35
Setting up a Synthesis
Special Case: Sequences Containing Asp-Gly (DG)
The Asp-Gly (DG) segment is particularly sensitive to base-catalyzed aspartimide formation. Simple addition
of HOBt or Oxyma to the standard deprotection solutions may not be sufcient to prevent this side reaction.
Therefore, less aggressive deprotection conditions should be used or a Dmb protected dipeptide that prevents this
side reaction. Below are a few additional approaches for combating aspartimide formation.
Supression Strategy Microwave Method
5% (w/v) Piperazine w/ 0.1 M Oxyma Pure in EtOH:NMP (10:90) Two step room temperature deprotection:
1. Conventional initial deprotection
2. Conventional deprotection
Use Fmoc-Asp(OtBu)-(Dmb)Gly-OH dipeptide and any deprotection cocktail applicable for
standard peptides
Standard
Use Fmoc-Asp(OMpe)-OH and any deprotection cocktail applicable for standard peptides. Standard
Activation Strategy Selection
General Strategy
The two most popular activation strategies for solid phase peptide synthesis are carbodiimide promoted coupling
(using a carbodiimide such as DIC with an additive such as HOBt or Oxyma to quickly convert the O-acylisourea
intermediate to a reactive ester) and onium salt promoted coupling (using a base such as DIEA to initiate
conversion of the amino acid to the activated ester through reaction with an onium salt such as HBTU). The table
below details CEM’s preferred strategies and recommended microwave conditions for each strategy.
CEM Preference Reagents Cycle Exceptions
1 AA/DIC/Oxyma
1
in DMF Single Coupling
2
Arg: Double Coupling
2 AA/HBTU
3
/DIEA
4
in DMF
90°C 1 min Deprotection
Wash (x4)
75°C 5 min Single Coupling
Cys: Single 50°C 10 min Coupling
His: Single 50°C 10 min Coupling
Arg:
1
st
Coupling: 25 min room temp, 75°C 2 min
2
nd
Coupling: 75°C 5 min
3 AA/DIC/Oxyma
1
in NMP
90°C 1 min Deprotection
Wash (x4)
75°C 10 min Coupling
Cys: Double 50°C 10 min Coupling
His: Double 50°C 10 min Coupling
Arg:
1
st
Coupling: 25 min room temp, 75°C 2 min
2
nd
Coupling: 75°C 5 min
4 AA/HBTU
3
/DIEA
4
in NMP
90°C 1 min Deprotection
Wash (x4)
75°C 5 min Single Coupling
Cys: Single 50°C 10 min Coupling
His: Single 50°C 10 min Coupling
Arg:
1
st
Coupling: 25 min room temp, 75°C 2 min
2
nd
Coupling: 75°C 5 min
1
HOBt, 6-Cl-HOBt, or HOAt can be used in place of Oxyma.
2
Recommended cycles assume employment of His(Boc). For other His derivatives, use Single 50°C 10 min Coupling cycle.
3
These conditions should be used for all onium activators (HBTU, 6-Cl-HBTU, HATU) and for the phosphonium activators (PyBOP,
PyAOP).
4
DIEA should be prepared in NMP.
NOTE
For assistance with cycle and microwave method programming, please contact CEM Applications Support
at peptide.support@cem.com.
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