Post-radiolabeling thioether oxidation to enhance the bioorthogonal reactivity of 18F-tetrazines

Radiolabeled 1,2,4,5-tetrazines are powerful heterocyclic agents for bioorthogonal PET imaging due to their fast cycloaddition with trans-cyclooctenes. However, fluorine-18 radiolabeling of highly reactive tetrazines is often not feasible due to limited compound stability. We demonstrate that post-radiolabeling oxidation of thioether functionalities is a viable and transferable strategy to avoid these limitations, enabling the synthesis of highly reactive 18F-tetrazines.


Introduction
1,2,4,5-Tetrazines (Tz) react rapidly and selectively with alkenes and alkynes in a cycloaddition/cycloreversion cascade reaction first described by Carboni and Lindsey [1].Exceptionally high second order rate constants can be reached using strained alkenes, such as trans-cyclooctene (TCO) [2].In 2008, the group of Joseph M. Fox demonstrated that the reactions between tetrazines and TCOs are biocompatible and can be used as bioorthogonal ligation reactions [3].
While this type of click chemistry has emerged as a valuable tool for a variety of applications [4,5], it is of particular interest to the field of radiolabeling and molecular imaging [6].Here, high reaction rates and selectivity are crucial, given the diminutive concentrations of radiolabeled compounds and short half-lives of commonly used radioisotopes, such as fluorine-18.Enabled by bioorthogonal chemistry, pretargeting strategies decouple accumulation kinetics from the physical half-life of radionuclides: a tagged marker compound is administered and given sufficient time to accumulate in the desired target tissues.Following accumulation and subsequent excretion of non-bound marker, a radiolabeled agent is administered that rapidly binds to the pre-administered compound via a bioorthogonal reaction [4,[7][8][9][10].Although a variety of bioorthogonal reactions have been reported, the ligation between 1,2,4,5-tetrazines and strained dienophiles proved to be especially suitable for radiolabeled agents owing to exceptionally high ligation rates of up to 10 7 M −1 s −1 [9,11].
Martin Wilkovitsch and Dennis Svatunek contributed equally.
Early radiofluorination attempts of tetrazines conducted by Fox and coworkers resulted in degradation and led to the conclusion that 1,2,4,5-tetrazines are unstable under typical 18 F-labeling conditions [12].In 2014, the first radiofluorinated tetrazine, [ 18 F]1, was developed in our lab [13] utilizing a dialkyl-scaffold that improves Tz stability to allow for direct fluorine-18 labeling.However, due to the lower reactivity the in vivo applicability of this probe proved to be limited.Since our first report of an 18 F-labeled Tz, several such compounds have been reported by us and others using different labeling methods and scaffolds [14][15][16][17][18].
Here we investigated a series of derivatives of our original fluorine-18 labeled tetrazine with improved reactivity.Limitations in radiolabeling, caused by high reactivity of some of the tetrazines, can be circumvented by post-radiolabeling modification that boosts the reactivity after the radiolabeling step.We translate this concept of post-radiolabeling activation to highly reactive mono-substituted tetrazines, resulting in an 18 F-labeled tetrazine with exceptional high reactivity.

