Ru(II) and Os(II) nucleosides and oligonucleotides: Synthesis and properties

Article abstract of DOI:10.1021/ja0123103

A general and versatile method for the site-specific incorporation of polypyridine Ru^II and Os^II complexes into DNA oligonucleotides using solid-phase phosphoramidite chemistry is reported. Novel nucleosides containing a [(bpy)₂

Full text of DOI:10.1021/ja0123103

Published on Web 03/13/2002
Ru(II) and Os(II) Nucleosides and Oligonucleotides: Synthesis
and Properties
Dennis J. Hurley and Yitzhak Tor*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of California,
San Diego, La Jolla, California 92093-0358
Received October 9, 2001. Revised Manuscript Received January 3, 2002
Abstract: A general and versatile method for the site-specific incorporation of polypyridine RuII and OsII
complexes into DNA oligonucleotides using solid-phase phosphoramidite chemistry is reported. Novel
nucleosides containing a [(bpy)2M(3-ethynyl-1,10-phenanthroline)]2+ (M ) Ru, Os) metal center covalently
attached to the 5-position in 2′-deoxyuridine are synthesized, and their electrochemical as well as
photophysical properties are studied. The RuII nucleoside exhibits a rather long-lived excited state in
phosphate buffer pH 7.0 (τ ) 1.08 µs) associated with a relatively high emission quantum efficiency (φ )
0.051). The solvent dependence of the absorption and emission spectra is consistent with an emissive
MLCT state where charge localization takes place on the extended heterocycle-linked phenanthroline. In
contrast, the OsII-containing nucleoside is quite nonemissive in aqueous environment (τ ) 0.027 µs, φ )
1 × 10-4). The metal-containing nucleosides are converted into their phosphoramidites and are utilized for
the high-yield preparation of modified oligonucleotides. The novel oligonucleotides, characterized by
absorption and emission spectroscopy, enzymatic digestion, and electrophoresis, form stable duplexes.
Circular dichroism spectra confirm that the global conformation of the double helix is not altered by the
presence of these polypyridyl complexes in the major groove. Metal-containing phosphoramidites with
predetermined absolute configuration at the octahedral coordination center are synthesized and utilized
for the synthesis of diasteromerically pure metal-containing DNA oligonucleotides. Emission spectroscopy
suggests a higher protection of the ∆ metal center from the bulk solvent and better accommodation within
the major groove.
complexation of a chelate-containing oligonucleotide,3c,4 or by
conjugation of a functionalized oligonucleotide with a suitably
activated metal complex.1,5 Most approaches facilitate the
incorporation of coordination complexes at the oligo’s termini,
while internal modification remains a challenge.6,7 A general
methodology for the incorporation of metal centers at any
Introduction
Oligonucleotides containing photo- and redox-active transition
metal complexes have become a unique tool for studying
photophysical processes in nucleic acids,1,2 as well as for the
assembly of DNA hybridization probes and sensors.3 The
incorporation of coordination compounds into oligonucleotides
has been traditionally accomplished by a postsynthetic metal
(4) Bashkin, J. K.; Frolova, E. I.; Sampath, U. J. Am. Chem. Soc. 1994, 116,
5981-5982. Matsumura, K.; Endo, M.; Komiyama, M. J. Chem. Soc.,
Chem. Commun. 1994, 2019-2020. Chen, C.-H. B.; Sigman, D. S. J. Am.
Chem. Soc. 1988, 110, 6570-6572.
(5) Magda, D.; Miller, R. A.; Sessler, J. L.; Iverson, B. L. J. Am. Chem. Soc.
1994, 116, 7439-7440. Hall, J.; Hu¨sken, D.; Pieles, U.; Moser, H. E.;
Ha¨ner, R. Chem. Biol. 1994, 1, 185-190. Hall, J.; Hu¨sken, D.; Ha¨ner, R.
Nucleic Acids Res. 1996, 24, 3522-3526.
(6) For approaches utilizing metal-containing phosphoramidites or H-phos-
phonates, see Manchanda, R.; Dunham, S. U.; Lippard, S. J. J. Am. Chem.
Soc. 1996, 118, 5144-5145. Schlipe, J.; Berghoff, U.; Lippert, B.; Cech,
D. Angew. Chem., Int. Ed. Engl. 1996, 35, 646-648. Music, R. C.; Herrlien,
M. K.; Mirkin, C. A.; Letsinger, R. L. Chem. Commun. 1996, 555-557.
Meggers, E.; Kusch, D.; Giese, B. HelV. Chim. Acta 1997, 80, 640-652.
Magda, D.; Crofts, S.; Lin, A.; Miles, D.; Wright, M.; Sessler, J. L. J. Am.
Chem. Soc. 1997, 119, 2293-2294. Lewis, F. D.; Helvoigt, S. A.; Letsinger,
R. L. Chem. Commun. 1999, 327-328. Wiederholt, K.; McLaughlin, L.
W. Nucleic Acids Res. 1999, 27, 2487-2493. For a photoaddition of RuII
complex to DNA, see Jacquet, L.; Davis, R. J. H.; Kirsch-De Mesmaeker,
A.; Kelly, J. M. J. Am. Chem. Soc. 1997, 119, 11763-11768. For an
overview article, see Beilstein, A. E.; Tierney, M. T.; Grinstaff, M. W.
Comments Inorg. Chem. 2000, 22, 105-127.
(1) For selected examples, see Murphy, C. J.; Arkin, M. R.; Jenkins, Y.; Ghatlia,
N. D.; Bossmann, S. H.; Turro, N. J.; Barton, J. K. Science 1993, 262,
1025-1029. Meade, T. J.; Kayyem, J. F. Angew. Chem., Int. Ed. Engl.
1995, 34, 352-354. Dandliker, P. J.; Holmlin, R. E.; Barton, J. K. Science
1997, 275, 1465-1468. Holmlin, R. E.; Tong, R. T.; Barton, J. K. J. Am.
Chem. Soc. 1998, 120, 9724-9725. Ortmans, I.; Content, S.; Boutonnet,
N.; Kirsch-De Mesmaeker, A.; Bannwarth, W.; Constant, J.-F.; Defrancq,
E.; Lhomme, J. Chem. Eur. J. 1999, 5, 2712-2721. Stemp, E. D. A.;
Holmlin, R. E.; Barton, J. K. Inorg. Chim. Acta 2000, 297, 88-97.
Schiemann, O.; Turro, N. J.; Barton, J. K. J. Phys. Chem. B 2000, 104,
7214-7220. Williams, T. T.; Odom, D. T.; Barton, J. K. J. Am. Chem.
Soc. 2000, 122, 9048-9049.
(2) For overview articles, see Netzel, T. L. J. Chem. Educ. 1997, 74, 646-
651. Diederichsen, U. Angew. Chem., Int. Ed. Engl. 1997, 36, 2317-2319.
Holmlin, R. E.; Dandliker, P. J.; Barton, J. K. Angew. Chem., Int. Ed. Engl.
