Towards echinomycin mimetics by grafting quinoxaline residues on glycophane scaffolds

Article abstract of DOI:10.1016/j.bmc.2010.12.009

Echinomycin is a natural depsipeptide, which is a bisintercalator, inserting quinoxaline units preferentially adjacent to CG base pairs of DNA. Herein the design and synthesis of echinomycin mimetics based on grafting of two quinoxaline residues onto a macrocyclic scaffold (glycophane) is addressed. Binding of the compounds to calf-thymus DNA was studied using UV-vis and steady state fluorescence spectroscopy, as well as thermal denaturation. An interesting observation was enhancement of fluorescence emission for the peptidomimetics on binding to DNA, which contrasted with observations for echinomycin. Molecular dynamics simulations were exploited to explore in more detail if bis-intercalation to DNA was possible for one of the glycophanes. Bis-intercalating echinomycin complexes with DNA were found to be stable during 20 ns simulations at 298 K. However, the MD simulations of a glycophane complexed with a DNA octamer displayed very different behaviour to echinomycin and its quinoxaline units were found to rapidly migrate out from the intercalation site. Release of bis-intercalation strain occurred with only one of the quinoxaline chromophores remaining intercalated throughout the simulation. The distance between the quinoxaline residues in the glycophane at the end of the MD simulation was 7.3-7.5 , whereas in echinomycin, the distance between the residues was ～11 , suggesting that longer glycophane scaffolds would be required to generate bis-intercalating echinomycin mimetics.

Full text of DOI:10.1016/j.bmc.2010.12.009

Bioorganic & Medicinal Chemistry 19 (2011) 826–835
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Towards echinomycin mimetics by grafting quinoxaline residues
on glycophane scaffolds
Dilip V. Jarikote a,à, Wei Li b,c, ,à, Tao Jiang , Leif A. Eriksson a,d, Paul V. Murphy a,
c
⇑
a
School of Chemistry, National University of Ireland, Galway, Ireland
b
School of Chemistry and Chemical Biology, Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Pharmacy, Ocean University of China, Qingdao 266003, China
School of Science and Technology, Örebro University, 701 82 Örebro, Sweden
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Echinomycin is a natural depsipeptide, which is a bisintercalator, inserting quinoxaline units preferen-
tially adjacent to CG base pairs of DNA. Herein the design and synthesis of echinomycin mimetics based
on grafting of two quinoxaline residues onto a macrocyclic scaffold (glycophane) is addressed. Binding of
the compounds to calf-thymus DNA was studied using UV–vis and steady state fluorescence spectros-
copy, as well as thermal denaturation. An interesting observation was enhancement of fluorescence
emission for the peptidomimetics on binding to DNA, which contrasted with observations for echinomy-
cin. Molecular dynamics simulations were exploited to explore in more detail if bis-intercalation to DNA
was possible for one of the glycophanes. Bis-intercalating echinomycin complexes with DNA were found
to be stable during 20 ns simulations at 298 K. However, the MD simulations of a glycophane complexed
with a DNA octamer displayed very different behaviour to echinomycin and its quinoxaline units were
found to rapidly migrate out from the intercalation site. Release of bis-intercalation strain occurred with
only one of the quinoxaline chromophores remaining intercalated throughout the simulation. The
distance between the quinoxaline residues in the glycophane at the end of the MD simulation was
Received 9 September 2010
Revised 23 November 2010
Accepted 3 December 2010
Available online 13 December 2010
Keywords:
Echinomycin
Peptidomimetics
Macrocycle
Scaffold
Carbohydrate
DNA
7
.3–7.5 Å, whereas in echinomycin, the distance between the residues was ꢀ11 Å, suggesting that longer
glycophane scaffolds would be required to generate bis-intercalating echinomycin mimetics.
Ó 2010 Elsevier Ltd. All rights reserved.
1
. Introduction
drug candidates in this area. Echinomycin, a natural depsipeptide,5
described as the prototype bisintercalator, which can insert two
6
A number of approaches aimed at regulation of biological
quinoxaline units adjacent to CG base pairs of DNA as shown by
X-ray crystallography, binds to a sequence recognised by hypoxia
7
processes at the level of the gene are currently being investigated.
These include the development of proteins that can inhibit the
expression of target genes1 or the use of RNA interference (RNAi)
inducible factor-1 (HIF-1), known as the hypoxia-responsive ele-
0
0
ment (HRE) which contains the core sequence 5 -(A/G)CGTG-3
2
8
applied at the post-transcriptional level. There are potential prob-
and has been recognised as a point for clinical intervention. A re-
lated peptide thiocoraline presumably has a similar bis-intercala-
5
lems with biological approaches in that the bioavailability and
delivery of such agents, proteins or nucleic acids can be barriers
to their development into useful therapeutics. Proteins and RNAi
can also be expensive. Small molecules, prepared by synthetic
chemistry, that modulate protein–protein or protein–DNA interac-
tions offer an alterative approach. The polyamides developed by
Dervan and co-workers, for example, are DNA-binding small mol-
tion mechanism but does not display any sequence selectivity for
9
10
DNA. Recent work by Melillo and co-workers, suggest that it
may be justified to propose a role for echinomycin in treatment
of cancer where HIF-1 activity is high.
For these reasons the development of mimetics of echinomycin
would be interesting. Our group and others have been active in the
3
11
ecules and have been shown to disrupt protein–DNA interactions
design and synthesis of peptidomimetics which incorporate a
in a sequence-selective manner.4 Screening small-molecule and
natural product libraries have led to the identification of potential
carbohydrate component.
12,13
These are generally considered to
be non-peptide peptidomimetics. Recently we have been involved
1
4
in developing glycophane scaffolds, which led us to apply these
scaffolds as a basis to generate bivalent inhibitors of carbohy-
⇑
Corresponding author. Fax: +353 91 525700.
E-mail address: paul.v.murphy@nuigalway.ie (P.V. Murphy).
Present address: School of Pharmacy, Jining Medicinal College, Rizhao 276826,
1
4i,j
drate–protein interactions at cell surfaces.
Herein, we describe
the synthesis of echinomycin mimetics (Chart 1) where quinoxa-
line units were grafted to glycophanes as well as a spectroscopic
study of the interactions of these compounds with DNA. The work
China.
à
These authors contributed equally.
