C-lysine conjugates: pH-controlled light-activated reagents for efficient double-stranded DNA cleavage with implications for cancer therapy

Article abstract of DOI:10.1021/ja902140m

Double-stranded DNA cleavage of light-activated lysine conjugates is strongly enhanced at the slightly acidic pH (<7) suitable for selective targeting of cancer cells. This enhancement stems from the presence of two amino groups of different basicities. The first amino group plays an auxiliary role by enhancing solubility and affinity to DNA, whereas the second amino group, which is positioned next to the light-activated DNA cleaver, undergoes protonation at the desired pH threshold. This protonation results in two synergetic effects which account for the increased DNA-cleaving ability at the lower pH. First, lysine conjugates show tighter binding to DNA at the lower pH, which is consistent with the anticipated higher degree of interaction between two positively charged ammonium groups with the negatively charged phosphate backbone of DNA. Second, the unproductive pathway which quenches the excited state of the photocleaver through intramolecular electron transfer is eliminated once the donor amino group next to the chromophore is protonated. Experiments in the presence of traps for diffusing radicals show that reactive oxygen species do not contribute significantly to the mechanism of DNA cleavage at the lower pH, which is indicative of tighter binding to DNA under these conditions. This feature is valuable not only because many solid tumors are hypoxic but also because cleavage which does not depend on diffusing species is more localized and efficient. Sequence-selectivity experiments suggest combination of PET and base alkylation as the chemical basis for the observed DNA damage. The utility of these molecules for phototherapy of cancer is confirmed by the drastic increase in toxicity of five conjugates against cancer cell lines upon photoactivation.

Full text of DOI:10.1021/ja902140m

Published on Web 07/28/2009
C-Lysine Conjugates: pH-Controlled Light-Activated Reagents
for Efficient Double-Stranded DNA Cleavage with Implications
for Cancer Therapy
Wang-Yong Yang,† Boris Breiner,† Serguei V. Kovalenko,† Chi Ben,† Mani Singh,†
Shauna N. LeGrand,‡ Qing-Xiang Amy Sang,† Geoffrey F. Strouse,†
John A. Copland,‡ and Igor V. Alabugin*,†
Department of Chemistry and Biochemistry, Florida State UniVersity,
Tallahassee, Florida 32306-4390, and Department of Cancer Biology, Mayo Clinic
ComprehensiVe Cancer Center, JacksonVille, Florida 32224
Received March 19, 2009; E-mail: alabugin@chem.fsu.edu
Abstract: Double-stranded DNA cleavage of light-activated lysine conjugates is strongly enhanced at the
slightly acidic pH (<7) suitable for selective targeting of cancer cells. This enhancement stems from the
presence of two amino groups of different basicities. The first amino group plays an auxiliary role by
enhancing solubility and affinity to DNA, whereas the second amino group, which is positioned next to the
light-activated DNA cleaver, undergoes protonation at the desired pH threshold. This protonation results in
two synergetic effects which account for the increased DNA-cleaving ability at the lower pH. First, lysine
conjugates show tighter binding to DNA at the lower pH, which is consistent with the anticipated higher
degree of interaction between two positively charged ammonium groups with the negatively charged
phosphate backbone of DNA. Second, the unproductive pathway which quenches the excited state of the
photocleaver through intramolecular electron transfer is eliminated once the donor amino group next to
the chromophore is protonated. Experiments in the presence of traps for diffusing radicals show that reactive
oxygen species do not contribute significantly to the mechanism of DNA cleavage at the lower pH, which
is indicative of tighter binding to DNA under these conditions. This feature is valuable not only because
many solid tumors are hypoxic but also because cleavage which does not depend on diffusing species is
more localized and efficient. Sequence-selectivity experiments suggest combination of PET and base
alkylation as the chemical basis for the observed DNA damage. The utility of these molecules for
phototherapy of cancer is confirmed by the drastic increase in toxicity of five conjugates against cancer
cell lines upon photoactivation.
pramolecular polymers,3 phase transitions in stimuli-sensitive
polymers for drug delivery,4 rotaxane switching,5 properties of
Introduction
luminescent devices based on organic fluorophores6 and metal
complexes,7 permeability of mesoporous materials,8 growth of
Control of chemical reactions becomes especially challenging
when chemical processes have to work in the complexity of
biological environments. This is one of the reasons why the
ability to design molecules with structure, reactivity, and
biological activity “switchable” via an externally controlled
factor continues to draw significant attention, from both the
practical and fundamental points of view. Possible applications
of such molecules include design of molecular machines and
switches, logic gate mimics, optical sensors, drug delivery
systems, etc. Since pH-dependent “switchable” molecules are
of particular use for processes that occur in biochemical systems
and in the environment, interesting pH-sensitive systems were
developed to control such diverse phenomena as strand orienta-
tion and exchange in peptide assemblies,1 charge densities in
self-assembled monolayers,2 encapsulation of guests in su-
(3) Xu, H.; Stampp, S. P.; Rudkevich, D. M. Org. Lett. 2003, 5, 4583.
(4) Choi, H. S.; Huh, K. M.; Ooya, T.; Yui, N. J. Am. Chem. Soc. 2003,
125, 6350.
(5) (a) Marty´nez-Dy´az, M.-V.; Spencer, N.; Stoddart, J. F. Angew. Chem.,
Int. Ed. Engl. 1997, 36, 1904. Saha, S.; Stoddart, J. F. Chem. Soc.
ReV. 2007, 36, 77. (b) Ashton, P. R.; Ballardini, R.; Balzani, V.; Baxter,
I.; Credi, A.; Fyfe, M. C. T.; Gandolfi, M. T.; Gomez-Lopez, M.;
Marty´nez-Dy´az, M.-V.; Piersanti, A.; Spencer, N.; Stoddart, J. F.;
Venturi, M.; White, A. J. P.; Williams, D. J. J. Am. Chem. Soc. 1998,
120, 11932. (c) Elizarov, A. M.; Chang, T.; Chiu, H.-S.; Stoddart,
J. F. J. Org. Chem. 2002, 67, 9175. (d) Silvi, S.; Arduini, A.; Pochini,
A.; Secchi, A.; Tomasulo, M.; Raymo, F. M.; Baroncini, M.; Credi,
A. J. Am. Chem. Soc. 2007, 129, 13378. (e) Leigh, D. A.; Thomson,
A. R. Org. Lett. 2006, 8, 5377.
(6) Fabbrizzi, L.; Gatti, F.; Pallavicini, P.; Parodi, L. New J. Chem. 1998,
1403. (f) de Silva, A. P.; Gunaratne, H. Q. N.; McCoy, C. P. Chem.
Commun. 1996, 2399. (g) Becuwe, M.; Cazier, F.; Bria, M.; Woisel,
P.; Delattre, F. Tetrahedron Lett. 2007, 48, 6186.
† Florida State University.
‡ Mayo Clinic Comprehensive Cancer Center.
(7) (a) Pallavicini, P.; Amendola, V.; Massera, C.; Mundum, E.; Taglietti,
A. Chem. Commun. 2002, 2452. (b) Gunnlaugsson, T.; Leonard, J. P.;
Senechal, K.; Harte, A. J. J. Am. Chem. Soc. 2003, 125, 12062. (c)
Tajc, S. G.; Miller, B. L. J. Am. Chem. Soc. 2006, 128, 2532.
(1) Schnarr, N. A.; Kennan, A. Org. Lett. 2005, 7, 395. Schnarr, N. A.;
Kennan, A. J. J. Am. Chem. Soc. 2003, 125, 6364.
(2) Rooth, M.; Shaw, A. M. J. Phys. Chem. C 2007, 111, 15363.
9
11458 J. AM. CHEM. SOC. 2009, 131, 11458–11470
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C-Lysine Conjugates
A R T I C L E S
nanomaterials,9 control of recognition-mediated reactions,10 and
the design of bioreactors.11 Life itself is a pH-sensitive
phenomenon, as many biochemical processes work only within
a very narrow pH window.
In this study, we provide the first example of a “switchable”
molecular system for pH-controlled double-stranded DNA
cleavage designed for selective targeting of cancer cells. The
more acidic extracellular environment of solid tumors, relative
to that of the normal cells, leads to a pH gradient that has a
dramatic effect on drug uptake in tumor cells12 and can be
explored in the design of tumor-specific DNA cleaving agents.13
It is known that hyperglycemia (e.g., glucose infusion) and/or
certain drugs, e.g., amiloride, nigericin, and hydralyzine, are
also able to lower the intracellular pH of cancer cells. For
example, administration of amiloride and nigericin at dosages
that do not affect the normal cells drops the intracellular pH in
a number of tumor cell types from 7.2 to 6.2-6.6.14-17
Moreover, hyperglycemia as well as hypoxia leads to an even
further acidification to pH as low as 5.5.18,19
Figure 1. Approaches to pH-activated enediynes based on acid-catalyzed
transformations of unreactive prodrugs.
Although research in this direction has been hampered by
the scarcity of suitable pH-dependent cytotoxic agents, a number
of approaches can be used for the rational design of such
molecules.20 An illustration of how these ideas can be applied
to a highly potent class of natural enediyne antibiotics either
through isomerization into more reactive functional groups or
through unlocking of structural constraints is given in Figure
1.21 In addition, there have been promising reports of acid-labile
drugs that either hydrolyze at lower pH to give toxic products22
or produce free radicals due to accelerated Co-R bond
homolysis.23 An interesting recent finding involves acid-
promoted DNA cleavage by natural antibiotic varacin C.24 The
authors found a 2-fold increase in the extent of single-stranded
(ss) DNA cleavage at pH 5.5 (47% at 5 µM antibiotic loading)
compared with that at pH 7 (23%). Unfortunately, this increase
only applies to the ss damage which is, unlike the double-
stranded (ds) damage, usually repairable by the cell chemical
machinery.
