The Chemo-enzymatic Assembly of Hydroxamic Acid Radiochemical Ligands with an Amine Chemical Handle

Abstract

Immuno-positron emission tomography (immuno-PET) imaging is a versatile diagnostic technique, used in oncology. The radionuclide 89Zr(IV) possesses a half-life that is closely matched to the circulation time of monoclonal antibodies. However, clinical studies using 89Zr(IV) immunoconjugates have suggested that the stability of metal-ligand complexes could be improved. One group of attractive clinical agents are octadentate macrocyclic chelators containing a chemical handle. Access to hydroxamic acid macrocyclic chelators, such as the bacterial natural product DFOT1, has been limited due to multi-step syntheses. The final enzyme responsible for the biosynthesis of DFOT1, DesD, has proven potential as a biocatalyst. This work aimed to test the capacity of DesD to accept non-native substrates to assemble DFOT1 derivatives with a chemical handle. This was achieved by evaluating reaction profiles with non-native monomeric and dimeric HSC analogues containing an amine chemical handle. The chelator pool was incubated with Zr(IV) and Ga(III) to evaluate metal binding properties by LC-MS.

Chemo-enzymatic reactions of non-native monomeric substrates generated chelators containing an amine chemical handle, albeit in low abundances, which limited addition evaluation. Chemo-enzymatic reactions of non-native dimeric substrates generated increased abundances of chelators containing an amine handle compared to monomeric substrate reactions. These chelators were generated in sufficient quantities to probe metal chelation characteristics and were observed to readily chelate Zr(IV) and Ga(III).

Overall, this work showed DesD is a viable biocatalyst for the generation of biocombinatorial mixtures of hydroxamic acid radiochemical ligands containing an amine handle. The elastic-like properties of DesD was leveraged to generate tetradentate, octadentate, and dodecadentate amine functionalized chelators with potential future application in immuno-PET imaging with 68Ga(III) and 89Zr(IV).