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Synthesis and applications of Rhodamine derivatives as fluorescent probes Mariana Beija, Carlos A. M. Afonso and Jose´ 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 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 yield 3 and polarization 4 ), pigments and as fluorescent probes to characterize the surface of polymer nanoparticles, 5,6 fluidity of lipid membranes, 7 as well as in the detection of polymer-bioconjugates, 8 studies of adsorption of oligonucleotides on latexes, 9,10 studies of structure and dynamics of micelles, 11 single-molecule imaging 12,13 and imaging in living cells. 14–16 Rhodamine derivatives have also been employed as molecular switches, 17 as a thermometer, 18,19 for surface modification of a virus 20 and particularly as chemosensors 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, Gonc¸alves reviewed the fluorescent labelling of biomolecules using organic probes, highlighting the importance of rhodamine derivatives for that application. 33 Fig. 1 Molecular structures of xanthene (A) and rhodamine dyes (B). Centro de Quı´mica-Fı´sica Molecular and IN–Institute of Nanoscience and Nanotechnology, Instituto Superior Te ´cnico, 1049-001, Lisboa, Portugal. E-mail: [email protected], [email protected], [email protected]; Fax: +351 218 464 455 Mariana Beija Mariana Beija was born in Sa ˜o 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 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. Carlos A. M. Afonso Carlos A. M. Afonso gradu- 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 (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 Agregac ¸a ˜o. His research focus is mainly on the development of more sustainable methodologies in asymmetric organic transformations. 2410 | Chem. Soc. Rev., 2009, 38, 2410–2433 This journal is c The Royal Society of Chemistry 2009 CRITICAL REVIEW www.rsc.org/csr | Chemical Society Reviews Downloaded by Universidade Tecnica de Lisboa (UTL) on 03 October 2012 Published on 27 April 2009 on http://pubs.rsc.org | doi:10.1039/B901612K View Online / Journal Homepage / Table of Contents for this issue
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Page 1: Synthesis and applications of Rhodamine …...Synthesis and applications of Rhodamine derivatives as fluorescent probes Mariana Beija, Carlos A. M. Afonso and Jose´ M. G. Martinho

Synthesis and applications of Rhodamine derivatives

as fluorescent probes

Mariana Beija, Carlos A. M. Afonso and Jose 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

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

polymer nanoparticles,5,6 fluidity of lipid membranes,7 as well

as in the detection of polymer-bioconjugates,8 studies of

adsorption of oligonucleotides on latexes,9,10 studies of structure

and dynamics of micelles,11 single-molecule imaging12,13 and

imaging in living cells.14–16

Rhodamine derivatives have also been employed as

molecular switches,17 as a thermometer,18,19 for surface

modification of a virus20 and particularly as chemosensors

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,

Goncalves reviewed the fluorescent labelling of biomolecules

using organic probes, highlighting the importance of

rhodamine derivatives for that application.33

Fig. 1 Molecular structures of xanthene (A) and rhodamine dyes (B).

Centro de Quımica-Fısica Molecular and IN–Institute of Nanoscienceand Nanotechnology, Instituto Superior Tecnico, 1049-001, Lisboa,Portugal. E-mail: [email protected], [email protected],[email protected]; Fax: +351 218 464 455

Mariana Beija

Mariana Beija was born inSao Paulo (Brazil) in 1981.She studied Chemistry inInstituto Superior Tecnico(Technical University ofLisbon, Portugal), where shereceived a school merit awardin 2000. In 2004, she startedher PhD in Chemistry jointlysupervised by Prof. Jose M. G.Martinho, in Centro deQuımica-Fısica Molecular(Instituto Superior Tecnico,Lisbon, Portugal), andDr Marie-Therese Charreyre,in Unite Mixte CNRS-

bioMerieux (Lyon, France). Her doctoral research consistedof the synthesis of novel dye-labelled thermoresponsive blockcopolymers by RAFT polymerization, involving the synthesis ofrhodamine-derived RAFT agents.

