Synthesis and characterisation of metallopolyamide complexes

Article abstract of DOI:10.1016/j.ica.2012.06.013

Platinum(II) and ruthenium(II)-based complexes that contain imidazole, pyrrole and β-alanine subunits, capable of recognising specific DNA base-pair sequences, have been synthesised. These polyamides, two platinum(II), [Cl(NH₃)₂Pt-L₆-β-Ala-Py-L ₄-Im]⁺ (HSP-2), and [Cl(NH₃) ₂Pt-L₆-β-Ala-PyPyPy-L₄-ImImIm] ⁺ (HSP-6) and two ruthenium, Δ and Λ-[β-Ala-Py- L₄-Im-β-dpq-Ru(phen)₂]²⁺ (Δ and Λ-RUP1), were designed to recognise DNA sequences up to seven base-pairs in length. They were obtained in good yield by a combination of solid and solution phase chemistries. The chirality of the ruthenium precursors Δ- and Λ-[Ru(phen)₂(phendo)]²⁺ was conserved throughout the synthesis. Characterisation was achieved using NMR, UV-Vis and ESI-MS and CD for Δ- and Λ-RUP1. Cytotoxicity was not determined for HSP-2 or HSP-6, due to insolubility, however the IC₅₀ values of Δ and Λ-RUP1 were confirmed to be greater than 40 μM (at an incubation time of 48 h). LD studies indicated that the ruthenium complexes interact with ct-DNA through a mixed binding mode, which is influenced by complex concentration and chirality.

Full text of DOI:10.1016/j.ica.2012.06.013

Inorganica Chimica Acta 393 (2012) 187–197
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis and characterisation of metallopolyamide complexes
Nikita Orkey a, Robin I. Taleb b,1, Janice R. Aldrich-Wright a,
⇑
a School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith, 2571 NSW, Australia
b School of Arts and Sciences, Lebanese American University, P.O. Box 36, Byblos, Lebanon
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 23 June 2012
Platinum(II) and ruthenium(II)-based complexes that contain imidazole, pyrrole and b-alanine subunits,
capable of recognising specific DNA base-pair sequences, have been synthesised. These polyamides, two
platinum(II), [Cl(NH3)2Pt-L6-b-Ala-Py-L4-Im]+ (HSP-2), and [Cl(NH3)2Pt-L6-b-Ala-PyPyPy-L4-ImImIm]+
Metals in Medicine Special Issue
(HSP-6) and two ruthenium,
designed to recognise DNA sequences up to seven base-pairs in length. They were obtained in good yield
by a combination of solid and solution phase chemistries. The chirality of the ruthenium precursors
and
-[Ru(phen)2(phendo)]2+ was conserved throughout the synthesis. Characterisation was achieved
using NMR, UV–Vis and ESI-MS and CD for - and -RUP1. Cytotoxicity was not determined for HSP-
2 or HSP-6, due to insolubility, however the IC50 values of and -RUP1 were confirmed to be greater
than 40 M (at an incubation time of 48 h). LD studies indicated that the ruthenium complexes interact
D and K (D and K-RUP1), were
-[b-Ala-Py-L4-Im-b-dpq-Ru(phen)2]2+
Keywords:
DNA recognition
Platinum
Ruthenium
Polyamides
D-
K
D
K
D
K
l
Sequence selective
with ct-DNA through a mixed binding mode, which is influenced by complex concentration and chirality.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
DNA. When imidazole (Im) and/or pyrrole (Py) heterocyclic rings,
b-alanine (b-ala) and/or amino butyric acid (L4 linker) residues
Cisplatin, carboplatin and oxaliplatin are used for the treatment
of a variety of human cancers including head, neck, ovarian, testic-
ular, lung and colorectal cancers [1]. When treatment by cisplatin
is poorly tolerated, carboplatin is used, whereas oxaliplatin is used
to treat specific cisplatin-resistant cancers [2]. Despite being effi-
cient drugs, their use is restricted by dose limiting side effects, such
as nephrotoxicity, neurotoxicity and ototoxicity, and intrinsic or
acquired resistance [1,3]. There are several mechanisms which pro-
duce resistance such as decreased cellular uptake/increased drug
efflux, increased drug deactivation arising from increased intracel-
lular glutathione concentration and enhanced DNA adduct repair/
tolerance [3a]. These limitations drive the researcher to search
for new compounds with which to treat cancer. Producing metallo-
drugs that can target specific sequences of DNA and deliver thera-
peutic effect is an area of research at the interface of biology and
chemistry that is proving to be a challenge.
(Fig. 1) are combined in specific combinations they can target spe-
cific sequences of DNA [4]. The base-pairing rules that have been
developed, relate to the anti-parallel association of the pyrrole–
imidazole polyamide to their predicted target DNA sequences
[4b]. Each pair of heterocyclic rings recognises a specific base-pair
in DNA: Im/Py and Py/Im pairings distinguish between G:C and C:G
base pairs respectively, while a combination of Py/Py, b-ala and the
L4 linker is selective for both A:T and T:A base pairs. The L4 linker
facilitates the formation of a hairpin by allowing it to fold and form
dimers with itself. The hairpin polyamides have a distinct advan-
tage in binding, achieving affinities and specificities comparable
to DNA-binding proteins.
Previously wehavesynthesisedtrans-chlorodiammine[N-(2-ami-
noethyl)-4-[4-(N-methylimidazole-2-carboxamido)-N-methylpyr-
role-2-carboxamido]-N-methylpyrrole-2-carboxamide]plat-
inum(II) chloride (DJ1953-2) and trans-chlorodiammine-[N-(6-
aminohexyl)-4-[4-(N-methylimidazole-2-carboxamido)-N-methyl-
pyrrole-2-carboxamido]-N-methylpyrrole-2-carboxamide]plati-
num(II) chloride (DJ1953-6) (Fig. 2) [5]. Circular dichroism (CD)
studies have shown that these complexes are capable of binding
the DNA sequence d(CATTGTCAGAC)2. Although neither complex af-
fected DNA melting temperatures, DJ1953-2 unwound the helix by
13°. DJ1953-2 was also found to inhibit RNA synthesis in vitro. The
cytotoxicity of both metal complexes in the murine leukaemia cell
Sequence selective polyamides, capable of targeting specific
DNA sequences, have been synthesised over the past ten years.
They comprise of heterocyclic groups linked by flexible bonds that
rotate, and thus conform to the contour of the minor groove of
DNA. This topological complementarity is stabilised by van der
Waals interactions between the polyamide and the groove of
⇑
Corresponding author. Tel.: +61 2 4620 3218; fax: +61 2 4620 3025.
