Mechanism of fluorescence quenching of tyrosine derivatives by amide group

Article abstract of DOI:10.1016/S0009-2614(01)00470-5

The difference between fluorescence lifetimes of the following amino acids: phenylalanine (Phe), tyrosine (Tyr), (O-methyl)tyrosine (Tyr(Me)), (3-hydroxy)tyrosine (Dopa), (3,4-dimethoxy)phenylalanine (Dopa(Me)₂) and their amides was used to tes

Full text of DOI:10.1016/S0009-2614(01)00470-5

15 June 2001
Chemical Physics Letters 341 /2001) 99±106
www.elsevier.nl/locate/cplett
Mechanism of ¯uorescence quenching of tyrosine derivatives
by amide group
Wiesøaw Wiczk a,*, Alicja Rzeska a, Joanna èukomska a, Krystyna Stachowiak a,
Jerzy Karolczak b, Joanna Malicka a, Leszek èankiewicz a
a
Faculty of Chemistry, University of Gdanꢀsk, Sobieskiego 18, 80-952 Gdanꢀsk, Poland
b
Quantum Electronics Laboratory, Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznanꢀ, Poland
Received 12 January 2001; in ®nal form 10 April 2001
Abstract
The di€erence between ¯uorescence lifetimes of the following amino acids: phenylalanine /Phe), tyrosine /Tyr), /O-
methyl)tyrosine /Tyr/Me)), /3-hydroxy)tyrosine /Dopa), /3,4-dimethoxy)phenylalanine ꢀDopaꢀMe† † and their amides
2
was used to testify the mechanism of ¯uorescence quenching of aromatic amino acids by the amide group. On the basis
of the Marcus theory of photoinduced electron transfer parabolic relationships between ln kET and ionization potentials
reduced by energy of excitation ꢀIP À E0Ã;0† for the above-mentioned amino acids were obtained. This ®nding indicates
the occurrence of photoinduced electron transfer from the excited chromophore group to the amide group. Ó 2001
Elsevier Science B.V. All rights reserved.
1. Introduction
model assumes the existence of well-de®ned rota-
mers about the Ca±Cb bond, whose interconver-
sion time is considerably longer than excited-state
lifetimes. This rotamer model has been further
extended to the Cb±Cc bond conformers [6±9]. The
di€erent lifetimes of the rotamers arise from
quenching interaction between the phenol ¯uoro-
phore and quenching groups. The quenching e-
ciency is controlled by the orientation of both
groups and is distance dependent. In the case of
tyrosine analogs and derivatives the rotamer
model showed that the rotamer in which the phe-
nol ring can come into the closest contact with the
carbonyl group had the shortest ¯uorescence life-
time. Both, Cowgill [10] and Tournon et al. [11]
suggested that the ¯uorescence quenching of aro-
matic amino acid by peptide /amide) group was
occurring by a charge transfer between the excited
aromatic chromophore /phenol ring), as a donor,
The ¯uorescence of aromatic amino acids
/phenylalanine, tyrosine and tryptophan) and their
residues incorporated into a peptide or protein
chain is a subject of extensive studies because of
their use as internal probes in conformational
analysis [1±3]. In the case of the tyrosine zwitterion
and tyrosine derivatives with an ionized a-carb-
oxyl group mono-exponential ¯uorescence decay
was observed. Conversion of the a-carboxyl group
to the corresponding amide or its protonation re-
sults in a complex ¯uorescence decay [1±4]. This
behavior can be partially explained in terms of the
Gauduchon and Whal rotamer model [5]. This
* Corresponding author. Fax: +48-58-3410357.
E-mail address: ww@chemik.chem.univv.gda.pl /W. Wiczk).
0009-2614/01/$ - see front matter Ó 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 9 - 2 6 1 4 / 0 1 ) 0 0 4 7 0 - 5

