Photooxygenation mechanisms in naproxen-amino acid linked systems

Article abstract of DOI:10.1039/c3pp50252j

The photooxygenation of model compounds containing the two enantiomers of naproxen (NPX) covalently linked to histidine (His), tryptophan (Trp) and tyrosine (Tyr) has been investigated by steady state irradiation, fluorescence spectroscopy and laser flash photolysis. The NPX-His systems presented the highest oxygen-mediated photoreactivity. Their fluorescence spectra matched that of isolated NPX and showed a clear quenching by oxygen, leading to a diminished production of the NPX triplet excited state (³NPX*-His). Analysis of the NPX-His and NPX-Trp photolysates by UPLC-MS-MS revealed in both cases the formation of two photoproducts, arising from the reaction of singlet oxygen (¹O₂) with the amino acid moiety. The most remarkable feature of NPX-Trp systems was a fast and stereoselective intramolecular fluorescence quenching, which prevented the efficient formation of ³NPX*-Trp, thus explaining their lower reactivity towards photooxygenation. Finally, the NPX-Tyr systems were nearly unreactive and exhibited photophysical properties essentially coincident with those of the parent NPX. Overall, these results point to a type II photooxygenation mechanism, triggered by generation of ¹O₂ from the ³NPX* chromophore. The Royal Society of Chemistry and Owner Societies.

Full text of DOI:10.1039/c3pp50252j

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Photooxygenation mechanisms in naproxen–
amino acid linked systems†‡
Cite this: Photochem. Photobiol. Sci.,
2014, 13, 224
Ignacio Vayá,a Inmaculada Andreu,b M. Consuelo Jiménez*a and
Miguel A. Miranda*a
The photooxygenation of model compounds containing the two enantiomers of naproxen (NPX) co-
valently linked to histidine (His), tryptophan (Trp) and tyrosine (Tyr) has been investigated by steady state
irradiation, fluorescence spectroscopy and laser flash photolysis. The NPX–His systems presented the
highest oxygen-mediated photoreactivity. Their fluorescence spectra matched that of isolated NPX and
showed a clear quenching by oxygen, leading to a diminished production of the NPX triplet excited state
(3NPX*–His). Analysis of the NPX–His and NPX–Trp photolysates by UPLC-MS–MS revealed in both cases
the formation of two photoproducts, arising from the reaction of singlet oxygen (1O2) with the amino acid
moiety. The most remarkable feature of NPX–Trp systems was a fast and stereoselective intramolecular
fluorescence quenching, which prevented the efficient formation of 3NPX*–Trp, thus explaining their
lower reactivity towards photooxygenation. Finally, the NPX–Tyr systems were nearly unreactive and
exhibited photophysical properties essentially coincident with those of the parent NPX. Overall, these
results point to a type II photooxygenation mechanism, triggered by generation of 1O2 from the 3NPX*
chromophore.
Received 30th July 2013,
Accepted 9th September 2013
DOI: 10.1039/c3pp50252j
www.rsc.org/pps
blocks.12–14 Thus, His reacts efficiently with O2, whereas Tyr
acts as a radical trap site, inducing a cascade of reactions ulti-
1
1. Introduction
The most mutagenic and carcinogenic component of solar mately leading to protein modifications. In the case of Trp,
radiation is UVB light, which is directly absorbed by bio- both singlet oxygen and radical mechanisms can be
molecules.1,2 In addition, photosensitised oxidation is at the involved.15,16
origin of lipid, protein or nucleic acid damage upon UVA-Vis
Nonsteroidal anti-inflammatory drugs (NSAIDs), parti-
excitation. It can be mediated by a variety of chromophores cularly those belonging to the 2-arylpropionic acid family, con-
(naphthalene, anthracene, etc.) and operates through a type I stitute typical examples of photosensitising drugs,17–19 which
(radical) or a type II (singlet oxygen, 1O2) mechanism.3–8
can induce oxidative damage to specific amino acids in pro-
Proteins are major targets for photosensitised oxidation, teins.20 Naproxen (NPX) is a NSAID containing a naphthalene
which is associated with loss of activity, denaturation, amino chromophore, whose photobehaviour has been characterised
acid oxidation, fragmentation, etc.9–11 In this context, amino in some detail.18,21 Indeed, it has recently been demonstrated
acid residues such as histidine (His), tryptophan (Trp) and that intermolecular photosensitisation of Trp by NPX can
tyrosine (Tyr) are among the most vulnerable protein building occur through a combination of both type I and type II
mechanisms.22
The use of tailored, well-defined linked systems has proven
to be an appropriate tool to investigate excited state inter-
actions involving fundamental processes such as energy or
electron transfer, exciplex formation, etc. In addition, these
aDepartamento de Química/Instituto de Tecnología Química UPV-CSIC, Universitat
Politècnica de València, Camino de Vera s/n, E-46022 Valencia, Spain.
