Rational design and synthesis of a novel class of highly fluorescent rhodamine dyes that have strong absorption at long wavelengths

Article abstract of DOI:10.1016/S0040-4039(03)00938-9

A novel class of strongly fluorescent rhodamine dyes were designed and synthesized by extending the π conjugation of chromophore with limited flexibility. These dyes were shown to have longer absorption in the range of 581 to 631 nm with quantum yields between 0.64 and 0.89.

Full text of DOI:10.1016/S0040-4039(03)00938-9

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 4355–4359
Rational design and synthesis of a novel class of highly
fluorescent rhodamine dyes that have strong absorption at
long wavelengths
Jixiang Liu,a,* Zhenjun Diwu,b Wai-Yee Leung,a Yixin Lu,a Brian Patcha and Richard P. Hauglanda
aMolecular Probes, Inc., 29851 Willow Creek Road, Eugene, OR 97402, USA
bMolecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089, USA
Received 4 February 2003; revised 10 April 2003; accepted 11 April 2003
Abstract—A novel class of strongly fluorescent rhodamine dyes were designed and synthesized by extending the p conjugation of
chromophore with limited flexibility. These dyes were shown to have longer absorption in the range of 581 to 631 nm with
quantum yields between 0.64 and 0.89. © 2003 Elsevier Science Ltd. All rights reserved.
Rhodamine dyes are highly fluorescent and have great
photostability. Additionally, their fluorescence spectra
are pH-independent from pH 4 to 10. Over the years a
variety of rhodamine dyes have been prepared and used
both as laser dyes1 and as fluorescent detection
reagents. For example, carboxyrhodamine 110 succin-
imidyl ester, carboxyrhodamine 6G succinimidyl ester,
carboxytetramethylrhodamine succinimidyl ester, tetra-
methylrhodamine isothiocyanate, Lissamine rhodamine
B sulfonyl chloride and sulforhodamine 101 sulfonyl
chloride (Texas Red® sulfonyl chloride) are among the
most widely used fluorescent reagents for labeling
proteins and oligonucleotides. In particular, researchers
commonly use these amine-reactive rhodamines to pre-
pare fluorescent antibodies and avidin conjugates for
immunochemistry applications.2
example, Drexhage, Arden-Jacob and their co-workers
developed a new class of long-wavelength pentacyclic
rhodamine dyes called ‘JA’ dyes.3,4 And recently, Ben-
son et al. reported the synthesis of dibenzorhodamine
derivatives with absorption beyond 600 nm.5
The additional conjugation of the p system, as well as
the formation of rigid rings, in organic dyes can shift
their absorption and emission maxima to longer wave-
lengths. Furthermore, the lack of molecular rotation6
restricts or diminishes the nonradiative deactivation
processes, which in turn increases the rigidity of the
molecular system and increases the fluorescence quan-
tum yield of the dye. Therefore, we predicted that
rhodamine derivatives with different aryl or heteroaryl
substituents on the 2% and 7% positions (made rigid by
dimethylmethylene bridges to the nitrogen atoms of
rhodamine) might exhibit the desired characteristics of
long-wavelength absorption and emission with high
fluorescence quantum yield. Based on this hypothesis,
we designed and synthesized a novel class of highly
fluorescent rhodamine derivatives with absorption
beyond 600 nm.
Because these rhodamine dyes have absorption maxima
that are well below 600 nm, their fluorescence detection
sensitivity is severely compromised by background sig-
nals caused by biological autofluorescence (e.g. from
serum, proteins and other macromolecular com-
pounds). The development of sensitive detectors, such
as CCD cameras, and of inexpensive lasers with long-
wavelength emission (e.g. He/Ne lasers, 633 nm), as
well as technological advances in multicolor labeling
and capturing images in the visible–near IR region, has
prompted researchers to invent long-wavelength rho-
damine dyes with absorption beyond 600 nm. For
These new rhodamine derivatives were prepared from
commercially available 4-bromo-3-nitroanisole and five
different arylboronic acids, as shown in Scheme 1. The
improved Suzuki cross-coupling reaction in water gave
a high yield of pure product 1.7 Catalytic hydrogena-
tion of compound 1 gave the aniline derivative 2, which
was converted to the amide 3 with acetic anhydride.
