Supporting Information. for. Angew. Chem. Int. Ed. Z Wiley-VCH 2002

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1 Supporting Information for Angew. Chem. Int. Ed. Z19311 Wiley-VCH Weinheim, Germany

2 A highly enantioselective receptor for N-protected glutamate and anomalous solvent-dependent binding properties Sara Rossi, Graham M. Kyne, David L. Turner and Jeremy D. Kilburn. Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K. ELECTRONIC SUPPLEMENTARY INFORMATION: i) 1 H NMR spectra of macrocycle 2 in a) d 6 -DMSO at 2 C b) CDCl 3 at 2 C c) CDCl 3 at -40 C ii) Binding data from NMR titration experiments. a) NMR titration data for N-Boc-L-Glu with macrocycle 2 in CD 3 CN b) NMR titration data for N-Boc-D-Glu with macrocycle 2 in CD 3 CN c) NMR titration data for N-Boc-L-Glu with macrocycle 2 in d 6 -DMSO d) NMR titration data for N-Boc-D-Glu with macrocycle 2 in d 6 -DMSO iii) Job plots a) for macrocycle 2 with N-Boc-L-Glu in CD 3 CN b) for macrocycle 2 with N-Boc-D-Glu in CD 3 CN iv) Binding data from isothermal calorimetry experiments a) ITC data for N-Boc-L-Glu with macrocycle 2 in CH 3 CN b) ITC data for N-Boc-D-Glu with macrocycle 2 in CH 3 CN c) ITC data for N-Boc-L-Glu with macrocycle 2 in DMSO v) Determination of the solution structure of macrocycle 2 by NMR.

3 a) b) c) (ppm) i) 1 H NMR spectra of macrocycle 2 a) in d 6 -DMSO at 2 C b) in CDCl 3 at 2 C c) in CDCl 3 at -40 C

4 ii) Binding data from NMR titration experiments. The binding constants were calculated by fitting the titration data, using NMRTit HG software, kindly provided by Prof C. A. Hunter, University of Sheffield (A. P. Bisson, C. A. Hunter, J. C. Morales, K. Young, Chem. Eur. J. 1998, 4, 84). Data was recorded using standard NMR titration protocols, recording the shift for various 1 H signals on addition of aliquots of guest. An association constant was determined using the data from each separate 1 H signal, and averaged to give a final association constant. The rather large discrepancies between association constants obtained from the data for different 1 H signals in CD 3 CN, in the same titration experiment, is not surprising in view of the fact that the data gives binding constants at the upper limit of detectability using NMR titrations ( M -1 ) (L. Fielding, Tetrahedron 2000, 6, 611; C. S. Wilcox, Frontiers in Supramolecular Organic Chemistry and Photochemistry (Eds.: H.-J. Schneider, H. Dürr), VCH, Weinheim, 1991, pp ; G. Weber, S. R. Anderson, Biochemistry, 196, 4, 1942.) In these cases it can be concluded that K a > 10 4 M -1, but more precise values cannot be reliably obtained from the NMR titration data.

5 a) NMR titration data for N-Boc-L-Glu with macrocycle 2 in CD 3 CN Solvent: CD 3 CN Starting volume of host solution: 600µL Concentration of host solution: 1. mm Concentration of guest solution: 13.6 mm Association constant: M -1 (ave) ie K 1:1 a > 10 4 M -1 Volume added /µl Amide proton /ppm Thiourea proton /ppm CH proton /ppm K a 1:

6 Titration curves for N-Boc-L-Glu with macrocycle 2 in CD 3 CN Boc-L-Glu in MeCN (CH protons) Boc-L-Glu in MeCN (thiourea protons) ppm ppm [guest] E E E E E-03 [guest] Boc-L-Glu in MeCN (amide protons) 9.92 ppm E E E E E-03 [guest]

7 b) NMR titration data for N-Boc-D-Glu with macrocycle 2 in CD 3 CN Solvent: CD 3 CN Starting volume of host solution: 600µL Concentration of host solution: 1. mm Concentration of guest solution: 13.0 mm Association const (2:1, guest:host): 1.4x10 M -1 (ave) ie K 2:1 a >10 4 M -1 Volume added /µl Amide proton /ppm Thiourea proton /ppm CH proton /ppm K a 2:

8 Titration curves for N-Boc-D-Glu with macrocycle 2 in CD 3 CN Boc-D-glu in MeCN (thiourea proton Boc-D-glu in MeCN (CH protons ppm ppm E+00.0E E-03 1.E E-03 2.E E-03 3.E-03 [guest].4 0.0E+00.0E E-03 1.E E-03 2.E E-03 3.E-03 [guest] Boc-D-glu in MeCN (amide protons) ppm E+00.0E E-03 1.E E-03 2.E E-03 3.E-03 [guest] Comparison of shift for macrocycle amide NH on titration with N-Boc-L- Glu and N-Boc-D-Glu incd 3 CN (1:1 host:guest is reached when [guest] = 1. mm): Boc-Glu in MeCN (amide hydrogens) Boc-Glu in MeCN (CH protons) ppm Boc-L-glu in MeCN Boc-D-glu in MeCN ppm Boc-L-glu in MeCN Boc-D-glu in MeCN [guest] [guest]