Carlos A. M. Afonso

Carlos A. M. Afonso gradu-ated from University ofCoimbra (1984) and receivedhis PhD in 1990 from NewUniversity of Lisbon. Heworked for one year as post-doctoral fellow at the ImperialCollege of Science Technologyand Medicine under thesupervision of Prof. W. B.Motherwell (1990) and onemore academic year ofsabbatical leave (1997/98) atthe University of Bath, UK(Prof. J. Williams) and atthe University of Toronto

(Professor R. Batey). In 2004 he moved to Instituto SuperiorTecnico as associate professor and in 2008 received his Agregacao.His research focus is mainly on the development of moresustainable methodologies in asymmetric organic transformations.

2410 | Chem. Soc. Rev., 2009, 38, 2410–2433 This journal is �c The Royal Society of Chemistry 2009

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

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Page 2: Synthesis and applications of Rhodamine …...Synthesis and applications of Rhodamine derivatives as fluorescent probes Mariana Beija, Carlos A. M. Afonso and Jose´ M. G. Martinho

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 40- or 50-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

steps will take place.

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 (lmaxabs , lmax

em , 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

(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 lmaxabs . 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.

Jose M. G. Martinho

J. M. G. Martinho, born inPortugal, in 1950, receivedhis PhD in ChemicalEngineering from InstitutoSuperior Tecnico (TechnicalUniversity of Lisbon, Portugal)in 1982. In 1985, he joinedProf. M. A. Winnik’s researchgroup as a postdoctoral fellowand in 1993 he was invitedProfessor at the OntarioCenter of Materials Researchof the University of Toronto.He is Full Professor ofChemistry and head of theresearch unit, Centro de

Quımica-Fısica Molecular, at IST (Lisbon). His major researchinterests are in the areas of polymers and colloids, photo-chemistry and photophysics and fast chemical kinetics.

This journal is �c The Royal Society of Chemistry 2009 Chem. Soc. Rev., 2009, 38, 2410–2433 | 2411

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Page 3: Synthesis and applications of Rhodamine …...Synthesis and applications of Rhodamine derivatives as fluorescent probes Mariana Beija, Carlos A. M. Afonso and Jose´ M. G. Martinho

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

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 40 and/or 50 or modification of the carboxylic

acid group (position 20).

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).

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

Table 1 Photophysical parameters of Rhodamine dyes

Rho 101 Rho B

Cationic42 Zwitterionic41 Lactone41 Cationic40 Zwitterionic41 Lactone43

lmaxabs /nm (solvent) 574 (EtOH) 568 (EtOH) 317 (THF) 553 (EtOH) 543 (EtOH) 311 (Et2O)

lmaxabs /nm (solvent) 599 (EtOH) 590 (EtOH) 522 (THF) 572 (EtOH) 563 (EtOH) 442 (Et2O)

eabs/105 M�1 cm�1 1.10 (EtOH) 0.95 (EtOH) 0.12 (THF) 1.17 (EtOH) 1.11 (EtOH) 0.16 (Et2O)

f 0.89 (EtOH) 0.98 (EtOH) 0.006 (THF) 0.53 (EtOH) 0.70 (EtOH) 0.022 (Et2O)t/ns 4.34 (EtOH) 4.37 (EtOH) 0.13 (THF) 2.42 (EtOH) 2.88 (EtOH) 5.8 (Et2O)

2412 | Chem. Soc. Rev., 2009, 38, 2410–2433 This journal is �c The Royal Society of Chemistry 2009

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Page 4: Synthesis and applications of Rhodamine …...Synthesis and applications of Rhodamine derivatives as fluorescent probes Mariana Beija, Carlos A. M. Afonso and Jose´ M. G. Martinho

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

(Table 3, entry 17)58 and detection of thiols (Table 3, entry 26).57

For this latter application, also a sulfonamide derivative was

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

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

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

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

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 40 and/or 50)

The positions 30 and 60 of the carboxyphenyl ring of

rhodamines are sterically hindered, and as a result only two

positions (besides the carboxylic acid group in 20 position) are

available to undergo a functionalization reaction: 40 and 50.

