An optimized strategy for the mild and efficient solution phase iodination of tyrosine residues in bioactive peptides

Margret Schottelius, Matthias Konrad, Theresa Osl, Andreas Poschenrieder, Hans-Jürgen Wester

29.07.2015 [Original Artikel]

Usually, the accessibility of 3-iodo-Tyr-containing peptides relies on the time-consuming de novo solid phase peptide synthesis. In this study, methods for the direct (mono)iodination of unprotected peptides were evaluated. The use of N-iodosuccinimide (NIS) in acetonitrile/water proved to be a particularly mild, fast (⩽5 min) and efficient method with broad applicability to structurally diverse peptides. NIS iodination therefore represents a very practicable tool for the generation of iodinated peptides on a small (<1 mg) to medium (1–20 mg) scale in reasonable isolated yields (28 ± 8%), providing easy and straightforward access to iodinated reference compounds, for example, for in vitro evaluation.



A shortcut to high-affinity Ga-68 and Cu-64 radiopharmaceuticals: one-pot click chemistry trimerisation on the TRAP platform

Zsolt Baranyai, Dominik Reich, Adrienn Vágner, Martina Weineisen, Imre Tóth, Hans-Jürgen Wester und Johannes Notni

05.05.2015 [Original Artikel]

Due to its 3 carbonic acid groups being available for bioconjugation, the TRAP chelator (1,4,7-triazacyclo-nonane-1,4,7-tris(methylene(2-carboxyethylphosphinic acid))) is chosen for the synthesis of trimeric bio-conjugates for radiolabelling. We optimized a protocol for bio-orthogonal TRAP conjugationviaCu(I)-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC), including a detailed investi-gation of kinetic properties of Cu(II)–TRAP complexes. TRAP building blocks for CuAAC, TRAP(alkyne)3 and TRAP(azide)3 were obtained by amide coupling of propargylamine/3-azidopropyl-1-amine, respect-ively. For Cu(II) complexes of neat and triply amide-functionalized TRAP, the equilibrium properties as wellas pseudo-first-order Cu(II)-transchelation, using 10 to 30 eq. of NOTA and EDTA, were studied by UV-spectrophotometry. Dissociation of any Cu(II)–TRAP species was found to be independent on the nature orexcess of a competing chelator, confirming a proton-driven two-step mechanism. The respective thermo-dynamic stability constants (logKML: 19.1 and 17.6) and dissociation rates (k: 38 × 10−6 and 7 × 10−6 s−1, 298 K, pH 4) show that the Cu(II) complex of the TRAP-conjugate possesses lower thermodynamicstability but higher kinetic inertness. At pH 2–3, its demetallation with NOTA was complete within severalhours/days at room temperature, respectively, enabling facile Cu(II) removal after click coupling by directaddition of NOTA trihydrochloride to the CuAAC reaction mixture. Notwithstanding this, an extrapolateddissociation half life of >100 h at 37 °C and pH 7 confirms the suitability of TRAP-bioconjugates for application in Cu-64 PET (cf. t1/2(Cu-64) = 12.7 h). To showcase advantages of the method, TRAP(DUPA-Pep)3,a trimer of the PSMA inhibitor DUPA-Pep, was synthesized using 1 eq. TRAP(alkyne)3, 3.3 eq. DUPA-Pep-azide, 10 eq. Na ascorbate, and 1.2 eq. Cu(II)-acetate. Its PSMA affinity (IC50), determined by the competition assay on LNCaP cells, was 18-times higher than that of the corresponding DOTAGA monomer(IC50: 2 ± 0.1vs. 36 ± 4 nM), resulting in markedly improved contrast in Ga-68-PET imaging. In conclusion, the kinetic inertness profile of Cu(II)–TRAP conjugates allows for simple Cu(II) removal after clickfunctionalisation by means of transchelation, but also confirms their suitability for Cu-64-PET as demon-strated previously (Dalton Trans., 2012,41, 13803).