Synthesis, DNA-binding affinities, and binding mode of berberine dimers

Article abstract of DOI:10.1016/j.bmc.2005.07.069

Six novel berberine dimers (3a-f) were synthesized in 37-84% yield from the reaction of berberrubine (2) with dihaloalkanes of varying lengths from two to seven carbons. Their interactions with calf thymus (CT) DNA and three double helical oligodeoxynucleotides, d(AAGAATTCTT)₂, d(AAGCATGCTT) ₂, and d(TAAGAATTCTTA)₂, were investigated by means of fluorometric titration and ethidium bromide (EB) displacement experiments. Compared with the monomeric parent berberine (1), these dimers' DNA-binding affinities increased up to approximately 100-fold, suggesting a cooperative interaction of the two berberine subunits in the molecules. Furthermore, these dimers linked by different spacers show a prominent structure-activity relationship when bound with oligodeoxynucleotides. The relative binding affinities are in the order of 3b > 3a > 3c > 3d > 3e > 3f with d(AAGAATTCTT)₂ and d(TAAGAATTCTTA)₂, and 3b > 3c > 3a > 3d > 3e > 3f with d(AAGCATGCTT)₂. Dimer 3b, linked with a propyl chain, exhibits the highest binding affinity. This suggests that a propyl chain may be the most suitable spacer to bridge the two berberine units for DNA binding. Spectrophotometric titration and competitive EB displacement of berberine (1) and dimer 3b indicate that both berberine and its dimers form intercalating complexes with duplex DNA. A larger redshift, a stronger hypochromic effect, and a much higher EB displacement ratio, observed in 3b, indicate that the dimer is in more intimate contact with DNA than berberine. In addition, no obvious binding of canadine (4), a hydrogenated product of berberine, with CT DNA was observed, suggesting critical roles of the quaternary ammonium cation and planar structure in the DNA-binding of berberine.

Full text of DOI:10.1016/j.bmc.2005.07.069

Bioorganic & Medicinal Chemistry 14 (2006) 25–32
Synthesis, DNA-binding affinities, and binding mode
of berberine dimers
Yong Qin,a Ji-Yan Pang,b Wen-Hua Chen,b,* Zongwei Caic and Zhi-Hong Jianga,*
aSchool of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
bSchool of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China
cDepartment of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong
Received 21 June 2005; revised 26 July 2005; accepted 27 July 2005
Available online 19 September 2005
Abstract—Six novel berberine dimers (3a–f) were synthesized in 37–84% yield from the reaction of berberrubine (2) with dihaloalk-
anes of varying lengths from two to seven carbons. Their interactions with calf thymus (CT) DNA and three double helical oligo-
deoxynucleotides, d(AAGAATTCTT)2, d(AAGCATGCTT)2, and d(TAAGAATTCTTA)2, were investigated by means of
fluorometric titration and ethidium bromide (EB) displacement experiments. Compared with the monomeric parent berberine
(1), these dimersÕ DNA-binding affinities increased up to approximately 100-fold, suggesting a cooperative interaction of the two
berberine subunits in the molecules. Furthermore, these dimers linked by different spacers show a prominent structure–activity rela-
tionship when bound with oligodeoxynucleotides. The relative binding affinities are in the order of 3b > 3a > 3c > 3d > 3e > 3f with
d(AAGAATTCTT)2 and d(TAAGAATTCTTA)2, and 3b > 3c > 3a > 3d > 3e > 3f with d(AAGCATGCTT)2. Dimer 3b, linked
with a propyl chain, exhibits the highest binding affinity. This suggests that a propyl chain may be the most suitable spacer to bridge
the two berberine units for DNA binding. Spectrophotometric titration and competitive EB displacement of berberine (1) and dimer
3b indicate that both berberine and its dimers form intercalating complexes with duplex DNA. A larger redshift, a stronger hypo-
chromic effect, and a much higher EB displacement ratio, observed in 3b, indicate that the dimer is in more intimate contact with
DNA than berberine. In addition, no obvious binding of canadine (4), a hydrogenated product of berberine, with CT DNA was
observed, suggesting critical roles of the quaternary ammonium cation and planar structure in the DNA-binding of berberine.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
rus drugs,3 and developing chemotherapeutic agents.4
Modification of natural products with established affin-
ities and binding modes with DNA has been
demonstrated to be a practicable approach. A typical
example is the rational structural modification of the
antibiotics netropsin and distamycin leading to the
development of lexitropsins.5
Controlling gene expression with small DNA-binding
molecules has been a challenge at the interface of medic-
inal chemistry and biology.1 To achieve this goal, a
number of chemical approaches have been investigated
to search for small molecules that can selectively bind
to DNA and either activate or inhibit gene expression.
This includes the examination of the specific and nonco-
valent interactions of small organic molecules with
DNA and RNA.2 Efforts have focused on the rational
design of ligands capable of binding tightly and specifi-
cally to any sequence of double-stranded DNA. These
agents would potentially have wide applications in eluci-
dating the mechanism of action of antitumor and antivi-
Alkaloids are well-known to be a group of important
natural products in medicinal chemistry due to their
extensive biological activities. Especially noteworthy
are the isoquinoline alkaloids that are widely distribut-
ed in several botanical families having myriad thera-
peutic applications.6 Protoberberine alkaloids, such as
berberine (1) and palmatine, possessing antimicrobial,
antileukemic, anticancer, and topoisomerase inhibitory
activities,7 are useful for the development of more effi-
cient DNA-binding agents. Berberine is known as a
DNA binder and its binding affinities have been exten-
sively characterized by employing several analytical
techniques including absorption, fluorescence, NMR,
Keywords: Berberine dimer; DNA-binding; Intercalation; Ethidium
bromide displacement; Fluorescence spectrometry.
