Synthesis of 5- and 6-carboxy-x-rhodamines

Article abstract of DOI:10.1021/ol801904k

(Equation Presented) An efficient route is reported to 5- and 6-carboxy-X-rhodamines (compounds 1 and 2) that contain multiple n-propylene or y,y-dimethylpropylene groups bridging terminal nitrogen atoms and the central xanthene core. Gram quantities of these dyes are synthesized from inexpensive starting materials. The isolated products are activated by selective transformation of the carboxylic acid group into N-hydroxysuccinimidyl esters in situ and then conjugated with an amino group of a molecule of interest.

Full text of DOI:10.1021/ol801904k

ORGANIC
LETTERS
2008
Vol. 10, No. 21
4799-4801
Synthesis of 5- and
6-Carboxy-X-rhodamines
Md. Jashim Uddin and Lawrence J. Marnett*
A. B. Hancock Jr. Memorial Laboratory for Cancer Research, Department of
Biochemistry, Chemistry and Pharmacology, Vanderbilt Institute of Chemical Biology,
Vanderbilt UniVersity School of Medicine, NashVille, Tennessee 37232-0146
larry.marnett@Vanderbilt.edu
Received August 14, 2008
ABSTRACT
An efficient route is reported to 5- and 6-carboxy-X-rhodamines (compounds 1 and 2) that contain multiple n-propylene or γ,γ-dimethylpropylene
groups bridging terminal nitrogen atoms and the central xanthene core. Gram quantities of these dyes are synthesized from inexpensive
starting materials. The isolated products are activated by selective transformation of the carboxylic acid group into N-hydroxysuccinimidyl
esters in situ and then conjugated with an amino group of a molecule of interest.
Rhodamine dyes are important because of their favorable
photochemical and photophysical properties.1 They have long
wavelength absorption maxima, high molar absorptivities,
and high quantum yields.2 Compared to the fluorescein dyes,
rhodamines are relatively resistant to photobleaching and
their fluorescence spectra are pH independent over a range
from 4 to 10.3 For these reasons, they have broad application,
not only in biotechnology for fluorescent labeling or single
molecule detection but also in medicine for imaging living
cells or live animals in preclinical research.6,7 In recent years,
a wide variety of rhodamine dyes have been commercialized
for conjugation with biomolecules.2 Of these, 5- and 6-car-
boxy-X-rhodamines (1 or 2) are of special interest because
of their highly intense absorption and emission. This is
possibly due to the presence of the added structural rigidity
introduced by multiple n-propylene bridges, which prevents
fluorescence deactivation by nonradiative processes. The high
cost of these dyes from commercial sources limits their use
in biomedical research. Although different methods are
available in the literature for the synthesis of diverse
rhodamine derivatives,8-11 no report describes the synthesis
of 5- and 6-carboxy-X-rhodamines. Therefore, as a part of
our ongoing program to develop fluorescent ligands, we
report the synthesis and structure of 5- and 6-carboxy-X-
rhodamine dyes that contain repetitive n-propylene or γ,γ-
(4) Kreis, T. E.; Geiger, B.; Schlessinger, J. Cell 1982, 29, 835.
(5) (a) Kwon, J. Y.; Jang, Y. J.; Lee, Y. J.; Kim, K. M.; Seo, M. S.;
Nam, W.; Yoon, J. J. Am. Chem. Soc. 2005, 127, 10107. (b) Yang, Y.-K.;
Yook, K.-J.; Tae, J. J. Am. Chem. Soc. 2005, 127, 16760.
(1) (a) He, J.; Ritalahti, K. M.; Yang, K.-L.; Koenigsberg, S. S. Loffler
Nature 2003, 424, 62. (b) George, A.; Mottola-Hartshorn, C.; Warner, A.;
Fields, B.; Chen, L. B. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 6579.
(2) Hougland, R. P. Handbook of Fluorescent Probes and Research
Products, 9th ed.; Molecular Probes Inc.: Eugene, OR, 2002.
(3) (a) Lakowicz, J. R. Principles of Fluorescnce Spectroscopy, 2nd ed.;
Plenum Publishing: New York, 1999. (b) Valeur, B. Molecular Fluores-
cence: Principles and Applications; Wiley-VCH: Weinheim, Germany,
2002.
(6) (a) Johnson, L. V.; Walsh, M. L.; Chen, L. B. Proc. Natl. Acad.
Sci. U.S.A. 1980, 77, 990. (b) Belmont, L. D.; Hyman, A. A.; Sawin, K. E.;
Mitchison, T. J. Cell 1990, 62, 579
(7) Marnett, L. J.; Uddin, M. J.; Crews, B. C. US Patent Application
WO/2007149546, 2007
(8) Scala-Valero, C.; Doizi, D.; Guillaumet, G. Tetrahedron Lett. 1999,
40, 4803
.
.
.
10.1021/ol801904k CCC: $40.75
Published on Web 10/07/2008
2008 American Chemical Society

