Small-molecule fluorophores with large stokes shifts: 9-iminopyronin analogues as clickable tags

Article abstract of DOI:10.1021/jo502213t

The design, synthesis, and both experimental and theoretical studies of several novel 9-(acylimino)- and 9-(sulfonylimino)pyronin derivatives containing either an oxygen or a silicon atom at position 10 are reported. These compounds, especially the Si analogues, exhibit remarkably large Stokes shifts (around 200 nm) while still possessing a high fluorophore brightness, absorption bands in the near-UV and visible part of the spectrum, and high thermal and photochemical stabilities in protic solvents. The reason for the observed large Stokes shifts is an intramolecular charge-transfer excitation of an electron from the HOMO to the LUMO of the chromophore, accompanied by elongation of the C9-N bond and considerable solvent reorganization due to hydrogen bonding to the solvent. Due to the photophysical properties of the studied compounds and their facile and high-yielding synthesis, as well as a simple protocol for their bioorthogonal ligation to a model saccharide using a Huisgen alkyne-azide cycloaddition, they represent excellent candidates for biochemical and biological applications as fluorescent tags and indicators for multichannel imaging. 9-(Acylimino)pyronins alter their optical properties upon protonation and may also be used as pH sensors.

Full text of DOI:10.1021/jo502213t

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pubs.acs.org/joc
Small-Molecule Fluorophores with Large Stokes Shifts:
9‑Iminopyronin Analogues as Clickable Tags
̌
̌
Peter Horvath, Peter Sebej, Tomas Solomek, and Petr Klan*
́
́
̌
́
Department of Chemistry and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
S
* Supporting Information
ABSTRACT: The design, synthesis, and both experimental and
theoretical studies of several novel 9-(acylimino)- and 9-
(sulfonylimino)pyronin derivatives containing either an oxygen or a
silicon atom at position 10 are reported. These compounds, especially
the Si analogues, exhibit remarkably large Stokes shifts (around 200
nm) while still possessing a high fluorophore brightness, absorption
bands in the near-UV and visible part of the spectrum, and high
thermal and photochemical stabilities in protic solvents. The reason for
the observed large Stokes shifts is an intramolecular charge-transfer
excitation of an electron from the HOMO to the LUMO of the
chromophore, accompanied by elongation of the C9−N bond and
considerable solvent reorganization due to hydrogen bonding to the
solvent. Due to the photophysical properties of the studied compounds
and their facile and high-yielding synthesis, as well as a simple protocol
for their bioorthogonal ligation to a model saccharide using a Huisgen alkyne−azide cycloaddition, they represent excellent
candidates for biochemical and biological applications as fluorescent tags and indicators for multichannel imaging. 9-
(Acylimino)pyronins alter their optical properties upon protonation and may also be used as pH sensors.
imaging.10 Large Stokes shifts are common for fluorescent
INTRODUCTION
■
proteins,11 metal complexes,12,13 quantum dots,14 or UV
Small-molecule organic fluorophores with tailored chemical and
photophysical properties have become an essential tool for
absorbing chromophores. However, these fluorophore families
lack one or more of the advantages that are connected to the
imaging and sensing in biochemistry, biophysics, molecular
biology, medicine, and material sciences.1−5 The major benefit of
small-molecule organic fluorophores mentioned above.
Excited-state geometry relaxation accompanied by solvent
utilizing synthetic fluorophores is the opportunity to carefully
design their chemical and spectroscopic properties using
reorganization are required to achieve a substantial Stokes shift.15
methods of modern organic chemistry.1,5 Various bioconjugation
This usually leads the molecule to explore a considerable part of
its excited state potential energy surface where conical
strategies that involve fluorescent dye markers have also been
developed.6
intersections can be encountered, and as a result, the excited
state can be deactivated via a radiationless internal conversion
Newly designed fluorophores for bioapplications should be
thermally and photochemically stable, nontoxic, sufficiently
decreasing the fluorescence quantum yield.16 Rotation of a π-
conjugated substituent in the molecular structure of a
soluble in water, absorb visible light with large molar absorption
fluorophore has been proposed to yield a large Stokes shift,17
coefficients, and emit light efficiently in the red or near-infrared
parts of the spectrum, where a higher signal-to-noise ratio due to
but it is rather difficult to predict the nature and the position of
such substituents in a fluorophore, because detailed guidelines,
low background autofluorescence and enhanced tissue pene-
though under development,18 are still incomplete. Other
tration (650−950 nm)7,8 are of a great benefit. High fluorophore
strategies based on Forster resonance energy transfer (FRET)
̈
brightness, proportional to the product of the decadic molar
absorption coefficient and the fluorescence quantum yield,
allows visualization at low fluorophore concentrations, which
suppresses their aggregation and improves optical resolution in
single-molecule microscopy.9 Aggregation of fluorophores in
solutions may result in self-quenching and loss of fluorescence
intensity.
do not entirely eliminate the self-quenching and inner-filter
effects.19
In this report, we describe the design, synthesis, and
photophysical properties of novel 9-(acylimino)- and 9-
(sulfonylimino)pyronin fluorophores containing oxygen and
silicon at position 10 that exhibit remarkably large Stokes shifts, a
high fluorophore brightness, and high thermal and photo-
Self-quenching occurs principally in fluorophores with small
Stokes shifts.9 Therefore, a large Stokes shift is a very important
attribute of a fluorophore. In addition, a cleverly designed set of
fluorophores with large Stokes shifts can enable multichannel
Received: September 26, 2014
© XXXX American Chemical Society
A
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chemical stability. An explanation for the observed Stokes shifts is
suggested and corroborated by quantum chemical calculations.
The effects of the structure modifications as well as of solution
pH are discussed. We also demonstrate that a facile ligation
strategy can be used to attach the 9-iminopyronin fluorophores
to a model saccharide.
(acylimino)pyronins) 1a, 2a, 2c, 2d, and 2f in methanol
max
possessed a major absorption band at λ
= 375−400 nm
abs
(Table 1; 2a: Figure 1), the position of which was insignificantly
affected by polar or protic properties of a solvent (the absorption
spectra of 2a in CH2Cl2, CH3CN, DMSO, and methanol are
shown in Figure S39, Supporting Information). These
compounds showed bright fluorescence at room temperature
with the quantum yields of Φf from 0.28 to 0.56 in methanol but
were barely or nonemissive in aprotic solvents. In addition,
Figure S40 shows the changes in the fluorescent spectra of 2a in
RESULTS
■
Synthesis of 1 and 2. The synthesis of the target 9H-
xanthene-3,6-diamine derivatives, 1a,b, and their analogues
containing silicon at position 10, 2a,b, is depicted in Scheme 1.
acetonitrile upon addition of small amounts of methanol; the
max
em
emission intensity increased significantly, and λ was shifted
bathochromically by about 30 nm. The emission band maxima of
Scheme 1. Synthesis of 1a,b and 2a−e
max
the Si-analogues 2a,c,d,f (λ ∼600 nm) were shifted bath-
em
max
em
ochromically relative to the oxygen analogue 1a (λ ∼510 nm).
The corresponding Stokes shifts were ∼200 nm (∼9090 cm−1)
for Si-analogues and 137 nm (7100 cm−1) for the O-analogue 1a.
The absorption and emission bands of the 9-(alkylsulfonyl)-
iminopyronins 1b, 2b, 2e, and 2g were bathochromically shifted
(λambasx = 422−452 nm; λmemax = 525−617) compared to those of the
9-(acylimino) derivatives (Table 1; Figure 1 and Figure S48,
Supporting Information), and the effect of the atom at position
10 was analogous to that observed in the acyl series. The
fluorescence quantum yields (Φf = 0.23−0.43) and the Stokes
shifts (103−173 nm) were slightly smaller than those of 9-
(acylimino)pyronins. The spectroscopic properties of the
xanthenones 5 and 6, also studied elsewhere,22 are shown in
Table 1 for comparison. The fluorescence decay of 2f and 2g
(Table 1) obeyed a single-exponential rate law with a lifetime
similar to that of fluorescein23 (τ = 4.0 ns).
Photophysical Properties in Aqueous Solutions. We
evaluated the acid−base properties of 2 in aqueous solutions. In
In the first step, 9-aminopyronin 3 and silicon-containing 9-
aminopyronin 4 were prepared from the xanthenone analogues 5
and 6 in very good yields (85−97%) according to a procedure
adopted from Wu and Burgess.20 The isolated 9-aminopyronins
were subsequently converted to the title 9-imino derivatives 1a,b
and 2a,b by a reaction with acyl or sulfonyl chlorides in 74−92%
yields. The overall yields over the two steps were 63−90%. A
more water-soluble derivative 2c was also prepared.
The alkynyl derivatives 2d and 2e synthesized in the same way
from 4 (Scheme 1) in 89 and 38% yield, respectively, were linked
to 2-azido-2-deoxy-D-glucose using a copper(I)-catalyzed azide−
alkyne cycloaddition reaction21 in excellent yields (90 and 86%,
respectively; Scheme 2). The resulting 1,2,3-triazoles 2f,g were
mixtures of two regioisomers in an approximately 55:45 ratio
(see the Experimental Section for further details).
max
max
general, both λ and λ were only slightly bathochromically
abs
em
shifted compared to those observed in methanol. The acid
dissociation constants of the water-soluble 9-(acylimino)-Si-
pyronin (2c) and 9-(sulfonylimino)-Si-pyronin (2b) derivatives
were obtained by spectrophotometric titration of their aqueous
solutions with 1% of DMSO as cosolvent. A series of UV−vis
spectra was measured by addition of 0.01 mol dm−3 HCl to a
solution of 2c or 2b in 0.01 mol dm−3 aqueous NaOH or 0.01
mol dm−3 NaOH to a solution of 2c or 2b in 0.01 mol dm−3
aqueous HCl. The acid dissociation constants (ionization
quotients at ionic strength I = 0.1 mol dm−3) resulting from a
global analysis of the spectra and fitting of a titration function
provided the pKas of 6.77 0.06 for 2c and approximately 2−3
for 2b (Table 2; see Figure S62 for details). The global analyses
provided the species spectra identical to those determined
spectrophotometrically (Table 2, Figure 2).
