A water-soluble β-cyclodextrin derivative possessing a fullerene tether as an efficient photodriven DNA-cleavage reagent

Article abstract of DOI:10.1016/j.tetlet.2005.01.181

A simple synthetic route for a water-soluble cyclodextrin-fullerene conjugate based on the Diels-Alder reaction between anthryl-cyclodextrin and fullerene has been presented. Aided by the fascinating biochemical functions of fullerene, the resultant cyclodextrin-C₆₀ conjugate displays a satisfactory DNA-cleavage ability under the visible-light irradiation.

Full text of DOI:10.1016/j.tetlet.2005.01.181

Tetrahedron
Letters
Tetrahedron Letters46 (2005) 2507–2511
A water-soluble b-cyclodextrin derivative possessing a
fullerene tether as an efficient photodriven DNA-cleavage reagent
Yu Liu,* Yan-Li Zhao, Yong Chen, Peng Liang and Li Li
Department of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, PR China
Received 1 September 2004; revised 18 January 2005; accepted 25 January 2005
Abstract—A simple synthetic route for a water-soluble cyclodextrin–fullerene conjugate based on the Diels–Alder reaction between
anthryl-cyclodextrin and fullerene has been presented. Aided by the fascinating biochemical functions of fullerene, the resultant
cyclodextrin-C60 conjugate displays a satisfactory DNA-cleavage ability under the visible-light irradiation.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Fullerene isgenerally considered asa powerful building
block in material sciences and medicinal chemistry
owing to the combination of itsseveral unique properties,
particularly with respect to its electron donor or accep-
tor capability aswell asitsphotophyiscal and photo-
chemical behavior.1–10 For example, C60 and its
derivatives are reported to be able to exhibit satisfactory
enzyme-inhibiting,11–13 radical-quenching,14 and DNA-
cleavage abilities.15–17 Notably among them, cyclodex-
trin (CD)-C60 conjugateshave attracted extenivse
interest of chemists and biologists.18–23 Compared with
other C60 derivatives, CD-C60 conjugates possess an
inherent advantage in the field of biochemistry due to
the capability of the CD cavity to recognize various
model substrates. Upon being associated with a biolog-
ical guest molecule, the CD cavity can selectively bind
with certain fragmentsof the guets, thusenabling the
site-specific interaction between C60 and the model
substrate.18–20 To combine the drug binding ability of
CD and photodriven DNA-cleavage propertiesof fuller-
ene,21 we prepare a water-soluble b-CD derivative pos-
sessing a [60]fullerene tether. Herein we report a very
simple method for synthesizing CD-C60 conjugate by
the Diels–Alder reaction of C60 with anthryl-b-CD.
Although [4+2] cycloaddition hasbeen proved to be
one of the most expeditious methods for selective func-
tionalization of [60]fullerene at the 6,6-ring junc-
tions,3,4,24–32 itsapplication to the ysntheisof CD-
fullerene conjugateshasnever been reported, to the best
of our knowledge. Our current work in thisdirection
presents a new avenue for the design and preparation
of water-soluble fullerene derivative. Moreover, benefit-
ing from the good biological property of C60, the ob-
tained water-soluble CD-C60 conjugate shows an
efficient DNA-cleavage ability under the visible-light
irradiation attributing to the sensitized singlet oxygen
(1O2) by the photoexcitation of C60 moiety.
CD-C60 233 isprepared by the Diel–sAlder reaction of
C60 with mono[6-(9-anthrylformamido)ethyleneamino-
6-deoxy]-b-CD 134 in DMF/toluene solution under
nitrogen according to the procedure shown in Scheme
1. Owing to the introduction of b-CD asa os lubilizer,
CD-C60 2 exhibitsa moderate water-oslubility up to
2.5 g LÀ1 at 25 ꢁC and can remain stable for several
weekswhen stored in a freezer ( À10 ꢁC). Notably, since
both 1 and 2 are readily soluble in water, the uncon-
sumed C60 can be easily reclaimed by filtration after
the reaction.