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,

Table 2 Commercially available Rhodamines

Structure h/ga

Rho B 0.45

Rho 6G 1.60

Rho 19 156

Rho 101 80

Rho 110 128

Rho 116 205

Rho 123 1650

a An average for common suppliers: Acros Organics, Aldrich, Alfa

Aesar, Fluka, Radiant dyes laser, Sigma.

Scheme 1 Usual methods for modification of amino groups of

xanthene ring of Rhodamine 110.

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Page 5: Synthesis and applications of Rhodamine …...Synthesis and applications of Rhodamine derivatives as fluorescent probes Mariana Beija, Carlos A. M. Afonso and Jose´ M. G. Martinho

Table

3Rhodaminederivatives

obtained

bymodificationofaminogroupsofxanthene

PrecursorRho

Entry

RR0

Reactionconditions

Yield

(%)

Application

Ref.

Rho110

1Cbz-Arg

HRho110,Cbz-L-A

rghydrochloride(1.5

eq.),

EDCI,DMF/Pyr(1:1)

13

Serineprotease

substance

44

2Cbz-Arg

Cbz-Arg

Rho110,Cbz-L-A

rghydrochloride(30eq.),EDCI,

DMF/Pyr(1:1)

83

3Cbz-a.a.-Arg

Cbz-a.a.-Arg

(Arg-N

H) 2Rho110,Cbz-a.a.,EDCI,DMF/Pyr(1:1)

45–85

45

(10examples)

4Gly-Pro

COCH

31.Rho110,Boc-Gly-Pro,NEM,EDCI,DMF

2.AceticanhydrideorR’Cl,DMF

96.3

46

5CO(C

H2) nCl;

n=

1–4

58–63

6CO(C

H2) 4Br

49.2

7CO(C

6H

4)C

H2Cl

18.7

8Gly-Pro

CO(C

H2) 3maleim

ide

1.Rho110,Boc-Gly-Pro,NEM,EDCI,DMF

2.R’O

H,NEM,EDCI,DMF

3.CH

2Cl 2,TFA

64.9

9CO(C

H2) 5maleim

ide

43.6

10

DEVD

DEVD

Rho110,N-Fmoc-Asp(O

tBu),EDCI,DMF/Pyr(1:1)

86(1

ststep)

Caspase

substrate

47

Successivedeprotection/reactionofa.a.stepsare

carried

outforconstructionofpeptidesequence

11

DEVDNHCO(C

H2) 5NH–CO(C

H2) 4CO

1.Rho110,protected

DEVD

n.d.

48

2.After

severaltransform

ations;adipoylchloride,

collidine,

THF

12

CH

3(C

H2) 7OCO

DEVD

1.Rho110,CH

3(C

H2) 7OCOCl,DIPEA,DMF

n.d.

49

2.Protected

DEVD,EDCI,DMF/Pyr(1:1)then

TFA/C

H2Cl 2

13

C6F5CO

DEVD

1.Rho110,RCl,DIPEA,DMF

2.Protected

DEVD,EDCI,DMF/Pyr(1:1)

3.CH

2Cl 2,TFA

11

50

14

C6F4HCO

551

15

N-M

orpholinecarbonyl

DEVD

1.Rho110,RCl,DIPEA,DMF

82(1

ststep)

Successivedeprotection/reactionofa.a.stepsare

carried

outforconstructionofpeptidesequence

16

NH

2-X

a.a.-Xa.a.

NH

2-X

a.a.-Xa.a.

1.Rho110,Boc-NH-X

aa-O

H,HATU,DIPEA,DMF

2.TFA/D

CE

3.Boc-NH-X

aa-O

H,HATU,DIPEA,DMF

4.TFA/D

CE

n.d.

CathepsinC

substrate

58

(10examples)

(10examples)

17

N-M

orpholinecarbonyl

NH

2-X

a.a.-Xa.a.

1.N-m

orpholinecarbonyl-Rho110,Boc-NH-X

aa-O

H,

HATU,DIPEA,DMF

2.TFA/D

CE

3.Boc-NH-X

aa-O

H,HATU,DIPEA,DMF

4.TFA/D

CE

(6examples)

18

Acetylated

Acetylated

Rho110,acetylatedtrim

ethyllock

(2eq.),

EDCI,DMF/Pyr(1:1)

29

Esterase

substrate

59

Trimethyllock

Trimethyllock

2414 | Chem. Soc. Rev., 2009, 38, 2410–2433 This journal is �c The Royal Society of Chemistry 2009

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Table

3(continued

)

PrecursorRho

Entry

RR0

Reactionconditions

Yield

(%)

Application

Ref.

