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CRITICAL REVIEW www.rsc.org/csr | Chemical Society Reviews

Synthesis and applications of Rhodamine derivatives

as fluorescent probes
Mariana Beija, Carlos A. M. Afonso and José M. G. Martinho
Received 26th January 2009
First published as an Advance Article on the web 27th April 2009
DOI: 10.1039/b901612k

Rhodamine dyes are widely used as fluorescent probes owing to their high absorption coefficient
and broad fluorescence in the visible region of electromagnetic spectrum, high fluorescence
quantum yield and photostability. A great interest in the development of new synthetic
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procedures for preparation of Rhodamine derivatives has arisen in recent years because for most
applications the probe must be covalently linked to another (bio)molecule or surface. In this
critical review the strategies for modification of Rhodamine dyes and a discussion on the variety
of applications of these new derivatives as fluorescent probes are given (108 references).

Introduction
Rhodamine dyes are fluorophores that belong to the family of
xanthenes along with fluorescein and eosin dyes. The general
structures of xanthene chromophore and rhodamine dyes are
represented in Fig. 1.
Due to their excellent photostability and photophysical
properties, rhodamines are used as laser dyes,1,2 fluorescence
standards (for quantum yield3 and polarization4), pigments
and as fluorescent probes to characterize the surface of Fig. 1 Molecular structures of xanthene (A) and rhodamine dyes (B).
polymer nanoparticles,5,6 fluidity of lipid membranes,7 as well
as in the detection of polymer-bioconjugates,8 studies of
Rhodamine derivatives have also been employed as
adsorption of oligonucleotides on latexes,9,10 studies of structure
molecular switches,17 as a thermometer,18,19 for surface
and dynamics of micelles,11 single-molecule imaging12,13 and
modification of a virus20 and particularly as chemosensors
imaging in living cells.14–16
used either in vitro as in vivo in detection of Hg(II), Cu(II),
Fe(III), Cr(III), thiols among other analytes.21–32 Recently,
Centro de Quı´mica-Fı´sica Molecular and IN–Institute of Nanoscience Gonçalves reviewed the fluorescent labelling of biomolecules
and Nanotechnology, Instituto Superior Te´cnico, 1049-001, Lisboa,
Portugal. E-mail: marianabeija@ist.utl.pt, carlosafonso@ist.utl.pt, using organic probes, highlighting the importance of
jgmartinho@ist.utl.pt; Fax: +351 218 464 455 rhodamine derivatives for that application.33

Mariana Beija was born in Carlos A. M. Afonso gradu-

São Paulo (Brazil) in 1981.
She studied Chemistry in
Instituto Superior Te´cnico
(Technical University of
Lisbon, Portugal), where she
received a school merit award
in 2000. In 2004, she started
her PhD in Chemistry jointly
supervised by Prof. Jose´ M. G.
Martinho, in Centro de
Quı´mica-Fı´sica Molecular
(Instituto Superior Te´cnico,
Lisbon, Portugal), and
Mariana Beija Dr Marie-The´re`se Charreyre,
in Unite´ Mixte CNRS-
bioMe´rieux (Lyon, France). Her doctoral research consisted
of the synthesis of novel dye-labelled thermoresponsive block
copolymers by RAFT polymerization, involving the synthesis of
rhodamine-derived RAFT agents.
ated from University of
Coimbra (1984) and received
his PhD in 1990 from New
University of Lisbon. He
worked for one year as post-
doctoral fellow at the Imperial
College of Science Technology
and Medicine under the
supervision of Prof. W. B.
Motherwell (1990) and one
more academic year of
sabbatical leave (1997/98) at
the University of Bath, UK
Carlos A. M. Afonso (Prof. J. Williams) and at
the University of Toronto
(Professor R. Batey). In 2004 he moved to Instituto Superior
Te´cnico as associate professor and in 2008 received his Agregação.
His research focus is mainly on the development of more
sustainable methodologies in asymmetric organic transformations.

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c The Royal Society of Chemistry 2009

Although for some of those applications the dye is used in
its free form, for most of them the probe must be attached to
another molecule (polymer, oligonucleotide, biomolecule, etc.)
or surface. In order to obtain these rhodamine conjugates,
usually a reaction between a nucleophilic functionality in the
molecule of interest and a 4 0 - or 5 0 -activated rhodamine
derivative [in Fig. 1(B): G = activated ester, an acyl chloride,
a sulfonyl chloride or a isothiocyanate functionality] is
carried out. Several of these activated dyes are commercially
available. However, either they are found as a mixture of
isomers or isomerically pure dyes have extremely high costs
(more than 40 000 h/g), which is prohibitive when there is a
need for several grams of product and when further synthetic
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steps will take place.
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Hence, in order to obtain derivatives of a Rhodamine dye in
a large amount, it is necessary to synthesise it. Aiming to do
that, the condensation reaction that leads to formation
of Rhodamine dyes has to be carried out using previously
functionalized reagents. Another possibility is to modify less
expensive unfunctionalized commercially available rhodamines.
Herein, the synthetic strategies for functionalization of
Rhodamine dyes will be reviewed and the reasons for the
choice of a particular synthetic pathway will be discussed. In
order to contextualize the potential applications, a brief
introduction on the photophysics of Rhodamine dyes is also
included.
Photophysical properties
Depending on the substituents R1, R2, R3, R4, G and even on
the counter ion X (usually Cl, Br or ClO4),1 the dye will
present different photophysical properties in solution, such as
absorption and emission maxima (lmax max
abs , lem , fluorescence
lifetime (t) and fluorescence quantum yield (f).
The major differences in the photophysical properties of
Rhodamines are explained by the non-radiative deactivation
by internal conversion. The internal conversion has both
activated and non-activated components.34 In rhodamine dyes
which carry none, only one alkyl substituent at each nitrogen
J. M. G. Martinho, born in
Portugal, in 1950, received
his PhD in Chemical
Engineering from Instituto
Superior Te´cnico (Technical
University of Lisbon, Portugal)
in 1982. In 1985, he joined
Prof. M. A. Winnik’s research
group as a postdoctoral fellow
and in 1993 he was invited
Professor at the Ontario
Center of Materials Research
of the University of Toronto.
He is Full Professor of
José M. G. Martinho Chemistry and head of the
research unit, Centro de
Quı´mica-Fı´sica Molecular, at IST (Lisbon). His major research
interests are in the areas of polymers and colloids, photo-
chemistry and photophysics and fast chemical kinetics.
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c The Royal Society of Chemistry 2009
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(these latter derivatives normally bear an alkyl group as R4) or
when the amino groups are rigidised, the activated process is
absent and the quantum yield of these dyes is very high and
independent of temperature.34,35 In opposition, rhodamine
dyes with two alkyl substituents at each nitrogen show
activated internal conversion and consequently the quantum
yield and fluorescence lifetime vary with temperature.
The activated process seems to be associated with a non-
fluorescent twisted intramolecular charge-transfer (TICT)36
state characterized by an electron transfer from the amino
groups to the xanthene ring followed by a rotation between
them.37 The energy of the TICT state is higher than the energy
of the first excited singlet state for the dyes without activated
processes and lower for those with activated internal
conversion. Then, the activated energy dissipation is explained
by the population of the TICT state that is non-emissive and
deactivates quickly to the ground state.38 The non-activated
process involves energy dissipation by C–H and N–H
streching modes coupled with high frequency vibration modes
of the solvent. The N–H vibration modes are found to be very
effective in the dissipation of the electronic energy to hydroxylic
solvents.1,2 Rhodamine 101 (Rho 101) and Rhodamine B
(Rho B) are among the most used rhodamines and present
an interesting behaviour with pH and solvent polarity (Fig. 2).
In acidic solutions, the carboxyl group is protonated and the
rhodamine dye is found in its cationic form. However, in basic
solution, dissociation occurs and the rhodamine dye is
converted into a zwitterion. Although both the cationic and
zwitterionic forms share the same chromophore, the negative
charge has an inductive effect on the central carbon atom of
xanthene chromophore, leading to a hypsochromic shift
of both absorption and fluorescence maxima and a slight
reduction of the extinction coefficient at lmax abs . The differences
in the specific dye-solvent interaction were also invoked to
explain the small differences in quantum yield and lifetime for
the cationic and zwitterionic forms.39 In less polar organic
solvents, the zwitterionic dye undergoes a reversible
conversion to a colorless lactone due to the interruption of
p–conjugation of the chromophore. Consequently, absorption
of lactones of rhodamine occurs in the UV spectral region and
the fluorescence quantum yield and lifetime are very low.1,40,41
Table 1 summarizes known photophysical parameters of all
forms of Rho 101 and Rho B. The very low quantum yield and
Fig. 2 Molecular structures of three forms of Rho 101 and Rho B in
equilibrium.
Chem. Soc. Rev., 2009, 38, 2410–2433 | 2411