The absorption and emission spectra of the 9-(acylimino)
derivative 2f measured at low and high pH values are shown in
Figures 2a,b, and the corresponding photophysical parameters
are listed in Table 2. The spectra obtained under physiological
conditions (PBS, pH = 7.4) are portrayed in the same figure for
comparison (Figure 2c). The λmabasx (401 nm) and λemmax (606 nm)
values as well as the Stokes shifts determined at pH above the
corresponding pKa (Figure 2a; pKa = 6.76 0.11) were similar to
those obtained in methanol (for example, 2f: λmabasx = 386 nm and
λmemax = 596 nm; Table 1, Figure S61), and this species is assigned
to the neutral base B (Scheme 3). The spectrum observed at low
Photophysical Properties in Methanol and Aprotic
Solvents. The absorption spectra of the O- and Si-atom
containing 9-(acylimino)-9H-xanthene-3,6-diamines (or 9-
Scheme 2. Copper(I)-Catalyzed Alkyne−azide Cycloaddition
of 2c,d with 2-Azido-2-deoxy-D-glucose To Give 2f,g (Only
One Regioisomer Is Shown for Clarity)
pH was assigned to the acid A. In this case, the absorption
max
abs
maxima are substantially bathochromically shifted (λ (2f) =
664 nm), whereas the emission maxima are shifted only slightly
compared to those of the base B. The corresponding Stokes shifts
B
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Table 1. Spectroscopic Properties of 1, 2, 5, and 6 in Methanol
a
b
compd
λmabasx (nm)
log ε (dm3 mol−1 cm−1
)
λmemax (nm)
Φf (τ/ns )
Stokes shift (nm) (cm−1
)
εΦ/103 (dm3 mol−1 cm−1
)
5
379
376
422
402
387
400
387
386
4.63
4.32
4.19
4.51
4.24
4.21
4.40
4.34
442
513
525
489
597
596
595
596
0.56 0.03
0.36 0.01
0.33 0.02
0.40 0.03
0.56 0.04
0.47 0.04
0.28 0.03
0.30 0.03
(2.31 0.01)
0.23 0.01
0.43 0.02
0.36 0.03
(3.06 0.01)
63 (3760)
137 (7100)
103 (4650)
87 (4430)
23.9
7.5
1a
1b
6
5.1
12.9
9.7
2a
2c
2d
2f
210 (9090)
196 (8220)
208 (9030)
208 (9130)
7.6
7.0
6.6
2b
2e
2g
444
452
448
4.17
4.30
4.18
617
617
616
173 (6370)
165 (5920)
168 (6090)
3.4
8.6
5.4
a
b
max
abs
Fluorescence lifetimes (τ). Brightness of the fluorophore (the product of the molar absorption coefficient at λ
and the corresponding
fluorescence quantum yield).
CD3OD/D2O (4:1, v/v) solution) exhibited a similar change of
the spectral properties and allowed us to estimate the pKa ∼ 6
(Figure S31). The exchange of the acidic proton (form A) is fast
enough to show only an average NMR signals instead of signals
of two species. The pKa of the 9-(alkylsulfonyl)imino derivative
2g could not be determined accurately because the compound is
hydrolyzed at pH below 3 in the dark within minutes. However,
the estimated value of pKa ∼ 2−3 ensures that only the base B is
present at pH = 7.4 (Figure 3). The fluorescence quantum yields
and fluorescence lifetimes for 2f,g (Table 2) in a PBS buffer are
similar to those determined in methanol.
A possible excited-state proton-transfer (ESPT)9 process was
investigated for 2g by measuring the absorption and fluorescence
spectra as a function of the acetate buffer concentration (Figure
S69). The pH of the solution was kept at ∼4.6, i.e., higher than
the corresponding pKa value (∼2−3; Table 2). The absorption
spectrum of 2g was not affected by the buffer concentration in
Figure 1. Absorption (black line) and normalized emission (red line)
spectra of 2a (solid lines; c = 7.4 × 10−5 mol dm−3; λexc = 389 nm) and 2b
(dashed lines; c = 6.2 × 10−5 mol dm−3; λexc = 443 nm) in methanol.
the range of 0.03 and 3 M. However, while only one emission
max
band (λ = 629 nm) dominated the spectrum at a low buffer
are relatively small (below 20 nm). Under physiological
conditions (PBS, pH = 7.4), the spectra revealed the presence
of both acid−base forms (Figure 2c) and are equal to those
observed under the extreme pH conditions. In addition, nearly
identical spectroscopic behavior was observed for 2c (Table 2).
em
concentration, a new band at λmemax = 693 nm appeared as a major
emission peak at a very high buffer concentration.
No change of the absorption spectra, usually associated with
dye aggregation in polar media,24,25 was observed in the case of 2f
and 2g in PBS/DMSO (99:1, v/v) in the concentration range
1
The analysis of series of H NMR spectra of 2a (in a buffered
Table 2. Spectroscopic Properties of 2 in Aqueous Solutions
a
b
f
e
compd
pKa
pH
λmabasx (nm) log ε (dm3 mol−1 cm−1
)
λmemax (nm)
Φf (τ/ns )
Stokes shift (nm) (cm−1
15 (330)
)
εΦ/103 (dm3 mol−1 cm−1
)
2c
6.77 0.06
3.0
11.0
3.0
666
418
664
401
4.92
4.11
5.09
4.25
681
0.28 0.02
(2.32 0.01)
0.46 0.01
(2.34 0.01)
0.26 0.04
(2.10 0.02)
0.35 0.03
(1.90 0.01)
n.d.
23.3
607
682
606
189 (7450)
18 (400)
5.9
32.0
6.2
2f
6.76 0.11
11.0
205 (8440)
c
2b
2g
2−3
2−3
7.4
3.0
7.4
469
667
469
n.d.
n.d.
4.04
630
692
631
161 (5450)
25 (540)
n.d.
n.d.
4.7
d
n.d.
0.43 0.03
(2.79 0.01)
157 (5470)
a
b
The pKa values were determined by titration (see the text). The values for 2b,g could not be determined precisely. I = 0.1 mol dm−3 adjusted by
c
KCl; 1% of DMSO was added as a cosolvent. pH of the solution was determined using a calibrated glass electrode. 10% of DMSO was added as a
cosolvent. These values were determined at pH = 3.0 at which the compound is sufficiently stable in the dark; both forms A and B (Scheme 3) were
present; an estimated amount of the form B was <20%. See footnote b in Table 1. See footnote a in Table 1.
d
e
f
C
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Scheme 3. Acid−base Equilibrium for 1 and 2
Figure 3. Absorption (black solid line; c = 2.7 × 10−5 mol dm−3),
normalized emission (red solid line, λexc = 401 nm), and normalized
excitation (λem = 632 nm; red dotted line) spectra of 2g in PBS/DMSO
(99:1, v/v) at pH = 7.4.
Chemical Stability. The chemical stability of 2f,g in the dark
was determined in various solvents (methanol, DMSO and PBS/
DMSO, 99:1, v/v). A plot of the dye absorbance λmabasx against time
was used to calculate the half-lives of the compounds by fitting
the data with a pseudo-first order kinetic equation. The
decomposition half-lives of 2f were τ1/2 = 33 h−1 and τ1/2 > 104
h−1 in PBS/DMSO and DMSO, respectively, at 20 °C.
Compound 2g was even more stable (τ1/2 = 750 h−1 in PBS/
DMSO; for details see the Supporting Information, page S2).
The compounds 2f (Figure 4) and 2a (Figure S38) exhibited a
full reversibility between the acid A and base B forms over five
cycles of periodic addition of aq HCl and NaOH between pH 3.0
and 11.0.
Both 2f and 2g decomposed in methanol and PBS/DMSO
upon irradiation using 32 high-energy LEDs (λem = 470 nm,
Figure S71) or a diode-laser (λem = 375 nm, Figure S70). The
decomposition was about 4-fold faster in aqueous solutions than
that in methanol with the half-life of ∼1 h for 2g in PBS solution
(Supporting Information).
Quantum-Chemical Calculations. We performed a
conformational analysis of the studied dyes 1a,b and 2a,b in
their ground electronic states. Two distinct conformations were
identified for each compound (Figure S72 and Table S4), and the
geometries of those with the lowest potential energies were then
chosen to represent the Franck−Condon points from which the
excited-state geometry optimizations started. The absorption
maxima of the pairs of conformers did not differ by more than 10
nm. Inspection of the optimized geometries revealed that the
bond between a C9 substituent and the C9 carbon atom (C9−N
Figure 2. (a) Absorption (black solid line; c = 1.9 × 10−5 mol dm−3),
normalized emission (λexc = 664 nm; red solid line), and normalized
excitation (λem = 682 nm; red dotted line) spectra of 2f in aqueous HCl/
DMSO (99:1, v/v; I = 0.1 mol dm−3, KCl) at pH = 3.0. (b) Absorption
(black solid line; c = 1.6 × 10−5 mol dm−3), normalized emission (λexc
=
401 nm; red solid line), and normalized excitation (λem = 606 nm; red
dotted line) spectra of 2f in aqueous KOH/DMSO (99:1, v/v; I = 0.1
mol dm−3, KCl) at pH = 11.0. (c) Absorption (black solid line, c ∼ 1.1 ×
10−5 mol dm−3), normalized emission (λexc = 401 nm; red solid line; λexc
= 664 nm; blue solid line) and normalized excitation (λem = 606 nm; red
dotted line; λem = 682 nm; blue dotted line) spectra of 2f in PBS/DMSO
(99:1, v/v) at pH = 7.4.
from 9 × 10−8 to 3 × 10−4 mol dm−3 (Figures S64 and S68). The
upper concentration value represents the limit of the solubility of
the dye in an aqueous solution (I = 0.1 mol dm−3).
D
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The computed wavelengths, which correspond to the vertical
excitation energies of absorption and fluorescence, and the
corresponding Stokes shifts, are compared to the experimental
values in Table 3.26
Table 3. Comparison of Experimental and Theoretical
Absorption and Emission Properties of the Studied Dyes
a
b
experiment
calculations
max
max
max
max
λ
λ
Stokes shift
λ
λ
em
Stokes shift
abs
em
abs
compd
1a
(nm)
(nm)
(cm−1
890
)
(nm) (nm)
(cm−1
1257
)
549
577
473
502
(form A)
1a
(form B)
1b
2a
(form A)
2a
(form B)
2b
376
513
7100
414
497
4051
Figure 4. Changes of absorbance (at λobs = 401 nm; black full square and
λobs = 664 nm; red full circle) of 2f (c = 6 × 10−6 mol dm−3) upon
repeating changes of pH (pH = 11.0, aq NaOH; pH = 3.0, aq HCl) in an
aq 0.1 mol dm−3 KCl/DMSO (99:1, v/v) solution. The solid line is
shown to guide the eye.