Rho110

19

N-M

orpholinecarbonyl

Acetylated

1.Rho110,RCl,NaH,DMF

2.Acetylatedtrim

ethyllock,EDCI,DMF,Pyr

23

53

Trimethyllock

20

4-M

aleim

idobutyryl

Acetylated

1.Rho110,Boc 2O,NaH,DMF

2.4-M

aleim

idobutyricacid,DPPA,DIPEA,THF

3.CH

2Cl 2,TFA

4.Acetylatedtrim

ethyllock,EDCI,DMF,Pyr

18

Trimethyllock

21

N-M

orpholinecarbonyl

Methylated

N-M

orpholynecarbonyl-Rho110,methylatedtrim

ethyl

lock

(Jones

reagent),EDCI,DMF/Pyr(3:2)

26

CytochromeP450

substrate

60

Trimethyllock

22

Allylcarbamate

Allylcarbamate

n.i.

n.d.

Catalysis

56

23

Quinoneacid

Quinoneacid

Rho110,quinoneacid(2

eq.),EDCI,DMF/Pyr(1:1)

38

DTdiaphorasesubstrate

55

24

6-H

eptynylurea

Acetylated

1.6-H

eptynoic

acid,DPPA,DIPEA,THF

2.CH

2Cl 2,TFA

3.Acetylatedtrim

ethyllock,EDCI,DMF,Pyr

38

Fluorogenic

polymer

54

Trimethyllock

25

2-M

ethylphenylboronic

acid

1.Rho110,2-form

ylphenylboronic

acid

81

Saccharides

and

tetraserinemotifs

chem

osensor

62,63

2.NaBH

4

26

H2N(C

H2) 2SS(C

H2) 2OCO

1.Rho110,N-Boc-protected

R–Cl

26

57

27

2,4-D

initrobenzenosulfonyl

Rho110,tB

uOK

(3eq.),

2,4-dinitrobenzenosulfonylchloride

10

61

Rho6G

28

m-Trifluoromethyphenyl

selenium

H1.KSeC

N,3-bromobenzotrifluoride,

DMF

2.Rho6G,Et 3N,CuI

n.d.

Detectionofthiols

(cellularglutathione)

27

Rho110:Rhodamine110;Rho6G:Rhodamine6G;Cbz:

benzyloxycarbonyl;Arg:arginine;

EDCI:1-(3-dim

ethylaminopropyl)-3-ethylcarbodiimidehydrochloride;

DMF:N,N

-dim

ethylform

amide;

Pyr:pyridine;a.a.:aminoacid(alanineorglutamineorglutamicacidorglycineorleucineormethionineorphenylalanineorprolineortryptophanorvalineor2-aminobutyricacidornorvaline,etc);

Gly:glycine;

Pro:proline;

NEM:N-ethylm

orpholine;

TFA:trifluoroacetic

acid;DEVD:Aspartic

acid

(Asp)-Glutamic

acid

(Glu)-Valine

(Val)-A

spartic

acid

(Asp);

Fmoc:

9H-fluoren-9-

ylm

ethoxycarbonyl;

THF:tetrahydrofuran;DIPEA:N,N

-diisopropylethylamine;

HATU:2-(1H-7-A

zabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium

hexafluorophosphate

methanaminium;

Boc:

tert-Butyloxycarbonyl;

DCE:1,2-dichloroethane;

DPPA:diphenylphosphorylazide;

Et 3N:triethylamine;

Xa.a:unspecified

orunknown

amino

acid;Boc 2O:di-tert-butyldicarbonate;

tBuOK:potassium

tert-butoxide;

n.d.notdetermined;n.i.:notindicated.