Table 1 Photophysical parameters of Rhodamine dyes
Rho 101
42 41
Cationic Zwitterionic
lmax
abs /nm (solvent) 574 (EtOH) 568 (EtOH)
lmax
abs /nm (solvent) 599 (EtOH) 590 (EtOH)
eabs/105 M1 cm1 1.10 (EtOH) 0.95 (EtOH)
f 0.89 (EtOH) 0.98 (EtOH)
t/ns 4.34 (EtOH) 4.37 (EtOH)
lifetime of lactones of Rhodamine B and Rhodamine 101 were
attributed to an electron-transfer reaction in the excited state
that generates a charge transfer excited state and the singlet
and triplet states of the dye in the zwitterionic form.41
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Modification of rhodamine dyes for use as
fluorescent probes
Three types of modification of Rhodamine derivatives can be
envisioned: modification of the amino groups of xanthene
moiety (positions 3 and 6); modification of the carboxyphenyl
ring at positions 4 0 and/or 5 0 or modification of the carboxylic
acid group (position 2 0 ).
Although in some cases rhodamine derivatives are prepared
directly through a condensation reaction using previously
functionalized reactants, most of the examples presuppose
modification of commercially available Rhodamine dyes. As
one can see in Table 2, Rho B and Rho 6G are the less
expensive dyes of this family and consequently they have been
the most employed for further applications.
In the following sections, the main developments for each
type of modification is reviewed.
Modification of the amino groups of xanthene moiety
(positions 3 and 6)
Usually, functionalization of the amino groups of xanthene
moiety of Rhodamine dyes can lead to severe changes in their
photophysical properties, causing in some cases even the total
loss of fluorescence. This property was found useful for the
synthesis of latent fluorophores (pro-fluorophores), with an
advantage over ‘‘conventional’’ fluorophores whose bulk
fluorescence can obscure valuable information in biological
experiments. Thus, in recent years, several groups have made
an effort to design new rhodamine derivatives to be used as
latent fluorophores in studies of enzymatic activity
(serine protease,44–46 caspase,47–51 esterase,52–54 DT diaphorase55),
of organometallic catalysis in living cells,56 in biomedical
imaging53,54 or in in vivo detection of small molecules
(thiols).27,57
Among all rhodamines, Rhodamine 110 (Rho 110) is the
most used for this purpose because it carries non-alkylated
and, consequently, more reactive amino groups. Generally,
they are modified either by reaction with an acyl chloride
(or chloroformate) or with a carboxylic acid using a carbodiimide
as a coupling agent. Both symmetric and asymmetric
modification of Rho 110 can be performed using this
procedure (Scheme 1). Depending on the application, different
types of substituents have been introduced, however variable
reaction yields were obtained in each case (vide Table 2).
2412 | Chem. Soc. Rev., 2009, 38, 2410–2433
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Rho B
41
Lactone Cationic40 Zwitterionic41 Lactone43
317 (THF) 553 (EtOH) 543 (EtOH) 311 (Et2O)
522 (THF) 572 (EtOH) 563 (EtOH) 442 (Et2O)
0.12 (THF) 1.17 (EtOH) 1.11 (EtOH) 0.16 (Et2O)
0.006 (THF) 0.53 (EtOH) 0.70 (EtOH) 0.022 (Et2O)
0.13 (THF) 2.42 (EtOH) 2.88 (EtOH) 5.8 (Et2O)
It should be noticed that unfavourable steric interactions
caused by the modification of both amino groups enhance the
nucleophilicity of the phenolic oxygen and lead to lactone
formation. As a result, the conjugation system of the
chromophore is disrupted and these rhodamine derivatives
are non-fluorescent.
In the first reported examples, Rho 110 was modified in
order to prepare synthetic fluorogenic amide substrates for
assays of serine proteases and caspases. Two important
criteria must be fulfilled by a pro-fluorophore that will be
used as a synthetic substrate: (1) easy detectability; (2) high
reactivity of the bond undergoing cleavage. Rho 110 appeared
as an excellent candidate since it is highly fluorescent at the
same time as the uncleaved substrate is nonfluorescent
(low background signal), it absorbs and emits in the visible
range of electromagnetic spectrum and it is a very good
leaving group because cleavage of the amide bond is accom-
panied by a large increase in the degree of conjugation
(and thus a large increase in stability). Hence, several bis-
substituted peptide derivatives of Rho 110 were synthesised
and used in enzymatic activity studies (Table 3, entries 1–3,
10–11 and 16).44,45,47,58 After this pioneering work in the
synthesis of pro-fluorophore compounds, several bis-
substituted peptide derivatives of Rho 110 began to be traded
by some chemical suppliers such as Aldrich or Molecular
Probes. Nowadays more than 10 different peptide sequences
are commercially available, with prices usually higher than
37 000 h/g. Nevertheless, it was very soon remarked that the
presence of two steps for the enzymatic hydrolysis process was
limiting the linear dynamic range of this substrate.47 In fact,
the first hydrolysis product becomes fluorescent; however
maximal fluorescence signal can only be achieved after
cleavage of both peptide groups. Thus, new rhodamine-based
substrates with only one cleavable amide bond were
synthesised. In addition, the other amino group was modified
with groups that could bind the dye onto the cell surface46
(Table 3, entries 4–9) or that could enhance cell penetration
and retention48–50 (Table 3, entries 11–15). Although mono-
substituted Rho 110 is less fluorescent than the parent dye, the
capping with an amide, carbamate or urea can preserve much
of its fluorescence, since its zwitterionic form remains more
stable than the spirolactone one.49,51
In recent years, a slightly different approach of this strategy
has been developed, in which the fluorescence of modified
rhodamine derivative is unmasked by a user-designated
chemical reaction. Chandran et al. first presented the use of
‘‘trimethyl lock’’ to mask Rho 110 fluorescence. The trimethyl
lock is an o-hydroxycinnamic acid derivative in which
unfavourable steric interactions between three methyl groups
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Table 2 Commercially available Rhodamines
Structure
Rho B
Rho 6G
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Rho 19
Rho 101
Rho 110
Rho 116
Rho 123
a
An average for common suppliers: Acros Organics, Aldrich, Alfa
Aesar, Fluka, Radiant dyes laser, Sigma.
induce rapid lactonization to form a hydrocoumarin. Thus, a
highly stable bis(acetylated trimethyl lock)Rho 110 pro-
fluorophore was synthesised and it was shown that Rho 110
fluorescence was unmasked by esterase catalysis in vitro or
in cellulo (Scheme 2) (Table 3, entry 18).59 Similarly, Yatzeck
et al. prepared an asymmetric trimethyl lock fluorogenic probe
to assay cytochrome P450 activity (Table 3, entry 21).60
Similar approaches were employed in the aim to synthesise
latent Rho 110 derivatives for biomolecular imaging (Table 3,
entries 19, 20 and 24),53,54 characterization of enzymatic
kinetics of DT diaphorase (Table 3, entry 23),55 monitoring
of the proteolytic activity of cathepsin C in live cells
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h/ga
0.45
1.60
Scheme 1 Usual methods for modification of amino groups of
xanthene ring of Rhodamine 110.
(Table 3, entry 17)58 and detection of thiols (Table 3, entry 26).57
For this latter application, also a sulfonamide derivative was
156
prepared by reaction of Rho 110 with a sulfonyl chloride
derivative through an identical mechanism as depicted in
Scheme 1 (Table 3, entry 27).61 On the other hand,
Kim et al. synthesised a rhodamine-derived bisboronic acid
for the detection of mono- and oligosaccharides62 which
can also be used as a fluorescent sensor for tetraserine motifs
80 in proteins as recently described by Halo and co-workers
(Table 3, entry 25).63
A completely different synthetic route was used by
Corrie et al. for the synthesis of photo-labile Rhodamine
derivatives suitable for labelling of proteins. Instead of
128 functionalizing the amino groups of the xanthene ring, a
modified asymmetric Rhodamine dye was prepared by a
condensation reaction between 2-(4-((2-acetamidoethyl)-
(methyl)amino)-2-hydroxy-benzoyl)benzoic acid and
m-((2-acetamidoethyl)(methyl)amino) phenol that were
previously functionalized (Scheme 3).52 Although the product
205
is obtained in a relatively good yield (60%), the reaction must
be carried out at high temperatures.
Another approach proposed by Tang et al. synthesised a
Rhodamine 6G (Rho 6G) derivative, containing a Se–N bond
designed for detecting thiols, by reaction of Rho 6G with
1650 3-bromobenzotrifluoride in the presence of KSeCN, CuI and
triethylamine (Table 3, entry 28).27
As shown in this section, several derivatives of rhodamine
dyes have been obtained by modification of amino groups of
xanthene moiety, especially after 2000. In Table 3, these
derivatives, their reaction conditions and aimed application
are summarized. It can be noticed that similar synthetic routes
are employed in most examples and usually Rho 110 is used as
the precursor dye.
Modification of the carboxyphenyl ring (positions 4 0 and/or 5 0 )
The positions 3 0 and 6 0 of the carboxyphenyl ring of
rhodamines are sterically hindered, and as a result only two
positions (besides the carboxylic acid group in 2 0 position) are
available to undergo a functionalization reaction: 4 0 and 5 0 .
Most of the commercially available rhodamine derivatives for
chemical immobilization present a reactive group in one or
both of these positions. Nonetheless, as previously remarked,
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Table 3 Rhodamine derivatives obtained by modification of amino groups of xanthene

