Structural variations of rosamine.

<p>(A) of the rosamine dyes variously functionalized at the <i>meso</i> position as previously reported by Lim et al. (Anticancer Drugs 2009, 20: 461–468), the ones shown here with <i>meso</i>- thiofuran or 4-iodophenyl had superior anticancer activities in cellular assays. (B) Second-generation targets featured in this work.</p

Schematic illustration of the synthesis of rosamines.

<p>(A) The starting material of xanthone ditriflate was prepared in solution by triflation of the phenols, followed by animation of the triflate with piperidine to give symmetrical cyclic amines substitution or; (B) by stepwise addition of piperidine and morpholine to give unsymmetrical cyclic amines substitution.</p

<i>In vivo</i> antitumor effects of rosamine.

<p>(A) The relative tumor volume (RTV) – time profile of 4T1 murine breast carcinoma in Balb/C mice following intravenous dosing of rosamine <b>5</b> or saline as vehicle control. Each point represents median ±95% confidence interval of RTV to staging day (n = 8). The terminal % T/C value for mice receiving 5 mg/kg and 3 mg/kg (q2d×6) of <b>5</b> were 72% and 66% respectively. The two doubling tumor growth delay (T-C)/C (dotted line, RTV = 4) for mice receiving a single bolus of 5 mg/kg and multiple doses of 3 mg/kg (q2d×6) of <b>5</b> were 22% and 38%, respectively. T and C refer to RTV for treatment and control groups, respectively. *Difference with <i>P</i>-value<0.05 compared to control animal. (B) Percentage of mean body weight of mice received 5 mg/kg or 3 mg/kg (q2d×6) of <b>5</b> compared to untreated mice. Body weight loss was observed in treatment groups but none of these mice experienced weight loss of more than 15%.</p

Inhibition of mitochondrial oxidative phosphorylation complexes.

<p>The dose-response inhibition of mitochondrial oxidative phosphorylation Complex 1 (A), Complex II (B), Complex IV (C) and ATP synthase (D) activities by rosamine <b>2</b> (solid line) and <b>5</b> (dotted line). The activity of Complex II was partially inhibited by <b>5</b> with IC₅₀ value of 9.6±0.1 µM whereas for <b>2</b>, inhibition was observed but with undetermined IC₅₀ value. Both <b>2</b> and <b>5</b> also inhibited the ATP synthase activities with IC₅₀ values of 3.9±0.3 and 3.0±0.8 µM respectively. The activity of Complex I and Complex IV were not affected by the rosamines at the treated concentrations (highest at 10 µM). IC₅₀ values depict concentration that inhibits the complexes activity by 50%. ND - indicate non-determined IC₅₀ values based on the concentration used.</p

Antiproliferative activities of rosamine analogs against a panel of cancer cell lines.

a<p>IC₅₀, the concentration of compound, which inhibits the viability by 50% as compared with control untreated cells. Values represent the mean ± SD of at least three determination assessed 48 h post-treatment using methylthiazolyldiphenyl-tetrazolium bromide assay.</p>b<p>Different symbols (†, ‡, and §) indicate statistically significant differences (P<0.05) among the mean values.</p

Summary of the structures of the BIPS derived probes prepared for the red-shifting of the SP and MC spectra.

<p>Summary of the structures of the BIPS derived probes prepared for the red-shifting of the SP and MC spectra.</p

Schematic representations of optical switching reactions and the modulation of MC fluorescence.

<p>(<b>A</b>), Optically-induced transitions between the SP and MC states of BIPS. (<b>B</b>), Modulation of the MC-fluorescence signal in response to orthogonal control of the SP and MC states.</p

Optical switching of C₁₂-TzBIPS (Fig. 3C) in living NBT-II cells.

<p><b>A</b>), Image montage showing MC fluorescence signal of MC-state of C₁₂-TzBIPS within a field of living cells over 3 cycles of optical switching using a low power objective; <b>B</b>), Higher magnification view of the optical switching of MC-fluorescence of C₁₂-TzBIPS within a single cell in the same sample; <b>C</b>), Intensity trace of MC-fluorescence corresponding to the yellow boxed region in (5A); <b>D</b>), Intensity trace of MC-fluorescence within the yellow boxed region in (5B). <b>E</b>), Image montage 6 cycles of rapid optical switching of C₁₂-TzBIPS in living cells within a narrow field of view; <b>F</b>), Intensity trace of the MC-fluorescence averaged over the entire image field (<b>E</b>).</p

Spectroscopic properties of BIPS probes in ethanol.

a<p>Reference compound 6-NO₂-BIPS;</p>b<p>Photochemistry of <b>1e</b> and <b>1g</b> is too low to be measured;</p>c<p>The MC-state of <b>2f</b> does not convert back to the SP-state even with light irradiation;</p>d<p>The MC-state of <b>1f</b> is thermally stable in the dark at room temperature but it converts back to SP state upon exposure to green light;</p>e<p>Colorability of the probe after exposure to 365 nm light for 30 s;</p>f<p>Colorability of the probe after exposure to 405 nm light for 60 s.</p

High-Contrast Fluorescence Imaging in Fixed and Living Cells Using Optimized Optical Switches

<div><p>We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID) imaging microscopy. A triazole-substituted BIPS (TzBIPS) is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP) and a far-red absorbing merocyanine (MC) state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C₁₂-TzBIPS) is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP) has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz) within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.</p></div