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• An intriguing example of how chirally enriched amino acids in the prebiotic world can generate sugars with Dconfiguration & with enantioenrichment: Cordova et al. Chem. Commun., 2005, 2047-2049 The Model: O L-proline OH O H H OBn BnO 2-4 days O OH + OBn OBn BnO OBn OBn 95-99% ee >99% ee hexose sugar L-proline: a 2° amine; popular as an organocatalyst because it forms enamines readily O N H L-proline OH Mechanism: enamine formation O + H OBn O + N O OH H N H OH OBn dilute O OH + N O OH OBn OBn O H OBn N OH OBn CO2H participates as acid OH OBn O OBn 1st aldol product (4C) OH O O OH N O OH + OBn OH OBn 2nd proline-mediated aldol reaction OBn BnO O OBn OBn OH OH BnO BnO OBn OBn benzyl protected allose BnO OBn O OBn OH Enantioenrichment % ee of sugar vs % ee of AA • Initially used 80% ee proline to catalyze reaction → >99% ee of allose • Gradually decreased enatiopurity of proline – Found that optical purity of sugar did not decrease until about 30% ee of proline! – Non-linear relationship! • 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? Ancient Amino Acids (i.e., meteorites) • Small peptides? Ancient Peptides Enzymes Catalysis by Small Peptides • Small peptides can also catalyze aldol reactions with enantioenrichment (See Cordova et al. Chem. Commun. 2005, 4946) H O O O OH Catalytic Peptide + i.e., NO2 L-ala-L-ala L-val-L-val L-val-L-ala NO2 81-96 % ee • 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! From Amino Acids Peptides • Peptides are short oligomers of AAs (polypeptide ~ 2050 AAs); proteins are longer (50-3000 AAs) O + H3N Ala H2O O CH3 + H3N O Cys CH3 O O N H + + H3N CH2SH O petide bond O CH2SH H2O • Reverse reaction is amide hydrolysis, catalyzed by proteases • 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): O .. C O N N C H sp2 C C H • Primary structure: AA sequence with peptide bonds • Secondary structure: local folding (i.e. -sheet & -helix) -sheet helix Amide bond: Formation & Degradation O + R O H OH N R' R H N H R' + H2O • Thermodynamics Overall rxn is ~ thermoneutral (Δ G ~ 0) Removal of H2O can drive reaction to amide formation In aqueous solution, reaction favors acid • Kinetics O Very slow reaction Forward: + R H + H N R' O Resonance stablilized anion -stable & not prone to nucleophilic attack X H Protonated--not a nucleophile O Reverse: R N H R' + resonance stabilized: most stable C=O derivative H2O X weak nucleophile T.I = tetrahedral intermediate O Reaction Coordinate Diagram: Large EA for forward reaction TS2 TS1 ΔG R OH NH2+ Charge separation EA T.I EA No resonance Large EA for reverse reaction HIGH ENERGY! How do we overcome the barrier? 1) Heat NH4+ + H2N -N C O O H2N First “biomimetic” synthesis Disproved Vital force theory But, cells operate at a fixed temperature! 2) Activate the acid: Activated acid acid + H2O • Activation of carboxylic acid e.g. O PCl5 acid chloride R Cl O R OH P2O5 -H2O O O anhydride R O R (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? • Recall carboxylic acid derivative reactivity: O R O > Cl R O O O > R R SR' O > R O O P O O > R O > OR' R O > OH NHR' R O increasing stability increasing reactivity • Depends on leaving group: O – Inductive effects (EWG) – Resonance in derivative – Leaving group ability Cl > O > S > N .. .. .. Cl .. N > O > S > Cl O + NHR Cl- > -OCOR > -SR > -OR > -NHR • Nature uses acyl phosphates, esters (ribosome) & thioesters (NRPS)—more on this later 3) Catalysis • • • Lowering of TS energy Usually a Lewis acid catalyst such as B(OR)3 Another problem with AA’s O H2N OH HN O HO O NH NH2 O • • • This doesn’t occur in nature Easy to form 6 membered ring rather than peptide Acid activation can give the same product • 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 HO i.e., O O H2N OH H N H2N OH O HO SERINE hydroxyl group is a good nucleophile & needs to be protected BEFORE we make peptide bond OH • Nature uses protection & activation as part of its strategy to make proteins on the ribosome: Nature uses an Ester to activate acid (protein synthesis): Adenylation O O H N H O Adenosine O O O P O P O P O O O O R Activation Formyl-AA (methionine) O H N H O O O P Adenosine O R (raises energy of CO2H) tRNA OH Primary amine is protected from further reaction 3'-OH terminus of specific tRNA sequence O H N H O O tRNA R ester: more reactive than an acid H N H O O O H2N O R O tRNA tRNA H R O H N O R R O N H O AA3 AA1 AA2 AA3 AA4...O H2O polypeptide Each AA is attached to its specific tRNA tRNA NH2 tRNA • A specific example: tyrosyl-tRNA synthase (from tyr) O + O NH3 Adenosine Good L.G. (PPi) O P O P O P O O O O O O + O O NH3 O anhydride-like P Adenosine O OH OH 3 potential nucleophiles! 3 potential reactive P's R one of 20 AA's tRNAtyr O B only! L-enantiomer only! OH R O + B 3'-OH only! O NH3 O OH OH tRNA Tyr OH • 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 Adenylation O O H2N H2N O P Adenosine O R O R O HS O NH2 NRPS S R Activated thioester NRPS good Lv group O potential Nu: NH2 NRPS S R O NRPS S NH2 Activated thioester O H N NRPS S R NH2 O hydrolysis nonribosomal peptide • 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 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 e.g. isopenicillin N: NH3+ -O2C • 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 O SH N H H N O CO2- L--aminoadipyl-L-cysteinyl-D-valine (ACV) isopenicillin N synthase NH3+ O H N -O2C S N O Isopenicillin N Plan for Synthesis: NH3+ O -O2C SH N H O H N CO2- -aminoadipic acid * NH + 3 -O2C * O * N Needs to be activated cysteine H O * Need protecting groups *SH H N CO2- * valine Protection of Carboxylic acid: OH Ph H2N CO2H H2N H+ O O Ph heat = OBn (benzyl) Val Selective Protection of R group (thiol): H2N CO2H BnCl H2N CO2H NaOH SH Cys S • 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 O O O O O O H2N CO2H SBn O H N CO2H SBn 2X protection = BOC H N CO2H SBn Now that we have our protected AA’s, we need to activate the carboxylate towards coupling O O H N H2N CO2H O SBn Ph O Activation & Coupling (see exp 6): + H N C N R BOCHN CO2- DCC Cy SBn O N C N Cy H good Lv group DCC = dicyclohexylcarbodiimide = Coupling reagent that serves to activate carboxylate towards nucleophilic attack