Light-induced tryptophan radical generation in a click modular assembly of a sensitiser-tryptophan residue

Article abstract of DOI:10.1039/c3pp50021g

Click chemistry was used as an efficient method to covalently attach a chromophore to an amino acid. Such easily prepared model systems allow for time-resolved studies of one-electron oxidation reactions by the excitation of the chromophore by a laser flash. The model complex ruthenium-tryptophan (Ru-Trp) has been synthesised and studied for its photophysical and electrochemical properties. Despite a small driving force of less than 100 meV, excitation with a laser flash results in fast internal electron transfer leading to the formation of the protonated radical (TrpH⁺). At neutral pH electron transfer is followed by deprotonation to form the neutral Trp radical with the rate depending on the concentration of water acting as the proton acceptor. The formation of the tryptophan radical was confirmed by EPR. The Royal Society of Chemistry and Owner Societies 2013.

Full text of DOI:10.1039/c3pp50021g

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Photobiological Sciences
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Light-induced tryptophan radical generation in a click
modular assembly of a sensitiser-tryptophan residue†
Cite this: DOI: 10.1039/c3pp50021g
a
a,b
a,c
b,c
Sujitraj Sheth, Aurélie Baron, Christian Herrero, Boris Vauzeilles,
a,c
a
Ally Aukauloo* and Winfried Leibl*
Click chemistry was used as an efficient method to covalently attach a chromophore to an amino acid.
Such easily prepared model systems allow for time-resolved studies of one-electron oxidation reactions
by the excitation of the chromophore by a laser flash. The model complex ruthenium–tryptophan
(
Ru–Trp) has been synthesised and studied for its photophysical and electrochemical properties. Despite a
Received 23rd January 2013,
Accepted 15th March 2013
small driving force of less than 100 meV, excitation with a laser flash results in fast internal electron transfer
+
leading to the formation of the protonated radical (Trp˙H ). At neutral pH electron transfer is followed by
DOI: 10.1039/c3pp50021g
deprotonation to form the neutral Trp˙ radical with the rate depending on the concentration of water
acting as the proton acceptor. The formation of the tryptophan radical was confirmed by EPR.
www.rsc.org/pps
The simplest approach is to study amino acid oxidation by
Introduction
9–11
intermolecular interaction with a photosensitiser.
Model
1
Electron transfer reactions are crucial processes in biology.
complexes with tryptophan or tyrosine residues covalently
Most significantly, the energy transduction pathways of respir- linked to an Ru–bipyridyl sensitiser via an amide bond have
1
2
ation and photosynthesis rely heavily on electron transfer reac- been previously reported. Such assemblies offer, among other
tions between well-organised cofactors in the protein matrices. advantages, (i) to fix the constitutive units at a given distance,
Examples of such cofactors include chlorophylls, cytochromes, (ii) exclude the energy needed for the encounter complex for
quinones, and metal centres, as well as redox active amino acid electron transfer processes, (iii) minimise deleterious intermole-
2–4
residues such as tyrosine and tryptophan. The study of radical cular pathways, and (iv) permit intramolecular electron transfer.
5,6
formation on amino acids is a field of growing interest. In However, the synthetic tactics for realising these assemblies is
general, electron transfer chain reactions involve the partici- quite restrictive and a more versatile approach leading to the
pation of a series of redox active units where tuning of their rela- synthesis of a library of molecular assemblies is still needed.
tive position, and redox properties, is crucial in order to
optimise and control charge flow over large distances. Further azide–alkyne cycloadditions (CuAAC), commonly termed as
complications arise from the coupling of redox events to proto- click chemistry, is a relatively easy synthetic method to form
We have recently shown that the use of copper catalysed
7
1
3
nation/deprotonation reactions. Studies concerning the mechan- modular assemblies composed of ruthenium–polypyridyl type
1
4,15
istic aspects of proton-coupled electron transfer, amino acid chromophores and functional ligands.
