Supramolecular Rotaxane‐Based Multi‐Modal Probes for Cancer Biomarker Imaging**

Abstract

Mechanically interlocked molecules present opportunities to construct therapeutic drugs and diagnostic imaging agents but harnessing supramolecular chemistry to make biologically active probes in water is a challenge. Here, we describe a rotaxane-based approach to synthesise radiolabelled proteins and peptides for molecular imaging of cancer biomarkers in vivo. Host-guest chemistry using β-cyclodextrin-and cucurbit-[6]uril-catalysed cooperative capture synthesis produced gallium-68 or zirconium-89 radiolabelled metallo-[4]rotaxanes. Photochemical conjugation to trastuzumab led to a viable positron emission tomography (PET) radiotracer. The rotaxane architecture can be tuned to accommodate different radiometal ion complexes, other protein-or peptide-based drugs, and fluorophores for optical detection. This technology provides a platform to explore how mechanical bonding can improve drug delivery, enhance tumour specificity, control radiotracer pharmacokinetics, and reduce dosimetry.

A rotaxane-based platform for tailoring the pharmacokinetics of cancer-targeted radiotracers

“…Full details on the synthesis and characterisation of the precursors and [4]pseudorotaxane 1, the photochemical activation step, the radiochemistry and characterisation of both the 68 Ga-and 89 Zr-radiolabelled compounds were reported (see also Supporting Figures S1). 26 Simultaneous 89 Zr-radiolabelling and light-induced bioconjugation to onartuzumaban engineered monovalent antibody that binds the extracellular domain of the human hepatocyte growth factor receptor (c-MET) 30,31 afforded the [ 89 Zr]Zr- [4]semirotaxane-azepin-onartuzumab intermediate. Subsequent capping with FeCl3 and reaction quenching with diethylenetriamine pentaacetic acid (DTPA) gave the bimetallic full rotaxane-mAb conjugate, [ 89 Zr]ZrFe- [4]rotaxane-azepin-onartuzumab (Scheme 1, [ 89 Zr]ZrFe-2).…”

“…S1). 26 Subsequent addition of protein to the reaction mixture (using an initial rotaxane-to-protein ratio of ~2:1), followed by pH adjustment, 32 and irradiation of the mixture with a powerful light emitting diode (LED, 395 nm, [ 89 Zr]ZrFe-2 was isolated with a d.c. RCY of 10.6%, an RCP of 90%, and an approximate 6-fold higher molar activity, with Am = 0.61 MBq nmol -1 of protein (6.176 MBq mg -1 of protein). The scaled-up reaction confirmed that the photoradiochemistry can be adapted to produce useful quantities of rotaxane-based radiotracers.…”

“…S1 †). 26 Simultaneous 89 Zr-radiolabelling and lightinduced bioconjugation to onartuzumaban engineered monovalent antibody that binds the extracellular domain of the human hepatocyte growth factor receptor (c-MET) 30,31 afforded the [ 89 Zr]Zr- [4]semirotaxane-azepin-onartuzumab intermediate. Subsequent capping with FeCl 3 and reaction quenching with diethylenetriamine pentaacetic acid (DTPA) gave the bimetallic full rotaxane-mAb conjugate, [ 89 Zr]ZrFe- [4]rotaxane-azepinonartuzumab (Scheme 1, [ 89 Zr]ZrFe-2).…”

“…Supramolecular systems and mechanically interlocked molecules (MIMs) present interesting opportunities in the design of diagnostic imaging agents and therapeutic drugs. 15,[18][19][20][21][22][23][24][25][26] Supramolecular platforms have been shown to be viable routes for the synthesis of disease-targeted imaging probes, with key features including synthetic flexibility, rapid synthesis, facile labelling, water solubility, and biocompatibility of the construct. Here, we used mechanical bonding to make a supramolecular rotaxane-based 89 Zr-radiolabelled mAb for PET imaging of the human hepatocyte growth factor receptor (c-MET) and evaluated the impact of the non-covalent design on the radiotracer pharmacokinetics in vivo.…”

“…aminoazepin linker leads to the formation of [ 89 Zr]ZrFe-[4]rotaxane-2-hydroxyazepin ([ 89 Zr]ZrFe-3, Figure9A), which is also an observed byproduct of the photoradiosynthesis of [ 89 Zr]ZrFe-2 that was synthesised, characterised and found to exhibit rapid hepatobiliary and renal clearance with t1/2(eff) value 0.58 ± 0.13 h (Figure9B, green diamonds and curve) 26. A small fraction (approximatively 6% of the total injectate) of [ 89 Zr]ZrFe-3 was found to be retained in the Page 28 kidneys as shown by the ex vivo biodistribution data collected at 24 h post-injection (Figure9C, green bars).…”