Merging Bambus[6]uril and Biotin[6]uril into an Enantiomerically Pure Monofunctionalized Hybrid Macrocycle

Bambus[6]urils and biotin[6]urils are macrocycles with an exceptional affinity for inorganic anions. Here, we investigated statistical condensation of 2,4-dibenzylglycoluril and d-biotin, monomers of the corresponding macrocycles, to prepare the enantiomerically pure macrocycle 1 containing a single d-biotin and five glycoluril units. Host–guest properties of 1 in chloroform solution and solid state were investigated. The macrocycle 1 bearing a single functional group was employed in the formation of [1]rotaxane utilizing reversible covalent bonds.


General Methods
All reagents and deuterated solvents were purchased from commercial suppliers and used without further purification.HPLC and deuterated solvents were further dried over 4 Å molecular sieves.Reaction mixtures were heated on DrySyn heating blocks, and the reaction temperatures stated refer to the settings of the magnetic stirrer.
NMR spectra were recorded on a Bruker Avance III 300 MHz, and Bruker Avance III 500 MHz spectrometer.Chemical shifts (in ppm) are referenced to residual solvent peaks of deuterated solvent.Standard abbreviations for multiplicity are used as follows: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, m = multiplet, and br = broad.Structural assignments were made with additional information from COSY, HSQC, HMBC, ROESY, and DOSY experiments.
ITC analysis was recorded on MicroCal VP-ITC from Malvern.
HRMS analysis was recorded on Agilent 6224 Accurate-Mass TOF LC-MS.Samples were ionized by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI).Matrix assisted laser desorption ionization with detection of time of flight (MALDI-TOF) mass spectra were measured on the MALDI-TOF MS UltrafleXtreme (Bruker Daltonics).Samples were ionized by Nd-YAG laser (355 nm) from 2,5-dihydroxybenzoic acid (DHB) matrix.Melting points were measured on Stuart SMP40 melting point apparatus.
Diffraction data were collected on a Rigaku MicroMax-007 HF rotating anode CCD diffractometer and the structures were solved by direct methods and refined by full matrix least-squares methods using SHELXT and SHELXL.
In situ Preparation of 5 [1]Rotaxane 5 was synthesized in situ in NMR tube, requiring only a few minutes of reaction time.The reaction was initiated by mixing a solution of 4 (0.5 mL, 5 mM, 1 equiv.) in CD3CN-d3 with the solution of 5 (5 mg, 2.5 µmol) in CD3CN-d3.S18

Isothermal Titration Calorimetry (ITC)
An analysis using isothermal titration calorimetry (ITC) was conducted utilizing a MicroCal VP-ITC instrument from Malvern.The experiments were carried out at 298.15 K in a chloroform solvent.The heat responses recorded during the titration process are illustrated in the upper graph of each figure within this section.Each peak on the graph corresponds to the introduction of a 10 μL salt solution into the cell containing 1 alone or complexed with the competitor.In general, the lower graph depicts the cumulative heat released as a function of the total concentration of the ligand.The solid red line on the graph represents the best-fit line obtained through a least-squares analysis of the data.To examine the integrated heat effects, a single-site model was employed in nonlinear regression analysis.The association constant Ka and the standard binding enthalpy ΔH° were determined using experimental data matched to a theoretical titration curve.The standard free energy ΔG° and standard entropy ΔS° were obtained by means of the equation: ΔG°= ΔH°-TΔS°=-RT lnKa, where T is the absolute temperature and R is the molar gas constant (8.3145J mol -1 K -1 ).

Crystallography
Colorless crystals were prepared by slow diffusion of diethyl ether vapor into chloroform solution of 1 (1mM) and tetrabutylammonium chloride (1mM).
Diffraction data were collected on a Rigaku MicroMax-007 HF rotating anode CCD diffractometer using Mo Kα radiation at 120 K. CrystalClear was used for data collection, CrysAlisPro for data reduction and absorption correction.The structures were solved by the direct methods procedure and refined by full matrix least-squares methods on F 2 using SHELXT and SHELXL.Crystal data and refinement parameters are gathered in Table S

Figure S24 .
Figure S24.Molecular structure of the Cl - 1 complex.Thermal ellipsoids are drawn at the 50% probability level.

Figure S25 .
Figure S25.Side view of the Cl - 1 complex.
#.The supplementary crystallographic data for this paper can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. TableS1.Crystallographic information for 1.