1
2
3
DNA Binding by Ru(II)-bis(bipyridine)-
4
Pteridinyl Complexes
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Shannon R. Dalton, Samantha Glazier, Belinda Leung, Sanda Win, Courtney Megatulski,
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Sharon J. Nieter Burgmayer 
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Shannon Dalton - Department of Chemistry, Bryn Mawr College, Bryn Mawr, PA 19010
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Samantha Glazier - Department of Chemistry, St. Lawrence University, Canton, NY 13617
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Belinda Leung- Department of Chemistry, Bryn Mawr College, Bryn Mawr, PA 19010
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Sanda Win- Department of Chemistry, Bryn Mawr College, Bryn Mawr, PA 19010
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Courtney Megatulski- Department of Chemistry, Bryn Mawr College, Bryn Mawr, PA 19010
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Sharon J. Nieter Burgmayer  - Department of Chemistry, Bryn Mawr College, Bryn Mawr,
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PA 19010, sburgmay@brynmawr.edu, FAX: 610-526-5086
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1
Abstract
2
The interactions of five bis(bipyridyl) Ru(II) complexes of pteridinyl-
3
phenanthroline ligands with calf thymus DNA have been studied. The pteridinyl
4
extensions were selected to provide H-bonding patterns complementary to the
5
purine and pyrimidine bases of DNA and RNA. The study includes three new
6
complexes [Ru(bpy)2(L-pterin)]2+, [Ru(bpy)2(L-amino)]2+ and [Ru(bpy)2(L-
7
diamino)]2+, two previously reported complexes [Ru(bpy)2(L-allox)]2+ and
8
[Ru(bpy)2(L-Me2allox)]2+, the well-known DNA intercalator [Ru(bpy)2(dppz)]2+
9
and the negative control [Ru(bpy)3]2+. Reported are the syntheses of the three
10
new Ru-pteridinyl complexes and the results of calf thymus DNA binding
11
experiments as probed by absorption and fluorescence spectroscopy, viscometry
12
and thermal denaturation titrations. All Ru pteridine complexes bind to DNA via
13
an intercalative mode of comparable strength. Two of these four complexes—
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[Ru(bpy)2(L-pterin)]2+ and [Ru(bpy)2(L-allox)]2+ —exhibit biphasic DNA melting
15
curves interpreted as reflecting exceptionally stable surface binding. Three new
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complexes—[Ru(bpy)2(L-diamino)]2+, [Ru(bpy)2(L-amino)]2 and [Ru(bpy)2(L-
17
pterin)]2+ —behave as DNA molecular “light switches”.
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Keywords
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Ruthenium, pterin, pteridine, , DNA binding, DNA intercalation, hydrogen bonding, viscometry
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1
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Introduction
Small molecule interactions with DNA continue to be intensely and widely studied for
3
their usefulness as probes of cellular replication and transcriptional regulation, and for their
4
potential as pharmaceuticals. [1-3] Intercalating molecules in particular have been targets of
5
many studies because intercalation distorts the helical shape of DNA, causing inhibition of
6
replication enzymes. In the burgeoning area of DNA intercalation studies, transition metal
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intercalators have been a rich source of experimental data for decades since their redox and
8
photophysical properties make it possible to utilize multiple techniques to study DNA
9
intercalation processes.[4-6] The great success of using Ru-polypyridyl complexes for probing
10
DNA binding is documented in a vast literature. [7-10] Among the many complexes studied, the
11
ruthenium(II)-bis(bipyridine) complex of dipyridophenazine, [Ru(bpy)2(dppz)]2+, was
12
demonstrated to have extraordinary properties, notably the ability to act as a “light switch” when
13
bound to DNA.[11] Although such results for [Ru(bpy)2(dppz)]2+ appeared nearly two decades
14
ago, this complex remains the subject of recent investigations which aim to clarify conditions
15
under which it demonstrates multiple binding modes. [12-14] Efforts to develop so-called
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“second generation” ligands based on the dipyridophenazine ligand (dppz) have been
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reported.[10, 15-18] Some studies have focused on the extension of the dppz pi-system to
18
promote tighter intercalative binding [17] and have added redox active quinone groups [15]
19
while others [18] have begun investigations into the effects of substituents on the electronic
20
structure and associated changes in binding strength.
21
Our experience with metal complexes of pteridines [19] prompted us to develop
22
syntheses of pteridine ligands derived from dppz and investigate how placing pteridinyl
23
appendages on transition metal complexes influences their interactions with DNA. The close
3
1
structural similarity between nucleic acids and certain pteridines, particularly between guanine
2
and pterin, suggested exploration of the consequences of pteridine-DNA interactions. Figure 1
3
illustrates the Watson-Crick C-G H-bonding motif mimicked by a C-pterin pair. This structural
4
similarity has been exploited by others through the construction of DNA with an abasic site and
5
the subsequent study of pterin binding in the vacant site. [20]
H
H
N
N
H
H
O
R
O
N
N
H
N
N
O
H N
H
6
N
N
R
C-G hydrogen bonding
R
N
N
H
N
N
O
H N
N
N
H
C-pterin hydrogen bonding
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Figure 1. Comparison of Watson Crick hydrogen bonding in a CG base pair and
8
between C and pterin.
9
10
We chose the bis(bipyridine)Ru(II) system on which to build pteridine extensions from a
11
phenanthroline chelate. Five different pteridinyl-phenanthroline ligands (L-pteridine) have been
12
synthesized from phenanthroline fused to pterin (L-pterin), 4-aminopteridine (L-amino), 2,4-
13
diaminopteridine (L-diamino), alloxazine (L-allox) and 1,3-dimethyalloxazine (L-Me2allox).
14
The different pteridine extensions were chosen for their potential to demonstrate varying H-
15
bonding interactions when their Ru(II) complexes interact with DNA. Four functional groups—
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pterin, alloxazine, aminopteridine and diaminopteridine — are capable of strong hydrogen
17
bonding analogous to those of the base pairs in DNA. The dimethyalloxazine was included in the
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group of new ligands to probe whether its two methyl groups precluding H-bonding interactions
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with DNA would result in lower affinity to DNA. These pteridinyl-phenanthroline ligands were
4
1
used to prepare five bis(bpy)-Ru complexes, where bpy is 2,2’-bipyridine, and these are shown
2
in Figure 2. [Ru(bpy)2(L-Me2allox)]2+ was first synthesized McGuire et al.[21] While our work
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was in progress, Gao et al. reported [22, 23] the synthesis and DNA binding characteristics of
4
[Ru(bpy)2(L-allox)]2+ and the previously reported complex [Ru(bpy)2(L-Me2allox)]2+.
N
N
N
N
N
NH2
N
NH
Ru
N
N
O
N
N
N
N
N
N
N
Ru
N
NH2
N
NH2
N
N
N
N
N
Ru
N
NH2
(b) [Ru(bpy)2(L-diamino)]2+
(a) [Ru(bpy)2(L-pterin)]2+
N
N
N
N
N
N
(c) [Ru(bpy)2(L-amino)]2+
N
N
N
N
N
N
5
N
N
NH
Ru
N
N
O
N
H
O
(d) [Ru(bpy)2(L-allox)]2+
O
N
N
N
N
N
Ru
N
N
N
CH3
O
CH3
(e) [Ru(bpy)2(L-Me2allox)]2+
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Figure 2. Shown are the five ruthenium pteridine complexes studied in this work: (a)
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[Ru(bpy)2(L-pterin)]2+, (b) [Ru(bpy)2(L-diamino)]2+, (c) [Ru(bpy)2(L-amino)]2+, (d)
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[Ru(bpy)2(Lallox)]2+, and (e) [Ru(bpy)2(L-Me2allox)]2+, all as hexafluorophosphate salts.