Thiophene or benzothiophene derivatives can poison
the Pd/C catalyst, so zinc dust/concentrated hydrochlo-
* Corresponding author. Fax: (+1) 541-344-6504; e-mail: jixiang.liu@
probes.com
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00938-9

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J. Liu et al. / Tetrahedron Letters 44 (2003) 4355–4359
ric acid (instead of hydrogenation) was used to
reduce compound 1 containing thiophene or benzo-
thiophene moieties. Bischler–Napieralski cyclization
Compound 4 was quaternized with p-TsOMe to give
compound 5. Compound 5 was reacted with the
MeMgCl Grignard reagent to afford the aniline
derivative 6.
of amide
3 with POCl3 afforded compound 4.
Scheme 1. Synthesis of the rhodamine derivatives 9a–i and their intermediates. Reaction conditions: (i) Pd(OAc)2, K2CO3,
š
n-BuN BrT, H2O, 80°C, 1–2 h, under N2, yield 85–91%; (ii) H2, 10% Pd(C), MeOH, 4 h, yield 100%; or Zn/con. HCl,
THF/MeOH, rt, 1 h, yield 82–90%; (iii) Ac2O, Py, THF, reflux, 1–2 h, yield 91–95%; (iv) POCl3, 80°C, 30 min, yield 62–84%; (v)
p-TsOMe, chlorobenzene, reflux, 2 days, yield 70–79%; (vi) MeMgCl (large excessive), THF, under N2, in the dark, yield 65–80%;
(vii) BBr3, CH2Cl2, 0°C“rt, 1 h, in the dark, yield 85–93%; (viii) a. phthalic anhydride, cat. p-TsOH·H2O, EtCO2H, reflux, 6–12
h, in the dark; b. 4-formyl-1,3-benzenedisulfonic acid, disodium salt, TFA, DMF, rt overnight and then 110–120°C, 7–10 h, in the
dark.

J. Liu et al. / Tetrahedron Letters 44 (2003) 4355–4359
4357
Scheme 2. Synthesis of rhodamine derivative 12. Reaction conditions: (i) MeI, K2CO3, THF, reflux, 1 day, yield 90%; (ii) BBr3,
CH2Cl2, 0°C“rt, 1 h, in the dark, yield 91%; (iii) phthalic anhydride, cat. p-TsOH·H2O, EtCO2H, reflux, 6–12 h, in the dark, yield
15%.
Table 1. The photophysical characteristics of the new rhod-
Note that the reaction of compound 5 with the
amine derivatives (compounds 12 and 9a–i) dissolved in
MeMgCl Grignard reagent should be carried out in the
dark. Otherwise, product 6 continues to react with
excess MeMgCl, and a significant amount of by-
product 7 is formed, most likely via a free radical
reaction.
MeOH
a
a,b
d
Compound
umax. abs
umax. em
bfc
‡
max
(nm)
(nm)
(M−1 cm−1
)
12
9a
9b
9c
9d
9e
9f
9g
9h
9i
548
582
581
596
631
628
627
600
610
616
571
604
605
620
648
649
645
627
629
633
0.25
0.75
0.85
0.71
0.64
0.66
0.76
0.66
0.85
0.89
82,000
125,000
130,000
126,000
141,000
140,000
124,000
132,000
130,000
143,000
Demethylation of compound 6 with BBr3 generated the
desired key aminophenol derivative 8. Finally, the novel
rhodamine derivatives 9a–i were obtained via condensa-
tion of the aminophenol derivative 8 with different
phthalic anhydrides or 4-formyl-1,3-benzenesulfonic
acid, disodium salt. Interestingly, only the rhodamine
dyes were obtained in the condensation of the
aminophenol derivative 8 with 4-formyl-1,3-benzenesul-
fonic acid, disodium salt, which indicated that the
dihydrorhodamine intermediates were readily oxidized
by air.
a Uncertainty is 1 cm.
b Emission spectra measured on the fluorescence spectrometer
Aminco SPF-500C are corrected.
c The fluorescence quantum yields of the new dyes are determined by
using the reference standards [rhodamine B (bf=0.50 in ethanol)9
for 12; rhodamine 101 (bf=0.96 in ethanol)9 for 9a–c, 9g–i and JA
22 (bf=0.90 in ethanol)3 for 9c–e as standards]. Uncertainty is
56%.
