enthalpy compensation in anion binding: Biotin[6]uril and

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Entropy/enthalpy compensation in anion binding: Biotin[6]uril and biotin-L-sulfoxide[6]uril reveal strong solvent dependency Nicolaj N. Andersen, Kristina Eriksen, Micke Lisbjerg, Mille E. Ottosen, Birgitte Olai Milhøj, Stephan P. A. Sauer, and Michael Pittelkow J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02797 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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The Journal of Organic Chemistry

Entropy/enthalpy compensation in anion binding: Biotin[6]uril and biotin-L-sulfoxide[6]uril reveal strong solvent dependency Nicolaj N. Andersen, Kristina Eriksen, Micke Lisbjerg, Mille E. Ottesen, Birgitte O. Milhøj, Stephan P. A. Sauer, Michael Pittelkow* University of Copenhagen, Department of Chemistry, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark ABSTRACT: Binding of anions using macrocyclic structures with a non-polar interior using the CH···anion interaction as the recognition motif has gained popularity in the past few years, and such receptors often rely on a subtle interplay between enthalpy and entropic factors. For these types of receptors solvation of both the anion and the binding pocket of the macrocyclic host play important roles in the overall energetic picture of the binding event. Systematic chemical modifications of synthetic receptors that are able to bind anions in a variety of solvents is an important tool to gain understanding of the factors that determine the supramolecular chemistry of anions. Here we present the chiral macrocyclic structure biotin-L-sulfoxide[6]uril as a host molecule that binds anions in both water and in organic solvents. Biotin-Lsulfoxide[6]uril is prepared in a highly diastereoselective one-pot synthesis from the macrocycle biotin[6]uril. We compare the binding properties with that of biotin[6]uril, also studied in acetonitrile and in aqueous buffer at neutral pH. The biotinL-sulfoxide[6]uril generally exhibits stronger recognition of anions in acetonitrile, but weaker binding in water as compared to the biotin[6]uril macrocycle. We have studied the binding events using a combination of NMR spectroscopy, isothermal titration calorimetry (ITC) and computational methods.

Introduction An important challenge in supramolecular chemistry is to predict how structural modifications in receptors affect their binding affinities towards specific guest molecules.1 This challenge becomes even more pronounced when changing the solvent from a non-polar solvent to a polar protic solvent (e.g. water), where binding affinity is often completely lost or significantly diminished.2 Molecular recognition events, especially in water, requires a number of issues to be considered. A particularly difficult concept to understand and to take into consideration when preparing a new supramolecular system is that of enthalpy/entropy compensation.3-7 Often it is observed that even when enthalpy factors are optimized for a particular binding event the entropy contribution appears to counteract the binding affinity (G=H-TS).3 As highlighted by Fox, Whitesides and coworkers in a recent review paper,3 at least nine factors must be considered when attempting to understand the binding of a ligand by a protein: the formation of protein-ligand contacts; the rearrangement of water initially solvating the protein; the rearrangement of water initially solvating the ligand; the formation of a hydration structure around the protein-ligand complex; changes in the conformation of the protein between bound and unbound states; changes in the conformation of the ligand; changes in the dynamics of the protein, i.e. the sampling of multiple protein conformations on multiple timescales; changes in the dynamics of the ligand; and changes in the organisation – and interactions associated with – buffer ions.

To address the topic of entropy/enthalpy compensation, detailed investigations of solvent effects are of critical importance.8 Fundamental lessons about design criteria learned from supramolecular systems can be directly applied in medicinal chemistry and chemical biology applications.9,10 A key advantage of studies using relatively ridged macrocyclic structures as opposed to proteins is that a number of the nine parameters can be excluded, especially those associated with the dynamic nature of a protein and its binding pocket. This still leaves many parameters to consider, and especially those associated with solvation/de-solvation of the host (highenergy water) and the guest (ligand) are difficult to predict.1113

Molecular recognition of anions both in non-polar solvents and in aqueous solution present particular sets of challenges, and the recognition of anions is substantially understudied compared to that of cations.1,4 Monoatomic anions are typically larger and more diffuse than their isoelectric cationic counterparts, rendering intermolecular interactions with polar moieties on host molecules less enthalpically favorable. Solvation/de-solvation of anions influences the ability to interact with non-polar surfaces, especially in proteins. This is reflected in the so-called Hofmeister series.14,15 A host possessing highly polar motifs in its binding pocket can show high binding affinity in organic solvents, but can fail to recognize its target guest in aqueous solution, as water recognition competes for the binding site. Instead, multiple interactions that are independently weak but collectively strong can be employed in a strength through numbers strategy.16,17 Non-covalent interactions that are comparatively