Another approach to pH-regulated DNA-cleaving agents
involves protonation of basic functional groups. Amine func-
tionality is one of the obvious choices, and several elegant
experimental designs based on the protonation of this functional
group appeared in the literature (Figure 2). In particular, the
groups of Kerwin, Chen, and Kraka and Cremer reported
(8) Fattakhova-Rohlfing, D.; Wark, M.; Rathousky, J. Chem. Mater. 2007,
19, 1640.
(9) Xue, C.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 2036.
(10) Turega, S. M.; Philp, D. Chem. Commun. 2006, 35, 3684.
(11) Broz, P.; Driamov, S.; Ziegler, J.; Ben-Haim, N.; Marsch, S.; Meier,
W.; Hunziker, P. Nano Lett. 2006, 6, 2349.
(12) Adams, D. J.; Dewhirst, M. W.; Flowers, J. L.; Gamcsik, M. P.; Colvin,
O. M.; Manikumar, G.; Wani, M. C.; Wall, M. E. Cancer Chemother.
Pharmacol. 2000, 46, 263. Gabr, A.; Kuin, A.; Aalders, M.; El-Gawly,
H.; Smets, L. A. Cancer Res. 1997, 57, 4811. Teicher, B. A.; Holden,
S. A.; Khandakar, V.; Herman, T. S. J. Cancer Res. Clin. Oncol. 1993,
119, 645. Wood, P. J.; Sansom, J. M.; Newell, K.; Tannock, I. F.;
Stratford, I. J. Int. J. Cancer 1995, 60, 264. Vukovic, V.; Tannock,
I. F. Br. J. Cancer 1997, 75, 1167. Wachsberger, P. R.; Burd, R.;
Wahl, M. L.; Leeper, D. B. Int. J. Hyperthermia 2002, 18, 153.
(13) Hoffner, J.; Schottelius, J.; Feichtinger, D.; Chen, P. J. Am. Chem.
Soc. 1998, 120, 376.
(21) Selected examples given below are limited to acid-catalyzed trans-
formations: Enediyne-eneyne-cumulene conversion: (a) Toshima,
K.; Ohishi, K.; Tomishima, M.; Matsumura, S. Heterocycles 1997,
45, 8. Naoe, Y.; Kikuishi, J.; Ishigaki, K.; Iitsuka, H.; Nemoto, H.;
Shibuya, M. Tetrahedron Lett. 1995, 36, 9165. Acid-catalyzed ring
opening: (b) Unno, R.; Michishita, H.; Inagaki, H.; Suzuki, Y.; Baba,
Y.; Jomori, T.; Moku, M.; Nishikawa, T.; Isobe, M. Biorg. Med. Chem.
Lett 1997, 5, 903. Allylic rearrangement: (c) Dai, W.-M.; Fong, K. C.;
Lau, C. W.; Zhau, L.; Hamaguchi, W.; Nishimoto, S.-I. J. Org. Chem.
1999, 64, 682. Enol-keto tautomerization: (d) Semmelhack, M. F.;
Gallagher, J. J.; Minami, T.; Date, T. J. Am. Chem. Soc. 1993, 115,
11618. A number of other intellectually interesting examples involve
activation at the basic pH: Toshima, K.; Ohta, K.; Ohashi, A.;
Nakamura, T.; Nakata, M.; Tatsuta, K.; Matsumura, S. J. Am. Chem.
Soc. 1995, 117, 4822. David, W. M.; Kumar, D.; Kerwin, S. M. Bioorg.
Med. Chem. Lett. 2000, 10, 2509. Swakyama, M.; Shigenaga, A.;
Nemoto, H.; Shibuya, M. Tetrahedron Lett. 2000, 41, 10019. Nicolaou,
K. C.; Hong, Y. P.; Dai, W. M.; Zeng, Z. J.; Wrasidlo, W. Chem.
Commun. 1992, 1542. Nicolaou, K. C.; Dai, W. M. J. Am. Chem.
Soc. 1992, 114, 8908. Kerwin, S. M. Tetrahedron Lett. 1994, 35, 1023.
Pal, R.; Basak, A. Chem. Commun. 2006, 2992.
(14) Adams, G. E.; Stratford, I. J. Int. J. Radiat. Oncol. Biol. Phys. 1994,
29, 231. Priyadarsini, K. I.; Dennis, M. F.; Naylor, M. A.; Stratford,
M. R. L.; Wardman, P. J. Am. Chem. Soc. 1996, 118, 5648, and
references therein.
(15) Stubbs, M.; Rodrigues, L.; Howe, F. A.; Wang, J.; Jeong, K. S.; Veech,
R. L.; Griffiths, J. R. Cancer Res. 1994, 54, 4011.
(16) Lyons, J. C.; Ross, B. D.; Song, C. W. Int. J. Radiat. Oncol. Biol.
Phys. 1993, 25, 95. Song, C. W.; Lyons, J. C.; Griffin, R. J.;
Makepeace, C. M. Radiother. Oncol. 1993, 27, 252. Song, C. W.;
Lyons, J. C.; Griffin, R. J.; Makepeace, C. M.; Cragoe, E. J., Jr. Cancer
Res. 1993, 53, 1599. Song, C. W.; Lyons, J. C.; Griffin, R. J.;
Makepeace, C. M.; Cragoe, E. J., Jr. Int. J. Radiat. Oncol. Biol. Phys.
1994, 30, 133. Song, C. W.; Kim, G. E.; Lyons, J. C.; Makepeace,
C. M.; Griffin, R. J.; Rao, G. H.; Cragoe, E. J., Jr. Int. J. Radiat.
Oncol. Biol. Phys. 1994, 30, 1161. Lyons, J. C.; Song, C. Radiat.
Res. 1995, 141, 216.
(17) Newell, K.; Wood, P.; Stratford, I.; Tannock, I. Br. J. Cancer 1992,
66, 311. Hasuda, K.; Lee, C.; Tannock, I. F. Oncol. Res. 1994, 6,
259.
(22) Tietze, L. F.; Fischer-Beller, A. Carbohydr. Res. 1994, 254, 169, and
references therein For an application of this idea to enediynes, see.
Nace, Y.; Kikuishi, J.; Ishigaki, K.; Iitsuka, H.; Nemoto, H.; Shibuya,
M. Tetrahedron Lett. 1995, 36, 9165.
(18) Osinsky, S. P.; Levitin, I. Y.; Bubnovskaya, L. N.; Ganusevich, I. I.;
Sigan, A. L.; Tsykalova, M. V.; Zagorujko, L. I. Exp. Oncol. 1999,
21, 216. Tannock, I. F.; Rotin, D. Cancer Res. 1989, 49, 4373. Wike-
Hooley, J. L.; Haveman, J.; Reinhold, H. S. Radiother. Oncol. 1984,
2, 343.
(23) Volpin, M. E.; Levitin, I.; Osinsky, S. Angew. Chem., Int. Ed. Engl.
1996, 35, 2395. (Angew. Chem. 1996, 108, 2516). Levitin, I. Y.;
Belkov, V. M.; Novodarova, G. N.; Shabarova, Z. A.; Volpin, M. E.
MendeleeV Commun. 1996, 153.
(19) von Andrenne, M. AdV. Pharmacol. 1972, 10, 339. Osinsky, S.;
Bubnovskaya, L. Arch. Geschwulstforsch. 1984, 54, 463.
(20) Kar, M.; Basak, A. Chem. ReV. 2007, 107, 2861–2890.
(24) Lee, A. H. F.; Chen, J.; Liu, D.; Leung, T. Y. C.; Chan, A. S. C.; Li,
T. J. Am. Chem. Soc. 2002, 124, 13972.
9
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Yang et al.
be protonated at a wider range of physiological conditions. This
auxiliary group plays three roles through (a) enhancing solubility
of the conjugates in water, (b) increasing their affinity to the
negatively charged backbone of DNA, and (c) modulating the
basicity of the pH trigger. The pH trigger itself corresponds to
the second amino group which should enable pH-switchable
behavior at the desired pH range.
The bifunctional pH-regulated part in our design is derived
from a diamino carboxylic acid (lysine) which is connected to
the DNA-cleaving moiety through the carboxyl group of lysine
(Figure 3). This mode of attachment is different from the most
common way of lysine incorporation into conjugates or proteins
through the formation of a peptide bond at the expense of the
R-amino group.29 Importantly, the use of the carboxyl group
for the assembly preserves the two amino groups, both of which
are essential for solubility, binding to DNA, and pH regulation
of DNA cleavage.
Figure 2. Literature examples of pH-controlled amino enediynes.
systems where protonation increased the efficiency of radical
damage through simultaneous acceleration of the H-abstraction
step and deceleration of p-benzyne diradical deactivation through
the retro-Bergman ring-opening.25 After detailed computational
studies indicated that properly positioned cationic groups
decrease the activation barrier for Bergman cyclization,26 Basak
and co-workers27 pursued an alternative approach based on
significant acceleration of cycloaromatization step imposed by
a spatially close ammonium moiety.
