophore. We reported that the binding of 6 registered during the addition of incremental amounts of L-Pro (up to 30 equiv. and 20 equiv., respectively) in dichloromethane solution: λ = 335 nm. Insets: plot of the in the cavity of the receptor produced the efficient quenching exc emission change at 426 nm (black circles) vs concentration of L-Pro. The red line of the fluorescence of the dansyl group through a FRET corresponds to the fit of the titration data to a 1:1 binding model considering two process.11 emissive species: the free cavitand and the 1:1 complex with the corresponding amino We calculated the spectral overlap between the absorption acid. spectrum of N-oxide 6 and the emission spectrum of 1in to be J = 2.5 ´ 10-9 cm6 mol-1 (Figure S28). This value is even larger This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 6 Please do not adjust margins Please do not adjust margins Journal Name ARTICLE than the one calculated for the FRET pair formed by 6 and the chemical shift timescale. The protons of the pyridyl-N-oxide phosphonate calix[4]pyrrole containing the dansyl residue of 6 were shifted upfield in comparison to the free fluorophore, which resulted in ca. 95% quenching of the counterpart, placing them within the aromatic walls of the receptor’s fluorescence in the 1:1 complex. The addition of 1 cavitand. Compound 6 is included in the cavity of 1in by equiv. of 6 to a 5 μM dichloromethane solution of 1in establishing four hydrogen bonds between the oxygen atom of produced a strong quenching of the emission band of the receptor at 422 nm (> 95%., lexc= 335 nm) (Figure S30).VV The the pyridyl-N-oxide knob and the four pyrrole NHs of the fluorescence quenching is the direct consequence of the FRET receptor in addition to π–π and CH–π interactions. We also process operating in the formed 1:1 inclusion complex. The observed a new set of signals in the downfield region of the phenyl-amino-naphthyl substituent of the 1in receptor acts as spectrum and we assigned them to the protons of free L-Pro. energy donor and the bound N-oxide 6 as the acceptor. We The addition of 1 equiv. of 6 induced the exclusive observation obtained analogous results for the equimolar mixture of 1out of the proton signals of the receptor in the 6Ì1in complex. and 6 (Figure S31). The quantitative formation of the Concomitantly, the signals of free L-Pro grew in intensity complexes 6⸦1in and 6⸦1out in the presence of 1 equiv. of (Figure 8c). This result is in complete agreement with the the N-oxide allowed us to estimate their binding constants as binding constant of L-ProÌ1in being two orders of magnitude larger than 107 M-1. We expected similar binding constant smaller (vide supra). It also demonstrates that the guest values for the two complexes owing to the lack of additional exchange process is fast on the human timescale (i.e. min.).rr interaction of guest 6 with the P=O group of the receptors. We performed a titration of 1in with 6 using more diluted Next, we studied the competitive displacement of the conditions ([1in] = 5 ´ 10-8 M, Figure 7) and determined the indicator N-oxide 6, involved in the 6Ì1in complex, by L-Pro accurate binding constant of the 1:1 complex to be K(6⸦1in) = and L-Pip at micromolar concentrations using emission 3.2 ´ 107 M-1.r The incremental addition of 6 to a 1µM solution spectroscopy. We prepared an equimolar dichloromethane of 7, used as reference of the fluorescent substituent of the solution of 6 and 1in (1 µM). At this concentration and taking receptors, did not produce noticeable emission changes. This in consideration the binding constant determined in the result demonstrated that the formation of the 1:1 complexes previous section, the 6Ì1in complex is present in 84% extent. induces the observation of the FRET-quenching process. Thus, the fluorescence observed for the ensemble is assigned to the 16% of dye 6 remaining free in solution (Figure 9). The titration of the solution with incremental amounts of L-Pro evidenced a gradual increase of the fluorescence emission. In the presence of 30 equiv. of L-Pro, the observed intensity for the maximum of the emission band was 2.5 times higher than the initial one (I0). We rationalized the noticed fluorescence “turn-on” by considering the displacement of 6, as