100
W. Wiczk et al. / Chemical Physics Letters 341 22001) 99±106
is the Coulomb attractive energy between the
produced ion pair.
It can be assumed that for a given series of
donors with the same acceptor and solvent the last
three terms in Eq. /2) are constant and it can be
rewritten as
and electrophilic units in the amino acid backbone
/carbonyl of amide bond), as an acceptor. This
mechanism of ¯uorescence quenching of tyrosine
derivatives was further supported by the depen-
dence of quenching eciency on the distance be-
tween the phenol of tyrosine residue and amide
group [12]. An assumption that the photoinduced
electron transfer process is responsible for the ar-
omatic amino acid ¯uorescence quenching by the
amide group means that the electron transfer rate
constant should depend on the ionization potential
of the excited chromophore /electron donor). In
this work we studied ¯uorescence quenching in a
series of amidated tyrosine derivatives as well as
phenylalanine amide to check the correctness of
mechanism of aromatic amino acid ¯uorescence
quenching by the amide group.
DG0 ˆ IP À E0Ã;0 ‡ const:
Substitution of Eq. /3) into Eq. /1) gives
ꢀ3†
2p
ꢁh
HR2P
kET
ˆ
1=2
ꢀ4pkkBT†
"
#
2
ꢀIP À E0Ã;0 ‡ const ‡ k†
4kkBT
 exp
À
:
ꢀ4†
ꢀ5†
Logarithmic transformation of Eq. /4) gives
2
3
ꢂ
ꢃ
2
IP À E0Ã;0 ‡ b
6
4
7
5
ln kET ˆ ln A À
;
c
2. Theory
1=2
where A ˆ ꢀ2p=ꢁh†ꢀHR2P=ꢀ4pkkBT† †, b ˆ const ‡ k
The electron transfer rate for a non-adiabatic
reaction constant according to Marcus theory [13±
16] can be expressed as [14±18]
and c ˆ 4kkBT .
Eq. /5) predicts that for the photoinduced
electron transfer process ln kET should be a para-
bolic function of the di€erence between ionizatioÃn
ꢀ
ꢁ
ˆ
2p
ꢁh
HR2P
DG
1
kET
ˆ
exp
À
;
ꢀ1†
potential and energy of the excited state of D .
1=2
kBT
ꢀ4pkkBT†
The values of E0Ã;0 were obtained from the overlap
of the normalized absorption and emission spectra
of the appropriate amino acid. When the chro-
mophore in an amino acid amide is raised to its
®rst excited singlet state, electron transfer occurs
from the chromophore to the amide group. The
electron transfer rate constant is usually obtained
from ¯uorescence lifetimes using the equation [17]
ˆ
where kB is the Boltzmann constant, DG ˆ
ꢀDG0 ‡ k†2=4k is the Gibbs activation energy in
Marcus theory, and DG0 is the Gibbs energy
change /driving force) for electron transfer reac-
tion. HRP is the electronic coupling matrix element
for the reactant /R) and product /P) and it is de-
termined in part by the distance between the
electron donor and acceptor, and in part by the
nature of the bridging group [19]. k is the Gibbs
reorganization energy of the reaction. Eq. /1) is
valid as long as the electron transfer reaction is in
the `normal' region [15,20]. For the photoinduced
electron transfer process the free energy change of
the reaction is given by [21]
1
1
kET
ˆ
À
;
ꢀ6†
s1 s2
where s1 and s2 are the ¯uorescence lifetimes of
amino acid amide and amino acid, respectively.
When the ¯uorophore displays a heterogeneous
¯uorescence decay, the ¯uorescence lifetime is ex-
pressed as an average over component lifetimes,
according to the equation [22]
DG0 ˆ IP À E0Ã;0 À EA À Dgs À e2=R;
ꢀ2†
Ã
P
i aisi
P
where E0Ã;0 is the energy of the excited donor ꢀ D †,
IP is the ionization potential of the donor, EA is
the electron anity of the acceptor, Dgs is the
solvation energy of the reduced ion pair and Àe2=R
hsi ˆ
;
ꢀ7†
1
i ai
where si is the decay time of the ith component
and ai is the pre-exponential factor obtained from

