Tetraaryl‑, Pentaaryl‑, and Hexaaryl-1,4-dihydropyrrolo[3,2‑<i>b</i>]pyrroles: Synthesis and Optical Properties

Efficient conditions for the synthesis of tetra-, penta-, and hexasubstituted derivatives of 1,4-dihydropyrrolo­[3,2-<i>b</i>]­pyrrole were developed. The tetraaryl derivatives were obtained in a novel one-pot reaction among aromatic aldehydes, aromatic amines, and butane-2,3-dione. After a thorough examination of various reaction parameters (solvent, acid, temperature) <i>p</i>-toluenesulfonic acid was identified as the crucial catalyst. As a result, 1,4-dihydropyrrolo­[3,2-<i>b</i>]­pyrroles were obtained in the highest yields reported to date. The scope and limitation studies showed that this new method was particularly efficient for sterically hindered aldehydes (yields 45–49%). Pentaaryl- and hexaaryl-1,4-dihydropyrrolo­[3,2-<i>b</i>]­pyrroles were prepared from tetraaryl-1,4-dihydropyrrolo­[3,2-<i>b</i>]­pyrroles via direct arylation by employing both electron-poor and electron-rich aromatic and heteroaromatic haloarenes. Strategic placement of electron-withdrawing substituents at the 2-, 3-, 5-, and 6-positions produced an acceptor–donor–acceptor type fluorophore. The resulting multiply substituted heteropentalenes displayed intriguing optical properties. The relationship between the structure and photophysical properties for all compounds were directly compared and thoroughly elucidated. All synthesized products displayed strong blue fluorescence and exhibited moderate to large Stokes shifts (3000–7300 cm^–1) as well as high quantum yields of fluorescence up to 88%. Two-photon absorption cross-section values measured in the near-IR region were surprisingly high (hundreds of GM), given the limited conjugation in these propeller-shaped dyes

Quantification of <i>BRAF</i> V600E mutation in cancer cell line DNA using fluorometry.^a

<p>^a For details of the <i>BRAF</i> copy number calculation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136720#sec005" target="_blank">Materials and Methods</a>. Thus, 50 fM ss DNA corresponds to 3x10⁶ molecules per 100 μL, or 1x10⁶ molecules of genomic DNA in 100 μL of the sample corresponds to the concentration 16.6 fM. Abundance value is determined using ratio of EvaGreen fluorescence obtained for individual analysis on wild-type and mutant specific capture probes as follows: I(<b>CP2m</b>)/(I(<b>CP2m</b>)+I(<b>CP2w</b>)).</p><p>* I (excitation/emission wavelength, nm); background signal is determined by fluorescence measurement of free single-stranded genomic DNA at the same concentration.</p><p>Quantification of <i>BRAF</i> V600E mutation in cancer cell line DNA using fluorometry.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136720#t002fn001" target="_blank">^a</a></p

Main principle of DNA detection by short LNA/DNA capture probes on solid support.

<p>Target binding specificity results from the difference in melting temperature (<i>T</i>_m) between fully-matched and mismatched capture probe:target complexes. CPG = controlled pore glass.</p

Detection of cancer DNA by fluorescence.

<p>(A) Visualization of <i>BRAF</i> V600E mutation on solid-support containing capture probe <b>CP2m</b>: <b>CP2m</b>:HT29 (2.5 pM, tube 1), <b>CP2m</b>:LS411N (2.5 pM, tube 6). Signal is obtained under laboratory UV-vis lamp (excitation at 365 nm) at 19°C using 10 pM signal-enhancing probe and 0.6X EvaGreen dye. (B) Quantification of genomic <i>BRAF</i> targets by fluorometry in solution. Target titration curves were obtained for fully complementary and mismatched complexes (blue and red lines, respectively) of LNA/DNA capture probes <b>CP2w</b> and <b>CP2m</b> with corresponding targets and signal-enhancing probe <b>P1</b>: 5’-GCT A+GA CCA +AAA TCA CCT A+TT TTT ACT GTG AG+G TCT TCA TGA AGA +AAT AT-3’. LNA nucleotides are marked with plus before the corresponding letter.</p

Sequences and thermal denaturation temperatures of LNA/DNA capture probes prepared in this study.^a

