Electrochemiluminescent peptide nucleic acid-like monomers containing Ru(II)-dipyridoquinoxaline and Ru(II)-dipyridophenazine complexes

Article abstract of DOI:10.1021/ic201911f

A series of Ru(II)-peptide nucleic acid (PNA)-like monomers, [Ru(bpy) ₂(dpq-L-PNA-OH)]²⁺ (M1), [Ru(phen)₂(dpq-L-PNA- OH)]²⁺ (M2), [Ru(bpy)₂(dppz-L-PNA-OH)]²⁺ (M3), and [Ru(phen)₂

Full text of DOI:10.1021/ic201911f

Article
pubs.acs.org/IC
Electrochemiluminescent Peptide Nucleic Acid-Like Monomers
Containing Ru(II)−Dipyridoquinoxaline and Ru(II)−
Dipyridophenazine Complexes
Tanmaya Joshi,† Gregory J. Barbante,‡ Paul S. Francis,‡ Conor F. Hogan,§ Alan M. Bond,†
and Leone Spiccia*,†
†ARC Centre of Excellence for Electromaterials Science and School of Chemistry, Monash University, Clayton, Victoria 3800,
Australia
‡School of Life and Environmental Sciences, Deakin University, Geelong, Victoria, 3217, Australia
§Department of Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Victoria, 3086, Australia
S
* Supporting Information
ABSTRACT: A series of Ru(II)−peptide nucleic acid (PNA)-
like monomers, [Ru(bpy)2(dpq-L-PNA−OH)]2+ (M1), [Ru-
(phen)2(dpq-L-PNA−OH)]2+ (M2), [Ru(bpy)2(dppz-L-
PNA−OH)]2+ (M3), and [Ru(phen)2(dppz-L-PNA−OH)]2+
(M4) (bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline, dpq-
L-PNA−OH = 2-(N-(2-(((9H-fluoren-9-yl)methoxy)-
carbonylamino)ethyl)-6-(dipyrido[3,2-a:2′,3′-c]phenazine-11-
carboxamido)hexanamido)acetic acid, dppz-L-PNA−OH = 2-
(N-(2-(((9H-fluoren-9-yl) methoxy)carbonylamino)ethyl)-6-
(dipyrido[3,2-f:2′,3′-h]quinoxaline-2-carboxamido)acetic acid)
1
have been synthesized and characterized by IR and H NMR spectroscopy, mass spectrometry, and elemental analysis. As is
typical for Ru(II)−tris(diimine) complexes, acetonitrile solutions of these complexes (M1−M4) show MLCT transitions in the
443−455 nm region and emission maxima at 618, 613, 658, and 660 nm, respectively, upon photoexcitation at 450 nm. Changes
in the ligand environment around the Ru(II) center are reflected in the luminescence and electrochemical response obtained
from these monomers. The emission intensity and quantum yield for M1 and M2 were found to be higher than for M3 and M4.
Electrochemical studies in acetonitrile show the Ru(II)−PNA monomers to undergo a one-electron redox process associated
with RuII to RuIII oxidation. A positive shift was observed in the reversible redox potentials for M1−M4 (962, 951, 936, and 938
mV, respectively, vs Fc0/+ (Fc = ferrocene)) in comparison with [Ru(bpy)3]2+ (888 mV vs Fc0/+). The ability of the Ru(II)−PNA
monomers to generate electrochemiluminescence (ECL) was assessed in acetonitrile solutions containing tripropylamine (TPA)
as a coreactant. Intense ECL signals were observed with emission maxima for M1−M4 at 622, 616, 673, and 675 nm,
respectively. At an applied potential sufficiently positive to oxidize the ruthenium center, the integrated intensity for ECL from
the PNA monomers was found to vary in the order M1 (62%) > M3 (60%) > M4 (46%) > M2 (44%) with respect to
[Ru(bpy)3]2+ (100%). These findings indicate that such Ru(II)−PNA bioconjugates could be investigated as multimodal labels
for biosensing applications.
chemical properties as well as introducing new functionalities
and spectroscopic characteristics. Thus, the field of PNA−metal
bioconjugates has been advancing rapidly since the concept was
first introduced.16−19 Incorporation of metal complexes into the
PNA oligomer allows tailoring of properties to achieve
particular desired objectives. PNA−metal bioconjugates present
themselves as very promising candidates for electrochemical,
radiochemical, and optical biosensing and can also be employed
to confer and/or influence hybridization stability in the formed
duplexes.16,18−24 Examples of metal−PNA oligomers/mono-
mers incorporating ferrocene, Re(II), Ru(II), Zn(II), Cu(II),
Zr(IV), Mn(III), Co(II) and 99mTc derivatives have been
INTRODUCTION
■
Peptide nucleic acids (PNA)artificial DNA analogues
assembled on the N-(2-aminoethyl)-glycine backbonehave
fascinated researchers throughout the past two decades due to
their potential therapeutic applications.1−9 They possess
inherent specificity and strong affinity for Watson−Crick base
pairing for cDNA or RNA strands, partly attributed to the lack
of electrostatic repulsion.2,10,11 The neutral polyamide back-
bone not only contributes to the hybridization stability but also
makes PNA resistant to both proteases and nucleases.4,12 These
properties are particularly attractive for antisense and antigene
therapies, as well as for diagnostic and analytical applications in
biosensing areas.7−9,13−15 To further diversify the applications
of PNA, the native molecule has been subjected to numerous
modifications aimed at improving the innate physical and
Received: September 1, 2011
Published: October 31, 2011
© 2011 American Chemical Society
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Figure 1. Structures of the dpq-L-PNA and dppz-L-PNA ligands and M1−M4.
reported in the literature.19,24−34 As first described by Metzler-
Nolte et al.,17,18 covalent attachment of metal complexes to the
reactive N-terminus of the PNA sequence represents the
classical approach for metal insertion, but it limits the degree of
flexibility achievable in metal−PNA oligomers. This restriction
has led to the development of new classes of metal-containing
PNA monomers for direct insertion into the desired location
within the PNA oligomers.19,28,35−37 However, while there are
many examples of such monomers in the literature, as far as we
are aware, the only successful insertion of a metal complex
within the desired PNA oligomer on a solid phase is contained
in reports by Metzler-Nolte and co-workers.32,38,39 In these
studies, ferrocenyl−PNA bioconjugates were prepared by
employing “click chemistry” between the alkyne-containing
PNA oligomer and the corresponding azido-ferocene deriva-
tive.32,38,39 Similarly, 99mTc tricarbonyl-containing PNA
oligomers were prepared using a related “click chemistry”
approach to create a metal complexation site on the oligomeric
unit which was converted into a 99mTc tricarbonyl derivative.34
To date, this approach for insertion of the metal complex is the
main route explored for the synthesis of ferrocenyl−PNA
oligomers. Significantly, the stability of the ferrocenyl group
and its large derivatives library make it an excellent choice for
electrochemical biosensors.40,41 Nevertheless, it is also desirable
to prepare PNA−metal bioconjugates that widen the scope of
biosensor applications by allowing multiple detection probes.
Polypyridyl−Ru(II) complexes, for example, provide attractive
electrochemical and photochemical properties and hence
represent suitable contenders for dual detection probes. Due
to their high chemical, thermal, and photochemical stability;
reversible redox behavior; intense UV−visible absorption with
long-lived MLCT excited states; and large Stoke shifts, Ru(II)
polypyridyl complexes play an important role in a variety of
biosensing applications based on photoluminescence, chem-
iluminescence (CL), and electrochemiluminescence
(ECL).42−59
In order to advance the dual sensing field, we have been
interested in introducing a robust framework of Ru(II)−PNA
monomers, which can be inserted into any selected position
within the PNA sequences.37 Our initial work showed that (as
expected) the required characteristic electrochemical and
photochemical properties of the Ru(II)−PNA monomers
remain more or less unperturbed when conjugated onto the
PNA backbone, but major synthetic and purification difficulties
were encountered. Previous synthetic shortcomings are now
addressed by designing new types of Ru(II)−polypyridyl-based
PNA-like monomers that allow easy insertion into the
oligomeric sequences. Photoluminescence and electrochemical
and ECL responses are then explored. In particular, the
synthesis of two novel PNA derivatives, dpq-L-PNA, dppz-L-
PNA, and their Ru(II)−bis(bipyridyl)/bis(phenanthroline)
complexes, M1, M2, M3, and M4 (Figure 1), is described.
