FULL PAPER This is the peer reviewed version of the following article: “Hydrolysis of Aliphatic Bis-isonitriles in the Presence of a Polar Super Aryl-Extended Calix[4]pyrrole Container”, which has been published in final form at https://doi.org/10.1002/chem.202101643. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving Hydrolysis of Aliphatic Bis-isonitriles in the Presence of a Polar Super Aryl-Extended Calix[4]pyrrole Container Qingqing Sun,[a][b]‡ Luis Escobar[a][b]‡ and Pablo Ballester[a][c]* Abstract: We report binding studies of an octa-pyridinium super challenging and troublesome. The improvement of the selectivity aryl-extended calix[4]pyrrole receptor with neutral difunctional in desymmetrization reactions should increase their application aliphatic guests in water. The guests have terminal isonitrile and in synthetic strategies. Molecular containers featuring non-polar formamide groups, and the complexes display an inclusion binding cavities have been used as reaction flasks to address the mono- geometry and 1:1 stoichiometry. Using 1H NMR titrations and ITC functionalization problem.9 For example, Rebek and co-workers experiments, we characterized the dissimilar thermodynamic and employed a water-soluble octa-methyl tetra-benzimidazolone kinetic properties of the complexes. The bis-isonitriles possess resorcin[4]arene cavitand for increasing the selectivity of: the independent reacting groups, however, in the presence of 1 equiv. of reduction of α,ω-bis-azides to mono-amines,13 the hydrolysis of the receptor the hydrolysis reaction produces mixtures of non- α,ω-bis-esters to mono-acids, in both acid and basic media,14 statistical composition and a significant decrease in reaction rates. and obtaining mono-epoxides from α,ω-bis-enes.15 In all cases, The selectivity for the mono-formamide product is specially the product distributions were exceptional, containing the mono- enhanced in the case of the bis-isonitrile having a spacer with five functionalized or mono-reacted compound in significantly larger methylene groups. The analysis of the kinetic data suggests that the extents than the statistically predicted upper limit (36.8%) observed modifications in reaction rates and selectivity are related to assuming identical rate constants, k1 = k2, for each site.14 the formation of highly stable inclusion complexes in which the In these applications, the function of the molecular container isonitrile is hidden from bulk water molecules. The concentration of doubles as supramolecular water-solubilizing and protecting the reacting substrates in the bulk solution is substantially reduced group. The binding of the water-insoluble symmetric substrates by binding to the receptor. In turn, the hydrolysis rates of the to the cavitand produces soluble 1:1 inclusion complexes. The isonitrile groups for the bound substrates are slower than in the bulk bound symmetric guests adopt folded J-conformations, which solution. The receptor acts as both a sequestering and interconvert rapidly on the 1H NMR chemical shift time scale, supramolecular protecting group. most likely, through a “yo-yo” motion.16,17 The J-shape of the bound guest provokes an alternative exposure of only one of the two reactive ends to the bulk solution. The exposed end reacts Introduction at the interface of the inclusion complex and water. Subsequently, the equilibrium between the reacted and unreacted ends of the bound guest is modified owing to their Molecular containers have been used to mediate and catalyze different hydrophobicities. Typically, the unreacted site (being chemical transformations, as well as to alter the selectivity of less polar) is preferentially hidden from the water/cavitand reactions occurring in bulk solution.1,2,3,4,5,6,7,8,9,10,11,12 In solution interface, and its further reaction is inhibited. The selectivity for the mono-functionalization of symmetric difunctional compounds the mono-functionalization reaction, compared to that in the featuring independent reacting groups, i.e., the chemical absence of the container, is increased. modification of one group has little effect on the reactivity of the other, yields statistical mixtures of