Dataset Information

Novel [¹⁸F]-labeled thiol for the labeling of Dha- or maleimide-containing biomolecules.

ABSTRACT:

Background

Prosthetic approach for the radiolabeling of biologics with fluorine-18 is a robust strategy and has been employed for many years. It requires fast, biocompatible and selective reactions suited to these fragile molecules. Michael addition of a nucleophilic thiol moiety on α,β-unsaturated carbonyl entities is an interesting compromise between simplicity of preparation of the prosthetic reagent and control of the selectivity of the addition. The α,β-unsaturated carbonyl entity of the biologic can easily be generated by addition of a maleimide function using adequate heterobifunctional linkers or generated by selective modification of a cysteine residue leading to a dehydroalanine moiety. We report here the design, synthesis and radiosynthesis of a new fluoropyridine-based thiol [¹⁸F]FPySH and its conjugation via Michael addition on model dehydroalanine- or maleimide-containing biologics.

Results

The preparation of cold reference and labeling precursor of [¹⁸F]FPySH was achieved and its radiosynthesis was fully automated, enabling production of the thiol prosthetic group with a 7 ± 2.1% radiochemical yield after two steps. The conjugation of [¹⁸F]FPySH to two model Dha-containing molecules was then carried out in reducing conditions, yielding the corresponding adducts in 30-45 min reaction time. Furthermore, [¹⁸F]FPySH was employed to radiolabel the maleimide-modified c(RGDfK) peptide, affording the radiofluorinated analogue in 15 min.

Conclusion

We have developed an original [¹⁸F]-labeled thiol for site-selective conjugation and radiolabeling of Dha or maleimide-containing biomolecules of interest. Labeling of three model compounds was successfully carried out and gave the expected radiofluorinated adducts in less than 45 min, thus compatible with fluorine-18 half-life.

SUBMITTER: Richard M

PROVIDER: S-EPMC8986957 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Publications

Novel [<sup>18</sup>F]-labeled thiol for the labeling of Dha- or maleimide-containing biomolecules.

Richard Mylène M Hinnen Françoise F Kuhnast Bertrand B

EJNMMI radiopharmacy and chemistry 20220406 1

<h4>Background</h4>Prosthetic approach for the radiolabeling of biologics with fluorine-18 is a robust strategy and has been employed for many years. It requires fast, biocompatible and selective reactions suited to these fragile molecules. Michael addition of a nucleophilic thiol moiety on α,β-unsaturated carbonyl entities is an interesting compromise between simplicity of preparation of the prosthetic reagent and control of the selectivity of the addition. The α,β-unsaturated carbonyl entity o ...[more]

PMID: 35384570

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Project description:The efficient radiosynthesis of biomolecules utilizing minute quantities of maleimide substrate is important for availability of novel peptide molecular imaging agents. We evaluated both 3-18F-fluoropropane-1-thiol and 2-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)ethane-1-thiol (18F-fluoro-PEG4 thiol) as prosthetic groups for radiolabeling under physiological conditions. The precursor employed a benzoate for protection of the thiol and an arylsulfonate leaving group. The radiofluorination was fully automated on an Eckert & Ziegler synthesis system using standard Kryptofix222/K2CO3 conditions. In order to minimize the amount of biological molecule required for subsequent conjugation, the intermediates, S-(3-18F-fluoropropyl) benzothioate and 18F-fluoro-PEG4 benzothioate, were purified by HPLC. The intermediates were isolated from the HPLC in yields of 37-47% and 28-35%, respectively, and retrieved from eluate using solid phase extraction. Treatment of the benzothioates with sodium methoxide followed by acetic acid provided the free thiols. The desired maleimide substrate in acetonitrile or phosphate buffer was then added and incubated at room temperature for 15 min. The final radiolabeled bioconjugate was purified on a separate HPLC or NAP-5 column. Maleimides utilized for the coupling reaction included phenyl maleimide, an Evans Blue maleimide derivative, a dimeric RGDfK maleimide (E[c(RGDfK)]2), two aptamer maleimides, and PSMA maleimide derivative. Isolated radiochemical yields (non-decay corrected) of maleimide addition products based on starting 18F-fluoride ranged from 6 to 22% in a synthesis time of about 90 min. 18F-thiol prosthetic groups were further tested in vivo by conjugation to E[c(RGDfK)]2 maleimide in a U87MG xenograft model. PET studies demonstrated similar tumor accumulation of both prosthetic groups. 18F-fluoro-PEG4-S-E[c(RGDfK)]2 displayed a somewhat favorable pharmacokinetics compared to 18F-fluoropropyl-S-E[c(RGDfK)]2. Bone uptake was low for both indicating in vivo stability.

