Botan et al. 10.1073/pnas.0701762104. |
Fig. 6. ORTEP drawing of the two independent molecules (A and B) in the X-ray diffraction structure of PAZ-Aib-L-Ala-(Aib)6-OMe with backbone and terminal groups atom numbering. H- atoms are omitted for clarity. Displacement ellipsoids are drawn at the 30% probability level. Intramolecular H-bonds are indicated by dashed lines.
Fig. 7. FTIR absorption spectra in the N-H stretching region of PAZ-L-Ala*-Aib7-OMe (1), PAZ-Aib-L-Ala*-Aib6-OMe (2), and PAZ-Aib3-L-Ala*-Aib4-OMe (4) in CDCl3 solution. Peptide concentration: 1 mM.
Fig. 8. Plot of NH proton chemical shifts in the 1H NMR spectra PAZ-L-Ala*-Aib7-OMe as a function of increasing percentages of DMSO (vol/vol) added to the CDCl3 solution. Peptide concentration: 1 mM.
Fig. 9. Plot of NH proton chemical shifts in the 1H NMR spectra PAZ-Aib-L-Ala*-Aib6-OMe as a function of increasing percentages of DMSO (vol/vol) added to the CDCl3 solution. Peptide concentration: 1 mM.
Fig. 10. Plot of NH proton chemical shifts in the 1H NMR spectra PAZ-Aib3-L-Ala*-Aib4-OMe as a function of increasing percentages of DMSO (v/v) added to the CDCl3 solution. Peptide concentration: 1 mM.
Fig. 11. Far-UV CD spectrum of PAZ-Aib3-L-Ala*-Aib4-OMe in 2,2,2-trifluoroethanol solution. Peptide concentration: 1 mM.
Fig. 12. Fraction of the 310-helix H-bonds of the PAZ-Aib8 (black) and PAZ-Aib3-Ala-Aib4 (red) obtained from 40-ns trajectories at equilibrium, as a function of hydrogen bond site.
Fig. 13. Time evolution of the mean kinetic energy of the several residues of the Aib8 peptide chain. The black and red lines are results obtained from: initial conformations with high average (4.7) and low (3.5) average number of 310-helix hydrogen bonds, respectively.
Fig. 14. Time evolution of the mean kinetic energy of the residues of the Aib8 peptide chain. Shown are the results of: kinetic energy of the backbone CONH atoms (black), and kinetic energy of the backbone with side-chain CONHCaCH3CH3 atoms (red).
SI Text
Chemical Characterization
TLC
: Merck 60 F254 plates. Rf1 = CHCl3 /EtOH 9:1. Rf2 = 1-butanol/AcOH/H2O 3:1:1. Rf3 = toluene/EtOH 7:1.
PAZ-L-Ala*-OH.
This amino acid derivative was prepared from PAZ-Cl and L-Ala*. mp = 130-131°C.
PAZ-Aib-OH.
This amino acid derivative was prepared from PAZ-Cl and Aib. mp = 127-128°C.
PAZ-L-Ala*-(Aib)7-OMe.
PAZ-L-Ala*-OH (70 mg, 0.2 mmol) was dissolved in dry CH2Cl2 (5 ml) at 0°C. Then, HOAt (27 mg, 0.2 mmol) and EDC (38 mg, 0.2 mmol) were added to the solution. This solution was slowly added to a solution of H-(Aib)7-OMe [obtained by catalytic hydrogenation of the corresponding Z-derivative (100 mg, 0.14 mmol) in MeOH] and DIEA (0.2 mmol, 35 ml) in CH2Cl2. The reaction was stirred at room temperature for 3 days. Then, the solvent was removed in vacuo and the residue dissolved in EtOAc. The organic solution was washed with 10% KHSO4, H2O, 5% NaHCO3 and H2O, dried over Na2SO4, and evaporated to dryness. The product was isolated by flash chromatography (eluant: CHCl3/EtOH 96:4). Yield: 84%. mp = 237-238°C (EtOAc/EP). Rf1 = 0.85; Rf2 = 0.95; Rf3 = 0.25. [a]D20 = - 9.0 (c = 0.5, MeOH). IR (KBr) : 3311, 1729, 1661, 1621, 1527 cm-1. 1H NMR (250 MHz, CDCl3): d 7.64, 7.62, 7.55, 7.52, 7.41 and 7.17 (6s, 6H, 6 NH), 6.73 and 5.87 (2d, 2H, 2 NH), 4.01 (m, 1H, a-CH), 3.68 (s, 3H, O-CH3), 1.60-1.30 (m, 45H, 15 b-CH3). Mass spectrometry: [M]+ calcd. 948.1770; [M+H]+ exptl. 949.4691.
