Synthesis of metal complexes of 2,9-bis(2-hydroxyphenyl)-1,10-phenanthroline and their DNA binding and cleaving activities

Article abstract of DOI:10.1039/a707941i

A series of metal complexes that combine the structure of phenanthroline and salen have been synthesized and characterized by electron paramagnetic resonance spectroscopy. The effects of the 2,9-bis(2- hydroxyphenyl)-1-10-phenanthroline compounds complexe

Full text of DOI:10.1039/a707941i

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Synthesis of metal complexes of 2,9-bis(2-hydroxyphenyl)-1,10-
phenanthroline and their DNA binding and cleaving activities
Sylvain Routier,a Valérie Joanny,a Anne Zaparucha,a Hervé Vezin,a
Jean-Pierre Catteau,a Jean-Luc Berniera and Christian Bailly†,b
a Laboratoire de Chimie Organique Physique associé à l’ENSCL, URA CNRS 351,
USTL Bât. C3, 59655 Villeneuve d’Ascq, France
b INSERM Unité 124 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar
Lambret, Institut de Recherches sur le Cancer, Place de Verdun, 59045 Lille, France
A series of metal complexes that combine the structure of phenanthroline and salen have been synthesized
and characterized by electron paramagnetic resonance spectroscopy. The effects of the 2,9-bis(2-
hydroxyphenyl)-1,10-phenanthroline compounds complexed with CuII, NiII, CoII or MnIII on the
temperature-dependent helix-to-coil transition of DNA have been measured. The interaction with DNA is
metal-dependent and the highest stabilization is observed with the Co complex. The DNA cleaving
activities have been studied with plasmid DNA and/or with a 32P-labelled duplex oligonucleotide
depending on the redox properties of the complexes. The Cu complex is inactive whereas the Co chelate
efficiently cleaves DNA in the presence of a reducing agent. Cleavage of DNA by the Mn complex can
occur either in the presence of a reducing agent via the production of oxygen radicals (which are detected
by EPR spectroscopy) or in the presence of an oxidant such as KHSO5. In both cases, the cleavage of
nucleic acids is very efficient whereas no cleavage is observed with the Ni complex. The complexes of
bis(hydroxyphenyl)phenanthroline with Mn and Co complement the tool-box of reagents available for
cleavage of DNA.
For a long time metallosalen catalysts have been used for select-
ive olefin epoxidation and hydrocarbon oxidation.1 Bis-
N
N
(salicylidene)ethylenediamine and other salen-type Schiff bases
can form stable square-planar complexes with metal ions such
as CuII, NiII, CoII and MnIII, forming four-membered chelate
rings via coordination of two oxygen and two nitrogen atoms.
In recent years, salen catalysis has been introduced in nucleic
acid chemistry, as a probe for investigating DNA and RNA
structure. We have previously shown that salen–copper com-
plexes interact with double-stranded DNA and can induce non-
selective cleavage in the presence of 2-mercaptopropionic acid.2
A reducing agent is also needed to promote DNA cleavage by
salen–cobalt complexes.3 In contrast, due to differing redox
properties, nickel- and manganese-bound salens produce DNA
cleavage in the presence of an oxidant.4–7
OH
HO
salen
N
N
phenanthroline
A wide range of metal complexes are available for the scis-
sion of nucleic acids. Peptides (e.g. GHK-Cu, VIHN-Ni),
polypyridyl (e.g. 2,2Ј-bipyridyl–Fe) and ethylenediamine com-
pounds can serve as chemical nucleases.8 One of the most
effective metal-based nucleases is the copper complex of 1,10-
phenanthroline which has been extensively used for mapping
protein and drug binding sites on DNA as well as for study-
ing DNA structure.8,9 It is also of considerable practical
importance as a RNA cleaver. These considerations have
prompted us to synthesize a new type of transition metal-based
DNA cleavage agent that combines the features of both salen
and phenanthroline ligand frameworks. The rationale behind
this molecular architecture is that the introduction of the
phenanthroline structure can reinforce the interaction with
DNA and may stabilize the metal complex so as to promote the
redox-dependent cleavage process. Here we report the synthesis
and DNA-binding and cleaving properties of a 2,9-bis(2-
hydroxyphenyl)-1,10-phenanthroline complexed with either
Cu, Ni, Co or Mn.
