Molecular Tweezers with Varying Anions - A Comparative Study SomDutt a, ConstanzeWilch a, Thomas Gersthagen a, Peter Talbiersky a, Kenny Bravo- Rodriguez b, MattiHanni c, Elsa Sánchez-García* b,christian chsenfeld* c, Frank-Gerrit Klärner* a, Thomas Schrader* a a University of Duisburg-Essen, Department of Chemistry, Universitätsstr. 7, 45117 Essen, Germany b Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 4547 Mülheim an der Ruhr, Germany c Chair of Theoretical Chemistry, Department of Chemistry, University of Munich (LMU), Butenandtstr. 7, 81377 Munich, Germany and Center for Integrated Protein Science (CIPSM) at the Department of Chemistry, University of Munich (LMU), Butenandtstr.5-13, 81377 Munich, Germany esanchez@mpi-muelheim.mpg.de; christian.ochsenfeld@uni-muenchen.de; frank.klaerner@uni-duisburg-essen.de; thomas.schrader@uni-due.de Supporting Information Table of Contents 1. Experimental Section: Page 1.1 1 H NMR and 13 C NMR spectra of the tweezers 1c, 1g, and 1d 2 1.2 Dimerisation of the molecular tweezers determined by NMR titration 8 1.3 Host-guest complex formation of the molecular tweezers with various amino acid and peptide guest molecules containing lysine or arginine moieties determined by fluorescence titration 11 1.4 Host-guest complex formation of the molecular tweezers with various amino acid and peptide guest molecules containing lysine or arginine moieties determined by 1 H-NMR titration 47 1.5 Solvent-dependent 1 H-NMR spectra of host-guest complexes 55 1.6 Host-guest complex formation of the molecular tweezers with various amino acid and peptide guest molecules containing lysine or arginine moieties determined by isothermal titration calorimetry (ITC) 57 2. Computational Section: 2.1 Calculation of host-guest complex structures by QM/MM 6 2.2 Calculation of 1 H NMR shifts of the guest signals by ab initio methods 64 1
1 Experimental Section 1.1 1 H NMR and 13 C NMR spectra of the tweezers 1c, 1g, and 1d 15 16 17 18 19 2 21 22 S 1 14 13 23 24 25 26 2 3 12 11 1 9 8 7 S 6 5 4 1 H-NMR in CD 3 D: 1c 7.99 7.25 6.773 4.854 4.482 4. 3.31 2.51 2.334 2.256 7.1 7. 6.9 6.8 ppm 2.55 2.5 2.45 2.4 2.35 2.3 2.25 ppm 7. 6.5 6. 5.5 5. 4.5 4. 3.5 3. 2.5 2. 1.5 1..5 ppm 4.5 3.999 4. 3.998 3.987 1.988 3.943 2.1 1 H-NMR in D 2 : 1c 7.18 7.28 6.66 4.447 4.22 2.478 2.389 2.346 7.15 7.1 7.5 7. ppm 2.5 2.45 2.4 2.35 2.3 ppm 7. 6.5 6. 5.5 5. 4.5 4. 3.5 3. 2.5 2. 1.5 1..5 ppm 3.926 4. 3.413 4.12 4.22 2.59 4.26 2.57 2
13 C-NMR in CD 3 D: 1c 3
3 CH 3 14 13 15 16 17 18 28 19 2 21 22 23 24 25 26 12 11 1 9 8 7 6 5 1 4 2 3 CH 3 1 H-NMRin CDCl 3 : 1g 4
13 C-NMRin CDCl 3 : 1g 5
Na 14 13 15 16 17 18 28 19 2 21 22 23 24 25 26 12 11 1 9 8 7 6 5 1 4 2 3 Na 1 H-NMR in D 2 : 1d 6
13 C-NMR in D 2 : 1d 7
1.2 Dimerisation of the molecular tweezers determined by NMR titration 1.2.1 Dilution titration of the phosphate tweezer 1a Receptor (R) 1a M R g/mol] 1a.19 Solvent Phosphate buffer m R (1a) [mg] 1.1 Substrate T [ C] Itself 25 V [ml] [R] [mm] 2 7.4 δ (5-H, 11-H, 16-H, 22-H) = 4.162 P 24 a i 4 Li δ (6-H, 1-H, 17-H, 21-H) = 7.26 18 19 i 25 a 9 2 δ (1-H, 4-H, 12-H, 15-H) = 7.142 8 7 17 1 P δ (2-H, 3-H, 13-H, 14-H) = 6.865 21 6 23 16 a i 22 11 15 5 i a 26 12 1 4 13 14 2 3 [1a] [mm] δ obs (2-H, 3-H, 13-H, 14-H) δ obs δ calc 7.4 6.12.745.771 3.7 6.341.524.51 1.85 6.541.324.298.92 6.679.186.166.58 6.775.91.19.29 6.82.46.57 1,,8 obs [ppm],6,4,2 K dim [M -1 ] = 6 ± 1 δ max (5-H, 11-H, 16-H, 22-H) = -.2 δ max (6-H, 1-H, 17-H, 21-H) = -.28 δ max (1-H, 4-H, 12-H, 15-H) =.37 δ max (2-H, 3-H, 13-H, 14-H) = 2.23,,,1,2,3,4,5 [R] [mol/l] 8
1.2.2 Dilution titration of the sufate tweezer 1c Receptor(R) 1c M R [g/mol] 77.78 Solvent Phosphate buffer m R [mg] 1.13 T[ o C] 25 V [ml] 2.3 Substrate Itself [R] [mm] 1.13 S 2Na 24 18 19 25 9 2 8 7 17 S 1 21 6 23 16 26 11 15 5 22 12 4 1 14 13 2 3 [R][mM] δ obs [ppm] δ monomercalc - δ obs = δ calc [ppm] 1.13 6.212.77.565 6.419.5.282 6.624.295.141 6.733.186.76 6.836.83.353 6.858.61 7, 6,8 obs [ppm] 6,6 6,4 6,2 K dim [M -1 ] = 37 ± 8 δ max [ppm] = 2.2 δ monomercalc [ppm] = 6.92 6,,,2,4,6,8,1,12 [R] [mol/l] 9
1.2.3 Concentration-dependent 1 H NMR spectra of tweezer 1a in aqueous phosphate buffer (7 mm, ph 7.2, 25 C) 1.2.3 Concentration-dependent 1 H NMR spectra of tweezer 1c in aqueous phosphate buffer (1 mm, ph 7.2, 25 C) 1
1.3 Fluorescence titrations 1.3.1 UV-Vis and Fluorescence spectra of molecular tweezers and 1,4-dimethoxybenzene 1,4-dimethoxybenzene, DMB 1f (R 1 =R 2 =Ac) 1a (R 1 =R 2 =P 3 2-2Li + ) Compound 1a (R 1 =R 2 = P 3 2-2Li + ) 1f:(R 1 =R 2 = Ac) 1,4-dimethoxybenzene Solvent H 2 CD 3 CN CD 3 CN Absorption max ε max [nm] [M -1 cm -1 ] 284-284 - 29 28 Emission max Ф em [nm] [ns] 336.1 3.1 318.11 3.8 32.11 2.5 Figure S1. UV-VIS and fluorescence spectra of the molecular tweezers substituted by phosphate or acetoxy groups in the central benzene bridge and of 1,4-dimethoxybenzene (determined by P. Ceroni, Dipartimento di ChimicaCiamicianUniversita' di Bologna Via Selmi, 2, 4126 Bologna, Italy). 11
1.3.2 Fluorescence titrations Fluorescence titrations for Ac Lys Me Titration of Ac Lys Me and tweezer 1b in phosphate buffer (2mM, ph 7.64) λ exc = 28 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1b.165 1.277 2.18 1-5 Guest Ac Lys Me HCl.38.66 2.4 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 336 ) I obs 7 2.19E-5.E+. 53.7.. 1 71 2.19E-5 3.4E-5 1.55 5.9 2.8 3.1 2 72 2.19E-5 6.7E-5 3.7 49.1 4.6 4.8 3 73 2.19E-5 9.91E-5 4.54 47.9 5.9 5.9 4 74 2.19E-5 1.3E-4 5.97 46.9 6.8 6.7 6 76 2.19E-5 1.9E-4 8.71 46. 7.7 7.6 8 78 2.19E-5 2.47E-4 11.32 45.3 8.4 8.2 1 8 2.19E-5 3.1E-4 13.79 44.9 8.8 8.5 12 82 2.19E-5 3.53E-4 16.15 44.7 9. 8.8 15 85 2.19E-5 4.26E-4 19.47 44.8 8.9 9.1 18 88 2.19E-5 4.93E-4 22.57 44.6 9.1 9.2 22 92 2.19E-5 5.77E-4 26.39 44.4 9.4 9.4 26 96 2.19E-5 6.53E-4 29.89 44.3 9.4 9.5 3 1 2.19E-5 7.24E-4 33.11 44.3 9.5 9.6 I calc 1 8 6 4 2 55 5 45 4 35 3 V(AcLysMe) = µl V(AcLysMe) = 1 µl V(AcLysMe) = 2 µl V(AcLysMe) = 3 µl V(AcLysMe) = 4 µl V(AcLysMe) = 6 µl V(AcLysMe) = 8 µl V(AcLysMe) = 1 µl V(AcLysMe) = 12 µl V(AcLysMe) = 15 µl V(AcLysMe) = 18 µl V(AcLysMe) = 22 µl V(AcLysMe) = 26 µl V(AcLysMe) = 3 µl 5 1 15 2 25 3 35 [AcLysMe]/[1b] 25 3 35 4 45 5 [nm] K a [M -1 ] = 14766 ± 1%, K d [µm] = 68 ± 1% 12
Titration of AcLysMe and tweezer 1c in phosphate buffer (2mM, ph 7.64) λ exc = 285 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.156 8.69 2.33 1-5 Guest Ac Lys Me HCl.41.673 2.55 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 337 ) I obs I calc 7 2.33E-5.E+. 918.114. 1 71 2.33E-5 3.59E-5 1.54 725.153 192.961 193.3159 2 72 2.33E-5 7.9E-5 3.4 642.548 275.566 272.211 3 73 2.33E-5 1.5E-4 4.5 611.539 36.575 39.9996 4 74 2.33E-5 1.38E-4 5.92 584.48 333.634 331.4145 6 76 2.33E-5 2.1E-4 8.65 565.857 352.257 354.424 8 78 2.33E-5 2.62E-4 11.23 551.1 367.113 366.4685 1 8 2.33E-5 3.19E-4 13.69 548.189 369.925 373.8478 12 82 2.33E-5 3.73E-4 16.3 541.879 376.235 378.8253 15 85 2.33E-5 4.5E-4 19.33 532.487 385.627 383.8455 18 88 2.33E-5 5.22E-4 22.4 527.754 39.36 387.2144 22 92 2.33E-5 6.1E-4 26.19 526.147 391.967 39.2914 26 96 2.33E-5 6.91E-4 29.66 525.849 392.265 392.4293 3 1 2.33E-5 7.66E-4 32.86 524.197 393.917 394.8 5 1 4 3 2 1 8 6 4 l l 2 l 3 l 4 l 6 l 8 l 1 l 12 l 15 l 18 l 22 l 3 l 5 1 15 2 25 3 35 [AcLysMe]/[1c] 2 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 36 ± 2 % K d [µm] = 28 ± 2 % 13
Titration of AcLysMe and tweezer 1d in phosphate buffer (2mM, ph 7.64) λ exc = 285 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1d.119 4. 4.94 1-5 Guest Ac Lys Me HCl.184.4 1.93 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 349 ) I obs I calc 7 4.9E-5.E+. 363.42.. 1 71 4.9E-5 2.71E-5.663 359.354 4.48 2.4168 2 72 4.9E-5 5.35E-5 1.38 359.55 4.347 4.4184 3 73 4.9E-5 7.92E-5 1.934 356.959 6.443 6.942 4 74 4.9E-5 1.4E-4 2.544 354.993 8.49 7.5123 6 76 4.9E-5 1.52E-4 3.716 353.948 9.454 9.7697 8 78 4.9E-5 1.98E-4 4.828 352.32 11.37 11.4768 1 8 4.9E-5 2.41E-4 5.884 351.464 11.938 12.87 12 82 4.9E-5 2.82E-4 6.889 35.378 13.24 13.8699 15 85 4.9E-5 3.4E-4 8.37 348.26 15.142 15.1132 18 88 4.9E-5 3.94E-4 9.628 347.229 16.173 16.644 22 92 4.9E-5 4.61E-4 11.256 346.654 16.748 17.34 26 96 4.9E-5 5.22E-4 12.749 345.19 18.212 17.7638 3 1 4.9E-5 5.78E-4 14.122 344.553 18.849 18.3391 I 2 15 1 5 I 4 3 2 1 V(Ac-Lys-Me) = L V(Ac-Lys-Me) = 1 L V(Ac-Lys-Me) = 2 L V(Ac-Lys-Me) = 3 L V(Ac-Lys-Me) = 4 L V(Ac-Lys-Me) = 6 L V(Ac-Lys-Me) = 8 L V(Ac-Lys-Me) = 1 L V(Ac-Lys-Me) = 12 L V(Ac-Lys-Me) = 15 L V(Ac-Lys-Me) = 18 L V(Ac-Lys-Me) = 22 L V(Ac-Lys-Me) = 26 L V(Ac-Lys-Me) = 3 L 5 1 15 2 25 [AcLysMe]/[1d] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 4434 ± 14% K d [µm] = 226 ± 14% 14