0
968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.12.009

D. V. Jarikote et al. / Bioorg. Med. Chem. 19 (2011) 826–835
827
O
Me
Me
N
O
N
N
N
NH
H
N
Me
O
O
O
S
O
N
N
O
S
O
N
H
O
O
Me
N
HN
N
O
Me
Me
O
Echinomycin 1
HO
O
O
HO
O
O
N
R
N
R
O
HO
O
N
HO
N
N
N
N
(
)n
(
)n
R = OH
(
O
)n
(
O
)n
N
N
N
O
O
HO
HO
R
R
N
N
HO
O
HO
O
2
3
n = 1
n = 3
4 n = 1
5 n = 3
Chart 1. Structures of 1–5.
has provided a basis for development of echinomycin and other
depsipeptidomimetics using glycophane and other macrocyclic
scaffolds.
dride gave the diamine 7. The reaction of 8/9 with oxalyl chloride
gave the respective acid chlorides and their subsequent reaction
with 7 gave 10/11. Ring closure metathesis (RCM) using the Grub-
1
bs-I catalyst and subsequent deacetylation gave 2/3. Both the
H
1
3
NMR and C NMR spectra of 2 and 3 showed more than one set
of signals. For 3 one major alkene isomer (trans isomer) is present
and the presence of multiple signal sets is due to the existence of
rotamers 3a and 3b which result from amide bond isomerisation
2
2
. Results and discussion
.1. Molecular modelling
(
Chart 2) and which exchange slowly enough that both isomers
The high resolution crystal structure of the bis-intercalation
complex of the echinomycin with (ACGTACGT) was used for the
2
can be observed by NMR. Hence for 3a both amides have Z-config-
uration, whereas for 3b one amide is Z and the other has the E-con-
first computationally based docking experiment. We first wanted
to establish whether the placement of the quinoxaline residues
onto the glycophane scaffold, such as in 2, could adopt a similar
spacing to that found in echinomycin 1 and potentially intercalate
without unfavourable steric interactions to DNA. A model was
therefore built using Macromodel (for both E and Z-isomers) of 2
so that the distance between the quinoxaline residues and their
respective orientation matched that found in the X-ray structure
1
7
figuration. For 2 two signal sets were also observed. However in
this case it is due to the presence of a mixture of both the cis (Z)
and trans (E) alkene isomers (1:1) from the RCM reaction. There
is no amide isomerism observed for 2. This is because 2 is smaller
and significantly more constrained than 3 and isomerisation of the
amides from ZZ to EZ in 2 would give rise to a more strained
macrocycle. This proposal is supported by conformational analysis
carried out using Macromodel which indicates the isomer of 2
with E and Z amides would be ꢀ12 kJ/mol less stable than the iso-
mer where the amides have both E configurations. Efforts to reduce
alkene of 2 by catalytic hydrogenation led to reduction of the imine
of the quinazoline residue and was not pursued for other com-
pounds. All subsequent biophysical studies with 2 were carried
out with the mixture of alkenes, which were not separable using
chromatography.
1
8
2
of echinomycin complexed to the (ACGT) sequence. Next this
conformation of 2 was manually docked into the site where echi-
nomycin was located; the quinoxaline residues were placed at
identical locations to those adopted by the same residues of echi-
nomycin. The echinomycin residue was then deleted from the
model, leaving 2 and then the energy minimization of the complex
2
of 2 with the (ACGT) was carried out giving the model shown in
Figure 1b. Having shown that 2 had potential to bisintercalate
we commenced with its synthesis as well as that of the more flex-
ible analogues such as glycophane 3 and acyclic analogues 4 and 5.
The direct deacetylation of 10/11 gave respectively 4/5. The
presence of multiple signals for 4 and 5 (e.g., d 100.3, 100.2 and
1
1
3
00.0 for the anomeric carbon for 5 in its C NMR spectrum) indi-
cated, similarly to 3, that 4/5 are mixtures of rotamers.
2
.2. Synthesis of echinomycin mimetics
2
.3. Spectroscopic based measurements
The synthesis of the glycophane derivatives was achieved from
glycosides 8 and 9 which were derived from glucuronic acid, as
previously described (Scheme 1).15 Thus the aldehyde 6 was re-
acted with p-xylylenediamine to give a diimine intermediate.
Subsequent reduction of the diimine with sodium cyanoborohy-
16
Obtaining samples of 2–5 provided the opportunity to study
their binding or interaction with DNA. Previous binding studies
with echinomycin have been carried out using NMR, DSC, CD and

8
28
D. V. Jarikote et al. / Bioorg. Med. Chem. 19 (2011) 826–835
7
Figure 1. (a) ACGT and echinomycin 1 bound fragment of the X-ray crystal structure of 1 (PDB ID: 2ADW ). (b) Two views of the initial model of 2 (trans alkene isomer)
docked to ACGT.
was first studied. Echinomycin was shown to exist as a monomer
before and after binding with calf thymus DNA at the concentra-
tions used as described previously. Addition of calf thymus
N
O
20
N
6
DNA to the echinomycin solution resulted in a gradual reduction
of the intensity of the broad band maximum wavelength
21
1
2
. 1,4-Xylylenediamine, EtOH, 95%
(ꢀ325 nm) in the absorbance spectra. The UV absorbance spectra
. NaBH CN, AcOH, MeOH, 72%
of free 2–5 were measured and they were found to have extinction
coefficients similar to echinomycin itself (Table 1); the absorbance
maximum of free 2 (321 nm) and 3 (319 nm) was blue-shifted by 4
and 6 nm, respectively, when compared to the free echinomycin.
As for echinomycin, the titration of calf thymus DNA to 2 and 3
caused a gradual decrease of intensity of the absorption band in
the visible region of the spectrum. The observed results were con-
sistent with the binding of the chromophores of both echinomycin
and the mimetics 2–5 to double stranded DNA.
3
N
N
N
H
N
H
N
N
7
HO C
2
O
AcO
AcO
(COCl) , DMF,
2
The properties of quinoxaline chromophores in various com-
pounds upon their interaction with DNA were next explored by
steady state fluorescence emission spectroscopy. It has been re-
ported that the fluorescence of echinomycin is gradually quenched
AcO
DIPEA, CH Cl2
2
O
(
)n
8
9
n = 1
n = 3
19f
upon titration with DNA duplexes. This observation was repli-
cated during this study (Fig. 2); in order to observe the emission
spectrum excitation was carried out at a wavelength of 285 nm
and at saturation no further change in fluorescence quenching
was recorded. The fluorescence spectra of 2–5 are shown in Figures
AcO
O
R
N
AcO
O
N
N
N
O
3
and 4, respectively, both in absence and presence of increasing
(
)n
concentrations of DNA. Before treatment with DNA, 2 showed a
strong fluorescence emission band at 420 and at 351 nm. Interest-
ingly when 2 was titrated with calf-thymus DNA fluorescence
enhancement was observed with a 5.5-fold fluorescence enhance-
ment noted at 320 nm. These observations are important as fluo-
rescence enhancement on binding with duplex DNA has
previously only been observed with particular intercalators such
R = OAc
(
)n
O
N
O
AcO
R
N
AcO
1
O
22
as thiazole orange derivatives and ethidium bromide.