N
N
OH HO
1,10-bis(2-hydroxyphenyl)phenanthroline
Synthesis
The key intermediate for the synthesis of the different transi-
tion metal complexes was the symmetrical compound 3. Ini-
tially, the protected ligand 2 was obtained by addition of 2-
methoxyphenyllithium to 1,10-phenanthroline 1, followed by
an oxidation with MnO2.10–12 We used 40 equivs. of aryllithium
to produce the disubstituted adduct 2. Deprotection of the
methoxy group by BBr3 at low temperature10–12 afforded ligand
3. Finally, different complexations of 3 were achieved using
copper,2 nickel,13 manganese14 or cobalt15 acetates to afford
compounds 4, 5, 6 and 7, respectively.
Results and discussion
The ability of the metal complexes to alter the thermal denatur-
† Fax: (ϩ33) 320 16 92 18; E-mail: bailly@lille.inserm.fr
J. Chem. Soc., Perkin Trans. 2, 1998
863

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a
OMe
, Li, Et2O
Br
N
O
N
O
b MnO2, CH2Cl2
N
N
1
2
BBr3
CH2Cl2
N
N
OH HO
3
a Cu(OAc)2, 1H2O, MeOH
b Ni(OAc)2, 4H2O, MeOH
c Mn(OAc)2, 4H2O, LiCl, MeOH
d Co(OAc)2, 4H2O, MeOH
a
c
d
b
N
N
N
N
N
N
Mn3+
N
N
Ni2+
Cu2+
Co2+
O–
–O
O–
–O
O–
–O
O–
–O
Cl–
4
5
6
7
ation profile of DNA can be used as an indication of their
propensity to bind to DNA. The complexes formed between
DNA and compounds 4–7 displayed in each case simple,
monophasic, melting curves (the transition remains sharp even
though the Tm changes). The effects of increasing the concen-
tration of compounds 4–7 on the Tm of the helix-to-coil transi-
tion of calf thymus DNA are shown in Fig. 1. The ∆Tm values
correspond to the difference between the Tm measured in
the presence of the ligand and that obtained with the free
DNA. A large increase in the Tm of nucleic acids is observed
for compound 7 and to a lower extent with compound 5
whereas the stabilisation of the duplex structure is weak with
compound 6 and very little effect was observed with the
copper complex 4. The stabilization of the DNA double helix
by binding of the Co complex increases smoothly with an
increasing molar ratio of drug to DNA-phosphate (D/P). The
stabilizing action of the Ni complex is substantial but always
smaller than that of the Co analogue. The complexes rank in the
order 7–Co > 5–Ni > 6–Mn > 4–Cu. The effect is manifestly
metal-dependent.
Due to different redox properties, the metal complexes were
studied separately depending on the requirement of a reducing
or an oxidizing cofactor to initiate the cleavage reaction. In
parallel to the DNA cutting studies, we performed a series of
electron paramagnetic resonance (EPR) measurements either to
identify the metal complex formation or to detect the produc-
tion of oxygen-based radicals by means of spin-trapping.
Fig.
1
Variation in difference melting temperature (∆Tm =
Tmligand-DNA Ϫ TmDNA) measured with the four metal complexes at
different ligand:DNA-phosphate molar ratios. Tm measurements were
performed in BPE buffer pH 7.1 (6 m Na2HPO4, 2 mM NaH2PO4,
1m EDTA) using 20 µ calf thymus DNA (nucleotide conc) in 3 ml
quartz cuvettes at 260 nm with a heating rate of 1 ЊC minϪ1. Each drug
concentration was tested in duplicate. The Tm of the DNA alone is
66 1 ЊC.