*
Corresponding authors. Tel.: +852 34112906; fax: +852 34112461;
e-mail addresses: cescwh@zsu.edu.cn; zhjiang@hkbu.edu.hk
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.069

26
Y. Qin et al. / Bioorg. Med. Chem. 14 (2006) 25–32
2. Results and discussion
O
O
N
2.1. Synthesis
OMe
OMe
The route of synthesis of compounds 3a–f is shown in
Scheme 1. Partial demethylation of berberine (1) at
190 ꢁC under vacuum for 15 min, according to the
reported protocols,10 gave berberrubine (2) in 60%
yield. Linking two berberrubine molecules with
dibromo-(3a–c and 3f) or diiodoalkanes (3d,e) of vary-
ing lengths from two to seven carbons, followed by
the exchange of all anions into chloride, afforded the
corresponding berberine dimers 3a–f in 37–84% yield.
Compounds 3a–f were fully characterized by ESI-
HRMS and 1H NMR spectral analysis. Each com-
Figure 1. Structure of canadine.
and electrospray ionization mass (ESI-MS) spectrome-
tries.8 However, its modest binding affinities with du-
plex DNA necessitate structural modifications to
produce novel DNA-binding agents with enhanced
affinities.
pound
afforded
two-charged
ESI-MS
peaks
([Mꢁ2Cl]2+) in the mass spectrum. The 1H NMR
spectra of these compounds showed only one set of
berberine moiety, indicating the presence of a symmet-
ric structure in the molecule. The ratios of the inte-
grated areas for the protons of berberine subunits to
those for the alkyl chain protons in the 1H NMR
spectra were in full accord with the linked berberine
dimeric structures.
We have previously reported the synthesis of five
dimeric berberines 3a–e linked with C2 ꢀ C6 alkyl
chains and described their greatly enhanced DNA-
binding affinities.9 The results strongly suggest that
these dimers 3a–e are exploitable as potentially strong
DNA-binders. Accordingly, these observations intensi-
fy our further efforts to establish the structure–activity
relationship and to clarify their DNA-binding modes,
by (i) synthesizing a series of dimeric berberines linked
with alkyl chains of varying lengths from two carbons
to six carbons, that is 3a–e, plus another dimer 3f
linked by a longer alkyl chain (i.e., heptyl group);
and (ii) evaluating the binding affinities of 3a–f toward
various DNA sequences, that is, three double-stranded
oligodeoxynucleotides, d(AAGAATTCTT)2, d(AAGC
ATGCTT)2, d(TAAGAATTCTTA)2, and calf thymus
(CT) DNA. In this paper, we present a full account
of the synthesis, DNA-binding affinities, and binding
mode of these dimeric berberines. In addition, to eval-
uate the effect of the quaternary ammonium cation of
berberine on DNA-binding, the interaction of canadine
(4, namely, tetrahydroberberine, Fig. 1) with CT DNA
was examined.
2.2. DNA-binding affinities of berberine dimers (3a–f)
Various techniques for precise determination of binding
constants have been developed and provide information
for understanding the nature of the complexes between
small molecules and biomacromolecules. Fluorescence
spectrometry is a very sensitive analytical technique,
which has been widely used in the investigation of non-
covalent complexes of small organic molecules with bio-
polymers, such as DNA.2a,b The complex formation is
reflected in the change, either enhancement or quench-
ing, of fluorescence intensities.
Berberine and dimeric berberines 3a–f studied in this
paper have strong absorbance peaks at around
O
O
Cl-
N
Cl-
190oC,Vacuum,
15min
N
O
O
OMe
OMe
OH
OMe
1
2
O
O
O
O
X
X
1)
n
DMF, 60oC, 12~15 h
2) Anion Exchange
Cl-
Cl- N+
N
3a: n = 0, X = Br (46%)
3b: n = 1, X = Br (37%)
O
3c: n = 2, X = Br (84%)
3d: n = 3, X = I (47%)
3e: n = 4, X = I (74%)
3f : n = 5, X = Br (40%)
O
n
MeO
OMe
Scheme 1. Synthetic route for berberine dimers 3a–f.

Y. Qin et al. / Bioorg. Med. Chem. 14 (2006) 25–32
27
350 nm, and relatively weak and broad absorbance
peaks above 400 nm. They weakly fluoresce at around
520 nm in aqueous solution when excited at 350 or
450 nm.8c,d,e Upon complex formation with DNA, their
fluorescences are greatly enhanced. These fluorescence
spectroscopic changes ensure the spectrofluorimetric
determination of the binding activities of berberine
and its dimers with DNA, such as CT DNA and
self-complementary double-stranded oligodeoxynucleo-
tides, d(AAGAATTCTT)2, d(AAGCATGCTT)2, and
d(TAAGAATTCTTA)2, which were described in this
paper. Representative spectrofluorimetric titration
spectra of 3b with d(TAAGAATTCTT A)2 are shown
in Figure 2. The weak fluorescence of 3b was dramat-
ically enhanced upon the addition of DNA, suggesting
an interaction of 3b with the added DNA. The 1:1
binding stoichiometry between 3b and d(TAA-
GAATTCTTA)2 was determined by molar ratio meth-
ods shown as the inset in Figure 2. The binding
stoichiometries between other dimeric berberines and
duplex oligodeoxynucleotides were determined by the
same method.
