ypical approaches in classical peptide synthesis: segment condensation CO - Q , Q - NH CO - Q , Q - NH CO-X CO – Q , H 2 N Q - NH CO-X CO - Q , H 2 N Q - NH Partial deprotection Partial deprotection / activation CO-X Prot - NH CO – Q , H 2 N Q - NH CO – Q , H 2 N COOH Deprotection
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Typical approaches in classical peptide synthesis: segment condensation
Typical approaches in classical peptide synthesis: stepwise elongation
Bruce R. Merrifield
The Origins
There is a need for rapid, quantitative, automatic method for the synthesis of
long peptides. A possible approach may be the use of chromatographic columns
Where the peptide is attached to the polymer packing and added to by an activated
amino acid followed by removal of protecting group, with repetition of the process
Until the desire peptide is built up. Finally the peptide must be removed from the
supporting medium.
R.B.Merrifield Laboratory note book (1959)
JACS 85, 2149 (1963)
Cleave, purify Elongate
Couple
AnchorFunctionalize
..
..
..
Resin
Resin
Resin
.
.
ResinResinX
The solid phase principle
Target peptide
S S
H2N
COOH
1. Peptide-polymer bond stable during synthesis2. Temporary protection of the -amino group 3. Permanent protection of the side chains
4. Efficient cleavage with simultaneous side chain removal
A combination of protecting groups (N-, C-, side chains) that ensures:B. The protection scheme
1. Contains reactive sites that allow functionalization2. Peptide-polymer bond must be cleaved efficiently3. Stable to physical and chemical synthesis conditions
4. Good accessibility of the growing peptide chain to solvents and reagents
A. The solid support
Essetial aspects of solid phase peptide synthesis
Solid phase synthesis on polystyrene-divinylbenzene
Functionalize AnchorDeprotect /Neutralize
1,4-divinylbenzene
Q-NH-CHR2-COOHCarboxyl activation
Repeat, n times
Cleave, purify, etc...
X
X
X
Q-NH-CHR1-CO
Q-NH-CHR1-CO
Q-NH-CHR1-CO
H2N-CHR1-CO
H2N-CHR1-CO
H2N-CHR1-CO
Q-NH-CHR2-CONH -CHR1-CO
Q -NH-CHR2-CONH -CHR1-CO
Q-NH-CHR2-CONH -CHR1-CO
Q-NH-CHRn-CO ...... NH-CHR 1-CO
Q-NH-CHRn-CO ...... NH-CHR 1-CO
Q NH-CHR n-CO ...... NH-CHR 1-CO
Q = N-terminal amino-protecting group
Protection in solid phase synthesis
R
ab
c
a. N- Protecting group ("temporary")b. Side-chain protecting groups ("permanent")c. Resin-peptide anchorage
Boc/benzyl chemistry - Merrifield method
CH3
CH3
CH3
OCO-NH CH
CH2
O
OCH2CO P
labile to mediumacid (TFA / CH2Cl2)
labile to strong acid (HF, TFMSA)
Robert C. Sheppard
Fmoc/t-butyl chemistry - Sheppard method
OCO-NH CH
CH2
O
OCH2 OCH2H
CH3 CH3
CH3
CO
Removed by base(piperidine / DMF)
P
Labile to TFA The para alkoxy substituentdecreases the acid resistance of the peptide-resin linkage