Cu and Co complexes
The Co complex acts as a potent DNA cleaver in the presence
of a reducing agent whereas, surprisingly, under identical con-
ditions the Cu complex is totally inactive. No sharp EPR signal
was observed with the copper complex attesting that the Cu
atom is not properly complexed. Only a broad and unresolved
spectrum was obtained from a frozen solution of 4. The
absence of a stable copper complex probably explains the lack
of hydroxyl radical production and DNA cleavage. In contrast,
the Co atom is perfectly complexed. Previous studies have
shown that Co complexes give rise to an EPR spectrum typical
864
J. Chem. Soc., Perkin Trans. 2, 1998

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Fig. 4 Cleavage of closed circular pUC12 DNA (form I) by com-
pound 6–Mn in the presence of ascorbic acid as reducing agent. Forms
II and III refer to the nicked and linear DNA forms respectively. The
drug concentration (µ) is indicated at the top of each gel lane. The
lanes marked DNA and asco refer to the plasmid DNA incubated
without drug in the absence and presence of ascorbic acid, respectively.
Fig. 2 EPR spectrum of CoII–O2 adduct complex at 77 K. Experi-
mental settings: 20 mW power, 10 G modulation amplitude, gain 104
and scan rate 4 min.
Fig. 3 Cleavage of closed circular pUC12 DNA (form I) by com-
pounds 4–Cu and 7–Co in the presence of 2-mercaptopropionic acid
(MPA) as reducing agent. Forms II and III refer to the nicked and
linear DNA forms respectively. The drug concentration (µ) is indi-
cated at the top of each gel lane. The lanes marked DNA and MPA
refer to the plasmid DNA incubated without drug in the absence and
presence of MPA, respectively. The DNA was incubated with the metal
complex for (A) 1 h or (B) 6 h.
Fig. 5 EPR signal of DMPO᎐OOH adduct with complex 6 (upper
trace) and DMPO control (lower trace) at room temperature. Experi-
mental settings: 20 mW power, 1 G modulation amplitude, gain
3.2 × 104 and scan rate 4 min.
of a planar four coordinated CoII type with nearly axial sym-
metry and an eight-line parallel hyperfine pattern due to inter-
action with the 59Co (I = 7/2) nucleus.16 Such a signal provides
when the reaction is longer (6 h, Fig. 3, panel B) whereas the
Cu complex remains inactive. The Coؒbis(hydroxyphenyl)phen-
anthroline derivative 7 complements the tool-box of reagents
which can be utilized to produce single-strand cleavage of
DNA.
Co
EPR parameters of g|| = 2.025 and g⊥ = 2.3 and A ≈ 100 G,
||
characteristic of a low-spin CoII form. Spin-trapping experi-
ments failed to provide evidence for the formation of oxygen
radicals. However, the Co complex reacts in the presence of
oxygen. Under oxygenation the EPR parameters are drastically
changed. As shown in Fig. 2, the spectrum of complex 7
exhibits the following EPR parameters: g|| = 2.098, g⊥ ≈ 2.0 and
a hyperfine splitting constant A of 23 G due to 59Co. Such EPR
parameters are consistent with an oxygen adduct complex17
and suggest that the unpaired spin density no longer resides on
the cobalt metal center but instead on the dioxygen moiety.18
For both the Cu and Co complexes, DNA cleavage was ana-
lysed by monitoring the conversion of supercoiled plasmid
DNA (form I) to the nicked circular molecules (form II) and
linear DNA (form III). The tests were performed under aerobic
conditions in the presence of 2-mercaptopropionic acid (MPA)
as a reducing agent. No cleavage occurs in the absence of MPA
or with uncomplexed Cu or Co (not shown). As shown in Fig.
3, the Co chelate efficiently cleaves DNA. Incubation of the
plasmid at 37 ЊC for 1 h with 50 µ of compound 7 causes the
complete conversion of form I to the nicked form II. We there-
fore conclude that the activation of the cobalt complex leads
mainly to single strand cleavage of duplex DNA. As the ligand
concentration increases the probability of double-strand scis-
sions is enhanced once the DNA has undergone a single-strand
break. This is manifested in the gel by the appearance of linear-
ized DNA molecules (form III). The percentage of linear DNA
molecules observed with the Co complex is much higher
Mn complex
In this case the cleavage reaction can be activated by the add-
ition of either ascorbic acid (reduction of MnIII to MnII) or an
oxidant such as KHSO5 (oxidation of MnIII to MnV). The two
chemical pathways were tested.
