Efficient plasmid DNA cleavage by copper(II) complexes of 1,4,7-triazacyclononane ligands featuring xylyl-linked guanidinium groups

Article abstract of DOI:10.1021/ic102301n

Three new metal-coordinating ligands, L¹, L², and L³, have been prepared by appending o-, m-, and p-xylylguanidine pendants, respectively, to one of the nitrogen atoms of 1,4,7-triazacyclononane (tacn). The copper(II) complexes of these ligands are able to accelerate cleavage of the P-O bonds within the model phosphodiesters bis(p-nitrophenyl) phosphate (BNPP) and [2-(hydroxypropyl)-p-nitrophenyl]phosphate (HPNPP), as well as supercoiled pBR 322 plasmid DNA. Their reactivity toward BNPP and HPNPP is not significantly different from that of the nonguanidinylated analogues, [Cu(tacn)(OH₂)₂]²⁺ and [Cu(1-benzyl-tacn) (OH₂)₂]²⁺, but they cleave plasmid DNA at considerably faster rates than either of these two complexes. The complex of L¹, [Cu(L¹H⁺)(OH₂)₂] ³⁺, is the most active of the series, cleaving the supercoiled plasmid DNA (form I) to the relaxed circular form (form II) with a k _obs value of (2.7 ± 0.3) × 10^-4 s ^-1, which corresponds to a rate enhancement of 22- and 12-fold compared to those of [Cu(tacn)(OH₂)₂]²⁺ and [Cu(1-benzyl-tacn)(OH₂)₂]²⁺, respectively. Because of the relatively fast rate of plasmid DNA cleavage, an observed rate constant of (1.2 ± 0.5) × 10^-5 s^-1 for cleavage of form II DNA to form III was also able to be determined. The X-ray crystal structures of the copper(II) complexes of L¹ and L ³ show that the distorted square-pyramidal copper(II) coordination sphere is occupied by three nitrogen atoms from the tacn ring and two chloride ions. In both complexes, the protonated guanidinium pendants extend away from the metal and form hydrogen bonds with solvent molecules and counterions present in the crystal lattice. In the complex of L¹, the distance between the guanidinium group and the copper(II) center is similar to that separating the adjacent phosphodiester groups in DNA (ca. 6 A). The overall geometry of the complex is also such that if the guanidinium group were to form charge-assisted hydrogen-bonding interactions with a phosphodiester group, a metal-bound hydroxide would be well-positioned to affect the nucleophilic attack on the neighboring phosphodiester linkage. The enhanced reactivity of the complex of L¹ at neutral pH appears to also be, in part, due to the relatively low pK_a of 6.4 for one of the coordinated water molecules.

Full text of DOI:10.1021/ic102301n

ARTICLE
pubs.acs.org/IC
Efficient Plasmid DNA Cleavage by Copper(II) Complexes
of 1,4,7-Triazacyclononane Ligands Featuring Xylyl-Linked
Guanidinium Groups
Linda Tjioe,† Anja Meininger,†,‡ Tanmaya Joshi,† Leone Spiccia,*,† and Bim Graham*,§
†School of Chemistry, Monash University, Clayton, Victoria 3800, Australia
§Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052,
Australia
S Supporting Information
b
ABSTRACT: Three new metal-coordinating ligands, L1, L2,
and L3, have been prepared by appending o-, m-, and p-
xylylguanidine pendants, respectively, to one of the nitrogen
atoms of 1,4,7-triazacyclononane (tacn). The copper(II) com-
plexes of these ligands are able to accelerate cleavage of the
PꢀO bonds within the model phosphodiesters bis(p-nitrophe-
nyl)phosphate (BNPP) and [2-(hydroxypropyl)-p-nitrophe-
nyl]phosphate (HPNPP), as well as supercoiled pBR 322
plasmid DNA. Their reactivity toward BNPP and HPNPP is
not significantly different from that of the nonguanidinylated analogues, [Cu(tacn)(OH2)2]2þ and [Cu(1-benzyl-tacn)(OH2)2]2þ
,
but they cleave plasmid DNA at considerably faster rates than either of these two complexes. The complex of L1,
[Cu(L1Hþ)(OH2)2]3þ, is the most active of the series, cleaving the supercoiled plasmid DNA (form I) to the relaxed circular
form (form II) with a kobs value of (2.7 ( 0.3) ꢁ 10ꢀ4 sꢀ1, which corresponds to a rate enhancement of 22- and 12-fold compared to
those of [Cu(tacn)(OH2)2]2þ and [Cu(1-benzyl-tacn)(OH2)2]2þ, respectively. Because of the relatively fast rate of plasmid DNA
cleavage, an observed rate constant of (1.2 ( 0.5) ꢁ 10ꢀ5 sꢀ1 for cleavage of form II DNA to form III was also able to be determined.
The X-ray crystal structures of the copper(II) complexes of L1 and L3 show that the distorted square-pyramidal copper(II)
coordination sphere is occupied by three nitrogen atoms from the tacn ring and two chloride ions. In both complexes, the
protonated guanidinium pendants extend away from the metal and form hydrogen bonds with solvent molecules and counterions
present in the crystal lattice. In the complex of L1, the distance between the guanidinium group and the copper(II) center is similar to
that separating the adjacent phosphodiester groups in DNA (ca. 6 Å). The overall geometry of the complex is also such that if the
guanidinium group were to form charge-assisted hydrogen-bonding interactions with a phosphodiester group, a metal-bound
hydroxide would be well-positioned to affect the nucleophilic attack on the neighboring phosphodiester linkage. The enhanced
reactivity of the complex of L1 at neutral pH appears to also be, in part, due to the relatively low pKa of 6.4 for one of the coordinated
water molecules.
’ INTRODUCTION
on the subminute-to-second time scale.13 Indeed, such enzymes
are crucial to life, with reactions involving cleavage of the
phosphodiesters or transfer of the phosphoryl groups under-
pinning many fundamental biological processes, e.g., cellular
signaling and regulation, energy storage and production, and
nucleic acid synthesis, degradation, and repair.7,14ꢀ16 These
enzymes have provided inspiration for the design of a large range
of synthetic mimics. By the same token, our understanding
of the relationship between enzyme structure and function
has benefited considerably from the study of such model
compounds.
Transition-metal complexes that are capable of cleaving the
phosphate ester backbone of DNA and/or RNA under physio-
logical conditions continue to be the subject of considerable
research activity, spurred on by the prospect of producing
tailormade artificial restriction enzymes and catalytic metallo-
drugs.1ꢀ12 Despite significant advances, achieving practically
useful rates of cleavage remains a major challenge. The PꢀO
bonds within the sugarꢀphosphate backbone of nucleic acids are
exceptionally resistant to hydrolytic cleavage because the nega-
tively charged backbone strongly repels the attack of potential
nucleophiles.6 Nature spectacularly demonstrates, however, that
it is possible to engineer molecules that can vastly reduce the
activation energy associated with scission of these bonds. It has
evolved a range of enzymes (nucleases, ribonucleases, and
phosphatases) to facilitate manipulation of the phosphate esters
A quite recent development in the field of artificial metallo-
nucleases is the synthesis of metal complexes featuring ancillary
Received: November 18, 2010
Published: April 19, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ic102301n Inorg. Chem. 2011, 50, 4327–4339
|

Inorganic Chemistry
ARTICLE
Figure 1. Ligands L1, L2, and L3 prepared in this study as tetrahydrochloride salts.
ammonium and guanidinium groups designed to mimic lysine
and arginine residues found at the active sites of many
metalloenzymes.17ꢀ21 These charged residues complement the
catalytic activity of metal ions by assisting with substrate binding
and activation, as well as stabilization of the transition states.22,23
The guanidinium-containing amino acid side chain of arginine, in
particular, is frequently employed by enzymes to help bind
anionic substrates.22,24 It forms especially strong charge-assisted
hydrogen bonds with phosphates and carboxylates.
’ EXPERIMENTAL SECTION
Materials and Chemicals. Chemicals and solvents were of
reagent or analytical grade, and were used as received unless otherwise
indicated. Distilled water and high-performance liquid chromatography
(HPLC)-grade chloroform were used throughout. Tetrahydrofuran
(THF) was dried over 4 Å molecular sieves and then freshly distilled
from sodium/benzophenone prior to use. 1,4-Bis(tert-butoxycarbonyl)-
1,4,7-triazacyclononane28 and the sodium salt of [2-(hydroxypropyl)-p-
nitrophenyl]phosphate (NaHPNPP)29,30 were prepared according to
literature procedures. pBR 322 plasmid DNA was purchased from
Promega Corp. Milli-Q water used for DNA cleavage was sterilized by
autoclaving, and all reaction solutions were prepared according to
standard sterile techniques. Deoxygenated water was prepared by
boiling distilled water under nitrogen for 4 h and cooling while bubbling
with nitrogen gas. High-purity nitrogen gas was used directly from a
reticulated system.
Instrumentation and Methods. IR spectra were recorded as
KBr disks using a Bruker Equinox FTIR spectrometer at 4.0 cmꢀ1
resolution, fitted with an attenuated total reflectance (ATR) platform.
Microanalyses were performed by Campbell Microanalytical Service,
Otago, New Zealand. 1H and 13C NMR spectra were recorded at 25 ꢀC
in D2O or CDCl3 (as listed) on a Bruker AC200, AM300, or DX400
spectrometer. Chemical shifts were recorded on the δ scale in parts per
million (ppm). The chemical shifts, δ, were calibrated using either
tetramethylsilane or signals from the residual protons of deuterated
solvents. Abbreviations for the resonances for 1H NMR spectra are as
follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
and br s (broad singlet). Low-resolution electrospray ionization mass
spectrometry (ESI-MS) spectra were obtained with a Micromass
Platform II quadrupole mass spectrometer fitted with an electrospray
source. The capillary voltage was at 3.5 eV and the cone voltage at 35 V.
Thin-layer chromatography was performed using silica gel 60 F-254
(Merck) plates with detection of the species present by UV irradiation
or KMnO4 oxidation. UVꢀvis spectra were recorded in 1 cm quartz
cuvettes using a Varian Cary Bio 300 or 5G spectrophotometer.
