Deletion peptides

Figure 1 Proportion of the Xaa(P)-Deleted Peptide, ASQGLEDPA-NH2, Determined in Crude Products Obtained from the PyBroP/DIPEA, BOP/HOBt/NMM, BOP/HOBt/DIPEA, HBTU/HOBt/DIPEA, HATU/ HOAt/DIPEA, and HATU/DIPEA Coupling of Fmoc-iyr[PO(OBzl)(OH)]-OH (Y), Fmoc-Ser[PO-(OBzl)-(OH)]-OH (S), and Fmoc-Thr[PO(OBzl)(OH)]-OH (T) (3 equiv) for 1 hJ4Sl b...

Commercially available Edman sequencers perform a fully automated sequencing and HPLC separation and provide the HPLC traces for each reaction cycle. The presence of deletion peptides is easily spotted qualitatively by the appearance of secondary PTH peaks in each reaction cycle. The method is limited to a-amino acids, but polypeptides containing exotic a-amino acids can also be sequenced provided the appropriate standard PTH is available. The method is very sensitive, and usually a single bead is enough to allow an accmate determination of the sequence of a peptide, but the quantitation of each amino acid is difficult because of their different stabihties and properties under the conditions of the cleavage cycle. Edman sequencing can be used in conjunction with other techniques such as MS. 

Assembly of H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH2 [ACP(65-74)] percent of products, including various deletion peptides [des-Xaa-ACP(65-74)]. Solid-phase synthesis was carried out in DMF in the presence of 1.5 equiv of Fmoc amino acid and coupling reagent and 3 equiv of DIPEA. Coupling time was 1.5 min. 

Obtaining quantitative yields in amino acid coupling steps and in the repetitive A -amino group deprotection is the main problem in stepwise solid-phase peptide synthesis (SPPS). Since purification is only possible in the last step accumulation of deleted peptides and truncated sequences occurs. Therefore these undesired compounds are present in the crude mixture after final cleavage from the solid support are usually closely related to the target peptide in their physical properties, resulting in difficult purification steps. 

For both of the above examples it was important that the method could identify byproducts. When the reaction did not go to completion, so called deletion peptides were generated that were identified from their mass differ-... 

The purification of the protected peptide intermediates is an important aspect of the CSPPS strategy to ensure homogeneous molecular species, free from single-residue deletion peptides and other impurities. However, a prerequisite for any chromatographic purification is adequate solubility of the material to be purified in a solvent compatible with the procedure. Protected peptides exhibit unpredictable, but generally poor, solubility in water and in most of the commonly used organic solvents, which makes them difficult to purify and causes some of the most serious problems in CSPPS. 

Normal-phase LC is a less potent alternative [132,133] and has been recommended only when impurities in the crude were derived from the loss of side-chain protecting groups. If the crude product exhibits a significant amount of deletion peptides, then purification by reversed-phase HPLC techniques is required [131]. In this case, Cg or even C4 columns are the most suitable for peptides of —10 residues. For longer peptides the less polar diphenyl-based phase is recommended. 

Peptide synthesis is quite regularly accompanied by undesired side reactions, for instance by the alkylation of side chains during deprotection. In addition to such general problems for which at least partial remedies have been found, also a special problem is encountered in solid phase syntheses the formation of deletion peptides and truncated sequences . Both incomplete acylation and incomplete deprotection result in peptide chains from which one or more amino acid residues are missing. A second kind of deletion, the loss of the C-terminal dipeptide sequence, is caused by diketopiperazine formation followed by acylation of the resulting hydroxymethyl polymer (Fig. 8). Premature chain termination (truncation) takes place if acetic acid or trifluoroacetic acid are not completely removed after deprotection and then co-activated in the following coupling step. 

The consequences of incomplete acylation are quite similar. If a fraction of the resin-bound dipeptide B-A is left unacylated during the incorporation of residue C, then after the following deprotection and chain lengthening with (blocked) residue D, the intermediate Y-D-C-B-A will be contaminated with Y-D-B-A, a deletion peptide . This kind of complexity is particularly likely to... 

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