dimethylpropylene bridging moieties. In addition, we de-
scribe an efficient route to activate and conjugate these dyes
with the amino group of a molecule of interest to provide
easy excess to fluorescent ligands that are useful for materials
and biomedical and clinical research.
Scheme 1
.
Synthesis of Rhodamine-Based Fluorescent Dyes
1a,b and 2a,b
The alkylation of m-anisidine (3) using 1-chloro-3-
methylbut-2-ene in the presence of K2CO3 gave 3-methoxy-
N,N-bis(3-methylbut-2-enyl)aniline (4) in 69% yield. The
treatment of compound 4 with concd HCl at 0 °C afforded
3-methoxy-N,N-bis(3-methylbut-2-enyl)aniline hydrochloride
(5) in 99% yield. Intramolecular cyclization of compound 5
using neat MeSO3H at 95 °C gave 1,1,7,7-tetramethyl-8-
methoxyjulolidine (6) in 65% yield, and O-desmethylation
of compound 6 using BBr3 gave the corresponding 1,1,7,7-
tetramethyl-8-hydroxyjulolidine (7b) in 80% yield. The
8-hydroxyjulolidine (7a) was synthesized using a reported
protocol.12 The synthesis of 5- or 6-carboxy-X-rhodamine
dyes (1 or 2) requires a symmetrical condensation of 2 equiv
of compound 7 with 1 equiv of 4-carboxyphthalic anhydride.
The overall process requires two sequential Friedel-Crafts-
type electrophilic aromatic substitution reactions for the
formation of the xanthene skeleton. We first tried a traditional
condensation protocol involving fusion of compound 7 (R
) H or Me) with 4-carboxyphthalic anhydride at high
temperature (180 °C). This reaction gave minimal yield,
possibly due to considerable sublimation of the starting
materials arround the vessel wall. Next, we used a catalytic
amount of Lewis acid (ZnCl2), or a protic acid (H2SO4) that
gave a poor yield of the desired products. However, the yield
was dramatically increased by using a high-boiling weakly
acidic solvent (n-PrCO2H, pKa 4.82) with a trace of 2 M
H2SO4 under reflux (Scheme 1).
The separation of the two isomers from the regioisomeric
mixture [1a, 2a (R ) H) or 1b, 2b (R ) Me)] was achieved
by flash chromatography. Extensive optimization was per-
formed to develop conditions for the separation of significant
quantities of 1 and 2. Silica gel 60 (230-450 mesh) was
slurry packed into a 10 in. column (1 × 15 in.) and eluted
with methanol/chloroform gradients at a flow rate of 5 mL/
min. The progress of separation was monitored with a
Spectroline ENF-280C UV detector (365 nm window). The
best yield was obtained using 0.5 g of crude sample with
relatively shallow gradients. In a typical experiment, we
obtained 34% and 32% isolated yields for compounds 1a
and 2a or 42% and 15% isolated yield for compounds 1b
and 2b, respectively.
The isomeric purity of each of the isolated compounds
was determined by HPLC analysis to be greater than 99%.
A complete structural analysis of compounds 1 and 2 was
conducted using 1D and 2D NMR spectroscopy. There are three
aromatic protons in each of these compounds that are charac-
teristic for their differentiation by 1H NMR. For compound 1a,
signals for two neighboring aryl protons at position-6 and
position-7 (Harom-6 and Harom-7) were assigned as two separate
doublets at 8.18 and 7.05 ppm, respectively, with a large vicinal
coupling constant (J ) 9.0 Hz). The assignment was confirmed
by a 1H-1H COSY experiment, where Harom-6 showed a strong
correlation with Harom-7 (Supporting Information). The proton
located at position-4 (Harom-4) was assigned at 8.27 ppm and
showed a weak correlation with Harom-6 in the COSY spectrum.
This weak correlation indicates that Harom-4 is located at least
four bonds from Harom-6 and supports the location of Harom-4
within two carboxyl groups of the aryl ring. In compound 2a,
the resonances for the two neigboring aryl protons (Harom-4 and
H
arom-5) appeared as two doublets at 7.77 ppm and 8.04 ppm
(9) Kojima, H.; Hirotani, M.; Urano, Y.; Kikuchi, K.; Higuchi, T.;
Nagano, T. Tetrahedron Lett. 2000, 41, 69.
(J ) 8.9 Hz), respectively. This assignment was confirmed by
a 1H-1H COSY spectrum, in which Harom-4 showed a strong
correlation with Harom-5. The sharp singlet located at 7.43 ppm
was assigned as Harom-7, that has a weak correlation with Harom-5
in the COSY spectrum. This indicates that Harom-7 is located
(10) Jiao, G.-S.; Castro, J. C.; Thoresen, L. H.; Burgess, K. Org. Lett.
2003, 5, 3675.
(11) Liu, J.; Giwu, Z.; Leung, W.-Y.; Lu, Y.; Patch, B.; Haugland, R. P.
Tetrahedron Lett. 2003, 44, 4355.
(12) Gompel, J. V.; Schuster, G. B. J. Org. Chem. 1987, 52, 1465.
4800
Org. Lett., Vol. 10, No. 21, 2008

