Protected peptide fragments purification

The purification of protected peptide fragments is described in Chapter 9, Protocol 4. 

These methodologies have been reviewed (22). In both methods, synthesis involves assembly of protected peptide chains, deprotection, purification, and characterization. However, the soHd-phase method, pioneered by Merrifield, dominates the field of peptide chemistry (23). In SPPS, the C-terminal amino acid of the desired peptide is attached to a polymeric soHd support. The addition of amino acids (qv) requires a number of relatively simple steps that are easily automated. Therefore, SPPS contains a number of advantages compared to the solution approach, including fewer solubiUty problems, use of less specialized chemistry, potential for automation, and requirement of relatively less skilled operators (22). Additionally, intermediates are not isolated and purified, and therefore the steps can be carried out more rapidly. Moreover, the SPPS method has been shown to proceed without racemization, whereas in fragment synthesis there is always a potential for racemization. Solution synthesis provides peptides of relatively higher purity however, the addition of hplc methodologies allows for pure peptide products from SPPS as well. 

In the case of the stepwise synthesis of peptide fragments starting from an amino acid without C-terminal protection, peptide bond forming methods are limited to the mixed anhydride method,the active ester method, and the azide methodJ l Although such peptide fragments can be directly used for the segment condensation reaction, the purification of the products after each coupling reaction is not so easy. 

The synthetic strategy based on the minimum protection philosophy Pl (Section 4.1.2) favors the use of unprotected hydroxy groups, which leads to more soluble peptides and milder deprotection procedures,but also to a certain degree to side reactions involving the free hydroxy groups. The resulting side-products have to be removed by careful purification of the intermediate fragments. 

---