27.14
The Strategy of Peptide
Synthesis
The Challenge of Peptide Synthesis
•Making peptide bonds between amino acids is
not difficult: there are numerous methods to
make amides from amines and carboxylic acids.
•The challenge is connecting amino acids in the
correct sequence.
•Random peptide bond formation in a mixture of
phenylalanine and glycine, for example, will give
four dipeptides.
•Phe—Phe
Gly—Gly
Phe—Gly
Gly—Phe
Amino Acids are Structurally Bifunctional
H
O
N
H
OH
H
H
O
N
H
OH
Glycine
L-Phenylalanine
Possible Products from the Condensation of
Phenylalanine and Glycine
O
H2N
H
OH
H
O
N
N
H
H2N
O
Gly-Gly
OH
O
Phe-Gly
O
H
N
H2N
OH
O
Gly-Phe
O
H
H
N
H2N
H
OH
O
Phe-Phe
H
Step 1 of Peptide Synthesis: Protection
• 1.Limit the number of possible reactions
by "protecting" the nitrogen of one amino
acid and the carboxyl group of the other.
H
O
N
PG
OH
H
H
O
PG
N
H
O
O-Protected
Glycine
N-Protected
L-Phenylalanine
C-Electrophile
PG
N-Nucleophile
protecting
group
Step 2 of Peptide Synthesis: Coupling
• 2. Couple the two protected amino acids.
H
O
N
OH
PG
H
H
O
PG
N
H
O
Peptide
Coupling
H
O
N
PG
O
H
N
H
PG
O
PG-Phe-Gly-PG
Peptide Synthesis, Step 3: Global
Deprotection
• 3.Deprotect the amino group at the Nterminus and the carboxyl group at the Cterminus. H O
N
PG
O
H
N
H
PG
O
Deprotection
O
H3N
O
H
N
H
O
Phe-Gly
Does the Requirement for Three Extra Steps
Outweigh the Need to Purify a Mixture?
O
H2N
H
OH
H
O
N
N
H
H2N
O
Gly-Gly
OH
O
Phe-Gly
O
H
N
H2N
OH
O
Gly-Phe
O
H
H
N
H2N
H
OH
O
H
Phe-Phe
Yes - synthesis is easier than purification
27.15
Amino Group Protection
What are we Trying to Achieve by Protecting
the Amino Group?
Amino groups can behave as nucleophiles and undergo
reaction with carboxylic acid derivatives. The nitrogen atom
in amides is much less nucleophilic. As a result, amide
derivatives of amines can be viewed as protecting groups.
Amine
Amide Derivative
O
H2N
O
OR
H
N
R2
OR
O
Nucleophilic
Atom
Non-Nucleophilic
Atom
Peptide Synthesis: Amine Protecting Groups
1. Amino groups are normally
converting them to amides.
atom in an amide does not
nucleophile and will not react
groups.
protected by
The nitrogen
behave as a
with carboxyl
2. Benzyloxycarbonyl (C6H5CH2O—) is a
common protecting group. It is abbreviated
as Z or Cbz.
3. Cbz-protection is carried out by treating an
amino acid with benzyloxycarbonyl chloride.
Amine Protecting Groups:
Benzyloxycarbonyl
O
O
R
O
Cl
Benzyloxycarbonyl
chloride
(Cbz-Cl)
O
N
H
Benzyloxycarbonyl
group
Amine Protecting Groups: Introduction of
Benzyloxycarbonyl Protecting Groups
O
O
O
H3N
O
1. NaOH
H2O
H
Cl
2. H3O+
O
H
N
O
OH
O
(85%)
H
Benzyloxycarbonyl is Abbreviated to Cbz or Z
O
O
H
N
H
N
O
Cbz
OH
O
H
H
N
OH
H
Cbz-Phe
H
O
Z
OH
Cleavage of Cbz Groups
•An advantage of the benzyloxycarbonyl
protecting group is that it is easily removed
by:
•a)catalytic hydrogenolysis under extremely
mild
conditions
•b) cleavage with HBr in acetic acid
•Both reagents cleave the relatively weak
benzylic carbon-oxygen ether bond, albeit by
Hydrogenolysis of Cbz Groups
O
H
N
O
OEt
H
O
N
H
O
ethyl ester is
stable to
hydrogenolysis
H2, Pd/C
solvent
O
H
N
HO
H2C
Toluene
(volatile)
H
O
OEt
H
N
H
O
Carbamic Acid
(Very Unstable)
Hydrogenolysis of Cbz Groups
O
H
N
HO
O
OEt
H
N
H
O
Spontaneous
decarboxylation
O
H2N
OEt
H
N
H
O
O
C
O
(100%)
Acid-Mediated Cleavage of Cbz Groups
O
H
N
O
OEt
H
O
N
H
O
HBr
acetic acid
O
Br H3N
H2C
Benzyl
bromide
(volatile)
Br
OEt
H
N
H
O
O
C
O
(82%)
Amine Protecting Groups: tertButyloxycarbonyl
O
O
O
O
R
O
N
H
O
tert-Butyloxycarbonyl
group
O
O
O
Di-tert-butyl dicarbonate
(Boc 'anhydride')
O
O
Cl
tert-Butyl chloride
(instablity limits use)
tert-Butyloxycarbonyl is Abbreviated to Boc
O
O
H
N
O
H
N
OH
O
H
Boc
OH
H
Boc-Phe
Cleavage of Boc Groups
The tert-butyloxycabonyl protecting group is readily
removed by treatment wit strong, anhydrous
BrØnsted acids:
a) cleavage with trifluoroacetic acid in methylene
chloride
b) cleavage with HBr in acetic acid
Both reagents cleave the quaternary carbonoxygen ether bond by an acid-mediated elimination
reaction.
