Versatile Diphosphine Chelators for Radiolabeling Peptides with 99mTc and 64Cu

We have developed a diphosphine (DP) platform for radiolabeling peptides with 99mTc and 64Cu for molecular SPECT and PET imaging, respectively. Two diphosphines, 2,3-bis(diphenylphosphino)maleic anhydride (DPPh) and 2,3-bis(di-p-tolylphosphino)maleic anhydride (DPTol), were each reacted with a Prostate Specific Membrane Antigen-targeted dipeptide (PSMAt) to yield the bioconjugates DPPh-PSMAt and DPTol-PSMAt, as well as an integrin-targeted cyclic peptide, RGD, to yield the bioconjugates DPPh-RGD and DPTol-RGD. Each of these DP-PSMAt conjugates formed geometric cis/trans-[MO2(DPX-PSMAt)2]+ (M = 99mTc, 99gTc, natRe; X = Ph, Tol) complexes when reacted with [MO2]+ motifs. Furthermore, both DPPh-PSMAt and DPTol-PSMAt could be formulated into kits containing reducing agent and buffer components, enabling preparation of the new radiotracers cis/trans-[99mTcO2(DPPh-PSMAt)2]+ and cis/trans-[99mTcO2(DPTol-PSMAt)2]+ from aqueous 99mTcO4– in 81% and 88% radiochemical yield (RCY), respectively, in 5 min at 100 °C. The consistently higher RCYs observed for cis/trans-[99mTcO2(DPTol-PSMAt)2]+ are attributed to the increased reactivity of DPTol-PSMAt over DPPh-PSMAt. Both cis/trans-[99mTcO2(DPPh-PSMAt)2]+ and cis/trans-[99mTcO2(DPTol-PSMAt)2]+ exhibited high metabolic stability, and in vivo SPECT imaging in healthy mice revealed that both new radiotracers cleared rapidly from circulation, via a renal pathway. These new diphosphine bioconjugates also furnished [64Cu(DPX-PSMAt)2]+ (X = Ph, Tol) complexes rapidly, in a high RCY (>95%), under mild conditions. In summary, the new DP platform is versatile: it enables straightforward functionalization of targeting peptides with a diphosphine chelator, and the resulting bioconjugates can be simply radiolabeled with both the SPECT and PET radionuclides, 99mTc and 64Cu, in high RCYs. Furthermore, the DP platform is amenable to derivatization to either increase the chelator reactivity with metallic radioisotopes or, alternatively, modify the radiotracer hydrophilicity. Functionalized diphosphine chelators thus have the potential to provide access to new molecular radiotracers for receptor-targeted imaging.

SPECT/CT imaging was accomplished using a pre-clinical nanoScan SPECT/CT Silver Upgrade instrument (Mediso), calibrated for technetium-99m.The SPECT scans were acquired by helical SPECT (4-head scanner with 4 × 9 pinhole collimators), and CT scans by helical CT (55 kVP X-ray source, 1000 ms exposure time in 180 projections over 9 min).1.0 mm pinhole collimators were used.SPECT acquisition was done in eight segments: the first segment was acquired at 15-30 min post injection (frame time of 12s; 9 min acquisition time), followed by seven imaging segments of 30 min each (frame time of 33 s; 24.75 min acquisition time) up until 4 h post-injection.At the end of the imaging procedure, the mouse was culled by cervical dislocation and a sample of the urine analysed by analytical HPLC (method 2).SPECT images were reconstructed at 0.3 mm isotropic voxel size with the HiSPECT (Scivis GmbH) reconstruction software package using standard reconstruction with 35% smoothing and nine iterations.The CT and SPECT images were further processed and analysed using VivoQuant software (inviCRO, USA).(dd, J = 10.9, 7.9 Hz, 4H, Hd/d'), 7.48 (dd, J = 10.9, 7.9 Hz, 4H, Hd/d'), 7.15 (ddd, J = 12.                                             6. Infra-red spectroscopy results:

Figure S2 :
Figure S2: Comparison of the 31 P{ 1 H} NMR signals of DP Ph and DP Tol in chloroform-d.

Figure S8 :
Figure S8: Comparative 1 H NMR spectrum of DP Tol -PSMAt and [Cu(DP Tol -PSMAt)2] + in DMF-d7 with DIPEA base.Coordination leads to broadening of signals corresponding to protons in close proximity to the Cu(I) center (aromatic signals, the two p-tolyl CH3 peaks at approx.2.3 ppm, and most PEGlinker protons).

Figure S10: 1 H
Figure S10: 1 H NMR spectrum of DP Tol in chloroform-d.

Figure S11: 13 C
Figure S11: 13 C NMR spectrum of DP Tol in chloroform-d.

Figure
Figure S14: 31 P{ 1 H} NMR spectrum of DP Tol in chloroform-d.