BackgroundOver the last years, several strategies have been reported to improve the metabolic stability of minigastrin analogs. However, currently applied compounds still reveal limited in vitro and in vivo stability. We thus performed a glycine scan at theN-terminus of DOTA-MGS5 (DOTA-d-Glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal) to systematically analyze the peptide structure. We substitutedN-terminal amino acids by simple PEG spacers and investigated in vitro stability in human serum. Furthermore, we evaluated different modifications on its tetrapeptide binding sequence (H-Trp-(N-Me)Nle-Asp-1-Nal-NH2).ResultsAffinity data of all glycine scan peptides were found to be in a low nanomolar range (4.2–8.5 nM). However, a truncated compound lacking thed-γ-Glu-Ala-Tyr sequence revealed a significant loss in CCK-2R affinity. Substitution of thed-γ-Glu-Ala-Tyr-Gly sequence of DOTA-γ-MGS5 (DOTA-d-γ-Glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH2) by polyethylene glycol (PEG) spacers of different length exhibited only a minor influence on CCK-2R affinity and lipophilicity. However, in vitro stability of the PEG-containing compounds was significantly decreased. In addition, we confirmed that the tetrapeptide sequenceH-Trp-Asp-(N-Me)Nle-1-Nal-NH2is indeed sufficient for high CCK-2R affinity.ConclusionWe could demonstrate that a substitution ofd-γ-Glu-Ala-Tyr-Gly by PEG spacers simplified the peptide structure of DOTA-MGS5 while high CCK-2R affinity and favorable lipophilicity were maintained. Nevertheless, further optimization with regard to metabolic stability must be carried out for these minigastrin analogs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13550-023-01016-y.