9 c) Binding data for N-Boc-L-Glu with macrocycle 2 in DMSO-d 6 Solvent: DMSO-d 6 Starting volume of host solution 600 µl Concentration of host solution: 2.3 mm Concentration of guest solution: 94.2 mm Association constant: 3.72x10 3 M -1 (average) Volume added /µl Amide proton /ppm Thiourea proton /ppm K a 1:1 3.2x x10 3

10 Binding data for N-Boc-D-Glu with macrocycle 2 in DMSO-d 6 Solvent: DMSO-d 6 Starting volume of host solution 600 µl Concentration of host solution: 2.3 mm Concentration of guest solution: 94.2 mm Association constant: the data could not be fitted to a simple 1:1 or 1:2 binding isotherm which implies that complex multiple equilibria are operating as borne out by the isothermal calorimetry results. Volume added /µl Amide proton /ppm Thiourea proton /ppm

11 Comparison of shift for macrocycle amide NH on titration with N-Boc-L- Glu and N-Boc-D-Glu in DMSO-d 6 (1:1 host:guest is reached when [guest] = 2.3 mm): Boc-Glu in DMSO (amide hydrogens) ppm L-Boc Glu in DMSO D-Boc Glu in DMSO E E E E E-03 [guest]

12 iii) Job plots a) for macrocycle 2 with N-Boc-L-Glu in CH 3 CN 0.8 Boc-L-Glu in MeCN Job plot Host mole fraction b) for macrocycle 2 with N-Boc-D-Glu in CH 3 CN Boc-D-Glu in MeCN - Job plot chem shift*mol fraction molar fraction

13 iv) Binding data from isothermal calorimetry experiments. a) Data for N-Boc-L-Glu and macrocycle 2 in CH 3 CN (data corrected by subtracting the background dilution data for a injection of the guest solution into the cell containing neat CH 3 CN). [ligand] [receptor] [ligand]/ cal/mol area injection (mm) (mm) [receptor] of (µcal) (µl) (before (before (after injected inj.) inj.) inj.) ligand

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15 N-Boc-L-Glu and macrocycle 2 in CH 3 CN Data fitted using two binding site model: K a 1:1 = ± 4820 M -1 H = ± 0.18 kjmol -1, S = Jmol -1 K -1 ) (K a 2:1 = ± 4.32 M -1 )

16 b) Data for N-Boc-D-Glu and macrocycle 2 in CH 3 CN (data corrected by subtracting the background dilution data for a injection of the guest solution into the cell containing neat CH 3 CN). [guest] [receptor] [guest]/ cal/mol area injection (mm) (mm) [receptor] of (µcal) (µl) (before (before (after injected inj.) inj.) inj.) ligand

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18 N-Boc-D-Glu and macrocycle 2 in CH 3 CN Data fitted using two binding site model: K 2:1 a = 4910 ± 67 M -1 H = ± kjmol -1, S = 67.8 Jmol -1 K -1 ) (K a 1:1 = 38.4 ± 1.66 M -1 )

19 c) Data for N-Boc-L-Glu and macrocycle 2 in DMSO (data corrected by subtracting the background dilution data for a injection of the guest solution into the cell containing neat DMSO). area (µcal) inject ion (µl) [guest] (mm) (before inj.) [receptor ] (mm) (before inj.) [guest]/ [recepto r] (after inj.) cal/mol of injected ligand

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21 N-Boc-D-Glu and macrocycle 2 in DMSO Data fitted using one binding site model: K a 1:1 = 2281 ± 28M -1 H = ± 0.32 kjmol -1, S = 28.6 Jmol -1 K -1

22 v) Determination of the solution structure of macrocycle 2 using torsion angle dynamics with NOE and scalar coupling constant constraints. P. Güntert, C. Mumenthaler, K. Wüthrich, J. Mol. Biol. 1997, 273, 283; L. Brennan, D. L. Turner, A. C. Messias, M. L. Teodoro, J. LeGall, H. Santos, A. V. Xavier, J. Mol. Biol. 2000, 298, 61. The molecule was constructed as two identical fragments linked across amide bonds. The C 2 symmetry revealed by the NMR spectrum implies that each NOE may arise from cross-relaxation between protons in the same or different fragments. Lower distance constraints (derived from the upper limit of each NOE volume) were applied to all four possible pairs with the standard DYANA type 1 target function. Upper distance constraints used the modified target function (Nilges, M.; O Donoghue, S. I. Prog. NMR Spectrosc., 1998, 32, ) b < d β + α : (d b) 2 d > β + α : α(3α-2β) α 2 (2α β)/(d b) + β(d b) where d is the interproton distance in the calculated structure and b is the constraint derived from the lower limit of the NOE volume. This function is quadratic for constraint violations up to α and then tends to a linear form with slope β. The parameters were set to 1. and 0.1 Å, with an overall relative weight of 0.1 for the upper limits. This allows large violations of incorrect constraints without distorting the structure. The ambiguities with respect to upper distance constraints were removed wherever possible during initial structure

23 calculations. The final structure was calculated with 9 lower limits, each applied to 4 proton pairs, and 4 upper limits of which 11 remained ambiguous.