Precursor Rho Entry R R0 Reaction conditions Yield (%) Application Ref.

Rho 110 1 Cbz-Arg H Rho 110, Cbz-L-Arg hydrochloride (1.5 eq.), 13 Serine protease 44
EDCI, DMF/Pyr(1:1) substance

2 Cbz-Arg Cbz-Arg Rho 110, Cbz-L-Arg hydrochloride (30 eq.), EDCI, 83

DMF/Pyr(1:1)

3 Cbz-a.a.-Arg Cbz-a.a.-Arg (Arg-NH)2Rho 110, Cbz-a.a., EDCI, DMF/Pyr(1:1) 45–85 45

(10 examples)
4 Gly-Pro COCH3 1. Rho 110, Boc-Gly-Pro, NEM, EDCI, DMF 96.3 46

2414 | Chem. Soc. Rev., 2009, 38, 2410–2433

5 CO(CH2)nCl; 2. Acetic anhydride or R’Cl, DMF 58–63
n = 1–4
6 CO(CH2)4Br 49.2
7 CO(C6H4)CH2Cl 18.7
8 Gly-Pro CO(CH2)3maleimide 1. Rho 110, Boc-Gly-Pro, NEM, EDCI, DMF 64.9
2. R’OH, NEM, EDCI, DMF
9 CO(CH2)5maleimide 3. CH2Cl2, TFA 43.6
10 DEVD DEVD Rho 110, N-Fmoc-Asp(OtBu), EDCI, DMF/Pyr(1:1) 86 (1st step) Caspase substrate 47
Successive deprotection/reaction of a.a. steps are carried
out for construction of peptide sequence
11 DEVDNHCO(CH2)5NH–CO(CH2)4CO 1. Rho 110, protected DEVD n.d. 48
2. After several transformations; adipoyl chloride,
collidine, THF
12 CH3(CH2)7OCO DEVD 1. Rho 110, CH3(CH2)7OCOCl, DIPEA, DMF n.d. 49
2. Protected DEVD, EDCI, DMF/Pyr(1:1) then
TFA/CH2Cl2
13 C6F5CO DEVD 1. Rho 110, RCl, DIPEA, DMF 11 50
2. Protected DEVD, EDCI, DMF/Pyr(1:1)
14 C6F4HCO 3. CH2Cl2, TFA 5 51
15 N-Morpholinecarbonyl DEVD 82 (1st step)

