A fluorescence study of (4-{(1E,3E)-4-[4-(dimethylamino)phenyl]-buta-1,3- dienyl}phenyl)methanol and its bioconjugates with bovine and human serum albumins

Article abstract of DOI:10.1039/b304951e

Electronic absorption and fluorescence properties of (4-{(1E,3E)-4-[4- (dimethylamino)phenyl]buta-1,3-dienyl}phenyl)methanol in different media, including organic solvents of varying polarity at 298 K, an ethanol-methanol matrix at 77 K and protein enviro

Full text of DOI:10.1039/b304951e

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P A P E R
A fluorescence study of (4-{(1E,3E)-4-[4-(dimethylamino)phenyl]-
buta-1,3-dienyl}phenyl)methanol and its bioconjugates with
bovine and human serum albumins
Anil K. Singh* and Manjula Darshi
Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076,
India. E-mail: retinal@chem.iitb.ac.in; Fax: þ91 22 2576 7152
Received (in Montpellier, France) 2nd May 2003, Accepted 9th September 2003
First published as an Advance Article on the web 3rd November 2003
Electronic absorption and fluorescence properties of (4-{(1E,3E)-4-[4-(dimethylamino)phenyl]buta-1,3-
dienyl}phenyl)methanol in different media, including organic solvents of varying polarity at 298 K, an ethanol–
methanol matrix at 77 K and protein environments, have been examined. The diene shows solvatochromic
fluorescence, which is correlated with empirical solvent polarity parameters. The red-shifted fluorescence
emission of the diene is attributed to a polar, conformationally relaxed intramolecular charge transfer excited
state. Further, the diene has been covalently attached to bovine serum albumin and human serum albumin and
the fluorescence probe properties of the protein-diene conjugates have been examined in phosphate buffer.
pany (USA). Diphosgene was procured from IICT (Hydera-
Introduction
bad, India). Sephadex-G50 used for gel filtrations was from
Pharmacia Biotechnology (Sweden). Solvents used for the
synthesis of dienes and for photophysical studies were from
Spectrochem India Ltd. (Mumbai) and were freshly dried
and distilled prior to use. Thin layer chromatography (TLC)
and column chromatography were run on silica gel and neutral
alumina, respectively. Deionised and double-distilled water
was used for the preparation of all aqueous solutions. Phos-
phate buffer used was 0.1 M (pH 7.4).
Melting points were determined on Veego melting point
apparatus by the capillary method and are uncorrected. UV-
vis measurements were carried out on a Shimadzu UV-160
spectrophotometer. IR spectra in KBr discs were recorded
on an Impact 400 Nicolet FTIR spectrophotometer. 1H-
NMR (300 MHz) spectra in CDCl3 as solvent and TMS as
internal standard were measured on a Varian VXR 300 FT
NMR spectrometer. Elemental analyses were done on a Ther-
moquest CE instrument-1112 series CHNS autoanalyser.
Steady state fluorescence measurements were carried out on
a DM1B microprocessor controlled Spex-112 Fluorolog spec-
trofluorimeter with slit widths of 1 and 1.5 mm for the excita-
tion and emission monochromators, respectively. The low
temperature fluorescence studies were done using an etha-
nol–methanol (1:1, v/v) glass at 77 K using a Spex-112 Fluor-
olog equipped with Spex-1932 F accessories. The fluorescence
quantum yields (Ff) at ambient temperature were determined
using quinine sulfate as the standard21 (Ff ¼ 0.55, the revised
value is 0.53).22 The low temperature Ff are relative to the qui-
nine sulfate Ff at ambient temperature.
The strategy of studying the stereoelectronic nature of biomo-
lecular binding sites by critically examining spectroscopic
changes in a probe when bound to the binding site is well-
known.1–9 Fluorescence spectroscopy and probe molecules
that exhibit solvatochromic fluorescence have been particu-
larly useful for this purpose.10–15 Since the intensity and the
maximum wavelength of the fluorescence emission also
depends on solvent polarity, compounds exhibiting solvato-
chromic dual fluorescence emission can be used as probes of
environmental polarity. We have recently reported that certain
diphenylbutadienes are capable of solvatochromic fluorescence
emission and that these can be used as fluorescence probes for
studying the microenvironment of organised assemblies.16–20
The large red shift in the fluorescence emission of such com-
pounds bearing strong donor or acceptor groups on the phenyl
ring has been attributed to conformational relaxation of the
excited state and intramolecular charge transfer.