PAZ-Aib-L-Ala*-(Aib)6-OMe.
PAZ-Aib-OH (78 mg, 0.2 mmol) was dissolved in dry CH2Cl2 (5 ml) at 0°C. Then, HOAt (27 mg, 0.2 mmol) and EDC (38 mg, 0.2 mmol) were added to the solution. This solution was slowly added to a solution of H-L-Ala*-(Aib)6-OMe [obtained by catalytic hydrogenation of the corresponding Z-derivative (90 mg, 0.14 mmol) in MeOH] and 1 equivalent of DIEA (0.2 mmol, 35 ml) in CH2Cl2. The reaction was stirred at room temperature for 3 days. Then, the solvent was removed in vacuo and the residue dissolved in EtOAc. The organic solution was washed with 10% KHSO4, H2O, 5% NaHCO3 and H2O, dried over Na2SO4, and evaporated to dryness. The product was isolated by flash chromatography (eluant: CHCl3/EtOH 96:4). Yield: 81%.
mp = 246-247°C (EtOAc /EP). Rf1 = 0.85; Rf2 = 0.95; Rf3 = 0.25. [a]D20 = - 7.8 (c = 0.5, MeOH). IR (KBr) : 3308, 1736, 1706, 1662, 1617, 1528 cm-1. 1H NMR (250 MHz, CDCl3): d 7.67, 7.55 and 7.52 (3s, 3H, 3 NH), 7.50 (d, 1H, 1 NH), 7.40 and 7.35 (2s, 2H, 2 NH), 6.85 (1d, 1H, 1 NH), 5.94 (s, 1H, 1 NH), 3.97 (m, 1H, a-CH), 3.67 (s, 3H, O-CH3), 1.60-1.30 (m, 45H, 15 b-CH3). Mass spectrometry: [M]+ calcd. 948.1770; [M+H]+ exptl. 949.4547.
PAZ-Aib-L-Ala-(Aib)6-OMe (unlabelled).
PAZ-Aib-OH (78 mg, 0.2 mmol) was dissolved in dry CH2Cl2 (5 ml) at 0°C. Then, HOAt (27 mg, 0.2 mmol) and EDC (38 mg, 0.2 mmol) were added to the solution. This solution was slowly added to a solution of H-L-Ala-(Aib)6-OMe [obtained by catalytic hydrogenation of the corresponding Z-derivative (90 mg, 0.14 mmol) in MeOH] and 1 equivalent of DIEA (0.2 mmol, 35 ml) in CH2Cl2. The reaction was stirred at room temperature for 3 days. Then, the solvent was removed in vacuo and the residue dissolved in EtOAc. The organic solution was washed with 10% KHSO4, H2O, 5% NaHCO3 and H2O, dried over Na2SO4, and evaporated to dryness. The product was isolated by flash chromatography (eluant: CHCl3/EtOH 96:4). Yield: 83%. mp = 244-245°C (EtOAc /EP). Rf1 = 0.85; Rf2 = 0.95; Rf3 = 0.25. [a]D20 = - 7.7 (c = 0.5, MeOH). IR (KBr) : 3314, 1729, 1660, 1531 cm-1. 1H NMR (250 MHz, DMSO): d 7.88, 7.65, 7.49, 7.43, 7.41 and 7.39, (6s, 6H, 6 NH), 7.37 (1s, 5H, 5 Ar) 7.35 and 7.33 (2s, 2H, 2 NH), 5.14 (m, 2 H, Z-CH2), 2.28 (m, 1H, 1 b-CH), 2.20-1.85 (7H, 7 b-CH), 1.40-1.20 (24H, 8 b-CH3), 1.05-0.75 (48 H, 16 g-CH3). Mass spectrometry: [M]+ calcd. 936.0925; [M+H]+ exptl. 937.5192.
