Synthesis of Fmoc-protected Asn^F9. Percentages represent the yields of the respective synthesis steps.

¹⁹F characteristics of peptide probes. A) ¹⁹F-NMR spectra of peptides P1–P6 at ¹⁹F concentration of 5.4 mm (0.2 mm of P1–P4 and 0.6 mm of P5–P6) in PBS buffer at pH 7.4 (left) and methanol (right). The narrowest peak width at half height for each condition is marked in red. B) ¹⁹F relaxation times of the peptides (5.4 mm ¹⁹F) in PBS and methanol. C) ¹⁹F-MRI of peptide solutions in 6 mm CuSO₄ (65 mm NaOH added in P1 and P2 solution for pH adjustment) at ¹⁹F concentration of 675 mm (P1–P4 at 25 mm and P5–P6 at 75 mm). The aqueous solutions were filled into capillaries with an inner diameter of 1.6 mm and an outer diameter of 2.0 mm, and fitted into a homemade microcoil.^[32] Images of coronal planes were acquired using a Fast Low Angle Shot Gradient Echo (FLASH) sequence with a recycle delay of 5.765 ms, an echo delay of 2.72 ms, and a flip angle of 45°. The images were processed using a smoothing algorithm (binning with an average shrink factor 32) of ImageJ. Scale bar: 1 mm.

Estimate of the limiting concentration needed for the acquisition of an ¹⁹F MRI image. The signal/noise ratios of 1024 repetitions were measured for peptides P1–P6 at different concentrations in PBS buffer (in 5 mm tubes) using solution ¹⁹F NMR at 7 T (300 MHz ¹H resonance frequency). A signal/noise ratio of ≈10 000 (marked by a dashed line) is estimated to be sufficient to collect a ¹⁹F MRI image (2D slice of 64 × 64 matrix) within 10 min.

In vitro biological properties of the ¹⁹F-peptide probes. A) Stability of peptides in bovine serum with increasing incubation time (0–7 days) at 37 °C. B) Viability of HeLa (left) and HEK293 (right) cell cultures exposed to various concentrations of peptide probes. The cell viability was obtained by monitoring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay), where the amount of the formazan product (quantified by optical absorbance) was plotted as cell viability. 100% cell viability refers to untreated cells.

Biodistribution of the rhodamine labeled peptides (Rho-P1 to Rho-P6) in zebrafish embryos. A,A´) Rho-P1, B,B´) Rho-P2, C,C´)Rho-P3, D,D´) Rho-P4, E,E´) Rho-P5, and f,F,F´) Rho-P6 were intravenously injected into 3 dpf Tg(kdrl:EGFP)s843Tg zebrafish embryos and 3D images from the head (A–F) and trunk (A´–F´) regions were acquired every hour for 19 h (n = 3 embryos for each peptide). f) A single focal plane in the brain region was selected to highlight the distribution of Rho-P6 along the radial glia fiber. Images shown are from 1 h-post-injection. Peptide distribution and blood vessels are shown in magenta and green, respectively. The fluorescence microscopy images are superimposed on bright-field images (grey) from a selected single plane. Lateral views of the embryos are oriented anterior left with dorsal up. a, anterior; p, posterior; d, dorsal; v, ventral; ca, caudal artery; cvp, caudal vein plexus; s, spinal cord. Scale bar: 50 µm.

Conjugation of fluorinated peptides with biomolecules, and ¹⁹F-NMR spectra of conjugates with GSH and BSA. A) Coupling of the peptides to a cross-linker via copper-catalyzed “click” reaction, followed by thiol conjugation with BSA and GSH in PBS. B) GSH-peptides and BSA-peptides in PBS were characterized using ¹⁹F-NMR. Peak linewidths at half height are indicated.