Unexpected side products in the conjugation of an amine-derivatized morpholino oligomer with p-isothiocyanate benzyl DTPA and their removal

In connection with pretargeting, an amine-derivatized morpholino phosphorodiamidate oligomer (NH2-cMORF) was conjugated conventionally with p-isothiocyanate benzyl-DTPA (p-SCN-Bn-DTPA). However, after 111In radiolabeling, unexpected label instability was observed. To understand this instability, the NH2-cMORF and, as control, the native cMORF without the amine were conjugated in the conventional manner. Surprisingly, the 111In labeling of the native cMORF conjugate was equally effective as that of the NH2-cMORF conjugate (>95%) despite the absence of the amine group. Furthermore, heating the radiolabeled NH2-cMORF and native cMORF conjugates resulted in a 35% loss and a complete loss of the label, respectively. Since the 111In labeled DTPA is known to be stable, the instability in both cases must be due to some unstable association of DTPA to the cMORF, presumably unstable association to some endogenous sites in cMORF. Based on this assumption, a postconjugation–prepurification heating step was introduced, and labeling efficiency and stability were again investigated. By introducing the heating step, the side products were dissociated, and after purification and labeling, the NH2-cMORF conjugate provided a stable label and high labeling efficiency with no need for postlabeling purification. The biodistribution of this radiolabeled conjugate in normal mice showed significantly lower backgrounds compared with the labeled unstable native cMORF conjugate. In conclusion, the conventional conjugation procedure to attach the p-SCN-Bn-DTPA to NH2-cMORF resulted in side product(s) that were responsible for the 111In label instability. Adding a postconjugation–prepurification heating step dissociated the side products, improved the label stability and lowered tissue backgrounds in mice.