Photochemical reaction of 2-(3-benzoylphenyl)propionic acid (ketoprofen) with basic amino acids and dipeptides

Article abstract of DOI:10.1021/jp405863e

Photoreaction of 2-(3-benzoylphenyl)propionic acid (ketoprofen, KP) with basic amino acids (histidine, lysine, and arginine) and dipeptides (carnosine and anserine) including a histidine moiety in phosphate buffer solution (pH 7.4) has been investigated w

Full text of DOI:10.1021/jp405863e

Article
pubs.acs.org/JPCB
Photochemical Reaction of 2‑(3-Benzoylphenyl)propionic Acid
(Ketoprofen) with Basic Amino Acids and Dipeptides
Tadashi Suzuki,*,† Mio Shinoda,† Yohei Osanai,‡ and Tasuku Isozaki†
†Department of Chemistry and Biological Science, Aoyama Gakuin University, Fuchinobe, Chuo-ku, Sagamihara, Kanagawa
252-5258, Japan
‡Department of Chemistry and Materials Science, Tokyo Institute of Technology, Ohokayama, Meguro-ku, Tokyo 152-8551, Japan
ABSTRACT: Photoreaction of 2-(3-benzoylphenyl)propionic
acid (ketoprofen, KP) with basic amino acids (histidine, lysine,
and arginine) and dipeptides (carnosine and anserine)
including a histidine moiety in phosphate buffer solution
(pH 7.4) has been investigated with transient absorption
spectroscopy. With UV irradiation KP− gave rise to a
carbanion through a decarboxylation reaction, and the
carbanion easily abstracted a proton from the surrounding
molecule to yield a 3-ethylbenzophenone ketyl biradical
(EBPH). The dipeptides as well as the basic amino acids
were found to accelerate the proton transfer reaction whereas
alanine and glycine had no effect on the reaction, revealing that
these amino acids having a protonated side chain act as a
proton donor. The formation quantum yield of EBPH was estimated to be fairly large by means of an actinometrical method with
benzophenone, and the bimolecular reaction rate constant for the proton transfer between the carbanion and the protonated
basic amino acids or the protonated dipeptides was successfully determined. It has become apparent that the bimolecular reaction
rate constant for the proton transfer depended on the acid dissociation constant for the side chain of the amino acids for the first
time. This reaction mechanism was interpreted by difference of the heat of reaction for each basic amino acid based on the
thermodynamical consideration. These results strongly suggest that the side chain of the basic amino acid residue in protein
should play an important role for photochemistry of KP in vivo.
the proton transfer reaction proceeds in the triplet potential
surface by time-resolved resonance Raman spectroscopy
coupled with quantum chemical calculations.19 The series of
the photochemical reaction will afford adducts of KP to a
protein, and finally cause photocontact dermatitis.
1. INTRODUCTION
Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to
be a drug providing analgesic, antipyretic, and anti-inflamma-
tory effects. Ketoprofen (2-(3-benzoylphenyl)propionic acid,
KP), one of the 2-arylpropionic acids with a benzophenone
chromophore (Figure 1), is a very potent NSAID and has been
used widely. However, serious side effects such as photocontact
dermatitis, which is divided into two categories, phototoxic and
photoallergic, have been reported in these decades.1−10
Photostability, photochemistry, and phototoxicity are key
factors in the perspective of pharmacology. The pathogenic
mechanism to cause the photocontact dermatitis by NSAIDs
with UV irradiation should be unveiled for development of new
drugs.
Photochemistry of KP has been extensively studied with
experimental and computational methods.11−19 After the UV
irradiation of KP in phosphate buffer solution (pH 7.4), where
KP exists as a deprotonated form due to its acidity (pKa 4.7),
KP− gives rise to a carbanion through a decarboxylation
reaction (Figure 2). The carbanion easily abstracts a proton
from the surrounding molecule to yield a 3-ethylbenzophenone
ketyl biradical (EBPH). The pump−probe experiment with a
two-laser system demonstrated that EBPH is produced as the
carbanion disappeared.20 Recently, Phillips’ group reported that
A protein is a polymer of amino acids joined head-to-tail in a
long chain. The chain composes a three-dimensional structure
that is unique to each type of protein. A NSAID molecule will
make a complex with a protein, and in the binding site, where a
NSAID molecule exists in a protein a stereoselective photo-
chemistry should take place.21−27 Monti et al. investigated the
interaction of R-(−)- and S-(+)-KP with bovine serum albumin
in buffer solution at neutral pH by circular dichroism, time-
resolved fluorescence, and transient absorption spectrosco-
py.21−23 They estimated the individual disappearance quantum
yields of the 1:1 and 2:1 diastereomeric KP:BSA complexes,
and discussed the photoreactivity in the BSA matrix on the
basis of diastereoselective photodecarboxylation in the two
main protein sites. It is essential to gain information on the
reactivity of NSAIDs with amino acids for understanding the
Received: June 13, 2013
Revised: July 17, 2013
Published: July 23, 2013
© 2013 American Chemical Society
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Figure 1. Molecular structures of KP, glycine, alanine, basic amino acids, and dipeptides including a histidine moiety.
