Discovery of a Cyclic 6 + 6 Hexamer of D-Biotin and Formaldehyde

The discovery of receptors using templated synthesis enables the selection of strong receptors from complex mixtures. In this contribution we describe a study of the condensation of D-biotin and formaldehyde in acidic water. We have discovered that halide anions template the formation of a single isomer of a 6 + 6 macrocycle. The macrocycle (biotin[6]uril) is water-soluble, chiral and binds halide anions (iodide, bromide and chloride) with selectivity for iodide in water, and it can be isolated on a gram scale in a one-pot reaction in 63% yield.


LC-MS
HPLC analyses were performed on a Dionex UltiMate 3000 system coupled to an UltiMate 3000 diode array UV/Vis detector.Separations were achieved using a Dionex Acclaim RSLC 120 C18 2.2 μm 120 Å 2.1 × 100 mm column maintained at 20 °C.The mobile phase solutions were 0.1 % formic acid in H 2 O and 0.1 % formic acid in MeCN.The water used as eluent was purified by a Millipore system.LC/MS analysis was carried out on a Bruker MicrOTOF-QII-system with an ESI-source with the following settings: nebulizer 1.2 bar, dry gas 8.0 L min -1 , dry temperature 200 °C, capillary 4500 V, end plate offset −500 V, funnel 1 RF 300.0 Vpp, ISCID energy 0.0 eV, funnel 2 RF 400.0 Vpp, hexapole RF 400.0 Vpp, quadrupole ion energy 5.0 eV, low mass 300.00 m/z, collision energy 10.0 eV, collision RF 630.0 Vpp, transfer time 120.0 μs, and pre puls storage 1.0 μs.The LC/MS data were processed using DataAnalysis v. 4.0 SP 5.
In the processing of HRMS measurements a sodium formate calibrant solution eluting in the first part of the LC-run was used to calibrate the system in each measurement.

NMR
1 H-NMR and 13 C-NMR spectra were recorded at 500 MHz and 126 MHz, respectively, on a Bruker Ultrashield Plus 500 spectrometer using residual non-deuterated solvent as the internal standard.
All NMR samples in D 2 O were equipped with a DMSO-d 6 lock tube and the spectra were calibrated according to the non-deuterated DMSO signal (marked with * in the spectra).All D 2 O samples contains 200 mM Na 2 CO 3 unless otherwise stated.
The 1 H-NMR titrations and the 1D-TOCSY experiments were recorded on a 500 MHz varian spectrometer, using a standard pentaprobe, except for the NaCl and NaBr titrations which were carried out on a Bruker Ultrashield Plus 500.The 1D-TOCSY was performed to confirm the spin system of the aliphatic sidechain on the Biotin[6]uril (2) and the experiment was carried out with different mixing times.

Isothermal Titration Calorimeter
A Microcal VP-ITC microcalorimeter was used for all titrations.The temperature was set at 30C and a stirring speed of 307 rpm was used.56 injections of 5 l of halide solution, with 240 s between each injection and an initial delay of 60 s were used for all titrations.

Elemental analysis
Elemental analysis for C, H, N and Cl was performed with a CE Instrument: FLASH 1112 series EA, at the microanalytical laboratory, University of Copenhagen.

Polarimeter
Optical rotation data were obtained on a Perkin Elmer 341 Polarimeter.

Diffractometer
All single-crystal X-ray diffraction data were collected at 122(1) K either on a Nonius KappaCCD areadetector diffractometer, equipped with an Oxford Cryostreams low-temperature device, using graphitemonochromated MoKα radiation, or a Bruker D8 Venture equipped with a IµS microfocus source, a KAPPA goniometer, a nitrogen cryostream cooling device and a PHOTON 100 detector, using MoKα radiation.The structures were solved using direct methods (SHELXS97) and refined using the SHELXL2013 software package.

Chemicals
Unless otherwise stated, all chemicals were purchased from commercial suppliers and used as received.Solvents were HPLC grade and used as received.

