Stereodifferentiation in the compartmentalized photooxidation of a protein-bound anthracene

Article abstract of DOI:10.1021/ol201209h

Encapsulation within transport proteins strongly reduces the photooxidation rate of (S)- and (R)-2-(9-anthracenyl)propanoic acid (1) and results in a significant stereodifferentiation. The most remarkable effects are observed within human serum albumin (HSA).

Full text of DOI:10.1021/ol201209h

ORGANIC
LETTERS
2011
Vol. 13, No. 15
3860–3863
Stereodifferentiation in the
Compartmentalized Photooxidation of a
Protein-Bound Anthracene
ꢀ
Rafael Alonso, M. Consuelo Jimenez,* and Miguel A. Miranda*
´
Departamento de Quımica/Instituto de Tecnologıa Quımica UPV-CSIC, Universidad
Politecnica de Valencia, Camino de Vera s/n, E-46022 Valencia, Spain
´
´
ꢀ
mcjimene@qim.upv.es; mmiranda@qim.upv.es
Received May 23, 2011
ABSTRACT
Encapsulation within transport proteins strongly reduces the photooxidation rate of (S)- and (R)-2-(9-anthracenyl)propanoic acid (1) and results in
a significant stereodifferentiation. The most remarkable effects are observed within human serum albumin (HSA).
Asymmetric organic photochemistry has emerged as an
important field in the past two decades.1 Chiral host
systems and synthetic assemblies such as cyclodextrins,2
modified zeolites,3 hydrogen-bonding templates,4 nano-
porous materials,5 or crystal lattices,6 as well as biomacro-
molecules, including DNA7 and proteins,8ꢀ10 have shown
potential in inducing stereoselective photoreactions.
Stereodifferentiating photoprocesses in the presence of
carrier proteins, such as serum albumins (SAs) and R-1-
acid glycoproteins (AAGs), are of special interest for the
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r
10.1021/ol201209h
Published on Web 06/24/2011
2011 American Chemical Society

