Photoinduced DNA cleavage by fullerene-lysine conjugate

Article abstract of DOI:10.1016/j.tetlet.2009.09.027

A novel water-soluble [60] fullerene-substituted lysine derivative 3 has been synthesized and characterized by elemental analysis, ¹H NMR, ¹³C NMR and FAB-MS. The synthetic procedure involved condensation of Boc-protected lysine with

Full text of DOI:10.1016/j.tetlet.2009.09.027

Tetrahedron Letters 50 (2009) 6526–6530
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Photoinduced DNA cleavage by fullerene–lysine conjugate
Anish Kumar a, Mandava V. Rao b, Shobhana K. Menon a,
*
a Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad 380 009, Gujarat, India
b Department of Zoology, School of Sciences, Gujarat University, Ahmedabad 380 009, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel water-soluble [60] fullerene-substituted lysine derivative 3 has been synthesized and character-
ized by elemental analysis, 1H NMR, 13C NMR and FAB-MS. The synthetic procedure involved condensa-
tion of Boc-protected lysine with terephthaldehyde followed by 1,3-dipolar cycloaddition reaction with
C60 in the presence of sarcosine and finally deprotection of the amino group using trifluoroacetic acid. The
synthesized compound 3 exhibited high DNA cleavage efficiency upon visible light irradiation in the pres-
ence of NADH.
Received 12 August 2009
Revised 3 September 2009
Accepted 4 September 2009
Available online 9 September 2009
Keywords:
DNA cleavage
Ó 2009 Elsevier Ltd. All rights reserved.
Lysine
1,3 Dipolar cycloaddition reaction
Fullerene
Recent years have seen the potential utility of fullerenes as
pharmaceuticals with a wide variety of applications such as anti-
bacterials,1 anti-HIV agents,2 anti-inflammatory3 and antiapoptosis
agents.4 We have recently reported some of the novel derivatives
of fullerene with potential application as antibacterials5 and anti-
TB agents.6 Of all the reported activities of C60, the DNA-cleaving
activity and lipid peroxidation, in particular, have attracted consid-
erable attention.7 Photoirradiation of C60 results in the formation of
that has piqued our interests due to the potential of using a fuller-
ene-based amino acid derivative for the systematic creation of
nano bio-conjugates with the application as DNA-photocleaving
agent. Amino acids are the most basic and essential building units
for living organisms at all levels. The incorporation of fullerene-
based amino acids into proteins, peptides or antibodies could lead
to new applications in medicinal chemistry. To date, several ap-
proaches have been taken towards synthesizing fullerene-based
amino acids.13
ꢀ
1
the singlet excited state C60, which undergoes efficient intersys-
ꢀ
8
3
tem crossing (ISC) to give the triplet excited state C60
.
There
In parallel L-lysine derivatives possessing a chromophore have
are several possible pathways for the DNA-cleaving process involv-
ing 3Cꢀ60 to occur (a) via superoxide anion, (b) via singlet oxygen or
(c) direct oxidation of DNA.9 Two reasons for the interest in the
incorporation of fullerene into molecular structures of biological
importance are the highly hydrophobic nature and unusual phys-
ico-chemical properties of fullerenes, making them ideal candi-
dates as interesting pharmacophores. Unfortunately, the direct
use of fullerenes in biological applications is limited by their poor
solubility in aqueous media.10 To overcome this obstacle, two dif-
ferent approaches have been adopted to increase the solubility. The
first strategy involves non-covalently encapsulating fullerene mol-
ecules into soluble polymeric or host molecules.11 The second
strategy relies on covalent functionalization of fullerene by the
introduction of hydrophilic groups by chemical modification.12
The latter approach is attracting more interest as it can not only al-
ter the physical and chemical properties of fullerene to readily
achieve the desired properties, but also provide useful building
blocks for further molecular constructions. It is this latter approach
been found to induce efficient and highly selective cleavage of dou-
ble stranded DNA upon photoexcitation.14 Previous efforts to de-
velop intercalator-based probes utilizing the diverse chemistry of
amino acids for novel DNA-binding reagents have yielded com-
pounds with nuclease activity. However, in most of the systems
described to date, the intercalating moiety functions solely to deli-
ver the peptides with low intrinsic binding affinity to DNA and
does not contribute to chemical reactivity. Hence if we attach ful-
lerene to lysine, DNA cleavage activity from both the photoexcited
C60 and appended amino acids can be conceived. To the best of our
knowledge, no examination of DNA cleavage by fullerene amino
acid conjugate has been performed. With these points in view
we have synthesized a novel fullerene lysine conjugate 3 by a sim-
ple and modular strategy. Moreover, the positive charge that com-
pound 3 carries can help in better interaction with the negatively
charged DNA.