1997, 36, 2714-1730. Netzel, T. L. J. Biol. Inorg. Chem. 1998, 3, 210-
214. Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem. ReV. 1999, 99, 2777-
2795. Grinstaff, M. W. Angew. Chem., Int. Ed. 1999, 38, 3629-3635.
Nu´n˜ez, M. E.; Barton, J. K. Curr. Opin. Chem. Biol. 2000, 4, 199-206.
(3) (a) Bannwarth, W.; Schmidt, D.; Stallard, R. L.; Hornung, C.; Knorr, R.;
Mu¨ller, F. HelV. Chem. Acta 1988, 71, 2085-2099. (b) Bannwarth, W.;
Schmidt, D. Tetrahedron Lett. 1989, 30, 1513-1516. (c) Telser, J.;
Cruickshank, K. A.; Schanze, K. S.; Netzel, T. L. J. Am. Chem. Soc. 1989,
111, 7221-7226. (d) Bannwarth, W.; Pfleiderer, W.; Mu¨ller, F. HelV. Chem.
Acta, 1991, 74, 1991-1999.
(7) Following the publication of our preliminary communication (ref 8), a
nonconjugated RuII-containing phosphoramidite (Khan, S. I.; Beilstein, A.
E.; Grinstaff, M. W. Inorg. Chem. 1999, 38, 418-419) and solid-support
modifications (Khan, S. I.; Grinstaff, M. W. J. Am. Chem. Soc. 1999, 121,
4704-4705) have been reported.
9
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J. AM. CHEM. SOC. 2002, 124, 3749-3762
3749

A R T I C L E S
Hurley and Tor
position along the DNA sequence using automated DNA
synthesizers is highly desirable. This capability is particularly
important when the systematic study of energy- and electron-
transfer processes in DNA is concerned: changing the distance
between the termini requires the synthesis of oligonucleotides
of variable lengths, while varying the internal positioning of
metal probes within a single DNA template will allow to
methodically study distance-dependency without sequence
perturbations.
A systematic approach to the modification of DNA with
photo- and redox-active metal centers needs to fulfill the
following requirements: (a) generality - the incorporation of
the metal complexes at any position along the DNA duplex
should be possible; (b) structural stability - the modification
should ensure minimal structural perturbation of the DNA
duplex; (c) placement - the complexes have to be directly
connected to the stacked heterocyclic bases; (d) versatility and
tunability - a family of novel bases with a range of redox
potentials and spectral characteristics, as well as tunable
electronic communication with the heterocycle, should be
accessible; and (e) simplicity - the building blocks should be
synthetically accessible and compatible with standard solid-
phase DNA synthesis.
In a preliminary account, we reported the first solid-phase
controlled synthesis of RuII- and OsII-containing oligonucle-
otides.8 Our approach involves the synthesis of metal-containing
phosphoramidites and their sequence-specific incorporation into
oligonucleotides using standard automated phosphoramidite
chemistry. In this paper, we outline the design criteria and the
experimental details involved in preparing, characterizing, and
studying metal-containing oligonucleotides. We describe the
synthesis and characterization of RuII- and OsII-containing
nucleosides, as well as their redox and photophysical charac-
teristics. We discuss the preparation and purification of the
modified phosphoramidites and their facile incorporation into
metal-containing DNA oligonucleotides. The analysis of the
novel oligonucleotides is outlined in detail, together with their
photophysical characteristics. We report, for the first time, the
synthesis of metal-containing phosphoramidites with predeter-
mined absolute configuration at the octahedral coordination
center, and their utilization for the synthesis of diasteromerically
pure metal-containing DNA oligonucleotides. The hybridization
and luminescence properties of the metal-modified oligonucle-
otides are discussed. These uniquely modified oligonucleotides
form stable DNA duplexes and are valuable tools for the study
of photophysical processes in nucleic acids.
Figure 1. Base-paired conjugated metal-containing deoxyuridine nucleo-
sides point toward the major groove of the double-helix. Note the potential
versatility in coordinated metal ions and co-ligands.
DNA.9 The rigid ethynyl linker ensures that the metal complex
does not back-intercalate into the duplex and produces substan-
tial electron delocalization, allowing for electronic communica-
tion between the base-pairing heterocycle and the metal
chelator.10 Polypyridine complexes of RuII and OsII were
selected due to their chemical stability, and favorable redox and
photophysical characteristics.11 The availability of 5-halogenated
pyrimidines together with numerous cross coupling methodolo-
gies provides multiple synthetic routes to novel modified
nucleobases.
Synthesis of Metal-Containing Nucleosides. We prepared
the metal-containing nucleosides 5a and 5b, employing func-
tionalized coordination compounds as synthetic building
blocks.12,13 The approach entails the synthesis of 5-ethynyldeoxy-
uridine 2, a known modified nucleoside prepared from the
commercially available 5-iododeoxyuridine 1,14,15 followed by
a Sonogashira cross-coupling reaction with coordination com-
plexes [(bpy)2M(3-bromo-1,10-phenanthroline)]2+(PF6-)2.12,13
Thus, 2 was cross-coupled to 4a and 4b in the presence of
(dppf)PdCl2‚CH2Cl2, CuI, and DMF/Et3N to afford the RuII-
and OsII-containing nucleosides 5a and 5b, respectively, in high
yields (Scheme 1).
The high reactivity of the functionalized metal complexes
4a and 4b in cross-coupling reactions12,13 enabled us to similarly
modify 5′-(4,4′-dimethoxytrityl)-protected nucleosides. Thus, 2
is first treated with 4,4′-dimethoxytrityl chloride (DMT-Cl) in
the presence of DMAP to provide the DMT-protected nucleoside
3. Cross coupling with 4a and 4b provides the 5′-protected
metal-containing nucleosides 6a and 6b, respectively.16 Phos-
(9) Ethynyl-linked substitutions at the 5 positions are particularly favored; see
Ahmadian, M.; Zhang, P.; Bergstrom, D. E. Nucleic Acids Res. 1998, 26,
3127-3135.
Results and Discussion
(10) Tzalis, D.; Tor, Y. Tetrahedron Lett. 1995, 36, 6017-6020.
(11) (a) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; Von
Zelewsky, A. Coord. Chem. ReV. 1988, 85, 85-277. (b) Sauvage, J.-P.;
Collin, J.-P.; Chambron, J.-C.; Guillerez, S.; Coudret, C.; Balzani, V.;
Barigelletti, F.; De Cola L.; Flamigni, L. Chem. ReV. 1994, 94, 993-1019.
(d) Balzani, V.; Juris, A.; Venturi, M.; Campagna, S.; Serroni, S. Chem.
ReV. 1996, 96, 659-833. (e) Barigelletti, F.; Flamigni, L.; Collin, J.-P.;
Sauvage, J.-P. Chem. Commun. 1997, 333-338.
