Solid-phase synthesis and spectroscopic studies of TRH analogues incorporating cis- and trans-4-hydroxy-L-proline

An efficient solid-phase synthesis of the TRH analogue Glp-His(Nim-Trt)-Hyp-OH is described. Na-Fmoc protected amino acids and DCC/HOBt activation were employed. The bulky and mild-acid-sensitive 2-chlorotrityl resin, utilised as the solid support, completely suppressed dioxopiperazine formation. The tripeptide is a key intermediate in the synthesis of TRH analogues incorporating cis- and trans-4-hydroxy-L-proline. The tripeptide was converted, with inversion of configuration at C-4 of the Hyp residue, to Glp-His(Nim-Trt)-cHyp lactone in the presence of triphenylphosphine-diethyl azodicarboxylate (TPP-DEAD). One-pot MeOH-TPP-DEAD transesterification of the lactone, followed by Nim-detritylation, provided Glp-His-cHyp-OMe. This ester gave the corresponding amide and acid on ammonolysis and saponification, respectively. A high-field 1H NMR investigation of Glp-His-cHyp-OH and its diastereomer Glp-His-Hyp-OH, obtained by Nim-detritylation of the key tripeptide, showed that the configuration at C-4 of the prolyl residues is critical for the determination of the preferred three-dimensional structure of the molecules.