Unfortunately, simple aliphatic amines are too basic for the
change in the protonation state to occur at the pH window
necessary for targeting hypoxic cancer cells. As a result, even
when protonation has been shown to accelerate the Bergman
cyclization of enediynes, the change in reactivity did not occur
in the optimal pH range. Nevertheless, the basicity of nitrogen
bases can be controlled in a number of ways, and thus the above
obstacle is not insurmountable. For example, anilines are
significantly less basic than aliphatic amines and can be fine-
tuned through substitution28 to accept the proton only at the
desired pH range. Amides,25b suggested by Chen, and
aldimines,25c designed by Kraka and Cremer, may offer an
excellent solution for fine-tuning the nitrogen basicity.
The present work describes the development of the first pH-
controlled system capable of inducing the much more thera-
peutically important double-stranded (ds) DNA cleavage.
Several challenges presented themselves at the beginning of this
study. First, the change in reactivity has to occur at a relatively
narrow and predefined pH point (ideally ∼ pH 7). Second, an
efficient DNA cleaver was needed which could operate within
the physiological pH range and be attached to a pH-sensitive
functionality without sacrificing its potency.
In order to solve the first problem, we utilized a simple but,
to the best of our knowledge, new strategy for the design of
pH-regulated molecules which is based on the presence of two
amino moieties (or related functional groups) with different
basicities. The first amino group should be sufficiently basic to
(29) Gengrinovitch, S.; Izakovich, E. WO 2005072061 A2 20050811, 2005,
73. Saito, I.; Takayama, M.; Sugiyama, H.; Nakatani, H. J. Am. Chem.
Soc. 1995, 117, 6406. Saito, I.; Takayama, M.; Kawanishi, S. J. Am.
Chem. Soc. 1995, 117, 5590. Plourde, G.; El-Shafey, A.; Fouad, F. S.;
Purohit, A. S.; Jones, G. B. Bioorg. Med. Chem. Lett. 2002, 12, 2985.
(30) Basicity of these groups is known to be sensitive to the environment.
See, for example: Highbarger, L. A.; Gerlt, J. A.; Kenyon, G. L.
Biochemistry 1996, 35, 41.
(31) Armitage, B. Chem. ReV. 1998, 98, 1171.
(32) Alabugin, I. V.; Kovalenko, S. V. J. Am. Chem. Soc. 2002, 124, 9052.
(33) For other photochemically activated enediynes, see: Tachi, Y.; Dai,
W.-M.; Tanabe, K.; Nishimoto, S.-I. Bioorg. Med. Chem. 2006, 14,
3199. Bhattacharyya, S.; Zaleski, J. M. Curr. Top. Med. Chem. 2004,
4, 1637. Schmittel, M.; Viola, G.; Dall’Acqua, F.; Morbach, G. Chem.
Commun. 2003, 5, 646. Karpov, G.; Kuzmin, A.; Popik, V. V. J. Am.
Chem. Soc. 2008, 130, 11771. Polukhtine, A.; Karpov, G.; Popik, V. V.
Curr. Top. Med. Chem. 2008, 8, 460. Yoshimura, N.; Momotake, A.;
Shinohara, Y.; Nishimura, Y.; Arai, T. Chem. Lett. 2008, 37, 174.
Sud, D.; Wigglesworth, T. J.; Branda, N. R. Angew. Chem., Int. Ed.
2007, 46, 8017. Karpov, G. V.; Popik, V. V. J. Am. Chem. Soc. 2007,
129, 3792. Poloukhtine, A.; Popik, V. V. J. Org. Chem. 2006, 71,
7417. O’Connor, J. M.; Friese, S. J.; Rodgers, B. L. J. Am. Chem.
Soc. 2005, 127, 16342. Kar, M.; Basak, A.; Bhattacharjee, M. Bioorg.
Med. Chem. Lett. 2005, 15, 5392. Zhao, Z.; Peacock, J. G.; Gubler,
D. A.; Peterson, M. A. Tetrahedron Lett. 2005, 46, 1373. Poloukhtine,
A.; Popik, V. V. J. Org. Chem. 2005, 70, 1297. Bhattacharyya, S.;
Zaleski, J. M. Curr. Top. Med. Chem. (Sharjah, United Arab Emirates)
2004, 4, 1637. Fouad, F. S.; Crasto, C. F.; Lin, Y.; Jones, G. B.
Tetrahedron Lett. 2004, 45, 7753. Jones, G. B.; Russell, K. C. CRC
Handbook of Organic Photochemistry and Photobiology, 2nd ed.; CRC
Press: New York, 2004; pp 29/1-29/21. Poloukhtine, A.; Popik, V. V.
J. Org. Chem. 2003, 68, 7833. Chandra, T.; Kraft, B. J.; Huffman,
J. C.; Zaleski, J. M. Inorg. Chem. 2003, 42, 5158. Benites, P. J.;
Holmberg, R. C.; Rawat, D. S.; Kraft, B. J.; Klein, L. J.; Peters, D. G.;
Thorp, H. H.; Zaleski, J. M. J. Am. Chem. Soc. 2003, 125, 6434. Banfi,
L.; Guanti, G.; Rasparini, M. Eur. J. Org. Chem. 2003, 7, 1319. Choy,
N.; Blanco, B.; Wen, J.; Krishan, A.; Russell, K. C. Org. Lett. 2000,
2, 3761. Jones, G. B.; Wright, J. M.; Plourde, G., II; Purohit, A. D.;
Wyatt, J. K.; Hynd, G.; Fouad, F. J. Am. Chem. Soc. 2000, 122, 9872.
Kaneko, T.; Takahashi, M.; Hirama, M. Angew. Chem., Int. Ed. 1999,
38, 1267. Evenzahav, A.; Turro, N. J. J. Am. Chem. Soc. 1998, 12,
1835. Ramkumar, D.; Kalpana, M.; Varghese, B.; Sankararaman, S.;
Jagadeesh, M. N.; Chandrasekhar, J. J. Org. Chem. 1996, 61, 2247.
Kagan, J.; Wang, X.; Chen, X.; Lau, K. Y.; Batac, I. V.; Tuveson,
R. W.; Hudson, J. B. J. Photochem. Photobiol. B 1993, 21, 135.
Nicolaou, V. K. C.; Dai, W. M.; Wendeborn, S. V.; Smith, A. L.;
Torisawa, Y.; Maligres, P.; Hwang, C. K. Angew. Chem. 1991, 103,
1034. Shiraki, T.; Sugiura, Y. Biochemistry 1990, 29, 9795. Falcone,
D.; Li, J.; Kale, A.; Jones, G. B. Bioorg. Med. Chem. Lett. 2008, 18,
934. Fouad, F. S.; Wright, J. M.; Plourde, G., II; Purohit, A. D.; Wyatt,
J. K.; El-Shafey, A.; Hynd, G.; Crasto, C. F.; Lin, Y.; Jones, G. B. J.
Org. Chem. 2005, 70, 9789. Poloukhtine, A.; Popik, V. V. Chem.
Commun. 2005, 5, 617. Kraft, B. J.; Coalter, N. L.; Nath, M.; Clark,
A. E.; Siedle, A. R.; Huffman, J. C.; Zaleski, J. M. Inorg. Chem. 2003,
42, 1663. Plourde, G.; El-Shafey, A.; Fouad, F. S.; Purohit, A. S.;
Jones, G. B. Bioorg. Med. Chem. Lett. 2002, 12, 2985. Basak, A.;
Bdour, H. M.; Shain, J. C.; Mandal, S.; Rudra, K. R.; Nag, S. Bioorg.
Med. Chem. Lett. 2000, 10, 1321.
(25) (a) David, W. M.; Kerwin, S. M. J. Am. Chem. Soc. 1997, 119, 1464.
(b) Hoffner, J.; Schottelius, M. J.; Feichtinger, D.; Chen, P. J. Am.
Chem. Soc. 1998, 120, 376. (c) Kraka, E.; Cremer, D. J. Am. Chem.
Soc. 2000, 122, 8245–8264.
(26) Alabugin, I. V.; Manoharan, M. J. Phys. Chem. A 2003, 107, 3363–
3371. Alabugin, I. V.; Manoharan, M.; Kovalenko, S. V. Org. Lett.
2002, 4, 1119–1122. For a general development of these ideas to other
substituents, see recent work on the ortho-effect in the Bergman
cyclization. Zeidan, T.; Kovalenko, S. V.; Manoharan, M.; Alabugin,
I. V. J. Org. Chem. 2006, 71, 962–975. Zeidan, T.; Manoharan, M.;
Alabugin, I. V. J. Org. Chem. 2006, 71, 954–961.
(27) Basak, A.; Kar, M. Bioorg. Med. Chem. 2008, 16, 4532. This system
shows promising pH dependency (more cleavage at pH 7.5 than at
pH 8). However, the change occurs at slightly basic conditions, which
is insufficient for differentiating normal and cancer cells.
(28) Alabugin, I. V.; Manoharan, M.; Buck, M.; Clark, R. J. THEOCHEM
2007, 813, 21.
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Figure 3. Two types of lysine conjugates.
Figure 5. Structures of lysine conjugates 1-5 and related control
compounds 6-8.
sequences of DNA.36 After a serendipitous discovery of the
photochemical removal of the terminal radioactive label from
32P-labeled oligonucleotides that suggested lysine conjugates are
capable of recognition of terminal phosphate groups, we utilized
this phenomenon for the design of reagents which can recognize
the DNA-damage sites (nicked or gapped ss cleavage). These
reagents allowed for the first time to achieve a controlled
photochemical transformation of the easily repairable ss DNA
cleavage to the much more therapeutically important ds cleav-
age.37
These considerations guided our design and prompted us to
investigate DNA-cleavage activity and binding to DNA of five
lysine conjugates 1-5 in the range of pH 5.5-8 which can be
used to differentiate between cancer and healthy cells (Figure
5). After testing whether the pH-dependent DNA cleavage based
on this molecular design is feasible, we will concentrate on the
mechanistic basis of this phenomenon through a combination
of photophysical techniques, NMR titrations, analysis of DNA
binding trends, and effect of chemical additives at the cleavage
efficiency.