Design Criteria. Several major factors were taken into
consideration in designing our first-generation metal-containing
nucleosides suitable for automated DNA synthesis: (1) the base
modification position, (2) the nature of the linking group, (3)
the kinetic stability of the metal centers and their compatibility
with standard conditions of automated DNA synthesis, as well
as (4) synthetic accessibility and versatility. We have chosen
to covalently attach RuII and OsII polypyridine complexes to
the 5-position of 2′-deoxyuridine (Figure 1). A modification at
this position is projected into the major groove of the double
helix and is likely to have little impact on the stability of duplex
(12) Tzalis, D.; Tor, Y. Chem. Commun. 1996, 1043-1044.
(13) Connors, P. J., Jr.; Tzalis, D.; Dunnick, A. L.; Tor, Y. Inorg. Chem. 1998,
37, 1121-1123.
(14) Robins, M. J.; Barr, P. J. J. Org. Chem. 1983, 48, 1854-1862.
(15) Hashimoto, H.; Nelson, M. G.; Switzer, C. J. Am. Chem. Soc. 1993, 115,
7128-7138.
(16) Attempts to protect the 5′-hydroxyl in the metalated nucleosides 5a and
5b as the DMT derivatives were unsuccessful. It is likely that the bulky
polypyridine complexes interfere with this reaction. See also Supporting
Information for additional evidence for the interaction between the 5′-DMT
group and the metal center at the 5 position.
(8) Hurley, D. J.; Tor, Y. J. Am. Chem. Soc. 1998, 120, 2194-2195.
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Ru(II) and Os(II) Nucleosides and Oligonucleotides
A R T I C L E S
Scheme 1. Synthesis of Phosphoramidites 7a and 7ba,b
a Reagents: (a) i. (CF3CO)2O; ii. Me3Si-C≡CH, (Ph3P)4Pd, CuI, DMF, Et3N, 95-98%; iii. K2CO3, MeOH, 75-92%; (b) 4,4′-dimethoxytrityl chloride
(DMT-Cl), DMAP, pyridine, Et3N, 92%; (c) (dppf)PdCl2‚CH2Cl2, CuI, DMF, Et3N, 72-84%; (d) (iPr2N)2POCH2CH2CN, (1H)-tetrazole, CH3CN, 75-92%.
b All metal-modified nucleosides were isolated as their PF6 salts.
-
Table 1. Redox and Photophysical Properties of Metal-Containing
Nucleosides 5a and 5ba
b
E
1/2
3+ 2+
d
e
comp (M / )
UV−Visc
λem
629
[633] [0.051]
749 0.0003
Φem
τ f
5a
5b
0.993 242 (4.0), 256 (3.9), 286
(6.9), 352 (2.6), 450 (1.3)
0.560 242 (3.8), 254 (3.6), 290
0.137
2.78
[1.08]
0.078
(6.4), 358 (2.6), 436 (1.1), [754] [0.0001] [0.027]
484 (1.1)g
a Measurements were performed with 1 × 10-5 M deoxygenated
acetonitrile solutions at room temperature. λem, Φem, and τ values in brackets
are for deoxygenated aqueous solutions. b Redox potentials in V as measured
in acetonitrile vs Ag/Ag+; E1/2 ) 0.085 V for Fc/Fc+ under these conditions.
c In acetonitrile. Absorption maxima are given in nm and extinction
coefficients (ꢀ × 10-4 in parentheses) in M-1 cm-1. In water, the
phenanthroline absorption bands shift from 352 (2.6) to 346 (3.2) for 5a
and from 358 (2.6) to 352 (2.8) for 5b. d Excitation at 450 nm. λem reported
in nm. e MLCT emission quantum yields were calculated from the integrated
spectra relative to Ru(bpy)3 in H2O (Φ ) 0.042). f Lifetimes are reported
in µs. Decays were monitored at the λem for each compound. Estimated
error is 3%. g Broad absorption tailing to >600 nm.
vs Ag/Ag+). The values obtained for the metalated nucleosides
are in good agreement with prototypical RuII and OsII polypy-
ridine complexes.11,13
Figure 2. Electrochemical and photophysical characteristics of metalated
nucleosides. (a) Square-wave voltammograms of 5a (solid line) and 5b
(dashed line) shown next to ferrocene (-‚-‚). All taken in degassed
acetonitrile solution containing 0.1 M [n-Bu4N+][PF6-] vs Ag/Ag+. (b) UV-
Vis absorption spectra of 5a (solid line) and 5b (dashed line) in acetonitrile.
(c) Steady-state emission spectra of 5a (solid line) and 5b (dashed line) in
deoxygenated acetonitrile. (d) Time-resolved luminescence decay of 5a
(solid line) and 5b (dashed line) in acetonitrile.
The absorption and emission characteristics of the RuII and
OsII nucleosides are shown in Figure 2b-d, and the data are
summarized in Table 1. Both nucleosides exhibit intense π-π*
transitions of the aromatic diimine ligands at 280 nm, a lower
energy transition, assigned to the extended phenanthroline at
350 nm,12,13 and typical metal-to-ligand charge-transfer (MLCT)
transitions in the visible region (ca. 450 nm for RuII, 440 and
500 nm for OsII). Excitation of an acetonitrile solution of 5a at
460 nm leads to an intense red emission centered at 630 nm
(Figure 2c). Using the method reported by Crosby,18 the
quantum efficiency of the Ru-based emission in acetonitrile was
determined to be 0.137. This value is twice as high as compared
to [Ru(bpy)3]2+ (0.062).19 The higher luminescence efficiency
is also manifested in the relatively long-lived excited state of
5a (τ ) 2.78 µs) when compared to [Ru(bpy)3]2+ (τ ) 0.87
µs).20 These differences are likely to result from the lower
symmetry of the metal complex and extended conjugation of
the phenanthroline ligand in 5a. In contrast, the OsII-containing
nucleosides 5b is quite nonemissive, giving rise to a weak
phitylation of the protected nucleosides with 2-cyanoethyl-
N,N,N′,N′-tetraisopropylphosphorodiamidite in the presence of
(1H)-tetrazole provided the corresponding metal-containing
phosphoramidites 7a and 7b (Scheme 1). These phosphoramid-
ites have been carefully purified by flash chromatography over
basic alumina using acetonitrile-water mixtures containing
saturated KPF6 (see experimental part). As discussed below,
high purity of the phosphoramidites was found to be critical
for ensuring high coupling efficiency during oligonucleotide
synthesis. These sensitive building blocks were therefore
1
thoroughly characterized by H, and 31P NMR together with
ESI mass spectrometry.
Redox and Photophysical Properties of Metal-Containing
Nucleosides. In addition to the routine analytical characterization
of the new metal modified nucleosides (see experimental part),
we have examined their redox and photophysical properties.