Acid-Mediated Cleavage of Boc Groups
O
H
N
O
OEt
H
O
N
H
O
O
F3C
OH
O
F3C
Butene
(volatile)
H
H
CH3
O
O
OEt
H3N
H
N
H
O
O
C
H3C
trifluoroacetic
acid
O
(high yield)
27.16
Carboxyl Group
Protection
Peptide Synthesis: Carboxyl Protecting
Groups
Carboxyl groups are normally protected as
esters.
Deprotection of methyl and ethyl esters is by
hydrolysis in base.
Benzyl esters
hydrogenolysis.
can
be
cleaved
by
Simultaneous Hydrogenolysis of
Cbz Group and Benzyl Ester
O
H
N
O
O
H
O
N
H
O
H2, Pd/C
solvent
O
H
N
HO
H2C
Toluene
(volatile)
H
O
OH
H
N
H
O
Carbamic Acid
(Very Unstable)
Simultaneous Hydrogenolysis of
Cbz Group and Benzyl Ester
O
H
N
HO
O
OEt
H
N
H
O
Spontaneous
decarboxylation
O
H2N
OEt
H
N
H
O
O
C
O
(87%)
27.17
Peptide Bond Formation
Peptide Synthesis: Forming Peptide Bonds
The two major methods are:
1. coupling of suitably protected amino
acids using N,N'dicyclohexylcarbodiimide (DCC)
2. via an active ester of the N-terminal
amino acid.
N,N'-Dicyclohexylcarbodiimide (DCC) is a
Powerful Dehydrating Agent
H
O
N
H
R1
H
O
R2
C
N
N,N'-dicyclohexylcarbodiimide
H
O
N
H
N
R1
O
H
Very high
temps
R2
-H2O
O
N
H
O
R1
'H2O'
N
H
R2
Amide
N
H
N,N'-dicyclohexylurea
Peptide Coupling is a Condensation
Reaction
• 2. Couple the two protected amino acids.
H
O
N
OH
PG
H
H
O
PG
N
H
O
Peptide
Coupling
H
O
N
PG
-H2O
O
H
N
H
PG
O
DCC-Mediated Peptide Coupling
• 2. Couple the two protected amino acids.
H
O
H
N
Cbz
OH
O
N
Et
H
O
H
DCC, CHCl3
H
O
N
O
Cbz
H
N
H
Et
O
(83%)
Mechanism of DCC-Promoted
Coupling
H
O
N
Cbz
OH
N
C
N
H
1,2-Addition
H
O
N
N
Cbz
O
H
N
H
O-Acylisourea
derivative
O-Acylisoureas are Powerful
Acylating Agents
The O-acylisourea
intermediate formed
by addition of the
Cbz-protected amino
acid to DCCI is similar
in structure to an acid
anhydride and acts as
an acylating agent.
H
O
N
N
Cbz
O
H
N
H
O-Acylisourea
Derivative
O
H3C
O
O
CH3
Acid
Anhydride
Mechanism of DCC-Promoted
Coupling
H
O
N
N
Cbz
O
N
H
H
O
N
Attack by the
amine function of
the carboxylprotected amino
acid on the
carbonyl group
leads to
nucleophilic acyl
substitution.