form EBPH, revealing that the protonated form of these amino
acids acts as a good proton donor. Especially, the relative
formation quantum yield of EBPH by HisH+ was ca. 40 times
larger than that by ArgH+ or LysH+. However, the reactivity
and the reaction mechanism of KP with amino acids have not
been understood sufficiently.
In this article, we estimated the formation quantum yield of
EBPH from KP carbanion by means of an actinometrical
method with benzophenone, and the bimolecular reaction rate
constant for the proton transfer reaction between the carbanion
and the basic amino acids or the dipeptides was successfully
determined. The reaction mechanism of KP with basic amino
acids will be discussed in detail.
2. EXPERIMENTAL SECTION
KP (Sigma-Aldrich; purity 99.9%), L-histidine (Wako Chem-
ical; GR grade), L-lysine, L-arginine, L-anserine, L-carnosine, L-
alanine, and glycine (Sigma-Aldrich; GR grade) were used as
received. Distilled water (Kanto Chemical; HPLC grade) was
used as a solvent from freshly opened bottles. All phosphate
buffer solutions were carefully prepared as the pH values kept
to 7.4 (the concentration was 1/15 mol L−1) and were
deaerated by argon gas (purity 99.9%) for half an hour before
use.
An experimental setup for the transient absorption spectrum
measurement has been described elsewhere.30−32 Briefly, a
XeCl excimer laser (Lambda Physik; COMPex102, 308 nm)
was used as an excitation light source, and a Xe short arc lamp
(Ushio; UXL-300DO, 300 W) was used as a monitoring light.
The monitoring light passing through a sample cell (Tosoh
Quartz; T514M-ES-10, 10 mm optical path length) was
detected with a monochromator (Nikon; P250)/photomulti-
plier tube (Hamamatsu Photonics; R928) system. The signal
was measured by a digital oscilloscope (Sony Tektronix;
TDS380P) and transferred to a personal computer. The signal
was averaged over 50 laser shots to improve the S/N ratio. The
sample solution constantly flowed in the cell to remove the
effect of photoproducts. All measurements were carried out at
room temperature.
Figure 2. Reaction scheme of excited KP− with a protonated basic
amino acid (RH+) in phosphate buffer solution (pH 7.4).
pathogenic mechanism causing the photocontact dermatitis by
NSAIDs with UV irradiation. In the previous papers, we studied
the photochemistry of KP with three basic amino acids,
histidine (His), lysine (Lys), and arginine (Arg), in the
phosphate buffer solution (pH 7.4).28,29 The basic amino
acids having the protonated side chain, HisH+, ArgH+, and
LysH+, were found to accelerate the proton transfer reaction to
Absorption spectra were measured with a double beam
spectrophotometer (Jasco; Ubest V-550).
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3. RESULTS AND DISCUSSION
Article
3.1. Disappearance of KP Carbanion and Formation of
EBPH by Amino Acids and Dipeptides. Figure 3a shows a
two-dimensional description of transient absorption of KP
(0.70 mM) in phosphate buffer solution (pH 7.4) in the
presence of 20 mM carnosine (Car). The spectrum between
450 and 700 nm immediately after the laser irradiation (Figure
3
3b) was assigned to KP carbanion, produced from KP−*
through decarboxylation. The quantum yield of the decarbox-
ylation was reported to be as large as 0.75.11 The spectrum at
1.7 μs has a peak at around 520 nm, which was safely assigned
to 3-ethylbenzophenone ketyl biradical (EBPH). The absorp-
tion and emission spectra of EBPH were similar to those of a
KP ketyl radical (just like a benzophenone ketyl type radical)
but slightly blue-shifted.20 The pump−probe experiment with a
two-laser system probed that EBPH was formed as the
carbanion disappeared because the rise time of the emission
of EBPH corresponded to the lifetime of the carbanion.