Monomers M1 and M2 are assembled around the dipyr-
idoquinoxaline (dpq) backbone using dpq-L-PNA, whereas
dppz-L-PNA, which bears the dipyridophenazine (dppz) unit, is
used in the cases of M3 and M4. The ligands are attached to
the PNA monomer backbone via a hexyl linker to provide steric
flexibility. While photophysical and electrochemical properties
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which comprised a glassy carbon working electrode (area = 0.0079
cm2), a large surface area Pt counter electrode, and a Ag/Ag+ (0.1 M
AgNO3 in CH3CN) reference electrode. The potential of the Ag/Ag+
reference electrode was frequently calibrated against that of the
ferrocene/ferrocinium (Fc0/+) redox couple by measuring the
reversible potential derived from oxidation of Fc under identical
conditions used for the voltammetric measurements on the Ru(II)
complexes. The working electrode was polished with an aqueous slurry
of aluminum oxide (0.3 μm), then rinsed with acetone and dried
before each voltammetric experiment.
of Ru(II)−PNA monomers/oligomers have previously been
reported,17,37 to the best of our knowledge, this is the first study
of the electrochemiluminescent properties of PNA-like
monomers with conjugated ECL active compounds.
EXPERIMENTAL SECTION
■
Chemicals. Unless otherwise stated, the chemicals were either of
reagent or analytical grade quality and were used as purchased from
the manufacturer. 2,3-Bis(tert-butoxycarbonylamino)propanoic acid
(1),60 ethyl-6-aminohexanoate hydrochloride,61 1,10-phenanthroline-
5,6-dione (9),62 tert-butyl-2-(2-(((9H-fluoren-9-yl)methoxy)-
carbonylamino)ethylamino) acetate hydrochloride (8),63 Ru-
Electrochemiluminescence experiments were undertaken with a
PGSTAT 12 Autolab electrochemical workstation (MEP Instruments,
North Ryde, NSW, Australia) in combination with General Purpose
Electrochemical Systems (GPES) software (version 4.9). In these
studies, the electrochemical cell consisted of a glass cell with a quartz
base. The cell was enclosed in a custom-built light-tight faraday cage. A
three-electrode configuration, consisting of a 3 mm-diameter glassy
carbon working electrode shrouded in Teflon (CH Instruments,
Austin, TX, USA), a 1 cm2 platinum gauze auxiliary electrode, and a
silver wire quasi reference electrode, was used. ECL spectra were
obtained using an Ocean Optics CCD spectrometer, model QE65000,
coupled to the cell with a 1 m fiber optic cable. The spectral
acquisition was triggered simultaneously with the electrochemical
experiment with the aid of an HR 4000 breakout box (Ocean Optics).
The Ru(II) complexes were prepared at a concentration of 0.1 mM in
freshly distilled acetonitrile, with 0.1 M nBu4NPF6 as the supporting
electrolyte and 0.1 M tripropylamine as the ECL coreactant. Prior to
each experiment, the working electrode was polished sequentially with
slurries of 0.3 and 0.05 μm alumina on a felt pad, sonicated in water (1
min), rinsed in freshly distilled acetonitrile, and dried with a stream of
nitrogen. The working electrode was then positioned ca. 2 mm from
the bottom of the cell for detection of the ECL signal, and the solution
was purged with argon for 3 min. ECL intensities are given relative to
[Ru(bpy)3](PF6)2 (100%) measured under the same conditions. To
generate the emission, the working electrode was pulsed for 2.0 s
between 0 V and a value sufficiently positive to generate the
ruthenium(III) complex in each case. The resulting ECL spectra were
collected over the duration of three such chronoamperometric cycles.
Synthesis. Ethyl-6-(2,3-bis(tert-butoxycarbonylamino)-
propanamido)hexanoate (2). DCC (0.255 g, 1.24 mmol), HOBt
(0.165 g, 1.20 mmol), and DMAP (20 mg) were added to a solution of
2,3-bis(tert-butoxycarbonylamino)propanoic acid (1; 0.250 g, 0.822
mmol) in THF (10 mL). The resulting suspension was stirred at room
temperature under nitrogen for 2 hours. Ethyl-6-aminohexanoate
solution (prepared by treating ethyl-6-aminohexanoate hydrochloride
(0.250 g, 1.30 mmol) with triethylamine (0.130 g, 1.60 mmol) in 10
mL dry acetonitrile) was then added dropwise, and stirring was
continued for 16 h under nitrogen. The reaction mixture was filtered
through a small celite pad. The celite pad was washed thoroughly with
dichloromethane and the filtrate concentrated under reduced pressure.
The solid residue was dissolved in dichloromethane (60 mL) and
filtered. The filtrate was washed with 10% citric acid (3 × 25 mL),
saturated NaHCO3 (3 × 25 mL), water (1 × 20 mL), and brine (1 ×
20 mL). The organic phase was then dried over anhydrous Na2SO4
and filtered and the filtrate evaporated to dryness to obtain 2 as a
white solid. Yield: 0.265 g (75%). Rf = 0.50 in 5% MeOH/DCM. IR
(KBr): ν 3375s (N−H), 3293s (N−H), 2939s (C−Haliph), 2866m
(C−Haliph), 1734s (CO), 1718s (CO), 1701s (CO), 1527m,
1459w, 1390w, 1369m, 1305m, 1281m, 1253m, 1198m, 1165s, 1098m,
64
(bpy)2Cl2,64 and Ru(phen)2Cl2 were synthesized according to
literature procedures. Characterization data were in agreement with
literature reports. When required, solvents for the synthetic work were
degassed by purging with dry, oxygen-free nitrogen for at least 30 min
before use. Acetonitrile was dried before use by standing over calcium
hydride overnight. Deionized water was used for all reactions
undertaken in aqueous media. HPLC-grade solvents were used for
all spectral studies. Tetrabutylammonium hexafluorophosphate
(nBu4NPF6, Fluka) was recrystallized prior to use as the electrolyte
for the electrochemical studies in MeCN.65
Instrumentation and Methods. A vacuum line and Schlenk
glassware were employed when reactions had to be carried out under
an atmosphere of dry, oxygen-free nitrogen. Protection from light was
provided if necessary by wrapping the assemblies with aluminum foil.
1H and 13C NMR spectra were obtained with Bruker AC200, AM300,
or DRX 400 spectrometers and referenced against tetramethylsilane
(TMS) or signals from the residual protons of deuterated solvents.66
The chemical shifts, δ, are reported in parts per million (ppm) relative
to the reference, and coupling constants, J, are given in Hertz. Peak
multiplicities are abbreviated as follows: s (singlet), d (doublet), t
(triplet), and m (multiplet). ESI mass spectra were recorded using a
Micromass Platform II mass spectrometer fitted with an ESI source
(capillary voltage 3.5 eV and cone voltage 35 V). The most intense m/
z value is listed. Accurate high-resolution mass spectra were obtained
with a Bruker BioApex II 47e FT-ICR MS instrument fitted with an
Analytica Electrospray Source. Samples were introduced by a syringe
pump at a rate of 1 μL min−1, and the capillary voltage was 200 V.
Infrared spectra were recorded from KBr disks or neat as indicated,
using a Perkin-Elmer 1600 Series FTIR spectrometer in the range
4000−500 cm−1 with a resolution of ±4.0 cm−1. Peak intensities are
given as broad (br), very strong (vs), strong (s), medium (m), and
weak (w). Microanalyses were carried out by the Campbell
Microanalytical Laboratory, University of Otago, New Zealand. UV/
vis spectra were recorded using Varian Cary Bio 300 or 5G
spectrophotometers. Photoluminescence spectra were obtained with
a Fluoromax-4 spectrofluorometer (Horiba Jobin Yvon Inc.), following
excitation at 450 nm, and were autocorrected for instrumental
response using a correction factor provided by the instrument
manufacturer. The excitation and emission slit width for recording
the emission spectra were set to 3.0 and 2.5 nm, respectively. UV/
visible and emission spectra were measured using a 10 μM acetonitrile
solution of each complex at 25 °C in 1 cm quartz cuvettes. All
solutions were degassed by purging with nitrogen. Thin layer
chromatography (TLC) was performed using silica gel 60 F-254
(Merck) plates with detection of spots achieved either by exposure to
iodine or UV light or by using ninhydrin stain. Column
chromatography was undertaken using silica gel 60 (0.040−0.063
mm mesh, Merck). Eluent mixtures are expressed as volume to volume
(v/v) ratios.