Analyses of fluorescence intensity against the DNA con-
centrations by nonlinear curve fitting methods11 were
used to determine the association constants (KaÕs) of
3a–f with CT DNA and three self-complementary dou-
ble-stranded oligodeoxynucleotides (Table 1). For com-
parison, the binding constants of 1 with CT DNA and
three self-complementary oligodeoxynucleotide duplex-
es were obtained in a similar way. The binding affinities
of 3a–f, relative to
1 with CT DNA, d(AA-
GAATTCTT)2, d(AAGCATGCTT)2, and d(TAA-
GAATTCTTA)2, are illustrated in Figure 3.
It is apparent from Figure 3 and Table 1 that the dimers
3a–f have higher affinities of binding than their mono-
meric parent compound 1. Dimers 3a–f bind to CT
DNA at least 10 times, to d(AAGAATTCTT)2 at least
1.5–20 times, to d(AAGCATGCTT)2 at least 1.5–7
times, and to d(TAAGAATTCTTA)2 at least 1.5–90
times greater than 1. The binding of 3b to d(TAA-
GAATTCTTA)2, with the association constant, is
approximately two orders of magnitude higher than 1.
These increases in the binding affinities may be due to
Figure 2. Fluorescence spectra of 3b (2.47 · 10ꢁ6 M) with d(TAAGAATTCTTA)2 of increasing concentrations (0–8.89 · 10ꢁ6 M) in 50 mM Tris–
HCl (pH 6.35) at room temperature, excitation 355 nm. The inset indicates the relationship between the fluorescence intensities (emission at
516.7 nm) and the molar ratio of d(TAAGAATTCTTA)2 to 3b.
Table 1. Association constants (KaÕs, Mꢁ1) of 1 and 3a–f with CT DNA, d(AAGAATTCTT)2, d(AAGCATGCTT)2, and d(TAAGAATTCTTA)2
a
Compound
CT DNA
d(AAGAATTCTT)2
RAb
d(AAGCATGCTT)2
Ka
RAb
d(TAAGAATTCTTA)2
Ka
RAb
Ka
RAb
Ka
1
(1.12 0.04) · 104
(1.45 0.22) · 105
(1.33 0.18) · 105
(1.60 0.05) · 105
(1.64 0.08) · 105
(1.27 0.03) · 105
(1.42 0.39) · 105
1
(1.24 0.12) · 104
(1.18 0.10) · 105
(2.46 0.07) · 105
(8.78 0.26) · 104
(5.77 0.13) · 104
(3.42 0.13) · 104
(2.10 0.13) · 104
1
(1.85 0.09) · 104
1
(2.93 0.31) · 104
1
3a
3b
3c
3d
3e
3f
12.9
11.9
14.3
14.6
11.3
12.7
9.5
19.8
7.1
4.7
2.8
1.7
(4.52 0.33) · 104
(1.30 0.09) · 105
(8.47 0.44) · 104
(3.63 0.23) · 104
(3.60 0.52) · 104
(2.71 0.14) · 104
2.4
7.0
4.6
2.0
1.9
1.5
(1.62 0.35) · 106
(2.76 0.37) · 106
(3.49 0.60) · 105
(3.21 0.20) · 105
(2.48 0.34) · 105
(4.10 0.37) · 104
54.9
94.2
11.9
11.0
8.5
1.4
a In 50 mM Tris–HCl (pH 6.35) at room temperature unless specified. These values were derived from the experimental data by nonlinear curve fitting
methods using KaleidaGraph (version 3.6).
b RA denotes relative affinity.

28
Y. Qin et al. / Bioorg. Med. Chem. 14 (2006) 25–32
100
80
60
40
20
0
CT DNA
d(AAGAATTCTT) 2
d(AAGCATGCTT)2
d(TAAGAATTCTTA)
2
1
3a
3b
3c
3d
3e
3f
Figure 3. Relative binding affinities of 3a–f with CT DNA, d(AAGAATTCTT)2, d(AAGCATGCTT)2, and d(TAAGAATTCTTA)2 in 50 mM Tris–
HCl (pH 6.35) at room temperature.
the cooperative interactions of the two berberine sub-
units in dimers 3a–f, demonstrating the usefulness of a
Ôdimeric analogÕ for refining the rational design process
to obtain promising candidates. The great enhancement
in binding abilities was demonstrated further by spectro-
photometric titration and competitive EB displacement
(vide infra). Second, compounds 3a–f show a prominent
structure–activity relationship and their binding abilities
with three self-complementary duplex DNAs can be
modulated by the lengths of the alkyl chains. Com-
pounds 3a–f show binding affinities in the order of
3b > 3a > 3c > 3d > 3e > 3f with d(AAGAATTCTT)2
and d(TAAGAATTCTTA)2, and 3b > 3c > 3a > 3-
d > 3e > 3f with d(AAGCATGCTT)2. In all cases, di-
mer 3b, linked by a propyl chain, exhibits the highest
binding affinity with all these self-complementary duplex
DNAs. This shows that the propyl is the most suitable
spacer of two berberine units in these studies and may
lead to future rational design efforts. However, no
prominent structure–activity relationship is observed
with CT DNA binding, perhaps because CT DNA is a
mixture of different DNA chains ranging from 200 to
6000 bases. Third, compounds 3a–f show higher binding
affinities with the 12-mer d(TAAGAATTCTTA)2 than
with the 10-mer d(AAGAATTCTT)2 and d(AAG
CATGCTT)2. For example, the binding constants of
3a,b with d(TAAGAATTCTTA)2 are about 5–20 times
higher than with d(AAGAATTCTT)2 or d(AAG
CATGCTT)2. This result suggests that dimeric berbe-
rines occupy a greater number of base pairs,12 with
potentially more stringent sequence recognition com-
pared with the monomeric berberine (1).
