Stereodifferentiation in the intramolecular singlet excited state quenching of hydroxybiphenyl-tryptophan dyads

Article abstract of DOI:10.1039/c3ob27278h

The photochemical processes occurring in diastereomeric dyads (S,S)-1 and (S,R)-1, prepared by conjugation of (S)-2-(2-hydroxy-1,1′-biphenyl-4-yl) propanoic acid ((S)-BPOH) with (S)- and (R)-Trp, have been investigated. In acetonitrile, the fluorescence spectra of (S,S)-1 and (S,R)-1 were coincident in shape and position with that of (S)-BPOH, although they revealed a markedly stereoselective quenching. Since singlet energy transfer from BPOH to Trp is forbidden (5 kcal mol^-1 uphill), the quenching was attributed to thermodynamically favoured (according to Rehm-Weller) electron transfer or exciplex formation. Upon addition of 20% water, the fluorescence quantum yield of (S)-BPOH decreased, while only minor changes were observed for the dyads. This can be explained by an enhancement of the excited state acidity of (S)-BPOH, associated with bridging of the carboxy and hydroxy groups by water, in agreement with the presence of water molecules in the X-ray structure of (S)-BPOH. When the carboxy group was not available for coordination with water, as in the methyl ester (S)-BPOHMe or in the dyads, this effect was prevented; accordingly, the fluorescence quantum yields did not depend on the presence or absence of water. The fluorescence lifetimes in dry acetonitrile were 1.67, 0.95 and 0.46 ns for (S)-BPOH, (S,S)-1 and (S,R)-1, respectively, indicating that the observed quenching is indeed dynamic. In line with the steady-state and time-resolved observations, molecular modelling pointed to a more favourable geometric arrangement of the two interacting chromophores in (S,R)-1. Interestingly, this dyad exhibited a folded conformation in the solid state.

Full text of DOI:10.1039/c3ob27278h

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Biomolecular Chemistry
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Stereodifferentiation in the intramolecular singlet
excited state quenching of hydroxybiphenyl–
tryptophan dyads†
Cite this: Org. Biomol. Chem., 2013, 11,
1958
Paula Bonancía,a Ignacio Vayá,a Dimitra Markovitsi,b Thomas Gustavsson,b
M. Consuelo Jiménez*a and Miguel A. Miranda*a
The photochemical processes occurring in diastereomeric dyads (S,S)-1 and (S,R)-1, prepared by conju-
gation of (S)-2-(2-hydroxy-1,1’-biphenyl-4-yl)propanoic acid ((S)-BPOH) with (S)- and (R)-Trp, have been
investigated. In acetonitrile, the fluorescence spectra of (S,S)-1 and (S,R)-1 were coincident in shape and
position with that of (S)-BPOH, although they revealed a markedly stereoselective quenching. Since
singlet energy transfer from BPOH to Trp is forbidden (5 kcal mol−1 uphill), the quenching was attributed
to thermodynamically favoured (according to Rehm–Weller) electron transfer or exciplex formation.
Upon addition of 20% water, the fluorescence quantum yield of (S)-BPOH decreased, while only minor
changes were observed for the dyads. This can be explained by an enhancement of the excited state
acidity of (S)-BPOH, associated with bridging of the carboxy and hydroxy groups by water, in agreement
with the presence of water molecules in the X-ray structure of (S)-BPOH. When the carboxy group was
not available for coordination with water, as in the methyl ester (S)-BPOHMe or in the dyads, this effect
was prevented; accordingly, the fluorescence quantum yields did not depend on the presence or absence
of water. The fluorescence lifetimes in dry acetonitrile were 1.67, 0.95 and 0.46 ns for (S)-BPOH, (S,S)-1
and (S,R)-1, respectively, indicating that the observed quenching is indeed dynamic. In line with the
steady-state and time-resolved observations, molecular modelling pointed to a more favourable geo-
metric arrangement of the two interacting chromophores in (S,R)-1. Interestingly, this dyad exhibited a
folded conformation in the solid state.