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1. Rho 110, RCl, DIPEA, DMF

c

Successive deprotection/reaction of a.a. steps are carried
out for construction of peptide sequence
16 NH2-Xa.a.-Xa.a. NH2-Xa.a.-Xa.a. 1. Rho 110, Boc-NH-Xaa-OH, HATU, DIPEA, DMF n.d. Cathepsin C 58
2. TFA/DCE substrate
(10 examples) (10 examples) 3. Boc-NH-Xaa-OH, HATU, DIPEA, DMF
4. TFA/DCE
17 N-Morpholinecarbonyl NH2-Xa.a.-Xa.a. 1. N-morpholinecarbonyl-Rho 110, Boc-NH-Xaa-OH,
HATU, DIPEA, DMF
2. TFA/DCE
(6 examples) 3. Boc-NH-Xaa-OH, HATU, DIPEA, DMF
4. TFA/DCE
18 Acetylated Acetylated Rho 110, acetylated trimethyl lock (2 eq.), 29 Esterase substrate 59
Trimethyl lock Trimethyl lock EDCI, DMF/Pyr(1:1)

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c

Table 3 (continued )
Precursor Rho Entry R R0 Reaction conditions Yield (%) Application Ref.
Rho 110 19 N-Morpholinecarbonyl Acetylated 1. Rho 110, RCl, NaH, DMF 23 53
Trimethyl lock 2. Acetylated trimethyl lock, EDCI, DMF, Pyr
20 4-Maleimidobutyryl Acetylated 1. Rho 110, Boc2O, NaH, DMF 18
Trimethyl lock 2. 4-Maleimidobutyric acid, DPPA, DIPEA, THF
3. CH2Cl2, TFA
4. Acetylated trimethyl lock, EDCI, DMF, Pyr

21 N-Morpholinecarbonyl Methylated N-Morpholynecarbonyl-Rho 110, methylated trimethyl 26 Cytochrome P450 60

Trimethyl lock lock (Jones reagent), EDCI,DMF/Pyr (3:2) substrate
22 Allylcarbamate Allylcarbamate n.i. n.d. Catalysis 56
23 Quinone acid Quinone acid Rho 110, quinone acid (2 eq.), EDCI, DMF/Pyr(1:1) 38 DT diaphorase substrate 55

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24 6-Heptynyl urea Acetylated 1. 6-Heptynoic acid, DPPA, DIPEA, THF 38 Fluorogenic 54
Trimethyl lock 2. CH2Cl2, TFA polymer
3. Acetylated trimethyl lock, EDCI, DMF, Pyr
25 2-Methylphenylboronic acid 1. Rho 110, 2-formylphenylboronic acid 81 Saccharides and 62,63
2. NaBH4 tetraserine motifs
26 H2N(CH2)2SS(CH2)2OCO 1. Rho 110, N-Boc-protected R–Cl 26 chemosensor 57
27 2,4-Dinitrobenzenosulfonyl Rho 110, tBuOK (3 eq.), 10 61
2,4-dinitrobenzenosulfonylchloride
Rho 6G 28 m-Trifluoromethyphenyl H 1. KSeCN, 3-bromobenzotrifluoride, DMF n.d. Detection of thiols 27
selenium 2. Rho 6G, Et3N, CuI (cellular glutathione)

Rho 110: Rhodamine 110; Rho 6G: Rhodamine 6G; Cbz: benzyloxycarbonyl; Arg: arginine; EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMF: N,N-dimethylformamide;
Pyr: pyridine; a. a.: amino acid (alanine or glutamine or glutamic acid or glycine or leucine or methionine or phenylalanine or proline or tryptophan or valine or 2-aminobutyric acid or norvaline, etc);
Gly: glycine; Pro: proline; NEM: N-ethylmorpholine; TFA: trifluoroacetic acid; DEVD: Aspartic acid (Asp)-Glutamic acid (Glu)-Valine (Val)-Aspartic acid (Asp); Fmoc: 9H-fluoren-9-
ylmethoxycarbonyl; THF: tetrahydrofuran; DIPEA: N,N-diisopropylethylamine; HATU: 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium;
Boc: tert-Butyloxycarbonyl; DCE: 1,2-dichloroethane; DPPA: diphenylphosphoryl azide; Et3N: triethylamine; Xa. a: unspecified or unknown amino acid; Boc2O: di-tert-butyldicarbonate;
tBuOK: potassium tert-butoxide; n.d. not determined; n.i.: not indicated.

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Scheme 2 Mechanism of fluorescence unmasking of bis(acetylated trimethyl lock) Rho 110 lead by esterase catalysis.
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Scheme 3 Condensation reaction for synthesis of modified Rhodamine as proposed by Corrie et al.52
they are extremely expensive and usually sold as a mixture of
isomers. For instance, succinimidyl ester, isothiocyanate and
maleimide derivatives of Rho 110, Sulforhodamine B
(SRho B) or Rhodamine 101 are offered by some suppliers
such as Sigma-Aldrich or Molecular Probes for dye-labelling
procedures (Table 4).
Although they are costly, these commercial rhodamine
derivatives have been extensively employed for fluorescent
labelling of biomolecules and other compounds, which can
be easily found in the literature. But when high quantities of
dye are needed for a target application, the use of these
commercial dyes is usually restricted by economical reasons.
The methods of synthesis of derivatives of Rhodamine dyes,
which are modified in the carboxyphenyl ring, are exclusively
reported in the patent literature. They have traditionally
been prepared by the condensation of a previously
functionalized phtalic anhydride with N-alkylated (or, in some
cases, non-alkylated) m-aminophenols in the presence of
concentrated sulfuric acid. In the particular case of succinimidyl
ester derivatives, the most usual synthetic route involves the
preparation of 4 0 ,(5 0 )-carboxyrhodamine dye from mellitic
anhydride, followed by esterification reaction with N-hydroxy-
succinimide (Scheme 4).
Menchen and Fung were the first to report the synthesis of
succinimidyl derivatives of tetramethylrhodamine (TMR)
and Rhodamine 101 by using, in the second step, di-N-
succinimidylcarbonate (DSC) and 4-dimethylaminopyridine
(DMAP) in DMF.64 Later, Cruickshank and Bittner proposed
a slight variation in step 2 by using N-hydroxysuccinimide
(NHS) and N,N 0 -diisopropylcarbodiimide (DIPC) as a
coupling agent with the aim of synthesising Rho 110
derivatives for labelling of nucleotides.65
It should be noticed that both 4 0 and 5 0 isomers are obtained
in the condensation reaction (step 1). Hence, in order to
prepare isomerically pure dyes, the mixture of isomers has to
be separated. The purification can be carried out after one of
both steps, but it is preferable before the esterification
reaction. Nevertheless, owing to the extreme resemblance
between the two obtained structures and the fact that
rhodamine dyes are cationic, the separation of the two isomers
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is cumbersome, requiring severely long and laborious
purification procedures.
On the other hand, the use of non-isomerically pure dyes
can provoke several problems in some applications. In fact, it
is very likely that different proportions of the two isomers are
obtained from different batches and, consequently, it can be
difficult to reproduce some results consistently and
accurately.66 Aiming to circumvent this problem, Corrie and
Craik provided an alternative method, comprising the
following sequence of reactions (Scheme 5).
Contrary to the first step in Scheme 4, only one equivalent
of m-dimethylaminophenol was used and, thus, a benzo-
phenone derivative was obtained. After reduction of the nitro
group and protection of the resulting amino group (step 2) the
final rhodamine structure is achieved by reaction with another
equivalent of m-dimethylaminophenol in the presence of a
catalyst (step 3). According to the authors, it is possible to
prepare an isomerically pure product if a separation procedure
by crystallisation is performed at the end of either step 1 or
step 2 (more conveniently between these two steps). After
deprotection, the amine derivative can be further converted
into bromo-, chloro- and iodoacetamide or maleimide
derivatives using the appropriate reaction conditions.66
A method for functionalization of sulforhodamine dyes was
also proposed by Jackson and co-workers. Using either
sulforhodamine B or sulforhodamine 101 (SRho 101), a
transformation of one or both sulfonate groups into sulfonyl
chloride groups were carried out using phosphorus oxychloride.
This derivative was then reacted with a diamine (with one
protected amino group) in order to obtain an amino
derivative that could be further converted into several other
functionalities by reaction with the appropriate reagent.
Hence, acyl halides, thiols, phtalimides, hydrazides, sulfonyl
halides and maleimides derivatives of SRho B and SRho 101
were prepared using this procedure. However, this method
presents a major disadvantage: during the first step, the sulfonyl
chloride can be formed either at position 2 0 or at position 4 0 or
both; so, usually a mixture of isomers is obtained.67
Recently, Uddin and Marnett reported an efficient
synthetic route for preparation of ridigised 4 0 and
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Table 4 Some commercially available reactive rhodamine derivatives

