Synthesis, chemical reactivity, and photophysical properties of 2′,7′ phenylated rhodamine dyes

Article abstract of DOI:10.1016/j.tetlet.2014.05.124

While exploring water soluble rhodamine based fluorescent polymeric systems for biological imaging applications we came across new rhodamine derivatives that possess interesting optical properties. We report the synthesis of three different 2′,7′-diphenylated rhodamine derivatives (1-3) with distinct photophysical properties. The three rhodamine derivatives differ by the number of methyl groups present on the nitrogens and their absorption maxima are red-shifted on increased methylation. We observed an unusual inertness of these compounds toward traditional DCC-DMAP esterification conditions, which we attribute to the ease of lactonization in the presence of even minute amounts of the nucleophile/base DMAP (pK_a = 9.2). Synthesis of acrylate esters was successfully accomplished using MSNT (1-(Mesitylene-2-sulfonyl)-3-nitro-1,2, 4-triazole) coupling conditions using a much milder nucleophile/base, for example, N-methyl imidazole (pK_a = 6.95).

Full text of DOI:10.1016/j.tetlet.2014.05.124

Tetrahedron Letters 55 (2014) 4222–4226
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis, chemical reactivity, and photophysical properties
of 20,70 phenylated rhodamine dyes
⇑
Arunkumar Natarajan , Eugene P. Boden, Andrew Burns, Patrick J. McCloskey, Michael J. Rishel
GE Global Research, 1 Research circle, Niskayuna, NY 12309, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
While exploring water soluble rhodamine based fluorescent polymeric systems for biological imaging
applications we came across new rhodamine derivatives that possess interesting optical properties.
We report the synthesis of three different 20,70-diphenylated rhodamine derivatives (1–3) with distinct
photophysical properties. The three rhodamine derivatives differ by the number of methyl groups present
on the nitrogens and their absorption maxima are red-shifted on increased methylation. We observed an
unusual inertness of these compounds toward traditional DCC–DMAP esterification conditions, which we
attribute to the ease of lactonization in the presence of even minute amounts of the nucleophile/base
DMAP (pKa = 9.2). Synthesis of acrylate esters was successfully accomplished using MSNT (1-(Mesity-
lene-2-sulfonyl)-3-nitro-1,2,4-triazole) coupling conditions using a much milder nucleophile/base, for
example, N-methyl imidazole (pKa = 6.95).
Received 22 April 2014
Revised 24 May 2014
Accepted 29 May 2014
Available online 17 June 2014
Keywords:
20,70-Diphenyl rhodamine dyes
Emission
Absorption
MSNT coupling
Lactonization
Ó 2014 Elsevier Ltd. All rights reserved.
fluorescent polymeric systems as tracer dyes and bio-imaging
applications we discovered some interesting chemical and photo-
physical properties in the 20,70-diphenylated rhodamine deriva-
tives (1–3) in terms of their, (i) unusual ease of lactonization and
therefore chemical inertness during DCC–DMAP esterification con-
ditions; and (ii) changes in absorption and emission spectra based
on the extent of methylation of the nitrogen groups.
Introduction
Rhodamine dyes belong to the fluorone dye family of
compounds and are widely used as laser gain media as well as
for contrast enhancement in biotechnological applications such
as fluorescence microscopy, flow cytometry, fluorescence correla-
tion spectroscopy, and enzyme-linked immunosorbent assay
(ELISA).1 Other uses include tracer dyes for fluorescent detection
applications in groundwater flow tracing,2 leak detection, septic,
and sewer inspections and similar applications.3 The rhodamine
family of dyes includes Rhodamine B, Rhodamine 6G, Rhodamine
123, Carboxytetramethylrhodamine (TAMRA), tetramethylrhod-
amine (TMR) and its isothiocyanate derivative (TRITC), sulforhoda-
mine 101 (and its sulfonyl chloride form Texas Red), and
Rhodamine Red and covers the spectral range from 450 to
650 nm emission with high quantum yields ranging from 0.65 to
0.95 depending on solvents and extinction coefficients of typically
Liu et al.,6 first reported the synthesis of tetramethyl derivative
of the 20,70-diphenylated rhodamine and used it as a benchmark
system to compare the properties of the extended conjugation rho-
damine based near-IR dyes they had synthesized and studied. We
followed a similar synthesis protocol as described in Liu et al.6 for
the synthesis of 20,70-diphenylated rhodamine derivative 3. How-
ever in addition to the tetramethyl derivative (3) we also observed
the trimethyl- and dimethyl-diphenylrhodamine products (2 and
1, respectively) in reasonably larger yields compared to compound
3 under our synthesis conditions. All three compounds were easily
purified using column chromatography and each of the products
possessed interesting and distinct photophysical properties. We
present here the five step synthesis procedure of 20,70-diphenylated
rhodamines, formation of three different 20,70-diphenylated rhoda-
mine products (tetra-, tri-, and di-methylated diphenylrhodamine)
obtained during the condensation step, their photophysical prop-
erties and an exploration of their chemical inertness to DCC–DMAP
(dicyclohexylcarbodiimide, dimethylaminopyridine) esterification
conditions. Finally, we demonstrate an alternative esterification
method using MSNT (1-(Mesitylene-2-sulfonyl)-3-nitro-1,2,4-tria-
zole)7which delivers high esterification yields.