Figure 4. Design of pH-dependent DNA cleavers based on different stages
of protonation of the lysine side chain. The dominant protonation pathway
is indicated by the bold arrows.
The auxiliary amino group is at the remote terminus whereas
the pH-switchable amine is next to the carbonyl group.30 We
were interested in determining whether the presence of a highly
polarized acceptor moiety can differentiate the basicities of the
two amino groups to the extent where distinct protonation states
exist at the biologically relevant pH regions (Figure 4). If this
approach is successful, sequential protonation of these groups
at different pH can be used to control the photophysical
properties of such molecules, their binding to DNA, and
ultimately the efficiency of ds DNA cleavage.
The pH-activated part was combined with light-activated
DNA cleavers in order to take advantage of the high degree of
spatial and temporal controls over reactivity inherent to the
photochemical activation.31 We based the choice of the photo-
chemically activated DNA part on our earlier discovery of the
C1-C5 cyclization of enediynes which results in the net transfer
of four hydrogen atoms to the enediyne moiety.32,33 In particular,
the tetrafluoropyridine (TFP)-substituted enediynes were found
to be both photochemically and thermally stable, unless pho-
toexcitation occurs in the proximity of a suitable electron donor,
such as DNA. We have recently shown that these molecules
are efficient reagents for the double-strand (ds) DNA photo-
cleavage34 and that they are amenable to two-photon activa-
tion.35 We have also shown that the lysine conjugates of
enediynes, as well as related fulvenes and acetylenes, perform
guanosine-specific (oxidative) cleavage at the ends of AT-rich
Results and Discussion
pH-Dependent DNA Cleavage. The ability of lysine conju-
gates to cleave DNA upon irradiation was investigated using
conversion of supercoiled plasmid DNA into the respective
relaxed circular and linear forms (forms II and III). The relative
amounts of the three DNA forms were determined by densito-
metric analysis38 of the gel electrophoresis bands. Although
enhancement of DNA-cleaving ability has been observed for
all lysine conjugates (see Figure 8), we will focus our discussion
below on compound 4.
The most remarkable observation is the dramatic increase in
efficiency of ds DNA cleavage caused by compound 4 when
pH changes from neutral to slightly acidic (Figure 6). While
control experiments clearly indicate that no additional DNA
cleavage is caused at this pH by the conjugate alone in the dark
(36) Interestingly, this selectivity is observed not only for the TFP-
substituted molecules but also for the Ph-substituted enediynes
expected to undergo the photochemical Bergman cyclization. Breiner,
B.; Schlatterer, J. C.; Kovalenko, S. V.; Greenbaum, N. L.; Alabugin,
I. V. Angew. Chem. 2006, 45, 3666.
(37) Breiner, B.; Schlatterer, J. C.; Kovalenko, S. V.; Greenbaum, N. L.;
Alabugin, I. V. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 13016.
(38) The cleavage was quantified by using SAFA software: Laederach,
R. D. A.; Pearlman, S.; Herschlag, D.; Altman, R. RNA 2005, 11,
344.
(34) Kovalenko, S. V.; Alabugin, I. V. Chem. Commun. 2005, 1444.
(35) Kauffman, J. F.; Turner, J. M.; Alabugin, I. V.; Breiner, B.; Kovalenko,
S. V.; Badaeva, E. A.; Masunov, A.; Tretiak, S. J. Phys. Chem. A
2006, 110, 241.
9
J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009 11461

A R T I C L E S
Yang et al.
Figure 6. Plasmid relaxation assays with conjugate 4 (15 µM) and pBR
322 plasmid DNA (30 µM/bp) in 20 mM sodium phosphate buffer after 3
min of irradiation. Form I ) intact supercoiled DNA, form II ) relaxed
form (ss cleavage), form III ) linear form (ds cleavage). Lanes 1-8: from
pH 5.5 to pH 9, lane 9: no compound + UV, lane 10: compound + no UV.
Cleavage data were quantified through densitometry. Reported values
represent the average of four experiments.
or by UV itself, the damage is amplified strongly when both
light and compound 4 interact with DNA at the lower pH. At
pH 6, only 16% of intact DNA (form I) remained after 3 min
of irradiation in contrast to 60% and 86% of unreacted DNA at
pH 7 and 8, respectively. Even more remarkable is that at the
concentrations where hardly any ds cleavage is observed at pH
7, the amount of double-stranded cleavage at the lower pH
reaches the 2:1 ss:ds ratio, which rivals DNA cleavage mediated
by the natural antibiotics calicheamicin γ39 and bleomycin!40
Higher concentrations of 4 lead to as much as 50% of ds
cleavage which, to the best of our knowledge, is unprecedented
for a small molecule-based DNA cleaver (natural or designed).
Interestingly, the dependences of all three forms of DNA
cleavage from pH follow the classic titration curves, which
afford the apparent pKa values of 6.6, 6.8, and 6.2, respectively
for forms I, II, and III (Figure 7).41 All of these values agree
that a significant change in the efficiency of DNA cleavage
occurs at the pH 6-7 window ideal for targeting cancer cells.
Although all lysine conjugates have similar pH-dependent
trends (Figure 8), conjugates 1 and 4 possess the greatest DNA-
cleaving ability. As a result, we focused our mechanistic
discussion on acetylene 4 which is not only less toxic than
enediyne 1 in the dark (vide infra) but also does not aggregate
and precipitate at the higher pH values (∼8). When necessary,
we incorporated data for other conjugates as well.
Figure 7. Percentage of unreacted DNA (form I), single-stranded cleavage
(form II), and double-stranded cleavage (form III) as a function of pH, and
the apparent pKa values corresponding to these plots.
In order to investigate the relative role of the two amino
groups in these conjugates, we extended this study to include
DNA cleavage activities of alanine and caproic acid conjugates
6 and 7. These compounds have only one amino group (at the
R- and ε-carbon, respectively) and, as expected, show different
behavior (Figure 9). Even though the monoamines 6, 7 are
comparable in reactivity to diamine 4 at pH 8, the two
Figure 8. Quantified plasmid relaxation assays with 15 µM of compounds
1, 2, 5 and 30 µM/bp of pBR 322 plasmid DNA in 20 mM sodium phosphate
buffer at pH 6, 7, and 8 after 3 min irradiation.
conjugates lacking one of the two amino groups rapidly fall far
behind in their efficiency at the lower pH values42 and do not
produce any ds cleavage even at pH 6.
The fact that only the lysine conjugates produce clear ds
cleavage under these conditions suggests that both amino groups
are required for this process to occur. Several explanations are
possible. For example, the remarkable ability of lysine to
recognize a nick in DNA and facilitate a break of the
complementary strand (the ssfds cleavage conversion) discov-
ered in our previous work37 may play some role in the high
(39) The ds/ss ratio is 1:2 for plasmid DNA and 1:3 for cellular DNA:
Elmroth, K.; Nygren, J.; Martensson, S.; Ismail, I. H.; Hammarsten,
O. DNA Repair 2003, 2, 363.
(40) About 10% of the DNA strand breaks induced by bleomycin are
double-strand breaks: Charles, K.; Povirk, L. F. Chem. Res. Toxicol.
1998, 11, 1580. Povirk, L. F.; Wubker, W.; Kohnlein, W.; Hutchinson,
F. Nucleic Acids Res. 1977, 5, 3573. See also Lloyd, R. S.; Haidle,
C. W.; Hewitt, R. R. Cancer Res. 1978, 38, 3191. Harsch, A.; Marzilli,
L. A.; Bunt, R. C.; Stubbe, J.; Vouros, P. Nucleic Acids Res. 2000,
28, 1978. Stubbe, J.; Kozarich, J. W. Chem. ReV. 1987, 87, 1107.
Burger, R. M. Chem. ReV. 1998, 98, 1153. Quada, J. C., Jr.; Zuber,
G. F.; Hecht, S. M. Pure Appl. Chem. 1998, 70, 307. Hecht, S. M. J.
Nat. Prod. 2000, 63, 158.
(42) Interestingly, monoamines possess a certain (but lesser) degree of pH
dependency and presence of an R-amine in 6 has a slightly larger
effect than the presence of an ε-amine in 7.
(41) Value for the ds cleavage (form III) is less accurate because of the
greater experimental error bars.
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11462 J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009

C-Lysine Conjugates
A R T I C L E S
Another possibility is illustrated by a model for stronger
binding of the diprotonated lysine moiety to DNA at lower pH,43
as shown in Figure 11b. At neutral pH, only the more basic,
remote amino group of the lysine moiety is protonated, leading
to a relatively loose binding to DNA. At lower pH, the
electrostatic interaction of positively charged ammonium groups
and negatively charged phosphate groups of DNA brings the
DNA-cleaving warhead close to the ribose backbone of DNA,
providing a possible mechanism for the increase in the extent
of DNA damage by this warhead.
Finally, we envisioned that a conformational change may
occur upon the second protonation (Figure 11c). Although it
was not clear a priori whether the coiled conformation may gain
sufficient importance in the strongly hydrogen bonding aqueous
environment, it is interesting to consider such possibility as a
protonation-induced shape change may lead to a change in the
DNA binding mode.