Titration of AcLysMe and tweezer 1a in phosphate buffer (1 mm, ph 7.6) λ exc = 285 nm λ em = 334 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1a.225 1.71 2.579 1-5 Guest AcLysMe HCl.263.78 4.573 1-4 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 334 nm ) I obs I calc 7 2.58E-5.E+. 594.451.. 1 71 2.58E-5 6.44E-6.25 55.838 43.613 42.321 2 72 2.58E-5 1.27E-5.49 515.279 79.172 78.336 3 73 2.58E-5 1.88E-5.73 483.59 11.861 17.795 4 74 2.58E-5 2.47E-5.96 459.677 134.774 131.58 6 76 2.58E-5 3.61E-5 1.4 433.532 16.919 162.749 8 78 2.58E-5 4.69E-5 1.82 416.212 178.239 181.512 1 8 2.58E-5 5.72E-5 2.22 43.594 19.857 193.191 14 84 2.58E-5 7.62E-5 2.95 389.476 24.975 26.378 18 88 2.58E-5 9.35E-5 3.63 382.237 212.214 213.434 24 94 2.58E-5 1.17E-4 4.53 373.44 221.47 219.364 3 1 2.58E-5 1.37E-4 5.32 369.385 225.66 222.793 6 5 4 3 2 l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 14 l 18 l 24 l 3 l 25 2 15 1 1 5 3 32 34 36 38 4 42 44 nm 1 2 3 4 5 6 [AcLysMe]/[1a] K a [M -1 ] = 113 ± 6 % K d [µm] = 9 ± 6 % I max = 24 (4 %) 15
Titration of AcLysMe and tweezer 1c in phosphate buffer (1 mm, ph 7..2) λ exc = 285 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.1 5.568 2.33 1-5 Guest Ac Lys Me HCl.354.581 2.55 1-3 Guest V (µl) 1 2 3 4 6 8 1 12 15 18 22 26 3 Receptor V (µl) 7 71 72 73 74 76 78 8 82 85 88 92 96 1 [Receptor] [Guest] [mol/l] [mol/l] 2.33E-5.E+ 2.33E-5 3.6E-5 2.33E-5 7.9E-5 2.33E-5 1.5E-4 2.33E-5 1.38E-4 2.33E-5 2.2E-4 2.33E-5 2.62E-4 2.33E-5 3.19E-4 2.33E-5 3.74E-4 2.33E-5 4.5E-4 2.33E-5 5.22E-4 2.33E-5 6.1E-4 2.33E-5 6.91E-4 2.33E-5 7.66E-4 [Guest]/ [Receptor]. 1.54 3.4 4.5 5.92 8.65 11.24 13.69 16.3 19.33 22.41 26.2 29.67 32.86 F I (I 338 ) 925.462 72.81 623.958 591.476 578.192 559.736 545.225 544.797 537.159 532.127 533.112 53.63 533.667 522.77 I obs. 222.661 31.54 333.986 347.27 365.726 38.237 38.665 388.33 393.335 392.35 394.832 391.795 43.385 I calc. 222.446 299.942 332.975 35.558 368.613 377.746 383.242 386.98 39.573 393.15 395.234 396.769 397.893 5 4 3 2 1 5 1 15 2 25 [AcLysMe]/[1c] 3 35 1 9 8 7 6 5 4 3 2 3 35 4 45 µl 1 µl 2 µl 3 µl 4 µl 6 µl 8 µl 1 µl 12 µl 15 µl 18 µl 22 µl 26 µl 3 µl K a [M -1 ] = 515 ± 3 % K d [µm] = 19 ± 3 % I max = 48 (44 %) 16
Titration of AcLysMe and tweezer 1d in phosphate buffer (1 mm, ph 7.2) λ exc = 285 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1d.492 7. 9.67 1-5 Guest Ac Lys Me HCl 1.59.8 7.9 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 348 ) I obs I calc 7 9.67E-5.E+. 715.684.. 1 71 9.67E-5 1.11E-4 1.151 676.845 38.839 33.723 2 72 9.67E-5 2.19E-4 2.269 648.935 66.749 59.5512 3 73 9.67E-5 3.25E-4 3.358 639.67 76.77 79.9818 4 74 9.67E-5 4.27E-4 4.416 619.84 95.844 96.1721 6 76 9.67E-5 6.24E-4 6.45 593.112 122.572 12.5213 8 78 9.67E-5 8.1E-4 8.38 586.929 128.755 137.6953 1 8 9.67E-5 9.88E-4 1.213 559.378 156.36 15.5497 12 82 9.67E-5 1.16E-3 11.956 557.382 158.32 16.5912 15 85 9.67E-5 1.39E-3 14.418 548.79 166.894 171.3778 18 88 9.67E-5 1.62E-3 16.712 535.738 179.946 179.9713 22 92 9.67E-5 1.89E-3 19.537 525.169 19.515 188.593 26 96 9.67E-5 2.14E-3 22.127 52.197 195.487 194.1389 3 1 9.67E-5 2.37E-3 24.51 515.458 2.226 198.8329 I 25 2 15 1 5 I 8 6 4 2 V(Ac-Lys-Me) = L V(Ac-Lys-Me) = 1 L V(Ac-Lys-Me) = 2 L V(Ac-Lys-Me) = 3 L V(Ac-Lys-Me) = 4 L V(Ac-Lys-Me) = 6 L V(Ac-Lys-Me) = 8 L V(Ac-Lys-Me) = 1 L V(Ac-Lys-Me) = 12 L V(Ac-Lys-Me) = 15 L V(Ac-Lys-Me) = 18 L V(Ac-Lys-Me) = 22 L V(Ac-Lys-Me) = 26 L V(Ac-Lys-Me) = 3 L 5 1 15 2 25 3 [AcLysMe]/[1d] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 1555 ± 7% K d [µm] = 643 ± 7% I max = 225 (36 %) 17
Fluorescence titrations for H Lys H Titration of H LysH and tweezer 1b in phosphate buffer (2 mm, ph 7.6) λ exc = 28 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1b.165 1.277 2.18 1-5 Guest H Lys H HCl.314.71 2.4 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 336 ) I obs 7 2.19E-5.E+. 54... 1 71 2.19E-5 3.41E-5 1.56 53.6.4.5 2 72 2.19E-5 6.72E-5 3.8 52.9 1..9 3 73 2.19E-5 9.95E-5 4.55 52.7 1.3 1.3 4 74 2.19E-5 1.31E-4 5.99 52.4 1.6 1.6 6 76 2.19E-5 1.91E-4 8.74 51.6 2.4 2.2 8 78 2.19E-5 2.48E-4 11.36 51.3 2.7 2.8 1 8 2.19E-5 3.3E-4 13.84 51. 2.9 3.2 12 82 2.19E-5 3.54E-4 16.21 5.6 3.4 3.6 15 85 2.19E-5 4.27E-4 19.54 49.8 4.2 4.1 18 88 2.19E-5 4.95E-4 22.65 49.4 4.7 4.5 22 92 2.19E-5 5.79E-4 26.48 49.1 4.9 4.9 26 96 2.19E-5 6.56E-4 29.99 48.4 5.6 5.4 3 1 2.19E-5 7.26E-4 33.23 48.5 5.5 5.7 I calc 6 55 5 4 3 2 1 5 45 4 35 3 V(HLysH) = µl V(HLysH) = 1 µl V(HLysH) = 2 µl V(HLysH) = 3 µl V(HLysH) = 4 µl V(HLysH) = 6 µl V(HLysH) = 8 µl V(HLysH) = 1 µl V(HLysH) = 12 µl V(HLysH) = 15 µl V(HLysH) = 18 µl V(HLysH) = 22 µl V(HLysH) = 26 µl V(HLysH) = 3 µl 5 1 15 2 25 3 35 [HLysH]/[1b] 25 3 35 4 45 5 [nm] K a [M -1 ] = 1144 ± 1% K d [µm] = 874 ± 1% 18
Titration of HLysH and tweezer 1c in phosphate buffer (1mM, ph 7.6) λ exc = 285 nm λ em = 338 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.154 8.58 2.33 1-5 Guest H LysH HCl.282.67 2.54 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 338 ) I obs I calc 7 2.33E-5.E+. 668.11.. 1 71 2.33E-5 3.58E-5 1.54 624.46 43.641 37.471 2 72 2.33E-5 7.6E-5 3.3 6.417 67.684 66.272 3 73 2.33E-5 1.5E-4 4.49 571.74 97.27 88.995 4 74 2.33E-5 1.37E-4 5.9 565.345 12.756 17.324 6 76 2.33E-5 2.1E-4 8.62 539.165 128.936 134.979 8 78 2.33E-5 2.61E-4 11.19 516.322 151.779 154.784 1 8 2.33E-5 3.18E-4 13.64 495.84 172.261 169.631 14 84 2.33E-5 4.24E-4 18.19 478.98 19.3 19.359 18 88 2.33E-5 5.2E-4 22.32 465.56 22.541 24.114 24 94 2.33E-5 6.49E-4 27.86 451.82 217.19 217.799 3 1 2.33E-5 7.63E-4 32.74 437.57 23.594 226.874 25 2 15 1 5 6 4 2 l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 14 l 18 l 24 l 3 l 5 1 15 2 25 3 35 [HLysH]/[1c] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 441 ± 6 % K d [µm] = 227 ± 6% 19
Titration of HLysH and tweezer 1d in phosphate buffer (1 mm, ph 7.2) λ exc = 285 nm λ em = 348 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1d.259 4. 8.91 1-5 Guest H LysH HCl.655.8 4.55 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 348 ) I obs I calc 7 8.91E-5.E+. 555.58.. 1 71 8.91E-5 6.41E-5.719 546.684 8.896 5.437 2 72 8.91E-5 1.26E-4 1.418 544.499 11.81 1.246 3 73 8.91E-5 1.87E-4 2.99 545.179 1.41 14.4953 4 74 8.91E-5 2.46E-4 2.76 541.476 14.14 18.358 6 76 8.91E-5 3.59E-4 4.31 528.748 26.832 24.998 8 78 8.91E-5 4.67E-4 5.237 523.192 32.388 3.4993 1 8 8.91E-5 5.69E-4 6.383 519.935 35.645 35.1385 12 82 8.91E-5 6.66E-4 7.473 513.713 41.867 39.962 15 85 8.91E-5 8.3E-4 9.11 512.214 43.366 44.476 18 88 8.91E-5 9.31E-4 1.445 58.936 46.644 48.994 22 92 8.91E-5 1.9E-3 12.211 54.988 5.592 52.4776 26 96 8.91E-5 1.23E-3 13.83 497.261 58.319 55.9979 3 1 8.91E-5 1.36E-3 15.319 497.562 58.18 58.8887 I 7 6 5 4 3 2 I 6 V(H-Lys-H) = L V(H-Lys-H) = 1 L V(H-Lys-H) = 2 L 5 V(H-Lys-H) = 3 L V(H-Lys-H) = 4 L V(H-Lys-H) = 6 L 4 V(H-Lys-H) = 8 L V(H-Lys-H) = 1 L V(H-Lys-H) = 12 L V(H-Lys-H) = 15 L 3 V(H-Lys-H) = 18 L V(H-Lys-H) = 22 L V(H-Lys-H) = 26 L 2 V(H-Lys-H) = 3 L 1 1 2 4 6 8 1 12 14 16 18 [HLysH]/[1d] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 855 ± 2% K d [µm] = 117 ± 2% 2
Fluorescence titration for peptide KAA Titration of KAA and tweezer 1a in phosphate buffer (2 mm, ph 7.6) λ exc = 285 nm Tweezer 1a KAA Amount [mg]: Volume [ml]: Concentration [mol/l]:.164 (2.18 1-4 mmol) 1 2.18 1-5.347 (1.2 1-3 mmol).5 2.4 1-3 V Guest V total [1a] [KAA] [KAA]/ I [ L] [ L] [mol/l] [mol/l] [1a] 334 nm I obs I calc 7 2.19 1-5 41.6. 1 71 2.19 1-5 3.39 1-5 1.55 38.1 3.4 3.4 2 72 2.19 1-5 6.69 1-5 3.6 36.7 4.9 4.8 3 73 2.19 1-5 9.89 1-5 4.53 36.1 5.5 5.5 4 74 2.19 1-5 1.3 1-4 5.95 35.6 5.9 5.9 6 76 2.19 1-5 1.9 1-4 8.69 35.3 6.3 6.4 8 78 2.19 1-5 2.47 1-4 11.29 34.8 6.7 6.7 1 8 2.19 1-5 3.1 1-4 13.77 34.9 6.7 6.8 12 82 2.19 1-5 3.52 1-4 16.12 34.6 7. 6.9 15 85 2.19 1-5 4.25 1-4 19.43 34.6 6.9 7. 18 88 2.19 1-5 4.92 1-4 22.53 34.4 7.1 7.1 22 92 2.19 1-5 5.76 1-4 26.33 34.4 7.2 7.2 26 96 2.19 1-5 6.52 1-4 29.82 34.3 7.2 7.2 3 1 2.19 1-5 7.22 1-4 33.4 34.3 7.3 7.2 8 6 4 2 44 42 4 38 36 34 32 3 28 V(KAA) = µl V(KAA) = 1 µl V(KAA) = 2 µl V(KAA) = 3 µl V(KAA) = 4 µl V(KAA) = 6 µl V(KAA) = 8 µl V(KAA) = 1 µl V(KAA) = 12 µl V(KAA) = 15 µl V(KAA) = 18 µl V(KAA) = 22 µl V(KAA) = 26 µl V(KAA) = 3 µl 5 1 15 2 25 3 35 [KAA]/[1a] 26 24 3 35 4 45 5 [nm] K a [M -1 ] = 3334 ± 3 % K d [µm] = 3 ± 3 % 21