W. Wiczk et al. / Chemical Physics Letters 341 22001) 99±106
101
the ¯uorescence intensity decay analysis /Eq. /8),
see experimental part).
compounds
a
time-correlated single-photon
counting apparatus /the pico/femtosecond laser
system, Ti:Sapphire `Tsunami' laser pumped with
an argon ion laser `BeammLok' 2060 and
R3809U-05 MCP-PTM; a half-width of the re-
sponse function was about 35 ps) at the Labora-
tory of Ultrafast Laser Spectroscopy, Adam
3. Experimental
3.1. Materials
ꢀ
Mickiewicz University, Poznan, Poland [25] was
used. The excitation wavelength was 258 nm for
Phe and its derivative or 277 nm for tyrosine and
its derivatives. The excitation /258 or 277 nm) and
emission wavelengths /280 nm for Phe or 315 nm
for Tyr) were selected by means of monochroma-
tors /about 10 nm bandwidth). The emission was
detected with a magic-angle polarizer. All mea-
surements were performed at 20°C.
To prepare the amides of the compounds
studied, ®rst the amino acids Phe, Tyr, /O-
methyl)tyrosine /Tyr/Me)), /3-hydroxy)tyrosine
/Dopa), all from Aldrich, were protected with Boc-
group according to the known typical procedure
[23]. The amides, Boc±Aaa±NH±R, where R ˆ H,
Me or Et, were prepared using mixed anhydride
/obtained from iso-butyl chloroformate) and am-
monia or appropriate amine /Me±NH2 or
Et±NH2). Boc protection was removed with 4 M
HCl in dioxane. Methylation of Dopa hydroxyls
was accomplished by alkylation with dimethyl
sulfate under phase-transfer conditions. All pre-
pared derivatives were puri®ed by reverse-phase
liquid chromatography /RP-HPLC) and the puri-
ties were assessed by analytical RP-HPLC and
mass spectrometry /FD or FAB).
Fluorescence decay data were ®tted by the it-
erative convolution to the sum of exponents
X
Iꢀt† ˆ
ai expꢀÀt=si†;
ꢀ8†
i
where ai and si are parameters de®ned in Eq. /7).
The adequacy of the exponential decay ®tting was
judged by visual inspection of the plots of weigh-
ted residuals and by the statistical parameter vR2
and shape of the autocorrelation function of the
weighted residuals and serial variance ratio /SVR).
In steady-state measurements, the sample concen-
tration was about 5 Â 10À4 M, whereas it was
1 Â 10À3 M in time-resolved experiments. All
measurements were performed in double deionized
water /Millipore) at pH ˆ 6 at room temperature.
3.2. Apparatus
Absorption spectra were recorded using a Per-
kin-Elmer Lambda 18 spectrophotometer.
Fluorescence spectra were recorded using a
Perkin-Elmer LS-50B spectro¯uorimeter with 4
nm bandwidth for excitation and emission. The
excitation wavelengths were 258 nm for the phe-
nylalanine derivative and 277 nm for tyrosine de-
rivatives. The optical density of the sample at this
excitation wavelength did not exceed 0.1. The
¯uorescence quantum yields were obtained by
comparing the integral intensity of the steady-state
emission spectra /corrected for absorbance) with
that of tyrosine in water, using a value of 0.14 for
the latest [24]. Fluorescence decays for phenylala-
nine and its amide were collected by the time-
correlated single photon counting technique on an
Edinburgh Analytical Instruments type CD-900
¯uorometer /a half-width of the response function
was about 1 ns) interfaced to an IBM PC as de-
scribed previously [4], whereas for the remaining
3.3. Procedure
Ionization potentials /IPs) were calculated as a
di€erence of total energy of ionized and neutral
molecules. We used methyl-substituted chromo-
phore instead of whole amino acid residue as a
model for calculations. Calculations were per-
formed using the PM3 method /MOPAC 6.0
package) [26]. The calculated IPs were scaled to
the experimental values obtained from literature:
8.83 eV [27] for toluene, 8.22 eV [28] for p-methyl-
phenol, 8.05 eV [29], 8.09 eV [28] for
p-methyl-anisole, and 7.91 eV [30] for 2-methyl-
naphthalene. The described dependence was
obtained with very good correlation coecient