The synthetic procedure for fullerene–lysine conjugate 315 in-
volved the condensation of Boc-protected lysine with terephthalal-
dehyde followed by prato’s 1,3-dipolar cycloaddition reaction with
C60 to form fulleropyrrolidine 2 and finally deprotection of amino
group by treating with trifluoroacetic acid to get fulleropyrrolidine
* Corresponding author. Tel.: +91 079 26302286; fax: +91 079 26308545.
E-mail address: shobhanamenon07@gmail.com (S.K. Menon).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.09.027

A. Kumar et al. / Tetrahedron Letters 50 (2009) 6526–6530
6527
3 (Scheme 1). The synthesized fulleropyrrolidine 3 showed good
solubility in water and also fluorescence quantum yield that are
comparable to C60. Fulleropyrrolidines 2 and 3 were characterized
by elemental analysis, FTIR, 1H NMR, 13C NMR and FAB-MS.
FTIR spectra of both fulleropyrrolidines 2 and 3 showed peaks
corresponding to fullerene moiety, azomethine linkage and car-
bonyl group. A single peak corresponding to N–H stretching was
observed for 2 while two broad peaks corresponding to N–H
stretching were obtained for 3 indicating the deprotection of ami-
no group in 3.
reaches saturation (see ESI). The concentration of 3 and NADH
has been investigated to obtain a better understanding of the
DNA cleavage by the synthesized fullerene–lysine conjugate 3.
The yield of nicked DNA improved drastically with the increase
in the concentration of 3. At a low concentration (1 ꢁ 10ꢂ6 M), only
a small amount of supercoiled plasmid DNA is converted to nicked
DNA, while the conversion ratio of supercoiled plasmid DNA is im-
proved dramatically as the concentration of 3 is increased to
1.0 ꢁ 10ꢂ5 M which is much lower than that reported for
c-cyclo-
dextrin-bicapped C60 (CD/C60) by Wang et al.7a The conversion ra-
tio of supercoiled plasmid DNA is quite small at a low NADH
concentration (1.0 ꢁ 10ꢂ5 M–1.0 ꢁ 10ꢂ4 M), while the proportion
of nicked DNA was substantially increased at a high NADH concen-
tration (1 mM), consistent with the previous results that NADH is
an important coagent for the photoinduced cleavage of DNA by ful-
lerenes. In order to analyze the role of positive charge on com-
pound 3 in cleavage activity, the same experiments were also
carried out with intermediate compound 2. Because of low solubil-
ity, a suspension of compound 2 in water was prepared for biolog-
ical studies. Under the same experimental conditions, that is, with
photoirradiation and in the presence of NADH, compound 2 was
also found to cleave DNA but only at concentration higher than
1 mM, which is about 100 times more than that required for com-
pound 3. This difference in activity clearly indicates the better
interaction of positively charged compound 3 with negatively
charged DNA.
