Enantioselective photochromism of diarylethenes in human serum albumin

Article abstract of DOI:10.1002/chem.201301459

Making light work of chromism: Enantioselective photochromic ring-closing reactions of three bisthienylethene compounds possessing either no or two hydroxy groups were carried out in the hydrophobic pockets of human serum albumin (HSA) in aqueous media. When 10 equivalents of HSA were used, 1,2-bis(5-hydroxymethyl-2-methyl-3-thienyl)hexafluorocyclopentene predominantly produced the S,S closed form (see scheme; O=open, C=closed), in 63 % ee at RT and 71 % ee at -4°C upon irradiation with 313 nm light. Copyright

Full text of DOI:10.1002/chem.201301459

DOI: 10.1002/chem.201301459
Enantioselective Photochromism of Diarylethenes in Human Serum Albumin
Mai Fukagawa, Izuru Kawamura, Takashi Ubukata, and Yasushi Yokoyama*[a]
Enantioselective photochromic ring-closing reactions of
three bisthienylethenes, possessing either no or two hydroxy
groups, were carried out in the hydrophobic pockets of
human serum albumin (HSA) in aqueous media. When
10 equivalents of HSA were used, the reaction of 1,2-bis(5-
hydroxymethyl-2-methyl-3-thienyl)hexafluorocyclopentene
predominantly produced the S,S closed form in 63% ee at
RT and 71% ee at À48C upon irradiation with 313 nm light.
Diarylethenes (DAEs) are photochromic compounds with
a high potential for applications in switches with functions
based on 6p-electrocyclization–cycloreversion reactions be-
tween a 1,3,5-hexatriene and a 1,3-cyclohexadiene.[1] We
have focused our efforts on achieving high diastereoselectiv-
ity in the photochromism of diarylethenes with the aid of
chiral factors such as asymmetric stereogenic carbon atoms
or an axially or facially chiral unit, introduced within the
molecules themselves.[2] So far, we have achieved completely
diastereoselective ring-closing reactions with several com-
pounds.[2a,b,d] However, although the highly enantioselective
photochromic reactions of di-
serum albumin peptides, we can convey less-water-soluble
photochromic compounds to even the farthest periphery of
biological systems by using serum albumin as the carrier in
aqueous media.
We report herein on the enantioselective photochromism
of diarylethenes incorporated in human serum albumin
(HSA) in buffered aqueous media to elucidate the effect, on
the photochromism of diarylethenes, of the chiral peptides
in HSA,[6] an abundant simple protein in human beings that
can incorporate a remarkably wide range of chemical spe-
cies within its hydrophobic pockets. Since HSA consists of
optically active amino acids, compounds introduced into the
HSA pockets are surrounded by a fully chiral environment.
However, although research into the induction of chirality
in photochemical dimerization products has been reported,[7]
photochromic reactions have yet to be carried out in HSA.
As a basic diarylethene, we employed 1,2-bis(2,5-dime-
AHCTUNGTREGtNNUN hyl-3-thienyl)hexafluorocyclopentene (1O), and prepared
its derivatives 2O and 3O (Scheme 1). These compounds
ACHTUNGTRENNUNGarylethenes in a chiral crystal-
line state have been reported,[3]
those in solution have yet to be
achieved.[4]
The application of photo-
chromic compounds to biologi-
cal systems has recently attract-
ed considerable attention.[5] To
apply photochromic compounds
based on 6p-electrocyclization
in a biological environment, the
solubility of the compounds in
water is essential. Therefore,
since water-insoluble materials
can be delivered to cells of
living creatures with the aid of
Scheme 1. Photochromism of bisthienylethene 1O and its derivatives 2O and 3O employed in this study.
were designed to be small enough for secure incorporation
into HSA. In addition, they gained the ability to form hy-
drogen bonds with the chiral peptide walls of the HSA
pockets in two different ways by the introduction of the hy-
droxy groups at different positions in 1O.
Photochromic reactions of 1O–3O in the absence of HSA
were first carried out in acetonitrile.[8] The conversion ratio
(c.r.) values to the closed form (C-form) of 1O–3O at their
photostationary states (pss) in acetonitrile were 68, 58, and
59%, respectively.
[a] M. Fukagawa, Dr. I. Kawamura, Dr. T. Ubukata, Prof. Y. Yokoyama
Department of Advanced Materials Chemistry
Graduate School of Engineering, Yokohama National University
79-5, Tokiwadai, Hodogaya, Yokohama 240-8501 (Japan)
Fax : (+81)45-339-3934
E-mail: yyokoyam@ynu.ac.jp
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201301459; including the syn-
thetic procedures for 1O, 2O, and 3O.
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COMMUNICATION
We then carried out photochromic reactions of 1O–3O in
the presence of HSA in buffered aqueous solution. They ex-
hibited photocyclization in the presence of HSA with
313 nm light. For all of the diarylethenes employed, we de-
tected two enantiomers of the C-form by use of an HPLC
machine equipped with a chiral column. Fortunately, we had
previously determined the absolute stereochemistry of opti-
cally resolved enantiomers of 2C by X-ray crystallographic
analysis[9] so that the slower moving major enantiomer of
2C was identified as the S,S configuration. Absorption spec-
tral changes starting from 2O in the presence of an equimo-
lar amount of HSA are shown in Figure 1.
the more HSA there is in the reaction medium, the more
2O binds to the pocket in which 2O can take the most
stable enantiomeric helical conformation. On the other
hand, for 3, the c.r. value decreases although the ee value in-
creases as the concentration of HSA increases.
Since the order of magnitude of the ee values was found
to be 3C (27%)<1C (42%)<2C (63%) when the HSA/
DAE ratio was 10, we have tentatively concluded that the
shape of the molecule is an important factor in determining
its conformation within the HSA pockets. Moreover, the hy-
drogen bonds formed between the hydroxy groups on the
diarylethenes and the inner walls of HSA can either in-
crease or decrease the stereoselectivity depending on the
position of the hydroxy groups.
To determine how 2O, which showed the best ee and c.r.
values, and HSA interact with each other in the aqueous sol-
ution, we used 19F NMR spectroscopy, which has previously
been used to investigate the interaction of macromolecules
with a 19F-labelled ligand due to its high sensitivity. Since
there are almost no 19F atoms in biological materials, the
background signals can be eliminated.[10]
Table 1 shows the enantiomeric excess (ee) and c.r. values
