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 José M. G. Martinho

Received 26th January 2009

First published as an Advance Article on the web 27th April 2009

DOI: 10.1039/b901612k

Rhodamine dyes are widely used as fluorescent probes owing to their high absorption coefficient

and broad fluorescence in the visible region of electromagnetic spectrum, high fluorescence

quantum yield and photostability. A great interest in the development of new synthetic

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,

Gonçalves 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 Quı́mica-Fı́sica Molecular and IN–Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, 1049-001, Lisboa, Portugal. E-mail: [email protected], [email protected], [email protected]; Fax: +351 218 464 455

Mariana Beija

Mariana Beija was born in São Paulo (Brazil) in 1981. She studied Chemistry in Instituto Superior Técnico (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. José M. G. Martinho, in Centro de Quı́mica-Fı́sica Molecular (Instituto Superior Técnico, Lisbon, Portugal), and Dr Marie-Thérèse Charreyre, in Unité Mixte CNRS-

bioMérieux (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 Técnico as associate professor and in 2008 received his Agregação. His research focus is mainly on the development of more sustainable methodologies in asymmetric organic transformations.

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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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 , l max 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