Facile synthesis of rhodamine esters using acetyl chloride in alcohol solution

Article abstract of DOI:10.1080/00397910600616818

The rhodamines are a highly fluorescent class of compound used in many different fields of research, from the lasing medium in dye lasers to biological stains and markers for cellular drug resistance. In this study, esters (2-7) of rhodamine 110 (1) were conveniently prepared via the addition of acetyl chloride to a solution of the free acid (1) in the appropriate alcohol. This method conferred several advantages over previous preparations, namely that for low boiling alcohols, simple evaporation of the solution afforded the ester in quantitative yield with no need for purification. For higher boiling point alcohols, a method has been developed which allows the separation of longer chain esters from the alcohol solvent. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397910600616818

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Facile Synthesis of Rhodamine Esters using
Acetyl Chloride in Alcohol Solution
Justin A. Ross a , Benjamin P. Ross b , Halina Rubinsztein‐Dunlop a & Ross P.
McGeary b c
a Centre for Biophotonics and Laser Science, School of Physical Sciences,
University of Queensland, Brisbane, Queensland, Australia
b School of Molecular and Microbial Sciences, University of Queensland,
Brisbane, Queensland, Australia
c School of Molecular and Microbial Sciences, and School of Pharmacy,
University of Queensland, Brisbane, Queensland, Australia
Version of record first published: 22 Aug 2006
To cite this article: Justin A. Ross, Benjamin P. Ross, Halina Rubinsztein‐Dunlop & Ross P. McGeary (2006):
Facile Synthesis of Rhodamine Esters using Acetyl Chloride in Alcohol Solution, Synthetic Communications: An
International Journal for Rapid Communication of Synthetic Organic Chemistry, 36:12, 1745-1750
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Synthetic Communicationsw, 36: 1745–1750, 2006
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910600616818
Facile Synthesis of Rhodamine Esters using
Acetyl Chloride in Alcohol Solution
Justin A. Ross
Centre for Biophotonics and Laser Science, School of Physical Sciences,
University of Queensland, Brisbane, Queensland, Australia
Benjamin P. Ross
School of Molecular and Microbial Sciences, University of Queensland,
Brisbane, Queensland, Australia
Halina Rubinsztein-Dunlop
Centre for Biophotonics and Laser Science, School of Physical Sciences,
University of Queensland, Brisbane, Queensland, Australia
Ross P. McGeary
School of Molecular and Microbial Sciences, and School of Pharmacy,
University of Queensland, Brisbane, Queensland, Australia
Abstract: The rhodamines are a highly fluorescent class of compound used in many
different fields of research, from the lasing medium in dye lasers to biological stains
and markers for cellular drug resistance. In this study, esters (2–7) of rhodamine
110 (1) were conveniently prepared via the addition of acetyl chloride to a solution
of the free acid (1) in the appropriate alcohol. This method conferred several advan-
tages over previous preparations, namely that for low boiling alcohols, simple evapor-
ation of the solution afforded the ester in quantitative yield with no need for
purification. For higher boiling point alcohols, a method has been developed which
allows the separation of longer chain esters from the alcohol solvent.
Keywords: Rhodamine, esterification, acetyl chloride, lipophilicity
Received in the USA December 19, 2005
Address correspondence to Ross P. McGeary, School of Molecular and Microbial
Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
Tel: þ 61-7-3365-3955; Fax: þ61-7-3346-3249;. E-mail: r.mcgeary@uq.edu.au
1745