quencher of the fluorescence of the phenyl-amino-naphthyl group in the 6⸦1in complex, by L-Pro. This displacement leads to the Figure 7. Normalized emission spectra registered for the titration of 1in (0.05 µM) with incremental amounts of 6 (up to 4 equiv.) in dichloromethane solution: λexc = 335 nm. Inset: plot of the emission change at 426 nm (black circles) vs concentration of 6. The red line corresponds to the fit of the titration data to a 1:1 binding model considering two emissive species: free receptor and 1:1 complex with 6. Competitive IDA experiments Initially, we probed the competitive binding of 6 and L-Pro with the 1in receptor using 1H NMR spectroscopy. We prepared an equimolar CD2Cl2 solution of 1in and L-Pro ([1in]=[L-Pro] = 2.0 mM) by performing a solid-liquid extraction experiment (Figure 8a). The 1H NMR spectrum of the obtained solution revealed the exclusive presence of the diagnostic signals for the quantitative formation of the L-Pro⸦1in complex. The subsequent addition of 0.5 equiv. of 6 produced the appearance of a separate set of signals (Figure 8b). We attributed the new set of signals to the protons of the receptor in the 6Ì1in complex. The observation of two separate sets of Figure 8. Top) Competitive IDA binding equilibrium. Bottom) 1H and 31P NMR spectra of signals for the receptor’s protons in the L-ProÌ1in and 6Ì1in a 2 mM solution of L-ProÌ1in in CD2Cl2 after the addition of 0 (a), 0.5 (b) and 1.0 (c) complexes indicated that its chemical exchange is slow on the equiv. of 6. F denotes the fluorophore: phenyl-amino-naphthyl group. This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 7 Please do not adjust margins Please do not adjust margins Journal Name ARTICLE formation of the fluorescent L-Pro⸦1in complex. It is worthy to note that the identical titration monitored using absorption spectroscopy produced negligible changes in the registered spectra. We analysed mathematically the titration data obtained in the emission IDA experiment using a theoretical binding model considering the competitive formation of two 1:1 complexes (6Ì1in and L-ProÌ1in). We assigned emissive properties to only two (free 1in and L-ProÌ1in) of the six species involved the binding model. ‡‡ We fixed the binding constant of the 6Ì1in complex to the previously determined value of K = 3.2 ´ 107 M-1 (vide supra). The fit of the experimental data to the model was good and returned the stability constant value for the L-ProÌ1in as K(L-ProÌ1in)IDA = 4.5 ´ 105 M-1. This value is in good agreement with the one determined previously using direct BBS titration experiments (K(L-ProÌ1in)BBS = 3.2 ´ 105 M- 1, vide supra). The fit of the experimental data also provided the calculated emission spectra for 1in and L-ProÌ1in. The calculation of the spectra of the emissive (a.k.a. coloured) species is one of the advantages of performing global fitting multivariate data analysis compared to the simpler single or even multiple wavelength fitting alternatives. Obtaining sensible calculated spectra for the coloured species is a necessary condition to support the quality and fit of the data Figure 9. Emission spectra registered during the competitive IDA experiments of 6Ì1in analysis. To our delight, the calculated spectra showed a nice complex (1 µM) with incremental addition of L-Pro (up to 30 equiv.) (a) and L-Phe (up agreement with the experimental ones registered in separate to 30 equiv.) (b) in dichloromethane solution; λexc = 335 nm. Insets: plots of the experiments. We also tested the performance of the ensemble emission change at 426 nm (black circles) vs concentration of the amino acid. The red of 6 and 1out in emission IDA experiments with L-Pro (Figure line corresponds to the fit of the titration data to a 1:1 theoretical binding model considering only two emissive species: the free cavitand and the 1:1 complex with the S32). Using this methodology, the calculated binding constant corresponding amino acid. for the L-ProÌ1out complex was K(L-ProÌ1out)IDA = 7.9 ´ 104 M-1. In short, the emission IDA experiments reflected that the We also determined the binding constant of L-phenylalanine difference in the binding constant values of the L-ProÌ1in and (L-Phe) with 1in using analogous competitive IDA experiments. L-ProÌ1out inclusion