Recognition and discrimination of DNA quadruplexes by acridine-peptide conjugates

Article abstract of DOI:10.1039/b814682a

We have explored a series of trisubstituted acridine-peptide conjugates for their ability to recognize and discriminate between DNA quadruplexes derived from the human telomere, and the c-kit and N-ras proto-oncogenes. Quadruplex affinity was measured as

Full text of DOI:10.1039/b814682a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Recognition and discrimination of DNA quadruplexes by acridine-peptide
conjugates†
James E. Redman,a J. M. Granadino-Rolda´n,b James A. Schouten,a Sylvain Ladame,a Anthony P. Reszka,b
Stephen Neidleb and Shankar Balasubramanian*a
Received 22nd August 2008, Accepted 22nd September 2008
First published as an Advance Article on the web 30th October 2008
DOI: 10.1039/b814682a
We have explored a series of trisubstituted acridine-peptide conjugates for their ability to recognize and
discriminate between DNA quadruplexes derived from the human telomere, and the c-kit and N-ras
proto-oncogenes. Quadruplex affinity was measured as the peptide sequences were varied, together with
their substitution position on the acridine, and the identity of the C-terminus (acid or amide). Surface
plasmon resonance measurements revealed that all compounds bound to the human telomeric
quadruplex with sub-micromolar affinity. Docking calculations from molecular modelling studies were
used to model the effects of substituent orientation and peptide sequence. Modelling and experiment
were in agreement that placement of the peptide over the face of the acridine is detrimental to binding
affinity. The highest degrees of selectivity were observed towards the N-ras quadruplex by compounds
capable of forming simultaneous contacts with their acridine and peptide moieties. The ligands that
bound best displayed quadruplex affinities in the 1–5 nM range and at least 10-fold discrimination
between the quadruplexes studied.
telomerase.24 However a major challenge is to design ligands with
specificity for individual quadruplexes amongst the multitude of
Introduction
Quadruplexes are four-stranded nucleic acid secondary structures
potential quadruplex sequences.
in which guanine bases form stacked planar tetrads stabilized
We describe here the development of a general quadruplex-
by hydrogen bonding and cation coordination.1 Folding rules
binding scaffold with substituents that confer affinities of 1–5 nM
predict many potential quadruplex sequences within the human
together with up to an order of magnitude discrimination between
genome, in addition to those at telomeric repeats.2 Sequences with
the quadruplexes studied. Molecular modelling has been used to
a high propensity to form quadruplexes are not distributed evenly
rationalize the binding to the telomeric quadruplex and to suggest
throughout the genome but are more likely to be found in gene
how discrimination between quadruplexes might occur.
promoters, proto-oncogenes and the first intron.3,4 Quadruplex-
Monomolecular quadruplexes display a wide range of topolo-
forming sequences have been found in genes including c-myc,5
gies differing in the nature and positions of the loops connecting
the strands, and thus in relative strand orientation. The loops
and peripheral grooves provide a pattern of hydrogen bonding,
c-kit,6,7 N-ras,8,9 Ki-ras,10,11 Rb,12 RET,13 VEGF,14 HIF-1a,15
BCL2,16 PDGF-A,17 and c-myb.18 We and others have shown that
quadruplex-binding ligands can regulate the expression of such
hydrophobic p surfaces, and negative charges which are unique
genes.11,19–22 This evidence, together with a genome wide analysis
to the sequence of a particular quadruplex. The guanine tetrads
of expression levels23 of genes containing putative quadruplex
themselves are conserved amongst quadruplexes hence ligand
forming sequences in the vicinity of the transcription start
motifs that interact predominantly with the tetrad should display
site suggests that quadruplexes may naturally act as regulatory
broad quadruplex affinity. It has been found that ligands can
show a degree of discrimination between the c-myc25 or c-
elements. Quadruplex-binding ligands will enable us to further
address this hypothesis and artificially regulate the expression of
genes of medical interest. The human telomeric quadruplex is
also a target for antitumour compounds that disrupt the telomere
nucleoprotein complex and/or inhibit the action of the enzyme
kit21,26,27 quadruplexes and human telomeric quadruplex or even
perturb quadruplex topology.27,28 Our strategy for the discovery
of high affinity ligands with selectivity between quadruplexes
employs a common core that targets the planar surface of a
G-tetrad, appended with variable substituents to contact the
loops and grooves which distinguish quadruplexes from each
other. As core units from which to build quadruplex binding
ligands according to this design template, we and others have
investigated various fused tricyclic planar heterocycles including
ethidium derivatives,29 quindolines,22 isoalloxazines,21 carbazoles30
and acridines.31–35 The dimension of the long axis of these planar
cores approximately matches the width of a G-tetrad, such that
substituents are expected to be positioned correctly to interact with
up to three of the quadruplex loops and grooves. Here we describe
aUniversity Chemical Laboratory, University of Cambridge, Lensfield Road,
Cambridge, CB2 1EW, UK. E-mail: sb10031@cam.ac.uk; Fax: +44 1223
336913; Tel: +44 1223 336347
bCancer Research UK Biomolecular Structure Group, The School of
Pharmacy, University of London, 29-39 Brunswick Square, London, WC1N
1AX, UK. E-email: stephen.neidle@pharmacy.ac.uk; Fax: +44 020 7753
5970; Tel: +44 7753 5969
† Electronic supplementary information (ESI) available: HPLC conditions,
amino acid analysis and mass spectral data for compounds 7–21. UV
thermal melt and CD spectrum of N-ras DNA quadruplex. See DOI:
10.1039/b814682a
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peptide-acridine conjugates designed according to these principles,
with structural variation to test the binding mode hypothesis. The
affinity of the compounds for three proto-oncogene quadruplexes
and the human telomeric quadruplex is compared, and interpreted
in terms of the compound structures and models of complexes with
the telomeric quadruplex.