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Page 7: Synthesis and applications of Rhodamine …...Synthesis and applications of Rhodamine derivatives as fluorescent probes Mariana Beija, Carlos A. M. Afonso and Jose´ M. G. Martinho

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 40,(50)-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,N0-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 40 and 50 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

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 20 or at position 40 or

both; so, usually a mixture of isomers is obtained.67

Recently, Uddin and Marnett reported an efficient

synthetic route for preparation of ridigised 40 and

Scheme 2 Mechanism of fluorescence unmasking of bis(acetylated trimethyl lock) Rho 110 lead by esterase catalysis.

Scheme 3 Condensation reaction for synthesis of modified Rhodamine as proposed by Corrie et al.52

2416 | Chem. Soc. Rev., 2009, 38, 2410–2433 This journal is �c The Royal Society of Chemistry 2009

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50-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 40 derivative is

obtained with 34% (n-propylene) and 32% (g,g-dimethyl-

propylene) yields and 50 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

Table 4 Some commercially available reactive rhodamine derivatives

Commercial name Structure Isomer h/ga

Rho 110 Rhodamine Greent, carboxylic acid,succinimidyl ester, hydrochloride

40 and 50 (mixed) 71 800

Rhodamine Greent-X, succinimidyl ester,hydrochloride

40 and 50 (mixed) 71 800

SRho B Rhodamine Redt-X, succinimidyl ester,hydrochloride

40 41 200

Rhodamine Redr C2, maleimide 20 and 40 (mixed) 52 100

Rho 101 X-rhodamine-isothiocyanate 40 and 50 (mixed) 27 000

Carboxy-X-rhodamine,Succinimidyl ester

40 and 50 (mixed) 8250

40 72 70050 58 000

a An average for suppliers such as Sigma-Aldrich or Molecular Probes.

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functionalize low-priced commercially available rhodamine

dyes. Their usual application is the dye-labelling of molecules

of interest.

Modification of the carboxylic acid group (position 20)

Although 20-position could be seen as the easiest to

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

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 20-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

Also using a strategy of activation for the carboxylic acid

group, Grechishnikova et al. prepared a bisteroid-Rho 101

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

(Table 5, entry 4).72 However, only after the pioneering work

reported by Czarnik’s group in 1997 and especially over the

past 3–4 years a more significant development in the synthesis

of 20-rhodamine derivatives has arisen, when the spirolactam

(nonfluorescent) to ring-opened amide (fluorescent) process of

rhodamine dyes was demonstrated to be attractive in the

conception of chemosensors of metal ions (Fig. 3).

Scheme 4 Usual synthetic route for the synthesis of succinimidyl

derivatives of Rhodamine dyes. DSC: di-N-succinimidylcarbonate,

DIPC: N,N0-diisopropylcarbodiimide; NHS: N-hydroxysuccinimide.

Scheme 5 Synthetic route for preparation of 40 and 50 TMR derivatives.

Fig. 3 Spirolactam ring-opening process of Rho B derivative.

2418 | Chem. Soc. Rev., 2009, 38, 2410–2433 This journal is �c The Royal Society of Chemistry 2009

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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 20-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,

BODIPY or fluorescein) that will behave as Forster 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

20-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 20-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 20-ester derivatives103 and 20-amide derivatives.104

In the former example, the lactone of Rho 6G was prepared

by pyrolysis and further reacted with activated alkyl halides

Scheme 6 Synthetic route for preparation of 40 and 50-carboxy-X-rhodamines containing g,g-dimethylpropylene bridging group as proposed by

Uddin and Marnett.68

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Table

5Rhodaminederivatives

obtained

bymodificationofthecarboxylicacidgroup

Entry

PrecursorRho

Structure

ReactionConditions

Yield

(%)Application

Ref.

1RhoB

1.RhoBethylester,methylamine(40%

inwater)

Light-screeningdyes

inphotographic

productsand

processes

69

2.Aceticacid,zinc

3.BuLi(1.6

Min

THF)

4.ClCO

2(C

H2) 2SO

2CH

3

5.Air(bubbling),iodine

n.d.

2Rho6G

Rho6G

carboxylicacid(3.5

eq.),benzylamine,

POCl 3(1.3

eq.)

n.d.