Received 23rd November 2012,
Accepted 11th January 2013
DOI: 10.1039/c3ob27278h
www.rsc.org/obc
study of dyads containing NSAIDs and the amino acid trypto-
phan (Trp), as valuable tools to provide relevant information
Introduction
Bichromophoric compounds incorporating two photoactive on drug–protein interactions within the binding sites.3 In this
moieties are useful models to gain insight into key mechanis- context, steady-state and time-resolved fluorescence spectro-
tic aspects of light-induced processes. In this context, intra- scopy has been widely used to address a variety of biological
molecular energy, proton or electron transfer, as well as problems,4 including the excited state interactions between
exciplex formation and photochemical reactivity, have received proteins and complexed ligands.5
considerable attention.1 These processes have been investi-
The photophysical behaviour of diastereomeric dyads con-
gated in a variety of systems, including dyads containing a taining the biphenyl-like NSAID flurbiprofen ((S)-FBP, Chart 1)
non-steroidal anti-inflammatory drug (NSAID) covalently and Trp has recently been described.6 Upon irradiation at
linked to a biomimetic substructure (for instance, an amino 266 nm, both chromophores are excited, but only Trp emission
acid or a nucleoside, as models for proteins or DNA).2 Specifi- (ES = 96 kcal mol−1) is detected. Moreover, stereoselective
cally, previous efforts have been devoted to the photophysical
aDepartamento de Química/Instituto de Tecnología Química UPV-CSIC, Universitat
Politécnica de València, Camino de Vera s/n, 46022 Valencia, Spain.
E-mail: mcjimene@qim.upv.es, mmiranda@qim.upv.es
bCNRS, IRAMIS, SPAM, Francis Perrin Laboratory, URA 2453, 91191 Gif-sur-Yvette,
France
†Electronic supplementary information (ESI) available. CCDC 662956, 910283,
and 910284. For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c3ob27278h
Chart 1
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intramolecular 1Trp* charge transfer quenching is observed,
accompanied by formation of exciplexes. Indeed, the absorp-
tion and emission properties of Trp are very sensitive to the
local environment7 and have been employed to investigate
structural changes of protein functions8 and to address the
protein binding of endogenous and exogenous agents (such as
drugs, fatty acids, metabolites).9
Attachment of a hydroxy group at the ortho position of a
biphenyl results in a significant decrease of its singlet energy
(ca. 10 kcal mol−1) and provides an additional deactivation
pathway, namely excited state intramolecular proton transfer
(ESIPT). As ESIPT is in general a reversible process,10 it has
found application for the design of photostabilizers, solar
filters, solar energy collectors, etc.11 In hydroxybiphenyls,
Fig. 2 Fluorescence spectra of (S)-BPOH (black), (S,S)-1 (red) and (S,R)-1 (blue)
at λexc = 267 nm, under air. Solid lines: MeCN; dashed lines: MeCN–H2O 4 : 1.
ESIPT occurs from the acidic phenolic proton to the sp2 hybrid- (S)-TrpMe), indicating the absence of significant interactions
ized 2′-carbon of the aromatic ring, giving rise to a reactive between the two moieties in the ground state.
quinone methide. In addition, intermolecular excited state
deprotonation is observed in the presence of bulk water, to after excitation at λ = 267 nm in MeCN–air, are shown in Fig. 2
give the corresponding phenolate.12
(solid lines) together with that of (S)-BPOH. All the emissions
The fluorescence spectra of (S,S)-1 and (S,R)-1, obtained
With this background, we have now investigated the photo- consisted in a structureless band centered at ca. 330 nm
physical processes occurring in diastereomeric dyads (S,S)-1 (Table 1). In the dyads, the incident photons are absorbed by
and (S,R)-1 (Chart 1), which contain a chiral 2-hydroxybiphenyl both the hydroxybiphenyl chromophore and the Trp moiety
derivative covalently linked to Trp.
(Fig. 1); however, no Trp contribution was observed in the fluo-
rescence spectra, which were coincident in shape and position
with that of (S)-BPOH. From the intersection between the
excitation (λem = 330 nm) and emission (λexc = 267 nm) normal-
ised bands (Fig. S4 in ESI†), a singlet energy value of ca.
91 kcal mol−1 was obtained for (S)-BPOH, (S,S)-1 and (S,R)-1.
In the dyads, the most remarkable observation was a dra-
matic quenching, which turned out to be stereoselective (see
Table 1). Since SSET from (S)-BPOH to Trp is thermodynami-
cally forbidden, the observed quenching was in principle
attributed to electron transfer or exciplex formation.