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less charged, such as the CH···anion interaction, may complement the diffuse charge of the anionic guest.

diastereoselective (99.4 % de per oxidation) yielding biotin-Lsulfoxide[6]uril in one simple step from biotin[6]uril. The purification can be achieved without chromatography. We have further prepared a biotin-L-sulfoxide[6]uril hexaester to enable studies of the new macrocycle in both water and in an organic solvent. Binding constants and enthalpies of binding were measured using isothermal calorimetry (ITC). The data enables us to compare the binding energetics with that of biotin[6]uril, and we discuss how interactions between the solvent and partial charges on host and guest contribute to recognition strength in organic and aqueous media. Our data strongly suggest that the cause of the enthalpy/entropy compensation is a consequence of changes in the solvation of the receptors and the anions.

Figure 1: Top: structure of the biotin[6]uril macrocycle and key features regarding the anion recognition properties of the hexa-carboxylic acid derivative and the corresponding hexaester derivatives. Bottom: structure of the biotin-Lsulfoxide[6]uril macrocycle described (both as hexacarboxylic acid and hexa-ester) in this manuscript and an overview of key features regarding anion recognition.

The past few years has witnessed an increased emphasis on the studies of the CH···anion interactions,18,19 and we have contributed to this research area by introducing a new type of macrocyclic anion receptor called biotin[6]uril (Figure 1).20-24 Biotin[6]uril can be prepared on a gram scale in high yield by reacting D-Biotin and paraformaldehyde in 7 M hydrochloric acid. Only one regioisomer of the chiral biotin[6]uril is isolated. The resulting water soluble macrocycle can be converted to the hexaester to achieve solubility in organic medium, enabling anion recognition studies both in water and in organic solvents. Anion recognition by biotin[6]uril, and of other members of the hemicucurbit[6]uril family of macrocycles,17,2535 employs up to twelve CH···anion interactions, two from each monomeric unit, to achieve high association constants in aqueous solution.36 The CH bonds are slightly polarized on account of inductive electron withdrawal of the urea moieties, but not to an extent that facilitates strong interaction with water or guest molecules. The monomers show no recognition of anions, exemplifying the strength through numbers strategy. In this contribution we explore the biotin[6]uril family of macrocycles to study the phenomenon of entropy/enthalpy compensation. We demonstrate a new strategy for functionalizing the biotin[6]uril macrocycle by oxidizing the sulfide moiety of each biotin monomer of the macrocycle (Figure 1) to give the hexa-sulfoxide. This introduces six new stereocentres, and we have found that the oxidation is highly

Scheme 1: Synthesis of biotin[6]uril 1, biotin-Lsulfoxide[6]uril 2, biotin[6]uril hexamethylester 3, and biotinL-sulfoxide[6]uril hexamethylester 4. Synthesis of compounds 1 and 3 have previously been reported.20,21

Results and discussion Synthesis of biotin[6]uril macrocycles We prepared biotin-L-sulfoxide[6]uril from biotin[6]uril20 using stoichiometric amounts (6 equiv.) of hydrogenperoxide in glacial acetic acid in 72 % yield on 2 g scale (Scheme 1). The product precipitated from the reaction mixture as a single diastereoisomer upon addition of ether,§ eliminating the need of chromatography. 13 % of a second diastereoisomer was initially present in the isolated product (HPLC-MS) when the reaction was run at 5 mM concentration of macrocycle, but dilution of the reaction mixture to 2.5 mM suppressed the formation of this diastereoisomer to 1.6 % (ESI). This is equivalent to 99.7 % diastereoselectivity for the oxidation of each monomer of the macrocycle. The selectivity is important,