422
658
525
678
4650
450
435
522
494
542
2766
687
387
597
9090
436
548
4694
444
617
478
482
442
489
6370
2230
7580
3760
4430
490
471
400
375
388
417
453
431
499
545
441
527
422
458
476
468
519
2881
2335
7689
2062
2163
1073
1822
3 (form A) 432
3 (form B) 353
bond length denoted as j; Figures S74−S84) has a partial double-
bond character.
5
379
402
547
499
636
6
The lowest energy electronic transition in all studied
compounds is well described by a transition of a single electron
between the frontier molecular orbitals. The excitation of the B
form of 1 and 2 is accompanied by a large charge redistribution
and can thus be described as an intramolecular charge-transfer
(ICT) transition. However, our gas-phase calculations suggest
that a locally excited (LE) state may become the lowest excited
state in a nonpolar environment. The LUMO possesses a
character of an antibonding orbital of π-symmetry of the C9−N
bond (Figure S73). The extent to which the pz orbital of the C9-
nitrogen atom contributes to the LUMO depends strongly on
the chemical character and the steric bulk of the whole C9
substituent. The major geometric change upon excitation is
associated with the length of the C9−N bond (j). The geometry
of 2a, which possesses the largest Stokes shift (in nm) in its
ground and first excited electronic states, is shown in Figure 5.
The corresponding geometries for 1a,b, 2b, and 5−8 are
compared in Figures S72−S81.
c
d
f
c
7a
7b
8
562
d
e
e
558
2120
290
f
648
792
c
a
b
In methanol. See the Experimental Section for details. From
Nagano and co-workers.28 Figure 6; R3 = diethylamino, R4
=
d
−(CH2)5−) in CH2Cl2; from Wu and Burgess.20 In CH2Cl2; modeled
e
within the PCM approach From Klan
́
and co-workers.22
f
The vibronic analysis of the absorption and fluorescence
spectra of 1 and 2 (forms B) within the Franck−Condon method
using the FCclasses program27 showed that the overlap of the
zero-point vibrational wave functions of the ground and the first
excited electronic states is very small. Inspection of the
Duschinsky matrices revealed that a number of normal modes
in the first excited state, that are related to vibrations of the C9-
substituent, are expressed by a large number of normal modes of
the ground electronic state. On the other hand, the vibronic
spectra could be easily obtained for 5 and 6.
DISCUSSION
■
The Stokes shifts of the pyronin analogues modified at positions
9 and 10 (Figure 6; e.g., X = O, CR2, SiR2; R3 = aryl, alkyl; R4 = H,
alkyl), in which the first absorption band is described by the
HOMO−LUMO electron transition, are typically very small, in
the range of 20−30 nm.28−32 However, shifts of up to ∼80 nm for
highly fluorescent 9-(alkylamino)- and 9-(dialkylamino)-
pyronins (7: X = O; R3 = dialkylamino; Figure 6) have been
reported by Burgess et al.20,33 A recent patent showed that
Figure 5. Structure of 2a (form B) in its ground (left) and first excited
electronic state (right) and the corresponding bond lengths (j−l). A
complete picture with all bond lengths can be found in Figure S78
(Supporting Information). The color coding is as follows: C (black), H
(white), N (light blue), O (red), and Si (gray).
Figure 6. Pyronin derivatives.
E
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further modifications of the 9-(alkylamino)pyronin (7: X = O,
CR2, S; Figure 6) aromatic core and the C3- and C6-amino
groups may lead to Stokes shifts of up to 180 nm.34 However, the
origin of these large Stokes shifts has not been elucidated.
Although only a few pyronin analogues bearing a substituted
nitrogen-atom at position 9, such as alkylamino, dialkylami-
no,20,35,36 or bis(trimethylsilyl)amino37 groups, have been
reported, 9-(dialkylamino)pyronins have already been used in
a recent study of protein−protein interactions.38 We therefore
assumed that pyronins substituted by nitrogen-atom containing
substituents at position 9 could be excellent candidates for
fluorophores that exhibit very large Stokes shifts.
Wu and Burgess have proposed that an acid−base equilibrium
is likely established by treating 9-(alkylamino)pyronin 9 (form
A) with triethylamine as a base to form a deprotonated species B
(Scheme 4).20 This form was reported to be nonfluorescent,
which is rather unexpected because the ketones 5 and 6 as
structural analogues are highly fluorescent (Table 1).
Scheme 4. Acid−Base Equilibrium in 9 Proposed by Wu and
Burgess20
A detailed inspection of the spectral data reported for 9-
(alkylamino)pyronins20 reveals that pyronins with monoalkyla-
mino groups at the position 9 possess slightly larger Stokes shifts
than those with dialkylamino groups. Because of the bulkier
nature of the secondary amino group, we hypothesized that the
N-alkyl groups tend to avoid the hydrogen atoms at positions 1
and 8 (Figure 6), thereby decreasing the coplanarity of the
chromophore found in the case of a monoalkylamino derivative
by single-crystal X-ray analysis.20 Such an interaction should lead
to a decreased overlap of the nitrogen lone electron pair with the
molecular orbitals (MOs) of the pyronin chromophore affecting
its spectral properties, such as Stokes shifts. The frontier MOs of
3 (forms A and B) and 7b calculated by DFT are shown in Figure
7 (see also Figure S73 for 5, 1a, and 8). It is obvious that the
nodal properties of the frontier orbitals are the same for all the
studied compounds but the overlap of the pz orbital of the C9
nitrogen atom in 7b with the C9 pz orbital in the LUMO is
smaller than that in 3, which confirms our hypothesis. A π−
bonding interaction increasing the C9−N bond order exists in
planar 3, which is evidenced by a much shorter C9−N bond
length than that in 7b (Figures S82−S84) or than is typical, for
example, in anilines (1.355 Å).39 The antibonding arrangement
between the C9 and nitrogen atoms in the LUMO suggests that
promotion of an electron from the HOMO to the LUMO upon
excitation will elongate the C9−N bond, and can be a reason for
the observed large Stokes shifts. Their magnitude will then be
closely related to the C9−N bond order and the overlap of the
C9 nitrogen pz orbital in the LUMO. Indeed, a comparison with
the spectral properties of the analogous ketones 5 and 6 (Table 1;
Figures S73, S80−S81) reveals that their Stokes shifts are
substantial, probably as a consequence of an elongation of the
C9−O bond.40 Unfortunately, no additional substituent can be
Figure 7. Frontier molecular orbitals (left: HOMO; right: LUMO) of 3
and 7b computed on the ground-state geometries (see the Experimental
Section).
installed on the oxygen atom to tune the dyes’ spectral properties
and preserving simultaneously the carbonyl character in 5 or 6.
Once we postulated that increasing the C9−N bond order
could lead to elevated Stokes shifts, we advanced to prepare
pyronin derivatives possessing a formal double C9−N bond.
Because the C9 nitrogen atom in the deprotonated form B must
be fairly basic due to a negative charge on nitrogen in one of the
mesomeric structures (Scheme 3), its conjugation with an
electron-withdrawing group, such as an acetyl and, particularly, a
sulfonyl group, was expected to stabilize the imino form B. To
date, there is only a single report on the preparation of a 9-
sulfonylaminopyronin derivative.41 In this patent, a base-
promoted rearrangement of 9-sulfamoylphenylpyronin in
methanol is shown to give 9-alkyl(benzenesulfonyl)-
aminopyronin which, nonetheless, cannot attain the imino
form B due to the presence of two substituents on the C9-
nitrogen atom. 9-Imino-substituted pyronins have thus never
been reported before.
For this work, a facile synthesis of Wu and Burgess20 was
modified to provide crystalline, colored 9-imino-O- (1a,b) and
Si-pyronins (2a−e) (Scheme 1) in high yields. All synthesized
pyronins are soluble in polar organic solvents, and the 9-
(sulfonylimino)pyronins 1b and 2b as well as the 9-(acylimino)-
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O-pyronin 1a are also partially soluble in aqueous solutions. The
9-acylimino-Si-pyronin analogues had to be modified to get a
more water-soluble derivative 2c. Our 1H−15N g-HMBC
examination confirmed the existence of a formal C9−N double
bond in 2 in the solution (Figures S32 and S43). The observed
15N shifts of 321 ppm (2a) and 281 ppm (2b) are outside the
region of chemical shifts typical for an amidic nitrogen (80−170
nm),42 and can be compared to those of the N-acetylacetophe-
none imines43 and 3-oxo-5-methylpyrazine derivatives.44
All compounds were found to be strongly fluorescent (Φf in
the range of 0.23−0.56) in protic solvents, apparently contra-
dicting the original observation that 9 (B) is nonfluorescent in
dichloromethane.20 Indeed, only barely detectable or no
fluorescence from the compounds 1 and 2 was observed in
aprotic solvents (Figure S40). These compounds exhibit
remarkably large Stokes shifts (Table 1 and 2): 103−137 nm
(4.4−7.1 × 103 cm−1) for O-pyronins and particularly 157−210
nm (5.5−9.1 × 103 cm−1) for Si-pyronins in methanol as well as
aqueous solutions when pH is kept above their pKa’s. The
positions of the major absorption and emission bands in 1 and 2
are markedly affected by the substitution at position 10. The
replacement of oxygen by silicon caused bathochromic shifts of
the band maxima in all solvents, which has also been previously
reported for various rhodamine dyes.28−30 Besides the large
Stokes shifts, it should be noticed that the λmabasx and λemmax values of
1 and 2 in protic methanol (Table 1, Figure 8) are similar to those
of the ketones 5 and 6, suggesting that the lowest excited state in
both types of chromophores may possess an analogous
intramolecular charge-transfer (ICT)9,45 character. In contrast,
the lack of fluorescence in aprotic solvents for 1 and 2 indicates
that a locally excited (LE) species without charge separation
should be the lowest, nonemissive excited state in the absence of
H-bonding stabilization, as was also supported by our gas-phase
calculations. Thus, a solvent reorganization must add signifi-
cantly to the overall Stokes shift. Unlike most cases where a
fluorescent compound is weakly fluorescent in protic solvents
and strongly fluorescent in aprotic media,9 this is an unusual
behavior, although a few other examples can also be found in the
literature.46,47 Our observation of a substantial increase in the
fluorescence intensity from 2a in acetonitrile upon addition of a
small amount of methanol (while keeping the solvent polarity as
low as possible at very low methanol concentrations) without
max
em
observing any pronounced bathochromic shift of λ (Figure
S40) supports our presumption that the appearance of the
emission peak is due to H-bonding stabilization48 of an ICT state
at the expense of an LE state.
