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:
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
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
• 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
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
• 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
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
• 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
..
• Primary structure: AA sequence with peptide bonds• Secondary structure: local folding (i.e. -sheet & -helix)
-sheet helix
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
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!
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
• 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
• 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> >> >
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
• 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
• Nature uses protection & activation as part of its strategy to make proteins on the ribosome:
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
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
• 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!
• 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
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
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:
• 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
-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:
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
• 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
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
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