Fig 1.
Synthesis scheme for the preparation of 3.
A protected form of HYNIC (1) was coupled to a commercially available tetrazine to form 2. The Boc group was removed prior to labelling by treatment with TFA in DCM to produce 3. Tz* = (4-(1,2,4,5-tetrazin-3-yl)phenyl) methanamine.
Fig 2.
Radiolabelling scheme for the HYNIC-tetrazine ligand 3 and HPLC chromatogram of the isolated product 4.
99mTc labelling of the HYNIC-tetrazine 3 (A). A γ-HPLC chromatogram of the purified final product 4 (B).
Fig 3.
Data are presented as the mean (± SEM) percent injected dose per gram (%ID/g) for selected tissues and fluids from CD1 mice at 0.5, 1, 2 and 6 h post injection (n = 3 per time point). Approximately 0.88 MBq were administered per mouse. Full biodistribution data can be found in the supporting information (S4 File).
Fig 4.
Synthesis scheme for the actively targeted derivative, 99mTc-HYNIC-tetrazine-TCO-BP (5).
The TCO derivative of the bisphosphonate, alendronate (TCO-BP) was mixed with 4 prior to administration to mice.
Fig 5.
Biodistribution data comparing active targeting to pretargeting.
Active targeting with 99mTc-HYNIC-tetrazine-TCO-BP (5) (black bars) is compared to pretargeting of TCO-BP administered 1 h prior to 4 (gray bars). Data are expressed as the mean (± SEM) %ID/g for selected tissues and fluids from Balb/c mice (n = 3 per time point). Tabulated biodistribution data can be found in the supporting information (S4 File).
Fig 6.
Synthesis scheme for 99mTc-HYNIC-tetrazine-TCO-vancomycin (7) from TCO-vancomycin (6) and 4.
Fig 7.
Binding of 4 to S. aureus in vitro using TCO-vancomycin 6.
S. aureus were pretreated with 6 in the absence (gray) or presence (black) of a 10-fold excess of vancomycin. The mean percentages (± SEM) of total radioactivity bound after 1 and 6 h incubation times with 4 are shown.
Fig 8.
Biodistribution data for active targeting of S. aureus infection using 99mTc-HYNIC-tetrazine-TCO-vancomycin (7).
Compounds 4 and 6 were combined prior to i.v. injection of Balb/c mice (n = 3 per time point). Select fluids and tissues were collected at 1 (gray bars) and 6 h (black bars) post injection, including the infected calf muscle (right), and the non-infected calf muscle (left). Data are expressed as the mean percent injected dose per gram (%ID/g) ± SEM. Tabulated biodistribution data can be found in the supporting information (S4 File).
Fig 9.
Biodistribution data for pretargeting using TCO-vancomycin (7) and 4.
Balb/c mice (n = 3 per time point) were given S. aureus infections in the right calf muscle, and 24 h later were treated with 7 administered 1 h prior to 4. Radioactivity was measured 1 and 6 h later in selected tissues and fluids. Data are expressed as the mean percent injected dose per gram (%ID/g) ± SEM. Tabulated biodistribution values can be found in the Supporting Information.