1
1025w, 870s cm−1. H NMR (300 MHz, CDCl3): δ 4.21−4.08 (m,
3H), 3.44−3.23 (m, 4H), 2.26−2.23 (m, 2H), 1.89−1.58 (m, 4H),
1.42 (s, 9H), 1.39−1.22 (m, 14H) ppm. 13C NMR (75 MHz, CDCl3):
δ 173.9, 170.8, 157.1, 80.7, 80.3, 60.6, 49.4, 39.5, 34.4, 29.4, 28.7, 28.6,
26.6, 25.9, 24.8, 14.5 ppm. MS (ESI+): m/z 468.3 [M + Na]+.
Ethyl-6-(2,3-diaminopropanamido)hexanoate (3). Ethyl-6-(2,3-
bis(tert-butoxycarbonylamino)propanamido)hexanoate (2; 0.223 g,
0.50 mmol) was dissolved in DCM/trifluoroacetic acid (1:1, 5 mL)
and the solution stirred at room temperature for 5 h. The solvent was
evaporated under reduced pressure, and the residue was triturated with
toluene and ether to obtain the trifluoroacetic acid salt of 3 as a brown
Electrochemical Measurements. Cyclic voltammetric measure-
ments were performed at (20 ± 2)°C in acetonitrile solutions
containing 0.1 M nBu4NPF6 as the supporting electrolyte, over the
scan rate range of 100−1000 mV s−1 using a BAS 100B (Bioanalytical
Systems) electrochemical workstation. Solutions used in electro-
chemical measurements were deoxygenated by purging with high
purity nitrogen for at least 10 min before commencing the
experiments. A conventional three electrode cell was employed
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solid. Yield: 0.230 g (98%). IR (KBr): ν 3328s (N−H), 2929s (C−
141.5, 141.2, 138.1, 134.0, 133.3, 133.1, 128.0 (maj) and 127.9 (min),
127.3, 126.9, 126.5, 125.4, 125.2, 124.6, 124.4 (min) and 124.3 (maj),
120.2 (min) and 120.1 (maj), 83.3 (min) and 82.5 (maj), 67.4 (maj)
and 66.1 (min), 50.0, 49.7 (maj) and 49.5 (min), 47.5 (min) and 47.4
(maj), 40.0 (min) and 39.8 (maj), 34.3, 32.8, 29.6, 28.4 (min) and 28.3
(maj), 26.9, 24.8 ppm. MS (ESI+): m/z 767.9 [M + H]+, 790.0 [M +
Na]+.
Haliph), 2851s (C−Haliph), 1693s (CO), 1653s (CO), 1578m,
1312m, 1272w, 1244m, 1205w, 1158w, 1088m, 892m cm−1. 1H NMR
(300 MHz, CD3OD): δ 4.22−4.18 (m, 1H), 4.09 (q, 3J = 7.1 Hz, 2H),
3
3.44−3.35 (m, 3H), 3.25−3.14 (m, 1H), 2.30 (t, J = 7.3 Hz, 2H),
3
1.71−1.51 (m, 4H), 1.36−1.31 (m, 2H), 1.22 (t, J = 7.1 Hz, 3H)
ppm. 13C NMR (75 MHz, CD3OD): δ 174.4, 165.9, 60.3, 48.6, 39.7,
33.6, 28.5, 26.2, 25.6, 24.4, 13.4 ppm. MS (ESI+): m/z 246.1 [M +
H]+.
3,4-Bis(tert-butoxycarbonylamino)benzoic Acid (5). To a solu-
tion of di-tert-butyl dicarbonate (7.20 g, 33.0 mmol) in DMF (15 mL)
was added 3,4-diaminobenzoic acid (2.00 g, 13.2 mmol) followed by
diisopropylethylamine (2.10 g, 16.0 mmol). The reaction mixture was
stirred at room temperature under nitrogen for 16 h and then poured
into 150 mL of water. The solution pH was adjusted to 6 using 1 M
HCl and extracted with EtOAc (3 × 125 mL). The combined organic
layers were washed with water (4 × 50 mL), dried over Na2SO4,
filtered, and concentrated under reduced pressure. The crude product
was recrystallized from chloroform to give 5 as a white solid. Yield:
2.60 g (56%). Rf = 0.25 in 80% EtOAc/hexane. IR (KBr): ν 3367m
(N−H), 2981s (C−Harom), 2934m (C−Harom), 1741s (CO), 1718s
(CO), 1689s (CO), 1609w, 1499m, 1420m, 1396s, 1369m,
6-(Pyrazino[2,3][1,10]phenanthroline-2-carboxamido)hexanoic
Acid (4). 1,10-Phenanthroline-5,6-dione (0.074 g, 0.350 mmol) was
added to absolute ethanol (10 mL) and the mixture refluxed to
complete the dissolution.
A solution of ethyl-5-(2,3-
diaminopropanamido)hexanoate (3; 0.212 g, 0.450 mmol) in absolute
ethanol (10 mL) was then added, and the reaction mixture was
refluxed. Monitoring by TLC indicated completion of the reaction
after 5 h. The reaction mixture was cooled to room temperature. The
small amount of yellow precipitate was filtered, and the filtrate was
evaporated to dryness to obtain a reddish orange solid (115 mg) as the
crude product. The crude product was dissolved in 8 mL of MeOH/
H2O (3:1) and solid NaOH (0.042 g, 1.10 mmol) added at 0 °C. After
stirring overnight at room temperature for 16 h, the volume of the
reaction mixture was reduced to 4 mL by rotary evaporation. The
alkaline solution was acidified to pH 3 using 1 M HCl and evaporated
to dryness. The resulting solid was purified by column chromatog-
raphy (SiO2, 10% MeOH/DCM) to obtain 4 as a white solid. Yield:
0.085 g (63%). Rf = 0.20 in 10% MeOH/DCM. IR (KBr): ν 3410w
(N−H), 3058w (C−Harom), 2929m (C−Haliph), 2855m (C−Haliph),
1720s (CO), 1670s (CO), 1533m, 1432w, 1402m, 1390m,
1376m, 1261w, 1246m, 1202s, 1159m, 1128w, 1076w, 940m, 823m,
1322s, 1248s, 1160s, 1050m, 1027m, 963w, 876w, 828w, 772m cm−1
.
1H NMR (300 MHz, DMSO-d6): δ 8.71 (s, 1H), 8.66 (s, 1H), 8.08 (s,
1H), 7.73−7.62 (m, 2H), 1.46 (s, 18H) ppm. 13C NMR (75 MHz,
DMSO-d6): δ 167.6, 154.0, 153.5, 135.2, 129.7, 126.4, 126.1, 125.9,
122.9, 80.8, 80.4, 28.8, 28.7 ppm. MS (ESI−): m/z 351.2 [M − H]−.
Ethyl-6-(3,4-bis(tert-butoxycarbonylamino)benzamido)-
hexanoate (6). Compound 6 was prepared in a manner similar to 2,
but using 3,4-bis(tert-butoxycarbonylamino)benzoic acid (5; 0.620 g,
1.75 mmol), Et3N (0.30 mL, 2.10 mmol), DCC (0.536 g, 2.60 mmol),
HOBt (0.311 g, 2.30 mmol), DMAP (25 mg), and ethyl-6-
aminohexanoate hydrochloride (0.552 g, 2.82 mmol) in dry DMF
(15 mL). The product 6 was isolated as viscous yellow oil. Yield: 0.660
g (76%). Rf = 0.35 in 50% EtOAc/hexane. IR (neat): ν 3365m (N−
H), 2979m (C−Harom), 2934m (C−Harom), 2866w (C−Haliph), 1740m
(CO), 1731m (CO), 1715m (CO), 1621w, 1518w, 1427m,
1393m, 1368m, 1311w, 1242m, 1159m, 1103w, 1050m, 1027m, 903m,
1
742s cm−1. H NMR (300 MHz, DMSO-d6): δ 9.56 (s, 1H), 9.40−
9.38 (m, 2H), 9.23−9.22 (m, 2H), 7.96−7.90 (m, 2H), 3.45−3.38 (m,
3
2H), 2.24 (t, J = 7.1 Hz, 2H), 1.63−1.51 (m, 4H), 1.47−1.39 (m,
2H) ppm. 13C NMR (75 MHz, DMSO-d6): δ 175.2, 163.4, 153.3,
153.0, 148.1, 147.8, 144.8, 144.0, 141.8, 138.6, 134.5, 133.6, 127.0,
126.8, 125.2, 124.9, 41.2, 34.4, 29.9, 26.9, 25.1 ppm. MS (ESI−): m/z
388.1 [M − H]−.