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The Journal of Organic Chemistry as isolation of the pure macrocycle would have been almost impossible due to the 16 different possible diastereoisomers that theoretically could have been formed by oxidation of the six sulfur atoms (Figure 2). It has been shown that oxidation of thianes and biotin occurs in the equatorial position (Lconfiguration)37-39 and the hexa-sulfoxide is assigned as the Lisomer, having the oxygen atoms positioned exo on the bicyclic monomers. This places the sulfoxide oxygen atoms towards the portals of the macrocycle. This assignment is supported by the specific rotation obtained for the isolated macrocycle ( [𝛼]25 𝐷 = -47 °), which compares better with biotin-L-sulfoxide ( 25 [𝛼]25 𝐷 = -40 °) than biotin-D-sulfoxide ([𝛼]𝐷 = 130 °). Attempts to unambiguously verify the stereochemistry by single crystal X-ray crystallography or by breaking down the macrocycle and comparing the product to monomeric biotin-D-sulfoxide and biotin-L-sulfoxide unfortunately failed. The methyl esters of both biotin[6]uril21 and biotin-L-sulfoxide[6]uril were obtained by means of esterification in methanol with catalytic amounts of hydrochloric acid and isolated by extraction into organic solvent.

Figure 2: 16 possible stereoisomers can theoretically form as combinations of L-oxidations (red) and D-oxidations (black) of biotin[6]uril. The six-petaled flower represents biotin[6]uril as it possesses both the one C3 and the three C2 axes of the macrocycle. The degeneracy of each product is indicated under the structure.

Computational studies Computational methods are often used to predict structures and binding geometries for new receptors and to rationalize how strong and selective binding motifs are.40 While this is a powerful tool for investigating binding events in the gas phase or in non-polar solvents, it provides less consistent results when predicting binding events in water, expecially when supramolecular systems are being considered.41,42 One reason for this is that it is difficult to reliably account for the energetic contributions associated with solvation/desolvation in water or specific hydrogen bonding interactions, and because these effect can be larger than the actual binding events (vide infra). At the outset of this work we speculated that oxidation of the sulfide moieties of biotin[6]uril to sulfoxides would lead to decreased electron density on the protons responsible for anion recognition. Following the logic from bambusuril and thiobambusuril this should lead to stronger affinity for small, unpolarisable (hard) anions. The sulfoxide moiety is considerably more electron withdrawing than sulfide (Hammet σ para values of 0.49 and 0.00 for SOMe and SMe, respectively)43 so a substantial difference in electron density was anticipated. We calculated electrostatic surface potentials

(Figure 3) at the CAM-B3LYP/6-311G(d) level of theory to support this hypothesis. Both biotin-D-sulfoxide[6]uril and biotin-L-sulfoxide[6]uril were investigated to assess the effect the different configurations at sulfur would have on the guest recognition. The pentanoic acid side chains were substituted for methyl groups to shorten the processing.

Figure 3: Electrostatic surface potentials of biotin[6]uril, biotin-D-sulfoxide[6]uril, and biotin-L-sulfoxide[6]uril at the CAM-B3LYP/6-311G(d) level of theory. Pentanoic acid side chains have been replaced with methyl groups to lower the computational cost. The macrocycles are shown top-down looking into the cavities. The corresponding truncated structures are shown below.

The parent biotin[6]uril displays a neutral exterior as visualized by the green color (Figure 3). The sulfur atoms carry some electron density (yellow) while the oxygen atoms of the urea moieties carry the highest electron density (red). The cyan cavity corresponds to a surface potential of approximately +130 kJmol-1 and correlates well with the electron deficiency required for anion recognition. Interestingly, the cavity potential of biotin-D-sulfoxide[6]uril does not increase substantially above +130 kJmol-1, but rather covers a larger area of the cavity, including both the α and β protons (Figure 4). Biotin-L-sulfoxide[6]uril displays substantial, localized electron deficiency in the cavity on the β protons with a surface potential of approximately +200 kJmol-1, but without electron deficiency on the α protons. We justify these differences between the two sulfoxides by considering how the SO bond may accept electrons from the bonds to the α protons: for the D-sulfoxide, the CH and SO bonds are parallel (Figure 4) favoring orbital overlap and justifying the electron deficiency seen on these protons. For the L-sulfoxide, the same bonds are almost orthogonal, preventing efficient orbital interaction. Either sulfoxide is nevertheless potentially a better anion hosts than the parent biotin[6]uril as both are more electron deficient in the cavity.



a more idealized C3 symmetry with encapsulated ethanol, water or iodide.20,22

Figure 4: Bond alignment in fragments of the D- and Lsulfoxides of biotin[6]uril. The CH and SO bonds discussed in the text are highlighted in black. Carbon (grey) oxygen (red) sulfur (yellow) hydrogen (white).