products. Generally, such mixtures arising from the reaction makes the purification and isolation of the desired mono-reacted (non-symmetric) product [a] Institute of Chemical Research of Catalonia (ICIQ), The Barcelona Institute of Science and Technology (BIST) Av. Països Catalans, 16, 43007, Tarragona, Spain E-mail: pballester@iciq.es [b] Universitat Rovira i Virgili, Departament de Química Analítica i Química Orgánica c/Marcel·lí Domingo, 1, 43007, Tarragona, Spain [c] Catalan Institution for Research and Advanced Studies (ICREA) Passeig Lluís Companys, 23, 08010, Barcelona, Spain ‡ These authors contributed equally. Supporting information for this article is given via a link at the end of the document. FULL PAPER component reactions,32 and are easily attached to biomolecules, making long-chain aliphatic α,ω-formamide-isonitriles valuable intermediates in organic and bio-organic synthesis. The efficient binding of primary formamides exhibited by water-soluble calix[4]pyrrole receptors and their supramolecular sequestering and protecting abilities suggested an improved selectivity in the hydrolysis reaction of symmetric aliphatic α,ω-bis-isonitriles; we report on these developments here. Initially, we examined the binding of SAE-C[4]P 1 in water to a series of difunctional aliphatic guests: bis-isonitriles 2, mono- formamides 3 and bis-formamides 4 (Figure 1b). We investigated the acid-catalyzed hydrolysis of a series of bis- isonitriles 2a-e in the absence and in the presence of receptor 1 (1 equiv.). The results showed that the presence of 1 induces yield enhancements for the mono-formamides (non-symmetric) 3a-e with respect to the statistical mixture of products obtained in the absence of the receptor. The largest yield enhancement was obtained for the mono-formamide 3b. The modifications in the composition of the reaction mixture and the observed diminution in reaction rates are related to the formation of stable inclusion complexes of the reacting substrates (2 and 3) with receptor 1. Figure 1. a) Chemical structure of SAE-C[4]P 1; b) Stepwise reaction scheme of the acid-catalyzed hydrolysis of bis-isonitrile 2 to give mono-formamide 3 Results and Discussion and bis-formamide 4. c) Line-drawing structure of 4-phenylpyridine N-oxide 5 used as competitive binding guest. The interaction of SAE-C[4]P 1 with bis-isonitrile 2b and mono- and bis-formamides, 3b and 4b, in D2O solution was followed 18 , 19 20 21 using 1H NMR spectroscopy (see SI). The 1H NMR spectrum of The groups of Gibb, Nitschke and Dalcanale, among others, 22 a millimolar D2O solution of free 1 shows broad proton signals have also reported beautiful examples of molecular due to aggregation or intermediate dynamics between containers able to sequester reactive groups from external conformational changes (cone and alternate). The addition of 1 reagents. Both polar and steric factors have strong impacts on equiv. of the mono-formamide 3b provoked the sharpening of the transition state’s energy of reactive groups sequestered in 23,24 the signals of receptor 1. In addition, the five methylene units of molecular containers. 3b appeared upfield shifted, locating them within the four We reported the synthesis and binding properties of water- 25 , 26 aromatic walls of SAE-C[4]P 1. The addition of more than 1 soluble aryl-extended (AE-C[4]P) and super aryl-extended 27 equiv. of 3b produced a separate set of signals corresponding to calix[4]pyrrole (SAE-C[4]P) receptors containing polar aromatic the protons of the free guest (Figure 2c). These results cavities. We demonstrated that in aqueous solution an AE-C[4]P 4 -1 indicated the formation of a 1:1 inclusion complex, 3bÌ1, for binds formamides with high affinity, Ka > 10 M . There is which we can estimate a stability constant value (Ka) larger than selectivity for the cis-isomer of primary and secondary 25 104 M-1, with a binding process displaying slow exchange formamides, despite the energetic preference for a trans- 28 dynamics on the 1H NMR chemical shift time scale. The five conformation when free in solution. Water-soluble SAE-C[4]Ps methylene signals of bound 3b showed quite different upfield were also effective in the binding of