Research ArticleChemistry

A metal-free blue chromophore derived from plant pigments

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Science Advances  03 Apr 2020:
Vol. 6, no. 14, eaaz0421
DOI: 10.1126/sciadv.aaz0421
  • Fig. 1 Semisynthesis and spectroscopic data of BeetBlue.

    (A) Acid-catalyzed coupling of betalamic acid (HBt) and 2,4-dimethylpyrrole (2,4-dmp) in ethyl acetate. The reaction, which takes less than 30 min to complete, is performed at room temperature (rt) under air, and the product can be purified by flash gel permeation chromatography using water as eluent. TFA, trifluoroacetic acid; BB, BeetBlue; Bn, betanin. (B) Chemical structures of BeetBlue and indocyanine green (ICG), atom numbering, and density functional theory (DFT)–optimized geometry of BeetBlue. NOESY, nuclear Overhauser effect spectroscopy; HMQC, heteronuclear multiple-quantum coherence; HSQC, heteronuclear single-quantum coherence. (C) 1H and 13C NMR spectra of BeetBlue (13 mM, 800 MHz/200 MHz, D2O at 288 K). (D) High-resolution mass spectrum of BeetBlue; m/z 289.1181, [M + H]+.

  • Fig. 2 Characterization of the electronic and physicochemical properties of BeetBlue in solution.

    (A) Comparison of the normalized ultraviolet-visible (UV-Vis) (filled) and fluorescence spectra of BeetBlue, betanin, and indicaxanthin (BtP) in water. FI, fluorescence. (B) Titration of BeetBlue within the range of pH 2 to pH 12 using Britton-Robinson buffer (40 mM). The top projection shows the dependence of the absorption at selected wavelengths on the pH; lines are a nonlinear fit of a sigmoidal function to the data for determining the pKa values. The right projection shows the absorption spectra at selected pHs (2, 2.9 [pKa1], 7, 9.6 [pKa2], and 12). For reference, the pKas of the carboxylic acid groups of betanin and indicaxanthin are roughly 3.5 (23). (C) Absorption spectra, magnification of the CIELUV (CIE 1976 L*, u*, v* color space) chromaticity diagram (2015 D65/10°), and MacAdam ellipses of diluted solutions of BeetBlue in acidified (0.1 mM p-toluenesulfonic acid) polar molecular solvents. Values of RAL (Reichs-Ausschub für Lieferbedingungen) color matching system and HSV (hue, saturation, value) and color names are given for referencing purposes. MeOH, acidified methanol; DMSO, dimethyl sulfoxide; TFE, trifluoroethanol; iPrOH, isopropanol; HFIP, hexafluoro-2-propanol. (D) Natural transition orbitals (NTOs) of BeetBlue at the SMD/PBE0/6-31+G(d,p)//SMD/B3LYP/6-31+G(d,p) level (isovalue, 0.002 e); orbital and relevant atom contribution to hole and particle states. LUMO, lowest unoccupied molecular orbital.

  • Fig. 3 Toxicological analyses of BeetBlue.

    (A) Viability of Huh-7, Hep-G2, and HepaRG cells treated with BeetBlue [1 mM to 1 nM, 8 hours, in phosphate-buffered saline (PBS)] measured by using the MTT assay. Negative controls were carried out using PBS; cells irradiated with UVC light (20 and 50 J/m2) were used as positive controls. The Research Resource Identifiers (RRIDs) for the cell lines are given for convenience. The letters indicate statistical significance (P < 0.05, one-way ANOVA, Tukey’s post hoc test, N > 10). (B) Quantitative Comet assay results in HepaRG cells treated with BeetBlue (10 μM) for up to 8 hours. Cells irradiated with UVC light (6 J/m2) were used as positive controls. DNA fragmentation was expressed as olive tail moment (OTM), viz., the product of the tail length and the fraction of total DNA in the tail. Untreated cells were used as a negative control group. AU, arbitrary units. (C) Viability of ARPE-19 cells (1 × 10−6 cells) incubated with PBS, hydrogen peroxide (50 mM, 5 hours, in PBS), or BeetBlue (10 μM, 24 hours, in PBS) measured by flow cytometry; percentages show the relative populations. (D) Aspect of zebrafish larvae in culture for 4 days in the presence of 10 μM BeetBlue versus negative control. (E) Survival of zebrafish in the presence and absence of BeetBlue (10 μM) for 4 days; N = 16.

  • Fig. 4 Photochemical and dying properties of BeetBlue.

    (A) Nanosecond transient absorption spectra of BeetBlue in water; pump wavelength, 580 nm. mOD (milli optical density). (B) Effect of laser excitation at 355 nm (2.23 J/s cm2) or 582 nm (0.45 J/s cm2) on the absorption spectrum of an aqueous solution of BeetBlue (18.5 μM) over time. Experiments were carried out at room temperature under magnetic stirring. (C) Accelerated photobleaching profile of BeetBlue monitored by change in absorption at 582 nm. Solid lines are the logistic regression fit. (D) BeetBlue encapsulated in maltodextrin (5% w/w) or incorporated in complex matrices. Samples were illuminated with white light, and pictures were not submitted to hue adjust. The noncolored precursor materials are shown for reference.

Supplementary Materials

  • Supplementary Materials

    A metal-free blue chromophore derived from plant pigments

    B. C. Freitas-Dörr, C. O. Machado, A. C. Pinheiro, A. B. Fernandes, F. A. Dörr, E. Pinto, M. Lopes-Ferreira, M. Abdellah, J. Sá, L. C. Russo, F. L. Forti, L. C. P. Gonçalves, E. L. Bastos

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