Our first focus was to equip Dp44mT and DpC with an azido functionality to enable bioconjugation to Tras by means of the azide-alkyne cycloaddition reaction. To preserve the backbone of the DpTs, we decided to functionalize one of the pyridyl rings with the desired azido functionality (Figure ) instead of employing a previously reported method of derivatizing thiosemicarbazones at the thiocarbonyl group via a transamidation reaction. Considering that the DpTs utilize Cu(II) and Fe(II) chelation as one of their primary modes of action, 3 we envisioned that positioning of the linker is crucial to retain biological activity. Therefore, we decided to place the azido linker at two different positions to compare the effects of the linker positioning on the activities of the modified toxins and resulting ADCs. We developed a versatile synthetic route that allows us to procure both isomers via similar approaches, as illustrated for the ortho derivatives in Scheme 1 below. Synthesis of the representative DpT-O commenced with the formation of 2-pyridylethanol O1 by treating 2-bromo-6-methylpyridine with DMF, followed by reduction of the resulting aldehyde using NaBH4. After protecting the alcohol with TBS, O2 was reacted with 2-cyanopyridine to yield ketone O3. During the acidic work-up, the alcohol was conveniently deprotected via acid hydrolysis, and O3 was then subjected to functional group interconversion to give rise to the azide O5 over two steps. Finally, the latter was condensed with the respective thiosemicarbazide to obtain the DpT-Os (1 and 2) in good yields (c.f. ESI). The corresponding para-substituted derivatives DpT-Ps (3 and 4) were similarly acquired using 2bromo-4-methylpyridine as the starting material in moderate yields (c.f. ESI). The final compounds 1-4 were each isolated as an inseparable mixture of E/Z isomers in approximately 1:1 ratio; 1 H NMR spectra of all synthesized DpTs exhibited twice the number of expected signals (e.g., Figure ). Of note, for compounds 1 and 2, the isomers could be distinguished and identified using NOESY and COSY experiments.