Increasing mixing time
Increasing mixing time To evaluate if there were aggregation or binding to the chloride dilution experiments of the Biotin[6]uril (2) at various concentration in water at and added NaCl was carried out (Figure S10).
H 2 SO 4 .The solids were washed with water (3  5 ml), followed by dissolving in Na 2 CO 3 and analysis by LC-MS (Figure S19 and Figure S20).The conc.H 2 SO 4 was analysed directly by LC-MS.6 Biotin[6]uril from H 2 SO 4 and NaBr Biotin (204 mg ; 0.84 mmol), para-formaldehyde (112 mg ; 3.7 mmol) and NaBr (5.19 g ; 50.4 mmol) was dissolved in 2.5 M H 2 SO 4 (10 ml) and the heterogeneous solution was heated to 60 C for 2 days.The mixture was cooled to room temperature and water (20 ml) was added.The solids were filtered and washed with water (3  5 ml).The solids were dissolved in 2 M NaOH, filtrated and precipitated with 5 M HBr followed by filtration.

General Job Plot Method
A 20 mM solution of NaX (Cl -, Br -or I -) in 200 mM Na 2 CO 3 (D 2 O) was mixed with a solution of 20 mM Biotin[6]uril in 200 mM Na 2 CO 3 (D 2 O) in the ratios shown in Table 1 and each sample was analyzed by 1 H-NMR.For KX a 2 mM solution of KX (X = Cl -, Br -or I -) in 20 mM Na 2 CO 3 was mixed with 2 mM of Biotin[6]uril in 20 mM Na 2 CO 3 in the same amounts as in Table 1.

Binding Constant Determination from NMR Titrations
From the Job plots a 1:1 stoichiometry between host and guest was found.Hence, the equilibrium constant for the host-guest complexation is given by Eq. 1.In this expression, the denominator is expanded by substitution Eq. 2 can be rearranged to the second order equation (Eq. 3) with [ ] as the unknown, and the general solution is given in Eq. 4. [ Only the solution where the last term is subtracted is chemically meaningful because the solution with a plus sign results in a concentration of complex that is higher than the smallest of the numbers [ ] and [ ] .
Eq. 4 gives an expression where the unknowns are [ ] and .The purpose is to find and 1 H-NMR was used to provide a measure of [ ]. Various amounts of ionic guests were titrated into a solution of the biotin[6]uril under conditions where the total concentration of host was constant and the movement of a host signal (denoted δ) was followed.
Under the used conditions the complexation was fast on the chemical shift time scale, and therefore the observed signal δ is as a weighted average of the signals (chemical shift of the proton in pure host) and (chemical shift of the proton in pure complex) with the mole fractions and as the weighting factors.This is expressed in Eq. 5 which via standard manipulations can be written as Eq. 6 δ (Eq.5) For each measurement in the titration, the change from to the observed δ was calculated and denoted .The unknown quantity, , indicates the maximal obtainable change in the titration and is denoted . With these notations, Eq. 6 can be rewritten to Eq. 7 and by substitution of Eq. 4 into Eq.7, the final fitting equation Eq. 8 is obtained.
In Eq. 8 the quantities and are unknown but linked to the measurable quantity and the known [ ] and [ ] .In Origin 8.6, and were determined by fitting Eq. 8 to the titration data.

NMR Titrations
The 1 H-NMR (500 MHz, D 2 O) titrations were carried out by the following method.Two solutions were used for the 1 H-NMR titrations one containing 5.01 mM of Biotin[6]uril in 200 mM Na 2 CO 3 (pH = 10.8)used for NaCl, and one with 0.505 mM Biotin[6]uril in 20 mM Na 2 CO 3 (pH = 10.8)used for NaBr, NaI and KX.The sodium or potassium halides were added while keeping the Biotin[6]uril concentration constant.

ITC Titrations
Biotin[6]uril was dissolved in Na 2 CO 3 buffer (Table 4: Samples for ITC), and the pH was adjusted to 10.8 with NaOH.The sodium halides were dissolved in NaHCO 3 (Table 4: Samples for ITC) and the pH was adjusted to 10.8 with NaOH.Dilution experiments for the Biotin[6]uril and the sodium halides were run under the same parameters, and subtracted from the raw data.The raw data were analyzed using Origin and fitted by the routines provided by MICROCAL.A low c fitting procedure with reduced χ 2 was used to fit the data. 1     The crystal of Biotin[6]uril, for single crystal x-ray diffraction, was produced by slow evaporation of ethanol.
When Biotin[6]uril was dissolved in MeOD-d 4 it was converted into the hexa-D 18 ester in two days, probably because of the inclusion of HCl which catalyse the esterification.Figures S1 to S10 corresponds to the fully esterified Biotn[6]uril-D 18 ester.