logemax >8.8 Mꢀ1 cmꢀ1 4
further application area in which rhodamine dyes are
.
A
commonly used is monitoring the residual levels of polymeric
coagulants in wastewater effluents. A common approach to moni-
toring the level of water soluble polymer coagulants is to blend
fluorescent dyes in small amounts and to use fluorescence of the
mixture to determine the concentration of the polymer in aqueous
systems.5 While exploring water soluble rhodamine based
⇑
Corresponding author. Tel.: +1 518 387 4772; fax: +1 518 387 5592.
E-mail address: natararu@ge.com (A. Natarajan).
http://dx.doi.org/10.1016/j.tetlet.2014.05.124
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

A. Natarajan et al. / Tetrahedron Letters 55 (2014) 4222–4226
4223
acetate/Hexane = 2:8 as elution solvent. The 1H NMR analysis con-
firmed the formation of three compounds 1–3. It is interesting to
note that the major difference between the three compounds is
seen on the N-methyl groups in 1H NMR, one signal at 2.88 ppm
(s, 6H0s) for compound 1, showing two signals for compound 2,
2.6 ppm (s, 6H0s), 2.87 ppm (s, 3H0s), and only one signal at
2.6 ppm (s, 12H0s) for compound 3.9 LC-MS analysis of the com-
pounds showed E+ with molecular mass 512 (for compound 1),
526 (for compound 2), and 540 (for compound 3) in the positive
ion mode when eluted using a reverse phase C18 column with
H2O–CH3CN solvent system.
The UV–vis absorption spectra of compounds 1–3 show that
each molecule has a distinct absorption spectrum (in methanol)
with the tetramethyl derivative (3) being the most red shifted kmax
at 578 nm (bathochromic shift) followed by compounds 2 and 1
which are 557 nm and 539 nm respectively. The trend in the shift
of absorption spectrum we see here is very similar to the trend
seen in the commercial molecules Rhodamine 6G (tetraethyl deriv-
ative) having kmax in ethanol at 540 nm (533 nm in methanol) and
Rhodamine B (dimethyl derivative) having kmax at 526 nm in
ethanol, respectively.10 We believe that the extended conjugation
conferred by diphenylation of derivatives (1–3) is responsible for
the red-shifts seen in their absorptions when compared to the
non-phenylated versions. As shown in Figure 1, the absorption
spectrum of Rhodamine 6G (tetraethyl derivative) has a similar
absorption spectrum to the dimethyl diphenyl rhodamine deriva-
tive (1) and the tetramethyl diphenyl rhodamine derivative is
red-shifted by ꢁ18 nm. This shows that the phenylated rhodamine
derivatives can effectively extend the spectral range of the rhoda-
mine compounds to access longer wavelengths.
Results and discussion
The rhodamine derivatives (1–3) were prepared from commer-
cially available 4-bromo-3-nitroanisole, as shown in Scheme 1.
The first step involved the synthesis of the 4-methoxy-2-nitrobi-
phenyl via Suzuki coupling of 4-bromo-3-nitroanisole with the
phenyl boronic acid and gave 85% yield.8 The second step was the
reduction of the nitro-group to amine using conventional Fe/con-
centrated HCl which produced an 80% yield of the 4-methoxybi-
phenyl-2-amine. The third step was the methylation of the amino
group using K2CO3/MeI to obtain 4-methoxy-N,N-dimethylbi-
phenyl-2-amine in 90% yield. It is important to emphasize here that
the excess MeI (5–7 equiv) was used and the reaction was moni-
tored by TLC until the N,N dialkylation was complete. We observed
that isolation of the product without base wash yielded the proton-
ated form (yellow crystals) of the N,N-dimethylbiphenyl-2-amine
due to HI by-product formation during the alkylation reaction.
