Development of ¹⁸F-Labeled hydrophilic trans-cyclooctene as a bioorthogonal tool for PET probe construction

Abstract

We report the development of a hydrophilic ¹⁸F-labeled a-TCO derivative [¹⁸F]3 (LogP = 0.28) through a readily available precursor and a single-step radiofluorination reaction (RCY up to 52%). We demonstrated that [¹⁸F]3 can be used to construct not only multiple small molecule/peptide-based PET agents, but protein/diabody-based imaging probes in parallel.

Bioorthogonal reactions are chemical reactions that occur in biological environments without interfering with existing biochemical processes.¹ As an inverse electron-demand Diels-Alder (IEDDA) cyclo-addition, ligation between 1,2,4,5-tetrazine and strained dienophiles represents one of the fastest types of bio-orthogonal reactions and has been shown to be a valuable tool in conjugation chemistry.^1,2 In particular, trans-cyclooctene (TCO) has risen to the forefront as a dienophile, as the TCO-tetrazine ligation is the fastest known bioorthogonal reaction with reaction rates typically ranging from 10⁴ to 10⁶ M⁻¹·s⁻¹.^2,3

Numerous applications of tetrazine ligation have been reported for intracellular imaging and chemical probe assembly, chemical proteomics, nuclear medicine, and drug release.^4-9 From a tumor imaging perspective, tetrazine ligation has been successfully used for radiolabeling of antibodies that are difficult to label directly with short-lived radioactive isotopes.^10,11 Tetrazine ligation has allowed for a pretargeted method of tumor imaging that significantly improves contrast.¹² Over the past decade, we and others have focused on the development of ¹⁸F-labeled PET agents based on tetrazine ligation with TCO. Efforts have been made to introduce ¹⁸F into both tetrazine and TCO derivatives. Some previous attempts at direct ¹⁸F labeling of tetrazine derivatives using [¹⁸F]Kryptofix or [¹⁸F]TBAF have been reported by Mikula’s group, Airaksinen’s group, and us.^13-16 Herth’s group reported their recent studies of direct ¹⁸F labeling of tetrazine derivatives and showed dramatically increased RCYs under less basic conditions or Cu-mediation.^17-20 Instead of direct fluorination of tetrazine, Keinänen et al. reported an indirect tetrazine labeling method by conjugation with 5-deoxy-5-[¹⁸F]fluoro-D-ribose ([¹⁸F]FDR) in almost quantitative yields.²¹ For ¹⁸F-labeled TCO derivatives, the radiofluorination procedure is relatively straightforward and high-yielding. ¹⁸F-labeled TCOs, such as strained trans-cyclooctene (s-TCO), trans-5-oxocene (oxo-TCO), and dioxolane-fused trans-cyclooctene (d-TCO) for the rapid construction of PET agents, has been developed (Fig. 1A), with [¹⁸F]s-TCO having the fastest reaction rates.^7,22,23 Despite these advances, the hydrophobic nature of the TCO motif can lead to high background and slow clearance in vivo.²² Although we can exploit the hydrophobic nature of the resulting PET agents to enhance tumor accumulation in some cases, improved hydrophilicity is often preferred to accelerate clearance from normal organs/tissues.^24,25 Previously, we reported [¹⁸F]oxoTCO, which displays greatly improved tumor-to-background contrast due to the increased hydrophilicity of its conjugates.^7,26 (Fig. 1B) Despite the promising results, the synthesis of the [¹⁸F]oxoTCO precursor is lengthy and has an overall low yield.

Fig. 1.

Example of our previous investigation of radiolabeled (A) [¹⁸F]s-TCO and (B) [¹⁸F]oxo-TCO in tetrazine ligation. (C) The development of ¹⁸F-labeled a-TCO for tetrazine ligation.

Recently, 5-axial-hydroxy-5-alkyl-trans-cyclooctene (a-TCO) derivatives have been developed and applied in live-cell fluorescence imaging and proteomic applications.²⁷ Compared to previous generations of TCO agents, the hydroxyl group on the a-TCOs significantly improves the physicochemical properties, making the analogs suitable for applications in biomolecular conjugation, such as protein modification.²⁷ Moreover, in terms of reaction kinetics, a-TCO maintains a high rate constant (k₂= 150,000 ± 8,000 M⁻¹s⁻¹), which is higher than the parent TCO.^22,27 Taking advantage of the synthetic scalability, fast reactivity, and improved physicochemical properties, we herein report the development of an ¹⁸F-labeled a-TCO as a bioorthogonal tool for the construction of positron emission tomography (PET) Imaging probes. (Fig. 1C)

To evaluate whether ¹⁸F-labeled a-TCOs could be prepared as a prosthetic group for PET probe construction, an a-TCO precursor with a tosylate leaving group for fluorination was first synthesized. Briefly, compound 1 was synthesized from amino-PEG₄-alcohol through amino protection, tosylation, and Boc deprotection. Conjugation with a-TCO NHS ester was then performed to obtain the a-TCO precursor 2 by simple amide 6more stable when stored in solution compared to neat. The compound was prepared as a stock solution in DCM at a concentration of 35 μg/μL and remained stable at −20 °C over three months. Repeated warm-up/freeze cycles did induce a small amount of degraded byproduct over time. The standard compound [¹⁹F]3 was synthesized by direct fluorination of 2 in the presence of Kryptofix222 and potassium fluoride at 45 °C.