A typical gel showing the cutting of DNA in the presence of
the Mn complex is shown in Fig. 4. As with the Co complex,
supercoiled DNA is easily converted into the nicked and linear
forms. No cleavage was seen in the control lane in the absence
of ascorbate which is required for the reduction of the
manganese.
The complex 6 MnIII redox state is an ‘EPR silent’ species due
to its spin state S = 2. Therefore no relevant EPR signal of the
metal could be detected. However, we employed EPR spec-
troscopy to monitor the production of oxygen radicals by spin-
trapping using 5,5Ј-dimethylpyrroline N-oxide (DMPO). Fig. 5
shows the signal of the DMPO᎐OOH spin adduct with the
characteristic hyperfine splitting constants aN = 14.0 and
aH = 12.1 G which reflect the production of the superoxide
anion radical O2 Ϫ by compound 6.
᝽
The interaction of ascorbate (AHϪ) with MnIII complex
resulted in two superimposed EPR signals (Fig. 6). The first one
is characterised by a doublet centered around a g value of 2.005
J. Chem. Soc., Perkin Trans. 2, 1998
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Ϫ
Fig. 6 EPR signal of DMPO᎐CH3 adduct and ascorbyl radical (A
)
᝽
with complex 6 in the presence of ascorbic acid at room tem-
perature. Experimental settings: 20 mW power, 1 G modulation
amplitude, gain 3.2 × 103 and scan rate 4 min.
Fig. 7 EPR signal of MnII reduced form of complex 6 with signal of
DMPO᎐CH3 adduct and ascorbyl radical in the presence of ascorbic
acid at room temperature. Experimental settings: 20 mW power, 5 G
modulation amplitude, gain 2 × 103 and scan rate 4 min.
and a hyperfine splitting constant aH = 1.8 G with a relative
intensity of 1:1 for both peaks. The second one corresponds to
the spin adduct DMPO᎐CH3 with hyperfine splitting constants
aN = 23.0 and aH = 16.1 G. For these spin-trapping experiments,
the metal complexes, including compound 6-Mn, were dis-
solved in pure DMSO at a concentration of 1 m. The
DMPO᎐CH3 spin adduct indicates the production of hydroxyl
radicals which then react with DMSO to give the methyl rad-
icals. No DMPO᎐OH signal was detected. The use of pure
DMSO explains the complete loss of the DMPO᎐OH spin
adduct. It must be noted that the spectrum lineshape is modi-
fied indicating that another paramagnetic species is overlapped.
This corresponds to the reduction of the MnIII metal center
upon interaction with ascorbate. The reduction of MnIII yields
the Kramer ion MnII (S = 5/2) detected by EPR spectroscopy.
The spectrum in Fig. 7 shows six lines centered around a g value
of 2.0 with hyperfine splitting constant A|| = 90 G. The cleavage
of DNA in the presence of compound 6 is therefore attributable
to the hydroxyl radicals produced upon the ascorbate-induced
reduction of the Mn ion. These hydroxyl radicals may arise via
autooxidation of the low valent state with consequent form-
ation of superoxide radicals and hence hydrogen peroxide.
Cleavage of DNA by salen ligands complexed with Ni and
Mn can be achieved in the presence of oxygen donor com-
pounds such as potassium monoperoxysulfate (KHSO5) and
magnesium monoperoxyphthalic acid (MMPP). Under these
conditions, the plasmid assay employed with the other com-
plexes is inapplicable because the DNA is partially cleaved
in the presence of the activator alone. We therefore tested
the cleavage activity of compounds 5 and 6 using a synthetic
Fig. 8 Cleavage of the 32P-labelled duplex oligonucleotides (2 µ) by
compounds 5 and 7 at 25 and 50 µ in the presence of KHSO5.
