The invention discloses a preparation method of Retatrutide, and belongs to the technical field of polypeptide synthesis. The invention relates to a preparation method of Retatrutide, which comprises the following steps: by taking Sieber
amide resin as a
coupling resin carrier, sequentially
coupling and inoculating Fmoc protected
amino acid and
dipeptide and
tripeptide protected
amino acid fragments, and then
cutting the resin to prepare
a peptide fragment I, namely an intermediate M1; cTC resin is used as a
coupling resin carrier, Fmoc protected
amino acid and
oligopeptide protected amino acid fragments are sequentially coupled and connected, then the resin is
cut, and
a peptide fragment II, namely an intermediate M2, is prepared; coupling the intermediate M1 and the intermediate M2 to prepare full-protection Retatrutide, namely an intermediate M3, and
cracking the intermediate M3 by using a
cracking agent to remove a protective functional group, so as to obtain a crude Retatrutide product; and dissolving, filtering, purifying, and freeze-
drying to obtain a Retatrutide refined product. Two long
peptide fragments and a plurality of protected amino acid fragments are used for preparing Retatrutide, the generation of impurities is reduced, the single
impurity is small, the purification difficulty is reduced, the product purity is ensured, and the purity of the obtained product is greater than 98.0%.