The structure of 2 is confirmed by elemental analysis,
UV–vis, FT-IR, and NMR spectra. The UV–vis absor-
bance (Fig. 1) observed for 1 (kmax/(loge)) in water are
384.0 (4.04), 364.5 (4.07), 346.5 (3.90), and 330.0
(3.62) nm, while 2 only exhibitstwo clear abosrbances
at 253.5 (5.15) and 329.5 (4.43) nm under the same con-
ditions. The disappearance of the corresponding charac-
teristic bands of anthracene above 330 nm indicates that
the conjugated system of anthracene is broken. In addi-
tion, a characteristic vibration band at 801 cmÀ1 as-
signed to the C60 unit is also observed in the FT-IR
spectrum of 2 (see Supplementary data). Moreover, we
Keywords: Cyclodextrin; Diels–Alder reaction; DNA-cleavage; Fullerene.
*
Corresponding author. Tel./fax: +86 22 2350 3625; e-mail:
yuliu@public.tpt.tj.cn
1
observe that the H NMR spectrum of 2 issignificantly
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.01.181

2508
Y. Liu et al. / Tetrahedron Letters 46 (2005) 2507–2511
OH
OH
O
HO
OH
O
7
β-CD
O
O
C
NH
HN
C
HN
N
H
DMF/Toluene
+
80 o
C
C60
2
1
Scheme 1. Synthetic route of CD-C60 conjugate 2.
The above information strongly supports the antici-
pated structure of 2.
3.5
3.0
2.5
The conformation of the CD-C60 conjugate 2 isinvesti-
gated by circular dichroism spectroscopy, since it has
been amply demonstrated that the inclusion of a chro-
mophoric achiral guest in a chiral host such as CDs pro-
duces induced circular dichroism (ICD) signals at the
absorbance wavelengths of the guest chromophore.36,37
From a comparative analysis based on the circular
dichroism spectra of hosts 1 and 2 (see Supplementary
data), we observe that the host 2 displays relatively weak
ICD signals in the examined wavelength range, which
2.0
A
1.5
1.0
2
0.5
0.0
1
indicatesthat the C
unit in 2 isnot included in the
60
300
400
500
600
700
800
CD cavity. Thisisexpected, because b-CD isknown to
be too small to accommodate the fullerene sphere. The
observed conformation will provide a C60-capped CD
cavity to selectively bind with certain biologic guests
and thusenable the interaction between C 60 and a model
substrate. It is noteworthy that the water-soluble CD-C60
conjugate 2 shows a satisfactory DNA-cleavage ability
under the visible-light irradiation at 298 K.38,39 As
shown in Figure 4, both 1 and 2 show no DNA-cleavage
ability in the absence of light (Fig. 4, lanesb and c,
respectively). However, under the visible-light irradia-
λ / nm
Figure 1. The UV–visspectra of 1 (1.4 · 10À5 M) and 2 (1.2 · 10À5 M)
in aqueoussolution at 25 ꢁC.
1
different from that of 1. Seen from Figure 2, the H
NMR spectrum of 2 shows three sets of signals centered
at 7.92, 7.59, and 5.98 ppm, respectively, assigned to the
Ha, Hb, and Hc protonsof the dihydroanthracene
group in 2, while the corresponding H1(8)/H4(5),
H2(7)/H3(6), and H9 protonsof the anthracene moiety
in 1 are centered at 8.05, 7.46, and 8.40 ppm.
