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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
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
Although for some of those applications the dye is used in (these latter derivatives normally bear an alkyl group as R4) or
its free form, for most of them the probe must be attached to when the amino groups are rigidised, the activated process is
another molecule (polymer, oligonucleotide, biomolecule, etc.) absent and the quantum yield of these dyes is very high and
or surface. In order to obtain these rhodamine conjugates, independent of temperature.34,35 In opposition, rhodamine
usually a reaction between a nucleophilic functionality in the dyes with two alkyl substituents at each nitrogen show
molecule of interest and a 4 0 - or 5 0 -activated rhodamine activated internal conversion and consequently the quantum
derivative [in Fig. 1(B): G = activated ester, an acyl chloride, yield and fluorescence lifetime vary with temperature.
a sulfonyl chloride or a isothiocyanate functionality] is The activated process seems to be associated with a non-
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carried out. Several of these activated dyes are commercially fluorescent twisted intramolecular charge-transfer (TICT)36
available. However, either they are found as a mixture of state characterized by an electron transfer from the amino
isomers or isomerically pure dyes have extremely high costs groups to the xanthene ring followed by a rotation between
(more than 40 000 h/g), which is prohibitive when there is a them.37 The energy of the TICT state is higher than the energy
need for several grams of product and when further synthetic of the first excited singlet state for the dyes without activated
steps will take place. processes and lower for those with activated internal
Hence, in order to obtain derivatives of a Rhodamine dye in conversion. Then, the activated energy dissipation is explained
a large amount, it is necessary to synthesise it. Aiming to do by the population of the TICT state that is non-emissive and
that, the condensation reaction that leads to formation deactivates quickly to the ground state.38 The non-activated
of Rhodamine dyes has to be carried out using previously process involves energy dissipation by C–H and N–H
functionalized reagents. Another possibility is to modify less streching modes coupled with high frequency vibration modes
expensive unfunctionalized commercially available rhodamines. of the solvent. The N–H vibration modes are found to be very
Herein, the synthetic strategies for functionalization of effective in the dissipation of the electronic energy to hydroxylic
Rhodamine dyes will be reviewed and the reasons for the solvents.1,2 Rhodamine 101 (Rho 101) and Rhodamine B
choice of a particular synthetic pathway will be discussed. In (Rho B) are among the most used rhodamines and present
order to contextualize the potential applications, a brief an interesting behaviour with pH and solvent polarity (Fig. 2).
introduction on the photophysics of Rhodamine dyes is also In acidic solutions, the carboxyl group is protonated and the
included. 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
Photophysical properties zwitterionic forms share the same chromophore, the negative
charge has an inductive effect on the central carbon atom of
Depending on the substituents R1, R2, R3, R4, G and even on
xanthene chromophore, leading to a hypsochromic shift
the counter ion X (usually Cl, Br or ClO4),1 the dye will
of both absorption and fluorescence maxima and a slight
present different photophysical properties in solution, such as
reduction of the extinction coefficient at lmax abs . The differences
absorption and emission maxima (lmax max
abs , lem , fluorescence
in the specific dye-solvent interaction were also invoked to
lifetime (t) and fluorescence quantum yield (f).
explain the small differences in quantum yield and lifetime for
The major differences in the photophysical properties of
the cationic and zwitterionic forms.39 In less polar organic
Rhodamines are explained by the non-radiative deactivation
solvents, the zwitterionic dye undergoes a reversible
by internal conversion. The internal conversion has both
conversion to a colorless lactone due to the interruption of
activated and non-activated components.34 In rhodamine dyes
p–conjugation of the chromophore. Consequently, absorption
which carry none, only one alkyl substituent at each nitrogen
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
J. M. G. Martinho, born in
forms of Rho 101 and Rho B. The very low quantum yield and
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- Fig. 2 Molecular structures of three forms of Rho 101 and Rho B in
chemistry and photophysics and fast chemical kinetics. equilibrium.