0 n = 1,70%
1 n = 3,48%
Untreated acyclic compounds 4 and 5 were strongly fluorescent
and were almost 4–5-fold more fluorescent when compared with
the more conformationally locked derivatives 2 and 3. Compounds
4 and 5 showed emission maxima at 370 and 391 nm, respectively.
The addition of DNA led to 2.8-fold fluorescence enhancements for
both 4 and 5, respectively. The different spectral shape (compare
Figs. 3 and 4) and comparatively low fluorescence enhancement
observed for the acyclic compounds may be due to these com-
pounds having a different binding mode to the DNA duplex, possi-
bly due to their flexibility.
1
1
. Grubbs-I
NaOMe
MeOH
CH Cl
2
2
2
. NaOMe
MeOH
2
3
60%
57%
4 60%
5 57%
Scheme 1. Synthesis of 2–5.
The thermal stabilities were next measured for the calf thymus
DNA duplex before and after hybridising with 2–5 and echinomy-
cin. The melting temp of calf thymus DNA duplex was recorded
fluorescence spectroscopy.19 Herein UV–vis and steady state fluo-
rescence and CD spectroscopy were applied. The thermal denatur-
ation of DNA was also studied in the absence and presence of the
compounds.
(T
with 2 (T
The calf thymus DNA showed high duplex stability upon binding
with 4 (T = 79 °C) and 5 (T = 81 °C) also. The value for echinomy-
M
= 76 °C). The melting temp of calf thymus DNA upon binding
M
= 78 °C) and 3 (T = 79 °C) had increased in both cases.
M
The effect of adding increasing amounts of DNA on the UV
absorbance spectrum of echinomycin (20
lM) in aqueous buffer
M
M

D. V. Jarikote et al. / Bioorg. Med. Chem. 19 (2011) 826–835
829
OH
O
HO
O
O
OH
HO
HO
O
N R
O
OH
O
N
N
N R
R =
O
N
R
N R
O
HO
O
O
HO
OH
O
O
HO
HO
OH 3a
3b
Chart 2. Rotamers of 3.
Table 1
Spectroscopic parameters of free echinomycin, 2, 3, 4 and 5
complexes of the (ACGTACGT)
2
sequence with two echinomycin
7
molecules (PDB ID: 2ADW ), and the corresponding initial model
with two glycophane molecules 2 docked in the place of echinomy-
cin, were explored by means of molecular dynamics (MD) simula-
a
b
ꢂ1
ꢂ1)
c
Substrate
k
max(abs) (nm)
e
(M cm
k
max(em) (nm)
Echinomycin
325
321
319
320
320
11,500
10,481
11,126
11,668
12,302
387
416
394
369
391
24
2
3
4
5
tions. All simulations were performed using the YASARA code,
2
5
and the Amber03 force field. The DNA–drug complexes were
soaked in a water box of approximate dimensions 55 ꢁ 31 ꢁ
3
0 Å, containing 1200–1300 TIP3P water molecules giving a total
a
b
c
Wavelength of absorbance maximum.
Extinction coefficient at absorbance maximum.
Wavelength of fluorescence emission maximum.
of 5100–5200 atoms. The cell was neutralized by replacing random
water molecules by counterions (Na ) and corrected to an overall
+
ionic strength corresponding to physiological conditions. Follow-
ing energy minimization based on steepest decent and simulated
annealing techniques, three sets of 20 ns MD simulations were
conducted for each system (i.e., echinomycin bound to the DNA se-
quence and 2 bound to the DNA sequence). These included (i) a
simulation at 298 K, starting from the energy minimized structure,
5
4
3
2
1
0
0
0
0
0
0
(
ii) a simulation of the same system at T = 150 K, and (iii) a simu-
lation at 298 K starting from the end point of the 20 ns simulation
at 150 K. In order to verify the DNA-water system to be stable, a
3
2 ns MD simulation of the pure DNA octamer system was also
conducted at T = 298 K for benchmarking purposes, using the same
force fields and neutralization techniques as above. The pure DNA
simulations showed the approach to function very well, giving a
system with only minor end-effects in that the outmost base pairs
to some degree starts to hydrogen bond to the aqueous environ-
ment. All simulations were conducted in the NPT ensemble
approximation with periodic boundary conditions, and used Parti-
cle Mesh Ewald (PME) summation for long-range Coulomb interac-
tions with cutoff at 7.86 Å. The simulations were conducted using
3
30 370 410 450 490
λ / nm
1
.25 fs time steps. Intermolecular forces were recalculated every
two simulation steps, and pressure control employed to maintain
a water density of 1.00 g/cm . The simulations showed that the
Figure 2. Fluorescence emission spectra of echinomycin 1 before (black) and after
addition of calf thymus DNA [2 (pink), 4 (violet), 6 (turquoise) and 8 M (blue)].
Measurement conditions: 20 in degassed buffer (100 mM NaCl, 10 mM
NaH PO at pH 7.0). Ex. 285 nm, Em. 318–550 nm Ex. Slit: 10 nm and Em. Slit.
0 nm.
3
l
lM
introduction of the bis-interacalating compounds at the minor
groove forces a partial unwinding of the DNA double helix. Instead
of the 10 base pair long full helix turn in crystalline DNA, the X-ray
structure of the echinomycin–DNA octamer complex reveals a
mere 1/4 turn rotation in eight base pairs. The reduced coiling is
also accompanied by a stretching of the DNA chain, as seen in
Figure 5. The same observation is noted for the DNA octamer com-
plexed with two glycophane molecules 2 (Fig. 5). The DNA-echino-
mycin structure remained very stable throughout each of the 20 ns
simulations; in Figure 6 we display the structures of the echinomy-
cin–DNA complexes (energy minimized) before and after the 20 ns
MD simulations at 298 K. As seen in the figure, the overall struc-
tures are well retained, albeit with some minor end effects in that
the terminal base pairs start interacting with and form hydrogen
bonds to the surrounding water molecules. The RMSD between
the two structures is 2.613 Å over all 782 atoms of the DNA–echi-
2
4
1
M
cin was T = 79 °C. The comparison of the melting temp in the ab-
sence and presence of the compounds suggests that 2–5 and echi-
nomycin are binding to the DNA duplex and inducing higher
duplex stability. The observed high stability of duplexes is similar
with the previously reported binding of daunomycin, cryptolepine
and chlorobenzylidine with DNA duplexes.23
2
.4. Molecular dynamics simulations
The use of computational methods to gain insights into possible
modes of binding or interaction of the glycophane based echino-
mycin derivatives was next explored. Both the bis-intercalation

8
30
D. V. Jarikote et al. / Bioorg. Med. Chem. 19 (2011) 826–835
A
B
5
0
0
0
0
0
0
50
increasing DNA
concentration
increasing DNA
concentration
4
3
2
1
40
30
20
10
0
3
18 373 428 483 538
318 373 428 483 538
λ / nm
Figure 3. Fluorescence emission spectra of (A) 2 and (B) 3 before (Black) and after addition of calf thymus DNA [2 (pink), 4 (violet), 6 (turquoise), 8 (blue), 10 (bright green),
2 (orange) and 14 (teal), 16 (red), and 18 M (grey)]. Measurement conditions: see caption of Figure 5.