Dyeingofpaper

stocks

70

3RhoI

RhoI,amine,

DPPA

(1.3

eq.),DMF

61

Biologicaldiagnostic

assay

71

4Rho101

Rho101(3

eq.),bisteroid

diol,DCC

(5.2

eq.),

4-pyrrolidinopyridine(5.9

eq.),CHCl 3

78

FRETstudiesin

model

and

biologicalmem

branes

72

5RhoB

1.RhoBbase,DCE,POCl 3(reflux)

2.CH

3CN,anhydroushydrazine(excess)

80

Cu(II)chem

osensor

73

6RhoB,hydrazinehydrate,MeO

H(reflux)

64

Peroxynitrite

chem

osensor

74

2420 | Chem. Soc. Rev., 2009, 38, 2410–2433 This journal is �c The Royal Society of Chemistry 2009

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Table

5(continued

)

Entry

PrecursorRho

Structure

ReactionConditions

Yield

(%)Application

Ref.

7RhoB

1.RhoB,hydrazinehydrate,EtO

H(reflux)

2.2-H

ydroxybenzaldehyde(4

eq.)

57

30

8RhoB

RhoBhydrazide,

2-form

ylphenylboronic

acid

(4eq.),EtO

H(reflux)

47

Cu(II)chem

osensor

76

9Rho6G

1.Rho6G,hydrazinehydrate,EtO

H(reflux)

2.Salicylaldehyde(5

eq.),EtO

H/C

H2Cl 2,

reflux,12h

73

75

10

Rho6G

Rho6G

hydrazide,

glucose,toluene/MeO

H2:6

(reflux),PTSA

17

Hg(II)chem

osensor

77

11

RhoB

1.RhoB,hydrazinehydrate,MeO

H(reflux)

2.KSCN,EtO

H/H

2O

2M

HCl,reflux,10h

55

Hg(II)chem

osensor

79

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Table

5(continued

)

Entry

PrecursorRho

Structure

ReactionConditions

Yield

(%)Application

Ref.

12

RhoB

1.RhoB,hydrazinehydrate,MeO

H(reflux)

2.1-Ferrocene-2-(quinolin-8-yloxy)ethanone

(0.7

eq.),dry

toluene(reflux)

53

Multisignalingoptical-

electrochem

icalsensor

forHg(II)

24

13

Rho6G

1.Rho6G,hydrazinehydrate,MeO

H(reflux)

2.phenylisothiocyanate,DMF

86

Hg(II)chem

osensor

32

14

RhoB

RhoBhydrazide,

n–butylisothiocyanate,CHCl 3,

reflux,3days

68

Cu(II)chem

osensor

78

15

Rho6G

1.Rho6G,hydrazinehydrate,MeO

H(reflux)

2.2-Pyridinecarbaldehyde

85

Hg(II)chem

osensor

26

16

RhoB

1.RhoB,hydrazinehydrate,MeO

H(reflux)

2.benzoylchloride,

THF

65

Hypoclorite

chem

osensor

80

17

1.RhoBbase,POCl 3,CH

2Cl 2

2.Benzoic

hydrazide,

CH

3CN

45

pH

probe

81

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Table

5(continued

)

Entry

PrecursorRho

Structure

ReactionConditions

Yield

(%)Application

Ref.

18

RhoII

1.RhoII,POCl 3(5.2

eq.),70–751C

2.Ethylisonipecotate

(5.5

eq.),DMF,Et 3N

34

Synthesisoffluorescence

quenchers

82

19

RhoB

1.R

hoBbase,DCE,POCl 3(reflux)

2.HO–NH

2.H

Cl,CH

3CN,Et 3N

49

—85

20

60

Cu(II)chem

osensor

83

21

Rho6G

1.Rho6G,NaOH,H

2O

2.DCE,POCl 3(reflux)

3.HO–NH

2.H

Cl,DCE,Et 3N

36

Hypochlorousacid

chem

osensor

84

22

RhoB

1.RhoBbase,DCE,POCl 3(reflux)

2.MeO

–NH

2.H

Cl,CH

2Cl 2,Et 3N

81

Fe(

III)chem

osensor

86

23

RhoB

RhoB,3-aminopropyltriethoxysilane,

CHCl 3,reflux

100

Silica-linked

rhodamine

probe

87

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Table

5(continued

)

Entry

PrecursorRho

Structure

ReactionConditions

Yield

(%)Application

Ref.