1H NMR spectra of 3 (Fig. 1) showed singlets at d 12.1, 8.23 and
7.80 corresponding to the carboxylic proton (H1), proton of azome-
þ
thine linkage (H2) and protons of NH3 (H3), respectively. Other
peaks such as doublets for aromatic protons and protons of pyrrol-
idine ring were also observed. All these peaks were also observed
þ
in compound 2 except the peak of NH3
.
In 13C NMR spectra of 3 (Fig. 2), apart from the peaks corre-
sponding to C of carboxylic and azomethine groups, 29 signals
were observed in the range of d 150 to 130 corresponding to sp2
C’s of C60. Two peaks at d 75.2 and 68.4 were also observed for
the sp3 C’s of C60
.
The DNA cleavage activity of 3 was investigated using pBR322
supercoiled plasmid DNA (form 1) in a 3-NADH system under vis-
ible light irradiation.16 As shown in Figure 3, buffer solution (pH
8.0) of 3-NaDH system was found to cleave pBR322 supercoiled
DNA into nicked DNA (form 2) after 3 h visible light irradiation
at 298 K (Lane 3). Under dark condition no DNA cleavage was ob-
served (Lane 5). When the same experiment was carried out with
the intermediate lysine-terephthalaldehyde conjugate 1, no DNA
cleavage was observed either on photoirradiation or in dark (Lanes
6, 7 and 8). In control experiments, it was found that no DNA cleav-
age was found to occur in dark or under photoirradiation in the ab-
sence of either 3 or NADH (Lane 1, 2, 4), showing the importance of
both NADH and compound 3 in the cleavage activity. Effect of vis-
ible light irradiation time on DNA cleavage was analyzed by chang-
ing the irradiation time from 1 to 6 h. The amount of nicked DNA
was found to increase with the prolongation of irradiation time.
After 6 h of irradiation the percentage of nicked DNA almost
The effect of pH on DNA cleavage was also analyzed by varying
the pH from 7 to 9. The DNA-cleaving efficiency was found to in-
crease from pH 7 to 8, reached its maximum at pH 8 and then de-
clined at pH 8–9. Therefore pH
8 is the optimum pH for
photoinduced DNA cleavage by 3-NADH system.
The photoinduced DNA cleavage depends greatly on the gener-
ation of reactive oxygen species (ROS), including singlet oxygen
(1O2), superoxide radical anion (O2Åꢂ) and hydroxyl radical (ÅOH),
which are proposed to be the actual active species forꢀthe photoin-
duced DNA cleavage. The triplet excited state C60 (3C60) generated
via the intersystem crossing from the singlet excited state of C60
,
which is produced upon light irradiation, is a key intermediate
Scheme 1. Synthetic route of fullerene–lysine conjugate 3.

6528
A. Kumar et al. / Tetrahedron Letters 50 (2009) 6526–6530
Figure 1. 1H NMR spectra of 3 in DMSO-d6 at 25 °C.
Figure 2. 13C NMR spectra of 3 in DMSO-d6 at 25 °C.
for the generation of ROS. Either the energy transfer process can
Zhang et al. have shown that larger aggregation destabilizes C60 an-
ion radical ꢀsuggesting that the C60 radical anion generated by
ꢀ
3
occur, where the energy is transferred from C60 to the oxygen
molecule to generate 1O2, or the electron transfer process can occur
in the presence of NADH, where 3C6ꢀ0 is reduced to produce C60 an-
ion radical (C60Åꢂ), followed by an electron transfer from C60Åꢂ to the
3
reducing C60 with NADH would be less stable in the more aggre-
Åꢂ
gated C60 solution, thus impairing the generation of O2 and
ÅOH. Water-soluble fullerenes are found to be easily aggregated
in aqueous solution, where the hydrophobic force between fuller-
ene surfaces is proposed to be the driving force for such aggrega-
tion. Wang et al.7a have shown that although the hydrophobic
force that drives the aggregation of C60 molecules can also enhance
oxygen molecule to give rise to O2 or ÅOH in a further step. How-
Åꢂ
ever, the aggregationꢀof C60 has been shown to significantly accel-
3
erate the decay of C60, resulting in less interaction time between
3Cꢀ60 and oxygen molecule, thus reducing the generation of 1O2.