Therefore, after the reaction went to completion, the product was
washed twice with NaHCO3 to deprotonate the product, ultimately
yielding N,N-dimethylbiphenyl-2-amine (yellow oil). By allowing
the reaction to run to completion and isolating only the dialkylated
product (based on 1H NMR analysis) we are confident that no
monomethylated product was carried forward to the reaction with
phthalic anhydride. The fourth step was the dealkylation of the
methoxy group using BBr3 to obtain 2-(dimethylamino)biphenyl-
4-ol in 88% yield. After the reaction was complete, the product
was washed thrice with NaHCO3 to prevent isolation of the
protonated form of the 2-(dimethylamino)biphenyl-4-ol. Finally,
condensation of 2-(dimethylamino)biphenyl-4-ol with 10 equiv of
phthalic anhydride, catalytic amount of p-toluenesulfonic acid,
and heating at 180 °C for 8 h in dichlorobenzene gave a mixture
of products (1–3) with an overall yield of 30% and individual yields
of 5.4%, 10.9%, and 13.7%, respectively. In contrast to Liu et al.,6
which reports only the tetramethyl rhodamine (3) in 15% yield,
we obtained three rhodamine derivatives (1–3) with overall yield
of 30%. Due to the differing polarities of the di, tri, and tetra-
methylated rhodamine derivatives, the products 1–3 eluted with
different Rf values of 0.7, 0.5, and 0.2 in 2/8 Ethyl acetate/Hexane
on silica gel TLC plate. The products were easily isolated in pure
forms using silica gel chromatography and the same Ethyl
Next, we measured the fluorescence of compounds (1–3) and
found that these compounds had distinct emission profiles and
quantum yields. Specifically, increasing methylation correlated
with decreases in quantum yield. Rhodamine 6G in methanol
was used as a standard for relative quantum yield analysis,11 each
dye was diluted to the same peak absorbance (0.25 OD) and
excited at their respective maximum absorbances (1 nm slit
width). The acid form of the dimethyl, diphenyl derivative (1)
exhibits an absolute quantum yield of >90% (comparable to Rhoda-
mine 6G) as shown in Figure 1, while the trimethyl derivative (2)
HO
OH
B
OCH3
OCH3
OCH3
excess
OH
OCH3
Br
BBr3,
MeI (5-7equs.),
5equ. K2CO3,
THF, reflux,
8eq. Fe,
CH2Cl2, 0°C to rt,
1 h, in the dark,
35equ. Conc.HCl,
EtOH, 6 h
N
O2N
H2N
N
1 day
O2N
K2CO3
NBuBr
yield 88%
yield 90%
yield 80%
H2O, 80°C, 1–2 h,
under N2,
O
O
yield 85%
cat. p-TsOH·H2O,
ODCB, 180oC, 8 h,
in the dark
10equs.O
H
N
H
H
N
O
N
N
O
N
N
O
-
-
-
CO2
CO2
CO2
1
2
3
yield 5.4%
yield 10.9%
yield 13.7%
1 3
Overall yield ( - ): 30%
Scheme 1. Synthesis route for 20,70-diphenyl rhodamine derivatives (1–3).

4224
A. Natarajan et al. / Tetrahedron Letters 55 (2014) 4222–4226
Figure 1. Absorption and emission profiles in methanol of 20,70-diphenyl rhodamine derivatives (1–3) compared to Rhodamine 6G (Rh6G).
exhibits a relative quantum yield of ꢁ4.5% and the tetramethyl
derivative (3) exhibits a relative quantum yield of approximately
1% or less. These quantum yields highlight the differences between
the diphenylated rhodamine systems and traditional rhodamine
dyes which generally exhibit quantum yields >90% across a range
of N-alkylations (e.g., Rhodamine B, Rhodamine 6G, etc.).11 How-
ever, we have not yet deciphered the reason for the dramatic
decrease in quantum yields for the tri- and tetra-methyl deriva-
tives, especially when the dimethyl derivative 1 shows fluores-
cence quantum yields close to Rhodamine 6G, though.