1746
J. A. Ross et al.
The rhodamines are a highly fluorescent class of compound used in many
different fields of research, from the lasing medium in dye lasers to biological
stains and markers for cellular drug resistance.[1–3] Rhodamine 123 (2), the
methyl ester of rhodamine 110 (1), is a fluorescent lipophilic compound[4]
with a positive charge delocalized across the xanthene rings. It is commonly
[5]
used as a biological marker for mitochondria
and as a substrate for
multidrug resistance transporter (MDT) assays.[6–9] The use of fluorescent
substrates as MDT markers forms the basis of a convenient method of
tracking the transport of substrates. We have synthesized a series of esters
(2–7) of rhodamine 110 with increasing lipophilicity. Thus a rhodamine
ester with similar lipophilic properties to a drug of interest could be chosen
to mimic the drug in MDT assays. In this article we report the facile
method by which esters 2–7 were prepared. The method conferred several
advantages over previous preparations, namely that for low boiling
alcohols, simple evaporation of the solution afforded the ester in quantitative
yield with no need for purification.
Pal and coworkers synthesized the methyl (2) and n-butyl (5) esters of
rhodamine 110 (1) by bubbling hydrogen chloride through a solution of
rhodamine 110 (1) in methanol or n-butanol respectively.[10] The reaction
mixtures were refluxed, and then the solvent was evaporated in vacuo to
afford the crude ester, which was purified by column chromatography using
silica gel. The disadvantages of this method are the requirement for gaseous
hydrogen chloride and the need for two silica flash columns to purify the product.
Ramos and coworkers prepared a variety of rhodamine esters, including
the methyl (2) and ethyl (3) esters of rhodamine 110 (1), under Fischer con-
ditions.[11] The rhodamine was dissolved in the appropriate alcohol solution
containing 3% sulfuric acid, and this solution was heated with stirring at
508C for 1–6 days. The solution was concentrated, and the ester was precipi-
tated as a bromide, iodide, or perchlorate salt by treatment with a 14% aqueous
solution of potassium iodide, potassium bromide, or sodium perchlorate
respectively. In our hands, the synthesis of rhodamine esters using this
method proved troublesome. Precipitation of the esters was not suitable for
longer-chain alcohols because the aqueous and alcoholic phases were immis-
cible. For shorter-chain alcohols the aqueous and alcoholic phases were
miscible; however, the addition of an aqueous phase to the reaction in
the presence of strong acid resulted in some hydrolysis of the ester to the
free carboxylic acid, which therefore contaminated the product.
We have developed an improved method for the preparation of rhodamine
esters that utilizes anhydrous hydrogen chloride generated in situ by the
addition of acetyl chloride (AcCl) to a mixture of the rhodamine free acid
(1) in the appropriate alcohol (Scheme 1). After heating the stirred solution
at 508C for 2–4 days, evaporation of the solution in vacuo afforded the
rhodamine esters (2–6) in quantitative yield without the need for purification.
For the synthesis of rhodamine 110 octyl ester (7), the solvent (n-octanol) was
insufficiently volatile (bp 1968C) for complete removal in vacuo, and therefore

Facile Synthesis of Rhodamine Esters
1747
Scheme 1.
7 was isolated by flash column chromatography using neutral alumina. The
use of silica for flash chromatography caused streaking of the product and
gave a poor recovery of the ester.
In summary, we have developed a facile method for the preparation of
rhodamine esters that utilizes anhydrous hydrogen chloride generated in situ
by the addition of AcCl to a mixture of the rhodamine free acid in the
appropriate alcohol. The method confers several advantages over previous
preparations, namely that for low boiling alcohols, simple evaporation of
the solvent affords the ester in quantitative yield with no need for purification.
The synthesis of compound 7 illustrates a convenient method that can be used
to isolate rhodamine esters from alcohols with high boiling points.
EXPERIMENTAL
Commercial reagents were used without further purification. Rhodamine
110 (1) was purchased from Fluka (Buchs, Switzerland) and acetyl chloride
was purchased from Aldrich Chemical Company (Milwaukee, WI). Thin-
layer chromatography (TLC) was performed on silica-gel 60 F254 aluminum
sheets (Merck, Darmstadt, Germany). The solvent for TLC was n-butanol–
water–ethanol, 9:2:1. Aluminum oxide 90 active neutral, 70–230 mesh
(Merck, Darmstadt, Germany) was used for flash column chromatography.
Reverse-phase High Performance Liquid Chromatography (HPLC) purifi-
cation and analysis was carried out with a Shimadzu HPLC system (SCL-
10A VP controller, LC-10AT VP pump, DGU-14A degasser, FCV-10AL
VP solvent mixer and a SPD-10A VP UV detector) using an analytical
(LiChrospherw 100, RP-18e, 250 ꢀ 4 mm, 5 mm) or semipreparative column
(LiChrospherw 100, RP-18e, 250 ꢀ 10 mm, 10 mm). The mobile phase was
solvent A: 0.025 M ammonium acetate in water; solvent B: 10% solvent A
and 90% acetonitrile. For analytical HPLC, a linear gradient of 30–100% B
over 70 min was employed at a flow rate of 1 mL/min. For semipreparative
HPLC, a linear gradient of 20–100% B over 80 min was used with a flow