A. Kumar et al. / Tetrahedron Letters 50 (2009) 6526–6530
6529
T.; Mochizuchi, M. Bioorg. Med. Chem. Lett. 1999, 9, 2959; (d) Da Ros, T.; Prato,
M.; Novello, F.; Maggini, M.; Banfi, E. J. Org. Chem. 1996, 61, 9070.
2. (a) Tanimoto, S.; Sakai, S.; Matsumura, S.; Takahashi, D.; Toshima, K. Chem.
Commun. 2008, 5767; (b) Marcorin, G.; Da Ros, T.; Castellano, S.; Stefancich, G.;
Borin, I.; Miertus, S.; Prato, M. Org. Lett. 2000, 2, 3955; (c) Friedman, S. H.;
Ganapathi, P. S.; Rubin, Y.; Kenyon, G. L. J. Med. Chem. 1998, 41, 2424; (d)
Sijbesma, R.; Srdanov, G.; Wudl, F.; Castoro, J. A.; Wilkins, C.; Friedman, S. H.;
DeCamp, D. L.; Kenyon, G. L. J. Am. Chem. Soc. 1993, 115, 6510.
3. (a) Huang, S. T.; Ho, C. S.; Lin, C. M.; Fang, H. W.; Peng, Y. X. Bioorg. Med. Chem.
2008, 16, 8619; (b) Huang, S. T.; Liao, J. S.; Fang, H. W.; Lin, C. M. Bioorg. Med.
Chem. Lett. 2008, 18, 99.
4. (a) Jensen, A. W.; Wilson, S. R.; Schuster, D. I. Bioorg. Med. Chem. 1996, 4, 767;
(b) Da Ros, T.; Prato, M. Chem. Commun. 1999, 663; (c) Wilson, S. R. In The
Fullerene Handbook; Kadish, K., Ruoff, R., Eds.; Wiely: New York, 2000; pp 437–
465.
5. Kumar, A.; Menon, S. K. Eur. J. Med. Chem. 2009, 44, 2178.
6. Kumar, A.; Patel, G.; Menon, S. K. Chem. Biol. Drug. Des. 2009, 73, 553.
7. (a) Wang, D.; Sun, L.; Liu, W.; Chang, W.; Gao, X.; Wang, Z. Environ. Sci.
Technol. 2009, 43, 5825; (b) Zhao, X. J. Phys. Chem. C 2008, 112, 8898; (c)
Ikeda, A.; Doi, Y.; Hashizume, M.; Kikuchi, J.; Konishi, T. J. Am. Chem. Soc.
2007, 129, 4140; (d) Liu, Y.; Zhao, Y. L.; Chen, Y.; Liang, P.; Li, L.
Tetrahedron Lett. 2005, 46, 2507; (e) Boutorine, A. S.; Takasugi, M.; Hélène,
C.; Tokoyuma, H.; Isobe, H.; Nakamura, E. Angew. Chem., Int. Ed. Engl.
1994, 33, 2462; (f) Yamakoshi, Y. N.; Yagami, T.; Sueyoshi, S.; Miyata, N. J.
Org. Chem. 1996, 61, 7236; (g) Tokuyama, H.; Yamago, S.; Nakamura, E. J.
Am. Chem. Soc. 1993, 115, 7918; (h) Ikeda, A.; Hatano, T.; Kawaguchi, M.;
Suenaga, H.; Shinkai, S. Chem. Commun. 1999, 1403.
8. (a) Lof, R. W.; van Veenendaal, M. A. K. B.; Jonkman, H. T.; Sawatzky, G. A. Phys.