The ¹⁸F labeling reaction (Scheme 2) conditions were first evaluated to obtain the highest radiochemical yield (RCY). Reaction solvent, reaction time, and temperature were investigated. For labeling reactions, the DCM solution of 2 was used directly without removing the solvent. Azeotropically dried [¹⁸F]TBAF was prepared according to the previous report and then redissolved in MeCN.²⁸ The optimal reaction time was 10 minutes at 80 °C in dichloromethane and acetonitrile (RCY = 52%, Table S1, entry 9). Increasing the reaction time to 30 min led to a significantly reduced yield and the formation of side products. We also performed a large-scale preparation of [¹⁸F]3 was performed in a hot cell with a semi-prep HPLC using the optimal condition. Briefly, 700 μg 2 in stock solution was mixed with 15.5 Gbq [¹⁸F]TBAF (approximately 100 μL in total volume). The mixture was heated at 80 °C for 10 minutes and then quenched with 1 mL of 10% acetonitrile aqueous solution. The reaction mixture was then passed through an alumina light cartridge to remove unreacted [¹⁸F]TBAF, and the radioactivity (8.62 Gbq) was then purified by semi-preparative HPLC. 1.96 Gbq [¹⁸F]3 was obtained with >95% radiochemical purity. The RCY was determined to be 27% (decay-corrected). The experimental LogP of [¹⁸F]3 is 0.28, which is indeed more hydrophilic than previously reported ¹⁸F-labeled TCOs, such as [¹⁸F]-oxoTCO (LogP = 0.57) using a partitioning experiment.^7,22

Scheme 2.

¹⁸F labeling reaction to obtain [¹⁸F]3.

Tetrazine-modified small molecules 4 and 5 were synthesized, targeting prostate-specific membrane antigen (PSMA), and fibroblast activation protein (FAP), respectively.^29,30 (Fig. 2A & B) The [¹⁸F]3 solution obtained from HPLC was added directly to the tetrazine-modified small-molecule ligand solutions. The mixtures were thoroughly mixed and then injected into HPLC within a couple of minutes to obtain pure PET imaging agents, [¹⁸F]6 and [¹⁸F]7. The RCYs of the ligation step ranged from 38% to 44%.

Fig. 2.

Ligation of [¹⁸F]3 with tetrazine-modified small molecules (A) 4 (targeting PSMA) to generate [¹⁸F]6 and (B) 5 (targeting FAP) to generate [¹⁸F]7. PET imaging of (C) [¹⁸F]6 at 0.5 h p.i. in PC3-PIP mouse model and (D) [¹⁸F]7 at 4.0 h p.i. in U87-MG mouse model.

We performed imaging studies and compared the results with some known compounds. Compound [¹⁸F]6 was injected into PC3-PIP tumor-bearing mice for in vivo PET imaging studies. (Fig. 2C & Table S3) The tumor/kidney ratio was 1.79 at 3 h p.i. For comparison, we also evaluated [⁶⁸Ga]Ga-PSMA-11 imaging on PC3-PIP tumor-bearing mouse models, and the tumor/kidney ratios are 0.135, 0.134, and 0.169 at 0.5, 1.5, and 4 h p.i., respectively.³¹ In another study, Banerjee et al. reported tumor/kidney ratios of about 0.2 for [⁶⁸Ga]Ga-PSMA-11 in the imaging of PC3-PIP tumor-bearing mouse models at 1, 2, and 3 h p.i., although the tumor uptake was about 26 %ID/g.³² The comparison of some PSMA agent tumor/kidney ratios is listed in Table S2. The comparison suggests that compared to [⁶⁸Ga]Ga-PSMA-11, [¹⁸F]7 has better tumor-to-kidney contrast, potentially partially due to easier wash-out from the kidneys after the tetrazine ligation with [¹⁸F]3. However, side by side comparison is needed before a conclusion is drawn. For [¹⁸F]7, we injected it in U87 tumor-bearing mice. (Fig. 2D & Table S4) The average tumor uptake of was 9.77 ± 4.41, 9.75 ± 3.88, and 8.10 ± 2.77 at 0.5, 1.5, and 4 h p.i., respectively. Our results indicate that [¹⁸F]7 has a longer retention time on U87 tumors than some known tracers, such as [¹⁸F]AlF-NOTA-FAPI-04. Tumor uptake of [¹⁸F]AlF-NOTA-FAPI-04 peaked at 1 h p.i. (15 %ID/g) and rapidly decreased to one-third of the peak value (approximately 5 %ID/g).³³ However, unlike [¹⁸F]AlF-NOTA-FAPI-04, [¹⁸F]7 has relatively high uptake and slow clearance in the kidney, liver, heart, and muscle, which may be caused by the long PEG linker. Further evaluation of the impact of PEG length on organ uptake is needed.