<p>^a Thermal denaturation temperatures <i>T</i>_m (°C)/change in <i>T</i>_m relative to corresponding reference duplex, Δ<i>T</i>_m (°C). <i>T</i>_m values measured as the maximum of the first derivatives of the melting curves (A₂₆₀ vs temperature). Reported Tm values were obtained in medium salt buffer and are averages of at least two measurements with resulting <i>T</i>_m ± 0.5°C. LNA nucleotides are marked with plus before the corresponding letter. Allele-specific nucleotide is underlined. 63Mer <i>BRAF</i> target sequences (SNP position is underlined): 5’-CATGAAGACCTCACAGTAAAAATAGGTGATTTTGGTCTAGC TACAG<u>T</u>GAAATCTCGATGGAGT-3’ (<b>T1</b>); 5’-CATGAAGACCTCACAGTAAAAATAGGTGATT TTGGTCTAGCTACAG<u>A</u>GAAATCTCGATGGAGT-3’ (<b>T2</b>).</p><p>Sequences and thermal denaturation temperatures of LNA/DNA capture probes prepared in this study.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136720#t001fn001" target="_blank">^a</a></p

Fluorescence images of <i>BRAF</i> DNA fragments from cell line HT29.

<p>(A) Complex of DNA with <b>CP2m</b>, signal-enhancing probe <b>P1</b> and EvaGreen dye, bright dots with intensities over 80 are seen. (B) Target DNA re-annealed with <b>CP2m</b> and EvaGreen dye in the absence of <b>P1</b>, darker dots are seen with counts up to about 20. (C) EvaGreen dye in 1xX PBS (0.06X solution), no signal is seen. Images were obtained using two photon laser scanning microscope (ex 535+35nm; laser@ 840); at 19°C using 1.5 fM cancer DNA and re-annealing with 10 pM signal-enhancing probe and 0.06X EvaGreen dye. The images were taken using the same instrument settings and adjusted using the same intensity threshold. The graph below the images shows a line plot of the line in the image.</p

Detection of cancer DNA using novel enzyme-free assay.

<p>The main steps of the LNA/DNA assay: 1) binding to gene-specific capture probe, 2) washing and cleavage from support, 3) adding signal-enhancing LNA/DNA probe and intercalating dye, 4) fluorescence detection. B = biotin, S = streptavidin, CPG = controlled pore glass, L = LNA.</p

Oscillations in the Generalized Polarization (GP) function of ACDAN in oscillating cells.

<p><b>Panel A</b>) Oscillations of the values correspond to the weighted difference of the intensity of emission at the maxima shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117308#pone.0117308.g001" target="_blank">Fig. 1</a>. Panel <b>B</b>) Power spectrum of the frequency of the GP oscillations. The GP was calculated as described in supplementary text (equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117308#pone.0117308.e001" target="_blank">1</a>). Note that the GP function indicates that the blue (440 nm) and green (490 nm) emission intensities oscillate in a correlated manner, as would be expected if both the B and G regions of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117308#pone.0117308.g001" target="_blank">Fig. 1</a> were oscillating synchronously. The same behavior was observed with PRODAN.</p

The effect of D₂O on NADH, ACDAN and PRODAN oscillations.

<p>The top panels show NADH oscillations in the presence of no D₂O (A), 10% D₂O (B) and 50% D₂O (C). The bottom panels show the power spectra of the oscillations of NADH (D), ACDAN (E) and PRODAN (F) with increasing concentrations of D₂O (black 0%, blue 10% and red 50%).</p

Oscillatory behavior of glycolysis and DAN probes in the fluorometer.

<p>Panel <b>A</b>) Oscillations of NADH. Panel <b>B</b>) Oscillations of ACDAN (red), PRODAN (blue) and LAURDAN (black). Panel <b>C</b>) Non-oscillatory behavior of ANS labeled cells. Panel <b>D</b>) Phase relationships: ACDAN and NADH are expressed as fluorescence intensity, ATP is plotted in concentration units (mM). The arrows in panels <b>A</b>), <b>B</b>), and <b>C</b>) indicate the time of addition of 30 mM glucose followed by 5 mM KCN.</p