Rev. Lett. 1992, 68, 3924; (b) Arbogast, J. W.; Darmanyan, A. P.; Foote, C. S.;
Rubin, Y.; Diederich, F. N.; Alvarez, M. M.; Anz, S. J.; Whetten, R. L. J. Phys. Chem.
1991, 95, 11; (c) Dimitrijevic, N. M.; Kamat, P. V. J. Phys. Chem. 1992, 96, 4811;
(d) Fraelich, M. R.; Weisman, R. B. J. Phys. Chem. 1993, 97, 11145.
9. (a) Sayes, C. M.; Fortner, J. D.; Guo, W.; Lyon, D.; Boyd, A. M.; Ausman, K. D.;
Tao, Y. J.; Sitharaman, B.; Wilson, L. J.; Hughes, J. B.; West, J. L.; Colvin, V. L. Nano
Lett. 2004, 4, 1881; (b) Dhawan, A.; Taurozzi, J. S.; Pandey, A. K.; Shan, W. Q.;
Miller, S. M.; Hashsham, S. A.; Tarabara, V. V. Environ. Sci. Technol. 2006, 40,
7394; (c) Isakovic, A.; Markovic, Z.; Todorovic-Markovic, B.; Nikolic, N.;
Vranjes-Djuric, S.; Mirkovic, M.; Dramicanin, M.; Harhaji, L.; Raicevic, N.;
Nikolic, Z.; Trajkovic, V. Toxicol. Sci. 2006, 91, 173.
Figure 3. Photocleavage of pBR322 supercoiled DNA by fullerene–lysine conjugate
3
analyzed by agarose gel electrophoresis. [3] = 1 ꢁ 10ꢂ5 M, [1] = 1 ꢁ 10ꢂ5 M,
[NADH] = 1 ꢁ 10ꢂ3 M.
the interactions between C60 and DNA since DNA bases are hydro-
phobic, smaller the size of aggregated particles more is the effi-
ciency in photoinduced DNA cleavage. Hence dynamic light
scattering (DLS) experiment was carried out to analyze the size
of the aggregated particles of 3 if any. The concentration for DLS
analysis was taken as 1.0 ꢁ 10ꢂ5 M, the same that was used for
DNA photocleavage. The average size of aggregated particles were
found to be 20 nm which is much smaller as compared to the
aggregates of
c-cyclodextrin-bicapped C60 (CD/C60) reported by
Wang et al.7a In order to understand the mode of DNA cleavage,
the same experiments of gel electrophoresis were carried out in
the presence of sodium azide and L-histidine, singlet oxygen scav-
engers. No change in DNA cleavage was observed in the presence of
either of these two singlet oxygen scavengers. However the cleav-
ing activity was clearly inhibited by the addition of superoxide dis-
10. Kadish, K. M.; Ruoff, R. S.. Fullerenes: Chemistry, Physics, and Technology; Wiley:
New York, 2000.
11. (a) Yamakoshi, Y. N.; Yagami, T.; Fukuhara, K.; Sueyoshi, S.; Miyata, N. J. Chem.
Soc., Chem. Commun. 1994, 517; (b) Atwood, J. L.; Koutsantonis, G. A.; Raston, C.
L. Nature 1994, 368, 229; (c) Andersson, T.; Nilsson, K.; Sundahl, M.; Westman,
G.; Wennerstrçm, O. J. Chem. Soc., Chem. Commun. 1992, 604.
Åꢂ
mutase (SOD), which quenches O2Åꢂ. This result suggested that O2
is a key intermediate for DNA-cleaving activity of 3 in the presence
of NADH.
12. Chiang, L. Y.; Bhonsle, J. B.; Wang, L.; Shu, S. F.; Chang, T. M.; Hwu, J. R.
Tetrahedron 1996, 52, 4963.
