Peptide recognition by a synthetic receptor at subnanomolar concentrations

This paper describes the discovery and characterization of a dipeptide sequence, Lys–Phe, that binds to the synthetic receptor cucurbit[8]uril (Q8) in neutral aqueous solution with subnanomolar affinity when located at the N-terminus. The thermodynamic and structural basis for the binding of Q8 to a series of four pentapeptides was characterized by isothermal titration calorimetry, NMR spectroscopy, and X-ray crystallography. Submicromolar binding affinity was observed for the peptides Phe-Lys-Gly-Gly-Tyr (FKGGY, 0.3 μM) and Tyr-Leu-Gly-Gly-Gly (YLGGG, 0.2 μM), whereas the corresponding sequence isomers Lys-Phe-Gly-Gly-Tyr (KFGGY, 0.3 nM) and Leu-Tyr-Gly-Gly-Gly (LYGGG, 1.2 nM) bound to Q8 with 1000-fold and 170-fold increases in affinity, respectively. To our knowledge, these are the highest affinities reported between a synthetic receptor and an unmodified peptide. The high-resolution crystal structures of the Q8·Tyr-Leu-Gly-Gly-Gly and Q8·Leu-Tyr-Gly-Gly-Gly complexes have enabled a detailed analysis of the structural determinants for molecular recognition. The high affinity, sequence-selectivity, minimal size of the target binding site, reversibility in the presence of a competitive guest, compatibility with aqueous media, and low toxicity of Q8 should aid in the development of applications involving low concentrations of target polypeptides.

Electrospray Ionization Time-of-Flight Mass Spectrometry.Mass spectra of purified peptides and their complexes with Q8 (Figures S18-25) were acquired by infusion using an Agilent 6230 TOF LC/MS mass spectrometer with an electrospray ion source in the positive ion mode.Samples were dissolved in pure water at a concentration of 100 µM purified peptide in the absence and presence of one molar equivalent of Q8.

Isothermal Titration Calorimetry (ITC).
Titrations were carried out in 10 mM pH 7.0 phosphatebuffered H2O at 300 K using either a VP-ITC calorimeter (Malvern, Inc.), or a nanoITC LV (low volume) calorimeter (TA Instruments) (Figure S1-S8).In a typical experiment, Q8 was in the sample cell at concentrations in the range of 30-50 µM.S5) The peptide solution was in the syringe in the concentration range 300-500 µM.The titration schedule consisted of 28 consecutive injections of 10 µL with at least a 200 s interval between injections.Heats of dilution, measured by titrating beyond saturation, were subtracted from each dataset.All solutions were degassed prior to titration.The data were analysed using Origin software and fit by non-linear regression to the binary equilibrium binding model (non-interacting sites) supplied with the software to determine molar enthalpy, equilibrium association constant, and binding stoichiometry.These values were used to calculate the free energies of binding and the entropic contributions to the binding free energies.