1
l
A
B
1
80
60
40
20
00
increasing DNA
concentration
180
160
140
120
100
80
increasing DNA
concentration
1
1
1
1
80
60
40
20
0
60
40
20
0
3
18 373 428 483 538
318 373 428 483 538
λ / nm
Figure 4. Fluorescence emission spectra of 4 (A) and 5 (B) before (black) and after addition of calf thymus DNA [2 (pink), 4 (violet), 6 (turquoise), 8 (blue), 10 (bright green),
2 (orange) and 14 (teal), 16 (red), and 18 M (grey)]. Measurement conditions: see caption of Figure 5.
1
l
nomycin complex; for the echinomycin moieties alone, the RMSDs
are 1.64 and 1.88 Å, respectively. No difference in interactions are
noted from the 20 ns at 150 K followed by 20 ns at 298 K simula-
tions, compared with running the simulation for 20 ns at 298 K
only.
The MD simulations of compound 2 complexed with the DNA
octamer however displayed very different behaviour to that of ech-
inomycin. In the 20 ns simulation at 298 K, several of the quinoxa-
line units rapidly migrate out from the intercalation sites, and
within 5 ns simulation only one of the total four chromophores
present at the beginning remained intercalating. Releasing the
bis-intercalation strain from the system furthermore allowed the
DNA double helix to twist back towards its natural coiled helix
structure. The system then remained having only one intercalating
unit throughout the simulation (see Fig. 6).
upon heating the system to 298 K for another 20 ns MD simulation,
the bis-intercalating entity very rapidly (within 1 ns) attained a
mono-intercalated conformation, which was retained for the
remainder of the simulation. The main rationale behind the diffi-
culty for glycophane 2 to stay bis-intercalated is to be found in the
length of the linker backbone. The distance between the nitrogens
connecting the scaffold to the quinoxaline rings is only 7.3–7.5 Å,
whereas in echinomycin, the corresponding linker length is approx-
imately 11 Å. Given that the linker length is restricted by the xylyl-
ene moiety, which is the same for all four systems synthesized
herein, a very similar behaviour can be expected also for compounds
3–5 (not simulated during this work).
2.5. Summary and conclusions
In the case of 20 ns simulation at 150 K, the system is far more
stable than at 298 K. One of the two peptidomimetic compounds
remains bis-intercalated throughout the simulation, and one is
mono-intercalated with the second quinoxaline ring aligned in a
Glycophane scaffolds to which chromophores are grafted pro-
vides a basis for the design compounds that can interact with
DNA, as is the case for natural depsipeptide backbones. This ex-
tends the applications of glycophanes as scaffolds in bioorganic
chemistry, as they can be modified with recognition groups, in this
‘
flat’ orientation with the p-system facing the DNA-stack. However,

D. V. Jarikote et al. / Bioorg. Med. Chem. 19 (2011) 826–835
831
Figure 5. (a) DNA (ACGTACGT)
2
octamer. (b) X-ray structure of two echinomycin molecules and (c) model of glycophane molecule 2, bound to samt DNA octamer. Top row:
DNA in stick model, echinomycin/glycophane in ball and stick. Bottom row: top view displaying the reduced coiling of the DNA backbone.
case quinoxaline residues, to give compounds that bind to nucleic
acids. Spectroscopic measurements and melting experiments indi-
cated the glycophane derivatives bind to DNA. Molecular dynamics
simulations indicated that the glycophanes complex with a DNA
octamer in a different manner to echinomycin; whereas echinomy-
cin bis-intercalates the glycophanes derived from p-xylylenedi-
amine backbone mono-intercalate. In order to obtain mimetics of
echinomycin (i.e., bis-intercalators) glycophanes which have a dis-
tance between the residues of ꢀ11 Å would be more optimal. The
high affinity of echinomycin for DNA and its sharp sequence selec-
tivity derive from the cyclic peptide portion which fits into the
minor groove of DNA, establishing contacts (H-bonds) with DNA.
This would also need to be considered in synthesis of mimetics
which would have high affinity and selectivity.
signals were assigned with the aid of COSY. Coupling constants
are reported in Hertz. FTIR spectra were recorded using either thin
film between NaCl plates or KBr discs, as specified. Low and high
resolution mass spectra were measured on either LC–MS/MS or
LC Time-of-flight (LCT) mass spectrometers and were measured
in positive and/or negative mode as indicated. TLC was performed
on aluminium sheets precoated with silica Gel and spots visualized
2 4 4
by UV and charring with H SO –EtOH (1:20) and/or PMA, KMnO ,
ninhydrine or mostaine solutions. Column chromatography was
carried out using Silica Gel (0.040–0.630 micron, Merck) and using
a stepwise solvent polarity gradient (starting from the conditions
indicated in each case and increasing the polarity) correlated with
TLC mobility. Reaction solvents were freshly dried and distilled
where stated: acetonitrile, toluene and dichloromethane from
calcium hydride; MeOH from magnesium turnings and tetrahydro-
furan from sodium wire. Alternatively, dichloromethane, tetrahy-
drofuran and MeOH were used as obtained from a Pure-Solv
solvent purification system. Anhydrous DMF and pyridine were
used as purchased from Sigma–Aldrich. Molecular sieves are acti-
vated 4 Å molecular sieves. Semi-preparative HPLC used reverse
3
3
. Experimental section
.1. General
Optical rotations were determined at the sodium D line at 20 °C.
phase YMC-Pack ODS-AQ (S5
l
m, 250 ꢁ 20 mm) unless otherwise
Chemical shifts in NMR spectra are reported relative to internal
O (d 4.80) for 1H and CDCl
(d
stated. The wavelength for detection of peaks using preparative
HPLC was 259 nm. All compounds subjected to the biophysical
study were purified by semi-preparative HPLC. The fluorescence
Me
4
Si in CDCl
3
(d 0.0), HOD for D
2
3
1
3
13
7
7.0) for C at 30 °C, unless otherwise stated C signals were
1
assigned with the aid of DEPT-135, HSQC and HMBC. H NMR

8
32
D. V. Jarikote et al. / Bioorg. Med. Chem. 19 (2011) 826–835
Figure 6. (A) Alignment of DNA octamer complexed with two echinomycin before (green) and after (red) 20 ns simulation at 298 K. (B) DNA octamer with two glycophanes 2,
after 20 ns simulation at 298 K. Intercalating unit indicated by an arrow. (C) As (B), after 20 ns simulation at 150 K, followed by 20 ns simulation at 298 K.
