Chemical Physics Letters 485 (2010) 258–261

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Chemical Physics Letters

journal homepage: www.elsevier.com/locate/cplett

A dramatic heavy-atom effect in the quenching of dichlorosubstituted

lucigenin fluorescence
Eamonn F. Healy *, Samuel Manzer, Jeffrey Gorman, Alicia Jones, Nicholas Cristea
Department of Chemistry, St. Edward’s University, Austin, TX 78704, USA

a r t i c l e i n f o a b s t r a c t

Article history: A dichloro derivative of lucigenin has been synthesized and found to exhibit almost no fluorescence. Cal-
Received 24 October 2009 culations using the graphical unitary group approach (GUGA), as implemented in the GAMESS program
In final form 15 December 2009 suite and using the full Brett-Pauli spin–orbit operator yields unusually large spin–orbit coupling
Available online 21 December 2009
(SOC) values. Comparison with experimental and theoretical results for similar systems identify the
intramolecular heavy-atom effect (HAE) observed for chlorine in this system to be comparable to that
observed for bromosubstituted polyaromatics in earlier studies.
Ó 2009 Elsevier B.V. All rights reserved.

1. Introduction derivative exhibits almost no fluorescence (Fig. 2), and theoretical

The quenching of lucigenin (Fig. 1) fluorescence by chloride has
found particular application in the measurement of intracellular
Cl concentrations. This in turn has proven useful in the investiga-
tion of a variety of biophysical systems, including disease states
such as cystic fibrosis [1]. The mechanism of quenching has been
traditionally explained as proceeding through the formation of a
transient charge transfer complex [2], and this can be written as
(1):
 
L2þ þ hm ! ðL2þ Þ þ Cl ! ðLþ Cl Þ ! L2þ þ Cl :

ð1Þ
Whereas the quenching efficiency of lucigenin fluorescence by
chloride, and other anions such as cyanide and thiocyanate, is di-
rectly correlated to the oxidation potential of the anion, quenching
by iodide and bromide is normally attributed to the heavy-atom
effect [3]. This relativistic phenomenon can be described qualita-
tively as an interaction between the spin magnetic moment and
the nuclear magnetic field caused by the electron’s orbital motion
[4], often termed spin–orbit coupling (SOC). The radiationless path
for decay from the excited state generated by such SOC is given in
(2), where kisc is the intersystem crossing rate:
kisc
L2þ þ hm ! ðL2þ Þ þ X ! 1 ðL2þ X Þ ! 3 ðL2þ X  Þ ! 3 ðL2þ Þ þ X  :
ð2Þ
While the original study did investigate, and discount, the effect
of collisional factors on chloride quenching, no other contributions
to quenching were evaluated. In an effort to quantify any heavy
atom contribution to the quenching of lucigenin fluorescence by
chloride we have synthesized a chloro-substituted lucigenin. This
* Corresponding author. Fax: +1 512 448 8492.
E-mail address: healy@stedwards.edu (E.F. Healy).
calculations yield large spin orbit SOC values. Though care must be
taken when extrapolating from an intramolecular to an external
heavy-atom effect, we feel that these results indicate that the hea-
vy atom component of the quenching of fluorophores such as luc-
igenin by chloride cannot totally be discounted.
2. Materials and methods
2.1. Synthesis
Use of diphenyliodonium carboxylate (DPIC) in the synthetic
scheme in Fig. 3 presents an excellent reagent for nucleophilic dis-
placement, and represents a modification of a previously reported
synthesis of lucigenin [5]. DPIC was synthesized according to liter-
ature procedure [6]. All analytical grade reagents were obtained
from Sigma–Aldrich. High-resolution mass spectra (HRMS) were
determined by the Mass Spectrometry Facility of the Department
of Chemistry and Biochemistry at the University of Texas at Austin,
and yielded monoisotopic 35Cl formulae with errors of <1 ppm.
Diphenyliodonium 2-carboxylate and Cu(OAc)2 were refluxed
under nitrogen, with 3-chloroaniline in isopropyl alcohol. After sol-
vent removal and washing with aqueous NaOH, HCl(aq) was added.
Cooling and vacuum filtration provided the substituted N-pheny-
lanthranilic acid (Fig. 3A) as a light yellow solid (79%). HRMS calcu-
lated for C13H10NO235Cl 247.0400, found 247.0400.
The dry N-phenylanthranilic acid derivative was dissolved in
conc. H2SO4 and heated in a bath of boiling water. Upon cooling
the bright yellow precipitate was isolated, suspended in a solution
of Na2CO3, and heated to boiling. After cooling 1-chloroacridone
and 3-chloroacridone (Fig. 3B) were isolated by vacuum filtration
as a 1:1 mixture of isomers (92%). HRMS calculated for
C13H8NO35Cl 229.0294, found 229.0292.