Photophysical properties of lysine conjugates with acceptor
substituents will help us to determine whether the choice of
photochemical DNA cleavers activated through photoinduced
electron transfer (PET) introduces a new component in the pH-
controlled behavior (Figure 11a). NMR analysis of different
protonated species will elucidate the possibility of conformation
change in Figure 11c whereas pH dependence in DNA binding
will help to determine whether tighter binding is achieved for
dications as illustrated in Figure 11b.
However, before comparing the relative importance in the
three possible pathways for the observed pH dependence of
DNA cleavage for the lysine conjugates, it is important to obtain
reliable answers regarding the basicities of the two amino groups
in the lysine conjugates. Is the presence of an acceptor moiety
next to the R-amino group and another already protonated amino
group sufficient to decrease the basicity of the R-amino group
to the extent where the change in its protonation will occur in
the vicinity of pH 6-7? If yes, how are these changes in the
protonation state transferred in the changes in the efficiency of
DNA binding? In order to address these questions, we proceeded
to determine pKas for the two amino groups of the lysine moiety
as well as binding affinities of the different protonated states to
DNA.
Figure 9. Plasmid relaxation assays for DNA photocleavage with 15 µM
of compounds 4, 6, and 7 and pBR322 plasmid DNA (30 µM/bp) in 20
mM sodium phosphate buffer (pH 6, 7, 8) after 10 min of UV irradiation.
Quantified cleavage data are presented on the top, and the original gels are
on the bottom.
efficiency of the ds cleavage. However, the enhancement of this
type of cleavage at the lower pH is an unprecedented phenom-
enon. This effect is likely to stem from the presence of a
protonated R-amino group in compound 4 which enhances the
efficiency of DNA cleavage through one of the three mecha-
nisms outlined in Figure 11 (vide infra).
The difference in kinetics of DNA photocleavage by conju-
gate 4 at different pH values provides another illustration of
the remarkable degree of pH control (Figure 10). DNA cleavage
at pH 6 is remarkably fast: plasmid DNA is completely
consumed within 5 min of irradiation, even though control
experiments clearly show that there is very little of DNA damage
by UV itself under these conditions. In a sharp contrast, DNA
cleavage at pH 8 is slow: even after 1 h of irradiation, ∼60%
of DNA remains unreacted (note, however, that this reaction is
still considerably faster than the damage by UV itself shown at
the bottom part of Figure 10). These observations indicate that,
depending on the pH, compound 4 can induce DNA damage in
two kinetically different ways. The DNA-damaging activity at
pH 7 can be described as a superposition of the two different
cleavage patterns described above. These experiments also
provide the control data to confirm that change in the pH per
se does not induce acceleration of DNA photocleavage (lower
part of Figure 10). Instead, the observed pH-dependence stems
from the properties of lysine conjugates.
Determination of pKa Values. Direct potentiometric pH
determination of both pKa values is inaccurate for most of the
conjugates because, at a pH where both ammonium groups are
deprotonated and these molecules lose all their charge, the
solubility in water becomes very low (<1 mM). The decrease
in solubility leads to the apparent aggregation and distortion of
potentiometric curves at the higher pH. This distortion prevents
accurate curve-fitting (see Figure S1, Supporting Information).
Thus, we had to resort to alternative spectral methods. Fortu-
nately, there is a selection of convenient procedures which
provide complementary information which can be used for
cross-checking the accuracy of the alternative approaches.
NMR Titrations. The advantage of NMR titrations44 is that
they provide direct structural information, allowing differentia-
tion of the two protonation events through observations of
Mechanistic Alternatives for the Observed pH-Dependence
of DNA Cleavage. Several possible mechanisms which could
lead to the observed pH dependency for the interaction of lysine
conjugates with DNA are outlined in Figure 11. Mechanism
(a) involves the control of deactivating intramolecular electron
transfer from the donor R-amino group to the excited chro-
mophore through protonation. Electron transfer intercepts the
excited state of the DNA cleaver, making it less active at higher
pH. In contrast, at lower pH, both amino groups are protonated
and the nitrogen lone pair is not available for deactivating the
reactive excited state.
(43) Standke, K. H. C.; Brunnert, H. Nucleic Acids Res. 1975, 2, 1839.
Ahmad, J.; Ashok, B. T.; Ali, R. Immunol. Lett. 1998, 62, 87. Tyler,
J. K.; Allen, K. E.; Everett, R. D. Nucleic Acids Res. 1994, 22, 270.
(44) Gao, G.; DeRose, E. F.; Kirby, T. W.; London, R. E. Biochemistry
2006, 45, 1785. Kakehashi, R.; Shizuma, M.; Yamamura, S.; Maeda,
H. J. Colloid Interface Sci. 2005, 289, 498. Rush, J. R.; Sandstrom,
S. L.; Yang, J.; Davis, R.; Prakash, O.; Baures, P. W. Org. Lett. 2005,
7, 135.
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A R T I C L E S
Yang et al.
Figure 10. Quantified plots of pBR 322 plasmid (30 µM/bp) relaxation assays for 10 µM of compound 4 in 20 mM sodium phosphate buffer at pH 6 (a),
7 (b), and 8 (c) as a function of the irradiation time. Plots on the bottom show the effect of UV on the DNA without the conjugate at different pH. Plots on
the top illustrate reactivity in the presence of compound 4.
Figure 12. 1H NMR spectra of R- and ε-hydrogens in compound 8 (5
mM) in D2O at pD 5, 6, 7, 8, 9, 10, 11, 12, 13, and chemical shift-pD
titration data for R- and ε-hydrogens in compound 8. These pKa values
change to 7.44 and 10.40 when correction for going from pKaH* to pKa is
applied.
of the lysine chain protons upon the protonation/deprotonation
of the two amino groups during the pH (pD) titration. The
titrations curves clearly showed that R-amino group is less basic
(pKa ) 7.56) than ε-amino group (pKa ) 10.75). To allow
comparison of these pKas with pKas obtained by other methods,
one has to take into account the difference between pKaH*, which
is obtained by a direct reading of the H2O-calibrated pH meter
in a D2O solution, and pKa (pKa ) 0.929pKaH* + 0.42).46
Figure 11. Three possible mechanisms for pH-regulated DNA modification
Interestingly, monitoring of protons on the ꢀ-, γ-, and
δ-carbons produced two protonation curves with their relative
sensitivity being directly proportional to the proximity to the
by lysine conjugates.
chemical shifts for hydrogens spatially close to one of the two
amino groups.45 Such differentiation can confirm unambiguously
whether the presence of a carbonyl group next to the amino
group leads to decrease in the pKa of the respective ammonium
ion.
Favorable solubility of N-phenyl amide 8 in D2O rendered
this molecule a particularly convenient substrate for the 1H NMR
studies. These studies followed changes in the chemical shifts
(45) As the control experiment, we tested two pKa values of ethylene
diamine with NMR titration. After correcting measured pKa values
with the equation (ref 42), the values of 7.03 and 9.99 are obtained
experimentally (R2 ) 0.999). These data are in good agreement with
the corresponding potentiometric literature values of 7.19 and 9.98.
Avdeef, A.; Kearney, D. L.; Brown, J. A.; Chemotti, A. R. Anal. Chem.
1982, 54, 2322.
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11464 J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009

C-Lysine Conjugates
A R T I C L E S
Figure 14. The pH-dependent lifetimes of the singlet excited state for
conjugates 4 and 6 (10 µM), measured in 20 mM sodium phosphate buffer.
Table 1. The pKa Values of Amino Groups in Lysine Conjugates 4,
8, and Related Monoamines 6, 7 According to Emission/
Absorption and NMR Spectra
compound
4
6
7
8
lysine
Figure 13. Fluorescence spectra of compound 4 (10 µM) at pH 4.1, 5.0,
6.1, 6.5, 6.9, 7.2, 8.0, 9.0, and 9.5. Insert: Quantified changes in fluorescence
intensity as a function of pH and their fit to the Henderson-Hasselbalch
equation for an acid-base equilibrium involving one amino group.
method emission/
absorption
pKa1
emission/
absorption
7.6/7.4
emission/
absorption
10.8/9.4
1H NMR
-
a
b
6.98/6.9
-/9.5
7.56 (7.44)
10.75 (10.40) 10.53
8.95
b
a
pKa2
-
-
a Data in parentheses include a correction factor from ref 46. b Data
from ref 49.
respective amino groups. The observation that R-hydrogens are
not sensitive to the change in protonation of the ε-amino group
suggests a lack of communication between the two amino
groups, thus eliminating the cyclic H-bonded structure in the
monoprotonated species where the ammonium group is hydro-
gen bonded to the other NH2 group of the same molecule (the
central structure in Figure 4 which corresponds to mechanism
(c) of pH regulation in Figure 11).47
Fluorometric Determination of pKa of the r-Amino Group.
Highly sensitive fluorescence methods are especially convenient
for compounds of limited solubility. We determined the basicity
of the R-amino group using intramolecular quenching of
fluorescence through photoinduced electron transfer (PET) from
the nitrogen lone pair to the excited chromophore. At the lower
pH, amino groups are protonated and the lone pair of electrons
at nitrogen is unavailable for inter- or intramolecular electron
transfer. However, as the ammonium group adjacent to the
chromophore loses the proton at the higher pH, electron transfer
becomes possible and quenches the fluorescence (Figure 13).
Although the validity of this approach is based on the assump-
tion that changes in intermolecular PET and other fluorescence
quenching pathways with the pH are insignificant relative to
intramolecular PET at the 10-5 M concentration, the advantage
of fluorescence is that it is more sensitive to changes in the
immediate vicinity of the chromophore and, thus, directly reports
protonation of the R-amino group.
state lifetimes for compounds 4 and 6 as a function of pH.