Results and discussion
To improve on our previously reported tetrazine scaffold, 3-(3-fluoropropyl)-6-methyl-1,2,4,5-tetrazine (1), we exchanged the methyl group against different aryl substituents to enhance click reactivity (Fig. 1a).A range of electron withdrawing and electron donating substituents were chosen to cover a range of different reactivities and polarities, both of which were shown to influence in vivo performance of tetrazines [16].
First, 19 F-labeled tetrazines 2-8 were synthesized applying the procedure developed by Devaraj and coworkers (Fig. 1b) [19].4-Fluorobutanenitrile (9) was used to directly produce the fluorinated compounds.Consecutively, their click reactivity with trans-cyclooctene (10) was investigated in anhydrous 1,4-dioxane at 25 °C using stopped-flow spectrophotometry.Second order rate constants were determined to range from 1.0 to 15 M −1 s −1 (Fig. 1c).As expected, the reactivity of aryl-substituted tetrazines modified with electron-donating groups (Me, OMe, SMe) was found to be similar to previously reported 1.In contrast, electron-withdrawing (trifluoromethyl)phenyl and 4-(methylsulfonyl)phenyl substituents accelerate the reaction by 3-to 5-fold.The highest reactivity is observed for the 2-pyridyl derivative where a combined electronic and distortion lowering effect is at play as recently revealed by our group [20].A computational investigation on the click reactivity of alkyl-aryl substituted tetrazines 2-8 was published previously [21].
Having investigated the reaction rates of the fluorinated aryl-alkyl tetrazines, we next turned to radiolabeling.For direct nucleophilic 18-fluorine labeling of the tetrazines we used the corresponding tosylated precursors 17-22 which were prepared from tetrazine alcohols 11-16 (Fig. 2a).Synthesis of 11-16 was carried out using the same methodology as used for 2-8 [19].Applying this method, the tosylated sulfone 24 could not be obtained.Therefore, this tetrazine was synthesized by oxidation of 21 to 23 using dimethyldioxirane (DMDO) [22] and subsequent tosylation.The p-tolyl tosylate 18 was used for optimization of the radiofluorination method.As the standard [2.2.2]cryptand/K 2 CO 3 method proved to be most suitable for the preparation of [ 18 F]1, the same procedure was applied for [ 18 F]3 resulting in 11.2% yield (determined by radio-HPLC).DMSO as solvent or the use of KHCO 3 instead of potassium carbonate resulted in lower yields (< 6%).When tetrabutylammonium (TBA) hydrogen carbonate in dry acetonitrile was used for labeling a significantly improved radiochemical yield of [ 18 F]3 (22.9%) was noted.Further optimization of the reaction solvent revealed an increased yield of 56% when a 1:1 (v/v) mixture of acetonitrile and 2,3-dimethyl-2-butanol (thexyl alcohol) was used as solvent, which is in agreement with previous reports documenting the positive effect of tertiary alcohols on fluorine-18 radiolabeling (Fig. 2b) [23,24].This method was transferred onto an automated synthesis module (TRACERlab FX FDG synthesis module, GE Healthcare) housed in a shielded hot cell, where in a non-optimized sequence 10 GBq of [ 18 F]3 could be obtained from 181 GBq cyclotron produced fluorine-18 (9.5% decay-corrected radiochemical yield).In this automated procedure, purification of the compounds was achieved by preparative RP-HPLC using 10 mM PO 4 -buffer (pH 6) and acetonitrile as eluents.The HPLC eluate was monitored in series for radioactivity and UV absorption, the product fraction was collected, diluted with water, and passed over a preactivated C18 Sep-Pak Plus cartridge (Waters, Milford, MA, USA).The cartridge was then eluted with 2.5 cm 3 ethanol to obtain the respective [ 18 F]tetrazines.
Applying the optimized radiolabeling conditions to phenyl-Tz-precursor 17, a radiochemical yield of 40% was achieved as determined by radio-TLC and radio-HPLC.As a result, 8.9 GBq of [ 18 F]2 with a specific activity of 99 GBq/ µmol were obtained using the automated synthesizer (6.3% isolated radiochemical yield).All attempts of nucleophilic fluorination with 4-CF 3 -phenyl Tz 19 as precursor failed to obtain at least 1% incorporation yield of [ 18 F]4.Starting from 20, [ 18 F]5 could be isolated in 15.8% decay-corrected radiochemical yield (18% yield as investigated by radio-HPLC) using the TBA-HCO 3 /thexyl-alcohol/acetonitrile system.Attempts to further optimize the reaction conditions for this particular derivative resulted in diminished yields.Using the automated synthesizer, 7.0 GBq of [ 18 F]5 could be obtained, corresponding to a decay-corrected radiochemical yield of 4.9%.For the thiomethyl derivative [ 18 F]6 up to 47.5% incorporation yield were measured by radio-HPLC.11.5 GBq of [ 18 F]6 (8.6% decay-corrected radiochemical yield) with high specific activity (230 GBq/µmol) were obtained using the automated TRACERlab FX FDG synthesis module.Attempts of preparing sulfone [ 18 F]7 by direct radiofluorination of tosylate 24 failed due to decomposition of the tetrazine observed by HPLC analysis.We assume that the high reactivity of this tetrazine derivative renders direct 18 F-labeling impossible.Despite excessive optimization attempts, we were not able to prepare the pyridyl derivative [ 18 F]8 by nucleophilic fluorination of precursor 22 (radiochemical yields < 1%).In addition to the successful preparation of novel radio-tetrazines [ 18 F]2, [ 18 F]3, and [ 18 F]4-6, the radiosynthesis of already described dialkyltetrazine [ 18 F]1 could also be significantly improved to a decay-corrected isolated radiochemical yield of 35% (vs.previously reported 5%, [4]) using the TBA-HCO 3 , thexyl alcohol, and acetonitrile labeling system (Fig. 2c).
In conclusion, while showing lower reactivity, more electron-rich tetrazines can be radiolabeled by direct nucleophilic 18 F-fluorination.Access to radiolabeled tetrazines with higher reactivity is not possible using this method.To address this limitation, we employed a different strategy in which the high reactivity of the tetrazine is established after the radiolabeling step.This post-radiolabeling activation allows stable but lower reactivity tetrazines to be used in the radiolabeling step, but still yields highly reactive radiolabeled compounds after additional activation.Using sulfide [ 18 F]6, which can be radiolabeled in good yields, we prepared the more reactive [ 18 F]7 by oxidation with DMDO (Fig. 3).A solution of [ 18 F]6 in ethanol, as obtained by automated radiosynthesis, was concentrated to dryness, and redissolved in acetone (1 cm 3 ) containing DMDO (~ 40 mM).After short reaction time (60-80 s) volatiles were removed on the rotary evaporator and the residue was purified using RP-HPLC to obtain [ 18 F]7 in 56% radiochemical yield and a specific activity of 56 GBq/µmol thus bypassing the inaccessibility through direct radiolabeling.
Having demonstrated the applicability of post-radiolabeling activation through sulfide oxidation, we turned towards even more reactive compounds.Recently, we described the synthesis of fluorine-18 labeled tetrazines using a two-step reaction sequence in which an azide is first radiofluorinated and then connected to an alkyne-modified tetrazine by copper-catalyzed azide-alkyne cycloaddition (Fig. 4a) [16].Using this strategy, highly reactive 18 F-labeled tetrazines Fig. 4 a Two-step radiolabeling approach applying copper-catalyzed azide-alkyne cycloaddition [16]; b Failed attempt for the synthesis of Tz 26 could be prepared with second order rate constants of up to 230 times higher than tetrazine 1.During that study we found that the copper-catalyzed azide-alkyne cycloaddition between the reactive sulfone-tetrazine 25 and 2-fluoroethylazide is not possible (Fig. 4b).However, we aimed to obtain the highly reactive sulfone Tz 26 by post-radiolabeling oxidation of sulfide 30 (Fig. 5).
The 19 F-labeled reference compound 30 was prepared using Pinner salt 28, formamidine acetate, and hydrazine monohydrate, followed by oxidation with sodium nitrite under acidic conditions to obtain 29 (Fig. 5a), which was modified by copper-catalyzed click reaction with 2-fluoroethylazide.30 could be selectively oxidized to the sulfoxide 31 and eventually to the sulfone 26 (Fig. 5b).As expected, reaction kinetics investigations revealed an increase in second order rate constants in correlation with increased sulfur oxidation state, ranging from 90 M −1 s −1 and 190 M −1 s −1 for 30 and 31, respectively, to 370 M −1 s −1 for 26 (Fig. 5c).
To test the post-radiolabeling activation strategy, 29 was coupled to fluorine-18 labeled [ 18 F]2-fluoroethylazide (Fig. 6a), which was obtained starting from 2-azidoethyl nosylate using a fully automated procedure previously described by our group [25].The highly volatile