Additionally, we
radical formation and its consequent migration in simpler have shown that the resulting triazole functional group formed
model systems are of great interest for the elucidation of struc- between the modules provides the right electronic coupling to
8
ture–function relationships in more complex biological systems.
allow for efficient electron transfer, while preserving the redox
and optical properties of the different modules. The com-
ponents used for the click reaction are an alkyne-derived Ru-
complex and a ligand bearing an azido group. Herein, we
present the synthesis and photophysical characterisation of a
dyad composed of a chromophore and a tryptophan amino
acid covalently linked via a triazole bridge.
1
6
a
CEA, iBiTec-S, UMR 8221, Service de Bioénergétique Biologie Structurale et
Mécanismes (SB2SM), F-91191 Gif-sur-Yvette, France. E-mail: winfried.leibl@cea.fr;
Fax: +33 (0) 16908 8717; Tel: +33 (0) 16908 5289
b
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles du CNRS,
UPR-2301, 1 Avenue de la Terrasse, F-91198 Gif-sur-Yvette, France
c
Institut de Chimie Moléculaire et des Matériaux d’Orsay, UMR-CNRS 8182,
Université de Paris-Sud XI, F-91405 Orsay, France. E-mail: ally.aukauloo@u-psud.fr;
Results and discussion
Fax: +33 (0) 169 154 756; Tel: +33 (0) 169 154 756
†
Electronic supplementary information (ESI) available: Details concerning syn-
The azide modified tryptophan derivative was synthesised fol-
lowing the method of Todd et al. Nucleophilic substitution
thesis, experimental procedures and additional figures mentioned in the text.
See DOI: 10.1039/c3pp50021g
17
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1
7,18
Scheme 1 Reagents and conditions: (i) NaN
, H
3 2
O (93%),
(ii) HBTU, DMAP,
17,18
DIPEA, CH
3
CN (99%).
DMAP = 4-(dimethyl-amino)-pyridine; HBTU = O-benzo-
triazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate; DIPEA
= N,N-
diisopropylethylamine.
Fig. 1 Cyclic voltammetry of 1 mM Ru–Trp vs. ferrocene in argon-saturated
acetonitrile with tetrabutyl ammonium hexafluoro phosphate as the supporting
electrolyte. Scan rate: 100 mV s−
1
.
triazole unit was detected in the potential window investigated
in these experiments. The pH dependence of the oxidation
potential of Ru–Trp was studied using differential pulse volta-
mmetry in water while varying the pH of the solution by the
Scheme 2 Reagents and conditions: (i) CuSO
CH Cl –H O (90%).
4 2
·5H O, sodium ascorbate,
2
2
2
of 2-bromo acetic acid in the presence of sodium azide (NaN3) addition of diluted solutions of HCl and NaOH. The potential
gave 2-azido acetic acid in high yield. The peptide linkage with was independent of pH below 3 and displayed a slope of
tryptophan methyl ester yielded the azido tryptophan deriva- −30 mV per pH unit in the range pH 3–8, corresponding to a
tive quantitatively (Scheme 1). The complex Ru–triazole–trypto- two-electron, one-proton process. These results are in agree-
1
1,20
phan (Ru–Trp) was obtained after performing a copper ment with those reported in similar systems.
A pK of
a
catalysed azide–alkyne cycloaddition reaction between the about 2.7 ± 0.5 can be estimated for the oxidised tryptophan
alkyne-derived complex, Ru–CCH, and the azido-derived which is at least one unit lower than typical values for an iso-
2
1,22
tryptophan (Scheme 2).
Ru–Trp shows ground state absorption and emission prop- the Ru(III)/Ru(II) potential with pH was observed.
erties very similar to those of the Ru–CCH and Ru(bpy) parent
Flash photolysis experiments were performed in order to
compounds. Its absorption spectrum is dominated by strong investigate the light-induced oxidation of the tryptophan
π–π* (bpy) transitions at 290 nm, and a metal to ligand charge residue. Excitation of Ru–Trp in a MeCN–H O (50 : 50) mixture
transfer (MLCT) absorption with a maximum at 458 nm with 460 nm laser flashes in the presence of 10 mM methyl
Fig. S1 in the ESI†). The emission maximum is found at viologen acting as a reversible electron acceptor showed
20 nm and emission kinetics shows monoexponential decay depletion of the Ru(II) signal as evidenced by a bleach in its
lated tryptophan (pK
∼ 4.3–4.7).