For this work, we also investigated whether the fluorophores
undergo an intermolecular excited-state proton transfer (ESPT)
by measuring the absorption and fluorescence spectra as a
function of the acetate buffer concentration, analogous to the
studies of ESPT in fluorescein and other xanthenone
derivatives.49−51 A change of the major emission band positions
max
em
for 2g at low and high buffer concentrations at a pH ∼ 4.6, λ
=
631 nm (cbuffer = 0.03 M) and 692 nm (cbuffer = 3 M),
corresponding to the emission from the forms B and A,
respectively (Table 2), suggests that a high buffer concentration
pronounces protonation of the basic form in the excited state,
thus pKb* < pKb. Relatively low buffer concentrations used in our
study did not apparently allow for an efficient ESPT within the
lifetime of the fluorophores; nevertheless, the relative intensity of
the emission could still affect some imaging applications,
especially when working with buffers at pHs close the pKa of
the probes.
Our calculations qualitatively reproduce the trends for the
experimental Stokes shifts (Table 3). They show that substantial
geometry reorganization, involving particularly a change in the
C9−N bond length (j), follows the photoexcitation of 1 and 2
(Figures S74−S81). As a result, the fluorescence of 1 and 2 is
similar to that of pyronins with simple 9-amino substituents (for
example, in 7b), although still hypsochromically shifted as a
consequence of the excess negative charge on the C9-nitrogen
atom. In addition, we found that the Franck−Condon factors for
the 0−0 transition in 1 and 2 are small and, as a result, the 0−0
transitions should have a relatively low intensity in the
experimental spectra (see, for example, in Figures 1−3). The
absorption and fluorescence maxima related to the transitions
from/to higher vibrational levels are therefore more separated,
elevating the Stokes shifts.
Figure 8. Comparison of the optical properties of known mono-
chromophoric transition-metal-free small-organic molecule fluoro-
phores and several common dyes with those of 9-iminopyronins. The
max
max
Dyes with very large Stokes shifts allow bioimaging
applications of one excitation source and several different
fluorescence channels without burden of spectral overlap
artifacts (bleed-through or crosstalk).52 However, such dyes
often exhibit low fluorescence quantum yields, although it has
been demonstrated that an intramolecular charge transfer
(ICT)53,54 or excited-state proton transfer55 can also be
responsible for bathochromic shifts in the absorption and
emission spectra as well as large Stokes shifts.18 The remarkably
large Stokes shift values up to 208 nm (9130 cm−1) in protic
solvents, together with high brightness, εΦ, in the range of 6.2
absorption (λ or νabs
̃
max) and emission (λ or νemmax) maxima (as the
̃
abs
em
ends of the abscissas), the fluorophore brightness, and solvents in which
the data were measured (blue: aqueous solution; red: methanol or
ethanol (only for ix); black: aprotic organic solvents; CH2Cl2 (iii, viii),
CHCl3 (iv), THF(xi)) are shown. The fluorophores: sulforhodamine
101 (i),56 fluorescein (ii),58,59 a 9-alkylaminopyronin derivative (iii),20
a
perylene derivative (iv),62 ATTO 430LS (v),61 a 9-(alkylamino)pyronin
analogue, AZ 312 (vi),34 ATTO 490LS (vii),61 a 9-dialkylamino-
pyronin derivative (viii),20 a 4-trifluoromethyl-7-aminocoumarin
derivative (ix),63 a naphthofluorescein derivative (x),64 and a 2,5-
bis(2-thienyl)pyrrole derivative (xi).60 The values of Stokes shifts for the
compounds studied in this work (depicted by Arabic numerals) are
shown in Tables 1−3; the Stokes shifts of the common dyes (depicted
by Roman numerals, i−iv, vi, and viii−xi) and their structures are
provided in Figure S1 and Table S3 (Supporting Information).
and 8.6 × 103 dm3 mol−1 cm−1 and absorption in the visible part
max
abs
of the spectrum at λ ∼ 450 nm (Tables 1 and 2), introduce 9-
imino-Si-pyronins as very promising candidates not only for
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common fluorescent labeling experiments but particularly for
multicolor confocal and STED microscopy applications. Special
optical properties of these compounds are apparent from their
comparison with those of the monochromophoric transition-
metal-free small-organic molecule fluorophores with, to our
knowledge, the largest Stokes shifts reported in the literature and
several well-known and widely used xanthene and pyronin dyes
(Figure 8). The emission properties of our pyronin derivatives in
the acidic form A (2f in Figure 8) are comparable to those of the
common very bright fluorophores (εΦ over 104 dm3 mol−1
cm−1), such as sulforhodamine 101 (i)56 (compare to Texas
red57) or fluorescein (ii),58,59 possessing only small Stokes shifts.
The majority of fluorophores with large Stokes shifts absorb in
the UV region, possess relatively low ε values, and are insoluble in
water or fluoresce with small quantum yields, for example, a N-
borylated pyrrol derivative xi.60 Figure 8 also presents
commercially available dyes ATTO 430LS (v), ATTO 490LS
(vii),61 and a pyronin analogue AZ 312 (vi).34 As a result, our 9-
imino-Si-pyronins (especially 2f, Figure 8) are, to our knowledge,
dyes with one of the largest Stokes shifts reported to date with
fluorophore brightness above 103 dm3 mol−1 cm−1.
Due to favorable spectral properties of our 9-imino-Si-
pyronins, we decided to closely investigate the Si-pyronin
analogues which, moreover, represent a new class of
fluorophores. The acid−base properties studied by optical
spectroscopy provided reliable pKa values to identify and assign
the corresponding acid−base forms A and B. An additional NMR
analysis in a buffered CD3OD/D2O mixture showed similar
results. A low pKa (∼3) of 9-(sulfonylimino)-Si-pyronins implied
that the compounds attain a neutral form B in aqueous solutions
at practically all biologically relevant values of pH. Only 9-
(acylimino)-Si-pyronins (2c,f) had a sufficiently high pKa to
allow us to observe a pure protonated form A. This form, with a
formal single C9−N bond in its ground state, is characterized by
the absorption and emission band maxima located in the red
region and, in agreement with our calculations, a small Stokes
shift (Table 3). The acid and base forms A and B were found to
have a robust chemical stability and can repeatedly be switched
from one to another by changing the solution pH (Figure 4 and
Figure S36).
other hand, 9-(sulfonylimino)-Si-pyronins are robust, stable and
reliable fluorescent tags which are characterized by a high
fluorophore brightness and outstanding and distinctive Stokes
shifts under a wide span of solution pH.
CONCLUSION
■
We designed, synthesized, and both theoretically and exper-
imentally studied several novel 9-(acylimino)- and 9-(sulfony-
limino)-O- and Si-pyronin fluorophores. These compounds,
especially the Si-analogues, exhibit remarkably high Stokes shifts
(around 200 nm) while possessing a high fluorophore brightness,
absorption bands in the near-UV and visible part of the spectrum,
and high thermal and photochemical stabilities in protic solvents.
We elucidated using quantum chemical calculations that the
observed large Stokes shifts are related to the excitation of an
electron from the HOMO to the LUMO of the chromophore
that leads both to elongation of the C9−N bond and significant
solvent reorganization. The photophysical properties of the
compounds, their facile and high-yielding synthesis, and a simple
protocol for their bioorthogonal ligation to a model saccharide
using a Huisgen alkyne−azide cycloaddition predestine 9-
iminopyronins for biochemical and biological applications as
fluorescent tags and indicators for multichannel imaging, and for
optoelectronic and dye laser applications. In addition, the
spectral properties of 9-acyliminopyronins are shown to be
alterable by solution pH, which is a significant value-added
potential for their applications as pH sensors.
EXPERIMENTAL SECTION
■
Materials and Methods. The reagents and solvents of the highest
purity available were used as purchased, or they were purified/dried
when necessary. Acetonitrile, dichloromethane and tetrahydrofuran
were dried by standard procedures, kept over activated 3 Å molecular
sieve (8−12 mesh) under dry N2; they were freshly distilled for each
experiment. Phosphate buffer saline (PBS) was prepared by a 1:10
dilution of the stock solution (prepared by direct weighting of NaCl (80
g), KCl (2.0 g), Na2HPO4·12H2O (23 g) and KH2PO4 (2.4 g) into a
volumetric flask which was filled with distilled water to 1 L). The
deuterated aqueous methanol-based phosphate buffers were prepared
by titration of the aliquots prepared as a mixture of 120 μL of a stock
solution (obtained by direct weighting of Na3PO4 (82.0 mg) into a
volumetric flask which was filled with D2O to 5 mL) and 480 μL of
CD3OD with DCl (either 0.1 mol dm−3 or 0.01 mol dm−3; I = 0.1 mol
dm−3, KCl). Synthetic steps were performed under ambient atmosphere
unless stated otherwise. All glassware was oven-dried prior to use when
water- and/or air-sensitive reagents were used. In selected cases, the
Schlenk techniques were used for work with air and/or moisture
sensitive chemicals. All column chromatography purification procedures
were performed on columns packed with silica.
Finally, two water-soluble 2-deoxy-D-glucose derivatives, 2f,g,
prepared by a straightforward copper(I)-catalyzed azide−alkyne
cycloaddition reaction (Scheme 2), were selected to demonstrate
the simplicity of their implementation as “clickable” fluorescent
probes.3,65 The spectral properties of the fluorophores attached
to a model saccharide were practically identical to those of the
model compounds 2a,b, thus the effect of a structural
modification on the 9-imino group is negligible. The chemical
NMR spectra were recorded on 300 or 500 MHz spectrometers in
stability of both 2f and 2g in DMSO in the dark was high (τ1/2
≫
acetonitrile-d3, chloroform-d, dimethyl sulfoxide-d6, methanol-d4, water-
1 month) allowing their long-term storage in stock solutions.
Their stability was lower in methanol and in a PBS buffer but still
quite sufficient for a convenient manipulation with the solutions
during the experiments (Table S1). Their good photostability
(e.g., τ1/2 ∼1 h for 2g with LED or laser light sources; Table S2)
in aqueous solutions fulfills an additional very important
criterion4 for utilizing these fluorophores under prolonged
irradiation, for example, in fluorescence microscopy applications.
Thanks to their pKa close to 7, the spectral properties of 9-
(acylimino)-Si-pyronins can be easily altered by pH, and offer
thus a potential for using these dyes as environmental pH
indicators in aqueous media. Intracellular pH strongly affects the
cell functions; therefore, it is of a great interest to measure pH
qualitatively, particularly using a single-molecule probe.66 On the
1
d2, or their mixtures. The signals in H and 13C NMR spectra were
referenced67 to the residual peak of the (major) solvent except for D2O,
while those of the 19F NMR were unreferenced and those of 29Si NMR
were referenced to TMS. The 15N shifts were obtained from 1H−15N g-
HMBC and were referenced to the nitrogen signal of acetonitrile-d3 as a
solvent (δ = 246 ppm).68 The deuterated solvents (except for D2O)
were kept over activated 3 Å molecular sieve (8−12 mesh) under dry N2.