High molecular weight biological macromolecules, such as single-stranded oligonucleotides, peptides, and proteins (including antibody fragments), are of great interest in the imaging field. [¹⁸F]3’s fast reaction rate, coupled with favorable physicochemical properties, may lead to unique applications in the generation of ¹⁸F-labeled proteins. Because proteins can be sensitive to organic solvents, we chose to use a C18 cartridge (Waters) to remove ACN from [¹⁸F]3 in the HPLC purification process. Like other reports³⁴, we observed radiolysis during the QC, which could be caused by the high activity concentration during the cartridge trapping step. We then dissolved [¹⁸F]3 in PBS buffer to form a stock solution that could be aliquoted to different tetrazine-modified proteins (mouse serum albumin or MSA 8, anti-CD8 diabody 9, anti-CD4 diabody 10, and a HER2-binding protein ligand 11) in parallel. The resulting radiolabeled proteins (¹⁸F labeled MSA [¹⁸F]12, ¹⁸F labeled anti-CD8 diabody [¹⁸F]13, ¹⁸F labeled anti-CD4 diabody [¹⁸F]14, and ¹⁸F labeled HER2-binding protein ligand [¹⁸F]15) were purified on PD-10 columns to remove unreacted small molecules. These radiolabeled proteins were then analyzed by SDS-PAGE (Fig. 3). Since the ¹⁸F labeled 13-15 has a high molar activity (47.69 – 85.06 GBq/μmol), unlabeled protein samples were added to a separate gel to detect the signal from Coomassie Blue. As shown in Fig 5, multiple ¹⁸F labeled proteins can be obtained in parallel with high purity. The autoradiography and Coomassie blue staining correlated well.

Fig. 3.

Coomassie blue stained SDS-PAGE gel (left) of tetrazine-modified proteins and their corresponding radiolabeled products. The autoradiography (right) was also performed for the SDS-PAGE gel for the radiolabeled products, which were barely visualized by Coomassie blue staining due to trace amounts.

As a working example, we investigated the infiltration of CD 8⁺ T cells into tumors using ¹⁸F-labeled anti-CD8 diabody. The [¹⁸F]13 was injected into B16F10 tumor-bearing mice for in vivo PET imaging study. (Fig. 4 & Table S5) As expected, like other anti-CD8 tracers,^35-37 high spleen uptake was observed with both [⁸⁹Zr]Zr-labeled anti-CD8 and [¹⁸F]13 imaging, indicating accumulation of CD 8⁺ T cells in the spleen. Compared with the previously reported [⁸⁹Zr]Zr-labeled anti-CD8 diabody³⁵, we did not observe high bone uptake with [¹⁸F]14 during the imaging course, suggesting that the labeling motif was stable in vivo. Radioactivity accumulation in tumor tissue was also observed due to the infiltration of CD 8⁺ T cells. The tumor was well visualized at 3 hours post-injection. The tumor/muscle ratios of CD 8⁺ cells for [¹⁸F]13 were 5.63, 4.28, and 6.18 at 0.5, 1.5, and 3 h p.i., respectively.

Fig. 4.

PET imaging of [¹⁸F]13 at 0.5 h p.i. in B16F10 mouse model. The spleen is indicated with a white arrow, and the tumor region is indicated with a red arrow.

In conclusion, we report the development of the compound [¹⁸F]3 as a versatile radiolabeling tool to construct PET agents on both small molecules and proteins. Radiochemical labeling was optimized, and large-scale radiolabeling was also tested, allowing for multi-dose preparations from a single batch of [¹⁸F]3. In vivo imaging demonstrated that the corresponding PET agents could achieve prominent tumor accumulation. Initial comparison with previously reported probes warrants further investigation of the newly constructed agent to understand its normal organ distribution, metabolism, toxicity, etc.

Scheme 1.

Synthesis of a-TCO precursor 2 and standard 3. Reaction conditions: (a) (Boc)₂O, DCM, room temperature, 1 h; (b) 4-DMAP, Et₃N, 4-toluenesulfonyl chloride, DCM, 0 °C to room temperature, overnight; (c) TFA, DCM, 25 min; (d) a-TCO NHS ester, Et₃N, DCM, 30 min, 97% yield based on 1; (e) KF, Kryptofix^® 222, K₂CO₃, ACN, 45 °C, 1 h, 61% yield by NMR.