PAZ-(Aib)3-L-Ala*-(Aib)4-OMe.
PAZ-Aib-OH (78 mg, 0.2 mmol) was dissolved in dry CH2Cl2 (5 ml) at 0°C. Then, HOAt (27 mg, 0.2 mmol) and EDC (38 mg, 0.2 mmol) were added to the solution. This solution was slowly added to a solution of H-(Aib)2-L-Ala*-(Aib)4-OMe [obtained by catalytic hydrogenation of the corresponding Z-derivative (90 mg, 0.14 mmol) in MeOH] and 1 equivalent of DIEA (0.2 mmol, 35 ml) in CH2Cl2. The reaction was stirred at room temperature for 3 days. Then, the solvent was removed in vacuo and the residue dissolved in EtOAc. The organic solution was washed with 10% KHSO4, H2O, 5% NaHCO3 and H2O, dried over Na2SO4, and evaporated to dryness. The product was isolated by flash chromatography (eluant: CHCl3/EtOH 96:4). Yield: 69%. mp = 243-244°C (EtOAc /EP). Rf1 = 0.85; Rf2 = 0.95; Rf3 = 0.25. [a]D20 = + 25.2 (c = 0.5, MeOH). IR (KBr) : 3314, 1728, 1661, 1622, 1528 cm-1. 1H NMR (250 MHz, CDCl3): d 7.75 and 7.55 (2d, 2H, 2 NH), 7.53, 7.48, 7.31, 7.27, 6.72 and 6.11 (6s, 6H, 6 NH), 4.03 (m, 1H, a-CH), 3.67 (s, 3H, O-CH3), 1.60-1.30 (m, 45H, 15 b-CH3). Mass spectrometry: [M]+ calcd. 948.1770; [M+H]+ exptl. 949.4636.
X-Ray diffraction.
Single crystals of PAZ-Aib-L-Ala-(Aib)6-OMe were grown by slow cooling of an acetonitrile solution.
Crystallographic data.
formula C46H68N10O11, Mr = 937.1, crystal dimensions 0.45 ´0.20 ´ 0.15 mm3, monoclinic, space group P21, unit cell dimensions a = 13.215(3) Å, b = 18.345(3) Å, c = 21.476(4) Å, b= 90.41(5)°, V = 5206.3(17) Å3, Z = 4, rcalcd = 1.196 Mg/m3, m = 0.711 mm-1, l = 1.54178 Å (CuKa), T = 293(2) K, 2qmax = 120°. In total 8797 reflections were collected, of which 8557 were independent (Rint = 0.051) and used for refinement. Data/restraints/parameters: 8557/1/1159. R1 = 0.0535 [on F ³4s(F)], wR2 = 0.1642 (on F2, all data); min/max residual electron density -0.40/0.27 e Å-3. Diffraction data were collected on a Philips PW1100 four-circle diffractometer. The structure was solved by direct methods of the SIR 2002 program [1], and refined by least-squares procedures on F2, with all non-H atoms anisotropic, by application of the SHELXL-97 program [2]. The asymmetric unit is composed of two independent peptide molecules (A and B). The phenyl rings of the PAZ moieties were constrained to the idealized geometry. Interestingly, in each molecule the two phenyl rings of the PAZ moiety are not co-planar. Conversely, a tilt of 25.3-24.8° is observed. In both independent molecules the PAZ moiety forms an angle of 55° with respect to the helix axis. H-atoms were calculated at idealized positions and refined using a riding model. The atomic coordinates have been deposited in the Cambridge Structural Database, Cambridge Crystallographic Data Centre, Cambridge CB2 1EZ, United Kingdom (CSD reference no. 637253) and can be obtained at www.ccdc.cam.ac.uk/data_request/cif.
1. Burla MC, Camalli M, Carrozzini B, Cascarano GL, Giacovazzo C, Polidori G, Spagna R (2003) J Appl Crystallogr 36:1103.
2. Sheldrick GM, Schneider TR (1997) Methods Enzymol 277:319-341.