Mass spectra were recorded on a GC-coupled (30 m DB-XLB column)
spectrometer in a positive mode with EI (70 eV). UV−vis spectra were
obtained with matched 1.000 cm quartz cells. Fluorescence was
measured on an automated luminescence spectrometer in 1.0 cm quartz
fluorescence cuvettes at 25 1 °C; the sample concentration was set to
keep the absorbance below 0.1 at λmax; each sample was measured five
times, and the spectra were averaged. Emission and excitation spectra are
normalized; they were corrected using standard correction files.
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Scheme 5. Synthesis of 10 and 13
Fluorescence quantum yields were determined as the absolute values in
1.0 cm quartz fluorescence cuvettes at 25 1 °C using an integration
sphere. For each sample, the quantum yield was measured five times, and
the values were averaged. Nanosecond flash lamp (filled with H2) was
used for measuring the fluorescence lifetimes. The data obtained were
reconvoluted from measured decay curves of the sample and the
instrumental response function. IR spectra were obtained on an FT-IR
spectrometer from the KBr pellets or using an ATR mode. Exact masses
were obtained using a spectrometer equipped with a time-of-flight mass
analyzer in positive ion mode. Melting points were obtained using a
noncalibrated Kofler’s hot stage melting point apparatus. The solution
pH values were determined using a glass electrode calibrated with
certified buffer solutions at pH = 4, 7, or 10. A global analysis software
was used for determination of the pKa values from a series of absorption
spectra measured in aqueous solutions (I was kept constant at 0.1 mol
dm−3 using KCl when necessary) of the compounds.
Quantum-Chemical Calculations. All calculations were carried
out with the Gaussian 09 suite of electronic structure programs.69 The
ground- and excited-state geometries were optimized with the (TD)-
PBE070 density functional and the 6-31+G(d) basis set, which was
shown to perform satisfactorily in a number of studies that modeled the
absorption and emission spectra as well as the Stokes shifts.71,72 A tighter
DFT integration grid was used in all calculations. The collective effects
of the surrounding solvent were modeled within the polarizable
continuum model (PCM) with methanol as a solvent. State-specific
nonequilibrium solvation was used in calculations of vertical excitation
energies, while equilibrium solvation was used in geometry
optimizations. Analytical and numerical frequency analysis was
performed to verify that all structures correspond to true minima on
the ground and the excited potential energy surfaces (PES), respectively,
and to provide the zero-point vibrational energy (ZPVE) corrections
that has not been scaled. Single point energies of the dyes on the ground
state PES were computed at the PBE0/6-311+G(2df,p) level of theory
in PCM to assess the energies of different conformations. The final
energies in the conformational analysis are the sum of the single point
energies and the ZPVEs. ZPVEs were also added to the TD-PBE0
vertical excitation energies to provide the energies of absorption and
fluorescence. Calculations of vibrationally resolved spectra were
performed using the FCclasses program27 with the default settings of
the program in Gaussian 09.
further purification. Yield: 0.80 g (83%). Brown oil. 1H NMR (300 MHz,
CDCl3): δ (ppm) 3.03 (t, 2H, J = 7.1 Hz), 2.47 (td, 2H, J1 = 7.1 Hz, J2 =
2.6 Hz,), 2.34 (s, 3H), 2.01 (t, 1H, J = 2.6 Hz, CC−H). 13C NMR
(75.5 MHz, CDCl3): δ (ppm) 19.6, 28.2, 30.7, 69.7, 82.2, 195.3. FTIR
(ATR, cm−1): 3291, 2941, 2120 (weak, CC), 1686 (CO), 1428,
1354, 1130, 974, 945, 619, 535, 466. MS (EI): 127 (33), 113 (100), 86
(47), 85 (36), 53 (24). This compound has also been characterized
elsewhere.75
But-3-yne-1-sulfonic Acid (12; Scheme 5). Aqueous solution of
hydrogen peroxide (30%, v/v, 3.84 mL, 37.6 mmol) was added dropwise
to a solution of S-but-3-yn-1-yl ethanethiolate (11, 0.80 g, 6.22 mmol) in
acetic acid (10 mL), and the reaction mixture was stirred at 60 °C for 1 h
until no starting material was observed by GC. The reaction mixture was
then cooled down to 20 °C, and the volatiles were removed under
reduced pressure. The traces of acetic acid were removed by azeotropic
distillation with n-heptane (2 × 10 mL). The resulting title product was
used in the next step without further purification. Yield: 0.70 g (84%).
Colorless oil. 1H NMR (300 MHz, D2O): δ (ppm) 2.39 (t, 1H, J = 2.6
Hz, CC−H), 2.63 (td, 2H, J1 = 7.4 Hz, J2 = 2.6 Hz), 3.08 (t, 2H, J = 7.4
Hz). This compound has also been reported elsewhere.76
But-3-yne-1-sulfonyl Chloride (13; Scheme 5). A solution of thionyl
chloride (2.7 mL, 37 mmol), but-3-yne-1-sulfonic acid (12, 0.73 g, 5.4
mmol) and dimethylformamide (0.1 mL) in dry dichloromethane (1.8
mL) was refluxed under N2 atmosphere for 5 h. The reaction mixture
was then cooled down to 20 °C, and the volatiles were removed under
reduced pressure. The resulting residue was distilled on a Kugelrohr
apparatus to give the pure title product which was used in the next step.
Yield: 0.11 g (13%). Colorless oil. Bp: 55−60 °C (1 mmHg) (lit.76 44−
45 °C, 0.5 mmHg). 1H NMR (300 MHz, CDCl3): δ (ppm) 2.17 (t, 1H, J
= 2.7 Hz, CC−H), 2.96 (td, 1H, J1 = 7.7, J2 = 2.7 Hz), 3.86 (t, 1H, J =
7.7 Hz). FTIR (ATR, cm−1) = 3296, 2995, 2934, 1737, 1368, 1164, 657,
592, 530, 474, 447. This compound has also been reported elsewhere.76
Scheme 6. Synthesis of 5
Synthetic Procedures. Hex-5-ynoyl Chloride (10; Scheme 5). This
procedure is a modification of that reported by Vollhardt.73 Oxalyl
chloride (8.5 g, 67 mmol) was added dropwise to a stirred solution of
hex-5-ynoic acid (5.0 g, 44.6 mmol) in benzene (50 mL) at 20 °C. The
resulting solution was stirred at 30 °C for 2 h. The solvent was removed
under reduced pressure, and the residue was distilled on a Kugelrohr
apparatus to give the title product. Yield: 3.71 g (64%). Colorless liquid
with pungent odor. Bp: 65 °C (1 mmHg) (lit.73 bp 50−55 °C). 1H NMR
(300 MHz, CDCl3): δ (ppm) 1.88−1.96 (m, 2H), 2.02 (t, 1H, J = 2.6
Hz, CC−H), 2.31 (dt, 2H, J1 = 6.7 Hz, J2 = 2.7 Hz), 3.07 (t, 2H, J = 7.2
Hz). 13C NMR (75.5 MHz, CDCl3): δ (ppm) 17.4, 23.9, 45.8, 70.2, 82.4,
173.6. This compound has also been characterized elsewhere.73,74
S-But-3-yn-1-yl Ethanethiolate (11; Scheme 5). A solution of 4-
bromobut-1-yne (1.0 g, 7.5 mmol) and potassium ethanethiolate (2.6 g,
22.6 mmol) in acetonitrile (50 mL) was stirred at 20 °C for 20 h. The
reaction mixture was filtered; the volatiles were removed under reduced
pressure to give a brown oil which was used in the next step without
9-Oxo-9H-xanthene-3,6-diyl Bis(trifluoromethanesulfonate) (14;
Scheme 6). Compound 14 was prepared according to previously
published procedure.77
3,6-Bis(dimethylamino)-9H-xanthen-9-one (5). Tetrakis-
(triphenylphosphino)palladium (100 mg, 0.142 mmol) was added in
one portion to a solution of 9-oxo-9H-xanthene-3,6-diyl bis-
(trifluoromethanesulfonate) (14, 1.00 g, 2.03 mmol) in dry tetrahy-
drofuran (20 mL) in a high-pressure glass tube, and the reaction mixture
was cooled to −78 °C. Liquid dimethylamine (4.0 mL, 60 mmol) was
subsequently added to the reaction mixture in one portion, and the
vessel was sealed. The reaction mixture was stirred overnight at 90 °C
and then cooled to 20 °C. Water (20 mL) was added, and
tetrahydrofuran was removed under reduced pressure. Yellow
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suspension of the crude product was filtered and subsequently washed
with cold water (5 mL) and cold n-hexane (3 × 2 mL). The filtrate was
dried under high vacuum and used in next step without any purification.
Yield: 423 mg (74%). Slightly yellow powder. Mp: 262−264 °C (lit.37
235−242 °C). 1H NMR (500 MHz, CDCl3): δ (ppm) 3.10 (s, 12H),
6.48 (d, 2H, J = 2.4 Hz), 6.90 (dd, 2H, J1 = 9.0 Hz, J2 = 2.4 Hz), 8.14 (d,
2H, J = 9.0 Hz) (Figure S2). 13C NMR (125 MHz, CDCl3): δ (ppm)
40.4, 97.3, 109.2, 112.3, 127.9, 154.6, 158.4, 176.4 (Figure S3). FTIR
(KBr, cm−1) = 2905, 2807, 1602, 1532, 1432, 1351, 1331, 1255, 1159,
1121, 808, 709, 668. MS (EI): 282 (100), 265 (15), 236 (6), 222 (4),
194 (3), 166 (4), 140 (17), 127 (19), 111 (9), 97 (14), 83 (15), 70 (16),
57 (18). UV−vis (CH3OH, c = 1.0 × 10−4 mol dm−3): λmax (log ε) = 379
(4.24) nm (dm3 mol−1 cm−1) (Figure S4). Fluorescence (CH3OH, A
(λmax (excitation)) ≤ 0.1): λmax (emission) = 442 nm (Figure S4).
HRMS (APCI+−TOF) m/z: [M + H]+ calcd for C17H19N2O2 238.1441;
found: 283.1442. This compound has also been characterized
elsewhere.37
Note: The noncatalyzed Buchwald−Hartwig amination of 9-oxo-9H-
xanthene-3,6-diyl bis(trifluoromethanesulfonate) (14) with piperidine
leads to 3,6-di(piperidin-1-yl)-9H-xanthen-9-one in high yield
(>90%).20,77 However, we found that dimethylamine does not react
under these conditions, and 5 is formed only upon addition of a Pd-
catalyst (in 74% yield using 7 mol % of Pd(PPh3)4 and in 54% yield using
7 mol % of PdCl2(PPh3)2).
