2. Chem 27 - Exam 1 Review Wednesday Feb. 22, 2006 Science Center Hall D K.C. O’Brien Carol Fang Walter Kowtoniuk

3. Outline of Topics 1) Conformational Analysis of amino acids 2) Protein Folding 3) Edman Degradation(-like) chemistry 4) Cyanogen bromide(-like) chemistry 5) Peptide Coupling/Synthesis 6) Biosynthesis of Proteins

4. Conformational Analysis K.C. O’Brien

5. Amino Acid Structure Amino acids are chiral molecules Stereochemistry at  -carbon always as shown (R group coming out) All natural amino acids have S configuration, except cysteine pKa’s: NH 3 + is about 9 COO- is about 2.2 Hydrophobic, polar and charged side chains

6. Staggered vs. Eclipsed Conformation Hyperconjugation  C-H   C-H Newman projections help visualize interactions

7. Gauche interactions

8. Cyclohexane Chair Conformations Ring flip changes groups from axial to equatorial Lower energy conformation has large groups equatorial A values are used to quantify the energy difference between the axial and equatorial positions

9. Syn-pentane Interaction Syn-pentane > 3.7 kcal /mol 1,3-diaxial groups generate a syn pentane interaction

10. A 1,3 Strain H is in the same plane as double bond If R=R’=R”=Me, A 1,3 =3.5 kcal/mol Minimize A 1,3 in amide bonds

11. Template Projection of Amino Acids Amino acid template projection is based on cyclohexane chair structure Add up gauche and syn-pentane interactions to find the lowest energy conformation R 1 >R 2 >R 3 is a good place to start, but consider other conformations Make sure you don’t invert the stereochemistry of the amino acid or its side chain!!!!

12. Protein Folding: Hydrogen Bonds 1-4 kcal/mol Directionality is important N-H-----O=C Stabilize  -helices,  -sheets and turns

13. Protein Folding:  -helix stabilized by hydrogen bonding 3.6 amino acids per turn

14. Protein Folding:  -sheet NH’s of one strand H-bond to C=O of next strand R groups alternate on opposite sides of the plane

15. Protein Folding:  -turn C=O and N-H are 10 atoms apart Changes the direction of the main chain

16. Protein Folding: Electrostatic Interactions Between oppositely charged amino acids Most important in the interior of the protein Neutralizes charges

17. Protein Folding: Hydrophobic Interactions: Hydrophobic amino acids pack into the interior of the protein Folding increases the disorder of the solvent Positive  H is overcome by positive  S Disulfide Bonds: Dihedral angle 90 o n s donates into  * S-R Two Cys oxidized to form a disulfide bond

18. Edman Degradation Carol Fang

19. Nucleophilic Amine (primary and secondary) E and Nu are 5 atoms apartRotatable bond Thiazolinone Derivative Kinetic product New N-terminal

20. Enol Formation Potential racemization PTH, to be detected by HPLC Thermodynamic Product Pre-note

21. Frame of Reaction When racemization is taken care of

22. Brain teasers: a)a peptide is not reactive to Edman Degradation b)After a round of Edman degradation, only one fragment is obtained c) After a round of Edman degradation, two PTH products are obtained d)Bicyclic PTH product from Edman Degradation c) Special case: Lysine No nucleophilic amine Cyclic peptide D05 Breaking the peptide bond does not break the molecule Presence of Nu amine; Cyclic D10, D12 2 Nu amines at both ends / 1 PTH end and 1 Nu amine end D10, D12 A ring before Edman degradation D02, D04 A more protonated amine D09

23. Cyanogen Bromide Cleavage

24. Nucleophilic S Nu and E 5 atoms apartRotatable bond Met (C)N cleaved

25. Brain teasers: 1)A peptide gives only one fragment after CNBr cleavage a)A cyclic peptide b)C-terminal Methionine 2) It is known that a peptide has n Met. It gives n pieces of fragments 3) How about (n+1) fragments?

26. How this reacts with CNBr? (2004 Exam 1)

27. Why S / C=O combo can be so different in these two reactions? Edman Degradation CNBr Cleavage C=S bond, S is Nucleophilic 3 C-S bond, S has an extra Covalent bond; adjacent C is ready for S N 2

28. Peptide Syntheses Walter Kowtoniuk

29. Amide Bond Synthesis - Synthesis of an amide bond using the corresponding carboxylic acid and amine. - Use DCC to both activate the acid and serve as a dehydrating agent

30. Amide Bond Synthesis

32. Protecting Groups Why do we need them?

33. Protecting Groups Lecture Notes pg33

34. Protecting Groups t-Boc Synthesis t-Boc Deprotection

35. Protecting Groups Cbz follows the same mechanism as shown for t-Boc

36. Protecting Groups Ts synthesis Ts Deprotection

37. Protecting Groups DNP synthesis DNP deprotection

38. C to N Directionality why not N to C?

39. Solid Phase Peptide Synthesis

41. Peptide Fragment Coupling Thioester True coupling

42. Determining Yield Synthesizing a 100mer requires 99rxns, thus n=99 If we factor in the initial coupling to the solid phase, the 100mer requires 100rxns, thus n=100 For convergent synthesis we are concerned with the longest linear sequence of steps. In this case the yield of each individual reaction is multiplied

43. Translation

44. Biological Carbonyl Activation

47. Ribosome

48. Role of A2486