understanding of drugꢀbiomolecule interactions. The
photobehavior of included guests can be modified as a
result of conformational restrictions imposed by the pro-
tein binding pockets. A number of examples of modified
photoreactivity within protein cavities have been reported,
including dehalogenation,9 dimerization, cyclization, or
isomerization.10
oxygen, generatingsinglet oxygen(1O2); subsequent[4 þ 2]
cycloaddition of this reactive species to the central anthra-
cene ring, followed by rearrangement of the resulting
endoperoxide and cleavage of the 9-substituent, would
lead to 2. The photoreaction of 1 was monitored by UV
absorption spectroscopy, following the decrease of the
characteristic long-wavelength bands of the anthracene
chromophore. In the absence of proteins, 1 was almost
completely consumed after 20 min of irradiation. The
reaction was also performed in the presence of equimolar
amounts of SAs and AAGs. Upon protein encapsulation,
photooxidation of 1 was much slower than in PBS. More-
over, when 1 was incorporated into HSA, a dramatic
stereodifferentiation in the reaction rate was observed,
with the photoreactivity being much higher for (S)-1 than
for (R)-1. This led to a kinetic resolution (Figure S1 in the
Supporting Information (SI)), with a clear enhancement of
the less reactive (R)-enantiomer (ee 34% after 120 min of
irradiation). The reverse was true for 1 encapsulated within
BSA, where a slower photooxidation of (S)-1 was observed
(Figure 1). Concerning the reactivity inside R-1-acid gly-
coproteins, no stereodifferentiation was detected in the
presence of HAAG; by contrast (S)-1 reacted faster in the
presence of BAAG (Figure S2 in SI).
Scheme 1. Photooxidation of 1 and Key Mechanistic Steps
1
The main photochemical pathways for anthracene deri-
vatives are dimerization11 and oxidation,12 depending on
the reaction conditions. In the case of 9-substituted an-
thracenes, regioselective control of photocycloaddition in
organic solvents and within confined media has been
reported.13 Moreover, stereoselective photodimerization
of 2-anthracenecarboxylate within serum albumins at low
protein/substrate ratios has also been investigated.8aꢀc We
have previously described the photobehavior of a water-
compatible anthracene derivative in solution and upon
protein encapsulation.14 Now, we wish to report on the
sterodifferentiation in the compartmentalized photooxi-
dation of (S)- and (R)-2-(9-anthracenyl)propanoic acid
((S)- and (R)-1) encapsulated within human and bovine
serum albumins (HSA, BSA) and R-1-acid glycoproteins
(HAAG, BAAG).
Long wavelength irradiation (λmax = 350 nm, Gaussian
distribution) of 1 in aerated phosphate buffered solution
(PBS) afforded anthraquinone (2) as the only product
(Scheme 1).
The mechanism for this type of process in anthracenes15
involves tripletꢀtriplet energy transfer to molecular
To assess the involvement of O2 in the process, the
irradiations were performed in D2O, where this reactive
oxygen species is much longer-lived than in H2O.16 As a
matter of fact, a remarkable acceleration of the photoox-
idation rate was observed in all media (Figure 1, as well as
Figure S2 in SI). As expected for a photooxidation process,
a significant enhancement was noticed under oxygen. In
addition, the photoreaction was found to be compartmen-
1
talized, i.e., O2 reacts with 1 in the same microenviron-
ment where it is generated. To investigate this issue,
photosensitization with (R)-propranolol ((R)-PPN) and
(R)-naproxen ((R)-NPX) was attempted, as these drugs do
not bind to HSA and HAAG, respectively.17 Furthermore,
they can be selectively excited at 318 nm in the presence of
1 (see Figure S3 in SI) and are appropriate sources of
1O2 (φΔ = 0.28 for NPX and 0.24 for PPN).18 Actually, the
photosensitized oxidation of 1 was efficiently achieved in
the bulk solution, with both (R)-PPN and (R)-NPX.
However, protein encapsulation provided an exceptional
microenvironment protecting 1 from the attack by exter-
1
nally generated O2, using the unbound photosensitizers
(Figure S4 in SI).
After the photobehavior of 1 in the presence of proteins
was established, the formation of 1@SA and 1@AAG
complexes was investigated. Their stoichiometry was es-
tablished by means of Job plot analysis.19 Thus, the
absorbance of 1/protein mixtures was plotted versus the
(11) (a) Bouas-Laurent, H.; Castellan, A.; Desvergne, J.-P.; Lapouyade,
R. Chem. Soc. Rev. 2001, 30, 248–263. (b) Bouas-Laurent, H.; Castellan, A.;
Desvergne, J.-P.; Lapouyade, R. Chem.Soc.Rev.2000,29, 43–55. (c) Becker,
H. D. Chem. Rev. 1993, 93, 145–172.
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Aspler, J.; Carlsson, D. J.; Wiles, D. M. Macromolecules 1976, 9, 691–
695. (c) Sugiyama, N.; Iwata, M.; Yoshioka, M.; Yamada, K.; Aoyama,
H. Chem. Commun. 1968, 1563–1563. (d) Lindig, B. A.; Rodgers,
M. A. J.; Schaap, A. P. J. Am. Chem. Soc. 1980, 102, 5590–5593.
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Tung, C.-H. Org. Lett. 2003, 5, 1075–1077. (b) Wu, D.-Y.; Zhang, L.-P.;
Wu, L.-Z.; Wang, B.-J.; Tung, C.-H. Tetrahedron Lett. 2002, 43, 1281–
1283. (c) Tung, C.-H.; Guan, J.-Q. J. Org. Chem. 1998, 63, 5857–5862.
(14) Alonso, R.; Yamaji, M.; Jimenez, M. C.; Miranda, M. A. J.
Phys. Chem. B 2010, 114, 11363–11369.
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1972, 94, 1030–1031.
(17) Perez-Ruiz, R.; Alonso, R.; Nuin, E.; Andreu, I.; Jimenez,
M. C.; Miranda, M. A. J. Phys. Chem. B 2011, 115, 4460–4468.
(18) (a) Jimenez, M. C.; Pischel, U.; Miranda, M. A. J. Photochem.
ꢀ
Photobiol. C 2007, 8, 128–142. (b) Sortino, S.; Petralia, S.; Bosca, F.;
Miranda, M. A. Photochem. Photobiol. Sci. 2002, 1, 136–140.
(19) (a) Huang, C. Y. Methods Enzymol. 1982, 87, 509–525. (b) Job,
P. Ann. Chim. 1928, 9, 113–203.
(15) Gilbert, A.; Baggot, J. Essentials of Molecular Photochemistry;
Blackwell Scientific Publications: Oxford, 1991.
Org. Lett., Vol. 13, No. 15, 2011
3861

