Photo-responsive catalysis by thymine-cyclodextrin conjugates

Article abstract of DOI:10.1039/a606245h

Primary hydroxy groups of β-cyclodextrin (β-CD) have been substituted with thymine (Thy) groups. By photo-irradiation with UV light at 280 nm, the introduced thymine groups adjacent to the CD cavity underwent reversible dimerization and the catalytic effi

Full text of DOI:10.1039/a606245h

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Photo-responsive catalysis by thymine–cyclodextrin conjugates†
Tomoyuki Nozaki, Michiko Maeda, Yasushi Maeda and Hiromi Kitano*
Department of Chemical and Biochemical Engineering, Toyama University, Toyama, 930 Japan
Primary hydroxy groups of β-cyclodextrin (β-CD ) have been substituted with thymine (Thy) groups. By
photo-irradiation with U V light at 280 nm, the introduced thymine groups adjacent to the CD cavity
underwent reversible dimerization and the catalytic efficiency (kcat/Kdiss) of the modified CD in the
hydrolyses of p-nitrophenyl acetate and m-nitrophenyl acetate increased. By further irradiation with light
at 240 nm, the catalytic efficiency decreased to that of the CD –Thy conjugate due to the photo-cleavage of
the thymine dimer. This phenomenon implies that the binding of guest molecules by the CD –Thy
conjugate and subsequent change in the catalytic efficiency of the conjugate occurred photo-responsively.
The steric effect on the acceleration or deceleration of the hydrolyses of phenyl esters by CD –Thy and its
derivatives is also discussed.
Introduction
Thymine was from Wako Pure Chemicals, Osaka, Japan.
p-Nitrophenyl acetate (PNPA), from Wako Pure Chemicals,
was recrystallized from benzene. m-Nitrophenyl acetate
The photochemical transformation of nucleic acids by UV light
has been extensively studied due to its lethal effect on biological
systems. Of the four nucleic acid bases in DNA, pyrimidine
bases (thymine and cytosine) undergo photodimerization on
exposure to UV light at 280 nm and revert back to their mono-
mers with UV light at 240 nm (Scheme 1), whereas purine bases
(MNPA) and 3-nitro-4-acetoxybenzoic acid (NABA) were pre-
1
pared by the reaction of acetic anhydride with m-nitrophenol
and 3-nitro-4-hydroxybenzoic acid (Fluka, Buchs, Switzer-
land), respectively. Other reagents were commercially available.
Deionized water was distilled before preparation of sample
solutions.
(
adenine and guanine) do not. Such a reversible photosensitiv-
ity has been applied to photoresists, etc.
2–5
1
2
Preparation of 1-(2-carboxyethyl)thymine
Thymine (10.0 g, 79.3 mmol) was coupled with methyl
acrylate (9.11 g, 106 mmol) in the presence of hydroquinone
(100 mg, polymerization inhibitor) in EtOH (70 ml) con-
taining NaOH (276 mg) for 50 h at the reflux temperature.
After evaporation of the solvent, the methyl ester of 1-(2-
carboxyethyl)thymine obtained was washed several times with
cold EtOH and dried in vacuo (8.90 g, 52.9% yield). The
methyl ester (6.49 g, 30.6 mmol) was hydrolysed by 5  HCl
(40 ml) for 3 h at 100 ЊC [Scheme 2(a)]. The pH of the solu-
tion was adjusted to 4, the solution mixture was cooled over-
night and then filtered and dried in vacuo to give a slightly
brownish powder (4.67 g, 62.4% yield). δ (400 MHz, D O)
Scheme 1 Photodimerization of 1-(2-carboxyethyl)thymine–CD con-
jugate (CD–Thy)
H
2
1.86 (s, 3 H, CH
), 2.69 (t, 2 H, CH COOH), 3.99 (t, 2 H,
3 2
Ϫ1
NCH ) and 7.51 (s, 1 H, C(6)H). ν(KBr)/cm 3433, 3304,
2
Enzymatic processes are highly selective and efficient due to
the accompanying formation of non-covalent enzyme-substrate
complexes before the onset of the reactions. In this regard,
study of enzyme-mimetic catalytic systems, which begin to act
after the formation of the host–guest complex, has received
much attention. One molecule which acts as a catalyst in such a
way is cyclodextrin (CD), a cyclic α-1,4-oligoglucopyranoside
having a cylindrical shape. Catalytic effects of CD and its
derivatives on many chemical reactions such as hydrolyses,
decarboxylations and substitutions have been extensively
3
4
5
051, 2964, 2927, 1679, 1560, 1419, 1264 and 1130 (Found C,
9.07; H, 5.09; N, 14.07. Calc. for C H N O : C, 48.48; H,
.09; N, 14.14%).