Within the error of measurement, all TFP-substituted compounds
show a clear decrease in the lifetime of the singlet excited state
at the higher pH. An illustration of the pH dependence for the
singlet excited state lifetimes of compound 4 and 6 is provided
in Figure 14.
The observed pH dependency in the lifetimes means that the
S1 state is less available for triggering DNA cleavage at the
higher pH, a factor which should contribute to the observed
decrease in activity under these conditions. At the lower pH,
both amino groups are protonated, and hence electron transfer
from the nitrogen lone pair does not deactivate the excited state,
exactly as it is described in Figure 11a.
UV-Absorption Titrations. Similar pKa values can be obtained
using pH-dependent changes in the UV-vis absorption spectra
of the conjugates. This method is less sensitive, as the changes
observed are much smaller (about 10% increase in absorption
upon transition from pH 11 to 5.5) than the analogous changes
in the fluorescence spectra discussed in the previous section.
In contrast to fluorescence, there are two titration curves for
compound 4 which correspond to absorption changes of almost
equal magnitude (Figure S4, Supporting Information). The pKa
for the first of these curves corresponds exactly to the pKa
obtained from the fluorescence titrations. Interestingly, pKa for
the ε-ammonium group of compound 7 is >1 pKa units lower
than that derived from the fluorescence changes. Although the
reasons for this interesting behavior of the terminal amino group
extend beyond the scope of this paper,48 this subtle difference
is irrelevant at the biologically relevant pH region where we
observe the enhancement of ds-DNA cleavage. It is clear that,
independent of the method, the pKa of the auxiliary ammonium
group is sufficiently high to allow more than 99% of the
respective amine to be protonated even at neutral pH. The above
Lifetimes. To gain further insight into the effect of protonation
on the excited state properties, we measured the singlet excited
(46) pKa values measured with pH* are assumed to be similar to the
corresponding values in H2O: Primrose, W. U. NMR of Macromol-
ecules. A Practical Approach; Poberts, G. C. K., Ed.; Oxford
University Press: Oxford, 1993; pp 22-23. Scheller, K. H.; Scheller-
Krattiger, V.; Martin, R. B. J. Am. Chem. Soc. 1981, 103, 6833.
Alternatively, one can use a formula for correlating pKa values
determined in D2O and H2O: Krezel, A.; Bal, Wojciech, A. J. Inorg.
Biochem. 2004, 98, 161.
(47) Another interesting observation is that chemical shifts of aromatic
hydrogens show small but measurable response to the changes in
pH in the vicinity of the pH 10 region (see the Supporting
Information), suggesting that there may some contribution from
cation-π interaction. Gallivan, J. P.; Dougherty, D. A. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 9459. Ma, J. C.; Dougherty, D. A. Chem.
ReV. 1997, 97, 1303. Mercozzi, S.; West, A. P., Jr.; Dougherty, D. A.
J. Am. Chem. Soc. 1996, 118, 2307. Dougherty, D. A. Science 1996,
271, 163.
(48) A possible explanation is that fluorescence indicates properties of an
excited molecule, whereas absorption indicates a ground-state coun-
terpart. Acidities of ground and excited states are often different: Tolbert,
L. M.; Solntsev, K. M. Acc. Chem. Res. 2002, 35, 19. The validity of
this explanation depends on the relative rates of protonation/depro-
tonation and the key photophysical events. This complicated question
is outside the scope of this paper.
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A R T I C L E S
Yang et al.
Table 2. NBO Analysis of Electronic Properties of R-and ε-Nitrogen Lone Pairs of Diamines 4 and 8 at the B3LYP/6-31G(d,p) Level of
Theory with the Polarizable Continuum Model (PCM)52 for Solvation in H2O
neutral
protonated at the ε-nitrogen atom
compound 4
occupancy, e
energy, a.u.
hybrid character, n in spn orbital
occupancy, e
energy, a.u.
hybrid character, n in spn orbital
R
1.92791
1.96564
occupancy, e
1.92941
1.96555
-0.30618
-0.29939
energy, a.u.
-0.30457
-0.29893
4.42
3.50
1.92765
-
occupancy, e
1.92906
-
-0.30724
-
energy, a.u.
-0.30557
-
4.49
ε
-
compound 8
R
ε
hybrid character, n in spn orbital
hybrid character, n in spn orbital
4.46
4.39
3.52
-
experiments do confirm unequivocally that the two amino groups
of lysine conjugates possess different basicities and that the less
basic R-group has the right properties to serve as a pH trigger
for selective targeting of cancer cells.
pH-Dependent DNA Binding. From a practical perspective,
the pKa value of 7.0 for the TFP-acetylene 4 suggests that this
compound exists mostly as the monocation at the slightly basic
pH, and it should be converted into >90% of dication at the
same pH region (pH < 6) where we observe the amplification
of the ds DNA cleavage. The following section concentrates
on interaction (binding) of DNA and lysine conjugates in the
different protonation states. We approached this question through
a combination of several methods outlined below. Note that
qualitative treatments of binding affinities in these systems are
difficult due to the presence of several species with different
spectroscopic properties (the three protonation states of di-
amines) and inherent complexity of calf thymus (CT) DNA
which offers numerous types of binding environments to the
DNA cleaver. As the result, we will limit ourselves in providing
sufficient information for illustrating the differences in binding
at the semiquantitative basis.
Summary of Experimental pKa Trends. The results for
conjugates 4, 6, 7, and 8 are summarized in Table 1. The R-pKa
for the lysine conjugates 4 is ∼0.5 units lower than the pKa for
the analogous alanine conjugate 6. This difference indicates that,
as expected, the presence of a second cationic moiety does play
some role in the decrease of R-amino moiety basicity. However,
the relatively close pKa values for the R-ammonium groups in
compounds 4 and 6 together with their large deviation from
the pKa values for the ε-ammonium groups in compounds 4
and 7 illustrate that the lowered basicity of the R-amino group
in lysine is mostly due to its proximity to the amide moiety. It
is also interesting that the presence of the acceptor tetrafluo-
ropyridinyl moiety in 4 leads to a noticeable (∼0.5-0.6 pKa
units) decrease in the basicity of the R-amino group relative to
that in a simple phenyl conjugate 8.
Absorbance and Fluorescence Titration. Titrations of 10 µM
solutions of compounds 1-5 with calf thymus (CT) DNA
Computational Analysis of pKa Trends. In order to provide
a theoretical rationale to the observed differences in basicity,
we investigated electronic properties of the two amino groups
in diamines 4 and 8. Natural Bond Orbital (NBO)50 analysis of
electronic structure of these compounds at the B3LYP/6-
31G(d,p) level clearly shows that the R-nitrogen lone pair is
lower in energy and has a significantly lower population than
the ε-nitrogen lone pair (see Table 2 and the Supporting
Information). Both of these factors render the R-nitrogen a less
efficient donor28,51 and a weaker base than the ε-nitrogen.
Proton affinities calculated at the B3LYP/6-31G(d,p) level
in water (Figure 15) support the above experimental findings
very well. For both compounds 4 and 8, proton affinity of the
ε-nitrogen is significantly higher than that of the R-nitrogen. In
agreement with the experimental pKa values for monoamines 6
and 7 relative to the two pKa values of the diamines 4 and 8,
the first protonation has some effect on the basicity of the second
amino group, but the effect is not dramatic (1.2-1.5 kcal/mol
difference in the proton affinity values). In addition, and again
in a full agreement with the experiment, the R-amino group in
compound 8 is noticeably more basic than the R-amino group
in compound 4 (1.2 kcal/mol difference).
Figure 15. Proton affinities (in kcal/mol) of R- and ε-nitrogen lone pairs
of diamines 4 and 8 at the B3LYP/6-31G(d,p) level of theory with PCM
solvation in H2O.
Figure 16. UV-vis absorbance titrations of 4 (10 µM) in 20 mM sodium
phosphate buffer with 0, 1, 2, 3 equiv of CT DNA (bp) at pH 5.5 (a), pH
7 (b), and pH 8 (c).
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11466 J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009

C-Lysine Conjugates
A R T I C L E S
Figure 17. Emission titrations of 4 (10 µM) in 20 mM sodium phosphate buffer with 0, 10, 20, 30 µM/bp of CT DNA at pH 5.5 (a), pH 7 (b), and pH 8
(c). The initial slopes of plots illustrating changes in fluorescence of 4 as a function of DNA concentration (d).
revealed a significant difference in absorbance and emission
changes at different pH values. Results for compound 4 are
discussed in more detail in this section. While little or no shift
of λmax and small hypochromicity (14%, 12%, respectively) are
observed for the titrations of conjugate chromophore with DNA
at pH 7 and 8, the λmax changes from 310 to 315 nm during the
titrations with the same amounts of DNA at pH 5.5 and 20%
hypochromicity is observed. The greater change of absorbance
at pH 5.5 indicates the larger interaction between the chro-
mophore and DNA.
Changes in fluorescence upon the DNA titrations followed a
similar pattern where greater changes were observed at the lower
pH, indicating a stronger binding affinity to DNA. Interestingly,
while emission of TFP-enediyne-lysine conjugate 1 was
quenched with DNA (Figure S6, Supporting Infromation), most
likely through an electron transfer pathway,53 we observed an
increase in fluorescence for compound 4. This finding is likely
to indicate that the DNA-binding modes for these two com-
pounds are different. In particular, the fluorescence increase
displayed by compound 4 suggests that it may be a DNA groove
binder.54 Although fluorescence increase is often observed for
Figure 18. Changes in fluorescence of ethidium bromide (10 µM, the
excitation wavelength ) 535 nm) in CT DNA (10 µM/bp) as a function of
displacement by compound 1.
intercalated chromophores as well,55 intercalation is unlikely
in this system because close proximity to the nucleobases should
quench fluorescence of 4 through electron transfer.