Titration of KAA and tweezer 1b in phosphate buffer (2 mm, ph 7.6) λ exc = 28 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1b.165 1.277 2.18 1-5 Guest KAA.36.52 2.4 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 334 ) I obs 7 2.19E-5.E+. 52.9.. 1 71 2.19E-5 3.38E-5 1.55 52.4.5.4 2 72 2.19E-5 6.67E-5 3.5 51.9.9.8 3 73 2.19E-5 9.87E-5 4.51 51.9.9 1.1 4 74 2.19E-5 1.3E-4 5.94 51.5 1.4 1.4 6 76 2.19E-5 1.9E-4 8.67 51. 1.9 2. 8 78 2.19E-5 2.46E-4 11.27 5.4 2.5 2.5 1 8 2.19E-5 3.E-4 13.73 49.9 2.9 2.9 12 82 2.19E-5 3.51E-4 16.8 49.6 3.3 3.3 15 85 2.19E-5 4.24E-4 19.38 49.2 3.7 3.7 18 88 2.19E-5 4.91E-4 22.47 48.9 4. 4.1 22 92 2.19E-5 5.74E-4 26.27 48.3 4.6 4.5 26 96 2.19E-5 6.5E-4 29.75 48. 4.9 4.9 3 1 2.19E-5 7.2E-4 32.95 47.7 5.2 5.2 I calc 6 55 5 4 3 2 1 5 45 4 35 3 V(KAA) = µl V(KAA) = 1 µl V(KAA) = 2 µl V(KAA) = 3 µl V(KAA) = 4 µl V(KAA) = 6 µl V(KAA) = 8 µl V(KAA) = 1 µl V(KAA) = 12 µl V(KAA) = 15 µl V(KAA) = 18 µl V(KAA) = 22 µl V(KAA) = 26 µl V(KAA) = 3 µl 5 1 15 2 25 3 35 [KAA]/[1b] 25 3 35 4 45 5 [nm] K a [M -1 ] = 115 ± 1% K d [µm] = 95 ± 1% 22
Titration of KAA and tweezer1c in phosphate buffer (1 mm, ph 7.6) λ exc = 285 nm λ em = 338 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.154 8.58 2.33 1-5 Guest KAA.37.53 2.55 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 338 ) I obs I calc 7 2.33E-5.E+. 671.428.. 1 71 2.33E-5 3.59E-5 1.54 644.313 27.115 32.63 2 72 2.33E-5 7.9E-5 3.4 66.546 64.882 57.917 3 73 2.33E-5 1.5E-4 4.5 595.53 75.925 79.153 4 74 2.33E-5 1.38E-4 5.92 575.253 96.175 96.878 6 76 2.33E-5 2.1E-4 8.64 544.662 126.766 124.722 8 78 2.33E-5 2.62E-4 11.23 525.733 145.695 145.552 1 8 2.33E-5 3.19E-4 13.69 514.4 157.424 1c.693 14 84 2.33E-5 4.25E-4 18.25 484.77 187.351 185.41 18 88 2.33E-5 5.22E-4 22.39 469.864 21.564 21.88 24 94 2.33E-5 6.51E-4 27.95 451.425 22.3 217.517 3 1 2.33E-5 7.65E-4 32.85 445.244 226.184 228.679 25 2 15 1 6 l 1 l 2 l 3 l 4 l 6 l 4 8 l 1 l 14 l 18 l 24 l 2 3 l 5 5 1 15 2 25 3 35 [KAA]/[1c] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 33 ± 5 % K d [µm] = 33 ± 5 % 23
Titration of KAA and tweezer 1d in phosphate buffer (1 mm, ph 7.2) λ exc = 285 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1d.215 4.5 6.65 1-5 Guest KAA 1.349.8 9.2 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 348 ) I obs I calc 7 6.57E-5.E+. 476.535.. 1 71 6.57E-5 1.3E-4 1.973 471.176 5.359 6.899 2 72 6.57E-5 2.56E-4 3.891 462.86 13.729 11.9661 3 73 6.57E-5 3.78E-4 5.756 454.449 22.86 17.6395 4 74 6.57E-5 4.98E-4 7.572 451.84 24.731 23.125 6 76 6.57E-5 7.27E-4 11.58 445.96 3.575 33.5432 8 78 6.57E-5 9.45E-4 14.367 433.475 43.6 43.338 1 8 6.57E-5 1.15E-3 17.59 417.927 58.68 52.4635 12 82 6.57E-5 1.35E-3 2.499 421.166 55.369 61.761 15 85 6.57E-5 1.63E-3 24.719 47.293 69.242 73.718 18 88 6.57E-5 1.88E-3 28.652 389.996 86.539 84.812 22 92 6.57E-5 2.2E-3 33.496 379.26 97.275 97.4253 26 96 6.57E-5 2.49E-3 37.937 365.539 11.996 19.451 3 1 6.57E-5 2.76E-3 42.22 355.788 12.747 12.344 I 14 12 1 8 6 4 2 1 2 3 4 5 [KAA]/[1d] I 7 6 5 4 3 2 1 3 32 34 36 38 4 42 44 [nm] V(KAA) = L V(KAA) = 1 L V(KAA) = 2 L V(KAA) = 3 L V(KAA) = 4 L V(KAA) = 6 L V(KAA) = 8 L V(KAA) = 1 L V(KAA) = 12 L V(KAA) = 15 L V(KAA) = 18 L V(KAA) = 22 L V(KAA) = 26 L V(KAA) = 3 L K a [M -1 ] = 3 ± 83% K d [µm] = 33333 ± 83% 24
I Fluorescence titrations for KLVFF Titration of KLVFF and tweezer1a in phosphate buffer (2 mm, ph 7.6) λ exc = 28 nm Tweezer 1a KLVFF Amount [mg]: Volume [ml]: Concentration [mol/l]:.172 (2.29 1-4 mmol) 1.487 2.18 1-5.217 (3.32 1-4 mmol).4 8.3 1-4 V Gast V gesamt [75] [KLVFF] I [KLVFF]/ [1a] [ L] [ L] [mol/l] [mol/l] 334 nm I obs I calc 7 2.19 1-5.. 43.3.. 1 71 2.19 1-5 1.17 1-5.54 41.3 2. 1.9 2 72 2.19 1-5 2.31 1-5 1.6 4.1 3.2 3.3 3 73 2.19 1-5 3.42 1-5 1.56 39.2 4.1 4.2 4 74 2.19 1-5 4.49 1-5 2.6 38.3 4.9 4.8 6 76 2.19 1-5 6.56 1-5 3. 37.6 5.7 5.7 8 78 2.19 1-5 8.52 1-5 3.9 37.4 5.9 6.1 1 8 2.19 1-5 1.4 1-4 4.75 36.9 6.4 6.4 12 82 2.19 1-5 1.22 1-4 5.56 36.7 6.6 6.6 15 85 2.19 1-5 1.47 1-4 6.71 36.4 6.6 6.8 18 88 2.19 1-5 1.7 1-4 7.78 36.3 6.9 6.9 22 92 2.19 1-5 1.99 1-4 9.9 36.3 6.9 7.1 26 96 2.19 1-5 2.25 1-4 1.3 35.9 7.4 7.2 3 1 2.19 1-5 2.49 1-4 11.41 35.9 7.4 7.3 8 45 6 4 2 4 35 3 V(KLVFF) = µl V(KLVFF) = 1 µl V(KLVFF) = 2 µl V(KLVFF) = 3 µl V(KLVFF) = 4 µl V(KLVFF) = 6 µl V(KLVFF) = 8 µl V(KLVFF) = 1 µl V(KLVFF) = 12 µl V(KLVFF) = 15 µl V(KLVFF) = 18 µl V(KLVFF) = 22 µl V(KLVFF) = 26 µl V(KLVFF) = 3 µl 25 2 4 6 8 1 12 [KLVFF]/[1a] 3 35 4 45 5 [nm] K a [M -1 ] = 49481 ± 5 % K d [µm] =2 ± 5 % 25
Titration of KLVFF and tweezer 1c in phosphate buffer (1 mm, ph 7.6) λ exc = 285 nm λ em = 338 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.113 6.724 2.18 1-5 Guest KLVFF.355.725 7.5 1-4 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 338 ) I obs I calc 7 2.18E-5.E+. 644.84.. 1 71 2.18E-5 1.6E-5.48 629.776 14.38 16.276 2 72 2.18E-5 2.8E-5.96 613.787 3.297 28.826 3 73 2.18E-5 3.8E-5 1.41 65.562 38.522 38.553 4 74 2.18E-5 4.5E-5 1.86 599.635 44.449 46.18 6 76 2.18E-5 5.92E-5 2.72 588.12 56.72 57.153 8 78 2.18E-5 7.69E-5 3.53 58.781 63.33 64.522 1 8 2.18E-5 9.38E-5 4.3 571.938 72.146 69.743 14 84 2.18E-5 1.25E-4 5.73 565.785 78.299 76.576 18 88 2.18E-5 1.53E-4 7.4 561.215 82.869 8.811 24 94 2.18E-5 1.92E-4 8.78 556.559 87.525 84.792 3 1 2.18E-5 2.25E-4 1.32 562.35 81.1b 87.36 1 8 6 4 2 2 4 6 8 1 12 [KLVFF]/[1c] 1 9 8 7 6 5 4 3 2 1 3 35 4 45 µl 1 µl 2 µl 3 µl 4 µl 6 µl 8 µl 1 µl 14 µl 18 µl 3 µl K a [M -1 ] = 262 ± 11 % K d [µm] =38 ± 11% λ ~ 2 nm,blue shift 26
Fluorescence titrations for KKLVFF Titration of KKLVFFand tweezer 1a in phosphate buffer (2 mm, ph 7.64) λ exc = 28 nm Tweezer1a KKLVFF Amount [mg]: Volume [ml]: Concentration [mol/l]:.172 (2.29 1-4 mmol) 1.487 2.18 1-5.26 (3.32 1-4 mmol).45 8.3 1-4 V Guest V total [1a] [KKLVFF] [KKLVFF]/ I [ L] [ L] [mol/l] [mol/l] [1a] 334 nm I obs I calc 7 2.19 1-5 43.3 5 75 2.19 1-5 5.89 1-6.27 41.9 1.4 1.5 1 71 2.19 1-5 1.17 1-5.54 4.4 2.9 2.8 15 715 2.19 1-5 1.74 1-5.8 39.3 4. 3.9 2 72 2.19 1-5 2.31 1-5 1.6 38.5 4.8 4.7 25 725 2.19 1-5 2.86 1-5 1.31 38.1 5.2 5.2 3 73 2.19 1-5 3.41 1-5 1.56 37.8 5.5 5.6 4 74 2.19 1-5 4.49 1-5 2.5 37.3 6. 5.9 6 76 2.19 1-5 6.56 1-5 3. 37.3 6. 6.3 8 78 2.19 1-5 8.52 1-5 3.9 36.9 6.4 6.4 1 8 2.19 1-5 1.4 1-4 4.75 36.9 6.4 6.5 12 82 2.19 1-5 1.22 1-4 5.56 36.9 6.3 6.6 15 85 2.19 1-5 1.47 1-4 6.7 36.7 6.6 6.6 18 88 2.19 1-5 1.7 1-4 7.77 36.6 6.7 6.6 22 92 2.19 1-5 1.99 1-4 9.8 36.5 6.8 6.7 26 96 2.19 1-5 2.25 1-4 1.29 36.5 6.8 6.7 3 1 2.19 1-5 2.49 1-4 11.4 36.5 6.8 6.7 8 6 4 2 45 4 35 3 V(KKLVFF) = µl V(KKLVFF) = 5 µl V(KKLVFF) = 1 µl V(KKLVFF) = 15 µl V(KKLVFF) = 2 µl V(KKLVFF) = 25 µl V(KKLVFF) = 3 µl V(KKLVFF) = 4 µl V(KKLVFF) = 6 µl V(KKLVFF) = 8 µl V(KKLVFF) = 1 µl V(KKLVFF) = 12 µl V(KKLVFF) = 15 µl V(KKLVFF) = 18 µl V(KKLVFF) = 22 µl V(KKLVFF) = 26 µl V(KKLVFF) = 3 µl 25 2 4 6 8 1 12 [KKLVFF]/[1a] 3 35 4 45 5 [nm] K a [M -1 ] = 27731 ± 1 % K d [µm] =4 ± 1 % 27
Titration of KKLVFF and tweezer 1b in phosphate buffer (2 mm, ph 7.64) λ exc = 28 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1b.165 1.277 2.18 1-5 Guest KKLVFF.497.765 8. 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 336 ) I obs 7 2.19E-5.E+. 53.7.. 1 71 2.19E-5 1.17E-5.35 52.3 1.3 1.2 2 72 2.19E-5 2.31E-5 1.5 51.3 2.4 2.2 3 73 2.19E-5 3.41E-5 1.56 5.7 2.9 2.9 4 74 2.19E-5 4.49E-5 2.5 49.9 3.7 3.6 6 76 2.19E-5 6.55E-5 3. 49.1 4.6 4.6 8 78 2.19E-5 8.51E-5 3.89 48.4 5.2 5.4 1 8 2.19E-5 1.4E-4 4.75 47.8 5.9 5.9 12 82 2.19E-5 1.21E-4 5.56 47.4 6.3 6.4 15 85 2.19E-5 1.46E-4 6.7 46.8 6.9 6.9 18 88 2.19E-5 1.7E-4 7.77 46.3 7.3 7.2 22 92 2.19E-5 1.98E-4 9.8 45.9 7.7 7.6 26 96 2.19E-5 2.25E-4 1.28 45.7 7.9 7.9 3 1 2.19E-5 2.49E-4 11.39 45.7 7.9 8.1 I calc 55 8 6 4 2 5 45 4 35 3 V(KKLVFF) = µl V(KKLVFF) = 1 µl V(KKLVFF) = 2 µl V(KKLVFF) = 3 µl V(KKLVFF) = 4 µl V(KKLVFF) = 6 µl V(KKLVFF) = 8 µl V(KKLVFF) = 1 µl V(KKLVFF) = 12 µl V(KKLVFF) = 15 µl V(KKLVFF) = 18 µl V(KKLVFF) = 22 µl V(KKLVFF) = 26 µl V(KKLVFF) = 3 µl 2 4 6 8 1 12 [KKLVFF]/[1b] 25 3 35 4 45 5 [nm] K a [M -1 ] = 1448 ± 1% K d [µm] = 71 ± 1 28