followed by alkylation with pyrrolidine and conversion to an
acridine by treatment with phosphoryl chloride according to
a literature route. Displacement of the 9-chloro group using
aminobenzoic acids (Scheme 1) afforded the acridine building
blocks required for conjugation at the N-terminus of a peptide to
yield compounds 10–21. Compounds 7–9 were prepared from bis-
acid 6 which was synthesised according to Scheme 2. Alkylation
of the chloropropionyl side chains of a substituted acridone with
sarcosine ethylester, supplied as a hydrochloride salt, was found to
proceed most effectively using an in situ deprotonation by dropwise
addition of sodium ethoxide. Conversion of 4 to acridine 5 using
POCl3 and reaction with N-dimethylphenyldiamine proceeded
smoothly. Careful hydrolysis of the ethyl esters of 5, to avoid
reformation of the acridone by hydrolytic loss of the 9-substituent,
Results and discussion
Ligand design
Previous studies have demonstrated that 3,6,9-substituted acridine
derivatives recognize quadruplexes,32,34 and it has been confirmed
by a co-crystal structure of a bimolecular telomeric sequence with
the compound BRACO-19 that the acridine stacks on a terminal
guanine tetrad.35 The ability of heterocyclic ligands to interact also
with quadruplex grooves has been suggested by circular dichroism
spectroscopy.36 We have previously shown37 that acridines bearing
peptides at the 3,6-positions, but lacking a 9-substituent, bind a
human telomeric monomolecular quadruplex with dissociation
constants in the range 0.1–10 mM. 9-Anilino substituents bearing
simple tertiary alkylamines have enhanced interactions with the
telomeric quadruplex relative to the parent compound with an N-
dimethylphenyldiamine substituent.38 Affinity for the telomeric
quadruplex, as judged by a FRET melting assay, was also
comparable or enhanced when a 9-anilino substituent was replaced
with extended and more flexible benzylamines.31 This substituent
provides a point for variation to adjust selectivity whilst main-
taining the constant tetrad binding motif and we anticipated that
a peptide would present a more complex recognition surface and
hence augment discrimination between quadruplexes. The effect of
a short peptide substituent at this position had not previously been
investigated. In the current work, we have therefore investigated
trisubstituted peptide acridine conjugates, varying the location
and orientation of the peptide substituent with respect to the
acridine core which is proposed to act as an anchor group by
stacking on to the terminal guanine tetrad.
Scheme 1 Reagents and conditions: (a) aminobenzoic acid, CHCl3, D.
In the first series of compounds, 7–9, the peptides are appended
to the 3- and 6-positions of the acridine core and the 9- substituent
is N-dimethylphenyldiamine from the parent compound BRACO-
19. In contrast to the approach of Carlson et al. in which an
acridine amino acid residue is incorporated into the backbone
of peptides,39 we have opted for a bis-carboxylic acid-substituted
acridine for symmetrical conjugation to the N-termini of two
peptides.40 Peptide sequences were chosen from our previous
screening experiments against the human telomeric quadruplex.41
In the second series, 10–21, pyrrolidine propionamide sub-
stituents at the 3,6-positions were retained from earlier optimal
designs34 and the peptide was conjugated through its N-terminus
to the 9-position via an aminobenzoyl linker. The linker permits
variation in the orientation of the peptide with respect to the
acridine, and ortho, meta and para variants were synthesized.
The peptide C-terminus is free, and amide and acid variants were
prepared in anticipation of charge effects on quadruplex affinity.
Ligand synthesis
Scheme 2 Reagents and conditions: (a) sarcosine ethylester hydrochloride,
NaI, DMF, NaOEt, D; (b) (i) POCl3, D (ii) N-dimethylphenyldiamine,
CHCl3, D; (c) LiOH, THF, H2O, rt.
The acridine core of the ligands was derived ultimately from
3,6-diaminoacridone by acylation with chloropropionyl chloride,
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Table 2 Substitution patterns and sequences of 9-peptide acridines
afforded the bis-carboxylic acid. All peptides were prepared by
Fmoc solid phase synthesis and after terminal Fmoc deprotection,
the acridines were conjugated to their N-termini using the same
reagents (PyBOP, HOBt, DIPEA) as for standard amino acid
couplings. This strategy was successfully employed to prepare the
3,6-bis peptide conjugates by one step intersite solid phase cross
linking.40
Binding studies
Compound
Substitution
Peptide
C-terminus
Surface plasmon resonance (SPR) was used to determine dissoci-
ation constants against various folded DNA targets that included
the human telomeric quadruplex (Htelo), quadruplexes from the
promoter of the human c-kit gene, c-kit1,6 c-kit2,7 and from the
5¢-untranslated region of the human N-ras gene.9 As a second mea-
sure of quadruplex interaction, the ability of ligands to stabilize a
telomeric quadruplex to thermal unfolding was determined,42 by
measuring the change in its melting temperature, Tm.
