Synthesis of a functionalized salen-copper complex and its interaction with DNA

Article abstract of DOI:10.1021/jo951840c

An original procedure for efficient synthesis of a functionalized salen·copper complex is reported. The mode of binding to DNA of the salen·Cu(II) complex was investigated by viscometry as well as by absorption, circular, and linear dichroism spectroscopy. The complex can induce DNA strand breakage in the presence of a reducing agent as revealed by a plasmid cleavage assay. The spectroscopic and biochemical data indicate that the salen·Cu(II) complex induces single-stranded breaks via an interaction within one of the grooves of the double helix.

Full text of DOI:10.1021/jo951840c

2326
J . Org. Chem. 1996, 61, 2326-2331
Syn th esis of a F u n ction a lized Sa len -Cop p er Com p lex a n d Its
In ter a ction w ith DNA
Sylvain Routier,† J ean-Luc Bernier,†,* Michael J . Waring,‡ Pierre Colson,§
,
Claude Houssier,§ and Christian Bailly *
Laboratoire de Chimie Organique Physique, URA CNRS 351, USTL Baˆt. C3, 59655 Villeneuve d’Ascq,
France, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ ,
England, Laboratoire de Chimie Macromole´culaire et Chimie Physique, Universite´ de Lie`ge, Lie`ge 4000,
Belgium, and Institut de Recherches sur le Cancer, INSERM U 124, Place de Verdun 59045 Lille, France
Received October 12, 1995X
An original procedure for efficient synthesis of a functionalized salen‚copper complex is reported.
The mode of binding to DNA of the salen‚CuII complex was investigated by viscometry as well as
by absorption, circular, and linear dichroism spectroscopy. The complex can induce DNA strand
breakage in the presence of a reducing agent as revealed by a plasmid cleavage assay. The
spectroscopic and biochemical data indicate that the salen‚CuII complex induces single-stranded
breaks via an interaction within one of the grooves of the double helix.
In tr od u ction
Resu lts a n d Discu ssion
Syn th esis (Sch em e 1). The key intermediate for the
synthesis of the functionalized salen‚copper complex 1
is the asymmetric R,â-diamine 5 which was synthesized
following a sequential method.5 The amide 3 was ob-
tained from the commercially available NR-Z-Nꢀ-BOC-L-
lysine 2 after treatment with ethyl chloroformate in
ammonia-saturated THF. Dehydration of the carboxa-
mide 3 with trifluoroacetic anhydride in the presence of
triethylamine furnished the nitrile 4 which was then
subjected to hydrogenation (over Raney-Ni) under high
pressure. The benzyloxycarbonyl protecting group was
cleaved during this reaction (hydrogenolysis). The R,â-
diamine 5 was then condensed with salicylaldehyde in
the presence of cuprous acetate monohydrate to give the
BOC-protected compound 6. Finally, deprotection under
acid conditions and purification afforded the salen‚Cu
complex 1 in 61% yield. The ESR parameters (g| ) 2.23
and A| ) 191 G) for the CuII‚salen complex 1 are typical
of a square planar complex. It is noteworthy that the
chemical procedure outlined in Scheme 1 is generally
applicable for the synthesis of other metal-salen-amino
acid complexes. Recently, the synthesis of Ni and Mn‚-
salen complexes has been successfully completed using
the same route.
There is continuing interest in DNA-binding metal
complexes. Cu‚phenanthroline, Fe‚EDTA, Mn‚porphyrin,
and Ru‚polypyridyl complexes are typical examples of
metal chelates capable of triggering single- and/or double-
stranded DNA cleavages after activation with appropri-
ate reducing agents.1 Studies on their DNA binding/
cleaving properties are of great importance for the design
of sequence-specific or stereospecific artificial nucleases.
Metal complexes of salen [bis(salicylidene)ethylenedi-
amine]2 can also induce DNA cleavage3 but, so far as we
are aware, their mechanism of binding to nucleic acids
has received little attention.4 In this paper, we report
an original procedure for efficient synthesis of a func-
tionalized salen‚Cu complex (1). Preliminary studies on
the DNA-binding and DNA-cleaving properties of this
copper complex using viscometry, measurements of ab-
sorption, circular and electric linear dichroism, and gel
electrophoresis are described.
Bin d in g to DNA. We set out to define the mode of
interaction between the copper‚salen complex 1 and calf
thymus DNA using a combination of spectroscopic and
hydrodynamic measurements.
† CNRS URA351.