Fluorescence titration of KKLVFFAK and tweezer 1a in phosphate buffer (2 mm, ph 7.64) λ exc = 28 nm Tweezer1a KKLVFFAK Amount [mg]: Volume [ml]: Concentration [mol/l]:.172 (2.29 1-4 mmol) 1.487 2.18 1-5.394 (4. 1-4 mmol).48 8.3 1-4 V Guest V total [1a] [KKLVFFAK] [KKLVFFAK]/ I [ L] [ L] [mol/l] [mol/l] [1a] 334 nm I obs I calc 7 2.19 1-5.. 43.8.. 1 71 2.19 1-5 1.17 1-5.54 4.1 3.7 3.3 2 72 2.19 1-5 2.32 1-5 1.6 38.1 5.6 5.5 3 73 2.19 1-5 3.43 1-5 1.57 37.1 6.7 6.7 4 74 2.19 1-5 4.51 1-5 2.6 36.5 7.3 7.4 6 76 2.19 1-5 6.58 1-5 3.1 36.2 7.6 8.1 8 78 2.19 1-5 8.55 1-5 3.91 35.9 7.9 8.4 1 8 2.19 1-5 1.4 1-4 4.77 35.6 8.2 8.6 12 82 2.19 1-5 1.22 1-4 5.58 35.3 8.5 8.7 15 85 2.19 1-5 1.47 1-4 6.73 35.1 8.7 8.8 18 88 2.19 1-5 1.7 1-4 7.8 34.7 9.1 8.9 22 92 2.19 1-5 1.99 1-4 9.12 34.5 9.3 8.9 26 96 2.19 1-5 2.26 1-4 1.33 34.3 9.5 9. 3 1 2.19 1-5 2.5 1-4 11.44 34.2 9.6 9.1 1 8 6 4 2 45 4 35 3 25 V(KKLVFFAK) = µl V(KKLVFFAK) = 5 µl V(KKLVFFAK) = 1 µl V(KKLVFFAK) = 15 µl V(KKLVFFAK) = 2 µl V(KKLVFFAK) = 25 µl V(KKLVFFAK) = 3 µl V(KKLVFFAK) = 4 µl V(KKLVFFAK) = 6 µl V(KKLVFFAK) = 8 µl V(KKLVFFAK) = 1 µl V(KKLVFFAK) = 12 µl V(KKLVFFAK) = 15 µl V(KKLVFFAK) = 18 µl V(KKLVFFAK) = 22 µl V(KKLVFFAK) = 26 µl V(KKLVFFAK) = 3 µl 2 4 6 8 1 12 [KKLVFFAK]/[1a] 3 35 4 45 5 [nm] K a [M -1 ] = 135848 ± 1 % K d [µm] = 7 ± 1 % 29
Fluorescence titration of H-KKKK-H and tweezer 1a in phosphate buffer (2 mm, ph 7.64) λ exc = 28 nm Tweezer1a H-KKKK-H Amount [mg]: Volume [ml]: Concentration [mol/l]:.172 (2.29 1-4 mmol) 1.487 2.18 1-5.26 (4.89 1-4 mmol).45 1.2 1-3 V Guest V total [1a] [KKKK] [KKKK]/ I [ L] [ L] [mol/l] [mol/l] [1a] 334 nm I obs I calc 7 2.19 1-5.. 42... 1 71 2.19 1-5 1.73 1-5.79 38.3 3.7 3.5 2 72 2.19 1-5 3.4 1-5 1.56 36.8 5.2 5.3 3 73 2.19 1-5 5.3 1-5 2.3 35.8 6.2 6.1 4 74 2.19 1-5 5.83 1-5 2.67 35.6 6.5 6.4 6 76 2.19 1-5 6.62 1-5 3.3 35.5 6.5 6.6 8 78 2.19 1-5 9.67 1-5 4.42 35.1 6.9 7.1 1 8 2.19 1-5 1.26 1-4 5.75 34.8 7.3 7.3 12 82 2.19 1-5 1.53 1-4 7. 34.7 7.3 7.5 15 85 2.19 1-5 1.79 1-4 8.2 34.8 7.3 7.5 18 88 2.19 1-5 2.16 1-4 9.89 34.6 7.4 7.6 22 92 2.19 1-5 2.51 1-4 11.46 34.3 7.8 7.7 26 96 2.19 1-5 2.93 1-4 13.4 34.2 7.8 7.7 3 1 2.19 1-5 3.32 1-4 15.18 34.1 7.9 7.8 8 6 4 2 I 44 42 4 38 36 34 32 3 28 26 V(H-KKKK-H) = µl V(H-KKKK-H) = 5 µl V(H-KKKK-H) = 1 µl V(H-KKKK-H) = 15 µl V(H-KKKK-H) = 2 µl V(H-KKKK-H) = 25 µl V(H-KKKK-H) = 3 µl V(H-KKKK-H) = 35 µl V(H-KKKK-H) = 4 µl V(H-KKKK-H) = 6 µl V(H-KKKK-H) = 8 µl V(H-KKKK-H) = 1 µl V(H-KKKK-H) = 12 µl V(H-KKKK-H) = 15 µl V(H-KKKK-H) = 18 µl V(H-KKKK-H) = 22 µl V(H-KKKK-H) = 26 µl V(H-KKKK-H) = 3 µl 2 4 6 8 1 12 14 16 18 [H-KKKK-H]/[1a] 24 3 35 4 45 5 [nm] K a [M -1 ] = 9661 ± 7 % K d [µm] = 1 ± 7 % 3
Fluorescence titrations for Ac ArgMe Titration of AcArgMe and tweezer 1a in phosphate buffer (2mM, ph 7.6) λ exc = 285 nm λ em = 335 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1a.17 8.96 2.33 1-5 Guest AcArgMe HCl.364.535 2.55 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 335 nm ) I obs I calc 7 2.33E-5.E+. 973.832.. 1 71 2.33E-5 3.59E-5 1.54 842.941 13.891 128.837 2 72 2.33E-5 7.9E-5 3.4 775.363 198.469 199.554 3 73 2.33E-5 1.5E-4 4.5 732.78 241.52 242.139 4 74 2.33E-5 1.38E-4 5.92 7.879 272.953 27.41 6 76 2.33E-5 2.1E-4 8.64 669.874 33.958 33.919 8 78 2.33E-5 2.62E-4 11.23 653.89 32.743 323.546 1 8 2.33E-5 3.19E-4 13.68 638.677 335.155 336.29 12 82 2.33E-5 3.73E-4 16.2 631.899 341.933 345.214 15 85 2.33E-5 4.5E-4 19.32 616.878 356.954 354.492 18 88 2.33E-5 5.22E-4 22.39 61.332 363.5 36.88 22 92 2.33E-5 6.1E-4 26.18 68.956 364.876 366.829 26 96 2.33E-5 6.91E-4 29.65 62.8 371.824 371.29 3 1 2.33E-5 7.65E-4 32.84 598.52 375.33 374.151 4 1 3 2 1 8 6 4 l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 12 l 15 l 18 l 22 l 26 l 3 l 5 1 15 2 25 3 35 [AcArgMe]/[1a] 2 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 164 ± 2 % K d [µm] = 6± 2 % 31
Titration of AcArgMeand tweezer 1c in phosphate buffer (2mM, ph 7.6) λ exc = 285 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.156 8.69 2.33 1-5 Guest Ac ArgMe HCl.34.499 2.55 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 337 ) I obs I calc 7 2.33E-5.E+. 921.74.. 1 71 2.33E-5 3.6E-5 1.54 863.162 58.542 56.863 2 72 2.33E-5 7.1E-5 3.5 825.328 96.376 98.541 3 73 2.33E-5 1.5E-4 4.51 795.25 126.454 13.157 4 74 2.33E-5 1.38E-4 5.93 765.497 156.27 154.846 6 76 2.33E-5 2.2E-4 8.66 723.468 198.236 19.741 8 78 2.33E-5 2.62E-4 11.24 73.545 218.159 215.465 1 8 2.33E-5 3.19E-4 13.7 691.642 23.62 233.478 12 82 2.33E-5 3.74E-4 16.4 675.124 246.58 247.162 15 85 2.33E-5 4.51E-4 19.35 662.666 259.38 262.445 18 88 2.33E-5 5.23E-4 22.42 654.232 267.472 273.656 22 92 2.33E-5 6.11E-4 26.22 638.21 283.683 284.648 26 96 2.33E-5 6.92E-4 29.69 625.181 296.523 292.749 3 1 2.33E-5 7.66E-4 32.89 619.139 32.565 298.964 35 1 3 25 2 15 1 5 8 6 4 l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 12 l 15 l 18 l 22 l 26 l 3 l 5 1 15 2 25 3 35 [AcArgMe]/[1c] 2 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 56 ± 4 % K d [µm] = 178 ± 4 % 32
Titration of AcArgMe and tweezer 1a in phosphate buffer (1mM, ph 7.6) λ exc = 285 nm λ em = 334 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1a.225 1.71 2.579 1-5 Guest AcArgMe HCl.11.828 4.573 1-4 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 334 nm ) I obs I calc 7 2.58E-5.E+. 593.577.. 1 71 2.58E-5 6.44E-6.25 556.958 36.619 37.456 2 72 2.58E-5 1.27E-5.49 525.313 68.264 69.73 3 73 2.58E-5 1.88E-5.73 493.88 99.769 95.421 4 74 2.58E-5 2.47E-5.96 473.99 119.587 117.216 6 76 2.58E-5 3.61E-5 1.4 443.24 15.337 15.75 8 78 2.58E-5 4.69E-5 1.82 421.96 171.671 172.777 1 8 2.58E-5 5.72E-5 2.22 45.99 187.587 188.925 14 84 2.58E-5 7.62E-5 2.96 386.727 26.85 29.81 18 88 2.58E-5 9.35E-5 3.63 373.825 219.752 222.433 24 94 2.58E-5 1.17E-4 4.53 359.4 234.573 233.984 34 14 2.58E-5 1.5E-4 5.8 345.525 248.52 244.467 6 5 4 3 2 l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 14 l 18 l 24 l 34 l 3 25 2 15 1 1 5 3 32 34 36 38 4 42 44 [nm] 1 2 3 4 5 6 7 [AcArgMe]/[1a] K a [M -1 ] = 56 ± 5 % K d [µm] = 2 ± 5 % ΔI max = 282 (47 %) 33
Titration of AcArgMe and tweezer 1c in phosphate buffer (1mM, ph 7.2) λ exc = 285 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.1 5.568 2.33 1-5 Guest Ac ArgMe HCl.354.52 2.55 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 338 ) I obs I calc 7 2.33E-5.E+. 96.244.. 1 71 2.33E-5 3.59E-5 1.54 827.374 78.87 76.58 2 72 2.33E-5 7.9E-5 3.4 782.379 123.865 12.955 3 73 2.33E-5 1.5E-4 4.5 753.14 153.14 149.585 4 74 2.33E-5 1.38E-4 5.92 739.276 166.968 169.129 6 76 2.33E-5 2.1E-4 8.65 713.5 192.744 193.83 8 78 2.33E-5 2.62E-4 11.23 7.13 26.114 28.68 1 8 2.33E-5 3.19E-4 13.69 691.374 214.87 218.432 12 82 2.33E-5 3.74E-4 16.3 684.85 221.439 225.413 15 85 2.33E-5 4.5E-4 19.33 679.489 226.755 232.761 18 88 2.33E-5 5.22E-4 22.41 668.375 237.869 237.873 22 92 2.33E-5 6.1E-4 26.19 662.42 243.842 242.675 26 96 2.33E-5 6.91E-4 29.67 656.113 25.131 246.88 3 1 2.33E-5 7.66E-4 32.86 65.395 255.849 248.638 1 8 6 4 2 3 32 34 36 38 4 42 44 [nm] l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 12 l 15 l 18 l 22 l 24 l 3 l 3 25 2 15 1 5 5 1 15 2 25 3 35 [AcArgMe]/[1c] K a [M -1 ] = 13 ± 5 % K d [µm] = 77 ± 5 % I max = 274 (3 %) 34
Titration of AcArgMe and tweezer 1d in phosphate buffer (2mM, ph 7.6) λ exc = 285 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1d.141 2. 9.7 1-5 Guest Ac ArgMe HCl.28.4 1.95 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 348 ) I obs I calc 7 9.7E-5.E+. 354.492.. 1 71 9.7E-5 2.75E-5.283 349.54 4.952 2.97 2 72 9.7E-5 5.42E-5.558 347.493 6.999 5.7261 3 73 9.7E-5 8.1E-5.826 345.364 9.128 8.2892 4 74 9.7E-5 1.5E-4 1.86 342.694 11.798 1.678 6 76 9.7E-5 1.54E-4 1.587 336.766 17.726 14.9956 8 78 9.7E-5 2.E-4 2.61 335.593 18.899 18.7888 1 8 9.7E-5 2.44E-4 2.512 334.62 2.43 22.1443 12 82 9.7E-5 2.85E-4 2.941 331.431 23.61 25.1314 15 85 9.7E-5 3.44E-4 3.546 325.565 28.927 29.44 18 88 9.7E-5 3.99E-4 4.111 323.95 3.587 32.395 22 92 9.7E-5 4.66E-4 4.86 318.624 35.868 36.1733 26 96 9.7E-5 5.28E-4 5.443 313.851 4.641 39.3446 3 1 9.7E-5 5.85E-4 6.29 311.581 42.911 42.394 I 5 4 3 2 1 I 4 3 2 1 V(Ac-Arg-Me) = L V(Ac-Arg-Me) = 1 L V(Ac-Arg-Me) = 2 L V(Ac-Arg-Me) = 3 L V(Ac-Arg-Me) = 4 L V(Ac-Arg-Me) = 6 L V(Ac-Arg-Me) = 8 L V(Ac-Arg-Me) = 1 L V(Ac-Arg-Me) = 12 L V(Ac-Arg-Me) = 15 L V(Ac-Arg-Me) = 18 L V(Ac-Arg-Me) = 22 L V(Ac-Arg-Me) = 26 L V(Ac-Arg-Me) = 3 L 2 4 6 8 1 [AcLysMe]/[1d] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 1134 ± 26% K d [µm] = 882 ± 26% 35