Herein we report a new fluorescence probe, namely (4-
{(1E,3E)-4-[4-(dimethylamino)phenyl]buta-1,3-dienyl}phenyl)-
methanol (6) that can be used to study the microenvironment
of proteins. The fluorescence probe properties of 6 are based
on its solvent polarity dependence and novel substituent-tuned
fluorescence emission behaviour. Its excited state is charac-
terised by charge transfer process. It shows large Stokes’ shifts
and its fluorescence quantum yield (Ff) is greater than that of
other known probe dienes (e.g., the nitro diphenyldienes),18
particularly in biologically significant aqueous medium. To
examine the usefulness of this diene as a fluorescence probe
for proteins, it has been covalently attached to bovine serum
albumin (BSA) and human serum albumin (HSA) and the
fluorescence characteristics of the resulting protein conjugates
8 and 9 have been investigated.
Syntheses
The synthetic routes for diene 6 and protein conjugates 8 and 9
are outlined in Scheme 1 and the details are described below.
Experimental
(4-{(1E,3E)-4-[4-(Dimethylamino)phenyl]buta-1,3-dienyl}phe-
nyl)methanol, 6. In a typical experiment, a mixture of 4-carbo-
methoxy benzyl bromide (1, 0.05 g, 0.23 mM) and
triethylphosphite (2, 0.21 g, 1.1 mM) was refluxed for 2 h to
give the corresponding phosphonate (3). The phosphonate
Materials and general procedures
Starting materials for the synthesis, BSA and quinine sulfate,
were procured from local suppliers and used as received.
Fatty-acid-free HSA was obtained from Sigma Chemical Com-
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
120
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 1 2 0 – 1 2 6

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Scheme 1
was slowly added to a stirred suspension of sodium hydride
(pre-washed with dry petroleum ether (60–80ꢀC)) in THF at
0 ꢀC under N2 atmosphere. 4-N,N-Dimethylamino cinnamal-
dehyde (4) taken up in dry THF was added dropwise to the
stirred reaction mixture. The reaction mixture was slowly
brought to room temperature and the stirring was allowed to
continue until most of the aldehyde had disappeared, as indi-
cated by TLC in 10% ethyl acetate in petroleum ether (60–
80ꢀC). After completion of the reaction, the reaction mixture
was quenched with brine and taken up in diethyl ether. The
ether layer was washed with water and dried over anhydrous
sodium sulfate. Removal of ether on a rotary evaporator
yielded solid material, which was purified by column chroma-
tography using 20% ethyl acetate in petroleum ether (60–80 ꢀC)
as eluent; methyl 4-{(1E,3E)-4-[4-(dimethylamino)phenyl]-
buta-1,3-dienyl}benzoate (5) was obtained in 62% yield. M.p.
209–210 ꢀC. UV-vis (MeOH) lmax/nm: 395 (e 26 644 molꢁ1
mixture was stirred at room temperature for 3 h. The progress
of the reaction was monitored by TLC in 40% ethyl acetate in
petroleum ether (60–80 ꢀC) and, after completion of the reac-
tion, the reaction mixture was cooled to 0 ꢀC and was slowly
neutralised with 50 mL saturated ammonium chloride solu-
tion. The quenched reaction mixture was extracted with ethyl
acetate and the organic layer was dried over anhydrous sodium
sulfate. Evaporation of ethyl acetate under reduced pressure
yielded a yellow solid, which was further purified by column
chromatography using 30% ethyl acetate in petroleum ether
(60–80ꢀC) as eluent; (4-{(1E,3E)-4-[4-(dimethylamino)phenyl]-
buta-1,3-dienyl}phenyl)methanol (6) was obtained in 67%
yield. M.p. 215–218 ꢀC. UV-vis (1,4-dioxane) lmax/nm: 372
(e 40 300 molꢁ1 cmꢁ1 l). IR (KBr) nmax/cmꢁ1: 3400–3500
(–OH), 1599 (–C C–), 1365 (–C–N str of –Ar–N). 1H
=
NMR (CDCl3 , 300 MHz) d: 2.98 (6H, s, NMe2), 4.67 (2H,
d, –CH2OH) 7.41 (2H, d, AB coupling, J ¼ 8.24, 2.01 Hz,
HOH2C–C6H4–), 7.31 (2H, d, AB coupling, J ¼ 8.24, 2.01
Hz, HOH2C–C6H4–), 7.35 (2H, d, AB coupling, J ¼ 8.97,
2.01 Hz, Me2N–C6H4–), 6.69 (2H, d, AB coupling, J ¼ 8.97,
2.01 Hz, NMe2–C6H4–), 6.95 (1H, dd, J ¼ 15.19, 8.08 Hz,
cmꢁ1 l). IR nmax/cmꢁ1: 1726 (–C O str), 1606 (–C C str),
1368 (–C–N str of –Ar–N), 958 (–C–H def of trans alkene).