3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-
one (6). This compound was prepared according to a previously
published procedure.22 29Si NMR (99.3 MHz, CDCl3): δ (ppm) −23.3
(s) (Figure S5). IR, see ref.22 (Figure S6). UV−vis (CH3OH, c = 4.4 ×
10−5 mol dm−3): λmax (log ε) = 402 (4.51) nm (dm3 mol−1 cm−1)
(Figure S10). Fluorescence (CH3OH, A (λmax (excitation)) ≤ 0.1): λmax
(emission) = 489 nm (Figure S10).
Synthesis of 9-Aminopyronin Analogues 3 and 4: A General
Procedure. Trifluoromethanesulfonic anhydride (2.2 g, 1.3 mL, 7.7
mmol) was added dropwise to a stirred solution of ketone (5 or 6, 1.54
mmol) in dry dichloromethane (15 mL) in a Schlenk flask under dry N2
atmosphere. The reaction mixture was stirred at 20 °C for 15 min, and
gaseous ammonia was then bubbled through this solution for 10 min
which was accompanied by a change of color. The reaction mixture was
further stirred under the ammonia atmosphere for 10 h. The mixture was
subsequently bubbled through with nitrogen to remove excess of
ammonia, and water (20 mL) was added in one portion. The reaction
mixture was acidified by careful addition of trifluoromethanesulfonic
acid to adjust the pH to ∼1, washed with water (2 × 20 mL), aq
NaHCO3 (5%, w/w, 2 × 20 mL) and brine (20 mL), dried over
anhydrous magnesium sulfate and filtered. The volatiles were removed
under reduced pressure, and the resulting colored crystalline solid was
chromatographed (CH3OH/CH2Cl2, 1:19, v/v) to give the pure
product.
nate (4). This compound was synthesized from 6 and ammonia.
Yield: 715 mg (98%). Red crystalline solid. Mp: 42.7−43.1 °C. 1H NMR
(500 MHz, CD3CN): δ (ppm) 0.46 (s, 6H, >Si(CH3)2), 3.14 (s, 12H),
6.89 (dd, 2H, J1 = 9.2 Hz, J2 = 2.6 Hz), 7.02 (d, 2H, J = 2.7 Hz), 7.90 (d,
2H, J = 9.2 Hz), 8.54 (bs, 2H, −NH2) (Figure S16). 13C NMR (126
MHz, CD3CN) δ (ppm) −1.6 (>Si(CH3)2), 40.4, 114.3, 117.5, 120.1,
121.0 (q, 1JC−F = 321 Hz), 131.3, 144.3, 154.1, 169.5 (Figure S17). 15
N
NMR (CD3CN) δ 71 (−N(CH3)2), 127 (−NH2) (ppm) (Figures S18
and S19). 19F NMR (282 MHz, CDCl3): δ (ppm) −78.6 (s). MS (EI):
m/z = 323 (100), 308 (85), 236 (23), 148 (32), 123 (21), 111 (36), 97
(67), 83 (84). FTIR (cm−1): 3332, 3180, 2920, 1692, 1579, 1356, 1256,
1146, 1111, 1057, 1033, 827, 759, 633, 553, 516 (Figure S20). HRMS
(APCI+−TOF) m/z: [M]+ calcd for C19H26N3Si 324.1891; found
324.1896.
Synthesis of 9H-Xanthene Amide-3,6-diamine Derivatives (1) and
their Si-Analogues 2: General Procedure. A solution of acyl chloride or
sulfonyl chloride (0.170 mmol) in dry dichloromethane (2 mL) was
added dropwise to a stirred solution of a 9-aminopyronin (3 or 4, 0.140
mmol) and triethylamine (0.072 g, 0.1 mL, 0.710 mmol) in dry
dichloromethane (3 mL) at 20 °C, and the reaction mixture was stirred
under dry N2 atmosphere for 2 h. The mixture was then diluted with
dichloromethane (10 mL), washed with water (2 × 10 mL), brine (10
mL), dried over anhydrous magnesium sulfate, and filtered. Volatiles
were removed under reduced pressure, and the resulting residue was
chromatographed (n-hexane/ethyl acetate, 5:1, v/v) to give the pure
product.
N-(3,6-Bis(dimethylamino)-9H-xanthen-9-ylidene)acetamide
(1a). This compound was synthesized from acetyl chloride and 3. Yield:
34 mg (74%). Red crystalline solid. Mp: 172−174 °C. 1H NMR (500
MHz, CDCl3): δ (ppm) 2.35 (s, 3H), 3.09 (s, 12H), 6.45 (d, 2H, J = 2.5
Hz), 6.63 (dd, 2H, J1 = 9.1 Hz, J2 = 2.5 Hz), 7.82 (d, 2H, J = 9.1 Hz)
(Figure S22). 13C NMR (126 MHz, CDCl3): δ (ppm) 25.6, 40.1, 97.3,
107.9, 109.5, 128.5, 147.3, 154.0, 156.3, 182.6 (Figure S23). MS (EI):
m/z = 323 (19), 308 (86), 292 (19), 280 (6), 264 (10), 236 (5), 154
(17), 43 (100). FTIR (cm−1): 1597, 1578, 1527, 1426, 1257, 1231, 1181,
1132, 1053, 919, 806, 697, 655, 603, 569, 507. UV−vis (CH3OH, c = 7.0
× 10−5 mol dm−3): λmax (log ε) = 202 (4.20), 237 (4.53), 278 (4.03), 376
(4.32) nm (dm3 mol−1 cm−1) (Figure S24). Fluorescence (CH3OH, A
(λmax (excitation)) ≤ 0.1): λmax (emission) = 513 nm (Figure S24). UV−
vis (1% HCl in CH3OH (v/v), c = 6.4 × 10−6 mol dm−3): λmax = 237,
253, 296, 359, 549 nm (Figure S25). Fluorescence (1% HCl in CH3OH
(v/v), A (λmax (excitation)) ≤ 0.1): λmax (emission) = 576 nm (Figure
S25). HRMS (ESI+): calcd for C19H22N3O2+ [M + H]+ 324.1707; found
324.1704.
N-(3,6-Bis(dimethylamino)-9H-xanthen-9-ylidene)methane-
sulfonamide (1b). This compound was synthesized from methane-
sulfonyl chloride and 3. Yield: 28 mg (84%). Orange crystalline solid.
1
Mp: >250 °C. H NMR (300 MHz, CDCl3): δ (ppm) 3.13 (s, 12H),
3.31 (s, 3H), 6.46 (d, 2H, J = 2.6 Hz), 6.74 (dd, 2H, J1 = 9.4 Hz, J2 = 2.6
Hz), 8.55 (d, 2H, J = 9.4 Hz) (Figure S26). 13C NMR (75.5 MHz,
CDCl3): δ (ppm) 40.3, 45.1, 96.7, 109.2, 109.9, 131.0, 154.7, 157.3,
157.7 (Figure S27). MS (EI): m/z = 359 (100), 344 (27), 294 (14), 281
(38), 252 (36), 236 (31), 140 (17), 127 (49), 42 (4). FTIR (cm−1):
2918, 2852, 1644, 1594, 1528, 1494, 1354, 1323, 1263, 1125, 1105, 966,
897, 844, 823, 802, 766, 658, 593, 510. UV−vis (CH3OH, c = 5.0 × 10−5
mol dm−3): λmax (log ε) = 422 (4.19) nm (dm3 mol−1 cm−1) (Figure
S28). Fluorescence (CH3OH, A (λmax (excitation)) ≤ 0.1): λmax
(emission) = 525 nm (Figure S28). HRMS (APCI+−TOF) m/z: [M
+ H]+ calcd for C18H22N3O3S 360.1376; found 360.1373.
N-(9-Amino-6-(dimethylamino)-3H-xanthen-3-ylidene)-N-meth-
ylmethanaminium Trifluoromethanesulfonate (3). This compound
was synthesized from 5 and ammonia. Yield: 473 mg (85%). Yellow
crystalline solid. Mp: >250 °C. 1H NMR (500 MHz, CD3CN): δ (ppm)
3.13 (s, 12H), 6.50 (d, 2H, J = 2.5 Hz), 6.90 (dd, 2H, J1 = 9.4 Hz, J2 = 2.5
Hz), 7.68 (bs, 2H, −NH2), 7.78 (d, 2H, J = 9.4 Hz) (Figure S11). 13
C
NMR (126 MHz, CD3CN) δ (ppm) 40.6, 97.2, 101.8, 112.6, 121.0 (q,
1JC−F = 321 Hz), 126.4, 156.0, 157.1, 157.7 (Figure S12). 15N NMR
(CD3CN) δ 75 (−N(CH3)2), 98 (−NH2) (ppm) (Figure S13). 19F
NMR (282 MHz, CDCl3): δ (ppm) −80.2 (s). MS (EI): m/z = 281
(75), 264 (15), 239 (15), 226 (15), 127 (30), 97 (40), 85 (65), 57 (95),
43 (100). FTIR (cm−1): 3419, 3363, 3270, 2920, 1638, 1588, 1532,
1482, 1440, 1342, 1181, 1134, 1052, 814, 610, 567, 506. UV−vis
(CH3OH, c = 5 × 10−5 mol dm−3): λmax = 237, 290, 342, 432 nm (Figure
S14). Fluorescence (CH3OH, A (λmax (excitation)) ≤ 0.1): λmax
(emission) = 478 nm (Figure S14). UV−vis (basic CH3OH, c = 5 ×
10−5 mol dm−3): λmax = 234, 273, 292, 353 nm (Figure S15).
Fluorescence (basic CH3OH, A (λmax (excitation)) ≤ 0.1): λmax
(emission) = 482 nm (Figure S15). HRMS (APCI+−TOF) m/z:
[M]+ calcd for C17H20N3O 282.1601; found 282.1597.
N-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-
9-ylidene)acetamide (2a). This compound was synthesized from acetyl
chloride and 4. Yield: 48 mg (92%). Green crystalline solid. Mp: 131−
133 °C. 1H NMR (500 MHz, CDCl3): δ (ppm) 0.48 (s, 6H,
>Si(CH3)2), 2.07 (s, 3H), 3.06 (s, 12H), 6.75 (dd, 2H, J1 = 8.9 Hz, J2
= 2.8 Hz), 6.85 (d, 2H, J = 2.8 Hz), 7.91 (d, 2H, J = 8.9 Hz) (Figure S29).
