Control of aspartate epimerization during the coupling of caspase specific tetrapeptides with aromatic amines by using N-[[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]-pyridin-1-yl]methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) as a coupl

During the synthesis of new caspase substrates, we have encountered extensive aspartate epimerization upon coupling of t-Bu-protected tetrapeptides Ac-IETD-OH or Ac-DMQD-OH with aromatic amines (aminocoumarins and aminoquinolines) by using aminium-based coupling reagent HATU in the presence of 2,4,6-trimethylpyridine (TMP). To study this reaction in more detail, an RP-HPLC method was developed that afforded the separation of the epimers. By carefully adjusting the reaction conditions, the epimerization could be reduced to very small levels (from 75% down to below 3%). A new, highly hindered base 2,4,6-tri-tert-butyl-pyridine (TBP), was found to be superior to the traditional TMP in the catalysis of this reaction. Moreover, the aspartate epimers were found to be interconvertable at elevated temperatures in an inert solvent, whereby the d-aspartate containing epimer was the thermodynamically controlled product. Over the course of the study, new side products of HATU coupling reactions, the N,N-dimethylamides of the aspartic residue of above tetrapeptides, were also identified.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slide