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Presented here are the synthesis of the three new complexes [Ru(bpy)2(L-pterin)](PF6)2 ,
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[Ru(bpy)2(L-amino)](PF6)2 and [Ru(bpy)2(L-diamino)](PF6)2 and binding studies of the five Ru-
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pteridine complexes depicted in Figure 2 to calf thymus DNA using the methods of absorption
13
and fluorescence spectroscopy, viscometry, and thermal denaturation experiments. Parallel
5
1
experiments with [Ru(bpy)2(dppz)](PF6)2 and [Ru(bpy)3](PF6)2 served as controls. This suite of
2
methods was used to differentiate between the DNA binding mechanisms of intercalation and
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surface or electrostatic binding. The combined data from these methods are consistent with the
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conclusion that all five Ru-pteridine complexes bind to DNA via an intercalative mode with
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similar binding strength. Biphasic behavior is observed in thermal denaturation experiments
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using [Ru(bpy)2(L-pterin)]2+ and [Ru(bpy)2(L-allox)]2+ and indicates the presence of additional
7
binding modes, such as surface or groove binding.
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Abbreviations used in this manuscript:
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L-amino = 4-amino-pteridino(6,7-f)phenanthroline
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L-diamino = 2,4-diamino-pteridino(6,7-f)phenanthroline
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L-pterin = 2-diamino-4(3H)-oxo-pteridino(6,7-f)phenanthroline
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L-allox = 2,4-diketo-pteridino(6,7-f)phenanthroline
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L- Me2allox = 1,3-dimethyl-2,4-diketo-pteridino(6,7-f)phenanthroline
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L-pteridine = any of the above five ligands
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Ru-dppz= [Ru(bpy)2(dppz)]2+
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EtBr = ethidium bromide
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CT DNA = calf thymus DNA
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Experimental Section
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Materials.
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Syntheses. Cis-dichloro-bis(bipyridyl)ruthenium(II) dihydrate was obtained from Alfa Aesar.
23
All other reagents were purchased from Sigma-Aldrich Co. Synthesis of 1,10-phenanthroline-
6
1
5,6-dione followed the procedure according to Yamada et al.[24] and synthesis of L- Me2allox
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and [(bpy)2RuII(L- Me2allox)] followed procedures of McGuire et al.[21] [Ru(bpy)2(dppz)](PF6)2
3
and [Ru(bpy)3](PF6)2 were gifts from Prof. Stefan Bernhard, Princeton University.
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Phenanthroline-alloxazine (L-allox). 1,10-phenanthroline-5,6-dione (0.833 g, 4.00 mmol) and
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5,6-diamino-2,4-dihydroxypyrimidine sulfate (0.654 g, 4.61 mmol) were combined in 90 mL
6
methanol and heated under reflux for two hours. The reaction solution was cooled then vacuum
7
filtered using a Buchner funnel. Yield 0.8402 g (66%). IR (KBr, cm-1): 1723, 1600, 1569. 1H-
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NMR (10% CF3CO2D in CDCl3-d6, ppm):  8.15 (m, 2H), 9.3 (d, 2H), 9.46 (d, 2H), 11.96
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(s, 1H), 12.42 (s, 1H). Anal. Calcd for C16H8N6O2 · 2H2O: C, 54.55; H, 3.43; N, 23.86. Found:
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C, 54.77; H, 2.76; N, 23.96.
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Phenanthroline-pterin (L-pterin). A solution of 6-hydroxy-2,4,5-triaminopyrimidine sulfate
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(0.383 g, 1.6 mmol) in 20 mL water was prepared by adding 2M NaOH dropwise until the pH
13
reached 10. This solution was added to a 20 mL solution of 1,10-phenanthroline-5,6-dione
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(0.544 g, 2.6 mmol) in methanol. The resulting brown reaction mixture was allowed to stir for
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approximately 20 hours at ambient temperature. The suspension was vacuum filtered and the
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dark yellow powdery solid washed in succession with 5mL water, methanol, and excess diethyl
17
ether. Yield 0.4126g (82.0%). IR (KBr, cm-1): 1702, 1643, 1575, 1540 cm-1. 1H-NMR (10%
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CF3CO2D in CDCl3-d6, ppm(Hz)): 7.07 (s, 1H), 8.15-8.20 (dd, J = 8.345, 8.31, 1H), 8.24-
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8.29 (dd, J = 8.41, 8.33, 1H), 9.24-9.27 (td, J = 4.69, 1.32, 2H), 9.64-9.68 (dd, J = 8.32, 1.29,
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1H), 9.96-10.00 (dd J= 8.29, 1.28, 1H). ESI-MS: m/z 316.1. Anal. Calcd for C16H9N7O.2.5
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H2O: C, 53.18; H, 4.18; N, 27.14. Found: C, 53.37; H, 3.68; N, 27.18.
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Phenanthroline-amino (L-amino). A solution of 4,5,6-triaminopyrimidine sulfate (0.2232 g,
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1.0mmol) in 20 mL water was prepared by adding 2M NaOH dropwise until the pH reached
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1
11.5. This solution was added to a 20 mL solution of 1,10-phenanthroline-5,6-dione (0.4202 g,
2
2.0 mmol) in methanol. The resulting red-brown reaction mixture was allowed to stir for
3
approximately 20 hours at ambient temperature. The suspension was vacuum filtered and the
4
bright yellow solid washed in succession with 5mL water, methanol, and excess diethyl ether and
5
dried under vacuum. Yield 0.2642g (88.1%). 1H- NMR (DMSO-d6, ppm):  9.90-9.87 (d, 1H),
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9.51-9.49 (d, 1H), 9.27-9.25 (d, 1H), 9.09 (s, 1H), 8.71 (s, 1H), 8.62 (s, 1H), 7.99-7.93 (m, 2H)
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Anal. Calcd for C16H9N7.H2O . 1.25 H2O: C, 59.72; H, 3.60; N, 30.48. Found: C, 59.68; H, 3.27;
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N, 30.24.
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Phenanthroline-2,4-diaminopteridine (L-diamino). This ligand was prepared similarly to L-
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pterin. A solution of 2,4,5,6-tetraaminopyrimidine sulfate (0.239 g, 1.0 mmol) in 20 mL water
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was prepared by adding 2M NaOH dropwise to pH 11.5. This solution was added to a 20 mL
12
solution of 1,10-phenanthroline-5,6-dione (0.427 g, 2.0 mmol) in methanol. The resulting
13
reaction mixture was allowed to stir for approximately 20 hours at ambient temperature. The
14
suspension was vacuum filtered and the bright yellow solid washed in succession with 5mL cold
15
water, methanol and excess diethyl ether. Yield 0.1921g (61%). IR (KBr, cm-1): 1638, 1584,
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1524, 1470 cm-1. 1H- NMR (DMSO-d6, ppm):  6.77-7.20(s, 2H), 7.85-7.92 (m, 2H), 8.06
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(s, 1H), 8.53 (s, 1H), 9.09-9.10 (dd, 1H), 9.19-9.20 (dd, 1H), 9.37-9.39 (dd, 1H), 9.72-
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9.74 (dd, 1H). ESI-MS: m/z 315.1. Anal. Calcd for C16H10N8.H2O,0.5CH3OH: C, 56.89; H, 4.05;
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N, 32.17. Found: C, 56.62; H, 3.69; N, 32.22.
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[(bpy)2RuII(L-amino)](PF6)2. L-amino (0.1795 g, 0.6 mmol) and Ru(bpy)2Cl2 (0.2422 g, 0.5
21
mmol) were combined in 16mL ethylene glycol and 4mL water. The dark purple reaction
22
mixture was refluxed under nitrogen at 120oC for five hours. The solution was cooled and 20mL
23
of water was added. The mixture was vacuum filtered and to the dark red filtrate, solid NH4PF6
8
1
was added until precipitation was complete. The suspension was vacuum filtered and the orange
2
solid washed in succession with 2mL cold water, cold absolute ethanol, chloroform and excess
3
diethyl ether. The resulting powder was dried under vacuum and further recrystallized from
4
acetonitrile-ether. Yield 0.5012g (99.6%). NMR (10% CF3CO2D in DMSO-d6, ppm) 7.37 (t,
5
2H), 7.61 (t, 2H), 7.74 (t, 2H), 7.81 (d, 2H), 8.10 (m, 5H), 8.25 (m, 4H), 8.86 (t, 5H), 9.12 (s,
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1H), 9.44-9.46 (dd, 1H), 9.93-9.96 (dd, 1H). Anal. Calcd for RuP2F12N11C36H25.2.5 H2O: C,
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41.27; H, 2.88; N, 14.71. Found: C, 41.54; H, 2.43; N, 14.35. ESI-MS: m/z 858 [M+(PF6)]+,
8
356.5 [M]2+.