The magnitudes of the dissociation constants of 3,6-bis pep-
tide acridines (7–9) and the previously reported 3,6-bis peptide
acridones for Htelo correspond well,37 indicating that inclusion
of the additional 9-dimethylphenyldiamine substituent has little
effect on the Htelo affinity (Table 3). The apparent stoichiometries
differ between compounds, with 8 and 9 adopting a 2:1 binding
mode whereas compound 7 adopts a 1:1 mode, suggesting that
10
11
12
13
14
15
16
17
18
19
20
21
o
m
p
FRHR
FRHR
FRHR
KRSR
KRSR
KRSR
FRHR
FRHR
FRHR
KRSR
KRSR
KRSR
NH2
NH2
NH2
NH2
NH2
NH2
OH
OH
OH
OH
OH
o
m
p
o
m
p
o
m
p
OH
the former compounds bind to both terminal tetrads raising the
possibility that the latter either distinguishes between them or
binds in a strongly negatively cooperative manner. Compounds
7–9 display no significant discrimination between the three
quadruplexes tested, binding to all with similar affinity indicating
that interactions occur primarily through features shared by the
quadruplexes such as the presence of the guanine tetrads. We
hypothesise that the relatively flexible linkers at the 3- and 6-
position are not conducive to the formation of structure specific
interactions between the peptides and quadruplex.
Table 1 Sequences of 3,6-bis peptide acridines
The affinities of the 9-peptide conjugates (10–21) for Htelo
are higher than the 3,6-bis-peptide acridine conjugates (7–9) by
approximately an order of magnitude confirming the importance
of the acridine substitution pattern for affinity. We have therefore
focussed our attention on these compounds. The orientation of the
9-aminobenzoyl substitution was varied between ortho, meta and
para in the two series of compounds 10–12 and 13–15. Dissociation
Compound
Peptide
7
8
9
FRHR
KRSR
RKKV
Table 3 Dissociation constants (Kd std. error) and stoichiometries (n) of compounds 7–21 with the Htelo, c-kit1, c-kit2 and N-ras quadruplexes,
determined by surface plasmon resonance
Htelo
c-kit1
c-kit2
N-ras
Compound
Kd/nM
n
Kd/nM
n
Kd/nM
n
Kd/nM
n
a
a
a
a
a
a
7
770 80
250 40
380 50
140 60
50 10
1.2
2.2
2.3
0.9
1.1
0.8
0.7
1.2
1.0
0.4
0.8
0.7
0.6
0.8
0.8
800 100
290 40
400 50
120 40
130 50
50 10
1.3
2.0
1.9
0.6
1.3
0.7
0.7
1.2
1.1
0.4
0.9
0.7
0.6
1.0
0.9
440 70
240 40
460 70
0.7
1.5
1.6
0.7
0.6
0.8
0.8
0.5
0.8
0.9
0.6
0.5
1.1
0.6
0.6
8
9
10
11
12
13
14
15
16
17
18
19
20
21
25
50 20
8
0.4
0.9
1.3
0.8
1.2
1.3
0.4
0.7
0.6
0.6
0.8
0.8
129
3
60 40
28
38
22
23
20
25
19
30
13
17
5
9
4
4
8
3
3
3
2
2
138
37
5
7
21
16
4
2
3
1
1
37
7
50 20
90 10
49
40 10
40 10
60 30
50 10
40 10
40 20
7
9
69.3 0.8
40 10
17
5
29
5
1
2
35
37
33
8
3
6
29
9
3.9 0.8
4.4 0.9
50 10
30 10
a Not determined.
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Table 4 Ligand concentration required to stabilize F21T quadruplex by
Table 5 Interaction energies of compounds 10–15 with the human
parallel telomeric quadruplex, determined by docking calculations and
Boltzmann weighting (at 300 K) of the 25 lowest-energy complexes
25 ◦C in a FRET melting assay
Ligand
[Ligand] for DTm of 25 ◦C/mM
Compound
Energy/kcal mol-1
10
11
12
13
14
15
16
17
18
19
20
21
1.16
0.36
0.32
0.89
0.10
0.17
3.27
0.34
0.29
1.06
0.09
0.12
10
11
12
13
14
15
-110.34
-127.32
-133.85
-117.34
-141.19
-142.38
diimide46 and BRACO-1935 ligands have shown that these ligands
bind to a parallel topology with its feature of exposed terminal
G-tetrads. Additionally it has been reported that the parallel
topology is stabilized in K+ solution relative to the hybrid forms
under the influence of molecular crowding induced by polyethylene
glycolwhichis intendedtomimic biologically relevantintracellular
conditions.47 Hence here we have used the parallel native crystal
structure44 as a basis for the modelling studies, especially since
parallel folds are also relevant to the c-kit and N-ras quadruplexes.
Based on circular dichroism spectra, the N-ras DNA quadruplex
appears to adopt a parallel fold with a single nucleotide loop,9
contrasting with Htelo in which all loops are three nucleotides in
length. The 1H-NMR of the c-kit1 quadruplex was reported to be
sharp and consistent with a well-defined single fold, which sub-
sequent studies have shown to correspond to a parallel topology
and structure, albeit with novel loop arrangements.6 The native c-
kit2 sequence forms a mixture of predominantly parallel-stranded
conformations with both single and five-nucleotide loops.7 These
major structural differences between quadruplexes can be expected
to provide a basis for ligand discrimination between them. The
Htelo quadruplex is the sole member of this group for which
extensive structural data are currently available, and so modelling
studies have been restricted to this one.