The sequence of the labelled strand is indicated.
with polynucleotide kinase. After hybridisation with the com-
plementary sequence and gel purification, the 20-mer DNA was
subjected to cleavage by compounds 5 and 6 in the presence of
KHSO5. The cutting sites were visualized on the autoradio-
grams after boiling the DNA samples with piperidine. Practic-
ally no cleavage can be detected without alkaline treatment of
the oxidized products. Also, there was absolutely no cleavage
with the uncomplexed Ni and Mn metals or with KHSO5 alone
(not shown). As shown in Fig. 8, the Ni complex turned out to
be inactive whereas the Mn complex cleaves DNA efficiently.
The cleavage, which is probably mediated by a [salen᎐MnV᎐O]ϩ
intermediate, is essentially non-selective. Apart from the two T
residues on the 3Ј side of the GG doublet which are weakly
cleaved, all the other bases are attacked by the Mn complex.
According to previous studies by Burrows and Rokita4 and
ourselves,13 salen–Ni complexes can only cleave DNA at access-
ible guanine residues. The lack of cleavage of the 20-mer oligo-
nucleotide by compound 5–Ni may be attributed to the duplex
structure that does not present unpaired guanines. However, we
also tested the single-strand oligonucleotide with two adjacent
unpaired guanine residues but no cleavage was observed with
the Ni complex (data not shown).
oligonucleotide
duplex.
The
oligonucleotide
5Ј-
In conclusion, we have designed a new type of chemical
nuclease based on the 2,9-bis(2-hydroxyphenyl)-1,10-phen-
CTCTTTTTGGTTATTCCC-3Ј was 32P-labelled at the 5Ј-end
866
J. Chem. Soc., Perkin Trans. 2, 1998

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anthroline structure. There is a good agreement between the
EPR data and the results of the DNA binding and cleaving
experiments. The geometry of the designed ligand is apparently
not well suited for complexation with Cu and Ni but is adequate
for complex formation with Co and Mn. The ligand forms
stable complexes with both Co and Mn. The Mn complex pro-
duces oxygen radicals that are responsible for the efficient
cleavage of DNA under reducing conditions. A different mech-
anism must take place with the Co complex which does not
generate detectable oxygen radicals but, however, behaves as a
potent DNA cleaver. More elaborate analyses are required to
define the nature of the intermediates and the exact mechanism
of cleavage by the Co complex. The Mn complex can also func-
tion under oxidative conditions. With compound 6, the object-
ive of designing a versatile chemical nuclease has been fulfilled;
this complex is now used as a footprinting probe for studying
drug binding to nucleic acids.
CH2Cl2, was added dropwise over 1 h. After addition, the reac-
tion mixture was stirred for 48 h. After hydrolysis was per-
formed with 25 cm3 of concentrated NaHCO3, the precipitate
was filtered and treated three times with 20 cm3 of MeOH
and ca. 30 mm3 H2SO4. Solvents were distilled under reduced
pressure. Crude material was purified by flash chromato-
graphy (CH2Cl2) to afford compound 3 as a white solid (46%,
0.220 g), mp 230–232 ЊC; νmax(KBr)/cmϪ1 3450, 2960, 1620;
m/z (MALDIϩ) 365.0 (M ϩ 1)ϩ, 387.0 (M ϩ Na)ϩ, 403.0
(M ϩ K)ϩ; Rf (CH2Cl2) 0.4; δH([2H6]DMSO) 7.00 (td, J 8.3, 1.2,
2H), 7.10 (d, J 8.1, 2H), 7.40 (t, J 7.0, 2H), 8.00 (s, 2H), 8.20 (d,
J 7.9, 2H), 8.55 (d, J 8.7, 2H), 8.65 (d, J 8.7, 2H), 10.15 (s, 2H);
δC([2H6]DMSO) 117.82 (CH), 119.09 (CH), 120.28 (Cq), 121.18
(CH), 126.20 (CH), 127.30 (Cq), 128.53 (CH), 131.94 (CH),
138.20 (CH), 141.45 (Cq), 157.04 (Cq), 159.09 (Cq). Anal. calc.
for C24H16N2O2: C, 79.09; H, 4.43; N, 7.69. Found: C, 79.15;
H, 4.35; N, 7.74%.