For comparison, the rhodamine derivative 12 was also
synthesized from compound 2 as shown in Scheme 2.
The alkylation of compound 2 with MeI/K2CO3 gave
compound 10, which was demethylated with BBr3 to
afford the aminophenol derivative 11. The condensa-
tion of compound 11 with phthalic anhydride gave the
rhodamine derivative 12. All compounds were con-
d Uncertainty is 55%.
1
firmed with their MS and H NMR spectra, their Rf
values were also measured on silica gel TLC.8
Absorption maxima (Abs), fluorescence maxima (Em),
fluorescence quantum yields (bf) and extinction coeffi-
cient (‡) for all of the rhodamine derivatives (com-
pounds 12 and 9a–i) are summarized in Table 1. In
general, all of the new rhodamine derivatives have
absorption and emission band shapes similar to that of
tetramethylrhodamine with a large bathochromic shift.
The absorption and emission spectra of compound 9d
was compared with those of tetramethylrhodamine in
Figure 1. As expected, the fluorescence spectra of all
the new rhodamine derivatives have approximate
mirror symmetry with their absorption spectra.
Figure 1. Normalized absorption and emission spectra of
compound 9d (—) and tetramethylrhodamine (-·-) in MeOH.
Compared with compound 12, compound 9a has
absorption and emission maxima that are remarkably

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J. Liu et al. / Tetrahedron Letters 44 (2003) 4355–4359
The photostabilities of representative compounds 9a,b
and 9c, as well as fluorescein and 5-(and-6)-carboxy-
tetramethylrhodamine (5-(and-6)-TAMRA), were
tested using glass capillary tubes filled with dyes in
MeOH under 100 W mercury arc lamp of the fluores-
cence microscope and shown in Figure 2. As expected,
compounds 9a,b and 9c have much higher photostabili-
ties than fluorescein, and have similar photostabilities
to 5-(and-6)-TAMRA.
In summary, we have reported the rational design and
synthesis of novel class of highly fluorescent rhodamine
dyes with long-wavelength absorption and emission.
These new rhodamine dyes can be readily converted to
reactive derivatives and used as fluorescent tags to
prepare various bioconjugates for biological applica-
tions.10 Both the methodology used to synthesize these
rhodamine dyes and the intermediate aminophenol
derivatives involved in that synthesis are significant
developments that should open new routes for prepar-
ing other fluorescent dyes and heterocyclic compounds.
Figure 2. Comparison of photostability of representative
dyes.
red-shifted by 34 nm; the fluorescence quantum yield of
compound 9a is also three times higher than that of
compound 12. These differences can be attributed to
the dimethylmethylene bridges, which provide a rigidity
that reduces rotation of the phenyl groups and nitrogen
atoms, thereby preventing the nonradiative process.
Some interesting observations were made by the com-
parison of compounds 9a,b and 9c. Compounds 9a and
9b have almost identical absorption and emission max-
ima and there is little difference between their fluores-
cence quantum yields; however, Compound 9c exhibits
much longer absorption and emission maxima. Com-
pounds 9b and 9c have thiophenyl substituents at posi-
tions 2% and 7%, whereas compound 9a has a simple
phenyl substituent. Compounds 9b and 9c differ only in
the position of the sulfur atoms, suggesting that the
sulfur atom in compound 9c also acts as an additional
polar substituent incorporated into the p conjugation
system.
References
1. Drexhage, K. H. Top. Appl. Phys. 1973, 1, 144–274.
2. For a sourcebook and comprehensive references, see:
Haugland, R. P. Handbook of Fluorescent Probes and
Research Products, 9th ed.; Molecular Probes, Inc.:
Eugene, OR, USA, 2002.
3. Sauer, M.; Han, K.-T.; Muller, R.; Nord, S.; Schulz, A.;
Seeger, S.; Wolfrum, J.; Arden-Jacob, J.; Deltau, G.;
Marx, N. J.; Zander, C.; Drexhage, K. H. J. Fluoresc.
1995, 5, 247–261.
4. Herrmann, R.; Josel, H. P.; Drexhage, K. H.; Arden-
Jacob, J. US Patent 5,750,409, 1998.
