*2.4. Biodistribution of [18F]6 and [18F]10*

The biodistribution of [18F]**6** was investigated in healthy 11 to 12 week-old female CD-1 mice. After intravenous administration into the lateral tail vein, [18F]**<sup>6</sup>** (14.4 <sup>±</sup> 0.5 MBq/animal in ~200 <sup>μ</sup>L of 10% EtOH and 0.5% Solutol HS 15 in 0.01 M PBS pH 7.4) exhibited hepatobiliary excretion and a fast clearance from the blood (Figure 4, Figure S13). The highest percentage of the injected dose per gram of tissue (%ID/g) for urine (229.5 ± 204.5) and gallbladder (143.9 ± 103.1) was found to be at 60 min post-injection. In addition to the high radioactivity detected in the urine, gallbladder, liver, and the feces, a high bone uptake of 18F<sup>−</sup> was observed 60 min post-injection (13.4 <sup>±</sup> 1.6% ID/g). No major passage of [18F]**<sup>6</sup>** through the blood–brain barrier was observed (0.7 <sup>±</sup> 0.2% ID/g) at 5 min post-injection. In order to study the metabolism of [18F]**6**, the blood samples were collected at (*t* = 5, 30, and 60 min) post-injection. The deproteinized plasma samples were analyzed through radio-HPLC and radio-TLC methods, which revealed the formation of a highly polar metabolite (Rf 0.00 on TLC) that was retained at the origin. On HPLC, a metabolite ([18F]**M1**) eluting at 7 min was also detected. Furthermore, there was no indication of more lipophilic metabolites (Figure S17).

**Figure 4.** Biodistribution of radioactivity after the intravenous injection of [18F]SiFA-Tz ([18F]**6**, *n* = 3), demonstrating a high bone uptake of radioactivity, 60 min post-injection. (S.M.—skeletal muscle; bone—tibia, S.I.—small intestine; L.I.—large intestine).

In order to investigate how the conjugation of [18F]**6** onto a macromolecule influences its in vivo defluorination rate, we used [18F]**6** to synthesize [18F]fluoroalbumin ([18F]**10**) and evaluated its biodistribution in CD-1 mice. Intravenously injected [18F]**<sup>10</sup>** (0.6 <sup>±</sup> 0.1 MBq/animal in ~100 <sup>μ</sup>L of 0.01 M PBS, pH 7.4) had a prolonged residence time in blood with 7.1 ± 0.3% ID/g, at 60-min post-injection and a plasma half-life of 49 min (Figure 5). The highest % ID/g for urine (54.7 ± 25.8) and gallbladder (275.3 <sup>±</sup> 185.6), after intravenous injection of [18F]**<sup>10</sup>** was shown to be at 60 min post-injection, for both tissues.

**Figure 5.** Biodistribution of radioactivity after the intravenous administration of [18F]fluoroalbumin. ([18F]**10**, *n* = 5) demonstrating a prolonged residence time of radioactivity in whole blood and reduced bone accumulation, indicating resistance to defluorination in vivo. (S.M.—skeletal muscle; bone—tibia; S.I.—small intestine; L.I.—large intestine).

Stability of [18F]**<sup>10</sup>** was further investigated by separating molecules with molecular weight of <sup>≥</sup><sup>30</sup> kDa from plasma samples collected after intravenous administration of [18F]**10** through ultrafiltration. The proportion of radiolabeled molecules with a MW of <sup>≥</sup>30 kDa (presenting intact [18F]**10**) was over 90% until 180 min post-injection, after which it decreased to 40% over the next 60 min (240 min incubation in total), as shown in Figure 6.

By comparing the bone uptake of 18F in these two biodistribution studies, it was seen that the 18F-Si bond in [18F]**10** was noticeably more stable than in the [18F]SiFA-Tz ([18F]**6**) alone (Figure 7). The bone uptake for both tracers peaked around 120 min after administration and the radioactivity persisted in the bone until the last time point of the biodistribution study, 240 min post-injection. This was an indication of the uptake of free [18F]fluoride, which was a result of defluorination of the SiFA moiety, in vivo [30].

**Figure 6.** The ex vivo distribution (%) of <sup>≥</sup>30 kDa 18F-radiolabeled molecules in mouse plasma after intravenous injection of [18F]fluoroalbumin ([18F]**10**), separated with a molecular weight cut-off filter revealed a highly stable albumin tracer until 2 h post-injection. At 240 min post-injection around 40% of radioactivity was of ≥30 kDa size, with almost 60% of the radioactivity being comprised of species of lower molecular weight.