Having demonstrated the optical properties of these dyes,
attempts were then made to attach a methacrylate monomer via
esterification in order to have a polymerizable moiety. Conven-
tional rhodamine molecules have been converted to methacrylate
esters in the literature using DCC–DMAP coupling chemistry via
the carboxylic acid with near quantitative yields. However, to our
surprise, when we used a similar synthesis protocol to make the
corresponding methacrylate esters of compounds 1–3, none of
the compounds would react with the 2-hydroxyethyl methacrylate
to form the desired products (Scheme 2). In all the cases, the 1H
NMR resonances indicated a chemical reaction had occurred, but
the proton resonances did not correspond to the desired ester
products. Instead, all of the 1H NMR spectra showed signs of lact-
onization from the open acid form, which were confirmed, based
on the Rhodamine B-lactone proton resonance as a model system.
Further, LC-MS study shows no new product (ester) formation.
Therefore we conducted UV–vis analysis of compounds 1–3
upon addition of DMAP alone without other reagents to see if
change in the basicity of the solution affected the diphenyl rhoda-
mine molecule’s absorption properties. As anticipated, upon excess
DMAP addition, the solution absorbance was seen to dramatically
decrease, virtually removing the visible wavelength absorptions
seen above in Figure 1a suggesting lactone formation. This phe-
nomenon indicates the lactonization of the dye which is known
to dramatically decrease the visible wavelength absorption of xan-
thene species.12
It became apparent that the ease of lactonization of the diphe-
nylated rhodamine derivatives (1–3) under even under mildly
basic conditions was the reason for the lack of esterification
reaction (Scheme 3). Use of DMAP as an acylation catalyst in the
DCC–DMAP coupling reaction with a conjugate acid pKa of 9.2
was enough to shift the equilibrium to the lactone and thereby
render the acid group unavailable for DCC coupling. In the case
of conventional Rh-B (without the phenyl derivatization at 20 and
70 position) type dyes, esterification happens easily with the
DCC–DMAP coupling conditions13,14 as lactonization requires a
much stronger base like NaOH or triethylamine (pKa = 10.78),
potassium tert-butoxide (pKa = 18).15
Finally, in order to accomplish the synthesis of the methacrylate
ester monomers of compounds 1–3, we sought a coupling reaction
that used a milder base for the esterification. Our hypothesis was
that if coupling reagents were available that used much milder
bases and/or nucleophilic acylation catalysts with pKa values lower
than that of DMAP (pKa = 9.2), then we could prevent lactonization
and thereby make the acid group available for esterification. We
found the 1-(Mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT)
coupling chemistry developed by Reese et al.7 which uses
N-methyl imidazole as base/acylation catalyst, which has a pKa of
6.95. We tested this coupling chemistry following a similar proce-
dure reported in Reese et al., wherein, one equivalent of diphenyl
O
O
N
H
Cl-
O
N
H
O
OH
OH
O
O
O
DCC/DMAP
0oC, CH2Cl2
O
X
O
O
N
O
N
H
H Cl-
N
H
O
N
H
Cl-
Scheme 2. DCC–DMAP mediated esterification 20,70-diphenyl rhodamine derivatives (1–3) reaction resulted only in lactonization (only the schematic for derivative 1 shown).

A. Natarajan et al. / Tetrahedron Letters 55 (2014) 4222–4226
4225
O
OH
NaOH
O
O
HCl
N
O
N
N
O
N
O
OH
mild base
HCl
O
O
N
H
O
N
H
N
H
O
N
H
mild base: DMAP
Scheme 3. Lactonization conditions for Rhodamine 6G and diphenylated rhodamine (1).
O
O
O
O
OH
OH
O
O
O
MSNT/N-MeIm
RT, 2h, CH2Cl2
N
H
N
H
O
N
H
O
1
( )
Cl-
N
H
Cl-
4
( ) >90%
O
O
O
O
O
O
O
O
N
O
N
Cl-
N
O
N
Cl-
H
(6)
(5)
Scheme 4. MSNT mediated esterification reaction conditions shown for 20,70-diphenyl rhodamine derivatives (1) ? (4). Similar protocols followed for synthesis of (5) and (6).