1H NMR (CDCl3 , 300 MHz) d: 2.99 (6H, s, NMe2), 3.91
(3H, s, –COOMe), 6.58 (1H, d, J ¼ 15.48, NMe2–C6H4–
=
=
=
=
CH CH–), 6.67 (1H, d, J ¼ 15.56 Hz, MeOOC–C6H4–
HOH2C–C6H4–CH CH–), 6.76 (1H, dd, J ¼ 15.19, 8.05 Hz,
=
=
CH CH–), 6.69 (2H, d, AB coupling J ¼ 8.79 Hz, NMe2–
Me2N–C6H4–CH CH–), 6.60 (1H, d, J ¼ 15.19 Hz,
C6H4–), 6.79 (1H, dd, J ¼ 15.65, 10.05 Hz, Me2N–C6H4–
HOH2C–C6H4–CH–), 6.56 (1H, d, J ¼ 15.19 Hz, Me2N–
C6H4–CH–). Anal. calcd for C19H21NO (279.39): C, 81.68;
H, 7.58; N, 5.01; found: C, 81.74; H, 7.51; N, 5.78.
=
=
CH CH–CH ), 7.05 (1H, dd, J ¼ 15.36, 9.92 Hz, MeOOC–
=
C6H4–CH CH–), 7.35 (2H, d, AB coupling J ¼ 8.79 Hz
Me2N–C6H4–), 7.45 (2H, d of AB coupling, J ¼ 8.24 Hz,
MeOOC–C6H4–), 7.98 (2H, dd of aromatic quartet,
J ¼ 8.24, 1.83 Hz, MeOOC–C6H4–). Anal. calcd for
C20H21NO2 (307.40): C, 78.15; H, 6.89; N, 4.56; found: C,
78.19; H, 6.74; N, 4.63.
Preparation of protein conjugates 8 and 9. The protein conju-
gates were prepared as described elsewhere for immunoglobu-
lin.23 Thus, diene 6 was treated with diphosgene and the
resulting chloro compound 7 was allowed to react with HSA
and BSA to obtain the desired conjugates. In a typical proce-
dure, 6 (0.010 g) was reacted with 15 mL diphosgene in 1 mL
dry 1,4-dioxane in the presence of 5 mL pyridine as a catalyst.
A white precipitate immediately appeared. After stirring for 30
min, TLC showed total disappearance of diene 6. Unreacted
materials were evaporated away under a stream of nitrogen.
The ester 5 prepared as above was converted to the corre-
sponding alcohol 6 by reducing it with lithium aluminium
hydride. In a typical reaction, lithium aluminium hydride
(0.036 g, 9.6 mM) was added into 10 mL dry THF under nitro-
gen atmosphere and stirred for 10 min. To this was added ester
5 (0.1 g, 3.2 mM) taken up in 10 mL dry THF and the reaction
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 1 2 0 – 1 2 6
121

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The residue by FTIR analysis showed a peak at 1775 cmꢁ1 cor-
responding to the carbonyl group of chloride 7. Ethanol (5
mL) was added to the mixture and this solution of 7 was used
for reaction with the proteins.
slightly red-shifted while in protic solvents like methanol and
water it experiences a blue shift. In 1,4-dioxane–water mixtures
a similar trend is observed, that is the absorption maximum
tends to decrease as the water content is increased. However,
the fluorescence excitation wavelength does not change. These
absorption spectral features can be due to ground state hydro-
gen bond interactions and aggregate formation, particularly in
water in which the diene is not freely soluble.