In conclusion a novel fullerene–lysine conjugate has been syn-
thesized and was found to cleave the supercoiled DNA under phot-
oirradiation in the presence of NADH. Although the mechanism of
action is not very clear, superoxide radical generated on photoirra-
diation seems to be the reactive oxygen species (ROS) behind the
DNA cleavage. This work opens up interesting prospects in the field
of DNA cleavage by this novel class of fullerene-based amino acids.
13. (a) Burley, G. A.; Keller, P. A. F.; Pyne, S. G.; Ball, E. J. Org. Chem. 2002, 67,
8316; (b) Maggini, M.; Scorrano, G.; Bianco, A.; Toniolo, C.; Sijbesma, R. P.;
Wudl, F.; Prato, M. J. Chem. Soc., Chem. Commun. 1994, 305; (c) Gan, L.;
Zhou, D.; Luo, C.; Tan, H.; Huang, C.; Lu, M.; Pan, J.; Wu, Y. J. Org. Chem.
1996, 61, 1954; (d) An, Y.-Z.; Anderson, J. L.; Rubin, Y. J. Org. Chem. 1993,
58, 4799; (e) Pellarini, F.; Pantarotto, D.; Da Ros, T.; Giangaspero, A.; Tossi,
A.; Prato, M. Org. Lett. 2001, 3, 1845; (f) Pantarottoab, D.; Tagmatarchisa,
N.; Bianco, A. Mini-Rev. Med. Chem. 2004, 4, 805; (g) Hu, Z.; Guan, W. C.;
Wang, W. Cell Biol. Int. 2007, 31, 798; (h) Pantarotto, D.; Bianco, A.;
Pellarini, F. J. Am. Chem. Soc. 2002, 124, 12543; (i) Yang, J. Z.; Alemany, L.
B.; Driver, J. Chem. Eur. J. 2007, 13, 2530; (j) Zhang, J.; Wang, Y. X.; Shao,
Y. Y.; Li, Z. J.; Yang, X. L. Chin. Chem. Lett. 2008, 19, 1159.
Acknowledgements
14. Saito, I.; Takayama, M.; Kawanishi, S. J. Am. Chem. Soc. 1995, 117, 5590.
A.K. would like to thank the Council of Scientific and Industrial
Research (CSIR), New Delhi for Senior Research Fellowship. Finan-
cial assistance from UGC, New Delhi is also gratefully acknowl-
edged. We would also like to thank Shiva S. Chettiar, Zoology
Department, Gujarat University for gel electrophoresis studies.
15. Synthesis of compound 3. The N-protected fulleropyrrolidine
2 (30 mg,
0.03 mmol) was dissolved in a 1:1 mixture of toluene/trifluoroacetic acid and
stirred for 12 h. The reaction was monitored by TLC (SiO2; toluene/propanol,
9:1). After completion of the deprotection, the solvents were evaporated, and
some MeOH was added and evaporated again. The residue was taken up in
CH2Cl2, and the solution was added dropwise to excess hexane. The
precipitated solid was separated by centrifugation, washed with
a small
amount of Et2O and then dried under high vacuum to obtain 3 as brownish
solid product. Yield 25 mg (83.3%) mp 251 °C; Anal. Calcd for C78O4H24N3F3: C,
83.35; H, 2.15; N, 3.74. Found: C, 83.26; H, 2.14; N, 3.76; IR(KBr; cmꢂ1) 528
(C60), 1600 (CH@N), 1659 (C@O stretching), 3257 (NH stretching), 3497 (NH
stretching); 1H NMR (400 MHz, DMSO-d6, Me4Si, 298 K) d 1.50–1.97 (m, 8H, –
CH2–), 2.82 (3H, s, N–CH3), 4.1(s, 1H, –CH–), 4.45 (d, 2J = 9.3 Hz, 1H, HHC–N–),
4.90 (s, 1H, HC–N–), 5.25 (d, 2J = 9.3 Hz, 1H, HHC–N–), 7.16 (d, 3J = 8.0 Hz, 2H,
ArH), 7.57(d, 3J = 8.0 Hz, 2H, ArH), 7.80 (s, 3H, NH3þ), 8.23 (s, 1H, CH@N),
Supplementary data
Supplementary data (general methods, synthetic procedure and
characterization data of intermediates 1 and 2. A copy of FAB-MS
and FTIR spectra of 3, dynamic light scattering data of compound
3. General method for DNA cleavage, effect of photoirradiation
time, concentration of 3 and concentration of NADH on DNA cleav-
age) associated with this article can be found, in the online version,
at doi:10.1016/j.tetlet.2009.09.027.
