Protected peptides purification

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. 

M Gain, P Lloyd-Williams, F Albericio, E Giralt. Convergent solid-phase peptide synthesis. 12. Chromatographic techniques for the purification of protected peptide segments. Int J Pept Prot Res 46, 119, 1995. 

The reduced form of Na+, K+-ATPase inhibitor-I (10) was obtained by treatment of the protected peptide synthesized by the soln procedure with HF, followed by reaction with Hg(OAc)2. After purification of the crude product on Sephadex G-25, the reduced peptide (110 mg) was dissolved in 0.1 M NH4OAc buffer (1L, pH 7.8) at a peptide concentration of 0.018 mM and then stirred at rt. After 24 h, the major peak in the HPLC, which coeluted with the natural product, corresponded to 55% of the product distribution. The mixture was acidified to pH 3 with AcOH and 10 was purified by RP-HPLC. When the oxidation was carried out in the presence redox reagents at a peptide/GSH/GSSG ratio of 1 100 10, after 24 h the major oxidation product increased to 69%. The mixture was acidified with AcOH and the product (10) isolated by preparative HPLC yield 20%. The product was characterized by MALDI-TOF-MS and amino acid analysis a combination of enzymatic peptide mapping and synthetic approaches were applied to assign the cystine connectivities. 

The mild acidolytic cleavage procedure noted above is used to deprotect various side chain protected peptides synthesized in solution or on chlorotrityl-resin with tyrosine O-sulfate synthons as listed in Table 4. The overall yields are significantly superior to those obtained by postsynthetic sulfation of the purified peptides, since they are typical for synthetic peptides after the final deprotection and purification step. The additional main advantage of this approach derives from a facile analytical characterization, since sulfonated byproducts at the tyrosine and tryptophan level, as well as oxidation of the methionine residues resulting from postsynthetic sulfation of tyrosine peptides are avoided. 

Protected Peptides with Built-In TVr(S03H) Deprotection Step Chromatographic Purification Peptide Yield (%) Ref... 

The helical peptide sequence [I] 59 was prepared on Rink amide resin by Fmoc SPPS with cysteine residues at the N- and C-terminus as described in Section 13.1.2.5.2. Briefly, starting from resin (606 mg, substitution level 0.33 mmol-g 1), the fully protected peptide-resin (1.03 g) was obtained. The fully protected peptide-resin (200 mg) was treated with 1M TMSBr/PhSMe in TFA (10 mL) containing 2% m-cresol and 2% EDT under ice-cooling for 1.5 h, then at 20 °C for 30 min. After precipitation with Et20 (40 mL), lyophilization, purification by Sephadex G-10, and subsequently HPLC, a white fluffy powder was obtained yield 25.5 mg (25% overall) LSI-MS mlz [M + H]+ calcd, 2571.1 found, 2570.7. For the... 

The main goal of this chapter is to describe the synthesis details of complex, orthogonally protected peptide constructs. Thus, major emphasis is placed on the peptide chain assembly design and practice and the alterations from the solid-phase synthesis of simple, nonmodified peptides. The technology for peptide purification and quality control is not significantly different from that of other peptides, and these methods will be just briefly described. Many chapters of this book focus on the optimization of HPLC and MALDI-MS procedures for peptide separation and analysis and illustrate the expected and/or acceptable quality control parameters. 

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. 

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