complexes is close to one order of We observed a 1.5 times fluorescence enhancement in the magnitude, in favour of the former. In addition, the binding presence of 30 equiv. of L-Phe (Figure 9b). This result hinted to constant values measured for the complexes using IDA and similar binding constant values for the L-Phe⸦1in and L- BBS experiments are fully consistent. Pip⸦1in complexes. The fit of the experimental data returned We carried out analogous IDA experiments with L-Pip. We a binding constant of K(L-PheÌ1in)IDA = 3.5 ´ 104 M-1. observed a 1.7 times fluorescence enhancement for the ensemble of 6 and 1in in the presence of 30 equiv. of L-Pip (i.e. Influence of the polar hydrogen bonding in the inclusion I30 = 1.7´I0) (Figure S33). The measured fluorescence complexes of 1in enhancement factor is reduced when compared to the 2.5-fold In previous studies, we learned that in water solution six- observed for L-Pro under identical conditions. The fit of the membered ring lactams produced more stable inclusion data of the IDA experiments to the same theoretical binding complexes with bis-phosphonate cavitand than the five- model used before for L-Pro returned a binding constant for L- membered analogues.16 These results were rationalized based PipÌ1in complex of K(L-PipÌ1in)IDA = 6.3 ´ 104 M-1. Again, this on the superior size and shape fit of the 6-membered ring, value is in line with the one obtained in direct BBS with respect to the receptor’s cavity, and its increased experiments. It is worthy to mention that under the used hydrophobicity. Considering our previous finding, we were experimental conditions for the IDA, and considering the surprised to calculate a larger binding constant for the L- calculated binding constant for the L-PipÌ1in complex, this Pro⸦1in complex, involving a 5-membered cyclic guest, than species is only formed in a 20% extent. The reduced extension for the analogous complex with L-Pip (6-membered ring). in which the L-PipÌ1in complex is formed diminishes the To gain some insight on the structures of the amino acid’s accuracy of the calculated binding constant value. inclusion complexes with receptor 1in, we computed their Unfortunately, the low solubility of L-Pip in dichloromethane energy-minimized structures at the BP8617,18/def2SVP level of hampered the formation of the L-PipÌ1in complex to a larger theory using GAUSSIAN 09.19 In all the computed inclusion extent. complexes, the calix[4]pyrrole core adopts the cone conformation by establishing four hydrogen bonding interactions with one of the oxygen atoms of the carboxylate This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 8 Please do not adjust margins Please do not adjust margins Journal Name ARTICLE group of the included guest (Figure 10 and Figure S34). It is Table 1. Binding constants Ka (M-1) at 298 K for the 1:1 inclusion complexes of the 1in also possible to infer the presence of multiple CH-π and O-π and 1out receptors in dichloromethane and free Gibbs energy ΔG (kcal mol-1) interactions in the complexes (Figure S35). Moreover, all 1in 1out ΔΔG(1in-1out) complexes display a charged hydrogen bonding interaction 3.2 ±0.6 ×105a 3.9 ±0.8 ×104a between the inwardly directed P=O group of the receptor and Ka 4.5 ±0.9 ×105b 7.9 ±1.5 ×104b the protonated amino group of the zwitterionic form of the L-Pro ~-1.1 -7.5 ±0.1a -6.3 ±0.1a bound amino acids (Figure S35). ΔG -7.7 ±0.1b -6.7 ±0.1b In the particular case of the L-Pip⸦1in complex, we computed 6.3 ±1.3 ×104a Ka 2.3 ±0.5 ×104a two different binding geometries. One of them involved the 6.3 ±1.3 ×104b L-Pip -0.5a energetically more favourable conformation of L-Pip, L-Pip -6.5 ±0.1a eq ΔG -6.0 ±0.1a (i.e. COO- substituent equatorial in the protonated piperidine -6.5 ±0.1b chair conformation). The other one considers the higher Ka 3.5 ±0.7 ×104b n.d. L-Phe n.d. energy conformer, L-Pipax (i.e. COO- substituent is axial in the ΔG -6.2 ± 0.1b n.d. protonated piperidine chair conformation). The results of the Ka and ΔG are the average values from two independent titrations. Errors are calculations indicated that both inclusion complexes were reported as standard deviation for Ka and propagated for ΔG. aDetermined by isoenergetic. direct BBS experiments. bDetermined by IDA experiments. n.d. Not