As illustrated in Figure 2a, square wave voltammetry in
acetonitrile shows a well-defined M2+/3+ wave at ca. +1.0 and
+0.56 V (vs Ag/Ag+) for nucleosides 5a and 5b, respectively.17
For calibration, the ferrocene/ferrocinium (Fc/Fc+) couple,
measured under the same conditions, is also shown (+85 mV
(17) Cyclic voltammetry measurements under identical conditions show revers-
ible, one-electron M2+/3+ waves for the metal-containing nucleosides 5a
and 5b (∆Ep = 60-90 mV, ia = ic).
(18) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991-1024.
(19) Casper, J. V.; Meyer, T. J. J. Am. Chem. Soc. 1983, 105, 5583-5590.
(20) Elfring, W. H., Jr.; Crosby, G. A. J. Am. Chem. Soc. 1981, 103, 2683-
2687. Kaizu, Y.; Ohta, H.; Kobayashi, K.; Kobayashi, H.; Takuma, K.;
Matsuo, T. J. Photochem. 1985, 30, 93-103.
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A R T I C L E S
Hurley and Tor
Figure 3. Model compounds utilized for solvatochromism studies.
Table 2. Solvent-Dependent Absorption and Emission Maximaa
5a
8
9
c
c
c
λmax
(nm)
λem
(nm)
λmax
(nm)
λem
(nm)
λmax
(nm)
λem
(nm)
solvent
ꢀb
ꢀb
ꢀb
H2O
MeOH
iPrOH
344 3.2 633 330 3.0 631 264 5.1 607
354 2.7 624 342 2.7 623 266 4.9 603
358 2.4 621 344 2.4 619 266 4.6 598
CH3CN 354 2.6 624 332 2.7 629 266 4.2 607
CH2Cl2 360 2.0 607 348 2.6 609 268 5.1 598
Figure 4. Solvent-dependent emission energies of nucleoside 5a (b) and
reference compounds 8 (4) and 9 (0) plotted against the experimentally
determined ET(30) values.
a All measurements were performed on nondeoxygenated 1 × 10-5
M
solutions. b Extinction coefficients are of the phenanthroline π-π* absorp-
tion band and are reported in 10-4 × M-1 cm-1
.
c Excitation at the MLCT
band (450 nm).
the bipyridine π-π* remain unaffected. Associated with this
bathochromic shift in the absorption maxima upon decreasing
solvent polarity is a concomitant hypochromic effect. The
phenanthroline’s π-π* extinction coefficient decreases from
3.2 × 104 in water to 2.0 × 104 M-1 cm-1 in dichloromethane.
Complex 8 shows very similar behavior, and the absorption band
is red shifted from 330 nm in water to 348 nm in dichlo-
romethane. This transition also suffers a hypochromic effect,
albeit to a lower extent (Table 2). In contrast, the parent complex
9 that lacks any extended conjugation shows very little
solvatochromic behavior with the higher energy phenanthroline
π-π* transition shifting from 264 nm in water to 268 nm in
dichloromethane.
In RuII-polypyridyl complexes, it is typically the low-lying
MLCT excited state that is emissive.11a Figure 4 shows a plot
of the emission energy of 5a, 8, and 9 against the solvent polarity
ET(30) value. Both the metal-containing nucleoside 5a and the
extended complex 8 exhibit significant positive solvato-
chromism, where their emission maxima are red-shifted with
increasing solvent polarity, from below 610 nm in dichlo-
romethane to above 630 nm in water. In contrast, a minimal
solvatochromic effect on the emission maxima is observed for
the reference unsubstituted complex 9.
emission centered at 750 nm with emission quantum efficiency
of ca. 3 × 10-4, and a very short excited-state lifetime (τ )
0.078 µs) in deoxygenated acetonitrile (Figure 2d and Table
1).21 As anticipated, both nucleosides are less emissive in
aqueous solutions, with emission quantum efficiencies of 0.051
and <1 × 10-4 for the RuII and OsII nucleosides, respectively.
This is also reflected in shorter excited lifetimes in degassed
buffered solution. The excited states for both the free RuII and
OsII nucleoside are characterized by single exponential decays,
regardless of the solvent employed.
Extending the conjugation of the rigid 1,10-phenanthroline
at the 3 and 8 positions has a substantial effect on the electronic
properties of the free ligands as well as the resulting transition
metal complexes.12,22 The electronic delocalization offered by
the aryl-ethynyl substitution at these positions results in red-
shifted ligand-centered transitions and emission bands, as well
as increased sensitivity to the environment when compared to
unmodified phenanthroline ligands or phenanthroline-containing
complexes.22 To learn about the excited-state properties of the
metal-containing nucleosides, the absorption and emission
spectra of the RuII-containing nucleoside were studied in
different solvents, ranging from water to dichloromethane. For
comparison, the spectra of [(bpy)2Ru(3-phenylethynyl-phen)]2+
8 and [(bpy)2Ru(phen)]2+ 9 were also recorded. Complex 8 has
a similar extended ring structure as found in the RuII nucleoside
5a, but lacks the hydrogen-bonding capability and 2′-deoxyri-
bose moiety of the nucleoside, and derivative 9 lacks any
extended ligands (see Figure 3 for structures).
Solvatochromism results from the differential solvation of
the ground and excited states, and is intimately associated with
changes in the molecular dipole moment upon electronic
excitation. The significant hypsochromic shift observed for the
ground-state absorption spectra of nucleoside 5a and the
analogous complex 8 upon increasing solvent polarity, together
with the corresponding bathochromic shift observed in the
emission spectra, suggest destabilization of the ground state and
stabilization of the emissive excited state. This contrasts the
relatively low sensitivity of the absorption and emission spectra
of the unsubstituted [(bpy)2Ru(phen)]2+ 9 to solvent polarity.
Taken together, these observations imply that the extended
complexes undergo significant changes in dipole moment upon
electronic excitation. We therefore suggest that the emissive
charge separated MLCT excited state in complexes 5a and 8 is
not delocalized over all polypyridyl ligands, but rather the
promoted electron is mainly localized on the extended phenan-
throline ligand.24,25
In the absorption spectra, solvatochromism is primarily
manifested in changes in intensity and absorption maxima of
the band around 350 nm, which is attributed to the π-π*
transition of the extended phenanthroline ligand (Table 2).22,23
In the nucleoside 5a, this transition is red-shifted from 344 nm
in water to 360 nm in dichloromethane, while the MLCT and
(21) As with the RuII nucleoside 5a, the excited-state lifetime of the OsII-
containing nucleoside 5b (Table 1) is also longer lived than the lifetime of
the “parent” Os(bpy)3 (τ ) 0.060 and 0.019 µs in acetonitrile and water,
respectively). See Kober, E. M.; Caspar, J. V.; Lumpkin, R. S.; Meyer, T.
J. J. Phys. Chem. 1986, 90, 3722-3734; Creutz, C.; Chou, M.; Netzel, T.
L.; Okumura, M.; Sutin, N. J. Am. Chem. Soc. 1980, 102, 1309-1319.
(22) Joshi, H. S.; Jamshidi, R.; Tor, Y. Angew. Chem. Int. Ed. 1999, 38, 2721-
2725.
