Absorption and emission UV-Vis spectra of the TRITC fluorophore

molecule in solution: a quantum mechanical study

Alfonso Pedone,*a Julien Bloino,ab Susanna Monti,c Giacomo Prampolinibd and

Vincenzo Baronea

Received 29th September 2009, Accepted 30th October 2009

First published as an Advance Article on the web 3rd December 2009

DOI: 10.1039/b920255b

The absorption and emission properties as well as the electronic structure in the ground (S0) and

excited (S1) states of tetramethylrhodamine isothiocyanate (TRITC) fluorophore molecule have

been investigated by time-dependent density functional theory (TD-DFT). The effect of water

and ethanol solvents on the structure and optical properties of the dye was taken into account

by using both explicit and implicit solvent models, as well as combinations of them.

Different hybrid and long range corrected functionals have been tested in reproducing absorption

and emission transition energies. It has been found that the B3LYP functional coupled with mixed

explicit/implicit solvent models reproduces correctly experimental data concerning both the solvent

and Stokes shifts. This work presents a first step to a more challenging project devoted to the

development of integrated multiscale approaches and protocols for studying optical properties

of fluoroprobes embedded in biological systems and/or encapsulated in nanoparticles of

technological interest.

Introduction

Tetramethylrhodamine isothiocyanate (TRITC) is an amine-

reactive derivative of rhodamine dye and finds a wide-ranging

application as antibody and probe label in fluorescence

microscopy, flow cytometry and immunofluorescence-based

assays.1,2

TRITC is functionalized with an isothiocyanate reactive

group (–NQCQS) at one of the meta or para positions on

the bottom ring of the structure (see Fig. 1) which makes it

reactive towards primary amine groups on proteins, peptides

and other biomolecules.3

Together with fluorescein, it is one of the most common

fluorophores used as labeling probes. Other labels include

fluorescent proteins such as the various forms of green

fluorescent protein (GFP) and the phycobiliproteins.4 While

having the ability to produce an intense fluorescent signal for

detection, fluorescent proteins can be difficult to optimize for

conjugation purposes and may create steric hindrance or

background signal issues in binding assays.

Although not as sensitive as enzymatic detection, the use of

fluorophore-conjugated probes in fluorescent detection

methods reduces chemical waste and has the added advantage

of multiplex compatibility; that is, using more than one

fluorophore in the same experiment.

The growing demand for multiplex assays has driven the

development of many new fluorescent dyes.5 A great effort has

been spent in the design and synthesis of new fluorophores

which are brighter and more photostable than the traditional

fluorescein and rhodamine molecules and give rise to a

broader range of non-overlapping spectral bands.

Moreover, recent studies regarding the encapsulation of

TRITC molecules in silica-based nanoparticles has paved the

route towards the development of a new class of highly

fluorescent and photostable core–shell nanoparticles.6 These

systems show tremendous promise as indicators and photon

sources for a number of biotechnological and information

technology applications.7

Information on the behavior of fluorophores in solution as

well as in more complex biological and nano materials can be

gained, in principle, by a number of spectroscopic techniques.

However, the interpretation of the rich indirect information

that can be inferred from the analysis of experimental

spectra is seldom straightforward without reference to some

theoretical model.

In this context, computational simulations are valuable

tools for the interpretation and prediction of optical properties

of fluorophores embedded in different environments and

effective methodologies for the design of new compounds

due to their capability to rapidly screen the photocenters

and determine the structure–property relationships of these

systems.8

However, the size of these systems still represents a great

challenge for the most advanced theoretical approaches and a

compromise between accuracy and computational cost needs

to be done.