8
10
2
4
Modification of CD with 1-(2-carboxyethyl)thymine
The (aminoethyl)aminocyclodextrin [940 mg; degree of sub-
stitution (DS) evaluated by H NMR, 7] was incubated with 1-
13
1
(2-carboxyethyl)thymine (1.52 g) in water (20 ml) at pH 5 in
the presence of 1-ethyl-3-(dimethylamino)propylcarbodiimide
hydrochloride (EDC; 1.60 g) for 24 h [Scheme 2(b)]. The β-CD
modified with thymine was separated from unreacted reagents
by GPC and lyophilized (CD–Thy, 683 mg, 37.4% yield; DS,
6
–11
investigated.
In this report, in order to obtain a catalyst having a photo-
responsiveness, we attempted to introduce thymine groups into
the CD molecule. The effect of photo-irradiation on the
catalysis of the hydrolyses of phenyl esters by the modified
CD has been examined.
7
2
3
3
). δ(D O) 1.75 (t, 2 H, CH NH), 1.81 [s, 3 H, CH (Thy)],
2
2
3
.61 (br s, 2 H, CH CONH), 2.87 (t, 2 H, CH NHCO), 3.13,
2
2
.32, 3.45, 3.66 [br s, 5 H, C(2)H, C(4)H, C(5)H, C(6)H (CD)],
.90 [br s, 1 H, C(3)H (CD)], 3.99 (t, 2 H, NCH ), 5.11 [br s, 1
2
Ϫ1
H, C(1)H (CD)] and 7.43 [s, 1 H, C(6)H (Thy)]. ν(KBr)/cm
3
Experimental
392, 3059, 2930, 2823, 1675, 1546, 1468, 1363, 1220, 1082 and
Materials
1042.
β-Cyclodextrin was from Nacalai Tesque, Kyoto, Japan.
Instrumentation
H NMR spectra were recorded on a JEOL JNM-A400 spec-
trometer. IR spectra were recorded on a Perkin Elmer system
1
†
Presented at the 45th Annual Meeting of the Society of Polymer
Science, Japan, at Nagoya in May 1996.
J. Chem. Soc., Perkin Trans. 2, 1997
1217

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Fig. 1 Double reciprocal plot of the concentration of CD–Thy vs.
reaction rate constant for the hydrolysis of PNPA before (᭹) and after
᭺) UV irradiation at 280 nm and 20 ЊC
(
Kinetic measurements
All reactions were followed with a UV–VIS spectrophotometer.
The pH of the reaction solution was determined by using a pH
meter (Model DS-12, Horiba Kyoto, Japan). In all measure-
ments, the pseudo-first-order conditions were maintained by
using an excess amount of cyclodextrin ([CD] = 0.4–1.5 m).
The hydrolyses of PNPA, MNPA and NABA were followed
using changes in the absorbance at 400, 390 and 408 nm,
respectively.
Determination of dissociation constants, Kdiss, and rate constants,
kcat, of the cyclodextrin–ester complex from the kinetic data
The hydrolytic reaction of the phenyl esters (S) catalysed by
cyclodextrin (CD) proceeds according to reaction (1), where
k
cat
CD ϩ S K CDؒS
CD ϩ P
(1)
diss
CDؒS and P are the cyclodextrin–substrate complex and the
product, respectively. The observed first-order rate constants
for hydrolyses of phenyl esters in the absence (k ) and in the
un
presence (kobs) of cyclodextrin were determined. By plotting
1
/(kobs Ϫ k ) vs. 1/[CD] (Fig. 1), a straight line was obtained
un
Scheme 2 Scheme of preparation of 1-(2-carboxyethyl)thymine–
cyclodextrin conjugate
having an x intercept of Ϫ1/Kdiss, where Kdiss is the dissociation
constant of the cyclodextrin–ester complex with a 1:1 stoichio-
metry; kcat, the rate constant for the reaction of the complexed
ester (= kobs Ϫ kun at infinite cyclodextrin concentration), was
estimated from the reciprocal of the y intercept in the plot.