E-mail: mcjimene@qim.upv.es, mmiranda@qim.upv.es; Fax: (+34)-963879349;
systems are convenient models for biologically relevant
Tel: (+34)-963877344
bUnidad Mixta de Investigación IIS La Fe-UPV, Hospital La Fe, Avda. Campanar 21,
entities.23–28
With this background, we report here a thorough mechani-
stic study on the photooxygenation of a series of naproxen–
amino acid linked systems (Chart 1). The study includes
steady-state irradiation, fluorescence, laser flash photolysis
(LFP) and product analysis by means of liquid chromatography
coupled to tandem mass spectrometry (UPLC-MS–MS).
46009 Valencia, Spain
†Dedicated to the memory of Prof. Nicholas J. Turro.
‡Electronic supplementary information (ESI) available: Additional X-ray struc-
tures, photodegradation kinetics, UV, fluorescence and transient absorption
data (8 pages). CCDC 662964, 662965, 662957, 662958, 662959 and 662960. For
ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c3pp50252j
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flows were 40 and 800 L h−1, respectively. The collision gas
flow and collision energy applied were 0.2 mL min−1 and 12 V,
respectively. All data collected in Centroid mode were acquired
using the Masslynx™ software (Waters Corp.). Leucine-enkeph-
alin was used at a concentration of 500 pg μL−1 as the lock
mass generating an [M + H]+ ion (m/z 556.2771) and fragment-
ing at m/z 120.0813 with a flow rate of 50 μL min−1 to ensure
accuracy during the MS analysis.
Fluorescence measurements
Emission spectra were recorded using a JASCO spectrofluoro-
meter system provided with a monochromator in the wavelength
range 200–900 nm. The solutions were placed into 10 × 10 mm2
quartz cells with a septum cap and were purged with nitrogen
or oxygen for at least 15 min before measurements. The absor-
bance of the samples at the excitation wavelength was kept
below 0.1. Time-resolved measurements were performed with a
Time Master fluorescence lifetime spectrometer (TM-2/2003)
from PTI by means of the stroboscopic technique. All the experi-
ments were carried out at room temperature (22 °C).
Chart 1 Chemical structure of the investigated naproxen–amino acid
linked systems.
2. Experimental section
Materials and solvents
(S)- and (R)-NPX, (S)-Trp methyl ester hydrochloride, (S)-His
methyl ester hydrochloride, (S)-Tyr methyl ester hydrochloride,
glycine methyl ester hydrochloride, 1-(3-dimethylaminopro-
pyl)-N-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxy-
benzotriazole (BtOH) were commercially available. Reagent or
spectroscopic grade solvents (hexane, ethyl acetate, aceto-
nitrile) were used without further purification.
Laser flash photolysis experiments
The LFP experiments were performed by using a Brilliant
Q-switched Nd:YAG laser (266 nm, 4 mJ per pulse, 5 ns fwhm)
coupled to a Luzchem mLFP-111 miniaturized equipment. All
transient spectra were recorded employing 10 × 10 mm2 quartz
cells with 4 mL capacity and were bubbled for 15 min with N2
or O2 before acquisition. The absorbance of the samples was
0.2 at the laser excitation wavelength. All the experiments were
General
Steady state absorption spectra were recorded with a Perkin- carried out at room temperature.
Elmer Lambda 35 UV/Vis spectrophotometer. The 1H NMR
Steady-state photolysis
and 13C NMR spectra were recorded in CDCl3 or CD3OD as a
solvent at 300 and 75 MHz, respectively, using a Varian Gemini Steady-state photolysis was performed by using a multilamp
instrument; chemical shifts are reported in δ (ppm). Combus- Luzchem photoreactor emitting at λmax = 300 nm (14 × 8 W
tion analyses were performed at the Instituto de Química Bio- lamps). Solutions (0.5 mg mL−1) were irradiated for different
Orgánica of the CSIC in Barcelona. The X-ray structures were times in acetonitrile, under N2, air or O2 through quartz.