Circular dichroism (CD) spectra of DNA were recorded at room
temperature on a Jasco J-815 spectropolarimeter with a continuous
flow of nitrogen purging the polarimeter, using 1-cm-path-length
quartz cuvettes. Each sample solution was scanned from 320 to
220 nm at a speed of 20 nm minꢀ1, and the buffer background
spectrum was automatically subtracted. Data were recorded at intervals
of 0.1 nm. The CD spectrum of calf thymus DNA (CT-DNA) alone
(116 μM) was recorded as a control, together with the CD spectra of
DNA in the presence of the complex at various [complex]/[DNA]
ratios (0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0).
Agarose gel electrophoresis of plasmid DNA cleavage products was
performed using a Biorad Mini-Protean 3 electrophoresis module.
Bands were visualized by UV light irradiation, fluorescence-imaged
using an AlphaImager, and photographed with a CCD camera. The gel
photographs were analyzed with the aid of the ImageQuaNT program,
version 4.1.
Our most recent contribution to this area of research con-
cerned the development of copper(II) complexes of 1,4,7-
triazacyclononane (tacn) derivatives featuring two guanidine
groups attached to the amines of the tacn ring via ethyl or propyl
chains.25 The complex incorporating propyl spacers was signifi-
cantly more active in cleaving two model phosphodiesters, bis(p-
nitrophenyl)phosphate (BNPP) and [2-(hydroxypropyl)-p-ni-
trophenyl]phosphate (HPNPP), than [Cu(tacn)(OH2)2]2þ but
only moderately more active than the copper(II) complexes of
N-trialkylated tacn ligands. This suggested that the enhanced
reactivity was largely due to, for example, the reduced formation
of inactive dihydroxo-bridged dimers rather than the guanidine
groups playing a major role in the cleavage mechanism. In
addition, the complex was found to be only marginally more
active in hydrolyzing plasmid DNA than [Cu(tacn)(OH2)2]2þ
,
possibly because of unfavorable steric interactions with the
double-helix structure negating any potential positive contribu-
tion from the guanidine groups toward substrate binding and
activation. Lastly, X-ray crystallography and speciation studies
showed that, for the ligand with ethyl spacers, binding of the
guanidine groups resulted in a very stable five-coordinate com-
plex that was unreactive toward BNPP and HPNPP.
Taking the above findings into consideration, we postulated
that better cleavage activity might be achieved with copper(II)
complexes of tacn ligands featuring single guanidine groups
attached via aromatic spacers. Our hypothesis was that more
rigid pendants would enhance the activity by (i) preventing
coordination of the guanidine group to the copper(II) center and
(ii) reducing the entropic penalty associated with the simulta-
neous binding of the copper(II) center and protonated guanidi-
nium group to the phosphodiester substrate, provided a spacer
with suitable geometry could be found.26,27 The incorporation of
a single pendant, rather than two, should reduce the possibility of
unfavorable steric interactions occurring upon attack of the
complex on a double-helix structure. Herein, we describe the
results of a study designed to test these hypotheses. This involved
the preparation of copper(II) complexes of three new tacn-based
ligands, L1, L2, and L3, featuring o-, m-, and p-xylylguanidine
pendant groups, respectively (Figure 1), and an examination of
the kinetics of cleavage of BNPP, HPNPP, and pBR 322 plasmid
DNA by the three complexes. The crystal structures of the
complexes of L1 and L3 were also determined, helping to shed
some light on the reasons for the observed differences in the
cleavage activity.
Caution! Although no problems were encountered in this work, perchlo-
rate salts are potentially explosive. They should be prepared in small
quantities and handled with care.
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dx.doi.org/10.1021/ic102301n |Inorg. Chem. 2011, 50, 4327–4339

Inorganic Chemistry
ARTICLE
Syntheses. 1-(o-Phthalimidoxylyl)-4,7-bis(tert-butoxycarbonyl)-
1,4,7-triazacyclononane (1). A solution of 2-[2-(bromomethyl)benzyl]-
isoindoline-1,3-dione (2.89 g, 8.78 mmol) in acetonitrile (50 mL) was
added dropwise to a mixture of 1,4-bis(tert-butoxycarbonyl)-1,4,7-
triazacyclononane (2.89 g, 8.78 mmol), K2CO3 (3.03 g, 22.0 mmol),
and KI (0.12 g) in acetonitrile (50 mL). The resulting mixture was
stirred for 1 h at room temperature and then refluxed for 3 days. After
cooling to room temperature, the inorganic salts were filtered off and the
solvent was removed from the filtrate under reduced pressure to yield 1
as a yellow oil. Yield: 4.06 g (80%). 1H NMR (300 MHz, CDCl3): δ 1.43
18H, tBu CH3), 1.95 (s br, 2H, NH2), 2.60ꢀ2.71 (m, 4H, tacn CH2),
3.20ꢀ3.22 (m, 4H, tacn CH2), 3.35ꢀ3.43 (m, 4H, tacn CH2), 3.70 (s,
2H, ethyl CH2), 3.83 (s, 2H, ethyl CH2), 7.12ꢀ7.31 (m, 4H, aromatic
CH). 13C NMR (75 MHz, CDCl3): δ 28.3 (tBu CH3), 43.8 (ethyl CH2),
t
48.2ꢀ53.3 (tacn CH2), 58.7 (ethyl CH2), 79.5 (quaternary Bu C),
126.5, 127.4, 128.0, 130.3 (aromatic CH), 136.7, 142.1 (aromatic C),
155.4 (CdO). IR (neat, cmꢀ1): 3369w (νNꢀH), 2974s (νCꢀH(aromatic)),
2864s (νCꢀH), 1694s (νCdO), 1455s, 1416s, 1367s, 1247s (νCꢀO),
1170s, 1027m, 998s, 860s, 772s. ESI-MS: m/z 449.2 (100%) [M þ H]þ.
1-(m-Aminoxylyl)-4,7-bis(tert-butoxycarbonyl)-1,4,7-triazacyclo-
nonane (5). Compound 5 was prepared following the same method as
that for 4 but using 2 (4.68 g, 8.09 mmol) and hydrazine monohydrate
(2.83 g, 56.7 mmol). Yield: 2.90 g (80%). 1H NMR (300 MHz, CDCl3):
δ 1.45 (s, 18H, tBu CH3), 1.68 (s br, 2H, NH2), 2.56ꢀ2.67 (m, 4H, tacn
CH2), 3.10ꢀ3.26 (m, 4H, tacn CH2), 3.39ꢀ3.48 (m, 4H, tacn CH2),
3.60 (s, 2H, ethyl CH2), 3.78 (s, 2H, ethyl CH2), 7.10ꢀ7.21 (m, 4H,
aromatic CH). 13C NMR (75 MHz, CDCl3): δ 28.4 (tBu CH3), 46.3
(ethyl CH2), 48.8ꢀ54.1 (tacn CH2), 60.8 (ethyl CH2), 79.3 (quaternary
tBu C), 125.5, 127.3, 127.5, 128.2 (aromatic CH), 140.1, 143.0
(aromatic C), 155.5 (CdO). IR (neat, cmꢀ1): 3375w (νNꢀH), 2974s
(νCꢀH(aromatic)), 2862s (νCꢀH), 1694s (νCdO), 1455s, 1416s, 1360s,
1250s (νCꢀO), 1155s, 1095m, 978s, 831s, 772s. ESI-MS: m/z 449.2
(100%) [M þ H]þ.
t
(s, 18H, Bu CH3), 2.73 (m, 4H, tacn CH2), 3.23ꢀ3.30 (m, 4H, tacn
CH2), 3.45ꢀ3.53 (m, 4H, tacn CH2), 3.96 (s, 2H, ethyl CH2), 5.07 (s,
2H, ethyl CH2), 7.16ꢀ7.19 (m, 4H, aromatic CH), 7.70ꢀ7.73 (m, 2H,
aromatic CH), 7.82ꢀ7.87 (m, 2H, aromatic CH). 13C NMR (75 MHz,
CDCl3): δ 28.4 (tBu CH3), 38.1 (ethyl CH2), 47.8ꢀ54.7 (tacn CH2),
59.8 (ethyl CH2), 79.7 (quaternary tBu C), 123.2, 127.4, 128.2, 130.5,
132.0, 133.9, 135.5, 137.1 (aromatic CH), 155.5 (CdO), 168.1 (CdO).
IR (neat, cmꢀ1): 3058w (νCꢀH(aromatic)), 2974s (νCꢀH), 1690s (νCdO),
1682s (νCdO), 1464s, 1393s, 1249s (νCꢀO), 1160s, 998w, 860w, 772w,
716w, 628w. ESI-MS: m/z 579.3 (100%) [M þ H]þ.
1-(m-Phthalimidoxylyl)-4,7-bis(tert-butoxycarbonyl)-1,4,7-tria-
zacyclononane (2). Compound 2 was prepared as a yellow oil in a
manner identical with that of 1 by the slow addition of 2-[3-(bromo-
methyl)benzyl]isoindoline-1,3-dione (3.47 g, 10.5 mmol) in acetonitrile
(50 mL) to a mixture of 1,4-bis(tert-butoxycarbonyl)-1,4,7-triazacyclo-
nonane (3.47 g, 10.5 mmol), K2CO3 (3.64 g, 26.4 mmol), and KI (0.12 g)
1-(p-Aminoxylyl)-4,7-bis(tert-butoxycarbonyl)-1,4,7-triazacyclono-
nane (6). Compound 6 was prepared via the method used to obtain 4
but using 3 (4.03 g, 6.97 mmol) and hydrazine monohydrate (2.44 g,
48.8 mmol). Yield: 2.48 g (79%). 1H NMR (300 MHz, CDCl3): δ 1.45
(s, 18H, tBu CH3), 1.54 (s br, 2H, NH2), 2.55ꢀ2.66 (m, 4H, tacn CH2),
3.10ꢀ3.25 (m, 4H, tacn CH2), 3.39ꢀ3.47 (m, 4H, tacn CH2), 3.59 (s,
2H, ethyl CH2), 3.78 (s, 2H, ethyl CH2), 7.12ꢀ7.28 (m, 4H, aromatic
CH). 13C NMR (75 MHz, CDCl3): δ 28.3 (tBu CH3), 46.0 (ethyl CH2),
1
in acetonitrile. Yield: 4.68 g (77%). H NMR (300 MHz, CDCl3): δ
1.40 (s, 18H, tBu CH3), 2.56ꢀ2.68 (m, 4H, tacn CH2), 3.15ꢀ3.27 (m,
4H, tacn CH2), 3.44ꢀ3.51 (m, 4H, tacn CH2), 3.63 (s, 2H, ethyl CH2),
4.80 (s, 2H, ethyl CH2), 7.18ꢀ7.33 (m, 4H, aromatic CH), 7.68ꢀ7.71
(m, 2H, aromatic CH), 7.81ꢀ7.84 (m, 2H, aromatic CH). 13C NMR (75
MHz, CDCl3): δ 28.4 (tBu CH3), 41.5 (ethyl CH2), 49.2ꢀ53.8 (tacn
CH2), 60.6 (ethyl CH2), 79.3 (quaternary tBu C), 123.2, 126.9, 128.3,
128.5, 128.8, 132.0, 133.9, 136.1, 140.5 (aromatic CH), 155.6 (CdO),
167.8 (CdO). IR (neat, cmꢀ1): 3058w (νCꢀH(aromatic)), 2974s
(νCꢀH), 1686s (νCdO), 1682s (νCdO), 1466m, 1394s, 1248m
(νCꢀO), 1158s, 955w, 859w, 772w, 712w, 530w. ESI-MS: m/z 579.3
(100%) [M þ H]þ.
t
48.7ꢀ53.9 (tacn CH2), 60.4 (ethyl CH2), 79.3 (quaternary Bu C),
126.6, 129.0 (aromatic CH), 138.3, 142.1 (aromatic C), 155.5 (CdO).
