As a payload for PDCs (and ADCs), analogues of the duocarmycins are attractive. Duocarmycin SA and yatakemycin rank among the most potent natural cytotoxins discovered. The cyclopropyl and prodrug seco forms are both naturally occurring and equipotent in most circumstances. Studies of the binding-driven bonding model of their interaction with DNA suggest that their utility will be enhanced when targeted to tumor cells. In fact, SYD985, an ADC that utilizes a peptide linker for a duocarmycin analogue to trastuzumab, has recently been progressed to phase III clinical trial.

The crucial steps during the synthesis of aminooxyacetylated peptides are the incorporation of aminooxyacetic acid (Aoa), including a protecting group, and the final cleavage and the working-up procedure of the Aoa-containing peptide derivatives. In most cases, Boc-protected Aoa is attached to a peptide chain in the last step of solid-phase peptide synthesis. It has been observed that over-acylation (additional Boc-Aoa-OH connected to the Aoa moiety) may occur as the main side reaction. Many approaches have been investigated to overcome this problem, including carbodiimide-mediated one-pot acylation without a base or the application of Boc-Aoa-OSu active ester as an acylating agent, as well as the use of a high excess (8 equiv) of Boc-Aoa-OH and coupling agents for a short acylation time (10 min). Nevertheless, the coupling of the diBoc-protected Aoa derivative has proved to be the best solution. However, the aminooxyacetyl moiety is very sensitive to molecules containing carbonyl groups, with the partial impact of the peptide sequence. Therefore, the free NH2-O-R group reacts often with these compounds during the working-up procedure after the final cleavage of Aoa-modified peptides from the resin. These carbonyl group-containing derivatives might come from the plastic tubes or residues of acetone used in a laboratory. This cannot be prevented even by using diBoc-protected Aoa or working in argon. We found a highly sensitive peptide to this side reaction; the synthesis of a somatostatin analog developed in Schallys laboratory elongated with Aoa (H-Aoa-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2) was unsuccessful. After several trials to optimize the reaction conditions, we elaborated the following procedure: The semi-protected peptide H-D-Phe-Cys-Tyr-D-Trp-Lys(Dde)-Val-Cys-Thr-NH2 was cleaved from Rink Amide MBHA resin and reacted with Boc-Aoa-OPcp to incorporate Aoa into the N-terminus in a solution. After the efficient coupling reaction, the Dde-protecting group was removed with 2% hydrazine in DMF. Surprisingly, during the cleavage of Dde, a cyclic peptide also formed (Boc-Aoa-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2). The Boc group was cleaved in 95% TFA solution in the presence of 10 equiv-free Aoa as a carbonyl capture reagent that could prevent the reaction of the peptide with any carbonyl derivative. The crude product was purified by RP-HPLC, and the solvent was evaporated, followed by direct ligation to daunomycin (Dau) in 0.2 M NaOAc solution at pH 5. This procedure proved to be very efficient to prepare oxime-linked Dau-peptide conjugates.