Development and characterisation of protecting groups that enhance the solubility of synthetic peptides : Methods to improve the aqueous solubility of hydrophobic peptides

Abstract: Chemical synthesis of peptides is generally performed by solid phase peptide synthesis (SPPS). Although SPPS is a rapid and convenient method to prepare peptides, the major methodological problem in SPPS is related to solvation/solubility. Poor solvation of the peptide during the synthesis on solid phase will frequently lead to deletion peptides and if the peptide is poorly soluble after it has been cleaved from the resin, it can be difficult to purify by liquid chromatography. In order to increase the solubility of peptides, two new amino acid derivatives and a modification of the 2-hydroxy-4-methoxy-benzyl (Hmb) group, a protecting group for the peptide bond, have been developed. The Boc-N-methyl-N-[(2-methylamino)ethyl]carbamoyl (Boc-Nmec) group was used for protection of aromatic hydroxyl groups of tyrosine and the Hmb group. The Boc-4-(N-methyl-amino)butanoyl (Boc-Nmbu) group was used for protection of the indole nitrogen on tryptophan. The new derivatives were introduced into model peptides by standard protocol for Fmoc chemistry. The Boc protection of the Nmec/Nmbu group is removed during the cleavage of the peptide from the resin but the Nmec/Nmbu group is still attached to the peptide. The Nmec/Nmbu group contains a secondary amino group that is protonated at low pH and thereby increases the solubility during purification and handling of the peptide in aqueous solutions. By raising the pH the Nmec (pH 8)/Nmbu (pH 9) group is cleaved by an intramolecular cyclization reaction to give the fully deprotected peptide. Amyloid b (1-42) is a large peptide that is very difficult both to assemble on solid phase and to purify by HPLC.  It is shown that by using the Boc-Nmec protected dipeptides, amyloid b (1-42) could be synthesized by SPPS with only traces of by-products and that the peptide was easy to purify by HPLC. It is likely that the new protecting groups would be very useful in synthesis and handling of hydrophobic peptides.