1,4‐Diazepine‐2,5‐dione ring formation during solid phase synthesis of peptides containing aspartic acid β‐benzyl ester

Abstract

The Fmoc-based SPPS of H-Xaa-Asp(OBzl)-Yaa-Gly-NH(2) sequences results in side reactions yielding not only aspartimide peptides and piperidide derivatives, but also 1,4-diazepine-2,5-dione-peptides. Evidence is presented to show that the 1,4-diazepine-2,5-dione derivative is formed from the aspartimide peptide. The rate of this ring transformation depends primarily on the tendency to aspartimide and piperidide formation, which is influenced by the nature of the amino acid following the aspartic acid beta-benzyl ester (Xaa). However the bulkiness of the amino acid side chain preceeding the aspartic acid beta-benzyl ester (Yaa) is also important. Under certain conditions the 1,4-diazepine-2,5-dione peptide derivative may even be formed dominantly, which is a highly undesirable side reaction in peptide synthesis, but which provides a new way for the synthesis of diazepine peptide derivatives with targeted biological or pharmacological activity.

The aspartimide problem persists: Fluorenylmethyloxycarbonyl‐solid‐phase peptide synthesis (Fmoc‐SPPS) chain termination due to formation of N‐terminal piperazine‐2,5‐diones

“…Hence, not only adjacent Yaa but also the residue Xaa has a certain contribution to the observed level of Asi at the Xaa‐Asp‐Yaa motif and consequently of N‐terminal DKP. A similar observation was previously described for DKD formation from Asp β ‐benzyl esters …”

“…Aspartimides are ubiquitous by‐products in peptides with the Asp‐Yaa motif synthesized via Fmoc‐SPPS. It is thus conceivable that both a seven‐membered DKD following pathways A or B in Scheme and/or a six‐membered DKP following pathway C can be formed . However, the formation of a seven‐membered DKD was not observed in this study.…”

“…The main characteristics of a six‐membered cycle is obtained from the 1 H‐ 13 C long range correlations of amide protons (Figure , where the correlations of H‐2 and H‐5 to carbons C‐1 and C‐4 confirm the presence of this cycle. For a seven‐membered ring (such as described in Süli‐Vargha et al), the correlation of H‐5 to C‐1 would not be observed. Although the long range correlation of H‐9 to C‐7 is rather weak, it is a further proof that the methylene group C7 must be exocyclic (for a full list of long range correlations, see the Supporting Information).…”

“…In the context of SPPS such Asi‐related cyclizations, possibly leading to either DKDs and/or DKPs, are so far exclusively known for Asp β ‐benzyl esters. To the best of our knowledge, there is no published study describing whether a six‐ or seven‐membered species or a mixture is formed and to what extent this side‐reaction occurs …”

Solid-Phase Synthesis of Seven-Membered Heterocycles with Two Nitrogen Atoms

“…The α-amino acids were initially immobilized on a Wang resin via a carboxylate group, and the desired scaffold was formed by cyclative cleavage upon acylation with β-amino acids. Similarly, it was found that using aspartic acid β-benzyl ester in Fmoc-based solid-phase peptide synthesis not only risks the formation of the aspartimide peptide but also results in its further transformation into a 1,4-diazepane-2,5-dione-peptide derivative [96].…”