Synthesis of DOTA-Conjugated Peptides

Peptide Ala-Thr-Trp-Leu-Pro-Pro-Arg (A7R) and peptidomimetic Lys(hArg)-Dab-Pro-Arg (K4R) as well as their conjugates with Ahx linker and DOTA chelator (DOTA-Ahx-A7R—conjugate 1, and DOTA-Ahx-K4R—conjugate 2) were synthesized manually by SPPS method on the preloaded Fmoc-Arg(Pbf)-Wang resin following the Fmoc chemistry with DIC/HOBt coupling method and active ester method for coupling DOTA-tris(tBu)-NHS [41 (link)]. In the case of conjugate 1 (DOTA-Ahx-A7R), a linear chain was built first, and then without cleavage peptide from the resin, the linker and chelator were attached to the N-terminus of the alanine residue (Path I in Scheme 1). Additionally, in the case of conjugate 2 (DOTA-Ahx-K4R containing branched K4R peptidomimetic) synthesis, the main linear chain was first built, and next it was extended by two branches: first at the N-terminus by adding lysine residue (as a Boc-Lys(Fmoc) building block), which was then guanylated after deprotection of the Fmoc group, and second in the 2,4-diaminobutyric acid (Dab) side chain, where Fmoc-Ahx-OH and DOTA-tris(tBu)-NHS ester were added (Path II in Scheme 1). All detailed information is thoroughly described in our previous publication [41 (link)]. Retaining the N-terminal fragment of Lys(hArg) unchanged was necessary to maintain high affinity binding to the NRP-1 co-receptor, according to data obtained in [38 (link)].