2,9-Bis(2-hydroxyphenyl)-1,10-phenanthroline copper(II)
complex 4
Experimental
Materials
The compound 3 (0.1 g, 0.27 mmol) was dissolved in 2.2 cm3 of
dry ethanol. Then 2 equivs. of NaOH (0.02 g, 0.54 mmol) in 0.5
cm3 of EtOH were added dropwise. After stirring for 15 min, a
solution of Cu(OAc)2ؒ1H2O (0.109 g, 0.54 mmol) in water (0.7
cm3) was added. The reaction mixture was refluxed for 1.5 h,
then concentrated under reduced pressure. After filtration, the
residue was washed with ethanol. Compound 4 (60 mg, 52%)
was obtained as a brown solid, mp >240 ЊC; νmax(KBr)/cmϪ1
2930, 2850, 1600, 1560, 1500; m/z (MALDIϩ) 427.0
(M ϩ 1)ϩ,448.1(M ϩ Na)ϩ, 492.2 (M ϩ K)ϩ, 852.9 (2M ϩ 1)ϩ;
Rf (CH2Cl2) 0.2. Anal calc. for C24H14N2O2Cu: C, 67.76; H,
3.32; N, 6.59. Found: C, 67.90; H, 3.29; N, 6.51%.
All chemicals were purchased from Aldrich Chemical Co. Solv-
ents were distillated under argon prior to use. The purity of all
1
compounds were assessed by TLC, H and 13C NMR spec-
troscopy, and by mass spectrometry. Kieselgel 60 (004–0063
mesh) was used for flash chromatography columns. TLC was
carried out using silica gel 60F-254 (0.25 mm thick) precoated
UV sensitive plate. Spots were visualized by inspection under
visible light or UV at 254 nm. Melting points were determined
using a hot plate microscope and are uncorrected. NMR spec-
tra were recorded on a Bruker AC 300 NB. Chemical shifts were
reported using trimethylsilane as an internal reference and are
given in δ units and coupling constants (J) are given in Hz.
MALDI (Mass Assisted Laser Desorption Ionisation) mass
spectra were determined on a Finigan MAT vision 2000
(Bremen). The matrix used was dihydroxybenzoic acid–water.
2,9-Bis(2-hydroxyphenyl)-1,10-phenanthroline nickel(II)
complex 5
Compound 3 (0.27 mmol, 0.1 g) was dissolved in 2.2 cm3 of dry
ethanol. Then 2 equivs. of NaOH (0.02 g, 0.54 mmol) in 0.5 cm3
of ethanol were added dropwise. After 15 min, Ni(OAc)2ؒ4H2O
(0.13 g, 0.54 mmol) was poured into the solution. The solvent
was removed under reduced pressure and the crude product was
washed with cold methanol. Compound 5 (40%, 44 mg) was
obtained as a orange powder, mp >240 ЊC; νmax(KBr)/cmϪ1
3050, 2930, 2850, 1600, 1580, 1500; m/z (MALDIϩ) 421.4
(M ϩ 1)ϩ, 443.4 (M ϩ Na)ϩ; Rf (CH2Cl2) 0.3; δH([2H6]DMSO)
7.03 (t, J 6.84, 2H), 7.09 (d, J 7.81, 2H), 7.41 (t, J 7.81, 2H), 8.09
(s, 2H), 8.28 (d, J 8.3, 2H), 8.61 (d, J 8.79, 2H), 8.71 (d, J 8.79,
2H); δC([2H6]DMSO) 117.83 (CH), 119.14 (CH), 120.63 (Cq),
121.30 (CH), 126.27 (CH), 127.36 (Cq), 128.59 (CH), 131.97
(CH), 138.27 (CH), 141.57 (Cq), 157.11 (Cq), 159.08 (Cq).
Anal. calc. for C24H14N2O2Ni: C, 68.56; H, 3.36; N, 6.67.
Found: C, 68.48; H, 3.30; N, 6.65%.
