Toward Efficient Zn(II)-Based Artificial Nucleases

Article abstract of DOI:10.1021/ja039465q

A series of cis-cis-triaminocyclohexane Zn(II) complex-anthraquinone intercalator conjugates, designed in such a way to allow their easy synthesis and modification, have been investigated as hydrolytic cleaving agents for plasmid DNA. The ligand structure

Full text of DOI:10.1021/ja039465q

Published on Web 03/19/2004
Toward Efficient Zn(II)-Based Artificial Nucleases
Elisa Boseggia,† Maddalena Gatos,| Lorena Lucatello,‡ Fabrizio Mancin,†
Stefano Moro,‡ Manlio Palumbo,*,‡ Claudia Sissi,‡ Paolo Tecilla,*,§
Umberto Tonellato,*,† and Giuseppe Zagotto‡
Contribution from the Dipartimento di Scienze Chimiche e Istituto CNR Tecnologia delle
Membrane-Sezione di PadoVa, UniVersita` di PadoVa, Via Marzolo 1, I-35131 PadoVa, Italy,
Dipartimento di Scienze Farmaceutiche, UniVersita` di PadoVa, Via Marzolo 5,
I-35131 PadoVa, Italy, Istituto CNR di Chimica Inorganica e delle Superfici, corso Stati Uniti 4,
I-35127 PadoVa, Italy, and Dipartimento di Scienze Chimiche, UniVersita` di Trieste,
Via Giorgeri 1, I-34127 Trieste, Italy
Received November 6, 2003; E-mail: manlio.palumbo@unipd.it; tecilla@dsch.univ.trieste.it; umberto.tonellato@unipd.it
Abstract: A series of cis-cis-triaminocyclohexane Zn(II) complex-anthraquinone intercalator conjugates,
designed in such a way to allow their easy synthesis and modification, have been investigated as hydrolytic
cleaving agents for plasmid DNA. The ligand structure comprises a triaminocyclohexane platform linked
by means of alkyl spacers of different length (from C4 to C8) to the anthraquinone group which may intercalate
the DNA. At a concentration of 5 µM, the complex of the derivative with a C8 alkyl spacer induces the
hydrolytic stand scission of supercoiled DNA with a rate of 4.6 × 10-6 s-1 at pH 7 and 37 °C. The conjugation
of the metal complex with the anthraquinone group leads to a 15-fold increase of the cleavage efficiency
when compared with the anthraquinone lacking Zn-triaminocyclohexane complex. The straightforward
synthetic procedure employed, allowing a systematic change of the spacer length, made possible to gain
more insight on the role of the intercalating group in determining the reactivity of the systems. Comparison
of the reactivity of the different complexes shows a remarkable increase of the DNA cleaving efficiency
with the length of the spacer. In the case of too-short spacers, the advantages due to the increased DNA
affinity are canceled due to the incorrect positioning of the reactive group, thus leading to cleavage inhibition.
few years.4,5 Hovewer, almost all these systems employ metal
ions different from those used by the natural catalysts and, in
Introduction
The phosphodiester bonds of DNA are exceptionally resistant
to hydrolysis under uncatalyzed physiological conditions. At
neutral pH and 25 °C, half-life times ranging from hundreds of
thousands to billions of years have been estimated for the
hydrolysis of phosphodiester bonds of DNA.1 Nucleases are able
to accelerate this reaction up to 1016-fold, thus making possible
the DNA manipulations that are essential to life.2 The enormous
effect of these enzymes most often depends on the presence in
their active sites of metal ions, particularly Zn(II), Mg(II), and
Fe(III), and on their ability to act as Lewis acids.3 Although
many of these enzymes are currently used in the laboratory
practice, there is an increasing interest in the realization of small
and robust artificial nucleases for their potential applications.4
As a matter of fact, several examples of metal complexes that
promote the hydrolysis of DNA have been reported in the past
particular, are often based on lanthanide ions5d-f,6 or Cu(II).5g-l
Among the physiologically relevant metal ions, Zn(II) is
probably the best suited metal ion for the development of
artificial metallonucleases. In fact, being a strong Lewis acid
and exchanging ligands very rapidly, it is an ideal candidate to
play the role of hydrolytic catalysts. Furthermore, it is character-
ized by a well-defined coordination chemistry, which allows
detailed studies of the reactivity of its complexes, and by the
absence of a relevant redox chemistry that, unlike Cu(II) based
systems, rules out competing oxidative cleavage pathways.
(5) Selected examples: (a) Rammo, J.; Hettich, R.; Roigk, A.; Schneider, H.-
J. Chem. Commun. 1996, 105-107. (b) Hettich, R.; Schneider, H.-J. J.
Am. Chem. Soc. 1997, 119, 5638-5647. (c) Dixon, N. E.; Geue, R. J.;
Lambert, J. N.; Moghaddas, S.; Pearce, D. A.; Sargeson, A. L. Chem.
Commun. 1996, 1287-1288. (d) Schnaith, L. H.; Hanson, R. S.; Que, L.,
Jr. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 569-573. (e) Ragunathan, K.
G.; Schneider H.-J. Angew. Chem., Int. Ed. Engl. 1996, 35, 1219-1221.
(f) Komiyama, M.; Takeda, N.; Shigekawa, H. Chem. Commun. 1999,
1443-1451. (g) Branum, M. E.; Tipton, A. K.; Zhu, S.; Que, L., Jr. J. Am.
Chem. Soc. 2001, 123, 1898-1904. (h) Chand, D. K.; Schneider, H.-J.;
Bencini, A.; Bianchi, A.; Giorgi, C.; Ciattini, S.; Valtancoli, B. Chem.-
Eur. J. 2000, 6, 4001-4008. (i) Deck, K. M.; Tseng, T. A.; Burstyn, J. N.
Inorg. Chem. 2002, 41, 669-677. (j) Ren, R.; Yang, P.; Zheng, W.; Hua,
Z. Inorg. Chem. 2000, 39, 5454-5463. (k) Sissi, C.; Mancin, F.; Palumbo,
M.; Scrimin, P.; Tecilla, P.; Tonellato, U. Nucleosides, Nucleotides &
Nucleic Acids 2000, 19, 1265-1271. (l) Sreedhara, A.; Freed, J. D.; Cowan,
J. A. J. Am. Chem. Soc. 2000, 122, 8814-8824. (m) Itoh, T.; Hisada, H.;
Sumiya, T.; Hosono, M.; Usui; Y.; Fujii, Y. Chem. Commun. 1997, 677-
678.
† Dipartimento di Scienze Chimiche, Universita` di Padova.
| Istituto di Chimica Inorganica e delle Superfici.
‡ Dipartimento di Scienze Farmaceutiche, Universita` di Padova.