Conclusion
We show that post-radiolabeling activation via rapid sulfur oxidation is a viable strategy for the synthesis of highly reactive 18 F-tetrazines, in particular if the respective Tz scaffold/precursor cannot be directly radiofluorinated.To demonstrate this concept, 18 F-labeled phenyl-substituted tetrazines were prepared, including Tz scaffolds with high bioorthogonal reactivity.

Experimental
Unless otherwise noted, reactions were carried out under an atmosphere of argon in air-dried glassware with magnetic stirring.Air-and/or moisture-sensitive liquids were transferred via syringe.All reagents were purchased from commercial sources and used without further purification.Dichloromethane, methanol, THF, diethyl ether, and 1,4-dioxane were dried using PURESOLV-columns (Inert Corporation, USA).Solvents used for flash column chromatography were purchased from Donau Chemie AG (Austria).Dry acetonitrile and dry DMF were obtained from Sigma-Aldrich (Germany) and ACROS Organics (Belgium), respectively, and stored under argon.Thin layer chromatography was performed using TLC plates on aluminum support (Merck, silica gel 60, fluorescent indicator 254).Column chromatography was performed using a BUCHI Sepacore Flash System (2 × BUCHI Pump Module C-605, BUCHI Pump Manager C-615, BUCHI UV Photometer C-635, and BUCHI Fraction Collector C-660) and a Reveleris ® X2 Flash Chromatography/Prep Purification Systems (BUCHI).Silica gel 60 (40-63 µm) was obtained from Merck.A Kinetex ® 5 µm C18 100 Å, AXIA LC column (100 × 30.0 mm, Phenomenex) was used for preparative HPLC.HPLC grade solvents were purchased from VWR (USA).
HRMS analysis was carried out using methanol solutions (concentration: 10 ppm) on an Agilent 6230 LC TOFMS mass spectrometer equipped with an Agilent Dual AJS ESI-Source.The mass spectrometer was connected to a liquid chromatography system of the 1100/1200 series from Agilent Technologies (Palo Alto, CA, USA).The system consisted of a 1200SL binary gradient pump, a degasser, column thermostat, and an HTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland).