Next, we performed docking experiments with the halide anions (Table 1). Good correlation was found to the surface potential calculations with docking strengths increasing in the order biotin[6]uril → D-sulfoxide → L-sulfoxide. All three macrocycles prefer docking the smaller halides. This is a reflection of the charge density of the halides and the corresponding stability of the naked ion in the gas phase. The same trend is seen in the gas phase interaction calculations between halide and methane, indicating that the calculated energies are primarily from CH···anion interactions.44 The highly electron deficient cavity of biotin-L-sulfoxide appears to contribute more favorable docking energies by approximately 22 kJmol-1 compared to the less electron deficient cavity of the biotin-D-sulfoxide[6]uril. Table 1: Gas phase interaction zero point energies at the CAM-B3LYP/6-311G(d) level of theory for the complexes of biotin[6]uril, biotin-D-sulfoxide[6]uril, biotin-Lsulfoxide[6]uril, and the halide anions F-, Cl-, Br-, and I-. Units given in kJmol-1. Anion

Sulfide

D-Sulfoxide

L-Sulfoxide

F-

-460

-486

-504

Cl-

-232

-258

-280

Br-

-220

-248

-270

I-

-169

-199

-222

We then modelled the binding interactions between biotinand iodide (Figure 5) at the CAM-B3LYP/6311G(d) level of theory to investigate whether the more electron deficient cavity would alter the binding motif. It did not, as the guest is still situated in the center of the cavity. The assembly deviates slightly from idealized C3 symmetry yielding an average CH···I- distance of 3.2 Å. This value is slightly smaller than the sum of the van der Waals radius of hydrogen (1.2 Å) and the ionic radius of iodine (2.1 Å), suggesting a snug fit with optimal bond lengths. One portal is expanded compared to the other by tilting the monomers pivoting around the methylene bridges. This gives rise to a somewhat conical structure of the macrocycle. In comparison, the previously published crystal structure of biotin[6]uril shows a similar binding pocket exhibiting 12 CH bonds to the encapsulated iodide, but adopts L-sulfoxide[6]uril

Figure 5: Iodide encapsulated in biotin-L-sulfoxide[6]uril at the CAM-B3LYP/6-311G(d) level of theory. The 12 CH···I- are highlighted in red. The pentanoic acid side chains have been substituted for methyl to lower the computational cost. The macrocycle is shown with the larger portal towards the viewer. Carbon (grey) oxygen (red) nitrogen (blue) sulfur (yellow) hydrogen (white) iodine (purple).

Anion recognition Anion recognition in aqueous medium. The polar nature of water makes it an effective competitor for binding sites of anions. Obtaining only association constants for anion recognition in water therefore reveals little of the nature of the binding interactions. NMR and ITC can instead both be employed to investigate the binding event. While both methods provide the association strength, NMR reveals details about how the host binds the guest, while ITC provides the enthalpy and entropy of binding. Association strengths and corresponding thermodynamic parameters of 2 to chloride, bromide, iodide, nitrate, azide, perchlorate, cyanate, selenocyanate, thiocyanate, tetrafluoroborate, dicyanoargentate, dicyanoaurate, and hexafluorophosphate were measured in aqueous phosphate buffer at pH 7.5 using ITC and association strengths validated using NMR (Table 2). The data was fitted to the one-site model with fixed stoichiometry. Phosphate buffer is appropriate for these measurements, as none of the biotin[6]urils have affinity for hydrogenphosphates. Association constants were also measured using NMR titrations in aqueous phosphate buffer at pD 7.6 following the bridgehead protons. Job plots (ESI, fig. S5) confirmed 1:1 binding stoichiometry in the binding events, and the protons most affected by the binding event were the bridgehead (β) protons, similar to what was observed for anion binding using 1 as the host. Little influence of the counter ion was found, as sodium and caesium salts of iodide gave comparable results (ESI, Table S1 and Figures S13 and S14).