VP-ITC: (Figure
nanoITC LV: (Figures S1, S3, S6, S8) The peptide solution was in the syringe at the concentration range 198-330 µM.The titration schedule consisted of 28 successive injections of 1.76 µL (41 injections of 1.2 µL in the case of H-YLA-NH2) and with at least 150 s between injections.Heats of dilution, measured by titrating beyond saturation, were subtracted from each dataset.All solutions were degassed prior to titration.The data were analysed using the proprietary nanoAnalyze software and fit by non-linear regression to the independent equilibrium binding model supplied with the software to determine molar enthalpy, equilibrium association constant, and binding stoichiometry.These values were used to calculate the free energies of binding and the entropic contributions to the binding free energies.
Competitive ITC titrations to determine affinities >10 7 M -1 were carried out on the nanoITC LV as above, except for the presence of 100 equiv. of a weak competitive guest (G weak ) in the cell along with Q8 (Figures S2, S4, S7).This titration yields ΔH obs , and Ka obs for the displacement.The binding constant of the weak competitor in the cell (Ka weak ) was previously determined by direct titration.The binding constant of the high-affinity guest (Ka strong ) was then determined by the the following equation: Experimental conditions that yielded Ka obs also gave observed molar binding enthalpies ΔH obs .This binding enthalpy is the sum of the binding enthalpy of the weak competitor (ΔH weak ) and of the strong guest (ΔH strong ) and is based on the thermodynamic cycle of the form: Thus, the molar enthalpy of binding of the strong guest is given by Nuclear Magnetic Resonance (NMR) Spectroscopy: (Figures 2, S9-S17) All 1-D and 2-D NMR spectra were collected in D2O (δ 4.790) at 21 °C using either a Varian 500 MHz instrument with an operating frequency of 499.6 MHz, or a Bruker Avance NEO 500 MHz instrument with an operating frequency of 500.13MHz.NMR spectral data were processed using MNova 14 (Mestrelab Research SL).Multiplicity abbreviations are as follows: s -singlet; t -triplet.Signal presaturation of residual protiated solvent was used as necessary.To aid in solubility, the peptides and their 1:1 complexes with Q8 were dissolved in 10 mM sodium phosphate-buffered D2O, pHapparent 7.1, 3 which was prepared as follows: NaH2PO4 (5.54 mmol) and Na2HPO4 (3.49 mmol) were dissolved in D2O (100 g, 90.334 mL) to yield a solution of 100 mM pH 7.0 (pHapparent 7.1) sodium phosphate-buffered D2O, which was diluted to 10 mM prior to use.
Correlation spectroscopy (COSY) spectra were acquired with a 2 s relaxation delay and with a spectral width of 8012 Hz.The COSY spectrum of the 1:1 Q8•KFGGY complex was recorded with 1024t1 * 1024t2 complex points.The 2-D nuclear Overhauser effect spectrum of free KFGGY and the rotating-frame Overhauser effect spectrum of the 1:1 Q8•KFGGY were acquired using a 2 s relaxation delay, a 500 ms mixing (spin-lock) time (250 ms for the free peptide), a spectral width of 7500 Hz, and was recorded with 1024t1 * 512t2 complex points.
Chemical shift values were referenced relative to TSP via the residual solvent resonance at 4.77 ppm.

