Discussion Section (Day, Time): TF:

Transcription

1 ame: Chemistry 27 Professor Gavin MacBeath arvard University Spring 2004 Final Exam Thursday, May 28, :15 PM - 5:15 PM Discussion Section (Day, Time): Directions: TF: 1. Do not write in red ink. othing in red ink will be graded. 2. Write your name on every page of the exam. 3. Provide your answers in the designated space. 4. The last two pages of the exam provide the structures of common amino acids, cofactors, and DA bases for you to use, as well as relevant pka data. othing written on these pages will be graded. 5. You should have 15 pages total. We have provided plenty of space for each answer. 6. The formation of all reaction intermediates must be drawn as distinct steps in mechanisms unless otherwise specified. You need not draw out proton transfers as distinct steps; you may abbreviate them by writing " " or "- ". 7. Molecular model kits are permitted. Question Score 1 /20 2 /15 3 /15 4 /20 5 /35 Subtotal 1 /105 6 /20 7 /35 8 /25 9 /15 Subtotal 2 /95 Total /200 Page 1 of 15

2 ame: 1. (20 pts) Please indicate in the box provided whether each statement is true (T) or false (F). A) (3 pts) The compound shown below can undergo an E2-elimination in the presence of base. Br B) (5 pts) Reaction 1 proceeds more rapidly than Reaction 2 Reaction 1 C 3 1) a, 2 2) 3 Reaction 2 C 3 1) a, 2 2) 3 C) (3 pts) The following amino acid analogue can get loaded onto tra by the aminoacyl-tra synthetase for valine: 2 D) (3 pts) Laboratory synthesis of DA is performed in the 3 to 5 direction to prevent racemization. E) (3 pts) Salt bridges between Glu and Arg are typically stronger in the interior of a protein than on its surface. F) (3 pts) An amide bond is thermodynamically more stable than an thioester bond. G) (0 pts) I would recommend Chem27 to my best friend. Page 2 of 15

3 ame: 2. (15 pts) Cephalosporin derivative, L-658,758, irreversibly inhibits serine proteases by forming a covalent enzyme-inhibitor adduct. Please propose an arrow pushing mechanism for this reaction. You must show all relevant amino acid sidechains in the enzyme that contribute to catalysis. RS S R Enz-is S R Ser-Enz L-658,758 Covalent enzyme-inhibitor adduct Page 3 of 15

4 ame: 3. (15 pts) Epoxomicin is an inhibitor of -terminal nucleophile hydrolases. Please propose an arrow pushing mechanism for the conversion shown below Thr R 1 2 R 1 epoxomicin morpholine-derivative Page 4 of 15

5 ame: 4. (20 pts) Molecule 1 is a substrate for a newly discovered enzyme. The mechanism of this enzyme-catalyzed reaction involves elements from both PLP and chorismate mutase. Br Molecule 1 Please propose an arrow pushing mechanism for the conversion of molecule 1 to the final product. Begin your mechanism with the substrate already loaded on PLP. You may use the abbreviated structure for PLP shown below. You do T need to show the mechanism for transimination or imine hydrolysis/aminolysis, but simply indicate above the arrow when these events occur. PLP Page 5 of 15

6 ame: 5. (35 pts) A current challenge in proteomics research is to identify every residue in every protein in a cell that is phosphorylated. To do this, one must first digest cellular proteins into shorter peptides and then separate the phosphorylated peptides from those that do not contain phosphates. arvard professor Steve Gygi has devised the following strategy: 1) digest the proteins into peptides with trypsin (recall that trypsin cleaves after arginine and lysine residues). 2) lower the p of the solution containing the peptides to p 2.4 3) separate the phosphorylated peptides from non-phosphorylated peptides using ion exchange chromatography, which separates molecules based on their net charge. This method can cleanly separate peptides that differ in their net charge by as little as one charge unit. A) (6 pts) The region of a protein shown below contains three trypsin cleavage sites. Draw the two middle peptide fragments resulting from trypsin cleavage at these sites, as they would exist at p 2.4. Clearly indicate the net charge of each peptide. (You may wish to refer to the pka table provided on the last page of the exam). Based on this answer, state in one sentence how Gygi separates phosphorylated peptides from non-phosphorylated peptides. 2 P 2 2 Page 6 of 15

7 ame: B) (5 pts) ne limitation of Gygi's method is that some naturally-occurring phosphorylated peptides get missed: they end up with the non-phosphorylated peptides during the ion-exchange chromatography step. What is it about these phosphorylated peptides that gives them this property? C) (5 pts) Another potential limitation of Gygi's method is that some non-phosphorylated peptides end up with the phosphorylated peptides after the ion-exchange chromatography step. What is it about these non-phosphorylated peptides that gives them this property? D) (8 pts) Suppose you used ion-exchange chromatography to isolate the following peptide. You now want to sequence the peptide by mass spectrometry. Please draw and label the b2 and y2 ions resulting from fragmentation of this peptide. Assume the sidechains are protonated as shown below. 2 P 2 Page 7 of 15