determined at Unidade de Raios X, at the Universidade de San-
Analytical instrumentation
tiago de Compostela. Crystallographic data (excluding struc-
ture factors) for the structures of (S,S)-2, (R,S)-2, (S,S)-3, (R,S)-3, The irradiated solutions were analysed using an analytical
(S,S)-4 and (R,S)-4 have been deposited at the Cambridge Crys- Waters HPLC system connected to a PDA Waters 2996 detector,
tallographic Data Centre as supplementary publication using an isocratic flux (0.7 mL min−1) of MeCN–water–MeOH–
numbers CCDC 662964, CCDC 662965, CCDC 662957, CCDC HOAc (25 : 25 : 50 : 0.1 v/v/v/v) as an eluent, and a C18 Kromasil
662958, CCDC 662959 and CCDC 662960, respectively. Ultra 100 column, 5 μm (25 × 0.4 cm).
Performance Liquid Chromatography (UPLC) was carried out
on an ACQUITY UPLC system (Waters Corp.) with a con-
Synthesis of the substrates
ditioned autosampler at 4 °C. The separation was accom- To a solution of 0.8 mmol of (S)- or (R)-NPX in acetonitrile
plished on an ACQUITY UPLC BEH C18 column (50 mm × (20 mL), 0.8 mmol of EDC and 0.8 mmol of BtOH were added
2.1 mm i.d., 1.7 μm), which was maintained at 40 °C. The ana- as solids. The mixture was maintained under stirring, and
lysis was performed using acetonitrile and water (60 : 40 v/v then 0.8 mmol of the corresponding amino acid in 2 mL of
containing 0.01% formic acid) as the mobile phase with a flow acetonitrile were added dropwise. After three hours, the
rate of 0.5 mL min−1 and an injection volume of 5 μL. The solvent was removed under vacuum; the crude solid was dis-
Waters ACQUITY™ XevoQToF Spectrometer (Waters Corp.) was solved in methylene chloride, washed consecutively with
connected to the UPLC system via an electrospray ionization diluted NaHCO3, 1 M HCl and brine. Final purification was
interface. This source was operated in positive ionization performed by preparative layer chromatography on silica gel
mode at 100 °C with the capillary voltage at 1.5 kV and a temp- Merck 60 PF254, using hexane–ethyl acetate as an eluent,
erature of desolvation of 300 °C. The cone and desolvation gas followed by recrystallization.
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N-[2-(S)-(6-Methoxy-2-naphthyl)propanoyl]glycine
methyl
ester. 1H NMR (CDCl3) δ: 7.75–7.70 (m, 3H), 7.41–7.39 (dd, J1 =
8.4 Hz, J2 = 1.8 Hz, 1H), 7.18–7.12 (m, 2H), 5.89 (m, 1H),
4.06–3.88 (m, 2H), 3.92 (s, 3H), 3.77 (q, 1H, J = 7.2 Hz), 3.70 (s,
3H), 1.62 (d, 3H, J = 7.2 Hz). 13C NMR (CDCl3) δ: 174.8, 170.6,
158.0, 136.2, 134.1, 129.5, 129.3, 127.9, 126.6, 126.5, 119.5,
105.9, 55.6, 52.6, 47.1, 41.6, 18.7. Anal. Calcd for C17H19NO4:
C 72.54; H 6.09; N 6.51. Found: C 72.26; H 6.10; N 6.34.
N-[2-(R)-(6-Methoxy-2-naphthyl)propanoyl]glycine
methyl
ester. 1H NMR (CDCl3) δ: 7.75–7.70 (m, 3H), 7.41–7.39 (dd, J1 =
8.4 Hz, J2 = 1.8 Hz, 1H), 7.18–7.12 (m, 2H), 5.89 (m, 1H),
4.06–3.88 (m, 2H), 3.92 (s, 3H), 3.77 (q, 1H, J = 7.2 Hz), 3.70 (s,
3H), 1.62 (d, 3H, J = 7.2 Hz). 13C NMR (CDCl3) δ: 174.8, 170.6,
158.0, 136.2, 134.1, 129.5, 129.3, 127.9, 126.6, 126.5, 119.5,
105.9, 55.6, 52.6, 47.1, 41.6, 18.7. Anal. Calcd for C17H19NO4:
C 72.54; H 6.09; N 6.51. Found: C 72.35; H 6.08; N 6.42.