determined. A simple visual inspection of the energy-minimized structures of the amino acid’s inclusion complexes revealed that those of Additional CH-π interactions also assist in the stabilization of L-Pro featured a superior match in size and shape between the both complexes. cavity of 1in and the included amino acid (Figure 10). Most On the other hand, the calculated free energy difference likely, the L-ProÌ1in inclusion complex establishes between the analogous complexes of L-Pip (ΔΔG(L-PipÌ1in- L- energetically more favourable dispersive, van der Waals and PipÌ1out) is reduced to just -0.5 kcal mol-1. Most likely, this is due hydrogen bonding intermolecular interactions than the other to the formation of a weaker polar hydrogen bond in the L- counterparts (L-PipÌ1in and L-PheÌ1in) (Figure S34). PipÌ1in complex in comparison to the L-Pro counterpart. The experimentally determined values for the association We measured a small free energy difference for the complexes constants of the complexes of the amino acids with receptors of L-Pro and L-Pip with the 1out receptor (~-6.3 and -6.0 kcal 1in and 1out are summarized in Table 1. We draw the mol-1, respectively) that we assign it to the better fit of the following conclusions from the tabulated data. former in the cavity of the latter. Taken together, these results On the one hand, the determined ΔG values for L-ProÌ1in and indicate that the polar intermolecular hydrogen bond L-ProÌ1out complexes assigned a free energy gain of ~-1.1 interaction N-H···O=P plays a pivotal role in the binding kcal mol-1 (ΔΔG - ) in favour to L-ProÌ1in selectivity featured by 1in for L-Pro over L-Pip, ΔΔG(L-ProÌ1in- L- (L-ProÌ1in L-ProÌ1out) -1 complex. We attributed this difference to the additional polar PipÌ1in) ~ 1.0 kcal mol . hydrogen bonding interaction established in the inclusion complex with 1in. This interaction involves the oxygen atom of Conclusions the P=O group inwardly directed towards the receptor’s cavity and the protonated amino group of the included L-Pro. Due to We report the synthesis of two unprecedented geometrical reasons, this interaction is not present in the L- diastereoisomeric mono-phosphonate calix[4]pyrrole ProÌ1out complex. Thus, receptor 1out binds L-Pro through cavitands featuring a N-phenyl-naphthalamine fluorophore the formation of only four hydrogen bonds involving the directly attached to its phosphorous atom. The two isomers carboxylate group of the L-Pro and pyrrole NHs of 1out. differ in the relative orientation (in/out) of the P=O bridging group with respect to its polar aromatic cavity. We characterized the two isomers in solution using NMR and optical (UV-Vis absorption and emission) spectroscopies and in the solid state by X-ray diffraction studies. We used the non- fluorescent model receptors 5in and 5out to perform a competitive binding experiment with L-Pro. The analyses of the mixtures using 1H and 31P NMR spectroscopy showed that the L-Pro was preferentially bound to the 5in isomer. We attributed the higher thermodynamic stability exhibited by the L-Pro⸦5in complex to the existence of an additional charged hydrogen bonding interaction between the protonated amino group of bound L-Pro and the inwardly directed bridging P=O function of the receptor. We developed two different Figure 10. Side and top views of the energy-minimized inclusion complexes L-ProÌ1in supramolecular approaches for the sensing of L-Pro and L-Pip (a) and L-PipeqÌ1in (b). The structures are energy minima at the BP86/def2-SVP level of theory. The receptors are shown in stick representation. Non-polar hydrogen atoms using the fluorescent receptor 1in: direct binding-based were removed for clarity. The included amino acids are depicted as CPK models. sensing (BBS) and a FRET-based indicator displacement assay (IDA). The BBS strategy produced a small reduction of the This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 9 Please do not adjust margins Please do not adjust margins Journal Name ARTICLE emission intensity of 1in upon binding L-Pro or L-Pip. The fit of § Solubility was calculated by preparing 1 mL saturated solutions the reduced emission changes to a simple 1:1 binding model of L-Pro and L-Pip in dichloromethane at 298 K. Evaporation of allowed us to determine the binding