Commercial name Structure Isomer h/ga

Rho 110 Rhodamine Greent, carboxylic acid, 4 0 and 5 0 (mixed) 71 800

succinimidyl ester, hydrochloride

Rhodamine Greent-X, succinimidyl ester, 4 0 and 5 0 (mixed) 71 800

hydrochloride
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SRho B Rhodamine Redt-X, succinimidyl ester, 40 41 200

hydrochloride

Rhodamine Redr C2, maleimide 2 0 and 4 0 (mixed) 52 100

Rho 101 X-rhodamine-isothiocyanate 4 0 and 5 0 (mixed) 27 000

Carboxy-X-rhodamine, 4 0 and 5 0 (mixed) 8250

Succinimidyl ester

40 72 700
50 58 000
a
An average for suppliers such as Sigma-Aldrich or Molecular Probes.

5 0 -carboxy-X-rhodamines containing g,g-dimethylpropylene
or n-propylene groups bridging terminal nitrogen atoms and
the xanthene core.68 Starting from m-anisidine, sequential
alkylation with 1-chloro-3-methylbut-2-ene, treatment with
conc. HCl, intramolecular cyclization using neat MeSO3H
and O-desmethylation using BBr3 yield the corresponding
1,1,7,7-tetramethyl-8-hydroxyjulolidine. This compound could
be converted into the aimed rhodamine derivative by Friedel–
Crafts condensation with 4-carboxyphthalic anhydride. Four
different protocols were attempted for this reaction but the use
of a high-boiling weakly acidic solvent (n-PrCO2H, pKa 4.82)
with a trace of 2 M H2SO4 under reflux has shown to be the
most efficient one (Scheme 6). Both isomers are obtained but
the authors have optimized conditions for their separation by
silica gel flash chromatography. Typically, the 4 0 derivative is
obtained with 34% (n-propylene) and 32% (g,g-dimethyl-
propylene) yields and 5 0 derivative is obtained with 42% and
15% isolated yield for n-propylene and g,g-dimethylpropylene
derivatives, respectively.
Besides, a conjugation reaction between those dyes and an
amino derivative was carried out by activation of carboxylic
acid moiety using N,N,N,N-tetramethyl-O-(N-succinimidyl)
uronium tetrafluoroborate (TSTU) and proven to be useful
for dye-labelling of molecules of interest.
In conclusion, the modification of the carboxyphenyl ring of
rhodamine dyes is very difficult to perform when aiming to
prepare isomerically pure derivatives. Generally, it is necessary
to synthesise the rhodamine chromophore and not simply

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spirolactam; (b) reduction with glacial Zn/acetic acid;

(c) deprotonation of the formed amide with a strong base;
(d) reaction with an acrylating agent and (e) oxidation of the
leuco form to originate the desired dye (Table 5, entry 1).69
Some years later, Mayer and Oberlinner suggested another
synthetic route for derivatization in 2 0 -position by formation
of an acyl chloride of Rho 6G with phosphorus oxytrichloride
(POCl3), followed by reaction with benzylamine (Table 5,
entry 2).70 In both reports, however, yields and purity of the
obtained compounds were not mentioned. An alternative
method for the attachment of secondary amines was proposed
by Arnost et al. where the activation of carboxylic acid group
was carried out by reaction with diphenylphosphoryl azide
(DPPA) (Scheme 7; Table 5, entry 3).71
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Scheme 4 Usual synthetic route for the synthesis of succinimidyl Also using a strategy of activation for the carboxylic acid
derivatives of Rhodamine dyes. DSC: di-N-succinimidylcarbonate, group, Grechishnikova et al. prepared a bisteroid-Rho 101
DIPC: N,N 0 -diisopropylcarbodiimide; NHS: N-hydroxysuccinimide. ester conjugate through the reaction of a bisteroid diol
derivative and Rho 101 in the presence of DCC to be used
in FRET studies of model systems of biological membranes
functionalize low-priced commercially available rhodamine (Table 5, entry 4).72 However, only after the pioneering work
dyes. Their usual application is the dye-labelling of molecules reported by Czarnik’s group in 1997 and especially over the
of interest. past 3–4 years a more significant development in the synthesis
of 2 0 -rhodamine derivatives has arisen, when the spirolactam
(nonfluorescent) to ring-opened amide (fluorescent) process of
Modification of the carboxylic acid group (position 2 0 )
rhodamine dyes was demonstrated to be attractive in the
Although 2 0 -position could be seen as the easiest to conception of chemosensors of metal ions (Fig. 3).
functionalize since it bears already a functional group
(a carboxylic acid or ester, depending on the rhodamine),
the methods for its modification only began to be reported
in the scientific literature after 2000. Nonetheless, a few earlier
reports can be found, particularly in the patent literature.
Cincotta and Foley have patented the first method for
amidation of the carboxylic acid group of Rho B. It was a
very complex procedure, involving 5 steps: (a) reaction of Rho
B ethyl ester with an alkyl- or phenylamine, yielding a Fig. 3 Spirolactam ring-opening process of Rho B derivative.

Scheme 5 Synthetic route for preparation of 4 0 and 5 0 TMR derivatives.

