High-resolution mass spectral analysis of the blue-colored product ( Fig. 1D ) exhibited a signal at mass/charge ratio (m/z) 289.1181 corresponding to the [M + H] + ion of BeetBlue (exact mass, 289.1183 Da). The fragmentation of this ion occurs via single and double decarboxylation leading to signals at m/z 245.1250 and 201.1384, respectively (fig. S7). The fragment at m/z 108.0800 results from double decarboxylation and loss of the pyrrolic portion of BeetBlue, supporting the presence of the C─C bond.

Nuclear magnetic resonance (NMR) analyses and quantum-mechanical gauge-independent atomic orbital (GIAO) calculations (figs. S1 to S6) show that the nucleophilic attack of C2 of 2,4-dmp on the carbonyl group of HBt forms a new C─C bond. Furthermore, these results increase knowledge of the structure of betalamic acid derivatives since their 13 C NMR and two-dimensional (2D) NMR spectra have been scarcely reported ( 28 , 29 ). The π-conjugation of the resulting diazapolymethine system is extended compared to that of betalains, resembling the chromophore of the widely used dye indocyanine green (ICG) ( Fig. 1B ). No evidence was found for the presence of a hydrogen atom at the α-position of the pyrrole ring, although the α carbon atoms (C9 and C12) were detected in the 13 C NMR spectrum ( Fig. 1C ). The large coupling constant between H7 and H8 ( 3 J H7,H8 = 15.2 Hz) is in the expected range for a trans alkene (14 to 16 Hz) but is larger than the values reported for betalains (roughly 10 to 12 Hz) ( 28 ), indicating C─C bond formation.

( 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 ) 1 H and 13 C NMR spectra of BeetBlue (13 mM, 800 MHz/200 MHz, D 2 O at 288 K). ( D ) High-resolution mass spectrum of BeetBlue; m/z 289.1181, [M + H] + .

BeetBlue was obtained by the irreversible dehydrative C─C coupling of betalamic acid (HBt) and 2,4-dimethylpyrrole (2,4-dmp) in acidified ethyl acetate ( Fig. 1A and movie S1). The product was isolated in 70% yield, which is three times higher than that reported for the semisynthesis of betalains ( 21 , 22 ). Betalamic acid can be extracted from base-hydrolyzed beetroot juice ( 20 , 23 ) or prepared by the enzymatic oxidation of l-dopa ( 24 – 27 ).

Blue hues of dyes often vanish or alter under acidic conditions, and molar absorption coefficients are quite low ( 5 ). That is not the case for BeetBlue, which maintains its blue color in a variety of acidified solvents ( Fig. 2C ) and becomes bluer in organic solvents such as dimethyl sulfoxide (DMSO) and trifluoroethanol (TFE). This minimal solvatochromism indicates that the hydrogen bond donation capacity of the solvent does not affect the charge distribution of the fully protonated form of BeetBlue. The perceived color of each solution is shown at the center of the magnified region of the chromaticity diagram; colors that are indistinguishable to the human eye are shown in the MacAdam ellipses ( Fig. 2C ) ( 31 ). Natural transition orbital (NTO) analysis shows that both the charge distribution of the ground state and the locally excited state are contained within the 1,11-diazaundecamethinium system, being shifted from the α,β-unsaturated enamine system formed by the atoms N1, C5, and C9 to the polymethine system (C4, C6, and C8) ( Fig. 2D ). The carboxylic acid moiety attached to the sp 2 carbon atom of the 2,3-dihydropyridine ring participates in the electronic transition, explaining the effect of its protonation state on the absorption spectrum of BeetBlue.

The spectrophotometric titration of BeetBlue reveals that its color does not depend on the pH within the range of 3 to 8 ( Fig. 2B ). It is noticeable that the protonation of the carboxylic acid moieties [pK a (the negative logarithm of the acid dissociation constant, K a ) = 2.9] under very acidic conditions redshifts the absorption spectra compared to neutral conditions. However, deprotonation of the N1 atom of the 2,3-dihydropyridine ring under alkaline conditions (pK a = 9.6) affects charge distribution in the chromophore, reversibly changing BeetBlue’s color from blue to yellow (fig. S9). This reversible behavior differs from that of natural betalains, which undergo further decomposition in alkaline media.

( 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 pK a values. The right projection shows the absorption spectra at selected pHs (2, 2.9 [pK a1 ], 7, 9.6 [pK a2 ], and 12). For reference, the pK a s 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.