Quantum mechanical methods represent undoubtedly the

main ingredients for a reliable evaluation of optical properties

of dyes9 but they have been essentially restricted to the

calculation of UV-Vis spectra of relatively small biomolecules

in the gas phase, mainly because of the difficulties in obtaining

a Scuola Normale Superiore, Piazza dei Cavalieri 7, 56100, Pisa,Italia. E-mail: alfonso.pedone@sns.it; Tel: +39 050509069

bUniversita di Napoli ‘‘Federico II’’, Complesso Universitario diMonte Sant’Angelo, Via Cintia, I-80126, Napoli, Italia

c Istituto per i Processi Chimico-Fisici, via G. Moruzzi 1, Pisa,56100, ITALIA

dDipartimento di Chimica, Universita di Pisa, via Risorgimento 35,56100, Pisa, ITALIA

1000 | Phys. Chem. Chem. Phys., 2010, 12, 1000–1006 This journal is �c the Owner Societies 2010

PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics

accurate descriptions of the excited electronic states of

polyatomic molecules. Recent developments in the electronic

structure theory for the excited states within the time-

dependent density functional theory (TD-DFT)10–12 and

resolution of the identity approximation of coupled cluster

theory have paved the route toward the simulation of spectra

for significantly larger systems.13,14

TD-DFT has been successfully used in a wide variety of

bio-chemical and physical problems.15–22 Several studies have

shown that the most common discrepancies on the excited

state energies depend on the type of the chromophoric unit, on

the nature of the excitation (singly-excited versus doubly

excited and n - p* versus p - p*), on the selected computa-

tional procedure (functional and basis set) and on the model

used to treat environment effects (e.g. implicit and explicit

solvent).15–22

Finally, environmental effects play a crucial role, not only

perturbing the spectroscopic parameters but also stabilizing

specific conformations. Glycine, which undergoes a chemical

equilibrium between its neutral form (stable in apolar solvents)

and the zwitterionic form (stable in polar solvents like water)

represents a typical example.23 Similarly, TRITC has two

isomeric forms stable in different environments (vide infra).24

In order to evaluate environmental effects (e.g. solvent,

biologically active macromolecules and nano particles) one

can resort to explicit and implicit models.25

However, it is not straightforward to well reproduce

both the structural and optical properties of large flexible

molecules in different environments and different integrated

computational approaches are mandatory.

In this paper, we explore intrinsic and environmental effects

on optical properties as well as the energetic of stabilization of

the excited state of a large flexible molecule (TRITC)

embedded in water and ethanol solvents using TD-DFT

calculations. We will explore the polarizable continuum model

extended with the inclusion of close solvent molecules, both in

the fully quantum-mechanical treatment as well as coupled

with QM/MM methods-like ONIOM.26–28

Computational methods

All calculations have been performed with a locally-modified

version of the Gaussian suite of programs.29 Except when

explicitly noted, the hybrid exchange–correlation functional

B3LYP30 coupled with the double-z N07D basis set31,32 has

been used. This basis set has been constructed by adding a

reduced number of polarization and diffuse functions to the

6-31G set which leads to performances comparable to those

of aug-cc-pVDZ basis set, with increased computational

efficiency.

Absorption spectra were simulated as follows. The S0 state

was optimized at the DFT level and the vibrational frequencies

have been evaluated by the analytical determination of the

Hessian matrix in order to check the absence of imaginary

frequencies. Then, excitation energies were computed by using

time dependent density functional theory (TD-DFT),10 which

has proven to be a robust and accurate method for describing

low-lying excited states.33,34

The recent implementation of analytical energy gradients11,12

in TD-DFT allows for the geometry optimization of molecules

in their excited states. This is an essential step for the

simulation of emission spectra. In this work the S1 - S0transition energy has been evaluated by performing a vertical

transition from the optimized S1 excited state.

The inclusion of environmental effects is crucial for

the correct calculation of electronic transition energies

especially if the polar groups of the molecule are exposed to

the solvent.

Fig. 1 (a) Numbering of the zwitterionic form of TRITC molecule optimized in PCM. (b) Closed lacton form of TRITC optimized in PCM.