Absor p tion . The electronic absorption spectrum of
compound 1 is significantly perturbed on binding to DNA.
Relative to the absorption spectrum of the DNA-free
compound, the absorption band centered at 346 nm is
red-shifted by 3 nm and displays 13% hypochromism
upon interaction of the ligand with DNA (not shown).
Attempts to determine binding isotherms were made by
titrating measured quantities of a stock solution of
compound 1 into a known volume of calf thymus DNA
solution and monitoring the resulting changes in the
absorption spectrum of the ligand. The bathochromic
shift proved too weak to permit a clear distinction
between free and bound molecules and consequently
‡ University of Cambridge.
§ Universite´ de Lie`ge.
INSERM U124.
* Corresponding authors: J . L. Bernier, Fax (+33) 20 43 65 61; C.
Bailly, Fax (+33) 20 52 70 83.
X Abstract published in Advance ACS Abstracts, March 15, 1996.
(1) (a) Barton, J . K. Science 1986, 233, 727-734. (b) Sigman, D. S.
Acc. Chem. Res. 1986, 19, 180-186. (c) Stubbe, J .; Kozarich, J . W.
Chem. Rev. 1987, 87, 1107-1136. (d) Sigman, D. S.; Mazumder, A.;
Perrin, D. M. Chem. Rev. 1993, 93, 2295-2316. (e) Pratviel, G.;
Bernadou, J .; Meunier, B. Angew. Chem., Int. Ed. Engl. 1995, 34, 746-
769.
(2) Pedersen, C. J . Science 1968, 241, 536-540.
(3) (a) Gravert, D. J .; Griffin, J . H. J . Org. Chem. 1993, 58, 820-
822. (b) Burrows, C. J .; Rokita, S. E. Acc. Chem. Res. 1994, 27, 295-
301. (c) Woodson, S. S.; Muller, J . G.; Burrows, C. J .; Rokita, S. E.
Nucleic Acids Res. 1993, 21, 5524-5525. (d) Bhattacharya, S.; Mandal,
S. S. J . Chem. Soc., Chem. Commun. 1995, 2489-2490.
(4) Sato, K.; Chikira, M.; Fujii, Y.; Komatsu, A. J . Chem. Soc., Chem.
Commun. 1994, 625-626.
(5) Houssin, R.; Bernier, J .-L.; He´nichart, J .-P. Synthesis 1988, 259-
260.
0022-3263/96/1961-2326$12.00/0 © 1996 American Chemical Society

Synthesis of a Functionalized Salen-Copper Complex
J . Org. Chem., Vol. 61, No. 7, 1996 2327
Sch em e 1
DNA induces significant changes in its CD spectrum
(Figure 2a). The band at 330 nm decreases as the DNA
concentration is raised; meanwhile the CD in the 350-
420 nm region increases significantly. The two isodich-
roic points at 318 and 354 nm and the constant spectral
proportions indicate the existence of a single binding
mode. Thus the binding appears geometrically homoge-
neous.
To evaluate the binding selectivity of compound 1 vis-
a`-vis GC or AT base pairs, further CD titration experi-
ments were performed using synthetic polynucleotides
having defined base compositions. As observed with calf
thymus DNA, addition of the alternating copolymer poly-
(dA-dT)‚poly(dA-dT) induces significant changes in the
CD spectrum of the salen‚Cu complex (Figure 2b).
However, the variation in CD intensity is less pronounced
than seen with calf thymus DNA which contains a nearly
equal proportion of AT and GC base pairs. By contrast,
the CD spectrum of the ligand is little affected by addition
of poly(dG-dC)‚poly(dG-dC) (Figure 2c), therefore sug-
gesting that compound 1 binds preferentially to AT
sequences rather than to GC sequences.6 A preference
for AT sequences has also been reported for MnIII‚salen
complexes.3a Substitution of inosine for guanosine does
not markedly promote binding of 1 to the polymer (Figure
2d). The CD spectra of complexes between coumpound
1 and either poly(dG-dC)‚poly(dG-dC) or poly(dI-dC)‚poly-
(dI-dC) at a polynucleotide/ligand ratio of 20 are almost
identical. In neither case does a positive CD band appear
at 390 nm, suggesting that the ligand has minimal
interaction with the GC- and IC-containing polynucle-
otides. In other words, unlike what we found recently
with AT-specific (e.g. netropsin, Hoechst 33258, berenil,
DAPI) and GC-specific (e.g. mithramycin, chromomycin)
minor groove binders and with intercalating drugs (e.g.
actinomycin, echinomycin),7 removal of the 2-amino group
of guanine (which is exposed in the minor groove of DNA)
has practically no influence on the binding of the salen‚-
Cu complex 1 to DNA.
