Practical synthetic route to functionalized rhodamine dyes

Article abstract of DOI:10.1021/ol035135z

(Matrix presented) An efficient method for the synthesis of functionalized rhodamine derivatives has been developed. Multigram quantities of these water-soluble fluorophores can be prepared from inexpensive precursors and purified without the use of chromatography. A series of protein-reactive functional groups has been installed through subsequent reactions, providing materials for biomolecule modification. For multicolor applications, a solid-phase purification strategy has been developed to afford rhodamine derivatives possessing a wide range of spectral properties.

Full text of DOI:10.1021/ol035135z

ORGANIC
LETTERS
2003
Vol. 5, No. 18
3245-3248
Practical Synthetic Route to
Functionalized Rhodamine Dyes
Trung Nguyen and Matthew B. Francis*
Department of Chemistry, UniVersity of California-Berkeley,
Berkeley, California 94720-1460, and Material Science DiVision,
Lawrence Berkeley National Labs, Berkeley, California 94720
francis@cchem.berkeley.edu
Received June 19, 2003
ABSTRACT
An efficient method for the synthesis of functionalized rhodamine derivatives has been developed. Multigram quantities of these water-soluble
fluorophores can be prepared from inexpensive precursors and purified without the use of chromatography. A series of protein-reactive
functional groups has been installed through subsequent reactions, providing materials for biomolecule modification. For multicolor applications,
a solid-phase purification strategy has been developed to afford rhodamine derivatives possessing a wide range of spectral properties.
The utility of fluorescent dyes spans many scientific disci-
plines. In biology, fluorescent probes have been used
extensively to track the locations of proteins in living cells,1
to detect specific protein functional groups,2 and to measure
intracellular ion concentrations.3 More recently, fluorescence
resonance energy transfer (FRET) has become a powerful
tool for measuring distance relationships in biomolecular
assemblies.4 In physics, fluorescent dyes are essential
components of many lasers, and in materials science,
fluorescent compounds have been used to create light-
harvesting materials5 and small-molecule sensors.6
Due to this utility, a variety of dye molecules have been
appended with reactive functional handles for further con-
jugation. However, the extremely high cost of these com-
pounds from commercial sources (typically >$30 000/g for
isomerically pure dyes) generally precludes their use in
materials science applications. In contrast, the unfunction-
alized analogues of these dyes are often available at low cost
(<$1/g), providing more economical precursors to reactive
chromophores if efficient functionalization chemistry can be
developed. For this purpose, we report herein a general
synthetic method for the direct modification of commercially
available rhodamines, affording multigram quantities of
water-soluble fluorescent dyes that can be conjugated to
virtually any substrate of interest. This method has been
carried out on several rhodamine derivatives, thus offering
(1) For a general review, see: Fluorescent and Luminescent Probes for
Biological ActiVity; Mason, W. T., Ed.; Academic Press: San Diego, 1999.
(2) (a) Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281,
269-271. (b) Adams, S. R.; Campbell, R. E.; Gross, L. A.; Martin, B. R.;
Walkup, G. K.; Yao, Y.; Llopis, J.; Tsien, R. Y. J. Am. Chem. Soc. 2002,
124, 6063. (c) Lemieux, G. A.; de Graffenried, C. L.; Bertozzi, C. R. J.
Am. Chem. Soc. 2003, 125, 4708.
(3) (a) Minta, A.; Kao, J. P. Y.; Tsien, R. Y. J. Biol. Chem. 1989, 264,
8171. (b) Minta, A.; Tsien, R. Y. J. Biol. Chem. 1989, 264, 19449.
(4) (a) Heyduk, T. Curr. Opin. Biotechnol. 2002, 13, 292. (b) Ha, T.;
Zhuang, X.; Babcock, H.; Kim, H.; Orr, J. W.; Williamson, J. R.; Bartley,
L.; Russell, R.; Herschlag, D.; Chu, S. The Study of Single Biomolecules
with Fluorescence Methods: Springer Series in Chemical Physics; Springer
Publishing: New York, 2001; Vol. 67, pp 326-337.
(5) (a) Adronov, A.; Fre´chet, J. M. J. Chem. Commun. 2000, 1701. (b)
Hecht, S.; Fre´chet, J. M. J. J. Am. Chem. Soc. 2001, 123, 6959. (c) Serin,
J. M.; Brousmiche, D. W.; Fre´chet, J. M. J. J. Am. Chem. Soc. 2002, 124,
11848.
(6) (a) Czarnik, A. W. Fluorescent Chemosensors for Ion and Molecular
Recognition; American Chemical Society: Washington, DC, 1993. (b)
Zhang, S.-W.; Swager, T. M. J. Am. Chem. Soc. 2003, 125, 3420.
10.1021/ol035135z CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/15/2003