IR (neat, cmꢀ1): 3373w (νNꢀH), 2975s (νCꢀH(aromatic)), 2864s (νCꢀH),
1694s (νCdO), 1463s, 1415s, 1366s, 1248s (νCꢀO), 1160s, 1092w,
979w, 860w, 772w. ESI-MS: m/z 449.2 (100%) [M þ H]þ.
1-{o-[bis(tert-Butoxycarbonyl)guanidino]xylyl}-4,7-bis(tert-buto-
xycarbonyl)-1,4,7-triazacyclononane (7). To a stirred solution of 4
(2.48 g, 5.53 mmol) in THF (20 mL) was added N,N0-Boc2-1H-
pyrazole-1-carboxamidine (1.72 g, 5.53 mmol) in THF (20 mL). The
resulting solution was stirred at room temperature for 2 days. The
solvent was evaporated under reduced pressure, and the residue was
dissolved with CH2Cl2 (50 mL). The organic layer was then washed
with 0.1 M NaOH (3 ꢁ 30 mL) and dried with Na2SO4, and the solvent
was removed under vacuum to produce a yellow oil. The crude product
was purified by silica gel column chromatography using 2% MeOH/
CHCl3 as the eluent. Rf = 0.25. Yield: 3.02 g (79%). 1H NMR (300 MHz,
CDCl3): δ 1.32 (s, 9H, tBu CH3), 1.39 (s, 27H, tBu CH3), 2.56ꢀ2.61
(m, 4H, tacn CH2), 3.15 (m, 4H, tacn CH2), 3.32ꢀ3.45 (m, 4H, tacn
CH2), 3.65 (s, 2H, ethyl CH2), 4.70 (m. 2H, ethyl CH2), 7.11ꢀ7.43 (m,
4H, aromatic CH), 8.45 (t br, NHBoc). 13C NMR (75 MHz, CDCl3): δ
27.9 (tBu CH3), 28.2 (tBu CH3), 28.4 (tBu CH3), 42.4 (ethyl CH2),
48.3ꢀ54.5 (TACN CH2), 59.5 (ethyl CH2), 79.2 (quaternary tBu C),
79.4 (quaternary tBu C), 82.9 (quaternary tBu C), 127.4 (aromatic CH),
128.5, 130.3 (aromatic CH), 136.2, 137.2 (aromatic C), 153.0 (CdO),
155.9 (CdO), 163.5 (CdO). IR (neat, cmꢀ1): 3334s (νNꢀH), 3138m
(νNꢀH), 2978s (νCꢀH(aromatic)), 1716s (νCdO), 1683s (νCdO), 1634s
(νCdO), 1557s (νCdN), 1456s, 1250s (νCꢀO), 1157s, 1058s, 999w,
927w, 869w. ESI-MS: m/z 691.4 (100%) [M þ H]þ.
1-(p-Phthalimidoxylyl)-4,7-bis(tert-butoxycarbonyl)-1,4,7-triazacy-
clononane (3). Compound 3 was prepared as a yellow oil following the
procedure described for 1 by the slow addition of 2-[4-(bromomethyl)-
benzyl]isoindoline-1,3-dione (3.01 g, 9.15 mmol) in acetonitrile (50 mL) to
a mixture of 1,4-bis(tert-butoxycarbonyl)-1,4,7-triazacyclononane (3.00 g,
9.15 mmol), K2CO3 (3.16 g, 22.9 mmol), and KI (0.12 g) in acetonitrile
(50mL). Yield:4.03g(76%). 1HNMR(300MHz, CDCl3):δ1.41 (s, 18H,
tBu CH3), 2.47ꢀ2.64 (m, 4H, tacn CH2), 3.10ꢀ3.31 (m, 4H, tacn CH2),
3.42ꢀ3.46 (m, 4H, tacn CH2), 3.56 (s, 2H, ethyl CH2), 4.77 (s, 2H, ethyl
CH2), 7.17ꢀ7.34 (m, 4H, aromatic CH), 7.61ꢀ7.68 (m, 2H, aromatic
CH), 7.75ꢀ7.80 (m, 2H, aromatic CH). 13C NMR (75 MHz, CDCl3): δ
28.4 (tBu CH3), 41.1 (ethyl CH2), 49.0ꢀ53.9 (tacn CH2), 60.2 (ethyl
t
CH2), 79.3 (quaternary Bu C), 123.1, 128.2, 129.1, 131.9, 133.7, 134.9,
139.5 (aromatic CH), 155.5 (CdO), 167.7 (CdO). IR (neat, cmꢀ1):
3057w (νCꢀH(aromatic)), 2975s (νCꢀH), 1702s (νCdO), 1687s (νCdO),
1478s, 1393s, 1247s (νCꢀO), 1160s, 938w, 860w, 772w, 715s, 624w.
ESI-MS: m/z 579.3 (100%) [M þ H]þ.
1-(o-Aminoxylyl)-4,7-bis(tert-butoxycarbonyl)-1,4,7-triazacyclono-
nane (4). Compound 1 (4.06 g, 7.01 mmol) was dissolved in ethanol
(50 mL), and hydrazine monohydrate (2.45 g, 49.1 mmol) was added.
The solution was heated at 50 ꢀC for 4 h. The white precipitate that
formed was removed by filtration. The filtrate was evaporated under
reduced pressure, and the residue was treated with CH2Cl2 (50 mL).
The organic layer was washed with 1 M NaOH (2 ꢁ 50 mL) and dried
with Na2SO4, and the solvent was removed under vacuum to produce a
yellow oil. Yield: 2.48 g (79%). 1H NMR (300 MHz, CDCl3): δ 1.46 (s,
1-{m-[bis(tert-Butoxycarbonyl)guanidino]xylyl}-4,7-bis(tert-buto-
xycarbonyl)-1,4,7-triazacyclononane (8). Compound 8 was prepared
via the method to obtain 7, by reacting 5 (2.90 g, 6.47 mmol) with N,N0-
Boc2-1H-pyrazole-1-carboxamidine (2.01 g, 6.47 mmol). Yield: 3.48 g
1
t
(78%). H NMR (300 MHz, CDCl3): δ 1.43 (s, 9H, Bu CH3), 1.49
4329
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Inorganic Chemistry
ARTICLE
(s, 27H, tBu CH3), 2.63ꢀ2.71 (m, 4H, tacn CH2), 3.16ꢀ3.30 (m, 4H,
tacn CH2), 3.44ꢀ3.52 (m, 4H, tacn CH2), 3.66 (s, 2H, ethyl CH2), 4.60
(m, 2H, ethyl CH2), 7.15ꢀ7.34 (m, 4H, aromatic CH), 8.53 (t br,
NHBoc). 13C NMR (75 MHz, CDCl3): δ 28.2 (tBu CH3), 28.6 (tBu
CH3), 28.8 (tBu CH3), 45.2 (ethyl CH2), 49.4ꢀ54.8 (tacn CH2), 60.9
1-(p-Guanidinoxylyl)-1,4,7-triazacyclononane Tetrahydrochloride
(L3 4HCl). Deprotection of 9 (2.91 g, 4.21 mmol), as for the synthesis
3
of L1 4HCl, yielded the product as a yellow solid. Yield: 0.85 g (70%).
3
Microanal. Calcd for C15H37N6Cl5: C, 34.2; H, 7.1; N, 16.0; Cl, 33.7.
Found C, 34.5; H, 6.8; N, 15.7; Cl, 33.2. 1H NMR (300 MHz, D2O): δ
2.95ꢀ2.98 (m, 4H, tacn CH2), 3.14ꢀ3.17 (m, 4H, tacn CH2), 3.55 (s,
4H, tacn CH2), 3.84 (s, 2H, ethyl CH2), 4.35 (s, 2H, ethyl CH2),
7.29ꢀ7.38 (m, 4H, aromatic CH). 13C NMR (75 MHz, D2O): δ 42.5
(tacn CH2), 43.9 (tacn CH2), 44.5 (ethyl CH2), 48.1 (tacn CH2), 59.0
(ethyl CH2), 127.7 (aromatic CH), 131.1 (aromatic CH), 135.1
(aromatic C), 136.5 (aromatic C), 157.2 (CdN). IR (KBr
disk, cmꢀ1): 3403s (νNꢀH), 2983s (νCꢀH(aromatic)), 2960s (νCꢀH),
1678s (νCdN), 1450m, 1263w, 1107w, 980w. ESI-MS: m/z 291.3
(100%) [M þ H]þ.
t
t
(ethyl CH2), 79.5 (quaternary Bu C), 79.7 (quaternary Bu C), 83.3
(quaternary tBu C), 126.6 (aromatic CH), 128.5 (aromatic CH), 128.9
(aromatic CH), 137.3, 140.8 (aromatic C), 153.2 (CdO), 156.2
(CdO), 163.8 (CdO). IR (neat, cmꢀ1): 3336s (νNꢀH), 3140m
(νNꢀH), 2978s (νCꢀH(aromatic)), 1720s (νCdO), 1694s (νCdO), 1639s
(νCdO), 1570s (νCdN), 1478s, 1250s (νCꢀO), 1157s, 1058s, 980w, 913w,
858w. ESI-MS: m/z 691.4 (100%) [M þ H]þ.