§ Universita` di Trieste.
(1) (a) Westheimer F. H. Science 1987, 235, 1173-1178. (b) Williams, N.
H.; Takasaki, B.; Wall, M.; Chin, J. Acc. Chem. Res. 1999, 32, 485-493.
(2) (a) Stra¨ter, N.; Lipscomb, W. N.; Klabunde, T.; Krebs, B. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 2024-2055. (b) Wilcox, D. E. Chem. ReV. 1996,
96, 2435-2458. (c) Cowan, J. A. Chem. ReV. 1998, 98, 1067-1088. (d)
Jedrzejas, M. J.; Setlow, P. Chem. ReV. 2001, 101, 608-618.
(3) (a) Blasko´, A.; Bruice, T. C. Acc. Chem. Res. 1999, 32, 475-484. (b)
Bashkin, J. K. Curr. Opin. Chem. Biol. 1999, 3, 752-758.
(4) Hegg, E. L.; Burstyn, J. N. Coord. Chem. ReV. 1998, 173, 133-165.
(6) Franklin, S. J. Curr. Opin. Chem. Biol. 2001, 5, 201-208.
9
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J. AM. CHEM. SOC. 2004, 126, 4543-4549
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A R T I C L E S
Boseggia et al.
However, its reactivity is somewhat lower than that of the other
commonly employed transition-metal ions,4 and this is probably
why the examples of Zn(II)-based artificial nucleases reported
to date are scarce.7-9 Among them, the most relevant are two
peptide-based systems: (i) a family of mononuclear Zn(II)-
binding peptides tethered to a rhodium complex as intercalator,
proposed by Barton and co-workers, which promotes plasmid
DNA cleavage at the rate of 2.5 × 10-5 s-1 at pH 6 and in the
presence of 5 µM complex concentration8 and (ii) a binuclear
Zn(II)-binding peptide conjugate with an acridine intercalator,
described by Scrimin and co-workers, which cleaves plasmid
DNA with a first-order rate constant of 1.0 × 10-5 s-1 at pH
7.0 and 3.6 µM complex concentration.9 These two peptide-
based catalysts, quite different in structure, share as a common
element the presence of the DNA-intercalating moiety that, as
in the case of some oxidative DNA cleaving agents,10 may be
at the source of the high reactivity observed. However, the effect
of conjugating hydrolytic metal complexes to intercalating
groups is not clearly established. On one hand, in the case of
Barton’s peptide system, the presence of the rhodium complex
intercalator seems to be fundamental for the activity of the
system, but its substitution with other intercalators leads to
unreactive systems.8b On the other hand, in Scrimin’s peptide
the removal of the acridine group does not affect substantially
the DNA cleavage efficiency of the system.9
Besides the two systems just discussed, examples of metal
complexes appended to intercalating groups as hydrolytic agents
are surprisingly rare. Schneider and co-workers appended two
naphthalene groups to an azacrown ligand via C6 alkyl spacers
and used it as a cofactor for Eu(III)-promoted DNA hydrolysis.5a
Again the reported results are puzzling because the conjugation
with the intercalating units led to an increase of the intrinsic
reactivity but to a decrease of the DNA affinity, resulting in an
overall decrease of the DNA cleavage rate at low concentration
of metal complex. Finally, an earlier report of Nakamura and
Hashimoto,11 who investigated the reactivity of a hydroxamic
acid linked to a phenanthridine intercalator in the presence of
different lanthanide ions, points attention to the length of the
spacer which tethers the intercalating unit to the catalytic group
as a key element for the cleavage activity. However, such
mechanistic investigations require systematic variations of the
catalyst structure, and therefore, a readily synthetically accessible
system is highly desired.
Figure 1. Structures of metal complex-anthraquinone conjugates (1-3)
and of reference compound 4.
Fujii5m and Cowan5l with Cu(II) complexes of triaminocyclo-
hexane and the aminoglycoside kanamycin A, respectively. Very
high DNA cleavage rate was obtained (plasmid DNA cleavage
rate constants of 4.8 × 10-5 s-1 for the Fujii complex and 3 ×
10-4 s-1 for the Cowan system at pH 8.1 and 7.3),12 and the
efficiency of the systems was attributed to specific ligand-
DNA interactions presumably via hydrogen bonds.
We report here the realization of a series of Zn(II) complex-
intercalator conjugates, whose structure features a cis-cis-
triaminocyclohexane chelating subunit linked to an anthraquino-
ne moiety through alkyl spacers of different length (Figure 1).
The hydrolytic subunit was chosen on the basis of our previous
studies which have shown that it is a rather efficient catalyst of
model phosphate esters cleavage,13 while the anthraquinone
intercalator was selected for the straightforward chemistry and
for its well-known ability to intercalate DNA base pairs.14 The
complexes have been designed to test their efficacy in the
hydrolytic cleavage of plasmid DNA and to get insight on the
role played by the intercalating group in determining the
reactivity of the systems. The latter point could be investigated
by systematically changing the length of the alkyl spacer using
a rather straightforward synthetic procedure.
Results and Discussion
The general synthetic route for the synthesis of the an-
thraquinone conjugates 5-7 is shown in Scheme 1. The di-
BOC-protected derivative of the triaminocyclohexane was
reacted with the proper Z-protected ω-bromoamine. Removal
of the Z protecting group, coupling with the chloride of the
anthraquinone-4-carboxylic acid, and final deprotection of the
BOC groups led to the desired compounds.
Zn(II) complexes 1-3 bind to dsDNA as highlighted by a
decrease of the anthraquinone absorbance at 332 nm, diagnostic
of the formation of a stable binary complex (see Supporting
Information). In the case of compound 2, we could evaluate a
binding constant of about 1 × 104 M-1, a value in the range
reported for similar intercalating units.14
Incubation of pBR 322 plasmid DNA with the complexes
1-4 at pH 7.0 for 24 h at 37 °C results in a different extent of
DNA cleavage depending on the nature and concentration of
the complex (Figure 2). DNA is converted from the supercoiled
form I to the nicked form II and eventually to the linearized
form III. On the other hand, control experiments have shown
that ligands 5-7, in the absence of Zn(II), are totally unreactive
(see Supporting Information).
Other strategies than conjunction to intercalating groups have
been explored to increase the DNA affinity of hydrolytic agents.
Again Schneider and co-workers, using Co(III) cyclen com-
plexes featuring alkylammonium chains of different length,
found an increase of the DNA affinity and, as a result, of the
overall reactivity as the spacer length was increased.5b Finally,
evidence of DNA binding was obtained also by the groups of
(7) (a) Basile, L. A.; Raphael, A. L.; Barton, J. K. J. Am. Chem. Soc. 1987,
109, 7550-7551. (b) Aka, F. N.; Akkaya, M. S.; Akkaya, E. U. J. Mol.