(23) See Supporting Information for additional experimental details.
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Ru(II) and Os(II) Nucleosides and Oligonucleotides
A R T I C L E S
To characterize the excited-state redox potential of the RuII
nucleoside 5a, its ground-state reduction waves were recorded
next to that of [(bpy)2Ru(phen)]2+ 9. The RuII nucleoside 5a
shows a reduction wave at -1.12 V, and is easier to reduce
than 9 which shows a corresponding wave at -1.28 V (vs SCE).
On the basis of these potentials and the E0-0 transition energies
(calculated at the 5% onset of emission), we have determined
the excited-state redox potentials in DMF at room tempera-
ture.23,26 Nucleoside 5a is thus slightly more oxidizing in the
excited state (*Ered ) + 1.05 V vs SCE) than the unsubstituted
complex 9 (*Ered ) + 1.03 V vs SCE).27
Oligonucleotide Synthesis and Purification. The novel
phosphoramidites 7a and 7b can be employed for the synthesis
of metal-containing oligonucleotides using the standard solid-
phase phosphoramidite DNA synthesis conditions. To control
the amount of reagents and reaction time, the coupling of the
modified base was performed manually (see experimental part).
It is important to note that the coupling efficiency of the metal-
containing phosphoramidites during DNA synthesis has been
found to be critically influenced by their purity and the amount
of (1H)-tetrazole utilized for their activation. When the highly
pure phosphoramidites 7a and 7b were coupled for 5 min in
the presence of 0.5 M (1H)-tetrazole, reaction efficiencies were
greater than 90%.28 Lower (1H)-tetrazole concentrations and
slight impurities would substantially decrease the reaction
efficiency to 50% or lower.
Figure 5. Sequences of oligonucleotides synthesized. The modified bases
are denoted as orange circles for 5a and olive-colored circles for 5b.
Numerous metal-containing oligonucleotides varying in length,
modified base position as well as composition have been
synthesized in good yields using the novel metal-containing
phosphoramidites 7a and 7b (Figure 5). Removal of the
completed oligonucleotides from the solid support using con-
centrated ammonium hydroxide was followed by incubation at
55 °C for 8-12 h to afford the crude oligonucleotides. The
metal-containing oligonucleotides were purified by denaturing
polyacrylamide gel electrophoresis and semipreparative HPLC.
HPLC analysis of the gel-purified oligonucleotides indicated
85-90% purity, which was satisfactory for many of the studies
reported below.23 Higher purity level (>95%) could be reached
by HPLC purification of a fully deprotected oligonucleotide
containing a 5′-DMT group. Acidic deprotection, followed by
an additional semipreparative HPLC purification, affords ana-
lytically pure metal-containing oligonucleotides (see Experi-
mental Section).
Figure 6. Denaturing 20% polyacrylamide gel used for the analysis of
metal-containing oligonucleotides. Lane 1 - control 20-mer 14; lane 2 -
RuII-containing oligonucleotide 15; lane 3 - OsII-containing oligonucleotide
16; lane 4 - RuII-containing oligonucleotide 17; lane 5 - dimetalated RuII-
and OsII-containing oligonucleotide 18; lane 6 - rectangles show the relative
location of the dye markers xylene cyanol and bromophenol blue.
Oligonucleotides were visualized using Stains-all.
analytical denaturing polyacrylamide gel comparing several
representative metal-containing 20-mer oligonucleotides to their
corresponding unmodified counterpart. Due to the increased
mass and reduced overall charge, the electrophoretic mobility
of the metal-containing oligonucleotides 15-17 is slower than
the corresponding unmodified oligonucleotide 14. Among the
metal-containing oligonucleotides, the internally modified DNA
oligos migrate slightly slower than the oligonucleotides with a
metal at the 5′-end. Oligonucleotides containing two metal
centers (e.g., 18) exhibit twice the retardation when compared
to a singly modified oligonucleotides 15 or 16.
Oligonucleotide Characterization. The synthesis of inter-
nally modified oligonucleotides (e.g., 11, 15) requires the
exposure of the metal-containing nucleotide to multiple coupling,
capping, and deprotection steps. The modified oligonucleotides
have therefore been carefully characterized. Figure 6 shows an
Enzymatic digestion reactions followed by HPLC analysis
of the nucleoside mixture were routinely employed to verify
the presence of the intact metal-containing nucleosides in the
modified oligonucleotides.23 Initially, a mixture of snake venom
phosphodiesterase and alkaline phosphatase was employed.
HPLC analysis indicated incomplete digestion, corresponding
to phosphodiester hydrolysis and dephosphorylation of the
natural nucleotides 3′-to the modification position. This was
confirmed by an independent digestion of an unmodified
oligonucleotide corresponding to the truncated sequence 3′ to
the metal-containing nucleoside. This may have resulted from
the inability of the phosphodiesterase, an exonuclease digesting
from the 3′-end of an oligonucleotide, to proceed past the bulky
(24) McCusker has recently demonstrated that even in Ru(bpy)3 the rapid initial
delocalization of the excited state is followed by localization of the
transferred charge into a single ligand. See Yeh, A. T.; Shank, C. V.;
McCusker, J. K. Science 2000, 289, 935-938.
(25) Additional support is obtained from transient absorption spectra taken
between 320 and 570 nm that show bleaching of the band at 350 nm,
associated with the ground-state absorption band of the conjugated
phenanthroline (see Figure S-2 in Supporting Information).
(26) Square wave voltammetry was measured in DMF containing 0.1 M
[n-Bu4N+][PF6-] as supporting electrolyte. DMF was used as it offers a
wider potential window at negative potentials that are required for the clear
recording of the reduction waves. Consequently, the emission spectra were
also taken in DMF.
(27) The value we determine for 9 is different than the value reported by Meyer
(*Ered ) + 0.78 V vs SCE).19 Note, however, that Meyer’s value was
determined by recording emission at 77 K in EtOH/MeOH glass.
(28) Coupling efficiencies were determined by quantifying the amount of trityl
cation release after each coupling step (see experimental part).
9
J. AM. CHEM. SOC. VOL. 124, NO. 14, 2002 3753

A R T I C L E S
Hurley and Tor
Table 3. Thermal Melting Temperatures (Tm) of Unmodified and
Metal-Containing DNA Duplexesa
DNA duplex
Tm (°C)
DNA duplex
Tm (°C)
10‚13
11‚13
12‚13
14‚20
15‚20
44
40
40
78
75
16‚20
17‚20
18‚20
15‚19
17‚19
75
79
70
75
78
a Tm values were determined from the first derivative of the melting curve
monitored at 260 nm. Solutions were 2 µM duplex in 100 mM NaCl, 10
mM phosphate buffer pH 7.0. Estimated error is ( 0.5 °C.
nm are attributed to the bipyridine π-π*, phenanthroline π-π*,
and the MLCT transition, respectively. To a first approximation,
this spectrum appears as a superposition of the absorption spectra
of the nucleoside and the unmodified oligonucleotide. However,
a slight red shift and a diminished intensity of the phenanthroline
π-π* band in the modified oligonucleotide 11 is observed.