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t/ns 4.34 (EtOH) 4.37 (EtOH) 0.13 (THF) 2.42 (EtOH) 2.88 (EtOH) 5.8 (Et2O)
lifetime of lactones of Rhodamine B and Rhodamine 101 were It should be noticed that unfavourable steric interactions
attributed to an electron-transfer reaction in the excited state caused by the modification of both amino groups enhance the
that generates a charge transfer excited state and the singlet nucleophilicity of the phenolic oxygen and lead to lactone
and triplet states of the dye in the zwitterionic form.41 formation. As a result, the conjugation system of the
chromophore is disrupted and these rhodamine derivatives
Modification of rhodamine dyes for use as are non-fluorescent.
In the first reported examples, Rho 110 was modified in
fluorescent probes
order to prepare synthetic fluorogenic amide substrates for
Three types of modification of Rhodamine derivatives can be assays of serine proteases and caspases. Two important
envisioned: modification of the amino groups of xanthene criteria must be fulfilled by a pro-fluorophore that will be
moiety (positions 3 and 6); modification of the carboxyphenyl used as a synthetic substrate: (1) easy detectability; (2) high
ring at positions 4 0 and/or 5 0 or modification of the carboxylic reactivity of the bond undergoing cleavage. Rho 110 appeared
acid group (position 2 0 ). as an excellent candidate since it is highly fluorescent at the
Although in some cases rhodamine derivatives are prepared same time as the uncleaved substrate is nonfluorescent
directly through a condensation reaction using previously (low background signal), it absorbs and emits in the visible
functionalized reactants, most of the examples presuppose range of electromagnetic spectrum and it is a very good
modification of commercially available Rhodamine dyes. As leaving group because cleavage of the amide bond is accom-
one can see in Table 2, Rho B and Rho 6G are the less panied by a large increase in the degree of conjugation
expensive dyes of this family and consequently they have been (and thus a large increase in stability). Hence, several bis-
the most employed for further applications. substituted peptide derivatives of Rho 110 were synthesised
In the following sections, the main developments for each and used in enzymatic activity studies (Table 3, entries 1–3,
type of modification is reviewed. 10–11 and 16).44,45,47,58 After this pioneering work in the
synthesis of pro-fluorophore compounds, several bis-
Modification of the amino groups of xanthene moiety
substituted peptide derivatives of Rho 110 began to be traded
(positions 3 and 6)
by some chemical suppliers such as Aldrich or Molecular
Usually, functionalization of the amino groups of xanthene Probes. Nowadays more than 10 different peptide sequences
moiety of Rhodamine dyes can lead to severe changes in their are commercially available, with prices usually higher than
photophysical properties, causing in some cases even the total 37 000 h/g. Nevertheless, it was very soon remarked that the
loss of fluorescence. This property was found useful for the presence of two steps for the enzymatic hydrolysis process was
synthesis of latent fluorophores (pro-fluorophores), with an limiting the linear dynamic range of this substrate.47 In fact,
advantage over ‘‘conventional’’ fluorophores whose bulk the first hydrolysis product becomes fluorescent; however
fluorescence can obscure valuable information in biological maximal fluorescence signal can only be achieved after
experiments. Thus, in recent years, several groups have made cleavage of both peptide groups. Thus, new rhodamine-based
an effort to design new rhodamine derivatives to be used as substrates with only one cleavable amide bond were
latent fluorophores in studies of enzymatic activity synthesised. In addition, the other amino group was modified
(serine protease,44–46 caspase,47–51 esterase,52–54 DT diaphorase55), with groups that could bind the dye onto the cell surface46
of organometallic catalysis in living cells,56 in biomedical (Table 3, entries 4–9) or that could enhance cell penetration
imaging53,54 or in in vivo detection of small molecules and retention48–50 (Table 3, entries 11–15). Although mono-
(thiols).27,57 substituted Rho 110 is less fluorescent than the parent dye, the
Among all rhodamines, Rhodamine 110 (Rho 110) is the capping with an amide, carbamate or urea can preserve much
most used for this purpose because it carries non-alkylated of its fluorescence, since its zwitterionic form remains more
and, consequently, more reactive amino groups. Generally, stable than the spirolactone one.49,51
they are modified either by reaction with an acyl chloride In recent years, a slightly different approach of this strategy
(or chloroformate) or with a carboxylic acid using a carbodiimide has been developed, in which the fluorescence of modified
as a coupling agent. Both symmetric and asymmetric rhodamine derivative is unmasked by a user-designated
modification of Rho 110 can be performed using this chemical reaction. Chandran et al. first presented the use of
procedure (Scheme 1). Depending on the application, different ‘‘trimethyl lock’’ to mask Rho 110 fluorescence. The trimethyl
types of substituents have been introduced, however variable lock is an o-hydroxycinnamic acid derivative in which
reaction yields were obtained in each case (vide Table 2). unfavourable steric interactions between three methyl groups
Structure h/ga
Rho B 0.45
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Rho 6G 1.60
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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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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
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.
