Transcript Solid-Phase Peptide Synthesis

The Strategy of Peptide Synthesis
General Considerations
Making peptide bonds between amino acids is
not difficult.
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
General Strategy
1. Limit the number of possibilities by
"protecting" the nitrogen of one amino acid
and the carboxyl group of the other.
N-Protected
phenylalanine
O
X
NHCHCOH
CH2C6H5
C-Protected
glycine
O
H2NCH2C
Y
General Strategy
2. Couple the two protected amino acids.
X
O
O
NHCHC
NHCH2C
Y
CH2C6H5
O
X
NHCHCOH
CH2C6H5
O
H2NCH2C
Y
General Strategy
3. Deprotect the amino group at the N-terminus
and the carboxyl group at the C-terminus.
X
O
O
NHCHC
NHCH2C
Y
CH2C6H5
O
+
H3NCHC
O
–
NHCH2CO
CH2C6H5
Phe-Gly
Amino Group Protection
Protect Amino Groups as Amides
Amino groups are normally protected by
converting them to amides.
Benzyloxycarbonyl (C6H5CH2O—) is a common
protecting group. It is abbreviated as Z.
Z-protection is carried out by treating an amino
acid with benzyloxycarbonyl chloride.
Protect Amino Groups as Amides
O
O
CH2OCCl
+
+
–
H3NCHCO
CH2C6H5
1. NaOH, H2O
2. H+
O
CH2OC
O
NHCHCOH
CH2C6H5
(82-87%)
Protect Amino Groups as Amides
O
CH2OC
O
NHCHCOH
CH2C6H5
is abbreviated as:
O
ZNHCHCOH
CH2C6H5
or Z-Phe
Removing Z-Protection
An advantage of the benzyloxycarbonyl
protecting group is that it is easily removed by:
a) hydrogenolysis
b) cleavage with HBr in acetic acid
Hydrogenolysis of Z-Protecting Group
O
CH2OC
O
NHCHCNHCH2CO2CH2CH3
CH2C6H5
H2, Pd
O
CH3
CO2
H2NCHCNHCH2CO2CH2CH3
CH2C6H5
(100%)
HBr Cleavage of Z-Protecting Group
O
CH2OC
O
NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
O
CH2Br
CO2
+
H3NCHCNHCH2CO2CH2CH3
–
CH2C6H5 Br
(82%)
The tert-Butoxycarbonyl Protecting Group
O
(CH3)3COC
O
NHCHCOH
CH2C6H5
is abbreviated as:
O
BocNHCHCOH
CH2C6H5
or Boc-Phe
HBr Cleavage of Boc-Protecting Group
O
(CH3)3COC
O
NHCHCNHCH2CO2CH2CH3
CH2C6H5
HBr
O
H3C
C
H3C
CH2
CO2
+
H3NCHCNHCH2CO2CH2CH3
–
CH2C6H5 Br
(86%)
Carboxyl Group Protection
Protect Carboxyl Groups as Esters
Carboxyl groups are normally protected as
esters.
Deprotection of methyl and ethyl esters is
by hydrolysis in base.
Benzyl esters can be cleaved by
hydrogenolysis.
Hydrogenolysis of Benzyl Esters
O
O
C6H5CH2OC
O
NHCHCNHCH2COCH2C6H5
CH2C6H5
H2, Pd
O
C6H5CH3
CO2
+
–
H3NCHCNHCH2CO
CH2C6H5
(87%)
CH3C6H5
Peptide Bond Formation
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.
DCCI-Promoted Coupling
O
O
ZNHCHCOH
+ H2NCH2COCH2CH3
CH2C6H5
DCCI, chloroform
O
ZNHCHC
O
NHCH2COCH2CH3
CH2C6H5
(83%)
Mechanism of DCCI-Promoted Coupling
O
+
ZNHCHCOH
C6H11N
C
CH2C6H5
H
C6H11N
O
C
C6H11N
OCCHNHZ
CH2C6H5
NC6H11
Mechanism of DCCI-Promoted Coupling
The species formed by addition of the Zprotected amino acid to DCCI is similar in
structure to an acid anhydride and acts as an
acylating agent.
Attack by the amine function of the carboxylprotected amino acid on the carbonyl group
leads to nucleophilic acyl substitution.
H
C6H11N
O
C
C6H11N
OCCHNHZ
CH2C6H5
Mechanism of DCCI-Promoted Coupling
O
H
C6H11N
C
O +
ZNHCHC
O
NHCH2COCH2CH3
CH2C6H5
C6H11NH
O
H2NCH2COCH2CH3
H
C6H11N
O
C
C6H11N
OCCHNHZ
CH2C6H5
The 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.
The Active Ester Method
O
ZNHCHCO
CH2C6H5
O
NO2 +
H2NCH2COCH2CH3
The Active Ester Method
O
O
ZNHCHCO
NO2 +
H2NCH2COCH2CH3
CH2C6H5
chloroform
O
ZNHCHC
O
NHCH2COCH2CH3 + HO
CH2C6H5
(78%)
NO2
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.
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
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.
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
Treating the polymeric support with
chloromethyl methyl ether (ClCH2OCH3) and
SnCl4 places ClCH2 side chains on some of the
benzene rings.
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
CH2Cl
The side chain chloromethyl group is a benzylic
halide, reactive toward nucleophilic substitution
(SN2).
The Solid Support
CH2
CH
CH2
CH
CH2
CH
CH2
CH
CH2Cl
The chloromethylated resin is treated with the Bocprotected C-terminal amino acid. Nucleophilic
substitution occurs, and the Boc-protected amino
acid is bound to the resin as an ester.
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
–
BocNHCHCO
R
CH
CH2
CH2Cl
CH
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
BocNHCHCO
Next, the Boc
protecting group is
removed with HCl.
R
CH
CH2
CH2
CH
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
H2NCHCO
DCCI-promoted
coupling adds the
second amino acid
R
CH
CH2
CH2
CH
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
BocNHCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
Remove the Boc
protecting group.
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
O
H2NCHC
R'
CH
O
CH2
CH
CH2
NHCHCO
R
Add the next amino
acid and repeat.
The 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
HBr in CF3CO2H
The Merrifield Procedure
CH2
CH
CH2
CH
CH2
CH
CH2
CH2Br
O
O
+
H3N peptide C NHCHC
R'
O
–
NHCHCO
R
CH
Peptide Synthesis
The Merrifield Method
Merrifield also 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
Nobel Prize in chemistry: 1984