The last step can occur intramolecularly, to give the macrocycle, or intermolecularly with another equivalent of linker, to give a linear product. We use a bis-BG and a BG-pNPP linker to join these fusion proteins into linear structures that can then react with a bis-pNPP linker that joins the ends into a cyclic product. This building block has N-terminal cutinase and C-terminal SnapTag domains that react irreversibly with p-nitrophenyl phosphonate (pNPP) and benzylguanine (BG) groups, respectively. The molecules are prepared through the reactions of a fusion protein building block with small molecule linkers that are terminated in irreversible inhibitors of enzyme domains present in the fusion. This paper describes the synthesis of giant cyclic molecules having diameters of 10–20 nm.
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