We have assumed a binding mode for the molecular modelling
in which the acridine stacks against the terminal G-tetrad of
the parallel form of the human monomolecular quadruplex. It
was reasoned that with the 9-aminobenzoyl substituent oriented
to avoid steric clash, meta and para substitution would direct
the peptide away from the tetrad to favour interactions with
loops and grooves. This would explain both the lower affinity
and selectivity of ortho-substituted compounds. In addition to
experimental measurements of association constants, we probed
this hypothesis using molecular modelling with the telomeric
quadruplex. Models of compounds 10–15 were docked to the
quadruplex, and the interaction energy (Table 5) was calculated as
a Boltzmann weighting of the 25 lowest-energy structures.
constants for the Htelo, c-kit1, c-kit2 and N-ras quadruplexes,
as determined by SPR, are given Table 3. The concentrations of
ligand required to induce a change in the melting temperature of
the telomeric quadruplex of 25 ◦C as determined in the FRET
melting assay42 are given in Table 4. Of the two peptide categories,
the KRSR compounds typically displayed higher affinity than
their FRHR counterparts. Compound 10 showed an almost 6-
fold greater affinity for the c-kit1 quadruplex compared to the
Htelo one, and similar discrimination against the c-kit2 and N-
ras quadruplexes. This selectivity is reversed for compounds 12
and 15, which have a 1:1 stoichiometry with both Htelo and c-
kit1 quadruplexes. Compounds 10–15 display little discrimination
between Htelo and c-kit2, although overall there is a slight (<3-
fold) bias towards Htelo.
The affinities for the N-ras quadruplex are some of the highest
reported to date for quadruplex complexes. There is a general
bias, with the exception of compound 16, for preferential binding
to the N-ras quadruplex over Htelo. The selectivity is greatest for
compound 14. Significant selectivities were observed with the two
c-kit quadruplexes; compound 14 shows an order of magnitude
discrimination for the N-ras quadruplex over both the c-kit1 and
c-kit2 ones. Compounds 15 and 21 also show selectivity for N-ras
over c-kit1 and to a lesser extent c-kit2, whereas compound 10 also
shows a five-fold preference for c-kit1 over N-ras. The highest (>5-
fold) selectivities towards N-ras over the two c-kit quadruplexes
are all achieved by ligands with a meta or para substitution pattern.
The ortho compounds show little or no selectivity for N-ras, and
in the case of 10 actually discriminates against this quadruplex in
favour of the c-kit1 one. Overall the KRSR peptide substituent
leads to greater N-ras selectivity than FRHR. It is interesting to
note that the apparent stoichiometry of binding in some instances
is less than 1:1. A possible interpretation of this phenomenon could
be heterogeneity in the folding of the quadruplex, with ligands
exhibiting preferential binding to a particular folded species out
of several simultaneously present.
The models were classified according to the binding mode
as “stacked” where the acridine core makes p–p contacts with
the G-tetrad, “groove” where there are interactions between the
acridine and the DNA groove, or “displaced” when both types
of interaction are absent. The resulting models predominantly
exhibited the “stacked” binding mode with the acridine group
stacked on the exposed tetrad, offset from the centre to locate
the pyrrolidine substituents in the vicinity of adjacent grooves
(Fig. 1). In these models the peptide typically either lies along a
single groove, or basic amino acid side chains contact adjacent
loops or grooves as shown in Fig. 1a and b respectively.
Molecular modelling
There is evidence from a considerable number of studies for
dynamic inter-converting parallel, anti-parallel and hybrid folds of
the human telomeric quadruplex.43,44 Cations, flanking sequences
and the presence of ligands have all been found to influence the
folding topology. Recent X-ray structural studies of co-crystals
of human telomeric quadruplexes with porphyrin,45 naphthalene
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Fig. 1 Representative models of peptide-acridine conjugates docked to the parallel human telomeric quadruplex (a) Compound 11, showing the acridine
and Phe groups stacking against a tetrad, with pyrrole and peptide contacts in three adjacent grooves. (b) Compound 15, showing the peptide side chains
making contacts with two adjacent grooves.
There is competition between the acridine, the 9-aminobenzoyl
substituent and a Phe side chain (of FRHR) for stacking on the
tetrad. In several models the 9-aminobenzoyl substituent, instead
of the acridine, stacks on the tetrad, as these two groups are
sterically inhibited from coplanarity. In some cases this leads to
complete displacement of the acridine from the tetrad and into the
quadruplex groove. Compound 11 showed the highest propensity
to adopt the “displaced” mode, with a weighted population of
32%. The models of ortho-substituted 13 indicate an especial
tendency towards acridine groove binding, exhibiting “stacked”
and “groove” binding modes of similar energy that could coexist
at room temperature with only 25% of the population in the
“stacked” binding mode, and the remainder in the groove.
A plot (Fig. 2) of the calculated interaction energy against
experimental log(Ka) for the Htelo quadruplex demonstrates the
trend that the ortho substitution pattern gives rise to the lowest
affinity compounds, which can be ascribed to the difficulty for
the quadruplex to simultaneously contact both the acridine and
peptide moieties. The greater affinity of the KRSR peptides for
Htelo relative to their FRHR counterparts is also reproduced
by the modelling. An explanation is that favourable hydrogen
bonding and electrostatic interactions from the lysine residue
contribute to binding to a greater extent than any hydrophobic
and p contacts formed by the phenylalanine, which must compete
for these interactions with the acridine and aminobenzoyl moieties.
The trends observed by SPR are reflected in data from the thermal
melting assay (Table 4), namely a higher concentration of ligand is
required to achieve DTm 25 ◦C for ortho-substituted ligands than
meta or para, and for FRHR-substituted ligands than their KRSR
analogues.