These changes are in accord with the spectral features of the
parent nucleoside 5a in solvents that less polar than water. This
suggests that even in a single-stranded oligonucleotide, stacking
interactions with neighboring heterocycles may shield the
coordinated phenanthroline ligand from the aqueous bulk
solvent.
Oligonucleotide Hybridization. Metal-containing oligo-
nucleotides have been hybridized with their unmodified or
metal-containing complements using standard denaturation-
reannealing cycles. An equimolar buffered solution of the two
single-stranded oligonucleotides is heated to 90 °C and then
cooled slowly to room temperature. Thermal denaturation studies
of the annealed duplexes done between 20 and 95 °C were used
to evaluate the relative stability of the metal-modified duplexes
with respect to their unmodified counterparts. The data are
summarized in Table 3.23 The presence of a metal-containing
nucleoside in the middle of the G-rich duplex 15‚20 slightly
destabilizes the duplex when compared to 14‚20, its unmodified
analogue (∆Tm ) -3 °C). Other singly modified duplexes show
similar behavior. Incorporating two metal-containing nucleosides
on the same strand (e.g., 18‚20) results in additional destabiliza-
tion (∆Tm ) -8 °C). On the other hand, a metal-containing
nucleoside at the 5′-end of the duplex 17‚20 is slightly stabilizing
(∆Tm ) +1 °C), and a duplex containing two modification,
one terminal and one internal (17‚19), is as stable as the parent
unmodified duplex (Table 3).29
Several factors may influence the relative stability of the
modified metal-containing oligonucleotides. These include
electrostatic, steric, and solvation effects. While the introduction
of a positively charged metal complex is expected to electro-
statically stabilize the duplex, the presence of large aromatic
ligands in the major groove might cause the opposite effect by
repealing water molecules and bound small cations. To a large
extent, these opposite effects appear to cancel one another. These
considerations are likely to be less important when a metal-
containing nucleosides is incorporated at the ends of the double-
stranded structures. In this situation, electrostatic stabilization
may be the dominating factor, as experimentally observed.
To determine the effect of metal modification on the overall
structural integrity of the DNA double helix, the CD spectra of
Figure 7. HPLC chromatograms of an enzymatic digestion reaction of a
RuII-containing oligonucleotide 15 (b) vs a mixture of nucleoside standards
(a). Chromatograms were monitored at 260 nm. Impurities found in a blank
digestion mixture are marked with an asterisk.
Figure 8. UV-Vis absorption spectra of a RuII-containing oligonucleotide
11 (solid line), the corresponding unmodified oligonucleotide 10 (dashed
line), and the RuII-containing nucleoside 5a (dotted line) in 10 mM
phosphate buffer, pH 7.0, and 100 mM NaCl.
base modification. To overcome this difficulty, we added
nuclease P1, an endonuclease, to our enzyme mixture. Thus,
incubation of the metal-modified oligonucleotides with snake
venom phosphodiesterase, alkaline phosphatase, and nuclease
P1 at 37 °C for 6 to 12 h, resulted in complete phosphodiester
hydrolysis and dephophorylation to yield a mixture of the natural
and modified nucleosides. Figure 7 depicts a reversed-phase
HPLC analysis of the nucleoside mixture obtained by digesting
oligonucleotide 15, a 20-mer containing a single modification
site. A comparison to the HPLC trace obtained for a standard
mixture of the natural and synthetic nucleosides verifies the
presence and correct stoichiometry of the intact metal-containing
nucleoside 5a.23
Figure 8 compares the absorption spectrum of a metal
containing oligonucleotide to that of an unmodified oligonucle-
otide of the same length and to the parent metal-containing
nucleoside. The spectrum of 11, a RuII-containing 8-mer,
exhibits the typical transitions of the heterocyclic DNA bases
around 260 nm. Additional bands around 300, 350, and 450
(29) The destabilization observed for singly modified duplexes is far smaller
than the effect of a single mismatch on duplex stability as previously
demonstrated (ref 8).
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Ru(II) and Os(II) Nucleosides and Oligonucleotides
A R T I C L E S
Figure 9. CD spectra of a 2 µM solution of an unmodified 20-mer duplex
14‚20 (solid line) and the RuII-containing 20-mer duplex 15‚20 (dotted line)
in 10 mM phosphate buffer, pH 7.0, and 100 mM NaCl.
Figure 11. CD spectra of Λ-5a (solid line), ∆-5a (dotted line), and epimeric
5a (dashed line) in isoabsorptive acetonitrile solutions.
Figure 10. Structures of diastereomerically pure nucleosides Λ-5a and
∆-5a and their corresponding phosphoramidites Λ-7a and ∆-7a.
the duplex 15‚20 and a control unmodified duplex 14‚20 were
compared (Figure 9). The spectrum of the metal-containing
duplex exhibits a negative band at 252 nm, a crossover at 266
nm, and a positive band at 286 nm. This spectrum is similar to
the spectrum obtained for the unmodified duplex. These spectra
indicate that metal-modification placed in the major groove does
not interfere with the formation of a B-form duplex DNA.30
Diastereomerically Pure Nucleosides and Oligonucleotides.
When an octahedral metal ion is coordinated to three bidentate
ligands, two enantiomeric complexes, Λ and ∆, are formed.
To control the absolute configuration at the metal center within
the modified DNA duplexes, we have synthesized the diaste-
reomerically pure nucleosides Λ-5a and ∆-5a and their corre-
sponding phosphoramidites Λ-7a and ∆-7a (Figure 10). We
have followed an identical synthetic scheme where 5-ethynyl-
deoxyuridine 2 was cross-coupled to the enantiomerically pure
Λ-4a and ∆-4a (Scheme 1).23,31 The CD spectra of the
diastereoisomeric nucleosides Λ-5a and ∆-5a are shown in
Figure 11 and compared to a 1:1 mixture of the two epimers
(i.e., 5a). As expected, the CD spectra are dominated by the
coordinated polypyridine chromophores, and the two epimeric
nucleosides give rise to essentially mirror-imaged patterns.
Typical excitonic interactions of the ligand-centered π-π*
transitions,32 and the strong Cotton effects observed for the
visible MLCT bands, confirm the assigned absolute configu-
ration at the metal centers.31,32 Importantly, the diastereomeric
metal-containing nucleosides Λ-5a and ∆-5a emit at the same
wavelength (632 nm) upon excitation at their MLCT bands and
display identical excited-state lifetimes (1.07 µs).
Figure 12. CD spectra of 20-mer DNA duplexes incorporating disatereo-
merically pure RuII nucleosides. Shown are duplexes Λ-15‚20 (solid line),
∆-15‚20 (dashed line), and the epimeric 15‚20 duplex containing the two
enantiomeric metal centers (-‚-‚) in 10 mM phosphate buffer, pH 7.0,
and 100 mM NaCl.