F igu r e 1. Viscometric titrations of pUC12 closed circular
duplex DNA with compound 1 (J ) and ethidium bromide (E).
The flow time is plotted as a function of the molar ratio of
drug added per DNA nucleotide.
attempts to estimate the binding constant from conven-
tional Scatchard analysis failed. As an alternative
procedure to examine the strength of the drug-DNA
interaction, we employed fluorescence spectroscopy to
investigate competition between the test drug and the
intercalating drug ethidium bromide for available binding
sites. The concentrations of compound 1 required to
reduce by 50% the fluorescence of the DNA-ethidium
complex under standard conditions (Q50) were 12 µM with
calf thymus DNA, 21 µM with poly(dA-dT).poly(dA-dT)
and >50 µM with poly(dG-dC)‚poly(dG-dC). The finding
that the drug diminishes the fluorescence of the ethidium-
poly(dA-dT)‚poly(dA-dT) complex more efficiently than
the ethidium-poly(dG-dC)‚poly(dG-dC) complex is con-
sistent with the CD data (see below) suggesting that the
drug interacts preferentially with AT rather than GC
sequences. It speaks for an affinity of the drug toward
the AT polynucleotide which is at least 2-fold higher than
that for the GC polymer.
(6) Footprinting experiments using DNAase I as cleaving agent have
failed to confirm the apparent preference for binding to AT sequences
inferred from the CD titration experiments. The lack of detectable
footprints may be due to the low affinity of the salen‚Cu complex for
DNA and possibly from the mechanism of recognition of DNA by
DNAase I. The nuclease binds to the minor groove of DNA whereas
compound 1 is suspected to interact within the major groove.
(7) (a) Waring, M. J .; Bailly, C. Gene 1994, 149, 69-79. (b) Bailly,
C.; Waring, M. J . Nucleic Acids Res. 1995, 23, 885-892. (c) Bailly, C.;
Waring, M. J . J . Am. Chem. Soc. 1995, 117, 7311-7316. (d) Bailly, C.;
Colson, P.; He´nichart, J .-P., Houssier, C. Nucleic Acids Res. 1993, 21,
3705-3709.
Viscom etr y. Compound 1 causes practically no change
in the viscosity of a supercoiled DNA solution whereas a
classical rise and fall in viscosity is observed with
ethidium bromide (Figure 1). Apparently, binding of the
salen‚Cu complex to DNA does not induce any significant
change in the winding of the helix.
Cir cu la r Dich r oism (CD). The CD spectrum of the
Cu‚salen chelate 1 in solution reflects the asymmetric
structure of the salen moiety. Addition of calf thymus

2328 J . Org. Chem., Vol. 61, No. 7, 1996
Routier et al.
F igu r e 2. Circular dichroism. Titration of compound 1 with (a) calf thymus DNA, (b) poly(dA-dT)‚poly(dA-dT). Panels c and d
show the CD spectra of compound 1 in the absence or presence of (c) poly(dG-dC)‚poly(dG-dC) and (d) poly(dI-dC)‚poly(dI-dC). In
panels a and b, the DNA-phosphate/drug ratio (P/D) increased as follows (bottom to top curves at 370 nm): 0, 1, 2.5, 5, 7.5, 10,
15, 20. In panels c and d, the P/D ratio is indicated. Solutions of drug with or without nucleic acids were scanned in 1 cm quartz
cuvettes using a J obin-Yvon CD6 dichrograph. Measurements were made by progressive addition of DNA to a ligand solution of
constant concentration in order to obtain the desired drug/DNA ratios.
F igu r e 3. Dependence of the reduced dichroism ∆A/A on (a) wavelength, (b) the DNA-phosphate to drug ratio (P/D), and (c)
electric field strength for compound 1-DNA complex (J ) and for DNA alone (E). Conditions: (a) P/D ) 20, 12.5 kV/cm, (b) 340
nm, 12.5 kV/cm, (c) 340 nm for 1, 260 nm for DNA, P/D ) 20 in 1 mM sodium cacodylate buffer, pH 6.5.
Electr ic lin ea r d ich r oism (ELD) represents a sensi-
tive method for investigating drug binding to DNA.8
Figure 3 illustrates a typical experimental data set
showing the dependence of the reduced dichroism on the
wavelength, the drug/DNA ratio and the electric field.