Project description:The accumulation of α-synuclein aggregates (α-syn) in the human brain is an occurrence common to all α-synucleinopathies. Non-invasive detection of these aggregates in a living brain with a target-specific radiotracer is not yet possible. We have recently discovered that the inclusion of a methylenedioxy group in the structure of diarylbisthiazole (DABTA)-based tracers improves binding affinity and selectivity to α-syn. Subsequently, complementary in silico modeling and machine learning (ML) of tracer-protein interactions were employed to predict surface sites and structure-property relations for the binding of the ligands. Based on this observation, we developed a small focused library of DABTAs from which 4-(benzo[d][1,3]dioxol-5-yl)-4'-(3-[18F]fluoro-4-methoxyphenyl)-2,2'-bithiazole [ 18 F]d 2, 6-(4'-(3-[18F]fluoro-4-methoxyphenyl)-[2,2'-bithiazol]-4-yl)-[1,3]dioxolo[4,5-b]pyridine [ 18 F]d 4, 4-(benzo [d][1,3]dioxol-5-yl)-4'-(6-[18F]fluoropyridin-3-yl)-2,2'-bithiazole [ 18 F]d 6, and 6-(4'-(6-[18F]fluoropyridin-3-yl)-[2,2'-bithiazol]-4-yl)-[1,3]dioxolo[4,5-b]pyridine [ 18 F]d 8 were selected based on their high binding affinity to α-syn and were further evaluated. Binding assay experiments carried out with the non-radioactive versions of the above tracers d 2, d 4, d 6, and d 8 showed high binding affinity of the ligands to α-syn: 1.22, 0.66, 1.21, and 0.10 nM, respectively, as well as excellent selectivity over β-amyloid plaques (Aβ) and microtubular tau aggregates (>200-fold selectivity). To obtain the tracers, their precursors were radiolabeled either via an innovative ruthenium-mediated (SNAr) reaction ([ 18 F]d 2 and [ 18 F]d 4) or typical SNAr reaction ([ 18 F]d 6 and [ 18 F]d 8) with moderate-to-high radiochemical yields (13% - 40%), and high molar activity > 60 GBq/μmol. Biodistribution experiments carried out with the tracers in healthy mice revealed that [ 18 F]d 2 and [ 18 F]d 4 showed suboptimal brain pharmacokinetics: 1.58 and 4.63 %ID/g at 5 min post-injection (p.i.), and 1.93 and 3.86 %ID/g at 60 min p.i., respectively. However, [ 18 F]d 6 and [ 18 F]d 8 showed improved brain pharmacokinetics: 5.79 and 5.13 %ID/g at 5 min p.i.; 1.75 and 1.07 %ID/g at 60 min p.i.; and 1.04 and 0.58 %ID/g at 120 min p.i., respectively. The brain uptake kinetics of [ 18 F]d 6 and [ 18 F]d 8 were confirmed in a dynamic PET study. Both tracers also showed no brain radiometabolites at 20 min p.i. in initial in vivo stability experiments carried out in healthy mice. [ 18 F]d 8 seems very promising based on its binding properties and in vivo stability, thus encouraging further validation of its usefulness as a radiotracer for the in vivo visualization of α-syn in preclinical and clinical settings. Additionally, in silico and ML-predicted values correlated with the experimental binding affinity of the ligands.

Dataset Information

Novel [¹⁸F]-labeled thiol for the labeling of Dha- or maleimide-containing biomolecules.

Background

Results

Conclusion

Publications

Novel [<sup>18</sup>F]-labeled thiol for the labeling of Dha- or maleimide-containing biomolecules.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Novel [18F]-labeled thiol for the labeling of Dha- or maleimide-containing biomolecules.

Background

Results

Conclusion

Publications

Novel [<sup>18</sup>F]-labeled thiol for the labeling of Dha- or maleimide-containing biomolecules.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Novel [¹⁸F]-labeled thiol for the labeling of Dha- or maleimide-containing biomolecules.