R.M. Pioli et al.
Dyes and Pigments 183 (2020) 108609
acid in ethyl acetate (9
μ
mol, 10 mL), the neat amine (2a-h) (45
μ
mol, 5
4.3. Absorption and fluorescence spectra
equiv.), and p-toluenesulfonic acid monohydrate (45
μmol, 5 equiv.).
The reaction mixture was stirred at room temperature for 30 min and
Absorption and fluorescence spectra were acquired using a 10-mm
light path quartz cuvette on a Varian Cary 50 Bio spectrophotometer
and on a Varian Cary Eclipse spectrofluorometer, respectively, both
equipped with water-cooled Peltier thermostatted cell holders; absorp-
◦
then kept at ꢀ 20 C for 2 days. The resulting suspension was centrifuged
◦
(
10,000×g, 10 min, 4 C) and the precipitate was washed with cold ethyl
acetate (2 × 8 mL) to remove any residual amount of catalyst and
unreacted starting material. The red/orange crude solid material was
solubilized in water (1–10 mL) and submitted to gel column chroma-
tography on Sephadex LH-20 using water as eluent. The red/orange-
colored fractions were combined and lyophilized (ꢀ 85 ◦C, 8 mbar)
and the final product was submitted to 1H NMR and HPLC-DAD-ESI
ꢀ
1
tion wavelength range: 300–750 nm, scan rate: 1200 nm min , at 25
◦
C; emission (EM) wavelength range: 480–800 nm (excitation (EX)
EX
ꢀ 1
wavelength (λ ) = 460 nm), scan rate: 600 nm min , EX and EM slits:
◦
20 nm bandwidth, photomultiplier voltage: 800 V, at 25 C.
1
3
(
+)-MS/MS analysis (see Supplementary Material). C NMR data could
not be obtained at 125 MHz, due to the time-limited persistence of these
compounds in D O and their low solubility in organic solvents.
mepBeet (3a). Red solid, obtained as a mixture of geometric isomers
4
.4. Molar absorption coefficients ( )
ε
2
The direct weighting of betalains is difficult due to the limited
amount of substance and their high hygroscopicity. Therefore, the molar
absorption coefficients ( ) of the mepBeets and the dipBeets were
determined using an endpoint method [28]. A stock solution of each
1
(
3:1); H NMR (D
2
O, 500 MHz) Major isomer δ 8.22 (br s, 1H), δ 7.53 (br
ε
s, 2H), δ 7.43 (br s, 3H), δ 6.31 (br s, 1H), δ 6.27 (br s, 1H), δ 4.50 (br s,
1
H), δ 3.63 (s, 3H), δ 3.40–3.37 (m, 1H), δ 3.17–3.15 (m, 1H). Minor
ꢀ 4
ꢀ 1
betalain (ca. 10 mol L ) was prepared in water. Next, working so-
isomer (different signals only) δ 8.32 (br s, 1H), δ 5.90 (br s, 1H), δ 5.69
lutions were prepared by diluting the stock solution 10 times in BR
+
(
br s, 1H); HRMS (ESI-TOF) m/z: [M] Calcd. for C16
H
17
N
2
O
4
301.1183,
◦
buffer pH 9.0 thermostatted at 25 C and the hydrolysis was monitored
found 301.1187 (1.3 ppm); Yield: 10%.
by absorption spectroscopy. The temporal change in absorbance at 424
1
dipBeet (3b). Orange solid; H NMR (D
2
O, 500 MHz) δ 8.38 (d, J =
nm was fitted to a monoexponential function to determine the maximum
1
5
3
3
3.0 Hz, 1H), δ 7.53 (br s, 4H), δ 7.38–7.33 (m, 6H), δ 6.12 (br s, 1H), δ
HBt
absorbance of betalamic acid (A
∞
). The molar absorption coefficient
.81 (d, J = 13.0 Hz, 1H), δ 4.37 (t, J = 7.5 Hz, 1H), δ 3.35 (br s, 1H), δ
S
of the sample at the maximum absorption wavelength (
ε
(λ)) was then
+
.22–3.17 (m, 1H); HRMS (ESI-TOF) m/z: [M] Calcd. for C21
63.1339, found 363.1350 (3.0 ppm); Yield: 17%.