1748
J. A. Ross et al.
rate of 6 mL/min. The eluent was monitored at 510 nm [for detection of the
product (7) and rhodamine-based impurities] and at 254 nm (for detection of
nonrhodamine impurities).[10] Mass spectra (ES-MS) were recorded on a
Perkin-Elmer Sciex API 3000 mass spectrometer operating in positive-ion
electrospray mode using 0.1% formic acid in 90% acetonitrile/10% water.
High-resolution mass spectra were run on a Finnigan MAT 900 XL-Trap
instrument with a Finnigan API III electrospray source using methanol as
solvent and polypropylene glycol as reference for accurate mass data
1
acquired by electric sector scan (HRMS). H NMR spectra were recorded
on a Bruker AV-500 instrument (500 MHz), and 13C NMR spectra were
recorded on a Bruker AV-300 instrument (75 MHz). The following abbrevi-
ations were used to indicate the peak multiplicity: s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet, m ¼ multiplet, and br ¼ broad.
General Procedure for the Preparation of Rhodamine Esters
The appropriate alcohol (7.5 mL) was added to rhodamine 110 (1, 30 mg,
0.082 mmol), and the mixture was stirred under an atmosphere of argon.
Acetyl chloride (375 mL, 5.27 mmol) was added dropwise, and the solution
was heated at 508C for 2–4 days. When the reaction was 70–90% complete
[analysis by TLC (n-butanol–water–ethanol 9:2:1)], additional acetyl
chloride (75 mL, 1.05 mmol) was added to drive the reaction to completion.
When TLC indicated no remaining starting material, the solution was evapor-
ated in vacuo (water bath temperature ,508C to avoid degradation of the
product) to afford the rhodamine ester (2–6), a hygroscopic amorphous
solid, in quantitative yield as determined by mass.
Data
Rhodamine 110 methyl ester (rhodamine 123) (2): Analytical HPLC
tR ¼ 17.6 min, purity 96% (510 nm), 95% (254 nm). TLC Rf 0.49; ES-MS,
m/z: 345 [M þ H]þ; 1H NMR (500 MHz, d6-DMSO) d 8.24 (l H, dd,
J ¼ 8.1 and 1.4 Hz), 8.10 (4H, br s), 7.90 (l H, dt, J ¼ 7.6 and 1.4 Hz), 7.82
(l H, dt, J ¼ 7.7 and 1.2 Hz), 7.49 (l H, dd, J ¼ 7.7 and 1.4 Hz), 6.96
(2 H, d, J ¼ 9.4 Hz), 6.84–6.82 (4 H, m), 3.57 (3 H, s); 13C NMR (75 MHz,
d6-DMSO) d 165.0, 159.4, 158.7, 157.3, 133.5, 133.1, 131.5, 130.7, 130.4,
130.3, 129.3, 116.9, 112.8, 97.0, 52.3; UV-visible (H2O): lmax 500 nm;
HRMS calcd. for [M þ H]þ 345.1239; found 345.1254.
Rhodamine 110 ethyl ester (3): Analytical HPLC tR ¼ 21.2 min, purity 97%
(510 nm), 96% (254 nm). TLC Rf 0.51; ES-MS, m/z: 359 [M þ H]þ; 1H NMR
(500 MHz, d6-DMSO) d 8.22 (1 H, dd, J ¼ 7.9 and 1.1 Hz), 8.14 (4 H, br s),
7.89 (1 H, dt, J ¼ 7.5 and 1.4 Hz), 7.82 (1 H, dt, J ¼ 7.7 and 1.4 Hz), 7.49