E-mail addresses: robin.taleb@lau.edu.lb (R.I. Taleb), j.aldrich-wright@uws.
line (L1210) was also determined, with DJ1953-6 (34
more active than DJ1953-2 (>50 M) [5].
lM) being
l
edu.au (J.R. Aldrich-Wright).
1
Tel.: +961 9 547 262x2429; fax: +961 9 546 262.
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.06.013

188
N. Orkey et al. / Inorganica Chimica Acta 393 (2012) 187–197
Fig. 1. The structures of the heterocyclic groups, Im and Py, together with b-alanine and
c
-aminobutyric acid linker.
N
H
N
N
H
N
O
NH3
N
H
N
O
Cl
Cl
Pt NH2
NH3
N
O
(a)
(b)
N
H
N
N
H
N
O
NH3
N
H
N
O
N
Pt NH2
NH3
O
O
N
NH
N
H
N
NH3
H
H
N
N
O
N
Cl
Pt NH2
NH3
(c)
(d)
O
O
O
N
O
NH
N
N
O
N
H
N
N
N
H
N
N
H
H
N
O
N
H
N
O
NH3
N
H
N
H
N
O
N
Cl
Pt NH2
NH3
O
O
2
O
O
N
HO
N
N
N
H
O
N
N
(e)
N
N
N
H
Ru
H
H
N
N
N
N
N
NH
O
O
N
O
Fig. 2. The polyamide complexes DJ1953-2 (a), DJ1953-6 (b), HSP-2 (c), HSP-6 (d) and RUP1(e).

N. Orkey et al. / Inorganica Chimica Acta 393 (2012) 187–197
189
There have been widespread efforts to utilise ruthenium com-
plexes in order to investigate the principle involved in DNA recog-
nition [6], act as artificial nucleases [7], and luminescent probes for
DNA [8] and to examine electron transfer mediated by DNA [9].
Many investigations have focussed upon metallointercalators –
that is, the insertion of a planar aromatic ring system between
the stacked bases of DNA. These complexes are generally of the
form [Ru(L)2(IL)]2+, where L is a ligand such as 2,20-bipyridine or
1,10-phenanthroline (phen), and IL is a ligand capable of insertion
between the base pairs of DNA, such as dipyrido[3,2-a:20,30-
c]phenazine (dppz) or dipyrido[3,2-d:2030-f]quinoxaline (dpq) [10].
Here we report the solid phase synthesis and characterisation of
four sequence selective hairpin metallopolyamides, two plati-
wise stated. 195Pt NMR were externally referenced to K2PtCl4
(ꢀ1631 ppm) [13].
Positive ion ESI mass spectra were acquired using a Micromass
(Wyntheshawe, UK) Quattro Micro™ spectrometer equipped with
a Z-spray probe. Solutions containing concentrations ranging be-
tween 10 and 50
lM were injected into the instruments at a flow
rate of 10
l
L minꢀ1. The source and desolvation temperatures were
150 and 120 °C, respectively. The capillary tip potential and cone
voltage were 2500 and 50 V, respectively. Between 10 and 50
acquisitions were summed to obtain spectra, which were cali-
brated against a standard CsI solution (750 mM) over the same
m/z range. CD and LD spectra were recorded on a Jasco J-810 spec-
tropolarimeter at room temperature. Samples were collected be-
tween 190 and 600 nm. Machine assisted synthesis was
performed on a Symphony Quartet 3.21 protein synthesiser on a
0.28 mmol scale. Each cycle of monomer addition involved a
DCM wash (ꢁ10 mL), a DMF wash (ꢁ10 mL) and deprotection with
20% piperidine/DMF (ꢁ10 mL). Activated acids were added manu-
ally to the reaction vessel at the end of a deprotection cycle. Once
the required molecule had been constructed, the resin was washed
with copious amounts of DMF and DCM and dried for 1 h under
num(II) complexes
and [Cl(NH3)2Pt-L6-b-Ala-PyPyPy-L4-ImImIm]+ (HSP-6) and two
ruthenium complexes and -[b-Ala-Py-L4-Im-b-dpq-Ru(-
phen)2]2+
and -RUP1) (Fig. 2). The platinum metallopolya-
–
[Cl(NH3)2Pt-L6-b-Ala-Py-L4-Im]+ (HSP-2),
–
D
K
(D
K
mides have the potential to covalently bind whereas the
ruthenium metallopolyamides are expected to interact by a combi-
nation of intercalation and groove binding. The cytotoxicity, gua-
nosine binding and interaction of
D and K-RUP1 with ct-DNA
have been investigated through linear dichroism (LD).
N2(g).
2.3. Synthesis of platinum(II) precursors
2. Experimental
2.3.1. Synthesis of Pt-L6-NHBoc
Transplatin (2.50 g, 8.33 mmol) and silver nitrate (1.27 g,
7.49 mmol) in DMF (100 mL) were stirred in the dark under N2(g)
for 12 h. The mixture was filtered and added to a solution of tert-
butyl-6-aminohexylcarbamate [14] (2.00 g, 9.26 mmol) in DMF
(30 mL). The solution was stirred in the dark under N2(g) for 18 h.
The solvent was removed under reduced pressure, acetone
(5 mL) was added and the mixture was kept at 4 °C overnight.
The precipitate that formed was filtered and re-suspended in
methanol (70 mL). This mixture was filtered and the filtrate was
evaporated under reduced pressure to obtain Pt-L6-NH.Boc
(2.74 g, 76%) as a yellow/white powder. 1H NMR 300 MHz (d2-
D2O): d 2.93 (t, 2H, J = 6.1 Hz), 2.55 (bm, 2H), 1.57 (bm, 2H), 1.3
(bp, 6H). 195Pt NMR 85 MHz (d7-DMF): ꢀ2423 (s).
2.1. Materials
N-Methyl-1H-imidazole-2-carboxylic acid was purchased from
Bachem. (9H-fluoren-9-yl)methyl chloroformate-(Fmoc),
c-ami-
no-butyric acid and Fmoc-b-alanine were purchased from Fluka.