of diarylethenes in HSA. These enantioselectivities were ap-
parently induced through the following processes: 1) the
Sharp 19F NMR signals due to the perfluorocyclopentene
moiety in 2O were observed at d=À109.0 (side 4F) and
À130.5 ppm (center 2F) in the phosphate buffer solution.
When five molar equivalents of HSA were added, the sig-
nals appeared at d=À107.8 and À129.8 ppm, respectively,
as shown in Figure 2. The signals broadened and shifted to
lower magnetic fields, which indicates that 2O is bound to
Figure 1. Absorption spectral changes of 2O in the presence of HSA
upon UV irradiation. HSA/2O: 1.0 (mol/mol) in phosphate buffer/aceto-
nitrile (99:1 v/v); concentration of 2O: 4.23ꢁ10À5 moldmÀ3; temperature:
RT; cell length: 1 cm; irradiation wavelength: 313 nm; light intensity:
0.322 mWcmÀ2; irradiation time: 0, 0.5, 1.5, 3.5, 6.5, 10.5, 16 min.
Table 1. Enantiomeric excess and conversion ratio of 1O–3O at the pss
with irradiation by 313 nm light in the presence of HSA in phosphate
buffer/acetonitrile (99:1 v/v).
HSA/DAE
1O to 1C
ee [%] c.r. [%]
2O to 2C
ee [%] c.r. [%]
3O to 3C
ee [%] c.r. [%]
10
5
1
42
40
33
8
67
46
22
17
63
61
45
18
57
57
53
27
27
21
7
12
14
25
31
Figure 2. 19F NMR spectra of 2O in phosphate buffer/acetonitrile (99:1
v/v for HSA/2O, 98:2 v/v for 2O only). Blue: 2O only; red: HSA/2O=1;
green: HSA/2O=5.
0.2
3
open-form molecules of diarylethene entered the HSA
pockets to avoid exposure to the aqueous medium; 2) due
to the shape and chirality of the HSA inner walls, a biased
distribution of the enantiomeric helical conformations of the
open form was induced; and 3) the UV irradiation “locked”
the enantiomeric ratio of the open form by generating its
enantiomeric C-form molecules.
As can be seen in Table 1, when the concentration of
HSA is increased, the ee and c.r. values increase for diaryl-
ACHTUNGTRENNUNG
the HSA pockets so that its movement is strongly dominat-
ed by the large HSA molecule.
The use of 19F NMR spectroscopy enabled us to obtain
the binding constants of 2O to HSA by titrating solutions of
4ꢁ10À4 moldmÀ3 2O with HSA. Since the change in chemi-
cal shifts and changes in ee and c.r. values are monotonic,
we carried out our analysis on the premise that there is one
major pocket that accepts 2O preferentially. The spectro-
scopic shifts of the 19F signals of 2O on addition of HSA
were plotted against the HSA concentration. A nonlinear
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Y. Yokoyama et al.
least-squares method by using KaleidaGraph software gave
a binding constant of 8.2ꢁ103 molÀ1 dm3 with HSA.[8]
To obtain information on the binding sites of 2O in HSA,
inhibition experiments were carried out by using warfarin
(War) and 4-iodobenzoic acid (4IB), both of which have
large binding constants to specific binding sites in HSA.
Warfarin binds to site I[6a,11] with a binding constant of
3.3ꢁ105 molÀ1 dm3, whereas 4-iodobenzoic acid binds to
site II[6a,11] with a binding constant of 1.8ꢁ105 molÀ1 dm3.[5a]
If the binding site of 2O is either site I or II, 2O will be
squeezed out of the binding site by the addition of an inhibi-
tor due to its comparatively small binding constant (8.2ꢁ
103 molÀ1 dm3) so that the ee and/or c.r. values may change
dramatically after UV irradiation. The results of these ex-
periments are shown in Table 2. Before the addition of an
inhibitor, the ee was 45% and the c.r. was 53%. When one
Table 2. Enantiomeric excess and conversion ratios of diarylethene 2O
in the presence of HSA and its inhibitors in phosphate buffer/acetonitrile
(99:1 v/v).
HSA/inhibitor[a]/2O
ee of 2C [%]
c.r. of 2C [%]
1:0:1
45
53
inhibitor=4IB
1:1:1
1:5:1
39
39
48
49
inhibitor=War
1:1:1
1:2:1
1:3:1
1:4:1
39
29
21
20
18
48
39
35
29
26
1:5:1
Figure 3. CD spectra of diarylethenes 1O and 2O at pss. Top: HSA/1O=
5 (ee: 40%: c.r.: 46%); bottom: HSA/2O=5 (ee: 61%, c.r.: 57%).
[a] 4IB: 4-iodobenzoic acid; War: warfarin.
equivalent of either of these inhibitors was added, both the
ee and c.r. values decreased slightly. However, although the
addition of more 4IB did not affect either the ee or the c.r.
values, the addition of more warfarin caused a continuous
and steady decrease of both the ee and the c.r. values. These
data indicate that 2O binds to neither site I nor II, but binds
to the site with which warfarin binds secondarily. The initial
decrease in ee and c.r. values on addition of the inhibitors
may be due to an allosteric effect.
We measured the CD spectra after UV irradiation to de-
termine the stereochemistry of the major C-forms of diaryl-
ACHTUNGTRENNUNGethenes that were formed in the HSA pockets (Figure 3).
For 2O, the spectrum showed a negative Cotton effect in
the visible region, indicating that the major enantiomer of
2C has an S,S configuration, as already mentioned.[9] On the
other hand, 1O showed a slight positive Cotton effect in the
visible region. Matsuda et al. have reported that the sign of
rotatory strength for an R,R enantiomer of the C-form avail-
able by the photoirradiation of 1,2-bis(5-substituted-2-me-
methyl groups in 1 and hydroxymethyl groups in 2, neither
of which are p-conjugated systems. Thus, the major enan-
tiomer of 1C has an R,R configuration,[13] unlike that of the
major enantiomer of 2C.
This brings into question whether 1O and 2O are accom-
modated in the same pocket in HSA. Thus a phosphate
buffer solution containing equimolar amounts of 1O, 2O,
and HSA was irradiated with 313 nm light and the ee values
were determined. Only a drastic decrease in the ee value of
1C was observed (Table 3). We, therefore, concluded that
1O and 2O competed to occupy the same pocket in HSA
and that the binding constant of 2O must be larger than
that of 1O.
Although 1O and 2O are incorporated in the same HSA
pocket, their major C-forms, formed by photochemical ring
closure, have opposite absolute stereochemistry. This means
Table 3. Enantiomeric excess values of 1C and 2C obtained as a result
of competition between 1O and 2O to occupy the pocket in HSA.
ACHTUNGTRENNUNGthylthienyl)hexafluorocyclopentenes is positive when the p-
DAE
ee [%]
conjugating substituent is no larger than a vinyl group,
whereas it is negative when the conjugating group is larger
than a vinyl group, such as in the case of phenyl or formyl
groups.[12] The substituents on both thiophene rings are
HSA/DAE=1:1
HSA/1O/2O=1:1:1
1O to 1C
2O to 2C
33
45
13
39
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Chem. Eur. J. 2013, 19, 9434 – 9437