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The Journal of Organic Chemistry Binding data previously obtained for 1, also in phosphate buffer are likewise shown in Table 2 for comparison.22 Both biotin[6]uril and biotin-L-sulfoxide[6]uril showed the strongest association with iodide (as compared with the other halides), and no binding to fluoride. This constitutes a reversal in binding affinities compared to the affinities predicted by the calculations (gas phase). The preference for the larger ions is often seen for ion recognition in water as strong solvation of the guest ion disfavor encapsulation in the cavity of the host. In contrast to the predictions of the computational study, 1 is a better host than 2 in water: small, hard anions (chloride, nitrate, azide, cyanate) are not recognized at all by 2, despite being recognized with moderate association constants by 1. The main contributors to this bias against small guests are ostensibly tightly bound water in the electron deficient cavity of 2 that must be displaced upon encapsulation of guest and charge-charge repulsion between the anionic guest and the partial negative charges on the sulfoxide oxygen atoms. Interestingly, 2 also does not recognize some large anions that are recognized by 1 (tetrafluoroborate, dicyanoargentate, hexafluorophosphate), hinting that charge diffuseness of the guest is not the only effect dictating association strength.

event: 2 exhibits stronger interactions to water than 1, and these interactions must be broken upon encapsulation of the guest. Consequently, the release of this water increases disorder in the bulk solvent, thus justifying the favorable shift in entropy. The solvation shell of the anion was also considered: the smallest anion that was found to bind, bromide, also displays the largest change towards favorable entropy and the largest change towards unfavorable enthalpy upon comparing 2 to 1. Due to these trends in enthalpy and entropy comparing 1 and 2, we suspected that the macrocycles might mutually exhibit enthalpy-entropy compensation.45,46 We therefore compared anion recognition of the two hexaacids by plotting (TΔS2 – TΔS1) against (ΔH2 – ΔH1) for each of the guests (Figure 6). This method allows for straightforward visual comparison of differences in ΔH and ΔS for the macrocycles. The line (TΔS2 – TΔS1) = (ΔH2 – ΔH1) separates the plot into two sections: the area above the line corresponds to parameter space where 2 is the better host, while the area below the line corresponds to parameter space where 1 is the better host.

Table 2: Binding constants and thermodynamic parameters obtained with 1H-NMR in phosphate buffer at pD 7.6 at 27 °C and ITC in a phosphat buffer of pH 7.5 at 30 °C for 1 and 2. ΔΗ and –TΔS are given in kJmol-1. Biotin[6]uril data previously reported.22 All errors are within 15 %. Biotin[6]uril 1 Anion

logKa NMR

logΚa ITC

ΔH

-TΔS

Br-

3.0

2.7

-37.5

21.6

I-

3.7

3.4

-42.8

23.0

ClO4-

2.7

2.4

-33.3

19.5

SeCN-

4.3

4.0

-37.7

14.5

4.5

4.1

-35.0

11.2

4.7

4.2

-37.1

12.6

SCNAu(CN)2

-

Biotin-L-sulfoxide[6]uril 2 Anion

logKa NMR

logΚa ITC

ΔH

-TΔS

Br-

1.5

1.6

-10.7

1.8

I-

2.8

2.7

-30.1

14.8

ClO4-

0.9

1.7

-24.1

14.3

SeCN-

2.3

2.4

-24.6

10.8

SCN-

1.9

2.2

-14.4

1.8

Au(CN)2-

1.8

2.0

-18.0

6.4

The anion recognition of 1 is driven by the non-classical hydrophobic effect: large favorable enthalpies are counteracted by smaller but substantial, unfavorable entropies. For 2, contributions from enthalpy and entropy are both attenuated compared to 1, but enthalpy to a higher extend leading to lower association strength for all the anions analysed. We propose molecules of water associate strongly to the partial charges of the sulfoxides moieties. This is supported by the decrease in favorably enthalpy for the recognition

Figure 6: Plot of TΔS2 – TΔS1 against ΔH2 – ΔH1. ● spherical anions and ClO4- (Slope = 0.82, R2 = 1.0). ○ linear anions (Slope = 0.36, R2 = 0.96). Measurements in phosphate buffer at pH = 7.5. The grey area corresponds to parameter space where 1 is a better anion receptor than 2.

Data points are consistently located below the diagonal, showing that 1 is the better host. The smallest guests are located farthest from the origin of the plot, indicating that the anion recognition of the two hosts differ most for the guest of highest charge density. All data points are also located in the first quadrant of the plot, showing that the anion recognition of 2 tends more toward the classical hydrophobic effect than 1 (favorable TΔS2 – TΔS1 and unfavorable ΔH2 – ΔH1), albeit both hosts still show recognition within the non-classical regime. We also noticed that the data divides into two groups, namely spherical (or near-spherical anions) and linear anions. Each group show near-perfect linear correlation, and the positive slopes demonstrates that the macrocycles exhibit enthalpy-entropy compensation mutually. For spherical anions, the slope is close to unity, reflecting that a loss in enthalpy is compensated by a gain in entropy. For the linear guests the slope of the correlation is more shallow, reflecting