Once incorporated into oligonucleotides, the subtle stereo-
chemical perturbation at the metal center is manifested at
different levels. The diastereomeric nature of the two duplexes
Λ-15‚20 and ∆-15‚20 is clearly reflected in their CD spectra
(Figure 12),33 where the strong excitonic interaction of the
coordinated ligands overlap with those derived from the DNA
bases. In the Λ-RuII-modified duplex, the strong positive
transition of the aromatic polypyridine ligands at 290 nm
overlaps with the transitions generated by the heterocyclic bases
in the same spectral region, resulting in an overall enhancement
of the band observed below 300 nm. In contrast, the ∆-RuII
modified 20-mer shows a unique pattern resulting from the
opposite excitonic transitions that overlap with the typical
B-DNA circular dichroism commonly observed for unmodified
duplexes. The mirrored CD effects of the Λ- and ∆-RuII
nucleosides can be distinctly seen in the visible region where
the DNA has no absorption. In regions where the circular
dichroism signals of the metal complexes are weak (e.g., below
250 nm), the typical CD spectrum of a B-form DNA predomi-
nates. This is particularly apparent at a crossover point in the
CD spectra of the diastereomerically pure metalated nucleosides
(e.g., at 280 nm). At this wavelength, the transitions of the
diastereoisomeric duplexes perfectly intersect that of the duplex
containing the racemic metal complexes.34
(30) As expected, no “spectral” interference is observed by the appended
polypyridine complexes since a 1:1 mixture of the ∆ and Λ metal centers
is present. This isolates the effect of the metal complex on the global
structure of the modified double helices.
(31) Tzalis, D.; Tor, Y. J. Am. Chem. Soc. 1997, 119, 852-853.
(32) Bosnich, B. Acc. Chem. Res. 1969, 2, 266-273.
(33) The prefix Λ or ∆ designates the absolute configuration at the metal center.
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J. AM. CHEM. SOC. VOL. 124, NO. 14, 2002 3755

A R T I C L E S
Hurley and Tor
Table 4. Spectroscopic Data for Metal-Containing Single Strands
and Duplexesa
c
samples
<τ>(µs)b
τ1(µs)
A1 (%)
τ1(µs)
A2(%)
λem
15
16
17
15‚20
Λ-15‚20
∆-15‚20
16‚20
15‚19
Λ-15‚19
∆-15‚19
1.17
0.032
1.23
1.12
1.10
1.13
0.027
0.43
0.39
0.42
1.54
0.08
1.63
1.91
2.03
1.78
0.08
1.02
1.07
0.94
55
15
49
28
20
40
10
37
35
37
0.72
0.024
0.85
0.83
0.87
0.71
0.021
0.08
0.07
0.08
45
85
51
72
80
60
90
63
65
63
632
740
627
629
633
622
740
624
629
616
a All measurements were performed with 2 µM deoxygenated solutions
in 100 mM NaCl, 10 mM phosphate, pH 7.0 at room temperature. Samples
15-17 and 15‚20 were excited at 450 nm. Emission decays were monitored
at 630 nm for RuII samples and 740 for OsII samples, and were analyzed
according to the biexponential function: Iem(t) ) A1exp(-t/τ1) + A2exp(-
t/τ2). Samples 15‚19 were excited at the isosbestic point for the absorption
of the RuII- and OsII-modified free nucleosides (467 nm). Emission decays
were monitored at 630 nm. b Average lifetimes <τ> were calculated from
<τ> ) A1τ1 + A2τ2. c Emission maxima were determined by steady-state
fluorescence.
Figure 13. Thermal denaturation curves of diastereomerically pure RuII-
modified DNA 20-mers in 10 mM phosphate buffer pH 7.0, 100 mM NaCl.
Shown are Λ-15‚20 (0), ∆-15‚20 (4), “racemic” 15‚20 (9), and the
unmodified control duplex 14‚20 (b).
Although the rigidly linked metal complexes are projected
into the major groove of the duplex, the ancillary bipyridine
ligands around the metal centers may influence the fit within
the groove. To test this plausible effect, the relative stability of
the diastereoisomeric duplexes have been examined. The thermal
melting curves of the diasteromerically pure RuII duplexes Λ-15‚
20 and ∆-15‚20 are very similar to that obtained for the
“racemic” duplex 15‚20 (Figure 13). All show a slight desta-
bilization of 2-3 °C when compared to the corresponding
unmodified 20-mer duplex 14‚20 (Tm ) 79 °C). Although nearly
identical Tm values for the diastereomerically pure duplexes are
obtained, subtle differences in the shape of the curves are likely
due to the chiral metal center. As discussed below, a better
insight into the differential accommodation of the enantiomeric
metal centers in the groove is obtained the investigating the
photophysical characteristics of the diastereomeric oligonucle-
otides.
Photophysical Properties of Metal-Containing Oligonucle-
otides. The absorption spectra of metalated single-stranded
oligonucleotides in degassed 10 mM phosphate buffer pH 7.0,
and 100 mM NaCl are dominated by an intense band at 260
nm resulting from the π-π* transitions of the heterocyclic DNA
bases, with a weak shoulder at 290 nm arising from the ligand-
centered transitions of the metal-containing nucleosides (see
Figure 8 for an example). Lower energy transitions at around
350 nm are characteristic of the extended conjugation of the
pyrimidine-linked phenanthroline ligand. The metalated oligo-
nucleotides also exhibit the characteristic MLCT bands of the
corresponding ruthenium or osmium metal complexes (450 and
440-500 nm, respectively).
cant differences between the excited-state behavior of the metal-
containing nucleosides and metalated oligonucleotides. The RuII-
nucleosides 5a and OsII-nucleoside 5b exhibit a monoexponen-
tial luminescence decay in aqueous solutions. In contrast, both
the RuII- and OsII-modified single strands 15 and 16, respec-
tively, display biexponential emission decays with a slightly
longer average luminescence lifetime than the nucleosides.35
Both single-stranded metal-DNA conjugates exhibit a long-
lived component of the excited state that comes closer to the
lifetime observed for the corresponding nucleosides in organic
acetonitrile solutions. This may suggest a certain degree of
protection of the complex-centered excited state from the bulk
aqueous solvent by neighboring bases.36,37
In RuII-containing oligonucleotides, hybridization to a comple-
mentary strand results in 8-15% decrease in the steady-state
emission intensity when compared to both the RuII-modified
nucleoside or single strand oligonucleotide (Figure 14). This
diminished luminescence depends on the sequence and position-
ing of the emitting nucleotide within the duplex. Time-resolved
studies show that the RuII center within the metalated duplexes
is also characterized by a biexponential luminescence decay,
accompanied by a slight decrease in the averaged excited-state
lifetime (Table 4). Unlike the single strands, the decay of both
the RuII and OsII-containing duplexes are dominated by the
short-lived component.38 Duplex formation also results in the
partial protection of the excited state from the bulk solvent. This
(35) Altering the position of the metal-complex within the oligonucleotide has
little effect on either the averaged excited-state lifetime of the relative
distribution of the biexponential components (compare 15 and 17 in Table
4).