No significant change of
a
3
2
(
6
with a lifetime of 1040 ns (Fig. S2 in the ESI†). This value is in 450 nm absorption band, and the concomitant formation of
+
agreement with the emission quantum yield of 0.074 which reduced methyl viologen (MV ˙) observed as an increase in the
was determined from steady state emission using Ru(bpy)
3
as absorption at 605 nm (Fig. 2). The recovery of the 450 nm Ru(II)
1
9
a reference. These data show that the photophysical proper- signal is very fast (350 ns) whereas the recovery of the oxidised
ties of the chromophore are unaltered in the Ru–triazole– methyl viologen occurs on a much longer time scale (≈300 μs,
tryptophan assembly, which is in agreement with previous inset in Fig. 2). These results clearly indicate that the Ru(II)
results obtained on other triazole-connected chromophore– ground state absorption is recovered by intramolecular elec-
1
4,15
ligand assemblies.
The fact that the emission is not tron transfer from the tryptophan ligand to the oxidised chro-
quenched shows the absence of either excited state energy mophore rather than by intermolecular charge recombination
transfer, or electron transfer between the ruthenium chromo- between Ru(III) and the reduced form of the electron acceptor.
phore and the ligand.
From a thermodynamic point of view, this process is in accord
The electrochemical properties of Ru–Trp in acetonitrile with the electrochemical data where the oxidation of Trp by
were studied by cyclic and differential pulse voltammetry the Ru(III) is expected to be exergonic by about 130 mV. A com-
using ferrocene as the reference. Besides the typical features parison of the emission lifetime in the presence of the electron
of ruthenium trisbipyridyl complexes, i.e. a quasi-reversible acceptor and the kinetics of recovery of Ru(II) reveals apparent
Ru(III)/Ru(II) oxidation wave at 0.82 V (ΔE = 110 mV) and three rates which are identical within the error range. This indicates
p
2
+
quasi-reversible reduction waves for the bipyridine ligands that, under the MV concentrations used (10 mM), the for-
(
−2.25, −1.99, −1.78 V; ΔE
p
= 90, 80, 60 mV), we observed an mation of the oxidised state of the chromophore by inter-mole-
additional irreversible wave at 0.69 V (Fig. 1) which is attribu- cular reduction of MV is rate-limiting for the subsequent
ted to the oxidation of the tryptophan. No redox activity of the ligand oxidation, and that the intrinsic rate of internal electron
2
+
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2
Fig. 2 Transient absorption traces for Ru–Trp in argon-saturated MeCN–H O
Fig. 4 Transient absorption spectra recorded 3 μs after the laser flash for
(
50 : 50) solutions at 605 nm (black) and 450 nm (blue) upon excitation with
3
+
2+
Ru–Trp in MeCN–H
2
O (50 : 50) with 20 mM [Ru(NH
3
)
6
]
as an electron acceptor
nanosecond laser flashes at 460 nm. 10 mM MV was used as an external elec-
and 5 mM citric acid as a buffer.
tron acceptor. Inset: decay of 605 nm transient at a longer time scale.
2
+
2
+
The use of MV as an external electron acceptor has the
advantage that its redox state can easily be followed due to the
strong and characteristic absorption of its reduced form at 390
and 605 nm. However, the same property represents a draw-
back when monitoring the changes in the visible range that
might arise from the oxidation of the ligand. Therefore, hexa-
mineruthenium(III) chloride, which shows negligible absorp-
tion changes in the visible region upon reduction, was used as
an alternative electron acceptor. Fig. 4 shows the transient
absorption spectra at 3 μs after the excitation flash of a
mixture of the complex Ru–Trp and 20 mM hexamineruthe-
nium(III) chloride upon light excitation at 460 nm light in an
acetonitrile–water mixture. Citric acid buffer was used to
adjust the pH of the solution at values above and below the
transfer is significantly faster. Using higher MV concen-
trations allows to accelerate the formation of the Ru(III) state to
about 20 ns, as detected by the emission decay kinetics. Under
these conditions the rate of internal electron transfer could be
7
−1
determined as 100 ns (10
50 nm kinetics. This value confirms the hypothesis of the
triazole ring providing a link which allows for efficient electron
s ) by kinetic analysis of the
4
1
4,15
transfer.