that loss in enthalpy is far from compensated by the gain in entropy. The less favorable binding enthalpy of the linear anions might be due to the charge-charge repulsion between the guest and the sulfoxide moieties on 2. This is not the case for the spherical anions as they are more compact and thus situated farther from the partially negatively charged sulfoxide moieties, minimizing repulsion. Anion recognition in organic medium. The association strengths of the tetraalkylammonium salts of chloride, bromide, and iodide to the hexamethyl ester 4 were measured in acetonitrile using ITC. The data fitted well to a one-to-one model (ESI, fig. S18-S20) with titrations giving sigmoidal curve shape. Table 3 shows the association constants and thermodynamic parameters obtained and association data previously found for 3.21 4 recognizes iodide and chloride better than 3. Bromide is recognized slightly better by 3. In organic medium, both enthalpy and entropy contribute favorably to the association for the halides under study. This is consistent with the charged halide being stabilized to a higher extend in the host cavity than in solution (enthalpy) and with solvent coordinated to host and especially guest being released to the bulk solvent upon encapsulation (entropy). 3 and 4 underwent the same enthalpy-entropy compensation study as the hexaacids. (TΔS4 – TΔS3) was plotted against (ΔH4 – ΔH3) for each of the three guests (Figure 7). Again, the diagonal (TΔS4 – TΔS3) = (ΔH4 – ΔH3) separates the plot into two sections: the area above this diagonal corresponds to parameters space where 4 is the better host, while the area below the line corresponds to parameters space where 3 is the better host. The smaller guests were again situated father from the origin, showing that the largest difference in enthalpy and entropy between the two hosts is for guests of high charge density. Through linear regression, a slope of 1.8 was found. The positive sign of the slope shows that the system does indeed exhibit enthalpy-entropy compensation. With respect to difference in enthalpy (ΔH4 – ΔH3), the smaller anions are preferentially bound. Here, 4 shows a gain of -5 kJmol-1 for the recognition of chloride and bromide compared to 3. Evidently, the higher cavity charge of 4 leads to encapsulating the guests of the most dense charge, as was found for the gas phase calculations. For the value of (TΔS4 – TΔS3), more complex behavior is observed: for iodide, the entropic contribution is favorable, indicating solvent is released to the bulk to a higher extend for 4 than for 3 upon guest encapsulation. For both chloride and bromide, 4 shows more unfavorable contribution from entropy than 3. Evidently, solvent is released to a lesser extend from the 4-guest associate than from the 3-guest associate. Table 3: Binding constants and thermodynanic parameters obtained with ITC in acetontrile at 30 °C for 1 and 2. ΔΗ and – TΔS given in kJmol-1. Biotin[6]uril data previously reported.20 All errors are within 25 %. Biotin[6]uril hexamethyl ester 3 Anion

logΚa

ΔH

-TΔS

Cl-

4.5

-12.4

-13.5

Br-

4.5

-12.5

-13.1

I-

3.0

Page 6 of 9 -8.1

-8.9

Biotin-L-sulfoxide[6]uril hexamethyl ester 4 Anion ClBrI-

logΚa

ΔH

-TΔS

4.7

-17.9

-9.3

4.4

-17.1

-7.9

3.6

-9.5

-11.3

Figure 7: Plot of TΔS4 – TΔS3 against ΔH4 – ΔH3. Slope = 1.8, R2 = 0.89. Measurements in acetonitrile. The grey area corresponds to parameter space where 3 is a better anion receptor than 4.

Conclusions We have synthesized biotin-L-sulfoxide[6]uril and its hexamethyl ester derivative and investigated their anion recognition in aqueous and organic media. The anion recognition event occurs using C-H···anion interactions. Our calculations predicted that biotin-L-sulfoxide[6]uril would be a better anion host than biotin[6]uril, which was true in acetonitrile but not in aqueous buffer. Inspection of thermodynamic parameters obtained from ITC titrations revealed enthalpy-entropy compensation for the systems, most prominently in aqueous solution but also in organic solvent. In a broader context, the work presented herein contributes to the understanding of how solvation/de-solvation of anions and of anion-binding cavities contributes to the energetics of binding. Of the nine suggested contributing factors suggested by Whitesides,3 the solvation of macrocyclic cavity before anion binding, the solvation of the anion before binding, and solvation of the host-guest complex appears to contribute most significantly to the binding events in the binding of anions to macrocycles with hydrophobic binding pockets. In the example presented in this paper, solvation causes a complete reversal in the binding selectivity of the anion binding receptor.