tion, CD-C60 conjugate
2 can effectively cleave
pGEX5X2 DNA (form I) into nicked DNA (form II)
and linear DNA (Fig. 4, lane e, form III), and about
70% of supercoiled DNA is converted to nicked and lin-
ear DNA, which isclose to the reported values(about
50–90%) by Yamakoshi et al.17c In order to explore the
DNA cleavage mechanism, the EPR spectrum40 of 2 with
2,2,6,6-tetramethyl-4-piperidone (TEMP) isperformed
under the visible-light irradiation at 298 K. It is well doc-
umented that, when the singlet oxygen (1O2) is sensitized
by the photoexcitation of fullerene, it can be detected by
From the 13C NMR data of 2 (Fig. 3), we observe that,
apart from the signals assigned to the methylene carbons
(47.1 and 45.9 ppm), the b-CD skeleton carbons (60.6,
68.8, 72.5, 73.1, 73.8, 81.9, 82.3, and 102.7 ppm), the
carbonyl carbon (172.3 ppm), the aromatic carbonsof
dihydroanthracene (127.5 and 128.6 ppm), and the
unsaturated sp2 carbonsof C 60 (140–150 ppm), there ap-
pears a new signal at 70.7 ppm assigned to the saturated
sp3 carbons. This signal indicates a closed conformation
of the C60 unit in 2, since the newly generated saturated
carbons in the closed structures will resonate at a higher
1
the EPR spin-trapping using a O2-trapping agent such
17b,21,41
asTEMP,
1
because TEMP can react with O2 to
give a O2-adduct, TEMPO. In the present case, three
characteristic EPR signals assigned to TEMPO are ob-
served from a phosphate buffer aqueous solution
1
field than normal unsaturated sp2 carbonsof C
usually appear in the range of d 130–160 ppm.24,25,35
that
60

Y. Liu et al. / Tetrahedron Letters 46 (2005) 2507–2511
2509
Figure 2. 1H NMR spectra of 1 (4.2 · 10À3 M) and 2 (4.4 · 10À3 M) in DMSO-d6 at 25 ꢁC.
Figure 3. The 13C NMR spectrum of 2 (2.1 · 10À3 M) in DMSO-d6 at 25 ꢁC.
neither 2 nor TEMP exhibitsthe appreciable EPR signals
in the same condition. Moreover, CD-C60 conjugate 2
shows no DNA cleavage ability in the absence of O2.
Therefore, we deduce that a singlet oxygen mechanism
should be responsible for the DNA cleavage reaction.
That is, the C60 moiety in 2 may be located close to the
guanosine position of DNA.16a Under the visible-light
irradiation, the singlet oxygen (1O2) is sensitized by the
photoexcitation of C60. Then, the sensitized singlet oxy-
gen reactswith the guanosinesof DNA by either [4+2]
or [2+2] cycloaddition to the five-membered imidazole
ring of the purine base and thus cleaves the DNA.42
Herein, the function of CD isto enhance the water-solu-
bility of fullerene and thusenablesthe DNA cleavage
reaction in aqueoussolution.
Figure 4. The DNA-cleavage ability of 2 in phosphate buffer (50 mM,
pH 7.4) after the visible-light irradiation with a 300 W reflector lamp
for 1 h at 298 K. (À: absence, +: presence) [1] = 4.2 · 10À5 M;
[2] = 4.4 · 10À5 M; [DNA] = 0.004 lg lÀ1
.
(pH 7.4, containing 5% DMF) of 2/O2 system under the
visible-light irradiation as shown in Figure 5, which indi-
catesthat 1O2 isgenerated. In the control experiments,
In conclusion, we have presented the preparation of a
water-soluble CD-C60 conjugate 2 using a simple

2510
Y. Liu et al. / Tetrahedron Letters 46 (2005) 2507–2511
5. Nakamura, E.; Isobe, H. Acc. Chem. Res. 2003, 36, 807–
815.
5k
4k
3k
2k
1k
0
6. Braun, T.; Schubert, A. P.; Kostoff, R. N. Chem. Rev.
2000, 100, 23–37.
7. (a) Curl, R. F. Angew. Chem., Int. Ed. 1997, 36, 1566–
1576; (b) Kroto, H. W. Angew. Chem., Int. Ed. 1997, 3(6),
1578–1593; (c) Smalley, R. E. Angew. Chem., Int. Ed. 1997,
36, 1594–1601.
8. Homann, K.-H. Angew. Chem., Int. Ed. 1998, 37, 2434–
2451.
9. Nierengarten, J.-F. Chem. Eur. J. 2000, 6, 3667–
3670.
-1k
-2k
-3k
-4k
-5k
10. Guldi, D. M. Chem. Soc. Rev. 2002, 31, 22–36.
11. Friedman, S. H.; DeCamp, D. L.; Sijbesma, R. P.;
Srdanov, G.; Wudl, F.; Kenyon, G. L. J. Am. Chem.