calation and groove binding are the two main binding
modes for small organic molecules that bind noncova-
lently to DNA.16,17 Binding modes can be convincingly
established by high-resolution structural studies, using
either X-ray diffraction methods or NMR. In the ab-
sence of high resolution data, binding modes may be in-
ferred from the results of solution studies,18 and
evidence for the intercalative mode of binding can be
obtained from a UV–vis spectrometric investigation.19
The spectrophotometric titration of berberine and 3b is
shown in Figure 4, in which spectra a and b represent
their UV–vis spectra before and after the addition of
CT DNA, respectively. The addition of CT DNA to
the solutions of berberine (1) and 3b of fixed concentra-
tions induces redshifts of 1 nm from 343 to 344 nm for
berberine (the redshift of berberine becomes more obvi-
ous from 343 to 345 nm when the ratio of DNA-phos-
phate/berberine reaches 8.6:1, figure not shown),20 and
5 nm from 342 to 347 nm for 3b, and hypochromic
responses around 5% at 343 nm for berberine, and
around 30% at 342 nm for 3b. These spectral variation
effects of berberine and 3b may be considered as the
characteristic features of aromatic chromophore (p–p*)
interactions with the nucleic acid bases.21
The bathochromic shifts and hypochromic effects of ber-
berine and 3b toward CT DNA strongly indicate their
interaction with DNA. The magnitudes of these spectral
alterations provide evidences for intercalative binding
because it was established that the p systems of berber-
ine or 3b are in intimate contact with those of the DNA
bases.19a,b The binding of berberine and 3b is proposed
to be through intercalation, which is consistent with the
previous studies on berberine and related protoberber-
ine alkaloids.7c,8h,22
2.3. DNA-binding mode of berberine dimers 3a–f
Some planar cationic molecules readily bind to nucleic
acids and display a variety of interesting biological
properties.13 A particular binding mode depends on
considerable factors including the DNA base pair com-
position, as well as ligand structural features.14,15 Inter-
The binding modes of berberine and 3b were confirmed
further by a competitive EB displacement assay that has
been employed to assess the binding affinities of DNA

Y. Qin et al. / Bioorg. Med. Chem. 14 (2006) 25–32
29
0.3
0.2
0.3
A
B
a
a
0.2
b
b
Abs
0.1
Abs
0.1
0
300
0
300
320
340
360
380
400
320
340
360
380
400
Wavelength [nm]
Wavelength [nm]
Figure 4. UV-vis absorption spectra of berberine (1.17 · 10ꢁ5 M, A) and 3b (9.26 · 10ꢁ6 M, B) in 50 mM Tris–HCl buffer (pH 6.35) before (a) and
after (b) the addition of CT DNA. The concentration ratios of DNA-phosphate/berberine and DNA-phosphate/3b are 4.4:1 and 5.6:1, respectively.
binders, especially for typical intercalators.23 Ethidium
bromide is a cationic dye that is widely used as a probe
for native DNA because the ethidium ion displays a dra-
matic increase in fluorescence efficiency when it interca-
lates into DNA. This technique, based on the decrease
of fluorescence resulting from the displacement of EB
from a DNA sequence, is an efficient and rapid method
to confirm the intercalating binding mode. The plots of
the relative fluorescence intensity (I/I0) of EB against the
concentrations of added berberine and 3b are shown in
Figure 5. It is obvious that both berberine and 3b can
decrease the fluorescence intensity of the complex of
EB and CT DNA, suggesting that berberine and 3b
can replace EB bound to CT DNA. Compound 3b
induces a much larger decrease in fluorescence intensity
than berberine, indicating that 3b is a much stronger
DNA intercalator than berberine.
DNA. It should be noted that both complexes possess
different spectral characteristics. Compared with berber-
ine, compound 3b shows a larger redshift and a stronger
hypochromic effect. Consistent with this spectrophoto-
scopic titration study, 3b shows a displacement ratio
higher than that of berberine in the competitive EB dis-
placement experiment, possibly because it is effected
through a binding mode of bis-intercalation.
To investigate the role of the quaternary ammonium
group of berberine in the DNA-binding activity, we
examined the interaction of canadine (4), a reductive
product of berberine (1), with CT DNA using spectro-
fluorimetric titration and competitive EB displacement
experimental techniques. In both experiments, no
change in the fluorescence intensity was observed after
the addition of CT DNA to canadine solution, indicat-
ing that no detectable binding occurred between them.
Compared to berberine, the most important structural
characteristic of canadine is the absence of a quaternary
ammonium cation. The loss of positive charge also
makes the planar structure of berberine having sp2
nitrogen transform into a tetrahedral structure of cana-
dine having sp3 nitrogen. Thus, these experiments indi-
cate that the quaternary ammonium cation and planar
structure may be critical factors for the binding of pro-
toberberine alkaloids with double-stranded DNA.10
Thus, DNA-binding modes of berberine and dimeric
berberine 3b are evident from these spectrophotometric
titration and competitive EB replacement experiments.
Under these specific experimental conditions, both ber-
berine and 3b form intercalating complexes with CT
1.0
A
0.9
0.8
3. Summary
Six novel berberine dimers (3a–f) have been successfully
synthesized in moderate- to high-yield from the reaction
of berberrubine with dihaloalkanes of varying lengths.
Their structures were fully characterized by ESI-HRMS
0.7
1
and H NMR.