polar neutral substrates shifts (Dd < 0), which reflect the dissimilar anisotropic shielding such as pyridyl N-oxides, Ka > 105 M-1.27 The cone conformation provided by the four walls of 1 along its aromatic cavity. In of AE-C[4]Ps and SAE-C[4]Ps, i.e., 1 (Figure 1a and Figure 3), contrast to the observations made in inclusion complexes of creates sizeable polar aromatic cavities. In water, their inclusion resorcin[4]arene cavitands,9 the magnitudes of the upfield shifts complexes are stabilized not only by the hydrophobic effect and are not directly related to the various depth of the guest’s alkyl CH-π, NH-π and π-π interactions29 but also by the establishment chain included in the SAE-C[4]P 1. For example, the formyl of four convergent hydrogen bonds between the pyrrole NHs of proton of bound 3b appeared at δ = 4.39 ppm (Δδ = - 3.60 ppm) the calix[4]pyrrole unit of the receptor and the oxygen atom of (see SI, Figure S59). We observed analogous complexation- the bound guest.30 31 induced shifts (CIS, Dd) in the binding of the cis-isomers of In bulk solution, the acid-catalyzed hydrolysis of long-chain phenyl and alkyl formamides with a water-soluble AE-C[4]P.25 aliphatic α,ω-bis-isonitriles provides the mono-hydrolyzed This places the cis-formamide group of 3b in the closed and formamide-isonitrile as a statistical mixture with the unreacted polar aromatic cavity of calix[4]pyrrole 1. 33 In turn, this starting material and the doubly-hydrolyzed compound (Figure arrangement fixes the geometry of the 3bÌ1 complex by placing 1b). Formamide and isonitrile (isocyanide) functional groups display orthogonal reactivity. Isonitriles participate in multi- FULL PAPER the isonitrile end of 3b close to the open rim of the receptor bound 4b. The lack of symmetry and the magnitude of the (Figure 3b). upfield shifts experienced by the methylene signals in the 4bÌ1 complex indicated that a cis-formamide end is bound by the calix[4]pyrrole unit of 1 while the other formamide end, in cis (15%) and trans (85%) conformation, occupies the upper non- polar aromatic cavity. We performed the assessment of the binding constant values for the 3bÌ1 and 4bÌ1 complexes using ITC experiments (Table 1). A bound symmetric difunctional guest, having five methylene groups and experiencing fast tumbling on the 1H NMR chemical shift time scale inside the cavity of 1, should lead to the observation of only three signals. At 313 K, we observed only three upfield shifted signals in the 1H NMR spectrum of mixtures of the symmetric bis-isonitrile 2b (0.5-1 equiv.) with 1 (Figure 2a,b). In this case, however, the binding process displayed fast exchange dynamics on the chemical shift time scale. This latter characteristic serves to explain the symmetric pattern of the proton signals of bound 2b without the need to invoke fast tumbling of the bound guest. The addition of more than 1 equiv. of 2b did not induce further changes in the proton signals of 1 Figure 2. Selected region of the 1H NMR spectra (500 MHz with cryoprobe, (Figure S45). This observation allows us to estimate that the D2O, 313 K) registered for the titration of SAE-C[4]P 1 with bis-isonitrile 2b: a) binding constant value of the 2bÌ1 complex is also larger than 0.5 and b) 1 equiv. added. Panels c) and d) display the identical region of the 104 M-1. The CIS of the methylene protons of bound 2b were 1H NMR spectra (298 K) of mixtures containing 1 with 2 equiv. of mono- formamide 3b and bis-formamide 4b, respectively. Primed numbers calculated by extrapolating the fit of its chemical shift changes correspond to the proton signals of bound species: 2b (in red), 3b (in blue) experienced in the titration of 1 using a theoretical 1:1 binding and 4b (in orange). model (Figure S46). The calculated values represent average chemical shifts of the two separate signals expected for both alpha- and beta-methylene protons in a 2bÌ1 complex The two methylene groups alpha (H1) and beta (H2) with respect experiencing slow dynamics on the chemical shift time scale for to the formamide end of 3b are significantly less shielded (Δδ = - its binding and tumbling processes (see CIS for 2bÌ1 in Figure 0.99 and -0.62 ppm, respectively). This