*4.1. Synthesis*

*Tert-butyl(2-{[4-(1,2,4,5-tetrazin-3-yl)ben-zyl]amino}-2-oxoethoxy)carbamate* (**3**). HATU (77 mg, 201 μmol) in DMF (0.5 mL) was added to a solution of **1** (32 mg, 168 μmol) in dry DMF (0.7 mL), under argon. The mixture was stirred at room temperature for 10 min, then **2** (25 mg, 112 μmol) in DMF (2.5 mL) was added, followed by DIPEA (49 μL, 281 μmol). After 20 h mixing at room temperature, ethyl acetate (15 mL) was added and the organic phase was extracted with ultrapure water (10 mL). The organic phase was extracted with 5% LiCl solution (3 × 10 mL, 1 × 20 mL), dried over MgSO4, and concentrated in vacuo. The crude product was purified through silica gel column chromatography (EtOAc:heptane) to afford **3** as a pink solid (26 mg, 72 μmol, 65% yield).

1H-NMR (300 MHz, CDCl3, Supplement Figure S1) δ 10.20 (d, *J* = 0.6 Hz, 1H), 8.79 (s, 1H), 8.61–8.49 (m, 2H), 7.72 (s, 1H), 7.60–7.51 (m, 2H), 4.63 (d, *J* = 6.1 Hz, 2H), 4.40 (s, 2H), 1.42 (d, *J* = 0.6 Hz, 9H). 13C-NMR (75 MHz, CDCl3, Supplement Figure S2) 169.29, 166.53, 158.19, 157.98, 144.09, 130.73, 128.00, 83.63, 68.00, 42.83, 28.27 ppm.

*N-[4-(1,2,4,5-tetrazin-3-yl)benzyl]-2-(aminooxy)acetamide hydrochloride* (**4**). 1 M HCl in diethyl ether (50 mL) was added to a solution of **3** (22 mg, 61 μmol) in MeOH (4 mL). After 24 h, the solution was concentrated in vacuo, to afford **4** as a pink solid. The solid was dissolved in 2 mL of methanol and 20 mL of cold diethyl ether was added to the solution. The closed flask was kept in +4 ◦C overnight, to facilitate the crystallization of the purified product. The recrystallized solid (10 mg, 39 μmol, 65% yield) was suction filtered and used as such in the next reaction step.

*(E,Z)-N-[4-(1,2,4,5-tetrazin-3-yl)benzyl]-2-({[4-(di-tert-butylfluorosilyl)benzylidene]-amino}oxy)acetamide* (**6**). Compound **5** (8.5 mg, 32 μmol) in ACN (0.5 mL) was added into a solution of **4** (4 mg, 15 μmol) in 0.3 M anilinium acetate buffer pH 4.6 (2 mL). After 15 min, ultrapure water (10 mL) was added to the reaction mixture, concentrated with two Sep-pak C18 Light cartridges (Milford, MA, USA) and eluted with ACN (5 mL). The crude product was purified through RP-HPLC (0.01 M H3PO4:CAN, 20:80, 3 mL/min) to afford **6** as a pink solid (7 mg, 14 μmol, 90% yield).

1H-NMR (300 MHz, CDCl3, Supplement Figure S3) δ 10.21 (s, 1H), 8.57 (d, *J* = 8.5 Hz, 2H), 8.21 (s, 1H), 7.69–7.49 (m, 6H), 4.76 (s, 2H), 4.68 (d, *J* = 6.1 Hz, 2H), 1.05 (d, *J* = 1.1 Hz, 18H). 19F-NMR (282 MHz, CDCl3 Supplement Figure S4) δ-189.13. 13C-NMR (126 MHz, CDCl3, Supplement Figure S5) δ 170.10, 166.58, 158.15, 151.49, 143.92, 134.80, 134.77, 132.32, 131.18, 129.02, 128.65, 126.67, 77.61, 77.56, 77.36, 77.11, 73.68, 42.93, 27.61, 20.64, 20.54. ESI-TOF MS: Calculated for C26H34FN6O2Si [M + H]<sup>+</sup> 509.24911 *m*/*z*, found 509.2147 *m*/*z*. Calculated [M + Na]<sup>+</sup> 531.22378 *m*/*z*, found 531.1967 *m*/*z*. Calculated [M + K]<sup>+</sup> 547.19771 *m*/*z*, found 547.1706 *m*/*z*.

Albumin trans-cyclooctene (albumin-TCO (**9**)). TCO-PEG4-NHS ester (5 mg, 9.7 μmol) in DMSO : 0.5 M borate buffer (1:1, 1 mL) pH 9 was added to a solution of bovine serum albumin **7** (3.5 mg, 53 nmol) in borate buffer pH 9.0 (1 mL). After 1 h, the reaction mixture was purified with a PD-10 size-exclusion column (GE Healthcare, Chicago, IL, USA), using ultrapure water as the eluent. The collected fractions were analyzed by a SEC-column HPLC, using 0.1 M PBS as an eluent, with a flow rate of 0.8 mL/min, for identifying the fractions containing albumin-TCO. The fractions containing albumin-TCO were lyophilized to afford **9** as white solid (*n* = 3). Albumin-TCO (1 mg) was dissolved in 1 mL of ultrapure water and analyzed with MALDI-TOF-MS (calculated for bovine serum albumin 66,338 Da, measured for albumin-TCO 82039-82265 Da).

#### *4.2. Radiochemistry*

No-carrier-added 18F-Fluoride was produced in-house with Cyclone 10/5 cyclotron (IBA, Louvain-la-Neuve, Belgium) through a 18O(*p,n*) 18F nuclear reaction, by bombarding H2 18O with 10 MeV protons. The radiosynthesis was carried out in a semiautomatic synthesis unit (DM Automation), with an integrated preparative HPLC system for the purification of the radiotracer. The nucleophilic 18F– was trapped on a Waters QMA Light ion-exchange cartridge, followed by elution with a basic K[18F]FK2.2.2-complex solution. Water residue was evaporated azeotropically by adding anhydrous ACN, followed by heating, under a 40-mL/min argon flow.