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[(bpy)2RuII(L-allox)](PF6)2. L-allox (0.111 g, 0.35 mmol) and Ru(bpy)2Cl2 (0.146 g, 0.35mmol)
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were combined in 20 mL ethylene glycol. The dark purple reaction mixture was refluxed at 200o
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C for three hours. The solution was cooled and 20 mL of water was added. The mixture was
12
vacuum filtered to remove any insoluble impurities and solid NH4PF6 was added to the dark red
13
filtrate until product precipitation was complete. The orange product was vacuum filtered,
14
washed in succession with 2mL cold water, cold absolute ethanol, chloroform and excess diethyl
15
ether and dried under vacuum. Yield 0.2933g (95.9%). IR (KBr, cm-1): C=O 1720, 1603, C=N
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1577 cm-1. 1H-NMR (DMSO-d6, ppm): 7.36 (t, 2H), 7.58 (t, 2H), 7.71 (d, 2H), 7.82 (d, 2H), 8.00
17
(m, 2H), 8.09
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(t, 2H), 8.15 (m, 2 H), 8.29 (d, 2H), 8.85 (d, 4H), 9.22 (d, 1H), 9.35 (d, 1H), 12.07 (s, 1H), 12.55
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(s, 1H). Calcd for RuP2F12O2N10C36H24 · 2H2O: C, 40.95; H, 2.67. Found: C, 40.95; H, 3.21.
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ESI-MS: m/z 875 [M+(PF6)]+, 365 [M]2+.
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[(bpy)2RuII(L-pterin)](PF6)2. L-pterin (0.1892 g, 0.6 mmol) and Ru(bpy)2Cl2 (0.2423 g, 0.5
22
mmol) were combined in ethylene glycol (16 mL)and water (4 mL). The dark purple reaction
23
mixture was refluxed under nitrogen at 120oC for five hours. The solution was cooled and 20 mL
9
1
of water was added. The mixture was vacuum filtered and solid NH4PF6 was added to the dark
2
red filtrate until precipitation was complete. The suspension was vacuum filtered and the orange
3
solid washed in succession with 2mL cold water, cold absolute ethanol, chloroform and excess
4
diethyl ether. The resulting powder was dried under vacuum and further recrystallized from
5
acetonitrile-ether. Yield 0.4257 g (83.6%). IR (KBr, cm-1): 1705, . 1H-NMR NMR
6
(DMSO-d6, ppm): 7.35 (m, 2H), t, 2H), 7.73(t, 2H), 7.80(d, 2H), 7.93(m, 2H), 8.15(t, 3H),
7
8.22(t, 3H), 8.84 (t, 4H), 9.32(d, 1H), 9.39(d, 1H). ESI-MS: m/z 875 [M+(PF6)]+, 365 [M]2+.
8
Anal. Calcd for RuP2F12ON11C36H25.3.5 H2O: C, 39.97; H, 2.98; N, 14.24. Found: C, 40.24; H,
9
2.91; N, 14.17.
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[(bpy)2RuII(L-diamino)](PF6)2. Preparation of this compound was similar to that for
11
[(bpy)2RuII(L-pterin)](PF6)2. Using L-diamino (0.1886 g, 0.6 mmol) and Ru(bpy)2Cl2 (0.2427 g,
12
0.5 mmol) , 0.5076g of an orange solid was produced (99.5%). IR (KBr, cm-1): 
13
cm-1. 1H-NMR (DMSO-d6, ppm): 7.34-7.38 (t, 3H),t, 2H), 7.69-7.73(t, 2H), 7.81 (d,
14
2H), 7.91-7.98(m, 2H), 8.04-8.14 (m, 3H), 8.82-8.88 (t, 4H), 9.39-9.42 (d, 1H), 9.79-9.81 (d,
15
1H). ESI-MS: m/z 874 [M+(PF6)]+, 364 [M]2+. Anal. Calcd for RuP2F12N12C36H26.4 H2O: C,
16
39.68; H, 3.15; N, 15.42. Found: C, 39.78; H, 2.67; N, 15.04.
17
Methods.
18
Instrumentation. 1H NMR spectra were taken on a Bruker 300 MHz FT-NMR Spectrometer.
19
Chemical shifts were reported in parts per million (ppm) from a standard internal
20
tetramethylsilane (TMS) reference. Fourier transform infrared (FTIR) spectra were obtained on
21
Perkin-Elmer Model 2000 FT-IR Spectrometer from samples that were prepared as KBr pellets.
22
All electronic absorption spectra were taken in a quartz crystal cuvette with an Aligent
23
Technologies UV/Vis Diode Array Spectrophotometer or a Perkin-Elmer Lambda Bio
10
1
UV/Visible spectrophotometer. Fluorescence emission spectra were measured on a SPEX
2
Fluoromax-3. ESI-MS data were obtained using an Agilent LS-MS instrument at Haverford
3
College, Haverford PA. Samples for mass spectral analysis were dissolved in acetonitrile under
4
aerobic conditions and directly injected, bypassing the liquid chromatography column.
5
Solutions. All aqueous solutions were prepared in buffers using sterile distilled water, with the
6
exception of the Ru complex solutions and phen-ligand solutions for the viscosity and
7
fluorescence titrations. Ru solutions for viscosity titrations were prepared in acetonitrile while
8
DMF was used to dissolve the free L-pteridine ligands. Ru solutions were stored in the dark in
9
order to prevent photodegradation. Double stranded calf thymus DNA was purchased from
10
Sigma-Aldrich Co. Solutions of CT DNA used for absorbance, fluorescence and viscometry
11
were made with 10 mM phosphate buffer and 50 mM NaCl at pH 7. Thermal denaturation
12
experiments used 1 mM phosphate buffer and 2 mM NaCl at pH 7 for CT DNA solutions. In a
13
typical preparation, DNA (Type 1 fibers) was added to a 10 mM phosphate buffer and 50 mM
14
NaCl then sonicated for 0.5 to 1.5 hours. The solution was vortexed, centrifuged for 12 minutes,
15
and the supernatant was vortexed again. DNA concentrations were determined by spectroscopy
16
employing the following extinction coefficients at 260 nm: CT DNA: 6,600 M-1cm-1(per
17
nucleotide), 13,100 M-1cm-1(per base pair) [25]. DNA concentrations used in the following
18
experiments are per base pair (bp) unless otherwise noted.
19
Viscometry. An Ostwald viscometer in a non-circulating water bath at 24-25 oC was used to
20
measure the relative viscosity of DNA solutions in the presence of EtBr or Ru complexes. The
21
concentrations of the Ru complex and CT DNA were chosen to minimize the volume of Ru
22
complex added to a solution of DNA concentrated enough to make changes in the slope
23
maximally distinguishable. A 2.5 mM stock solution of each Ru complex was prepared in
11
1
acetonitrile due to its limited solubility in aqueous buffer. A 0.30 mM (bp) solution of CT DNA
2
was titrated with Ru complex over the range 0 – 0.2 [Ru] / [DNA]. Upon each addition of Ru
3
complex, the solution in the viscometer was bubbled with nitrogen gas to aid mixing. The
4
solution was then drawn up through the capillary portion of the viscometer using a pipette bulb,
5
and a stopwatch was used to time the downward flow of the solution. In accordance with the
6
theory of Cohen and Eisenberg,[26] the resulting data were plotted as intrinsic viscosity, (η/ηo)1/3
7
versus the ratio of complex to DNA. The ratio (η/ηo)1/3 was calculated based on equation 1,
8
where is the viscosity of DNA in the presence of the complex, tf is the flow time of the
9
experimental trial in seconds, to is the flow time of buffer in seconds, and  o is the viscosity of
10
11
DNA solution.

t f  to
to
(1)
12
Thermal Denaturation. Studies were carried out using a Beckman-Coulter DU800
13
spectrophotometer equipped with a Peltier temperature-controlling programmer. The samples
14
were all prepared using 1 mM phosphate buffer with 2 mM NaCl at pH 7, and contained in
15
Beckman Tm stoppered cuvettes. The absorbance at 260 nm was continuously monitored for
16
solutions of CT-DNA (100 M per nucleotide) in the presence and absence of the ruthenium
17
compounds (6.7 M). The temperature of the samples was increased by 5 oC min-1 from 25o to
18
45o, and 1 oC min-1 from 45o to 94o. Data points were collected every 5 oC min-1 from 25o to 45o,
19
and 1 oC min-1 from 45o to 94o. The Tm was obtained by fitting the raw data to a sigmoidal
20
function using SigmaPlot 10.0 then assigning the inflection point to Tm. All fitted data plots
21
presented have R2 values of greater than 0.99.