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Scheme 6 Synthetic route for preparation of 4 0 and 5 0 -carboxy-X-rhodamines containing g,g-dimethylpropylene bridging group as proposed by
Uddin and Marnett.68
Czarnik and colleagues synthesised a Rho B hydrazide in
80% yield by reaction of Rho B with POCl3 in dichloroethane
(80 1C) followed, without purification, by reaction with
anhydrous hydrazine and they demonstrated its use as a
chemodosimeter for Cu(II) (Table 5, entry 5).73
Some years later, Yang et al. synthesised the same molecule
by a one-step reaction of Rho B with hydrazine hydrate in
methanol under reflux (68% yield) and showed its potentiality
as fluorogenic probe for determination of peroxynitrite
(Scheme 8; Table 5, entry 6).74
Thereafter, a great increase in number of publications
concerning 2 0 -derivatives of rhodamines as chemosensors
was observed, that were recently reviewed by Kim et al.22
Not only rhodamine hydrazide derivatives were further
modified by reaction with aldehydes (Table 5, entries
7–10),26,30,75,76 including aldoses such as glucose (Table 5,
entry 11),77 ketones (Table 5, entry 12)24, isothiocyanates
(Table 5, entries 13–15)32,78,79 or acyl chloride (Table 5,
entry 16)80 to prepare other chemosensors but also novel
compounds were obtained by reaction of Rho derivatives with
benzoic hydrazide (Table 5, entry 17),81 ethylpenicotate
(Table 5, entry 18),82 hydroxylamine (Table 5, entries 19–21),83–85
O-methylhydroxylamine (Table 5, entry 22),86 3-aminopropyl
triethoxysilane (Table 5, entry 23),87 2-((bis(2-(ethylthio)ethyl)-
amino)methyl)aniline (Table 5, entry 24),88 2-aminopyridine
(Table 5, entry 25),89 2-bromoethylamine (Table 5, entries
26–27),90,91 ethylenediamine (Table 5, entries 28–30),23,92
diethylenetriamine (Table 5, entries 31–32),29,31 tris(2-amino-
ethyl)amine (tren) (Table 5, entries 33–35),21,29,93 cystamine
dihydrochloride (Table 5, entry 36)94 and 2-aminoethanol
(Table 5, entry 37)95 through this same synthetic route,
followed (or not) by subsequent modification reactions. Thus,
new rhodamine-based chemosensors for Cu(II),21,23,30,76,78,79
Hg(II),24,29,32,77,83,85,90–94,96,97 Pb(II),98 Fe(III),31,86,90 Ag(I),95
hypochlorite anion80 and hypochlorous acid84 as well as for
in vivo evaluation of intracellular pH81 have been continuously
developed in recent years. In addition, fluorophore
dyads comprising another fluorophore (naphtalimide, dansyl,
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BODIPY or fluorescein) that will behave as Förster resonance
energy transfer (FRET) donor have been synthesised in
order to produce FRET-based chemosensors (Table 5,
entries 38–41). After addition of a specific metal ion [Cr(III),14
Cu(II)21 or Hg(II)99,100] a spirolactam opening process takes
place and rhodamine emission is observed upon excitation of
the donor. On the other hand, solid-supported chemodosimeters
can also be prepared using this strategy such as the platinum-
film immobilized Rhodamine based chemodosimeter for Cu(II)
recently reported by Kim et al. (Table 5, entry 34).101
In 2000, Adamczyk et al. proposed a method of preparation
of rhodamine conjugates by directly reacting rhodamine
2 0 -esters with primary amines (Table 5, entries 42, 43).102
The authors suggested that primary amines (on primary
carbons) could undergo reversible reactions at the 9-position
of non-alkylated or mono-N-alkylated rhodamine ester
derivatives. This addition would be followed by intra-
molecular trapping of the amine intermediate with the ester
functional group in 2 0 -position, originating subsequently a
fluorescent rhodamine amide derivative by ring opening of
the spirolactam intermediate (Scheme 9).
Several Rho 110 and Rho 6G amide conjugates were
prepared either by using an excess of Rhodamine ester or of
the amine substrate. Reactions with both simple amines
[benzyl 6-aminohexanoate, 1-(4-aminophenyl)ethylamine,
4-amino methylpiperidine, 4-aminobutanol] or more complex
ones (lysine, normetanephrine, amino-containing steroids)
were carried out and it was noticed that the amine was the
limiting reagent. In fact, when 3 eq. of amine was used per
1 eq. rhodamine ester, the reaction was complete in 12 h and
higher yields were attained comparatively to when 2 eq. of
rhodamine ester was used per 1 eq. of the amine
(reaction time 96 h).
In 2003, Afonso et al. and Nguyen and Francis reported,
respectively, novel synthetic routes for the preparation of
rhodamine 2 0 -ester derivatives103 and 2 0 -amide derivatives.104
In the former example, the lactone of Rho 6G was prepared
by pyrolysis and further reacted with activated alkyl halides
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Table 5 Rhodamine derivatives obtained by modification of the carboxylic acid group

Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.

1 Rho B 1. Rho B ethyl ester, methylamine (40% in water) Light-screening dyes in 69

2. Acetic acid, zinc photographic products and
3. BuLi (1.6 M in THF) processes
4. ClCO2(CH2)2SO2CH3
5. Air (bubbling), iodine n.d.

2 Rho 6G Rho 6G carboxylic acid (3.5 eq.), benzylamine, n.d. Dyeing of paper stocks 70

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POCl3 (1.3 eq.)

3 Rho I Rho I, amine, DPPA (1.3 eq.), DMF 61 Biological diagnostic assay 71

4 Rho 101 Rho 101 (3 eq.), bisteroid diol, DCC (5.2 eq.), 78 FRET studies in model and 72

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4-pyrrolidinopyridine (5.9 eq.), CHCl3 biological membranes

c

5 Rho B 1. Rho B base, DCE, POCl3 (reflux)
2. CH3CN, anhydrous hydrazine (excess) 80 Cu(II) chemosensor 73

6 Rho B, hydrazine hydrate, MeOH (reflux) 64 Peroxynitrite chemosensor 74

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Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.

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7 Rho B 1. Rho B, hydrazine hydrate, EtOH (reflux)
2. 2-Hydroxybenzaldehyde (4 eq.) 57 30

8 Rho B Rho B hydrazide, 2-formylphenylboronic acid 47 Cu(II) chemosensor 76

(4 eq.), EtOH (reflux)

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9 Rho 6G 1. Rho 6G, hydrazine hydrate, EtOH (reflux)
2. Salicylaldehyde (5 eq.), EtOH/CH2Cl2, 73 75
reflux, 12 h

10 Rho 6G Rho 6G hydrazide, glucose, toluene/MeOH 2:6 17 Hg(II) chemosensor 77

(reflux), PTSA

11 Rho B 1. Rho B, hydrazine hydrate, MeOH (reflux)

2. KSCN, EtOH/H2O 2M HCl, reflux, 10 h 55 Hg(II) chemosensor 79

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Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.

12 Rho B 1. Rho B, hydrazine hydrate, MeOH (reflux)

2. 1-Ferrocene-2-(quinolin-8-yloxy)ethanone 53 Multisignaling optical- 24
(0.7 eq.), dry toluene (reflux) electrochemical sensor
for Hg(II)

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13 Rho 6G 1. Rho 6G, hydrazine hydrate, MeOH (reflux)
2. phenyl isothiocyanate, DMF 86 Hg(II) chemosensor 32

14 Rho B Rho B hydrazide, n–butyl isothiocyanate, CHCl3, 68 Cu(II) chemosensor 78

reflux, 3 days

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15 Rho 6G 1. Rho 6G, hydrazine hydrate, MeOH (reflux)
2. 2-Pyridinecarbaldehyde 85 Hg(II) chemosensor 26

16 Rho B
1. Rho B, hydrazine hydrate, MeOH (reflux)
2. benzoyl chloride, THF 65 Hypoclorite chemosensor 80

17 1. Rho B base, POCl3, CH2Cl2

45 pH probe 81

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2. Benzoic hydrazide, CH3CN
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Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.