Further evidence for a significant difference in binding modes
of enediyne 1 and acetylene 4 is provided by the ethidium
bromide displacement assays (Figure 18). Only compound 1
displaces intercalated ethidium bromide from its complex with
DNA, whereas addition of compound 4 does not lead to the
displacement. This observation indicates again that compound
4 is not an intercalator. Nevertheless, despite the above
differences in the DNA-binding mode, both lysine conjugates
display similar pH dependence in their DNA cleavage.
Overall, both compounds show a much greater spectral
response at the lower pH. As pH increases, the changes become
(49) Carey, F. A. Organic Chemistry, 6th ed.; McGraw Hill: New York,
2006.
(50) Reed, A. E.; Curtiss, L. A.; Weinhold, F. Chem. ReV. 1988, 88, 899.
(51) Alabugin, I. V.; Manoharan, M.; Zeidan, T. A. J. Am. Chem. Soc.
2003, 125, 14014. Alabugin, I. V.; Manoharan, M. J. Comp. Chem.
2007, 28, 373.
(52) (a) Miertus, S.; Tomasi, J. Chem. Phys. 1982, 65, 239. (b) Miertus,
S.; Scrocco, E.; Tomasi, J. Chem. Phys. 1981, 55, 117.
(53) Both singlet and triplet excited states of DNA are too high in energy
to allow for efficient quenching by energy transfer.
(54) For major groove binders: Kashanian, S.; Gholivand, M. B.; Ahmadi,
F.; Taravati, A.; Colagar, A. Spectrochim. Acta, Part A 2007, 67A,
472. Vijayalakshmi, R.; Kanthimathi, M.; Subramanian, V.; Nair, B. U.
Biochim. Biophys. Acta 2000, 1475, 157. Tysoe, S. A.; Morgan, R. J.;
Baker, A. D.; Strekas, T. C. J. Phys. Chem. 1993, 97, 1707. For minor
groove binders: Karlsson, H. J.; Eriksson, M.; Perzon, E.; Akerman,
B.; Lincoln, P.; Westman, G. Nucleic Acids Res. 2003, 31, 6227.
Tawar, U.; Jain, A. K.; Chandra, R.; Singh, Y.; Dwarakanath, B. S.;
Chaudhury, N. K.; Good, L.; Tandon, V. Biochemistry 2003, 42,
13339. Adhikary, A.; Buschmann, V.; Mueller, C.; Sauer, M. Nucleic
Acids Res. 2003, 31, 2178. Rucker, V. C.; Foister, S.; Melander, C.;
Dervan, P. B. J. Am. Chem. Soc. 2003, 125, 1195. Baliga, R.; Crothers,
D. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7814. Kumar, C. V.;
Turner, R. S.; Asuncion, E. H. J. Photochem. Photobiol. A 1993, 74,
231.
(55) Li, J.; Wei, Y.-X.; Wei, Y.-L.; Dong, C. J. Lumin. 2007, 124, 143.
Melvin, M. S.; Ferguson, D. C.; Lindquist, N.; Manderville, R. A. J.
Org. Chem. 1999, 64, 6861. Spillane, C. B.; Dabo, M. N. V.; Fletcher,
N. C.; Morgan, J. L.; Keene, F. R.; Haq, I.; Buurma, N. J. J. Inorg.
Biochem. 2008, 102, 673. Fuerstenberg, A.; Deligeorgiev, T. G.;
Gadjev, N. I.; Vasilev, A. A.; Vauthey, E. Chem.sEur. J. 2007, 13,
8600. Bhadra, K.; Maiti, M.; Kumar, G. S. Biochim. Biophys. Acta
2007, 1770, 1071. Erve, A.; Saoudi, Y.; Thirot, S.; Guetta-Landras,
C.; Florent, J.-C.; Nguyen, C.-H.; Grierson, D. S.; Popov, A. V. Nucleic
Acids Res. 2006, 34, e43/1. Marinic, M.; Piantanida, I.; Rusak, G.;
Zinic, M. J. Inorg. Biochem. 2006, 100, 288. Zipper, H.; Brunner,
H.; Bernhagen, J.; Vitzthum, F. Nucleic Acids Res. 2004, 32, e103/1.
Piantanida, I.; Palm, B. S.; Zinic, M.; Schneider, H.-J. J. Chem. Soc.,
Perkin Trans. 2 2001, 9, 1808.
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A R T I C L E S
Yang et al.
Figure 19. Summary of possible mechanistic alternatives for the observed DNA cleavage by amino acid/acetylene conjugates.
less pronounced, indicating a lower degree of interaction
between the compounds and DNA at the higher pH. This trend
was also shown in measurement of binding affinity of compound
4 to CT DNA by isothermal titration calorimetry (ITC; see the
Supporting Information for details). These observations are
consistent with the protonation behavior of these diamines
discussed in the previous sections. At the lower pH, both amino
groups are protonated to provide a higher degree of attraction
between the two positive charges of the ammonium groups and
the negative charges of the DNA phosphate backbone.
Mechanistic Considerations and pH Dependence of the
Involvement of Diffusing Radicals in the DNA Cleavage. The
potential complexity of chemical mechanisms which can be
involved in DNA photocleavage by lysine acetylene conjugates
is illustrated in Figure 19. A priori, a number of mechanisms
may be considered as an explanation for the observed photo-
chemical DNA cleavage by compound 4.56 In the first step,
photochemical excitation leads to the formation of an acetylene
excited singlet state. After this necessary step, the list of
diverging possibilities includes a number of choices such as
generation of reactive oxygen species like singlet oxygen,
superoxide, and OH radical;57 direct hydrogen abstraction from
the DNA sugar backbone by photogenerated radicals, or singlet
and triplet excited states;58 oxidative damage due to electron
transfer from DNA nucleobases;59 and photoinduced DNA
alkylation.60
and G selectivity for the formation of reactive species through
PET.36 Both the selectivity and the fact that the lesions in DNA
oligomers required piperidine treatment clearly indicated that
PET is involved in the damage. However, purely oxidative
damage could not be the only mechanism because several
compounds including 4 caused noticeable damage at a single
G inside of a 12-bp AT tract. This observation suggested that
these photocleavers may damage DNA through base alkylation
as well. Subsequent work confirmed that compound 4 is capable
of localized DNA cleavage, and the damage does not dissipate
through hole-hopping when the same single G site is chosen as
a target using phosphate-lysine recognition.37 Furthermore, we
also observed frank cleavage in 5′-(AATT)nGG(AT)mG-
G(AAATTT)l-3′ oligomers. The cleavage is amplified by heat
which is indicative of either base pair alkylation or abstraction
of a C1′-hydrogen by reactive radical species.40 We have
confirmed that triplet excited states of TFP-acetylenes behave
as strongly electrophilic radicals which can alkylate π-systems.61
Taken together, these observations suggest base pair alkylation
is likely to complement direct PET damage as an important
mechanism for the DNA damage, especially at the lower pH
and tighter binding.
Further mechanistic details are unclear at the moment, and a
number of possible scenarios can be suggested. In particular,
intersystem crossing (ISC) may convert this excited state into
a triplet state of the TFP-acetylene moiety, shown to behave
as a highly electrophilic diradical.61 In addition, the triplet state
of the photocleaver may sensitize the formation of singlet
oxygen. Alternatively, PET from DNA can lead to the formation
of acetylene radical-anion and electron hole at one of the DNA
bases. Electron transfer from acetylene radical-anion to dissolved
oxygen would form superoxide anion which can be transformed
We have shown previously that cleavage by the enediyne
and acetylene derivatives discussed in the work proceeds with
selectivity for guanine-rich parts of DNA which flank AT tracts
because of a compromise between AT selectivity for binding
(56) Saito, I.; Nakatani, K. Bull. Chem. Soc. Jpn. 1996, 69, 3007. Hiraku,
Y.; Ito, K.; Hirakawa, K.; Kawanishi, S. Photochem. Photobiol. 2007,
83, 205.
(60) Nielsen, P. E.; Jeepsen, C.; Egholm, M.; Buchardt, O. Nucleic Acids
Res. 1988, 16, 3877. Chatterjee, M.; Rokita, S. E. J. Am. Chem. Soc.
1990, 112, 6397. Henriksen, U.; Larsen, C.; Karup, G.; Jeepsen, C.;
Nielsen, P. E.; Buchardt, O. Photochem. Photobiol. 1991, 53, 299.
Chatterjee, M.; Rokita, S. E. J. Am. Chem. Soc. 1994, 116, 1690. Saito,
I.; Takayama, M.; Sakurai, T. J. Am. Chem. Soc. 1994, 116, 2653.
Nakatani, K.; Shirai, J.; Tamaki, R.; Saito, I. Tetrahedron Lett. 1995,
36, 5363.
(57) Devasagayam, T. P. A.; Steenken, S.; Obendorf, M. S. W.; Schulz,
W. A.; Sies, H. Biochemistry 1991, 30, 6283.
(58) Saito, I. Pure Appl. Chem. 1992, 64, 1305. Quadda, J. C.; Levy, M. J.;
Hecht, S. M. J. Am. Chem. Soc. 1993, 115, 12171.