12
1
Isothermal Binding Titrations. The absorption titrations were performed at room temperature
2
using a constant concentration of ruthenium complex in each sample, and increasing the
3
concentration of CT DNA. The samples were all prepared using 10 mM phosphate buffer with
4
50 mM NaCl at pH 7. The concentration of each metal compound was between 12.5 and 23 M
5
and CT DNA was added until the [DNA]/[Ru] ratio was in the range 6-9, depending on
6
compound. Samples were incubated at room temperature for 10 minutes before absorption
7
spectra were recorded. The binding constant K and the site size, s, were extracted from the
8
absorption data fitted [27] to equations 2 and 3:
9
10
(a – f)/(b – f) = (b - (b2 – 2K2C[DNA]/s))1/2/2KC
(2)
b = 1 + KC + K[DNA]/2s
(3)
11
where C is the constant total concentration of Ru species, [DNA] is the total concentration of
12
added DNA as M base pairs, a is the apparent absorption in the presence of DNA, f is the
13
extinction coefficient of free Ru complex in the buffer and b is the extinction coefficient of the
14
DNA-bound Ru complex. The value of f was obtained from a Beer’s plot of the Ru complex
15
while the value of b was obtained from the absorbance of a saturated Ru-DNA sample divided
16
by the concentration C.
17
Fluorescence binding titrations were performed at room temperature under the same
18
buffer conditions as the absorption titrations. A 10 M of Ru solution was titrated with DNA
19
from 0 to 120 M. The excitation wavelength for Ru complexes was at the MLCT absorption
20
maximum, typically around 450 nm, resulting in peak emission intensity around 600 nm. The
21
data were fit using equations (2) and (3) to calculate Kb and s for each complex with the
22
modification that fluorescence intensity, I, replaced the  values used for the absorption
23
titrations.
13
1
X-Ray Crystal Determination of [(bpy)2RuII(L-Me2allox)](PF6)2. Crystals of [(bpy)2RuII(L-
2
Me2allox)](PF6)2 were growh from diethyl ether vapor diffusion into an acetonitrile solution of
3
the Ru complex forming bright orange plates. C42H34N12P2O2F12Ru, crystallizes in the triclinic
4
_
space group P1 with a=11.7468(8)Å, b=13.7747(7)Å, c=15.0494(12)Å, =78.043(7)°,
5
=85.315(8)°, =68.603(5)°, V=2218.0(3) Å3, Z=2 and dcalc=1.692 g/cm3. X-ray intensity data
6
were collected on a Rigaku Mercury CCD area detector employing graphite-monochromated
7
Mo-K radiation (=0.71073 Å) at a temperature of 143K. Preliminary indexing was performed
8
from a series of twelve 0.5° rotation images with exposures of 30 seconds. A total of 598 rotation
9
images were collected with a crystal to detector distance of 35 mm, a 2swing angle of -12°,
10
rotation widths of 0.5° and exposures of 75 seconds: scan no. 1 was a -scan from 157.5° to
11
367.5° at  = 10° and  = 20°; scan no. 2 an -scan from -20° to 5° at  = -90° and  = 315°;
12
scan no. 3 was an -scan from -20° to 4° at  = -90° and  = 135°; scan no. 4 was an -scan
13
from -20° to 20° at  = -90° and  = 225°. Rotation images were processed using CrystalClear
14
[28], producing a listing of unaveraged F 2 and (F 2) values which were then passed to the
15
CrystalStructure [28] program package for further processing and structure solution on a Dell
16
Pentium III computer. A total of 17270 reflections were measured over the ranges 5.2 250
17
°, -12 h 13, -16 k 16, -17 l 17 yielding 7728 unique reflections (Rint = 0.0586). The
18
intensity data were corrected for Lorentz and polarization effects and for absorption using
19
REQAB (minimum and maximum transmission 0.779, 1.000).
20
The structure was solved by direct methods (SIR97). Refinement was by full-matrix least
21
squares based on F 2 using SHELXL-97. All reflections were used during refinement (F 2 ’s that
22
were experimentally negative were replaced by F 2 = 0). The weighting scheme used was
14
1
w=1/[2(F 2o )+ 0.0594P 2 + 12.1843P] where P = (F 2o + 2F 2c )/3 . Non-hydrogen atoms were
2
refined anisotropically and hydrogen atoms were refined using a “riding” model. Refinement
3
converged to R1=0.0722 and wR2=0.1514 for 5440 reflections for which F > 4(F) and
4
R1=0.1111, wR2=0.1806 and GOF = 1.033 for all 7728 unique, non-zero reflections and 641
5
variables. The maximum / in the final cycle of least squares was 0.000 and the two most
6
prominent peaks in the final difference Fourier were +1.229 and -0.815 e/Å3.
7
Crystallographic data (without structure factors) for the structure reported in this paper have been
8
deposited with the Cambridge Crystallographic Data Centre as supplementary publication no
9
CCDC 678380. Copies of the data can be obtained free of charge from the CCDC (12 Union
10
Road, Cambridge CB2 1EZ, UK; tel (+44) 1223-336-408; fax (+44) 1223-336-003; e-mail:
11
deposit@ccdc.cam.acc.uk.
12
Results
13
Syntheses and characterization of ruthenium pteridine complexes. All phenantholine-
14
pteridinyl ligands are synthesized via a condensation reaction of 1,10-phenanthroline–5,6-dione
15
with the appropriate diaminopyrimidine. The ligands where phenanthroline is fused to 4-
16
aminopteridine (L-diamino), 2,4-diaminopteridine (L-diamino) [29] and to pterin (L-pterin) are
17
prepared in 60-90% yields. Bis-bipyridine-Ru(II) complexes of these ligands were prepared by
18
refluxing Ru(II)(bpy)2Cl2 and one equivalent of ligand in ethylene glycol for five hours followed
19
by addition of excess ammonium hexafluorophosphate to precipitate the complexes as di-
20
hexafluorophosphate salts (Fig. 3).
21
15
1
2
Figure 3. A representative synthetic route to pterinyl-phenanthroline ligands and their
3
Ru(II) complexes as illustrated for (L-pterin) and [Ru(bpy)2(L-pterin)]2+.
4
5
All ruthenium L-pteridine complexes used in this study were characterized by ESI-MS,
6
1
7
complexes, both the cation [M](PF6)+ and the dication [M]2+ could be observed.
8
Crystal Structure Determination of [Ru(bpy)2(L-Me2allox)](PF6)2. Crystal structures for all
9
of the Ru-pteridine complexes are desired to compare intermolecular H-bonding interactions.
10
Thus far only two complexes, Ru-L-Me2allox and Ru-L-diamino, have crystallized. The tiny
11
needle-shaped crystals of Ru-L-diamino forms clusters of tiny crystals too small for diffraction
12
data collection. A small orange plate of Ru-L-Me2allox was successfully used for a single crystal
13
_
X-ray determination. The compound crystallized in the triclinic group P1 from vapor diffusion
14
of diethyl ether into an acetonitrile solution of the complex. The unit cell contains two Ru
15
complexes, four PF6- counterions, and two molecules of acetonitrile. An ORTEP drawing of the
16
Ru-L-Me2allox cation is shown in Figure 4A while a view of the unit cell is shown in Figure 4B.
H NMR, FT-IR and electronic spectroscopy. In the ESI-MS spectra of all ruthenium
16
1
All six Ru-N bonds fall within a narrow range of 2.054(5) to 2.064(5) Å, the same range reported
2
for [Ru(bpy)3]2+ [30], and likewise the N-Ru-N bond angles within the bpy and L-Me2allox
3
4
5
Figure 4. (A) ORTEP drawing of the cation in [Ru(bpy)2(L-Me2allox)](PF6)2 with 20%
6
probability thermal ellipsoids. (B) The unit cell illustrating the coplanar L-Me2allox
7
ligands of symmetry-related molecules, the acetonitrile molecules aligned in the plane of
8
L-Me2allox and the packing of the hexafluorophosphate anions.