**Figure 7.** Comparison of the radioactivity accumulated in bone (tibia) in the biodistribution studies of [ 18F]SiFA-Tz ([18F]**6**) and [18F]fluoroalbumin ([18F]**10**). A significant enhancement in stability was seen for the albumin-bound [18F]SiFA group in [18F]**10 (**\*\* *<sup>p</sup>* <sup>≤</sup> 0.01, \*\*\* *<sup>p</sup>* <sup>≤</sup> 0.001), compared to [18F]**6**.

Furthermore, the rate of defluorination was substantially diminished when [18F]**6** was used to chemoselectively radiolabel albumin in vitro through the IEDDA ligation. The plasma protein radiotracer [18F]**10**, which acted as a model protein in this study, demonstrated a significantly (*p* <sup>≤</sup> 0.0001) longer blood circulation time in healthy CD-1 mice than the 18F-labeled tetrazine [18F]**6** alone (Figure 8), with a biological half-life similar to what has been reported for other [18F]SiFA-radiolabeled serum albumins.

**Figure 8.** Radioactivity (%ID/g) in ex vivo blood samples at selected time–points, post-injection for [18F]SiFA-Tz ([18F]**6**) and [18F]fluoroalbumin ([18F]**10**), demonstrated a significantly prolonged circulation time for the plasma protein tracer ([18F]**10**) (\*\*\*\* *<sup>p</sup>* <sup>≤</sup> 0.0001).

#### **3. Discussion**

Two PET-tracer candidates, [18F]SiFA-Tz ([18F]**6**) and [18F]fluoroalbumin ([18F]**10**) were synthesized and evaluated in vivo. Compound [18F]**6** was synthesized with two different synthesis methods, from which the two-step approach was selected, due its higher RCY and good reproducibility. In the one-step radiolabeling approach, the RCY of the product was observed to decrease rapidly as a function of time, indicating decomposition of the precursor in the alkaline reaction mixture. In the two-step method, aminooxy tetrazine **4** was introduced into the reaction mixture at pH 4.6, which was found to be an advantage to avoid any unnecessary decomposition of the tetrazine group. In addition to the two-step method presented in this study, alterative elution protocols with milder reagents, such as copper salts or weak base solutions, as described by Scott et al., should be investigated for the radiolabeling of base sensitive precursors [31]. The in vivo metabolic profile of [18F]**6** displayed hepatobiliary elimination, which is characteristic for compounds with low hydrophilicity. Nevertheless, no observable passage of [18F]**<sup>6</sup>** through the blood–brain barrier was detected (0.7 <sup>±</sup> 0.2% ID/g) at 5 min post-injection, despite the favorable lipophilicity of the tracer (Log*D* = 1.56 ± 0.20). Based on the radio-HPLC metabolite analysis (Supplement. Figure S17) from ex vivo blood samples and the detection of radioactivity in the bone, we concluded that [**18**F]**6** underwent rapid biotransformation, generating highly polar metabolites, one of which was most likely free [**18**F]fluoride detached from the radiotracer. Furthermore, the accumulation of radioactivity in bone is a characteristic indication of fast defluorination in vivo. After defluorination, the free fluoride is sequestered rapidly from circulation and either binds into the surface of the bone or accumulates irreversibly into the hydroxyapatite Ca10(PO4)6(OH)2, forming fluorapatite (Ca10(PO4)6F2) [30]. Thus, it was evident that unexpected

and relatively fast defluorination was observed in vivo. Free fluoride was also excreted into the urine, in vivo. Defluorination of 18F-radiolabeled tracer [18F]**6** could be detected in the bone as early as 10–20 min after injection [30]. The observed rapid defluorination in vivo limited the utility of [ 18F]**6** for pretargeted PET imaging, and further structural optimization was warranted to stabilize the structure towards the defluorination. However, since the stability of biomacromolecular SiFA conjugates has been reported to be good, the possibility of using [18F]**6** as a prosthetic group for the in vitro bioorthogonal radiolabeling of proteins was investigated through administration of [18F]**10**, to healthy mice. Stability of the [18F]SiFA-Tz group against in vivo defluorination was dramatically improved when the group was bound to albumin (13.4 <sup>±</sup> 1.6% ID/g in bone for [18F]**<sup>6</sup>** vs. 3.4 <sup>±</sup> 1.5% ID/g in bone for [18F]**10**, at 60 min post-injection). Furthermore, the blood circulation half-life was 48 min, which is in the order of the reported plasma half-life of 60 min, for the bovine serum albumin in mice [32,33].