is due to their location in 3a). The calculated average CIS values for the two separate the cavity region of 1 surrounded by the alkynyl linkers, which alpha- and beta-methylene proton signals observed for 3bÌ1 or impart a reduced magnetic shielding. In striking contrast, the the symmetric bis-formamide 4b in the 4bÌ1 complex are in line remaining three methylene units, specially the one alpha to the with those of the bis-isonitrile 2b in the 2bÌ1 complex. This isonitrile end (H5), although being less deep in the cavity of 1, result suggests that 2b also adopts an extended conformation experienced more intense shielding (Δδ = -2.42 ppm). These when included in the cavity of 1. In this conformation, one of the larger CIS derive from the placement of the methylene groups in two isonitrile groups of 2b is more accessible to water molecules the upper non-polar aromatic cavity of 1. In this location, they in the interface between the open end of the SAE-C[4]P 1 and are exposed to the strong shielding effect exerted by the four the bulk solution. ITC experiments provided a more accurate terminal phenyl substituents of the receptor. value for Ka (2bÌ1) (Table 1). We calculated large and negative We did not observe additional signals that could be assigned to heat capacity values (DCp) for all binding processes. This result the hydrogen atoms of the “reverse” binding geometry of the indicates that the formation of the complexes, 2b-4bÌ1, is 3bÌ1 complex (Figure 3c). Remarkably, the chemical exchange mainly driven by the hydrophobic effect.34,35 between free and bound 3b was fast on the EXSY time scale Based on the above, we hypothesized that in the 2bÌ1 complex, (tmix = 0.3 s). The observed chemical exchange cross-peaks the rate of the hydrolysis reaction producing the mono- were used for unequivocal assignment of the proton signals of formamide 3b should be different from that in the bulk solution. bound 3b. The exclusive observation of i + 2 NOE peaks between the methylene proton signals of bound 3b supports the extended conformation of its alkyl chain. The binding of the symmetric bis-formamide 4b with the SAE- C[4]P 1 produced identical results (see Figure 2d and SI for details). That is, a 1:1 inclusion complex of 4b with Ka > 104 M-1, and its slow bound/free exchange dynamics on the 1H NMR chemical shift time scale. Fast exchange with the free counterpart on the EXSY time scale was detected, but not tumbling of the bound guest on the same time scale. We observed five separate signals for the methylene protons of FULL PAPER Assuming that the reaction occurs preferentially at the water/SAE-C[4]P interface (isonitrile end located at the open cavity of the container), the resulting mono-formamide 3b will locate the non-reacted isonitrile end buried in the polar cavity of 1 protected from bulk water. This would result in the “reverse” binding geometry of the 3bÌ1 complex (Figure 3c). However, as mentioned above, the 1H NMR spectrum of the 3bÌ1 complex (Figure 2c) did not show additional proton signals hinting to the presence of this isomer in solution. Nevertheless, we cannot rule out its existence to a reduced extent (< 10-4 M). Our expectations were that the inclusion of the bis-isonitrile 2b and the mono-formamide 3b in the cavity of 1 might produce a reduction in the hydrolysis reaction rates of their isonitrile ends exposed at the water/container interface in comparison to the bulk solution. Possible reasons of this putative effect include: a limited exposure to bulk water molecules and a reduced solvation of the transition state. It is worth mentioning that the superior binding properties of SAE-C[4]P 1 for the mono- formamide 3b will reduce its concentration in solution to a large extent.39 In order to test our hypotheses, we studied the hydrolysis reaction of bis-isonitrile 2b chaperoned by the synthetic container 1 in water. In doing so, we also wanted to evaluate the potential application of 1 in addressing the mono- functionalization problem of 2b. We performed the hydrolysis reaction by adding 5 equiv. of citric acid as a solution (0.5 M in D2O) to a NMR tube containing an equimolar mixture (1 mM) of 2b and 1 in 0.5 mL of D2O (final