N-[2-(S)-(6-Methoxy-2-naphthyl)propanoyl]-(S)-histidine methyl
ester. 1H NMR (CD3OD) δ: 7.71–7.67 (m, 3H), 7.50 (br s, 1H),
7.38–7.35 (dd, J1 = 8.4 Hz, J2 = 1.5 Hz, 1H), 7.19 (d, J = 2.4 Hz,
1H), 7.12–7.08 (dd, J1 = 9.0 Hz, J2 = 2.4 Hz, 1H), 6.74 (br s, 1H),
4.66–4.61 (dd, J1 = 8.7 Hz, J2 = 5.4 Hz, 1H), 3.89 (s, 3H), 3.79 (q,
J = 7.2 Hz, 1H), 3.59 (s, 3H), 3.12–3.06 (dd, J1 = 14.7 Hz, J2 =
5.4 Hz, 1H), 3.01–2.93 (dd, J1 = 14.7 Hz, J2 = 8.7 Hz, 1H), 1.45
(d, J = 7.2 Hz, 3H). 13C NMR (CD3OD) δ: 177.5, 173.7, 159.5,
138.0, 136.7, 135.6, 130.8, 130.6, 128.4, 127.8, 127.3, 120.2,
107.0, 56.1, 54.5, 53.0, 47.5, 30.3, 19.1. Anal. Calcd for
C21H23N3O4: C 66.13; H 6.08; N 11.02. Found: C 66.40; H 6.03;
N 10.77.
Fig. 1 X-ray structures of (S,S)-2 (A), (S,S)-3 (B) and (S,S)-4 (C).
Irradiation was performed in a multilamp photoreactor
(λmax = 300 nm, MeCN) under N2, air or O2 atmospheres. The
course of photodegradation was followed by HPLC (detection
at λ = 254 nm). As anticipated, the reference compound (S)-1
was fairly photostable under all conditions (Fig. S2 of ESI‡).
The same was true for the Tyr derivatives (S,S)- and (R,S)-4.
By contrast, the behaviour of the histidine and tryptophan
analogues exhibited different patterns (see Fig. 2 and Fig. S3
of ESI‡).
N-[2-(R)-(6-Methoxy-2-naphthyl)propanoyl]-(S)-histidine methyl
ester. 1H NMR (CD3OD) δ: 7.69–7.65 (d + d, J = 9.0 and 8.7 Hz,
2H), 7.61 (br s, 1H), 7.37 (br s, 1H), 7.30–7.26 (dd, J1 = 8.7 Hz,
J2 = 1.8 Hz, 1H), 7.18 (d, J = 2.4 Hz, 1H), 7.12–7.08 (dd, J1
9.0 Hz, J2 = 2.4 Hz, 1H), 6.56 (br s, 1H), 4.66–4.62 (dd, J1
=
=
8.7 Hz, J2 = 5.4 Hz, 1H), 3.88 (s, 3H), 3.79 (q, J = 7.2 Hz, 1H),
3.69 (s, 3H), 3.08–3.02 (dd, J1 = 14.7 Hz, J2 = 5.4 Hz, 1H),
2.95–2.87 (dd, J1 = 14.7 Hz, J2 = 8.7 Hz, 1H), 1.48 (d, J = 7.2 Hz,
3H). 13C NMR (CD3OD) δ: 177.5, 173.9, 159.5, 138.1, 136.5,
135.6, 130.8, 130.6, 128.5, 127.6, 127.2, 120.3, 107.0, 56.1, 54.4,
53.2, 47.4, 30.4, 18.9. Anal. Calcd for C21H23N3O4: C 66.13;
H 6.08; N 11.02. Found: C 66.18; H 6.27; N 10.77.
3. Results and discussion
3.1 Steady-state photolysis
Synthesis of compounds 1–4 (see Chart 1) was performed by
coupling of NPX with the appropriate amino acid methyl ester.
They were fully characterised by spectroscopic techniques and
X-ray diffraction (see Fig. 1 for the (S,S)-diastereomers and
Fig. S1 of ESI‡ for the (R,S)-analogues). The glycine derivative
(S)-1 was selected as a model system, since it contains the pep-
tidic bond that could modulate the photoreactivity and the
photophysical properties of the naphthalene moiety. Blocking
the carboxy group of NPX in (S)-1 was expected to avoid the for-
mation of photoproducts derived from decarboxylation of the
parent drug.19
Fig. 2 Top: photoreactivity of (S)-1 (black), (S,S)-2 (blue), (S,S)-3 (green)
and (S,S)-4 (red). Middle: oxygen-mediated photoreactivity of the same
compounds (values under air minus values under nitrogen). Bottom:
photoreactivity of (S,S)-2 under different conditions. Codes: solid circles
(N2), half solid circles (air) and open circles (O2).