Aliquots of an ethanolic solution of the chloride 7 (20–80
mL) were added to 2 mg of the proteins taken up in 1 mL
aq. NaHCO3 (0.1 M) solution. The mixture was kept overnight
at 4 ꢀC. After this, the reaction mixture was passed through a
Sephadex-G50 column to remove unreacted diene. The pro-
tein-diene conjugates were further dialysed for 6 h against 1
L of phosphate buffer at 4 ꢀC to ensure total removal of
unreacted diene. The mixture was diluted to 5 mL with phos-
phate buffer and then its UV-vis absorption spectrum was
measured. The average number of diene residues bound to
the protein was estimated by known procedures from the con-
centration of the protein and the concentration of the diene in
the conjugates using the following equation:24 D/P ¼ Cd/Cp
where Cd ¼ Al /ed , Cp ¼ [A280 ꢁ (Al F)]/ep , and F ¼
In contrast to a very moderate solvent polarity effect on the
absorption maximum and almost no effect on the fluorescence
excitation spectrum, there is a marked influence of the solvent
polarity on the fluorescence emission behaviour of the diene
(Fig. 1). The fluorescence emission gets red-shifted by 91 nm
when the diene is in a polar protic medium like water as com-
pared to that in a non-polar solvent like heptane. In the rela-
tively non-polar media of heptane, 1,4-dioxane and THF, the
diene shows two fluorescence bands centred at around 430
and 470 nm. With increase in solvent polarity the peak at
430 nm disappears and there is red shift in the longer wave-
length peak. Even though in water there is large blue shift in
the absorption maximum, the fluorescence emission maximum
is highly red-shifted. The fluorescence excitation maximum in
organic solvents is similar to the absorption maximum in the
same solvents. However, in water the excitation maximum is
considerably red-shifted as compared to its absorption maxi-
mum in water, and it is similar to that of the excitation max-
imum observed in organic solvents. The excitation spectrum
was found to be independent of emission wavelength in all
the media studied. Similarly, the emission spectrum is also
independent of excitation wavelength except that the fluores-
cence intensity is reduced when excitation is done at a wave-
length other than the absorption maximum.
max
max
Ad(280)/Ad . In this equation D/P is the number of chromo-
phores bound to the protein, Cd is the concentration of chro-
mophore in the conjugate (mol Lꢁ1), Cp is the concentration of
protein in the conjugate (mol Lꢁ1), Al is the absorbance of
max
chromophore in the conjugate at lmax ( ¼ 325 nm), ed is the
absorption coefficient due to diene chromophore at 325 nm,
Ad(280) is absorbance of free chromophore at 280 nm, Ad is
the absorbance of diene chromophore at 325 nm, A280 is the
absorbance of protein in conjugate at 280 nm, and ep is the
extinction coefficient of protein at 280 nm.
Five conjugates (8, 8a–8d, and 9, 9a–9d) of each protein hav-
ing a different number of diene units were prepared. The num-
ber of diene units bound to the proteins depended on the
aliquot of diene solution added to the protein during the cou-
pling reaction. Conjugate 8 and 9 were found to contain a
maximum of 4.6 and 4.25 units of the diene chromophore.
The other protein conjugates were found to contain the follow-
ing concentrations of the diene units: 8a, 1.9 ꢂ 10ꢁ5 M (D/P
ꢃ0.99); 8b, 2.7 ꢂ 10ꢁ5 M (D/P ꢃ1.42); 8c, 5.0 ꢂ 10ꢁ5 M (D/
P ꢃ2.7); 8d, 7.7 ꢂ 10ꢁ5 M (D/P ꢃ4.1); 9a, 2.1 ꢂ 10ꢁ5 M (D/
Ff decreases with increase in solvent polarity. The change in
the excited state dipole moment of 6 was determined from Lip-
pert–Mataga analysis using the equation:25,26 na ꢁ nf ¼
{[2(me ꢁ mg)2/hca3]F(D,n)}, where na ꢁ nf is the Stokes’ shift,
me ꢁ mg ¼ Dm is the change in dipole moment, h is Planck’s
constant, c is the velocity of light, a is the Onsagar radius
P ꢃ1.27); 9b, 2.8 ꢂ 10ꢁ5 M (D/P ꢃ1.7); 9c, 5.0 ꢂ 10ꢁ5
(D/P ꢃ03.03); 9d, 6.4 ꢂ 10ꢁ5 M (D/P ꢃ3.88).
M
and
where D is the dielectric constant and n is the refractive index
of the solvent. The Onsager radius has been taken as approxi-
F(D,n) ¼ Df ¼ (D ꢁ 1)/(2D þ 1) ꢁ (n2 ꢁ 1)/(2n2 þ 1),
˚
mately 9 A, a value that has been used for a similar diene, 1-p-
Results and discussion
N,N-dimethylaminophenyl-4-p-nitrophenylbuta-(1E,
3E)-
diene.27 The Lippert–Mataga correlation plot is characterised
by a straight line with a slope of 10 634 and an R2 value of
0.9528, where R is the linear correlation. The change in excited
state dipole moment (Dm) is determined to be 22.54 D, indicat-
ing the polar nature of the fluorescent excited state of 6.