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18 Rho II 1. Rho II, POCl3 (5.2 eq.), 70–75 1C
2. Ethyl isonipecotate (5.5 eq.), DMF, Et3N 34 Synthesis of fluorescence 82
quenchers

19 Rho B 1.Rho B base, DCE, POCl3 (reflux)

2. HO–NH2.HCl, CH3CN, Et3N 49 — 85

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20 60 Cu(II) chemosensor 83

21 Rho 6G 1. Rho 6G, NaOH, H2O

2. DCE, POCl3 (reflux)
3. HO–NH2.HCl, DCE, Et3N 36 Hypochlorous acid 84
chemosensor

22 Rho B 1. Rho B base, DCE, POCl3 (reflux)

2. MeO–NH2.HCl, CH2Cl2, Et3N 81 Fe(III) chemosensor 86

23 Rho B Rho B, 3-aminopropyl triethoxysilane, 100 Silica-linked rhodamine 87

CHCl3, reflux probe

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Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.

24 Rho B 1. Rho B base, DCE, POCl3 (reflux)

2. CH3CN, Et3N
2-((bis(2-(ethylthio)ethyl) amino)methyl)aniline 26 Hg(II) chemosensor 88

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25 Rho B Rho B, 2-aminopyridine, POCl3 (cat) 60 Chemosensor for transition 89
metals cations

26 Rho B 1. Rho B, POCl3 (reflux)

2. Bromoethylenamine hydrobromide,
Et3N, CH3CN
3. Aza-18-crown-6, CH3CN, DIPEA 7.4 Fe(III) and Hg(II) selective 90
dual chemosensor

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27 Rho B 1. Rho B, POCl3
2. 2-Bromoethylamine hydrobromide, Et3N
3. Cyclen, toluene (reflux) 18 91

28 Rho B 1. Rho B, ethylenediamine, EtOH (reflux)

2. 1-Isocyanate-4-nitrobenzene, toluene (reflux) 32 Hg(II) chemosensor

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Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.

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29 Rho B 1. Rho B, ethylenediamine, EtOH (reflux) 92
2. m-Xylenediisocyanate (0.5 eq.), toluene (reflux) 18

30 Rho B 1. Rho B, ethylenediamine, EtOH

2. t-Boc-protected a-bromoacetic acid, DIPEA,
NaI, CH3CN (reflux)

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3. TFA, CH2Cl2 49 Cu(II) chemosensor 23

31 Rho B 1. Rho B, diethylenetriamine, MeOH (reflux)

2. TsCl, Pyr, CHCl3 58 Hg(II) chemosensor 29

32 Rho B 1. Rho B base, DCE, POCl3 (reflux)

2. Diethylenetriamine, CH3CN 18 Fe(III) chemosensor 31

33 Rho B 1. Rho B, tren, MeOH (reflux)

2. TsCl, Pyr, CHCl3 68 Hg(II) chemosensor 29

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Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.

34 Rho B 1. Rho B-tren conjugate, 6-bromohexanoyl chloride,

Et3N, 2 days
2. Thiourea, EtOH, reflux. 5 h
3. NaOH, water 81 Platinum film immobilized 101
Cu(II) chemosensor

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35 Rho B 1. Rho B, tren, MeOH (reflux)
2. 3-(Triethoxysilyl)propyl isocyanate, toluene, 43 Mesoporous silica 93
80 1C. immobilized Hg(II)
chemosensor

36 Rho B 1. Rho B base, DCE, POCl3 (reflux)

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2. CH3CN, cystamine dihydrochloride (0.56 eq.), 56 Hg(II) chemosensor 94

c

Et3N

37 Rho B 1. Rho B, EtOH, 2-aminoethanol, 120 1C

2. MsCl, Et3N, CH2Cl2
3. NaI, acetone (reflux) chemosensor 84 Ag(I) chemosensor 95

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Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.

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38 Rho B 1. Rho B hydrazide
2. 8-hydroxylquinoline-2-aldehyde, EtOH (reflux)
3. 2-hydroxyethyl-4-(6-morpholin-4-yl-1H, 40 Cr(III) chemosensor 14
3H-benzo[de])-isoquinoline, anhydrous THF

The Royal Society of Chemistry 2009

39 Rho B 1. Rho B, tren, MeOH (reflux)
2. Dansyl chloride, Et3N, CHCl3 40 Cu(II) chemosensor 21

40 Rho B Rho B hydrazide, 1-ethynyl-BODIPY- 53 Hg(II) chemosensor 99

4-isothiocyanate benzene, DMF, 50 1C

41 Rho B Rho B hydrazide, fluorescein isothiocyanate, 89 100

DMF

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Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.

42 Rho 6G Rho, amine (2–3 eq.), DMF, DIPEA 54–92 Rhodamine conjugates 102
43 Rho 110 (for neutralization of amine salts),

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44 Rho 6G 1. Rho 6G, 265–275 1C (pyrolysis)
2. DIPEA (1.2 eq.), NaI (cat), CH3CN,
BrCH2CO2CH2Ph or a,a 0 -dichloro-p-xylene
3. Further modification 69–86 Fluorescent probes for 103
conjugation to amino
acids and peptides

45 Rho B 1. AlMe3, piperazine, CH2Cl2

2. Rho base (reflux) 70 Rhodamine probes 104

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46 Rho 6G 48
47 Rho 101 6

48 Rho 101 Rho 101, amine (1.3 eq.), HATU (2 eq.), Et3N, 66–80 Reversible red fluorescent 17
CH2Cl2 molecular switches

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Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.

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49 Rho B Rho B hydrazide or Rho B hydroxylamide, 19 Hg(II) chemosensor 85
Lawesson’s reagent

50 Rho B 1. Rho B base, POCl3 (reflux) 97

2. Thiourea, Et3N 45

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51 1. Rho B base, DCE, POCl3 (reflux) 105
2. Na2S (aq) 85

52 Rho B base, Lawesson’s reagent 65 105

53 Rho 6G 1. Rho 6G, NaOH (aq), EtOH, reflux

2. POCl3, DCE, reflux
3. Et3N/THF, thiourea in water 51 96

Rho B: Rhodamine B; Rho 6G: Rhodamine 6G; Rho 101: Rhodamine 101; BuLi: butyllithium; BODIPY: boron–dipyrromethene; THF: tetrahydrofuran; DPPA: diphenylphosphoryl azide;
DMF: N,N-dimethylformamide; DCC: N,N 0 -dicyclohexylcarbodiimide; Et3N: triethylamine; DIPEA: N,N-diisopropylethylamine; EtOH: ethanol; MeOH: methanol; PTSA: p-toluene sulfonic acid;
MsCl: methane sulfonyl chloride; TsCl: tosyl chloride; Pyr: pyridine; t-Boc: tert-Butyloxycarbonyl; TFA: trifluoroacetic acid; tren: tris(2-aminoethyl)amine; HATU: 2-(1H-7-Azabenzotriazol-1-yl)-
1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium; DCE: 1,2-dichloroethane; tren: tris(2-aminoethyl) amine.