Figure S10 :
Figure S10: 1 H-NMR (500 MHz, D 2 O) of Biotin[6]uril-HCl at different concentrations.The signals (b and f) move due to binding of chloride.The signals are set according the triplet at 2.15 ppm.

Figure S18 :
Figure S18: 1 H-NMR (500 MHz, D 2 O) Biotin[6]uril at different concentrations.No signals move which confirms that the chloride is removed, and that there is no aggregation.*External reference of DMSO-d 6 .

Figure S19 :
Figure S19: Total Ion Chromatogram of the solids from the reaction between biotin and para-formaldehyde at different concentration of HCl.The asterisk (*) indicates Biotin[6]uril all other peaks are linear or cyclic oligomers.LC-MS method B was used.

Figure S20 :
Figure S20: Total Ion Chromatogram of the solids from the reaction between biotin and para-formaldehyde at different concentration of H 2 SO 4 .All peaks are linear or cyclic oligomers, no Biotin[6]uril was detected for any of the samples.LC-MS method B was used.

Figure S25 :
Figure S25: Total Ion Chromatogram of the solids from the reaction between biotin and para-formaldehyde at different concentration of HBr.The asterisk (*) indicates Biotin[6]uril all other peaks are linear or cyclic oligomers.LC-MS method B was used.

Figure S26 :
Figure S26: Total Ion Chromatogram of the solids from the reaction between biotin and para-formaldehyde at different concentration of HI.The asterisk (*) indicates Biotin[6]uril all other peaks are linear or cyclic oligomers.LC-MS method B was used.

Figure S29 :
Figure S29: Job plot of NaBr and biotin[6]uril, where the proton b is followed.The red dots are estimated results, because the proton signal is under another signal.

Figure S30 :Figure S31 :
Figure S30: Job plot ofNaI and biotin[6]uril, where the proton f is followed.The 0.6 is omitted as the signal is under the water signal.

Figure S32 :
Figure S32: Job plot of KCl and Biotin[6]uril, were the proton f is followed.

Figure S36 :Figure S37 :
Figure S36: Plot showing experimental data for 1 H-NMR titration of NaCl following proton f together with the fitted curve (red).

Figure S42 :
Figure S42: Plot showing experimental data for 1 H-NMR titration of NaI following proton b together with the fitted curve (red).

Figure S44 :
Figure S44: Plot showing experimental data for 1 H-NMR titration of KCl following proton f together with the fitted curve (red).

Figure S45 :
Figure S45: Plot showing experimental data for 1 H-NMR titration of KCl following proton b together with the fitted curve (red).

Figure S48 :
Figure S48: Plot showing experimental data for 1 H-NMR titration of KBr following proton b together with the fitted curve (red).

Figure S50 :
Figure S50: Plot showing experimental data for 1 H-NMR titration of KI following proton f together with the fitted curve (red).

Figure S51 :
Figure S51: Plot showing experimental data for 1 H-NMR titration of KI following proton b together with the fitted curve (red).

Figure 56 :
Figure 56: Structure of Biotin[6]uril with a ethanol molecule in the cavity, other solvent molecules, disordered atoms and hydrogen atoms is omitted for clarity.Yellow: sulphur; Blue: nitrogen; Gray: carbon; Red: oxygen;.

4 Chloride free Biotin[6]uril
The solution was stirred overnight at room temperature.The reaction mixture was filtered and acidified with conc.H 2 SO 4 .The product was filtered off and washed with water (3  10 ml).

Table 1 :
Sample composition of Job plot.

Table 2 :
Titration data for proton b.
Figure S27: Job plot of NaCl and biotin[6]uril, it is the b proton which is followed.Figure S28: Job plot of NaBr and biotin[6]uril, where proton f s followed.

Table 4 :
Samples for ITC