2
000 FTIR spectrometer. GPC was carried out using a Sepha-
dex G-10 column (id 2 × 40 cm) with water as eluent. Products
were detected at 270 nm using a Soma Optics Model S-310A
UV monitor.
Results and discussion
Photodimerization of CD–Thy
Dimerization of thymine groups in CD–Thy
Photodimerization of the thymine moiety was carried out in
a 10 mm quartz cell filled with 3 ml of aqueous solution of
CD–Thy at room temperature. A UV lamp (HB-251A, Ushio,
Tokyo, Japan) and a diffraction grating (Model 356, Hitachi,
Tokyo, Japan) were used to obtain UV lines at 240 and 280 nm.
Aqueous CD–Thy (1 m) was photo-irradiated for 2 h at room
temperature.
It was previously reported that the photodimer of thymine is
not readily produced by UV irradiation of a thymine monomer
model with only one thymine residue, whereas for models which
contain two thymine residues the photodimerized thymine is
1
4
produced reversibly. Kita et al. observed a gradual decrease in
the absorption at 275 nm accompanying the formation of
photodimers upon photo-irradiation of polymer compounds
1
5
Measurements of photodimerization
which had many thymine residues as side chains.
The absorbance of solution of CD derivatives from 220 to 300
nm at 20 ± 0.5 ЊC was followed with a UV–VIS spectro-
photometer (Ubest–35, Japan Spectroscopic Co., Tokyo, Japan).
The observation cell was thermostatted by a Peltier device
By irradiation of a solution of CD–Thy with UV light at 280
nm, the absorption at 271 nm corresponding to the thymine
moiety decreased, but the absorbance was increased again by
the further irradiation at 240 nm (Fig. 2). However, reversible
photodimerization of CD–Thy by repeated exposure to UV
light at 240 and 280 nm was incomplete, probably because of
the unavoidable photodecomposition of the thymine rings or
the formation of stable photodimers.
(EHC-441, Japan Spectroscopic Co.). The quantitative estima-
tion of the photodimerization of thymine groups was carried
1
out by H NMR spectroscopy (400 MHz, D O). The GPC
2
analysis of CD–Thy before and after the photo-irradiation were
performed using a Waters Model 440 (column, Wakobeads-F
G-30; mobile phase, water).
1
H NMR measurements were also made before and after UV
irradiation at 280 nm in order to confirm the relationship
1
218
J. Chem. Soc., Perkin Trans. 2, 1997

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attack of the secondary amino group of CD–Thy derived from
ethylenediamine partly participates in the hydrolysis of the
phenyl ester. Since dependence of the catalytic efficiency of
CD–Thy on pH was similar to that of unmodified CD and the
efficiency drastically reduced below pH 10 (pK of the second-
a
1
8
ary hydroxy group of CD is 12.3 ), the catalytic effect of the
secondary amine might be small. The rate constant (kcat) for the
CD modified with thymine before photo-irradiation (CD–Thy)
was about ca. 40% of that for unmodified CD, whereas Kdiss
was about ca. 25%. These phenomena might be induced by
the increase in hydrophobic microenvironment around the CD
cavity due to a high local concentration of thymine, which
would enhance the hydrophobic interaction between PNPA and
CD–Thy. To be effectively hydrolysed, the carbonyl group of
the substrate has to approach one of the dissociated secondary
hydroxy groups located on the rim of the CD cavity. The
nucleophilic attack of the ester by the dissociated hydroxy
group of CD–Thy was, however, sterically hindered by the too
deep penetration of the substrate into the cavity (which
Fig. 2 Absorption change of CD–Thy at 271 nm by exposure to UV
light at 280 and 240 nm. The photo-irradiation was performed in a
quartz cell containing aqueous CD–Thy solution (0.02 mM) at pH 7: ᭺,
before photo-irradiation; ᭹, after UV irradiation at 280 nm; ᭺, after
Њ
UV irradiation at 240 nm.
Table 1 Hydrolysis of PNPA in the presence of CD derivatives at
a
2
0 ЊC
decreases the kcat value), giving a slight increase in kcat/Kdiss.
