Chromatographic Studies on the Racemization of Thiopeptides

It was found by chromatographic, CD and NMR methods, that the thionation of piperazine-2,5-diones [cyclo(Aaa(1)-Aaa(2)) --> cyclo (Aaat(1)-Aaat(2) (Aaa = -NH-CHR-CO-; Aaat = -NH-CHR-CS-)] or piperazine-2,5-onthiones [cyclo(Aaat(1)-Aaa(2)) --> cyclo (Aaat(1)-Aaat(2))] and, occasionally, 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 diastereoisomers were separated by semipreparative HPLC and their quantitative product distribution was determined by an optimized HPLC method on Hypersil-silica column with CH2Cl2-EtOAc eluent mixtures. Isocratic RP-HPLC on ODS-Hypersil column and pre-column derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) were used to monitor the racemization of Ala and Pro residues and to determine the ratio of enantiomers. Thionation of urethane protected dipeptide esters or dethionation of the corresponding endothiodipeptide derivatives were not found to result in significant racemization. However, during the thionation of cyclic dipeptides or thiopeptides or isolation of piperazine-2,5- mono- or dithiones a partial or complete racemization could always be detected. Moreover, the acidic hydrolysis of thiopeptides was also accompanied by racemization and resulted in partially racemized amino (oxo)acids.