The reduced dichroism ∆A/A is always negative in sign
even in the 300-420 nm absorption band where there is
no contribution from the DNA molecules. When com-
pound 1 is fully bound to DNA (at P/D g 20) the DNA
bases and the salen‚copper complex display comparable
reduced dichroism values. The fact that ∆A/A depends
similarly upon field strength at 260 nm for the DNA
bases and at 340 nm for compound 1 indicates that the
planar copper complex is tilted close to the plane of the
DNA bases.
The spectroscopic and hydrodynamic data reported
above provide key information about the binding geom-
etry of the Cu‚salen complex 1 when associated with
DNA. The copper chelate almost certainly binds to DNA
with a single prefered geometry as judged from the CD
spectra. The ELD data indicate that the Cu‚salen
complex is oriented roughly parallel to the plane of the
base pairs (within 10°). At first sight, such an orientation
would be consistent with an intercalative mode of binding
as recently proposed for structurally related Cu‚salen
derivatives.4 But the viscometric results are hard to
(8) Colson, P.; Bailly, C.; Houssier, C. Biophys. Chem. 1996, 58, 125-
140.

Synthesis of a Functionalized Salen-Copper Complex
J . Org. Chem., Vol. 61, No. 7, 1996 2329
reconcile with any binding model based on intercalation,
even partial, of compound 1 into the DNA double helix.
The manifest lack of effect of the Cu‚salen derivative 1
on the viscosity of supercoiled DNA is frankly incompat-
ible with an intercalative binding mode, given that all
intercalating drugs provoke significant unwinding of the
double helix.9 The intense and positive CD signals
obtained with calf thymus DNA and poly(dA-dT)‚poly-
(dA-dT) are also in conflict with intercalation.10 External
binding of the ligand at the surface of the double helix is
also unlikely since such a process would be expected to
be completely nonspecific as regards orientation whereas
the CD spectra attest that the binding is geometrically
homogeneous. The possibility that the chelate fits into
one of the grooves of DNA has been examined and
appears most likely at first sight, though insertion of a
ligand into the minor groove of DNA gives rise to large
positive ELD signals8 whereas those observed for the
complex between compound 1 and calf thymus DNA are
always negative. We considered the possibility of a
binding site within the major groove of DNA. This would
probably not require unwinding of the helix, and the
major groove is certainly large enough to accommodate
the Cu‚salen chelate oriented parallel to the base pairs.
Insertion between the major groove edges of the bases
has been proposed for certain metal‚trisphenanthroline
complexes.11 Moreover, the binding data obtained with
compound 1 are reminiscent of those reported for planar
alkaloids12 and the phenothiazinium dye methylene blue
which is believed to bind in the major groove of poly(dA-
dT)‚poly(dA-dT).13 Our finding that the substitution of
inosine for guanosine residues has no effect on the
binding of compound 1 to DNA would be compatible with
major groove binding, since all drugs which interact with
DNA via the minor groove have been found to be sensitive
to the presence or absence of the guanine 2-amino group,
even if they do not interact with it directly.7 The only
drugs we have found so far whose binding to DNA is not
dependent on the placement of the 2-amino group of
guanine are bis-naphthalimide derivatives which are
believed to interact in the major groove of the double
helix.14 However, the case for major groove binding of 1
is far from proved, and the hypothesis must at present
be considered speculative, though binding of some sort
into either or both of the helical grooves seems most
probable.
F igu r e 4. Cleavage of closed circular pUC12 DNA (form I)
by compound 1 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
(µM) is indicated at the top of each gel lane. Control lanes
marked cont + and cont - refer to the plasmid DNA incubated
without drug in the presence and absence of MPA, respectively.
DNA. As the salen concentration increases the prob-
ability of double-strand scissions is enhanced once the
DNA has undergone a single-strand break. This is
manifested in the gel by the appearance of linearized
DNA molecules (form III). The reactive species (presum-
ably oxygen-based radicals)15 have free access to the site
of first cleavage. High resolution affinity cleavage stud-
ies, to be reported in detail elsewhere (manuscript
submitted for publication), reveal that the reaction is
essentially non-sequence-specific. The Cu‚salen deriva-
tive 1 complements the tool box of reagents which can
be utilized to produce single-strand cleavage of DNA.
