**1. Introduction**

Fluorine-18 is an ideal radionuclide for labeling of radiopharmaceuticals for positron emission tomography (PET), due to its nuclear and physical characteristics, including the relatively long half-life (109.7 min), the low energy levels of emitted positrons (*E*max = 0.635 MeV), and high positron decay probability (97%) [1]. Fluorine-18 can be produced via the 18O(p,n)18F reaction, by irradiating 18O-enriched water with protons, yielding high molar activity [18F]fluoride, in aqueous solutions. Several advances in 18F-fluorinations, such as metal-mediated (e.g., Pd, Cu, Ag) aromatic, aliphatic, and aryl boronic ester radiofluorinations, as well as TiO2-catalyzed 18F-fluorinations in aqueous

media, have been recently presented [2–4]. The incorporation of nucleophilic [18F]fluoride into molecules generally requires alkaline conditions and elevated temperatures. Faster, milder, and more selective radiolabeling methodologies are desired, especially for the radiolabeling of compounds sensitive to temperature and higher pH. Fast and efficient catalyst-free click-reactions, such as the inverse electron-demand Diels-Alder (IEDDA) reaction, have been applied as effective tools for the selective incorporation of radiolabels, such as 18F, into bio- and macromolecules, via small radiolabeled prosthetic groups. Other widely known click-reactions, such as the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and the stainpromoted azide-alkyne cycloaddition (SPAAC) have been the starting point for the modification of chemoselective biomolecules [5]. The CuAAC reaction was first reported in 2002 by Sharpless et al. [6] and Meldal et al. [7]. The aim of these studies was to utilize the CuAAC reaction for the formation of an enormous variety of five-membered heterocycles, triazoles, and peptide derivatives. The use of CuAAC led to the investigation on the utility of these highly efficient reactions for the labeling of biomolecules in living systems. However, the toxicity of copper limited the feasibility of CuAAC in biological applications. SPAAC was developed in 2004 by Bertozzi et al. who demonstrated the chemical modification of live Jurkat cells with an azide-modified sugar for the subsequent conjugation of alkyne-biotin for fluorescent labeling with FITC-avidin, without any apparent decline in cell viability [8]. Since then, SPAAC has served as a catalyst-free alternative to overcome the cytotoxicity concerns of the CuAAC reaction. However, both CuAAC and SPAAC have relatively slow reaction kinetics, which renders the reactions unsuitable for labeling of biomolecules in a living system, for applications lile in vivo pretargeting. In 1959, Lindsey et al. reported the ability of tetrazines to react chemoselectively with unsaturated compounds through a 1,4-cycloaddition reaction [9]. These findings introduced a new and highly reactive bioorthogonal IEDDA click-reaction as a pivotal tool for synthetic modification of biomolecules.

The bioorthogonal IEDDA-reaction has been successfully used for various pretargeted in vivo radiolabeling applications [10–12]. Pretargeted imaging has found exceptional utility with imaging agents with slow pharmacokinetics, such as antibodies and nanomaterials, which when directly radiolabeled would require the use of long-lived radioisotopes (such as 89Zr or 111In, both with ~3-day physical half-lives), to track their biodistribution in vivo. In the pretargeted approach, the targeting vector (such as an IgG antibody) is first modified by one reactant of the IEDDA reaction, and allowed to distribute in the body after administration. Next, it is tracked using the other reactant that is radiolabeled with a short-lived radioisotope, with improved image contrast and lower radiation burden to the subject [13,14]. The fastest IEDDA-reaction reported so far is from the conjugation between tetrazine (Tz) and trans-cyclooctene (TCO) (*<sup>k</sup>* <sup>≈</sup> <sup>10</sup><sup>6</sup> <sup>M</sup>−<sup>1</sup> <sup>s</sup><sup>−</sup>1) [5], rendering this reactive pair of utmost interest in the fields of chemical biology, nuclear imaging, and radiotracer development. However, the sensitivity of tetrazines towards alkaline conditions and elevated temperature renders the direct radiolabeling of tetrazines with fluorine-18 challenging. Therefore, the use of prosthetic groups for radiolabeling, such as the glycoconjugate [18F]-5-fluoro-5-deoxyribose (FDR) or Al[18F]F [15–17], is necessary to ensure that radiolabeling conditions preserve the reactivity of the Tz.

The silicon-fluoride acceptor (SiFA) chemistry relies on 19F/ 18F-isotopic exchange for introducing fluorine-18 into radiotracers, and has emerged as a fast and mild radiolabeling tool, especially for sensitive molecules [18]. The small lipophilic SiFA compounds mainly utilized in the radiolabeling of larger constructs such as peptides, proteins, and nanoparticles, in most cases have demonstrated excellent stability against in vivo defluorination [19–23]. The lipophilic character of the SiFA-derivatives can be used to tailor the pharmacokinetics of biomolecular tracers. To our knowledge, there is only one compound containing SiFA bound to a tetrazine, SiFA-OTz, which has been reported to date, but its enzymatic stability in vitro and in vivo has not yet been studied [24]. Nevertheless, the tracer SiFA-OTz demonstrated good stability under the radiolabeling conditions. We have reported the development of highly stable and highly hydrophilic 18F-tetrazines from sugar analogues, such as [ 18F]fluorodeoxyribose ([18F]FDR-Tz) and 2-deoxy-2-[18F]fluoro-*D*-glucose ([18F]FDG-Tz) [17,25]. We

also previously studied the feasibility of utilizing the 18F-FDR-Tz for the in vivo IEDDA pretargeting of antibodies and nanoparticles and were able to prove this approach to be highly successful [26,27].