19 2 4
H N O
calculated according to Eq. (1):
S
A
p-MeO-mepBeet (3c). Red solid, obtained as a mixture of geometric
pH 7
εS(λ) = εHBt, 424 nm
(1)
1
HBt
isomers (2:1); H NMR (D
Hz, 1H), δ 7.43–7.37 (m, 2H), δ 7.13–7.07 (m, 2H), δ 6.26 (s, 1H), δ 6.20
d, J = 11.6 Hz, 1H), δ 4.38 (br s, 1H), δ 3.87 (s, 3H), δ 3.58 (s, 3H), δ
2
O, 500 MHz) Major isomer δ 8.12 (d, J = 11.6
A
∞
HBt,424 nm
ꢀ 1
ꢀ 1
(
where
ε
= 27,000 L mol cm [29] and the absorbance of the
S
3
.33–3.29 (m, 1H), δ 3.15–3.11 (m, 1H). Minor isomer (different signals
sample at the maximum absorption wavelength (A pH 7) was acquired at
pH 7.0 to prevent change in concentration due to hydrolysis.
only) δ 8.27 (d, J = 11.6 Hz, 1H), δ 5.88 (s, 1H), δ 5.65 (d, J = 11.6 Hz,
+
1
3
H), δ 3.66 (s, 3H); HRMS (ESI-TOF) m/z: [M] Calcd. for C17
19 2 5
H N O
31.1288, found 331.1290 (0.6 ppm); Yield: 16%.
4
.5. Fluorescence quantum yields
1
p-MeO-dipBeet (3d). Red solid; H NMR (D
2
O, 500 MHz) δ 8.36 (br
s, 1H), δ 7.50 (br s, 2H), δ 7.31 (br s, 5H), δ 7.13 (br s, 2H), δ 6.10 (br s,
Fluorescence quantum yields (ΦFl) were determined in water using
1
H), δ 5.82 (br s, 1H), δ 4.36 (t, J = 7.4 Hz, 1H), δ 3.89 (s, 3H) δ 3.33 (br
rhodamine B (ethanol solution; n
D
= 1.3616; ΦFl = 0.51) [47] and
+
ꢀ 1
s, 1H), δ 3.19 (dd, J = 17.1, 7.4 Hz, 1H); HRMS (ESI-TOF) m/z: [M]
fluorescein (solution in 0.1 mol L NaOH(aq); n
D
= 1.3325; ΦFl = 0.95)
Calcd. for C22
2%.
H
21
N
2
O
5
393.1445, found 393.1450 (1.3 ppm); Yield:
[
48] as the secondary fluorescence standards [51]. Fluorescence spectra
1
of the standards and the betalains were taken under identical spectro-
1
EX
p-NC-mepBeet (3e). Orange solid; H NMR (D
2
O, 500 MHz) δ 8.13
metric conditions; excitation (EX) wavelength (λ ): 460 nm, emission
(
d, J = 12.9 Hz, 1H), δ 7.86 (d, J = 8.9 Hz, 2H), δ 7.55 (d, J = 8.9 Hz,
(
EM) wavelength range: 480–800 nm, EX and EM slits: 20 nm band-
2
H), δ 6.40 (s, 1H), δ 6.15 (d, J = 12.9 Hz, 1H), δ 4.39 (dd, J = 8.1, 6.8
◦
width, photomultiplier voltage: 800 V, at 25 ± 1 C. Due to the low
fluorescence of these betalains and some experimental limitations, all
fluorescence spectra were fitted to multiple Gaussian functions using the
Fityk software [52], and the area under the emission curve (S) of the
resulting fitted spectra was used to calculate the ΦFl according to Eq. (2);
Hz, 1H), δ 3.56 (s, 3H), δ 3.34 (dd, J = 17.6, 6.8 Hz, 1H), δ 3.17 (dd, J =
+
1
3
7.6, 8.2 Hz, 1H); HRMS (ESI-TOF) m/z: [M] Calcd. for C17
H
16
N
3
O
4
26.1135, found 326.1135 (0.0 ppm); Yield: 11%.