0009-2614/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.cplett.2009.12.049
 E.F. Healy et al. / Chemical Physics Letters 485 (2010) 258–261 259

2.2. Spectroscopy

Absorption spectra (Fig. 4) for lucigenin (Sigma–Aldrich) and

dichlorolucigenin were obtained using freshly prepared aqueous
solutions (ultra-pure H2O) on a Varian Cary 100 double-beam
UV–Visible spectrometer. A small, near-uniform red-shift of less
than 5 nm was observed for the dichloro derivative. Emission spec-
tra (Fig. 2) were obtained using a Varian Cary Eclipse spectrofluo-
rometer, using a kexc of 435 nm for lucigenin, and a kexc of 439 nm
for dichlorolucigenin.

2.3. Theory

The intersystem crossing rate, or kisc, for the non-radiative tran-

Fig. 1. Lucigenin.
sition responsible for quenching can be computed with Eq. (3):

4p2 1
kisc ¼ h WjHSO j3 Wi2  ½FC  ðD13 Þ1 ; ð3Þ
h
where h1 WjHSO j3 Wi is the expectation value of the spin–orbit cou-
pling term for the non-radiative transition S1 ! T 1 , [FC] is the
Franck–Condon weighted density of states, which characterizes
the overlap of the vibrational wave functions, and ðD13 Þ1 is the en-
ergy difference between the initial and final states. The full Brett-
Pauli Hamiltonian [7] for spin–orbit coupling used for these calcu-
lations can be written as (4):
" #
a2 electron X
X nucleus ZA X 1
electron
HSO ¼ LiA S i  Lij ðSi þ 2S j Þ ; ð4Þ
2 i A
r 3iA ij
r 3iA

where a is fine structure constant, L is the orbital momentum oper-

Fig. 2. Emission spectrum for lucigenin (top, inset right) and dichlorolucigenin
(bottom, inset left).
Chloroacridone was dissolved in hot ethanolic KOH, the solution
concentrated, and N,N-dimethylformamide and methyl iodide
added. The reaction mixture was heated and the solution poured
into water. 1-Chloro-N-methylacridone and 3-chloro-N-methylac-
ridone (Fig. 3C). were recrystallized from aqueous ethanol (81%).
HRMS calculated for C14H10NO35Cl 243.0451, found 243.0453.
A suspension of the N-methylacridone derivative in ethanolic
HCl was heated to reflux. After adding zinc powder and refluxing,
the hot solution was added to water. After cooling the bis-acridini-
um intermediate was isolated by vacuum filtration and suspended
in aqueous HNO3. After heating the dark orange reaction mixture
was filtered hot, and orange crystals of dichlorolucigenin (mixture
of isomers) (Fig. 3D) were collected by vacuum filtration (28%).
HRMS calculated for C28H20N235Cl2 454.09926, found 454.09974.
ator, S is the spin momentum operator, and ZA is the nuclear charge.
The full operator in (4) can be partitioned into separate one-elec-
tron and two-electron contributions to the full SOC term. Neglecting
the second term and compensating for the omission by replacing
the true nuclear charge with a semiempirical parameter, Zeff or
effective nuclear charge, yields the commonly used one-electron
SO Hamiltonian.
Spin orbit configuration interaction (SO-CI) calculations in the
GAMESS program suite [8] are formulated using the graphical uni-
tary group approach (GUGA) [9]. The GUGA-based CI calculations
yield spin-adapted configuration state functions (CSFs), expansions
of type w ¼ u1 ð2Þu2 ð1Þu3 ð1Þ, where u, in this case, represents a
common set of HF/6-31G** orbitals and 1 and 2 denote singly
and doubly-occupied orbitals respectively. The orbitals u1 , u2
and u3 represent the CI active space, with all other doubly-occu-
pied orbitals comprising a frozen core. For all calculations in this
study u3 is the lowest unoccupied molecular orbital (LUMO),
shown as L0 in Fig. 5. The orbitals u1 and u2 were chosen as either
the near-degenerate highest occupied molecular orbitals (H0 and
H00 ), or the (H-2) near-degenerate pair, or (H-4)0 and (H-4)00 , or fi-
nally (H-5)0 and (H-5)00 (see Fig. 5). The choice of these orbitals
for the CI active space is discussed below. Addition of the full
Brett-Pauli spin–orbit operator then gives SOC values, in cm1,

