• An intriguing example of how chirally enriched amino acids in the prebiotic world can generate sugars with D configuration & with enantioenrichment: Cordova et al.

Chem. Commun

.,

2005

, 2047-2049 The Model: O OBn H L-proline 2-4 days OH O OBn OBn H 95-99% ee + BnO O OH BnO OBn OBn >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 OBn H + O N H dilute OH H + N O OH OBn OH + N O OH OBn OBn OH O OBn OBn 1st aldol product (4C) O OBn H N O OH OBn CO 2 H participates as acid

OH O OBn OBn + OBn N O OH 2nd proline-mediated aldol reaction OH OH O OBn OBn OBn BnO BnO O OH OBn OBn benzyl protected allose OH O BnO BnO OH OBn

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!

•  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 + NO 2 Catalytic Peptide i.e., L-ala-L-ala L-val-L-val L-val-L-ala 81-96 % ee NO 2 • 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 ~ 20 50 AAs); proteins are longer (50-3000 AAs) O + H 3 N H 2 O O Ala CH 3 + Cys O + H 3 N O CH 2 SH H 2 O O + H 3 N N H CH 3 petide bond CH 2 SH O O • 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

:

– RCO 2 -ve charge is stabilized by resonance – Amides are also delocalized &  carbon & nitrogen are sp 2 (unlike an sp 3 N in an amine): O C ..

N C H sp 2 O C N 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 OH + H H N R' R O N H R' + H 2 O • Thermodynamics Overall rxn is ~ thermoneutral ( Δ G ~ 0) Removal of H 2 O can drive reaction to amide formation In aqueous solution, reaction favors acid • Kinetics Very slow reaction Forward: R O O Resonance stablilized anion -stable & not prone to nucleophilic attack + H + H N H R' X Protonated--not a nucleophile

Reverse: ΔG

Large E A for forward reaction

E A O R N H R' resonance stabilized: most stable C=O derivative Reaction Coordinate Diagram: TS 1 T.I

+ H 2 O weak nucleophile X TS 2 E A T.I = tetrahedral intermediate O

Large E A for reverse reaction

R OH NH 2 +

Charge separation No resonance



HIGH ENERGY!

How do we overcome the barrier?

1) Heat -N C O H 2 N NH 4 + + First “biomimetic” synthesis Disproved Vital force theory But, cells operate at a fixed temperature!

H 2 N O + H 2 O 2) Activate the acid:

Activated acid acid

• Activation of carboxylic acid e.g.

O PCl 5 R O Cl acid chloride R OH P 2 O 5 -H 2 O O O R O R (Inorganic compound raises energy of acid) anhydride 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: R O Cl > R O O O R > R O SR' > R increasing stability O O O P O O > R O OR' > R O OH > R O NHR' • Depends on leaving group: increasing reactivity O Cl – Inductive effects (EWG)

Cl

> O >

S

> N – Resonance in derivative ..

N

> ..

O

> ..

S > ..

Cl O + NHR – Leaving group ability 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 H 2 N O OH H O O NH 2 O H N NH 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  side reactions potential for many i.e., H O O O H 2 N OH H 2 N H N OH O H O SERINE OH hydroxyl group is a good nucleophile & needs to be protected BEFORE we make peptide bond

• 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 H O H N O O R Formyl-AA (methionine) Primary amine is protected from further reaction Adenosine O P O O O P O O O P O O

Activation

(raises energy of CO 2 H) H O H N R O O O P O Adenosine 3'-OH terminus of specific tRNA sequence tRNA OH H H N O R O O tRNA ester: more reactive than an acid

H O H N O O tRNA R H 2 N R O O tRNA H O H N R O R N H O O tRNA AA 3 NH 2 AA 1 AA 2 AA 3 AA 4 ...O

tRNA H 2 O polypeptide Each AA is attached to its specific tRNA

• A specific example: tyrosyl-tRNA synthase (from tyr) Good L.G. (PP i ) + NH 3 O O Adenosine O P O O O P O O O P O O anhydride-like + NH 3 O O O P O Adenosine OH OH 3 potential nucleophiles!

3 potential reactive P's R one of 20 AA's tRNA tyr only!

O B L-enantiomer only!

OH

OH R O B + NH 3 O 3'-OH only!

O

OH tRNA Tyr OH

• Control!

– Only way to ensure specificity is to orient desired nucleophile (i.e., CO 2 ) adjacent to desire electrophile (i.e., P) What about Nonribosomal Peptide Synthase (NRPS)?

– Uses thioesters H 2 N R O O Adenylation H 2 N R O O O P O Adenosine H S NRPS O NH 2 NRPS S R Activated thioester

O NH 2 NRPS S R Activated thioester good Lv group NRPS S O NH 2 potential Nu: NRPS S O H N R O NH 2 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: NH 3 + O SH • To study enzyme IPNS, we need to synthesize tripeptide (ACV) • Small molecule → use solution technique • Synthesis (in sol n ) can be long & low yielding • But, can still produce enough for study -O 2 C L  -aminoadipyl-L-cysteinyl-D-valine (ACV) isopenicillin N synthase -O 2 C NH 3 + N H O O H N H N CO 2 S N O Isopenicillin N

Plan for Synthesis: -O 2 C NH 3 + O SH N H O H N CO 2  -aminoadipic acid -O 2 C * * NH 3 + * Need protecting groups Needs to be activated O N H * O * SH cysteine H N CO 2 * valine

Protection of Carboxylic acid: H 2 N CO 2 H Val Ph H + heat OH H 2 N O O Ph = OBn (benzyl) Selective Protection of R group (thiol): H 2 N CO 2 H Cys SH BnCl NaOH H 2 N CO 2 H 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) 2 O = an anhydride O O O O O H 2 N CO 2 H SBn O O H N CO 2 H 2X protection SBn = BOC H N CO 2 H SBn

Now that we have our protected AA’s, we need to activate the carboxylate towards coupling O O H N CO 2 H H 2 N O O Ph SBn Activation & Coupling (see exp 6): H + BOCHN CO 2 SBn N C N DCC R O Cy N C N H Cy good Lv group DCC =

d

i

c

yclohexyl

c

arbodiimide = Coupling reagent that serves to activate carboxylate towards nucleophilic attack