Titration of AcArgMe and tweezer 1d in phosphate buffer (1mM, ph 7.2) λ exc = 285 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1d.492 7. 9.67 1-5 Guest Ac ArgMe HCl 1.43.8 6.7 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 348 ) I obs I calc 7 9.67E-5.E+. 714.662.. 1 71 9.67E-5 9.44E-5.976 75.219 9.443 19.797 2 72 9.67E-5 1.86E-4 1.925 686.177 28.485 32.9222 3 73 9.67E-5 2.75E-4 2.848 665.925 48.737 41.9554 4 74 9.67E-5 3.62E-4 3.745 664.865 49.797 48.4348 6 76 9.67E-5 5.29E-4 5.47 656.591 58.71 56.9735 8 78 9.67E-5 6.87E-4 7.17 65.534 64.128 62.2789 1 8 9.67E-5 8.38E-4 8.661 642.645 62.574 65.8686 12 82 9.67E-5 9.81E-4 1.14 643.352 71.31 68.4499 15 85 9.67E-5 1.18E-3 12.228 639.91 75.571 71.1925 18 88 9.67E-5 1.37E-3 14.173 637.916 76.746 73.1148 22 92 9.67E-5 1.6E-3 16.569 64.94 73.722 74.93 3 1 9.67E-5 2.1E-3 2.787 635.733 78.929 77.1957 I 1 8 6 4 2 I 8 V(Ac-Arg-Me) = L V(Ac-Arg-Me) = 1 L V(Ac-Arg-Me) = 2 L V(Ac-Arg-Me) = 3 L V(Ac-Arg-Me) = 4 L 6 V(Ac-Arg-Me) = 6 L V(Ac-Arg-Me) = 8 L V(Ac-Arg-Me) = 1 L V(Ac-Arg-Me) = 12 L V(Ac-Arg-Me) = 15 L 4 V(Ac-Arg-Me) = 18 L V(Ac-Arg-Me) = 22 L V(Ac-Arg-Me) = 26 L V(Ac-Arg-Me) = 3 L 2 5 1 15 2 25 [AcArgMe]/[1d] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 3558 ± 18% K d [µm] = 281 ± 18% I max = 91 (13 %) 36
Fluorescence titrations for HArgH Titration of HArgH and tweezer 1c in phosphate buffer (1mM, ph 7.6) λ exc = 285 nm λ em = 338 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.154 8.58 2.33 1-5 Guest H Arg H HCl.282.67 2.54 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 338 ) I obs I calc 7 2.33E-5.E+. 663.928.. 1 71 2.33E-5 3.58E-5 1.54 651.777 12.151 1.66 2 72 2.33E-5 7.7E-5 3.3 64.915 23.13 2.121 3 73 2.33E-5 1.5E-4 4.49 633.35 3.578 28.573 4 74 2.33E-5 1.38E-4 5.9 629.736 34.192 36.168 6 76 2.33E-5 2.1E-4 8.62 69.553 54.375 49.252 8 78 2.33E-5 2.61E-4 11.2 62.31 61.618 6.118 1 8 2.33E-5 3.18E-4 13.65 595.15 68.823 69.282 14 84 2.33E-5 4.24E-4 18.2 587.86 76.842 83.879 18 88 2.33E-5 5.21E-4 22.34 573.167 9.761 94.982 24 94 2.33E-5 6.5E-4 27.88 555.393 18.535 17.43 3 1 2.33E-5 7.63E-4 32.76 543.114 12.814 116.533 14 7 12 1 8 6 4 6 5 4 3 2 l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 14 l 18 l 24 l 3 l 2 1 5 1 15 2 25 3 35 [HArgH]/[1c] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 143 ± 15 % K d [µm] = 699 ± 15 % 37
Titration of HArgH and tweezer 1d in phosphate buffer (1mM, ph 7.2) λ exc = 285 nm λ em = 348 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1d.259 4. 8.91 1-5 Guest H Arg H HCl.774.8 4.59 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 338 ) I obs I calc 7 8.91E-5.E+. 547.49.. 1 71 8.91E-5 6.47E-5.726 529.854 17.195 7.4316 2 72 8.91E-5 1.28E-4 1.432 531.17 16.32 13.636 3 73 8.91E-5 1.89E-4 2.118 523.134 23.915 18.8764 4 74 8.91E-5 2.48E-4 2.786 524.54 22.995 23.35 6 76 8.91E-5 3.63E-4 4.69 518.176 28.873 3.563 8 78 8.91E-5 4.71E-4 5.286 512.63 34.986 36.975 1 8 8.91E-5 5.74E-4 6.442 59.52 37.997 4.474 12 82 8.91E-5 6.72E-4 7.542 56.257 4.792 44.47 15 85 8.91E-5 8.1E-4 9.95 53.272 43.777 48.1879 18 88 8.91E-5 9.39E-4 1.542 492.28 55.21 51.4265 22 92 8.91E-5 1.1E-3 12.324 496.235 5.814 54.7516 26 96 8.91E-5 1.24E-3 13.958 488.97 58.952 57.316 3 1 8.91E-5 1.38E-3 15.461 482.845 64.24 59.318 I 7 6 5 4 3 2 I 6 5 4 3 2 V(H-Arg-H) = L V(H-Arg-H) = 1 L V(H-Arg-H) = 2 L V(H-Arg-H) = 3 L V(H-Arg-H) = 4 L V(H-Arg-H) = 6 L V(H-Arg-H) = 8 L V(H-Arg-H) = 1 L V(H-Arg-H) = 12 L V(H-Arg-H) = 15 L V(H-Arg-H) = 18 L V(H-Arg-H) = 22 L V(H-Arg-H) = 26 L V(H-Arg-H) = 3 L 1 1 2 4 6 8 1 12 14 16 18 [HArgH]/[1d] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 1643 ± 27% K d [µm] = 69 ± 27% 38
Fluorescence titrations for HArgMe Titration of HArgMe and tweezer 1c in phosphate buffer (1mM, ph 7.6) λ exc = 285 nm λ em = 338 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.154 8.575 2.33 1-5 Guest H ArgMe 2HCl.429.644 2.55 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 338 nm ) I obs I calc 7 2,33E-5,E+, 668,334,, 1 71 2,33E-5 3,59E-5 1,54 68,912 59,422 51,48 2 72 2,33E-5 7,9E-5 3,4 571,524 96,81 88,21 3 73 2,33E-5 1,5E-4 4,5 552,344 115,99 115,571 4 74 2,33E-5 1,38E-4 5,92 536,594 131,74 136,61 6 76 2,33E-5 2,1E-4 8,64 55,163 163,171 166,659 8 78 2,33E-5 2,62E-4 11,23 482,611 185,723 186,984 1 8 2,33E-5 3,19E-4 13,68 47,43 198,291 21,6 14 84 2,33E-5 4,25E-4 18,25 451,324 217,1 221,16 18 88 2,33E-5 5,22E-4 22,39 435,475 232,859 233,623 24 94 2,33E-5 6,51E-4 27,95 418,363 249,971 245,633 3 1 2,33E-5 7,65E-4 32,84 411,15 257,184 253,393 3 25 2 15 1 6 4 2 l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 14 l 18 l 24 l 3 l 5 5 1 15 2 25 3 35 [HArgMe]/[1c] 3 32 34 36 38 4 42 44 [nm] K a = 626 ± 6 % K d [µm] = 16 ± 6 % 39
Titration of RGD and tweezer 1a in phosphate buffer (2mM, ph 7.6) λ exc = 28 nm Tweezer 1a H ArgGly Asp H Amount [mg]: Volume [ml]: Concentration [mol/l]:.164 (2.18 1-4 mmol) 1 2.18 1-5.696 (2.1 1-3 mmol).825 2.4 1-3 V Guest V t [75] [RGD] [RGD]/ I [ L] [ L] [mol/l] [mol/l] [1a] 334 nm I obs I calc 7 2.19 1-5.. 42.7.. 1 71 2.19 1-5 3.43 1-5 1.57 4.4 2.2 2.2 2 72 2.19 1-5 6.77 1-5 3.1 39.1 3.6 3.6 3 73 2.19 1-5 1. 1-4 4.58 38.4 4.2 4.5 4 74 2.19 1-5 1.32 1-4 6.2 37.4 5.3 5.1 6 76 2.19 1-5 1.92 1-4 8.8 36.8 5.9 5.9 8 78 2.19 1-5 2.5 1-4 11.43 36.1 6.6 6.4 1 8 2.19 1-5 3.4 1-4 13.93 35.8 6.9 6.8 12 82 2.19 1-5 3.56 1-4 16.31 35.7 7. 7. 15 85 2.19 1-5 4.3 1-4 19.67 35.3 7.4 7.3 18 88 2.19 1-5 4.98 1-4 22.8 35.3 7.3 7.5 22 92 2.19 1-5 5.82 1-4 26.65 35.1 7.6 7.6 26 96 2.19 1-5 6.6 1-4 3.19 35.1 7.6 7.7 3 1 2.19 1-5 7.31 1-4 33.44 34.8 7.9 7.8 8 6 4 2 1 2 3 [RGD]/[1a] V(NAD + ) = µl 4 V(NAD + ) = 1 µl V(NAD + ) = 2 µl V(NAD + ) = 3 µl V(NAD + ) = 4 µl V(NAD + ) = 6 µl 35 V(NAD + = 8 µl V(NAD + ) = 1 µl V(NAD + ) = 12 µl V(NAD + ) = 15 µl 3 V(NAD + ) = 18 µl V(NAD + ) = 22 µl V(NAD + ) = 26 µl V(NAD + ) = 3 µl 25 3 35 4 45 5 [nm] K a [M -1 ] = 1166 ± 1 % K d [µm] = 86 ± 1 % 4
Titration of crgdfl and tweezer 1a in phosphate buffer (1 mm, ph 7.4) Receptor Tweezer1a Solvent Phosphate buffer ph 7.4 T [ C] 25 Substrate cgrgdfl m R [mg].12 m S [mg].25 [R] [M] 3.7 1-5 [S] [M].8 1-3 V R [ml] 4. V S [ml].4 V Guest [ L] V Total [ L] [R][mol/L] [S][mol/L] [S]/[R] I335 nm I obs 7 3.68E-5.E+. 439.525. 1 71 3.68E-5 1.13E-5.31 46.75 33.45 2 72 3.68E-5 2.24E-5.61 38.365 59.16 3 73 3.68E-5 3.31E-5.9 357.329 82.196 4 74 3.68E-5 4.36E-5 1.18 344.975 94.55 6 76 3.68E-5 6.36E-5 1.73 327.138 112.387 8 78 3.68E-5 8.26E-5 2.24 314.131 125.394 1 8 3.68E-5 1.1E-4 2.73 35.119 134.46 12 82 3.68E-5 1.18E-4 3.2 297.448 142.77 15 85 3.68E-5 1.42E-4 3.86 29.326 149.199 18 88 3.68E-5 1.65E-4 4.48 285.31 154.494 22 92 3.68E-5 1.93E-4 5.23 276.13 163.512 26 96 3.68E-5 2.18E-4 5.93 271.499 168.26 3 1 3.68E-5 2.42E-4 6.56 272.71 167.454 18 I 16 14 12 1 8 6 4 2 1 2 3 4 5 6 7 [S]/[R] I obs 5 4 3 2 1 3 32 34 36 38 4 42 44 [nm] V s = µl V s = 1 µl V s = 2 µl V s = 3 µl V s = 4 µl V s = 6 µl V s = 8 µl V s = 1 µl V s = 12 µl V s = 15 µl V s = 18 µl V s = 22 µl V s = 26 µl V s = 3 µl [cgrgdfl]/[1a] K a = 3876 ±3125 K d [µm] =26 ± 8 % 41
Titration of crgdfv and tweezer 1ain phosphate buffer (1mM, ph 7.4) Receptor 1a Solvent Phosphate buffer ph 7.4 T [ C] 25 Substrate crgdfv m R [mg].892 m S [mg] 4.97 [R] [M] 9.95 1-5 [S] [M] 1.12 1-3 V R [ml] 11. V S [ml] 7.6 V Guest [ L] V Total [ L] [R][mol/L] [S][mol/L] [S]/[R] I334 nm I obs 4 9.95 1-5. 242.896. 1 41 9.95 1-5 2.74 1-5.275 226.9 16.887 2 42 9.95 1-5 5.34 1-5.537 213.434 29.462 3 43 9.95 1-5 7.83 1-5.787 26.67 36.829 5 45 9.95 1-5 1.24 1-6 1.253 198.764 44.132 1 5 9.95 1-5 2.24 1-6 2.255 179.812 63.84 15 55 9.95 1-5 3.1 1-6 3.75 168.613 74.283 25 65 9.95 1-5 4.31 1-6 4.336 162.722 8.174 3 7 9.95 1-5 4.81 1-6 4.832 162.128 8.768 45 85 9.95 1-5 5.94 1-6 5.969 161.568 81.328 55 95 9.95 1-5 6.5 1-6 6.527 161.5 81.396 65 15 9.95 1-5 6.95 1-6 6.98 16.293 82.63 75 115 9.95 1-5 7.31 1-6 7.353 16.532 82.364 85 125 9.95 1-5 7.63 1-6 7.667 16.693 82.23 95 135 9.95 1-5 7.9 1-6 7.934 16.17 82.789 15 145 9.95 1-5 8.13 1-6 8.164 16.624 82.272 1 I 8 6 4 2 2 4 6 8 1 [S]/[R] I obs 3 25 2 15 1 5 3 35 4 45 5 55 6 [nm] V S = µl V S = 1 µl V S = 2 µl V S = 3 µl V S = 5 µl V S = 1 µ V S = 15 µ V S = 25 µ V S = 3 µ V S = 45 µ V S = 55 µ V S = 65 µ V S = 75 µ V S = 85 µ V S = 95 µ V S = 15 [crgdfv][1a] K a = 16791 ± 1817, K d [µm] = 59 ± 11 % 42