2,9-Bis(2-methoxyphenyl)-1,10-phenanthroline 2
A solution of 2-bromoanisole (24 g, 128 mmol) in 12 cm3 of dry
diethyl ether, was added dropwise under argon to a suspension
of lithium (1.97 g, 256 mmol) in 10 cm3 of dry diethyl ether.
After addition, the solvent was refluxed for 1 h to complete the
reaction. The reaction mixture was cooled to room temperature
and a solution of 1,10-phenanthroline (1.12 g, 6.4 mmol) in 30
cm3 of toluene was added dropwise. The reaction mixture was
then refluxed for 3 h and stirred for an additional 15 h at room
temperature. The solution was cooled at 0 ЊC and quenched by
addition of 64 cm3 of dry methanol and 64 cm3 of water. The
reaction mixture was concentrated under reduced pressure and
the aqueous solution extracted three times with 70 cm3 of
CH2Cl2. The combined organic phases were mixed with MnO2
(50 g, 0.57 mol) for 3 h. After drying with MgSO4 and filtration
under Celite, the solvent was removed by distillation under
reduced pressure. The product was purified by flash chrom-
atography [light petroleum (bp 45–65 ЊC)–CH2Cl2 1:1] to
afford compound 4 (1.04 g, 52%) as a pale yellow solid, mp
104–106 ЊC; νmax(KBr)/cmϪ1 2965, 1590, 1240; m/z (MALDIϩ)
393.1 (M ϩ 1)ϩ; Rf (Et2O–CH2Cl2 90:10) 0.65; δH(CDCl3) 3.90
(s, 6H), 7.05 (d, J 8.3, 2H), 7.20 (td, J 7.5, 1, 2H), 7.40 (td, J 7.5,
1.7, 2H), 7.80 (s, 2H), 8.20 (d, J 1.7, 4H), 8.30 (dd, J 7.5, 1.7,
2H); δC(CDCl3) 55.79 (CH3), 111.56 (CH), 120.56 (CH), 124.86
(CH), 125.84 (CH), 128.00 (Cq), 129.81 (Cq), 130.25 (CH),
132.46 (CH), 135.28 (CH), 146.24 (Cq), 156.26 (Cq), 157.57
(Cq). Anal. calc. for C26H20N2O2: C, 79.56; H, 5.14; N, 7.14.
Found: C, 79.61; H, 5.09; N 7.18%.
2,9-Bis(2-hydroxyphenyl)-1,10-phenanthroline manganese(III)
chloride complex 6
Under argon, compound 3 (0.1 g, 0.27 mmol) was dissolved in
2.2 cm3 of dry ethanol. Then 2 equivs. of NaOH (0.02 g, 0.54
mmol) in 0.5 cm3 of freshly distilled ethanol were added drop-
wise. After 15 min, the solution was refluxed and the complex
was obtained by addition of Mn(OAc)2ؒ4H2O (0.134 g, 0.54
mmol) over 1.5 h. LiCl (0.104 g, 0.54 mmol) was then added to
the reaction mixture which was placed in air and refluxed for
an additional 30 min. The solvent was removed under vacuum.
Water (25 cm3) was added and the precipited was filtered and
washed with cold ethanol. Compound 6 (58%, 72 mg) was
obtained as a brown powder, mp >240 ЊC; νmax(KBr)/cmϪ1
3030, 2900, 2830, 1595, 1560, 1550, 1490; m/z (MALDIϩ)
417.2 (M Ϫ Cl)ϩ, 833.8 (2M Ϫ 2Cl)2ϩ, m/z (MALDIϪ) 34.9
(ClϪ), 36.9 (ClϪ); Rf (CH2Cl2) 0.0. Anal. calc. for C24H14-
N2O2MnCl: C, 63.72; H, 3.12; N, 6.20. Found: C, 63.68; H,
3.08; N, 6.15%.