1
878w, 826w cm−1. H NMR (300 MHz, CDCl3): δ 7.65 (br s, 1H),
tert-Butyl-2-(N-(2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
ethyl)-6-(dipyrido[3,2-f:2′,3′-h]quinoxaline-2-carboxamido)-
hexanamido)acetate (dpq-L-PNA). 6-(Pyrazino[2,3][1,10]-
phenanthroline-2-carboxamido)hexanoic acid (4; 0.120 g, 0.308
mmol) was suspended in dry DMF (5 mL) under nitrogen. HBTU
(0.243 g, 0.616 mmol) and DMAP (20 mg) were added to the
suspension, and the reaction mixture was stirred at room temperature
for 30 min. tert-Butyl-2-(2-(((9H-fluoren-9-yl)methoxy)-
carbonylamino)ethylamino)acetate hydrochloride (0.277 g, 0.640
mmol) was added, and stirring continued at room temperature for a
further 15 min. Diisopropylethylamine (0.11 mL, 0.60 mmol) was
added dropwise at 0 °C. After the reaction mixture was stirred for 30
min at 0 °C, it was stirred at room temperature for a further 18 h.
Water (25 mL) was then added, resulting in the formation of a
precipitate which was collected by filtration. The precipitate was
dissolved in dichloromethane, the solution dried over Na2SO4, filtered,
and concentrated to obtain the crude product, which was purified by
column chromatography (SiO2, gradual change of eluent polarity from
100% EtOAc to 10% MeOH/DCM) to afford dpq-L-PNA as a pale
yellow solid. Yield: 0.165 g (73%). Rf = 0.45 in 10% MeOH/DCM. IR
(KBr): ν 3067w (C−Harom), 3014w (C−Harom), 2930s (C−Haliph),
2855m (C−Haliph), 1730s (CO), 1655s (CO), 1535br, 1459m,
1399m, 1377w, 1252s, 1235m, 1199w, 1156s, 1079w, 1035w, 1017w,
7.54−7.40 (m, 3H), 7.27 (d, 3J = 7.7 Hz, 1H), 7.106.92 (m, 1H), 4.03
(q, 3J = 7.1 Hz, 2H), 3.29−3.24 (m, 2H), 2.23−2.19 (m, 2H),
1.571.48 (m, 4H), 1.43 (s, 9H), 1.41 (s, 9H), 1.33−1.28 (m, 2H), 1.16
3
(t, J = 6.7 Hz, 3H) ppm. 13C NMR (75 MHz, CDCl3): δ 174.0,
167.2, 154.6, 153.8, 134.2, 130.9, 129.2, 124.3, 124.2, 122.8, 81.0, 60.5,
40.1, 34.4, 29.4, 28.5, 26.7, 24.8, 14.5 ppm. MS (ESI+): m/z 516.1 [M
+ Na]+.
Ethyl-6-(3,4-diaminobenzamido)hexanoate (7). Deprotection of
ethyl-6-(3,4-bis(tert-butoxycarbonylamino)benzamido)hexanoate (6;
0.365 g, 0.740 mmol) as for 3 gave 7 as a brown solid. Yield: 0.380
g (98%). IR (neat): ν 3361s (N−H), 3075w (C−Harom), 2932s (C−
H
arom), 2858m (C−Haliph), 1716s (CO), 1664s (CO), 1557w,
1463w, 1456m, 1394w, 1373m, 1323s, 1286w, 1191m, 1158s, 1031m,
973m, 892w, 835m cm−1. H NMR (300 MHz, CDCl3): δ 7.86−7.31
1
(m, 3H), 6.98 (d, 3J = 7.7 Hz, 1H), 4.12 (q, 3J = 7.1 Hz, 2H), 3.463.42
(m, 2H), 2.37−2.30 (m, 2H), 1.661.56 (m, 4H), 1.38−1.34 (m, 2H),
1.20 (t, 3J = 6.7 Hz, 3H) ppm. 13C NMR (75 MHz, CDCl3): δ 176.5,
168.6, 139.1, 136.4, 134.7, 132.1, 126.8, 120.7, 61.7, 41.1, 34.4, 28.6,
26.2, 24.5, 13.9 ppm. MS (ESI+): m/z 294.1 [M + H]+.
6-(Dipyrido[3,2-a:2′,3′-c]phenazine-11-carboxamido)hexanoic
Acid (8). 1,10-Phenanthroline-5,6-dione (0.120 g, 0.571 mmol) was
heated at reflux in 10 mL of absolute ethanol until dissolution was
complete; ethyl-6-(3,4-diaminobenzamido)hexanoate (7; 0.387 g,
0.742 mmol) dissolved in ethanol (10 mL) was added and the
reaction mixture heated at reflux. Conversion to the product was
complete after 5 h of reflux (monitored by TLC). The solvent was
evaporated under reduced pressure to obtain the crude product, which
was dissolved in 8 mL of MeOH/H2O (3:1). Solid NaOH (0.100 g,
2.5 mmol) was then added at 0 °C, and after continuous stirring at
room temperature for 16 h, the volume was reduced to 4 mL by rotary
evaporation. The pH solution was adjusted to pH 3 using 1 M HCl
and the suspension cooled overnight at −10 °C to precipitate the
product, which was collected by filtration, washed with MeOH, and
1
848m, 760m, 743s cm−1. H NMR (300 MHz, CDCl3): mixture of
rotamers δ 9.74−9.71 (m, 1H), 9.49−9.26 (m, 4H), 8.08−7.86
(rotamers, m, 1H), 7.83−7.73 (m, 2H), 7.68−7.66 (m, 1H), 7.55−
7.46 (m, 3H), 7.33 (t, 3J = 7.4 Hz, 1H), 7.25−7.15 (rotamers, m, 3H),
3
4.33−4.30 (m, 2H), 4.12 (q, J = 7.4 Hz, 1H), 3.97−3.93 (rotamers,
m, 2H), 3.66−3.38 (m, 6H), 2.46−2.25 (m, 2H), 1.75−1.70 (m, 4H),
1.50−1.43 (m, 11H) ppm. 13C NMR (75 MHz, CDCl3): mixture of
rotamers δ 174.5, 173.8, 170.0 (min) and 169.1 (maj), 163.3 (min)
and 163.2 (maj), 156.9, 153.3 (min) and 153.2 (maj), 153.0, 152.9,
148.1, 148.0, 144.2 (maj) and 144.1 (min), 144.0, 143.9, 143.6, 142.5,
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air-dried to obtain 8 as a light brown solid. Yield: 0.110 g (43%). IR
(KBr): ν 3408m (N−H), 3285s (O−H), 3082w (C−Harom), 3064w
(C−Harom), 2928s (C−Haliph), 2860m (C−Haliph), 1718s (CO),
1689s (CO), 1638s, 1549m, 1461w, 1419m, 1405m, 1389m, 1377w,
1363m, 1218m, 1191m, 1169w, 1129w, 1072s, 1032m, 892m, 819m,
3425br (O−H), 3081m (C−Harom), 3067w (C−Harom), 2930m (C−
H
aliph), 2857w (C−Haliph), 1703s (CO), 1653s (CO), 1544w,
1446m, 1414m, 1390w, 1373w, 1244m, 1164m, 1121m, 1082w,
1
1051w, 1020w, 843vs, 763s, 741m, 730s cm−1. H NMR (400 MHz,
acetone-d6): mixture of rotamers δ 9.88−9.72 (rotamers, m, 1H), 9.54
(s, 1H), 9.52−9.47 (m, 1H), 9.02−8.96 (min) and 8.84−8.78 (maj)
(rotamers, m, 4H), 8.57−8.49 (m, 2H), 8.24−8.21 (m, 2H), 8.16−
8.14 (m, 4H), 8.08−7.98 (m, 5H), 7.727.70 (m, 1H), 7.63−7.48 (m,
5H), 7.42−7.37 (m, 2H), 7.30−7.17 (rotamers, m, 4H), 4.20−4.18
1
741s cm−1. H NMR (300 MHz, DMSO-d6): δ 9.44−9.39 (m, 2H),
9.18 (dd, 3J = 4.2 Hz, 4J = 1.5 Hz, 2H), 8.96 (t, 3J = 5.6 Hz, 1H), 8.76
3
(br s, 1H), 8.38−8.31 (m, 2H), 7.90−7.87 (m, 2H), 2.26 (t, J = 7.2
Hz, 2H), 1.66−1.56 (m, 4H), 1.451.39 (m, 2H) ppm. Two proton
signals are masked by residual water from DMSO-d6. 13C NMR (75
MHz, DMSO-d6): δ 175.0, 165.5, 153.0, 152.9, 148.4, 148.3, 142.9,
142.0, 141.8, 141.4, 136.8, 133.6, 133.3, 129.9, 129.6, 128.4, 127.1,
125.0, 124.9, 39.9, 34.2, 29.2, 26.6, 24.8 ppm. MS (ESI−): m/z 438.1
[M − H]−.