N-Methyl-1H-imidazole, N,N0-diisopropylethylamine (DIEA) and
transplatin were purchased from Sigma–Aldrich. 2-Chloro-chloro-
trityl resin, Fmoc-lysine-(Fmoc)-OH and 2-(1H-benzotriazole-1-
YL)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) were
purchased from Auspep, Australia. Boc-L-2,4-diaminobutyric acid-
(Fmoc) was purchased from PepTech. N-Methyl-1H-pyrrole was
purchased from AlfaAesar. Potassium tetrachloroplatinate(II) and
ruthenium(III) chloride hydrate were purchased from Precious
Metals Online. Fmoc-protected amino-caproic acid (L6) and
2.3.2. Synthesis of Pt-L6-NH2
Fmoc-c-aminobutyric acid (L4) were bought from Merck while iso-
Pt-L6-NH.Boc (0.20 g, 0.39 mmol) was dissolved in methanol
(8 mL) before HCl (4 M, 7 mL) was added and the mixture stirred
for 48 h. The solvent was evaporated under reduced pressure to ob-
tain a yellow solid. Salt conversion was carried out using Dowex-
OH to obtain the product as a white solid (0.14 g, 87%). 1H NMR
300 MHz (d6-DMSO): d 7.85 (s, 2H), 5.42 (s, 2H), 4.01(s, 6H), 2.75
(t, 2H, J = 6.1 Hz), 2.56 (bp, 2H), 1.56 (bp, 4H), 1.30 (bp, 4H); 195Pt
NMR 85 MHz (d7-DMF): ꢀ2415 (bs).
propanol was bought from Baxter. Ethyl acetate and anhydrous
methanol were purchased from Mallinckrodt Chemicals. ct-DNA
was purchased from Invitrogen. Dowex Monosphere 550A (OH)
was purchased from Sigma. All other solvents were of analytical
grade and were used as received from Biolab. Silica gel 60
(0.003–0.040 mm) was bought from Silicycle (Canada). Dimethyl-
sulfoxide (D6, 99.9%), acetonitrile (D3, 96–97%), acetone (D6,
99.9%), dimethylformamide (D7, 99.9%) and deuterium oxide (D2,
99.9%) were purchased from Cambridge Isotope Laboratories. The
heterocyclic N-methylimidazole and N-methylpyrrole rings for
the polyamide were synthesised then activated as previously de-
scribed [11]. 1,10-Phenanthroline-5,6-dione (phendo) was syn-
thesised using an adaptation of the method by Yanagida et al.
[12] (0.88 g, 76%). 1H NMR 400 MHz (d6-DMSO): d 9.05 (d, 2H,
J = 3.23 Hz); 8.45 (d, 2H, J = 7.47 Hz); 7.74 (t, 2H, J = 5.88 Hz). Full
method is in the Supplementary data. All other reagents and sol-
vents were used as received unless otherwise stated.
2.4. Synthesis and resolution of ruthenium(II) precursors
2.4.1. Synthesis of [Ru(phen)2Cl2]
An adaptation of the method by Hiort was used [15] to obtain
the black crystallised product (1.3 g, 26%). 1H NMR 400 MHz (d3-
acetonitrile): d 8.69 (d, 4H, J = 7.51 Hz); 8.31 (s, 4H, J = 7.51 Hz);
8.10 (d, 4H, J = 7.51 Hz), 7.62 (t, 4H, J = 5.53 Hz).
2.4.2. Synthesis of [Ru(phen)2phendo]Cl2
An adaptation of the method by Hiort et al. [15] was used to
synthesise this complex (0.8 g, 94%). 1H NMR 400 MHz (d6-ace-
tone): d 8.9 (d, 1H, J = 8.15 Hz); 8.84 (d, 1H, J = 8.15 Hz); 8.74 (d,
1H, J = 4.88 Hz); 8.68 (d, 1H, J = 7.87 Hz); 8.49 (q, 2H, J = 15.32,
6.38 Hz); 8.38 (q, 2H, J = 12.75, 7.36 Hz); 8.05 (q, 1H, J = 8.32,
3.08 Hz); 7.83 (q, 1H, J = 8.22, 2.98 Hz); 7.75 (q, 1H, J = 7.86,
2.2. Instruments and physical measurements
NMR spectra were obtained on a 300 MHz Varian Mercury or a
400 MHz Bruker Avance spectrometer, either in d6-DMSO, d7-DMF
or D2O, referenced internally to the solvent. NMR experiments
were run at 35 °C for DMSO and 25 °C for D2O/DMF unless other-
2.24 Hz). Electronic spectra kmax nm (
e
molꢀ1 dmꢀ3 cmꢀ1, 50%

190
N. Orkey et al. / Inorganica Chimica Acta 393 (2012) 187–197
CH3CN in H2O): 220 (68000), 260 (16794), 290 (25950), 435
(15996).
(4 mL) was added and the mixture cooled to 4 °C for 1.5 h to pre-
cipitate a yellow solid. The solvent was decanted and water
(3 mL) was added to the solid before the solution was lyophilised
to yield the desired polyamide.
2.4.3. Resolution of [Ru(phen)2(phendo)](PF6)2
The
D- and K-isomers of [Ru(phen)2(phendo)](PF6)2 were syn-
thesised using the method by Hiort et al. [15].
2.5.5. Synthesis of b-Ala-Py-L4-Im
D
-[Ru(phen)2(phendo)](PF6)2 (0.18 g, 39%). CD (H2O:CH3CN,
The polyamide was cleaved from the resin, as previously de-
scribed, to obtain a pale yellow powder (0.08 g, 67%). 1H NMR
300 MHz (d6-DMSO): d 9.74 (s, 1H), 8.41 (t, 1H, J = 5.8 Hz), 7.99
(t, 1H, J = 5.5 Hz), 7.31 (s, 1H), 7.07 (s, 1H), 6.95 (s, 1H), 6.62 (s,
1H), 3.92 (s, 3H), 3.75 (s, 3H), 3.34 (q, 2H, J = 7.0 Hz), 3.24 (q, 2H,
J = 7.2 Hz), 2.42 (t, 2H, J = 7.2 Hz), 2.22 (t, 2H, J = 7.4 Hz), 1.77 (m,
2H, J = 7.1 Hz).
1.5 ꢂ 10ꢀ6 M, RT.): k nm (
De mdeg M cm): 256 (65.2), 267
(ꢀ113.2), 296 (ꢀ61.3), 410 (9.2), 460 (ꢀ7.1).
K
-[Ru(phen)2(phendo)](PF6)2 (0.10 g, 22%). CD (H2O:CH3CN,
1.5 ꢂ 10ꢀ6 M, RT.): k nm (
D
e
mdeg M cm): 256 (ꢀ54.6), 267
(94.7), 296 (51.9), 410 (ꢀ7.8), 4604 (6.0).