The feasibility of formation of radical ion pairs or exciplexes
from the singlet excited state can be estimated by application
of the Rehm–Weller equations.14 Taking into account the cor-
responding oxidation (EOX) and reduction (ERED) potentials,15
the appropriate dielectric constants (ε)16 and the (S)-BPOH
singlet energy (ES = 91 kcal mol−1), a favourable thermo-
dynamics was anticipated for charge transfer quenching via
both electron transfer and exciplex formation (see Table S1 on
p. S6 of ESI†). These processes are more likely to occur in
folded conformations, since close contact is required between
the donor and acceptor moieties. As a matter of fact, the (S,R)-
diastereomer exhibited a folded conformation in the solid
state, as shown by X-ray diffraction (Fig. 3). Interestingly,
quenching was more efficient in this dyad than in (S,S)-1.
To check whether ESIPT from the acidic phenolic proton to
the sp2 hybridised 2′-carbon of the neighbouring aromatic ring
could play a role in these systems, in competition with charge
transfer quenching, 20% of water was added (dashed lines in
Fig. 2). This water content should be low enough as to prevent
intermolecular excited state deprotonation to bulk water,
giving the corresponding phenolate.
Results and discussion
The required (S)-2-(2-hydroxy-1,1′-biphenyl-4-yl)propanoic acid
((S)-BPOH) was readily obtained by irradiation of (S)-FBP in
aqueous media, through a photonucleophilic substitution.13
The dyads (S,S)-1 and (S,R)-1 were prepared by conjugation of
((S)-BPOH) with the (S)- or (R)-methyl ester of tryptophan
(TrpMe), using a carbodiimide as the activating agent. The
chemical structures are shown in Chart 1.
The UV-Vis absorption spectrum of (S,S)-1 in MeCN is
shown in Fig. 1, together with those of (S)-FBP and (S)-BPOH
for comparison. It can be observed that replacement of fluor-
ine by hydroxyl induces a bathochromic shift of the longer
wavelength band. The spectra of (S,S)-1 and (S,R)-1 were coinci-
dent and matched that obtained upon addition of the absorp-
tion spectra of the isolated chromophores ((S)-BPOH and
In this solvent mixture, the emission maxima were red-
shifted, due to the enhanced medium polarity. Remarkably,
Fig. 1 UV-Vis absorption spectra of (S)-FBP (grey), (S)-BPOH (black), (S)-TrpMe
(green) and (S,S)-1 (red) in acetonitrile at 5 × 10−5 M concentration.
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Table 1 Relevant photophysical parameters for (S)-BPOH, (S)-BPOHMe, (S,S)-1 and (S,R)-1a
MeCN
MeCN–H2O
(S)-BPOH
(S)-BPOH
(S)-BPOHMe
(S,S)-1
(S,R)-1
(S)-BPOHMe
(S,S)-1
(S,R)-1
em
max
b
λ
ϕF
(nm)
331
0.241
1.67
1.43
—
334
0.290
2.10
1.38
—
330
332
338
0.200
1.27
1.57
—
340
0.280
1.80
1.55
—
339
339
0.116
0.95
1.22
4.5
0.52
0.43
0.042
0.46
0.91
15.8
0.83
0.68
0.139
1.07
1.29
1.5
0.31
0.16
0.060
0.57
1.05
9.7
0.70
0.55
τF (ns)c
kF × 108 (s−1 d
kQ × 108 (s−1 e
ϕQ(ov)
ϕQ(dyn)
)
)
f
—
—
—
—
—
—
—
—
g
b
a The absorbance of the samples was 0.2 at the excitation wavelength. λexc = 267 nm, air, (S)-Trp in water as standard (ϕstd = 0.13).
c λem = 330 nm. d kF = ϕF/τF. e kQ = (1/τF(dyad) − 1/τF(BPOH)). f ϕQ(ov) = [1 − (ϕF(dyad)/ϕF(BPOH))]. g ϕQ(dyn) = ϕF(dyad)kQ(dyad)/kF(dyad)
.