Fluorescence titrations of the tweezers and the amino acid guests in methanol and mixture of methanol-buffer Fluorescence titration of the tweezer 1a with AcLysMe in methanol λ exc = 285 nm λ em = 322 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1a.12 6. 2.46 1-5 Guest Ac Lys Me HCl.382 1. 1.6 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 322 ) - I obs - I calc 7 2.46E-5.E+. 68.731.. 1 71 2.46E-5 2.25E-5.92 615.971 7.24 7.651 2 72 2.46E-5 4.45E-5 1.81 621.386 12.655 12.889 3 73 2.46E-5 6.58E-5 2.68 625.74 17.9 16.594 4 74 2.46E-5 8.65E-5 3.52 626.25 17.519 19.38 6 76 2.46E-5 1.26E-4 5.15 632.937 24.26 22.964 1 8 2.46E-5 2.E-4 8.15 636.981 28.25 26.873 18 88 2.46E-5 3.27E-4 13.33 639.343 3.612 3.135 22 92 2.46E-5 3.83E-4 15.59 638.661 29.93 3.962 26 96 2.46E-5 4.33E-4 17.65 639.838 31.17 31.555 35 7 3 6 25 2 15 1 5 4 3 2 l 1 l 2 l 3 l 4 l 6 l 1 l 18 l 22 l 26 l 5 1 2 4 6 8 1 12 14 16 18 2 [AcLysMe]/[1a] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 152 ± 11 % K d [µm] = 66 ± 11 % ΔI max = -37 (6 %) 43
Fluorescence titration of the tweezer 1a with AcArgMe in 1:9 mixture of methanol/buffer (1 mm, ph 7.6) λ exc = 285 nm λ em = 322 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1a.178 9. 2.43 1-5 Guest Ac ArgMe HCl.318.75 1.59 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 329 ) I obs I calc 7 2.43E-5.E+. 577.521.. 1 71 2.43E-5 2.24E-5.92 559.216 18.35 17.616 2 72 2.43E-5 4.42E-5 1.82 545.347 32.174 31.65 3 73 2.43E-5 6.53E-5 2.69 534.2 43.51 43.31 4 74 2.43E-5 8.59E-5 3.54 521.926 55.595 52.412 6 76 2.43E-5 1.25E-4 5.17 58.4 69.121 66.898 8 78 2.43E-5 1.63E-4 6.72 51.594 75.927 77.513 1 8 2.43E-5 1.99E-4 8.18 495.321 82.2 85.598 14 84 2.43E-5 2.65E-4 1.91 482.86 94.661 97.59 18 88 2.43E-5 3.25E-4 13.39 473.636 13.885 14.77 24 94 2.43E-5 4.6E-4 16.72 467.25 11.271 112.518 3 1 2.43E-5 4.77E-4 19.64 454.493 123.28 117.696 14 6 12 1 8 6 4 5 4 3 2 l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 14 l 18 l 24 l 3 l 2 1 5 1 15 2 25 [AcArgMe]/[1a] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 638 ± 8 % K d [µm] = 157 ± 8 % ΔI max = 158 (27 %) 44
Fluorescence titration of the tweezer 1c with AcLysMe in methanol λ exc = 285 nm λ em = 32 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.118 7. 2.19 1-5 Guest Ac Lys Me.459.754 2.55 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 32 ) - I obs - I calc 7 2.19E-5.E+. 341.665.. 1 71 2.19E-5 3.59E-5 1.64 491.668 15.3 147.138 2 72 2.19E-5 7.8E-5 3.24 541.67 2.5 197.765 3 73 2.19E-5 1.5E-4 4.79 558.559 216.894 219.535 4 74 2.19E-5 1.38E-4 6.3 572.238 23.573 231.212 6 76 2.19E-5 2.1E-4 9.21 58.712 239.47 243.285 8 78 2.19E-5 2.62E-4 11.96 586.192 244.527 249.429 1 8 2.19E-5 3.19E-4 14.58 593.864 252.199 253.137 14 84 2.19E-5 4.25E-4 19.43 599.1 257.435 257.39 18 88 2.19E-5 5.22E-4 23.85 64.29 262.625 259.757 24 94 2.19E-5 6.51E-4 29.77 61.297 259.632 261.83 3 1 2.19E-5 7.65E-4 34.98 613. 271.335 263.74 3 25 6 2 15 1 5 4 2 l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 14 l 18 l 24 l 3 l 1 2 3 4 [AcLysMe]/[1c] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 498 ± 6 % K d [µm] = 2 ± 6 % ΔI max = -27 (44 %) 45
Fluorescence titration of the tweezer 1c with AcArgMe in methanol λ exc = 285 nm λ em = 32 nm Amount [mg]: Volume [ml]: Concentration [mol/l]: Receptor 1c.118 7. 2.19 1-5 Guest Ac ArgMe.484.711 2.55 1-3 Guest Receptor [Receptor] [Guest] [Guest]/ F I V (µl) V (µl) [mol/l] [mol/l] [Receptor] (I 32 ) - I obs - I calc 7 2.19E-5.E+. 342.695.. 1 71 2.19E-5 3.59E-5 1.64 351.154 8.459 9.834 2 72 2.19E-5 7.9E-5 3.24 359.956 17.261 17.635 3 73 2.19E-5 1.5E-4 4.8 369.16 26.465 23.956 4 74 2.19E-5 1.38E-4 6.31 372.464 29.769 29.172 6 76 2.19E-5 2.2E-4 9.21 379.892 37.197 37.254 8 78 2.19E-5 2.62E-4 11.97 385.463 42.768 43.213 1 8 2.19E-5 3.19E-4 14.59 387.77 45.12 47.78 14 84 2.19E-5 4.25E-4 19.45 398.23 55.58 54.36 18 88 2.19E-5 5.22E-4 23.87 41.469 58.774 58.74 24 94 2.19E-5 6.52E-4 29.8 46.335 63.64 63.235 3 1 2.19E-5 7.66E-4 35.1 49.9 66.314 66.264 7 5 6 5 4 3 2 1 4 3 2 1 l 1 l 2 l 3 l 4 l 6 l 8 l 1 l 14 l 18 l 24 l 3 l 1 2 3 4 [AcArgMe]/[1c] 3 32 34 36 38 4 42 44 [nm] K a [M -1 ] = 362 ± 7 % K d [µm] = 276 ± 7 % ΔI max = -91 (22 %) 46
1.4 1 H NMR titrations NMR titrations of the phosphate tweezer 1a Titration of tweezer 1a andaclysme in phosphate buffer (7 mm, ph 7.2) Receptor 1a M R [g/mol] 1a.19 Solvent Phosphate buffer M S [g/mol] 238.11 T [ C] 25 m R [mg] 3.61 Substrat Ac Lys Me m S [mg] 1.39 H 3 N X 6 5 4 3 2 HN CH 3 CH 3 δ (2-H) [ppm] = 4.379 δ (6-H) [ppm] = 2.98 δ (-NAc) [ppm] = 2.31 δ (-CMe) [ppm] = 3.752 V [ml] 2 [R] [mm] 2.41 [S] [mm] 2.49 X - 2- : im Phosphate buffer HP 3 [R] [mm] [S] [mm] obs (NAc-H) obs calc 2.41 2.49 2.325 -.294 -.293 1.2 1.24 2.313 -.282 -.283.6.62 2.3 -.269 -.269.3.31 2.281 -.25 -.252.15.16 2.258 -.228 -.228.8.8 2.232 -.21 -.199. obs [ppm] -.5 -.1 -.15 -.2 -.25 -.3 K a [M -1 ] = 5835 ± * δ max (2-H) [ppm] =.51 δ max (6-H) [ppm] = 3.91 δ max (-NAc) [ppm] = -.32 δ max (-CMe) [ppm]= -.23 -.35..1.2.3 [R] [M] * Limit of 95% - confidence interval from the nonlinear regression of the signals of the Protons NAc-H. 47
Titration of tweezer 1a and HLysH in phosphate buffer (7 mm, ph 7.2) Receptor 1a M R [g/mol] 1a.19 Solvent Phosphate buffer M S [g/mol] 182.8 T [ C] 25 m R [mg] 5. Substrat H Lys H m S [mg] 1.39 H 3 N 6 5 4 3 2 NH 3 X - : in Phosphate buffer HP 3 2- X δ (2-H) [ppm] = 3.741 δ (3-H) [ppm] = 1.713 δ (5-H) [ppm] = 1.891 δ (6-H) [ppm] = 3.9 V [ml] 2 [R] [mm] 2.82 [S] [mm] 3.24 [R] [mm] [S] [mm] obs (2-H) obs calc 2.82 3.24 3.545.196.196 1.41 1.62 3.553.188.188.71.81 3.564.177.177.35.41 3.581.16.1c.18.2 3.61.14.142.9.1 3.622.119.118.25 obs [ppm].2.15.1.5 K a [M -1 ] = 256 ± 189* δ max (2-H) [ppm] =.24 δ max (3-H) [ppm] =.76 δ max (5-H) [ppm] = 4.47 δ max (6-H) [ppm] = 4.51...1.2.3 [R] [M] * Limit of 95% - confidence interval from the nonlinear regression of the signals of the Protons 2-H. 48
Titration of tweezer 1a and KAA in phosphate buffer (7 mm, ph 7.2) Receptor 1a M R [g/mol] 1a.19 Solvent Phosphate buffer M S [g/mol] 288.35 T [ C] 25 m R [mg] 3.57 Substrat KAA m S [mg] 1.75 H 3 N 5 H 6 7 N H 8 9 3 CH 3 H 4 H N 2 H CH 3 1 X δ (1-H) [ppm] = 1.414 δ (3-H) [ppm] = 1.336 δ (4-H) [ppm] = 4.97 δ (5-H) [ppm] = 3.966 δ (6-H) [ppm] = 1.95 δ (7-H) [ppm] = 1.463 δ (8-H) [ppm] = 1.713 δ (9-H, 9 -H) [ppm] = 3.13 V [ml] 2 [R] [mm] 2.38 [S] [mm] 2.13 NH 3 X - 2- : im Phosphate buffer HP 3 [R] [mm] [S] [mm] obs (5-H) obs calc 2.38 2.13 3.773.193.194 1.19 1.6 3.776.19.19.59.53 3.782.184.183.3.27 3.793.173.173.15.13 3.87.159.16.7.7 3.823.144.143 obs [ppm].25.2.15.1.5...1.2.3 [R] [M] K a [M -1 ] = 983 ± 8423 δ max (1-H) [ppm] = -.13 δ max (3-H) [ppm] = -. δ max (4-H) [ppm] = -.17 δ max (5-H) [ppm] =.2 δ max (6-H) [ppm] = 1.9 δ max (7-H) [ppm] = 2.28 δ max (8-H) [ppm] = 3.22 δ max (9-H, 9 -H) [ppm] = 5.82, 5.92 * Limit of 95% - confidence interval from the nonlinear regression of the signals of the Protons 5-H. 49
Titration of tweezer 1a and AcArgMe in phosphate buffer (1 mm, ph 7.2) Receptor 1a M R [g/mol] 77.78 Solvent Phosphate buffer M S [g/mol] 266.7 T[ o C] 25 m R [mg].8 Substrate Ac ArgMe m S [mg].511 H 2 N X NH 2 C N H 5 X : HP 4 2-4 3 2 HN CH 3 CH 3 δ (2-H) [ppm] = 4.421 δ (5-H) [ppm] = 3.226 δ (4-H) [ppm] = 1.663 δ (-NAc) [ppm] = 2.52 δ (-C 2 Me) [ppm] = 3.775 [R] [mm] [S] [mm] δ (5-H) [ppm] Δδ obs [ppm] Δδ calc [ppm] 3. 3..198 3.424 3.442 1.5 1.5.113 3.338 3.323.75.75.58 3.168 3.162.375.375.274 2.952 2.947.188 1.88.563 2.663 2.671 4 obs [ppm] 3 2 1,,5,1,15,2,25,3,35 [R] [mol/l] K a [M -1 ] = 45939 ± 265 K d [µm] = 22 ± 4 % Δδ max (5-H) [ppm] = 3.748 ±.16 Δδ max (4-H) [ppm] = 2.54 ±.5 Δδ max (3-H) [ppm] = 1.229 ±.7 Δδ max (2-H) [ppm] =.63 ±.2 * Limit of 95% - confidence interval from the nonlinear regression of the signals of the Protons 5-H. 5