Surprisingly, the carboxyl-terminated compounds 16–21 bound
to Htelo as tightly as the corresponding amides, and induced
similar shifts in Tm. It had been anticipated that at pH 7.4 the
carboxyl terminus would be deprotonated, leading to electrostatic
repulsions with the oligonucleotide. This view was supported by
docking calculations on 17 and 20 with Htelo which revealed
that the weighted interaction energies of the deprotonated forms
were 15 and 23 kcal mol-1 less favourable than the protonated
forms respectively. The experimentally-observed binding affinities
of acids 16–21 relative to the amides could arise from protonation
of the carboxylates due to an environmental perturbation in the
pKa value owing to the close proximity of the oligonucleotide,
or intramolecular interactions involving the peptide terminus
which preorganize the conformation of the peptide for quadruplex
binding.
Conclusion
This study has demonstrated the principle that it is possible to
devise molecules with a common G-tetrad interacting acridine
core and variable peptide substituents that have the ability to
discriminate between different quadruplex types. The substitution
pattern and orientation of the peptide substituent with respect to
the acridine appears to be important for affinity and selectivity of
the ligands. Just a single peptide at the 9-position when combined
with 3,6-pyrrolidine propionamide substituents is sufficient to
result in ligands with high affinity and selectivity for particular
quadruplexes. Molecular modelling suggested that the most
successful compounds permit the acridine core to simultaneously
stack on a tetrad and the peptide to make loop and groove
contacts. Compounds with meta and para substitution typically
exhibited selectivity towards a parallel quadruplex derived from
the human N-ras gene in preference to the human telomeric
quadruplex. In terms of both selectivity and affinity, the peptide
substituent KRSR proved marginally superior to FRHR. These
Fig. 2 Plot of Ka
shown on a logarithmic scale against calculated interaction energies, for
compounds 10–15.
( std. error) for binding to the Htelo quadruplex,
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results demonstrate the feasibility of constructing high-affinity
quadruplex selective ligands by appending a variable substituent
to a generic quadruplex binding scaffold. It is hoped that the future
availability of structural data on non-telomeric quadruplexes will
enable rational design to reinforce these conclusions and permit
further enhancements to selectivity to be achieved.
110.4, 105.3, 54.0, 49.9, 33.0, 23.1; m/z (ES+) [M + H]+ requires
595.3033, found 595.2957.
3-[3,6-Bis-(3-pyrrolidin-1-yl-propionylamino)-acridin-9-ylamino]-
benzoic acid 2. Prepared analogously to 1, substituting with 3-
aminobenzoic acid instead of anthranilic acid. HPLC purification
yielded 2 as an orange solid (24 mg, 24%). Found: C, 48.6; H, 4.3;
N, 8.4. C34H38N6O4·3TFA·3H2O requires C, 48.5; H, 4.8; N, 8.5%;
dH(400 MHz, D2O) 8.02 (2 H, d, J 2, Acr 4,5-H), 7.88 (1 H, d, J
8, Ar 6-H), 7.67 (3 H, m, Ar 2-H, Acr 1,8-H), 7.44 (1 H, t, J 8, Ar
5-H), 7.29 (1 H, d, J 8, Ar 4-H), 7.07 (2 H, dd, J 9 and 2, Acr
2,7-H), 3.67 (4 H, m, pyrrolidine NCH), 3.55 (4 H, t, J 7, CH2),
3.11 (4 H, m, pyrrolidine NCH), 2.97 (4 H, t, J 7, CH2), 2.13 (4 H,
m, pyrrolidine CH), 1.99 (4 H, m, pyrrolidine CH); dC(126 MHz,
D2O) 170.6, 170.2, 162.9 (q, J 35), 152.9, 143.2, 141.1, 140.4,
133.0, 130.2, 128.1, 128.0, 126.3, 124.4, 117.4, 116.2 (q, J 292),
110.1, 105.6, 54.4, 50.1, 32.3, 22.6; m/z (ES+) [M + H]+ requires
595.3033, found 595.3051.
Experimental
DNA Sequences
Desalted biotinylated oligonucleotides were supplied by Invitro-
gen.
Htelo 5¢ Biotin GTT AGG GTT AGG GTT AGG GTT AGG
GTT AGG 3¢
c-kit1 5¢ Biotin AGA GGG AGG GCG CTG GGA GGA GGG
GCT G 3¢
c-kit2 5¢ Biotin CCC GGG CGG GCG CGA GGG AGG GGA
GG 3¢
4-[3,6-Bis-(3-pyrrolidin-1-yl-propionylamino)-acridin-9-ylamino]-
benzoic acid 3. Prepared analogously to 1, substituting with
4-aminobenzoic acid instead of anthranilic acid. The product was
obtained as an orange solid after HPLC purification (18.5 mg,
18%). Found: C, 48.25; H, 4.4; N, 8.4. C34H38N6O4·3TFA·3H2O
requires C, 48.5; H, 4.8; N, 8.5%; dH(400 MHz, D2O, 45 ◦C) 8.30
(2 H, d, J 2, Acr 4,5-H), 8.09 (2 H, d, J 9, Ar H), 7.95 (2 H,
d, J 9, Acr 1,8-H), 7.33 (4 H, m, Ar H and Acr 2,7-H), 3.89
(4 H, br m, pyrrolidine NCH), 3.76 (4 H, t, J 7, CH2), 3.33 (4 H,
br m, pyrrolidine NCH), 3.19 (4 H, t, J 7, CH2), 2.34 (4 H, br,
pyrrolidine CH), 2.20 (4 H, br, pyrrolidine CH); dC(126 MHz,
D2O) 170.5, 169.6, 162.9 (q, J 35), 152.1, 144.8, 143.4, 141.1,
131.2, 127.7, 126.3, 117.7, 116.2 (q, J 292), 110.8, 105.4, 54.4,
50.1, 32.3, 22.6; m/z (ES+) [M + H]+ requires 595.3033, found
595.2984.