5. Benson, S. C.; Lam, J. Y. L.; Menchen, S. M. US Patent
5,936,087, 1999.
6. Forster, T. Fluoreszenz Organischer Verbindungen; Van-
denhoeck and Ruprecht: Gottingen, 1951.
Compounds 9d and 9e have essentially identical photo-
physical properties, and their absorption and emission
maxima are about 30–35 nm longer than those of
compound 9c. This observation supports the hypothesis
that the substitution of protons (on the bottom carb-
oxyphenyl moiety) by electron-accepting atoms such as
Cl or F can decrease the excitation energy of the
rhodamine dyes. As predicted, the additional ‘benzo’
groups in compounds 9f and 9g shift the maxima of
absorption and emission to longer wavelengths.
Analogous to the effect of sulfonate groups on Lis-
samine rhodamine B and sulforhodamine 101,2 the
sulfonate groups of the newly reported rhodamines 9h
and 9i also cause a bathochromic shift in emission
wavelength and an increase in fluorescence quantum
yields compared with the corresponding carboxylated
analog compounds 9c and 9g. As expected, some of the
new rhodamine dyes display absorption beyond 600 nm
with strong fluorescence as shown in Table 1. These
dyes also possess high extinction coefficients. For exam-
ple, the extinction coefficients of compounds 9d and 9i
were determined to be 141,000 and 143,000 M−1 cm−1,
respectively, in MeOH.
7. Badone, D.; Baroni, M.; Cardamone, R.; Ielmini, A.;
Guzzi, U. J. Org. Chem. 1997, 62, 7170–7173.
8. 9a: blue solid, Rf=0.29 CHCl3/MeOH (v/v 10:1), yield
68%; mp 118–120°C; 1H NMR (400 MHz, [D4]MeOH):
l=8.24 (d, H; aromatic H), 7.80 (t, 1H; aromatic H),
7.32 (t, 1H; aromatic H), 7.65 (s, 2H; aromatic H), 7.50
(d, 2H; aromatic H), 7.45 (d, 2H; aromatic H), 7.35 (m,
3H; aromatic H), 7.28 (t, 2H; aromatic H), 6.94 (s, 2H;
aromatic H), 3.10 (s, 6H; CH3), 1.60 (s, 6H; CH3), 1.56 (s,
6H; CH3); MS (EI): m/z (%): 591 (100) [M++H]. 9b: blue
solid, Rf=0.26 CHCl3/MeOH (v/v 10:1), yield 70%; mp
177–180°C; 1H NMR (400 MHz, [D4]MeOH): l=8.22 (d,
1H; aromatic H), 7.78 (t, 1H; aromatic H), 7.72 (t, 1H;
aromatic H), 7.39 (d, 2H; heteroaromatic H), 7.34 (d, 1H;
aromatic H), 7.31 (s, 2H; aromatic H), 7.06 (d, 2H;
heteroaromatic H), 6.93 (s, 2H; aromatic H), 3.12 (s, 6H;
CH3), 1.73 (s, 6H; CH3), 1.69 (s, 3H; CH3); MS (EI): m/z
(%): 603 (100) [M++H]. 9c: blue solid, Rf=0.14/0.34
CHCl3/MeOH (v/v 5:1), yield 67%; mp >300°C; 1H NMR
(400 MHz, [D4]MeOH): l=8.93 (s, 5-isomer; aromatic
H), 8.37 (m, 5- and 6-isomer; aromatic H), 8.32 (d;
6-isomer; aromatic H), 8.00 (s, 6-isomer; aromatic H),
7.39 (m, 2H; heteroaromatic H), 7.08 (m, 2H; heteroaro-

J. Liu et al. / Tetrahedron Letters 44 (2003) 4355–4359