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Photodegradation of the investigated (S,S)- systems, under
air, is shown in Fig. 2 (top). Clearly, the highest photoreactivity
was observed for (S,S)-2 followed by (S,S)-3. To clarify the role
of oxygen in the overall photoreactivity, the extent of the photo-
degradation under nitrogen was subtracted from that obtained
under air (Fig. 2, middle). Although this constitutes a simpli-
fied approach, it suggests that (S,S)-2 is the substrate exhibit-
ing the highest oxygen-mediated photoreactivity. In this
context, the photobehaviour of (S,S)-2 under N2, air and O2
atmospheres is compared in Fig. 2 (bottom). While photo-
degradation under N2 was nearly negligible, this was not the
case in oxygenated media. Interestingly, the photoreactivity in
oxygen-saturated medium was lower than that observed for air-
equilibrated solutions.
It is well known that photooxidation of His basically occurs
through a type II mechanism, while Tyr mainly photoreacts
through the formation of radical intermediates.13,14,20,29,30 In
addition, it has been previously demonstrated that NPX is
able to photosensitise the formation of 1O2 with a quantum
yield ϕΔ = 0.27.31 This would be consistent with photo-
degradation of 2 in aerobic media and with the lack of reacti-
vity of the Tyr analogues (Fig. S4 of ESI‡). Interestingly,
although Trp can, in principle, be photooxidised through both
type I and type II mechanisms,9,22,32 a very low oxygen-
mediated photoreactivity was noticed for 3 (Fig. 2, middle). In
this context, to gain a better understanding of the involved
photooxidation processes, photophysical studies by means of
fluorescence and laser flash photolysis measurements were
performed.
Fig. 3 (A) Emission spectra of (S,S)-2 (λexc = 266 nm, MeCN) in N2, air or
O2. (B) Transient absorption spectrum of (S,S)-2 in MeCN/N2 upon laser
flash photolysis (λexc = 266 nm), obtained 2.9 μs after the laser pulse.
Insets: Stern–Volmer plots for quenching of the singlet and triplet
excited states by oxygen.
oxygen, shown in the Fig. 3B inset, a rate constant value of
8.6 × 109 M−1 s−1 was obtained.
3.2 Photophysical studies
Interestingly, photodegradation in
a saturated oxygen
medium occurs to a lesser extent than in the aerated solution
(Fig. 2, bottom), where 1O2 is more efficiently formed as a com-
bined result of poor quenching of the singlet and efficient
quenching of the triplet excited states. A similar behaviour was
observed for the (R,S)-analogue (Fig. S3 of ESI‡). The photo-
physical results are in agreement with a type II photooxidation
mechanism for the O2-mediated photoreactivity of NPX–His
systems.
As regards the Trp and Tyr-based systems (3 and 4), their
emission properties upon excitation at 266 nm (where both
chromophores absorb) have been recently reported.23 In the
present work, fluorescence measurements have been per-
formed at 310 nm, to achieve selective excitation of the NPX
chromophore. In agreement with previous observations, a
stereoselective charge transfer quenching was noticed for (S,S)-
and (R,S)-3, whose fluorescence quantum yields were 0.09 and
0.04, respectively (compared to 0.45 for (S)-1).
As stated above, photoreactivity was found to be highly depen-
dent on the presence of oxygen. Hence, the singlet and triplet
excited state behaviour of the systems was investigated in the
presence and absence of O2.
The UV-vis absorption spectra of 1–4 were identical to the
added spectra of isolated NPX and the corresponding amino
acid subunits at the same concentration (Fig. S5 of ESI‡). This
revealed the absence of any significant intramolecular ground-
state interaction between the two moieties.
The emission spectra of (S,S)- and (R,S)-2 were nearly identi-
cal to that of (S)-1, revealing the lack of quenching of the
excited singlet state (1NPX*) by His (Fig. S6 of ESI‡). However,
a clear oxygen effect on the fluorescence intensity was
observed (Fig. 3A). From the slope of the Stern–Volmer plot
(Fig. 3A inset), taking into account the singlet lifetime (τF
=
11.5 ns), the quenching rate constant by oxygen was deter-
mined as 2.1 × 1010 M−1 s−1 for (S,S)-2. This process competes
with intersystem crossing, resulting in a diminished formation
of the triplet excited state.