Soc. 1993, 115, 6506–6509.
12. Toniolo, C.; Bianco, A.; Maggini, M.; Scorrano, G.;
Prato, M.; Marastoni, M.; Tomatis, R.; Spisani, S.; Palu´,
R.; Blair, E. D. J. Med. Chem. 1994, 37, 4558–4562.
13. Marcorin, G. L.; Ros, T. D.; Castellano, S.; Stefancich,
G.; Bonin, I.; Miertus, S.; Prato, M. Org. Lett. 2000, 2,
3955–3958.
3460
3480
3500
3520
3540
3560
[G]
Figure 5. The EPR spectrum of a mixture of 2 (1.5 · 10À4 M) with
TEMP (9.2 · 10À3 M) after the visible-light irradiation in an aqueous
phosphate buffer solution (pH 7.4, containing 5% DMF) at 298 K.
14. Chiang, L. Y.; Lu, F.-J.; Lin, J.-T. J. Chem. Soc., Chem.
Commun. 1995, 1283–1284.
synthetic route, which will be useful for the design of
functional fullerene derivatives. Furthermore, the results
obtained also show that the above water-soluble CD-C60
conjugate can act asan efficient photodriven DNA-
cleavage reagent, which may serve as a potential photo-
active medicine precursor in the biochemical field. Fur-
ther endeavorsto explore the effect of the tether ꢀs
length of the CD-C60 conjugate are currently in
progress.
15. (a) Tokuyama, H.; Yamago, S.; Nakamura, E.; Shiraki,
T.; Sugiura, Y. J. Am. Chem. Soc. 1993, 115, 7918–7919;
(b) Nakamura, E.; Isobe, H.; Tomita, N.; Sawamura, M.;
Jinno, S.; Okayama, H. Angew. Chem., Int. Ed. 2000, 39,
4254–4257.
16. (a) An, Y.-Z.; Chen, C.-H. B.; Anderson, J. L.; Sigman, D.
S.; Foote, C. S.; Rubin, T. Tetrahedron 1996, 52, 5179–
5189; (b) Bernstein, R.; Prat, F.; Foote, C. S. J. Am. Chem.
Soc. 1999, 121, 464–465.
17. (a) Yamakoshi, Y.; Sueyoshi, S.; Fukuhara, K.; Miyata,
N.; Masumizu, T.; Kohno, M. J. Am. Chem. Soc. 1998,
120, 12363–12364; (b) Yamakoshi, Y.; Umezawa, N.;
Ryu, A.; Arakane, K.; Miyata, N.; Goda, Y.; Masumizu,
T.; Nagano, T. J. Am. Chem. Soc. 2003, 125, 12803–12809;
(c) Yamakoshi, Y. N.; Yagami, T.; Sueyoshi, S.; Miyata,
N. J. Org. Chem. 1996, 61, 7236–7237.
18. (a) Samal, S. K.; Geckeler, E. Chem. Commun. 2000,
1101–1102; (b) Murthy, C. N.; Geckeler, K. E. Chem.
Commun. 2001, 1194–1195.
Acknowledgements
This work was supported by NNSFC (Nos. 90306009,
20272028, and 20402008) and the Tianjin Natural Sci-
ence Foundation (No 043604411), which are gratefully
acknowledged. We thank Dr. Hameer Ruparel and
Dr. Xiao-Peng Bai (Columbia University) for their help
in English polishing. We also thank the referees for their
highly valuable suggestions regarding revision.
19. Filippone, S.; Heimann, F.; Rassat, A. Chem. Commun.
2002, 1508–1509.
20. Liu, Y.; Wang, H.; Liang, P.; Zhang, H.-Y. Angew. Chem.,
Int. Ed. 2004, 43, 2690–2694.
21. Nakanishi, I.; Fukuzumi, S.; Konishi, T.; Ohkubo, K.;
Fujitsuka, M.; Ito, O.; Miyata, N. J. Phys. Chem. B 2002,
106, 2372–2380.
