CHROMATOGRAPHIC STUDIES ON THE RACEMIZATION OF THIOPEPTIDES

It was found by chromatographic, CD and NMR methods, that the thionati on of piperazine-2,5-diones [cyclo(Aaa(1)-Aaa(2)) --> cyclo (Aaat(1)-A aat(2) (Aaa = -NH-CHR-CO-; Aaat = -NH-CHR-CS-)] or piperazine-2,5-onth iones [cyclo(Aaat(1)-Aaa(2)) --> cyclo (Aaat(1)-Aaat(2))] and, occasio nally, even the spontaneous cyclization of endothiodipeptide esters [H -Aaat(1)-Aaa(2) OR] result in enantiomeric (Aaa(1) or Aaa(2) = Gly) or diastereomeric mixtures of piperazine monothiones or dithiones. The d iastereoisomers were separated by semipreparative HPLC and their quant itative product distribution was determined by an optimized HPLC metho d on Hypersil-silica column with CH2Cl2-EtOAc eluent mixtures. Isocrat ic RP-HPLC on ODS-Hypersil column and pre-column derivatization with 1 -fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) were us ed to monitor the racemization of Ala and Pro residues and to determin e the ratio of enantiomers. Thionation of urethane protected dipeptide esters or dethionation of the corresponding endothiodipeptide derivat ives were not found to result in significant racemization. However, du ring the thionation of cyclic dipeptides or thiopeptides or isolation of piperazine-2,5- mono- or dithiones a partial or complete racemizati on could always be detected. Moreover, the acidic hydrolysis of thiope ptides was also accompanied by racemization and resulted in partially racemized amino (oxo)acids.