Synthesis and Characterization of Novel Fluoro-glycosylated Porphyrins that can be Utilized as Theranostic Agents

Abstract

Small molecules with modalities for a variety of imaging techniques as well as therapeutic activity are essential, as such molecules render opportunities to simultaneously conduct diagnosis and targeted therapy, so called theranostics. In this regard, glycoporphyrins have proven useful as theranostic agents towards cancer, as well as noncancerous conditions. Herein, the synthesis and characterization of heterobifunctional glycoconjugated porphyrins with two different sugar moieties, a common monosaccharide at three sites, and a 2-fluoro-2-deoxy glucose (FDG) moiety at the fourth site are presented. The fluoro-glycoconjugated porphyrins exhibit properties for multimodal imaging and photodynamic therapy, as well as specificity towards cancer cells. We foresee that our findings might aid in the chemical design of heterobifunctional glycoconjugated porphyrins that could be utilized as theranostic agents.

Keywords: cancer; glycoporphyrins; imaging; photodynamic therapy; photosensitizers.

Figure 1

A) Schematic drawing showing desirable properties of a porphyrin‐based theranostic agent. B) A porphyrin scaffold featuring two different alkyne‐functionalized handles (blue and red) for consecutive click‐reactions (left). The 2‐azidoethyl β‐d‐glycosides that was conjugated to the blue and red sites, respectively, on the porphyrin scaffold (right).

Scheme 1

General conditions and reagents: i) HSO₃Cl, room temperature; ii) propargylamine, DIPEA, DCM, room temperature; iii) Zn(OAc)₂ ^. 2 H₂O, DCM, MeOH, room temperature, 40 % over three steps; iv) LiCl, DMF, MW, 160 °C, 77 %.

Scheme 2

General conditions and reagents: i) CuSO₄, sodium l‐(+)‐ascorbate, THF, t‐BuOH, H₂O, MW, 85 °C, 59 % (13), 89 % (14), 84 % (15), 62 % (19), 55 % (20), 78 % (21); ii) 1. NHS, EDC, DMF, room temperature; 2‐propargylamine, DIPEA, room temperature, 85 % (16), 61 % (17), 52 % (18); iii) NaOH, MeOH, H₂O, room temperature, 99 % (1), 80 % (2), quant. (3).

Figure 2

A) Chemical structure of fluoro‐glycosylated porphyrins 1, 2, and 3. B) Photophysical characteristics of fluoro‐glycosylated porphyrins 1 (left), 2 (middle), and 3 (right). Absorption spectra are shown as red solid lines, whereas emission spectra are shown as green solid lines (excitation at 430 nm) or blue dotted lines (excitation at 561 nm). The fluoro‐glycosylated porphyrins were dissolved in DMSO to a concentration of 1.5 mm and further diluted in PBS to a final concentration of 15 μm prior to measurements.

Figure 3

Photophysical characterization of 1′: A) Absorption spectrum; B) emission spectrum (excitation at 419 nm); C) triplet excited‐state absorption spectrum (excitation at 355 nm); D) transient luminescence at 1270 nm of singlet oxygen generated by 1′ (excitation at 440 nm). For all the measurements, THF was used as solvent and argon gas was used to remove oxygen from the solvent.

Figure 4

Confocal microscopy 3D images of A) fibroblasts and B) melanoma cells stained with porphyrins (red) and DAPI (nuclei staining; seen in blue). The cells were incubated with the porphyrin variants (20 μm, 24 h) and, thereafter, fixated. Images were collected in z‐stack spectral mode (excitation at 405 and 561 nm) with the dimensions x=135 μm, y=135 μm and z=20 μm. Scale bar 20 μm. C) Flow cytometry analysis of cells exposed to porphyrin variants (20 μm, 24 h) in complete cell culture medium. Fluorescence intensities are compared in fibroblasts and melanoma cells stained with unglycosylated 24, FDG glycoporphyrins 1, 2, and 3, and non‐fluorinated glucoporphyrin 23. Diagram shows the combined results of four replicate measurements. Significant differences were determined by ANOVA and p≤0.05 are indicated with asterisks. D) Cell viability, determined by ATP content, in cell cultures exposed to porphyrin variants (20 μm, 24 h). Data is presented as mean + SD (n=4). Statistical evaluation using ANOVA demonstrate no significant alterations in cell viability compared to unstained controls. Final concentration of DMSO added to cells was 0.13%.