Catal. A 2001, 165, 291-294. (c) Ichikawa, K.; Tarani, M.; Uddin, M. K.;
Nakata, K.; Sato, S. J. Inorg. Biochem. 2002, 91, 437-450.
(8) (a) Fitzsimons, M. P.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 3379.
(b) Copeland, K. D.; Fitzsimons, M. P.; Houser, R. P.; Barton, J. K.
Biochemistry 2002, 41, 343-356.
Complex 4, lacking the intercalating subunit, has been used
as reference compound and shows a much lesser hydrolytic
(9) Sissi, C.; Rossi, P.; Felluga, F.; Formaggio, F.; Palumbo, M.; Tecilla, P.;
Toniolo, C.; Scrimin, P. J. Am. Chem. Soc. 2001, 123, 3169-3170.
(10) See for example: (a) Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem.
ReV. 1999, 99, 2777-2795. (b) Chen, C.-B.; Milne, L.; Landgraf, R.;
Perrin, D. M.; Sigman, D. S. ChemBioChem 2001, 2, 735-740.
(11) (a) Hashimoto, S.; Nakamura, Y. J. Chem. Soc., Chem. Commun. 1995,
1413-1414. (b) Hashimoto, S.; Nakamura, Y. J. Chem. Soc., Perkin Trans.
1 1996, 2623-2628.
(12) Calculated from refs 5l and 5m.
(13) Bonfa´, L.; Gatos, M.; Mancin, F.; Tecilla, P.; Tonellato, U. Inorg. Chem.
2003, 42, 3943-3949.
(14) (a) Lown, J. W.; Morgan, A. R.; Yen, S. F.; Wang, Y. H.; Wilson, W. D.
Biochemistry 1985, 24, 4028-4035. (b) Gatto, B.; Zagotto, G.; Sissi, C.;
Cera, C.; Uriarte, E.; Palu’, G.; Capranico, G.; Palumbo, M. J. Med. Chem.
1996, 39, 3114-3122.
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Toward Efficient Zn(II)-Based Artificial Nucleases
A R T I C L E S
Scheme 1
activity: after incubation of DNA in the presence of 180 µM
of 4, only about 50% of form I has been nicked. The same
extension of conversion from form I to II is obtained at
concentrations 75 and 36 µM of complexes 2 and 3, respectively.
This indicates a much more pronounced DNA cleavage ef-
ficiency for the anthraquinone coniugates. However, in the
concentration range investigated, no such reactivity has been
reached using complex 1. Thus, the presence of the an-
thraquinone group leads to a significant modulation of the
reactivity depending upon the length of the spacers.
The use of Zn(II) complexes allows us to discard the
possibility of metal ion-driven oxidative cleavage processes,
while the absence of cleavage observed with free ligands 5-7
and with complex 1 indicates that the possibility of an
anthraquinone-driven oxidative process can also be ruled out.
The results of DNA cleavage experiments performed in anaero-
bic conditions with complex 3 (see Supporting Information),
which show that the reactivity of the complex does not change
in the absence of oxygen, further support such conclusions.
To better define the behavior of the system, we carried out
kinetic measurements of the DNA cleavage reaction using
different concentrations of 3. The kinetic profiles obtained by
densitometric quantification of the agarose gels were (Figure
3, left) analyzed using two simplified kinetic models. The first
one assumes the occurrence of two consecutive, independent
first-order processes in which form II is first produced from
form I (single-strand cleavage, k1) and then is degraded to form
III (random double-strand cleavage, k2). The second one implies
three independent first-order processes in which form III can
be produced directly either from form I (nonrandom double-
strand cleavage, k3) or from form II (random double-strand
cleavage, k2), which in turn is the product of the cleavage of
form I (single-strand cleavage, k1). The fitting of the kinetic
data gave satisfactory results only with the first model, indicating
that any relevant nonrandom double-strand clavage, leading to
the direct conversion of form I to form III, can be ruled out. At
a 45 µM concentration of complex, form I cleavage rates are
3.2 × 10-5 s-1 and 2.0 × 10-6 s-1 in the presence of 3 and 4,
respectively. Therefore, a 15-fold acceleration in the reaction
rate is generated by the introduction of the intercalating subunit.
The rate of conversion of form I to form II increases with
the concentration of the complex showing (Figure 3, right) a
dose-dependent saturation behavior. This is admittedly not so
well-defined since the relatively low solubility of the complexes
did not allow us to explore a wider concentration range. At any
rate, fitting of the available data with the Michaelis-Menten
equation gives a formation constant of approximately 1 × 104
M-1, in agreement with the results of the UV-vis binding
experiments. The effective intercalation of the anthraquinone
Figure 3. Left: kinetic profiles for plasmid DNA cleavage in the presence
of 60 µM 3 (HEPES 20 mM, pH 7, 37 °C). The curves were generated by
fitting the data with the kinetic model described in the text. Right: cleavage
rate of pBR 322 DNA (forms I to II) as a function of the concentration of
3. The curve shows the fitting with the Michaelis-Menten equation.
Figure 2. Agarose gel electrophoresis of pBR 322 DNA (12 µM b.p.)
after 24 h incubation with the indicated concentration of complexes 1-4
in 20 mM HEPES, pH 7.0, 37 °C. Lane C is DNA incubated in the absence
of complexes.
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A R T I C L E S
Boseggia et al.
be substantially attributed to a still low DNA affinity, which
could be increased by selecting more efficient intercalating
groups. The plainness of the synthetic procedure involved and
the modular design of the system will allow the easy modifica-
tion and optimization of the systems to obtain more efficient
and selective catalysts.
Conclusion
In conclusion, we have realized a simple Zn(II)-based artificial
nuclease with a remarkable DNA cleavage activity. The source
of the enhanced reactivity is the conjugation of the metal
complex with a DNA targeting unit, which leads to a 15-fold
increase of the cleavage efficiency with respect to the metal
complex alone. In particular, the importance of a careful design
of the system and the choice of the appropriate spacer has been
highlighted. In fact, as the comparison of the reactivity of
complexes 1-4 clearly shows, a too-short spacer may decrease
or even cancel the advantages due to the increased DNA affinity.
The availability of a system that is easy to synthesize and, hence,
to modify is of paramount importance to reach the optimum
efficiency.
Figure 4. Calculated structures (MMFF94) of the intercalation complexes
between complexes 1 (A) and 3 (B) and DNA.
conjugates 1-3 in plasmid DNA is further confirmed by agarose
gel electrophoresis performed in the absence of SDS in the
medium (see Supporting Information). The considerable smear-
ing and decrease of the plasmid mobility observed clearly
indicates an increase of the hydrodynamic volume of the
negative supercoiled plasmid, as a result of the formation of
the intercalation complexes.
