Macrocyclization of Peptide Side Chains by Ugi Reaction: Achieving Peptide

Transcription

1 Macrocyclization of Peptide Side Chains by Ugi Reaction: Achieving Peptide Folding and Exocyclic N-Functionalization in One Shot Aldrin V. Vasco,, Carlos S. Pérez, Fidel E. Morales, Hilda E. Garay, ǁ Dimitar Vasilev, José A. Gavín, ǂ Ludger A. Wessjohann,, * and Daniel G. Rivera,, * Center for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y G, 10400, La Habana, Cuba. Facultad de Ingeniería Química. Instituto Superior Politécnico José Antonio Echeverría, CUJAE, Calle 114 # 11901, 11500, La Habana, Cuba. ǁ Synthetic Peptides Group, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, La Habana, Cuba. ǂ Instituto Universitario de Bioorgánica Antonio González and Departamento de Química Orgánica, Universidad de La Laguna, La Laguna, Tenerife, Spain. Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D , Halle/Saale, Germany. Corresponding Authors: * s: dgr@fq.uh.cu; wessjohann@ipb-halle.de S1

2 Table of contents NMR Structure determination Molecular Dynamics Simulations Figure 1. HPLC chromatogram of crude peptide 3. Figure 2. ESI-MS of peptide 3. Figure 3. HPLC Chromatogram of crude peptide 5. Figure 4. ESI-MS of peptide 5 Figure 5. HPLC Chromatogram of pure cyclic peptide 2. Figure 6. ESI-MS of cyclic peptide 2. Figure 7. HPLC Chromatogram of pure cyclic peptide 4. Figure 8. Mass spectrum of cyclic peptide 4. Figure 9. HPLC Chromatogram of pure cyclic peptide 6. S3 S4 S5 S5 S6 S6 S7 S7 S8 S8 S9 Figure 10. Mass spectrum of cyclic peptide 6. S9 Figure MHz 1 H-NMR spectrum in DMSO-d6 of cyclic peptide 2. S10 Figure MHz 13 C-NMR spectrum in DMSO-d6 of cyclic peptide 2. S11 Figure 13. TOCSY spectrum in DMSO-d6 of cyclic peptide 2. S12 Figure 14. HSQC spectrum of in DMSO-d6 cyclic peptide 2. S13 Figure 15. HMBC spectrum in DMSO-d6 of cyclic peptide 2. S14 Figure 16. ROESY spectrum in DMSO-d6 of cyclic peptide 2. S15 Figure MHz 1 H-NMR spectrum in DMSO-d6 of cyclic peptide 4. S17 Figure MHz 13 C-NMR spectrum in DMSO-d6 of cyclic peptide 4. S18 Figure 19. TOCSY spectrum in DMSO-d6 of cyclic peptide 4. S19 Figure 20. HSQC spectrum in DMSO-d6 of cyclic peptide 4. S20 S2

3 Figure 21. HMBC spectrum in DMSO-d6 of cyclic peptide 4. S21 Figure 22. ROESY spectrum in DMSO-d6 of cyclic peptide 4. S22 Figure MHz 1 H-NMR spectrum in DMSO-d6 of cyclic peptide 6. S23 Figure MHz 13 C-NMR spectrum in DMSO-d6 of cyclic peptide 6. S24 Figure 25. TOCSY spectrum of in DMSO-d6 cyclic peptide 6. S25 Figure 26. HSQC spectrum in DMSO-d6 of cyclic peptide 6. S26 Figure 27. HMBC spectrum in DMSO-d6 of cyclic peptide 6. S27 Figure 28. ROESY spectrum in DMSO-d6 of cyclic peptide 6. S28 Figure MHz 1 H-NMR spectrum in acetone-d6 of cyclic peptide 8. S29 Figure MHz 13 C-NMR spectrum acetone-d6 of cyclic peptide 8. S30 Figure MHz 1 H-NMR spectrum in CDCl 3 of cyclic peptide 10. S31 Figure MHz 13 C NMR spectrum in CDCl 3 of cyclic peptide 10. S32 References S33 NMR Structure determination NMR structure determination was performed in Xplor-NIH 1,2 through simulated annealing regularization and refinement in torsion angle space, using experimental data as inter-proton distances and dihedral angles restraints. Simulations were based on examples scripts distributed in the /eginput folder within Xplor-NIH package. For cyclic peptide 2, φ angles from the 3 J NHCHα of residues Leu1, Lys2, Phe3, Val4 and Glu5 as well as χ 1 angle from the Phe3 residue were used as dihedral constraints. For cyclic peptide 4, φ angles from the 3 J NHCHα of residues Val1, Ile2, Lys6 and Phe7 as well as χ 1 angle from the Phe7 residue were used as dihedral constraints. For cyclic peptide 6, φ angles from the 3 J NHCHα of residues Phe1 and Lys2 were used as dihedral constraints. For simulated annealing regularization 200 starting structures were randomly generated. Geometric bond, angle and improper energy contributions were scaled to 1.0, 0.4, and 0.1 respectively. Non-bonded Van der Waals contributions were set to and experimental NOE S3