In conclusion, the results suggest that the functional-
ized salen‚CuII complex 1 studied here induces DNA
cleavage via an interaction within one of the grooves of
the double helix. Both the peculiar mode of binding to
DNA and the DNA-cleaving properties entreat further
exploration into the use of metal‚salen complexes as tools
for investigating DNA structure and for the active
interest in developing metal-containing artificial nu-
cleases. The newly introduced butylamino side chain
may ultimately allow tailoring of the chelate to facilitate
cellular transport and DNA recognition. In particular,
it would be interesting to tether the functionalized salen
derivative 1 to an oligonucleotide for sequence-specific
recognition of DNA via triple-helix formation along the
lines already attempted with Mn‚porphyrin and Cu‚-
phenanthroline conjugates.16 Such efforts have now been
initiated.
DNA clea va ge was analyzed by monitoring the con-
version 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. As shown in Figure 4, compound 1 is able to
catalyze oxidative cleavage of DNA. Incubation of the
plasmid at 37 °C for 1 h with 50 µM compound 1 causes
the complete conversion of form I to the nicked form II.
We therefore conclude that the activation of the copper
complex leads mainly to single strand cleavage of duplex
Exp er im en ta l Section
Syn th esis. The purity of all compounds was assessed by
TLC, 1H- and 13C-NMR, 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
(15) The formation of free-radical species generated from oxygenated
solutions of compound 1 in the presence of MPA was monitored by
spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide
(9) Waring, M. J . J . Mol. Biol. 1970, 54, 247-279.
(10) (a) Lyng, R.; Ha¨rd, T; Norde´n, B. Biopolymers 1987, 26, 1327-
1345. (b) Lyng, R.; Rodger, A.; Norde´n, B. Biopolymers 1991, 31, 1709-
1720. (c) Lyng, R.; Rodger, A.; Norde´n, B. Biopolymers 1992, 32, 1201-
1215.
(11) (a) Haworth, I. S.; Elcock, A. H.; Freeman, J .; Rodger, A.;
Richards, W. G. J . Biomol. Struct. Dyn. 1991, 9, 23-44. (b) Terbrue-
ggen, R. H.; Barton, J . K. Biochemistry 1995, 34, 8227-8234.
(12) Caprasse, M.; Houssier, C. Biochimie 1983, 65, 157-167.
(13) Tuite, E.; Norde´n, B. J . Am. Chem. Soc. 1994, 116, 7548-7556.
(14) Bailly, C.; Bran˜a, M.; Waring, M. J . Eur. J . Biochem. 1996,
submitted.
(DMPO). ESR signals characteristic of DMPO-OH (AN ) AH ) 14.9G)
•-
were recorded suggesting the production of superoxide anions O2
.
The postulated mechanism of DNA cleavage by the salen‚Cu complex
involves reduction of 1‚CuII to 1‚CuI which reacts with O2 to give O2
•-
followed by the formation of hydrogen peroxide. H2O2 would decompose
upon reaction with DNA-bound 1‚CuI to yield OH• radicals capable of
reacting with the deoxyribose residues in DNA.
(16) (a) Thuong, N. T.; He´le`ne, C. Angew. Chem., Int. Ed. Engl. 1993,
32, 666-690. (b) Bigey, P.; Pratviel, G.; Meunier, B. J . Chem. Soc.,
Chem. Commun. 1995, 181-182.

2330 J . Org. Chem., Vol. 61, No. 7, 1996
Routier et al.
mm thick) precoated UV sensitive plates. Spots were visual-
ized by inspection under visible light or UV at 254 nm.
Melting points were determined in a hot plate microscope and
are uncorrected. 1H-NMR spectra were recorded on a Bruker
AM 300 WB. Chemical shifts were reported from tetrameth-
ylsilane as an internal reference and are given in δ (ppm)
units. IR spectra were obtained using KBr pellets and only
the principal sharp peaks are given. ESR spectra were
recorded at 77 K, with a maximum modulation amplitude of
8 G in a dual cavity operating in the TE104 mode. Samples
were frozen in liquid nitrogen into 4 mm diameter cylindrical
quartz tubes. The g factor measurements were related to the
“strong pitch”, g ) 2.0028. FAB mass spectra were determined
on a mass spectrometer arranged in EBE geometry. Samples
were bombarded using a beam of xenon with a kinetic energy
of 7 keV. The mass spectrometer was operated at 8 kV
accelerating voltage with a mass resolution of 3000. IE mass
spectra were determined on a quadrupolar spectrometer with
a kinetic energy of 70 eV.