B
Spectrofluorometric titration suggests that six dimers
show a prominent structure–activity correlation when
bound with short double-stranded DNA sequences. They
show similar relative binding affinities in the order of
3b > 3a > 3c > 3d > 3e > 3f with d(AAGAATTCTT)2
and d(TAAGAATTCTTA)2, and 3b > 3c > 3a > 3-
d > 3e > 3f with d(AAGCATGCTT)2. In all cases, dimer
3b linked by a propyl chain exhibits the highest affinity,
0.6
0
10
20
30
40
[berberine or 3b](uM)
Figure 5. Fluorescence decrease of EB (3.03 · 10ꢁ6 M) induced by the
competitive binding of (A) berberine and (B) 3b to CT DNA
(2.40 · 10ꢁ6 M) in 50 mM Tris–HCl buffer (pH 6.35) at room
temperature (excitation 491 nm, emission 592 nm).

30
Y. Qin et al. / Bioorg. Med. Chem. 14 (2006) 25–32
indicating that propyl chain may be the most suitable link-
er to bridge the two berberine units. Spectrophotometric
titration and competitive EB replacement assays indicate
that both berberine (1) and its dimeric derivatives (3a–f)
bind with duplex DNA in an intercalating mode.
4.2. Preparation of berberrubine 2
The synthetic procedures of berberrubine have been de-
scribed previously.9 1H NMR (CD3OD–CDCl3,
300 MHz): d 3.16 (t, 2H, J = 6.2 Hz, 6-H), 3.93 (s, 3H,
OCH3), 4.62 (t, 2H, J = 6.2 Hz, 5-H), 6.06 (s, 2H,
OCH2O), 6.82 (s, 1H, 4-H), 6.92 (d, 1H, J = 8.4 Hz,
12-H), 7.41 (s, 1H, 1-H), 7.53 (d, 1H, J = 8.4 Hz,
11-H), 8.00 (s, 1H, 13-H), 9.28 (s, 1H, 8-H); FAB MS:
m/z 322 ([MꢁCl]+). The data of 1H NMR were in accor-
dance with those reported by Iwasa et al.10
Additionally, we clarified that the quaternary ammoni-
um cation and planar structure are important for the
binding ability of protoberberine alkaloids with dou-
ble-stranded DNA.
4. Experimental
4.1. General
4.3. Synthesis of dimeric berberines 3a–f
General procedures: to a solution of berberrubine 2
(0.12 mmol) in DMF (4 mL) was added dihaloalkane
(0.05 mmol). The reaction was monitored by TLC. After
stirring at 60 ꢁC for 12–15 h, the solvent was removed
under reduced pressure. The obtained residue was
washed by 95% ethanol for several times and subject
to anion exchange into chloride form. The solvents were
evaporated and the residues were dried in vacuum to af-
ford the target compounds 3a–f as a yellow powder.
1H NMR spectra were recorded on a VARIAN UNITY
INOVA-300 spectrometer at 300 MHz and tetrameth-
ylsilane was used as an internal standard. FAB-MS
was carried on a JEOL JMS-700 mass spectrometer.
High-resolution mass spectra were measured on a mass
spectrometer API QSTAR PULSAR i. Fluorescence
spectra were obtained on a Perkin-Elmer LS55 Lumi-
nescence Spectrometer equipped with a fluorescence free
quartz cuvette of 2 mm path length. UV–vis absorption
spectra were measured on a Jasco UV-530 UV/vis spec-
trophotometer in matched quartz cuvettes of 2 mm path
length.
4.3.1. Compound 3a. Obtained yield 46%. 1H NMR
(DMSO-d6, 300 MHz): d 3.17 (s, 4H, 5-H), 4.02 (s,
6H, OCH3), 4.79 (s, 4H, OCH2), 4.85 (s, 4H, 6-H),
6.16 (s, 4H, OCH2O), 7.08 (s, 2H, 4-H), 7.75 (s, 2H, 1-
H), 7.98 (d, 2H, J = 9.6 Hz, 12-H), 8.19 (d, 2H,
J = 9.6 Hz, 11-H), 8.91 (s, 2H, 13-H), 9.83 (s, 2H, 8-
H); HRMS (ESI) for C40H34N2O8 ([Mꢁ2Cl]2+) Calcd
335.1152. Found: 335.1129.
Berberine chloride was obtained from the National
Institute for the Control of Pharmaceutical and Biolog-
ical Products, Beijing, China. Tris(hydroxymethyl)
aminomethane (Tris, ultrapure) was from USB Corpo-
ration, Cleveland, OH, USA. Silica gel 60 (0.063–
0.200 mm, 70–230 mesh) used for flash chromatography
was from Merck, and TLC was carried out on silica gel
60 F254 (Merck) aluminum-backed commercial plates.
Milli-Q water (Millipore Co.) was used in all solutions.
All other reagents were purchased from either Aldrich
Chemical Company or Sigma Chemical Company and
used without further purification unless otherwise
stated.
4.3.2. Compound 3b. Obtained yield 37%. 1H NMR
(CD3OD–CDCl3, 300 MHz):
d
2.65 (m, 2H,
CH2CH2O), 3.23 (t, 4H, J = 6.3 Hz, 5-H), 4.09 (s, 6H,
OCH3), 4.76 (t, J = 6.3 Hz, 4H, OCH2CH2), 4.92 (t,
J = 6.3 Hz, 4H, 6-H), 6.09 (s, 4H, OCH2O), 6.91 (s,
2H, 4-H), 7.59 (s, 2H, 1-H), 7.98 (d, 2H, J = 9.0 Hz,
12-H), 8.07 (d, 2H, J = 9.0 Hz, 11-H), 8.65 (s, 2H, 13-
H), 9.87 (s, 2H, 8-H); HRMS (ESI) for C41H36N2O8
([Mꢁ2Cl]2+) Calcd 342.1231. Found: 342.1213.