3
(m, 2H), 4.12 (q, J = 7.4 Hz, 1H), 4.06−3.98 (m, 2H), 3.59−3.30
(m, 6H), 2.41−2.25 (m, 2H), 1.60−1.56 (m, 4H), 1.41−1.33 (m, 2H)
ppm. MS (ESI+): m/z 562.4 [M]2+.
[Ru(phen)2(dpq-L-PNA−OH)](PF6)2·4H2O (M2). Complex M2 was
obtained as an orange solid employing the procedure described for
M1, using dpq-L-PNA (0.120 g, 0.16 mmol) and Ru(phen)2Cl2 (0.053
g, 0.10 mmol) in EtOH/H2O (1:1, 8 mL). Yield: 0.094 g (60%). Anal.
Calcd for C64H61F12N11O10P2Ru (%): C, 50.07; H, 4.00; N, 10.04.
Found: C, 50.02; H, 3.86; N, 10.20. IR (KBr): ν 3450br (O−H),
3085w (C−Harom), 3063w (C−Harom), 2928s (C−Haliph), 2856m (C−
tert-Butyl-2-(N-(2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-
ethyl)-6-(dipyrido[3,2-a:2′,3′-c]phenazine-11-carboxamido)-
hexanamido)acetate (dppz-L-PNA). 6-(Dipyrido[3,2-a:2′,3′-c]-
phenazine-11-carboxamido)hexanoic acid (8; 0.068 g, 0.150 mmol)
was suspended in dry DMF (5 mL) under nitrogen. HBTU (0.095 g,
0.250 mmol) and DMAP (0.020 g) were added to the suspension, and
the reaction mixture was stirred at room temperature for 30 min. tert-
Butyl-2-(2-(((9H-fluoren-9-yl)methoxy)carbonylamino)ethylamino)-
acetate hydrochloride (0.108 g, 0.250 mmol) was then added and
stirring continued for a further 15 min. The reaction mixture was
cooled to 0 °C, and diisopropylethylamine (40.0 μL, 0.250 mmol) was
then added dropwise. After stirring, the mixture at 0 °C for 30 min, it
was stirred at room temperature for a further 18 h. Water (10 mL) and
DCM (25 mL) were added and the mixture stirred for 2 h. The
organic layer was separated, and the aqueous layer was further
extracted with DCM (2 × 15 mL). The DCM layers were combined,
dried over Na2SO4, filtered, and concentrated to obtain the crude
product, which was purified by column chromatography (SiO2, eluent
polarity gradually changed from 100% EtOAc to 10% MeOH/DCM)
to afford dppz-L-PNA as a yellow solid. Yield: 0.101 g (80%). Rf =
0.40 in 10% MeOH/DCM. IR (KBr): ν 3068w (C−Harom), 3014w
(C−Harom), 2930s (C−Haliph), 2865m (C−Haliph), 1734s (CO),
1718s (CO), 1687m (CO), 1642s, 1539m, 1461m, 1407m,
1390w, 1365m, 1250m, 1155s, 1074m, 1035m, 847s, 760w, 741m
H
aliph), 1707m (CO), 1650s (CO), 1637s, 1542w, 1445m,
1428m, 1407m, 1227w, 1165m, 1056w, 841vs, 763m, 742m, 723s
cm−1. H NMR (400 MHz, acetone-d6): mixture of rotamers δ 9.79−
1
9.73 (rotamers, m, 1H), 9.659.53 (m, 1H), 8.82−8.75 (m, 4H),
8.628.51 (rotamers, m, 2H), 8.50−8.48 (m, 1H), 8.43−8.38 (m, 7H),
8.32−8.27 (m, 1H), 7.99−7.92 (m, 1H), 7.87−7.73 (m, 6H), 7.69−
7.45 (m, 4H), 7.34−7.21 (m, 2H), 7.19−7.17 (m, 2H), 4.27−4.24 (m,
3
2H), 4.15 (q, J = 7.4 Hz, 1H), 4.07−4.04 (m, 2H), 3.58−3.48 (m,
4H), 3.39−3.34 (maj) and 3.25−3.24 (min) (rotamers, m, 2H), 2.46−
2.43 (maj) and 2.33−2.30 (min) (rotamers, m, 2H), 1.73−1.64 (m,
4H), 1.51−1.42 (m, 2H) ppm. MS (ESI+): m/z 586.4 [M]2+.
[Ru(bpy)2(dppz-L-PNA−OH)](PF6)2·2.5H2O (M3). Complex M3
was synthesized by means analogous to those used for preparation
of M1, using dppz-L-PNA (0.082 g, 0.10 mmol) and Ru(bpy)2Cl2
(0.039 g, 0.08 mmol) in EtOH/H2O (1:1, 6 mL). The desired
complex was isolated as an orange solid. Yield: 0.075 g (62%). Anal.
Calcd for C64H60F12N11O8.5P2Ru (%): C, 50.90; H, 4.00; N, 10.20.
Found: C, 50.73; H, 3.81; N, 10.42. IR (KBr): ν 3425br (O−H),
3075w (C−Harom), 2931s (C−Haliph), 2862m (C−Haliph), 1720s (C
O), 1706s (CO), 1638m, 1535m, 1463m, 1447m, 1408w, 1358w,
1245m, 1184w, 1121w, 1079m, 1048w, 949w, 844vs, 763s, 741m,
1
cm−1. H NMR (400 MHz, DMSO-d6): mixture of rotamers δ 9.17−
9.06 (m, 4H), 8.88 (m, 1H), 8.57 (m, 1H), 8.28−8.26 (rotamers, m,
3
1H), 8.10 (t, J = 8.0 Hz, 1H), 7.83−7.74 (rotamers, m, 4H), 7.64−
1
730m cm−1. H NMR (400 MHz, acetone-d6): mixture of rotamers δ
7.58 (rotamers, m, 2H), 7.38−7.21 (m, 4H), 4.30−4.26 (m, 2H),
4.18−4.10 (m, 2H), 3.91 (m, 1H), 3.20−3.11 (rotamers, m, 2H),
2.41−2.21 (m, 2H), 1.66−1.59 (m, 4H), 1.44−1.39 (m, 11H) ppm.
9.64−9.61 (min) and 9.58−9.48 (maj) (rotamers, m, 2H), 8.86−8.80
(m, 4H), 8.58−8.50 (m, 3H), 8.26−8.21 (m, 4H), 8.18−8.12 (m, 6H),
8.05−7.98 (m, 2H), 7.71−7.61 (rotamers, m, 3H), 7.57−7.55 (m,
Four proton signals are masked by residual water from DMSO-d6. 13
C
NMR (100 MHz, DMSO-d6): mixture of rotamers δ 173.4 (min) and
173.0 (maj), 173.8, 169.6 (min) and 169.1 (maj), 165.4, 156.7 (maj)
and 156.6 (min), 152.8, 152.7, 148.0, 147.9, 144.3, 144.2, 143.0, 142.6,
141.5, 141.3, 141.1, 139.8, 137.8, 136.6, 133.5, 133.1, 129.7, 129.4,
129.3, 128.3, 128.0 (min) and 127.9 (maj), 127.7, 127.4, 126.9, 125.5,
125.4, 124.8, 121.8, 120.5, 120.4, 81.9 (min) and 81.0 (maj), 65.9,
51.2, 49.1 (maj) and 48.8 (min), 47.2, 39.9, 38.9, 32.6 (maj) and 32.2
(min), 29.3, 28.2 (maj) and 28.1 (min), 26.7, 24.8 ppm. MS (ESI+):
m/z 818.4 [M + H]+.
[Ru(bpy)2(dpq-L-PNA−OH)](PF6)2·1.5H2O (M1). dpq-L-PNA
(0.120 g, 0.16 mmol) was stirred in a 1:1 solution of TFA/CH2Cl2
(5 mL), at room temperature for 5 h. The solvent was evaporated
under reduced pressure to yield an oily residue, which was triturated
with toluene, CHCl3, and ether. The yellow solid obtained was
dissolved in degassed EtOH/H2O solution (1:1, 8 mL), Ru(bpy)2Cl2
(0.058 g, 0.12 mmol) added, and the solution refluxed under a N2
atmosphere for 4.5 h. The solvent was removed under reduced
pressure, and the residue was diluted with water (10 mL) and filtered.
