Beside high affinity and selectivity towards their distinct biological targets, the pharmacokinetic properties of drug candidates are crucial for their successful application in vivo. For theranostic radioligands, fast blood clearance and metabolism can strongly limit the enrichment of the compounds in the tumour tissue, even though the functional target is sufficiently available. A well-known strategy to increase the bioavailability of substances is conjugation to human serum albumin (HSA). In this context, N^ε-4-(4-iodophenyl)butanoyllysine (N^ε-IPB-lysine) was recently discovered as potent albumin binder. However, conjugation of N^ε-IPB-lysine via its α-amino group to proteins, peptides or small molecules is challenging, as a free α-carboxyl group is required to maintain good binding to HSA.

Here we present a novel approach for the late-stage introduction of N^ε-IPB-lysine via Cu-catalyzed azide-alkyne cycloaddition (CuAAC) using N^ε-IPB-lysine derivatives with azide/alkyne bearing moieties at the α-amino group. For the selective acylation of lysine, a solid-phase synthesis concept was established starting from Fmoc-Lys(Alloc)-OH. For upscaling, a synthesis in solution starting from Boc-Lys-OH was elaborated, which provides the building blocks in three steps.

Characterization of HSA binding was performed using microscale thermophoresis and a fluorescence-based competition assay. All azide/alkyne containing compounds showed similar or even better binding affinity to HSA (K_d<10 µM) as the originally reported N^α-acetyl-N^ε-IPB-lysine.

To demonstrate the suitability of the novel albumin binders, N^α-5-azidopentanoyl-N^ε-IPB-lysine was coupled to the somatostatin receptor subtype 2 agonist NODAGA-Pra-O2Oc-TATE by on-resin CuAAC. The conjugate showed significantly improved binding to HSA compared to NODAGA-TATE. The resulting effects on biodistribution and tumour uptake will be studied preclinically using the ⁶⁴Cu-labelled analog.