EXPERIMENTALS


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The Journal of Organic Chemistry The NMR spectra were recorded on a Bruker Ultrashield 500 Plus at 500.13 MHz for proton detected spectra and 125.77 MHz for carbon detected spectra. All spectra were recorded at 298 K. 1H chemical shifts were referenced to the solvent resonance. The 1H NMR spectra were recorded with spectral width 10330 Hz, acquisition time 3.17 s, and relaxation delay 1.0 s, collecting 16 FIDs, each consisting of 32k data point, zero filled to 64k data point with line broadening of 0.3 Hz. The 13C NMR spectra were recorded with spectral width 32000 Hz, acquisition time 0.91 s and relaxation delay 2.0 s, collecting 256 FIDs, each consisting of 32k data point, zero filled to 64k data points with line broadening of 1 Hz.

Author Contributions

Biotin-L-sulfoxide[6]uril. Biotin[6]uril (2.07 g; 1.35 mmol) was dissolved in glacial acetic acid (540 mL) by gentle heating. Upon full dissolution, the mixture was cooled to room temperature and 35 % H2O2 (749 μl; 8.08 mmol) was added. The reaction mixture was stirred overnight at room temperature. Ether (500 ml) was added and the precipitate was centrifuged and separated from the fluids. The white solid was washed with ether (3 · 100 ml) and heptane (3 · 100 ml), after which it was dried in vacuum. Yield: 1.59 g, 72 %. Melting point: > 199 °C (decomp.) Optical Rotation: [α]D20 = -59.35 (c = 1; 0.1 M sodium phosphate buffer at pH 7.5). 1H-NMR (500 MHz, DMSO-d6): δ = 12.00 (s, 6H), 4.78 (s, 12H), 4.49 (s, 6H), 4.10 (s, 6H), 3.61 (d, J = 14 Hz, 6H), 3.37 (m, 6H), 3.04 (m, 6H), 2.23 (t, J = 7.1 Hz, 12H), 1.60 - 1.25 (m, 36H). 13C-APT-NMR (126 MHz, DMSO-d6) δ 174.3, 159.9, 60.5, 59.0, 54.7, 54.6, 51.0, 46.1, 33.4, 28.5, 27.4, 24.2. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C66H97N12O24S6 1633.5057; Found 1633.5083. Anal. Calcd for C66H96N12O24S6+3H2O: C, 46.96; H, 6.09; N, 9.96. Found: C, 47.04; H, 5.91; N, 9.78.

§

Biotin-L-sulfoxide[6]uril hexamethylester. Biotin-Lsulfoxide[6]uril (0.301 mg; 184.2 μ mol) was dissolved in methanol (10 ml; 246.6 mmol) and one drop of conc. hydrochloric acid was added. The solution was stirred over night at room temperature. 2 M NaOH was added to adjust the pH to 7. The neutralised solution was ﬁltered through a plug of basic aluminium oxide, and the solvent was removed by a nitrogen flow. Yield: 250 mg, 79 %. Melting point: >125 °C (decomp.). 1H-NMR (500 MHz, CD3CN with 5 % CD3OD) δ 4.97 (m, 6H), 4.76 (m, 12H), 4.71 (s, 6H), 3.61 (s, 18H), 3.51 (m, 6H), 3.32 (dd, J = 14.4, 7.3 Hz, 6H), 3.15 (dd, J = 14,4, 5.9 Hz, 6H), 2.32 (t, J = 7.0 Hz, 12 H), 1.70-1.46 (m, 30H), 1.20-1.07 (m, 6H). 13C{1H}-NMR (126 MHz, CD CN with 5 % CD OD) δ 174.9, 160.7, 3 3 68.5, 60.3, 56.9, 53.7, 52.1, 51.3, 48.5, 34.2, 28.4, 25.6, 25.1. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C72H109N12O24S6 1717.5996; Found: 1717.5913.

ASSOCIATED CONTENT Supporting Information. Computational data and details regarding the ITC and NMR studies are available. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author

The manuscript was written through contributions of all authors.

ACKNOWLEDGMENT This work was supported by the Lundbeck Foundation and the Danish Council for Independent Research (Sapere Aude, DFF 4148-002606).

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