Figure 1. Kinetics for direct photooxidation of 1 (λexc = 350 nm)
in protein media. (S)-1: (A) HSA/D2O, (B) HSA/PBS, (E) BSA/
D2O, (F) BSA/PBS. (R)-1: (C) HSA/D2O, (D) HSA/PBS,
(G) BSA/D2O, (H) BSA/PBS.
protein molar fraction. In all cases, a maximum at χprotein
=
0.5 was reached, revealing the formation of 1:1 complexes
(see Figure S5 in SI).
Figure 2. Fluorescence spectra of 1 in the presence of increasing
amounts of HSA, up to a 1:1.5 molar ratio for (a) (S)-1 and
(b) (R)-1.
The fluorescence behavior of 1@SAs was markedly
different from that of free 1. For HSA, addition of increas-
ing amounts of protein to a PBS solution of 1 resulted in a
significant decrease of the fluorescence intensity. This pro-
cess was clearly configuration dependent, with a more
pronounced effect for (R)-1 (Figure 2). Similar results were
found for 1@BSA complexes; by contrast, in the case of
AAGs changes were less important (see Figure S6 in SI).
Laser flash photolysis (LFP) experiments were per-
formed to explore the possibility of using the triplet excited
states of 1 (1* (T1)) as reporters for its binding to proteins.
As in the case of 9-anthraceneacetic acid,14 1 showed a very
complex transient absorption spectrum in aqueous solu-
tion (Figure 3, inset); however, for equimolar 1/protein
mixtures, onlytheband attributedtothefirst triplet excited
state was observed (see Figure 3a for the case of (S)-1 in
HSA). Under aerobic conditions, the triplet lifetimes
(τT) of both enantiomers, monitored at420 nm, werefound
to be markedly longer in the protein media (see Figure 3b
and Table 1, as well as Figure S7 in SI). The observed
τT lengthening can be attributed to a slower deactivation of
the species inside the protein pockets, where the micro-
environment provides protection from attack by oxygen.20
As expected, triplet lifetimes were markedly longer
under an inert atmosphere (see Table 1). Interestingly, in
the case of SAs, two intraprotein microenvironments were
revealed by two different triplet lifetimes; this was ex-
plained taking into account that SAs are reported to
containtwo mainbindingsites for small organicmolecules,
namely sites I and II.21 Assignment was achieved based on
the presence of a Trp residue at site I of the albumins. In
PBS solution this amino acid was found to quench 1* (T1)
.
(Figure S8 in SI), with a rate constant of 1.6 ꢁ 108 Mꢀ1 sꢀ1
Therefore, the longer lifetime in the presence of SAs was
ascribed to 1 located in site II, while the shorter one was
safely assigned to 1 within the Trp containing site I. By
contrast, only one lifetime was needed to fit the decays in
the presence of AAGs, indicating only one binding site, in
agreement with the expectations from the literature.22
According tothe photooxidation mechanism (Scheme 1)
a number of processes (iꢀv) should in principle be taken
into account to anticipate the reactivity of 1. The inter-
system crossing quantum yields (ΦT) and rate constants of
quenching by oxygen (kq O2) would provide quantitative
data related to steps iii and iv, respectively. The values
obtainedfor these parameters are alsogiven in Table 1. It is
interesting to note that the highest ΦT was found for PBS.
In general, the presence of proteins resulted in a reduced
ΦT, which correlates well with the lower photoreactivity.
As regards the accessibility of oxygen to1 in protein media,
quenching of 1* (T1) by oxygen was also investigated.
Thus, the decay traces obtained at 420 nm under a nitro-
gen, air, and oxygen atmosphere were fitted to obtain the
corresponding triplet lifetimes. The oxygen quenching
constants (kq O2), obtained by SternꢀVolmer analysis,23
(22) (a) Kopecky, V., Jr.; Ettrich, R.; Hofbauerova, K.; Baumruk, V.
Biochem. Biophys. Res. Commun. 2003, 300, 41–46. (b) Rojo-Domin-
guez, A.; Hernandez-Arana, A. Protein Seq. Data Anal. 1993, 5, 349–
355. (c) Schmid, K.; Nimberg, R. B.; Kimura, A.; Yamaguchi, H.;
Binette, J. P. Biochim. Biophys. Acta 1977, 492, 291–302. (d) Aubert,
J. P.; Loucheux-Lefebvre, M. H. Arch. Biochem. Biophys. 1976, 175,
400–409. (e) Schmid, K.; Kaufmann, H.; Isemura, S.; Bauer, F.; Emura,
J.; Motoyama, T.; Ishiguro, M.; Nanno, S. Biochemistry 1973, 12, 2711–
2724. (f) Schmid, K. J. Am. Chem. Soc. 1953, 75, 60–68.
(20) (a) Perez-Ruiz, R.; Bueno, C. J.; Jimenez, M. C.; Miranda, M. A.
J. Phys. Chem. Lett. 2010, 1, 829–833. (b) Bueno, C. J.; Jimenez, M. C.;
Miranda, M. A. J. Phys. Chem. B 2009, 113, 6861–6865. (c) Vaya, I.;
Bueno, C. J.; Jimenez, M. C.; Miranda, M. A. Chem.;Eur. J. 2008, 14,
11284–11287. (d) Jimenez, M. C.; Miranda, M. A.; Vaya, I. J. Am. Chem.
Soc. 2005, 127, 10134–10135.
(23) IUPAC Compendium of Chemical Terminology, 2nd ed.;
McNaught, A. D., Wilkinson, A., Eds.; Royal Society of Chemistry:
Cambridge, 1997.
(21) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1975,
11, 824–832.
3862
Org. Lett., Vol. 13, No. 15, 2011