Table 1
Active space orbitals used for the three orbital, four electron GUGA CI calculations; configuration state function (CSF) coefficients; SOC values partitioned into one-electron, and
all-electron terms.

w1 ¼ u1 ð2Þu2 ð1Þu3 ð1Þ W 1 ¼ c 1 w1 þ c 2 w2 h1 W1 jHSO j3 W1 i h1 W1 jHSO j3 W2 i

w2 ¼ u1 ð1Þu2 ð2Þu3 ð1Þ W 2 ¼ c 2 w1  c 1 w2 (cm1) (cm1)
u1 u2 u3 c1 c2 Full 1e Full 1e
0 00 0
Isomer A H H L 0.69 0.72 13.3 16.2 9.3 11.4
Isomer A (H-3)0 (H-3)00 L0 0.18 0.98 20.8 25.1 15.0 18.5
Isomer A (H-5)0 (H-5)00 L0 0.79 0.62 111.8 136.4 85.4 104.3
Isomer B H0 H00 L0 0.43 0.90 0.7 0.8 0.4 0.5
Isomer B (H-3)0 (H-3)00 L0 0.82 0.58 11.6 14.3 3.1 3.9
Isomer B (H-4)0 (H-4)00 L0 0.0 1.0 43.2 52.9 19.9 24.3
260 E.F. Healy et al. / Chemical Physics Letters 485 (2010) 258–261

Fig. 3. Synthetic scheme for chloro-substituted lucigenin.

for coupling between singlet and triplet state systems, expressed w1 ¼ u1 ð2Þu2 ð1Þu3 ð1Þ and w2 ¼ u1 ð1Þu2 ð2Þu3 ð1Þ, large one-cen-
as W ¼ c1 w1 þ c2 w2 with optimized coefficients c1 and c2 as listed ter SOC terms will result from calculations using orbitals where
in Table 1. From Eq. (3) above the expected quenching due to u1 and u2 both have large halogen coefficients. In addition the
non-radiative singlet to triplet transition will vary as the square position of the chlorine proximate to the acridinium rings in isomer
of the SOC. All geometries were optimized with the AM1 semiem- A means that this system should result in a significantly larger
pirical Hamiltonian [10]. two-center SOC term than that observed for the di-halogenated
systems characterized in [11]. This can be shown by simplifying
Z Cl
3. Results and discussion the one-electron component of (4) to r3
LiCl and reducing
iCl
1 3 Z Cl
h WjHSO j Wi to hu 1 j r3 LiCl ju2 i. The largest one-center and two-cen-
In an analysis of the radiative and non-radiative properties of iCl