2.5. Synthesis of HSP-2, HSP-6 and RUP1
2.5.6. Synthesis of HSP-2
2.5.1. Synthesis of polyamides (general method) [11a]
b-Ala-Py-L4-Im (0.05 g, 0.12 mmol) in DMF (100 mL) was acti-
vated using HBTU (0.04 mg, 0.12 mmol) and DIEA (0.06 mL,
0.35 mmol). The solution was stirred for 5 min before Pt-L6-NH2
(0.025 mg, 0.07 mmol) was added and the mixture stirred over-
night under N2(g). The solvent was removed under reduced pres-
sure before the addition of acetone to precipitate the product as
a slightly yellow coloured powder (0.04 mg, 72.9%). 1H NMR
300 MHz (d6-DMSO): d 9.73 (s, 1H), 8.40 (t, 1H, J = 5.8 Hz), 7.92
(t, 1H, J = 5.5 Hz), 7.83 (t, 1H, J = 5.6 Hz), 7.31 (s, 1H), 7.07 (s, 1H),
6.96 (s, 1H), 6.61 (s, 1H), 5.41 (bp, 2H), 4.00 (s, 6H), 3.92 (s, 3H),
3.75 (s, 3H), 3.34 (q, 2H, J = 7.0 Hz), 3.24 (q, 2H, J = 7.2 Hz), 3.02
(q, 2H, J = 6.7 Hz), 2.50 (bm, 2H), 2.42 (t, 2H, J = 7.2 Hz), 2.22 (t,
2H, J = 7.4 Hz), 1.77 (p, 2H, J = 7.1 Hz); 195Pt NMR 85 MHz (d7-
DMF): -2425 (bs). Anal. Calc. for C24H44Cl2N10O4PtꢃH2O: C, 35.12;
H, 5.65; N, 17.07%; Found: C, 35.44; H, 5.64; N, 16.67%. ESI-MS Calc.
for C24H44ClN10O4Pt [MꢀClꢀ] 767.2 m/z; found 767.2 m/z. Elec-
Fmoc-b-alanine-chlorotrityl resin was prepared by the addition
of Fmoc-b-alanine-OH to 2-chloro-chlorotrityl resin. The next step
of the reaction was undertaken by machine assisted protocol. The
Fmoc-protected amino group of the b-alanine resin was removed
by the addition of 20% piperidine in DMF (10 mL). This was fol-
lowed by the addition of an activated monomer to the resin, allow-
ing a mix time from 3.5 to 9 h depending upon the monomer used.
The resin was filtered and washed with DMF (2 ꢂ 10 mL), DCM
(10 mL) and DMF (10 mL). The amino group was deprotected and
the cycle of addition, mixing and deprotection commenced and
continued until the desired polyamide was formed.
2.5.2. Resin preparation
Fmoc-b-alanine-OH (0.15 g, 0.48 mmol) was added to DIEA
(0.35 mL) in anhydrous DCM (4 mL) and the solution was stirred
for 5 min. It was then added to a suspension of 2-chloro-chlorotri-
tyl resin (0.50 g, 0.50 mmol) in anhydrous DCM (3 mL) and stirred
gently for 5.5 h. MeOH (2.5 mL) was added and the suspension was
stirred for a further 0.5 h. The solution was filtered and the solid
resin was air dried for 12 h.
tronic spectra kmax nm (e
molꢀ1 dmꢀ3 cmꢀ1, 50% CH3CN in H2O):
220 (68000), 260 (16794), 290 (25950), 435 (15996).
2.5.7. Synthesis of b-Ala-PyPyPy-L4-ImImIm
b-Ala-PyPyPy-L4-ImImIm was cleaved from the resin using the
general procedure described for chlorotrityl resin polyamide cleav-
age. The procedure was carried out twice to obtain a pale yellow
powder (0.20 g, 80%). 1H NMR 300 MHz (d6-DMSO): d 10.00 (s,
1H), 9.84 (s, 1H), 9.83 (s, 1H), 9.78 (s, 1H), 9.57 (s, 1H), 8.30 (t,
1H, J = 5.8 Hz), 7.96 (t, 1H, J = 5.4 Hz), 7.63 (s, 1H), 7.51 (s, 1H),
7.43 (s, 1H), 7.21 (d, 1H, J = 1.4 Hz), 7.17 (d, 1H, J = 1.4 Hz), 7.15
(d, 1H, J = 1.4 Hz), 7.07 (s, 1H), 7.03 (d, 1H, J = 1.4 Hz), 6.88 (d,
1H, J = 1.6 Hz), 6.85 (d, 1H, J = 1.6 Hz), 4.03 (s, 3H), 4.00 (s, 3H),
3.96 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 3.39 (t, 2H,
J = 7.0 Hz), 3.37 (q, 2H, J = 7.3 Hz), 2.47 (t, 2H, J = 7.1 Hz), 2.29 (t,
2H, J = 7.4 Hz), 1.82 (m, 2H, J = 7.1 Hz).
2.5.3. Activation of monomers and coupling (general method)
The monomer or linker of choice was dissolved in a mixture of
DMF and NMP followed by the addition of the coupling agent,
HBTU. DIEA was added and the solution was stirred for 10 min.
The activated monomer was added manually to the resin and the
suspension was mixed for any time between 3.5 - 9 h, whilst being
aspirated with N2(g). The resin was filtered, washed with DMF
(10 mL), treated with 20% piperidine in DMF (10 mL) and washed
with DCM (2 ꢂ 10 mL) and DMF (3 ꢂ 10 mL). The specific mono-
mers, activation conditions and coupling times for specific DNA
base-pair sequences are shown in Tables 1–3.
2.5.8. Synthesis of HSP-6
2.5.4. Polyamide cleavage (general method)
b-Ala-PyPyPy-L4-ImImIm (0.07 g, 0.18 mmol), in DMF was acti-
vated using HBTU (0.03 mg, 0.07 mmol) and DIEA (0.04 mL,
0.22 mmol). The solution was stirred for 5 min before Pt-L6-NH2
(0.02 mg, 0.05 mmol) was added and the mixture stirred overnight
under N2(g). The solvent was removed under reduced pressure be-
fore acetone was added to precipitate the product. HSP-6 (59 mg,
86.4%) was obtained as a slightly yellow coloured powder. 1H
NMR 300 MHz (d6-DMSO): d 9.98 (s, 1H), 9.84 (s, 1H), 9.83 (s,
Once prepared, the polyamides were cleaved from the resin by
adding a solution of DCM (10 mL), trifluoroethanol (TFE, 2.4 mL)
and acetic acid (1.2 mL) and shaken gently for 1.5 h. The resin
was removed by filtration and washed with TFE:DCM (1:4, 6 mL)
before it was subjected to the cleaving conditions twice more.
The yellow/brown filtrate was collected and the solvent was re-
moved under reduced pressure to ꢁ2 mL. Cold diethyl ether
Table 1
The activation conditions used for the monomers for the synthesis of b-Ala-Py-L4-Im.