NMR titrations of sulfate tweezer 1c Titration of tweezer 1c andaclysme in phosphate buffer (1 mm, ph 7.2) Receptor 1c M R [g/mol] 77.78 Solvent Phosphate buffer M S [g/mol] 238.7 T[ o C] 25 m R [mg].672 Substrate AcLysMe m S [mg].41 5 H 3 N 6 4 X X : HP 4 2-3 2 HN CH 3 CH 3 δ (2-H) [ppm] = 4.398 V [ml] 3. δ (6-H) [ppm] = 2.999 [S ][mm].5725 δ (-NAc) [ppm] = 2.5 δ (-C 2 Me) [ppm] = 3.77 V [ml] [R][mM] δ (2-H) [ppm] Δδ obs [ppm] Δδ calc [ppm].6 1.4548 4.313.367.3667.66 1.3225 4.332.3651.3658.72 1.2123 4.34.3643.3648.78 1.1191 4.34.3643.3637.84 1.391 4.359.3624.3624.9.9699 4.367.3616.369.96.993 4.374.369.3592 1.2.8558 4.45.3578.3572 1.8.882 4.443.354.3549 1.18.7397 4.51.3482.35 1.28.6819 4.57.3413.3436 1.4.6235 4.689.3294.3337 1.5.5819 4.761.3222.3235 1.7.5135 4.898.385.2997.. 4.3983..,4,3 K a [M -1 ] = 79536 ± 8889 [ppm],2,1 K d [µm] = 12 ± 11 % Δδ max (2-H) [ppm] =.37 Δδ max (3-H) [ppm] = 1.29 Δδ max (4-H) [ppm] = 2.64,,,2,4,6,8,1,12,14,16 [R][mol/l] Δδ max (6-H) [ppm] = 3.75 Δδ max (5-H) [ppm] = 4.41 * Limit of 95% - confidence interval from the nonlinear regression of the signals of the Protons 2-H. 51
Titration of tweezer 1c and AcArgMe in phosphate buffer (1 mm, ph 7.2) Receptor 1c M R [g/mol] 77.78 Solvent Phosphate buffer M S [g/mol] 266.7 T[ o C] 25 m R [mg].8 Substrate AcArgMe m S [mg].511 H 2 N X NH 2 C N H 5 X : HP 4 2-4 3 2 HN CH 3 CH 3 δ (2-H) [ppm] = 4.421 V [ml] 3.42 δ (5-H) [ppm] = 3.226 [S] [mm].562 δ (4-H) [ppm] = 1.663 δ (-NAc) [ppm] = 2.52 δ (-C 2 Me) [ppm] = 3.775 V [ml] [R] [mm] δ (5-H) [ppm] Δδ obs [ppm] Δδ calc [ppm].6 1.7298 -.3837 3.696 3.69.65 1.5968 -.3578 3.5837 3.5719.7 1.4827 -.3216 3.5475 3.5416.75 1.3839 -.295 3.529 3.511.8 1.2974 -.2534 3.4793 3.4773.85 1.2211 -.2227 3.4486 3.4433.9 1.1532 -.1865 3.4124 3.479.95 1.925 -.141 3.3669 3.3714 1. 1.379 -.926 3.3185 3.3338 1.1.9436 -.113 3.2372 3.2553 1.2.8649.957 3.132 3.1729 1.3.7984.17 3.559 3.876 1.4.7414.262 2.9639 3.4 1.5.6919.2955 2.934 2.9119 1.7.615.4491 2.7768 2.7358 1.9.5463.697 2.6162 2.5658.. 3.2259.. 4 ppm 3 2 1,,2,4,6,8,1,12,14,16,18,2 [R][mol/l] K a [M -1 ] = 11342 ± 561 K d [µm] = 88 ± 5 % Δδ max (2-H) [ppm] =.42 Δδ max (3-H) [ppm] = 1.32 Δδ max (4-H) [ppm] = 2.51 Δδ max (5-H) [ppm] = 3.86 * Limit of 95% - confidence interval from the nonlinear regression of the signals of the Protons 5-H. 52
NMR titrations of the carboxymethyl tweezer 1d Titration of tweezer 1d andaclysme in phosphate buffer (1 mm, ph 7.2) Receptor 1d M R [g/mol] 726.72 Solvent Phosphate buffer (74 mm) M S [g/mol] 238.7 T[ o C] 25 m R [mg].91 Substrate AcLysMe m S [mg].552 5 3 H 3 N 2 CH 3 6 4 HN CH 3 X X : HP 4 2- δ (3b-H) [ppm] = 1.682 V [ml] 3. δ (6-H) [ppm] = 2.984 [S ][mm].771 δ (-NAc) [ppm] = 2.38 δ (-C 2 Me) [ppm] = 3.763 V [ml] [R][mM] δ (3b-H) Δδ obs [ppm] Δδ calc [ppm] [ppm].6 2.87 1.236.446.4524.65 1.9265 1.247.435.4351.7 1.7889 1.25.432.4188.75 1.6696 1.276.46.436.8 1.5653 1.33.379.3893.85 1.4732 1.34.378.3759.9 1.3913 1.316.366.3634.95 1.3181 1.332.35.3515 1. 1.2522 1.343.339.344 1.1 1.1384 1.354.328.3199 1.2 1.435 1.387.295.316 1.3.9632 1.46.276.2853 1.4.8944 1.414.268.275 1.5.8348 1.425.257.2571 1.6.7826 1.427.255.2449.. 1.682..,5,4 ppm],3,2,1,,,5,1,15,2,25 [R][mol/L] K a [M -1 ] = 859 ± 11% Δδ max (3a-H) [ppm] =.52 Δδ max (3b-H) [ppm] =.77 Δδ max (4-H) [ppm] =.4 Δδ max (5-H) [ppm] =.54 Δδ max (6-H) [ppm] =.94 * Limit of 95% - confidence interval from the nonlinear regression of the signals of the Protons 3b-H. 53
Titration of tweezer 1d and AcArgMe in phosphate buffer (1 mm, ph 7.2) Receptor 1d M R [g/mol] 726.72 Solvent Phosphate buffer (74 mm) M S [g/mol] 266.7 T[ o C] 25 m R [mg].77 Substrate AcArgMe m S [mg].552 H 2 N X NH 2 C N H 5 X : HP 4 2-4 3 2 HN CH 3 CH 3 δ (2-H) [ppm] = 4.414 V [ml] 3. δ (5-H) [ppm] = 3.213 [S] [mm].6898 δ (3-H) [ppm] = 1.656 δ (-NAc) [ppm] = 2.41 δ (-C 2 Me) [ppm] = 3.762 V [ml] [R] [mm] δ (5-H) [ppm] Δδ obs [ppm] Δδ calc [ppm].6 1.7659 2.744.469.4846.65 1.631 2.735.478.4632.7 1.1514 2.75.463.4436.75 1.4127 2.787.426.4254.8 1.3244 2.84.49.486.85 1.2466 2.82.393.393.9 1.1773 2.842.371.3784.95 1.1153 2.862.351.3649 1. 1.596 2.876.337.3522 1.1.9632 2.881.332.3293 1.2.8829 2.91.33.391 1.3.815 2.924.289.2912 1.4.7568 2.934.279.2752 1.5.764 2.941.272.268 1.6.6622 2.955.258.2478.. 3.213..,6,5 ppm,4,3,2,1 K a [M -1 ] = 718 ± 18% Δδ max (5-H) [ppm] =.96 Δδ max (4b-H) [ppm] =.62 Δδ max (4a-H) [ppm] =.48 Δδ max (3-H) [ppm] =.52,,,2,4,6,8,1,12,14,16,18,2 [R][mol/L] * Limit of 95% - confidence interval from the nonlinear regression of the signals of the Protons 5-H. 54
1.5 Solvent-dependent 1 H NMR spectra of the complexes Figure S2. 1 H-NMR spectra of 1:1 mixture of the phosphate tweezer 1a and AcLysMe in different polarity medium methanol/phosphate buffer (1mM, ph 7.2) mixture. Lysine side-chain protons are assigned by numbers. 1:1 complex in CD3D:PB (2:1) 1:1 complex in CD3D:PB (1:2) 1:1 complex in CD3D:PB (1:9) 1:1 complex in PB Figure S3. 1 H-NMR spectra of 1:1 mixture of the phosphate tweezer 1a and AcArgMeeach at 1. mm concentration in different polarity medium methanol/phosphate buffer (PB, 1mM, ph 7.2) mixture. The spectrum in methanol/buffer (1:9) was measured at.5 mm concentration of both host and guest. Arginine side chain protons are assigned by numbers. 55
Figure S4. 1 H-NMR spectra of 1:1 mixture of the sulfate tweezer 1c and AcLysMe, each at 1. mm concentration.signals of lysine side-chain are shifted upfieldin the similar range both in buffer and in methanol. 6-H protons of lysine shifted upfield by ~ 3.4 ppm in phosphate buffer (1 mm, ph 7.2) and by ~ 3. ppm in methanol. 1:1 complex in PB Figure S5. 1 H-NMR spectra of 1:1 ratio of the sulfate tweezer 1c and AcArgMe, each at 1. mm concentration.signals of the arginine side chain protons areshifted upfield strongly in buffer but only weakly in methanol. The signal of the arginine protons 5-H is shifted upfield by ~ 2.7 ppm in buffer and by ~.7 ppm in methanol. 56
1.6 Isothermal titration calorimetry (ITC) studies of thehost-guest complex formation of tweezers 1a and 1c. ITC experiments of the phosphate tweezer 1a complexes ITC titration of the tweezer 1a (.1 mm) with AcLysMe (1. mm) in phosphate buffer (1 mm, ph 7.6) Nr. K a [M -1 ] n H[kcal/mol] -T S[kcal/mol] G[kcal/mol] 1 693 ± 37.679 ±.9-5.552 ±.11-1.5-6.6 2 679 ± 32.648 ±.8-5.69 ±.95 -.99-6.6 Time (min) Time (min) -1 1 2 3 4 5 6 7 8 9 1 11 12 13.2-1 1 2 3 4 5 6 7 8 9 1 11 12 13.2.. -.2 -.2 -.4 -.4 µcal/sec -.6 -.8 µcal/sec -.6 -.8-1. -1. -1.2-1.2-1.4-1.4-1 1 2 3 4 5 6 7 8 9 1 11 12 13 kcal/mole of injectant -2 kcal/mole of injectant -2-4 -4..5 1. 1.5 2. 2.5 Molar Ratio..5 1. 1.5 2. 2.5 Molar Ratio ITC titration of the tweezer 1a (.1 mm) with AcArgMe (1. mm) in phosphate buffer (1 mm, ph 7.6) Nr. K a [M -1 ] n H[kcal/mol] -T S[kcal/mol] G[kcal/mol] 1 317 ± 11.625 ±.1-6.85 ±.145.67-6.14 2 275 ± 17.613 ±.2-7.249 ±.35 1.19-6.6 57
.2 Time (min) -1 1 2 3 4 5 6 7 8 9 1 11 12 13.2 Time (min) -1 1 2 3 4 5 6 7 8 9 1 11 12 13.. -.2 -.2 -.4 -.4 µcal/sec -.6 -.8 µcal/sec -.6 -.8-1. -1. -1.2-1.2-1.4 kcal/mole of injectant -2 kcal/mole of injectant -2-4 -4..5 1. 1.5 2. 2.5 Molar Ratio..5 1. 1.5 2. 2.5 Molar Ratio ITC titration of the tweezer 1a (.1 mm) with KLVFF (.65 mm) in phosphate buffer (1 mm, ph 7.6) Nr. K a [M -1 ] n H[kcal/mol] -T S[kcal/mol] G[kcal/mol] 1 658 ± 26 1.2 ±.5-6.293 ±.55 -.28-6.57 2 653 ± 33.983 ±.7-6.466 ±.84 -.1-6.57 Time (min) -1 1 2 3 4 5 6 7 8 9 1 11 12 13 Time (min) -1 1 2 3 4 5 6 7 8 9 1 11 12 13.. -.2 -.2 -.4 -.4 µcal/sec -.6 µcal/sec -.6 -.8 -.8-1. -1. kcal/mole of injectant -2-4 kcal/mole of injectant -2-4 -6-6..5 1. 1.5..5 1. 1.5 Molar Ratio Molar Ratio 58
ITC experiments of the sulfate tweezer 1c complexes ITC titration of the tweezer 1c (.1 mm) with AcLysMe (1. mm) in phosphate buffer (1 mm, ph 7.6) Nr. K a [M -1 ] n H[kcal/mol] -T S[kcal/mol] G[kcal/mol] 1 362 ± 16.823 ±.11-8.171 ±.159 1.96-6.21 2 33 ± 18.782 ±.15-8.43 ±.226 2.24-6.16 µcal/sec. -.2 -.4 -.6 -.8-1. -1.2-1.4-1.6-1.8 Time (min) -1 1 2 3 4 5 6 7 8 9 1 11 12 13.2 µcal/sec -1 1 2 3 4 5 6 7 8 9 1 11 12 13.2. -.2 -.4 -.6 -.8-1. -1.2-1.4-1.6 Time (min) -2-2 kcal/mole of injectant -4 kcal/mole of injectant -4-6 -6..5 1. 1.5 2. 2.5 Molar Ratio..5 1. 1.5 2. 2.5 Molar Ratio The ITC titration of the tweezer 1c (.1 mm) with AcArgMe (1. mm) in phosphate buffer (1 mm, ph 7.6) Nr. K a [M -1 ] n H[kcal/mol] -T S[kcal/mol] G[kcal/mol] 1. 113 ± 1.94 ±.48-5.92 ±.45.387-5.53 2. 949 ± 11.816 ±.71-6.75 ±.796 1.317-5.43 59