N-ras 5¢ Biotin TGT GGG AGG GGC GGG TCT GGG TGC
3¢
F21T 5¢ FAM-GGG TTA GGG TTA GGG TTA GGG-
TAMRA 3¢
Synthetic Protocols
N-[9-Chloro-6-(3-pyrrolidin-1-yl-propionylamino)-acridin-3-yl]-
3-pyrrolidin-1-yl-propionamide and 3-chloro-N-[6-(3-chloro-pro-
pionylamino)-9-oxo-4a,9,9a,10-tetrahydro-acridin-3-yl]-propio-
namide were prepared according to published procedures.33,34
Resins for peptide synthesis were supplied by Novabiochem.
HPLC was performed using Phenomenex C18 Luna columns
(250 ¥ 4.6 mm for analytical, 250 ¥ 10 mm for preparative) unless
stated otherwise. HPLC solvent A = water, 0.1% TFA, solvent B =
MeCN, 0.1% TFA. NMR spectra were recorded on Bruker Avance
500 (1H 500 MHz, 13C 126 MHz) or DRX-400 (1H 400 MHz, 13
C
[(2 - {6 - [3 - (Ethoxycarbonylmethyl - methyl - amino) - propionyl -
amino]-9-oxo-9,10-dihydro-acridin-3-ylcarbamoyl}-ethyl)-methyl-
amino]-acetic acid ethyl ester 4. 3-Chloro-N-[6-(3-chloro-propio-
nylamino)-9-oxo-4a,9,9a,10-tetrahydro-acridin-3-yl]-propiona-
mide (200 mg), sodium iodide (145 mg), sarcosine ethylester
hydrochloride (1.14 g) were mixed in DMF (14 mL). The
suspension was heated with stirring to 80 ◦C while a solution
of sodium ethoxide (566 mg) in ethanol (20 mL) was added over
20 h using a syringe pump. Heating was continued for a further
10 h, after which the suspension was cooled to r.t., filtered and the
solvent evaporated. The residues were triturated with Et2O, and the
washings discarded. The remaining solids were purified by HPLC
to afford 4 as a pale yellow solid (146 mg, 39%). HPLC conditions:
Phenomenex C18 Luna (250 ¥ 10 mm) column, gradient 0–2.5–
11 min, 13–13–32% solvent B at 4.6 mL min-1, retention time
11.3 min. Found: C, 48.65; H, 4.95; N, 8.6. C29H37N5O7·2TFA·H2O
requires C, 48.7; H, 5.1; N, 8.6%; dH(400 MHz, D2O) 7.74 (2 H,
d, J 9, Acr 1,8-H), 7.10 (2 H, d, J 2, Acr 4,5-H), 6.80 (2 H, dd,
J 9 and 2, Acr 2,7-H), 4.27 (4 H, q, J 7, OCH2), 4.17 (4 H, s,
CH2CO2), 3.56 (4 H, t, J 7, NCH2), 3.00 (6 H, s, NCH3), 2.83
(4 H, t, J 7, CH2), 1.23 (6 H, t, J 7, CH2CH3); dC(101 MHz, D2O)
175.7, 168.9, 166.3, 162.6 (q, J 36), 141.5, 140.6, 126.0, 116.3 (q,
J 293), 115.4, 113.6, 104.7, 63.7, 56.1, 52.4, 41.5, 30.3, 13.1; m/z
(ES+) [M + H]+ requires 568.2771, found 568.2785.
101 MHz) spectrometers. J values are given in Hz.
2-[3,6-Bis-(3-pyrrolidin-1-yl-propionylamino)-acridin-9-ylamino]-
benzoic acid 1. A suspension of anthranilic acid (26 mg) and
N-[9-chloro-6-(3-pyrrolidin-1-yl-propionylamino)-acridin-3-yl]-
3-pyrrolidin-1-yl-propionamide in chloroform (5 mL) were
refluxed for 24 h. Methanol (0.5 mL) was added and the red
solution stirred for a further 2 d at r.t. The solvent was evaporated
and the product purified by HPLC on a Phenomenex C18 Luna
(250 ¥ 10 mm) column, eluting with a gradient 0.5–2.5–13–
13.5 min, 10–10–30–95% solvent B at 4.6 mL min-1. The product
eluted at r.t. 13.7 min and was obtained as an orange solid
after lyophilization (49 mg, 49%). Found: C, 49.3; H, 4.5; N,
8.6. C34H38N6O4·3TFA·2H2O requires C, 49.4; H, 4.7; N, 8.6%;
dH(500 MHz, DMSO) 13.87 (1 H, br, OH), 11.14 (1 H, br, NH),
10.99 (2 H, s, NH), 9.87 (2 H, br, NH), 8.50 (2 H, d, J 2, Acr
4,5-H), 8.06 (1 H, dd, J 8 and 2, Ar 6-H); 7.98 (2 H, d, J 9, Acr
1,8-H), 7.63 (1 H, td, J 8 and 2 Hz, Ar 4-H), 7.50 (1 H, t, J 8, Ar
5-H), 7.37 (1 H, d, J 8, Ar 3-H), 7.31 (2 H, dd, J 9 and 2, Acr
2,7-H), 7.11 (1 H, t, J 51, NH), 3.07 (2 H, m, pyrrolidine NCH),
2.94 (4 H, t, J 7, CH2), 2.02 (4 H, m, pyrrolidine CH), 1.87 (4 H,
m, pyrrolidine CH); dC(126 MHz, DMSO) 169.8, 167.5, 153.4,
144.6, 142.3, 141.4, 134.1, 132.2, 127.7, 127.1, 126.2, 125.9, 117.4,
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triisopropylsilane. After concentration of the TFA by nitrogen
blow-down, the products were recovered by Et2O precipitation.