Figure 4. Time profiles of the transient absorption monitored at 600
nm of KP in phosphate buffer solution (pH 7.4) without (red) and
with 20 mM of Car (blue). Inset: Plots of the decay rate constant of
carbanion (kcarbanion) against concentration of Car added.
Figure 5 shows time profiles of the transient absorption
monitored at 525 nm of KP without (red) and with 20 mM of
Car (blue). Two decaying components were observed: The fast
one is due to the carbanion and the slow one is due to EBPH.
By adding Car, the yield of EBPH obviously increased and the
decay of the carbanion was accelerated, revealing that Car
should enhance the proton transfer reaction of the carbanion to
afford EBPH.
Figure 5. Time profiles of the transient absorption monitored at 525
nm of KP in phosphate buffer solution (pH 7.4) without (red) and
with 20 mM of Car (blue). Atotal and Aslow are the total intensity
immediately after the laser irradiation and the pre-exponential factor of
the slow component, respectively.
Figure 3. (a) Two-dimensional description of transient absorption of
KP (0.70 mM) with Car (20 mM) in phosphate buffer solution (pH
Car, which is a dipeptide composed of β-alanine and His, was
found to be a proton donor for KP carbanion. The candidates
for the proton donating site of Car in the phosphate buffer
■
●
7.4) and (b) the spectra obtained at 0 ns ( , red), 40 ns ( , orange),
◆
▲
100 ns ( , green), and 1.7 μs ( , blue) after the laser.
+
solution (pH 7.4) are (i) the protonated N terminal (NH3 ),
(ii) the imidazole NH of the side chain, and (iii) the
protonated imidazole NH+, which is a minor species in the
acid−base equilibrium due to the acid dissociation constant of
the His moiety (pKa = 6.76 for Car33) (see Figures 1 and 2).
Although the same experiments with alanine and glycine
instead of Car were carried out, no effect on both the lifetime of
the carbanion and the quantity of EBPH was observed. In the
previous work on His, the protonated side chain NH+, not
the imidazole NH, was shown to be a proton-donating
site.28 Hence, it was concluded that the protonated imidazole
side chain of Car should be a proton-donating site for the KP
carbanion.
Figure 4 shows time profiles of the transient absorption
monitored at 600 nm without (red) and with 20 mM of Car
(blue) in phosphate buffer solution (pH 7.4). In the presence
of Car, the lifetime of the carbanion obviously became shorter,
indicating that some reaction took place between the carbanion
and Car. The plots of the decay rate constant of the carbanion
(kcarbanion) against the concentration of Car added are shown in
the inset of Figure 4. The apparent bimolecular quenching rate
constant, kqapp, was successfully determined to be 4.8 × 108 M−1
s−1 from the slope of the straight line obtained by the least-
squares fitting.
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Since the minor species of Car having the protonated His
side chain, CarH+, is a proton donor for the KP carbanion, the
apparent quenching rate constant of the carbanion should be
corrected with its concentration. The ratio of [CarH+] to [Car]
was estimated to be 1/4.37 in phosphate buffer solution of pH
7.4 based on the acid dissociation constant of the side chain for
Car (pKa = 6.76) with the following equation:
[Car]
log
= pH − pKa
[CarH+]
(1)
The net bimolecular quenching rate constant, kqnet, was
successfully estimated to be 2.6 × 109 M−1 s−1 (= (1 + 4.37)
kaqpp). The results are listed in Table 1. In a similar fashion, the
Table 1. Apparent and Net Quenching Rate Constants of KP
Carbanion by Amino Acids or Dipeptides and Formation
Rate Constants of EBPH
Figure 6. Plots of absorbance for BP in acetonitrile and KP in
phosphate buffer solution (pH 7.4) observed immediately after the
laser against incident laser power.
amino acids or
a
b
dipeptides
pKa
kaqpp /M−1 s−1 knqet /M−1 s−1 kr /M−1 s−1
phosphate buffer solution (pH 7.4) measured immediately after
the laser irradiation against incident laser power. A good linear
relation for both plots was observed. The εcarbanion value was
given by
c
c
His
Car
Ans
Lys
Arg
6.04
6.76
2.9 × 108
4.8 × 108
9.4 × 108
1.1 × 108
1.1 × 108
6.7 × 109
2.6 × 109
3.1 × 109
1.1 × 108
1.1 × 108
4.6 × 109
1.9 × 109
1.8 × 109
1.2 × 108
1.3 × 108
b
b
d
d
b
b
d
d
7.04
10.67
12.10
A
A
KP/IL ΦTBP
3
ε
=
·
·ε BP
carbanion
*
BP/IL
Φ
a
(3)