102
W. Wiczk et al. / Chemical Physics Letters 341 22001) 99±106
Table 1
Photophysical properties of amino acid and their derivatives
v2Ra
1 exp 2 exp 3 exp
Compound
Quantum yield
Pre-exponential
factor a
Lifetime s
/ns)
Phe
0.020
0.015
0.14
7.21b
1.0000
1.0000
1.0000
1.03
Phe±NH2
Tyr
5.14
1.09
3.35c
1.05
Tyr±NH2
0.042
1.39
0.43
0.5641
0.4359
8.67 1.10
Tyr/Me)
0.201
0.038
4.81
1.0000
1.04
TyrꢀMe†±NH2
1.46
0.18
0.5671
0.4329
1.36 1.03
Dopa
0.083
0.033
1.55
0.89
0.1250
0.8750
2.25 1.08
Dopa±NH2
1.14
0.23
0.54
0.047
0.558
0.395
7.0 1.46 1.15
DopaꢀMe†
0.155
0.050
1.85
1.0000
1.12
2
DopaꢀMe†2±NH2
Tyr±NHMe
0.42
0.70
0.4710
0.5290
4.80 1.12
0.055
0.083
1.01
1.78
0.4000
0.6000
4.67 1.05
5.32 1.03
Tyr±NHEt
1.14
2.25
0.7140
0.286
a For 1, 2 or 3 exponential ®t.
b From [33].
c From [34].
R ˆ 0:9951. For Dopa derivatives the IPs were
established from linear dependence between ex-
perimental and calculated IPs /Table 1).
4. Results and discussion
Absorption spectra of all compounds studied
/not shown) indicated that amidation of carboxylic
group in amino acid did not a€ect shapes and
positions of the spectra. Fluorescence spectra of
the compounds of interest are shown in Fig. 1. As
shown, amidation of carboxylates decreased the
¯uorescence intensities without changing the band
positions. The ¯uorescence quantum yields of all
compounds studied are summarized in Table 1.
Moreover, amidation of amino acids caused
shortening of ¯uorescence lifetimes and changed in
Fig. 1. Fluorescence spectra of phenylalanine /Phe), /O-me-
thyl)-tyrosine /Tyr/Me)), /3-hydroxy)tyrosine /Dopa) and /3,4-
dimethoxy)phenylalanine ꢀDopaꢀMe† † /solid lines) and their
2
amides /dashed lines) in water, pH ˆ 6 at room temperature.