the triplet state of haloaromatics it has been previously shown that ter contributions to this one-electron integral will come from the
when the halogen is coplanar with the aromatic ring, the mixing LCAO expanded terms hxu1Cl j rZ3Cl LiCl jyu2Cl i and hxu1Cl j rZ3Cl LiCl jzu2C i
between the singlet and triplet (p, p*) states is as vanishingly small
iCl iCl

as that for the parent hydrocarbon [11]. Singlet–triplet mixing in

such systems is postulated to occur as a result of either one of
the coupling models shown in (5):

a L
1ðr; r Þ ! 1ðr; p Þ ! 3ðp; p Þ
L a
or 1ðr; r Þ ! 3ðr; p Þ ! 3ðp; p Þ; ð5Þ
a L
where ! describes vibronic coupling and ! represents SO coupling
between the singlet and triplet states. Expanding the spatial compo-
nent of the one-electron HSO operator as a linear combination of
atomic orbitals (LCAO) confirms that the relatively small value for
the halogen coefficient in the p or p* orbital will limit the size of
any one-center SOC contribution, while the r13 dependence of the
SOC term limits the magnitude of the two-center contribution for
such coupling [12].
The calculated orbitals shown in Fig. 5 for the dichlorolucigenin
isomers identifies a system that overcomes these limitations. For
SOC between state systems W ¼ c1 w1  c2 w2 , where Fig. 4. Absorption spectrum for lucigenin and dichlorolucigenin.
 E.F. Healy et al. / Chemical Physics Letters 485 (2010) 258–261
Fig. 5. The 6-31G** orbitals for the two chloro-substituted lucigenin isomers used for the SOC calculations delineated in Table 1.
respectively, where x and y are the halogen coefficients for active
space orbitals u1 and u2 , and z are the coefficients of the ring car-
bons in u1 and u2 proximate to the chlorine. Thus the largest SOC
values should come from calculations where the CI active space is
constructed from those highest occupied molecular orbitals with
the greatest chlorine composition. As predicted by this analysis
the SOC terms calculated for isomers A and B, and listed in Table
1, range as high as 43 cm1 for isomer B and 112 cm1 for isomer
A.
Using fluorescence lifetimes and quantum yields, experimental
values of kisc (S1 ! T 1 ) have been obtained for a series of halonaph-
thalenes [13]. For 1-chloronapthalene the intersystem crossing rate
has been observed to increase by a factor of 21.6 over that measured
for naphthalene. This experimental result has been matched by a
theoretical value of a 21-fold enhancement calculated using
INDO/S calculations [14] and a vibronically induced spin–orbit
(VISO) coupling model [15]. This theoretical non-radiative transi-
tion rate was in turn computed from a calculated SOC term of
10.1 cm1. The SOC values calculated for dichlorolucigenin, and
listed in Table 1, are up to eleven times larger than the value re-
ported in [15], and since the kisc varies as the square of the SOC va-
lue this indicates that S1 ! T 1 non-radiative transitions, and thus
heavy atom quenching, is approximately 100 times more efficient
in this system than that observed for 1-chloronapthalene. Interest-
ingly as the halogen was changed from chlorine to bromine in the
experimental study of 1-halonapthalene a 23-fold increase in the
rate of kisc (S1 ! T 1 ) was observed. This would indicate that for
dichlorolucigenin, even allowing for the presence of two substitu-
ents as opposed to only one in reference [13], the intramolecular
heavy-atom effect observed here is comparable to that of bromo-
substituted aromatic systems. Quenching due to intramolecular
261
heavy-atom effects for bromosubstituted fluoranthene and fluo-
ranthene derivatives has been previously observed [16].
Acknowledgements
The authors are grateful to the W.M. Keck Foundation and the
Educational Advancement Foundation for their generous support
of this work. EFH, JG, AJ and NC also wish to thank the Welch Foun-
dation (Grant# BH-0018) for its continuing support of the Chemis-
try Department at St. Edward’s University. We would also like to
thank the staff of the Mass Spectrometry Facility at UT Austin,
for their assistance in the preparation of this manuscript.
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