(S)-N1,N2-Bis(sa licylid en e)-6-(ter t-bu tyloxyca r bon yl)-
1,2,6-tr ia m in oh exa n e Cop p er Com p lex (6). A solution of
5 (1.3 mmol, 300 mg) and salicylaldehyde (6.10 mmol, 650 mL)
in dry EtOH (25 mL) and cuprous acetate monohydrate (3.05
mmol, 610 mg in 5 mL of water) was refluxed under argon for
4 h. The solvent was distilled off under reduced pressure, and
the residue was triturated with CH2Cl2. After filtration, the
solvent was removed by distillation under reduced pressure.
Compound 6 was recrystallized from ethanol/water (1:1) (55%,
0.36 g): mp 135-136 °C; [R]25 -25 (c 4 × 10-4, MeOH); IR
D
(KBr, cm-1) ν 3400, 3370, 2950, 1715, 1615; MS (FAB+) 501-
(M + 1)+, 1002 (2 × (M + 1))+; Rf (MeOH) 0.00; ESR A| 192.5
G, g| 2.22.
(S)-N1,N2-Bis(sa licylid en e)-1,2,6-tr ia m in oh exa n e, Tr i-
flu or oa ceta te, Cop p er Com p lex (1). Trifluoroacetic acid
(11.41 mmol, 1.01 mL) was added dropwise at room temper-
ature to a solution of 6 (0.19 mmol, 100 mg) in 15 mL of CH2-
Cl2 containing anisole (4.6 µmol, 5 µL). The resulting mixture
was stirred for 30 min and then evaporated to dryness. The
crude residue was dissolved in ethanol, and the solvent was
removed by distillation under reduced pressure. The final
compound was recrystallized from EtOH as green solid (61%,
60 mg): mp 158-160 °C; IR (KBr, cm-1) ν 3200, 1620; MS
(FAB+) 401(M)+, 801 (2 × (M))+; Rf (MeOH) 0.00; ESR A|
191.25 G, g| 2.23.
Nr-Z-NE-BOC-L-lysin a m id e (3). A solution of NR-Z-Nꢀ-
BOC-L-lysine (2) (5.26 mmol, 2 g), NEt3 (5.31 mmol, 740 mL),
and ethyl choroformate (5.75 mmol, 550 mL) in dry THF (30
mL) was stirred under argon at -10°C for 45 min, 30 mL of
ammonia-saturated THF were added, and the mixture was
stirred for 1 h at -10 °C and then overnight at room
temperature. The solvent was removed by distillation under
reduced pressure, and the residue was dissolved in ethyl
acetate (150 mL). The organic layer was washed with 1 N
Na2CO3 (100 mL) and water (100 mL). After drying over Na2-
SO4 and filtration, the solvent was removed by distillation
under reduced pressure. Compound 3 was obtained as a white
Gen er a l. Ethidium bromide was purchased from Boe-
hringer (Mannheim, Germany); stock solutions were prepared
in water. A 100 mM stock solution of compound 1 was
prepared in DMSO since it is poorly soluble in water. This
solution was stored at -20°C in the dark and diluted to a
working concentration with water. All other chemicals were
analytical grade reagents, and all solutions were prepared
using doubly deionized, Millipore-filtered water. DNA from
calf thymus and the double-stranded polymers poly(dA-dT)‚-
poly(dA-dT), poly(dG-dC)‚poly(dG-dC), and poly(dI-dC)‚poly-
(dI-dC) were from Sigma Chemical Co. (La Verpillie`re, France).
Calf thymus DNA was deproteinized with sodium dodecyl
sulfate (protein content <0.2%) and all nucleic acids were
dialyzed against 1 mM sodium cacodylate buffer pH 6.5.