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Scheme 7 Modification of rhodamine dye through DPPA carboxylic acid activation.

(a-halo esters or benzyl halides) in the presence of DIPEA and
a catalytic amount of NaI in refluxing acetonitrile, leading to
lactone ring opening and formation of ester derivatives
(Table 5, entry 44) in high yields (66% to 88%). In the latter
example, a method for the preparation of tertiary amide
derivatives of Rhodamine dyes was developed. As already
remarked, secondary amides of rhodamines are usually
found as non-fluorescent spirolactams, except under acidic
conditions or in the presence of metal cations (vide Fig. 3),
preventing their use in biological experiments. Hence, the
authors decided to synthesise a piperazine amide derivative
from Rho B through exposure of Rho B lactone to 4 eq. of
piperazine and 2 eq. of AlMe3, in refluxing CH2Cl2, obtaining
the desired compound in 70% yield (Scheme 10).
Subsequently, the secondary amine group of this Rho B
derivative was converted in several other functional groups

Scheme 8 Synthetic routes for the preparation of Rho B hydrazide.

Scheme 9 Proposed mechanism for reaction of primary amines with non-alkylated or mono-N-alkylated Rhodamine dyes.

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Scheme 10 Synthesis of Rho B piperazine amide derivative.
through alkylation of the amine and common organic
chemistry transformation reactions (Table 5, entry 45). For
instance, it was used for surface modification of tobacco
mosaic virus20 but also for dye-labelling of polymers. For this
latter application, Geng and co-workers synthesised a
methacrylate monomer derivative for preparation of
Rhodamine-labelled glycopolymers106 and in our laboratory
a Rhodamine-labelled dithiobenzoate was prepared to be used
as a chain transfer agent in radical addition–fragmentation
chain transfer (RAFT) polymerization.107 Nguyen and
Francis have also prepared piperazine amide derivatives from
Rho 6G and Rho 101 (Table 5, entries 46 and 47). However,
lower yields were achieved (48% and 6%, respectively) and
purification of these derivatives showed to be very demanding
and inefficient.
Bossi et al. have prepared an amide derivative of Rho 101 in
high yields (66%–80%) by activating the carboxylic group
with HATU (Scheme 11; Table 5, entry 48).17 HATU is a
peptide coupling reagent from the family of uranium salts,
known to be very efficient in difficult sterically hindered
couplings.108
In Table 5, several examples of Rhodamine derivatives,
reported in the literature, that have been synthesised by
modification of a carboxylic acid group of a carboxyphenyl
ring are summarized. It can be noticed that for the most part,
Rho B has been used as the precursor dye, even if some
examples with Rho 6G or Rho 101 are also present. Either
way, it is remarkable that most of reported derivatives have
been synthesised only after 2000 and in the last 2–3 years a
‘‘boom’’ in the number of publications concerning new
chemosensors based on the spirolactam ring-opening process
Scheme 11 Synthesis of Rho 101 amide derivative using HATU as coupling agent.
This journal is 
c The Royal Society of Chemistry 2009
View Online
have entrained a development in the synthesis of novel
rhodamine derivatives.
Conclusions
When aiming to prepare a rhodamine derivative, several
aspects have to be taken in consideration in order to design
a synthesis strategy. First of all, the intended application of
such a derivative will restrict the possible synthetic pathways.
In fact, functionalization in positions 3 and 6 (amino groups of
xanthene rings) leads to loss of fluorescence of the Rho
derivative. Thus, this synthetic route is only interesting if
one wants to obtain a latent fluorophore. Analogously,
modification of position 2 0 (carboxylic acid group of
carboxyphenyl ring) through a secondary amide bond
formation will result in the synthesis of a non-fluorescent
dye that becomes fluorescent only in acidic conditions or in
the presence of metal cations. This can be very attractive for
the development of chemosensors or not particularly
prejudicial if the studied system can tolerate acidic conditions,
but it would be completely useless as a fluorescent probe for
studying a biological system. On the other hand, these latter
could be examined using rhodamine dyes functionalized at
2 0 -position with an N,N 0 -dialkylamide or ester derivative or,
as the most part of available commercial functionalized
rhodamine dyes, at positions 4 0 and/or 5 0 . Nevertheless, when
large quantities of product are needed, the use of these
commercial dyes becomes unaffordable due to their high price.
In fact, these latter years little development has been
observed in the synthesis of isomerically pure rhodamine dyes
modified at position 4 0 or 5 0 . Since it is still prepared through
Friedel–Crafts condensation, both isomers are obtained,
demanding complicated purification procedures. An alter-
native synthetic route yielding only one isomer is still
unknown even though its great importance for dye-labelling
of (bio)molecules and subsequent applications in in vitro or
in vivo diagnosis. Besides, the positions 4 0 and 5 0 are less
sterically hindered than position 2 0 , affording rhodamine-
(bio)conjugates in higher yield. In addition, for a strict use
as a fluorescent tag for biological imaging, the intramolecular
cyclization (spirolactam or spirolactone) should be avoided
aiming to maximize the fluorescent signal. Thus, if a
modification in position 2 0 is chosen, reaction with a secondary
amine has to be carried out since ester derivatives can suffer
hydrolysis in biological media. However, all reported procedures
for this kind of modification still imply severe reaction
conditions or the use of expensive coupling agents as HATU.
Chem. Soc. Rev., 2009, 38, 2410–2433 | 2431

Conversely, modification of amino groups of xanthene ring
and the preparation of spirolactam derivatives have met an
enormous progress. Derivatization of Rho 110 may be
considered as a very robust method for preparation of
pro-fluorophores. Analogously, reaction of Rho B and Rho
6G with primary amines either by reflux in ethanol or by
formation of acyl chloride with POCl3 can at present be
considered as a standard method for the preparation of metal
chemosensors. Nonetheless, these methods have not been
applied for the synthesis of Rho 101 derivatives. This rigidised
rhodamine derivative has a fluorescence quantum yield of near
one and its photophysical properties are insensitive to the
environment, which could be very interesting for some
Downloaded by Universidade Tecnica de Lisboa (UTL) on 03 October 2012
applications.
Published on 27 April 2009 on http://pubs.rsc.org | doi:10.1039/B901612K
In conclusion, due to these late developments on the
synthetic methods for derivatization of rhodamine dyes it is
today possible to envisage attaching this dye to almost every
molecule of interest, taking advantage of their outstanding
photophysical properties.
Acknowledgements
The authors thank Fundação para a Ciência e Tecnologia
(POCI 2010) and FEDER (POCI/QUI/61045/2004) for
financial support. Mariana Beija thanks FCT for a PhD grant
SFRH/BD/18562/2004.
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