As regards the transient absorption experiments, (S,S)-2 dis-
played the typical naphthalene-like triplet–triplet absorption
band corresponding to 3NPX* (Fig. 3B). The triplet lifetime (τT)
was identical to that obtained for (S)-1, revealing the absence
of any significant intramolecular interaction in the excited
triplet state. From the Stern–Volmer plot for quenching by
Expectedly, triplet formation in (S,S)- and (R,S)-3 was much
less effective than that for (S)-1 (Fig. 4B), which can be attribu-
ted to quenching of the singlet precursor. This is consistent
with the low reactivity of 3 in oxygenated media (Fig. 2,
1
middle), where the quantum yields of O2 would be very low.
Here, no shortening of the τT values was observed, indicating
the absence of any intramolecular quenching of the NPX
triplet excited state.
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Fig. 4 Photophysical measurements on (S)-1 (black), (S,S)-3 (dark
green) and (R,S)-3 (light green) in N2-purged acetonitrile solutions. (A)
Fluorescence spectra (λexc = 310 nm) and (B) triplet excited state decays
at 420 nm (λexc = 266 nm). Inset shows the triplet–triplet absorption
bands 2.9 μs after the laser pulse.
Fig. 5 Assigned structures and MS–MS spectra of the main photo-
products 2a (top) and 2b (bottom) obtained from (S,S)-2.
Finally, neither fluorescence nor laser flash photolysis
measurements exhibited noticeable differences between the
unreactive (S,S)- and (R,S)-4 and the model compound (S)-1
(Fig. S7 of ESI‡), indicating the lack of any excited state
interaction.
tentatively assigned to the N-formylkynurenine 3a (top) and
3-hydroxypyrroloindole 3b (bottom) derivatives. Exact mass
determination led to the molecular formulae C26H26N2O6 and
C26H26N2O5, respectively. The fragmentation patterns were
fully consistent with this assignment.
3.3 Photoproduct analysis
Attention was focused on the NPX–His system as it showed the
highest oxygen-mediated photoreactivity (Fig. 2, middle). It is
generally accepted that the photooxygenation of His involves
reaction with the singlet oxygen.14 The primary products are
4. Conclusions
unstable bicyclic endoperoxides,33 which break down to give The photooxygenation mechanism of linked systems contain-
the final products.
ing (S)- or (R)-naproxen and His, Trp or Tyr has been investi-
combination of steady-state photolysis and
The photomixtures obtained with (S,S)-2 were submitted to gated by
a
UPLC-MS-MS. This led to the detection of the major photo- photophysical measurements. The highest oxygen-mediated
products 2a and 2b; the attributed structures are shown in photoreactivity was observed for NPX–His, which gave rise to
Fig. 5.
the aminosuccinic acid derivatives 2a and 2b, typical His-
Structural assignment was based on the exact mass values, derived singlet oxygen products. Photodegradation was faster
indicating molecular formulae C19H22N2O5 and C19H21NO6. As in air equilibrated than in fully oxygenated solutions, in agree-
regards the fragmentation patterns, both photoproducts ment with the diminished production of 3NPX*–His under the
shared a common naproxen-derived C13H13O ion (denoted as latter conditions. The most remarkable feature of NPX–Trp
R in Fig. 5), together with diagnostically important C5-frag- systems was a fast and stereoselective intramolecular fluo-
ments corresponding to the oxidation of His (RI and RII in rescence quenching, which prevented the efficient population
Fig. 5).
of 3NPX*–Trp, thus resulting in a lower reactivity towards
Although the NPX–Trp systems reacted only sluggishly, photooxygenation. However, analysis of the NPX–Trp photo-
UPLC-MS–MS analysis of the photolysates also revealed the lysates by UPLC-MS–MS allowed the detection of N-formylky-
presence of typical Trp singlet oxygenation products5,34 in nurenine 3a and 3-hydroxypyrroloindole 3b, presumably
low amounts. Thus, Fig.
6
shows the MS–MS spectra arising from the reaction of a singlet oxygen (1O2) with the Trp
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Financial support from the Spanish Government (CTQ2010-
14882, JCI-2011-09926, Miguel Servet CP11/00154), from the
EU (PCIG12-GA-2012-334257), from the Universitat Politècnica
de València (SP20120757) and from the Consellería de Educa-
ció, Cultura i Esport (PROMETEOII/2013/005, GV/2013/051) is
gratefully acknowledged.
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