Scheme 2. Conjugate Chemistry
1
Figure 1. H NMR spectra of compounds 1a and 2a dissolved in
DMSO-d6 at 500 MHz.
between the carboxyl-group and rhodamine scaffold. The
chemical shifts for the two rhodaminyl/vinylic CH protons were
assigned at 6.0 ppm. These two protons (H-8 and H-9) are
equivalent. The CO2H proton on the aryl ring of compound 1a
or 2a appeared at 13.22 ppm as a singlet. The chemical shift
resonances and coupling patterns for the aliphatic protons of
compounds 1a and 2a are almost identical. Details are described
in the Supporting Information section.
) 583 nm (ε ) 4.1 × 104 M-1 cm-1), λemit ) 606 nm (φ )
0.92)] demonstrated similar photophysical properties. Also, their
absorption and emission maxima are essentially identical with
those of their conjugates 11 [λexit ) 581 nm (ε ) 3.6 × 104
M-1 cm-1), λemit ) 604 nm (φ ) 0.92)]. Compared with a
rhodamine dye [rhodamine B: λexit ) 554 nm (9.7 × 104 M-1
s-1), λemit ) 573 nm (φ ) 0.36)]13 that lacks such rigidity,
compounds 1, 2, or 11 exhibited red-shifted (30-40 nm)
absorption and emission maxima and higher fluorescence
quantum yields. Octamethyl substitution (1a,2a f 1b,2b)
modestly increases the molar absorptivity and quantum yields.
In summary, we report the first synthesis of 5- and
6-carboxy-X-rhodamines having multiple n-propylene or γ,γ-
dimethylpropylene bridging moieties that provide rigidity for
preventing nonradiative fluorescence deactivation processes.
Synthesis of gram quantities of these fluorescent dyes is
conducted from inexpensive starting materials. We describe
the structural analysis and methodology for activation
followed by conjugation of the dye with a molecule of
interest. These dyes and their conjugates exhibit red-shifted
absorption and emission maxima and high quantum yields
for fluorescence. The availability of a facile and inexpensive
route to these fluorophores should enable their adoption for
a broad range of applications in material sciences, biotech-
nology, and biomedical imaging.
We synthesized 4-methylbenzoylpiperazine hydrochloride
(10) using a protocol described in the Scheme 2. Briefly,
acylation of N-BOC piperazine by 4-methylbenzoyl chloride
(8) in the presence of N,N-diisoproylethylamine gave 4-(4-
methylphenylcarbonyl)piperazine-1-carboxylate (9) in 79%
yield. The 4-(4-methylphenylcarbonyl)piperazine-1-carboxy-
late (9) was then deprotected to compound 10 using hydrogen
chloride gas. This reaction proceeded in quantitative yield.
The fluorescent compound 1a or 1b was activated using
N,N,N,N-tetramethyl-O-(N-succinimidyl)uronium tetrafluo-
roborate (TSTU). The reaction was performed in the presence
of g2 equiv of base, such as triethylamine, to give the
corresponding N-hydroxysuccinimidyl ester which was gen-
erated in situ. The ester was then reacted with the amino
group of compound 10. This coupling reaction proceeded
efficiently with only a slight excess of TSTU. Air-free
techniques were used for both activation and coupling
reactions. The succinimidyl esterification was performed in
an activator vessel under an inert atmosphere, and the
activated dye was transferred via a cannula to the coupling
vessel that contained compound 10 in dimethyl sulfoxide.
The crude product was obtained by removal of solvent, and
purification was conducted by a silica gel flash column
chromatography using an isocratic elution of 35:7:1 CHCl3/
MeOH/concd NH4OH. This procedure afforded 64% yield
for compound 11. The structures were confirmed by 1H NMR
and mass spectrometry. Compound 11 is a model for targeted
small molecule-carboxy-X-rhodamine conjugates.
Acknowledgment. We are grateful to Brenda C. Crews
and Dr. Carol Rouzer of the Department of Biochemistry
for critical readings of this manuscript and to Gert-Jan
Kremers of the Department of Molecular Physiology and
Biophysics for quantum yield measurements. We gratefully
acknowledge the National Institutes of Health (U54CA105296)
for financial support.
Supporting Information Available: Complete description
of the experimental details and product characterization. This
material is available free of charge via the Internet at
http://pubs.acs.org.
The rigid rhodamine dyes 1a,b and 2a,b exhibited strong
fluorescence properties in aqueous buffer. Compounds 1a,2a
[1a: λexit ) 580 nm (ε ) 3.6 × 104 M-1 cm-1), λemit ) 604 nm
(φ ) 0.94); 2a: λexit ) 581 nm (ε ) 3.6 × 104 M-1 cm-1),
λemit ) 605 nm (φ ) 0.96)] and 1b,2b [1b: λexit ) 582 nm (ε
) 4.1 × 104 M-1 cm-1), λemit ) 605 nm (φ ) 0.91); 2b: λexit
OL801904K
(13) Nguyen, T.; Francis, M. B. Org. Lett. 2003, 5, 3245.
Org. Lett., Vol. 10, No. 21, 2008
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