pD ~ 3). We monitored the progress of the reaction at 313 K using 1H NMR spectroscopy Figure 3. Gas-phase energy-minimized structures at the BP8636,37-Def2SVP- (see SI, Figure S129). The distribution of the species as a D338 level of theory of simplified 1:1 inclusion complexes: a) 2bÌ1; b) 3bÌ1; c) function of time was determined using the integral values of the “reverse” binding geometry for 3bÌ1 and d) 3cÌ1. The complexation-induced shifts (CIS, Δδ) experienced by all the non-polar proton signals of the bound methylene proton signals in the corresponding inclusion 40 guests, 2b and 3b, and the dynamics of their in/out chemical exchange complexes. Alternatively, we added 1.2 equiv. of 4- process on the 1H NMR chemical shift time scale are indicated. Receptor 1 is phenylpyridine N-oxide 5 (Figure 1c) to the reaction mixture at shown in line-stick representation with non-polar hydrogen atoms omitted for different time intervals (Figure S132). N-oxide 5 is a competitive clarity. Bound guests are depicted as CPK models. The water-solubilizing guest27 groups at the upper and lower rims of 1 are pruned to methyl groups. See that displaces the reacting species bound in the cavity of Figure 1 for the line-drawing structures of the compounds. 1 to the bulk solution. Next, we used the integral values of the proton signals of the displaced guests to determine their concentrations. The two methodologies produced similar results. The formation of the mono-formamide 3b was evident from the Table 1. Binding constant values (Ka) determined using ITC experiments for the 1:1 complexes of receptor 1 with guests 2b-4b in water at 313 K and their loss of symmetry of its inclusion complex. Please note that we corresponding free energies (DG). Error values in Ka are reported as standard assigned a preferred binding geometry for the 3bÌ1 complex deviations and propagated to ΔG. The heat capacities (DCp) were calculated fixing the formamide group in the calix[4]pyrrole unit and having using the DH values of ITC experiments performed at 298 and 313 K. an extended conformation for the alkyl spacer (vide supra). After Guest K (M-1) x 10-5 DG (kcal·mol-1) DC (cal·mol-1·K-1) ca. 2 h, the composition of the reaction mixture contained the a p mono-formamide 3b in an amount close to 80% (Figure 4a). 2b 1.3 ± 0.1 7.3 ± 0.1 -240 This result suggests that the remaining isonitrile group in the 3bÌ1 complex is less accessible to bulk water. It also represents 3ba 6.9 ± 0.9 8.3 ± 0.1 -193 a significant improvement of the theoretical 36.8% predicted by 4ba 9.2 ± 0.1 8.5 ± 0.1 -233 Rebek and co-workers in the mono-functionalization of symmetrical difunctional substrates having independent reacting a The reported Ka values are apparent (Kapp) because only the cis-isomer of the groups.14 The isolation of the mono-formamide 3b was possible guests binds the receptor. Ka and Kapp have similar magnitudes when the by extracting it from the neutralized aqueous solution using ethyl percentage of the cis-isomer in solution is significant, as is the case here. See acetate (Figure S139). reference 25 for a more detailed analysis of the ratio between these constant values. In analogy to the Rebek’s report,14 we fit the experimental concentration data vs time to a theoretical kinetic model of two FULL PAPER consecutive first-order irreversible reaction steps, Eqs. 1 and 2 The selectivity of the reaction for the mono-formamide 3b (model 1), using COPASI kinetic modeling software. reached a maximum of 50%, in agreement with the theoretical statistical distribution for identical microscopic reaction rate constants. As could be expected from a protective/sequestering effect of the container, the calculated rate constant values in the absence of 1 increased more than four-fold with respect to those From the fit of the data, we obtained the rate constant values k determined above. For this reason, the 50% maximum 1 = 1.7 x 10-2 min-1 and k = 2.3 x 10-3 min-1 (Figure 4a, dashed concentration of 3b was obtained after 20 min of reaction 2 lines). Based on this model, the determined rate constant values compared to ca. 2 h needed to produce 3b in an extent of 80% for the hydrolysis reactions in the