Laser flash photolysis results suggest that light excitation of
Ru–Trp in the presence of an external electron acceptor leads
to oxidation of the ligand, i.e. the formation of a tryptophan
radical. We performed EPR control experiments on a sample
frozen during continuous illumination for 10 seconds in the
presence of 4-nitrobenzenediazonium tetrafluoroborate as a
sacrificial, irreversible electron acceptor. As depicted in Fig. 3,
we observed the formation of a narrow signal at g ∼ 2.00
width 14 G) typical of an organic radical. This signal is attrib-
uted to the formation of the tryptophanyl radical. The broad
signal at g ∼ 2.72 is attributed to Ru species formed after
additional oxidation of the ruthenium–tryptophanyl species.
pK of the oxidised Trp. The two spectra are characteristic of
a
the protonated (pH 2.4) and deprotonated (pH 5) forms of the
tryptophan radical which show a broad absorption maximum
at 530–600 nm for the former, and a sharper maximum at
00 nm for the latter. At time = 3 μs after the excitation flash
the two species are fully formed and decay in some hundred
(
2
3
III
5
microseconds due to charge recombination with the reduced
form of the electron acceptor.
+
At pH > pK
a
(Trp˙H ) oxidation of Trp is expected to be
accompanied by deprotonation. Time-resolved spectra with a
sample in water (neutral pH) show indeed transition from the
protonated form of the Trp radical to the deprotonated form
on a time scale of 1 μs (Fig. 5).
From the spectra of the two forms (Fig. 4 and 5) it appears
that the deprotonation reaction could be followed in the
500–600 nm spectral region. Absorption transients at 570 nm
and 510 nm are shown in Fig. 6. At both wavelengths the
prompt rise in absorption is due to the flash-induced for-
mation of the Ru excited state which decays quickly in about
2
5 ns due to quenching by the electron acceptor. At 570 nm a
Fig. 3 X-band EPR spectrum (10 scans) of a mixture of 500 μM Ru–Trp with
slower decay phase is detected which is well described by a
single exponential with a time constant of 410 ns. This transi-
ent is attributed to the fast (<100 ns) formation of the Trp˙H
radical by internal electron transfer and its subsequent dis-
appearance due to deprotonation to form Trp˙ which has
2
5 mM 4-nitrobenzenediazonium tetrafluoroborate in acetonitrile after 10
second illumination with 450 nm light. Experimental conditions: microwave fre-
quency 9.494 GHz, microwave power 0.100 mW, modulation amplitude 10 G,
temperature 50 K, time constant 1.28 ms, scan width 5000 G, modulation fre-
quency 100 kHz. Inset: zoom on the g ∼ 2.00 radical signal.
+
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Fig. 5 Transient absorption spectra of Ru–Trp in water with 40 mM [Ru-
2
Fig. 7 Kinetic traces at 570 nm in MeCN with different concentrations of H O;
3+
2+
(NH
3
)
6
]
as an electron acceptor measured at 200 (purple), 300, 500, 700,
20 mM MV was present as an electron acceptor.
1
000 ns (red) after laser flash.
percolating network of hydrogen-bonded molecules. Addition
of organic solvents has been reported to break this structure
2
5
and lead to a decrease in the rate of deprotonation reactions.