Experimental Section
General. Solvents were purified by standard methods. All com-
mercially available reagents and substrates were used as received. TLC
analyses were performed using Merck 60 F254 precoated silica gel glass
plates. Column chromatography was carried out on Macherey-Nagel
silica gel 60 (70-230 mesh). NMR spectra were recorded using a
1
Bruker AC250F operating at 250 MHz for H and 62.9 MHz for 13C.
The reactivity trend of complexes 1-3 (Figure 2) highlights
the remarkable effect due to the length of the alkyl spacer. While
complex 1, with a C4 spacer, is virtually ineffective at every
concentration, the reactivity progressively increases using
complexes 2 and 3 with C6 and C8 spacers. Such behavior
indicates that the length and flexibility of the spacer play a
fundamental role in the interaction between the catalytic subunit
and the phosphate backbone. After intercalation of the an-
thraquinone moiety, the spacer must be free to fold in such a
way as to position the metal complex close to a phosphate group,
and a too-short spacer may prevent the correct folding. This is
confirmed by molecular mechanics calculations: as shown in
Figure 4, only in the intercalation complex between 3 and DNA
can the metal complex reach the phosphate backbone and
promote its cleavage. In the case of complex 1, the spacer is
too short and does not allow this interaction to occur, so that
the intercalation process leads to inhibition rather than enhance-
ment of reactivity of the metal complex, because the metal
complex is hampered in interacting with the phosphate groups.
The present results may help to explain the apparently
contradictory results obtained by Scrimin,8 Barton,9 and
Schneider5a by appending intercalators to hydrolytic metal
complexes. The proper distance between the two subunits is a
main factor, and the catalyst efficacy results from a subtle
balance between increased DNA affinity and decreased mobility
of the metal complex.
Chemical shifts are reported relative to internal Me4Si. Multiplicity is
given as follow: s ) singlet, d ) doublet, t ) triplet, q ) quartet, qt
) quintet, m ) multiplet, br ) broad peak. UV-vis absorption
measurements were performed on a Perkin Helmer Lambda 16
spectrophotometer equipped with a thermostated cell holder. Zn(NO3)2
was an analytical grade product. Metal ion stock solutions were titrated
against EDTA following standard procedures. The buffer 4-(2-hy-
droxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma) was used
as supplied by the manufacturers. cis,cis-1,3,5-Triaminocyclohexane
(8),15 8-amino-1-octanol,16 and 9,10-dioxo-9,10-dihydro-anthracene-2-
carbonyl chloride17 were prepared as reported.
Procedure for DNA Cleavage Experiments. Stock solutions of the
anthraquinone conjugates 5-7 and model ligand 8 (0.5-1 mmol) were
prepared in H2O/CH3OH, 4:1 (ligands 5,6, and 8) and H2O/CH3OH,
2:3 (ligand 7). The ligand was dissolved in the proper solvent mixture,
1 equiv of Zn(NO3)2 in water was added, and the pH was corrected to
7 by addition of NaOH. In the case of ligand 7, the solubility at neutral
pH in the absence of the metal ion was very low, and stable stock
solutions of the free ligand could not be obtained. The methanol content
in the final reaction mixtures was always lower than 4%; control
experiments confirmed that the presence of such an amount of methanol
has no effect on the reaction rates.
DNA cleavage experiments were performed using pBR 322 (Gibco
BRL) in 20 mM HEPES, pH 7.0. Reactions were performed incubating
DNA (12 µM base pairs) at 37 °C in the presence and absence of
increasing amounts of metal complex for the indicated time. Reaction
products were resolved on a 1% agarose gel in TAE buffer (40 mM
TRIS base, 20 mM, acetic acid, 1 mM EDTA) containing 1% SDS to
dissociate the ligands from DNA. The resolved bands were visualized
by ethidium bromide staining and photographed. The relative amounts
The kinetic data obtained allow us to estimate, for a 5 µM
concentration of 3, a first-order rate constant of 4.6 × 10-6 s-1
for the cleavage of plasmid form I. This value compares well
with those reported for the others Zn(II)-based system, although
obtained under somewhat different experimental conditions, and
also with the Schneider lanthanide and Co(III)-based agents (see
infra). The slightly lower reactivity of the present systems can
(15) (a) Bowen, T.; Planalp, R. P.; Brechbiel, M. W. Bioorg. Med. Chem. Lett.
1996, 6, 807. (b) Ninomiya, K.; Shioiri, T.; Yamada, S. Tetrahedron 1974,
30, 2151.
(16) Bell, R. A.; Faggiani, R.; Hunter, H. N.; Lock, J. L. Can. J. Chem. 1992,
70, 186.
(17) Langhals, H.; Saulich, S. Chem.-Eur. J. 2002, 8, 5630
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Toward Efficient Zn(II)-Based Artificial Nucleases
A R T I C L E S
of different plasmid structures were quantified using a BioRad Gel Doc
1000 apparatus interfaced to a PC workstation.
specified number of independent docking runs (55 in our specific case)
and writes the resulting conformations and their energies to a molecular
database file. The resulting DNA-anthraquinone intercalated complexes
were subjected to MMFF94 all-atom energy minimization until the rms
of conjugate gradient was <0.1 kcal mol-1 Å-1. Also in these steps,
all bond distances involving Zn2+ metal ion were frozen during the
energy optimization. To model the effects of solvent more directly, a
set of electrostatic interaction corrections was used. MOE suite
implements a modified version of GB/SA contact function described
by Still and coauthors.24 These terms model the electrostatic contribution
to the free energy of solvation in a continuum solvent model. The
interaction energy values were calculated as the energy of the complex
The DNA degradation experiments in anaerobic conditions were
performed following a modification of the protocol reported by Burstyn
and co-workers.5i Deoxygenated 10 mM HEPES buffer at pH 7.0 was
prepared by five freeze-pump-thaw cycles; before each cycle, the
solution was equilibrated with argon under sonication to favor the
deoxygenation process. Deaerated methanol was prepared by sonication
under argon. Deoxygenated buffer and methanol were used immediately
after preparation. All anaerobic stock solutions were prepared in a
nitrogen-filled glovebag. Reaction mixtures were immediately prepared
by addition of the appropriate amounts of stock solutions to the reactions
tubes and incubated at 37 °C for 24 h in the same nitrogen-filled
glovebag. All other conditions were the same as reported in the previous
paragraph.