1-{p-[bis(tert-Butoxycarbonyl)guanidino]xylyl}-4,7-bis(tert-buto-
xycarbonyl)-1,4,7-triazacyclononane (9). Compound 9 was prepared
in a manner identical with that of 7, by reacting precursor 6 (2.48 g,
5.53 mmol) with N,N0-Boc2-1H-pyrazole-1-carboxamidine (1.72 g,
5.53 mmol). Yield: 2.91 g (76%). 1H NMR (300 MHz, CDCl3):
δ 1.40 (s, 9H, tBu CH3), 1.47 (s, 27H, tBu CH3), 2.62ꢀ2.65 (m, 4H,
tacn CH2), 3.13ꢀ3.28 (m, 4H, tacn CH2), 3.40ꢀ3.49 (m, 4H, tacn
CH2), 3.60 (m, 2H, ethyl CH2), 4.56 (m, 2H, ethyl CH2), 7.16ꢀ7.54
(m, 4H, aromatic CH), 8.52 (t br, NHBoc). 13C NMR (75 MHz,
CDCl3): δ 28.0 (tBu CH3), 28.2 (tBu CH3), 28.5 (tBu CH3), 44.7 (ethyl
CH2), 48.2ꢀ54.3 (tacn CH2), 60.4 (ethyl CH2), 79.2 (quaternary
[Cu(L1Hþ)Cl2]Cl 0.25DMF 0.25Et2O 1.5H2O (C1). To a stirred aque-
3
3
3
3
ous solution of L1 4HCl (0.096 g, 0.19 mmol) (4 mL) was added
Cu(ClO4)2 6H2O (0.084 g, 0.23 mmol) dissolved in water (4 mL). The
3
pH of the solution was adjusted to 9 with 1 M NaOH, resulting in a
deepening of the blue color and precipitation of a small amount of
Cu(OH)2, which was removed by filtration. The solution was evaporated
to dryness and the residue redissolved in N,N0-dimethylformamide
(DMF). The slow diffusion of diethyl ether into this solution produced
blue crystals of the product. Yield: 0.036 g (38%). Microanal. Calcd for
Cu1C17H34Cl3N6O2: C, 38.3; H, 6.6; N, 16.7. Found: C, 38.4; H, 6.3; N,
16.5. UVꢀvis (H2O): λmax (nm) [εmax (Mꢀ1 cmꢀ1)]: 613 [69], 926
[24]. Selected IR bands (ATR, cmꢀ1): 3251s (νNꢀH), 3151s, 2939m
(νCꢀH), 1638m (νCdN), 1488w, 1089w, 1006w, 940w, 727w.
t
t
tBu C), 79.5 (quaternary Bu C), 83.0 (quaternary Bu C), 127.4
(aromatic CH), 128.9 (aromatic CH), 129.2 (aromatic CH), 135.8,
139.4 (aromatic C), 153.0 (CdO), 155.6 (CdO), 163.5 (CdO). IR
(neat, cmꢀ1): 3336s (νNꢀH), 3138m (νNꢀH), 2978s (νCꢀH(aromatic)),
1722s (νCdO), 1683s (νCdO), 1640s (νCdO), 1557s (νCdN), 1458s,
1250s (νCꢀO), 1158s, 1058s, 1000w, 927w, 869w. ESI-MS: m/z 691.4
(90%) [M þ H]þ, 713.4 (10%) [M þ Na]þ.
[Cu(L2Hþ)Cl2]Cl (C2). Attempts to crystallize this complex from
solutions of equimolar amounts of L2 and Cu(ClO4)2 6H2O using
3
different crystallization techniques were unsuccessful. The copper(II)
1-(o-Guanidinoxylyl)-1,4,7-triazacyclononane Tetrahydrochloride
complex solution of C2 was prepared in situ by mixing equimolar
(L1 4HCl). A solution of the Boc-protected amine 7 (3.02 g, 4.37 mmol)
amounts of the desired ligand and Cu(ClO4)2 6H2O in water and
3
3
was dissolved in a mixture of 1:1 (v/v) TFA/CH2Cl2 (10 mL) and the
solution stirred at room temperature overnight. The solvent was then
removed under reduced pressure and the residual brown oil dissolved in
a mixture of EtOH (5 mL) and concentrated HCl (2 mL). The addition
of diethyl ether (5 mL) produced a white precipitate, which was filtered,
dissolved in a small amount of distilled water, and then freeze-dried to
yield the product as a white solid. Yield: 0.87 g (68%). Microanal. Calcd
for C15H36N6Cl4: C, 35.9; H, 7.3; N, 16.8; Cl, 30.4. Found: C, 36.0; H,
7.0; N, 16.7; Cl, 30.7. 1H NMR (300 MHz, D2O): δ 3.11ꢀ3.14 (m, 4H,
tacn CH2), 3.30ꢀ3.33 (m, 4H, tacn CH2), 3.75 (s, 4H, tacn CH2), 4.00
(s, 2H, ethyl CH2), 4.54 (s, 2H, ethyl CH2), 7.44ꢀ7.61 (m, 4H, aromatic
CH). 13C NMR (75 MHz, D2O): δ 43.0 (tacn CH2), 43.1 (ethyl CH2),
44.7 (tacn CH2), 49.2 (tacn CH2), 56.1 (ethyl CH2), 128.9 (aromatic
CH), 129.1 (aromatic CH), 131.3 (aromatic CH), 134.8 (aromatic C),
134.9 (aromatic C), 157.1 (CdN). IR (KBr disk, cmꢀ1): 3334s (νNꢀH),
2986s (νCꢀH(aromatic)), 2960s (νCꢀH), 1678s (νCdN), 1450m, 1263w,
1107w, 980w. ESI-MS: m/z 291.3 (100%) [M þ H]þ.
adjusting the pH to 7 with the addition of NaOH. UVꢀvis (H2O): λmax
(nm) [εmax (Mꢀ1 cmꢀ1)]: 630 [59], 987 [20].
[Cu(L3Hþ)Cl2]Cl 0.25MeOH 1.5H2O (C3). As for C1, L3 4HCl
3
3
3
(0.076 g, 0.14 mmol) (4 mL) was reacted with Cu(ClO4)2 6H2O
3
(0.064 g, 0.17 mmol) dissolved in water (4 mL). The product obtained
by evaporation of the filtrate was dissolved in methanol. Diffusion of
diethyl ether into this solution crystallized the product. Yield: 0.016 g
(22%). Microanal. Calcd for Cu1C15H31Cl3N6O2: C, 36.9; H, 6.3; N,
16.9. Found: C, 36.6; H, 5.8; N, 16.7. UVꢀvis (H2O): λmax (nm) [εmax
(Mꢀ1 cmꢀ1)]: 618 [64], 926 [20]. Selected IR bands (ATR, cmꢀ1):
3241s (νNꢀH), 2931m (νCꢀH), 1641m (νCdN), 1487w, 1352w, 1152w,
1002w, 942w, 824w.
Solution Speciation Studies. pH titrations of C1ꢀC3 were
performed by adding 1 μL aliquots of 10 M HCl to solutions of each
complex ([complex] = 7.5 mM, total volume = 10 mL). Stock complex
solutions were prepared by mixing equimolar amounts of the desired
ligands and Cu(ClO4)2 6H2O in water and adjusting the pH to 12 using
3
5 M NaOH. The solutions were stirred for 1 min after each addition to
ensure that the pH was stable, and the background-corrected UVꢀvisꢀ
NIR spectrum was measured. The apparent pKa values of the coordi-
nated water molecules in C1ꢀC3 were determined experimentally from
variation in the absorbance with [Hþ] at selected wavelengths of
maximum change (625, 900, and 1000 nm). Variation in the absorbance
versus [Hþ] over the pH range 4.0ꢀ8.0 was fitted using an equation
derived assuming a single deprotonation/protonation process (depro-
tonation of a coordinated water molecule) occurring in the pH range of
study:
1-(m-Guanidinoxylyl)-1,4,7-triazacyclononane Tetrahydrochloride
(L2 4HCl). Deprotection of compound 8 (3.48 g, 5.04 mmol) following
3
the procedure described for L1 4HCl yielded the product as a white
3
solid. Yield: 0.98 g (67%). Microanal. Calcd for C15H36N6Cl4: C,
36.1; H, 7.3; N, 16.8; Cl, 30.2. Found: C, 36.1; H, 6.8; N, 16.7; Cl, 30.5.
1H NMR (300 MHz, D2O): δ 3.06ꢀ3.10 (m, 4H, tacn CH2),
3.26ꢀ3.29 (m, 4H, tacn CH2), 3.68 (s, 4H, tacn CH2), 3.97 (s, 2H,
ethyl CH2), 4.49 (s, 2H, ethyl CH2), 7.38ꢀ7.53 (m, 4H, aromatic
CH). 13C NMR (75 MHz, D2O): δ 42.6 (tacn CH2), 44.0 (tacn CH2),
44.7 (ethyl CH2), 48.0 (tacn CH2), 59.2 (ethyl CH2), 127.2 (aromatic
CH), 128.9 (aromatic CH), 129.7 (aromatic CH), 130.1 (aromatic
CH), 136.2 (aromatic C), 137.1 (aromatic C), 157.2 (CdN). IR (KBr
disk, cmꢀ1): 3338s (νNꢀH), 2986s (νCꢀH(aromatic)), 2960s (νCꢀH),
1619s (νCdN), 1450m, 1263w, 1110w, 954w. ESI-MS: m/z 291.3
(100%) [M þ H]þ.