9
10
chelates are identical within a one degree (range of 78.9(2) to 79.6(2) o). All O, N, and C atoms
11
within the L-Me2allox ligand are coplanar with deviations ≤ 0.1 Å except for the largest
12
deviation of 0.25 Å for methyl C24 and 0.42 Å for carbonyl O2. Selected bond distances and
13
angles within the inner coordination sphere of ruthenium are given in [31] and a complete listing
17
1
is available in the electronic supplemental material (ESM). One molecule of acetonitrile lies
2
coplanar to the L-Me2allox ligand at a distance of 3.8 Å such that its C=N bond roughly bisects
3
the N11-C24 bond axis. The second acetonitrile is coplanar to one bpy ligand also at a distance
4
of 3.8 Å such that its methyl group sits about the centroid of the pyridine ring formed by N36-
5
C31-C35. The two cations of [Ru(bpy)2(L-Me2allox)]2+ pack within the unit cell so that the L-
6
Me2allox ligands are coplanar at a distance of 3.5 ± 0.1 Å where the centroids of the middle
7
pyrazine ring offset by 6.6Å.
8
Electronic Spectroscopy. The electronic absorption and luminescent properties of the five Ru-
9
pteridine complexes are similar to the parent complex Ru-dppz but not identical. An overlay of
10
the electronic spectra for the five Ru-L-pteridine complexes included in this study is shown in
11
Figure 5A. Previous spectral analysis of Ru-dppz has assigned the broad asymmetric low energy
12
absorption at 450 nm to overlapping MLCT transitions between a Ru-based HOMO and bpy-
13
and dppz- LUMO orbitals. [32, 33] The two narrower bands centered at 360nm are assigned to
14
overlapping -* transitions localized on the dppz ligand while the strong high energy
15
absorption at 270 nm comprises overlapping -* transitions from both bpy and dppz ligands.
16
Of the five Ru-L-pteridine complexes, the spectrum of Ru-L-Me2allox is most similar to that of
17
Ru-dppz in band shape with the exception of its two equal intensity absorptions between 350-400
18
nm that are shifted ~10 nm to lower energy. Since these have been assigned to -* transitions
19
localized on the dppz portion of the ligand, it appears the additional heteroatoms of L- Me2allox
20
cause a decrease in the energy difference between the orbitals. The Ru complex of L-allox has
21
transitions at similar energies to Ru-dppz, but the band shapes are distinctly altered. In particular
22
the ligand centered -* absorptions are less resolved while the MLCT band is both more
23
asymmetric and better resolved with higher absorptivity on the low energy side.
18
1
2
3
Figure 5. Electronic absorption spectra for all Ru complexes at 298 K in 10 mM
4
phosphate buffer (A) at pH 7; (B) over the pH range 1-13 for [Ru(bpy)2(L-pterin)]2+ and
5
(C) [Ru(bpy)2(L-diamino)]2+
6
7
This suggests the lower energy transition may be from the Ru to the pteridine ligand while the
8
higher energy shoulder represents the Ru to bpy ligand transition. The Ru complexes of L-pterin
9
and L-diamino are significantly different from the other complexes in the series. The most
19
1
prominent difference is the lack of absorptions for the -* transitions between 350 -400 nm.
2
They both show a broad absorption in the MLCT region.
3
4
Table 1. Extinction Coefficients for Ru-L-pteridine complexes in pH 7 phosphate buffer
Compound in buffer pH 7
Ru(bpy)2dppz
Ru(bpy)2(L-allox)
Ru(bpy)2(L-Me2allox)
Ru(bpy)2(L-pterin)
Ru(bpy)2(L-amino)
Ru(bpy)2(L-diamino)

 (M-1cm-1)
356
17973
369
17879
440
16200
382
15200
425
17500
450
18000
373
17432
386
17600
445
14700
380
16800
420
24317
440
23074
455
21900
410
17100
445
14800
427
22251
463
17556
5
20
1
Pteridine molecules can be amphoteric, so the pteridine portion of the L-pteridine ligands
2
in these complexes can participate in acid/base reactions in aqueous buffer solution.[34] Figures
3
5B and 5C shows the change in the electronic absorption spectra of [Ru(bpy)2(L-pterin)]2+ and
4
[Ru(bpy)2(L-diamino)]2+ in phosphate buffer over the pH range 1-13. Under the conditions of
5
the DNA binding experiments at pH 7, the spectrum of [Ru(bpy)2(L-diamino)]2+ is essentially
6
the same as spectra recorded at pH 10-13 and indicates the L-diamino ligand is unprotonated. It
7
is intriguing that in acidic solution the spectrum of Ru-L-diamino is nearly identical with that for
8
Ru-L-Me2allox and more similar to that for Ru-dppz. The spectrum of [Ru(bpy)2(L-pterin)]2+ at
9
pH 7 is intermediate between its spectrum at pH 13 and at pH 1 suggesting a distribution of
10
protonated, neutral and deprotonated L-pterin ligands on the Ru. The spectral changes of
11
[Ru(bpy)2(L-pterin)]2+ were compared with the spectral changes exhibited by the uncoordinated
12
ligand L-pterin over the pH range 1-14 (data not shown). Both the uncoordinated ligand L-pterin
13
and [Ru(bpy)2(L-pterin)]2+ exhibit a bathochromic shift of the low energy absorption that is due
14
to pterin deprotonation above pH 13. From this we conclude that the L-pterin ligand within the
15
[Ru(bpy)2(L-pterin)]2+ is largely in its neutral form under the conditions of DNA binding studies
16
at pH 7 and is only deprotonated under highly basic conditions ~pH 14.
17
Ruthenium polypyridyl complexes in acetonitrile solution typically show emission near
18
600 nm when excited at ~400 nm whereas when the complexes are dissolved in aqueous solution
19
emission is partly or fully quenched by solvent interactions with water. Intercalation into DNA
20
by such Ru complexes results in greatly enhanced emission. Ru-dppz in particular is the
21
prototype “molecular light switch” complex with quenched emission in buffer (i.e. the “off”
22
state), but a strong emission (i.e. the “on”state) when DNA is added because intercalation of the
23
dppz ligand isolates it from interactions with water. The five Ru-L-pteridine complexes studied
21
1
in this work show fluorescent emission between 602 - 608 nm when excited at ~450 nm in
2
acetonitrile solution (data not shown) and all five Ru-L-pteridine complexes have enhanced
3
emission in the presence of DNA in phosphate buffer. Three Ru-L-pteridine complexes, Ru-L-
4
amino, Ru-L-diamino and Ru-L-pterin, show “light switch“ type behavior where they exhibit no
5
visible fluorescence in buffer but fluoresce brightly after DNA is added. Relative emission
6
enhancement is listed in Table 3 as a summary of the ratio Ib / If of fluorescence intensities Ib of
7
bound Ru (Ru complex + DNA) to fluorescence intensities If of free Ru complex for all the Ru –
8
L-pteridine complexes in addition to Ru-dppz. Fig. 6 illustrates the emission enhancement
9
observed for Ru-L-amino, the Ru complex exhibiting the largest increase in emission intensity.
10
We note that Ru-L-allox [22], but not Ru-L-Me2allox [23], has been previously reported to show
11
“light switch “ behavior in Tris buffer, but enhanced “light switch” emission for Ru-L-allox was
12
not observed in our experiments.
13
14
Figure 6. Emission spectra for [Ru(bpy)2(L-amino)]2+illustrating the enhanced emission,
15
or “light switch” behavior, when the Ru complex is in the presence of DNA. (A) free
22
1
complex at a concentration of 10 M; (B) in the presence of DNA at a ratio of DNA:Ru =
2
12 where all Ru is bound. The emission intensity increases by a factor of 15.
3
4
5
DNA Binding Studies
Fluorescence spectroscopy provided the initial evidence of possible intercalation
6
interactions between calf thymus DNA and the [Ru(bpy)2(L-pteridine)]2+ complexes.
7
Viscometry and thermal denaturation experiments were done to further probe the nature of DNA
8
interaction and differences between the Ru(bpy)2(L-pteridine) complexes. Quantitation of the
9
strength of DNA binding was obtained by absorption and fluorescence titrations to evaluate Kb.