13C NMR (126 MHz, CDCl3): δ (ppm) −1.4 (>Si(CH3)2), 25.7, 40.2,
113.3, 115.0, 129.6, 130.6, 138.4, 150.7, 159.4, 184.0, (Figure S30). 15N
NMR (CD3CN) δ (ppm) 54 (−N(CH3)2), 321 (NCOCH3) (Figure
S32). MS (EI): m/z = 365 (19), 351 (29), 350 (100), 334 (13). FTIR
(cm−1): 2951, 2922, 2805, 1658, 1575, 1493, 1445, 1354, 1296, 1223,
N-(10-Amino-7-(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-
3(5H)-ylidene)-N-methylmethanaminium Trifluoromethanesulfo-
J
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The Journal of Organic Chemistry
Featured Article
1175, 1150, 1060, 836, 780, 757, 701, 661, 644, 611, 556, 470, 441
(Figure S33). UV−vis (CH3OH, c = 7.4 × 10−5 mol dm−3): λmax (log ε)
= 215 (4.48), 254 (4.22), 387 (4.24) nm (dm3 mol−1 cm−1) (Figure
S36). Fluorescence (CH3OH, A (λmax (excitation)) ≤ 0.1): λmax
(emission) = 597 nm (Figure S36). UV−vis (1% HCl in CH3OH (v/
v), c = 2.4 × 10−5 mol dm−3): λmax = 322, 428, 658 nm (Figure S37).
Fluorescence (1% HCl in CH3OH (v/v), A (λmax (excitation)) ≤ 0.1):
λmax (emission) = 679 nm (Figure S37). HRMS (APCI+−TOF) m/z:
[M + H]+ calcd for C21H28N3OSi 366.1996; found 366.1994.
N-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-
ylidene)but-3-yne-1-sulfonamide (2e). This compound was synthe-
sized from but-3-yne-1-sulfonyl chloride (13) and 4. Yield: 23 mg
(38%). Green crystalline solid. Mp: 86.7−88.1 °C. 1H NMR (500 MHz,
CDCl3): δ (ppm) 0.48 (s, 6H, >Si(CH3)2), 1.97 (t, 1H, J = 2.7 Hz), 2.84
(ddd, 2H, J1 = 8.3 Hz, J2 = 6.1 Hz, J3 = 2.7 Hz), 3.09 (s, 12H), 3.43 (m,
2H), 6.77 (dd, 2H, J1 = 9.0 Hz, J2 = 2.8 Hz), 6.82 (d, 2H, J = 2.8 Hz), 8.24
(d, 2H, J = 9.0 Hz) (Figure S56). 13C NMR (126 MHz, CDCl3): δ
(ppm) −1.6 (>Si(CH3)2), 14.8, 40.1, 54.6, 69.8, 81.2, 112.3, 114.8,
129.8, 133.0, 139.6, 151.4, 174.8 (Figure S57). MS (EI): m/z = 442 (61),
392 (100), 377 (63), 293 (14), 281 (46), 182 (43), 154 (11). FTIR
(cm−1): 3286, 2921, 2249 (weak, CC), 1581, 1550, 1486, 1363, 1294,
1179, 1115, 1063, 869, 838, 762, 731. UV−vis (CH3OH, c = 4.8 × 10−5
mol dm−3): λmax (log ε) = 213 (4.48), 247 (4.17), 392 (4.21), 452 (4.30)
nm (dm3 mol−1 cm−1) (Figure S58). Fluorescence (CH3OH, A (λmax
(excitation)) ≤ 0.1): λmax (emission) = 617 nm (Figure S58). HRMS
(APCI+−TOF) m/z: [M + H]+ calcd for C29H30N3O2SSi 440.1823;
found 440.1819.
N-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-
ylidene)methanesulfonamide (2b). This compound was synthesized
from methanesulfonyl chloride and 4. Yield: 35 mg (82%). Orange
1
crystalline solid. Mp: 219−221 °C. H NMR (500 MHz, CDCl3): δ
(ppm) 0.49 (s, 6H, >Si(CH3)2), 3.08 (s, 12H), 3.19 (s, 3H), 6.77 (dd,
2H, J1 = 9.0 Hz, J2 = 2.8 Hz), 6.83 (d, 2H, J = 2.8 Hz), 8.26 (d, 2H, J = 9.0
Hz) (Figure S41). 13C NMR (126 MHz, CDCl3): δ (ppm) −1.6
(>Si(CH3)2), 40.1, 44.6, 112.3, 114.8, 129.0, 132.8, 139.4, 151.3, 174.0
(Figure S42). 15N NMR (CD3CN) δ (ppm) 58 (−N(CH3)2), 281 (
NSO2CH3) (Figure S43). MS (EI): m/z = 401 (91), 386 (60), 372 (7),
336 (59), 322 (100), 307 (53), 292 (29), 278 (28), 263 (19), 249 (7),
161 (33), 147 (31). FTIR (cm−1): 2934, 1584, 1548, 1476, 1365, 1182,
1120, 1063, 968, 836, 760, 575, 526 (Figure S44). UV−vis (CH3OH, c =
6.2 × 10−5 mol dm−3): λmax (log ε) = 213 (4.34), 248 (4.03), 388 (4.05),
444 (4.17) nm (dm3 mol−1 cm−1) (Figure S47). Fluorescence (CH3OH,
A (λmax (excitation)) ≤ 0.1): λmax (emission) = 617 nm (Figure S47).
HRMS (APCI+−TOF) m/z: [M + H]+ calcd for C20H28N3O3SSi
402.1666; found 402.1668. pKa (nonbuffered aqueous solution, I = 0.1
mol dm−3) ∼ 2.8 0.2.
Azide−Alkyne Huisgen Cycloaddition of 2d and 2e with Azide: A
General Procedure. The procedure of Kim and co-workers was
utilized.21
N-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-
ylidene)-4-(1-((4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)-
tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)butanamide (2f).
This compound was synthesized from 2d and 2-azido-2-deoxy-D-
glucose. Yield: 73 mg (91%). Orange crystalline solid. Mp: 127.7−128.5
°C. 1H NMR (500 MHz, CD3OD): δ (ppm) 0.45 (s, 6H, >Si(CH3)2),
1.94 (p, 2H, J = 7.3 Hz), 2.45 (t, 2H, J = 7.0 Hz), 2.70 (t, 2H, J = 7.7 Hz),
3.05 (s, 12H, −N(CH3)2), 3.41−3.55 (m, 1.5H), 3.72−3.82 (m, 1.2H),
3.85 (dd, 0.6H, J1 = 11.4 Hz, J2 = 2.1 Hz), 3.89−3.98 (m, 1.1H), 4.00−
4.11 (m, 1.0H), 4.19−4.25 (m, 0.6H), 4.56 (dd, 0.6H, J1 = 11.0 Hz, J2 =
3.2 Hz), 4.80 (bs, 4H, −OH) 5.05 (d, 0.45H, J = 7.7 Hz, regioisomer a),
5.25 (d, 0.55H, J = 2.8 Hz, regioisomer b), 6.80 (dd, 2H, J1 = 8.7 Hz, J2 =
2.4 Hz), 6.93 (d, 2H, J = 2.4 Hz), 7.54 (s, 0.45H, N−C(CH2)CH−
N(glu)−, regioisomer a), 7.74 (s, 0.55H, N−C(CH2)CH−
N(glu)−, regioisomer b), 7.78 (d, 2H, J = 8.7 Hz) (Figure S59). 13C
NMR (126 MHz, CDCl3): δ −1.5 (>Si(CH3)2), 25.9 (−CH2−), 26.4
(−CH2−), 38.6 (NCOCH2−), 40.2 (−N(CH3)2), 62.6 (glu:
−CH2OH, anomer 1), 62.7 (glu: CH2OH, anomer 2), 66.8 (glu:
>CHOH, anomer 1), 69.6 (glu: >CHOH, anomer 2), 71.7 (glu:
>CHOH, anomer 1), 72.2 (glu: >CHOH, anomer 2), 72.5 (glu:
>CHOH, anomer 1), 73.3 (glu: >CHOH, anomer 1), 75.5 (glu:
>CHOH, anomer 2), 78.2 (glu: >CHOH, anomer 2), 92.8 (glu:
−CH(OH)−O−, anomer 1), 96.2 (glu: −CH(OH)−O−, anomer 2),
114.2 (CarH), 116.3 (CarH), 122.6 (>CCH−N(glu)−, regioisomer
b), 124.5 (>CCH−N(glu)−, regioisomer a), 131.7 (Car(q)), 140.3
(CarH), 142.6 (>CCH−N(glu)−, regioisomer a), 142.8 (>CCH−
N(glu)−, regioisomer b), 152.5 (Car(q)), 161.1 (>CN−C(O)−),
186.6 (>CO) (ppm) (Figure S60). MS (EI): m/z = 623 (M+, not
observed), 406 (<1), 337 (6), 323 (100), 308 (69), 294 (16), 281 (5),
193 (4), 160 (5), 148 (30), 108 (7), 96 (13), 53 (6). FTIR (cm−1): 3342,
2924, 2897, 1623, 1583, 1497, 1446, 1361, 1303, 1247, 1229, 1178,
1121, 1062, 840, 781, 758, 700, 668, 562. UV−vis (CH3OH, c = 4.2 ×
10−5 mol dm−3): λmax (log ε) = 254 (4.32), 386 (4.34) nm (dm3 mol−1
cm−1) (Figure S61). Fluorescence (CH3OH, A (λmax (excitation)) ≤
0.1): λmax (emission) = 595, 676 (two forms were observable) nm
(Figure S61). UV−vis (PBS/DMSO, 99:1, v/v, c ∼ 1.1 × 10−5 mol
dm−3): λmax = 664, 619, 401, 328 nm (Figure 2c). Fluorescence (PBS/
DMSO, 99:1, v/v, A (λmax (excitation)) ≤ 0.1): λmax (emission) = 606,
682 nm (Figure 2c). HRMS (APCI+−TOF) m/z: [M + H]+ calcd for
C31H43N6O6Si 623.3008; found 623.3012. pKa (nonbuffered aqueous
solution, I = 0.1 mol dm−3) = 6.76 0.11.