Oligodeoxynucleotides and calf thymus DNA were
purchased from Invitrogen Life Technologies and
Amersham Pharmacia Biotech, respectively. Oligode-
oxynucleotides were purified by open ODS column
chromatography before use. The elution solution ran-
ged from 5% to 11% acetonitrile aqueous solution
containing 10 mM ammonium acetate. Double-strand-
ed DNA was formed by heating the solutions of sin-
gle-stranded oligodeoxynucleotides in 50 mM Tris–
HCl (pH 6.35) buffer at 95 ꢁC in a water bath for
4 min and then chilling to room temperature slowly
over several hours. CT DNA was used without further
purification. The concentrations of single-stranded
self-complementary oligodeoxynucleotides were deter-
mined from UV absorbance at 260 nm, supposing
the molar extinction coefficients of A, T, G, and C
4.3.3. Compound 3c. Obtained yield 84%. 1H NMR
(DMSO-d6, 300 MHz): d 2.14 (s, 4H, CH2CH2O),
3.15–3.24 (m, 4H, 5-H), 4.04 (s, 6H, OCH3), 4.40 (t,
4H, J = 5.6 Hz, OCH2CH2), 4.93 (s, 4H, 6-H), 6.17 (s,
4H, OCH2O), 7.08 (s, 2H, 4-H), 7.78 (s, 2H, 1-H),
7.98 (d, 2H, J = 9.0 Hz, 12-H), 8.19 (d, 2H, J = 9.0 Hz,
11-H), 8.92 (s, 2H, 13-H), 9.78 (s, 2H, 8-H); HRMS
(ESI) for C42H38N2O8 ([Mꢁ2Cl]2+) Calcd 349.1309.
Found: 349.1318.
4.3.4. Compound 3d. Obtained yield 47%. 1H NMR
(DMSO-d6, 300 MHz):
d
1.65–1.80 (m, 2H,
CH2CH2CH2O), 1.99 (m, 4H, CH2CH2CH2O), 3.20
(m, 4H, 5-H), 4.04 (s, 6H, OCH3), 4.32 (t, 4H,
J = 6.3 Hz, OCH2CH2), 4.93 (t, 4H, J = 6.4 Hz, 6-H),
6.16 (s, 4H, OCH2O), 7.07 (s, 2H, 4-H), 7.75 (s, 2H, 1-
H), 7.97 (d, 2H, J = 8.7 Hz, 12-H), 8.18 (d, 2H,
J = 8.7 Hz, 11-H), 8.90 (s, 2H, 13-H), 9.75 (s, 2H, 8-
H); HRMS (ESI) for C43H40N2O8 ([Mꢁ2Cl]2+) Calcd
356.1387. Found: 356.1385.
to be 16,000, 9600, 12,000, and 7000 molꢁ1 dm3 cmꢁ1
,
respectively, and the molar extinction coefficient for
CT DNA was approximated with A260 being
13,200 molꢁ1 dm3 cmꢁ1 in base pair.

Y. Qin et al. / Bioorg. Med. Chem. 14 (2006) 25–32
31
4.3.5. Compound 3e. Obtained yield 74%. 1H NMR
4.6. Spectrophotometric titration experiments
(DMSO-d6, 300 MHz): d 1.60 (s, 4H, CH2CH2CH2O),
1.90–2.00 (m, 4H, CH2CH2CH2O), 3.20 (m, 4H, 5-H),
4.03 (s, 6H, OCH3), 4.29 (t, 4H, J = 5.8 Hz, OCH2CH2),
4.92 (t, 4H, J = 6.9 Hz, 6-H), 6.16 (s, 4H, OCH2O), 7.07
(s, 2H, 4-H), 7.75 (s, 2H, 1-H), 7.97 (d, 2H, J = 9.3 Hz,
12-H), 8.18 (d, 2H, J = 9.3 Hz, 11-H), 8.90 (s, 2H, 13-
H), 9.73 (s, 2H, 8-H); HRMS (ESI) for C44H42N2O8
([Mꢁ2Cl]2+) Calcd 363.1471. Found: 363.1504.
To the solution of alkaloids (1.17 · 10ꢁ5 and
9.26 · 10ꢁ6 M for berberine and 3b, respectively) in
50 mM Tris–HCl buffer (pH 6.35) were added aliquots
of CT DNA (1.60 · 10ꢁ3 M) solution containing the
same concentrations of alkaloids in 50 mM Tris–HCl
buffer (pH 6.35). The mixing was achieved by turning
the cuvette up and down for 5 min. After the measuring
solution was allowed to stand for 1 min, the correspond-
ing absorption spectra in the region from 200 to 500 nm
were measured at room temperature.
4.3.6. Compound 3f. Obtained yield 40%. 1H NMR
(CD3OD–CDCl3, 300 MHz): d 1.53–1.66 (m, 6H,
CH2CH2CH2CH2CH2O),
1.94–2.03
(m,
4H,
CH2CH2O), 3.24 (m, 4H, 5-H), 4.07 (s, 6H, OCH3),
4.42 (t, 4H, J = 5.8 Hz, OCH2CH2), 5.00 (t, 4H,
J = 6.9 Hz, 6-H), 6.07 (s, 4H, OCH2O), 6.84 (s, 2H, 4-
H), 7.49 (s, 2H, 1-H), 7.92 (d, 2H, J = 9.3 Hz, 12-H),
7.95 (d, 2H, J = 9.3 Hz, 11-H), 8.49 (s, 2H, 13-H), 9.73
4.7. EB displacement experiment
Competitive EB displacement experiments were carried
out in matched quartz cuvettes of 2 mm path length. Ali-
quots of the alkaloidsÕ (2.0 · 10ꢁ4 M) solutions contain-
ing CT DNA (2.40 · 10ꢁ6 M) and EB (3.03 · 10ꢁ6 M) in
50 mM Tris–HCl buffer (pH 6.35) were added to the
solution of CT DNA (2.40 · 10ꢁ6 M) and EB
(3.03 · 10ꢁ6 M) in 50 mM Tris–HCl buffer (pH 6.35).