Viscosity m ea su r em en ts were carried out in a capillary
viscometer submerged in a 45 L water bath which was
maintained at 25 ( 0.1 °C. Flow times were measured at least
in triplicate to an accuracy of (0.1 s with a stopwatch, and
the average time was calculated. The pUC12 plasmid DNA
was isolated by a standard sodium dodecyl sulfate-sodium
hydroxide lysis procedure and purified by banding twice in
CsCl-ethidium bromide gradients. This procedure yields pure
covalently closed circular supercoiled DNA suitable for viscos-
ity measurements. Aliquots (1-5 µL) of the test drug solution
(1-2 mM) were titrated directly into the viscometer containing
2 mL of a 250 µM solution of the plasmid. After each addition
the solutions were carefully mixed with a small flow of air
through the dilution bulb of the viscometer and the flow times
measured. Experiments were conducted in buffer containing
10 mM Tris-HCl (pH 7.0) and 10 mM NaCl. The system was
calibrated using ethidium bromide as a control.17
solid (1.7 g; 85%), mp 141-142 °C; [R]25 -1.75 (c 4 × 10-2
,
D
MeOH); IR (KBr, cm-1) ν 3370-3310, 3200, 2965, 1680, 1655,
1
1535; EIMS 380 (M + 1)+; Rf (MeOH/CHCl3 20:80) 0.74; H-
NMR (CDCl3) δ 1.4 (s, 9H), 1.47 (m, 4H), 1.62 (q, 2H), 3.06
(m, 2H), 4.15 (m, 1H), 5.10 (s, 2H), 5.75 (m, 2H), 6.40 (m, 2H);
13C-NMR (CDCl3) δ 22.6 (CH2), 28.40 (CH3), 29.43 (CH2), 40.02
(CH2), 44.38 (CH2), 64.64 (CH2), 77.32 (CH), 78.96 (Cq), 126.88
(CH), 127.23 (CH), 141.41(Cq), 156.33 (Cq). Anal. Calcd for
C19H29N3O5: C, 60.16; H, 7.65; N, 11.08. Found: C, 60.1; H,
7.5; N, 10.9.
(S)-1-Cya n o-N1-(ben zyloxyca r bon yl)-N5-(ter t-bu tyloxy-
ca r bon yl)-1,5-d ia m in op en t a n e (4). A solution of 3 (4.21
mmol, 1.6 g) and NEt3 (10.04 mmol, 1.4 mL) in THF (20 mL)
was stirred under argon at 0 °C. Trifluoroacetic anhydride
(4.95 mmol, 0.7 mL) was added dropwise, and the mixture was
stirred for 1 h at 0 °C and then overnight at room temperature.
The solvent was removed by distillation under reduced pres-
sure, and the residue was dissolved in Et2O (150 mL). The
organic layer was washed in turn with 0.1 N HCl (100 mL),
0.1 N NaOH (100 mL), and water (100 mL). After drying over
Na2SO4, the solvent was removed by distillation under reduced
pressure. Compound 4 was obtained as a white solid (1.26 g;
83%): mp 81-82 °C, [R]25 -27 (c 4 × 10-2, MeOH), IR (KBr,
D
cm-1) ν 3370, 2965, 1715, 1690, 1540; EIMS 361 (M + 1)+; Rf
1
(MeOH/CHCl3 20:80) 0.85; H-NMR (CDCl3) δ 1.38 (m, 11H),
Absor p tion Sp ectr oscop y. Absorption spectra were re-
corded on a Perkin-Elmer Lambda 5 spectrophotometer using
a 10 mm optical pathlength. Titrations of the drugs with DNA,
covering a large range of drug/DNA-phosphate ratios (D/P),
were performed by adding aliquots of a concentrated DNA
solution to a drug solution at constant ligand concentration
(10 µM).
1.47 (m, 4H), 3.10 (m, 2H), 4.53 (m, 1H), 5.14 (s, 2H), 5.76 (m,
2H), 7.34 (m, 5H); 13C-NMR (CDCl3) δ 21.27 (CH2), 28.37 (CH3),
29.38 (CH2), 32.11 (CH2), 39.42 (CH2), 42.65 (CH), 67.63 (CH2),
79.49 (Cq), 118.61 (Cq), 128.35 (CH), 128.59 (CH), 135.66 (Cq),
156.38 (Cq), 155.39 (Cq). Anal. Calcd for C19H27N3O4: C, 63.16;
H, 7.48; N, 11.63. Found: C, 63.1; H, 7.5; N, 11.5.
(S)-6-(ter t-Bu tyloxycar bon yl)-1,2,6-tr iam in oh exan e (5).
A solution of 4 (1.38 mmol, 500 mg) containing Raney Ni (1 g)
in MeOH (20 mL) saturated with ammonia was stirred under
50 atm hydrogen at 50 °C for 24 h. The reaction mixture was
filtered, and the solvent was removed under reduced pressure
to give compound 5 as a pink solid (93%, 0.3 g): mp 46-48
°C; IR (KBr, cm-1) ν 3200-3600, 2800-2900, 1690, 1540,
EISM 232 (M + 1)+; Rf (MeOH/CHCl3 40:60) 0.15; 1H-NMR
(CDCl3) δ 1.3-1.6 (m, 15H) 1.88 (m, 2H) 2.7-3.1 (m, 3H) 5.23
(m, 4H) 7.32 (m, 1H); 13C-NMR (CDCl3) δ 22.28 (CH2), 23.74
(CH2), 28.43 (CH3), 29.43 (CH2), 32.66 (CH2), 39.78 (CH2), 50.50
(CH), 79.04 (Cq), 156.25 (Cq). Anal. Calcd for C11H25N3O2: C,
57.14; H, 10.82; N, 18.18. Found: C, 57.1; H, 10.9; N, 18.2.