The dependence of the Stokes’ shifts on solvent polarity has
also been correlated with the empirical solvent polarity para-
meters ET(30) and p*. ET(30) is a solvent polarity parameter
proposed by Dimroth, Reichardt and coworkers, based on
the transition energy for the longest wavelength solvatochro-
mic absorption band of the pyridinium-N-phenoxide betain
dye.28,29 The ET(30) value for a solvent is defined as the transi-
tion energy of the dissolved betaine dye, measured in kcal
molꢁ1, and is recognised as one of the most comprehensive
Electronic absorption and fluorescence properties of 6 in organic
solvents and 1,4-dioxane–water mixtures
The UV-vis absorption and fluorescence spectral data of diene
6 in homogeneous media (organic solvents) and in 1,4-diox-
ane–water mixtures are summarised in Tables 1 and 2. For
comparison purposes the absorption and fluorescence spectral
data of the parent diene N,N-dimethyl-N-[4-phenylbuta-
(1E,3E)-dienyl]phenylamine in organic solvents are also given
in Table 1, while the data in 1,4-dioxane–water mixtures was
already reported.20 Similar to the parent diene, no significant
changes are observed in the absorption and fluorescence exci-
tation spectra of 6 in the various solvents examined, except
that in aprotic polar solvents the absorption maximum is
Table 1 UV-vis absorption and fluorescence spectral data of diene 6 in organic solvents. The values for the parent diene N,N-dimethyl-N-[4-phe-
nylbuta-(1E,3E)-dienyl]phenylamine are given in parentheses
Solvent
labs max/nm
lf max/nm
lex max/nm
Stokes’ shift/cmꢁ1
Ff
Heptane
1,4-Dioxane
THF
367 (364)
372 (368)
374 (369)
369 (371)
354 (370)
376 (378)
310
430, 457sh (424)
441sh, 458 (440)
446sh, 477 (474)
491 (492)
366 (366)
369 (367)
370 (372)
367 (362)
368 (369)
370 (376)
365
3992.(3887)
4205 (4446)
5995 (6003)
6733 (6628)
7881 (6365)
6311 (6293)
12 252
0.167 (0.125)
0.065 (0.055)
0.059 (0.052)
0.046 (0.028)
0.031 (0.029)
0.037 (0.045)
0.009
Acetonitrile
Methanol
DMF
491 (484)
493 (496)
521
Water
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
122
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 1 2 0 – 1 2 6

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Table 2 UV-vis absorption and fluorescence spectral data of diene 6
in 1,4-dioxane–water mixtures
tion at a number of sites, including carbon-carbon bonds
between the aromatic centres as well as the bond between
the donor dialkylamino group and the aromatic ring.
% Water in Dielectric
1,4-dioxane constant10
labs
lf
lex
Stokes’
max/nm max/nm max/nm shift/cmꢁ1 Ff
e
Low temperature fluorescence studies
0
2
5
372
371
372
370
368
365
368
364
458
478
486
491
496
499
503
504
369
369
370
370
370
368
370
370
4205
6033
6305
6660
7012
7357
7293
7631
0.065
0.051
0.047
0.041
0.038
0.034
0.033
0.030
The fluorescence spectral features of 6 in a 1:1 (v/v) ethanol–
methanol mixture at 298 and 77 K are shown in Fig. 2 and the
corresponding data are summarised in Table 3. An examina-
tion of this data reveals that as compared to emission at 298
K, the emission of 6 is blue-shifted by 56 nm at 77 K. At
298 K, the diene exhibits fluorescence emission at 485 nm with
a shoulder peak at 433 nm whereas at 77 K there is total dis-
appearance of longer wavelength emission and the spectrum is
characterised by well-defined structure with a maximum at 429
nm. Furthermore, as compared to 298 K, there is an approxi-
mately eight-fold increase in Ff at 77 K. The low temperature
fluorescence observations point towards conformational
relaxation in the excited state of diene 6. In a rigid matrix at
77 K, molecular motions and related geometrical changes in
the fluorophores are expected to be reduced. This will result
in a minimisation of solvent effects on the excited state. In
the absence of solvent effects, a blue shift in the fluorescence
emission is expected. At low temperature, non-radiative transi-
tions are expected to be weak, hence the increase in Ff . It may,
however, be mentioned that several other factors like a change
in solvent density, a decrease in the population of some vibra-
tional states, etc. may also need to be considered for a com-
plete description of the blue shift observed at low temperature.
It is also interesting to note that in heptane and 1,4-dioxane
at 298 K the intensity of the shorter wavelength fluorescence
emission band is greater than that of the longer wavelength
fluorescence emission band. In THF and ethanol–methanol
mixtures, however, the intensity of the longer wavelength
fluorescence emission band becomes much greater. Also, the
fluorescence emission spectrum in methanol is characterised
by the presence of only the CT band whereas that in 1:1 etha-
nol–methanol is characterised by the presence of dual fluores-
cence emission bands.