4 and dihedral constraints were scaled to 2 and 10 respectively. An initial 100 steps conjugated gradient Powell minimization was performed. A 100 ps molecular dynamics simulation at 3500 K was performed with a timestep of 3 fs. Before the cooling stage, the energy function due to experimental dihedral restraints was scaled to 200. The system was cooled from 3500 to 25 K, with a temperature step of 12.5 K. At each temperature step, 0.2 ps of molecular dynamics simulation was performed. During this stage the Van der Waals energy term was progressively scaled from to 4, and the experimental NOE potential was climbed to 30. Angle and improper terms were both progressively scaled to 1.0. A 500 steps torsion angle minimization was performed and finally the system was optimized by means of 500 steps conjugated gradient Powell cartesian minimization. The refinement protocol consisted in a slow cooling simulated annealing from the regularized structures. The initial weights of the energy functions was 0.4, 0.1 and 1.0 for the angle, improper, and bond terms; 10 and 2 respectively for the NOE and dihedral experimental restraints; and for the non-bonded Van der Waals term. A 10 ps molecular dynamics simulation at 3000 K was achieved with a time-step of 3 fs, afterwards dihedral restraints contribution was set to 200. The system was cooled with a temperature step of 12.5 K and a simulation time of 0.2 ps at each temperature. During this simulation the Van der Waals energy term was scaled from to 4. During this stage the Van der Waals energy term was progressively scaled from to 4, and the experimental NOE potential was climbed to 30. Angle and improper terms were both progressively scaled to 1.0. A 500 torsion angle minimization was performed afterwards a second 500 steps minimization was achieved in cartesian coordinates. A finally 1000 steps Powell minimization with an energy function nondependent of experimental restraints was executed. Final RMSD were calculated with the plugin RMSD calculator within VMD 1.9.2, 3 excluding N- and C-terminal residues in the calculation. Molecular Dynamics Simulations Solvent boxes were generated with the Solvate plugin within VMD Trajectories analysis of interatomic distances were performed in VEGA ZZ 4 while time distribution of ϕ and ψ angles (Ramachandran Plots) were generated within VMD. 3 The analysis of hydrogen bonds in time was performed within VMD using a cut-off criteria of 20 0 and a donor-acceptor distance criteria lesser than 3Å. S4

5 Figure 1. HPLC chromatogram of crude peptide 3. fidel-1 5 (0.108) AM (Cen,2, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (1:53) TOF MS ES+ 2.00e % m/z Figure 2. ESI-MS of peptide S5

6 Figure 3. HPLC Chromatogram of crude peptide 5. fidel-2 85 (1.643) AM (Cen,2, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (2:86) % Figure 4. ESI-MS of peptide 5 S6

7 Figure 5. HPLC Chromatogram of pure cyclic peptide 2. Figure 6. ESI-MS of cyclic peptide 2. S7

8 Figure 7. HPLC Chromatogram of pure cyclic peptide 4. favv361-pico2 17 (0.326) AM (Cen,2, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (2:23) A % A Figure 8. Mass spectrum of cyclic peptide 4. S8

9 Figure 9. HPLC Chromatogram of pure cyclic peptide 6. avv (2.100) AM (Cen,2, 80.00, Ht,5000.0,0.00,1.00); Sm (Mn, 2x3.00); Cm (2:119) % Figure 10. Mass spectrum of cyclic peptide 6. S9

10 Figure MHz 1 H-NMR spectrum in DMSO-d6 of cyclic peptide 2. S10

11 Figure MHz 13 C-NMR spectrum in DMSO-d6 of cyclic peptide 2. S11

12 Figure 13. TOCSY spectrum in DMSO-d6 of cyclic peptide 2. S12

13 Figure 14. HSQC spectrum of in DMSO-d6 cyclic peptide 2. S13

14 Figure 15. HMBC spectrum in DMSO-d6 of cyclic peptide 2. S14

15 Figure 16. ROESY spectrum in DMSO-d6 of cyclic peptide 2. S15

16 S16

17 Figure MHz 1 H-NMR spectrum in DMSO-d6 of cyclic peptide 4. S17

18 Figure MHz 13 C-NMR spectrum in DMSO-d6 of cyclic peptide 4. S18

19 Figure 19. TOCSY spectrum in DMSO-d6 of cyclic peptide 4. S19

20 Figure 20. HSQC spectrum in DMSO-d6 of cyclic peptide 4. S20

21 Figure 21. HMBC spectrum in DMSO-d6 of cyclic peptide 4. S21

22 Figure 22. ROESY spectrum in DMSO-d6 of cyclic peptide 4. S22

23 Figure MHz 1 H-NMR spectrum in DMSO-d6 of cyclic peptide 6. S23

24 Figure MHz 13 C-NMR spectrum in DMSO-d6 of cyclic peptide 6. S24

25 Figure 25. TOCSY spectrum of in DMSO-d6 cyclic peptide 6. S25

26 Figure 26. HSQC spectrum in DMSO-d6 of cyclic peptide 6. S26

27 Figure 27. HMBC spectrum in DMSO-d6 of cyclic peptide 6. S27

28 Figure 28. ROESY spectrum in DMSO-d6 of cyclic peptide 6. S28

29 Figure MHz 1 H-NMR spectrum in acetone-d6 of cyclic peptide 8. S29

30 Figure MHz 13 C-NMR spectrum acetone-d6 of cyclic peptide 8. S30

31 Figure MHz 1 H-NMR spectrum in CDCl 3 of cyclic peptide 10. S31

32 Figure MHz 13 C NMR spectrum in CDCl 3 of cyclic peptide 10. S32

33 References (1) Schwieters, C. D.; Kuszewski, J. J.; Clore, G. M. Prog. Nucl. Magn. Reson. Spectrosc. 2006, 48, 47 (2) Schwieters, C. D.; Kuszewski, J. J.; Tjandra, N.; G. M. Clore J. Magn. Reson. 2003, 160, 65. (3) Humphrey, W.; Dalke, A.; Schulten, K. J. Molec. Graphics 1996, 14, 33. (4) Pedretti, A.; Villa, L.; Vistoli, G. J. Mol. Graph. 2002, 21, 47. S33