An intriguing example of how chirally enriched amino acids in the prebiotic world can generate sugars with D-configurati

1. Cordova et al. Chem. Commun., 2005, 2047-2049 • An intriguing example of how chirally enriched amino acids in the prebiotic world can generate sugars with D-configuration & with enantioenrichment: The Model: L-proline: a 2° amine; popular as an organocatalyst because it forms enamines readily

2. Mechanism: enamine formation CO2H participates as acid

4. Enantioenrichment % ee of sugar vs % ee of AA • Initially used 80% ee proline to catalyze reaction → >99% ee of allose • Gradually decreased enatio-purity of proline • Found that optical purity of sugar did not decrease until about 30% ee of proline! • Non-linear relationship!

5.  chiral amplification • % ee out >> % ee in! • Suggests that initial chiral pool was composed of amino acids • Chirality was then transferred with amplification to sugars → “kinetic resolution” • Could this mechanism have led to different sugars diastereomers? • Sugars →→ RNA world →→ selects for L-amino acids? • Small peptides?

6. Catalysis by Small Peptides • Small peptides can also catalyze aldol reactions with enantioenrichment (See Cordova et al. Chem. Commun. 2005, 4946) • Found to catalyze formation of sugars • It is clear that amino acids & small peptides are capable of catalysis i.e., do not need a sophisticated protein!

7. From Amino Acids  Peptides • Peptides are short oligomers of AAs (polypeptide ~ 20-50 AAs); proteins are longer (50-3000 AAs) • Reverse reaction is amide hydrolysis, catalyzed by proteases

8. At first sight, this is a simple carbonyl substitution reaction, however, both starting materials & products are stable: • RCO2- -ve charge is stabilized by resonance • Amides are also delocalized &  carbon & nitrogen are sp2 (unlike an sp3 N in an amine):

9. Primary structure: AA sequence with peptide bonds • Secondary structure: local folding (i.e. -sheet & -helix) -sheet  helix

10. Amide bond: Formation & Degradation • Thermodynamics Overall rxn is ~ thermoneutral (Δ G ~ 0) Removal of H2O can drive reaction to amide formation In aqueous solution, reaction favors acid • Kinetics Very slow reaction Forward:

11. Reverse: T.I = tetrahedral intermediate Reaction Coordinate Diagram: TS2 TS1 ΔG Charge separation No resonance  HIGH ENERGY! T.I Large EA for forward reaction EA EA Large EA for reverse reaction

12. How do we overcome the barrier? • Heat First “biomimetic” synthesis Disproved Vital force theory But, cells operate at a fixed temperature! • Activate the acid: Activated acid acid

13. Activation of carboxylic acid e.g. (Inorganic compound raises energy of acid) Activation of carboxylic acid (towards nucleophilic attack) is one of the most common methods to form an amide (peptide) bond---in nature & in chemical synthesis! • Why is the energy (of acid) raised?

14. Recall carboxylic acid derivative reactivity: • Depends on leaving group: • Inductive effects (EWG) • Resonance in derivative • Leaving group ability • Nature uses acyl phosphates, esters (ribosome) & thioesters (NRPS)—more on this later

15. Catalysis • Lowering of TS energy • Usually a Lewis acid catalyst such as B(OR)3 • Another problem with AA’s • This doesn’t occur in nature • Easy to form 6 membered ring rather than peptide • Acid activation can give the same product

16. With 20 amino acids  chaos! • How do we control reaction to couple 2 AAs together selectively & in the right sequence? & at room temp (in vivo)? • Biological systems & synthetic techniques employ protection & activation strategies! • For peptide bond formation • Many different R groups on amino acids  potential for many side reactions i.e.,

17. Nature uses protection & activation as part of its strategy to make proteins on the ribosome:

18. Nature uses an Ester to activate acid (protein synthesis): Adenylation

19. Each AA is attached to its specific tRNA

20. A specific example: tyrosyl-tRNA synthase (from tyr)

21. Control! • Only way to ensure specificity is to orient desired nucleophile (i.e., CO2-) adjacent to desire electrophile (i.e., P) What about Nonribosomal Peptide Synthase (NRPS)? • Uses thioesters

22. Once again, we see selectivity in peptide bond formation • As in the ribosome, the NRPS can orient the reacting centres in close proximity to eachother, while physically blocking other sites

23. Chemical Synthesis of Peptides • Synthesis of peptides is of great importance to chemistry & biology • Why synthesize peptides? • Study biological functions (act as hormones, neurotransmitters, antibiotics, anticancer agents, etc) • Study potency, selectivity, stability, etc. • Structural prediction • Three-dimensional structure of peptides (use of NMR, etc.) • How? • Solution synthesis • Solid Phase synthesis • Both use same activation & protection strategy

24. e.g. isopenicillin N: • To study enzyme IPNS, we need to synthesize tripeptide (ACV) • Small molecule → use solution technique • Synthesis (in soln) can be long & low yielding • But, can still produce enough for study

25. Plan for Synthesis:

26. Protection of Carboxylic acid: Selective Protection of R group (thiol):

27. Both the amino group & carboxylate of cysteine need to couple to another AA • But, we can’t react all 3 peptides at once (must be stepwise) •  we protect the amino group temporarily, then deprotect later Protection of the Amine: (BOC)2O = an anhydride

28. Now that we have our protected AA’s, we need to activate the carboxylate towards coupling Activation & Coupling (see exp 6): DCC = dicyclohexylcarbodiimide = Coupling reagent that serves to activate carboxylate towards nucleophilic attack