10
20
30
40
50
60
70
10
16
25
32
42
50
2solvent polarity scales. The p* scale, proposed by Kamlet,
Abboud and Taft, is derived from solvent effects on the p–p*
transition of a variety of nitroaromatics.29,30 These solvent
polarity correlations were made in various organic solvents,
including 1,4-dioxane–water mixtures. When ET(30) is plotted
against the Stokes’ shifts in nonpolar, polar aprotic and polar
protic solvents, good correlations are not obtained. However,
good correlations are obtained when ET(30) is plotted against
the Stokes’ shifts in 1,4-dioxane–water mixtures. This can be
due to the specific hydrogen bond interactions between protic
solvents and excited diene. The ET(30) scale is known to show
a strong dependence on hydrogen bond interactions. This can
be the reason for the relatively poor correlations observed
when ET(30) is plotted against the Stokes’ shifts in apolar
and protic solvents together. The ET(30) correlation plot for
eight data points is characterised by a straight line slope of
162.7 and an R2 value of 0.9874. On the other hand, reason-
able correlations with p* in aprotic as well as in protic solvents
is observed. The p* correlation plot for eleven data points is
characterised by a straight line slope of ꢁ0.0004 and an R2
value of 0.8326. Apparently, the fluorescent excited state of 6
has polar character and it undergoes hydrogen bond inter-
actions with protic polar solvents.
The blue-shifted fluorescence emission of 6 in non-polar sol-
vents can be from the locally excited state, while the longer
wavelength emission can be from an intramolecular charge
transfer excited state, which is more polar and lower in energy
than the locally excited state. The intramolecular charge trans-
fer excited state can involve conformational relaxation of the
single bonds connecting the donor group to the acceptor
group. Hence, the polar solvents stabilise the excited state,
resulting in red-shifted fluorescence emission. The conforma-
tional relaxation of the excited state is possible by bond rota-
It seems that the protic solvents influence the excited species
more strongly when compared to the aprotic solvents. In pro-
tic solvents the hydrogen bond interactions may be important,
affecting the fluorescence excited state and, hence, the fluores-
cence emission spectra. At low temperature in an ethanol–
methanol (1:1) matrix the excited state of diene is expected
to have conformationally restricted geometries. On the other
hand, at ambient temperature the excited diene can undergo
conformational relaxation and occur in geometries that are dif-
ferent from the excited state geometries at low temperature.
Therefore, the interactions between solvent and excited diene
Fig. 1 Fluorescence spectra of diene 6 in different organic solvents
and water at 298 K: (a) heptane, (b) 1,4-dioxane, (c) THF, (d) MeCN,
(e) MeOH, (f) DMF and (g) water. Spectra are normalised at the
maximum fluorescence intensity.
Fig. 2 Fluorescence spectra of diene 6 in 1:1 (v/v) methanol-ethanol
matrix at (a) 77 K and at (b) 298 K.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 1 2 0 – 1 2 6
123

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Table 3 Fluorescence spectral data for 6 in a 1:1 (v/v) ethanol–
methanol mixture at 298 and 77 K
T/K
lex max/nm
lf max/nm
Ff
298
77
356
366
485, 433sh
429
0.052
0.425
are expected to be different at the two temperatures. This may
also contribute to the observed fluorescence excitation and
emission features of 6.
Furthermore, in solvents having a dielectric constant (e)
greater than 30, such as methanol (e ¼ 33), acetonitrile
(36.6), DMF (38.3), and water (80) only the longer wavelength
fluorescence emission band is seen for 6. However, two fluor-
escence bands for 6 are observed in a 1:1 ethanol–methanol
mixture. This may be due to the intermediate polarity of the
mixed alcohols. The dielectric constant of ethanol is 24 and
that of methanol is 33. So, a 1:1 mixture of ethanol and metha-
nol is expected to have dielectric constant between 24 and 33.
Thus, while in solvents of high dielectric constant only the CT
band is observed, in solvents of relatively lower dielectric con-
stant (ꢃ<30) both CT as well as non-CT bands appear.
Fig. 4 Fluorescence emission spectra of BSA and the corresponding
protein conjugate in phosphate buffer: (a) pure BSA and (b–e)
protein conjugates. The concentration of diene in the conjugates
is (b) 2.7 ꢂ 10ꢁ5, (c) 5.0 ꢂ 10ꢁ5, (d) 7.7 ꢂ 10ꢁ5 and (e) 8.8 ꢂ 10ꢁ5 M.
(lex ¼ 295 nm.)
said that in the protein conjugates 8 and 9, the coupled diene
6 is partially exposed to a relatively protic polar environment
where it can participate in hydrogen bond interactions, result-
ing in a blue shift of its absorption maximum. This is also evi-
dent from the absorption maximum of 6 in methanol (354 nm)
in which there is possibility of hydrogen bonding between the
diene and the solvent.