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 is cumbersome, requiring severely long and laborious
isomers. For instance, succinimidyl ester, isothiocyanate and purification procedures.
maleimide derivatives of Rho 110, Sulforhodamine B On the other hand, the use of non-isomerically pure dyes
(SRho B) or Rhodamine 101 are offered by some suppliers can provoke several problems in some applications. In fact, it
such as Sigma-Aldrich or Molecular Probes for dye-labelling is very likely that different proportions of the two isomers are
procedures (Table 4). obtained from different batches and, consequently, it can be
Although they are costly, these commercial rhodamine difficult to reproduce some results consistently and
derivatives have been extensively employed for fluorescent accurately.66 Aiming to circumvent this problem, Corrie and
labelling of biomolecules and other compounds, which can Craik provided an alternative method, comprising the
be easily found in the literature. But when high quantities of following sequence of reactions (Scheme 5).
dye are needed for a target application, the use of these Contrary to the first step in Scheme 4, only one equivalent
commercial dyes is usually restricted by economical reasons. of m-dimethylaminophenol was used and, thus, a benzo-
The methods of synthesis of derivatives of Rhodamine dyes, phenone derivative was obtained. After reduction of the nitro
which are modified in the carboxyphenyl ring, are exclusively group and protection of the resulting amino group (step 2) the
reported in the patent literature. They have traditionally final rhodamine structure is achieved by reaction with another
been prepared by the condensation of a previously equivalent of m-dimethylaminophenol in the presence of a
functionalized phtalic anhydride with N-alkylated (or, in some catalyst (step 3). According to the authors, it is possible to
cases, non-alkylated) m-aminophenols in the presence of prepare an isomerically pure product if a separation procedure
concentrated sulfuric acid. In the particular case of succinimidyl by crystallisation is performed at the end of either step 1 or
ester derivatives, the most usual synthetic route involves the step 2 (more conveniently between these two steps). After
preparation of 4 0 ,(5 0 )-carboxyrhodamine dye from mellitic deprotection, the amine derivative can be further converted
anhydride, followed by esterification reaction with N-hydroxy- into bromo-, chloro- and iodoacetamide or maleimide
succinimide (Scheme 4). derivatives using the appropriate reaction conditions.66
Menchen and Fung were the first to report the synthesis of A method for functionalization of sulforhodamine dyes was
succinimidyl derivatives of tetramethylrhodamine (TMR) also proposed by Jackson and co-workers. Using either
and Rhodamine 101 by using, in the second step, di-N- sulforhodamine B or sulforhodamine 101 (SRho 101), a
succinimidylcarbonate (DSC) and 4-dimethylaminopyridine transformation of one or both sulfonate groups into sulfonyl
(DMAP) in DMF.64 Later, Cruickshank and Bittner proposed chloride groups were carried out using phosphorus oxychloride.
a slight variation in step 2 by using N-hydroxysuccinimide This derivative was then reacted with a diamine (with one
(NHS) and N,N 0 -diisopropylcarbodiimide (DIPC) as a protected amino group) in order to obtain an amino
coupling agent with the aim of synthesising Rho 110 derivative that could be further converted into several other
derivatives for labelling of nucleotides.65 functionalities by reaction with the appropriate reagent.