Ka
½CuðLHÞðOH2Þ2þꢂ
s
½CuðLHÞðOH2ÞðOHÞꢂþ þ Hþ
F
s
R
The Ka value was determined by fitting the data to the equation
Abs = [Cu]total[εCu þ εCuꢀOH(Ka/Hþ)]/[1 þ (Ka/Hþ)], where
4330
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Inorganic Chemistry
ARTICLE
Scheme 1. Synthesis of Hydrochloride Salts of Ligands L1, L2, and L3a
a (i) 2 equiv of Boc-ON, NEt3, CHCl3, RT, 8 h; (ii) o-, m-, or p-bromoxylylphthalimide, K2CO3, KI, CH3CN, reflux, 3 days; (iii) N2H4.H2O, EtOH,
50 ꢀC, 4 h; (iv) N,N0-Boc2-1H-pyrazole-1-carboxamidine, THF, RT, 2 days; (v) (a) 1:1 TFA/CH2Cl2, RT, o/n, (b) concentrated HCl.
Abs = absorbance at a particular wavelength, [Cu]total = total copper(II)
complex concentration, and εCu and εCuꢀOH = molar extinction
coefficients for the initial and monoprotonated copper(II) complexes,
respectively. The choice of the model was supported by the fact that this
equation fitted the experimental data extremely well.
Cleavage of Model Phosphate Esters. Bis(p-nitrophenyl)-
phosphate (BNPP). These experiments were conducted using estab-
lished procedures.31,32 Briefly, the rate of cleavage of BNPP by the
copper(II) complexes was measured at pH 7.0 (HEPES buffer) and
pH 9.0 (CHES buffer) and T = 50 ꢀC, by following the formation of a
p-nitrophenoxide ion spectrophotometrically (λmax = 400 nm, ε =
18 700 Mꢀ1 cmꢀ1) in solutions containing 0.1 mM BNPP, 2 mM
copper(II) complex, and0.15 M NaClO4, over a period of 8000 min, with a
reading taken every 5 min. Because the complex was in large excess
compared to BNPP, the appearance of NP (and cleavage of BNPP)
respectively. The differential absorbance was obtained using the
relationship ΔAbs = θ/32.982, where θ = degrees of ellipticity.
DNA Cleavage Experiments. Electrophoresis experiments were
performed using pBR 322 plasmid DNA according the established
procedures.25 The cleavage of pBR 322 supercoiled plasmid DNA (38
μM base pair concentration) was accomplished by the addition of
copper(II) complexes (75, 112.5, 150, 225, and 300 μM) dissolved in a
40 mM buffer (HEPES) at pH 7.0. The mixtures were incubated in a
water bath at 37 ꢀC for periods of up to 48 h. The reactions were
quenched and the resulting solutions stored at ꢀ20 ꢀC until just prior to
analysis. The analysis involved loading of the solutions onto 1% agarose
gels containing 1.0 μg dmꢀ3 ethidium bromide, and the DNA fragments
separated by gel electrophoresis (70 V for 2 h in standard Tris-acetate-
EDTA (TAE) buffer, pH 8). Ethidium-stained agarose gels were
imaged, and densitometric analysis of the visualized bands was used to
determine the extent of supercoiled DNA cleavage, as reported in the
literature.17
followed a first-order dependence. Observed rate constants were deter-
obst
mined by fitting the data to the equation Abs = A þ Beꢀk
.
For all of the complexes except C1, the DNA cleavage data were fit
[2-(Hydroxypropyl)-p-nitrophenyl]phosphate (HPNPP). These ex-
periments were carried out in a manner similar to that of the BNPP
experiments. The rate of cleavage of HPNPP by the copper(II)
complexes was measured at pH 6.0 (MES), 7.0 (HEPES), and pH 9.0
(CHES) and T = 25 ꢀC, in solutions containing 0.1 mM HPNPP, 2 mM
copper(II) complex, and 0.15 M NaClO4. The observed rate constants
were determined as indicated above.
obst
to a first-order expression, %DNA = A þ Beꢀk , yielding the first-
order rate constant, kobs, for cleavage of form I DNA to produce form
II. In the case of C1, kinetic profiles were fitted using a single-strand
cleavage model derived by Kishikawa et al.,33 which accounts for not
only conversion of form I to form II (rate constant k1) but also
subsequent nicking of form II to produce form III (rate constant k2)
and further cleavage of form III to produce undetected forms of
DNA (rate constant k3 represents all processes that cleave form III
DNA). The rate equations for this model are as follows:
DNA Binding Assays. The binding of complexes C1ꢀC3 to CT-
DNA was studied by CD spectroscopy. The measurements were
performed at pH 7.0 and T = 20 ꢀC, using 116 μM solutions of CT-
DNA and increasing [complex]/[DNA] ratios (0.0, 0.1, 0.2, 0.4, 0.6,
0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0) dissolved in a 5 mM Tris-HCl/
50 mM NaCl buffer. UVꢀvis spectrophotometry could not be used
to monitor the interaction between small molecules and nucleic
acids because these complexes absorb in regions similar to those of
CT-DNA at 260 nm. The apparent binding constant, Kb, was
determined from variation in the differential absorbance with
[complex], using the equation ΔAbs = {ΔA1 þ ΔA2Kb[Cu]}/{1 þ
Kb[Cu]}, where ΔA1 and ΔA2 are the differential absorbances with
no complex added and with complex fully bound to DNA,
d½Iꢂ
dt
d½IIꢂ
dt
d½IIIꢂ
dt
¼ ꢀ k1½Iꢂ;
¼ k1½Iꢂ ꢀ k2½IIꢂ;
¼ k2½IIꢂ ꢀ k3½IIIꢂ
Integrating these expressions yields
½Iꢂ ¼ C0eꢀk t
1
ꢀ
ꢁ
k1C0
k1C0
1
2
½IIꢂ ¼
eꢀk t
þ
N0 ꢀ
eꢀk t
k2 ꢀ k1
k2 ꢀ k1
½IIIꢂ ¼ Reꢀk t þ βeꢀk t þ ðL0 ꢀ R ꢀ βÞeꢀk t
1
2
3
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Inorganic Chemistry
ARTICLE
k1k2C0(k2 ꢀ k1)ꢀ1(k3 ꢀ k1)ꢀ1 and
where R =
ꢀ
ꢁ
ꢀk1C0
k2 ꢀ k1
β ¼
þ N0 ðk3 ꢀ k2Þꢀ1
where C0, N0, and L0 are the initial values of [I], [II], and [III],
respectively.
Cleavage of pBR 322 plasmid DNA was also carried out in the
presence of standard radical scavengers. Aliquots (5 μL) of aqueous
solutions of scavenging agents (30 mM KI, DMSO, tBuOH, or NaN3 in a
40 mM HEPES buffer at pH 7.0) were added to solutions of supercoiled
DNA (5 μL, 113.5 μM base-pair concentration) prior to the addition of
complexes C1, C2, and C3. The reaction conditions were 150 μM
copper(II) complex, 10 mM scavenging agents, and 38 μM base-pair
concentration for supercoiled plasmid DNA. Each solution was incu-
bated at 37 ꢀC for 6 h, quenched, and analyzed according to the
procedure described above.
Experiments under anaerobic conditions were performed following
the protocol reported by Hegg and Burstyn34 and our previous work.25
The concentrations for the reaction mixtures were 150 μM for com-
plexes, 40 mM HEPES buffer, and 38 μM base-pair concentration for
supercoiled DNA.
Figure 2. Thermal ellipsoid representation of the complex unit in C1
showing the distorted SP copper(II) geometry and the hydrogen-
bonding interactions formed by the charged guanidinium group
(ellipsoids drawn at 50% probability; hydrogen atoms on carbon atoms
and solvent molecules have been omitted for clarity).
For complex C1, DNA cleavage experiments were also conducted
using a fixed complex concentration of 50 μM and varying the
concentration of pBR 322 plasmid DNA (base-pair concentration of
25ꢀ200 μM) at pH 7.0 (40 mM HEPES) and T = 37 ꢀC.
Table 1. Selected Bond Lengths [Å] and Angles [deg] for C1
Cu(1)ꢀN(1)
Cu(1)ꢀN(2)
Cu(1)ꢀN(3)
Cu(1)ꢀCl(1)
Cu(1)ꢀCl(2)
2.033(2)
2.037(2)
2.273(2)
2.2936(8)
2.2637(8)
N(2)ꢀCu(1)ꢀN(1)
N(1)ꢀCu(1)ꢀCl(2)
N(2)ꢀCu(1)ꢀCl(2)
N(1)ꢀCu(1)ꢀN(3)
N(2)ꢀCu(1)ꢀN(3)
Cl(2)ꢀCu(1)ꢀN(3)
N(1)ꢀCu(1)ꢀCl(1)
N(2)ꢀCu(1)ꢀCl(1)
Cl(2)ꢀCu(1)ꢀCl(1)
N(3)ꢀCu(1)ꢀCl(1)
83.1(1)
88.42(7)
165.61(7)
82.04(9)
82.02(9)
108.33(6)
169.99(8)
90.20(7)
96.63(3)
104.40(6)
The ionic strength dependence of the DNA cleavage activity of
complex C1 was evaluated over a range of NaCl concentrations (0, 20,
100, 250, and 500 mM). DNA cleavage experiments were performed
with a 38 μM base-pair concentration of pBR 322 plasmid DNA and
150 μM copper(II) complex in a 40 mM HEPES buffer at pH 7.0 and
T = 37 ꢀC .
X-ray Crystallography. The intensity data for blue crystals of C1
(0.20 ꢁ 0.20 ꢁ 0.05 mm) and C3 (0.35 ꢁ 0.25 ꢁ 0.10 mm) were
measured at 123 K on a Bruker Apex II CCD fitted with graphite-
monochromated Mo KR radiation (0.710 73 Å). The data were col-
lected to a maximum 2θ value of 55ꢀ (60ꢀ for C3) and processed using
the Bruker Apex II software package. The crystal parameters and details
of the data collection are summarized in Table S01 in the Supporting
Information. Each structure was solved by direct methods and expanded
using standard Fourier routines in SHELX-97.35,36 All hydrogen atoms
were placed and refined in idealized positions, except for the hydrogen
atoms on the nitrogen atoms, which were located on the Fourier
difference map. All non-hydrogen atoms were refined anisotropically.
had been adjusted to pH 9. Microanalytical data were consistent
with the proposed formulas, and the IR spectra of the complexes
showed bands due to the NH stretches of the guanidine group in
the region of 3150ꢀ3250 cmꢀ1, as well as sharp bands in the
range of 1640ꢀ1640 cmꢀ1, attributable to the ν(CdN) bands of
the guanidine pendant groups. The electronic spectra of C1 and
C3 exhibited broad absorption bands centered at 600 and
985 nm, typical for copper(II) complexes with square-pyramidal
(SP) geometry.37 Attempts to isolate the copper(II) complex of
ligand L2 from solution were unsuccessful. The UVꢀvis spec-
trum of this complex also exhibited bands indicative of a SP
copper(II) geometry.
