W12: SPPS and protein ligation

C-terminal activation

reason: carboxylic acids are normally deprotonated, less negative charge at C, O2- cannot act as a leaving group

—> introduction of a leaving group is necessary

Coupling agents:

(only N-terminal protection; there are also C-terminal protection strategies such as benzyl (H/Pd) or tBu (acidic))

Formation of Boc protection:

Boc removal:

Disadvantage of Boc:

• cleavage strategy is HF - difficult and dangerous to handle, requires special equipment because it reacts with glass

• limited scale (due to HF)

• needs additional neutralization step in the reaction cycle

• difficult to monitor spectroscopically

Formation of Fmoc protection:

Fmoc removal:

Side reactions

(aspartate is followed by glycine)

Problematic sequences

General principle

Ligation approaches

• non-native peptide linkages (result in a non-peptide bond between the two peptides)

  • example: thioester/thioether ligation

• native chemical ligation

  • N-terminal cysteine-S attacks C-terminus of other peptide, then rearrangement so that a native peptide bond is formed

C-terminus needs a thioester!:

Additional remarks on NCL:

Avoiding cysteine:

1. Getting rid of cysteine after coupling.

1. Using an auxiliary factor with a thiol group for the nucleophilic attack which is then cleaved after ligation.

1. Using modified side chains that can make the nucleophilic attack.

1. Traceless Staudinger ligation