Fig. 3 X-ray structure of compound (S,R)-1.
the fluorescence quantum yield of (S)-BPOH was clearly lower
than in dry MeCN, while only minor changes were observed
for the dyads (Table 1). A similar trend has been reported for
the model compound 2-hydroxybiphenyl under related con-
ditions (acetonitrile containing 10 M water), although fluo-
rescence quenching occurs to a lesser extent.12 The interesting
effect observed with (S)-BPOH in the presence of water can be
explained by enhancement of the excited phenol acidity,
associated with bridging of the carboxy and hydroxy groups by
water. As a matter of fact, this situation is clearly observed in
the X-ray structure of (S)-BPOH, which actually crystallizes with
a water molecule (Fig. 4A). This effect should be, in principle,
avoided by preventing water-mediated bridging between
Fig. 4 Fragment of the unit cell for A (S)-BPOH and B (S)-BPOHMe.
carboxy and hydroxy groups.
With this purpose, we synthesized the methyl esther (S)-
BPOHMe. As anticipated, the fluorescence quantum yields of
this model remained practically constant in the presence and
absence of water (Table 1). Accordingly, water molecules were
absent in the X-ray structure of (S)-BPOHMe (Fig. 4B). In the
case of the dyads, the acidity of the phenolic group is not
enhanced as in (S)-BPOH, because the carboxy group is
employed in the construction of the amide linker and is not
available for coordination with water. In fact, no water mol-
ecules were found in the crystalline samples.
The fluorescence lifetimes were determined by the time-cor-
related single photon counting (TCSPC) technique (Fig. 5). In
dry acetonitrile, τF ((S)-BPOH) was found to be 1.67 ns, while τF
values of 0.95 and 0.46 ns were measured for (S,S)-1 and (S,R)-1,
respectively (Table 1). This indicates that most of the overall
quenching observed in the steady-state experiments is indeed
Fig. 5 Kinetic decay traces (λexc = 267 nm, λem = 330 nm, A267 = 0.2) for
(S)-BPOH (black), (S,S)-1 (red) and (S,R)-1 (blue) under air, in MeCN (solid line)
and MeCN–H2O 4 : 1 (open circles).
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steady-state and time-resolved experimental results, molecular
modelling points to a more favourable geometric arrangement
of the two interacting chromophores in (S,R)-1 than in (S,S)-1;
this is validated by the X-ray analysis, indicating that the
former dyad exhibits a folded conformation in the solid state.
Fig. 6 Geometry optimised (HyperChem Release 8.0.3 for Windows Molecular
Model System, PM3) structure for (S,S)-1 (A) and (S,R)-1 (B).
Materials and methods
Commercial (S)-FBP, (S)- and (R)-TrpMe, 1-(3-dimethylamino-
propyl)-N-ethylcarbodiimide hydrochloride (EDC) and 1-hydro-
xybenzotriazole (BtOH) were purchased from Sigma-Aldrich.
Their purity was checked by 1H NMR and HPLC analysis. Spec-
trophotometric HPLC or reagent grade solvents were obtained
from Scharlab and used without further purification. Solutions
of phosphate-buffered saline (PBS) (0.01 M, pH 7.4) were pre-
pared by dissolving phosphate-buffered saline tablets (from
Sigma) in Milli-Q water. The 1H-NMR and 13C-NMR spectra
were recorded in CDCl3 at 300 and 75 MHz, respectively, using
a Varian Gemini instrument; chemical shifts are reported in
ppm. Exact mass values were obtained in Servicio de Espec-
troscopía de Masas de la Universidad de Valencia. The X-ray
structures were determined at Unidade de Raios X, at the Uni-
versidade de Santiago de Compostela. Crystallographic data
(excluding structure factors) for the structures of (S)-BPOH,
(S)-BPOHMe and (S,R)-1 have been deposited at the Cambridge
Crystallographic Data Centre as supplementary publication
numbers CCDC 662956, CCDC 910283 and CCDC 910284,
respectively. Isolation and purification were done by conven-
tional column chromatography on silica gel Merck 60
(0.063–0.200 mm), or by preparative layer chromatography on
silica gel Merck 60 PF254, using hexane–ethyl acetate as the
eluent.
dynamic, as expected for a charge transfer process (compare
the two sets of values in Table 1). In the presence of water,
a decrease of the (S)-BPOH fluorescence lifetime was noticed,
confirming again a dynamic quenching, in agreement with
enhanced participation of the ESIPT route. As discussed above,
this deactivation channel does not operate in the dyads, where
the carboxylate function is involved in formation of the amide
bond.