’ RESULTS AND DISCUSSION
Syntheses. The three new tacn ligand derivatives, L1, L2, and
L3, featuring xylylguanidine pendants, were prepared as their
tetrahydrochloride salts following the route shown in Scheme 1.
Following conversion of tacn to its di-Boc-protected derivative,28
the remaining free nitrogen was functionalized by reaction with
o-, m-, or p-bromoxylylphthalimide. Removal of the phthalimide
protecting group with hydrazine then exposed an amine group
within the newly introduced pendant, which was subsequently
converted to a Boc-protected guanidine via treatment with N,N0-
Boc2-1H-pyrazole-1-carboxamidine. Global Boc deprotection
using trifluoroacetic acid (TFA) yielded the target ligands, which
were isolated as their tetrahydrochloride salts. The new com-
Crystallography. The crystal structures of complexes C1 and
C3 were determined to ascertain the coordination environment
of the copper(II) centers and the relative orientations of the
guanidinium pendants.
Complex C1. The molecular structure of C1 consists of
[Cu(L1Hþ)Cl2]þ cationic units (Figure 2), noncoordinated
chloride anions, DMF, ether, and water molecules. The copper-
(II) center resides in a distorted SP geometry, with the basal
planes defined by the two chlorine atoms and two secondary
nitrogen atoms, N(1) and N(2), from the tacn macrocycle. The
apical position is occupied by the tertiary nitrogen atom, N(3),
bearing the protonated o-xylylguanidinium pendant. The degree of
distortion (τ) fromtheidealSPgeometryto the trigonal-bipyramidal
(TBP) geometry can be defined as a function of the two largest basal
angles, θand Φ:τ=[(θꢀ Φ)/60)] ꢁ 100, with τ=0%fortheideal
1
pounds were characterized by H and 13C NMR spectroscopy,
ESI-MS spectrometry, IR spectroscopy, and microanalysis.
The copper(II) complexes of L1, C1, and L3, C3, were
crystallized by diffusion of diethyl ether into either DMF or
methanolic solutions of the ligand and Cu(ClO4)2 6H2O, which
3
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Inorganic Chemistry
ARTICLE
Table 2. Hydrogen-Bonding Interactions in C1 [Å and deg]a
DꢀH
A
d(DꢀH) d(H A) d(D A) —DHA
3 3 3
3 3 3
3 3 3
N(2)ꢀH(2N) Cl(2)#1
0.86(4)
0.74(4)
2.62(4)
2.00(4)
2.43(4)
2.44(4)
2.46(4)
2.48(4)
3.303(3) 137(3)
2.731(3) 168(4)
3.224(3) 151(3)
3.222(3) 162(3)
3.197(3) 160(4)
3.280(3) 152(3)
3 3 3
N(4)ꢀH(4N) O(1)#2
3 3 3
N(6)ꢀH(6AN) Cl(3)#3 0.87(4)
3 3 3
N(6)ꢀH(6BN) Cl(3)#4 0.82(4)
3 3 3
N(5)ꢀH(5BN) Cl(3)
0.78(4)
3 3 3
N(5)ꢀH(5AN) Cl(3)#3 0.87(4)
3 3 3
a Symmetry transformations used to generate equivalent atoms: #1, ꢀx
Figure 4. Stick representation showing a head-to-tail arrangement
of the adjacent [Cu(L3Hþ)Cl2]þ units and the hydrogen bonding
(dashed bonds) between the charged guanidinium arm, cocrystallized
methanol molecules and chloride counteranions present in the crystal
lattice of C3.
1
1
3
1
1
þ 1, y ꢀ /2, ꢀz þ /2; #2, x, ꢀy þ /2, z ꢀ /2; #3, ꢀx, y ꢀ /2, ꢀz ꢀ
1
1
1/2; #4, x, ꢀy þ /2, z þ /2.
Figure 3. Thermal ellipsoid plot of the complex unit in C3 showing the
distorted SP copper(II) geometry (ellipsoids drawn at 50% probability;
hydrogen atoms on carbon atoms, solvent molecules and counteranions
have been omitted for clarity).
Table 3. Selected Bond Lengths [Å] and Angles [deg] for C3
Cu(1)ꢀN(1)
Cu(1)ꢀN(2)
Cu(1)ꢀN(3)
Cu(1)ꢀCl(1)
Cu(1)ꢀCl(2)
2.012(1)
2.032(1)
2.283(1)
2.3199(4)
2.2741(4)
N(2)ꢀCu(1)ꢀN(1)
N(1)ꢀCu(1)ꢀCl(2)
N(2)ꢀCu(1)ꢀCl(2)
N(1)ꢀCu(1)ꢀN(3)
N(2)ꢀCu(1)ꢀN(3)
Cl(2)ꢀCu(1)ꢀN(3)
N(1)ꢀCu(1)ꢀCl(1)
N(2)ꢀCu(1)ꢀCl(1)
Cl(2)ꢀCu(1)ꢀCl(1)
N(3)ꢀCu(1)ꢀCl(1)
83.35(5)
175.29(4)
92.01(4)
82.48(4)
82.72(4)
97.76(3)
90.74(4)
168.11(3)
93.69(1)
106.80(3)
Table 4. Hydrogen-Bonding Interactions in C3 [Å and deg]a
DꢀH
A
d(DꢀH) d(H A) d(D A) —DHA
3 3 3
3 3 3
3 3 3
N(1)ꢀH(1) Cl(1)#1
0.83(2)
0.86(2)
0.83(2)
0.83(2)
2.60(2)
2.40(2)
2.03(2)
2.48(2)
2.41(2)
2.30(2)
2.30
3.321(1) 145.6(2)
3.215(2) 158.1(2)
2.810(2) 156.8(2)
3.262(2) 156(2)
3.218(2) 167.1(2)
3.114(1) 169.3(2)
3.141(1) 177.3
3 3 3
N(5)ꢀH(5NB) Cl(3)
3 3 3
N(5)ꢀH(5NA) O(2)
3 3 3
N(6)ꢀH(6NB) Cl(3)
3 3 3
N(6)ꢀH(6NA) Cl(1)#2 0.83(2)
3 3 3
O(2)ꢀH(2O) Cl(3)#3
0.82(2)
3 3 3
O(1)ꢀH(1O) Cl(3)
0.84
3 3 3
a Symmetry transformations used to generate equivalent atoms: #1, ꢀx,
1
1
ꢀy þ 1, ꢀz; #2, ꢀx, y þ /2, ꢀz þ /2; #3, x, y ꢀ 1, z.
Figure 5. Change in the UVꢀvisꢀNIR spectrum of copper(II) com-
plexes (7.5 mM) observed in the pH range 8.0ꢀ4.0. The top spectrum
was recorded at pH 8.0 and the bottom spectrum at pH 4.0. Arrows
indicate the decrease in the absorbance upon moving from pH 8 to 4.
SP geometry and 100% for the ideal TBP geometry.38 The τ value of
8% for C1 indicates a geometry close to regular SP.
The mean deviation of the basal atoms from their least-squares
plane is 0.036 Å, with a 0.19 Å out-of-plane displacement for
copper(II), directed toward the apical nitrogen atom [N(3)]. As
expected, the CuꢀN distances in the basal plane are shorter than
the CuꢀN(apical) distance and the intraring NꢀCuꢀN angles
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are all <90ꢀ (Table 1). Coordination of the macrocycle also
causes the CuꢀN(apical) bond to be bent toward the basal
plane. The CuꢀN and CuꢀCl distances are similar to those
measured for [Cu(tacn)Cl2].37
The protonated guanidinium group lies on the side of the
aromatic ring opposite to the tacn macrocycle and is engaged in
charge-assisted hydrogen bonding with the chloride counteran-
ion and hydrogen bonds to the DMF molecule present in the
lattice (Figure 2 and Table 2).
Complex C3. The crystal structure of C3 is composed of
discrete cationic [Cu(L3Hþ)Cl2]þ units (Figure 3) and non-
coordinated chloride anions and methanol molecules. The
copper(II) center resides in a distorted SP environment, with
the degree of distortion from the ideal SP geometry measured as
τ = 12%.38 The CuꢀN distances and angles describing coordina-
tion of L3 to the copper(II) center are almost identical to those
observed in C1 (Table 3).
The halide ligand Cl(1) with the longer CuꢀCl bond distance
participates in intermolecular hydrogen bonding with the pro-
tonated guanidinium group of an adjacent complex unit
(Table 4). This results in a head-to-tail arrangement of the
[Cu(L3Hþ)Cl2]þ units within the crystal lattice of C3. In
addition, hydrogen-bonding interactions between the guanidi-
nium groups, the cocrystallized methanol molecules, and chlor-
ide counteranions link the alternate guanidinium pendants to
each other (Figure 4).