experiments were performed using Varian Cary Eclipse spectropho-
tometer. A Varian Cary 50 Bio-UV/vis spectrophotometer was used
2 4
10 mM NaH PO at pH 7.0 was degassed using vacuum. The calf
thymus DNA and compounds were then mixed and the solutions
adjusted to the final required concentration. The samples were
heated to 65 °C and cooled down to 20 °C before commencing
the experiment. The samples were then heated to 95 °C at a rate
for the optical analysis and
a Varian Cary 100 Bio-UV/vis
spectrophotometer was used for the thermal denaturation experi-
ments, respectively. Circular-dichroism spectroscopy was carried
out using a JASCO 810 CD spectrometer in phosphate buffer at
M
of 1 °C/min. Melting temps (T ) were defined as the maximum of
2
0 °C. All chemicals and DNA were purchased from Sigma-Aldrich.
M
the first derivative of the melting curve. The T values reported
in the text are an average of two experiments.
Ò
Water was purified with a Milli-Q Ultrapure Water Purification Sys-
tem and Millipore Corp. The concentration of DNA solution was
determined by using the average extinction coefficient of
3.4. Fluorescence measurements
ꢂ1
ꢂ1
e260 = 6600 M cm
in water. The echinomycin concentration
was determined using the molar extinction coefficient of
e
325
=
The fluorescence experiments were performed with samples
previously prepared for absorbance study. The fluorescence mea-
surements were carried out at 25 °C with excitation at 285 nm
(excitation slit width: 10 nm; emission slit width: 10 nm). Solvent
background signals were subtracted.
ꢂ1
ꢂ1
1
1,500 M cm in aq buffer.
3
.2. Absorbance measurements
The titration experiments were carried out by adding a stock
solution of compounds to a fluorescence quartz cuvette and diluted
with aq degassed buffer (100 mM NaCl, 10 mM NaH PO at pH 7.0)
to a final concentration of 20 M. The absorbance spectrum was
3.4.1. N,N’-[1,4-Phenylenebis(methylene)]-bis-(1-(quinoxalin-2-
2
4
yl)methanamine) 7
2
6
l
Quinoxaline-2-carbaldehyde 6
(1.58 g, 10 mmol) was dis-
recorded at 25 °C. DNA from stock solutions was added as required
to obtaining the specified concentration. After each addition the
solution was heated at 65 °C and it was cooled to room temp
slowly (5 °C/min). After 20 min the spectra were recorded at the
specified temp.
solved in EtOH (150 mL) and 1, 4-xylylenediamine (0.63 g,
4.6 mmol) was then added and mixture was stirred at 75 °C for
3 h. The mixture was then cooled to room temp and the precipitate
was filtered and washed with EtOH (ꢁ3) to give the intermediate
diimine as an off-white solid (1.82 g, 95%); mp = 185–186 °C. The
diimine (1.81 g, 4.3 mmol) was dissolved in MeOH (50 mL) and
then cooled over an ice bath. Sodium cyanoborohydride (273 mg,
3
.3. Thermal denaturation study
4
.3 mmol) and acetic acid (4 mL) were added to the cooled solution
Melting analysis was carried out using UV spectroscopy. The
thermal denaturation was carried out whilst observing changes
in absorbance at 260 nm. The buffer solution of 100 mM NaCl,
and the suspended solid dissolved gradually whilst the mixture
was stirred at room temp for 3 h. The volatile components were
evaporated under diminished pressure. Ethyl acetate was then

D. V. Jarikote et al. / Bioorg. Med. Chem. 19 (2011) 826–835
833
added to residue and this was then washed with aq NaHCO
aq layer was further extracted with EtOAc (3 ꢁ 10 mL). The com-
bined organic layer was dried (MgSO ), filtered and the solvent
was removed. Chromatography of the residue (EtOAc–MeOH,
:1) gave 7 as a dark red solid (1.31 g, 72%); mp = 116–117 °C;
3
. The
3.4.4. N,N’-Bis(quinoxalin-2-ylmethyl)-N,N’-[1,4-phenylenebis
(methylene)]-2-butene-1,4-diyl-bis-a-D-glucopyranosiduro-
4
namide 2
A degassed solution of 10 (132 mg, 0.12 mmol) in dry dichloro-
methane (120 mL, 1 mM) and under argon was treated with
Grubbs I catalyst (31 mg, 25%) for 42 h. The solvent was evaporated
and chromatography of the residue (dichloromethane–EtOAc, 1:3)
gave the protected intermediate (129 mg, 71%) as a grey solid;
R = 0.15 (dichloromethane–EtOAc, 1:2). To this intermediate
f
(80 mg, 0.074 mmol) in dry methanol (10 mL) and dry dichloro-
methane (5 mL) that was pre-cooled over ice, sodium methoxide
2
1
H NMR (CDCl
3
, 500 MHz): d = 8.88 (s, 2H), 8.05-8.09 (m, 4H),
7
2
1
.70–7.76 (m, 4H), 7.35 (s, 4H), 4.16 (s, 4H), 3.92 (s, 4H), 2.23 (s,
1
3
3
H, NH); C NMR (125 MHz, CDCl ) d 154.9 (C), 145.3, 141.9,
41.8 (each CH), 138.7 (C), 130.0, 129.3, 129.2, 129.0, 128.4 (each
+
CH), 53.4, 52.8 (each CH
2
). HRMS (ESI): Found 443.1960 [M+Na] ,
26 24 6
C H N Na requires 443.1968.