24

RhoB

1.RhoBbase,DCE,POCl 3(reflux)

2.CH

3CN,Et 3N

2-((bis(2-(ethylthio)ethyl)amino)m

ethyl)aniline

26

Hg(II)chem

osensor

88

25

RhoB

RhoB,2-aminopyridine,

POCl 3(cat)

60

Chem

osensorfortransition

metalscations

89

26

RhoB

1.RhoB,POCl 3(reflux)

2.Bromoethylenaminehydrobromide,

Et 3N,CH

3CN

3.Aza-18-crown-6,CH

3CN,DIPEA

7.4

Fe(

III)andHg(II)selective

dualchem

osensor

90

27

RhoB

1.RhoB,POCl 3

2.2-Bromoethylaminehydrobromide,

Et 3N

3.Cyclen,toluene(reflux)

18

91

28

RhoB

1.RhoB,ethylenediamine,

EtO

H(reflux)

2.1-Isocyanate-4-nitrobenzene,

toluene(reflux)

32

Hg(II)chem

osensor

2424 | Chem. Soc. Rev., 2009, 38, 2410–2433 This journal is �c The Royal Society of Chemistry 2009

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Table

5(continued

)

Entry

PrecursorRho

Structure

ReactionConditions

Yield

(%)Application

Ref.

29

RhoB

1.RhoB,ethylenediamine,

EtO

H(reflux)

92

2.m-X

ylenediisocyanate

(0.5

eq.),toluene(reflux)

18

30

RhoB

1.RhoB,ethylenediamine,

EtO

H2.t-Boc-protected

a-bromoaceticacid,DIPEA,

NaI,CH

3CN

(reflux)

3.TFA,CH

2Cl 2

49

Cu(II)chem

osensor

23

31

RhoB

1.RhoB,diethylenetriamine,

MeO

H(reflux)

2.TsC

l,Pyr,CHCl 3

58

Hg(II)chem

osensor

29

32

RhoB

1.RhoBbase,DCE,POCl 3(reflux)

2.Diethylenetriamine,

CH

3CN

18

Fe(

III)chem

osensor

31

33

RhoB

1.RhoB,tren,MeO

H(reflux)

2.TsC

l,Pyr,CHCl 3

68

Hg(II)chem

osensor

29

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Table

5(continued

)

Entry

PrecursorRho

Structure

ReactionConditions

Yield

(%)Application

Ref.

34

RhoB

1.RhoB-trenconjugate,6-bromohexanoylchloride,

Et 3N,2days

2.Thiourea,EtO

H,reflux.5h

3.NaOH,water

81

Platinum

film

immobilized

Cu(II)chem

osensor

101

35

RhoB

1.RhoB,tren,MeO

H(reflux)

2.3-(Triethoxysilyl)propylisocyanate,toluene,

801C.

43

Mesoporoussilica

immobilized

Hg(II)

chem

osensor

93

36

RhoB

1.RhoBbase,DCE,POCl 3(reflux)

2.CH

3CN,cystaminedihydrochloride(0.56eq.),

Et 3N

56

Hg( II)chem

osensor

94

37

RhoB

1.RhoB,EtO

H,2-aminoethanol,1201C

2.MsC

l,Et 3N,CH

2Cl 2

3.NaI,acetone(reflux)chem

osensor

84

Ag(I)chem

osensor

95

2426 | Chem. Soc. Rev., 2009, 38, 2410–2433 This journal is �c The Royal Society of Chemistry 2009

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Table

5(continued

)

Entry

PrecursorRho

Structure

ReactionConditions

Yield

(%)Application

Ref.