N-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-
ylidene-2-(2-(2-methoxyethoxy)ethoxy)acetamide (2c). This com-
pound was synthesized from 2-(2-(2-methoxyethoxy)ethoxy)acetyl
1
chloride and 4. Yield: 68 mg (83%). Green highly viscous oil. H
NMR (500 MHz, CDCl3): δ (ppm) 0.47 (s, 6H, >Si(CH3)2), 3.04 (s,
12H), 3.32 (s, 3H), 3.46−3.44 (m, 2H), 3.54−3.50 (m, 4H), 3.62−3.59
(m, 2H), 4.08 (s, 2H), 6.72 (dd, 2H, J1 = 8.9 Hz, J2 = 2.8 Hz), 6.82 (d,
2H, J = 2.8 Hz), 7.91 (d, 2H, J = 8.9 Hz) (Figure S49). 13C NMR (126
MHz, CDCl3): δ (ppm) −1.4 (>Si(CH3)2), 40.0, 59.0, 70.4, 70.5, 70.8,
71.4, 71.9, 113.1, 114.9, 129.2, 130.9, 138.7, 150.8, 161.7, 181.8 (Figure
S50). MS (EI): m/z = 484 (11), 470 (10), 350 (100), 334 (82), 323
(28), 320 (21), 309 (21), 292 (16), 263 (10), 175 (35), 102 (4), 59 (22).
FTIR (cm−1): 2875, 2812, 1643 (CO), 1578, 1495, 1445, 1357, 1296,
1246, 1176, 1104, 1059, 960, 926, 838, 782, 759, 700, 677, 577, 505, 445.
UV−vis (CH3OH, c = 3.3 × 10−5 mol dm−3): λmax (log ε) = 400 (4.42)
nm (dm3 mol−1 cm−1) (Figure S51). Fluorescence (CH3OH, A (λmax
(excitation)) ≤ 0.1): λmax (emission) = 596 nm (Figure S51). HRMS
(ESI+−TOF) m/z: [M + H]+ calcd for C26H38N3O4Si 484.2626; found
484.2623. pKa (nonbuffered aqueous solution, I = 0.1 mol dm−3) = 6.77
0.06.
N-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-
ylidene)hex-5-ynamide (2d). This compound was synthesized from
1
hex-5-ynoyl chloride (10) and 4. Yield: 53 mg (89%). Green wax. H
NMR (500 MHz, CDCl3): δ (ppm) 0.49 (s, 6H, >Si(CH3)2), 1.83 (p,
2H, J = 7.2 Hz), 1.84 (t, 1H, J = 2.6 Hz), 2.20 (td, 2H, J1 = 7.1 Hz, J2 = 2.6
Hz), 2.42 (t, 2H, J = 7.4 Hz), 3.06 (s, 12H), 6.74 (dd, 2H, J1 = 9.0 Hz, J2 =
2.8 Hz), 6.84 (d, 2H, J = 2.8 Hz), 7.90 (d, 2H, J = 9.0 Hz) (Figure S53).
13C NMR (126 MHz, CDCl3): δ (ppm) −1.4 (>Si(CH3)2), 18.2, 24.3,
37.2, 40.2, 68.7, 84.0, 113.2, 115.0, 129.7, 130.7, 138.5, 150.7, 159.3,
185.4 (Figure S54). MS (EI): m/z = 417 (9), 350 (73), 324 (100), 309
(76), 293 (24), 193 (16), 175 (15), 148 (36), 43 (21). FTIR (cm−1):
3291, 2901, 2809, 2116 (weak, CC), 1620, 1581, 1496, 1446, 1358,
1297, 1247, 1229, 1176, 1126, 1062, 961, 926, 768, 642, 576, 442. UV−
vis (CH3OH, c = 4.0 × 10−5 mol dm−3): λmax (log ε) = 387 (4.40) nm
(dm3 mol−1 cm−1) (Figure S55). Fluorescence (CH3OH, A (λmax
(excitation)) ≤ 0.1): λmax (emission) = 595 nm (Figure S55). UV−vis
(PBS/DMSO, 99:1, v/v, c = ∼ 1 × 10−5 mol dm−3): λmax = 408 nm (dm3
mol−1 cm−1). Fluorescence (PBS/DMSO, 99:1, v/v, A (λmax
(excitation)) ≤ 0.1): λmax (emission) = 605 nm. HRMS (APCI+−
TOF) m/z: [M + H]+ calcd for C25H31N3OSi 418.2309; found
418.2304.
Note: A mixture of two regioisomers in the ratio of ∼55:45 was
obtained and clearly distinguished by NMR; two anomers of the
saccharide were also distinguished in the NMR spectra (Figure S59).
N-(3,7-Bis(dimethylamino)-5,5-dimethyldibenzo[b,e]silin-10(5H)-
ylidene)-2-(1-((4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxymethyl)-
tetrahydro-2H-pyran-3-yl)-1H-1,2,3-triazol-4-yl)ethanesulfonamide
(2g). This compound was synthesized from 2e and 2-azido-2-deoxy-D-
glucose. Yield: 29 mg (86%). Orange crystalline solid. Mp: 123.5−128.5
K
dx.doi.org/10.1021/jo502213t | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Featured Article
1
°C. H NMR (500 MHz, CDCl3): δ (ppm) 0.47 (s, 6H, >Si(CH3)2),
REFERENCES
■
3.10 (s, 12H, −N(CH3)2), 3.41−3.52 (m, 1.9H), 3.55−3.60 (m, 2.4H),
3.71−3.79 (m, 1.3H), 3.64 (d, 0.6H, J = 2.6 Hz), 3.82−3.94 (m, 1.7H),
4.06−4.10 (m, 1H), 4.23 (t, 0.7H, J = 9.7 Hz), 4.57 (d, 0.3H, J = 2.6 Hz),
4.59 (d, 0.3H, J = 2.6 Hz), 5.08 (d, 0.5H, J = 7.1 Hz) 5.26 (d, 0.6H, J = 2.8
Hz), 6.81 (d, 2H, J = 8.8 Hz), 6.94 (br s, 2H), 7.86 (s, 0.45H, N−
C(CH2)CH−N(glu)−, regioisomer b), 7.97 (s, 0.55H, N−
C(CH2)CH−N(glu)−, regioisomer a), 8.21 (d, 2H, J = 8.7 Hz)
(Figure S65). 13C NMR (126 MHz, CDCl3): δ (ppm) −1.8
(>Si(CH3)2), 40.1 (−N(CH3)2), 56.4, 56.5 (both −CH2−), 62.6, 62.7
(both −CH2−), 67.0, 69.8, 71.8, 72.1, 72.5, 73.3, 75.6, 78.2, 92.7 (glu:
−CH(OH)−O−, anomer 1), 96.2 (glu: −CH(OH)−O−, anomer 2),
113.3 (CarH), 116.1 (CarH), 123.8 (N−C(CH2)CH−N(glu)−,
regioisomer a, only observed in 1H−13C HSQC), 125.5 (, N−
C(CH2)CH−N(glu)−, regioisomer b) 130.6 (Car(q)), 134.0 (CarH),
141.2 (Car(q)), 153.1 (Car(q)), 176.4 (>CN−SO2−) (Figure S66). MS
(EI): m/z = 645 (M+, not visible), 460 (<1), 429 (1), 369 (4), 323 (40),
308 (29), 295 (12), 281 (8), 221 (13), 194 (8), 168 (14), 148 (19), 110
(24), 97 (33), 84 (100), 55 (46). FTIR (cm−1): 3295, 2925, 2109, 1581,
1363, 1253, 1062, 1019, 956, 839, 762. UV−vis (CH3OH, c = 4.3 × 10−5
mol dm−3): λmax (log ε) = 218 (4.32), 245 (4.13), 391 (4.10), 447 (4.18)
nm (dm3 mol−1 cm−1) (Figure S67). Fluorescence (CH3OH, A (λmax
(excitation)) ≤ 0.1): λmax (emission) = 616 nm (Figure S67). UV−vis
(PBS/DMSO, 99:1, v/v, c = 2.7 × 10−5 mol dm−3): λmax = 469, 404, 246
nm (dm3 mol−1 cm−1) (Figure 3). Fluorescence (PBS/DMSO, 99:1, v/
v, A (λmax (excitation)) ≤ 0.1): λmax (emission) = 631 nm (Figure 3).
HRMS (ESI+−TOF) m/z: [M + H]+ calcd for C29H41N6O7SSi
645.2521; found 645.2525. pKa (nonbuffered aqueous solution, I =
0.1 mol dm−3) ∼ 2.7 0.7.
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ASSOCIATED CONTENT
■
S
* Supporting Information
̌
̌
(22) Pastierik, T.; Sebej, P.; Medalova, J.; Stacko, P.; Klan, P. J. Org.
́
́
Photophysical properties of selected fluorophores, NMR, IR,
Raman, UV/vis, fluorescence spectra, quantum chemical
calculations data, and stability determination experiments. This
material is available free of charge via the Internet at http://pubs.
acs.org.
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transition energies for absorption and fluorescence, which we computed
by the TD-PBE0 functional, do not vary systematically and depend on
the character of the substituent in the position 9 of the dye (Table 3).
Several other functionals (B3LYP, BMK, CAM-B3LYP, M06, and M06-
2X) have been tested but provided similar or even lower quality results.
The DFT methods seem to underestimate the vertical excitation
energies for the dyes that have a formal exo double bond on C9, while
they overestimate the excitation energies when a single bond between
C9 and N is present. This can be nicely demonstrated by comparing the
absorption and emission properties of the two acid−base forms (A and
B) of 1a with those of 5 and 7a, respectively. We decided to inspect the
behavior of various DFT methods for a larger set of related pyronin
molecules, which is, however, beyond the scope of the current work, and
it will be presented in a forthcoming study. The computed Stokes shifts
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AUTHOR INFORMATION
■
Corresponding Author
* E-mail: klan@sci.muni.cz. Phone: +420-54949-4856. Fax:
+420-54949-2443.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Support for this work was provided by the Grant Agency of the
Czech Republic (13-25775S), the Czech Ministry of Education
(LO1214) (P.K.), and the project “Employment of Best Young
Scientists for International Cooperation Empowerment”
(CZ.1.07/2.3.00/30.0037) cofinanced from the European Social
̌
Fund and the state budget of the Czech Republic (P.S.). P.H. was
supported by the AXA Fund. The authors express their thanks to
́
́ ̌ ́ ̌ ̌
Maier, Miroslava Bittova, Lubomir Prokes and Zdenek
Lukas
Moravec for their help with the NMR measurements, and mass
and Raman spectrometry. Jan Krausko is acknowledged for his
help with the fluorescence measurements, and Jakob Wirz and
Lukas Maier for fruitful discussions. We thank Tomas Pastierik
́
́
̌
́
̌
for his help with synthesis of some precursors. The computa-
tional facilities of University of Fribourg are acknowledged for
the computational resources.
2009, 38, 2410.
L
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