10
Viscometry. The measurement of viscosity change as molecules intercalate into DNA is viewed
11
as one of the best indications of intercalative binding.[35] Titrations of DNA by intercalating
12
molecules will show increased viscosity due to DNA lengthening in the presence of an
13
intercalating complex and this is commonly presented as a plot of (/0)1/3 vs. the ratio of
14
intercalator to DNA. [15, 35, 36] Figure 7 shows the plots of viscosity titration results for the
15
five Ru-L-pteridine complexes in addition to the their parent complex Ru-dppz, the positive
16
control ethidium bromide [37-39] and the negative control [Ru(bpy)3]2+. Positive slopes are
17
observed for all the Ru complexes with values only slightly less than that of the intercalator
18
ethidium bromide whereas the known non-intercalator [Ru(bpy)3]2+ produces a zero slope line.
19
These results are strong evidence supporting DNA intercalation by all the Ru-L-pteridine
20
complexes. The similarity in the viscosity plot slopes of the Ru-L-pteridine complexes to that of
21
Ru-dppz suggests they have comparable binding strength to that documented for Ru-dppz by
22
viscometry. [40-41] No change in viscosity (results not shown) was observed in titrations of each
23
1
of the five uncomplexed L-pteridine ligands with CT DNA indicating an absence of significant
2
binding by the pteridine ligands.
3
4
Figure 7. Viscosity titration of CT DNA and each of the five Ru-L-pteridine
5
complexes, the negative control [Ru(bpy)3]2+ and positive controls [Ru(bpy)2(dppz)]2+
6
and EtBr. Conditions: 10 mM phosphate buffer pH 7, 50 mM NaCl, [Ru] 2.5 mM. 
7
Ru-dppz, ▲ Ru-L-pterin,  Ru-L-diamino,  Ru-L-Me2allox,  Ru-L-allox, X
8
[Ru(bpy)3]2+,  Ru-L-amino, ▲ ethidium bromide. 
9
10
Viscosity titrations were repeated under conditions of high (200 mM NaCl) and low (2
11
mM NaCl) salt to evaluate the effect of ionic strength on intercalation as indicated by changes in
12
viscosity plot slopes. Fig. 8 summarizes the results of the viscosity titration plots under variable
13
salt concentrations. When the salt concentration is decreased to 2 mM, all the slopes increase,
14
except for the Ru-L-diamino and Ru-L-amino complexes. The increase is greatest for Ru-L-
15
Me2allox, Ru-L-allox and Ru-L-pterin. When the salt concentration is increased to 200 mM, the
24
1
slopes all decrease for the complexes having nucleic acid-like H-bonding substituents. The
2
complexes Ru-L-Me2allox, Ru-L-allox and Ru-L-pterin again show the greatest decrease.
3
4
Figure 8. Comparison of viscosity titration slopes under conditions of high (200 mM
5
NaCl), medium (50 mM NaCl) and low (2 mM NaCl) salt in phosphate buffer. Error bars
6
signify deviation from calculated average slope for multiple data sets.
7
8
Thermal Denaturation. Among the effects of intercalating molecules bound to the DNA helix
9
is a stiffening of the helix, increasing its rigidity and stabilizing the double helix. When
10
increasing concentrations of an intercalating molecule are added, this added stability effect
11
causes an increase in the temperature needed to induce duplex dissociation, called the melting
12
temperature, Tm. Melting curves at a constant ratio [DNA]:[Ru] of 15 in pH 7.1, 1 mM
13
phosphate buffer and 2 mM NaCl are shown in Figure 9. The melting points extracted from
14
these curves based on triplicate trials are listed in Table 2. All of the Ru-pteridine complexes
15
increased the melting temperature of CT DNA and these results are consistent with intercalation
16
by the [Ru-L-pterin]2+, [Ru-L-amino]2+ and [Ru-L-diamino]2+ complexes and duplicate the
25
1
previous results for Ru-L-allox and Ru-L-Me2allox . Based on the Tm values in Table 2, the
2
apparent relative binding strength can be estimated as follows: [Ru(bpy)2(L-amino)]2+ ~
3
[Ru(bpy)2(L-diamino)]2+ ~ [Ru(bpy)2(L-Me2allox)]2+ > [Ru(bpy)2(L-pterin)]2+ ~
4
[Ru(bpy)2(dppz)]2+ > [Ru(bpy)2(allox)]2+, a series largely consonant with equilibrium binding
5
constants, Kb (below).
The melting curve for [Ru(bpy)2(L-pterin)]2+ in Fig. 9A has a striking biphasic shape
6
7
distinguished by a marked increase in absorbance at temperatures above 80 oC.
8
emphasizes the difference between the normal sigmoidal curve of Ru-dppz (grey line) and the
9
biphasic curve of Ru-L-pterin (red line) when data are obtained at a lower [DNA]:[Ru] ratio of
Fig. 9B
10
10. Under these conditions, the melting curve for Ru-L-allox (dashed line) also shows a slightly
11
biphasic aspect. Further evidence for DNA melting distinctions between Ru-L-pterin and Ru-
12
dppz is shown in the inset of Figure 9B. Here, the second melting phase over the region 75-95
13
o
14
450 nm absorption of Ru-L-pterin increases steadily with addition of DNA, whereas no change
15
in absorbance is observed for Ru-dppz with DNA. This result suggests that the unusual biphasic
16
portion of the Tm plot is directly related to an interaction of Ru-L-pterin and DNA which does
17
not occur between Ru-dppz and DNA.
C is monitored using the strong MLCT absorption of Ru-L-pterin and Ru-dppz at 450 nm. The
26
1
2
Figure 9. Melting curves generated from measuring absorbance at 260 nm. (A) CT
3
DNA in pH 7.1 phosphate buffer and 2 mM NaCl, in buffer alone and after the addition
4
of each Ru-L-pteridine complex and the control [Ru(bpy)2(dppz)]2+. Data were collected
5
at a [CT DNA per nucleotide]:[Complex] ratio of 15. X CT DNA, ▲ Ru-dppz,  Ru-L-
6
pterin,  Ru-L-diamino,  Ru-L-Me2allox,  Ru-L-allox,  Ru-L-amino. (B) Melting
7
curves showing biphasic curve of Ru-L-pterin (red line) and Ru-L-allox (dashed line) for
8
data collected at a [CT DNA per nucleotide]:[Complex] ratio of 10. Inset shows relative
9
absorbance at 450 nm of Ru-L-pterin with DNA (red line) and Ru-dppz with DNA (gray
10
line) over the high temperature range 70-95 oC .
11
12
Table 2. Melting temperatures obtained from the melting curves shown in Figure 9A.
Ru Complex
Tm (Co)
Free CT DNA
62.5
[Ru(bpy)3]2+
64.5
[Ru(bpy)2(L-pterin)]2+
67.3a
27
[Ru(bpy)2(L-amino]2+
70.4
[Ru(bpy)2(L-diamino]2+
69.7
[Ru(bpy)2(L-allox)]2+
66.0
[Ru(bpy)2(L-Me2allox)]2+
69.4
[Ru(bpy)2(dppz)]2+
67.0
1
Values were obtained by taking the average of three trials that agreed within 0.8 oC.
2
a
3
values from 25 to 78 oC were used to calculate the Tm of 67.3 oC.
4
Evaluation of Binding Constants. Quantitative measure of DNA binding by polypyridyl
5
ruthenium complexes can be obtained by monitoring changes in electronic spectroscopy. Using
6
fluorescence spectroscopy, the enhanced emission intensity as a function of DNA added to Ru
7
can be used to generate isothermal binding curves. In absorption spectroscopy, Ru complex
8
binding to DNA has been correlated with hypochromic and bathochromic shifts in the MLCT
9
absorption.[42] We have used both spectroscopic methods to obtain values of equilibrium
In order to compare the Tm with those of the monophasic melting curves, only absorbance
10
binding constants and examples of the titrations using absorption spectroscopy and florescence
11
emission data is shown in Figures 10 and 11 respectively. Values of Kb, and site sizes, s, were
12
extracted from the data by fitting the data to the equations first derived by Bard and coworkers
13
[27] and used widely by others. [15-18] The fitting results from titrations on the five Ru-L-
14
pteridine complexes and the control Ru-dppz are listed in Table 3.