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Accurate simulations of bulk effects for both non-polar and

polar solvents can been obtained by polarizable continuum

models (PCMs).25 In PCM, the model is divided into a solute

part, the dye, lying inside a cavity, surrounded by the solvent

part represented as a structureless material characterized by its

macroscopic properties, i.e. dielectric constants and solvent

radius. This method reproduces well solvent effects when no

specific interaction (such as hydrogen bonds) takes place

between the solute and the solvent molecules. When this is

the case, as in the present work, the best compromise is

represented by mixed models, where all molecules involved

in the interactions (i.e. the first solvation shell) are explicitly

considered in the electronic calculations and then embedded in

a PCM to simulate bulk effects.

The so-called non-equilibrium and equilibrium PCM solutions

have been selected for the study of absorption and for the

excited state optimization, respectively.16 The solvatocromic

shift between water and ethanol has been studied by using

both pure PCM and considering explicit solvent molecules

(treated both with QM and MM methods) solvating the

carboxylate group.

Results and discussions

Energy stability: lactone vs. zwitterionic form

In order to discuss the structural and electronic properties of

TRITC we shall refer to the atom numbering displayed in

Fig. 1.

TRITC is commercialized as powders in several isomeric

forms depending on the position of the thiocyanate moieties.

Among these, two forms, namely a zwitterionic form

(see Fig. 1a) and a closed lactonic form in which O2 bonds

to C6 forming a 5-member ring (see Fig. 1b) can be found.

Geometry optimization of both structures at the B3LYP/

N07D level shows that the lactonic form is the only one

present in vacuum. The situation changes when solvent

effects are introduced by the PCM model, in such a case the

zwitterionic form being 1.72 kcal mol�1 more stable than the

closed one. This is a lower bound since, neglecting the explicit

H-bond interactions of the carboxylate group with the

surrounding water molecules leads to an overestimation of

the stability of the lactonic form. Although it is gratifying that

the PCM approach alone is able to stabilize the zwitterionic

form we are not interested in the right stabilization energy.

Moreover, since the zwitterionic form of TRITC showed

brightness enhancement when embedded in silica nano-

particles, we will focus our investigation on the excited states

and optical properties of this form.

Geometry and electron distribution of the S0 and S1

optimized states

The ground and excited states of the zwitterionic form of the

TRITC molecule in water and ethanol have been studied in

two different scenarios. In the first one, the molecule is placed

in a polarizable continuum solvent, whereas in the second one

explicit solvent molecules, interacting with the carboxylate

group, are included in the model and treated, like TRITC, at

the QM level.

The number of water molecules coordinating the

carboxylate group was determined by comparing the solute-

water binding energies due to the addition of one molecule

at a time with respect to the binding energy of the water–

water pair. It was found that 4 water molecules (two per

oxygen) are bound to the carboxylate group in the first

solvatation shell. Instead, in the case of ethanol, only two

molecules (one per oxygen) have been considered because of

steric hindrance.

Preliminary results obtained through classical molecular

dynamics simulations confirmed our approach. The discussion

of the results of the classical simulations is outside the scope of

the present work and will be reported in a future paper.35

The optimized S0 geometry of TRITC embedded in

PCM-water is showed in Fig. 1. No changes are observed

when the optimization was performed in ethanol. Indeed, the

maximum difference on bond lengths and angles was about

0.001 A and 0.21, respectively.

The molecule is composed of two mostly rigid aromatic

parts connected by a single bond (C6–C9) of 1.496 A, the

plane of the benzenoid ring is approximately perpendicular to

the plane of the phenolic rings and the dihedral angle between

these two planes (defined as C8–C6–C9–C12) is about 94.61

and and 61.11 in the S0 and S1 states, respectively.

The upper part of the molecule carries two ternary amine

groups with C19–N5 and C18–N4 bond lengths (1.37 A)

intermediate between typical single C–N (1.47 A) and double

CQN (1.27 A) bonds for both ground and excited states.