8 ame: A completely different approach has been used to separate peptides with phosphorylated serines or threonines from non-phosphorylated peptides following digestion with trypsin. Peptides are first treated with a base (Ba() 2 ) to form Intermediate A. Intermediate A is then treated with a derivitized thiol (R-S) to form the final product. The final products can then be purified by exploiting the R-group attached to sulfur. P - Ba() 2 Intermediate A P 4 3- R-S SR - E) (4 pts) Please provide an arrow-pushing mechanism for the formation of Intermediate A. F) (3 pts) Please describe the stereoelectronic requirements for elimination of the phosphate. Use a diagram that includes relevant molecular orbitals in your answer. G) (4 pts) Provide an arrow-pushing mechanism for the conversion of Intermediate A to the final product. Page 8 of 15

9 ame: 6. (20 pts) Compound A is a newly developed anti-cancer agent that is activated by bioreduction and converted into a diradical species via a cumulene intermediate (C=C=C=C). Please provide an arrow-pushing mechanism for the conversion of A into the diradical product. You do not need to show the mechanism of reduction, but simply write reduction over the arrow when the event occurs. Sugar Sugar Compound A Diradical product Page 9 of 15

10 ame: 7. (35 pts) The polyketide Discodermolide shows potent immunosuppressive and microtubulestabilizing activity. Discodermolide A) (14 pts) Please fill in the gene table for the first 6 modules and the last module of the polyketide synthase responsible for the biosynthesis of Discodermolide. Module Starting material ACP AT KS KR D ER TE B) (12 pts) In the box provided, please state what domain in what module is responsible for the stereochemistry at the indicated carbon. 2 Discodermolide Page 10 of 15

11 ame: C) (9 pts) Inversion of stereochemistry at C17 results in a compound (C17 epimer) that no longer has biological activity. Please draw a ewman projection across the C16-C17 bond for the lowest energy conformations of Discodermolide and of its C17 epimer. Using these ewman projections, speculate why the C17 epimer has no biological activity. C16 C17 2 Discodermolide Page 11 of 15

12 ame: 8. (25 pts) Trichodiene synthase is a sesquiterpene cyclase that catalyzes the formation of trichodiene, an intermediate in the biosynthesis of antibiotics and mycotoxin. PP Trichodiene synthase T-2 Toxin Farnesyl pyrophosphate trichodiene A) (20 pts) Please propose an arrow-pushing mechanism for the reaction leading to trichodiene. Make sure you draw out all intermediates. Your mechanism does not need to account for stereochemistry in trichodiene. ote: Your mechanism should include one 1,4-hydride shift. There may be other 1,2-hydride or 1,2-methyl shifts. B) (5 pts) Divalent metal cations (Mg 2 ions) in the active site of the trichodiene synthase are crucial for catalysis. Please speculate on the role of the Mg 2 ions. Page 12 of 15

13 ame: 9. (15 pts) Molecule A is a powerful new drug that, when taken 3 times a week for 3 months, imparts absolute understanding of the rganic Chemistry of Life. Please propose a concerted mechanism of cyclization to form Molecule A. You must show the 3 dimensional conformation that the substrate adopts in the enzyme active site that accounts for the observed stereochemistry of Molecule A. cyclase Molecule A Page 13 of 15

14 ame: 3 2- P 3-2 C 3 3 C P P P alanine glutamate PLP C P 2 ()C - 3 arginine glycine ATP PAP 2 C 3 - asparagine histidine - 3 C 3 C 2-2 C 3 C 3 3 C 3 - aspartate isoleucine - S 3 3 P P - - C 3 cysteine leucine - 3 C 3 - FAD P P - - ADP C() glutamine lysine P C S 3 methionine proline - 3 phenylalanine 3 serine - 3 C 3-3 threonine tryptophan tyrosine C C valine Template for side chain conformations C 2 adenine guanine cytosine thymine uracil Page 14 of 15

15 ame: Residue Structure pka Threonine 16 Serine 16 Arginine Lysine Tyrosine Cysteine S 8.5 istidine 7.0 Glutamic acid 4.7 Aspartic acid 4.7 Peptide -terminus Structure Approximate pka R Peptide C-terminus Phosphate Group (diprotonated) Phosphate Group (monoprotonated) R 3.7 P P C 1.2 R R Page 15 of 15