carbanion
pKa = −log Ka. Ka denotes acid dissociation constant of the side chain
of amino acids or dipeptides for aqueous solution at 298 K. Reference
where AKP and ABP are absorbance monitored at 525 nm
immediately after the laser irradiation for KP and BP,
respectively, IL is intensity of the incident laser shot, ΦBTP is
intersystem crossing yield of BP (1.0034), Φcarbanion is formation
quantum yield of the carbanion through decarboxylation
b
c
d
33. This work. Reference 28. Reference 29.
net quenching rate constant by anserine (Ans), which is a
dipeptide composed of β-alanine and N-methyl-His, was also
obtained. The results with basic amino acids and dipeptides are
summarized in Table 1. The knqet values varied widely from the
diffusion-controlled rate constant of water (6.5 × 109 M−1 s−1
at 293 K34) to 1 × 108 M−1 s−1, but it was found that the rate
constant strongly depends on the pKa value of the side chain in
the amino acids and the dipeptides. In the following section, the
formation quantum yield of EBPH and the proton transfer rate
constant for the formation of EBPH are estimated.
3.2. Determination of Quantum Yield and Proton
Transfer Rate Constant for EBPH Formation. As shown in
the previous section, increment of EBPH, produced from KP
carbanion through proton transfer, was clearly shown by
addition of basic amino acids and dipeptides (Figure 5). To
obtain the proton transfer rate constant between the carbanion
and amino acids or dipeptides, the quantum yield of EBPH was
estimated. Since the formation rate constant of EBPH, which
equals the disappearance rate constant of carbanion, was two
orders larger than the disappearance rate constant of EBPH, the
EBPH formation quantum yield can be expressed as
11
3
(0.75 ), and ε BP* is molar absorption coefficient at 525 nm
of 3BP* (6250 M−1 cm−1, ref 34). From the ratio of the slopes
in Figure 6 [(AKP/IL)/(ABP/IL)=0.37 0.01], the εcarbanion value
was determined to be (3100 100) M−1 cm−1. By assuming
that the εEBPH value equals that of the benzophenone ketyl
radical (BPK) at 545 nm (4600 M−1 cm−1, ref 35) because the
spectral features of BPK and EBPH are quite similar, the value
of ΦEBPH can be obtained. The ΦEBPH value in the absence of
Car was successfully estimated to be 0.50. Figure 7 shows the
formation quantum yield of EBPH against CarH+ concen-
tration. It became clear that CarH+ leads increment of EBPH.
The proposed reaction scheme for KP carbanion in the
presence of the basic amino acid is shown in Figure 8. The
formation quantum yield of EBPH from the carbanion can be
estimated from the following equation based on the reaction
scheme.
[EBPH]0
[carbanion]0
Aslow ε
Atotal εEBPH
carbanion
ΦEBPH
=
=
·
(2)
where [carbanion]0 and [EBPH]0 are yields of the carbanion
and EBPH with an incident laser power, Atotal and Aslow are total
and slow-component absorption intensities at t = 0 monitored
at 525 nm, and εcarbanion and εEBPH are molar absorption
coefficients of the carbanion and EBPH at 525 nm, respectively.
We measured the laser power dependence of absorption
intensity at 525 nm to estimate the molar absorption coefficient
of carbanion by the relative actinometrical method with
benzophenone (BP) in acetonitrile as a standard. Figure 6
shows the plots of absorbance for BP in acetonitrile and KP in
Figure 7. Plots of the Φr value against CarH+ concentration.
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Figure 8. Proposed reaction scheme of KP carbanion.
k1 + kr[amino acid]
ΦEBPH
=
k1 + k2 + kq[amino acid]
(4)
kq = kr + k3
Here, k1 and kr denote the rate constants to form EBPH
without and with amino acid, respectively, and k2 and k3 are the
rate constants for another reaction (for example 2-ethyl-
benzophenone production) without and with amino acid,
respectively. In the case of Car, the concentration of amino acid
is [CarH+].
The experimental results of the ΦEBPH value against the
CarH+ concentration shown in Figure 7 were analyzed by eq 4
with (k1 + k2) = 1.0 × 107 s−1, kq = 2.6 × 109 M−1 s−1, k1, and kr
used as fitting parameters. The value of kr by CarH+ was
successfully obtained to be 1.9 × 109 M−1 s−1. This large value
of kr in comparison with (k1 + k2) and kq indicates that the
proton transfer from the protonated histidine moiety in Car to
KP carbanion to afford EBPH is a quite efficient reaction, and
that the protonated histidine moiety is a good proton donor. In
addition, the reaction to produce EBPH not 3-ethylbenzophe-
none is the major process, suggesting that the reaction should
proceed on the triplet energy surface. This agrees well with the
experimental results obtained by time-resolved Raman spec-
troscopy, which suggested the carbanion would have a triplet
multiplicity.19
The results obtained by Car as well as other basic amino
acids and Ans are summarized in Table 1. Figure 9 shows the
logarithmic plots of the kr value against pKa. It was found that
the kr value largely depended on the pKa.