12.1(s, 1H, –COOH) ppm; 13C NMR(125 MHz, DMSO-d6, Me4Si, 298 K)
d
177.1(1C, –COO), 164.3(1C, CH@N), 154.2(2C, C60), 153.4(2C, C60), 151.6(2C,
C60), 150.7(2C, C60), 150.5(2C, C60), 149.6(2C, C60), 149.2(2C, C60), 148.9(2C,
C60), 148.3(2C, C60), 147.7(2C, C60), 147.4(2C, C60), 146.9(2C, C60) 146.2(2C, C60),
145.9(4C, C60), 145.8(2C, C60), 145.6(2C, C60), 145.4(2C, C60), 144.6(2C, C60),
143.2(1C, ArCq), 142.8(2C, C60), 142.2(2C, C60), 141.8(2C, C60), 141.7(2C, C60),
141.5(2C, C60), 140.7(2C, C60), 139.8(1C, ArCq), 139.5(2C, C60), 138.9(2C, C60),
136.4(2C, C60), 135.9(2C, C60), 135.9(2C, C60), 130.1(2C, ArCH), 129.8(2C, ArCH),
83.5(1C, NCH of the pyrrolidine ring), 75.2(1C, sp3 C– of C60), 69.1(1C, NCH2 of
pyrrolidine ring), 68.4(1C, sp3 C– of C60), 67.9 (1C, –CH–COOH), 66.5 (1C, –CH2–
NH3þ), 44.8(1C, –CH2), 42.3(1C, CH3 linked to N of the pyrrolidine ring),
References and notes
1. (a) Spesia, M. B.; Milanesio, A. E.; Durantini, E. N. Eur. J. Med. Chem. 2008, 43,
853; (b) Bosi, S.; Da Ros, T.; Castellano, S.; Banfi, E.; Prato, M. Bioorg. Med. Chem.
Lett. 2000, 10, 1043; (c) Mashino, T.; Okuda, K.; Hirota, T.; Hirobe, M.; Nagano,

6530
A. Kumar et al. / Tetrahedron Letters 50 (2009) 6526–6530
31.5(1C, –CH2), 30.7(1C, –CH2), 22.5(1C, –CH2) ppm; FAB-MS m/z (%) 1011
conditions. The samples were incubated under irradiation with visible
light for 3 h at 298 K, mixed with 10 of loading buffer (0.1%
bromophenol blue and 30% glycerol in TBE buffer) and loaded onto
1% agarose gel containing ethidium bromide (1
g mLꢂ1). The gels were
run at constant voltage of 70 V for 2 h in TBE buffer, washed with
(100) [MꢂCF3COO]+.
lL
16. DNA cleavage procedure: Thirty microlitres of aqueous solution of DNA
a
pBR322 (0.50
l
g
l
Lꢂ1) were diluted by adding 270
l
L of water. Typically,
L of aqueous solution
of tris-EDTA buffer
a micro test tube under dark
l
10
l
L of aqueous solution of 3 (1.0 ꢁ 10ꢂ5 M), 10
l
a
of DNA pBR322, NADH (5
l
L, 0.126 M) and
8
l
L
distilled water, visualized under a UV transilluminator and photographed
using an instant camera.
(10
lL, 150 ꢁ TE, pH 8.0) were mixed in