This operation ensured that the concentrations of alka-
loids increased gradually from 0 to 4.0 · 10ꢁ5 M, while
the concentrations of CT DNA and EB were kept con-
stant. Then, the corresponding fluorescence spectra were
measured at room temperature (excitation at 491 nm,
emission at 592 nm).
(s, 2H, 8-H); HRMS (ESI) for C45H44N2O8 ([Mꢁ2Cl]2+
)
Calcd 370.1549. Found: 370.1521.
4.4. Preparation of canadine (4)
To a solution of berberine (200 mg, 0.54 mmol) in
refluxing MeOH (15 mL) was added NaBH4 powder
(43 mg, 1.13 mmol). The reaction mixture was stirred
for 20 min at 70 ꢁC and then at room temperature for
4 h. The formed light yellow precipitates were collected
and recrystallized from MeOH to yield canadine (4,
126 mg, 63%). FAB MS: m/z 339 ([MꢁCl]+). The data
of 1H NMR were essentially identical to those
published.24
Acknowledgments
This work was financially supported by the Faculty Re-
search Grants from Hong Kong Baptist University and
Research Grants Council, University Grants Committee
of Hong Kong.
4.5. Spectrofluorimetric titration experiments
To the solutions of alkaloids (2.0 · 10ꢁ6 M) in 50 mM
Tris–HCl buffer (pH 6.35) were added aliquots of dou-
ble-stranded DNA (CT DNA, 8.0 · 10ꢁ4 M; d(AAGA
ATTCTT)2 and d(AAGCATGCTT)2, 4.0 · 10ꢁ4 M;
d(TAAGCATGCTTA)2: 4.0 · 10ꢁ5 M) solution con-
taining the same concentration of drugs in 50 mM
Tris–HCl buffer (pH 6.35). This operation ensured that
the concentration of DNA increased gradually from 0
to 2.0 · 10ꢁ4 M (0–2.0 · 10ꢁ5 M for d(TAAGC
ATGCTTA)2), while the concentrations of drugs were
kept constant at 2.0 · 10ꢁ6 M. The mixing was achieved
by turning the cuvette up and down for 5 min. Then, the
corresponding fluorescence spectra were measured at
room temperature (20 ꢁC) without degassing (ex
355 nm).
References and notes
1. (a) Gottesfeld, J. M.; Turner, J. M.; Dervan, P. B. Gene
Expression 2000, 9, 77; (b) Dervan, P. B. Bioorg. Med.
Chem. 2001, 9, 2215; (c) Gutierrez, A. J.; Matteucci, M.
D.; Grant, D.; Matsumura, S.; Wagner, R. W.; Froehler,
B. C. Biochemistry 1997, 36, 743; (d) Mercola, D.; Cohen,
J. S. Cancer Gene Therapy 1995, 2, 47.
2. (a) Fox, K. R. Drug–DNA Interaction Protocols; Humana
Press: Totowa, NJ, 1997; (b) Chaires, J. B. Drug–Nucleic
Acid Interactions; Academic Press: New York, 2001; (c)
Jiang, Z.-H.; Hwang, G.-S.; Xi, Z.; Goldberg, I. H. J. Am.
Chem. Soc. 2002, 124, 3216; (d) Chiang, S.-Y.; Azizkhan,
J. C.; Beerman, T. A. Biochemistry 1998, 37, 3109; (e)
Bailly, C.; Chaires, J. B. Bioconjugate Chem. 1998, 9, 513.
3. Wolkenberg, S. E.; Bogor, D. L. Chem. Rev. 2002, 102,
2477.
4. (a) Barton, J. K. Science 1986, 233, 727; (b) Chrisey, L. A.;
Bonjar, G. H. S.; Hecht, S. M. J. Am. Chem. Soc. 1988,
110, 644; (c) Zein, N.; Sinha, A. M.; McGarhren, W. J.;
Ellestad, G. A. Science 1988, 240, 1198.
5. Lown, J. W. Anti-Cancer Drug Des. 1988, 3, 25.
6. Schneller, T.; Latz-Brunng, B.; Wink, M. Phytochemistry
1997, 46, 257.
The association constants (KaÕs) were derived from an
analysis of the relationship between relative fluorescence
intensity (I/I0) and the DNA concentrations by a nonlin-
ear curve fitting to the equation I/I0 = 1 + ((I1 ꢁ I0)/(2I0
[alkaloid]0)) · {([DNA]0 + [alkaloid]0+1/Ka) ꢁ (([DNA]0 +
[alkaloid]0 + 1/Ka)2 ꢁ 4[DNA]0 [alkaloid]0)1/2}, wherein
I0, I, and I represent the fluorescence intensities of
1
alkaloid alone, the sample and alkaloid totally bound,
respectively. [DNA]0 and [alkaloid]0 are the initial ana-
lytical concentrations of DNA and alkaloids, respective-
ly. KaleidaGraph version 3.6 (Synergy Software, USA)
was applied for the nonlinear curve fitting.
7. (a) Creasey, W. A. Biochem. Pharmacol. 1979, 28, 1081;
(b) Kumar, G. S.; Debnath, D.; Maiti, M. Anticancer Drug
Des. 1992, 7, 305; (c) Kumar, G. S.; Debnath, D.; Sen, A.;

32
Y. Qin et al. / Bioorg. Med. Chem. 14 (2006) 25–32
Maiti, M. Biochem. Pharmacol. 1993, 46, 1665; (d)
Birdsall, T. C.; Kelly, G. S. Altern. Med. Rev. 1997, 2, 94.