H
1,2-Addition
then
Proton Transfer
OEt
H
OH
N
N
Cbz
O
N
H
O
N
H
Unstable
Intermediate
OEt
Mechanism of DCC-Promoted
Coupling
H
OH
N
Unstable
Intermediate
N
Cbz
O
N
H
N
H
Attack by the
amine function of
the carboxylprotected amino
acid on the
carbonyl group
leads to
nucleophilic acyl
substitution.
Elimination
O
OEt
H
O
N
OEt
Cbz
O
N
H
H
N
H
N,N'-dicyclohexylurea
N
H
O
Dipeptide
Peptide Synthesis: Forming Peptide Bonds
The two major methods are:
1. coupling of suitably protected amino
acids using N,N'dicyclohexylcarbodiimide (DCCI)
2. via an active ester of the N-terminal
amino acid.
Peptide Synthesis: Active Ester Method
A p-nitrophenyl ester is an example of an "active
ester.”
p-Nitrophenyl is a better leaving group than
methyl or ethyl, and p-nitrophenyl esters are
more reactive in nucleophilic acyl substitution.
O
N+
O
O–
O
is a more powerful
acylating agent than......
Alkyl
O
O
Alkyl Ester
4-Nitrophenyl
(PNP) Ester
Peptide Synthesis: Active Ester Method
H
NO2
O
N
Cbz
O
H
O
N
H
1,2-Addition
then
Proton Transfer
OEt
H
NO2
OH
N
Cbz
O
N
H
O
Unstable
Intermediate
OEt
Peptide Synthesis: Active Ester Method
H
NO2
OH
N
Cbz
Unstable
Intermediate
O
N
H
Elimination
O
OEt
H
O
N
OEt
Cbz
H
O2N
N
H
O
OH
para-Nitrophenol
Dipeptide
27.18
Solid-Phase Peptide
Synthesis:
The Merrifield Method
Solid Phase Peptide Synthesis
In solid-phase synthesis, the starting material is
bonded to an inert solid support.
Reactants are added in solution.
Reaction occurs at the interface between the solid
and the solution. Because the starting material is
bonded to the solid, any product from the starting
material remains bonded as well.
Purification involves simply washing the byproducts
from the solid support.
Polystyrene is the Basis for the Solid
Support
H
H H
C
H H
C
C
H
H H
C
C
H
H H
C
C
H
H
C
C
C
H
•The solid support is a copolymer of styrene
and divinylbenzene.
It is represented
above as if it were polystyrene. Cross-linking
with divinylbenzene simply provides a more
rigid polymer.
Functionalization of Polystyrene
•Treating the polymeric support with
chloromethyl methyl ether (ClCH2OCH3) and
SnCl4 places ClCH2 side chains on some of
the benzene rings.
Chloromethylation of Polystyrene
Cl
O
C
H2
Cl
CH2
Cl
CH2
Me
SnCl4
Solid Phase Peptide Synthesis
Cl
CH2
Cl
CH2
The side chain chloromethyl group is a benzylic
halide, reactive toward nucleophilic substitution
(SN2).
Solid Phase Peptide Synthesis
Cl
CH2
Cl
CH2
The chloromethylated resin is treated with the
Boc-protected
C-terminal
amino
acid.
Nucleophilic substitution occurs, and the Bocprotected amino acid is bound to the resin as an
ester.
Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
–
BocNHCHCO
R
CH
CH2
CH2Cl
CH
Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
BocNHCHCO
Next, the Boc
protecting group is
removed with HCl.
R
CH
CH2
CH2
CH
Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
DCCI-promoted
coupling adds the
second amino
acid
H2NCHCO
R
CH
CH2
CH2
CH
Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
BocNHCHC
R'
O
CH
CH2
CH
CH2
NHCHCO
R
Remove the Boc
protecting group.
Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
H2NCHC
R'
O
CH2
CH
CH
CH2
NHCHCO
R
Add the next
amino
acid
and repeat.
Merrifield Procedure
CH2
CH
CH2
O
CH
CH2
O
+
H3N peptide C NHCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
• Remove the
peptide from
the resin with
Merrifield Procedure
CH2
CH
CH2
CH
CH2
CH
CH2
CH2Br
O
O
+
H3N peptide C NHCHC
R'
O
–
NHCHCO
R
CH
Merrifield Procedure
•Merrifield automated his solid-phase
method.
•Synthesized a nonapeptide (bradykinin)
in 1962 in 8 days in 68% yield.
•Synthesized ribonuclease (124 amino
acids) in 1969.
369 reactions; 11,391 steps