rhodamine acid derivative was dissolved in 2 mL of dry CH2Cl2 and
mixed with 2 mL of dry CH2Cl2 containing 2-hydroxyethyl methac-
rylate/MSNT reagent/N-methyl imidazole (in molar ratios 2/2.2/2.2
with respect to the rhodamine acid). The solution was left to stir
overnight at room temperature. This esterification chemistry
generated quantitative reaction yields with compounds 1–3 to pro-
duce the 2-hydroxyethyl methacrylate derivatives 4–6 without
producing the corresponding lactones (Scheme 4). This suggests
that a N-methyl imidazole being a weaker base does not shift the
equilibrium to the lactone form at least for the amounts used in
our study thereby allowing the esterification reaction to proceed
quantitatively. However, excess amounts of esterification reagents
and the alcohol were needed because the competitive reaction of
etherification of the alcohol by MSNT to form the sulfonate may
occur otherwise. The crude reaction mixture was purified using
column chromatography using silica gel and using 95% CH2Cl2
and 5% MeOH as eluents. The products were characterized using
1H NMR9 and LC-MS analysis. LC-MS analysis of the compounds
4, 5, and 6 showed molecular masses of 623, 637, and 651 in the
E+ positive ion mode when eluted using a reverse phase C18 column
with H2O–CH3CN solvent system.
ease of lactonization in the presence of even minute quantities of
DMAP, a nucleophile/base with pKa = 9.2. We also demonstrated
that these compounds could be easily esterified by using MSNT
coupling conditions that use a much milder base, in this case
N-methyl imidazole (pKa = 6.95). We believe that the presence of
20,70-diphenyl substitution has a critical role to play in the
reduction of quantum yields in 2 and 3. Intriguingly, tri- and
tetra-methyl substituted cases (2 and 3) shows markedly lower
quantum yields of fluorescence while dimethyl substituted deriva-
tive (1) shows high quantum yields. We are currently pursuing fur-
ther studies to understand the reasons for this behavior and would
be included as a part of future publication.
References and notes
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In summary, we have reported the synthesis and purification of
three different 20,70-diphenylated rhodamine derivatives (1–3)
exhibiting distinct photophysical properties. Additionally, these
compounds showed unusual inertness toward ester formation
using traditional DCC–DMAP esterification conditions due to their
5. Moa, J.; Hwangb, J.-E.; Jegalb, J.; Kima, J. Dyes Pigm. 2007, 72, 240–245.
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9. 1H NMR (400 MHz, CDCl3): Compound 1: 2.88 (s, 6H), 4.0–4.6 (br s, 2H), 6.5 (s,
2H), 6.56 (s, 2H), 7.2–7.4 (m, 11H), 7.53 (t, 1H), 7.63 (t, 1H), 7.96 (d, 1H).
Compound 2: 2.6 (s, 6H), 2.87 (s, 3H), 4.1–4.25 (br s, 1H), 6.44 (s, 1H), 6.52 (s,
1H), 6.56 (s, 1H), 6.89 (s, 1H), 7.2–7.4 (m, 11H), 7.54 (t, 1H), 7.63 (t, 1H), 7.95 (d,
1H). Compound 3: 2.6 (s, 12H), 6.56 (s, 2H), 6.88 (s, 2H), 7.2–7.4 (m, 11H), 7.55
(t, 1H), 7.62 (t, 1H), 7.96 (d, 1H). Compound 4: 2.1 (s, 3H), 3.1 (s, 6H), 4.05 (m,
2H), 4.32 (m, 2H), 5.5 (s, 1H), 5.9 (s, 1H), 6.82 (s, 2H), 6.88 (s, 2H), 7.2–7.3 (m,
6H), 7.4–7.5 (m, 7H), 7.53 (t, 1H), 7.63 (t, 1H), 8.24 (d, 1H). Compound 5: 2.1 (s,
3H), 2.95 (s, 6H), 3.1 (s, 3H), 4.05 (m, 2H), 4.3 (m, 2H), 5.5 (s, 1H), 5.9 (s, 1H),
6.85 (s, 1H), 6.9 (s, 1H), 6.92 (s, 1H), 7.1 (s, 1H), 7.2–7.4 (m, 11H), 7.78 (t, 1H),
8.05 (t, 1H), 8.25 (d, 1H). Compound 6: 2.1 (s, 3H), 2.95 (s, 12H), 4.05 (m, 2H),
4.3 (m, 2H), 5.52 (s, 1H), 5.91 (s, 1H), 6.85 (s, 1H), 6.9 (s, 1H), 6.92 (s, 1H), 7.1 (s,
1H), 7.2–7.4 (m, 11H), 7.78 (t, 1H), 8.05 (t, 1H), 8.25 (d, 1H).