Electronic absorption and fluorescence properties of the protein
conjugates 8 and 9
The UV-vis absorption and fluorescence spectra of BSA and
the corresponding protein conjugate 8 with diene 6 are pre-
sented in Figs. 3 and 4, respectively. The fluorescence excita-
tion spectra of diene 6 and its conjugate with BSA (8) are
shown in Fig. 5. HSA showed similar absorption and fluores-
cence spectra. These absorption and fluorescence data are sum-
marised in Table 4.
Conjugates 8 and 9 show absorption bands at around 280
nm (due to the protein part) and at 325 with a shoulder at
around 365–368 nm due to the diene moiety. Thus, the absorp-
tion of the diene in the protein medium is blue-shifted as com-
pared to its absorption in both organic solvents. We have
noted that the absorption of diene 6 in aqueous medium is
drastically blue-shifted. The relatively short wavelength
absorption of 6 in water (310 nm as compared to an ꢃ370
nm absorption by 6 in organic solvents) can be due to the poor
solubility of 6 in water and also due to ground state hydrogen
bond interactions between 6 and water. Since the diene is not
freely soluble in water, there is a chance of aggregation, which
can reduce the absorption wavelength. Therefore, it can be
The number of diene molecules coupled to BSA and HSA
was determined by following the absorbance of the modified
protein at 325 nm (e 18 400 molꢁ1 cmꢁ1 l in 80% phosphate
buffer þ 20% methanol) due to the diene moiety and 280 nm
due to the protein residues. The absorption coefficients, e, for
BSA and HSA in phosphate buffer at 280 nm are 39 080 and
36 600 molꢁ1 cmꢁ1 l, respectively. The relative concentrations
of diene and protein moieties were calculated as discussed in
the Experimental and it was found that a maximum of about
four diene units could be coupled to BSA and HSA. It may
be mentioned here that the spectra of the conjugates were
taken in pure buffer, but the reference spectra with extinction
coefficients are taken in a mixed solvent (20% methanol). As
the compound is not properly soluble in water, its extinction
coefficient in pure water could not be calculated. The extinc-
tion coefficient, therefore, was obtained using water containing
20% methanol, in which 6 is properly soluble. As said earlier
there is a strong dependence of the absorption on alcohol mix-
ture and therefore the values may not be the real ones that one
expects in pure water.
Fig. 3 UV-vis absorption spectra of (a) BSA and (b–e) protein con-
jugates in phosphate buffer. The concentration of diene in the conju-
gates is (b) 2.7 ꢂ 10ꢁ4
8.8 ꢂ 10ꢁ5 M.
,
(c) 5.0 ꢂ 10ꢁ5
,
(d) 7.7 ꢂ 10ꢁ5 and (e)
Fig. 5 Excitation spectra of (a) diene 6 and (b) protein conjugate 8 in
phosphate buffer (emission wavelength 478 nm).
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
124
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 1 2 0 – 1 2 6

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Table 4 UV-vis absorption and fluorescence spectral dataa of protein
conjugates 8 and 9 in phosphate buffer
diene is located in a relatively non-polar environment in the
protein. The fluorescence emission of diene in the protein con-
jugates is similar to that observed for the diene in medium
polar solvents like THF in which the diene fluoresces at
around 475 nm with a shoulder at 445 nm.
Protein
Conjugate max/nm
labs
lex/nm lf max/nm lex max/nm Ff
BSA and HSA are homologous proteins composed of single
polypeptide chains with 583 and 585 amino acids, respectively,
with a similar sequence and a similar conformation.31 The
three-dimensional structure of HSA reveals that it contains
three homologous domains that assemble to form a heart-
shaped molecule.32–34 Each domain is the product of two
sub-domains (A and B) that possess common structural
motifs. It is also believed that the principle regions of ligand
binding to HSA are located in hydrophobic cavities of sub-
domains IIA and IIIA. However, the well-known difference
between BSA and HSA is that while BSA has two tryptophans,
HSA has only one tryptophan unit.
The tryptophan residue plays an important role as a chro-
mophore and a fluorophore in optical studies of proteins.