(
0.1 mL from a freshly prepared 1 M solution in MeOH) was added
3
.4.2. N,N’-Bis(quinoxalin-2-ylmethyl)-N,N’-[1,4-phenylenebis
-glucopyran-
and the reaction mixture was stirred at 0 °C for 30 min. The solvent
was removed under reduced pressure to give a yellow solid resi-
due. This was dissolved in water and the solution was acidified
(
methylene)]-bis-(allyl 2,3,4,-tri-O-acetyl-
a-D
osiduronamide) 10
+
The acid 8 (307 mg, 0.79 mmol) was dissolved in dry dichloro-
to pH 6 by addition of amberlite resin (H ). The resin was removed
methane (10 mL) and then cooled over ice and placed under nitro-
by filtration and the filtrate was lyophilized to give the macrocyclic
gen. Dry DMF (100
then added and the mixture stirred over ice for 15 min and at room
temperature for 1 h. The reaction mixture was again cooled over
l
L) and oxalyl chloride (100
l
L, 1.2 mmol) were
product as a white solid (52.2 mg, 85%); [
3 3
CH OH); H NMR (CDCl , 400 MHz): d = 8.97 and 8.95 (each s,
a
]
D
= +30.0 (c 1.07,
1
2H), 8.05–8.08 (m, 4H), 7.79–7.86 (m, 4H), 7.29 (s, 2H), 7.23 (s,
2H), 5.53 (d, J = 15.7 Hz, 1H), 5.45 (t, J = 3.4 Hz, 1H), 5.40 (d,
J = 16.2 Hz, 2H), 5.33 (t, J = 3.4 Hz, 1H), 5.22 (d, J = 17.5 Hz, 2H),
4.71 (d, J = 3.7 Hz, 1H), 4.66–4.69 (m, 2H), 4.53–4.64 (m, 6H),
3.77–3.81 (m, 2H), 3.64–3.72 (m, 8H), 3.50 (dd, J = 3.4 Hz and
ice and the amine 7 (147 mg, 0.35 mmol) and dry DIPEA (200
lL,
1
.14 mmol) in dry dichloromethane (10 mL) were transferred to
the flask. The mixture was stirred over ice for 15 min and subse-
quently at room temp for 15 h. The reaction mixture was then di-
luted with dichloromethane (10 mL) and washed with HCl (10 mL
1
3
3
9.3 Hz, 2H), 3.45 (dd, J = 3.7 Hz and 9.7 Hz, 2H); C NMR (CDCl ,
of 0.1 M) and satd NaHCO
further with dichloromethane (3 ꢁ 10 mL) and the combined ex-
tracts were dried (MgSO ), filtered and the solvent was removed
under reduced pressure. Chromatography (cyclohexane–EtOAc,
:3) gave 10 (270 mg, 70%); R = 0.34 (cyclohexane–EtOAc, 1:3);
mp = 100–102 °C; [ = +56.5 (c 1.04, CHCl
00 MHz): d = 8.82 (br s, 1H), 8.71 and 8.72 (each s, 1H), 8.01–
3
(10 mL). The aq phase was extracted
100 MHz): d = 173.4, 162.7 (each CO), 154.1, 153.8 (each C),
146.3, 146.1 (each CH), 143.0, 142.9, 142.7, 137.7, 137.51 (each
C), 131.8, 131.8, 131.3, 131.3, 129.9, 129.8, 129.8, 129.80, 129.49,
128.7, 128.1, 128.0, 127.7 (each CH), 100.9, 100.8 (each C), 74.8,
4
1
f
74.5, 73.8, 73.1, 72.9, 69.8, 69.1 (each CH), 69.0, 65.6 (each CH
52.8, 52.5, 52.4, 52.3 (each CH ); HRMS (ESI): Found 825.3065
45 6
[M+H] , C42H N O12 requires 825.3095.
2
)
1
a
]
D
3
); H NMR (CDCl
3
,
2
+
4
8
2
4
4
2
1
1
1
1
1
.14 (m, 4H), 7.73–7.82 (m, 4H), 7.09–7.31 (4H), 5.60–5.74 (m,
H) overlapping 5.47–5.50 (m, 4H), 5.09–5.16 (m, 4H) overlapping
.91–5.10 (m, 4H) overlapping 4.77–5.16 (m, 4H) overlapping
.59–4.78 (m, 4H), 3.94–4.05 (m, 2H) overlapping 3.81–3.96 (m,
3.4.5. N,N’-Bis(quinoxalin-2-ylmethyl)-N,N’-[1,4-phenylenebis
(methylene)]-4-octene-1,8-diyl-bis-a-D-glucopyranosiduro-
namide 3
H), 1.90–2.05 (m, 18H); 13C (CDCl
Ring closing metathesis of 11 and subsequent de-O-acetylation
as described for 10 gave 3 (29 mg, 57%) as an off-white solid;
3
, 100 MHz) d = 171.1, 170.3,
69.9, 168.7, 167.3 (each CO), 166.9, 166.8 (each NC@O), 151.7,
51.0 (C), 145.3, 143.4 (CH), 141.7, 141.8, 141.9 (each C), 136.3,
35.9, 135.7, 135.2 (C), 132.6, 132.4 (CH), 130.7,130.2, 130.1,
29.7, 129.3, 129.1, 129.0, 128.7, 127.8, 127.5 (each CH), 118.3,
R
f
= 0.5 (EtOAc–MeOH, 2:1); mp = 163–165 °C; [
3 3
a
]
D
= +15.6 (c 1.0,
1
CH OH); H NMR (CDCl , 500 MHz): d = 8.78 and 8.82 (each s,
2H), 8.01–8.04 (m, 4H), 7.70–7.80 (m, 4H), 7.26–7.28 (m, 4H),
5.14–5.16 (m, 1H), 5.08–5.09 (m, 1H), 4.95–4.98 (m, 2H), 4.84–
4.86 (m, 2H), 4.68–4.71 (m, 2H), 4.65–4.67 (m, 2H), 4.49 (dd,
J = 5.4 Hz and 15.6 Hz, 2H), 3.83–3.87 (m, 2H), 3.69–3.75 (m, 2H),
3.59 (dd, J = 8.6 and 15.3 Hz, 2H), 3.48–3.53 (m, 2H), 3.40–3.44
17.9 (CH
each CH), 50.4, 50.2, 49.2, 49.8 (each CH
COCH ). HRMS (ESI): Found 1105.4046 [M+H] , C56
quires 1105.4042.
2
), 95.8, 95.7 70.3 (CH), 69.7 (CH) 69.9 (CH
2
), 66.5, 66.7
(
2
), 21.0, 20.7, 20.6 (each
+
3
61 6
H N O18 re-
(
m, 2H), 3.31–3.32 (m, 3H), 1.78–1.84 (m, 2H), 1.66–1.73 (m,
13
3
.4.3. N,N’-Bis(quinoxalin-2-ylmethyl)-N,N’-[1,4-phenylenebis
2H), 1.49–1.57 (m, 2H), 1.40–1.47 (m, 2H);
3
C (CD OD,
(
methylene)]-bis-(pent-4-enyl 2,3,4,-tri-O-acetyl- -gluco-
a
-
D
125 MHz): d = 170.5 (C@O), 152.6, 144.7, 141.4, 141.0, 135.7 (each
pyranosiduronamide) 11
C), 130.2, 129.8, 129.7, 129.5, 129.1, 128.4, 128.3, 127.8, 127.4,
Reaction of the acid 9 (582 mg, 1.5 mmol) as described for 8
gave 11 (390 mg, 48%) as a yellow solid; R = 0.5 (CH Cl –EtOAc,
); H NMR (CDCl
100.2, 72.9, 72.3, 72.1 (each CH), 68.7 (CH
29.1, 28.6, 23.2 (each CH ); HRMS (ESI): Found 881.3690 [M+H] ,
46 53 6
C H N O12 requires 881.3721.