Table 5. Apparent pKa Values of Coordinated Water
Molecules in Copper(II) Complexes, Calculated from the
Decrease in the Absorbance Intensities at Selected
Wavelengths (i.e., 625, 900, and 1000 nm)
compound
pKa (coordinated water)
ref
[Cu(tacn)(OH2)2]2þ
7.3
34
[Cu(L1)(OH2)2]2þ (C1)
[Cu(L2)(OH2)2]2þ (C2)
[Cu(L3)(OH2)2]2þ (C3)
6.36 ( 0.17
6.95 ( 0.18
7.08 ( 0.08
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Solution Speciation Studies. To investigate the acidꢀbase
properties of complexes C1ꢀC3, a series of spectrophotometric
pH titrations were carried out that monitored changes in the
electronic spectrum of each complex as a function of the pH (see
Figure 5). Analysis of the systematic decrease in the absorbance
as the pH was decreased from 8.0 to 4.0 allowed determination of
the apparent pKa of the coordinated water molecules (see Figures
S01ꢀS03 in the Supporting Information and Table 5). C2 and
C3 were found to have pKa values of ca. 7, close to that previously
measured for the nonfunctionalized CuIItacn complex,34 while
that for C1 is substantially lower (6.4), indicating that the
coordinated water molecule in this complex is significantly more
acidic. This could be due to the closer proximity of the charged
guanidine pendant in C1, inductively lowering the pKa of the
coordinated water, as previously proposed for related complexes
with guanidinium pendants.39,40
Cleavage of Model Phosphodiesters. The rates of cleavage
of the simple model phosphodiesters BNPP and HPNPP by
complexes C1ꢀC3 were measured at a series of different pH
values, together with those of the nonguanidinium analogues,
[Cu(Bntacn)(OH2)2]2þ and [Cu(tacn)(OH2)2]2þ (Tables 6
and 7). At all pHs and for both substrates, each of the complexes
enhanced the rate of cleavage significantly above background
levels, particularly for the less reactive DNA mimic, BNPP
(HPNPP, like RNA, features a 20-OH group on the ribose ring
that may act as an internal nucleophile). In addition, the cleavage
activity increased with the pH in each case, consistent with the
Table 6. First-Order Rate Constants for Hydrolysis of BNPP
by Copper(II) Complexesa
kobs (ꢁ10ꢀ6 sꢀ1
)
compound
BNPP only
pH 7.0
pH 9.0
ref
0.0003
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31
[Cu(tacn)(OH2)2]2þ
[Cu(Bntacn)(OH2)2]2þ
[Cu(L1)(OH2)2]2þ (C1)
[Cu(L2)(OH2)2]2þ (C2)
[Cu(L3)(OH2)2]2þ (C3)
[Cu(A)]2þ
[Cu(B)(OH2)2]2þ
[Cu(Me3tacn)(OH2)]2þ
[Cu(iPr3tacn)(OH2)]2þ
a Conditions: [complex] = 2 mM, [BNPP] = 0.1 mM, [HEPES] or
[CHES] = 50 mM, [I] = 0.15 M, and T = 50 ꢀC). Abbreviations: Bntacn =
1-benzyl-1,4,7-triazacyclononane, A = 1-ethyl-4,7-bis(2-guanidinoethyl)-
1,4,7-triazacyclononane, B = 1-ethyl-4,7-bis(3-guanidinopropyl)-1,4,7-tria-
zacyclononane, Me3tacn = 1,4,7-trimethyl-1,4,7-triazacyclononane and
iPr3tacn = 1,4,7-triisopropyl-1,4,7-triazacyclononane.
1.71 ( 0.01
0.86 ( 0.05
1.65 ( 0.03
2.36 ( 0.03
2.39 ( 0.04
0.0100 ( 0.0003
72.4 ( 0.8
37
6.331
2.55 ( 0.03
3.07 ( 0.08
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7.02 ( 0.07 this work
7.49 ( 0.05 this work
25
25
31
41
43
Table 7. First-Order Rate Constants for Hydrolysis of HPNPP by Copper(II) Complexesa
kobs (ꢁ10ꢀ6 sꢀ1
)
compound
HPNPPb only
pH 6.0
0.019
pH 7.0
pH 9.0
0.807
ref
0.012 ( 0.007
3.58 ( 0.2
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25
[Cu(tacn)(OH2)2]2þb
[Cu(Bntacn)(OH2)2]2þ
[Cu(L1)(OH2)2]2þ (C1)
[Cu(L2)(OH2)2]2þ (C2)
[Cu(L3)(OH2)2]2þ (C3)
[Cu(A)]2þb
0.037
8.69
0.086 ( 0.0002
0.754 ( 0.007
1.51 ( 0.02
0.936 ( 0.006
1.73 ( 0.02
1.88 ( 0.03
2.74 ( 0.02
2.30 ( 0.03
0.13 ( 0.06
32.0 ( 3
2.63 ( 0.05
3.27 ( 0.09
10.1 ( 0.6
9.55 ( 0.01
[Cu(B)(OH2)2]2þ
25
a Conditions: [complex] = 2 mM, [HPNPP] = 0.1 mM [MES], [HEPES] or [CHES] = 50 mM, [I] = 0.15 M, and T = 25 ꢀC). Abbreviations: Bntacn =
1-benzyl-1,4,7-triazacyclononane, A = 1-ethyl-4,7-bis(2-guanidinoethyl)-1,4,7-triazacyclononane and B = 1-ethyl-4,7-bis(3-guanidinopropyl)-1,4,7-
triazacyclononane. b Data were analyzed using the initial rate method, yielding kobs directly.
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Table 8. Apparent Binding Constants for Interaction of
Copper(II) Complexes with CT-DNA in 5 mM Tris-HCl/
50 mM NaCl Buffer (pH 7.0)
compound
Kb (ꢁ103 Mꢀ1
)
[Cu(L1)(OH2)2]2þ (C1)
[Cu(L2)(OH2)2]2þ (C2)
[Cu(L3)(OH2)2]2þ (C3)
6.09 ( 0.70
33.0 ( 2.8
34.5 ( 1.7
Figure 6. CD spectra for CT-DNA in the presence of increasing
amounts of copper(II) complexes, C1ꢀC3 (aꢀc). Conditions:
[complex]/[DNA] = 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5
and 4.0), and [CT-DNA] = 0.116 mM in 5 mM Tris-HCl/50 mM NaCl
buffer. Arrows indicate the decrease in the 275 nm band with increasing
amounts of complexes. Insets show the change in the absorbance at the
initial peak maximum with increasing concentrations of complexes.
postulated hydrolytic mechanism for CuIItacn-based complexes,
which features a copper(II)-bound hydroxide nucleophile as the
active species.7 At neutral or basic pH, the rates of phosphodi-
ester cleavage by the three complexes were similar to those for
[Cu(Bntacn)(OH2)2]2þ and [Cu(tacn)(OH2)2]2þ, suggesting
that the guanidinium groups do not directly interact with the
phosphodiester groups and/or are not active participants in the
cleavage mechanism. At pH 6, the complexes were found to be
substantially more reactive toward HPNPP than either
[Cu(tacn)(OH2)2]2þ or [Cu(Bntacn)(OH2)2]2þ, suggesting
some contribution from the guanidinium groups toward the
cleavage activity at this particular pH.
The cleavage activities of complexes C1ꢀC3 are substantially
lower than those measured previously at pH 7 for the analogue
featuring two propylguanidine pendants, as well as the copper
complexes of the simple N-alkylated tacn ligands (also listed in
Tables 6 and 7). For these latter systems, the enhanced activity
relative to [Cu(tacn)(OH2)2]2þ has been ascribed to the
N-linked substituents inhibiting the formation of inactive dihy-
droxo-bridged dimers. This suggests that the single pendants in
C1ꢀC3 may be relatively ineffectual at inhibiting the formation
of such dimers at pH 7.
Figure 7. Agarose gel images showing cleavage of pBR 322 plasmid
DNA (38 μM bp) incubated with [Cu(tacn)(OH2)2]2þ, [Cu(Bntacn)-
(OH2)2]2þ and C1ꢀC3 (150 μM) in a HEPES buffer (40 mM, pH 7.0)
at 37 ꢀC for various time intervals. C = DNA control.
Interaction of Complexes with DNA. CD spectroscopy is a
useful technique for monitoring changes in the DNA conforma-
tion in solution and provides information about binding inter-
actions with DNA. Free helical DNA exhibits the so-called right-
handed B form CD spectrum, with a positive band at 275 nm due
to base stacking and a negative band at 245 nm due to helicity.42
Changes in the CD signals of DNA observed upon interaction
with small molecules have often been assigned to the correspond-
ing changes in the DNA structure.43,44 The intercalation of small
molecules to DNA is known to stabilize the right-handed B
conformation of CT-DNA, resulting in an increase in the
intensities of both bands. In contrast, electrostatic interactions
between small molecules with DNA generally result in little or no
change in the intensities of the two bands.45 Titration of complex
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Table 9. Observed Rate Constants for Single-Strand Clea-
vage of pBR 322 Plasmid DNA by Copper(II) Complexesa
compound
kobs (ꢁ10ꢀ5 sꢀ1
)
ref
[Cu(tacn)(OH2)2]2þ
[Cu(tacn)(OH2)2]2þb
[Cu(Bntacn)(OH2)2]2þ
[Cu(L1)(OH2)2]2þ (C1)
1.2 ( 0.4
1.5
25
34
2.2 ( 0.5
k1 = 27 ( 3
k2 = 1.2 ( 0.5
k3 = 11.2 ( 0.5
8.2 ( 1.0
6.7 ( 0.3
1.58 ( 0.05
2.53 ( 0.04
3.0
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[Cu(L2)(OH2)2]2þ (C2)
[Cu(L3)(OH2)2]2þ (C3)
[Cu(A)]2þ
[Cu(B)(OH2)2]2þ
[Cu(iPr3tacn)(OH2)]2þb
[Cu(TACI)(OH2)2]2þc
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25
34, 41
47
k1 = 230
k2 = 6.2
Figure 8. Time course showing conversion of the supercoiled form
(red) of pBR322 plasmid DNA to the nicked (green) and linear (blue)
forms in the presence of C1. The solid lines show the fit of the
experimental data (squares) to the single-strand cleavage model of
Kishikawa et al.33
[Cu(C)(OH2)2]2þd
85
48
a Conditions: [complex] = 150 μM, [pBR 322 plasmid] = 38 μM bp,
[HEPES] = 40 mM (pH 7.0 at 37 ꢀC). Abbreviations: Bntacn =
1-benzyl-1,4,7-triazacyclononane, A = 1-ethyl-4,7-bis(2-guanidinoeth-
yl)-1,4,7-triazacyclononane, B = 1-ethyl-4,7-bis(3-guanidinopropyl)-
i
1,4,7-triazacyclononane, Pr3tacn = 1,4,7-triisopropyl-1,4,7-triazacyclo-
nonane, TACI = all-cis-2,4,6-triamino-1,3,5-trihydroxycyclohexane and
C = 5,50-bis(guanidinomethyl)-2,20-bipyridyl. b [complex] = 25 μM,
[pBluescript II ks(ꢀ) supercoiled] = 25 nM bp at 50 ꢀC, pH 7.8.
c [complex] = 48 μM, [pBR 322 plasmid] = 12 μM bp at 37 ꢀC, pH 8.1.
d [complex] = 150 μM, [pBR 322 plasmid] = 38 μM bp at 37 ꢀC, pH 7.2.