General procedure A for the synthesis of compounds 1-6 and 8
A well-blended mixture of 4-fluorobutanenitrile (9, 1-2 eq.), a nitrile (1-7 eq.), and Zn(OTf) 2 (5 mol%) or NiCl 2 (5 mol%) was treated dropwise with hydrazine monohydrate (6-36 eq.) while cooled in an ice bath.The mixture was allowed to warm up to room temperature and stirred at the specified temperature for the specified time.The crude reaction mixture was poured onto ice-water (50 cm 3 ).After addition of NaNO 2 (4 eq.) the solution was acidified with aqueous 2 N HCl solution.The mixture was extracted with EtOAc or Et 2 O, dried over MgSO 4 , filtered and concentrated.The crude product was purified by column chromatography.

General procedure B for the synthesis of compounds 11-16
A well-blended mixture of a nitrile (1 eq.), 4-hydroxybutanenitrile (2 eq.), and NiCl 2 (5 mol%) was treated dropwise with hydrazine monohydrate (9-12 eq.) while cooling in an ice bath.The mixture was allowed to warm up to room temperature and stirred at the specified temperature for the specified time.The crude reaction mixture was poured onto ice-water (50 cm 3 ).After addition of NaNO 2 (4-6 eq.) the solution was acidified with aqueous 2 N HCl solution.The mixture was extracted with EtOAc, dried over MgSO 4 , filtered and concentrated.The crude product was purified by column chromatography.

General procedure C for the synthesis of compounds 17-22 and 24
A mixture of respective butanol-tetrazine (1 eq.) and tosyl chloride (2-4 eq.) was dissolved in dry DCM and cooled in an ice-bath.Dry pyridine (2-4 eq.) was added dropwise, and the mixture was stirred at room temperature for the specified time.The crude reaction mixture was diluted with DCM, washed with water and aqueous 1 N HCl followed by aqueous NaHCO 3 solution.Combined organic layer was dried over MgSO 4 , filtered and concentrated.The crude product was purified by column chromatography.

Click kinetics
Stopped-flow measurements were performed using an SX20-LED stopped-flow spectrophotometer (Applied Photophysics) equipped with a 535 nm LED (optical pathlength 10 mm, full width half-maximum 34 nm) to monitor the characteristic tetrazine visible light absorbance (520-540 nm).The reagent syringes were loaded with tetrazine and TCO (10) solutions and the instrument was primed.Subsequent data were collected in triplicate to sextuplicate for each tetrazine.Reactions were conducted at 25 °C and recorded automatically at the time of acquisition.
Data sets were analyzed by fitting an exponential decay using Prism 6 (Graphpad) to calculate the observed pseudo-first order rate constants that were converted into second order rate constants by dividing through the concentration of excess TCO.TCO (10) was dissolved in dry 1,4-dioxane to reach an approximate concentration of 4 mM.The exact concentration was determined by absorbance titration with DMT tetrazine (extinction coefficient 510 M −1 cm −1 at 520 nm) [30], quantifying the decrease in tetrazine absorbance upon reaction with TCO.

General procedure for manual radiolabeling of [ 18 F]1, [ 18 F]2, [ 18 F]3, [ 18 F]5, and [ 18 F]6
A QMA light cartridge (Waters) was preconditioned with 0.5 M K 2 CO 3 solution (7 cm 3 ) followed by 15 cm 3 water.Cyclotron produced 18 F-fluoride in water (0.5-2.5 cm 3 ) was passed over the cartridge, and the cartridge was eluted with a solution 0.075 M tetrabutylammonium hydrogen carbonate solution, followed by 1 cm 3 of acetonitrile.Volatiles were removed in a stream of argon at 105 °C and residual water was removed by addition and subsequent evaporation of 700 mm 3 dry acetonitrile.The azeotropic drying step was repeated two more times.Precursor was added in the labelling solvent, and the reaction mixture was heated to the indicated temperature for the indicated time span.After cooling of the reaction contents the incorporation yield was investigated by radio-HPLC and/or radio-TLC.In several cases the fluorine-18 tetrazine derivatives were additionally isolated using preparative HPLC.