An aqueous solution of HPF6 (60%) was then added dropwise to the
filtrate, until no further precipitation was observed. The orange
precipitate was collected by filtration, dissolved in acetonitrile,
reprecipitated by the addition of diethylether, filtered, and dried
under high vacuum conditions to give M1 as an orange powder. Yield:
0.111 g (66%). Anal. Calcd for C60H56F12N11O7.5P2Ru (%): C, 49.97;
H, 3.91; N, 10.68. Found: C, 49.89; H, 3.74; N, 10.76. IR (KBr): ν
3
2H), 7.53−7.49 (m, 2H), 7.47−7.42 (m, 2H), 7.27 (t, J = 7.4 Hz,
1H), 7.21−7.12 (rotamers, m, 3H), 4.30−4.22 (m, 2H), 4.21−4.15
3
(rotamers, m, 2H), 4.09 (q, J = 7.4 Hz, 1H), 3.61−3.55 (m, 2H),
3.43−3.38 (m, 2H), 3.30−3.25 (m, 2H), 2.49−2.31 (m, 2H), 1.66−
1.56 (m, 4H), 1.44−1.38 (m, 2H) ppm. m/z 587.7 [M]2+.
[Ru(phen)2(dppz-L-PNA−OH)](PF6)2·2.5H2O (M4). Using the
method described for the synthesis of M1, [Ru(phen)2(dppz-L-
PNA−OH)](PF6)2 was prepared from dppz-L-PNA (0.197 g, 0.26
mmol) and Ru(phen)2Cl2 (0.111 g, 0.21 mmol). This afforded M4 as
an orange powder. Yield: 0.174 g (55%). Anal. Calcd for
C68H60F12N11O8.5P2Ru (%): C, 52.41; H, 3.88; N, 9.89. Found: C,
52.37; H, 3.65; N, 9.90. IR (KBr): ν 3448br (O−H), 3065w (C−
H
arom), 2929s (C−Haliph), 2864m (C−Haliph), 1720s (CO), 1701s
(CO), 1651m (CO), 1560w, 1544m, 1445w, 1428m, 1410m,
1239w, 1145m, 1108w, 1079m, 1054w, 841vs, 775m, 762m, 742m,
723s cm−1. H NMR (300 MHz, CD3CN): mixture of rotamers δ
1
9.56−9.53 (m, 1H), 9.52−9.44 (m, 1H), 8.75−8.71 (m, 4H), 8.64−
8.58 (m, 1H), 8.43−8.36 (rotamers, m, 6H), 8.27−8.25 (m, 2H),
8.24−8.17 (m, 2H), 8.16−8.12 (m, 2H), 7.85−7.80 (m, 3H), 7.79−
7.76 (m, 2H), 7.75−7.66 (rotamers, m, 3H), 7.50−7.45 (rotamers, m,
3H), 7.32−7.20 (m, 1H), 7.17−7.10 (m, 3H), 4.30−4.21 (romaters,
m, 2H), 4.13−4.08 (m, 3H), 3.57−3.51 (m, 2H), 3.37−3.27 (m, 4H),
2.35−2.21 (rotamers, m, 2H), 1.73−1.59 (m, 4H), 1.49−1.40 (m, 2H)
ppm. MS (ESI+): m/z 611.7 [M]2+.
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a
Scheme 1. Synthesis of M1 and M2
a
Reagents and conditions: (a) ethyl-6-aminohexanoate hydrochloride, HOBt, DCC, DMAP, Et3N, dry THF, rt, 16 h, 75%. (b) TFA/CH2Cl2 (1:1),
rt, 5 h, 98%. (c) 1: 1,10-phenanthroline-5,6-dione, EtOH, Δ, 5 h. 2: NaOH, MeOH/H2O (3:1), 0 °C−rt, 16 h, 63%. (d) tert-butyl N-[2-(N-9-
fluorenylmethoxycarbonyl)-aminoethyl]glycinate, HBTU, Et3N, dry DMF, DIPEA, rt, 18 h, 73%; (e) 1: TFA/CH2Cl2 (1:1), rt, 5 h. 2: Ru(bpy)2Cl2,
EtOH/H2O (1:1), Δ, 4.5 h, 66%; (f) 1: TFA/CH2Cl2 (1:1), rt, 5 h. 2: Ru(phen)2Cl2, EtOH/H2O (1:1), Δ, 4.5 h, 60%.
PNA−monomer esters, dpq-L-PNA and dppz-L-PNA, and
RESULTS AND DISCUSSION
■
1
the intermediate derivatives were characterized using H and
Synthesis. The synthesis of the Ru(II)−PNA monomers
13C NMR spectroscopy, IR spectroscopy, and ESI-MS. In the
1H NMR spectra for 4 and 8, peak integration in the aromatic
region (7.00−9.00 ppm) corresponding to 8H and 10H,
respectively, along with the disappearance of upfield proton
signals between 4.00 and 4.20 ppm and 1.13−1.24 ppm
confirmed the hydrolysis of the ester. The IR spectrum of 4 and
8 showed two CO stretching vibrations between 1720 cm−1
and 1670 cm−1, assigned to amide and carboxylic acid
functionalities. [M − H]− peaks at m/z = 388.0 (4) and
438.1 (8) in the ESI-MS further confirmed the formation of the
products. The identity of the modified PNA monomer esters,
dpq-L-PNA and dppz-L-PNA, obtained in good yields (73−
M1, M2, M3, and M4, described in detail in Schemes 1 and 2,
followed a similar approach to that reported by Achim et al.21
involving the preparation of the bipyridine-modified PNAs,
which were then incorporated into Ru(II) complexes by
reaction with either [Ru(bpy)2Cl2] or [Ru(phen)2Cl2]. The
metal chelating scaffolds, dpq-L-PNA and dppz-L-PNA, were
first prepared by reacting the respective carboxamido
derivatives, 3 and 7, with 1,10-phenanthroline-5,6-dione, in
situ base mediated hydrolysis to form the dipyridoquinoxaline
(dpq) and dipyridophenazine (dppz) carboxylic acid deriva-
tives, 4 and 8, and their subsequent coupling to the protected
aminoethyl-glycine-based PNA monomer backbone. The
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a
Scheme 2. Synthesis of M3 and M4
a
Reagents and conditions: (a) di-tert-butyl dicarbonate, DIPEA, DMF, rt, 16 h, 56%. (b) ethyl-6-aminohexanoate hydrochloride, HOBt, DCC,
DMAP, Et3N, dry DMF, rt, 16 h, 76%. (c) TFA/CH2Cl2 (1:1), rt, 5 h, 98%. (d) 1: 1,10-phenanthroline-5,6-dione, EtOH, Δ, 5 h. 2: NaOH, MeOH/
H2O (3:1), 0 °C−rt, 16 h, 43%. (e) tert-butyl N-[2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]glycinate, HBTU, Et3N, dry DMF, DIPEA, rt, 18 h,
80%. (f) 1: TFA/CH2Cl2 (1:1), rt, 5 h. 2: Ru(bpy)2Cl2, EtOH/H2O (1:1), Δ, 4.5 h, 62%. (g) 1: TFA/CH2Cl2 (1:1), rt, 5 h. 2: Ru(phen)2Cl2,
EtOH/H2O (1:1), Δ, 4.5 h, 55%.
80%), was confirmed by ESI-MS, which showed peaks at m/z =
767.9 (dpq-L-PNA) and 818.4 (dppz-L-PNA) corresponding
to the [M + H]+ ions. The 1H NMR spectrum of the
monomers exhibited the expected signals due to the Fmoc and
the tert-butyl group in addition to resonances similar to those
found for the carboxylic acid precursors, 4 and 8. The IR
spectrum showed overlapping CO stretching vibrations due to
ester and amide functionalities (1655−1703 cm−1) as well as
aromatic C−H (3014−3068 cm−1) and aliphatic C−H (2856−
2930 cm−1) stretches.
