an intriguing example of how chirally enriched amino acids in the prebiotic world can generate...
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• An intriguing example of how chirally enriched amino acids in the prebiotic world can generate sugars with D-configuration & with enantioenrichment:
H
O
OBn
H
O
OBn
OH
OBn
O OH
OBn
OBn
BnO
BnOL-proline
2-4 days
+
95-99% ee >99% ee
hexose sugar
L-proline: a 2° amine; popular as an organocatalyst because it forms enamines readily
NH OH
O
L-proline
Cordova et al. Chem. Commun., 2005, 2047-2049
The Model:
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Mechanism: enamine formation
H
O
OBnNH OH
ON
OH
O
OBn
H
NOH
O
OBn
H
O
OBnN
OH
O
OBn
OH
OBn
OBn
OH
OBn
O
+
+
+
1st aldol product (4C)
dilute
CO2H participates as acid
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OBn
OH
OBn
ON
OH
O
OBnOBn
OH
OBn
OH O
OBn
OOH
OH
BnO
OBnBnO
O OH
OBn
OBn
BnO
BnO
+2nd proline-mediatedaldol reaction
benzyl protected allose
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• 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!
% ee of sugar vs % ee of AA
Enantioenrichment
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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?
Ancient Amino Acids(i.e., meteorites)
Ancient Peptides Enzymes
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• 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!
Catalysis by Small Peptides
OOH
NO2
OH
NO2
O
+Catalytic Peptide
L-ala-L-alaL-val-L-valL-val-L-ala
i.e.,
81-96 % ee
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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
H3N
O
CH3
O
H3N
O
CH2SH
O
NH
O
CH2SH
OH3N
O
CH3
+
+
+
Ala
Cys
+
H2O
H2O
petide bond
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• 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):
N
O
C H
CN
O
C H
C
sp2
..
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• Primary structure: AA sequence with peptide bonds• Secondary structure: local folding (i.e. -sheet & -helix)
-sheet helix
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Amide bond: Formation & Degradation
R OH
O
NH
H
R'R N
H
O
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 Very slow reaction
Forward:R O
O
NH
H
R'
H+ +
X
Resonance stablilizedanion -stable & notprone to nucleophilic attack
Protonated--not anucleophile
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Reverse: R NH
O
R' H2O+ X
resonance stabilized:most stable C=O derivative
weak nucleophile
ΔGTS1
TS2
T.I
T.I = tetrahedral intermediate
EA EA Large EA for forward reaction
Large EA for reverse reaction
Reaction Coordinate Diagram:R OH
NH2+
O
Charge separation
No resonance
HIGH ENERGY!
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How do we overcome the barrier?
1) Heat
First “biomimetic” synthesis
Disproved Vital force theory
But, cells operate at a fixed temperature!
2) Activate the acid:
NH4+ -N C O O
NH2
NH2
+ + H2O
acid
Activated acid
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• Activation of carboxylic acide.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?
R OH
OR Cl
O
R O
O
R
O
PCl5
P2O5
-H2O
acid chloride
anhydride
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• 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
R SR'
O
R O
O
P
O
O
O
R OH
O
R Cl
O
R O
O
R
O
R OR'
O
R NHR'
O>> > >> >
increasing stability
increasing reactivity
N O S Cl.. .. ..
> >>..
NO SCl > >>Cl
O
O
NHR+
Cl- -OCOR -SR -OR -NHR> >> >
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3) 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
NH
NH
O
O
NH2
O
OH
NH2
O
OH
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• 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.,
NH2
O
OH
OH
NH2
O
OH
NH
O
OH
OHSERINE
hydroxyl group is a good nucleophile& needs to be protectedBEFORE we make peptidebond
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• Nature uses protection & activation as part of its strategy to make proteins on the ribosome:
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O
O
R
NH
O
H
P
O
O
Adenosine O P
O
O
O OP
O
ONH
O
O
R
P
O
O
AdenosineH
O
tRNA OH
NH
O
O
R
H
O
tRNA
ActivationFormyl-AA
(methionine)(raises energy of CO2H)
3'-OH terminus of specific tRNAsequence
ester: more reactivethan an acid
Primary amine is protectedfrom further reaction
Nature uses an Ester to activate acid (protein synthesis):
Adenylation
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NH
O
O
R
H
O
tRNANH2
O
O
R
tRNA
NH
O
O
R
tRNANH
O
R
H
O
AA3 NH2
AA1 AA2 AA3 AA4...O tRNA
polypeptide
H2O
Each AA is attached to its specific tRNA
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• A specific example: tyrosyl-tRNA synthase (from tyr)
NH3
O
O
OH
P
O
O
Adenosine O P
O
O
O OP
O
O
NH3
O
O
OH
P
O
Adenosine
O
O
R
OHOH
B
O
R
OHO
B
NH3
O
OH
tRNA Tyr
+
+
+
tRNAtyr
only!
3'-OHonly!
anhydride-like
3 potential reactive P's
Good L.G. (PPi)
3 potentialnucleophiles!
one of 20 AA's
L-enantiomer only!
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• 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
NH2S
O
R
NRPS
NRPSSH
NH2
O
O
R
P
O
O
AdenosineNH2
O
O
R
Adenylation
Activated thioester
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NH2S
O
R
NRPS
S
O
NH2NRPS
O
NH2
NH
S
O
R
NRPS
Activated thioester
potential Nu:
goodLv group
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
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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
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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
-O2C NH
NH3+ O
O
SH
NH
CO2-
-O2C NH
NH3+ O
N
S
O
L--aminoadipyl-L-cysteinyl-D-valine (ACV)
isopenicillin N synthase
Isopenicillin N
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-O2C NH
NH3+ O
O
SH
NH
CO2-
NH
CO2-
NH
O
SH-O2C
NH3+ O*
*
**
*
Need protecting groups
Needs to be activated
*
valine
cysteine
-aminoadipic acid
Plan for Synthesis:
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NH2
CO2H
OHPh NH2
O O Ph
Val
H+
= OBn(benzyl)
heat
Protection of Carboxylic acid:
Selective Protection of R group (thiol):
NH2
SH
CO2H NH2
S
CO2H
Cys
BnCl
NaOH
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• 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:
NH2
SBn
CO2H
O O
O
O
O
NH
SBn
CO2H
O
O NH
SBn
CO2H= BOC
2X protection
(BOC)2O = an anhydride
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Now that we have our protected AA’s, we need to activate the carboxylate towards coupling
Activation & Coupling (see exp 6):
NH
SBn
CO2H
O
ONH2
O O Ph
BOCHN
SBn
CO2-
N C N
H+
N C NH
CyCy
OR
DCC
good Lvgroup
DCC = dicyclohexylcarbodiimide = Coupling reagent that serves to activate carboxylate towards nucleophilic attack