General procedure for manual radiolabeling of [ 18 F]2, [ 18 F]3, [ 18 F]5, and [ 18 F]6
A remote-controlled synthesis module (TRACERlab™ FXFDG, General Electric Healthcare, Uppsala, Sweden) with a 3 cm 3 glass reactor housed in a hot cell was used for automated labeling experiments.Cyclotron produced no carrier added [ 18 F] fluoride was trapped on a preconditioned (5 cm 3 0.5 M K 2 CO 3 followed by 15 cm 3 water) Waters QMA light cartridge.The radioactivity was eluted with 500 mm 3 0.075 M TBA-HCO 3 solution followed by 1 cm 3 acetonitrile.Volatiles were removed in vacuo at a temperature of 60-120 °C.After cooling to 50 °C a solution of precursor in a mixture of dry acetonitrile and thexyl alcohol was added.The reaction mixture was heated to 93 °C for 8 min followed by cooling to 45 °C and consequent transfer into a vial.The reactor was rinsed with 2 cm 3 DMSO:water = 1:1 (v/v).Combined solutions were injected into a preparative HPLC column (Macherey-Nagel EP 250/16 100-7 C-18, 10 µm, 16 × 250 mm) and eluted using a gradient of phosphate buffer (10 mM pH 6) and acetonitrile at a flow rate of 5 cm 3 /min (0-6 min 10% acetonitrile, 6.01 min 30% acetonitrile, 6.01 → 40 min 30% → 80% acetonitrile).The product fraction was diluted with 50 cm 3 water, prior to passage over a preconditioned (5 cm 3 acetonitrile followed by 15 cm 3 H 2 O) tc18 plus SepPak cartridge (Waters).The cartridge was eluted with 1.5 cm 3 ethanol. 18F-species prepared by automated labeling matched HPLC retention times with authentic standards.

Preparation of [ 18 F]30
First, a solution for the copper catalyzed azide-alkyne cycloaddition (CuAAC) was prepared: An aqueous solution of CuSO 4 •5H 2 O (10 mm 3 100 mg/cm 3 ) was mixed with an aqueous solution of sodium ascorbate (10 mm 3 , 300 mg/ cm 3 ).When the color of the mixture turned yellow a solution of Na 2 BPDS (40 mm 3 , 50 mg/cm 3 ) in water was added.
Following distillation and a 5 min reaction time at room temperature, the mixture was diluted with DMSO/water (1:1, 2 cm 3 ) and purified by semi-preparative HPLC.The collected HPLC fraction was trapped on a Sep-Pak C18 plus short cartridge, which was eluted with 1 cm 3 acetonitrile.41.4 mCi of [ 18 F]30 could be obtained (1.07%non-decaycorrected radiochemical yield) and synthesis duration was 103 min.

Preparation of [ 18 F]26 and [ 18 F]31
Volatiles of the [ 18 F]30 solution in acetonitrile were removed by rotary evaporation, and the residue was treated with 0.5 cm 3 DMDO solution in acetone.After 60 s reaction time, volatiles were removed, and the residue was taken up in 1 cm 3 acetonitrile.Fractions pre-and post-oxidation were analyzed using radio-TLC (SiO 2 , 5% MeOH in DCM).

[ 18 F
]-fluoroethylazide was distilled into a vial already containing tetrazine 29 and a catalytic mixture of copper sulfate, sodium ascorbate, and disodium bathophenanthroline disulfonate (Na 2 BPDS) in dry DMF.The reaction was carried out for 103 min at room temperature until the mixture was diluted with DMSO/water (v/v, 1/1), purified by HPLC and the collected fraction was trapped on a Sep-Pak C18 plus cartridge which was eluted with acetonitrile.41.4 mCi of [ 18 F]30 were obtained, corresponding to 1.1% non-decaycorrected radiochemical yield.Further optimization of the click radiolabeling step was not carried out, as we focused on showing subsequent post-radiolabeling oxidation to the respective sulfone [ 18 F]26 and sulfoxide [ 18 F]31.Therefore, [ 18 F]30 was treated with DMDO solution in acetone (~ 60 mM).After 1 min reaction time at room temperature, the mixture was concentrated, and the residue redissolved in acetonitrile.Radio-TLC revealed that the radiofluorinated sulfide [ 18 F]30 already contained traces of the sulfoxide and sulfone tetrazines [ 18 F]31 and [ 18 F]26, respectively, most likely due to air oxidation.Analysis of the DMDO-treated mixture confirmed complete conversion of the sulfide

[ 18 F
]31 to a mixture of the sulfoxide [ 18 F]31 and the sulfone [ 18 F]26 after a reaction time of 1 min (Fig.6b).