22. Yoshida, Z.-i.; Takekuma, H.; Takekuma, S.-i.; Matsu-
bara, Y. Angew. Chem., Int. Ed. Engl. 1994, 33, 1597–
1599.
Supplementary data
Circular dichriom, 13C NMR, and FT-IR spectra of 1
and 2. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.tetlet.2005.01.181.
23. Yuan, D.-Q.; Koga, K.; Kourogi, Y.; Fujita, K. Tetrahe-
dron Lett. 2001, 42, 6727–6729.
24. Murata, Y.; Kata, N.; Fujiwara, K.; Komatsu, K. J. Org.
Chem. 1999, 64, 3483–3488.
References and notes
25. Mikami, K.; Matsumoto, S.; Tonoi, T.; Okubo, Y.
Tetrahedron Lett. 1998, 39, 3733–3736.
1. Fullerene and Related Structures; Hirsch, A., Ed.;
Springer: Berlin, 1999.
2. Jenekhe, S. A.; Chen, X. L. Science 1998, 279, 1903–1907.
3. (a) Prato, M.; Maggini, M. Acc. Chem. Res. 1998, 31, 519–
526; (b) Guldi, D. M.; Prato, M. Acc. Chem. Res. 2000, 33,
695–703.
4. (a) Diederich, F.; Kessinger, R. Acc. Chem. Res. 1999, 32,
537–545; (b) Diederich, F.; Thilgen, C. Science 1996, 271,
´ ´
317–324; (c) Diederich, F.; Gomez-Lopez, M. Chem. Soc.
26. Schlueter, J. A.; Seaman, J. M.; Taha, S.; Cohen, H.;
Lykke, K. R.; Wang, H. H.; Williams, J. M. J. Chem. Soc.,
Chem. Commun. 1993, 972–974.
27. Tsuda, M.; Ishida, T.; Nogami, T.; Kurono, S.; Ohashi,
M. J. Chem. Soc., Chem. Commun. 1993, 1296–1298.
28. Takaguchi, Y.; Tajima, T.; Ohta, K.; Motoyoshiya, J.;
Aoyama, H.; Wakahara, T.; Akasaka, T.; Fujitsuka, M.;
Ito, O. Angew. Chem., Int. Ed. 2002, 41, 817–819.
29. Suzuki, T.; Li, Q.; Khemani, K. C.; Wudl, F. J. Am.
Chem. Soc. 1992, 114, 7301–7302.
Rev. 1999, 28, 263–277.

Y. Liu et al. / Tetrahedron Letters 46 (2005) 2507–2511
2511
30. Qian, W.; Chuang, S.-C.; Amador, R. B.; Jarrosson, T.;
Sander, M.; Pieniazek, S.; Khan, S. I.; Rubin, Y. J. Am.
Chem. Soc. 2003, 125, 2066–2067.
31. Liu, J.-H.; Wu, A.-T.; Huang, M.-H.; Wu, C.-W.; Chung,
W.-S. J. Org. Chem. 2000, 65, 3395–3403.
36. Kra¨utler, B.; Muller, T.; Maynollo, J.; Gruber, K.;
¨
Kratky, C.; Ochsenbein, P.; Schwarzenbach, D.; Burgi,
¨
H.-B. Angew. Chem., Int. Ed. Engl. 1996, 35, 1204–1206.
´
37. Kajtar, M.; Horvath-Toro, C.; Kuthi, E.; Szejtli, J. Acta
Chim. Acad. Sci. Hung. 1982, 110, 327–355.
32. (a) Wilson, S. R.; Yurchenko, M. E.; Schuster, D. I.;
Khong, A.; Saunders, M. J. Org. Chem. 2000, 65, 2619–
2623; (b) Zhou, Z.; Schuster, D. I.; Wilson, S. R. J. Org.
Chem. 2003, 68, 7612–7617.