2
), 68.3 (CH), 51.2, 49.5,
+
f
2
2
2
1
1
5
7
2
1
:2); mp = 84–86 °C; [
a
]
D
= +56.5 (c 1.0, CHCl
3
3
,
00 MHz): d = 8.82 (br s, 1H), 8.72 (br s, 2H), 8.01-8.14 (m, 4H),
.72–7.83 (m, 4H), 7.09–7.31 (4H), 5.45–5.71 (m, 6H), 5.10 (m,
H), 4.56–4.95 (m, 16H), 3.22–3.60 (m, 4H), 2.01–2.05 (m, 18H),
3.4.6. N,N’-Bis(quinoxalin-2-ylmethyl)-N,N’-[1,4-phenylenebis
(methylene)]-bis-(allyl -glucopyranosiduronamide) 4
De-O-acetylation of 10 (59 mg, 0.053 mmol) gave 4 (21 mg,
46%) as an off-white solid; R
= 0.5 (EtOAc–MeOH, 2:1); 1H NMR
(CDCl , 400 MHz): d = 8.83 (s, 1H), 8.71 and 8.70 (each s), 8.05–
a-D
1
3
.90–1.91 (m, 4H), 1.49–1.58 (m, 2H), 1.37–1.44 (m, 2H);
, 125 MHz) d = 170.4, 170.0, 169.9, 168.8, 168.7,
67.3, 167.0, 166.9 (each C@O), 151.7, 151.1, 145.3, 143.3 (each
C), 142.0, 141.9, 141.7 (each C), 137.3, 137.2 (each CH), 136.3,
C
NMR (CDCl
3
f
1
3
8.08 (m, 4H), 7.79–7.85 (m, 4H), 7.26 (m, 2H), 7.16–7.32 (m, 2H),
5.70–5.77 (m, 2H), 5.53 (d, J = 15.7 Hz, 1H) overlapping 5.45 (t,
J = 3.4 Hz, 1H) overlapping 5.41 (d, J = 16.2 Hz, 1H), 5.30–5.36 (m,
2H) overlapping 5.20–5.26 (m, 1H) overlapping 5.09–5.17 (m,
1H), 4.99–5.06 (m, 2H), 4.93–4.97 (m, 1H), 4.53–4.72 (m, 8H),
1
1
1
6
4
2
35.8, 135.6, 135.1 (each C), 130.7, 130.6, 130.2 130.1, 129.7,
29.4, 129.3, 129.1, 129.0, 128.7, 127.9, 127.5 (each CH), 115.3,
15.1 (each CH
9.3, 69.1 (each CH
9.1, 48.9, 48.8, 29.8, 29.7, 29.6, 29.5, 28.5, 28.4, 28.3 (each CH
0.7, 20.6, 20.5 (each COCH ). HRMS (ESI): Found 1161.4613
18 requires 1161.4668.
2
), 96.7, 96.6, 70.5, 70.0, 69.9, 69.8 (each CH),
), 66.8, 66.7 (each CH), 50.4, 50.2, 49.9, 49.2,
),
2
3.82–3.97 (m, 2H), 3.65–3.81 (m, 6H), 3.44–3.52 (m, 2H); 13
C
2
3
3
NMR (CDCl , 100 MHz): d = 171.4, 170.8 (each amide CO), 152.6,
+
[
M+H] , C60
H
69
N
6
O
144.6, 143.7, 141.3, 141.1, 135.8, 135.2, 133.6, 133.1, 130.4,

8
34
D. V. Jarikote et al. / Bioorg. Med. Chem. 19 (2011) 826–835
+
1
9
6
C
30.2, 129.9, 129.7, 128.3, 128.0, 127.4, 126.9, 126.2, 116.3, 115.3,
replacing random water molecules by counterions (Na ) and cor-
rected to an overall ionic strength corresponding to physiological
conditions. Following energy minimization based on steepest de-
cent and simulated annealing techniques, sets of 20 ns MD simula-
tions were conducted for each system as described in the main
text. All simulations were conducted in the NPT ensemble approx-
imation with periodic boundary conditions, and used Particle Mesh
Ewald (PME) summation for long-range Coulomb interactions with
cutoff at 7.86 Å. The simulations were conducted using 1.25 fs time
steps. Intermolecular forces were recalculated every two simula-
tion steps, and pressure control employed to maintain a water den-
8.9, 98.7, 73.3, 73.0, 72.6, 72.2, 71.6, 68.9, 68.5, 68.2, 67.6, 67.5,
+
4.2, 51.1, 50.4, 49.9, 49.6. HRMS (ESI): Found 853.3440 [M+H] ,
44
49 6
H N O12 requires 853.3408.
3
.4.7. N,N’-Bis(quinoxalin-2-ylmethyl)-N,N’-[1,4-phenylenebis
-glucopyranosiduronamide)
(
methylene)]-bis-(pent-4-enyl
a
-D
5
De-O-acetylation of 11 gave 5 (23 mg, 49%) as a white solid and
as a mixture of E and Z -isomers, R = 0.5 (EtOAc–MeOH, 2:1);
mp = 118–120 °C; [ = +8.4 (c 1.0, CH OD,
OH); 1H NMR (CD
f
a
]
D
3
3
3
5
00 MHz): d = 8.83 (br s, 1H) 8.73 and 8.72 (each s, 1H), 8.02–
sity of 1.00 g/cm .
8
.08 (m, 4H), 7.77–7.85 (m, 4H), 7.16–7.32 (m, 4H), 5.62–5.72
(
5
4
(
4
1
m, 1H), 5.39–5.49 (m, 1H) overlapping 5.28–5.35 (m, 1H), 4.93–
.11 (m, 3H) overlapping 4.83–4.91 (m, 2H) overlapping 4.81–
.83 (m, 4H), 4.75–4.78 (m, 2H), 4.50–4.71 (m, 6H), 3.75–3.84
Acknowledgements
The authors are grateful to Science Foundation Ireland
m, 2H) overlapping 3.64–3.70 (m, 2H) overlapping 3.34–3.54 (m,
(
RFP/06/CHEO32), NUI Galway, and the European Commission
H), 3.12–3.21 (m, 2H), 1.83–2.02 (m, 2H), 1.61–1.68 (m, 1H),
.46–1.57 (m, 2H), 1.13–1.16 (m, 2H); 1 C (CD
3
(Marie Curie EIF Grant No. 220948), the Chinese Ministry of Educa-
tion and the Chinese Scholarship Council for funding.