Molecular Mechanics Calculations. All calculations were run on
SGI Octane R12000 workstation. cis,cis-1,3,5-Triaminocyclohexane‚
Zn2+ (4) structure was optimized using Hartree-Fock calculations with
the 6-311++G(d,p) basis set. A water molecule coordinated to the
apical position of the Zn2+ ion was used. This water molecule was
used to saturate, at least partially, the coordination sphere of the zinc
ion. Quantum chemistry calculations were carried out with Gaussian
98. Harmonic vibrational frequencies were obtained from RHF/6-
311++G(d,p) calculations and used to characterize local energy minima
(all frequency real). Atomic charges were calculated by fitting to
electrostatic potential maps (CHELPG method).18
minus the energy of the ligand, minus the energy of DNA: ∆Einter
E(complex) - (E(L) + E(rDNA)).
)
all-cis-1,3-N,N-(Di-tert-butyloxycarbonyl)-1,3,5-triaminocyclohex-
ane (9). all-cis-1,3,5-Triaminocyclohexane (1.04 g, 8.0 mmol) was
dissolved in 400 mL of dry CH3OH, and triethylamnine (TEA, 1.9 mL,
13.6 mmol) was added. To the resulting solution, being stirred at room
temperature, was added dropwise a solution of di-tert-butyl dicarbonate
(BOC2O, 3.0 g, 13.6 mmol) in 70 mL of dry CH3OH during 7 h. After
the addition was completed, the reaction mixture was stirred at room
for 2 days, protected from moisture with a CaCl2 drying tube, following
the reaction by TLC (silica gel, eluant CHCl3/CH3OH/NH3, 9:1:0.1, Rf
(9) ) 0.25). The solvent was then evaporated, and the crude product
was dissolved in CH2Cl2 (500 mL) and washed first with a 5% aqueous
solution of NaHCO3 (3 × 100 mL) and then with water (2 × 100 mL).
The evaporation of the dried solvent (Na2SO4) afforded a crude product
which was purified by column chromatography (silica gel, eluant
CHCl3/CH3OH/NH3, 9:1:0.1), yielding 1.05 g (40%) of pure 9 as a
white solid. mp 214-215 °C.
Structures of metal complex-anthraquinone conjugates (1-3) were
built using the “Builder” module of Molecular Operation Environment
(MOE, version 2002.03).19 Conjugate complexes were minimized using
MMFF94 force field. All bond distances involving Zn2+ metal ion were
frozen during the optimization step. Charges for the cis,cis-1,3,5-
triaminocyclohexane Zn(II) structure were imported from the Gaussian
output files.
1H NMR (CDCl3) δ: 0.90 (m, 3H), 1.43 (s, 18H), 2.18 (m, 3H),
2.85 (brt, 1H), 3.54 (m, 2H), 4.43 (m, 2H). 13C NMR (CDCl3) δ: 28.4,
39.3, 42.5, 46.7, 47.2, 79.3, 154.9.
The present study involved the use of consensus dinucleotide
intercalation geometries d(ApT) and d(GpC) initially obtained using
Nucleic Acid MOdeling Tool (NAMOT2) software.20 d(ApT) and
d(GpC) intercalation sites were contained in the center of a deca-
nucleotide duplex of sequences d(5′-ATATA-3′)2 and d(5′-GCGCG-
3′)2, respectively. Decamers in B-form were built using the “DNA
Builder” module of MOE. Decanucleotides were minimized using
Amber94 all-atom force field,21 implemented by MOE modeling
package, until the rms value of the truncated Newton method (TN)
was <0.001 kcal mol-1 Å-1. The dielectric constant was assumed to
be distance-independent with a magnitude of 4. The metal complex-
anthraquinone conjugates (1-3) were docked into both intercalation
sites using flexible MOE-Dock methodology. The purpose of MOE-
Dock was to search for favorable binding configurations between a
small, flexible ligand and a rigid macromolecular target. Searching was
conducted within a user-specified 3D docking box, using “tabu` search”
protocol22 and MMFF94 force field.23 MOE-Dock performs a user-
N-Benzyloxycarbonyl-ω-amino-1-alcohol. The ω-amino-1-alcohol
(17 mmol) was dissolved in 10 mL of CH3OH, and TEA (5.45 mL,
39.2 mmol) was added. To this solution, stirred at room temperature,
was added dropwise a solution of benzyloxycarbonyl chloride (ZCl,
3.2 g, 18.77 mmol) in 5 mL of CH3OH. After the addition was
completed, the reaction mixture was stirred at room temperature for
10 h and protected from moisture with a CaCl2 drying tube. The solvent
was then evaporated, and the crude product was dissolved in CH2Cl2
(150 mL) and washed first with a 5% aqueous solution of KHSO4 (2
× 50 mL) and then with water (2 × 50 mL). The evaporation of the
dried solvent (Na2SO4) afforded the product as a white solid pure
enough to be used in the following steps.
N-Benzyloxycarbonyl-4-amino-1-butanol.25 Yield 91%, mp 82-
1
83 °C, Rf: 0.7 (silica gel, CHCl3/MeOH, 9:1). H NMR (CDCl3) δ:
1.56 (m, 4H), 2.36 (br s, 1H), 3.20 (m, 2H), 3.62 (m, 2H), 4.9 (br s,
1H), 5.08 (s, 2H), 7.32 (m, 5H). 13C NMR (CDCl3) δ: 26.4, 29.5,
40.7, 62.1, 66.5, 128.0, 128.4, 136.5, 156.6. ESI-MS (m/z): 223.9[M+
+ H+], 245.9[M+ + Na+], 262.0[M+ + K+].
(18) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M.
A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann,
R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin,
K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi,
R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.;
Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.;
Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz,
J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng,
C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.;
Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon,
M.; Replogle, E. S.; Pople, J. A. Gaussian 98, revision A.6; Gaussian,
Inc.: Pittsburgh, PA, 1998.
(19) Molecular Operating Environment (MOE version 2002.02), Chemical
Computing Group, Inc., 1255 University St., Suite 1600, Montreal, Quebec,
Canada, H3B 3X3.
(20) NAMOT2 (Nucleic Acid Modeling Tool); Los Alamos National Labora-
tory: Los Alamos, New Mexico, 1997.
(21) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.;
Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P.
A. J. Am. Chem. Soc. 1995, 117, 5179.
N-Benzyloxycarbonyl-6-amino-1-hexanol.26 Yield 82%, mp 81-
1
82 °C, Rf: 0.6 (silica gel, CHCl3/MeOH, 9:1). H NMR (CDCl3) δ:
1.24-1.56 (m, 8H), 3.18 (m, 2H), 3.62 (t, J ) 6.6 Hz, 2H), 4.69 (br,
1H), 5.09 (s, 2H), 7.35 (m, 5H).
