*2.3. In Vitro Studies of [18F]6 and [18F]10.*

The stability of [18F]**6** in 0.01M PBS pH 7.4 was shown to be excellent at 90 min, with minimal detachment of fluorine-18 (<1%) during the incubation (Figure S10). Plasma protein binding and metabolic stability of [18F]**6** were also evaluated by incubating the radiotracer in plasma and analyzing the deproteinized samples through radio-TLC and radio-HPLC methods, at selected time-points after incubation (Figure 2).

**Scheme 3.** Radiosynthesis of [18F]SiFA-Tz ([18F]**6**) by a two-step method. Reagents and conditions: (**a**) ACN, K[1 8F]F-[K2.2.2], (RCY 99%) and (**b**) anilinium acetate buffer pH 4.6, 25 ◦C, 15 min (RCY 43%).

**Figure 1.** Radiochemical yield (RCY) of [18F]fluoroalbumin ([18F]**10**) during titration of [18F]SiFA-Tz ([18F]**6**), with albumin-TCO revealed a maximal radiochemical yield of >99% with 0.26 nmol of albumin-TCO.

Through radio-TLC analysis, [18F]**6** demonstrated good stability in plasma with up to 6% detachment of the radiolabel over 180 min (94.9 ± 1.6 % intact tracer, *n* = 2). The slight difference in the stability profiles between radio-TLC and radio-HPLC analysis can be explained due to their different ability to quantify free fluoride from the samples. The retention of free fluoride on the silica-based C18 HPLC-column material makes the quantification of free fluoride with HPLC less accurate. Representative radio-TLC and radio-HPLC chromatograms from the experiment are shown in the Supplementary Data (Figures S15 and S16). A radio-HPLC analysis revealed no other radiometabolites of [18F]**6** during the 180 min incubation (Figure 3), in addition to a highly polar component, most likely

the free fluoride. Despite the observed minor defluorination, the enzymatic stability was found to be sufficient for proceeding into in vivo evaluation.

**Figure 2.** Free fraction of the radiotracer [18F]SiFA-Tz ([18F]**6**) during 240 min incubation in human and mouse plasma demonstrated comparable profiles with ~50% of the radioactivity in the free fraction at the end of incubation.

**Figure 3.** In vitro stability of [18F]SiFA-Tz ([18F]**6**) after incubation in human plasma at 37 ◦C, demonstrated excellent stability with ~95% intact tracer at 180 min after start of incubation. The amount of parent [18F]**6** was measured through radio-TLC and radio-HPLC analysis.

The Log*D*pH7.4 of [18F]**<sup>6</sup>** (1.6 <sup>±</sup> 0.2, *<sup>n</sup>* <sup>=</sup> 9) was determined by the shake-flask method, as previously reported [16]. It has been shown that lipophilic compounds tend to exhibit a higher plasma-protein binding than the more hydrophilic compounds [29], which support the findings of the measured Log*D*-value and the value obtained for the plasma protein-bound [18F]**6**, in this study.