Furthermore, by exposure to UV light at 280 nm, which
induced the photodimerization of the thymine moiety (CD–
Thy–D), the catalytic efficiency was about four times larger
than that of CD–Thy. The kcat of CD–Thy–D was ca. 77% of
that for CD–Thy, whereas Kdiss was ca. 20%. The large decrease
in Kdiss would imply that the capping effect was induced by the
photodimerization of thymine residues on the rim of the cyclo-
dextrin. Since the new hydrophobic field would be afforded
adjacent to the CD cavity with the photodimerization of thy-
mine groups (substituted to the primary OH groups at the 6
position), the capacity of hydrophobic cavity of CD–Thy–D
would be larger than those of unmodified CD and CD–Thy
2
Ϫ1
4
Ϫ1 Ϫ1
Host
kcat/10 s
Kdiss/10
M
(kcat/Kdiss)/M
s
β-CD
18.5 (±0.8)
6.6 (±0.4)
5.1 (±0.05)
6.4 (±0.9)
60.2 (±3.1)
15.7 (±0.2)
3.1 (±0.1)
11.6 (±3.2)
31 (±3)
42 (±12)
162 (±6)
55 (±18)
CD–Thy
CD–Thy–D
CD–Thy–M
b
c
a
Hydrolysis was carried out at pH 11.6 (±0.1) using a sodium carbon-
ate buffer (0.2 M). [PNPA] = 0.1 mM. The first-order rate constants for
the hydrolysis of PNPA in the absence of cyclodextrin (kun) was
Ϫ2 Ϫ1
b
2
.96 × 10 sc . After the exposure of CD–Thy to UV light at 280 nm
for 120 min. After the further exposure of CD–Thy–D to UV light at
2
40 nm for 120 min.
(Scheme 1). The substrate PNPA having a substituent group at
between the actual percentage of photodimerized thymine
groups and the absorption change observed. By irradiation at
the para position would be included more deeply and tightly
into the cavity of the CD–Thy–D than unmodified CD and
2
80 nm, the absorption at 271 nm corresponding to the thymine
CD–Thy, which results in the decrease in both kcat and Kdiss.
moiety decreased by 14%, whereas the peak area corresponding
By exposure to UV light at 240 nm, which causes the photo-
dimerized thymine to revert to monomeric thymine, both kcat
and Kdiss increased, but the values did not reach those of the
starting CD–Thy. These results show that some photodimerized
thymine groups did not revert to the monomeric form (CD–
Thy–M) by photo-irradiation, which is in agreement with the
result of absorption measurements (Fig. 2).
1
6
to the proton at the C(6) position (7.43 ppm) and the methyl
proton (1.81 ppm) of the thymine ring decreased by 22%. This
result shows that the observed absorption change was smaller
than the decrease in the actual concentration of monomeric
thymine groups, which might be partly due to the overlap of the
absorption of dimeric species with that of monomeric ones, and
partly due to the extremely large local concentration of thymine
groups (Beer’s law was not obeyed). Therefore, by irradiation
at 280 nm for 120 min, 1.5 thymine groups per CD molecule
would make photodimerization on average (three photodimers
per four CD molecules).
Hydrolyses of MNPA and NABA in the presence of CD
derivatives
In the hydrolysis of NABA, CD accelerated the reaction
Ϫ1
(kcat = 0.12 s and Kdiss = 6.6 m), whereas CD–Thy showed an
The GPC measurements showed that the molecular size of
CD–Thy did not change before or after photo-irradiation,
which shows that intramolecular dimerization of thymine
groups occurred. These phenomena suggest that thymine
groups binding to CD molecules made the intramolecular
photodimerization and photodissociation partly reversible by
UV irradiation.
inhibitory effect (Fig. 3). This unexpected result is probably due
to the steric hindrance of substituents (both nitro and carboxy
groups) restricting the inclusion of NABA in the CD cavity
to make the ester group approach to the secondary hydroxy
groups in the modified CD: it seemed to be sterically very
difficult for the substrate to penetrate into the cavity of CD–
Thy through the inlet at the C(6) side. For catalytic hydrolysis,
therefore, the phenyl group of the substrate has to penetrate
into the cavity through the inlet at the secondary hydroxy group
side, which would be largely hindered by the bulky substituents
at the meta and para positions.
Hydrolysis of PNPA in the presence of cyclodextrin derivatives
Hydrolysis of p-nitrophenyl acetate (PNPA) in the presence of
cyclodextrin derivatives were carried out. The pseudo-first-
order rate constants (k , kcat) and dissociation constants (Kdiss
)
The inhibition constant, K , estimated for CD–Thy (1.1 m)
un
i
were estimated from measurements of absorbance of the prod-
uct, the p-nitrophenolate (PNP) anion, and compiled in Table 1.