15
16
Table 3. DNA Binding Constants for Ru-L-pteridine and Ru-dppz Complexesa
Absorption Titration Data
Fluorescence Titration Data
28
complex
Kbb
Sb
%Hc
Kbb
Sb
Ib /
If d
Ru-dppz
2.9 x 106
0.84
38% (369 nm)
1.0 x 107
0.95
99
Ru-L-pterin
1.1 x 106
1.4
21% (420 nm)
7.2 x 105
2.0
7
Ru-L-diamino
5.1 x 106
0.88
21% (427 nm)
9.9 x 106
1.1
15
Ru-L-amino
2.6 x 106
0.76
20% (410 nm)
6.7x105
1.8
13
1.8 x 106
5.0
1
1.3 x 106
5.0
2
9% (450 nm)
Ru-L-allox
4.7 x 105
0.67
28% (369 nm)
17% (450 nm)
Ru-L-Me2allox
1.3 x 106
0.80
35% (386 nm)
1
a
Conditions: 10 mM phosphate pH 7.1, 50 mM NaCl, 12.5 - 23 M Ru-L .
2
b
Error in Kb and s values from absorption titrations are ± 0.8 x 106 and ± 0.08, respectively, and
3
± 0.14 x 106 and ± 0.1, respectively, from fluorescence titrations.
4
c
Percent hypochromicity at specified wavelength in the absorption spectrum
5
d
Ratio of fluorescence intensity of (Ru complex + DNA) to fluorescence intensity of Ru
6
complex in buffer
7
8
9
The change in absorption spectra as Ru-L-Me2allox and Ru-L-allox are titrated with
DNA is nearly identical to that previously reported [22, 23] with a large hypochromicity of 27%
10
and 28% in the LL band and smaller hypochromicity of 14% and 16% in the MLCT band.
No
11
bathochromicity occurs in the MLCT band of either Ru-L-Me2allox or Ru-L-allox but both
12
exhibit a ~ 10 nm shift in the LL region. The strong multi-component absorptions for Ru-L-
13
pterin and Ru-L-diamino at 420 and 428 nm shift to 428 and 440 nm, respectively, and undergo a
29
1
similar decrease in absorbance over the entire region, with a maximum decrease of 21% at 420
2
and 427 nm. No resolution of the multi-component absorption occurred during the titrations
3
with DNA. The spectral changes for Ru-L-amino during a DNA titration are unique in
4
exhibiting a change in the absorption profile. In addition to the typical bathochromic shift of 13
5
nm in the LL band with 13% hypochromicity of and 9% hypochromicity in the MLCT band at
6
450 nm, the shoulder near 380 nm disappears.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Figure 10. Absorption titrations of [Ru(bpy)2(L-amino)]2+ (top left), Ru(bpy)2(L-
22
diamino)]2+ (top right), Ru(bpy)2(L-allox)]2+ (bottom left) and [Ru(bpy)2(L-pterin)]2+
23
(bottom right) with increasing amounts of calf thymus DNA under conditions listed in
30
1
Table 3. For each Ru-pteridine complex, the spectral series show a progression of
2
decreasing intensity with increasing [DNA]/[Ru].
3
4
A typical saturation curve and fitted data obtained in a fluorescence titration are shown in
5
Figure 11 for [Ru(bpy)2(L-pterin)]2+ titrated with CT DNA. Increased emission intensity is
6
observed as the complex emission becomes incrementally more protected from the solvent due to
7
a close association between the complex and the DNA helix. Similar titration plots and data fits
8
were produced for all other Ru-L-pteridine complexes as well as the control Ru-dppz and are
9
available in the Electronic Supplemental Material.
10
11
Figure 11. Fluorescence titration of 10 M [Ru(bpy)2(L-pterin)]2+ with increasing
12
amounts of calf thymus DNA. The spectra show a progression of increasing intensity
13
with concentration. The inset shows the saturation curve of the fluorescence intensity
31
1
monitored at an emission wavelength of 610 nm at ratios of [DNA]:[Ru(bpy)2(L-allox)]
2
between 0 and 12.
3
4
Values of equilibrium binding constants, Kb, obtained from absorption and fluorescence
5
titrations are in reasonable agreement with those obtained from CT DNA titrations of similar
6
Ru(bpy)2(phen-derivative) complexes in the literature. [12-18, 22, 43] The magnitude of Kb
7
values obtained from absorption titrations span a range less than an order of magnitude (~5 x 105
8
to 5 x 106) while those from fluorescence titrations have a slightly larger range (~7 x 105 to 107).
9
The Kb values for Ru-dppz and Ru-L-diamino are the largest of the set by both methods and the
10
rest of the complexes show variation between the two methods so that no further ranking is
11
attempted. Given that precise measurement of Kb values near and greater than 106 by such
12
titrations have been questioned, [43] the attempt to make fine distinctions among the set of
13
complexes is probably not valid. The binding site size, s, obtained from absorbance titrations is
14
consistently between 0.8 and 1.4 corresponding with a binding stoichiometry of about one Ru-
15
pteridine complex per base pair site. Site sizes from fluorescence titrations remain small (s = 1-
16
2) for Ru-dppz, Ru-pterin, Ru-L-amino and Ru-L-diamino but peculiarly are quite large, s = 5,
17
for Ru-allox and Ru-L-Me2allox. A wide range of site sizes has been reported among complexes
18
that might be expected to have similar steric demands based on their structures, but an
19
explanation for such a range has not been forthcoming. We note that in an early study by
20
Chaires et al. [12] on the binding of Ru-dppz to CT DNA , they reported two binding modes
21
having stoichiometries of 3 DNA bp per Ru and 0.8 DNA bp per Ru.
22
Discussion
23
24
Complexes of ruthenium(II) coordinated by pteridinyl-phenanthroline ligands L-pterin,
L-diamino, L-amino, L-allox and L- Me2allox were designed expressly to probe the influence of
32
1
the pteridinyl groups on DNA interactions. The ligands have a molecular framework that may
2
be viewed as modification of the well-studied intercalating ligand dipyridophenazine (dppz)
3
(Figure 12). The different H-bonding patterns provided by the various pteridine groups slightly
4
extend the planar, conjugated structure derived from dppz. The heterocyclic pyrimidine terminus
5
adjusts the electronic structure to that of a nucleic acid. The initial goals of this study were to
6
evaluate what effect, if any, the H-bonding ability of the pteridinyl-substituted phenanthroline
7
ligands would have on the DNA intercalation as compared to [Ru(bpy)2(dppz)]2+ and to compare
8
the differences in DNA binding interactions among the four Ru-pteridine complexes. Interest in
9
the effects of H-bonding and functional groups on DNA binding affinity is not without
10
precedent. M. Palaniandavar et al. showed that intramolecular H- bonding could extend the
11
aromatic rings in tetrammine ruthenium(II) complexes of modified 1,10-phenanthrolines and
12
enhance DNA binding affinity.[42] The amino groups on ethidium bromide were determined to
13
play a significant role in the energetics and the fluorescent properties of DNA binding.[44]
14
While our study was in progress, separate reports of DNA binding studies of Ru-L-allox and Ru-
15
L-Me2allox were independently made by Gao and coworkers.[22,23] Consideration of their
16
results and conclusions will be included in discussion of our results.
N
N
N
N
N
NH
Ru
N
N
O
N
N
N
N
17
(a) [Ru(bpy)2(L-pterin)]2+
NH2
N
N
N
N
Ru
N
N
(b) [Ru(bpy)2(dppz)]2+
18
Figure 12. Comparison of the chemical structures of [Ru(bpy)2(L-pterin)]2+ and
19
[Ru(bpy)2(dppz)]2+.