It should be noticed that both 4 0 and 5 0 isomers are obtained Hence, acyl halides, thiols, phtalimides, hydrazides, sulfonyl
in the condensation reaction (step 1). Hence, in order to halides and maleimides derivatives of SRho B and SRho 101
prepare isomerically pure dyes, the mixture of isomers has to were prepared using this procedure. However, this method
be separated. The purification can be carried out after one of presents a major disadvantage: during the first step, the sulfonyl
both steps, but it is preferable before the esterification chloride can be formed either at position 2 0 or at position 4 0 or
reaction. Nevertheless, owing to the extreme resemblance both; so, usually a mixture of isomers is obtained.67
between the two obtained structures and the fact that Recently, Uddin and Marnett reported an efficient
rhodamine dyes are cationic, the separation of the two isomers synthetic route for preparation of ridigised 4 0 and
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 silica gel flash chromatography. Typically, the 4 0 derivative is
or n-propylene groups bridging terminal nitrogen atoms and obtained with 34% (n-propylene) and 32% (g,g-dimethyl-
the xanthene core.68 Starting from m-anisidine, sequential propylene) yields and 5 0 derivative is obtained with 42% and
alkylation with 1-chloro-3-methylbut-2-ene, treatment with 15% isolated yield for n-propylene and g,g-dimethylpropylene
conc. HCl, intramolecular cyclization using neat MeSO3H derivatives, respectively.
and O-desmethylation using BBr3 yield the corresponding Besides, a conjugation reaction between those dyes and an
1,1,7,7-tetramethyl-8-hydroxyjulolidine. This compound could amino derivative was carried out by activation of carboxylic
be converted into the aimed rhodamine derivative by Friedel– acid moiety using N,N,N,N-tetramethyl-O-(N-succinimidyl)
Crafts condensation with 4-carboxyphthalic anhydride. Four uronium tetrafluoroborate (TSTU) and proven to be useful
different protocols were attempted for this reaction but the use for dye-labelling of molecules of interest.
of a high-boiling weakly acidic solvent (n-PrCO2H, pKa 4.82) In conclusion, the modification of the carboxyphenyl ring of
with a trace of 2 M H2SO4 under reflux has shown to be the rhodamine dyes is very difficult to perform when aiming to
most efficient one (Scheme 6). Both isomers are obtained but prepare isomerically pure derivatives. Generally, it is necessary
the authors have optimized conditions for their separation by to synthesise the rhodamine chromophore and not simply
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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 BODIPY or fluorescein) that will behave as Förster resonance
80% yield by reaction of Rho B with POCl3 in dichloroethane energy transfer (FRET) donor have been synthesised in
(80 1C) followed, without purification, by reaction with order to produce FRET-based chemosensors (Table 5,
anhydrous hydrazine and they demonstrated its use as a entries 38–41). After addition of a specific metal ion [Cr(III),14
chemodosimeter for Cu(II) (Table 5, entry 5).73 Cu(II)21 or Hg(II)99,100] a spirolactam opening process takes
Some years later, Yang et al. synthesised the same molecule place and rhodamine emission is observed upon excitation of
by a one-step reaction of Rho B with hydrazine hydrate in the donor. On the other hand, solid-supported chemodosimeters
methanol under reflux (68% yield) and showed its potentiality can also be prepared using this strategy such as the platinum-
as fluorogenic probe for determination of peroxynitrite film immobilized Rhodamine based chemodosimeter for Cu(II)
(Scheme 8; Table 5, entry 6).74 recently reported by Kim et al. (Table 5, entry 34).101