Time (min) -1 1 2 3 4 5 6 7 8 9 1 11 12 13 Time (min) -1 1 2 3 4 5 6 7 8 9 1 11 12 13.. -.2 -.2 µcal/sec -.4 µcal/sec -.4 -.6 -.6 -.8 -.8 kcal/mole of injectant -2 kcal/mole of injectant -2..5 1. 1.5 2. 2.5 Molar Ratio..5 1. 1.5 2. 2.5 Molar Ratio 2. Computational Section: 2.1 Calculation of host-guest complex structures by QM/MM: Material and Methods The models of the inclusion complexes between the anionic tweezers 1a -d and amino acids or short peptides were built by using the VMD program. 1 Since the phosphate tweezers 1a is partially protonated in buffered aqueous solution at almost neutral ph value, we also used the mono- and diprotonated structures 1a and 1a for the calculations (Figure 7). The neutralized initial structures were then submitted to energy minimizations with the CHARMM c33b1 program. 2 After that, the system was placed in a water sphere of 3 Å of radii centered on one of the central carbon atoms of the tweezers. To ensure a correct water distribution twelve hydration-minimization cycles were performed. To prevent the water molecules from vaporing off, a four order polynomial potential was applied to all water oxygen atoms. After this, the hydrated systems were submitted to 1 ns MD simulations at 3 K for which the program CHARMM c33b1 with the CHARMM22 force field and the TIP3P model for water were used. 3,4 The parameters for the tweezers and tosyl terminal group were generated using the Swissparam server and tested by us (unpublished data). 5 Randomly selected snapshots from the MD simulations were then submitted to QM/MM optimization. The QM/MM optimizations were performed with the program ChemShell v3.2. 6 The Turbomole 5.1 program was used to handle the QM region and DL_PLY, as driver of the 6
CHARMM22force field, to treat the MM part. 7,8 The QM part which includes all atoms of the tweezers and part of the lateral chain of Lys or Arg (see Figure S) was described using the B3LYP density functional with empirical dispersive energy correction (B3LYP-D2) and the SVP basis set from the Turbomole basis set library. 9,1,11 pen valencies at the QM/MM border were saturated using hydrogen link atoms. 12 An electrostatic embedding scheme was used for the interactions between QM and MM regions. 13 To avoid overpolarization of the QM region at the boundary a charge shift scheme was applied. 14 No electrostatic cutoffs were used. The optimization was performed with the HDLC optimizer. 15 The active region consisted of a water sphere of 13 Å of radii centered on one of the central carbon atoms of the tweezers. All atoms within the active region were allowed to move in each optimization step. The optimization was finished when the maximum gradient component was below.45 a.u. Figure S6. A Distances (black dotted lines) and angle (blue dotted line) used to describe the interaction between the molecular tweezers and the amino acid and peptides models (Y = P, S, or C atom of the CH 2 C - 2 group of the tweezers, X = the N atom or the central C atom of the guanidinium moiety in the lateral chain of Lys or Arg respectively). B Atoms of the lateral chains of Arg and Lys included in the QM region used for the QM/MM optimizations are highlighted with a sphere representation. Table S1. Representative distances and the C 1 -C 2 -X angle for the isolated tweezers and the inclusion complexes between the tweezers and different Lys/Arg/peptide models (o stands for the guest not inserted in the tweezers cavity and i when the guest is threaded inside the tweezers). Depending on the systems X is the P, S or the C atom of the carboxylate group of the tweezers, while Y is the N or the central C atom of the guanidinium moiety in the lateral chain of Lys or Arg respectively. π H-cis the distance between the orto hydrogen atom of the last benzene ring and the center of the opposite benzene ring (see Figure 6). Values in 61
parentheses are for QM/MM optimized structures. All distances are in Å and the angle in degrees. System C p -C p C m -C m X-Y C 1 -C 2 -X π H-c 1a 5.51 ±.2 3.92 ±.2 4.49 ±.25 - - (5.24) (3.51) (3.81) 1a 5.71 ±.21 3.91 ±.2 4.7 ±.24 - - (5.43) (3.8) (4.13) 1a H Lys H 5.51 ±.21 4.11 ±.2 3.57 ±.15 85 ± 3 (5.24) (3.71) (3.39) (88) - 1a KAA 5.8 ±.21 4.67 ±.22 3.32 ±.1 92 ± 3 (5.32) (3.85) (3.29) (89) - 1a Ac Lys Me 6.24 ±.22 4.98 ±.22 3.82 ±.1 95 ± 3 (5.63) (4.24) (3.91) (98) - 1a Ac ArgMe 5.62 ±.18 3.96 ±.18 3.91 ±.7 91 ± 3 (5.52) (3.76) (4.) (89) - 1b 1b Ac Lys Me 1b TsArgMe 6.49 ±.24 (5.94) 7.23 ±.22 (7.83) 5.53 ±.18 (5.33) 4.96 ±.26 (4.33) 6.29 ±.21 (6.82) 3.86 ±.17 (3.83) - - 3.9 ±.12 (4.6) 3.88 ±.1 (3.95) 65 ± 3 (65) 86 ± 3 (82) 5.66 ±.31 (4.76) - - 1c 1c Ac Lys Me 1c Ac ArgMe 5.81 ±.22 (5.49) 5.69 ±.22 (5.48) 5.58 ±.17 (5.16) 3.96 ±.2 (3.89) 4.82 ±.22 (4.52) 3.72 ±.15 (3.48) - - 4.12 ±.1 (4.6) 4.1 ±.1 (4.28) 64 ± 3 (62) 82 ± 3 (78) 4.14 ±.25 (4.5) - - 1d 1d Ac Lys Me (i) 1d Ac Lys Me (o) 1d Ac ArgMe (i) 1d Ac Arg Me (o) 5.54 ±.2 (5.25) 5.3 ±.19 (4.97) 5.48 ±.19 (5.54) 5.55 ±.18 (5.2) 5.67 ±.22 (5.4) 3.73 ±.17 (3.51) 3.77 ±.16 (3.53) 3.78 ±.18 (3.68) 3.79 ±.16 (3.42) 3.97 ±.21 (3.67) - - 3.2 ±.19 (3.48) 3.32 ±.14 (3.2) 3.64 ±.1 (3.6) 3.84 ±.1 (3.9) 74 ± 3 (72) 64 ± 3 (62) 77 ± 3 (77) 69 ± 3 (65) 4.38 ±.25 (3.87) - - - - References (1) Humphrey, W., A. Dalke, and K. Schulten, VMD: Visual Molecular Dynamics. J. Mol. Graphics, 1996. 14: p. 33-38. (2) Brooks, B.R., et al., CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem.1983. 4(2): p. 187-217. 62
(3) Jorgensen, W.L., et al., Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics, 1983. 79(2): p. 926-935. (4) Mackerell, A.D., Jr, M. Feig, and C.L. Brooks, III, Extending the Treatment of Backbone Energetics in Protein Force Fields: Limitations of Gas-Phase Quantum Mechanics in Reproducing Protein Conformational Distributions in Molecular Dynamics Simulations. J. Comput. Chem., 24. 25: p. 14-1415. (5) Zoete, V., et al., SwissParam: A Fast Force Field Generation Tool for Small rganic Molecules. J. Comp. Chem., 21. 32: p. 2359-2368. (6) Chemshell, a Computational Chemistry Shell (www.chemshell.org). (7) Ahlrichs, R., et al., Electronic Structure calculation on workstation computers: The program system TURBMLE. Chem. Phys. Lett., 1989. 162: p. 165-169. (8) Smith, W. and T.R. Forester, DL-PLY _2.: a general-purpose parallel molecular dynamics simulation package. J. Mol. Graphics, 1996. 14(3): p. 136-141. (9) Lee, C., W. Yang, and R.G. Parr, Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B, 1988. 37: p. 785-789. (1) Grimme, S., Semiempirical GGA-type density functional constructed with a long-range dispersion correction. J ComputChem, 26. 27(15): p. 1787-1799. (11) Schäfer, A., H. Horn, and R. Ahlrichs, Fully optimized contracted Gaussian basis sets for atoms Li to Kr. J. Chem. Phys., 1992. 97: p. 2571-2577. (12) Antes, I. and W. Thiel, n the treatment of link atoms in hybrid methods. ACS Symp. Ser., 1998. 712(Combined Quantum Mechanical and Molecular Mechanical Methods): p. 5-65. (13) Bakowies, D. and W. Thiel, Hybrid Models for Combined Quantum Mechanical and Molecular Mechanical Approaches. J. Phys. Chem., 1996. 1: p. 158-1594. (14) de Vries, A.H., et al., Zeolite Structure and Reactivity by Combined Quantum-Chemical- Classical Calculations. J. Phys. Chem. B, 1999. 13(29): p. 6133-6141. (15) Billeter, S.R., A.J. Turnera, and W. Thiel, Linear scaling geometry optimisation and transition state search in hybrid delocalised internal coordinates.phys. Chem. Chem. Phys.2. 2: p. 2177-2186. 63
2.2 Calculation of 1 H NMR shifts of the guest signals by ab initio methods The solvent effects were modelled using optimized QM/MM structures with an explicit, static 4-Å water layer around both (the host-guest complex and the pure guest molecule as well). Figure S7 shows the compexstructures used for the computation of 1 H NMR shifts without the water layer: 1a H Lys H, 1 1a H Lys H, 2 1a H Lys H 1a H Lys H 1a H Lys H 1a Ac Lys Me, 1 64
1a Ac Lys Me, 2 1a Ac Lys Me 1a Ac Lys Me 1a KAA, 1 1a KAA, 2 1a KAA 65
1a KAA 1a H Arg H, 1 1a H Arg H, 2 1a H Arg H 1a H Arg H 1a Ac ArgMe, 1 66
1a Ac ArgMe, 2 1a Ac ArgMe 1a Ac ArgMe Figure S7. Host-guest complex structures of the differently protonated phosphate tweezers 1a ( 1: R 1 = P 2-3, R 2 = P(H) - 2 ), 1a ( 1: R 1 = R 2 = P(H) - 2 ), 1a ( 1: R 1 = R 2 = P 2-3 ) with lysine and arginine derivatives (without counterions) optimized by QM/MM calculations, each structure contains a 4Å water layer. In each host-guest complex of the tweezer1a there are two conformers, one with the positively charged lysine ammonium or arginine guanidinium end group pointing toward the doubly negatively charged phosphate group (1) and the other one pointing away from the doubly negatively charged phosphate group (2). 67