However, it is generally difficult to examine the behaviour of
individual tryptophans in relation to the total conformational
change of the protein, which contains the tryptophan(s), since
quite a few tryptophans are located at random in most of the
proteins. In this way, since there is only one tryptophan (Trp-
214) unit present in HSA, which has been proposed to be
located in a cleft or hydrophobic fold of sub-domain IIA, it
provides a unique opportunity to study the energy transfer
interactions and binding sites of the protein. In addition, one
of the two tryptophans in BSA (Trp-213) is anticipated to be
located in a similar environment as the single tryptophan resi-
due (Trp-214) in HSA, while the additional tryptophan residue
(Trp-134) has been proposed to be located on the surface of
the molecule. The diene moiety can be located in the hydro-
phobic environment of sub-domain IIA of HSA, and in view
of the observed similarity between the fluorescence character-
istics of protein conjugates 8 and 9, it can be proposed that
the diene moiety in BSA is also located in sub-domain IIA
of the protein.
8
9
280
295
342, 478
477
478
280, 372
372, 280
372, 280
279, 372
373, 280
373, 280
0.098
325, 365sh 325
365
277
295
342, 476
475
476
0.070
325, 368sh 325
365
a
The values correspond to the maximum number of diene molecules
bound to the protein, i.e., 1:4.1 for 8 and 1:3.88 for 9.
Both the protein conjugates show similar fluorescence spec-
tral characteristics, indicating that the diene is located in the
same region in both the proteins. There is no change in the
fluorescence emission or excitation maximum with increase
in the concentration of the diene in the conjugates. However,
there are changes in the fluorescence intensity. The fluores-
cence spectra of conjugates 8 and 9 were recorded by exciting
the conjugates at 295, 325 and 365 nm. The 295 nm excitation
(instead of 280 nm) was chosen since the excitation at 280 nm
(an absorption band shown by the conjugates due to protein)
can result in fluorescence emission by tyrosine residues of the
protein as well. Excitation of conjugates 8 and 9 at 295 nm
resulted in fluorescence emissions at 342 and 476–478 nm.
Similarly, excitations of the conjugates at 325 and 375 nm
resulted in fluorescence emission with maximum in the range
of 475–478 nm. Thus, BSA and HSA both show fluorescence
emissions at 342 nm when excited at 295 nm. Therefore, it
can be said that the fluorescence emission of protein conjugates
8 and 9 at 342 nm is from the tryptophan residue of the mod-
ified proteins and the fluorescence emission at 478 nm is due to
the diene moiety. However, at 295 nm the diene has consider-
able absorption and hence it can fluoresce. Therefore, the
fluorescence emission of the diene was examined by exciting
at 275 nm where it has a minimum in the absorption. Under
these excitation conditions, two emission bands located at
342 and 478 nm were observed. This points towards the possi-
bility of energy transfer interactions between tryptophan and
diene moieties in the protein conjugates.
With an increase in the concentration of the diene moieties
in the protein conjugate, there is a partial quenching of the
intrinsic fluorescence of the protein at 342 nm (due to trypto-
phan residues) and an increase in the fluorescence emission at
478 nm (due to diene moieties) when excited at 295 nm. Since
the diene also shows some absorption at 295 nm, an increase in
the fluorescence intensity at 478 nm with increase in the con-
centration of diene molecules can be attributed to the excita-
tion of the diene. However, there is a decrease in the
fluorescence intensity of tryptophan with increase in the con-
centration of diene. This indicates that the diene causes
quenching of the fluorescence due to tryptophan residues. To
determine whether energy transfer was taking place from the
tryptophan residues to the diene units, the fluorescence excita-
tion spectrum of the protein conjugates 8 and 9 (obtained at
478 nm fluorescence emission) was compared with that of
the fluorescence excitation spectrum of the diene in phosphate
buffer (Fig. 5). The excitation spectrum of diene 6 is charac-
terised by a band at 370 nm. However, the excitation spectrum
of the protein conjugates featured bands at 280 as well as 370
nm, clearly indicating the involvement of the protein residue.
These observations indicate the possibility of energy transfer
from the tryptophan to diene moieties in both the conjugates.
Compared to the spectra in aqueous medium, the diene in
the protein environments shows blue-shifted fluorescence emis-
sion and an increase in Ff . These observations indicate that the
Conclusions
In conclusion, diene 6 exhibits solvatochromic fluorescence
emission due to a polar, conformationally relaxed, intramole-
cular charge transfer excited state. The fluorescence behaviour
of 6 can be used to investigate the micropolarity, binding site
and energy transfer interactions in proteins. Thus, the present
studies provide new directions for the development of fluores-
cence probes, based on charge transfer diphenylpolyenes, for
organised structures in chemistry and biology.
Acknowledgements
Research grant 37/7/95-R&D-II/559 from the Board of
Research in Nuclear Sciences, Department of Atomic Energy,
Government of India is gratefully acknowledged. We thank
Dr. V. J Rao for providing diphosgene. We are also gratefully
thankful to the reviewers of this paper for their valuable sug-
gestions.
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