38. Examination of DNA-cleavage ability: 10 lL of aqueous
solution of DNA pGEX5X2 (0.51 lg lÀ1) isdiluted by
adding 90 lL of water. Then, 10 lL of aqueoussolution of
2 (1.6 · 10À4 M), 12 lL of aqueousoslution of DNA
pGEX5X2 (0.051 lg lÀ1), and 8 lL of phosphate buffer
(250 mM, pH 7.4) are mixed in a microtest tube under
dark conditions to give the sample solution. Sample
(20 lL) isincubated under irradiation with a 300 W
reflector lamp for 1 h at 298 K, then mixed with 4 lL of
6 · loading buffer, and loaded onto a 1% agarose gel
containing ethidium bromide (10 lg mLÀ1). The gelsare
run at a constant voltage of 80 V for 2 h in TAE buffer,
washed with distilled water, visualized under a UV
transilluminator, and photographed using an instant
camera.
33. Synthesis of 2: A DMF solution (30 mL) of 1 (30 mg,
0.021 mmol) is added dropwise to a toluene solution
(30 mL) of C60 (20 mg, 0.028 mmol), and the resultant
mixture istsirred at 80 ꢁC for 48 h under nitrogen. The
initial purple color of the solution becomes red as the
reaction proceeds. Then the solvent is removed under a
reduced pressure, and the precipitate formed is dried
in vacuo. The precipitate is dissolved in water (30 mL), and
hence the unconsumed C60 isremoved by filtration. The
filtrate ispurified on a Sephadex G-25 column with water
as eluent. After the residue is dried in vacuo, a pure sample
2 (dark brown color) isobtained in 14% yield. UV–vis
39. NMR experimental results show that 2 do not decompose
after the visible-light irradiation for 1 h.
40. EPR measurements: In a typical experiment of the EPR
k
max (H2O, nm)/(loge) 253.5 (5.15), 329.5 (4.43). 1H NMR
(300 MHz, DMSO-d6 TMS, ppm): d 2.73–2.90 (m, 4H),
3.35–3.62 (m), 4.45–4.62 (m), 4.82–4.84 (m, 7H), 5.66–5.94
(m, 14H), 6.04 (s, 1H), 7.55–7.67 (m, 4H), 7.92–7.97 (m,
4H). 13C NMR (300 MHz, DMSO-d6 TMS, ppm): d
172.3, 151.7, 147.9, 146.8, 145.9, 144.7, 144.1, 143.2, 141.9,
141.2, 140.1, 128.6, 127.5, 102.7, 82.3, 81.9, 73.8, 73.1,
72.5, 70.7, 68.8, 60.6, 47.1, 45.9. FT-IR (KBr, cmÀ1): m
3318, 2960, 2925, 1658, 1560, 1431, 1367, 1261, 1154, 1081,
1032, 944, 863, 801, 757, 704, 665, 608, 578, 527. Elemental
analysis calcd for C119H84O35N2Æ4H2O: C 65.75, H 4.27, N
1.29. Found: C 65.69, H 4.31, N 1.24.
1
measurement for the detection of O2, a phosphate buffer
aqueousoslution (pH 7.4, containing 5% DMF) of
2
(1.5 · 10À4 M) with TEMP (9.2 · 10À3 M) isintroduced
into an EPR sample tube. The sample is then irradiated
with
a 300 W reflector lamp for 2 min. The EPR
spectrum is measured with a BRUKER EMX-6/1 spec-
trometer and recorded for 170 sat 298 K. The magnitude
of the modulation is chosen to optimize the resolution
and the signal-to-noise ratio (S/N) of the observed
spectra.
34. Liu, Y.; Zhao, Y.-L.; Yang, E.-C.; Zhang, H.-Y. J.
Inclusion Phenom. Macrocycl. Chem. 2004, 50, 3–11.
35. Sternfeld, T.; Thilgen, C.; Chen, Z.; Siefken, S.; Schleyer,
P. V. R.; Thiel, W.; Diederich, F.; Rabinovitz, M. J. Org.
Chem. 2003, 68, 4850–4854.
41. Rion, Y.; Delmelle, M.; van de Vorst, A. Nature 1974, 263,
442–443.
42. (a) Sheu, C.; Foote, C. S. J. Am. Chem. Soc. 1993, 115,
10446–10447; (b) Sheu, C.; Foote, C. S. J. Am. Chem. Soc.
1995, 1(17), 474–477.