Enantioselective Photochromism of Diarylethenes
COMMUNICATION
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In conclusion, we have succeeded in transferring the chi-
ACHTUNGTRENNUNGrality of human serum albumin effectively to diarylethene
2O in aqueous media. Diarylethene 2O showed an excellent
enantiomeric excess of 63% to form (S,S)-2C predominant-
ly at RT with a HSA/2O ratio of 10. When the operation
was carried out at a temperature as low as À48C, the enan-
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the underlying mechanisms behind the incorporation phe-
nomena and enantioselective photocyclization reaction were
investigated.
Experimental Section
Photochromic reactions: The photochromic reactions of diarylethenes in
the presence of HSA were carried out in phosphate buffer solution
(pH 6.8) with different HSA/diarylethene ratios (0.2:1 to 10:1) and con-
stant concentrations of diarylethene (4.23ꢁ10À5 moldmÀ3) by irradiation
with 313 nm light for a constant period (16 min, 0.3 mWcmÀ2). Conver-
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spectra and the enantiomeric excesses were determined by use of an
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Acknowledgements
This work was supported by the MEXT Grant-in-Aid for Scientific Re-
search on Priority Areas (Photochromism, No. 471, 19050004), and JSPS
Grant-in-Aid for Scientific Research (B; 23350096). The authors are in-
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Keywords: chirality
enantioselectivity · human serum albumin · photochromism
transfer
·
diarylethenes
·
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Received: April 17, 2013
Published online: June 19, 2013
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9437