water/container interface show in the presence of 1. that the first step (Eq. 1) is seven-fold faster than the second Having determined the rate constants for the hydrolysis reaction one (Eq. 2). This result is difficult to reconcile with the of 2b in the bulk solution and the binding constants of the experimental observation made for the 3bÌ1 complex, which inclusion complexes of 1 with all the species at 313 K, we preferentially locates the reactive isonitrile end at the open cavity decided to mathematically analyze the hydrolysis data in the of the container not providing an increased protection for its presence of 1 using a more elaborate kinetic model. This model hydrolysis. includes two consecutive irreversible reactions occurring in the bulk solution, Eqs. 3 and 4, and the reversible formation of three complexes: 2bÌ1, 3bÌ1 and 4bÌ1, Eqs. 5-7 (model 3). It also assumes that the rates of the hydrolysis reaction of the bound substrates are negligible. Fixing k3 and k4 to the values determined in the absence of 1, and Ka (2bÌ1), Ka (3bÌ1) and Ka (4bÌ1) to the measured magnitudes (Table 1), within experimental error, also produced a reasonable fit to the experimental distribution of the species for the time course of the hydrolysis reaction of 2b chaperoned by 1 (Figure 4c, solid lines). Remarkably, the theoretical kinetic models 1 and 3 produced quite different results in the simulations of the time course for the hydrolysis reaction of 2b chaperoned by 1.5 equiv. of 1. Model 1 considers that the hydrolysis reactions take place exclusively in Figure 4. the bound substrates and forecasts no changes in reaction Plots of the concentration of the species vs time for the acid- rates.41 catalyzed hydrolysis of bis-isonitrile 2b: a) with 1 equiv. of 1; b) without Conversely, model 3 assuming that only the free species container; c) with 1 equiv. of 1 and d) with 1.5 equiv. of 1. Dashed lines are hydrolyzed in the bulk aqueous solution projects a significant represent fit/simulation of the kinetic data to model 1 in a) and d). Solid lines reduction in reaction rates owing to the increased sequestering represent fit/simulation of the kinetic data to model 2 in b) and model 3 in c) effect of the free reacting species by complexation with the and d). Error bars are standard deviations. additionally added container. In order to evaluate which of the two models was most reliable, Moreover, model 1 disregards that the reactions can also take we experimentally undertook the hydrolysis of 2b in the place in the bulk solution by assuming that the rates are very presence of 1.5 equiv. of 1. Figure 4d displays the experimental slow and the concentration of the free substrates negligible. time course of the reaction (points). The dashed and solid lines However, the bis-isonitrile 2b and the mono-formamide 3b are represent the simulated kinetic profiles using models 1 and 3, perfectly soluble in water at millimolar concentrations. respectively. The simulation of the experimental data to model 3 Using identical conditions, we performed the hydrolysis is good, suggesting that it better explains the observed experiment of the bis-isonitrile 2b in the absence of container 1. selectivity enhancement in the acid-catalyzed hydrolysis of 2b. The reaction profile showed also a good fit to two consecutive In short, container 1 sequesters 2b and 3b offering protection to first-order irreversible reactions, Eqs. 3 and 4 (model 2), with k their isonitrile end groups towards the hydrolysis reaction. The 3 = 7.0 x 10-2 min-1 and k = 3.5 x 10-2 min-1 (Figure 4b, solid lines). hydrolysis of the substrates occurs mainly as the free species 4 present in the bulk solution. Because the concentrations of the free reactants are very low, their hydrolysis reaction rates are reduced. This is specially the case for the mono-formamide 3b, FULL PAPER whose concentration free in solution becomes relevant when are not related to its depth in the aromatic cavity of 1. The close to 80% of 2b has reacted. addition of 1 equiv. of container 1 to the acid-catalyzed To evaluate the scope and generality of the sequestering and hydrolyses of the bis-isonitriles modifies the reaction selectivity