Noninvasive imaging of immune responses.

Synthesis of (Gly) 3 -Tetrazine. The tetrapeptide GGGC was synthesized by standard solid-phase peptide synthesis. Maleimide-tetrazine (ClickChemistryTools) was dissolved in 0.1 M phosphate buffer (PB), pH 7. The tetrapeptide GGGC was added and left to stir at room temperature for 3 h until TLC (1:1 Hex:EtOAc vol/vol) indicated near-complete conversion to the product. The solution was filtered and purified by reverse-phase HPLC with a semipreparative column (C 18 column, Gemini, 5 µm, 10 × 250 mm; Phenomenex) at a flow rate of 5.0 mL/min: solvent A, 0.1% TFA in H 2 O; solvent B, 0.1% TFA in CH 3 CN. (G) 3 -Tetrazine eluted at 30–35% (vol/vol) solvent B. Fractions containing pure product were collected and lyophilized. LC-MS calculated for C 37 H 54 N 11 O 13 S[M+H] + was 892.362, found 892.370. Synthesis of (Gly) 3 -NOTA. Maleimide-NOTA (Macromolecules) was dissolved in 0.1 M PB, pH 7. The tetrapeptide GGGC was added at room temperature for 3 h until TLC (1:1 Hex:EtOAc vol/vol) indicated almost complete conversion to the product. The solution was purified by RP-HPLC on a semipreparative column (C 18 column, Gemini, 5 µm, 10 × 250 mm; Phenomenex) at a flow rate of 5.0 mL/min: solvent A, 0.1% TFA in H 2 O; solvent B, 0.1% TFA in CH 3 CN. The desired product eluted from 15% to 20% (vol/vol) solvent B. Fractions containing pure product were collected and lyophilized. LC-MS calculated for C 27 H 45 N 10 O 11 S [M+H] + was 717.298, found 717.305. Enzymatic Incorporation of Substrates into Proteins Using Sortase. The penta-mutant sortase A, with an improved k cat , was used ( 35 ). Reaction mixtures (1 mL) contained Tris · HCl (50 mM, pH 7.5), CaCl 2 (10 mM), NaCl (150 mM), triglycine-containing probe (500 µM), LPETG-containing probe (100 µM), and sortase (5 µM) ( 11 , 36 ). After incubation at 4 °C with agitation for 2 h, reaction products were analyzed by LC-MS, with yields generally >90%. When the yield was below 90%, the reaction was allowed to proceed for an additional 2 h, with addition of sortase to 10 µM and triglycine-containing probe to 750 µM. Ni-NTA beads were added to the reaction mixture with agitation for 5 min at 25 °C, followed by centrifugation to remove sortase and any remaining unreacted His-tagged substrate. The final product—either the tetrazine-labeled protein, NOTA-labeled protein, or fluorophore-labeled protein—was purified by size-exclusion chromatography in PBS or Tris · HCl (50 mM, pH 7.5). The labeled protein was stored at −20 °C with 5% (vol/vol) glycerol and was stable for up to 3 mo. Synthesis and Characterization of 18 F-VHHs. In a typical reaction, a 1.5-mL centrifuge tube was loaded with VHH7-Tz in 1× PBS (40 μL, 150 μM), 1× PBS (300 μL), and 18 F-TCO in DMSO [4.0 mCi (148.0 MBq), 100 μL]. The tube was sealed and shaken at room temperature for 20 min. The mixture was analyzed by radio-TLC [instant thin layer chromatography (ITLC), 100% MeCN, R f 18 F-TCO = 0.9, R f 18 F-VHH7 = 0.0] showing 90% conversion to 18 F-VHH7. The reaction mixture was loaded onto a PD-10 size-exclusion cartridge (GE Healthcare), and elution with 1× PBS provided 2.3 mCi (85.1 MBq) of 18 F-VHH7 in 75.8% decay-corrected radiochemical yield. Starting with 5.3 mCi (196.1 MBq) 18 F-TCO, 18 F-VHHDC13 was prepared following the same procedure as described for 18 F-VHH7 to give 2.8 mCi (103.6 MBq) after size-exclusion chromatography, a 69.7% decay-corrected radiochemical yield. Synthesis and Characterization of 64 Cu-VHHs. In a typical reaction, a 1.5-mL centrifuge tube was loaded with VHH7-NOTA [400 μL, 20 μM in 200 mM NH 4 OAc buffer (pH 6.5)] and 64 CuCl 2 (5.7 mCi, 210.8 MBq) in 200 mM NH 4 OAc buffer (75 μL, pH 6.5). The tube was sealed and shaken at 37 °C for 20 min. The mixture was analyzed by radio-TLC (ITLC, 50 mM EDTA, pH 7, R f 64 Cu/EDTA = 1.0, R f 64 Cu-VHH7 = 0.0) showing 98% conversion to 64 Cu-VHH7. At this time, the mixture was loaded onto a PD-10 size-exclusion cartridge, and elution with 1× PBS provided 5.2 mCi (192.4 MBq) of 64 Cu-VHH7 in 94.2% decay-corrected radiochemical yield. Starting with 3.5 mCi (129.5 MBq) 64 CuCl 2 , 64 Cu-VHHDC13 was prepared following the same procedure as described for 64 Cu-VHH7 to give 3.1 mCi (114.7 MBq) after size-exclusion chromatography, a 92.3% decay-corrected radiochemical yield. Before injection, both 64 Cu-VHH7 and 64 Cu-VHHDC13 were analyzed by radio-TLC (ITLC, 50 mM EDTA, pH 7, R f 64 Cu/EDTA = 1.0, R f 64 Cu-VHH7 and 64 Cu-VHHDC13 = 0.0) and were found to have 99.6% and 99.8% radiochemical purity, respectively. PET-CT Imaging. All procedures and animal protocols were approved by the Massachusetts General Hospital subcommittee on research animal care.

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