F lu or escen ce measurements were made using a 10 mm
lightpath cuvette in a 0.01 M ionic strength buffer (9.3 mM
NaCl, 2 mM Na acetate, 0.1 mM EDTA) using 20 µM DNA or
polynucleotide and 2 µM ethidium bromide. The DNA-
ethidium complex was excited at 546 nm and the fluorescence
measured at 595 nm.18
Cir cu la r d ich r oism (CD) measurements were recorded on
a J obin-Yvon CD6 dichrograph interfaced to a microcomputer.
(17) Waring, M. J .; Henley, S. M. Nucleic Acids Res. 1975, 2, 567-
586.
(18) Baguley, B. C.; Denny, W. A.; Atwell, G. J .; Cain, B. F. J . Med.
Chem. 1981, 24, 170-177.

Synthesis of a Functionalized Salen-Copper Complex
J . Org. Chem., Vol. 61, No. 7, 1996 2331
Solutions of drugs and/or nucleic acids were scanned in 1 cm
quartz cuvettes. Measurements were made by progressive
addition of DNA or polynucleotide to a pure ligand solution to
obtain the desired drug/nucleic acid ratios.
dichroism is ∆A/A ) (A| - A )/A, where A is the isotropic
absorbance of the sample measured in the absence of electric
field at the same wavelength and using the same pathlength.
DNA Clea vin g Activity. Each reaction mixture contained
4 µL of supercoiled pUC12 DNA (3 µg), 5 µL of compound 1 (2
to 500 µM), and 1 µL of MPA (25 mM) to initiate the reaction.
After 1 h incubation at 37 °C, 1 µL of loading buffer (0.25%
bromophenol blue, 0.25% xylene cyanol, 30% glycerol in H2O)
was added to each tube and the solution was loaded on to a
0.9% agarose gel. The electrophoresis was carried out for
about 2 h at 100 V in TBE buffer (89 mM Tris-borate pH 8.3,
1 mM EDTA). Gels were stained with ethidium bromide (1
µg/µL) and then destained for 30 min in water prior to being
photographed under UV light.
Electr ic Lin ea r Dich r oism (ELD). The theory and
practice for measurements of electric linear dichroism have
already been the subject of detailed reports.19 The optical set-
up for a high sensitivity T-jump instrument equipped with a
Glan polarizer was used under the following conditions:
bandwidth 3 nm, sensitivity limit 0.001 in ∆A/A, response time
3 µs. All experiments were conducted at 20 °C with a 10 mm
path length Kerr cell having 1.5 mm electrode separation, in
1 mM sodium cacodylate buffer, pH 6.5. The conductivity of
the solutions, measured with a Metrohm conductimeter Model
E527, ranged from 0.8 to 1.2 mS. The DNA samples were
oriented by rectangular electric pulses at 13 kV/cm, and the
drug under test was present at 10 µM together with the DNA
or polynucleotide at 100 µM, unless otherwise stated. Linear
dichroism ∆A is defined as the difference between the absor-
bance for light polarized parallel (A|) and perpendicular (A )
to the applied field at a given wavelength. The reduced
Ack n ow led gm en t. This work was done under the
support of research grants (to J .L.B.) from the CNRS;
(to M.J .W.) from the Wellcome Trust, CRC, AICR and
the Sir Halley Stewart Trust; (to P.C. and C.H.) from
the FRFC convention 2.4501.91 and the Te´le´vie-FNRS
contract 7.4526.94; and (to C.B.) from the INSERM.
Support by the “convention INSERM-CFB” is
acknowledged.
(19) (a) Houssier, C. In Molecular Electro-Optics; Krause, S., Ed.;
Plenum Publishing Corp.: New York, 1981; pp 363-398. (b) Bailly,
C.; He´nichart, J .-P.; Colson, P.; Houssier, C. J . Mol. Recognit. 1992,
5, 155-171.
J O951840C