There are some examples of small molecular SiFA derivatives and a SiFA-conjugated peptide that have exhibited detectable in vivo defluorination, but not at the level observed in our study [34,35]. Rat serum albumin (RSA) radiolabeled with [18F]SiFA, through isothiocyanate modification of lysine residues has been shown to be relatively stable with only a low rate of defluorination, until 90 min after administration [36]. It has also been shown that the conjugation position of the [18F]SiFA-moiety on the albumin could have an influence on the rate of defluorination in vivo. A more stable maleimido-[18F]SiFA conjugated to RSA via thiol groups is an example of the enhanced stability of the radiolabel in a [18F]SiFA-radiolabeled serum albumin [37]. Thus, this radiolabeling system could be further improved by using a more selective conjugation chemistry (maleimide over N-hydroxysuccinimide) for the addition of the TCO to albumin, while simultaneously optimizing the TCO:albumin ratio and availability of the TCO moiety to the IEDDA reaction, with [18F]**6**. Nevertheless, our results demonstrated the feasibility of using the highly selective and rapid bioorthogonal reaction strategy for the radiolabeling of biomacromolecules with fluorine-18, under mild reaction conditions.

#### **4. Materials and Methods**

All reagents and solvents were purchased from commercial providers and used as received without further purification. Hyox-18 18O–enriched water (98%) was purchased from Rotem Industries Limited (Arava, Israel). Ultrapure water (18.0 MΩ) was produced with a Milli-Q Integral Water Purification System (Merck Millipore, Burlington, MA, USA). HATU, DMF, DIPEA, DMSO, LiCl, methanol, aniline, Kryptofix 2.2.2, 1 M HCl in diethylether, formic acid, ethylacetate and boc-aminooxy acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). MgSO4 was purchased from Merck Millipore (Darmstadt, Germany). TCO-PEG4-NHS ester was purchased from either Jena Biosciences (Jena, Germany) or Conju-Probe (San Diego, CA, USA). Tetrazine amine was purchased from either BroadPharm (San Diego, CA, USA) or Conju-Probe (San Diego, CA, USA). DNA synthesis quality anhydrous acetonitrile (max. 10 ppm water) was purchased from Merck (Kenilworth, NJ, USA). SiFA-aldehyde was purchased from Enamine (Monmouth, NJ, USA). Bovine serum albumin was purchased from Merck (Kenilworth, NJ, USA). Moisture or air sensitive reactions were carried out under an argon atmosphere in oven-dried glassware. Reactions were monitored by TLC Silica gel 60 F254 Merck Millipore (Darmstadt, Germany). Silica gel TLC-plates were run in EtOAc:heptane (7:3) as eluent. [18F]SiFA-Tz ([18F]**6**) R*<sup>f</sup>* = 0.59, [18F]fluoroalbumin ([18F]**10**) R*<sup>f</sup>* = 0.00.

1H-, 13C-, and 19F-NMR spectra were acquired with a Varian Mercury spectrometer (300 MHz, 500 MHz, 600 MHz) (Palo Alto, CA, USA). Chemical shifts (δ) are reported in ppm units, using the solvent residual signal as a reference. Coupling constants (*J*) are expressed in hertz (Hz). The purities of radiolabeled compounds were determined through RP-HPLC with photodiode array (PDA)-, and radiodetector and through silica TLCs analyzed with a Fujifilm FLA 5100 scanner (Fujifilm Life sciences, Cambridge, MA, USA). The excised tissue samples were measured with 1480 Wallac Wizard® 3" (PerkinElmerTM Life Sciences, Waltham, MA, USA) gamma counter for 60 s per sample.

High performance liquid chromatography was carried out with a Shimadzu HPLC system consisting of a DGU-20A degasser, an LC-20AD UPLC LC unit, a SIL-20A HT autosampler, a CTO20 AC column oven, a CBM-20A communications bus module, a Scionix Holland scintillation detector with a 51 BP 51/2 NaI(Tl) crystal and an SPD-M20A diode array detector. For the [18F]SiFATz, a Waters Symmetry semi-preparative C18 column (300 × 7.8 mm, 7 μm) was used, with 0.01 M H3PO4:ACN (20:80, 3 mL/min) as the eluent. Phenomenex BioSep SEC s3000 size exclusion column was used, with 0.1 M phosphate buffer pH 7 (0.8 mL/min) as the eluent, to analyze the conjugated albumin-TCO and radiolabeled protein tracer [18F]fluoroalbumin ([18F]10).

Preparative high performance liquid chromatography was carried out using a system consisting of a Phenomenex DegassexTM DG-4400 degasser, Merck LaChrom L-7100 pump, in-house prepared remote-controlled injection system, Amersham pharmacia biotech REC 112 dual channel chart recorder, Carroll & Ramsey Associates 101-H-DC3 multi-channel radiation detector, and a Knauer Azura UVD 2.1S detector. A waters Symmetry semi-preparative C18 column (300 × 7.8 mm, 7 μm), with 0.01 M H3PO4:ACN (20:80, 3 mL/min flowrate) as the eluent was used for the preparative purification of the [ 18F]SiFA-Tz radiotracer ([18F]6).