The Ru(II)−bis(bipyridyl) complexes, M1 and M3, were
prepared by reaction of the boc-deprotected dpq-L-PNA and
dppz-L-PNA monomers with Ru(bpy)2Cl2 and isolated as
hexafluorophosphate salts. Similar reactions of dpq-L-PNA and
dppz-L-PNA with Ru(phen)2Cl2 gave the complexes M2 and
M4, respectively. The Ru(II)-based PNA monomers were
isolated as a rotameric mixture in reasonable yields (55−70%)
and in high purity with no further chromatographic purification
required. Signals at m/z values of 562.4 (M1), 586.4 (M2),
587.7 (M3), and 611.7 (M4) for the corresponding [M]2+ ion
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observed in ESI-MS and elemental analysis confirmed the
successful synthesis of the target complexes. Coordination of
the dipyridoquinoxaline and dipyridophenazine units to the
Table 1. Complexes M1−M4 are classical Ru(II) complexes of
the form [Ru(L)2L′]2+, where L and L′ are both polypyridyl
ligands. As for related complexes,37,42,48,67−69 the absorption
spectra for M1−M4 exhibit broad overlapping MLCT (metal
to ligand charge transfer) bands at ca. 450 nm due to dπ−π*
transitions from Ru(II) center into the mixed π* orbitals of the
ligands (L(π*) and L′(π*)). More intense absorption bands
ascribable to the intraligand π−π* transitions are observed
between 250 and 300 nm in each case. Characteristic π−π*
transitions localized on the dppz ligand are also in seen in the
spectra of M3 and M4 at 372 and 375 nm, respectively. The
energy and intensity of the associated transition bands depend
on ligand L′ (dppz or dpq), giving rise to subtle differences in
the absorption maxima for the phenanthroline complexes, M2
and M4, compared with the bipyridyl complexes, M1 and M3.
Photoluminescence Spectroscopy. Steady state emis-
sion spectra for 10 ± 1 μM acetonitrile solutions of M1−M4
were recorded with excitation of the MLCT band at 450 nm
(Figure 3). The profiles are consistent with the 3MLCT
1
Ru(II) center was established by comparison of the H NMR
spectra of the free ligand and complexes. The 1H NMR spectra
of complexes M1−M4 showed additional aromatic signals for
the introduced bipyridine and phenanthroline moieties. The
disappearance of the singlet between 1.50 and 1.39 ppm for the
tert-butyl CH3 groups confirmed the hydrolysis of the t-boc
functionality in the parent ligand. As expected, the IR spectrum
retained overlapping CO stretching vibrations for the carbonyl
stretching vibration in ester and amide functionalities (1650−
1703 cm−1) as well as aromatic C−H (3063−3085 cm−1) and
aliphatic C−H (2856−2930 cm−1) stretches. Thus, the
spectroscopic data indicate that the modular approach
employed to prepare M1−M4 successfully overcomes the
previously described problems of purity and isolation of similar
Ru(II)−polypyridyl complexes attached onto the PNA
monomeric backbone.37 In addition, the monomeric PNA
scaffolds, dpq-L-PNA and dppz-L-PNA, can be conveniently
used to prepare libraries of transition metal−PNA monomers
and oligomeric PNA scaffolds for transition metal ions.
Electronic Absorption Spectra. The UV−visible spectra
of M1−M4, measured in acetonitrile at (10 ± 1) μM, are as
shown in Figure 2, and the spectral data are summarized in
Figure 3. Photoluminescence emission spectra recorded on excitation
of 10 ± 1 μM acetonitrile solutions of M1−M4 at 450 nm.
Table 2. Summary of Emission Spectral Data Measured for
M1−M4 (10 ± 1 μM Solutions in Acetonitrile) Following
Excitation at 450 nm
complex
λmax (nm)
Is/Iref (CH3CN)
ΦR
M1
618
613
658
660
615
1.18
1.08
1.21
1.23
1.00
0.078
0.058
0.068
0.057
0.062
Figure 2. Absorption spectra for M1−M4 (10 ± 1 μM) measured in
acetonitrile.
M2
M3
M4
[Ru(bpy)3]2+ 68
Table 1. UV−Vis Spectroscopic Data Obtained for 10 ± 1
μM Acetonitrile Solutions of M1−M4
emission commonly observed in Ru(II) polypyridyl com-
plexes.42 As listed in Table 2, the Ru(II) dpq complexes, M1
and M2, exhibit emission maxima at 618 and 613 nm,
respectively, similar to the [Ru(bpy)3]2+ emission wavelength
(615 nm). On the contrary, dppz-based Ru(II)−PNA
monomers, M3 and M4, show a red shift in their emission
maxima to 658 and 660 nm, respectively. The integrated
emission intensities, measured as the total number of emitted
photons between 500 and 800 nm, showed that the dpq
complex, M1, has a similar emission efficiency to that of the
dppz derivatives (M3 and M4), while that for M2 was slightly
lower (Table 2). The differences in the emission maxima
indicate significant influence from the dpq and dppz ligands on
the HOMO and/or LUMO energy levels. The relative
λmax (nm)
[LC]
εmolar (M−1
λmax (nm)
[MLCT]
εmolar (M−1
complex
cm−1
)
cm−1
)
M1
258
283
262
298
254
284
372
264
280
375
53100
55400
97800
26700
46900
89300
15600
105000
77900
18200
421
453
443
10100
11700
14000
M2
M3
448
449
14300
17000
M4
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Inorganic Chemistry
Article
positioning of the emission maxima (M1 and M2 vs M3 and
M4) suggests low-lying LUMO levels in the dppz complexes,
relative to [Ru(bpy)3]2+ or the dpq complexes.
The emission quantum yields (ΦR) for M1−M4 (Table 2)
were determined assuming Φref for [Ru(bpy)]3+ to be 0.062
and using the equation:
compared with [Ru(bpy)3]2+. On the other hand, the red shifts
in emission observed for M3 and M4 may be rationalized by a
relatively greater stabilization of the dppz-localized π* LUMO
energy levels compared to the small stabilization of the HOMO
indicated by the positive shift in Em relative to [Ru(bpy)3]2+.
The very small changes in Em for the Ru2+/3+ couple observed
on changing dpq to dppz (M3−M1 = 26 mV, M4−M2 = 13
mV) suggests that the energy of the HOMO is relatively
unaffected by the introduction of the phenyl ring. This means
that the emission spectra for the two dppz complexes, M3 and
M4, are red-shifted as a result of stabilization of the LUMO.
However, this direct correlation between the photophysical and
electrochemical properties can only be validated by studying
the ligand-based reduction process for the HOMO−LUMO
gap. Unfortunately, the ligand-based reduction steps are not
simple reversible processes, so the required thermodynamic
data are not available.
The diffusion controlled nature of the oxidation process was
confirmed by the linear dependence of the oxidation peak
current (ipox) on the square root of the scan rate (ν). The
diffusion coefficients calculated from the slope of these linear
plots and use of the Randles−Sevcik relationship71−73 are M1
(1.89 ± 0.1) × 10−5 cm2 s−1; M2 (1.18 ± 0.1) × 10−5 cm2 s−1;
M3 (1.35 ± 0.1) × 10−5 cm2 s−1; and M4 (1.34 ± 0.1) × 10−5
cm2 s−1. The values are comparable to those reported for other
Ru(II) polypyridyl complexes as well as their PNA−monomer
derivatives.37,68
where Is and Iref are the integrated emission intensities
calculated from the area under the emission spectrum of the
sample and reference, respectively, and As and Aref are the
absorbances of the sample and reference, respectively.48,67
The quantum yield for M1 was slightly higher than for the
reference compound, [Ru(bpy)3]2+, whereas it was similar for
M2, M3, and M4. In general, the bis(bipyridyl) Ru(II)
complexes, M1 and M3, showed higher quantum yields than
the bis(phenanthroline) derivatives, M2 and M4. However, no
such differentiation based on ancillary ligands in the Ru(II)
coordination sphere could be made in the relative emission
intensities and the emission maxima for the complexes (vide
supra).
Electrochemistry. Cyclic voltammograms for the oxida-
tion of complexes M1−M4 recorded in acetonitrile at a scan
rate of 100 mV s−1 are presented in Figure 4.
Electrochemiluminescence. The ECL properties of
M1−M4 in acetonitrile with 0.1 M tripropylamine (TPA) as
a coreactant were investigated. To the best of our knowledge,
the potential of Ru(II)−PNA-like monomers as the basis for
ECL biosensors remains unexplored. No studies appear to have
been reported on the ECL of such compounds or, for that
matter, any other PNA-based conjugates. Low complex
concentrations and high coreactant levels were chosen for the
ECL experiments, which provide relevance to practical
biosensing applications of these Ru(II)−PNA-like monomers.
The measured ECL emission intensities were as illustrated in
Figure 5. Emission maxima for M1−M4 were 622, 616, 673,
and 675 nm, respectively. No ECL signal was detected in
similar experiments using only the PNA backbone itself,
indicating that it does not interfere with these measurements.