38

RhoB

1.RhoBhydrazide

2.8-hydroxylquinoline-2-aldehyde,

EtO

H(reflux)

3.2-hydroxyethyl-4-(6-m

orpholin-4-yl-1H,

3H-benzo[de])-isoquinoline,

anhydrousTHF

40

Cr(

III)chem

osensor

14

39

RhoB

1.RhoB,tren,MeO

H(reflux)

2.Dansylchloride,

Et 3N,CHCl 3

40

Cu(II)chem

osensor

21

40

RhoB

RhoBhydrazide,

1-ethynyl-BODIPY-

4-isothiocyanate

benzene,

DMF,501C

53

Hg(II)chem

osensor

99

41

RhoB

RhoBhydrazide,

fluoresceinisothiocyanate,

DMF

89

100

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Table

5(continued

)

Entry

PrecursorRho

Structure

ReactionConditions

Yield

(%)Application

Ref.

42

Rho6G

Rho,amine(2–3eq.),DMF,DIPEA

(forneutralizationofaminesalts),

54–92

Rhodamineconjugates

102

43

Rho110

44

Rho6G

1.Rho6G,265–2751C

(pyrolysis)

2.DIPEA

(1.2

eq.),NaI(cat),CH

3CN,

BrC

H2CO

2CH

2Phora,a0 -dichloro-p-xylene

3.Further

modification

69–86

Fluorescentprobes

for

conjugationto

amino

acidsandpeptides

103

45

RhoB

1.AlM

e 3,piperazine,

CH

2Cl 2

2.Rhobase

(reflux)

70

Rhodamineprobes

104

46

Rho6G

48

47

Rho101

6

48

Rho101

Rho101,amine(1.3

eq.),HATU

(2eq.),Et 3N,

CH

2Cl 2

66–80

Reversible

redfluorescent

molecularsw

itches

17

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Table

5(continued

)

Entry

PrecursorRho

Structure

ReactionConditions

Yield

(%)Application

Ref.

49

RhoB

RhoBhydrazideorRhoBhydroxylamide,

Lawesson’sreagent

19

Hg(II)chem

osensor

85

50

RhoB

1.RhoBbase,POCl 3(reflux)

97

2.Thiourea,Et 3N

45

51

1.RhoBbase,DCE,POCl 3(reflux)

105

2.Na2S(aq)

85

52

RhoBbase,Lawesson’sreagent

65

105

53

Rho6G

1.Rho6G,NaOH

(aq),EtO

H,reflux

2.POCl 3,DCE,reflux

3.Et 3N/THF,thioureain

water

51

96

Rho

B:RhodamineB;Rho

6G:Rhodamine6G;Rho

101:Rhodamine101;BuLi:

butyllithium;BODIPY:boron–dipyrromethene;

THF:tetrahydrofuran;DPPA:diphenylphosphorylazide;

DMF:N,N

-dim

ethylform

amide;

DCC:N,N0 -dicyclohexylcarbodiimide;

Et 3N:triethylamine;

DIPEA:N,N

-diisopropylethylamine;

EtO

H:ethanol;MeO

H:methanol;PTSA:p-toluenesulfonic

acid;

MsC

l:methanesulfonylchloride;

TsC

l:tosylchloride;

Pyr:pyridine;

t-Boc:

tert-Butyloxycarbonyl;TFA:trifluoroaceticacid;tren:tris(2-aminoethyl)amine;

HATU:2-(1H-7-A

zabenzotriazol-1-yl)-

1,1,3,3-tetramethyluronium

hexafluorophosphate

methanaminium;DCE:1,2-dichloroethane;

tren:tris(2-aminoethyl)amine.

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

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.

2430 | Chem. Soc. Rev., 2009, 38, 2410–2433 This journal is �c The Royal Society of Chemistry 2009

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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

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 20 (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

20-position with an N,N0-dialkylamide or ester derivative or,

as the most part of available commercial functionalized

rhodamine dyes, at positions 40 and/or 50. 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 40 or 50. 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 40 and 50 are less

sterically hindered than position 20, 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 20 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.

Scheme 10 Synthesis of Rho B piperazine amide derivative.

Scheme 11 Synthesis of Rho 101 amide derivative using HATU as coupling agent.

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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

applications.

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 Fundacao para a Ciencia 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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