A New Method for the Detection of Racemization during Peptide Synthesis: Stereoselective hydrolysis of diastereomeric peptides by leucine aminopeptidase
✍ Scribed by Hans Rudolf Bonsshard; Israel Schechter; Arieh Berger
- Publisher
- John Wiley and Sons
- Year
- 1973
- Tongue
- German
- Weight
- 525 KB
- Volume
- 56
- Category
- Article
- ISSN
- 0018-019X
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✦ Synopsis
Abstract
A suitably protected dipeptide of configuration L‐D, e.g. Z‐L‐Ala‐D‐Ala is coupled with an all L alanine peptide, e.g. L‐Ala‐L‐Ala‐ONbAbbreviations according to the IUPAC‐IUB rules, ‘Symbols for Amino‐Acid Derivatives and Peptides, Recommendations (1971)’'. see e.g. J. biol. Chemistry 247, 977 (1972). In particular the following abbreviations have been used: Z = benzyloxycarbonyl‐, ‐ONb = p‐nitrobenzyloxy‐, ‐ONSu = succinimido‐oxy‐. Additional abbreviations are LAP = leucine aminopeptidase, DCCI = N,N′‐dicyclohexylcarbodiimide, DMF = dimethylformamide.
. The blocking groups are removed and the free peptide hydrolyzed by leucine amino peptidase (E.C. 3.4.1.1). This enzyme shows absolute L‐specificity for the penultimate peptide bond from the amino end and therefore cleaves only the all L peptide formed through racemization. The amount of free alanine determined by amino acid analysis gives a multiple of the degree of racemization. The sensitivity of the test allows 0.1% of (L‐Ala)~4~ to be detected in the synthesis of L‐Ala‐D‐Ala‐L‐Ala‐LAla. Coupling of Z‐L‐Ala‐D‐Ala and Z‐L‐Ala‐D‐Phe with di‐ and trialanine peptides has been studied using DCCI and DCCI + 1‐hydroxybenzotriazole as coupling reagents. The degree of racemization was around 80% for the coupling by DCCI in DMF but was reduced to 0.2–0.4% in the presence of 2 equivalents of 1‐hydroxybenzotriazole. Coupling using the succinimide esters Z‐L‐Ala‐D‐Ala‐ONSu and Z‐L‐Ala‐D‐Phe‐ONSu resulted in 0.8 to 10% racemization, depending on the solvent and base used.