constants of the inclusion the solvent and accurate weight of the resulting solids returned the solubility of L-Pro and L-Pip in dichloromethane as 8 x 10-4 complexes of 1in with L-Pro and L-Pip as, K(L-ProÌ1in)BBS = 3.2 and 5 x 10-4 mol L-1, respectively. ´ 105 M-1 and K(L-PipÌ1in)BBS = 6.3 ´ 104 M-1, respectively. Analogous titration experiments of the 1out isomer with the §§ We did not find significant differences in the fluorescence two amino acids resulted in greater decreases of the emission emission of 1in and 1out in dichloromethane. intensity of the free receptor. In contrast, the binding constant values determined for the complexes of 1out with L-Pro and L- V We did not see the formation of any precipitate after the Pip are close to one order of magnitude lower than those of addition of L-Pip to a micromolar dichloromethane solution of 1out as observed in the analogous experiments at millimolar 1in. The increased binding affinity of the amino acids for the concentration (i.e. NMR experiments). We consider that at 1in receptors is assigned to the charged hydrogen-bonding micromolar concentration the L-PipÌ1out complex is soluble in interaction established between the protonated amino group dichloromethane. Therefore, we conclude that the decrease in of the bound guest and the bridging P=O function inwardly fluorescence intensity is not related to the precipitation of the directed with respect to the polar aromatic cavity of the complex. receptor. VV Slit width of the monochromator as 1 nm. The sensing of amino acids (e.g. L-Pro, L-Pip and L-Phe) using IDA experiments with the ensemble composed by 1in and N- r These experimental conditions forced us to modify the slit oxide 6 produced more significant emission changes than width of the monochromator settings (from 1 nm to 5 nm). those of the direct BBS strategy. Remarkably, the IDA These new parameters did not allow us to consider the 6Ì1in experiments induced fluorescence “turn on” instead of host-guest complex non-emissive as occurred with the previous quenching. The binding constant values determined for the instrument set-up. Hence, the titration data was fit to a theoretical 1:1 binding model that considered two emissive amino acid’s inclusion complexes with 1in using IDA species (free and bound 1in). experiments were in complete agreement with those derived from the direct BBS counterparts. Receptor 1in showed rr The competitive displacement of guest 6 from the cavity of binding selectivity for L-Pro over the other amino acids tested. 1in by L-Pro in CD2Cl2 was not possible at millimolar Conversely, receptor 1out did not feature a noticeable concentration due to the poor solubility of L-Pro in this solvent. selectivity in the amino acids’ binding. We assign the dissimilar receptors’ binding selectivity to the existence of an ‡‡ The 6Ì1in complex was considered non-emissive under the experimental conditions and the instrument set up used for the intermolecular hydrogen bond interaction between the bound titration (slit width of the monochromator = 1 nm). amino acid and the P=O group of the receptor. For geometrical reasons, the required arrangement of functional groups is only possible for the 1in diastereoisomer. The obtained results 1 Z. Lou, L. Wang and G. 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Dalcanale, Molecular recognition at the gas– CTQ2017-84319-P and CEX2019-000925-S), the CERCA solid interface: a powerful tool for chemical sensing, Chem. Soc. Programme/Generalitat de Catalunya, and AGAUR (2017 SGR Rev., 2007, 36, 695-706. 1123) for financial support. We thank Dr. Eduardo C. Escudero- 7 M. Ciardi, F. Tancini, G. Gil-Ramírez, E. C. Escudero Adán, C. Adán, the X-ray Diffraction Unit of ICIQ, for help with the Massera, E. Dalcanale and P. Ballester, Switching from Separated to Contact Ion-Pair Binding Modes with Diastereomeric analysis of the X-ray crystallographic data. Calix[4]pyrrole Bis-phosphonate Receptors, J. Am. Chem. Soc., 2012, 134, 13121-13132. 8 M. Ciardi, A. Galán and P. Ballester, Tetra-phosphonate Notes and references Calix[4]pyrrole Cavitands as Multitopic Receptors for the ‡ We performed an addition solid-liquid extraction competitive Recognition of Ion Pairs, J. Am. Chem. 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