Monomer/linker
PyE
Mass (mg)
Moles (mmol)
DMF (mL)
NMP (mL)
HBTU (mg)
DIEA (mL)
Time (h)
86.97
87.21
78.08
0.24
0.27
0.22
3
3
4
3
3
2
83.28
83.28
83.28
0.12
0.12
0.12
3.5
4
5
Fmoc-c-aminobutyric acid linker
ImE

N. Orkey et al. / Inorganica Chimica Acta 393 (2012) 187–197
191
Table 2
The activation conditions used for the monomers for the synthesis of b-Ala-PyPyPy-L4-ImImIm.
Monomer/linker
Mass (mg)
Moles (mmol)
DMF (mL)
NMP (mL)
HBTU (mg)
DIEA (mL)
Time (h)
PyE
PyE
PyE
Fmoc-
ImE
86.97
86.97
86.97
87.21
78.08
78.08
78.08
0.24
0.24
0.24
0.27
0.22
0.22
0.22
3
3
3
3
4
4
4
3
3
3
3
2
2
2
83.28
83.28
83.28
83.28
83.28
83.28
83.28
0.12
0.12
0.12
0.12
0.12
0.12
0.12
3.5
3.5
3.5
3.5
5
c-aminobutyric acid linker
ImE
ImE
9
9
Table 3
The activation conditions used for the monomers for the synthesis of
D
- and -RUP1.
Monomer/linker
PyE
Mass (mg)
Moles (mmol)
DMF (mL)
NMP (mL)
HBTU (mg)
DIEA (mL)
Time (h)
86.97
87.21
78.08
74.72
131.65
200.00
0.24
0.27
0.22
0.24
0.25
0.21
3
3
4
4
3
5
3
3
2
2
3
–
83.28
83.28
83.28
83.28
83.28
–
0.12
0.12
0.12
0.12
0.12
–
3.5
4
4.5
4
7
5
Fmoc-c-aminobutyric acid linker
ImE
Fmoc-b-alanine linker
Fmoc-DAP(Fmoc)-OH
K- or
D
-[Ru(phen)2(phendo)](PF6)2
1H), 9.78 (s, 1H), 9.57 (s, 1H), 8.28 (t, 1H, J = 5.8 Hz), 7.93 (t, 1H,
J = 5.4 Hz), 7.81 (t, 1H, J = 5.4 Hz), 7.61 (s, 1H), 7.49 (s, 1H), 7.42
(s, 1H), 7.21 (d, 1H, J = 1.4 Hz), 7.17 (d, 1H, J = 1.4 Hz), 7.15 (d,
1H, J = 1.4 Hz), 7.07 (s, 1H), 7.03 (d, 1H, J = 1.4 Hz), 6.87 (d, 1H,
J = 1.6 Hz), 6.83 (d, 1H, J = 1.6 Hz), 5.19 (bp, 2H), 4.03 (s, 3H), 4.98
(s, 3H), 3.94 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.78 (s, 3H), 3.39 (t,
2H), 3.37 (q, 2H, J = 7.3 Hz), 3.02 (q, 2H, J = 7.1 Hz), 2.47 (t, 2H,
J = 7.1 Hz), 2.29 (m, 4H, J = 7.4 Hz), 1.82 (p, 2H, J = 7.1 Hz); 195Pt
NMR 85 MHz (d7-DMF): ꢀ2420 (bs). Anal. Calc. for
ing (CC) assay. These tests were conducted at the Andrew Durant
drug testing facility at the Peter MacCullum Cancer Institute. The
metallopolyamide complexes were dissolved in DMSO prior to
use. L1210 cells were exposed to the complexes for 48 h and the
experiments were carried out in duplicate.
2.6.2. LD studies of DNA binding affinity
Couette flow cell was used to measure the interactions between
ct-DNA and the ruthenium complexes. Phosphate buffer (10 mM,
pH 7) with sodium fluoride (20 mM) was used throughout the LD
experiments. Sodium fluoride was chosen as it does not absorb at
low wavelengths (<200 nm) like sodium chloride [16]. To calculate
the concentration of ct-DNA, a molar extinction value at 260 nm of
C46H66Cl2N20O8Ptꢃ6H2Oꢃ2.5C3H7NO: C, 40.57; H, 6.08; N, 19.90%;
Found: C, 40.84; H, 5.76; N, 19.66%. ESI-MS Calc. for
C
46H66ClN20O8Pt [MꢀClꢀ]+ 1257.6 m/z; found 1257.4 m/z.
e
= 13200 molꢀ1 cmꢀ1 was used [17]. ct-DNA (700
lL, 50 lM) was
2.5.9. Synthesis of
D- and -RUP1
-RUP1: 1H NMR 400 MHz (d6-DMSO):
d 10.05 (d, 1H,
placed in a Couette flow cell and the inner cylinder was rotated
(2 V) to orient ct-DNA and a LD spectrum was recorded. Fifteen
samples were prepared, with increasing metal complex and a con-
stant ct-DNA concentration (as shown in Supplementary data), and
allowed to equilibrate overnight at 25 °C. The LD of these samples
both spinning and non-spinning was measured. The non-spinning
measurement was subtracted from the spinning to correct for
background noise. The LD spectrum of ct-DNA was subtracted from
the sample spectrum to obtain a difference LD for each sample. All
binding experiments were performed in duplicates.
D
J = 8.03 Hz); 9.86 (s, 1H); 9.81 (s, 1H); 9.58 (d, 1H, J = 8.81 Hz);
8.85 (s, 2H); 8.46 (s, 2H); 8.29 (s, 2H); 8.13 (s, 1H); 8.06 (s, 1H);
7.99 (s, 1H); 7.84 (s, 2H); 7.44 (s, 1H); 7.13 (s, 1H); 6.67 (s, 1H);
3.96 (s, 1H); 3.82 (s, 3H); 3.39 (s, 2H); 3.32 (s, 2H); 2.41 (s, 2H);
2.31 (s, 2H); 1.83 (s, 2H). ESI-MS Calc. for C60H52N16O7Ru expected
(m/z): 605.30; found (m/z): 605.12. CD (H2O:CH3CN, 1.5 ꢂ 10ꢀ6 M,
RT.): k nm (
D
e
mdeg M cmꢀ1): 256 (86.3), 267 (ꢀ156.3), 296
(ꢀ35.6), 410 (5.8), 460 (ꢀ6.7). Electronic spectra kmax nm (
e
molꢀ1 dmꢀ3 cmꢀ1
,
50% CH3CN in H2O): 263 (1.2 ꢂ 105), 444
(1.7 ꢂ 104).