(22) Baxter, C. A.; Murray, C. W.; Clark, D. E.; Westhead, D. R.; Eldridge, M.
D. Proteins: Struct., Funct., Genet. 1998, 33, 367.
(23) (a) Halgren, T. A. J. Comput. Chem. 1996, 17, 490. (b) Halgren, T. A. J.
Comput. Chem. 1996, 17, 520. (c) Halgren, T. A. J. Comput. Chem. 1996,
17, 553. (d) Halgren, T. A. J. Comput. Chem. 1996, 17, 587. (e) Halgren,
T. A; Nachbar, R. J. Comput. Chem. 1996, 17, 616. (f) Halgren, T. A. J.
Comput. Chem. 1999, 20, 72. (g) Halgren, T. A. J. Comput. Chem. 1999,
20, 730.
(24) Qiu, D.; Shenkin, S.; Hollinger, F. P.; Still, W. C. J. Phys. Chem. 1997,
101, 3005.
(25) Mallams, A. K.; Morton, J. B.; Reichert, P. J. Chem. Soc., Perkin Trans.
1 1981, 2186.
(26) Ammann, H.; Dupuis, G. Can. J. Chem. 1988, 66, 1651.
9
J. AM. CHEM. SOC. VOL. 126, NO. 14, 2004 4547

A R T I C L E S
Boseggia et al.
N-Benzyloxycarbonyl-8-amino-1-octanol.27 Yield 42%, mp 87-
88 °C, Rf: 0.7 (silica gel, CHCl3/MeOH, 9:1). H NMR (CDCl3) δ:
1.49 (m, 12H), 3.17 (t, J ) 6.6 Hz, 2H), 3.63 (t, J ) 6.6 Hz, 2H), 4.75
(br s, 1H), 5.10 (s, 2H), 7.34 (m, 5H). 13C NMR (CDCl3) δ: 25.6,
26.6, 29.1, 29.2, 29.9, 32.7, 54.8, 62.9, 69.6, 128.3, 128.5, 128.6, 136.6,
157.0. ESI-MS (m/z): 280.2 [M+ + H+], 297.2 [M+ + NH4+], 302.1
[M+ + Na+], 318.1 [M+ + K+].
(CDCl3) δ: 27.9, 28.4, 39.5, 40.9, 46.6, 46.7, 53.7, 66.5, 79.4, 128.1,
1
128.2, 128.4, 128.5, 136.7, 154.9, 156.4. ESI-MS (m/z): 536.2 [M+
+
H+].
all-cis-3,5-N,N-Di-tert-butyloxycarbonyl-1-N-[6-N-(benzyloxycar-
bony)aminohexyl]-1,3,5-triaminocyclohexane (11b). Yield 38%. White
solid, mp 172-173 °C. Rf ) 0.6 (silica gel CHCl3/MeOH/NH3, 9.5:
1
0.5:0.1). H NMR (CDCl3) δ: 0.87 (dd, J ) 11.6 Hz, 3H), 1.37 (m,
N-Benzyloxycarbonyl-ω-bromo-1-alkylamine 10a-c. The above
protected amino alcohol (2 mmol) was dissolved in 15 mL of CH2Cl2,
and CBr4 (0.990 g, 2.98 mmol) and Ph3P (0.783 g, 2.98 mmol) were
added to the stirred solution at 0 °C. The solution was warmed at room
temperature, and the reaction was followed by TLC (petroleum ether/
EtOAc, 7:3). After 90 min at room temperature, water was added in
the reaction flask. The solution was diluted with 110 mL of CH2Cl2,
and the organic phase was washed twice with water and brine and dried
over Na2SO4. The solvent was evaporated to dryness under vacuum.
The crude material was purified by flash chromatography (SiO2,
petroleum ether/AcOEt, 90:10, gradient 2%) to give the bromide as a
transparent oil.
26H), 2.16 (dd, J ) 11.6 Hz, 3H), 2.58 (m, 3H), 3.17 (q, J ) 6.3 Hz),
3.54 (br, 2H), 4.42 (br, 2H), 4.78 (br, 1H), 5.09 (s, 2H), 7.33 (m, 5H).
13C NMR (CDCl3) δ: 26.5, 26.8, 28.4, 29.8, 30.1, 39.7, 40.9, 46.6,
47.1, 53.6, 66.5, 79.3, 128.0, 128.1, 128.4, 136.6, 154.9, 156.4. ESI-
MS (m/z): 563.2 [M+ + H+], 585.3 [M+ + Na+], 601.2 [M+ + K+].
all-cis-3,5-N,N-Di-tert-butyloxycarbonyl-1-N-[8-N-(benzyloxycar-
bony)aminooctyl]-1,3,5-triaminocyclohexane (11c). Yield 36%. White
solid, mp 164-165 °C. Rf ) 0.6 (silica gel CHCl3/MeOH/NH3, 9.5:
0.5:0.1). Rf ) 0.6 (CHCl3/MeOH/NH3, 9.5:0.5:0.1). 1H NMR (CDCl3)
δ: 0.88 (dd, J ) 11.5 Hz, 3H), 1.42 (m, 30H), 2.20 (dd, J ) 11.5 Hz,
3H), 2.58 (m, 3H), 3.18 (q, J ) 6.6 Hz, 2H), 3.55 (m, 2H), 4.48 (br,
2H), 4.82 (br, 1H), 5.09 (s, 2H), 7.32 (m, 5H). 13C NMR (CDCl3) δ:
26.6, 27.2, 28.4, 29.1, 29.3, 29.9, 30.2, 39.7, 41.0, 46.6, 47.3, 53.6,
66.5, 79.3, 128.0, 128.1, 128.4, 136.6, 154.9, 156.3. ESI-MS (m/z):
591.4 [M+ + H+].
all-cis-3,5-N,N-Di-tert-butyloxycarbonyl-1-N-(ω-aminoalkyl)-1,3,5-
triaminocyclohexane (12a-c). Compound 11 (0.4 mmol) was dis-
solved in MeOH, and 25 mg of Pd/C 5% was added to the stirred
solution. The mixture was kept under H2 atmosphere for 5 h, and the
removal of the CBz group was followed by TLC (silica gel, CHCl3/
MeOH/NH3, 9:1:0.1). The catalyst was filtered off, the solution was
evaporated to dryness, and the residue was coevaporated twice with
CH3CN, giving compound 12 which was used without further purifica-
tion.
