Fig 1.
Synthetic route of tetrazine-containing amino acid (S)-3-(4-(1, 2, 4, 5-tetrazin-3-yl) phenyl)-2-aminopropanoic acid 2 and protected tetrazine-containing amino acid 5 and 6, and the IED-DA reaction of 2 with 5-norbornene-2-carboxylic acid 3 give 4.
a) (i) Formamidine acetate (4 eq), N2H4 (40 eq), S (1 eq), r.t, 22 h; (ii) CH3COOH, NaNO2 (5 eq), 0°C, 30 min, 64.9%. b) Phosphate buffered solution (PBS), r.t, 30 min. c) Fmoc-OSu (1.5 eq), 1: 1 Dioxane/H2O (V/V), NaHCO3 (2.5 eq), 0°C, overnight, 64.5%. d) Boc2O (2 eq), 1: 1 Dioxane/H2O (V/V), NaHCO3 (2.5 eq), 0°C, 8h, 72.3%.
Fig 2.
The compound 2 reacted with norbornene to label cancer cells.
Cancer cells A549, which overexpressed EGFR, were exposed to the Cetuximab antibodies modified with norbornene and 5-carboxyfluorescein (green). In the next step, the pretargeted cells were labeled with a tetrazine bearing a fluorophore such as VT680 (red).
Fig 3.
Fluorescent microscope images of A549 lung cancer cells after pretargeting Cetuximab antibodies modified with 5-carboxyfluorescein and norbornene and subsequent labeling with tetrazine-VT680.
(A) Rhodamine channel. (B) Near-IR channel (tetrazine-VT680). Images of control experiments were also taken in near infrared channel. (C) Control experiment with unlabeled cetuximab and tetrazine-VT680. (D) Control experiment with norbornene-cetuximab and unlabeled VT680.
Fig 4.
Peptides 7 and 8 (both with 10 μM) for small intestine contraction with acetylcholine (10 μM) as positive control.