2,9-Bis(2-hydroxyphenyl)-1,10-phenanthroline 3
In a 50 cm3 flask flushed with argon, compound 4 (0.5 g, 1.3
mmol) was dissolved in 12 cm3 of dry CH2Cl2 and cooled to
Ϫ78 ЊC. A solution of BBr3 (4.46 g, 18 mmol) in 5 cm3 of
J. Chem. Soc., Perkin Trans. 2, 1998
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2,9-Bis(2-hydroxyphenyl)-1,10-phenanthroline cobalt(II)
complex 7
ATP (6000 Ci mmolϪ1) and T4 polynucleotide kinase and then
mixed with the complementary oligonucleotide sequence. The
20-mer duplex formed after cooling was purified on a 15% non-
denaturing polyacrylamide gel. DNA samples were treated with
the metal complex in the presence of 50 µ KHSO5 for 30 min
at room temperature and then precipitated with cold ethanol.
The DNA pellet was resuspended in 30 µl of 1  piperidine,
boiled for 12 min at 90 ЊC and then lyophilized. Cleavage prod-
ucts were resuspended in 5 µl of 80% formamide containing 10
m EDTA and 0.1% tracking dyes. Samples were heated to
90 ЊC for 4 min and then chilled in an ice-bath just before being
loaded on a sequencing gel (15% polyacrylamide, 7  urea).
Under argon, compound 3 (0.05 g, 0.13 mmol) was dissolved in
25 cm3 of deoxygenated methanol. The solution was stirred
vigorously and Co(OAc)2ؒ4H2O (0.035 g, 0.14 mmol) was then
added. The solution was refluxed for 3 h. After cooling, the
precipitate was filtered off and washed three times under argon
with dry methanol. Complex 7 was obtained as a red powder
(83%, 0.045 g), mp > 240 ЊC; νmax(KBr)/cmϪ1 3020, 2950, 1600,
1590, 1540, 1510, 1490; m/z (MALDIϩ) 421.3 (M) ϩ, 422.5
᝽
(M ϩ 1)ϩ, 444.5 (M ϩ Na)ϩ, 460.5 (M ϩ K)ϩ; Rf (CH2Cl2) 0.3.
Anal. calc. for C24H14N2O2Co: C, 68.40; H, 3.35; N, 6.65.
Found: C, 68.45; H, 3.29; N, 6.70%.
Acknowledgements
This work was carried out under the support of research grants
(to J. L. B.) from the CNRS and (to C. B.) from the Ligue
Nationale Contre le Cancer (comité du Nord).
Melting temperature studies
Melting curves were measured using a Uvikon 943 spectro-
photometer coupled to a Neslab RTE111 cryostat. For each
series of measurements, 12 samples were placed in a thermo-
statically controlled cell-holder (10 mm pathlength) and the
quartz cuvettes were heated by circulating water. The measure-
ments were performed in BPE buffer pH 7.1 (6 m Na2HPO4, 2
m NaH2PO4, 1 m EDTA). The temperature inside the
cuvette was monitored by using a thermocouple in contact with
the solution. The absorbance at 260 nm was measured over the
range 20–100 ЊC with a heating rate of 1 ЊC minϪ1. The ‘melt-
ing’ temperature Tm was taken as the mid-point of the hyper-
chromic transition.
References
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Product analysis by EPR experiments
The X-band EPR spectra of the metal complexes (1 m in
DMSO) were recorded at 77 K or at room temperature using a
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strong pitch (g = 2.0028) as a standard. For spin trapping
experiments 5 m 5,5Ј-dimethylpyrroline N-oxide (DMPO)
were added to the metal complex solution at 1 m.
DNA cleaving activity
Experiments with plasmid DNA on agarose gels. Each reac-
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µg), 5 µl of the tested compound at the desired concentration
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glycerol in H2O) was added to each tube and the solution was
loaded on to a 1% agarose gel. The electrophoresis was carried
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8.3, 1 m EDTA). Gels were stained with ethidium bromide
(1 µg mlϪ1) then destained for 30 min in water prior to being
photographed under UV light.
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Paper 7/07941I
Received 4th November 1997
Accepted 29th January 1998
Experiments with 32P-labelled DNA on sequencing gels. The
synthetic oligonucleotide was labelled at the 5Ј-end with γ-[32P]-
868
J. Chem. Soc., Perkin Trans. 2, 1998