Abstract:
Chemical modification of biomolecules like the introduction of metal-chelators into proteins can lead
to heterogeneous product formation. The nature and extend of the modification is important in
interpreting the biological properties of the bioconjugate, given their possible influence on the
pharmacokinetics as well as on the binding affinity to the target. The present study describes the
synthesis and analytical characterization of somatropin modified on its lysine’s ε-amino groups with
the acylating chelator S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-
SCN-Bn-NOTA). Direct separation and identification techniques (i.e. RP-MS and CE-MS) and
peptide mapping after trypsin and chymotrypsin digestion demonstrated that the use of higher amounts
of p-SCN-Bn-NOTA during synthesis leads to a complex product composition with higher order
substitution degrees (i.e. multiple NOTA-moieties per somatropin molecule), as well as the presence
of different position isomers. From the nine lysine (Lys) residues in somatropin, Lys-70 was
experimentally found to be the modification hotspot under our synthesis conditions (pH=9.0). This
was supported by the in silico calculated lowest pKa value of 8.3 for Lys-70. Based on the crystal
structure of somatropin in complex with the extracellular parts of the growth hormone receptor, the
Lys-70 residue is positioned outside the binding pockets and will therefore not directly interfere with
receptor binding. Gallium chelation by NOTA-somatropin resulted in a 100% complexation. The
synthesis of NOTA-somatropin using p-SCN-Bn-NOTA and somatropin under our operational
conditions is therefore a suitable synthesis procedure for the production of a target-specific
radiopharmaceutical for further investigation towards treatment and visualization of growth hormonespecific
cancers.