Thereafter, a great increase in number of publications In 2000, Adamczyk et al. proposed a method of preparation
concerning 2 0 -derivatives of rhodamines as chemosensors of rhodamine conjugates by directly reacting rhodamine
was observed, that were recently reviewed by Kim et al.22 2 0 -esters with primary amines (Table 5, entries 42, 43).102
Not only rhodamine hydrazide derivatives were further The authors suggested that primary amines (on primary
modified by reaction with aldehydes (Table 5, entries carbons) could undergo reversible reactions at the 9-position
7–10),26,30,75,76 including aldoses such as glucose (Table 5, of non-alkylated or mono-N-alkylated rhodamine ester
entry 11),77 ketones (Table 5, entry 12)24, isothiocyanates derivatives. This addition would be followed by intra-
(Table 5, entries 13–15)32,78,79 or acyl chloride (Table 5, molecular trapping of the amine intermediate with the ester
entry 16)80 to prepare other chemosensors but also novel functional group in 2 0 -position, originating subsequently a
compounds were obtained by reaction of Rho derivatives with fluorescent rhodamine amide derivative by ring opening of
benzoic hydrazide (Table 5, entry 17),81 ethylpenicotate the spirolactam intermediate (Scheme 9).
(Table 5, entry 18),82 hydroxylamine (Table 5, entries 19–21),83–85 Several Rho 110 and Rho 6G amide conjugates were
O-methylhydroxylamine (Table 5, entry 22),86 3-aminopropyl prepared either by using an excess of Rhodamine ester or of
triethoxysilane (Table 5, entry 23),87 2-((bis(2-(ethylthio)ethyl)- the amine substrate. Reactions with both simple amines
amino)methyl)aniline (Table 5, entry 24),88 2-aminopyridine [benzyl 6-aminohexanoate, 1-(4-aminophenyl)ethylamine,
(Table 5, entry 25),89 2-bromoethylamine (Table 5, entries 4-amino methylpiperidine, 4-aminobutanol] or more complex
26–27),90,91 ethylenediamine (Table 5, entries 28–30),23,92 ones (lysine, normetanephrine, amino-containing steroids)
diethylenetriamine (Table 5, entries 31–32),29,31 tris(2-amino- were carried out and it was noticed that the amine was the
ethyl)amine (tren) (Table 5, entries 33–35),21,29,93 cystamine limiting reagent. In fact, when 3 eq. of amine was used per
dihydrochloride (Table 5, entry 36)94 and 2-aminoethanol 1 eq. rhodamine ester, the reaction was complete in 12 h and
(Table 5, entry 37)95 through this same synthetic route, higher yields were attained comparatively to when 2 eq. of
followed (or not) by subsequent modification reactions. Thus, rhodamine ester was used per 1 eq. of the amine
new rhodamine-based chemosensors for Cu(II),21,23,30,76,78,79 (reaction time 96 h).
Hg(II),24,29,32,77,83,85,90–94,96,97 Pb(II),98 Fe(III),31,86,90 Ag(I),95 In 2003, Afonso et al. and Nguyen and Francis reported,
hypochlorite anion80 and hypochlorous acid84 as well as for respectively, novel synthetic routes for the preparation of
in vivo evaluation of intracellular pH81 have been continuously rhodamine 2 0 -ester derivatives103 and 2 0 -amide derivatives.104
developed in recent years. In addition, fluorophore In the former example, the lactone of Rho 6G was prepared
dyads comprising another fluorophore (naphtalimide, dansyl, by pyrolysis and further reacted with activated alkyl halides
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Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.
2 Rho 6G Rho 6G carboxylic acid (3.5 eq.), benzylamine, n.d. Dyeing of paper stocks 70
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
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5 Rho B 1. Rho B base, DCE, POCl3 (reflux)
2. CH3CN, anhydrous hydrazine (excess) 80 Cu(II) chemosensor 73
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
Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.