This effect is also observed in our complex as demonstrated by
the results shown in Fig. 7 where the concentration of water is
varied. Methyl viologen was used in these experiments because
of the insufficient solubility of hexamineruthenium(III) chlor-
+
ide in acetonitrile. The deprotonation of the Trp˙H radical
can be followed by the distinct decay of absorption on top of
+
the larger absorption due to the MV ˙ radical, which decays
on the hundreds of microseconds time scale. With decreasing
concentration of water, the rate of deprotonation is drastically
slowed down. For 10 vol% the corresponding time constant is
5
μs and for 3% it is 74 μs. For concentrations below 3%,
Fig. 6 Kinetic traces at 510 nm (black) and 570 nm (red) with 100 mM [Ru-
3+
(NH
3
)
6
]
as an electron acceptor in MeCN–H
2
O (50 : 50). The smooth lines rep- deprotonation can no longer be observed as its rate
+
resent best fits with monoexponential functions for t > 100 ns.
approaches the rate of charge recombination between MV ˙
and Trp˙H .
+
lower absorption at 570 nm. From the initial signal amplitude
−
3
23
+
(
3
2.6 × 10 ) and using an extinction coefficient for Trp˙H of
000 M− cm , an initial concentration of 0.9 μM could be
1
−1
Conclusions
estimated. This value compares well with the concentration of
the reduced electron acceptor (which equals the concentration In summary, we have used click chemistry as an efficient tool
of Ru(III)) formed under our experimental conditions (compare to perform the modular assembly of a chromophore and an
Fig. 2) and shows that oxidation of the Trp is quantitative. At amino acid. Such easily prepared model systems allow for
5
10 nm an exponential rise in absorption is apparent, time-resolved studies of one-electron oxidation reactions by
−
1
described by a rate of 645 ns . Similar increases in absorption creating a strongly oxidising state on the chromophore by exci-
are observed at shorter wavelengths down to 495 nm and we tation with a laser flash in the presence of external electron
assign this absorption rise to the formation of Trp˙, the depro- acceptors.
tonated form of the Trp radical, which has a slightly higher
Kinetic transient absorption studies in aqueous solution
extinction coefficient than the protonated form in this region. upon excitation with a nanosecond laser flash revealed fast
The similar kinetics observed at 570 nm and 510 nm show intramolecular electron transfer leading to the formation of a
+
that under these conditions oxidation of the tryptophan protonated tryptophanyl (Trp˙H ) species detected by its
6
−1
+
residue is followed by deprotonation with a rate of 2 × 10 s
The deprotonation of the tryptophan residue at neutral pH absorption at 570 nm disappears in less than one microsecond
is thermodynamically driven by the low pK of its oxidised with a concomitant increase in absorption at 510 nm, typical
.
a
characteristic absorption at 570 nm. At pH > pK (Trp˙H ), the
a
form. However, from a kinetic point of view proton dis- of the deprotonated form of Trp radicals. This observation
sociation is controlled by preformation of a favorable configur- demonstrates that electron transfer from Trp to Ru(III) is fol-
ation between the acid and a suitable base acting as the lowed by deprotonation to yield the neutral Trp˙ radical
2
4
proton acceptor. With water as the proton acceptor success- species. The kinetics of the deprotonation reaction is strongly
ful propagation of the dissociating proton requires
a
dependent on the water concentration.
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From a thermodynamic point of view, the system studied in
this work is characterised by a moderate driving force for
internal electron transfer from the ligand to Ru(III) resulting in
the formation of a weak acid. The value of ΔG = −130 meV
deduced from the difference of redox potentials of the chromo-
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a
) has to be considered as
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the maximum possible value for the electron transfer step.
Corrections for electrostatic interactions would bring this
value close to, or even below, 100 meV. Despite this low
driving force, the rate of internal electron transfer is fast
7
−1
(
>10 s ). This fast rate is taken to indicate good electronic
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2
6–28
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B. Vauzeilles and A. Aukauloo, Chem. Commun., 2011, 47,
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the triazole link, this approach seems to be an optimal alterna- 16 The reverse possibility, reacting an alkyne-derived ligand
tive for site-specific and selective post modification of pro-
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stability of the latter.
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Acknowledgements
2
2
This work was supported by the Agence Nationale de la
Recherche (ANR) (project ANR 2010 BLANC 0926), LABEX
CHARMMMAT and the Conseil Général d’Essonne. S. S. is sup-
ported by the IRTELIS training programme of the CEA. We
would like to thank Dr Annamaria Quaranta for helpful
discussion.
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1
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