The optimization of the S1 excited state leads to small

changes which are mainly localized on the bond angles of

the carboxylate group (O2–C23–O3 bends from 127.31 to

111.81 and the O2–C23–C12 increases from 115.61 to 125.71)

and the dihedral angles of the phenyl and carboxylate groups

(O2–C23–C12–C9 increases from 01 to 18.51). Additionally, it

is worth underlying the coupling between the former dihedrals:

as the C8–C6–C9–C12 dihedral deviates from its ground state

value of B901, the carboxylate group loses its co-planarity

with the benzenoid bottom ring.

Major differences of 0.02–0.05 A have been found in the

bond lengths for a few bonds, i.e., C19–N5 (1.357/1.375 A for

S0/S1 states) C6–C7/8 (1.414/1.435 A), C6–C9 (1.496/1.476 A)

and C12–C23 (1.536/1.487 A).

The key geometrical parameters of the optimized structures

of TRITC embedded PCM–water as well as in the different

explicit solvents are reported in Table 1.

As can be inferred by inspection of Table 1, the introduction

of explicit solvent molecules does not affect the bond lengths

and angles of the solute, the only differences found between S0and S1 states in both solvents being due to the flexibility of the

C8–C6–C9–C12 dihedral angle.

As mentioned previously, the inclusion of explicit water or

ethanol molecules (see Fig. 2 and 3 for their geometrical

arrangements) does not change the structural features of the

carboxylate group in the ground states. However, while the

valence angles related to the carboxylate group show a

significant change between the ground and excited states in

the implicit solvent (with a decrease of 161 for the O2–C23–O3

angle for example), this is not the case when the explicit

solvent molecules are included.

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The lengths of the bonds participating in hydrogen bonding

between the TRITC molecule and the solvent molecules are

shown in Table 2. The structural changes close to the explicit

solvent molecules are very small.

By analyzing the Mulliken charges of the backbone of the

TRITC molecule both in the ground and in the excited

electronic states, and both with and without the inclusion of

solvent molecules we can gain some insight into the charge

reorganizations due to the electronic excitations.

In Fig. 4, the charge distribution of the TRITC molecule

embedded in implicit solvent (PCM–water) is schematically

reported. Positive charges are localized on the C10, C11, C12,

C18 and C19 carbon atoms while negative charges are located

on the O1-3 and N4-5 atoms.

In most cases, the effect of excitation is to increase the

charges on C7–10 atoms (they become less negative) and

reduce the charges on C18, C19 and C23 atoms (they become

less positive).

Absorption and emission spectra

The S1 ’ S0 and S1 - S0 transition energies of the TRITC

molecule have been calculated in vacuum, (referred as

TRITC-V, i.e. single point calculation on the zwitterionic

structure previously optimized in PCM-water), with 4 QM

water molecules (TRITC-4QMW), embedded in implicit

solvent (TRITC-PCM-W), in interaction with 4 explicit

solvents and embedded in PCM (TRITC-4QMW-PCM).

The results are summarized in Table 3.

The energies show an increasing red shift when passing from

TRITC-V to TRITC-4QMW-PCM. The Table also shows

that the PCM accounts for the major contribution to

this shift (about 48 nm) with respect to explicit molecules

(about 13 nm).

The difference between the transition energies of TRITC-

4QM-PCM and the experimental ones are of 0.31 eV and 0.25 eV

for the absorption and emission spectra, respectively. These

discrepancies are in agreement with several theoretical studies

that revealed a typical mean absolute error of 0.2–0.3 eV for

TD-DFT/B3LYP investigations in organic chromogens.36,37

The analysis of the orbital compositions of the computed

singlet excited state shows that the lowest energy absorption is

due to the first dipole-allowed p- p* transition from HOMO

to LUMO with the largest oscillator strength.

These orbitals, which are depicted in Fig. 5, are localized on

the upper aromatic part of the molecule.