Table 1. Relevant Photophysical Parameters Obtained for the Two Enantiomers of 1 under Different Conditions
a
a
substrate
protein
ΦF
τ
F (ns) a
ΦT
τ
T (μs) b
τ
T (μs) a
kq O2 ꢁ 106 (Mꢀ1 sꢀ1
)
(S)-1
(S)-1
(S)-1
(S)-1
(S)-1
(R)-1
(R)-1
(R)-1
(R)-1
(R)-1
None
HSA
0.40
0.25
0.31
0.39
0.39
0.40
0.10
0.34
0.37
0.39
10.5
9.8
0.51
0.35
0.39
0.44
0.33
0.51
0.25
0.39
0.45
0.36
115
2.8
1100
110, 19
110, 16
120
50, 379
28, 237
98
18, 107
20, 126
27
BSA
11.6
11.8
11.2
10.5
8.1
HAAG
BAAG
None
HSA
124
28
120
115
2.8
1100
140, 23
55, 10
120
44, 311
32, 312
99
20, 109
24, 139
29
BSA
12.1
12.2
13.6
HAAG
BAAG
121
33
130
a Measurements performed under air. b Measurements performed under nitrogen.
Furthermore, different quenching constant values were
obtained within sites I and II for SAs; the photoprocess
was in general much slower in the latter (see Figure S9 in SI).
In summary, a remarkable stereodifferentiation is ob-
served for the photooxidation of 1 encapsulated within
serum albumins and R-1-acid glycoproteins. The observed
photoreactivity is markedly compartmentalized, with
higher reaction rates in the bulk solution than within the
protein microenvironments. The reaction is mediated by
1O2 andonlyoccursinthecompartment wherethisreactive
oxygen species is generated. Finally, quenching of the
triplet excited state of 1 by molecular oxygen, required to
produce 1O2, is much faster in site I than in site II of serum
albumins.
Acknowledgment. Financial support from the MI-
CINN (Grant CTQ2010-14882), the Generalitat Valenci-
ana (Prometeo Program, ref 2008/090), and the Carlos III
Institute of Health (Red RETICS) is gratefully acknowl-
edged. We also thank Dr. A. Llamas-Saiz and Servicio de
Raios X from Universidad de Santiago for his advice and
skillfull technical assistance. Dedicated to the memory of
Prof. Rafael Suau.
Figure 3. Laser flash photolysis in PBS, under air. (a) Transient
absorption spectra of (S)-1 in the presence of HSA, at 1:1 molar
ratio, recorded 2.9 μs after laser pulse. Inset: Spectrum obtained
in the absence of protein, 1 μs after the laser pulse. (b) Normalized
decays monitored at 420 nm for (S)-1 and (R)-1, in the absence
and presence of HSA at a 1:1 molar ratio.
Supporting Information Available. Characterization
data and experimental procedures for the synthesis of 1,
irradiation procedures, Job plot diagrams, fluorescence
measurements, laser flash photolysis experiments, Sternꢀ
Volmer plots for oxygen quenching, and X-ray crystal-
lographic data for the 2-bromophenyl ester of (S)-1.
This material is available free of charge via the Internet
at http://pubs.acs.org.
were in the range 1.0 ꢁ 107ꢀ1.4 ꢁ 108 Mꢀ1 sꢀ1, markedly
lower thanin PBS solution (Table 1). This is alsoconsistent
with the lower photooxidation rate inside the proteins.
Org. Lett., Vol. 13, No. 15, 2011
3863