The non-ribosomal peptide synthetase-independent siderophore synthetase DesD has been demonstrated to generate hydroxamic acid chelators with potential applications in metal-based radiopharmaceuticals and sequestering toxic or commodity metals.[1-2] DesD has been used as a biocatalyst with native substrates to generate diverse chelators varying size and denticity; however, there has been little focus on processing non-native substrates.[3-5] This study focused on masking the minimally active substrate N-hydroxy-N-glutarylcadaverine (2) by forming a heterodimer with the native DesD substrate N-succinyl-N-succinylcadaverine (1). Recombinant DesD from Salinispora tropica CNB-440 (StDesD) was probed with homo- and heterodimers of 1 and 2 (3–6), including N-to-C positional isomers. Chemoenzymatic reactions of the heterodimers of 1 and 2 (5, 6) showed similar substrate consumption and product profile to the native 1 homodimer (3), validating the viability of using non-native–native heterodimers to engineer structurally modified chelators from non-native substrates. Product profiles were further influenced by phenomena unique to using dimeric substrates. First, dimeric substrate prevented the ability of StDesD to generate the odd-numbered trimeric hexadentate macrocycle desferrioxamine E (DFOE), the established major product. Through the elimination of this product sink, StDesD generated the even-numbered tetrameric macrocycle of each dimeric substrate as the major product. Leverage of this dimeric substrate system could increase access to this useful class of octadentate chelator that has shown promise in the chelation of 89Zr(IV).[6] Second, while the upper limit of substrate units able to be assembled by StDesD was similar for both monomeric and dimeric substrates, use of the latter generated chelators with unprecedented cavity sizes and denticities, including an icosadentate chelator that formed a 3:1 metal:ligand complex with Ga(III).