As reported in Table 3, the experimental shift (Dl = 25 nm)

of the emission spectra with respect to the absorption one is

Table 1 Structural features of the S0 and S1 states of TRITC embedded in implicit and explicit solvents. Bond lengths are in A and angles in deg

PCM–water Explicit-water–PCM Explicit-ethanol–PCM

S0 S1 S0 S1 S0 S1

Bond lengths

O1–C10/11 1.361 1.373 1.360 1.364 1.360 1.372C8/7–C11/10 1.423 1.424 1.426 1.423 1.424 1.422C11/10–C16/15 1.383 1.386 1.381 1.386 1.383 1.383C16/15–C19/C18 1.418 1.418 1.419 1.418 1.418 1.426C18/19–N4/5 1.357 1.375 1.354 1.363 1.356 1.368N4/5–Me 1.463 1.458 1.464 1.460 1.463 1.459C19/18–C22/21 1.439 1.423 1.440 1.433 1.439 1.426C14/13–C22/21 1.371 1.385 1.370 1.377 1.370 1.380C7/8–C13/14 1.425 1.420 1.427 1.426 1.425 1.423C6–C8/7 1.414 1.435 1.412 1.438 1.412 1.431C6–C9 1.496 1.476 1.493 1.471 1.496 1.485C9–C12 1.409 1.423 1.412 1.423 1.410 1.411C9–C17 1.406 1.413 1.405 1.414 1.406 1.411C12–C20 1.398 1.400 1.398 1.396 1.398 1.400C17–C25 1.391 1.388 1.391 1.388 1.391 1.390C24–C20 1.400 1.397 1.401 1.403 1.401 1.400C24–C25 1.404 1.408 1.404 1.407 1.404 1.405C12–C23 1.536 1.487 1.522 1.521 1.530 1.533C23–O2 1.260 1.268 1.264 1.265 1.260 1.258C23–O3 1.257 1.256 1.260 1.261 1.259 1.262C24–N51 1.377 1.374 1.376 1.373 1.376 1.377N51–C30 1.181 1.184 1.183 1.182 1.181 1.181C30–S52 1.609 1.607 1.606 1.610 1.608 1.610Bond angles

O2–C23–O3 127.3 111.8 125.8 125.3 127.1 126.3O2–C23–C12 115.6 125.7 117.2 117.5 116.2 116.9C23–C12–C9 121.7 124.2 122.5 122.9 122.1 122.2C11–O1–C10 121.2 120.7 121.5 120.3 121.3 120.0Me–N4/5-Me 118.9 118.6 118.9 118.8 118.9 118.7C24–N51–C30 179.9 168.2 169.7 176.1 176.5 177.6C7–C6–C8 118.5 117.9 118.8 116.6 118.7 117.3Dihedral angles

O2–C23–C12–C9 0 18.5 37.7 39.4 0.9 2.4C23–C12–C9–C6 0 �5.5 3.3 7.2 �0.4 0.5C8–C6–C9–C12 94.6 61.1 112.0 125.7 94.5 107.6

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well reproduced when the molecule is solvated with 4 explicit

water molecules treated at the ab initio level (Dl = 32.4 nm).

The inclusion of explicit solvent molecules is also needed if

the effect of different solvents on the absorption and emission

spectra must be reproduced. In fact the spectra of TRITC

embedded in water and ethanol solvents treated as a

continuum by means of the PCM approach are essentially

identical. Conversely, the inclusion of explicit solvent

molecules reproduces correctly a red shift of 13.6 nm from

ethanol to water, the experimental one being of 10 nm.6

The absorption and emission spectra of the TRITC

molecule interacting with explicit solvent molecules and

embedded in PCM are reported in Fig. 6.