3
OD, 125 MHz):
d = 171.4, 170.8 (each C@O), 152.6, 152.1, 144.6, 143.7, 141.5,
41.3, 141.1 (each C), 137.8, 137.5 (each CH), 136.3, 135.8, 135.4
each C), 130.5, 130.2, 129.9, 129.8, 128.7, 128.5, 128.4, 128.3,
1
(
1
1
6
Supplementary data
2
28.1, 127.5, 126.9 (each CH), 114.0, 113.9, 113.6 (each CH ),
00.0, 99.7, 73.2, 73.0, 72.7, 72.3, 71.7, 71.6, 68.6, 68.5 (each CH),
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.12.009.
7.9, 67.8, 51.1, 50.5, 50.3, 49.8, 49.7, 49.6, 29.8, 29.4, 28.4, 28.0
+
(
each CH
2
). HRMS (ESI): Found 909.4047 [M+H] , C48
H
57
N
6
O
12 re-
quires 909.4034.
References and notes
3
.5. Initial molecular modeling docking and minimization
1
.
.
Blancafort, P.; Segal, D. J.; Barbas, C. F. Mol. Pharmacol. 2004, 66, 1361. and cited
references.
Fire, A.; Xu, S. Q.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Melillo, C. C.
The high resolution crystal structure of the bis-intercalation
2
15
complex of the echinomycin with (ACGTACGT)
2
obtained from
Nature 1998, 391, 806.
the Protein Databank (http://www.rcsb.org/pdb/; PDB ID: 2ADW)
was used. A fragment of the X-ray structure selected using Macro-
model. Thus all atoms were deleted with the exception of an
3. Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215.
4.
Nickols, N. G.; Jacobs, C. S.; Farkas, M. E.; Dervan, P. B. ACS Chem. Biol. 2007, 2,
61. and cited references.
5
5
.
(a) Corbaz, R.; Ettlinger, L.; Gaumann, E.; Keller-Schierlein, W.; Kradolfer, F.;
Neipp, L.; Prelog, V.; Reusser, P.; Zahner, H. Helv. Chim. Acta 1957, 40, 199; (b)
Yoshida, T.; Katagiri, K.; Yokozawa, S. J. Antibiot. 1961, 14, 330.
Dawson, S.; Malkinson, J. P.; Paumier, D.; Searcey, M. Nat. Prod. Rep. 2007, 24, 109.
Cuesta-Seijo, J. A.; Sheldrick, G. M. Acta Crystallogr., Sect. D 2005, 61, 442. and
cited references.
(
2
ACGT) fragment complexed to echinomycin. All bonds and
hybridizations of atoms were then checked and hydrogen atoms
were then added in Macromodel. This led to phosphate residues
being converted to phosphonic acids for the purpose of the model-
ling. A model of 2 (trans isomer) was then built in Macromodel so
that distance between the quinoxaline residues projected from the
nitrogen atoms of the scaffold and their respective orientation
matched that found in the X-ray structure of echinomycin com-
6
7
.
.
8
9
.
.
Cohen, H. T.; McGovern, F. J. N. Eng. J. Med. 2005, 353, 2477.
Boger, D. L.; Ichikawa, S.; Tse, W. C.; Hedrick, M. P.; Jin, Q. J. Am. Chem. Soc. 2001,
1
23, 561.
10. Kong, D.; Park, E. J.; Stephen, A. G.; Calvani, M.; Cardellina, J. H.; Monks, A.;
Fisher, R. J.; Shoemaker, R. H.; Melillo, G. Cancer Res. 2005, 65, 9047.
1
1
1
1. For recent reviews on peptidomimetic development see: (a) Robinson, J. A.;
2
plexed to the (ACGT) sequence. Next this structure 2 was manu-
DeMarco, S.; Gombert, F.; Moehle, K.; Obrecht, D. Drug Discovery Today 2008,
ally docked into the site where echinomycin was located using
the quinoxaline residues as a guide. Thus the quinoxaline residues
of the model of 2 were superimposed so that they were overlayed
with identical atoms of the quinoxaline residues of echinomycin.
The echinomycin residue was then deleted. Subsequently the com-
1
3, 944; (b) Clynen, E.; Baggerman, G.; Husson, S. J.; Landuyt, B.; Schoofs, L.
Expert. Opin. Drug Discov. 2008, 3, 425; (c) Vagner, J.; Qu, H. C.; Hruby, V. J. Curr.
Opin. Chem. Biol. 2008, 12, 292.
2. Hirschmann, R.; Nicolaou, K. C.; Pietranico, S.; Leahy, E. M.; Salvino, J.; Arison,
B. H.; Cichy, M. A.; Spoors, P. G.; Shakespeare, W. C.; Sprengeler, P. A.; Hamley,
P., ; Smith, A. B., III; Reisine, T.; Raynor, K.; Maechler, L.; Donaldson, C.; Vale,
W.; Freidinger, R. M.; Cascieri, M. A.; Strader, C. D. J. Am. Chem. Soc. 1993, 115,
2
plex of 2 and (ACGT) which remained was energy minimized to
12550.
remove any unfavourable interactions that existed from the dock-
ing. The OPLSAA force field in Macromodel was applied in the en-
ergy minimization. The pdb file of this minimized structure has
been included with Supplementary data.
3. For recent reviews on peptidomimetics derived from carbohydrates see: (a)
Murphy, P. V.; Dunne, J. L. Curr. Org. Synth. 2006, 3, 403; (b) Velter, I.; La Ferla,
B.; Nicotra, F. J. Carbohydr. Chem. 2006, 25, 97; (c) Murphy, P. V. Eur. J. Org.
Chem. 2007, 4177; (d) Murphy, P. V.; Velasco-Torrijos, T. In Glycoscience-
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3
.6. Molecular dynamics simulations
1
4. For synthesis and application of glycophanes, see: (a) Bukownik, R. R.; Wilcox,
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2
The bis-intercalation complex of the (ACGTACGT) sequence
with two Echinomycin molecules (PDB ID 2ADW), and the corre-
sponding model with two glycophane molecules 2 docked in the
place of echinomycin, were explored by means of molecular
dynamics (MD) simulations. All simulations were performed using
2
4
25
the YASARA code, and the Amber03 force field. The DNA–drug
complexes were soaked in a water box of approximate dimensions
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5
5 ꢁ 31 ꢁ 30 Å, containing 1200–1300 TIP3P water molecules
2009, 74, 9010.
giving a total of 5100–5200 atoms. The cell was neutralized by

D. V. Jarikote et al. / Bioorg. Med. Chem. 19 (2011) 826–835
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