Competition Experiments Using 1 H NMR spectroscopy. The competition experiment to
determine the binding affinity of H-KFGGY-OH to Q8 was performed according to the Isaacs competition method in order to provide a benchmark for the competitive ITC titration experiments (Figure S14). 4 Briefly, to a mixture of known concentrations of (ferrocenylmethyl)trimethylammonium chloride (FcNMe3) and H-KFGGY-OH in 10 mM sodium phosphate-buffered D2O, pHapparent 7.1, was added a limiting molar quantity of Q8.The mixture was subjected briefly to 1-3 cycles of sonication and heating at 60 °C to fully solubilize the Q8.
NMR spectra were acquired with a relaxation delay of 30 s to improve the accuracy of signal integration.The total concentration of peptide in the mixture ([peptide]o) was determined by UV spectroscopy (ε275 = 1420 M -1 cm -1 ).Therefore, the total integration of the aromatic signals of peptide was used as the benchmark for determining the concentrations of other species in the mixture via relative integration.The total concentration of Q8 ([Q8]o) in the mixture was determined using the following equation: where IQ8 is the integration value of a Q8 signal; NQ8 is the number of hydrogen atoms corresponding to that Q8 signal (i.e., 16); IAr is the total integration value of all aromatic signals; and NAr is the total number of hydrogen atoms corresponding to all signals in the aromatic region (i.e., 9).The same method was used to find the equilibrium concentrations of unbound peptide ([peptide]eq) and bound peptide ([peptide•Q8]eq).Using the conservation of mass, the equilibrium concentration of bound FcNMe3 ([FcNMe3•Q8]eq) is given by Similarly, the equilibrium concentration of free FcNMe3 ([FcNMe3]eq) can be found by mass balance These values can be used to determine the equilibrium constant for the competition reaction which is represented by the following equilibrium expression and is also the ratio of the equilibrium dissociation constant (Kd) values for the FcNMe3•Q8 and peptide•Q8 complexes.
Therefore, the Kd value for the peptide•Q8 complex was determined as
Iodomethane (2.5 mmol, 0.156 mL) was then added in one portion, and the mixture was allowed to stir at room temperature for 90 min.Another portion of iodomethane was added (2.5 mmol, 0.156 mL), and the mixture was allowed to stir for an additional 90 min.Thin-layer chromatography was used to monitor the reaction (Rf = 0.38, 10:1 DCM/MeOH).Anhydrous diethyl ether (20 mL) was then added to the mixture to induce precipitation of the iodide salt of the product.The suspension was then filtered, and the filter cake was washed with additional diethyl ether until the filtrate was colourless.The solids were dried under high vacuum for 30 min to yield (ferrocenylmethyl)trimethylammonium iodide as an orange powder.The solids were then dissolved in ultra-pure H2O (1 mL), passed through a short column loaded with AmberChrom 1X4 chloride form ion-exchange resin, and eluted using ultra-pure water until the eluate was colourless.
The eluted solution was then flash-frozen in liquid nitrogen and lyophilized to dryness to yield (ferrocenylmethyl)trimethylammonium chloride as a deep yellow powder (0.265 g, 0.902 mmol, 90%).Spectroscopic data agree well with the literature.and dry Q8 (0.9 equiv., 2.2 mg) in a scintillation vial.To each suspension was added 10 mM sodium phosphate-buffered D2O, pHapparent 7.1, to a final complex concentration of 1.0 mM.Each suspension was subjected to two cycles of sonication and heating to 70 °C for 20 minutes and cooling to room temperature, during which time plate-like crystals formed that were coloured under linearly polarized light.
Reflection data were collected at 100 K on a XtaLAB Synergy, Dualflex HyPix four-circle diffractometer using CuKα radiation (λ = 1.54184Å).All data were integrated with CrysAlisPro 6 and corrected for absorption using ABSPACK. 6The structures were solved by dual methods with SHELXT and refined by full-matrix least-squares methods against F 2 using SHELXL. 7,8 ll nonhydrogen atoms were refined with anisotropic displacement parameters.Most of the hydrogen atoms of the investigated structure were located from difference Fourier maps.Finally, their positions were placed in geometrically calculated positions and refined using a riding model including those bound to heteroatoms.When the hydrogen atom location was not obvious from difference maps hydrogen atoms were placed in logical hydrogen bonding geometries.Isotropic thermal parameters of the placed hydrogen atoms were fixed to 1.2 times the U value of the atoms they are linked to (1.5 times for methyl groups, NH groups, OH groups, and H2O).For YLGGG, a solvent mask was calculated and 164 electrons were found in a volume of 440 Å 3 in 1 void per unit cell.This is consistent with the presence of 8 water molecules per formula unit which account for 160 electrons per unit cell.The structures were also treated as a two-component inversion twin resulting in a BASF of 0.14(10).For LYGGG, a solvent mask was calculated and 476 electrons were found in a volume of 1278 Å 3 in 1 void per unit cell.This is consistent with the presence of 44 water molecules per formula unit which account for 440 electrons per unit cell.The structures were also treated as a two-component inversion twin resulting in a BASF of 0.14(10).Calculations and refinement of the structure was carried out using Olex2 software. 9The data were deposited to the Cambridge Crystallographic Data Center (deposition numbers 2312293 and 2314758) (Tables S1 and S2).

Figure S24 :
Figure S24: ESI-MS of a 100 µM aqueous solution of H-LYGGG-OH collected in positive mode.

Figure S25 :S29Figure S26 :
Figure S25: ESI-MS of a 100 µM aqueous solution of an equimolar mixture of H-LYGGG-OH and Q8 collected in positive mode.

Figure S27 :
Figure S27: ESI-MS of a 100 µM aqueous solution of an equimolar mixture of H-YLGGG-OH and Q8 collected in positive mode.

Table S1 .
Crystal Data and Structure Refinement for Q8•YLGGG

Table S2 .
Crystal Data and Structure Refinement for Q8•LYGGG