4359
matic H), 6.99 (m, 4H; aromatic H), 3.29, 3.28 (2×s, 6H;
CH3), 1.76, 1.75, 1.74 (3×s, 12H; CH3); MS (EI): m/z (%)
645 (100) [M+−H]. 9d: blue solid, Rf=0.22 CHCl3/MeOH
(v/v 10:1), yield 26%; mp 240°C (dec.); 1H NMR (400
MHz, CDCl3) l=7.10 (d, 2H; heteroaromatic H), 6.85
(d, 2H; heteroaromatic H), 6.65 (s, 2H; aromatic H), 6.40
(s, 2H; aromatic H), 2.97 (s, 6H; CH3), 1.60 (s, 12H;
CH3); MS (EI): m/z (%) 740 (100) [M+]. 9e: blue solid,
Rf=0.10 CHCl3/MeOH (v/v 10:1), yield 18%; mp 250°C
(dec.); 1H NMR (400 MHz, CDCl3) l=7.10 (d, 2H;
heteroaromatic H), 6.85 (d, 2H; heteroaromatic H), 6.70
(s, 2H; aromatic H), 6.45 (s, 2H; aromatic H), 2.98 (s,
6H; CH3), 1.60 (s, 12H; CH3); MS (EI): m/z (%) 675
(100) [M++H]. 9f: blue solid, Rf=0.21 CHCl3/MeOH (v/v
aromatic H), 7.78 (d, 2H; aromatic H), 7.34 (m, 4H;
aromatic H), 7.07, 7.04 (2×s, 2H; aromatic H), 6.90, 6.85
(2×s, 2H; aromatic H), 3.37, 3.27 (2×s, 6H; CH3), 2.00,
1.99, 1.96, 1.90 (4×s, 12H; CH3); MS (EI): m/z (%) 745
(100) [M+−H]. 9h: blue solid, Rf=0.09 CHCl3/MeOH
(v/v 5:1), yield 85%; mp >300°C; 1H NMR (400 MHz,
[D6]DMSO): l=8.34 (s, 1H; aromatic H), 7.79 (d, 1H;
aromatic H), 7.52 (d, 2H; heteroaromatic H), 7.24 (m,
3H; aromatic and heteroaromatic H), 6.94 (s, 2H; aro-
matic H), 6.82 (s, 2H; aromatic H), 3.25 (s, 6H; CH3),
1.72 (s, 6H; CH3), 1.70 (s, 6H; CH3); MS (EI): m/z (%):
719 (100) [M++H]. 9i: purple–blue solid, Rf=0.21 CHCl3/
1
MeOH (v/v 5:1), yield 88%; mp >300°C; H NMR (400
MHz, [D6]DMSO): l=8.37 (s, 1H; aromatic H), 8.03 (m,
4H; aromatic H), 7.83 (d, 1H; aromatic H), 7.38 (m, 4H;
aromatic H), 7.28 (d, 1H; aromatic H), 6.94 (s, 2H;
aromatic H), 6.89 (s, 2H; aromatic), 3.27 (s, 6H; CH3),
2.00 (s, 6H; CH3), 1.98 (s, 6H; CH3); MS (EI): m/z (%):
818 (100) [M+].
1
10:1), yield 30%; mp 275°C (dec.); H NMR (400 MHz,
CDCl3) l=8.14 (d, 2H; aromatic H), 7.72 (d, 2H; aro-
matic H), 7.65 (d, 2H; aromatic H), 7.52 (m, 2H; aro-
matic H), 7.40 (m, 4H; aromatic H), 7.01 (s, 2H; aromatic
H), 6.41 (s, 2H; aromatic H), 3.02 (s, 6H; CH3), 2.00 (s,
12H; CH3); MS (EI): m/z (%) 834 (100) [M+]. 9g: blue
solid, Rf=0.14/0.41 CHCl3/MeOH (v/v 5:1), yield 61%;
mp >300°C; 1H NMR (400 MHz, [D4]MeOH): l=8.90
(s, 5-isomer; aromatic H), 8.38 (d, 6-isomer, aromatic H),
8.28 (m, 5- and 6-isomer; aromatic H), 8.09 (s, 6-isomer;
aromatic H), 7.93 (d, 5-isomer; aromatic H), 7.89 (d, 2H;
9. Arden, J.; Deltau, G.; Huth, V.; Kringel, U.; Peros, D.;
Drexhage, K. H. J. Luminesc. 1991, 48 and 49, 352–358.
10. For bioconjugates and applications, see: Diwu, Z.; Liu,
J.; Haugland, R. P.; Gee, K. R. PCT Int. Appl. WO 02
12,195, 2002.