protection effect delivered by the synthetic chaperone 1 in towards the mono-formamides and the reaction rates compared improving the selectivity for the acid-catalyzed hydrolysis of to those in the bulk aqueous solution. The mathematical analysis symmetric aliphatic α,ω-bis-isonitriles, we used a series of of the kinetic data suggests that the influence of the container 1 shorter and longer homologues of 2b (Figure 1b). In the case of is mainly related to sequestration, due to complexation, of the the bis-isonitrile 2a, having a spacer with three methylene reacting substrates from solution. In addition, the hydrolysis groups, the reaction selectivity for the mono-formamide 3a was reaction of the isonitrile group in the bound substrates occurs reduced to 70% and the reaction rate was just slightly with rate constants that are smaller than in solution. The diminished compared to that in the absence of 1. The maximum protection level offered by 1 to the hydrolysis reaction of the percentage of 3a was obtained after 40 min of reaction instead bound guests depends on their length. For substrates having a of ca. 2 h required for 3b. Taking into account the smaller spacer of five methylene groups (2b and 3b), their hydrolysis binding affinities of the shorter substrates 2a and 3a for 1 (see reaction seems to be inhibited in the bound state. The right SI), compared to the longer counterparts 2b and 3b, the combination of binding affinity and protection offered by 1 leads, obtained results can be simply explained by invoking an after ca. 2 h of reaction time, to a maximum of 80% selectivity attenuation in the sequestering effect of the container. Also in for the mono-formamide 3b in the hydrolysis of the bis-isonitrile this case, model 3 provides a good fit to the obtained hydrolysis 2b. To the best of our knowledge, the effect exerted by a kinetic data (see SI, Figure S128). synthetic container, featuring a polar aromatic cavity (1), on the Longer bis-isonitriles 2c-e, having seven to nine methylene chemical selectivity of a desymmetrization reaction in water groups as spacers, and their corresponding mono-formamides solution and its proposed function mechanism are 3c-e (Figure 1b) display higher affinity for receptor 1 (Tables unprecedented.44 S12 and S13). As mentioned above, the hydrophobic effect is an important component in the binding of the homologous series of difunctional aliphatic guests by 1. The selectivity of the mono- Experimental Section formamides 3c-e in the hydrolysis reactions of 2c-e (1% DMSO/D2O)42 reaches values of 55-65%. The achievement of Experimental procedures and characterization data of the synthesized these selectivities require extensive reaction times (3 – 8 h). On compounds, NMR titration spectra, ITC experiments, plots of the kinetic the other hand, the hydrolysis reactions of 2c-e in the absence data for the hydrolysis reactions, protocols used in the GC-FID analyses of 1 (see SI) produced similar results to those discussed above of the reaction mixtures and energy-minimized structures of selected for 2b.43 Taking together, these results suggest that the longer inclusion complexes are contained in the Supporting Information for this symmetric difunctional substrates being more hydrophobic are article. present in reduced concentrations in the bulk solution. On the other hand, the formed inclusion complexes with 1 might provide a decrease in the hydrolysis rate of the bound isonitrile end Acknowledgements group that is located close to the open cavity of the container but not inhibition (total protection). This is due to the increased We thank Gobierno de España MCIN/AEI/FEDER, UE (projects protrusion of the isonitrile end groups of these complexes into CTQ2017-84319-P and CEX2019-000925-S), the CERCA the water/container interface (see Figure 3d for the energy- Programme/Generalitat de Catalunya, and AGAUR (2017 SGR minimized structure of the 3cÌ1 complex). 