Here the molar extinction coefficients of the simulated

spectra have been calculated by using a Gaussian model:21

eðlÞ ¼XNi¼1

fi

sexp �2:773 ðl� liÞ2

s2

!

where fi is the oscillator strength of the ith transition, N is the

number of transitions (here one because we studied the

S1 ’ S0 and S1 - S0 transitions for the absorption

and emission spectra, respectively), li is the wavelength of

electronic transition energies in nm and s is the half-

bandwidth which has been taken equal to 50 nm.

It is worth to note that our calculations show that the

oscillator strengths of the absorption and emission spectra of

the zwitterionic form of TRITC in water are 0.90 and 0.76,

respectively. The measured spectra were normalized to unity

and a direct comparison of the relative intensities is not

possible.

In Table 4 the absorption and emission energies of TRITC

embedded in explicit solvents treated at the QM level are

compared with those calculated by considering the solvent

molecules at the MM level within the ONIOM approach.

A two-layer model has been employed, performing QM/MM

computations with the so-called ‘electronic embedding’ scheme,

namely including the point charges of the MM layer in the QM

Hamiltonian of the model system according to the scheme:

HelðQM : MMÞ ¼ HelðQMÞ �XYi

XXJ

qJ

riJ

Fig. 2 Ground state optimized structure of TRITC interacting with

4 water molecules optimized at QM level.Fig. 3 Ground state optimized structure of TRITC interacting with

2 ethanol molecules optimized at the QM level.

Table 2 Lengths (in A) of hydrogen bonds between the carboxylategroup of TRITC with explicit water and ethanols molecules in S0 andS1 states

Explicit-water–PCM

S0 S1

W1 1.75 1.74W2 1.79 1.78W3 1.81 1.79W4 1.84 1.83

Explicit-ethanol–PCM

S0 S1

Et1 1.74 1.74Et2 1.76 1.76

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Where Hel(QM:MM) and Hel(QM) are the electronic

Hamiltonians for the QM region with and without the external

electrostatic field, Y is the number of electrons in the model

system and X is the number of point charges in the MM

region. This scheme allows the QM wave function to be

properly polarized by the electrostatic properties of the

surroundings. The Rappe and Goddard scheme has been

employed to calculate the atomic charges of the solvent

molecules.38

The two approaches provided very close results,

demonstrating that solvent effects on the p - p* vertical

transition are essentially of electrostatic nature.

As mentioned above, the B3LYP functional underestimates

lmax of the absorption spectra by 0.31 eV. Therefore, in

Table 5, we have compared the accuracy of different

hybrid (B3LYP30 and M06-2X39) and long-range corrected

(CAM-B3LYP,40 WB97XD41 and LC-WPBE42) functionals

when water molecules are considered as point charges.

The Table justifies the use of B3LYP functional, which

performs better than the long-range corrected ones and is

Fig. 4 Schematic representation of the Mulliken charge distribution

of TRITC in the ground state. Coloring scheme: green (more positive

charges), red (more negative charges).

Table 3 Vertical S1 ’ S0 and S1 - S0 excitation energies(eV in parenthesis) in nm with different models

lS1-S0 lS1’S0 DlStokes

TRITC-V — 419.3 (2.96) —TRITC-4QMW — 436.3 (2.84) —TRITC-PCM-W 509.4 (2.43) 466.8 (2.66) 42.6TRITC-4QMW-PCM 512.6 (2.42) 480.2 (2.58) 32.4Exp.6 572.0 (2.17) 547.0 (2.27) 25.0

Fig. 5 HOMO (left) and LUMO (right) molecular orbitals of TRITC-PCM-W at the B3LYP/N07D level of theory.

Fig. 6 Calculated absorption (continuous line) and emission

(dotted line) spectra of TRITC interacting with 4 explicit molecules

and embedded in PCM solvents compared to the experimental spectra

taken from ref. 6.

Table 4 Vertical S1 ’ S0 and S1 - S0 excitation energies in nm withdifferent explicit solvents treated at the QM or MM level

lS1-S0 QM/MM lS1’S0 QM/MM

TRITC-2Et-PCM 498.1/496.2 466.6/465.5TRITC-4W-PCM 512.6/510.1 480.2/478.7Dlsolvatochromic 14.5/13.9 13.6/13.2

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comparable to the hybrid M06-2X functional which has an

error of 0.30 eV.