Facile Synthesis of Rhodamine Esters
1749
(1 H, dd, J ¼ 7.6 and 1.2 Hz), 6.97 (2 H, d, J ¼ 9.5 Hz), 6.86–6.84 (4 H, m),
3.94 (2 H, q, J ¼ 7.1 Hz), 0.84 (3 H, t, J ¼ 7.1 Hz); 13C NMR (75 MHz, d6-
DMSO) d 164.7, 159.4, 158.5, 157.3, 133.1, 132.9, 131.5, 130.7, 130.3,
129.7, 116.9, 112.9, 97.0, 60.9, 13.3; UV-visible (H2O): lmax 501 nm;
HRMS calcd. for [M þ H]þ 359.1381; found 359.1396.
Rhodamine 110 propyl ester (4): Analytical HPLC tR ¼ 25.6min, purity 99%
(510 nm), 99% (254nm). TLC Rf 0.53; ES-MS, m/z: 373 [M þ H]þ; 1H NMR
(500 MHz, d6-DMSO) d 8.23 (1H, dd, J ¼ 7.9 and 1.1 Hz), 8.11 (4 H, br s), 7.89
(1 H, dt, J ¼ 7.5 and 1.3 Hz), 7.82 (1 H, dt, J ¼ 7.7 and 1.3 Hz), 7.49 (1 H, dd,
J ¼ 7.5 and 1.1 Hz), 6.98 (2H, d, J ¼ 9.0 Hz), 6.86–6.82 (4 H, m), 3.86 (2 H, t,
J ¼ 6.4 Hz), 1.23 (2 H, sextet, J ¼ 6.9 Hz), 0.59(3 H, t, J ¼ 7.4 Hz); 13C NMR
(75 MHz, d6-DMSO) d 165.0, 159.5, 158.6, 157.4, 133.1, 131.6, 130.8, 130.4,
129.8, 117.0, 112.9, 97.0, 66.7, 21.1, 10.0; UV-visible (H2O): lmax 501 nm;
HRMS calcd. for [M þ H]þ 373.1552; found 373.1550.
Rhodamine 110 butyl ester (5): Analytical HPLC tR ¼ 29.7 min, purity 99%
(510 nm), 99% (254 nm). TLC Rf 0.56; ES-MS, m/z: 387 [M þ H]þ; 1H NMR
(500 MHz, d6-DMSO) d 8.22 (1 H, dd, J ¼ 7.8 and 1.1 Hz), 8.15 (4 H, br s),
7.88 (1 H, dt, J ¼ 7.5 and 1.3 Hz), 7.82 (l H, dt, J ¼ 7.7 and 1.3 Hz), 7.49
(l H, dd, J ¼ 7.5 and 0.98 Hz), 6.97 (2 H, d, J ¼ 9.2 Hz), 6.86–6.84
(4 H, m), 3.89 (2 H, t, J ¼ 6.2 Hz), 1.15 (2 H, m), 0.91 (2 H, m), 0.69 (3 H, t,
J ¼ 7.3 Hz); 13C NMR (75 MHz, d6-DMSO) d 165.1, 159.5, 158.4, 157.3,
133.0, 132.9, 131.6, 130.9, 130.4, 129.8, 117.0, 112.9, 97.0, 65.0, 29.8,
18.5, 13.4; UV-visible (H2O): lmax 502 nm; HRMS calcd. for [M þ H]þ
387.1709; found 387.1715.
Rhodamine 110 pentyl ester (6): Analytical HPLC tR ¼ 34.6 min, purity
96% (510 nm), 95% (254 nm). TLC Rf 0.59; ES-MS, m/z: 401 [M þ H]þ;
1H NMR (500 MHz, d6-DMSO) d 8.22 (1 H, dd, J ¼ 7.8 and 1.1 Hz), 8.16
(4 H, br s), 7.88 (1 H, dt, J ¼ 7.5 and 1.3 Hz), 7.82 (l H, dt, J ¼ 7.7 and
1.2 Hz), 7.480 (1 H, dd, J ¼ 7.5 and 0.98 Hz), 6.98 (2 H, d, J ¼ 9.0 Hz),
6.87–6.84 (4 H, m), 3.87 (2 H, t, J ¼ 6.2 Hz), 1.14 (2 H, m), 1.07 (2 H, m),
0.84 (2 H, m), 0.74 (3 H, t, J ¼ 7.4 Hz); 13C NMR (75 MHz, d6-DMSO)
d 165.0, 159.5, 158.3, 157.3, 132.9, 132.8, 131.5, 130.8, 130.3, 130.3,
129.8, 116.9, 112.9, 97.0, 65.2, 30.6, 27.4, 21.7, 13.6; UV-visible (H2O):
lmax 502 nm; HRMS calcd. for [M þ H]þ 401.1865; found 401.1871.
Rhodamine 110 octyl ester (7). Compound 7 was prepared by following the
general procedure except the solvent (n-octanol) was not sufficiently volatile
(bp 1968C) for evaporation in vacuo. Instead, compound 7 was isolated by
flash column chromatography using neutral alumina. The reaction mixture
was loaded onto the column, and the n-octanol was eluted with ethyl acetate
and then 5% methanol in ethyl acetate. Further elution with methanol
afforded the product (7), which was contaminated with a small amount of n-
octanol. The molar ratio of rhodamine 110 octyl ester (7): n-octanol was ꢀ4:l

1750
J. A. Ross et al.
by 1H NMR, and the yield was 82% (corrected for n-octanol). A sample of this
material was further purified by semipreparative reverse-phase HPLC to afford
the product (7), an amorphous solid: analytical HPLC tR ¼ 49.1min, purity 98%
(510nm), 96% (254nm). TLC Rf 0.62; ES-MS, m/z: 443 [M þ H]þ; 1H NMR
(500MHz, d6-DMSO) d 8.21 (lH, dd, J ¼ 7.9 and 1.2 Hz) overlapping with
8:21 (4 H, br s), 7.87 (lH, dt, J ¼ 7.5 and 1.4 Hz), 7.81 (1 H, dt, J ¼ 7.7 and
1.3 Hz), 7.47 (1 H, dd, J ¼ 7.6 and 1.2 Hz), 6.94 (2 H, d, J ¼ 9.1 Hz), 6.84–
6.80 (4 H, m), 3.88 (2 H, t, J ¼ 6.1 Hz), 1.28–1.01 (12 H, m), 0.86 (3 H, t,
J ¼ 7.3 Hz); UV-visible (H2O): lmax 503 nm; HRMS calcd. for [M þ H]þ
443.2335; found 443.2329.
ACKNOWLEDGMENTS
We thank Graham MacFarlane (School of Molecular and Microbial Sciences,
University of Queensland) for accurate mass measurements. This work was
supported by funds from the Centre for Biophotonics and Laser Science,
School of Physical Sciences, University of Queensland.
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