W. Wiczk et al. / Chemical Physics Letters 341 22001) 99±106
103
some cases the character of ¯uorescence decay.
For example, the ¯uorescence decays of tyrosine
amides are heterogeneous /bi-exponentials). The
graphs of experimental ¯uorescence intensity de-
cay for Dopa and Dopa±NH2 are shown in Figs. 2
and 3, respectively. The approximately equal ratio
of the ¯uorescence quantum yields and the ratio of
average ¯uorescence lifetimes of the parent mole-
cule and its amide indicate that the quenching
process of the ¯uorophore takes place in excited
state only, without formation of the `dark com-
plex' in the ground state. The calculated electron
transfer rate constant, based on Eq. /6) and using
the average lifetime calculated based on Eq. /7) for
the heterogeneous decays, are in the range from
5:4  107 sÀ1 for phenylalanine /IP ˆ 8.82 eV), up
to 1:5  109 sÀ1 for Dopa /IP ˆ 7.91 eV) deriva-
tives, respectivelyÃ/Table 2). The relation between
ln kET and IP À E0;0 is presented in Fig. 4. As pre-
dicted by Eq. /5) this relation is parabolic with
very good correlation coecient /R ˆ 0:9892)
when all experimental points are used, and the
correlation is even better if the value for Dopa is
excluded /R ˆ 0:9985). The obtained parabolic
correlation between ln kET and IP À E0Ã;0 suggests
that the occurrence of photoinduced electron
transfer process from excited ¯uorophore /phenyl
or phenol ring) to the amide group is responsible
for the ¯uorescence quenching of the aromatic
amino acids by the amide /peptide) group. The
Gibbs reorganization energy ꢀk† calculated using
constant c, obtained from the ®t to Eq. /5), is equal
Fig. 2. Fluorescence intensity decay of Dopa together with the apparatus response function, best ®t /solid line) and the residuals /lower
panels) obtained for di- and mono-exponential ®t to the experimental decay.

104
W. Wiczk et al. / Chemical Physics Letters 341 22001) 99±106
Fig. 3. Fluorescence intensity decay of Dopa±NH2 together with the apparatus response function, best ®t /solid line) and the residuals
/lower panels) obtained for di- and mono-exponential ®t to the experimental decay.
Table 2
Fluorescence lifetimes, rate constant for electron transfer /ket) and ionization potentials obtained for model compounds studied
a
a
b
Compound
Average lifetime hsi
ket
ln kET
IPscal
/eV)
/ns)
/10À8 sÀ1
)
Phe
Phe±NH2
Tyr
7.21
5.14
3.35
0.973
4.81
0.906
0.98
0.395
1.85
0.57
0.541
17.806
20.407
20.613
21.136
20.917
9.09
8.53
8.42
8.30
8.17
7.29
8.96
Tyr±NH2
Tyr/Me)
Tyr/Me)±NH2
Dopa
15.11
12.14
Dopa±NH2
DopaꢀMe†
DopaꢀMe†2 À NH2
2
P
P
a For multi-exponential ®t calculated as hsi ˆ i aisi= i ai.
b Obtained from linear relation between experimental and calculated values /see Section 3).

W. Wiczk et al. / Chemical Physics Letters 341 22001) 99±106
105
decrease of its electron anity is additional proof
of the proposed mechanism.
5. Conclusions
The parabolic dependence between ln kET and
IP À E0Ã;0 obtained for the aromatic amino acid as
well as the reasonable values of the Gibbs reor-
ganization energy and electronic coupling matrix
element suggest the occurrence of photoinduced
electron transfer from excited ¯uorophore to the
amide group, which causes the ¯uorescence
quenching by the amide group. The hydration of
the whole molecule as well as the mutual spatial
orientation of the electron donor and electron ac-
ceptor and the substituent on the amide nitrogen
atom seem to play an essential role in this process.
Fig. 4. Correlation between ln ket and IP À E0Ã;0 for tyrosine
derivatives.
to 1.55 eV and similar to the value obtained by
Aoudia et al. [31] /k ˆ 1:60 Æ 0:06 eV) for photo-
induced electron transfer from tyrosine-containing
oligopeptides to metalloporphyrins in water. The
electronic coupling matrix element for the reactant
and product ꢀHRP† is equal to 0.3 meV. This value
justi®es the non-adiabatic theoretical framework
exploited in this work and use of Eq. /1) [32].
Moreover, it re¯ects the small overlap between
orbitals of the donor and acceptor involved in the
reaction.
Acknowledgements
This work was supported by Grant 0369/T09/
98/15 from the State Committee for Scienti®c
Research /KBN Poland).
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two hydroxyl groups as well as both factors men-
tioned above can play a signi®cant role.
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