The quenching rate constants (kQ), determined from the
fluorescence lifetimes τF, were in the range 108–109 −1; as
s
regards the radiative rate constants kF, they were obtained from
the values of τF and ϕF and found to be in the order of 108 s−1
(Table 1).
In order to get insight into the geometrical arrangement of
the two chromophores in the dyads, which is closely related
with the fluorescence quenching by charge transfer, simple
molecular modelling (PM3) was performed with both (S,S)-1
and (S,R)-1 (see Fig. 6).
Thus, (S,R)-1 (Fig. 6B) presented a more favourable geo-
metric arrangement of the two interacting chromophores com-
pared to the (S,S)-dyad (Fig. 6A), whose geometry is more
distorted, with the indole chromophore nearly orthogonal to
the biphenyl moiety, which makes the interchromophoric
interaction more difficult. This is in good agreement with the
steady-state and time-resolved observations of lower ϕF and
shorter τF values for the (S,R)-diastereomer.
Synthesis of the new compounds
(S)-BPOH. A solution of (S)-FBP (100 mL, 5 mM) in PBS was
irradiated for 9 h through quartz, inside a Luzchem multilamp
photoreactor, with the light from ten 8 W lamps emitting
mainly at 254 nm. The photomixture was acidified with HCl,
extracted with methylene chloride and dried over MgSO4. The
Conclusions
The (S)-BPOH chromophore dominates the fluorescence organic phase was evaporated in vacuo and purified by prepara-
spectra of diastereomeric dyads (S,S)-1 and (S,R)-1 in aceto- tive layer chromatography, using hexane–ethyl acetate 40/60
nitrile, which reveal a substantial intramolecular charge trans- (v/v) as the eluent, affording (S)-BPOH as a white powder (60%
fer quenching with remarkable stereodifferentiation. In the yield).
presence of water (20%), the fluorescence quantum yield of
(S)-BPOHMe. A solution of (S)-BPOH (59 mg, 0.23 mmol)
(S)-BPOH decreases; by contrast, only minor changes are and SOCl2 (2.4 mL) in MeOH (10 mL) was maintained under
observed for (S,S)-1 and (S,R)-1. (S)-BPOH being a phenol, this reflux for 2 h. The crude was rotavaporated, dissolved in
is attributed to a water-assisted enhancement of its excited methylene chloride and washed consecutively with saturated
state acidity, a hypothesis that is supported by the presence of NaHCO3, 1 M HCl and brine, then dried over MgSO4. The
water molecules linking the carboxy and hydroxy groups in the solvent was evaporated in vacuo. Purification was done by pre-
crystalline structure. This effect is prevented in the dyads, parative layer chromatography (hexane–ethyl acetate, 70/30,
whose fluorescence quantum yields do not depend on the v/v), affording (S)-BPOHMe as a white powder in nearly quanti-
presence or absence of water. The dynamic nature of the tative yield.
observed quenching is evidenced by a configuration-depen-
Dyads (S,S)-1 and (S,R)-1. To a solution of (S)-BPOH (56 mg,
dent shortening of the fluorescence lifetimes in (S,S)-1 0.23 mmol) in acetonitrile (20 mL), 0.23 mmol of EDC and
and (S,R)-1 compared to (S)-BPOH. In agreement with the 0.23 mmol of BtOH in acetonitrile were added. The mixture
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was maintained under stirring, and then 0.23 mmol of (S)- or pulses with a repetition rate of 4.75 MHz. A Schott WG 295
(R)-TrpMe in 2 mL of acetonitrile were added. After 3 h, the filter was placed in front of a SPEX monochromator. The detec-
solvent was evaporated; the crude was dissolved in methylene tor was a microchannel plate (R1564 U Hamamatsu) providing
chloride and washed consecutively with diluted NaHCO3, 1 M an instrumental response function of 60 ps (fwhm). The
HCl and brine, then dried over MgSO4. Final purification was average laser power (0.1 mW) was measured with a Melles
performed by preparative layer chromatography (methylene Griot broadband powermeter. Solutions were contained in a
chloride–ethyl acetate, 60/40, v/v), followed by recrystallization. 10 mm × 10 mm quartz cell and continuously stirred. Succes-
The yields were 50% and 47% for (S,S)-1 and (S,R)-1, sive recordings with the same sample gave identical decays
respectively.
