Synthesis and photophysical properties of thioglycosylated chlorins, isobacteriochlorins, and bacteriochlorins for bioimaging and diagnostics

Abstract

The facile synthesis and photophysical properties of three nonhydrolyzable thioglycosylated porphyrinoids are reported. Starting from meso-perfluorophenylporphyrin, the nonhydrolyzable thioglycosylated porphyrin (PGlc₄), chlorin (CGlc₄), isobacteriochlorin (IGlc₄), and bacteriochlorin (BGlc₄) can be made in 2-3 steps. The ability to append a wide range of targeting agents onto the perfluorophenyl moieties, the chemical stability, and the ability to fine-tune the photophysical properties of the chromophores make this a suitable platform for development of biochemical tags, diagnostics, or as photodynamic therapeutic agents. Compared to the porphyrin in phosphate buffered saline, CGlc₄ has a markedly greater absorbance of red light near 650 nm and a 6-fold increase in fluorescence quantum yield, whereas IGlc₄ has broad Q-bands and a 12-fold increase in fluorescence quantum yield. BGlc₄ has a similar fluorescence quantum yield to PGlc₄ (<10%), but the lowest-energy absorption/emission peaks of BGlc₄ are considerably red-shifted to near 730 nm with a nearly 50-fold greater absorbance, which may allow this conjugate to be an effective PDT agent. The uptake of CGlc₄, IGlc₄, and BGlc₄ derivatives into cells such as human breast cancer cells MDA-MB-231 and K:Molv NIH 3T3 mouse fibroblast cells can be observed at nanomolar concentrations. Photobleaching under these conditions is minimal.

Figure 1

Top: UV-Visible spectra of the compounds, 2 μM in ethanol. Bottom: emission spectra of the compounds in ethanol; excitation at 512 nm where the absorbance is ca. 0.05 for each compound.

Figure 2

Fluorescence microscopy of K:MOLV NIH 3T3 cells treated with 2.5 μM PGlc₄ (1b), CGlc₄ (2b), and IGlc₄ (3b). K:Molv NIH 3T3 cells were incubated for 20 hrs with porphyrinoids, followed by removal of unbound dye from the cell culture by repeated rinsing with PBS, and the cells were imaged under identical microscope settings and not enhanced; magnification 10X.

Figure 3

K:Molv NIH 3T3 cells were incubated with 10 μM BGlc₄ for 24 hrs., rinsed three times with PBS buffer, and fixed with 4% paraformaldehyde solution. Confocal microscope excitation at 514 nm, emission monitored with a 710–750 band pass filter. No images are observed using a 610–650 nm emission band pass filter, so fluorescence does not arise from the one of the other dye systems. The image is not enhanced, magnification is 60x.

Figure 4

Fluorescence image of a MDA-MB-231 breast cancer cell treated with 25 nM IGlc₄, and rinsed three times with BPS buffer to remove unbound dye (see Supporting Information for comparisons to the other porphyrinoids).

Scheme 1

Synthesis of PGlc₄ from tetrakis(pentafluorophenyl)porphyrin (TPPF₂₀)

Scheme 2

Synthesis of CGlc₄, IGlc₄ and BGlc₄

Scheme 3

CGlc₄ diastereomers (see Supporting Information).