1
p-NC-dipBeet (3f). Orange solid; H NMR (D
2
O, 500 MHz) δ 8.33 (d,
2
J = 13.0 Hz, 1H), δ 7.80 (d, J = 8.6 Hz, 2H), δ 7.62 (t, J = 7.6 Hz, 2H), δ
the non-linear regression coefficient of determination (R ) in all cases
7
.55 (t, J = 7.6 Hz, 1H), δ 7.35 (s, 2H), δ 6.21 (s, 1H), δ 5.80 (d, J = 13.0
was higher than 0.999 and the area of the spectrum determined by
non-linear data fitting was inferior to 15%.
Hz, 1H) δ 4.44 (dd, J = 8.4, 6.6 Hz, 1H), δ 3.41 (dd, J = 17.8, 6.6 Hz,
+
1
H), δ 3.21 (dd, J = 17.8, 8.4 Hz, 1H); HRMS (ESI-TOF) m/z: [M]
(
)
S
2
A
S
n
n
D
Calcd. for C22
H
18
N
3
O
4
388.1291, found 388.1291 (0.2 ppm); Yield: 7%.
S
ΦFl = Φ
(2)
1
Fl
A SS
S
bis(p-MeO)-dipBeet (3g). Red solid; H NMR (D
2
O, 500 MHz) δ 8.31
D
(
br s, 1H), δ 7.29 (br s, 4H), δ 7.15 (br s, 2H), δ 7.06 (br s, 2H), δ 6.06 (br
where A is the absorbance at 460 nm, S is the area under the emission
curve, and n is the refractive index of the solvent; S superscript refers to
the standard.
s, 1H), δ 5.84 (br s, 1H), δ 4.35–4.32 (m, 1H), δ 3.89 (s, 6H), δ 3.34 (br s,
1
+
H), δ 3.18 (br s, 1H); HRMS (ESI-TOF) m/z: [M] Calcd. for
23 23 2 6
C H N O 423.1551, found 423.1554 (0.8 ppm); Yield: 10%.
1
p-MeO-p-NC-dipBeet (3h). Orange solid; H NMR (D
.33 (d, J = 13.0 Hz, 1H), δ 7.78 (d, J = 8.5 Hz, 2H), δ 7.34 (d, J = 8.5
Hz, 2H), δ 7.30 (d, J = 8.9 Hz, 2H), δ 7.18 (d, J = 8.9 Hz, 2H), δ 6.21 (s,
2
O, 500 MHz) δ
8
4.6. Kinetics of hydrolysis
1
H), δ 5.79 (d, J = 13.0 Hz, 1H), δ 4.43 (dd, J = 8.4, 6.6 Hz, 1H), δ 3.91
To a 10-mm cuvette containing Britton-Robinson buffer (BR buffer,
ꢀ 1
◦
(
s, 3H), δ 3.39 (dd, J = 18.0, 6.6 Hz, 1H), δ 3.20 (dd, J = 18.0, 8.4 Hz,
2 mL, pH 5.0, 40 mmol L ) thermostatted at 60 C was added an aliquot
of betalain solution in water (20 L) to produce a solution with initial
+
1
H); HRMS (ESI-TOF) m/z: [M] Calcd. for C23
H
20
N
3
O
5
418.1397,
μ
found 418.1403 (1.4 ppm); Yield 11%.
absorbance at the maximum absorption wavelength of around 0.9. The
absorption spectra were registered from 300 to 750 nm at every 5 min
for 10 h. The observed rate constants (kobs) were calculated by fitting the
5