Natural betalains have yellow-orange or red-violet color depending on the electronic properties of the substituents attached to the imine/iminium (N9) nitrogen atom. The extended π-conjugation of the 1,11-diazaundecamethinium chromophore of BeetBlue induces a redshift of both its absorption and fluorescence spectra in water relative to the standard betalains betanin and indicaxanthin ( Fig. 2A ). The maximum molar absorption coefficient (ε) of BeetBlue at 582 nm is 54,000 liter mol −1 cm −1 (fig. S8), which is within the 40,000 to 70,000 liter mol −1 cm −1 range typical of betalains ( 30 ).

Toxicity and potential application

The application of new dyes requires safety assessment. BeetBlue was tested for cytotoxicity to human hepatic and retinal pigment epithelial cells in culture and for in vivo toxicity to zebrafish embryos (Danio rerio Hamilton, 1822). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the viability of the immortalized human hepatic cell lines Huh-7, Hep-G2, and HepaRG treated with BeetBlue for 8 hours (Fig. 3A). Cells incubated with the dye (0.1 mM to 1 nM) remained 100% viable, and 1 mM BeetBlue was required to reduce cell viability by 20 to 30%. Although these cell lines have been widely used in toxicological studies (32), hepatocyte-like HepaRG cells express a large set of liver-specific functions and, therefore, were selected to investigate the genotoxicity of BeetBlue. Cells were treated with the dye (10 μM) for up to 8 hours, and DNA fragmentation did not differ from the untreated negative control, as measured by single-cell gel electrophoresis (Comet assay; Fig. 3B). The cytotoxicity of BeetBlue was also verified by determining the viability of ARPE-19 cells using 7-aminoactinomycin D (7-AAD) as the fluorescent marker for membrane integrity (Fig. 3C). This human pigment epithelial cell line has been used as a model to investigate the toxicity and phototoxicity of dyes due to its response to factors related to photoinduced macular degeneration, including photosensitized singlet oxygen formation (33). After incubation with the dye (10 μM) for 24 hours, a single population of cells is found, in contrast with the at least two distinct cell populations observed when cells were incubated with 50 mM hydrogen peroxide for 5 hours (Fig. 3C). The fish embryo acute toxicity test was performed according to the guidelines of the Organization for Economic Cooperation and Development (OECD) (34). To test the effect of BeetBlue on the development of zebrafish embryos, we kept the animals in culture in E2 medium in the presence of the dye (10 μM) for 4 days. The morphology of the zebrafish larvae during growth in the presence of BeetBlue is identical to that of the negative control in the absence of the dye (Fig. 3D). The death of embryos and larvae incubated with BeetBlue along the experiment does not differ from the negative control (Fig. 3E).

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.

The toxicity of dyes is often related to their ability to produce singlet oxygen, which has been found to promote cell damage and death (35). Dyes able to undergo efficient intersystem crossing (ISC) from the singlet-excited state to the triplet manifold can transfer energy to molecular oxygen, producing singlet oxygen. Nanosecond transient absorption spectra of BeetBlue excited at 580 nm in the presence and absence of oxygen are identical (Fig. 4A). The spectra are dominated by the ground state bleaching and a positive absorption band centered at 615 nm, both with a 3.7-μs half-life, calculated from single-exponential curve fitting (fig. S10). The positive absorption could be assigned to triplet state absorption; however, the signal’s insensitivity to the presence of O 2 and the lack of the characteristic chemiluminescence emission of singlet (1Δ g ) oxygen at 1270 nm suggest otherwise. Since the formation of triplet-excited states of betalains via ISC is inefficient (36), the most logical assignment to the positive signal centered at 615 nm is the absorption of the cis-form of BeetBlue formed by ultrafast photoisomerization, as observed on cyanines and rhodopsins (37, 38). Laser excitation of the dye for up to 140 min at 355 or 582 nm using a power density of 2.23 and 0.45 J/s cm2, respectively, decreased the absorption at 582 nm concomitantly with a subtle increase at around 450 nm (Fig. 4B). The accelerated photobleaching of 18.5 μM BeetBlue shows a dose-response profile and required an input fluence of 1.5 kJ/cm2 at 582 nm or 12 kJ/cm2 at 355 nm to halve the concentration of the dye (Fig. 4C). For comparison, 7 kJ/cm2 is required to completely photobleach 0.2 mM ICG (39).

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.

To showcase some potential applications of BeetBlue, this prototype quasibetalain was incorporated into complex matrices—such as cellulose, cotton fabric, yogurt, hair, and Bombyx mori silk cocoon—and encapsulated in maltodextrin (Fig. 4D). Depending on the matrix, the hue differed from that measured in aqueous solution, but under magnification, a uniform blue color distribution was observed, thus reaffirming the applicability of BeetBlue as a metal-free blue colorant.