33
1
2
Four methods were employed for this investigation of DNA binding by the series of four
3
Ru-pteridinyl-phenanthroline complexes, where each method reports on a unique aspect of the
4
Ru molecules’ interactions with DNA. Viscometry is a simple, inexpensive and reliable method
5
for monitoring the hydrodynamic changes in solution as a result of DNA intercalation. DNA
6
solution viscosity will increase when a molecule intercalates between base pairs, pushing them
7
apart, causing an overall lengthening of the helix. Hence, an increase in solution viscosity is an
8
unambiguous result of molecules that intercalate DNA.[35] An increase in viscosity is not
9
observed for electrostatic binding of molecules within a groove or on the surface that does not
10
disturb the shape of the helix. Thermal denaturation studies, or DNA melting, provide a measure
11
of the change in DNA duplex stability under conditions of increasing temperature. Researchers
12
have attributed an increase in melting temperature of DNA to stabilization of base stacking by
13
intercalated molecules.[45] Spectroscopic probes of electronic structure changes at DNA and at
14
the interacting molecule can be exploited for determination of equilibrium binding constants.
15
Absorption spectroscopy reports changes in absorptivity and energetics of observed transitions.
16
Common consequences of DNA interactions with Ru-polypyridyl complexes, especially
17
intercalation, are a decrease in absorptivity (hypochromicity) and a shift to lower energies
18
(bathochromicity). Fluorescence emission enhancement is a frequently observed characteristic
19
of ruthenium-based intercalators possessing inherent fluorescence that is quenched in aqueous
20
buffers. Based on the results from viscometry, thermal denaturation, absorption and
21
fluorescence spectroscopy, all five Ru-L-pteridine complexes intercalate into calf thymus DNA
22
as tightly or nearly as tightly as the parent complex Ru-dppz.
34
1
Our results from binding studies of Ru-L-allox and Ru-L-Me2allox confirm in part the
2
previously reported [22,23] observation that both complexes intercalate into CT DNA.
3
However, our results show subtle differences from the published work. We find that L-Me2allox
4
intercalates somewhat more strongly than the related Ru-L-allox. Three methods provide data
5
consistent with this interpretation where a greater slope in viscosity plots, a larger Tm value and
6
larger binding constants are obtained for L-Me2allox as compared to Ru-L-allox.
7
results we infer that the presence of the methyl groups at the pyrimidine terminus does not
8
present a steric interference to L-Me2allox intercalation between stacked base pairs, in contrast to
9
conclusions reached in the previous study.[23] It should be noted that their DNA studies were
From these
10
conducted in Tris buffer, whereas the results reported here are from phosphate buffered
11
solutions. A dependence of Kb on buffer has been previously investigated where it was found
12
that higher Kb values were obtained from Tris buffered solutions than from phosphate buffer
13
solution.[46]
14
There are distinct differences observed in the DNA binding interactions among the five
15
Ru-L-pteridine complexes that are apparent in thermal denaturation and viscometry experiments.
16
DNA melting in the presence of Ru-L-pterin produces biphasic curves suggestive of a two step
17
DNA duplex dissociation. Under certain conditions (low ratios of [DNA] to [Ru]), the melting
18
curve of Ru-L-allox also exhibits some biphasic character (both illustrated in Fig. 9) Chaires et
19
al. reported a nearly identical biphasic melting curve obtained from a study of daunomycin
20
binding to DNA.[47] The two phases were attributed to distinct melting processes where the
21
lower temperature process was the melting of regions of DNA without daunomycin and the
22
second, higher temperature phase was to due to melting of regions containing daunomycin. The
23
two regions were presumed to result from redistribution of daunomycin on the DNA surface
35
1
during the melting process causing clustering of daunomycin on the unmelted surface. This
2
hypothesis was supported by theoretical modeling studies.[48] If we adopt their argument, a
3
reason is needed to explain why such biphasic melting is observed primarily for Ru-L-pterin and,
4
to a lesser extent, Ru-L-allox but is not observed using the other L-pteridine complexes. While
5
the five L-pteridine ligands all contain the same bicyclic pteridine structure, they differ in their
6
H-bonding capabilities. The placement of the hydrogen bond donors and acceptors of the L-
7
pterin ligand are the same as those of cytosine and guanosine, and likewise, hydrogen bond
8
donors and acceptors for the L-alloxazine ligand are in the same placement as those of thymine.
9
Both the pterin and the alloxazine termini are capable of binding to the surface of the major
10
groove of DNA in a manner analogous to the interactions involved in triplex DNA formation.
11
This is illustrated in Figure 13 where H-bonding of a triplex base grouping of CGC is compared
12
to a triple interaction in [Ru(bpy)2(L-pterin)]2+-G-C. This H-bonding in the major groove may
13
be a binding mode contributing to the exceptionally stable clustering of Ru-L-pterin and Ru-L-
14
allox on the surface of DNA that is responsible for biphasic melting curves. H-bonding sites of
15
suitable orientation also exist in the minor groove, however steric problems between the Ru
16
complex and phosphate backbone probably disfavor binding in this location.
36
N
N
Ru
N
N
N
CG-Ru-L-pterin interaction
at major groove
CGC triplex interaction
at major groove
R
H N
N
N
H
H
1
N
N
N
N
R
H
N
H
N
N
H
H
H
O
N
H
O
R
N
N
N
N
O
N
H
N
O
N
R
H
H
H
H
O
N
N
N
N
N
N
O
H
R
N
H
2
Figure 13. Comparison of C-G-C hydrogen bonding in triplex DNA (left) with
3
analogous H-bonding of [Ru(bpy)2(L-pterin)]2+ to base paired G-C (right).
4
5
Further differences among the five Ru-L-pteridine complexes when interacting with
6
DNA are indicated by the effect of ionic strength on the degree of intercalation. Thorp and
7
others have previously investigated the effect of ionic strength on intercalation and reported that
8
the intercalation binding constant increases as ionic strength decreases.[46] Under typical
9
conditions of 50 mM NaCl, the viscosity plots of all five pteridine complexes as well as that for
10
Ru-dppz have very similar slopes (see Fig. 7). When the salt concentration is decreased to 2
11
mM or increased to 200 mM, distinct differences among the Ru-L-pteridine complexes emerge.
12
Those pteridines bearing carbonyl groups—Ru-L-Me2allox, Ru-L-allox and Ru-L-pterin—show
13
the greatest sensitivity to salt concentration. If the viscosity slope is assumed to parallel DNA
14
intercalation binding strength, a relative decrease in slope would suggest decreased DNA
15
intercalation. The low salt results are consistent with the previously reported inverse correlation
37
1
between ionic strength and the strength of DNA intercalation where Kb for intercalation was
2
observed to increase under low ionic strength (2 mM) conditions.
3
Conclusion
4
The combination of viscometry, thermal denaturation, fluorescence and absorption
5
spectroscopy data show that all five Ru-L-pteridines complexes in this work intercalate into
6
DNA with binding strengths comparable within an order of magnitude. Favorable H-bonding
7
possible by [Ru(bpy)2(L-pterin)]2+ and [Ru(bpy)2(L-allox)]2+ increases the stability of surface
8
binding in the major grove. Further study will determine if there is base sequence specificity for
9
the binding of Ru(bpy)2(L-pterin)]2+, Ru(bpy)2(L-amino)]2+, Ru(bpy)2(L-diamino)]2+ and
10
[Ru(bpy)2(L-allox)]2 and what role their H-bonding capabilities play in their binding to DNA.
11
In light of a recent report [49] of hydrolytic cleavage of DNA by a ruthenium-bis(bipyridyl)
12
complex with H-bonding acceptor and donor substituents, future studies will also probe whether
13
the complexes possess the ability to cleave the DNA backbone via a hydrolytic or photoinitiated
14
mechanism, as well as investigate sequence specificity of cleavage reactions.
15
Acknowledgements. We wish to thank Pat Carroll and the Facility for X-ray Crystallography in
16
the Chemistry Department of the University of Pennsylvania and Alanna Albano and Lindsay
17
Alaishuski for their contributions to early development of this project.
18
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4
Me2allox)](PF6)2. Bond distances (Å): Ru1-N48 2.054(5), Ru1-N36 2.057(6), Ru1-N37
5
2.061(5), Ru1-N25 2.063(5), Ru1-N1 2.063(5), Ru1-N20 2.064(5). Bond angles (o) N48-Ru1-
6
N36 173.5(2), N48-Ru1-N37, 79.0(2), N36-Ru1-N37, 96.1(2), N48-Ru1-N25, 96.6(2), N36-
7
Ru1-N25, 78.9(2), N37-Ru1-N25, 89.9(2), N48-Ru1-N1, 88.0(2), N36-Ru1-N1, 97.0(2), N37-
8
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9
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