1123) for financial support. Q. S. thanks the Chinese Research Council for a predoctoral fellowship (2017-06870013). L. E. thanks MECD for a predoctoral fellowship (FPU14/01016). Conclusions Keywords: Amides • Host-Guest Systems • Isonitriles • Molecular Containers • Mono-Functionalization In summary, we demonstrated that, in water solution, the octa- pyridinium SAE-C[4]P 1 forms highly stable 1:1 inclusion complexes with a series of symmetric and non-symmetric difunctional aliphatic guests: bis-isonitriles, bis-formamides and 1 J. Kang, J. Rebek, Nature 1997, 385, 50-52. formamide-isonitriles. The cis-conformation of the formamide 2 M. Yoshizawa, M. Tamura, M. Fujita, Science 2006, 312, 251-254. end group is preferentially included in the deep aromatic cavity 3 M. D. Pluth, R. G. Bergman, K. N. Raymond, Science 2007, 316, 85-88. of 1. Four hydrogen bonds are established between the oxygen 4 V. Ramamurthy, Acc. Chem. Res. 2015, 48, 2904-2917. atom of the cis-formamide and the four pyrrole NHs of the 5 W. Cullen, M. C. Misuraca, C. A. Hunter, N. H. Williams, M. D. Ward, Nat. calix[4]pyrrole unit of 1. The large and negative heat capacity Chem. 2016, 8, 231-236. values measured for the complexation processes indicate that the hydrophobic effect is important for binding. Remarkably, the CIS experienced by the methylene groups in the bound guests FULL PAPER 6 A. Palma, M. Artelsmair, G. L. Wu, X. Y. Lu, S. J. Barrow, N. Uddin, E. Rosta, 29 L. M. Salonen, M. Ellermann, F. Diederich, Angew. Chem., Int. Ed. 2011, 50, E. Masson, O. A. Scherman, Angew. Chem., Int. 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Soc. 2020, 142, 2396-2403. 136, 5264-5266. 40 If all binding constant values of the complexes have similar magnitudes (see 17 Y.-S. Li, L. Escobar, Y.-J. Zhu, Y. Cohen, P. Ballester, J. Rebek, Y. Yu, Proc. Table 1), this methodology can be used to calculate the total Natl. Acad. Sci. U. S. A. 2019, 116, 19815-19820. concentration of the reacting species (free + bound) because their 18 S. Liu, H. Gan, A. T. Hermann, S. W. Rick, B. C. Gibb, Nat. Chem. 2010, 2, distribution is reflected by the relative concentrations of the complexes. 847-852. 41 The simulation of the time course for the hydrolysis reaction of 2b in the 19 K. Wang, J. H. Jordan, B. C. Gibb, Chem. Commun. 2019, 55, 11695-11698. presence of 1.5 equiv. of 1 using a kinetic model composed of Eqs. 1, 2, 20 M. M. J. Smulders, J. R. Nitschke, Chem. Sci. 2012, 3, 785-788. 5, 6 and 7 produced a similar result to that obtained using model 1. 21 R. M. Yebeutchou, E. Dalcanale, J. Am. Chem. Soc. 2009, 131, 2452-2453. 42 The hydrolysis reaction of longer bis-isonitriles 2c-e in the presence of 22 A. Galan, P. Ballester, Chem. Soc. Rev. 2016, 45, 1720-1737. container 1 was performed in 1% DMSO/D2O due to solubility reasons. 23 P. Mal, B. Breiner, K. Rissanen, J. R. Nitschke, Science 2009, 324, 1697- 43 Due to the overlap of proton signals and reduced solubility, we used GC-FID 1699. to monitor the reaction kinetics in the control hydrolysis experiments of 24 C. M. Hong, M. Morimoto, E. A. Kapustin, N. Alzakhem, R. G. Bergman, K. the longer bis-isonitriles 2c-e. The addition of 1% of DMSO did not N. Raymond, F. D. Toste, J. Am. Chem. Soc. 2018, 140, 6591-6595. produce a modification of the maximum selectivity of the mono- 25 L. Escobar, A. Diaz-Moscoso, P. Ballester, Chem. Sci. 2018, 9, 7186-7192. formamide. 26 G. Peñuelas-Haro, P. Ballester, Chem. Sci. 2019, 10, 2413-2423. 44 Previous reports on the use of molecular containers in controlling the 27 L. Escobar, P. Ballester, Org. Chem. Front. 2019, 6, 1738-1748. selectivity of desymmetrization reactions in water made exclusive use 28 V. P. Manea, K. J. Wilson, J. R. Cable, J. Am. Chem. Soc. 1997, 119, 2033- of non-polar binding cavities. 2039. FULL PAPER Entry for the Table of Contents (Please choose one layout) Layout 1: FULL PAPER A super aryl-extended calix[4]pyrrole Qingqing Sun, Luis Escobar and Pablo acts as both a sequestering and Ballester* supramolecular protecting group, enhancing the selectivity for the Page No. – Page No. mono-formamide product in the Hydrolysis of Aliphatic Bis-isonitriles hydrolysis of aliphatic bis-isonitriles. in the Presence of a Polar Super Aryl- Extended Calix[4]pyrrole Container Layout 2: FULL PAPER Author(s), Corresponding Author(s)* ((Insert TOC Graphic here; max. width: 11.5 cm; max. height: 2.5 cm)) Page No. – Page No. Title Text for Table of Contents