Purification by HPLC afforded compounds 7–21 (Tables 1, 2)
which were dissolved in water to form stock solutions. Peptides
were characterized by LCMS and stock solutions standardized by
amino acid analysis (Department of Biochemistry, University of
Cambridge). Retention times and amino acid analyses are given
in the supporting information.
[(2-{9-(4-Dimethylamino-phenylamino)-6-[3-(ethoxycarbonyl-
methyl-methyl-amino)-propionylamino]-acridin-3-ylcarbamoyl}-
ethyl)-methyl-amino]-acetic acid ethyl ester 5. Phosphoryl chlo-
ride (15 mL) was added to 4 (140 mg) and the suspension heated
◦
to 100 C for 1 h under N2. After cooling to room temperature
the orange suspension was poured into Et2O (200 mL), cooled to
-20 ◦C and the yellow solids collected by filtration and washed
with Et2O. To the solids were added N-dimethylphenyldiamine
(1.20 g) followed by chloroform (20 mL) and the dark solution
stirred at r.t. for 24 h. The solvent was evaporated to afford a
brown oil to which Et2O (100 mL) was added. After standing at
-20 ◦C for 1 h, the precipitate was collected by filtration, washed
with Et2O and dried in vacuo. The residues were dissolved in water
(5 mL) acidified with TFA (330 mL) and passed in 500 mL aliquots
through an Isolute C18EC cartridge (1 g) eluting with water (0.1%
TFA, 10 mL), 10% MeCN (0.1% TFA, 20 mL), 20% MeCN
(0.1% TFA,10 mL). The 10% MeCN and 20% MeCN eluents were
combined and evaporated. The residues were purified by HPLC
on a Phenomenex C18 Jupiter column (250 ¥ 21.2 mm) with a
gradient 0–2.5–32.5 min, 5–5–100% solvent B at 10 mL min-1,
retention time 16.5 min. Lyophilization afforded the product as
a dark red solid (153 mg, 84%). Found: C, 48.2; H, 4.65; N,
8.9. C37H47N7O6·3TFA·2H2O requires C, 48.5; H, 5.1; N, 9.2%;
dH(400 MHz, D2O) 7.91 (2 H, d, J 2, Acr 4,5-H), 7.64 (2 H, d, J 9,
Acr 1,8-H), 7.54 (2 H, d, J 9, Ar H), 7.26 (2 H, d, J 9, Ar H), 7.05
(2 H, dd, J 9 and 2, Acr 2,7-H), 4.21 (4 H, q, J 7, OCH2), 4.15
(4 H, s, CH2CO2), 3.62 (4 H, br m, CH2), 3.21 (6 H, s, NCH3),
3.00 (10 H, m, CH2 and NCH3), 1.17 (6 H, t, J 7, CH2CH3);
dC(101 MHz, D2O) 170.3, 166.4, 162.5 (q, J 36), 152.7, 143.5,
142.4, 141.1, 139.9, 126.3, 125.1, 122.2, 117.7, 116.2 (q, J 293),
110.6, 105.5, 63.7, 56.2, 52.4, 46.4, 41.7, 30.8, 13.1; m/z (ES+)
[M + H]+ requires 686.3661, found 686.3677.
Surface plasmon resonance
Surface plasmon resonance was performed on a Biacore 2000
instrument, using degassed phosphate running buffer (50 mM
potassium phosphate, 100 mM KCl, pH 7.4). Sensor chips
(Type SA, Biacore) were loaded with approximately 500 RU of
biotinylated oligonucleotides. Serial dilutions of compound were
injected at a flow rate of 20 mL min-1 and the equilibrium response
determined relative to the baseline.
3,6 Bis-peptide-substituted acridines (7–9) were varied over a
nominal range of concentration of 10–0.08 mM, whereas acridines
with a peptide substitution on the 9-position (10–21) were varied
over the concentration range 2.5–640 nM. Compounds 10–21 were
handled in glass vials as they were found to adsorb to plastic
tubes. Injections were each performed in duplicate on two separate
occasions. Between injections the sensor surface was refreshed
with injections of 1 M KCl and buffer. After subtraction of the
background response from the blank flow cell, the responses
were fitted to a BET isotherm (Eq. 1) using the BIAevaluation
software. The BET equation permits binding of a monolayer
with association constant Ka followed by multiple bindings with
association constant Km. The monolayer dissociation constant
-1
Kd = Ka is analogous to the dissociation constant of the
simpler Langmuir model which is often used for fitting SPR data.
The multilayer constant Km permits modelling of non-specific
adsorption which was observed at higher ligand concentrations.
The stoichiometry of binding was estimated from the response
corresponding to a monolayer coverage relative to the DNA
loading of the chip.
({2-[6-[3-(Carboxymethyl-methyl-amino)-propionylamino]-9-(4 -
dimethylamino-phenylamino)-acridin-3-ylcarbamoyl]-ethyl}-methyl-
amino)-acetic acid 6. To a solution of 5 (153 mg) in a mixture
of water (3.8 mL) and THF (3.8 mL) was added LiOH (34 mg).