N-Benzyloxycarbonyl-4-bromo-1-butylamine (10a).28 Yield 78%,
1
Rf: 0.6 (silica gel, petroleum ether/AcOEt, 7:3). H NMR (CDCl3) δ:
1.65 (qt, J ) 6.7 Hz, 2H), 1.86 (qt, J ) 6.7 Hz), 3.21 (q, J ) 6.7 Hz,
2H), 3.41 (t, J ) 6.7 Hz, 2H), 4.83 (br s, 1H), 5.09 (s, 2H), 7.33 (m,
5H). 13C NMR (CDCl3) δ: 28.6, 29.8, 33.1, 40.1, 66.6, 128.0, 128.1,
128.5, 136.5, 156.4. ESI-MS (m/z): 286.1, 288.2 [M+ + H+], 308.0,
310.0 [M+ + Na+].
N-Benzyloxycarbonyl-6-bromo-1-hexylamine (10b).29 Yield 84%,
1
Rf: 0.6 (silica gel, petroleum ether/AcOEt, 7:3). H NMR (CDCl3) δ:
1.5 (m, 6H), 1.85 (qt; J ) 6.8 Hz, 2H), 3.19 (q, J ) 6.8 Hz, 2H), 3.39
(t, J ) 6.8 Hz, 2H), 4.76 (br s, 1H), 5.1 (s, 2H), 7.33 (m, 5H). 13C
NMR (CDCl3) δ: 25.8, 27.7, 29.7, 32.5, 33.7, 40.8, 66.5, 128.0, 128.1,
128.4, 136.6, 156.3. ESI-MS (m/z): 314.1, 316.2 [M+ + H+], 331.1,
all-cis-3,5-N,N-Di-tert-butyloxycarbonyl-1-N-(4-aminobutyl)-1,3,5-
triaminocyclohexane (12a). Yield 77%. White solid. Rf ) 0.1 (silica
gel CHCl3/MeOH/NH3, 9:1:0.1). 1H NMR (CD3OD) δ: 1.01 (dd, J )
11.8 Hz, 3H), 1.42 (s, 18H), 1.58 (m, 4H), 2.06 (dd, J ) 11.8 Hz, 3H),
2.63 (m, 3H), 2.83 (m, 2H), 3.41 (br, 2H). ESI-MS (m/z): 302.3 [M+
- BOC], 401.3 [M+ + H+].
333.2 [M+ + NH4+], 335.8, 337.8 [M++ Na+], 351.9, 353.9 [M+
+
K+].
N-Benzyloxycarbonyl-8-bromo-1-octylamine (10c). Yield 88%,
1
Rf: 0.7 (silica gel, petroleum ether/AcOEt, 7:3). H NMR (CDCl3) δ:
1.53 (m, 10H), 1.84 (qt, J ) 6.9 Hz, 2H), 3.18 (q, J ) 6.9 Hz, 2H),
3.39 (t, J ) 6.9 Hz, 2H), 4.74 (br s, 1H), 5.09 (s, 2H), 7.33 (m, 5H).
13C NMR (CDCl3) δ: 26.6, 28.0, 28.6, 29.0, 29.8, 29.9, 33.9, 41.0,
66.6, 128.0, 128.1, 128.5, 136.6, 155.7. ESI-MS (m/z): 342.1, 344.2
[M+ + H+], 364.1, 366.1 [M+ + Na+], 380.1, 382.1 [M+ + K+].
all-cis-3,5-N,N-Di-tert-butyloxycarbonyl-1-N-(6-aminohexyl)-1,3,5-
triaminocyclohexane (12b). Yield 78%. White solid. Rf ) 0.1 (silica
1
gel CHCl3/MeOH/NH3, 9:1:0.1). H NMR (CDCl3) δ: 1.06 (m, 2H),
1.4 (m, 27H), 2.2 (m, 3H), 2.76 (m, 5H), 3.49 (br, 2H), 4.72 (br, 2H).
ESI-MS (m/z): 229.2 [M+ - 2BOC], 329.3 [M+ - BOC], 429.4 [M+
+ H+], 451.3 [M+ + Na+].
all-cis-3,5-N,N-Di-tert-butyloxycarbonyl-1-N-(8-aminooctyl)-1,3,5-
triaminocyclohexane (12c). Yield 88%. White solid. Rf ) 0.1 (silica
gel CHCl3/MeOH/NH3, 9:1:0.1). 1H NMR (CD3OD) δ: 0.95 (dd, J )
11.7 Hz, 3H), 1.34 (m, 30H), 2.04 (dd, J ) 11.7 Hz, 3H), 2.56 (m,
3H), 2.71 (m, 2H), 3.35 (br, 2H). ESI-MS (m/z): 457.4 [M+ + H+].
9,10-Dioxo-9,10-dihydro-anthracene-2-carboxylic Acid [ω-(cis-
3,5-(N,N-Di-tert-butyloxycarbonyl)diamino-cyclohexylamino)alkyl]-
amide (13). To a solution of the above amine 12 (0.16 mmol) in dry
THF (5 mL) were added first TEA (0.025 mL, 0.16 mmol) and then
9,10-dioxo-9,10-dihydro-anthracene-2-carbonyl chloride (53 mg, 0.019
mmol). The reaction mixture was stirred at room temperature protected
from moisture for 28 h following the reaction by TLC (CHCl3/CH3-
CH2OH, 10:0.5). The solvent was evaporated, and the crude material
was purified by two subsequent column chromatography using as eluant
first CHCl3/CH3CH2OH, 10:0.5 and then CHCl3/CH3OH/NH3, 10:1:
0.1.
all-cis-3,5-N,N-Di-tert-butyloxycarbonyl-1-N-[ω-N-(benzyloxycar-
bony)aminoalkyl]-1,3,5-triaminocyclohexane (11a-c). The bromide
10 (1 mmol) was dissolved in 10 mL of dry CH3CN, and a solution of
9 (0.25 g, 0.76 mmol) in 20 mL of dry CH3CN was added dropwise
under stirring. Diisopropylethylamine (DIPEA, 0.13 mL, 0.76 mmol)
was then added to maintain the pH around 8. The mixture was heated
at reflux for 8 days following the reaction by TLC (CHCl3/MeOH/
NH3, 9.5:0.5:0.1). The solvent was evaporated under reduced pressure,
and the residue was dissolved in 40 mL of CH2Cl2 and washed with
NaHCO3 5% (3 × 20 mL) and water (2 × 20 mL). The organic layer
was dried over Na2SO4, and the solvent was removed under vacuum.