Here, we investigated the use of a SiFA as a reaction strategy for the [18F]fluorination of tetrazines, under mild reaction conditions, with the possibility to yield a more hydrophobic tetrazine variant for the regulation of the pharmacokinetics of biomolecules labeled with [18F]SiFA-Tz. The aim of this study was to investigate [18F]SiFA-Tz as a standalone tracer, to reveal its potential for pretargeted imaging and its applicability for the rapid in vitro radiolabeling of TCO-containing biomolecules, under physiological conditions.

#### **2. Results**

#### *2.1. Chemistry*

SiFA-Tz (**6**) was synthesized via a three-step route, providing good yields for each step (Scheme 1) [14]. The first step comprised of an amide coupling reaction under argon, between a carboxylic acid and an amine, forming the t-Boc protected aminooxy-tetrazine **3**, in 65% yield after silica gel column chromatography purification. The next reaction step was the deprotection of **3** with hydrochloric acid in methanol, to give **4** as a pink solid in cold diethyl ether in 65% yield. This hydrochloric salt intermediate was used such for the next reaction, the oxime bond formation between compounds **4** and **5** generating an imine double bond such as *E*- and *Z*-isomers of SiFA-Tz (**6**) [28]. The reaction mixture was purified with semi-preparative HPLC, providing the desired product **6** in 90% yield. 1H-NMR, 13C-NMR, 19F-NMR spectra and ESI-TOF-MS were acquired for characterization of the final product, SiFA-Tz (**6**).

**Scheme 1.** Synthesis of the precursor SiFA-Tz (**6**). Reagents and conditions: (**a**) HATU, DIPEA, DMF, room temperature, 20 h, Argon (65%), (**b**) 1 M HCl Et2O 25 ◦C, 24 h, MeOH (65%), and (**c**) Aniliniumacetate-buffer pH 4.6, 25 ◦C, 15 min (90%). (HATU; 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate, DIPEA; N,N-Diisopropylethylamine, DMF; Dimethylformamide).

Bovine serum albumin (**7**) was functionalized with *N*-hydroxysuccinimide (NHS) ester of transcyclooctene (TCO) (200 eq), from the lysine residues available in the protein structure (Scheme 2). The analysis of the TCO:albumin ratio was carried out with MALDI-MS, which revealed a TCO:albumin ratio 40:1. The MALDI-MS analysis indicates that 65% of the total 62 lysine residues were functionalized with a TCO in one albumin molecule. The IEDDA cycloaddition product of SiFA-Tz (**6**) and albumin-TCO (**9**), fluoroalbumin (**10**), was synthesized as a reference for the radiolabeling studies by mixing the two reagents together at room temperature.

**Scheme 2.** Functionalization of bovine serum albumin (**7**) with trans-cyclooctene (**8**) to form albumin- trans-cyclooctene (TCO) (**9**), followed by the inverse-electron demand Diels-Alder (IEDDA) cycloaddition with SiFA-Tz (**6**) to form fluoroalbumin (**10**) (molar ratio 1:1.5 SiFA-Tz:fluoroalbumin).

#### *2.2. Radiochemistry*

Two different synthetic sequences were investigated for the radiosynthesis of [18F]**6**—a one–step direct radiolabeling of compound **6** and a two–step method where we first radiolabeled the SiFA moiety **5**, before linking it to the tetrazine **4** (Scheme 3). The one-step radiolabeling of **6**, resulted in 21% radiochemical yield (*n* = 1), but the yield was detected to decrease rapidly as a function of time, indicating decomposition of the precursor **6** in the reaction mixture, under the alkaline conditions (pH 8.5–9). In the two-step method, the radiolabeling of the SiFA-moiety **5** resulted in incorporation yields ranging from 89 to 99.6 ± 0.5% (*n* = 4), at the optimal time point (2 min) analyzed by radio-TLC. The radiochemical impurities (maximum 11% of total radioactivity) formed in the first step were analyzed by radio-HPLC and are shown in the Supplementary Data (Figure S6). The SiFA radiolabeling was followed by an oxime bond formation between the [18F]SiFA ([18F]**5**) and tetrazine oxyamine **4** at 42.7 <sup>±</sup> 14.2% (*<sup>n</sup>* <sup>=</sup> 8) yield in the reaction mixture (Figure S7). Radio-TLC analysis of product [18F]**<sup>6</sup>** revealed the formation of two isomers, with the (*E*)-isomer of [18F]**6** being predominant with the amount of the radiolabeled **<sup>6</sup>** (*Z*)-isomer only 1.45 <sup>±</sup> 0.35% (*n* = 16). The final product [18F]**<sup>6</sup>** was isolated at >98% radiochemical purity (Figure S9, radio-TLC) and was subsequently used as a prosthetic group to chemoselectively radiolabel the TCO-functionalized albumin in vitro. The [18F]fluorinated albumin [ 18F]**<sup>10</sup>** was radiolabeled at 99.1 <sup>±</sup> 0.2% radiochemical yield (RCY) (*n* = 3) (Figure S12, radio-TLC) and good molar activity (1.1 ± 0.2 GBq/μmol, *n* = 2). To achieve 99% RCY and the total consumption of added [18F]**6** (0.26 nmol), minimum of 0.27 nmol of albumin-TCO was to be used. This resulted in 2.5% of the TCOs in the albumin-TCO labeled with [18F]**6**. A radiochemical yield of >99% of [ 18F]**10** was achieved by incubating increasing amounts of albumin–TCO with [18F]**6** (0.27 nmol) (Figure 1). The radio-HPLC chromatograms of the purified tracers [18F]**6** and [18F]**10** are presented in the Supplementary Data (Figures S8 and S12).