which were eventually merged to improve the signal-to-noise
ratio. Such a procedure allowed us to ensure that the measured
signals were not altered during the measurements due to a
Characterisation of the new compounds
All new compounds were characterised by 1H and 13C NMR possible accumulation of photoproducts.
spectroscopy, as well as by high resolution mass spectrometry
(HRMS). Their purity was confirmed by gas chromatography
(GC) and high performance liquid chromatography (HPLC).
A summary of the most relevant data follows.
Acknowledgements
Methyl (S)-2-(2-hydroxy-1,1′-biphenyl-4-yl)propanoate. 1H-NMR
(CDCl3) (δ, ppm) 1.53 (d, 3H, J = 8.0 Hz), 3.70 (s, 3H), 3.73 (q,
1H, J = 8.0 Hz), 5.19 (s, 1H), 6.92–6.94 (m, 1H), 7.8–7.21 (m,
1H), 7.37–7.51 (m, 6H). 13C-NMR (CDCl3) (δ, ppm) 18.5, 45.2,
52.1, 114.9, 119.4, 127.0, 127.9, 129.0, 129.3, 130.8, 136.8,
141.8, 152.5, 174.7. Exact Mass (ESI) [MH+] Calcd for
C16H17O3: 257.1172; Found: 257.1181.
Financial support from the Spanish Government (CTQ2010-
14882, BES-2008-003314, JCI-2011-09926, PR2011-0581), from
the Generalitat Valenciana (Prometeo 2008/090) and from the
Universitat Politècnica de València (PAID 05-11, 2766) is grate-
fully acknowledged.
N-[2-(S)-(2-Hydroxy-1,1′-biphenyl-4-yl)propanoyl]-(R)-tryptophan
methyl ester. 1H-NMR (CDCl3) (δ, ppm) 1.46 (d, 3H, J = 7.1 Hz),
3.13–3.26 (m, 2H), 3.45 (q, 1H, J = 7.1 Hz), 3.64 (s, 3H), 4.90
(m, 1H), 6.05 (d, 1H, J = 8.0 Hz), 6.28 (s, 1H), 6.50 (d, 1H, J =
2.4 Hz), 6.80 (m, 2H), 7.01–7.23 (m, 4H), 7.36–7.53 (m, 10H),
8.11 (bs, 1H). 13C-NMR (CDCl3) (δ, ppm) 18.3, 27.5, 46.8, 52.6,
52.8, 109.5, 111.4, 115.1, 118.6, 119.7, 120.2, 122.2, 123.2,
127.5, 127.9, 129.0, 129.2, 130.9, 136.1, 137.5, 142.2, 153.3,
172.5, 173.9. Exact Mass (EI) [M+] Calcd for C27H26N2O4:
442.1893; Found: 442.1897.
N-[2-(S)-(2-Hydroxy-1,1′-biphenyl-4-yl)propanoyl]-(S)-trypto-
phan methyl ester. 1H-NMR (CDCl3) (δ, ppm) 1.47 (d, 3H, J =
7.2 Hz), 3.19–3.32 (m, 2H), 3.47 (q, 1H, J = 7.2 Hz), 3.61 (s, 3H),
4.85 (m, 1H), 6.05 (d, 1H, J = 7.8 Hz), 6.12 (s, 1H), 6.73–6.79
(m, 3H), 7.05–7.47 (m, 9H), 8.13 (bs, 1H). 13C-NMR (CDCl3) (δ,
ppm) 18.1, 27.4, 46.7, 52.4, 52.9, 109.9, 111.2, 115.1, 118.6,
119.7, 120.0, 122.2, 122.3, 127.2, 127.6, 127.7, 129.1, 130.7,
136.0, 137.1, 141.7, 152.9, 172.3, 173.3. Exact Mass (EI) [M+]
Calcd for C27H26N2O4: 442.1893; Found: 442.1891.
Notes and references
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Fluorescence measurements
Emission spectra were recorded on a spectrofluorometer
system which was provided with a monochromator in the wave-
length range of 200–900 nm. The solutions were placed in
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