14. Wilson, W. D. Intercalation Chemistry, Academic Press:
New York, 1982; Chapter 14, pp 445.
8. (a) Krey, A. K.; Halm, F. E. Science 1969, 166, 755; (b)
Saran, A.; Srivastava, S.; Coutinho, E.; Maiti, M. Indian
J. Biochem. Biophys. 1995, 32, 74; (c) Li, W.-Y.; Lu, H.;
Xu, C.-X.; Zhang, J.-B.; Lu, Z.-H. Spectrosc. Lett. 1998,
31, 1287; (d) Li, W.-Y.; Lu, Z.-H. Microchem. J. 1998,
60, 84; (e) Gong, G.-Q.; Zong, Z.-X.; Song, Y.-M.
Spectrochim. Acta Part A 1999, 55, 1903; (f) Mazzini, S.;
Bellucci, M. C.; Mondelli, R. Bioorg. Med. Chem. 2003,
11, 505; (g) Kumar, G. S.; Das, S.; Bhadra, K.; Maiti, K.
Bioorg. Med. Chem. 2003, 11, 4861; (h) Kluza, J.;
Baldeyron, B.; Colson, P.; Rasoanaivo, P.; Mambu, L.;
Frappier, F.; Bailly, C. Eur. J. Pharm. Sci. 2003, 20, 383;
(i) Park, H.; Kim, E. H.; Sung, Y.-H.; Kang, M. R.;
Chung, I. K.; Cheong, C.; Lee, W. Bull. Korean Chem.
Soc. 2004, 25, 539; (j) Chen, W.-H.; Chan, C.-L.; Cai, Z.;
Luo, G.-A.; Jiang, Z.-H. Bioorg. Med. Chem. Lett. 2004,
14, 4955; (k) Chen, W.-H.; Qin, Y.; Cai, Z.-W.; Chan,
C.-L.; Luo, G.-A.; Jiang, Z.-H. Bioorg. Med. Chem.
2005, 13, 1859.
9. Chen, W.-H.; Pang, J.-Y.; Qin, Y.; Peng, Q.; Cai, Z.-W.;
Jiang, Z.-H. Bioorg. Med. Chem. Lett. 2005, 15, 2689.
10. Iwasa, K.; Kamigauchi, M.; Ueki, M.; Taniguchi, M. Eur.
J. Med. Chem. 1996, 31, 469.
11. (a) Schneider, H.-J.; Yatsimirski, A. K. In Principles and
methods in supramolecular chemistry, John Wiley: New
York, 2000, pp 137–143; (b) Xi, Z.; Jones, G. B.; Qabaja,
G.; Wright, J.; Johnson, F.; Goldberg, I. H. Org. Lett.
1999, 1, 1375.
15. Denny, W. A.; Baguley, B. In Molecular Aspects of Anti-
Cancer Drug–DNA Interaction; Neidle, S., Warning, M.,
Eds.; Macmillan: London, 1994.
16. Waring, M. J. Ann. Rev. Biochem. 1981, 50, 159.
17. Wilson, W. D. In Nucleic Acids in Chemistry and Biology;
Blackburn, G. M., Gait, M., Eds.; IRL Press: Oxford,
1990; pp 295–336.
18. Suh, D.; Chairs, J. B. Bioorg. Med. Chem. 1995, 3, 723.
19. (a) Wall, R. K.; Shelton, A. H.; Bonaccorsi, L. C.; Bejune,
S. A.; Dube, D.; McMillin, D. R. J. Am. Chem. Soc. 2001,
123; (b) Sundquist, W. I.; Lippard, S. J. Coord. Chem. Rev.
1990, 100, 293.
20. Pang, J.-Y.; Qin, Y.; Chen, W.-H.; Luo, G.-A.; Jiang,
Z.-H. Bioorg. Med. Chem. 2005, 13, 5835.
21. Yadav, R. C.; Kumar, G. S.; Bhadra, K.; Giri, P.;
Sinha, R.; Pal, S.; Maiti, M. Bioorg. Med. Chem. 2005,
13, 165.
22. (a) Maiti, M.; Chaudhuri, K. Indian J. Biochem.
Biophys. 1981, 18, 245; (b) Debnath, D.; Kumar, G.
S.; Nandi, R.; Maiti, M. Indian J. Biochem. Biophys.
1989, 26, 201.
23. (a) Antony, T.; Thomas, T.; Shirahata, A.; Thomas, T.
J. Biochemistry 1999, 38, 10775; (b) Chen, W.; Turro,
N. J.; Tomalia, D. A. Langmuir 2000, 16, 15; (c)
Izumrudov, V. A.; Zhiryakova, M. V.; Goulko, A. A.
Langmuir 2002, 18, 10348; (d) Shim, Y. H.; Arimondo,
P. B.; Laigle, A.; Garbesi, A.; Lavielle, S. Org. Biomol.
Chem. 2004, 2, 915; (e) Rege, K.; Hu, S. H.; Moore, J.
A.; Dordick, J. S.; Cramer, S. M. J. Am. Chem. Soc.
2004, 126, 12306.
12. Leng, F.; Priebe, W.; Chaires, J. B. Biochemistry 1998, 37,
1743.
13. Wilson, W. D. Nucleic Acids in Chemistry and Biology,
IRL Press: New York, 1990; Chapter 8, pp 297.
24. Pinho, P. M. M.; Pinto, M. M. M.; Kijjoa, A.; Pharadai,
K.; Diaz, J. G.; Herz, W. Phytochemistry 1992, 31, 1403.