Figure 1.
BTP derivatives having different excitation and emission spectra.
A, Fluorescence views of BTP2, BTP3 and BTP4 in ACSF (upper) and on filter papers under 365 nm UV light (lower) are shown. B–D, Excitation (blue dots and lines) and emission (red dots and lines) spectra of BTP2 (B), BTP3 (C) and BTP4 (D) were measured in ACSF (pH 7.3). Blue and red numbers represent wave length (nm) at peak fluorescence intensity. Abbreviations: rF, relative fluorescence intensity.
Figure 2.
Synthesis of BTP2-Neu5Ac, BTP3-Neu5Ac and BTP4-Neu5Ac.
Conditions: (a) Amberlite IR-120(H+), dry MeOH, overnight, 92% yield. (b) AcCl-AcOH, overnight, quant. (c) BTP2∼4, NaH, THF-DMF, room temperature, overnight. (d) NaOMe, dry MeOH, 6 hr, room temperature, then NaOH aq., MeOH, room temperature, 2 days.
Figure 3.
Hydrolysis of BTP-Neu5Ac with bacterial sialidase.
A–D, Relative fluorescence intensities proportionally increased with increasing amounts of AUSA in 10 µM BTP2-Neu5Ac (A), BTP3-Neu5Ac (B) and BTP4-Neu5Ac (C) but not in 10 µM Res-Neu5Ac (D). Each bar and line represent the mean ± S.E.M. (n = 3). The fluorescence intensities of each black bar were set to 105. Res: 10 µM resorufin. E, AUSA blotted on PVDF membranes was stained with 10 µM BTP4-Neu5Ac or X-Neu5Ac. The PVDF membranes were observed under UV light for BTP4-Neu5Ac or visible light for X-Neu5Ac. The blue color caused by staining of AUSA with 100 µM X-Neu5Ac is shown at the right of the dotted line.
Figure 4.
Visualizing of sialidase activities in rat brain slices with BTP-Neu5Ac.
A–C, Sialidase activity was imaged in acute coronal slices of adult rat brains with BTP2-Neu5Ac (A), BTP3-Neu5Ac (B) and BTP4-Neu5Ac (C) at pH 7.3. Abbreviations: cc, corpus callosum; ec, external capsule; fi, fimbria; hip, hippocampus; ic, internal capsule. D, Brain slices were stained with various concentrations (1–1000 µM) of BTP4-Neu5Ac. E, Sialidase activity was imaged with 10 µM BTP4-Neu5Ac or 10 µM BTP4-Neu5Ac containing 1 mM DANA, a sialidase inhibitor. Emission filters that transmit above 420 and 510 nm were used in A–D and E, respectively. Scale bars in each panel represent 2 mm.
Figure 5.
Undetectable cytotoxicity of BTP-Neu5Ac and BTP for 16-h exposure.
MDCK cells were exposed to a serum-free medium (SFM) containing 10 or 100 µM BTP-Neu5Ac (A) or BTP (B) for 16 hr and released LDH was measured. LDH release is shown as relative to complete LDH release (100%) by treatment with lysis buffer.
Figure 6.
Detection of orthotopic colon cancer with BTP4-Neu5Ac in mouse colon tissue.
A, One week after orthotopic colon implantation of Colon26 NL-17 cells, living colon tissues were stained with BTP4-Neu5Ac. Arrowhead indicates the cancer region. B and C, Inflammatory (B) or normal (C) colons were also stained with BTP4-Neu5Ac. Left, middle and right panels in panel A–C show bright field, merged and fluorescent views, respectively. D and E, Enlarged image of cancer (D) and normal (E) region stained with BTP4-Neu5Ac. F, Background fluorescence level of panels D and E is shown. G, Immunohistochemical staining (red fluorescence) by using rabbit anti-CD71 antibody and PE-conjugated goat anti-rabbit IgG antibody and nuclear staining with DAPI (blue fluorescence) were performed to detect colon cancer in cross sections of the mouse colon tissues that were used for sialidase activity imaging in panels A and D. Arrows indicate the regions showing intense fluorescence of BTP in panel A and D. Scale bar in panel C represents 2.5 mm and is common in panels A and B. Scale bar in panel F represents 0.5 mm and is common in panels D and E. Scale bar in panel G represents 0.2 mm.
Figure 7.
Detection of human colon cancer with BTP4-Neu5Ac.
A, A human colon cancer specimen (region enclosed with a white line) was obtained from surgical cancer tissue (UICC T classification: T3). B–D, The colon tissue was stained with BTP4-Neu5Ac. B, C and D show photographic, merged and fluorescent images, respectively. E–G, Enlarged fluorescence images of normal (E) and cancer (F and G) regions were acquired with a fluorescent microscope. H and I, A longitudinal slice of colon tissue was prepared at the dotted line in panel B. Non-fluorescence and fluorescence regions in panel C were stained with hematoxylin-eosin and are shown in panels H and I, respectively. Scale bars in panel B, E and H represent 10 mm (common in panels B–D), 500 µm (common in panels E–G) and 250 µm (common in panels H and I), respectively.