(59) Steenken, S. Chem. ReV. 1989, 89, 503. Dunn, D. A.; Lin, V. H.;
Kochevar, I. E. Biochemistry 1992, 31, 11620. Sugiyama, H.;
Tsutsumi, K.; Fujimoto, K.; Saito, I. J. Am. Chem. Soc. 1993, 115,
4443. Cadet, J.; Berger, M.; Bunchko, G. W.; Joshi, R. C.; Raoul, S.;
Ravanet, J.-L. J. Am. Chem. Soc. 1994, 116, 7403. Armitage, B.;
Changjun, Y.; Devadoss, C.; Schuster, G. B. J. Am. Chem. Soc. 1994,
116, 9847.
(61) Zeidan, T.; Kovalenko, S. V.; Manoharan, M.; Clark, R. J.; Ghiviriga,
I.; Alabugin, I. V. J. Am. Chem. Soc. 2005, 127, 4270. Zeidan, T.;
Clark, R. J.; Kovalenko, S. V.; Ghiviriga, I.; Alabugin, I. V.
Chem.sEur. J. 2005, 11, 4953.
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C-Lysine Conjugates
A R T I C L E S
Figure 20. Effect of hydroxyl radical/singlet oxygen scavengers (10 mM) upon the efficiency of DNA cleavage at pH 6, 7, and 8 by conjugates 1 (a) and
4 (b) after 5 min irradiation. Color coding: light blue, form I; dark purple, form II; light yellow, form III.
into other reactive oxygen species. Depending on the relative
rates of hole-hopping and hole-trapping, the DNA electron hole
can either be trapped at a suitable location to produce an
oxidized DNA base (likely, a guanine) or transfer a proton to
the DNA-cleaver radical-anion. This proton transfer (which can
be classified as proton-assisted electron transfer)62 would
transform the charge separated ion-pair into two new radicals,
one at DNA and another at the partially reduced triple bond.
The latter vinyl radical can be also formed when the intermediate
radical anion of the DNA cleaver receives a proton (possibly
through a solvent-mediated mechanism) from the adjacent
Figure 21. Quantified cleavage data of pBR 322 plasmid (30 µM/bp)
R-ammonium group (Figure 19). Independent of the formation
pathway, this vinyl radical can either abstract hydrogen from
the deoxyribose backbone or alkylate an adjacent base.
In order to get further insight into the mechanism of the DNA
relaxation assays of compound 4 (10 µM) at different concentrations of
sodium phosphate buffer at pH 5.5, 7, and 8. Color coding: light blue, form
I; dark purple, form II; light yellow, form III.
cleavage by the conjugates, we used the plasmid relaxation
assays for the cleavage with enediyne conjugate 1 and
TFP-acetylene conjugate 4 in the presence of hydroxyl radical
total DNA cleavage activity of compound 4 by 48%, 40%, 34%
at pH 5.5, 7, and 8, respectively.63 This result suggests that the
electrostatic interaction between the positive charges of am-
(glycerol, DMSO) and singlet oxygen (NaN3) scavengers. The
monium group of compound 4 and the negative charges of DNA
results are summarized in Figure 20.
backbone is an important factor for the cleavage. Again, this
For compound 1, a small protecting effect of the hydroxyl
observation indicates relatively close binding between the DNA
radical scavengers is observed at all pH values. The protecting
cleaver and its target which should be particularly important
effect seems to be slightly lower at pH 6 than at the higher pH
for the DNA-cleavage mechanisms based on alkylation and
values. It is noteworthy, however, that the ds cleavage at pH 6
hydrogen abstraction. In contrast, the cleavage at pH 8 has
is not affected by any of the additives. Singlet oxygen scavengers
greater contributions from processes not affected strongly by
the ionic strength, such as the generation of diffusing reactive
oxygen species.
show no effect upon the efficiency of DNA damage by
compound 1 at pH 6 and 7.
Remarkably, neither of the additives is able to protect DNA
from damage by acetylene-lysine conjugate 4 at pH 6. Some
protecting effect is observed only at the higher pH. These results
clearly indicate that reactive oxygen species are not involved
in the mechanism of DNA cleavage at the lower pH, perhaps
due to the tighter binding under these conditions. This observa-
tion provides additional support for a direct mechanism of DNA
damage such as base pair alkylation. From a practical perspec-
tive, this is valuable not only because many solid tumors are
hypoxic but also because cleavage which does not depend on
diffusing species is more localized and efficient.
Cancer Cell Proliferation Assays. The ability of compounds
1-5 to inhibit cell proliferation in three human clear cell renal
cell carcinoma (RCC) cell lines was tested in the dark and under
photoactivation (Figure 22). No difference was detected in the
control experiments between cells not exposed to UV versus
those that were exposed to UV at 350 nm for 10 min in any of
the three RCC cell lines tested (Figure 22a-c; first two
columns). On the other hand, all five compounds inhibited
growth in each of the three cell lines but to varying degrees
both in the dark and after UV radiation. Although UV treatment
always enhanced the ability of conjugates 1- 3 to inhibit cell
proliferation, these compounds had strong growth inhibitory
activity even without photoactivation in all three cell lines
The final insight on the nature of lysine conjugates/DNA
interaction came from the effect of ionic strength on the
efficiency of DNA cleavage (Figure 21). Increasing concentra-
tion of phosphate buffer from 10 mM to 50 mM decreases the
(62) Reece, S. Y.; Hodgkiss, J. M.; Stubbe, J.; Nocera, D. G. Phil. Trans.
R. Soc. B 2006, 361, 1351.
(63) The total percentage of cleavage for each pH was calculated as (2 ×
[form III] + [form II])/([form III] + [form II] + [form I]).
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A R T I C L E S
Yang et al.
Figure 22. Cell proliferation assays using three different RCC cell lines: (a) UMRC3, (b) UMRC6, and (c) 786-O, and compounds 1-5. RCC cells were
resuspended in media and treated with UV radiation (350 nm) for 10 min with and without indicated compounds. Control cells were exposed to the indicated
compound 1-5 at final concentration of 10 µM. Cell numbers determined after 72 h as described in the Supporting Information.
(Figure 22a-c) with a range of 62-94% inhibition.64 On the
other hand, compounds 4 and 5 were considerably less cytotoxic
in the dark. Compound 4 seems to be an especially promising
lead for the future development of anticancer agents because
the decrease of cell numbers in control UV-irradiated cells as
compared to control without UV irradiation in UMRC3 and
UMRC6 cells was not statistically significant (Figure 22a,b),
but when exposed to UV radiation and the conjugate, these cells
displayed mortality of 94% in UMRC3 and 90% in UMRC6
cells.
Encouraged by these results, we studied the concentration
effects of compound 4 at the proliferation of in LNCaP human
Figure 23. LNCaP cell proliferation assays with varying concentrations
of compound 4. The cells were exposed to UV for 10 min. Cells were
prostate adenocarcinoma cells in the dark and under photoac-
tivation conditions in more detail (Figure 23). Remarkably, even
at the low 10 nM concentration, this system destroys more than
90% of LNCaP cells in one treatment whereas almost no toxicity
is observed without light.
counted 48 h after the exposure. Full experimental details are described in
the Supporting Information.
way and attack DNA through different chemical mechanisms.
Cleavage at the lower pH for the conjugate 4 is not affected by
the reactive oxygen species scavengers, suggesting possible
value of this DNA cleaver for targeting hypoxic cancer tissues.
Cancer cell proliferation assays further confirm remarkable
potential of these molecules for the development of light-
activated anticancer agents.
Conclusion
Hybrid molecules which combine pH-regulated lysine moiety
with a powerful DNA photocleaver display dramatically in-
creased DNA-cleaving reactivity at the lower pH. Synergistic
effects associated with the protonation of the R-amino group,
such as pH-dependent changes in photophysical properties as
well as in pH-dependent DNA binding (Figure 11), account for
the increase in reactivity. Deactivation of the internal quenching
pathway (Figure 11a) increases the lifetime of reactive singlet
state while transformation of the conjugates into their dicationic
form (Figure 11b) changes their binding mode with the
polyanionic backbone of DNA.
Remarkably, the observed switch from negligible to record-
breaking efficiency of the most therapeutically useful form of
DNA cleavage (the ds DNA cleavage) occurs upon a relatively
small change in the pH which can be used to differentiate
healthy cells from hypoxic cancer tissues. The amplification of
DNA-cleaving activity seems to be quite general and manifests
itself for DNA-cleaving agents which bind DNA in different
Acknowledgment. I.A. is grateful to the National Science
Foundation (CHE-0848686) for partial support of this research.
J.A.C. is funded in part by NIH/National Cancer Institute grant
R01CA104505 and Dr. and Mrs. Ellis Brunton Rare Cancer
Research Fund. I.A. and W.-Y.Y. acknowledge partial support from
NIH grant RO1EB006158 and Professor J. Schlenoff for helpful
discussions. Q.X.S. was funded in part by DOD grants DAMD 17-
02-1-0238 and W81XWH-07-1-0225. W.-Y.Y. and A.I. are grateful
to C. Callahan and S.A. Marrone for their help at different stages
of this project.
Supporting Information Available: Details for experimental
procedures, synthesis of DNA photocleavers and photophysical
experiments, extended survey of DNA binding studies, Cartesian
coordinates and energies of different protonation states of lysine
1
conjugates, and H and 13C NMR spectra. This material is
(64) We currently have no clear explanation for activity of the conjugates
in the dark, but for compounds and 2, it is unlikely to be associated
with thermal Bergman reaction which is too slow under these
conditions.
available free of charge via the Internet at http://pubs.acs.org.
JA902140M
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