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. 2023 Dec 18;62(50):20608-20620.
doi: 10.1021/acs.inorgchem.3c00426. Epub 2023 Mar 27.

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

Ingebjørg N Hungnes  1 , Truc Thuy Pham  1 , Charlotte Rivas  1 , James A Jarvis  2 , Rachel E Nuttall  1   3 , Saul M Cooper  4 , Jennifer D Young  1 , Philip J Blower  1 , Paul G Pringle  3 , Michelle T Ma  1
Affiliations

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

Ingebjørg N Hungnes et al. Inorg Chem. .

Abstract

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.

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Conflict of interest statement

The authors declare the following competing financial interest(s): A PCT application describing chemical technology included in this manuscript has recently been filed.

Figures

Scheme 1
Scheme 1. Preparation of [Cu(DPPh-Bn)2]+
(i) CuCl; (ii) 64CuCl2.
Scheme 2
Scheme 2. Preparation of DPPh
Scheme 3
Scheme 3. Mo Complexes of DPPh and DPTol Derivatives
Scheme 4
Scheme 4. Preparation and Complexation of DP-PSMAt Conjugates
(i) [ReO2I(PPh3)2] in DMF; (ii) [NtBu4][99gTcOCl4] in DMF; (iii) 99mTcO4–, SnCl2, sodium tartrate, in water (pH 8).
Figure 1
Figure 1
31P{1H} NMR spectra of (a-i) DPPh-PSMAt, (a-ii) DPTol-PSMAt, (b-i) [natReO2(DPPh-PSMAt)2]+, (b-ii) [natReO2(DPTol-PSMAt)2]+, (c-i) [natCu(DPPh-PSMAt)2]+, and (c-ii) [natCu(DPTol-PSMAt)2]+. Signals corresponding to cis-[natReO2(DPPh-PSMAt)2]+ and cis-[natReO2(DPTol-PSMAt)2]+ are highlighted in blue.
Figure 2
Figure 2
DP-PSMAt derivatives reacted with [NtBu4][99gTcOCl4] to yield [99gTcO2(DP-PSMAt)2]+), which consists of both cis and trans isomers. (a-i) UV chromatogram of [99gTcO2(DPPh-PSMAt)2]+); (a-ii) MS chromatogram of [99gTcO2(DPPh-PSMAt)2]+; (b-i) UV chromatogram of [99gTcO2(DPTol-PSMAt)2]+; (b-ii) MS chromatogram of [99gTcO2(DPTol-PSMAt)2]+. For HPLC method 8, see the SI.
Figure 3
Figure 3
Putative cis and trans isomers of (a) [99mTcO2(DPPh-PSMAt)2]+ and (b) [99mTcO2(DPTol-PSMAt)2]+, separated on a shallow analytical C18 HPLC gradient. The radioactive signals were coincident with the UV signals of characterized (c) [natReO2(DPPh-PSMAt)2]+ and (d) [natReO2(DPTol-PSMAt)2]+. For HPLC method 10, see the SI.
Figure 4
Figure 4
Maximum intensity projections of healthy male SCID Beige mice injected with (a-i) [99mTcO2(DPPh-PSMAt)2]+ and (b-i) [99mTcO2(DPTol-PSMAt)2]+ from 15 min to 4 h postinjection. Regions of interest were selected on VivoQuant (inviCRO, LLC, Boston, MA), and percentages of injected dose per milliliter (% ID/mL) were calculated for each of (a-ii) [99mTcO2(DPPh-PSMAt)2]+ (n = 1) and (b-ii) [99mTcO2(DPTol-PSMAt)2]+ (n = 1). K = kidneys; B = bladder.
Figure 5
Figure 5
Radio-HPLC analysis of urine from healthy male SCID Beige mice intravenously administered with either (a) [99mTcO2(DPPh-PSMAt)2]+ or (b) [99mTcO2(DPTol-PSMAt)2]+. Radio-HPLC shows that both radiotracers are highly metabolically stable and are excreted intact. For HPLC method 2, see the SI.
Scheme 5
Scheme 5. Reaction of DP-PSMAt Conjugates with Cu+
(i) [Cu(MeCN)4][PF6] in mixtures of water and acetonitrile; (ii) solutions of 64Cu2+ with a large excess of DP-PSMAt conjugate in an aqueous solution.
Figure 6
Figure 6
HPLC chromatograms of (a) [Cu(DPPh-PSMAt)2]+ and (b) [Cu(DPTol-PSMAt)2]+. DP-PSMAt derivatives were reacted with solutions of either [natCu(MeCN)4][PF6] (blue traces) or 64Cu2+ (red traces), with UV signals for [natCu(DP-PSMAt)2]+ derivatives coincident with radioactive signals for [64Cu(DP-PSMAt)2]+ (with slight differences in the retention times a result of the configuration of the UV and scintillation detectors in series). Analytical radio-HPLC analysis revealed that both radiotracers were stable in serum over 24 h (black traces). For HPLC method 2, see the SI.
See this image and copyright information in PMC

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