These results show the remarkable effects of the modification
of cyclodextrin with thymine residues and the dimerization of
thymine residues on the rate of the hydrolysis.
was reduced by photo-irradiation at 280 nm (0.53 m for CD–
Thy–D) and slightly increased by the subsequent irradiation at
240 nm (0.69 m for CD–Thy–M). The increase in the inhibit-
ory effect by the dimerization is probably due to the disadvant-
ageous cooperative effect of (i) the closure of the inlet at the
C(6) side, (ii) the increase in capacity of the hydrophobic cavity
of CD–Thy–D (the substrate once included might be too deeply
penetrated into the cavity) and (iii) the steric hindrance of the
substituents for the substrate to penetrate into the CD cavity.
Observations made in the hydrolysis of MNPA were similar
By modifying CD with thymine, the catalytic efficiency (kcat
/
Kdiss, the apparent second-order rate constant that describes the
rate in terms of the concentration of free catalyst and free
1
7
substrate) increased due to the decrease in the dissociation
constant (Kdiss). There is another possibility that nucleophilic
J. Chem. Soc., Perkin Trans. 2, 1997
1219

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in CD–Thy, PNPA and MNPA molecules could be tightly
included into the CD cavity and the catalytic efficiency of CD–
Thy was promoted. Such cyclodextrin derivatives modified with
thymine groups would be useful to obtain information for
designing highly functional photoresponsive compounds.
Acknowledgements
This work was supported by a Grant-in-Aid from the Ministry
of Education, Science and Culture (08246222). We are indebted
to Dr Y. Inaki, Osaka University, for his helpful suggestions.
References
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2
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Fig. 3 Effect of cyclodextrin derivatives on kobs for the hydrolysis of
NABA at 20 ЊC and pH 11.6 (0.2 M sodium carbonate buffer): ᭺, CD;
᭺Њ, CD–Thy; ᭢, CD–Thy–D
3
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a
6 M. L. Bender and M. Komiyama, Cyclodextrin Chemistry, Springer,
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2
0 ЊC
2
Ϫ1
4
Ϫ1 Ϫ1
7 F. Cramer and W. Kampe, J. Am. Chem. Soc., 1965, 87, 1115.
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kcat/10 s
Kdiss/10
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(kcat/Kdiss)/M
s
8
R. L. VanEtten, J. F. Sebastian, G. A. Clowes and M. L. Bender,
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β-CD
46.4 (±3.2)
18.8 (±0.7)
15.8 (±0.7)
16.5 (±0.5)
98.3 (±8.6)
11.5 (±1.1)
6.9 (±0.4)
10.1 (±0.9)
49 (±8)
9
H. Kitano and T. Okubo, J. Chem. Soc., Perkin Trans. 2, 1977, 432.
CD–Thy
CD–Thy–D
CD–Thy–M
164 (±21)
231 (±20)
162 (±20)
b
10 R. Breslow, M. F. Czarniecki, J. Emert and H. Hamaguchi,
c
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1
1
a
Hydrolysis was carried out at pH 11.6 (±0.1) using a sodium carbon-
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Ϫ2 Ϫ1
b
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1
.18 × 10 sc . After the exposure of CD–Thy to UV light at 280 nm
for 120 min. After the further exposure of CD–Thy–D to UV light at
1
1
2
40 nm for 120 min.
to those for the hydrolysis of PNPA, i.e. the introduction of the
thymine group increased the catalytic efficiency and the photo-
irradiation at 280 nm further promoted the efficiency, whereas
the subsequent irradiation at 240 nm caused a reduction in effi-
ciency (Table 2). The size of the increase caused by photo-
dimerization at 280 nm was, however, smaller than that for
PNPA, probably because of the similar, but much smaller steric
effect than that in the hydrolysis of NABA. This might be due
to the smaller number of substituents than NABA, which
resulted in the acceleration effect on the hydrolysis of MNPA.
In conclusion, by photodimerization of the thymine groups
1
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1
Paper 6/06245H
Received 10th September 1996
Accepted 19th February 1997
1
220
J. Chem. Soc., Perkin Trans. 2, 1997