K
-RUP1: 1H NMR 400 MHz (d6-DMSO):
d 10.03 (d, 1H,
3. Results and discussion
J = 8.03 Hz), 9.87 (s, 1H), 9.78 (s, 1H), 9.60 (d, 1H, J = 7.81 Hz),
8.85 (d, 2H, J = 7.74 Hz), 8.46 (s, 2H), 8.31 (s, 2H), 8.26 (s, 1H),
8.13 (s, 1H), 8.00 (m, 2H), 7.84 (s, 2H), 7.47 (s, 1H), 7.13 (s, 1H),
6.69 (s, 1H), 3.95 (s, 1H), 3.83 (s, 3H), 2.95 (s, 2H), 2.83 (s, 2H),
2.49 (s, 2H), 2.31 (s, 2H), 1.84 (m, 2H). ESI-MS Calc. for
3.1. Synthesis and characterisation of HSP-2
The synthesis of b-Ala-Py-L4-Im (shown in Fig. 3) was achieved
using Fmoc-b-alanine-chlorotrityl resin. The polyamide was
cleaved from the resin using acetic acid and TFE before it was
lyophilized to obtain a yellow powder (67%). HSP-2 was produced
by combining the polyamide, b-Ala-Py-L4-Im at Pt-L6-NH2 through
solution phase chemistry. The product was obtained in a yield of
67.2% (see Fig. 4).
C
60H52N16O7Ru expected (m/z): 605.30; Found (m/z): 605.12%. CD
(H2O:CH3CN, 1.5 ꢂ 10ꢀ6 M, RT.): k nm (
De
mdeg M cmꢀ1): 256
(ꢀ82.8), 267 (143.5), 296 (33.9), 410 (ꢀ5.6), 460 (6.4). Electronic
spectra kmax nm (e
molꢀ1 dmꢀ3 cmꢀ1, 50% CH3CN in H2O): 263
(1.2 ꢂ 105), 444 (1.7 ꢂ 104).
The b-alanine monomer attached to the resin was deprotected
by the addition of 20% piperidine in DMF. This removed the
Fmoc-protecting group, resulting in a free –NH2. In a separate ves-
sel, PyE was dissolved in DMF/NMP mixture (1:1 v/v) and activated
by the addition of HBTU and DIEA. HBTU itself needs to be acti-
vated by the addition of DIEA [18]. This mixture was stirred for
ꢁ5 min. HBTU forms a temporary bond with PyE by displacing
2.6. Biological studies
2.6.1. Cytotoxicity
IC50 testing was conducted on the metallopolyamide complexes
using the L1210 murine luekemia cell line are the results for
growth inhibition studies of compounds using the Coulter Count-

192
N. Orkey et al. / Inorganica Chimica Acta 393 (2012) 187–197
Fig. 3. The summary of the synthesis of HSP-2. The reaction conditions used were (i) DIEA, Fmoc-b-alanine-OH, 5 h; (ii) 20% piperidine in DMF, PyE, 3.5 h; (iii) 20% piperidine
in DMF, Fmoc- -aminobutyric acid, 4 h; (iv) 20% piperidine in DMF, Im, 4.5 h; (v) TFE/acetic acid/DCM (2:1:7 v/v) mixture, 2 h; (vi) activated b-Ala-Py-L4-Im was stirred with
Pt-L6-NH2 overnight.
c
3
O
N
1
NH 4
N
2
5
6
8 H
24
NH3
N
7
15
12
H
11
H
17
21
19
14
N
N
O
9
N
Cl
Pt
NH2
22
16
20
18
13
O
O
10
HDO
11 × CH2
NH3
23
H3 & H10
H23 & H24
DMSO
NH
1,2,9,11
3 × NH
H22
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm(t1)
Fig. 4. The chemical structure of HSP-2 along with the 195Pt and 1H NMR spectrum in d6-DMSO at 35 °C with labelled proton resonances.
the –OH group. The activated solution of PyE was added to the re-
sin and the suspension was stirred for 3.5 h under N2(g). This en-
sured that most of the activated PyE would react with the b-
alanine group on the resin. The free amine group on the b-alanine
displaced the HBTU group to form a peptide bond between PyE and
b-alanine.
One amide singlet, at 9.73 ppm, was observed for the proton la-
belled 8 in the 1H NMR of HSP-2. Three amide triplets at 8.40, 7.92
and 7.83 ppm correspond to the protons labelled 4, 12 and 16,
respectively. The four aromatic protons 1, 2, 9, and 11 were as-
signed to the four singlets at 7.31, 7.07, 6.96 and 6.61 ppm. The
NH2 directly coordinated to the platinum(II) centre was observed
as a broad peak at 5.41 ppm, while the two platinum(II)-bound
NH3 groups were observed together as
a broad singlet at
4.00 ppm. The two singlets at 3.92 and 3.75 ppm were assigned
as the two methyl groups labelled 3 and 10. The eleven CH2 groups
were observed in the aliphatic region between 1.77 and 3.34 ppm.
The peak of the platinum(II) was observed at ꢀ2425 ppm as a

N. Orkey et al. / Inorganica Chimica Acta 393 (2012) 187–197
193
broad singlet. ESI-MS gave a parent peak corresponding to the sin-
gly charged ion HSP-2+ in aqueous solution. The purity of HSP-2
was assessed by elemental analysis; with the analysis indicating
that HSP-2 contains one H2O of hydration.
cannot be observed. The 11 ꢂ –CH2 groups resonate in the aliphatic
region between 3.39 and 1.82 ppm. The 195Pt NMR spectrum of
HSP-6 shows a single platinum resonance at ꢀ2420 ppm. The
chemical shift is consistent with similar metal complexes with
PtN3Cl coordination spheres [4b,19]. Both HSP-2 and HSP-6 dis-
played poor solubility in aqueous solutions; as a result both com-
plexes were dissolved in 1% DMSO in water/ACN (1:1, v/v) for the
purposes of mass spectrometry. In the ESI mass spectrum of HSP-6,
there is one prominent ion peak which occurs at m/z 1,257.4 and
corresponds to the parent metal complex with the 1+ charge. Frag-
mentation ions are also observed. The peak at m/z 993.4 can be as-
signed to the metal complex minus the trans-chlorodiamine
platinum group.