a convenient series of compounds for multicolor fluorescence
applications.
carbodiimide coupling agents failed to produce appreciable
yields of the desired amide or led to the formation of
unidentified side products.
As the common condensation route7 used to prepare
xanthene dyes introduces a carbonyl group in the 2′ position,
this location has been targeted previously for modification
through amide bond formation.8 However, it has been
observed that secondary amides of rhodamines, such as 1,
rapidly cyclize to form nonfluorescent lactams under all but
the most acidic conditions, Scheme 1. This prevents the use
of these compounds for most biological labeling experi-
ments.9
After quenching the aluminum salts, compound 5 can be
purified using a simple workup procedure. Due to the high
water solubility of 5, lactone 4 can be extracted selectively
from basic aqueous solution with EtOAc. After acidification
and saturation with NaCl, 5 can then be extracted from the
i
aqueous layer with PrOH/CH2Cl2 (2:1). This procedure
routinely affords multigram quantities of 5 in 70% overall
yield and >95% purity (as determined by 1H NMR, MALDI-
MS, and HPLC, Figure 1). For most applications, this
Scheme 1. Cyclization of Rhodamine Amides
As a simple way to avoid this cyclization pathway, we
have developed a convenient method for the preparation of
tertiary amide 5 from rhodamine B (3).10 Exposure of lactone
4 (which is commercially available or can be prepared in
quantitative yield from 3) to 4 equiv of piperazine and 2
equiv of AlMe3 in refluxing CH2Cl2 results in clean conver-
sion to tertiary amide 5, Scheme 2.11 We have found these
Figure 1. Purification of rhodamine 5. Reversed-phase HPLC
analysis (monitored at 565 nm) of the reaction mixture (a) before
workup and (b) after extraction. HPLC and 1H NMR analysis
indicate >95% purity. (c) UV/vis spectra of 3 and 5.
Scheme 2. Synthesis of Rhodamine-Tertiary Amide
material requires no further purification. Upon conjugation,
the absorption and emission spectra 5 of are red-shifted by
10 nm, relative to compound 3, Figure 1c. This material is
highly soluble in aqueous solution and retains its fluorescence
emission under a wide range of pH conditions (Figure 2).
Derivatives
conditions to be uniquely effective for this transformation,
as numerous other procedures employing Lewis Acids and
(7) Scala-Valero, C.; Doizi, D.; Guillaumet, G. Tetrahedron Lett. 1999,
40, 4803 and references therein.
(8) (a) Adamczyk, M.; Grote, J. Bioorg. Med. Chem. Lett. 2000, 10, 1539.
(b) Adamczyk, M.; Grote, J. Synth. Commun. 2001, 31, 2681.
(9) An elegant solution to this problem has been provided by combining
fluorescein and rhodamine chromophores in a single fluorescent probe. The
resulting compound fluoresces at all pH values, although significant changes
in the emission spectra are observed. Adamczyk, M.; Grote, J. Bioorg. Med.
Chem. Lett. 2003, 13, 2327.
Figure 2. Fluorescence emission of 5 as a function of pH. Each
sample consists of 2.5 µM 5 in 5 mM phosphate buffer adjusted to
the appropriate pH with HCl or NaOH.
3246
Org. Lett., Vol. 5, No. 18, 2003