3.3. Reaction Dynamics of Excited KP with Basic
Amino Acids and Dipeptides. In the previous section, it was
shown for the first time that the bimolecular reaction rate
constant for the proton transfer strongly depended on the acid
dissociation constant for the side chain of the amino acids. The
reaction mechanism will be considered by difference of the heat
of reaction for each basic amino acid based on the
thermodynamical consideration.
Figure 9. Plots of the kr value against the pKa value of the side chain of
the amino acids or the dipeptides. The estimated α value was
determined to be 0.25 0.04.
logarithm of k against the logarithm of Ka for a series of acids
give a straight line with slope α. Horiuchi and Polanyi led the
following result from the reaction potential curve with the
activation energy Ea and the reaction enthalpy ΔH (see Figure
10),
ΔEa = αΔ(ΔH)
(7)
where ΔEa and Δ(ΔH) are differences of the activation energy
and the reaction enthalpy between reaction curves P1 and P2,
respectively, and α is the proportionality factor (0 < α < 1).
The ΔEa value equals the product of α and Δ(ΔH). The factor
α obviously depends on the relative slopes of curves R and P at
their points of intersection. The proportionality constant α
denotes the extent of reaction at the transition state. Reactions
that have low values for α are considered to have a transition
state closely resembling the reactant with little proton transfer,
while reactions for nearly unity α are to have a transition state
closely resembling the product.
The plots of log kr against pKa for the side chain of the amino
acids and dipeptides (Figure 9) clearly show a linear
correlation. This result reveals that the proton transfer reaction
between carbanion and protonated amino acids or dipeptides
obeys the Brønsted correlation. From the slope of this straight
line obtained with the least-squares analysis, the α value was
determined to be 0.25 0.04. This indicates that the proton
would be located near the side chain of the amino acid in the
The Brønsted catalysis equation is known to give information
about a reaction mechanism. Many general acid catalyzed
reactions in the ground state are found to obey the Brønsted
catalysis law,36,37
α
k ∝ Ka
(5)
log k = α log Ka + constant
(6)
where k is the catalytic reaction rate constant and Ka is the
corresponding acid dissociation constant. The plots of the
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the side chain of the basic amino acid residue in protein should
play an important role for the photochemistry of KP in vivo.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +81 427596232. Fax: +81 427596415. E-mail: suzuki@
chem.aoyama.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The present work was financially supported in part by a Grant-
in-Aid for Scientific Research (C22550007) (KAKENHI) from
Japan Society for the Promotion of Science (JSPS).
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carbanion with basic amino acids and dipeptides strongly
depends on the acidity of the side chain of the amino acids,
resulting from energy and the structure of the transition state.
Furthermore, the reaction to produce EBPH not 3-ethyl-
benzophenone is the major process, suggesting that the
reaction should proceed on the triplet energy surface. To
obtain further information on the reaction dynamics, quantum
chemical calculation on the reaction potential is in progress.
4. CONCLUSION
Photoreaction of KP− with basic amino acids and dipeptides
(carnosine and anserine) including a histidine moiety in a
phosphate buffer solution (pH 7.4) has been investigated. The
dipeptides as well as the basic amino acids were found to
accelerate the proton transfer reaction. The amino acids having
a protonated side chain act as a proton donor. The formation
quantum yield of EBPH was estimated by means of an
actinometrical method with benzophenone, and the bimolec-
ular reaction rate constant for the proton transfer was
successfully determined. It was found that the reaction of KP
carbanion with basic amino acids and dipeptides strongly
depends on the acidity of the side chain of the amino acids,
resulting from energy and the structure of the transition state.
Since the proportionality factor α was determined to be 0.25
0.04, the proton would be located near the side chain of the
amino acid in the transition state. Furthermore, the reaction to
produce EBPH not 3-ethylbenzophenone is the major process,
suggesting that the reaction should proceed on the triplet
energy surface. To obtain further information on the reaction
dynamics, quantum chemical calculation on the reaction
potential is in progress. These results strongly suggest that
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