The crude product was purified by flash chromatography (SiO2, CH2-
Cl2 100%, NH3 1%, gradient 0.5% MeOH), giving the product 11.
all-cis-3,5-N,N-Di-tert-butyloxycarbonyl-1-N-[4-N-(benzyloxycar-
bony)aminobutyl]-1,3,5-triaminocyclohexane (11a). Yield 61%. Pale
1
yellow oil. Rf ) 0.6 (silica gel, CHCl3/MeOH/NH3, 9.5:0.5:0.1). H
NMR (CDCl3) δ: 0.86 (dd, J ) 11.6 Hz, 3H), 1.40 (m, 22H), 2.15
(dd, J ) 11.6 Hz, 3H), 2.59 (m, 3H), 3.17 (q, J ) 6 Hz, 2H), 3.51 (br,
2H), 4.36 (br, 2H), 5.08 (s, 2H), 5.74 (br, 1H), 7.33 (m, 5H). 13C NMR
9,10-Dioxo-9,10-dihydro-anthracene-2-carboxylic Acid [4-(cis-3,5-
(N,N-Di-tert-butyloxycarbonyl)diamino-cyclohexylamino)butyl]-
amide (13a). Yield 32%. Rf ) 0.3 (silica gel CHCl3/CH3CH2OH,
10:0.5). 1H NMR (CDCl3) δ: 0.78-0.99 (m, 3H), 1.41 (s, 18H), 1.61-
1.77 (m, 4H), 2.17-2.20 (m, 3H), 2.61-2.75 (m, 3H), 3.49-3.53 (m,
4H), 4.52 (br s, 2H), 7.85-8.53 (m, 7H).
(27) Heckendorn, R.; Allgeier, H.; Baud, J.; Gunzenhauser, W.; Angst, C. J.
Med. Chem. 1993, 36, 3721.
(28) Wei, W.; Tomohiro, T.; Kodaka, M.; Okuno, H. J. Org. Chem. 2000, 65,
8979.
(29) Giardina, D.; Brasili, L.; Gregori, M.; Massi, M.; Picchio, M. T.; Quaglia,
V.; Melchiorre, C. J. Med. Chem. 1989, 32, 50.
9
4548 J. AM. CHEM. SOC. VOL. 126, NO. 14, 2004

Toward Efficient Zn(II)-Based Artificial Nucleases
A R T I C L E S
9,10-Dioxo-9,10-dihydro-anthracene-2-carboxylic Acid [6-(cis-3,5-
(N,N-Di-tert-butyloxycarbonyl)diamino-cyclohexylamino)hexyl]-
amide (13b). Yield 65%. Rf ) 0.3 (silica gel CHCl3/CH3CH2OH,
10:0.5). 1H NMR (CDCl3) δ: 0.78-0.99 (m, 3H), 1.41 (s, 18H), 1.68-
1.80 (m, 8H), 2.19-2.24 (m, 3H), 2.88-2.96 (m, 3H), 3.49-3.51 (m,
2H), 4.49 (br, 2H), 7.77-8.63 (m, 7H).
9,10-Dioxo-9,10-dihydro-anthracene-2-carboxylic Acid [8-(cis-3,5-
(N,N-Di-tert-butyloxycarbonyl)diamino-cyclohexylamino)octyl]-
amide (13c). Yield 24%. Rf ) 0.3 (silica gel CHCl3/CH3CH2OH,
10:0.5). 1H NMR (CDCl3) δ: 1.07-1.67 (m, 33H), 1.85-2.35
(m, 3H), 2.90-3.90 (m, 7H), 4.62 (m, 2H), 7.77-8.54 (m, 7H).
9,10-Dioxo-9,10-dihydro-anthracene-2-carboxylic Acid [ω-(cis-
3,5-(N,N-Diamino-cyclohexylamino)alkyl)]amide (5-7). The above
protected compound 13 (0.07 mmol) was dissolved in dry CH2Cl2 (2
mL), and trifluoroacetic acid (2 mL) was added. The resulting solution
was stirred at room temperature and protected from moisture, following
the reaction by TLC (silica gel, CH2Cl2/CH3CH2OH, 15:1). Evaporation
of the solvent gave in quantitative yield the title compound as
trifluoroacetate salt.
9,10-Dioxo-9,10-dihydro-anthracene-2-carboxylic Acid [6-(cis-3,5-
(N,N-Diamino-cyclohexylamino)hexyl)]amide (6). Yield 96%. Rf )
0 (silica gel, CH2Cl2/CH3OH, 10:2). H NMR (CD3OD) δ: 1.17 (m,
3H), 1.36-1.55 (m, 8H), 2.39 (m, 3H), 2.97 (t, 2H), 3.32 (m, 5H),
7.76-8.55 (m, 7H). 13C NMR (D2O) δ: 26.7, 27.0, 29.6, 32.5, 33.8,
34.0, 41.5, 46.5, 46.7, 53.2, 127.8, 128.4, 128.5, 129.0, 134.0, 134.3,
136.4, 140.6, 177.3, 183.3, 183.4. ESI-MS (m/z): 463 [M+ + H+].
1
9,10-Dioxo-9,10-dihydro-anthracene-2-carboxylic Acid [8-(cis-3,5-
(N,N-Diamino-cyclohexylamino)octyl)]amide (7). Yield 92%. Rf)0
1
(silica gel, CH2Cl2/CH3OH, 10:2). H NMR (CD3OD) δ: 1.09-1.57
(m, 15H), 2.21-2.33 (m, 3H), 3.36 (m, 5H), 7.78-8.50 (m, 7H). 13C
NMR (D2O) δ: 26.5, 27.1, 29.6, 30.1, 32.4, 33.5, 34.1, 41.3, 46.4,
46.9, 53.1, 127.3, 128.6, 128.8, 129.2, 134.1, 134.5, 136.1, 140.1, 177.0,
183.2, 183.3. ESI-MS (m/z): 491 [M+ + H+].
Acknowledgment. Financial support for this research has
been partly provided by the Ministry of Instruction, University
and Research (MIUR Contracts 2001038212 and 2002031238)
and by University of Padova (Young Researchers Grant
CPDG022585).
9,10-Dioxo-9,10-dihydro-anthracene-2-carboxylic Acid [4-(cis-3,5-
(N,N-Diamino-cyclohexylamino)butyl)]amide (5). Yield 78%. Rf )
1
0 (silica gel, CH2Cl2/CH3OH, 10:2). H NMR (D2O) δ: 1.45-1.60
Supporting Information Available: DNA cleavage experi-
ments by compounds 1-4 (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
(m, 7), 2.33-2.47 (m, 3H), 3.06 (t, 1H), 3.28 (t, 2H), 3.37-3.41 (m,
4H), 7.44-7.67 (m, 7H). 13C NMR (D2O) δ: 26.7, 29.1, 34.6, 36.1,
42.7, 48.3, 48.8, 55.5, 128.6, 130.4, 130.8, 135.4, 135.6, 136.0, 137.4,
138.6, 141.5, 170.0, 186.1, 186.3. ESI-MS (m/z): 435 [M+ + H+].
JA039465Q
9
J. AM. CHEM. SOC. VOL. 126, NO. 14, 2004 4549