3.2. Synthesis and characterisation of HSP-6
3.2. Synthesis and characterisation of HSP-6
The synthesis of HSP-6 was achieved in an overall yield of 69.1%
in a similar stepwise assembly as shown for HSP-2 in Fig. 3. The 1H
NMR spectrum of HSP-6 (Fig. 5) shows five amide singlets between
9.98 and 9.57 ppm which correspond to protons labelled 7, 17/21,
4, and 13, respectively. Three amide triplets at 8.28, 7.93 and
7.81 ppm correspond to 9, 25 and 38, respectively. The ten sing-
lets/doublets between 7.61 and 6.83 ppm correspond to the ten
aromatic protons. The terminal –NH2 group is observed as a broad
peak at 5.19 ppm while the six singlets between 4.03 and 3.78 ppm
correspond to the 6 ꢂ N–CH3 groups. The 2 ꢂ –NH3 groups reso-
nate at the same frequency as the N–CH3 groups and therefore
3.3. Synthesis and characterisation of
D- and K-RUP1
[Ru(phen)2(phendo)]2+ was chosen as the ruthenium precursor
because the resolution of this complex had already been reported
by Hiort et al. [15]. It was also essential that a ruthenium(II) inter-
mediate that included a phendo ligand be used (resolved) to attach
8
O
N
6
O
9
NH
3
N
N
10
O
5
N
H
7
13
11
N
N
H
1
N
H
4
N
12
17
16
N
2
H
N
14
O
21
20
N
15
H
37
NH3
N
18
O
24
28
25
N
34
32
30
H
H
27
N
N
22
O
N
19
Cl
Pt
NH2
26
33
31
29
35
O
O
23
NH3
36
H2O
3 × NH
10 × Ar H
d5-DMSO
11 × CH2
8.25
8.00
7.75
7.50
7.25
7.00
6.75
2 × NH3
6 × N-CH3
5 × NH
10.10 10.00 9.90 9.80 9.70 9.60 9.50 9.40
NH2
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
ppm(t1)
Fig. 5. The 1H NMR spectrum of HSP-6 (Fig. 4.5) in d6-DMSO at 35 °C.

194
N. Orkey et al. / Inorganica Chimica Acta 393 (2012) 187–197
a polyamide chain utilising solid phase synthesis [20]. The
K
D
and
do)](PF6)2 to the resin. Once the desired polyamide metal complex
was synthesised, the resin was thoroughly washed with DCM,
DMF, diethylether, H2O, acetone and EtOH, to remove all unreacted
materials, and dried under N2(g). The complex was then cleaved
from the resin by the addition of TFE/acetic acid/DCM (2:1:7, v/v)
mixture. The orange product was characterised by 1H NMR, NOESY
and ESI/MS.
-[Ru(phen)2(phendo)]2+ complexes isolated using the published
method were in this instance of greater enantiomeric purity that
had previously reported [15]. RUP1 was synthesised by solid phase
chemistry using the chlorotrityl resin in a stepwise procedure as
shown in Fig. 6. The synthesis was initiated with the preparation
of the resin.
The monomers/linkers, Fmoc-c-aminobutyric acid linker, ImE,
From the 1H NMR in Fig. 7 it can be seen that H4 and H7, and H5
and H6 are in the same chemical environment and correspond to
the two signals; a doublet at 8.84 ppm (H4 and H7) and a singlet
8.46 ppm (H5 and H6). The methyl groups on the Im and Py rings
resonate as singlets at 3.95 and 3.82 ppm respectively. The methyl
Fmoc-b-alanine-OH and Fmoc-DAP-(Fmoc)-OH were added to the
resin in a similar manner, after being activated with HBTU after
the previous attached monomer/linker had been deprotected. The
final step saw the addition of either
K
- or
D
-[Ru(phen)2(phen-
NH.Fmoc
Cl
Cl
O
i
Cl
ii
H
Cl
NH.Fmoc
O
N
N
O
O
N
O
O
NH.Fmoc
NH
N
O
iv
iii
Fmoc.HN
O
NH
H
NH
Cl
O
Cl
O
H
N
N
O
N
N
O
O
O
Fmoc.HN
Fmoc.HN
Fmoc.HN
O
O
O
N
HN
HN
O
O
N
N
v
vi
NH
HN
O
O
O
N
Cl
NH
NH
O
Cl
O
O
N
H
NH
N
O
O
N
H
N
N
N
N
N
N
N
N
N
Ru
N
Ru
N
N
N
N
N
O
HN
O
HN
N
N
O
O
vii
viii
N
N
O
HN
HN
O
O
NH
O
O
N
N
NH
HO
O
Cl
NH
NH
O
O
O
N
H
N
H
N
N
Fig. 6. The summary of the synthesis of
piperidine in DMF, Fmoc- -aminobutyric acid, 4 h; (iv) 20% piperidine in DMF, ImE, 4.5 h; (v) 20% piperidine in DMF, Fmoc-b-alanine-OH, 4 h; (vi) 20% piperidine in DMF,
Fmoc-DAP-(Fmoc)-OH, 7 h; (vii) 20% piperidine in DMF, - or -[Ru(phen)2(phendo)](PF6)2, 5 h; (viii) TFE/acetic acid/DCM (2:1:7 v/v) mixture, 2 h.
K- and D-RUP1. The reaction conditions used were (i) DIEA, Fmoc-b-alanine-OH, 5 h; (ii) 20% piperidine in DMF, PyE, 3.5 h; (iii) 20%
c
D
K

N. Orkey et al. / Inorganica Chimica Acta 393 (2012) 187–197
195
4
(PF6)2
5
3
24
O
6
O
26
28
HO
2
3'
N
N
N
H
23
2'
N
4'
7
O
27
N
25
N
N
N
8
N
H
21
10
9
11
14
Ru
N
22
H
N
H
N
13
20
N
16
N
19
12
NH
18
O
O
15
N
O
17
H3’
H24
HDO
H25
H19
H18
H2’
DMSO
H27
H17
H26
H3/8
H22
H4’
H5/6
H21
H20
H4/7
H2/9
H15 H23
H10
H11
H14
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
ppm
Fig. 7. The chemical structure of
K
-RUP1 along with the 1H NMR spectrum in d6-DMSO at 35 °C with labelled proton resonances.
singlets give a NOE signal (Supplementary data) to protons H15
and H23 (at 7.4 and 7.1 ppm), which correspond to the aromatic
spectra, produced by subtracting the UV of [Ru(phen)2(dpq)]2+
from that of RUP1. The method used to obtain the extinction co-
efficient for RUP1 is shown in the Supplementary data ⁄⁄(Figure
S3 and Table 1).
protons on the Im and Py rings respectively.
D-RUP1 was charac-
terised using 1H NMR and NOESY and is identical to the
(Supplementary data).
K-isomer
The chirality of the metal precursors,
do)]2+ was conserved and is apparent in the CD of the products
and -RUP1. As the structure of either - or
-[Ru(phen)2(dpq)]2+
make up the metal complexes component of the polyamide, their
CD spectra were compared with to - and -RUP1. Slight changes
D- or K-[Ru(phen)2(phen-
ESI/MS experiments for the
K
- and
D
-RUP1 isomers showed
D-
that both complexes contain the parent ions carrying a 2+ charge
of 605.3 (m/z), which was consistent with the theoretical value cal-
culated (605.12 (m/z). When the UV spectra of RUP1 was compared
to [Ru(phen)2(dpq)]2+ (Fig. 8), it was evident that that both com-
plexes have similar transitions, with a broad metal centred transi-
tion at 450 nm, a sharp n–p⁄ transition at 265 nm with a shoulder
at approximately 300 nm [21]. The contribution of the polyamide
to these transitions was made evident from a difference UV
K
D
K
D
K
in the wavelengths of the peaks were observed but more signifi-
cantly there are changes in the intensities of these peaks which
can be attributed to the influence of the polyamide (Fig. 9).