After stirring 1 h at r.t., TFA (350 mL) was added and the
solution concentrated to 1.5 mL. The product was purified by
HPLC on a Phenomenex C18 Luna column (250 ¥ 10 mm)
with gradient 0–2.5–11 min, 5–5–30% solvent B, 4.6 mL min-1,
retention time 10.6 min. Lyophilization afforded the product
as an orange solid (62 mg, 42%). Found: C, 45.4; H, 4.2; N,
9.25. C33H39N7O6·3TFA·3H2O requires C, 45.7; H, 4.7; N, 9.6%;
dH(400 MHz, D2O) 7.98 (2 H, d, J 2, Acr 4,5-H), 7.74 (2 H, d, J 9,
Acr 1,8-H), 7.59 (2 H, d, J 9, Ar H), 7.33 (2 H, d, J 9, Ar H), 7.10
(2 H, dd, J 9 and 2, Acr 2,7-H), 4.01 (4 H, s, CH2CO2), 3.62 (4 H,
br, CH2), 3.26 (6 H, s, NCH3), 3.04 (4 H, t, J 7, CH2), 2.90 (6 H, s,
NCH3); dC(101 MHz, D2O) 170.2, 168.0, 162.5 (m), 143.4, 142.2,
140.9, 139.9, 126.1, 125.1, 122.1, 117.6, 116.1 (q, J 293), 110.3,
105.3, 56.3, 52.3, 46.3, 41.5, 30.7; m/z (ES+) [M + H]+ requires
630.3040, found 630.3028.
RmoncKa
Req
=
(1)
(1- Kmc)(1- Kmc + Kac)
Req is the equilibrium response, Rmon is the response for a
single monolayer, Ka is the association constant for the first
monolayer, Km is the association constant for multilayers, and c is
the concentration of ligand. Data have been expressed in terms of
-1
the dissociation constant Kd = Ka
.
FRET melts
The FRET melting assay was conducted using the dual la-
belled (fluorescein, tetramethylrhodamine) oligonucleotide F21T
according to our modification48 of the procedure of Mergny et al.42
Peptide synthesis. Peptides were prepared on Rink amide
(amide terminated) or preloaded Wang (acid terminated) resins
using Fmoc protected amino acids and PyBOP, HOBt and DIPEA
in N-methylpyrrolidone for couplings and 50% piperidine in
DMF for deprotections. The final acridine residue was cou-
pled using identical reagents. Deprotection and cleavage from
the resin was performed using 95% TFA, 2.5% H2O, 2.5%
Molecular modelling
The crystal structure44 of the G-quadruplex formed from the
22-mer human telomeric DNA sequence d(AGGG[TTAGGG]3)
(PDB code 1KF1) was used as the binding host to the various
acridine-peptide conjugates. Ligands were built using the Insight
II package (Accelrys Inc.) on an SGI workstation. Partial charges
82 | Org. Biomol. Chem., 2009, 7, 76–84
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for the central scaffold were derived by fitting the HF/6-31G*
electrostatic potential obtained with the GAMESS ab initio
software49 to the atomic centres with the RESP program.50 Atomic
parameters for the peptide side chains were taken directly from
the Amber force field,51 as implemented within Insight II. The
protonation state of the amino acid side chains was determined
based on an assumed physiological pH of 7.0. The N10 position
of the acridine was also protonated with a +1 charge based on
its role as a hydrogen bond donor in a quadruplex-ligand crystal
structure (1L1H).52
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Supplementary Information for details.
Docking was performed with the Affinity Docking module of
Insight II using a previously-described protocol.36 In this way,
a multi-phase docking protocol was used. First the ligand was
manually displaced along the binding host while interactively
calculating their interaction energy. Once a favourable initial
relative conformation was found a binding pocket which included
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˚
hydrogen atoms 5 A from the ligand was defined, and then
the ligand was randomly orientated with respect to the binding
host 200 times. Van der Waals radii were set to 10% of the full
value, charges were not considered and non-bonded cut-offs were
˚
set to 8 A. The system was minimized for 300 steps using the
conjugate gradient method. The maximum allowable change for
succeeding structures was set to 10000 kcal mol-1 and the energy
range was set to 40 kcal mol-1. The 75 lowest-energy structures
were used for the second phase of the modelling. The second
phase of the docking protocol consisted of a simulated annealing
procedure in which the van der Waals radii were adjusted to
their full values, charges were included with a distance-dependent
˚
dielectric of 4*rij and the non-bonded cut-off was set to 18 A.
Each of the 75 lowest-energy structures was again minimized for
300 steps of conjugate-gradient minimization, and then molecular
dynamics calculations were performed, starting at a temperature
of 500 K and cooling the system to 300 K over 10 ps. The
resulting structures were minimized for 1000 steps of conjugate
gradients and the 25 structures with lowest total energy were used
for further evaluation. These 25 filtered structures were further
refined in order to perform an optimal conformational sampling of
the peptide side-chains while maintaining the ligand orientation.
Hence, the structures were subjected to another run of simulated
annealing in which the binding pocket was extended to contain all
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˚
atoms 5 A from the ligand while the bases of the G-quartet were
tethered to their original position. The system was cooled down
over 10 ps from 500 K to 300 K, and the resulting structures were
subjected to a 1000 steps of conjugate gradient minimization.
Acknowledgements
We thank Cancer Research UK for programme funding (to SB
and to SN).
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