Scheme 3. Rhodamine Derivatives for Protein
Scheme 4. Synthesis of Rhodamine Derivatives for Multicolor
Functionalizationa
Labeling Applications
a Reaction conditions: (a) Succinic anhydride, Et3N, DMAP,
CH2Cl2, 72%. (b) 3-Bromopropanol, DIPEA, DMF, 71%. (c) N,N′-
Disuccinimidyl carbonate, pyridine. (d) BnNH2, pyridine/H2O, 87%
from 7. (e) Chloroacetyl chloride, pyridine, CH2Cl2, 95%. (f) NaI,
MeOH, acetone, 88%. (g) NaSAc, DMF, 64%.
materials provide attractive alternatives for the modification
of a range of protein reactive groups.
The piperazine coupling protocol described above has also
proven to be effective for other rhodamine substrates. We
have found that both rhodamine 6G (13) and rhodamine 101
(15) react cleanly under these conditions to afford piperazine
derivatives 14 and 16, respectively, Scheme 4. However,
these reactions generally proceed with lower levels of
conversion, and although the starting materials for these
reactions are similarly inexpensive, the unreacted dyes are
difficult to remove. Extractions have been ineffective for the
purification of these derivatives due to solubility changes of
the dyes, and while silica gel chromatography can be used
to isolate these compounds, the high polarity of the dyes
renders this process difficult and low yielding.
As an alternative purification strategy, we have found that
the piperazine derivatives can be isolated using a solid-phase
capture strategy. After the aluminum salts are quenched, the
reaction mixtures are exposed to nitrophenyl carbonate-
functionalized12 Wang resin (polystyrene functionalized with
4-benzyloxybenzyl alcohol), Scheme 4. As shown in Figure
3a,b, this exposure selectively removes the piperazine
derivative from solution. After thorough rinsing, the desired
chromophore can be released from the dark-red beads
through exposure to a TFA/CH2Cl2 (1:9) mixture. HPLC
The secondary amine of 5 can be further elaborated to
provide common functional groups used in bioconjugation
reactions. For lysine modification, acid 6 can be prepared
through exposure to succinic anhydride, NEt3, and DMAP,
Scheme 3. A variety of functionality can be introduced
through alkylation of the amine, as exemplified by 7. The
hydroxyl group of this derivative can be transformed readily
to NHS-carbonate 8, which has been coupled to both small-
molecule amines and proteins (see Supporting Information
for a protein labeling protocol). Cysteine-reactive compounds
can be prepared through acylation of the amine with
chloroacetyl chloride, followed by conversion to iodoacet-
amide 11 using Finkelstein conditions. Chloroacetamide 10
can also be converted to thioacetate 12 using a similar
protocol. All of these derivatives can be obtained as pure
compounds without the use of chromatography. The low cost,
high purity, and excellent spectral properties of these
(10) Successful formation of rhodamine tertiary amides using TBTU as
a coupling agent has been reported: (a) Torneiro, M.; Still, W. C. J. Am.
Chem. Soc. 1995, 117, 5887. (b) Torneiro, M.; Still, W. C. Tetrahedron
1997, 53, 8739. Other approaches have also been described in the patent
literature; see: (c) Haugland, R. P.; Singer, V. L.; Yue, S. T. US Patent
6,399,392. Mayer, U.; Oberlinner, A. US Patent 4,647,675. For an example
using fluoresceins, see: (d) Gao, J.; Wang, P.; Giese, R. Anal. Chem. 2002,
74, 6397.
(11) Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett. 1977, 48,
4171.
(12) Francis, M. B.; Jacobsen, E. N. Angew. Chem., Int. Ed. 1999, 38,
937.
Org. Lett., Vol. 5, No. 18, 2003
3247

Figure 4. Spectral properties of 5, 14, and 16. Quantum yields
were determined as described in Supporting Information.
of 14 resembles that of unmodified rhodamine 6G, but 30
and 38% reductions are observed for 5 and 16, respectively.13
Nonetheless, it is anticipated that the high degree of spectral
overlap between 5, 14, and 16 will provide useful pairs of
chromophores for FRET applications in biology and for the
construction of light-harvesting systems.
In summary, we have reported an efficient method for the
conversion of inexpensive rhodamine derivatives into readily
functionalizable chromophores for biological labeling and
materials science applications. For single-color applications,
rhodamine B derivatives can be easily prepared on a
multigram scale. Using a solid-phase purification strategy,
additional rhodamine derivatives can be prepared to access
a range of spectral properties.
Figure 3. Purification of rhodamines 14 and 16 using a resin-
capture strategy. (a) Reversed-phase HPLC analysis of the crude
reaction mixture for 13. Compound 14 can be detected at 3.3 min.
(b) HPLC analysis of the supernatant after exposure to solid-phase
resin 17. Compound 14 has been completely removed from the
solution. (c) HPLC analysis of the supernatant after exposing resin
18 to TFA/CH2Cl2 (1:9). HPLC and NMR analysis indicate >95%
purity for compound 14. (d) UV/vis spectra of 13 and 14. (e) HPLC
analysis of the supernatant after exposing resin 19 to TFA/CH2Cl2
(1:9). HPLC and NMR analysis indicate >95% purity for compound
16. (f) UV/vis spectra of 15 and 16.
Acknowledgment. The authors gratefully acknowledge
the Department of Chemistry at the University of California,
Berkeley, and the Lawrence Berkeley National Laboratory,
Materials Science Division, for financial support (U.S.
Department of Energy Contract No. DE-AC03-76SF00098).
analysis of the material obtained using this method indicates
exceptionally high purity, Figure 3c,e. While this isolation
procedure is less suited for multigram applications, it can
nonetheless provide 100-200 mg quantities of highly pure
dye per gram of resin. Through additional experiments, we
have found that piperazine derivatives 14 and 16 can be
converted to compounds for protein conjugation in a fashion
similar to compound 5.
Although all of these dyes exhibit strong fluorescence in
aqueous buffer, conjugation to the piperazine moiety alters
the optical properties in somewhat unpredictable ways.
Lower extinction coefficients were observed for 14 and 16
(Figure 4) relative to that observed for 5. The quantum yield
Supporting Information Available: Full experimental
procedures and characterization data for compounds 5-12,
14, and 16. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL035135Z
(13) See Supporting Information for a tabulated comparison of these
values to those of the parent dyes.
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Org. Lett., Vol. 5, No. 18, 2003