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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
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
Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.
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27 Rho B 1. Rho B, POCl3
2. 2-Bromoethylamine hydrobromide, Et3N
3. Cyclen, toluene (reflux) 18 91
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
Table 5 (continued )
Entry Precursor Rho Structure Reaction Conditions Yield (%) Application Ref.
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2. CH3CN, cystamine dihydrochloride (0.56 eq.), 56 Hg(II) chemosensor 94
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Et3N
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
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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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
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
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.
(a-halo esters or benzyl halides) in the presence of DIPEA and conditions or in the presence of metal cations (vide Fig. 3),
a catalytic amount of NaI in refluxing acetonitrile, leading to preventing their use in biological experiments. Hence, the
lactone ring opening and formation of ester derivatives authors decided to synthesise a piperazine amide derivative
(Table 5, entry 44) in high yields (66% to 88%). In the latter from Rho B through exposure of Rho B lactone to 4 eq. of
example, a method for the preparation of tertiary amide piperazine and 2 eq. of AlMe3, in refluxing CH2Cl2, obtaining
derivatives of Rhodamine dyes was developed. As already the desired compound in 70% yield (Scheme 10).
remarked, secondary amides of rhodamines are usually Subsequently, the secondary amine group of this Rho B
found as non-fluorescent spirolactams, except under acidic derivative was converted in several other functional groups
Scheme 9 Proposed mechanism for reaction of primary amines with non-alkylated or mono-N-alkylated Rhodamine dyes.
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
Published on 27 April 2009. Downloaded by Pennsylvania State University on 1/27/2021 11:15:06 PM.
Scheme 11 Synthesis of Rho 101 amide derivative using HATU as coupling agent.
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c The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38, 2410–2433 | 2431
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Conversely, modification of amino groups of xanthene ring 19 Y. Shiraishi, R. Miyamoto and T. Hirai, J. Photochem.
and the preparation of spirolactam derivatives have met an Photobiol., A, 2008, 200, 432–437.
20 T. L. Schlick, Z. B. Ding, E. W. Kovacs and M. B. Francis, J. Am.
enormous progress. Derivatization of Rho 110 may be Chem. Soc., 2005, 127, 3718–3723.
considered as a very robust method for preparation of 21 M. H. Lee, H. J. Kim, S. Yoon, N. Park and J. S. Kim, Org. Lett.,
pro-fluorophores. Analogously, reaction of Rho B and Rho 2008, 10, 213–216.
6G with primary amines either by reflux in ethanol or by 22 H. N. Kim, M. H. Lee, H. J. Kim, J. S. Kim and J. Yoon, Chem.
Soc. Rev., 2008, 37, 1465–1472.
formation of acyl chloride with POCl3 can at present be 23 X. Zhang, Y. Shiraishi and T. Hirai, Org. Lett., 2007, 9,
considered as a standard method for the preparation of metal 5039–5042.
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chemosensors. Nonetheless, these methods have not been 24 H. Yang, Z. G. Zhou, K. W. Huang, M. X. Yu, F. Y. Li, T. Yi
and C. H. Huang, Org. Lett., 2007, 9, 4729–4732.
applied for the synthesis of Rho 101 derivatives. This rigidised 25 J. S. Wu, I. C. Hwang, K. S. Kim and J. S. Kim, Org. Lett., 2007,
rhodamine derivative has a fluorescence quantum yield of near 9, 907–910.
one and its photophysical properties are insensitive to the 26 D. Y. Wu, W. Huang, C. Y. Duan, Z. H. Lin and Q. J. Meng,
environment, which could be very interesting for some Inorg. Chem., 2007, 46, 1538–1540.
27 B. Tang, Y. L. Xing, P. Li, N. Zhang, F. B. Yu and G. W. Yang,
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In conclusion, due to these late developments on the 28 J. Mao, L. Wang, W. Dou, X. L. Tang, Y. Yan and W. S. Liu,
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