Fig. 8. The UV–Vis spectrum of RUP1 (A) and rac-[Ru(phen)2(dpq)]2+ (B) the insert
shows a spectrum where UV (B) is subtracted from UV (A) this represents the
polyamide contribution to the spectrum (C) in H2O:acetonitrile (1:1 v/v) mixture at
Fig. 9. The CD spectrum of
K
-RUP1 (A) and
D
-RUP1 (B), in comparison to
K-
[Ru(phen)2(phendo)]2+ (A0) and
H2O:acetonitrile (1:1 v/v) mixture.
D
-[Ru(phen)2(phendo)]2+ (Bh) at 10
lM in
room temperature. The concentration of both complexes was 10 lM.

196
N. Orkey et al. / Inorganica Chimica Acta 393 (2012) 187–197
Fig. 10. (A) LD spectra of LD-DNA titration with
D-RUP1 (left) and K-RUP1 (right). (B) LD spectra where the DNA spectrum has been subtracted from each LD-DNA titration
spectrum of
D
-RUP1 (left) and
K-RUP1 (right).The wavelength (nm) is plotted on the X-axis, while the MC complex concentration (M) on the Y- and the LD (dOD) on the Z-
axis respectively.
3.4. Biological studies
similar signals but with greater intensity at 380 and 480 nm (Sup-
3.4.1. In vitro testing. In vitro testing was conducted using L1210
murine leukaemia cells however HSP-2 and HSP-6 were not suffi-
ciently soluble, even when dissolved in a small amount of DMSO or
DMF, and precipitated out of the aqueous medium used for assays.
As a result of this, the IC50 values of the platinum metallopolya-
mide complexes could not be determined. Ruthenium(II) com-
plexes are not largely known for their cytotoxicity [22]. However,
these tests were performed to evaluate the effect of the sequence
selective ‘tail’ part of the molecule on overall cytotoxicity. Neither
plementary data Fig. S4).
The LD transitions for
D
-RUP1 when bound to DNA, shows a de-
crease in intensity at 258 nm and a small positive at 264 nm which
could indicate groove binding (Fig. 10A and B, left). Two small LD
bands in the visible region are also evident with both the positive
band centered at 380 nm and the negative band at 480 nm of sim-
ilar magnitude like
in Fig. 10B, clearly shows the relative changes. When bound to
DNA, -RUP1 also shows a decrease in intensity in the LD spec-
trum at 268 nm with a shoulder at 288 nm and a positive signal
at 325 nm (Fig. 10A and B, right). The signals due to MLCT of
RUP1 grow in intensity with increasing concentration and are lar-
ger than seen for -RUP1. Moreover the broad positive band at
D
-[Ru(phen)2(dpq)]2+. The difference LD shown
K
D
nor
K-RUP1 exhibited cytotoxicity with IC50 values >40 lM re-
quired to inhibit cell growth.
K
-
D
3.4.2. LD studies of DNA binding affinity. Previously, a 1H NMR study
380–430 nm is larger in intensity relative to the negative band at
480 nm. This evidence would suggest that groove binding is the
mode of interaction for both
conducted by Greguric et al. [23] reported that the
D-[Ru(-
phen)2(dpq)]2+ complex interacted with a hexanucleotide through
the minor groove by intercalation. Linear dichroism studies con-
ducted by Lincoln et al. [24] indicated that the analogous [Ru(-
phen)2(dppz)]2+ complex intercalated within the DNA base pairs.
D- and K-RUP1.
4. Conclusion
The LD spectra of ct-DNA with the
and - and -isomers of RUP1 were measured and the resulting
spectra compared. The initial LD spectrum of DNA shows a distinc-
tive negative peak at 260 nm which is indicative of the
p⁄ tran-
sitions of the DNA. Changes in the LD spectra that are produced
upon the titration of
-[Ru(phen)2(dpq)]2+ into DNA shows that
D- and K
-[Ru(phen)2(dpq)]2+
D
K
Four metallopolyamide complexes, two platinum(II) complexes
– [Cl(NH3)2Pt-L6-b-Ala-Py-L4-Im]+ (HSP-2) and [Cl(NH3)2Pt-L6-b-
Ala-PyPyPy-L4-ImImIm]+ (HSP-6), along with two ruthenium enan-
p–
tiomers,
D and K (D and K-
-[b-Ala-Py-L4-Im-b-dpq-Ru(phen)2]2+
D
RUP1), were successfully synthesised in good yield by a combina-
tion of solid and solution phase chemistries. Characterisation was
achieved using NMR and ESI-MS. CD for
the negative intensity below 300 nm becomes more positive.
Whereas the positive peak at 380 nm and the negative peak at
480 nm, due to MLCT of the metal complex, grow in intensity with
increasing concentration (Supplementary data Fig. S4). The LD
D
- and
K
-RUP1. Con-
- and
firmed that the chirality of the ruthenium precursors
D
K-
[Ru(phen)2(phendo)]2+ was conserved throughout the synthesis.
Cytotoxicity and binding interactions were not determined for
spectra of the titration of
K
-[Ru(phen)2(dpq)]2+ with DNA shows

N. Orkey et al. / Inorganica Chimica Acta 393 (2012) 187–197
197
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HSP-2 or HSP-6, which although isolated, were poorly soluble in
aqueous solution inhibiting testing. Since the IC50 value of and
-RUP1 were greater than 40 M they have potential to be used
as DNA probes. LD studies indicated that the ruthenium complexes
interact with ct-DNA through a mixed binding mode, which is
influenced by complex concentration and chirality.
D
K
l
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Recent Research Developments in Inorganic Chemistry 1 (1998) 13;
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42 (2004) 323;
Nikita Orkey and Robin Taleb were supported by an Australian
Postgraduate Award from the University of Western Sydney and
the School of Science and Health for Conference Support. We also
wish to acknowledge the University of Western Sydney for funding
through internal research grants and International Research Initia-
tive Scheme. Aspects of this research was supported were sup-
ported by Australian Government, through the International
Science Linkages (ISL) program. We thank Dr. Chris Chandler from
Auspep for his help in solid phase synthesis.
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2012.06.013.
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