Conclusions

The effect of the environment on optical properties of

TRITC fluorophore has been studied by means of TD-DFT

calculations by using different approaches. The B3LYP

functional coupled with mixed explicit/implicit solvent models

reproduces well the red shift between water and ethanol

solvents as well as the Stokes shift between absorption and

emission spectra.

It has been demonstrated that the explicit solvent molecules

can be safely treated at the MM level as point charges since

solvent effects on the p - p* vertical transition are essentially

of electrostatic nature.

This work presents a first step to a more challenging project

devoted to the development of integrated multiscale

approaches and protocols for studying optical properties of

important spectroscopic probes such as TRITC in biological

systems and encapsulated in nanoparticles of technological

interest.

Acknowledgements

The authors thank ‘Telecom Italia’ and ‘Compagnia di San

Paolo’ for financial support through grants, and Village-Na

(http://village.unina.it) for computational resources.

References

1 M. Miki and C. G. dos Remedios, J. Biochem., 1988, 104, 232–235.2 L. Larsson, CRC, Boca Raton, 1988, vol. 77–83, pp. 224–225.3 J. Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, London, 3rd edn, 1996.

4 K. F. Sullivan and S. A. Kay, Green Fluorescent Proteins,Academic Press, 1999.

5 J. E. Berlier, A. Rothe, G. Buller, J. Bradford, G. D. R.,B. J. Filanoski, W. G. Telford, S. Yue, J. Liu, C.-Y. Cheung,W. Chang, J. D. Hirsch, J. M. Beechem, R. P. Haugland andR. P. Haugland, J. Histochem. Cytochem., 2003, 51, 1699–1712.

6 H. Ow, D. R. Larson, M. Srivastava, B. A. Baird, W. W. Webband U. Wiesner, Nano Lett., 2005, 5, 113–117.

7 E. Herz, A. Burns, S. Lee, P. Sengupta, D. Bonner, H. Ow,C. Liddell, B. A. Baird and U. Wiesner, in Colloidal QuantumDots for Biomedical Applications, Proc. SPIE, 2006, vol. 6096,pp. 1–12.

8 D. Jacquemin, E. A. Perpete, I. Ciofini and C. Adamo, Acc. Chem.Res., 2009, 42, 326–334.

9 V. Barone, R. Improta and N. Rega, Acc. Chem. Res., 2008, 41,605–611.

10 E. Runge and E. K. U. Gross, Phys. Rev. Lett., 1984, 52, 997–1000.11 C. van Caillie and R. D. Amos, Chem. Phys. Lett., 1999, 308,

249–255.

12 G. Scalmani, M. J. Frisch, B. Mennucci, J. Tomasi, R. Cammi andV. Barone, J. Chem. Phys., 2006, 124, 094107.

13 B. O. Roos, in Computational Photochemistry, ed. M. Olivucci andJ. Michl, Elsevier, Amsterdam, 2005, vol. 1.

14 J. Paldus, The beginnings of coupled-cluster theory: An eyewitnessaccount, in Theory and Applications of Computational Chemistry:The First Forty Years, Elsevier, Amsterdam, 2005.

15 C. Adamo and V. Barone, Chem. Phys. Lett., 2000, 330, 152–160.16 M. Cossi and V. Barone, J. Chem. Phys., 2001, 115, 4708–4717.17 C. Jamorski-Jodicke and H. P. Luthi, J. Am. Chem. Soc., 2003,

125, 252.18 S. Zalis, N. Ben Amor and C. Daniel, Inorg. Chem., 2004, 43,

7978–7985.19 R. Improta and V. Barone, J. Am. Chem. Soc., 2004, 126, 14320.20 E. A. Perpete, J. Preat, J.-M. Andre and D. Jacquemin, J. Phys.