C1 into CT-DNA induced a slight decrease in the intensity of the
band at 275 nm (Figure 6). For complexes C2 and C3, however,
more pronounced reductions in the intensity were observed,
suggesting a greater degree of distortion of the initial DNA
structure. The apparent binding constants determined for each
complex (as indicated in the Experimental Section) are summar-
ized in Table 8. Significantly higher Kb values were found for
complexes C2 and C3 compared to C1. This difference in affinity
can be ascribed to differences in the charge of the complexes; viz.,
C1 is predominantly deprotonated at pH 7.0 (2þ charge, pKa =
6.36), whereas C2 and C3 exist as a roughly equimolar mixture of
protonated and deprotonated forms at this pH.
Cleavage of Plasmid DNA. The ability of complexes C1ꢀC3
to cleave DNA was assessed by incubating them with plasmid
pBR 322 DNA under physiological conditions (i.e., pH 7.0 and
T = 37 ꢀC). Conversion of the supercoiled form (form I) of the
DNA to the nicked circular form (form II) and linear form (form
III) was monitored by gel electrophoresis (Figure 7). In each
case, a decrease in the intensity of the band due to form I with the
incubation time was accompanied by the appearance and in-
tensification of a band corresponding to form II of the plasmid
DNA (as well as form III in the case of C1). Control experiments
revealed that no measurable DNA cleavage occurred when pBR
322 plasmid DNA was incubated with either 150 μM of the
nonmetalated ligands or 150 μM CuCl2 (Figure S22 in the
Supporting Information), clearly indicating that the observed
cleavage was due to the metal complexes. Experiments carried
out at variable concentrations of each complex revealed a
maximum rate of DNA cleavage at a concentration of 150 μM
(for typical DNA electrophoresis gels see Figures S04ꢀS06 and
Table S02 in the Supporting Information). The lower cleavage
rates observed above this concentration may be due to the
Figure 9. Stick model showing the plausible mode of interaction
between complex C1 and DNA and the subsequent attack of a copper-
(II)-bound hydroxide nucleophile on a phosphodiester linkage. Charge-
assisted hydrogen bonding between the protonated guanidine and
phosphate backbone is represented by dashed bonds (stick model for
the B-DNA duplex generated using the PDB entry: 3IXN, using the
UCSF Chimera Software Package).49
increased formation of dihydroxo-bridged dimers, which bind
to the DNA through electrostatic interactions and block access of
the cleavage-active monomeric species.31,32,41,46 A predomi-
nantly hydrolytic (as opposed to redox-mediated) mode of
cleavage was confirmed through experiments performed in the
presence of various scavengers for reactive oxygen species
(Figures S13ꢀS18 in the Supporting Information), as well as
under anaerobic conditions (Figures S19ꢀS21 in the Supporting
Information), consistent with earlier observations for other
CuIItacn-based complexes.25,34
For all of the complexes except C1, the DNA cleavage data
were satisfactorily fitted to a first-order expression, yielding the
first-order rate constant kobs for cleavage of form I DNA to
produce form II (Table 9). A single-strand cleavage model
derived by Kishikawa et al.33 was used to fit the kinetic profiles
for C1. This model accounts for not only the conversion of form I
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Figure 10. (A) Saturation kinetics of pBR 322 DNA cleavage using 50 μM C1 with different concentrations of pBR 322 DNA (25ꢀ200 μM) at 37 ꢀC in
a 40 mM HEPES buffer (pH 7.0). (B) Ionic strength dependence profile for DNA cleavage promoted by 150 μM C1 in the presence of different
concentrations of NaCl (0, 20, 100, 250, and 500 mM). The lines show the general data trends only.
to form II (k1) but also the subsequent nicking of form II to
produce form III (k2) and further cleavage of form III to produce
undetected forms of DNA (k3; see the Experimental Section for
rate equations). Figure 8 shows the best fit of the experimental
cleavage data for C1, with the corresponding values of k1, k2, and
k3 reported in Table 9.
The greater rates of DNA cleavage observed in the presence of
complexes C1ꢀC3 compared to the nonguanidinium analogues,
[Cu(Bntacn)(OH2)2]2þ and [Cu(tacn)(OH2)2]2þ, contrast
dramatically with the findings for the BNPP and HPNPP
substrates. This is especially the case for C1, which was
found to cleave BNPP and HPNPP at rates similar to
[Cu(tacn)(OH2)2]2þ, whereas plasmid DNA cleavage was
ca. 22-fold faster. These results indicate that the enhanced
nuclease activity of C1ꢀC3 could be due to them having greater
affinity for plasmid DNA than the nonguanidinylated complexes.
This conclusion is supported by the fact that these complexes
were shown to bind strongly to CT-DNA (vide supra). In this
context, the charged guanidinium pendants interact with neigh-
boring phosphodiester groups in the DNA backbone rather than
acting in concert with the copper(II) center to activate the
phosphodiester linkage undergoing cleavage. We note that it is
possible to position the X-ray crystal structure of C1 next to a
DNA double helix such that the guanidinium group forms strong
charge-assisted hydrogen bonds with a phosphodiester group,
while the copper(II) center coordinates to the oxygen atom of an
adjacent phosphodiester, with a cis-disposed hydroxo ligand
well-poised to carry out nucleophilic attack on the phosphorus
atom (Figure 9). The significantly higher activity of C1 com-
pared to C2 and C3 can also be partly rationalized in terms of the
greater acidity of this complex (pKa = 6.4 compared to 7.0 for C2
and C3), which means that, under the conditions used for the
DNA cleavage experiments, a higher concentration of the con-
jugate base, [Cu(L1Hþ)(OH)(OH2)]2þ, would be present,
leading to a faster rate of cleavage.
Figure 11. Examples of copper(II) complexes exhibiting higher nucle-
ase activity than C1.47,48
decrease the DNA cleavage reactivity (Figures 10B and S11 in the
Supporting Information), consistent with a “quenching” of
electrostatic interactions between [Cu(L1Hþ)(OH)(OH2)]2þ
(the predominant form of C1 existing at pH 7.0) and the
negatively charged phosphate backbone of DNA. Furthermore,
variation of log(kobs) with I1/2, where I = ionic strength, was
found to be linear (Figure S12 in the Supporting Information),
indicative of a bimolecular reaction between charged reactants.
Complex C1 exhibits the fastest rate of plasmid DNA cleavage
within the family of CuIItacn complexes reported so far.25,34 It is
considerably more active than the analogues featuring two
alkylguanidine pendants, as well as the copper complexes of
simple N-alkylated tacn ligands (Table 9). However, more active
synthetic nucleases have been reported, such as the copper(II)
complexes of all-cis-2,4,6-triamino-1,3,5-trihydroxycyclohexane
(TACI)47 and a 2,20-bipyridine derivative featuring guanidinium
substituents48 (Figure 11), which are 3 and 9 times more active
than C1, respectively (Table 9; it should be noted, however, that
the activity of the TACI complex was measured at pH 8.1). For
both of these complexes, secondary interactions between ancil-
lary groups and the DNA backbone have also been shown to
make an important contribution to the observed cleavage
activity. Their higher reactivities compared to that of C1 are
likely to reflect an enhanced capacity of the copper(II) centers to
access the sugarꢀphosphate backbone of the plasmid DNA
because the supporting ligand structures are less sterically
demanding than that in C1. The better DNA cleavage properties
of the CuIITACI complex also extends to conversion of the
relaxed circular DNA to linear DNA, with the reported k2 value
being 5-fold higher than that determined here for C1.
In order to help assess whether the enhanced reactivity of the
guanidinium-bearing complexes toward DNA was a reflection of
enhanced binding, we examined the dependence of the cleavage
activity on the substrate concentration and ionic strength (for
complex C1 only). Cleavage experiments performed using a
constant C1 complex concentration (50 μM) and variable DNA
concentration (25ꢀ200 μM; Figures 10A and S10 in the
Supporting Information) revealed the expected increase in the
observed cleavage rate with increasing DNA concentration, while
increasing ionic strength (adjusted by adding NaCl) was found to
’ CONCLUSION
Attachment of o-, m-, and p-xylylguanidinium pendants to the
tacn macrocycle results in copper(II) complexes with enhanced
nuclease activity compared to the “control” compounds,
[Cu(Bntacn)(OH2)2]2þ and [Cu(tacn)(OH2)2]2þ. However,
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the complexes are no more reactive toward the model com-
pounds, BNPP and HPNPP, containing single phosphodi-
ester linkages, which indicates that the guanidinium groups most
likely do not interact directly with the phosphodiester group
undergoing cleavage but rather enhance the concentration of the
complex in the vicinity of plasmid DNA by forming favorable
interactions with an adjacent phosphodiester group. For the
complex of the o-xylyl derivative (C1), a greater cleavage activity
compared to those of the m- and p-xylyl derivatives (C2 and C3)
can be ascribed to the higher acidity of coordinated water, which
generates a higher concentration of the deprotonated complex
involved in hydrolysis at neutral pH. This study further highlights
the potential to improve the activity of simple model systems
through the introduction of charged auxiliary groups but indi-
cates that a more rigid positioning of the guanidine group is
desirable to better mimic the cooperativity between metal ions
and arginine residues that occurs within the active sites of many
metallonucleases.
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Supporting Information. Crystallographic files in CIF
b
format, table of crystallographic data (Table S01), absorbance
versus [Hþ] graphs for solutions of Cu(II) complexes (Figures
S01ꢀS03), agarose gel images of DNA cleavage and kinetic
profiles (Figures S04ꢀS22 and Tables S02ꢀS11), and graphs
showing the extent of DNA cleavage in the presence of scaveng-
ing agents and under aerobic/anaerobic conditions (Figures S14,
S16, S18, and S23). This material is available free of charge via the
Internet at http://pubs.acs.org.
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Corresponding Author
*E-mail: Bim.Graham@monash.edu. (B.G.), Leone.Spiccia@
monash.edu (L.S.). Fax: þ61 3 9903 9582 (B.G.), þ61 3 9905
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Present Addresses
‡School of Chemistry, Ruprecht-Karls-Universit€at, Heidelberg,
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’ ACKNOWLEDGMENT
This work was supported by the Australian Research Council
through the Discovery Program. L.T. is the recipient of a Monash
Graduate Scholarship and Postgraduate Publication Award.
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