The oxidation−reduction mode of coreactant ECL for
[Ru(L)3]2+ and tripropylamine (TPA) is mechanistically
described in eqs 1−5:74−77
Figure 4. Cyclic voltammograms obtained at a glassy carbon electrode
using a scan rate of 100 mV s−1 for the oxidation of 1 mM Ru(II)
complexes in CH3CN (0.1 M nBu4NPF6).
All complexes exhibited a single essentially reversible
oxidation process associated with the RuII/RuIII couple. The
formal potentials (Ef° = Em) for the one electron oxidation
process, calculated over the scan rate range of 100−1000 mV
s−1 by averaging the oxidation and reduction peak potentials to
give the midpoint potential Em, are given in Table 3 (with
representative voltammograms shown in Figures S5−S8,
Supporting Information). The Ef° values determined for
M1−M4 were 962, 951, 936, and 938 mV, respectively, vs
Fc0/+. Relative to the Ef° values for the [Ru(bpy)3]2+/
[Ru(bpy)3]3+ system (888 mV vs Fc0/+),68 this indicates a
positive shift in potential for the Ru(II)−PNA monomers M1−
M4 of up to 74 mV. The electrochemical data in conjunction
with the emission profile of the complex can be used to probe
changes in molecular orbital levels. The small blue shift of the
emission maxima and a relatively positive reversible potential
indicate stabilization of the HOMO in M1 and M2 and
widening of the HOMO−LUMO gap for the emission process
(1)
(2)
(3)
(4)
(5)
However, it should be noted that the oxidation of the amine
described in reaction2 may also occur via homogeneous
reaction with [Ru(L)3]3+.74−76
Figure 5 compares the ECL spectra for the four complexes
with that of the [Ru(bpy)3]2+ standard. That the ECL emission
maxima (622, 616, 673, and 675 nm for M1−M4) were
12180
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Inorganic Chemistry
Article
Table 3. Cyclic Voltammetric Data Obtained As a Function of Scan Rate (ν) at a Glassy Carbon Electrode As Derived from
Oxidation of 1.0 mM CH3CN Solutions of M1−M4 in CH3CN (0.1 M nBu4NPF6) at (20 ± 2) °C
ab
,
ab
,
ab
,
ab
,
ac
red ,
ν (mV s−1
)
Ep (mV)
Epred(mV)
ΔEp (mV)
Em (mV)
|ipox/ip
|
ox
complex
M1
100
200
300
400
500
700
1000
100
200
300
400
500
700
1000
100
200
300
400
500
700
1000
100
200
300
400
500
700
1000
988
990
992
996
996
996
998
983
986
986
986
986
988
988
966
966
966
966
966
968
970
970
973
973
973
974
978
978
935
932
932
934
932
932
934
918
918
916
916
919
916
918
905
907
905
905
905
903
903
906
906
906
906
906
906
903
53
58
60
62
64
64
64
65
68
70
70
67
72
70
61
59
61
61
61
65
67
64
67
67
67
68
72
75
962
961
962
965
964
964
966
951
952
951
951
953
952
953
936
937
936
936
936
936
937
938
940
940
940
940
942
941
0.91
0.93
0.94
0.93
0.94
0.94
0.93
0.93
0.94
0.94
0.94
0.94
0.94
0.93
0.88
0.89
0.90
0.90
0.91
0.91
0.90
0.78
0.78
0.81
0.81
0.80
0.81
0.82
M2
M3
M4
a
Oxidation peak potential = Epox; reduction peak potential = Epred; midpoint potential = Em = (Epox + Epred)/2 versus Fc0/+; oxidation peak current =
b
c
ipox; reduction peak current = ipred. Peak potentials have an error of ±5 mV versus Fc0/+. The ratios of peak currents associated with the oxidation
and reduction peak currents were calculated according to the empirical method derived by Nicholson.70
integrated intensities for ECL generated from the PNA
monomers follow the order M1 (62%) ≥ M3 (60%) > M4
(46%) ≥ M2 (44%). The ECL intensities for the complexes are
only moderately lower than that of [Ru(bpy)3]2+, which may be
regarded as the benchmark ECL emitter. These data suggest
that the complexes are promising candidates for ECL-based
biosensing applications.
ECL intensities are governed by the ECL efficiency, ΦECL
(photons emitted per electron transferred in reaction 4 above),
which is strictly limited by the quantum yield, ΦR. Therefore, a
correlation might be expected between ECL intensity and
quantum yield. That such a correlation was not observed in this
study reflects the inherent complexity of ECL sys-
tems.47,48,51,74−77 Apart from ΦR, the ECL intensity may also
Figure 5. ECL spectra for Ru(II)−PNA monomers and [Ru(bpy)3]2+
be influenced by the oxidation potential of the complex (insofar
in acetonitrile (0.1 M nBu4NPF6). The ECL was generated using a 3
mm diameter glassy carbon electrode with 0.1 mM M1−M3 and
[Ru(bpy)3]2+ or 0.2 mM M4, containing 0.1 M TPA as a coreactant.
as it dictates the thermodynamics of reaction 4 above), or by
side reactions competing with reaction 1 or 4. Since the
oxidizing power of the complexes is approximately constant, the
most likely explanation for the observed trend is different
susceptibilities to parasitic side reactions between the
bipyridine- and phenanthroline-based complexes. This is in
agreement with published work that showed that phenanthro-
line-based ruthenium complexes were relatively unstable in the
oxidized form compared with [Ru(bpy)3]3+ 78 and is borne out
observed at slightly longer wavelengths compared with the
photoluminescence (PL) in each case may be simply explained
by considering the different detectors used, i.e., a PMT for PL
and a CCD for ECL. The ECL λmax data may be regarded as
closer to the true values because of the relatively small variation
in responsivity with wavelength associated with CCD
detectors.78 Under the conditions described above, the
red
(to an extent) in the ipox/ip data presented in Table 3.
12181
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Inorganic Chemistry
Article
(5) Good, L.; Nielsen, P. E. Antisense Nucleic Acid Drug Dev. 1997, 7,
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CONCLUSION
■
The synthesis of four new Ru(II)-containing PNA-like
monomers, [Ru(bpy)2(dpq-L-PNA−OH)]2+ (M1), [Ru-
(phen)2(dpq-L-PNA−OH)]2+ (M2), [Ru(bpy)2(dppz-L-
PNA−OH)]2+ (M3), and [Ru(phen)2(dppz-L-PNA−OH)]2+
(M4) has been achieved. This new approach for preparing such
Ru(II)−PNA monomers overcomes significant difficulties
encountered in previously described syntheses. In addition,
the key synthons, dpq-L-PNA and dppz-L-PNA, prepared en
route can either be used directly for preparing other transition
metal-PNA monomers for their subsequent incorporation at
any chosen position within the PNA oligomer sequence or be
employed for designing PNA templates to study metal
coordination effects on the stability of such inorganic−nucleic
acid duplexes, for nanotechnology applications. The UV/vis
absorbance and emission spectra showed profiles characteristic
of Ru(II) polypyridyl complexes, viz., a MLCT band centered
around 450 nm and distinctive emission maxima (610−660
nm). Electrochemical studies on M1−M4 showed them to
undergo a reversible one-electron RuII/RuIII oxidation process
at a positive potential of ca. 950 mV (vs Fc0/+). The
measurements showed the Ru(II)−PNA monomers to be
ECL-active luminophores. All of the monomers exhibited
reasonably intense ECL emission in the presence of a TPA
coreactant, with the bipyridine-based complexes M1 and M3
showing the highest ECL intensity. The data highlight the fact
that compounds having the highest photoluminescence
efficiency will not necessarily produce the most intense ECL
because other, non-photophysical parameters may come into
play. Overall, the photoluminescence and redox properties as
well as the efficient ECL signal obtained from the prepared
Ru(II)−PNA monomers identifies their scope and utility in
designing multimodal Ru(II)−PNA oligomeric labels for
biosensing applications.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra of M1−M4 (Figures S1−S4) and cyclic
voltammograms of M1−M4 as a function of scan rate (Figures
S5−S8). This material is available free of charge via the Internet
at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Fax: +61 3 9905 4597. E-mail: Leone.Spiccia@monash.edu.
■
Commun. 2008, 3675.
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ACKNOWLEDGMENTS
■
This work was supported by the Australian Research Council
through the Australian Centre of Excellence for Electro-
materials Science (L.S.). T.J. is a recipient of Monash Graduate
Scholarship and Monash International Postgraduate Research
Scholarship. PSF acknowledges funding from the Australian
Research Council (Future Fellowship) and Deakin University.
(38) Husken, N.; Gasser, G.; Koster, S. D.; Metzler-Nolte, N.
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