Chem. A, 2006, 110, 5629–5635.21 X.-H. Zhang, L.-Y. Wang, G.-H. Zhai, Z.-Y. Wen and

Z.-X. Zhang, J. Mol. Struct. (THEOCHEM), 2009, 906, 50–55.22 O. Clemens, M. Basters, M. Wild, S. Wilbrand, C. Reichert,

M. Bauer, M. Springborg and G. Jung, J. Mol. Struct.(THEOCHEM), 2008, 866, 15–20.

23 J. P. Greenstein and M. Winitz, Chemistry of the Amino Acids,John Wiley & Sons, New York, 1961.

24 U. K. A. Klein and F. W. Hafner, Chem. Phys. Lett., 1976, 43,141–145.

25 J. Tomasi, B. Mennucci and R. Cammi, Chem. Rev., 2005, 105,2999–3094.

26 F. Maseras and K. Morokuma, J. Comput. Chem., 1995, 16, 1170.27 M. Svensson, S. Humbel, R. D. J. Froese, T. Matsubara, S. Sieber

and K. Morokuma, J. Phys. Chem., 1996, 100, 19357.28 S. Humbel, S. Sieber and K. Morokuma, J. Chem. Phys., 1996,

105, 1959.29 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,

M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li,H. R. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng,J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda,J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,H. Nakai, T. Vreven, J. A. P. J. R. Montgomery Jr, F. Ogliaro,M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin,V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari,A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi,N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross,V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts,R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi,C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma,V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg,S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz,J. Cioslowski and D. J. Fox, GAUSSIAN DV, Revison H.05,Gaussian, Inc., Wallingford CT, 2009.

30 A. D. Becke, J. Chem. Phys., 1993, 98, 5648–5652.31 V. Barone, P. Cimino and E. Stendardo, J. Chem. Theory Comput.,

2008, 4, 751–764.32 V. Barone and P. Cimino, Chem. Phys. Lett., 2008, 454, 139.33 G. Onida, L. Reining and A. Rubio, Rev. Mod. Phys., 2002, 74,

601–659.34 N. T. Maitra, A. Wasserman and K. Burke, in Electron

Correlations and Materials Properties, ed. A. Gonis, A. Kioussisand M. Ciftan, Kluwer, Dordrecht, 2003, vol. 2.

35 S. Monti, G. Prampolini, A. Pedone and V. Barone, to besubmitted.

36 Y. S. Xue, X. D. Gong, H. M. Xiao and T. He, Acta Sin. Chim.,2004, 62, 963–968.

37 C. Joamorski-Jodicke and M. E. Casida, J. Phys. Chem. B, 2004,108, 7132–7141.

38 A. K. Rappe and W. A. Goddard III, J. Phys. Chem., 1991, 95,3358.

39 Y. Zhao and D. G. Truhlar, Theor. Chem. Acc., 2008, 120,215–241.

40 T. Yanai, D. Tew and N. Handy, Chem. Phys. Lett., 2004, 393,51–57.

41 J.-D. Chai and M. Head-Gordon, Phys. Chem. Chem. Phys., 2008,10, 6615–6620.

42 Y. Tawada, T. Tsuneda, S. Yanagisawa, T. Yanai and K. Hirao,J. Chem. Phys., 2004, 120, 8425.

Table 5 Vertical S1 ’ S0 and S1 - S0 excitation energies in nm withdifferent functional

lS1-S0 lS1’S0 DlStokes

B3LYP 510.1 478.7 31.4M06-2X 506.5 484.9 21.6WB97XD 489.9 471.52 18.4CAM-B3LYP 494.7 475.00 19.7LC-WPBE 472.89 458.65 14.2Exp. 572.0 547.0 25.0

1006 | Phys. Chem. Chem. Phys., 2010, 12, 1000–1006 This journal is �c the Owner Societies 2010