Substituent effects on aromatic interactions in water

Molecular recognition in water involves contributions due to polar functional group interactions, partial desolvation of polar and non-polar surfaces and changes in conformational flexibility, presenting a challenge for rational design and interpretation of supramolecular behaviour. Conformationally well-defined supramolecular complexes that can be studied in both water and non-polar solvents provide a platform for disentangling these contributions. Here 1 : 1 complexes formed between four different calix[4]pyrrole receptors and thirteen different pyridine N-oxide guests have been used to dissect the factors that govern substituent effects on aromatic interactions in water. H-bonding interactions between the receptor pyrrole donors and the guest N-oxide acceptor at one end of the complex lock the geometrical arrangement of a cluster of aromatic interactions at the other end of the complex, so that a phenyl group on the guest makes two edge-to-face and two stacking interactions with the four aromatic side-walls of the receptor. The thermodynamic contribution of these aromatic interactions to the overall stability of the complex was quantified by chemical double mutant cycles using isothermal titration calorimetry and 1H NMR competition experiments. Aromatic interactions between the receptor and a phenyl group on the guest stabilise the complex by a factor of 1000, and addition of substituents to the guest phenyl group further stabilises the complex by an additional factor of up to 1000. When a nitro substituent is present on the guest phenyl group, the complex has a sub-picomolar dissociation constant (370 fM). The remarkable substituent effects observed in water for these complexes can be rationalised by comparison with the magnitude of the corresponding substituent effects measured in chloroform. In chloroform, the double mutant cycle free energy measurements of the aromatic interactions correlate well with the substituent Hammett parameters. Electron-withdrawing substituents increase the strength of the interactions by a factor of up to 20, highlighting the role of electrostatics in stabilising both the edge-to-face and stacking interactions. The enhanced substituent effects observed in water are due to entropic contributions associated with the desolvation of hydrophobic surfaces on the substituents. The flexible alkyl chains that line the open end of the binding site assist the desolvation of the non-polar π-surfaces of polar substituents, like nitro, but at the same time allow water to interact with the polar H-bond acceptor sites on the substituent. This flexibility allows polar substituents to maximise non-polar interactions with the receptor and polar interactions with the solvent, leading to remarkably high binding affinities.


General methods
All the reagents and materials used in the synthesis of the compounds described below were obtained from commercial sources and used without prior purification. Compounds 1-4 were prepared as reported in literature. 1-3 Thin layer chromatography was carried out using Silica gel 60F on glass plates. Flash chromatography was carried out on an automated system (Combiflash Companion, Combiflash Rf+ or Combiflash Rf Lumen) using prepacked cartridges of silica (25 μm or 50μm PuriFlash® Columns). 1 H and 13 C NMR spectra were recorded on Bruker 400 MHz DPX400, 400 MHz AVIII400, 500 MHz DCH cryoprobe or 500 MHz TCI Cryoprobe spectrometer at 298.0 ± 0.1 K unless specifically stated otherwise. Residual solvent was used as an internal standard for referencing. In chloroform-d, 1 H spectra were referenced to δ 7.26 ppm and 13 C spectra to δ 77.06 ppm for the solvent signal. In dimethyl sulfoxide-d6, 1 H spectra were referenced to δ 2.50 ppm and 13 C spectra to δ 39.52 ppm. In deuterium oxide, 1 H spectra were referenced to δ 4.79 ppm. All chemical shifts are quoted in ppm on the δ scale and the coupling constants expressed in Hz. Signal splitting patterns are described as follows: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), m (multiplet). FT-IR spectra were recorded on a PerkinElmer Spectrum One FT-IR spectrometer equipped with an ATR cell. The LCMS analysis of samples was performed using a Waters Acquity H-Class UPLC coupled with a single quadrupole Waters SQD2 or a Waters Xevo G2-S bench top QTOF machine. Melting points were measured on a Mettler Toledo MP90 melting point apparatus. ITC titrations were carried out on a Malvern MicroCal VP-ITC MicroCalorimeter.

General experimental procedure for the synthesis of 4-arylpyridine N-oxides 5-14
To a solution of 4-chloro pyridine N-oxide (0.50 mmol, 1 equiv.) in dioxane (4 mL) under nitrogen, the corresponding aryl boronic acid (1.00 mmol, 2 equiv.), palladium tetrakis triphenylphospine (0.03 mmol, 0.05 equiv.) and sodium carbonate (2 M solution in water, 1 mL) were added. The mixture was stirred at 80 °C for 16 h. Ethyl acetate (10 mL) and water (5 mL) were then added. The black precipitate was filtered off through Celite® and the filtrate was extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with brine, dried over anhydrous MgSO4 and the solvents evaporated under reduced pressure. The crude product was purified by flash column chromatography on silica gel (methanol in dichloromethane 0 to 5 % in 20 minutes) to yield the corresponding 4-arylpyridine N-oxides 5-14.

Isothermal titration calorimetry (ITC) experiments
In a typical ITC experiment, the host (1-4) was dissolved in HPLC grade water or chloroform with a concentration 30-40 times the expected dissociation constant, and the solution was loaded into the sample cell of the microcalorimeter. A 7-10 times more concentrated solution of guest (5-17 and PNO) was loaded into the injection syringe. The number of injections was 35, and the volume of the injections was 8 μL. The thermogram peaks were integrated and thermodynamic parameters were calculated using the MicroCal PEAQ-ITC Analysis Software which uses the least-squares minimisation to obtain globally minimised parameters. In all cases the data fitted well to a simple 1:1 binding model.

Complex C of the DMC
The C-value for this experiment is 141, so the data could be used to determine both K and ∆H°. Figure S27. ITC data for titration of PNO (0.28 mM) into 1 (0.04 mM) in water at 298 K. The raw data for each injection is shown (differential power, DP), along with the least-squares-fit of the enthalpy change per mole of guest (ΔH) to a 1:1 binding isotherm.

Complex A of the DMC
The C-values for these experiments are in the range 10 5 -10 8 , so the data could only be used to determine ∆H°. The values of K were determined separately by NMR competition experiments.

Complex D of the DMC
The C-value for this experiment is 13, so the data could be used to determine both K and ∆H°.

Complex B of the DMC
The C-values for these experiments are in the range 37 -54, so the data could be used to determine both K and ∆H°.

Complex C of the DMC
The C-value for this experiment is 223, so the data could be used to determine both K and ∆H°.

Complex A of the DMC
The C-values for these experiments are in the range 10 3 -10 5 so the data could only be used to determine ∆H°. The values of K were determined separately by NMR competition experiments

Tetrachloro-aryl-extened calix[4]pyrrole 4 3.4.1. Complex D of the DMC
The C-value for this experiment is 39, so the data could be used to determine both K and ∆H° Figure S69. ITC data for titration of PNO (0.30 mM) into 4 (0.03 mM) in chloroform at 298 K. The raw data for each injection is shown (differential power, DP), along with the least-squares-fit of the enthalpy change per mole of guest (ΔH) to a 1:1 binding isotherm.

Complex B of the DMC
The C-values for these experiments are in the range 16 -97, so the data could be used to determine both K and ∆H°.

Pairwise 1 H NMR competitive titrations
Competitive titration experiments were performed using calix[4]pyrroles 1 and 2, and pyridine Noxides 5-17 and PNO in non-buffered deuterium oxide and deuterochloroform solutions. The association constant ratios between two competing complexes were determined by integrating selected proton signals in the acquired 1 H NMR spectra.