Table 4 summarizes the steady-state and time-resolved
luminescence data for representative oligonucleotides. Excitation
of the metal-modified single strands at 450 nm leads to a strong
emission centered at 632 nm for the RuII-oligonucleotides, and
a substantially weaker emission at 740 nm for the OsII-
containing strands. The emission intensity of the polypyridyl-
metal centers in the nucleosides remains relatively unchanged
upon incorporation into single stranded oligonucleotides. Time-
resolved luminescence spectroscopy shows, however, signifi-
(36) The presence of a substantially shorter-lived component in the RuII-
containing oligonucleotides may suggest the involvement of an intrastrand
reductive quenching mechanism by guanine bases. The redox potential for
G oxidation in DNA has been estimated to be around 1.15 V vs SCE
(Johnston, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem. Soc. 1995,
117, 8933-8938; see also Leconte, J.-P.; Kirsch-De Mesmaeker, A.;
Feeney, M.; Kelly, J. M. Inorg. Chem. 1995, 34, 6481-6491). On the basis
of our measurements, nucleoside 5a displays an uphill driving force for G
oxidation of only 0.1 V, which is significantly lower than might have been
anticipated. Preliminary Stern-Volmer titrations of the nucleoside 5a with
GMP have not shown, however, appreciable quenching of the RuII-based
emission.
(37) The short-lived component of the OsII-containing oligo 16 is very close to
the lifetime of the free nucleoside 5b. Since the excited state of the OsII is
likely to be less redox active than that of the RuII, this observation further
supports the partial involvement of reductive quenching processes in RuII-
containing single stranded oligonucleotides.
(34) The typical excitonic interactions of the polypyridyl π-π* transitions and
the strong Cotton effects observed for the visible MLCT bands confirm
the retention of absolute configuration at the metal centers during automated
DNA synthesis.
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3756 J. AM. CHEM. SOC. VOL. 124, NO. 14, 2002

Ru(II) and Os(II) Nucleosides and Oligonucleotides
A R T I C L E S
Figure 14. Steady-state emission spectra of isoabsorptive solutions of RuII-
containing nucleoside 5a (solid line), RuII-containing single-strand oligo-
nucleotide 15 (dashed line), and the RuII-containing duplex 15‚20 (dotted
line) in 10 mM phosphate buffer, pH 7.0, and 100 mM NaCl. Excitation
wavelength: 450 nm.
is manifested by the presence of a long-lived component of the
excited state. In the Ru-duplex 15‚20, this component of the
excited state lifetime is nearly 2-fold longer than that of the
RuII nucleoside 5a in solution. The OsII duplex 16‚20 exhibits
the same behavior, with an excited state lifetime component
nearly 3-fold longer than the OsII nucleoside 5b (Table 4).
Through the incorporation of octahedral RuII centers with
predetermined absolute configuration, it is possible to more
closely probe the interaction between the metal complexes and
the DNA duplex. The photophysical data for the diastereomeri-
cally pure duplexes Λ-15‚20 and ∆-15‚20 were compared to
those of the epimeric 15‚20 duplex (Table 4). When these
diastereomeric duplexes were irradiated at 450 nm, steady-state
luminescence revealed a 10-nm difference in their emission
wavelengths (Table 4 and Figure 15). The Λ-duplex exhibits a
strong emission at 633 nm, while the ∆-duplex showed a blue-
shifted emission of equal intensity at 622 nm. The emission of
the epimeric mixture 15‚20 was centered around 629 nm, thus
falling between those of the diastereomerically pure duplexes
(Figure 15). As discussed above, the metal-based emission of
the free RuII nucleoside is sensitive to solvent polarity. The
metal-based emission of the Λ-RuII center at 633 nm, when
attached to the DNA via the ethynyl linker, is indicative of a
more polar environment that would stabilize the charge separated
excited state. In contrast, the 622 nm emission of the ∆-RuII
center, when incorporated in DNA, suggests a less polar
environment for the complex. Importantly, the emission wave-
lengths of the diastereomerically pure free nucleosides in
aqueous solution are identical to that of the “racemic” nucleo-
side, all emitting at 632 nm (Figure 15).
Figure 15. Top: Steady-state emission spectra of isoabsorptive solutions
of duplexes Λ-15‚20 (solid line), ∆-15‚20 (dotted line) and epimeric 15‚
20 (dashed line). Bottom: Steady-state emission spectra of isoabsorptive
solutions of nucleosides Λ-5a (solid line), ∆-5a (dotted line), and epimeric
5a (dashed line). All spectra were taken in 10 mM phosphate buffer, pH
7.0, and 100 mM NaCl. Excitation wavelength: 450 nm.
distribution of the components of the biexponential decay is
nearly the average of the results obtained with the diastereo-
merically pure duplexes, with τ1 ) 1.91 µs (28%) and τ2 )
0.83 µs (72%).39 The larger contribution of the long-lived
component in the excited ∆-Ru duplex, together with its shorter
emission wavelength, suggest that the ∆-Ru complex fits more
tightly into the major groove than does the Λ-Ru complex.40,41
Summary
A novel and powerful approach for the site-specific incor-
poration of polypyridylmetal complexes into synthetic oligo-
nucleotides using automated phosphoramidite chemistry has
been established. The methodology is based on metal-containing
nucleosides, where the complexes [(bpy)2M(3-ethynyl-1,10-
phenanthroline)]2+ (M ) Ru, Os) are covalently attached to the
5-position in 2′-deoxyuridine. These RuII and OsII building
(39) It is important to note that in all duplexes discussed, the absorption bands
at 350 nm are equally suppressed when compared to the nucleoside 5a in
solution. This suggests that the differences in the excited-state lifetimes in
the diastereomerically pure oligonucleotides are not due to differential
shielding of the excited complex from solvent, but rather can be attributed
to a different rotamer population around the ethynyl linkage enforced by
the groove walls.
As seen in the time-resolved luminescence studies of the
metal-modified single strands, the duplexes 15‚20, Λ-15‚20, and
∆-15‚20 also exhibit biexponential decay kinetics (Table 4).
Although the average luminescence lifetime found for the Λ-
and ∆-RuII duplexes are nearly equal (1.10 and 1.13 µs,
respectively), the relative contributions of the long- and short-
lived decay components associated with the diastereomerically
pure duplexes are distinctly different. In the Λ-RuII duplex, the
RuII excited state decays with τ1 ) 2.03 µs (20%) and τ2 )
0.87 µs (80%). The ∆-RuII duplex decays with τ1 ) 1.78 µs
(40%) and τ2 ) 0.71 µs (60%). In the racemic RuII duplex, the
(40) The observation that the ∆ isomer is better protected from solvent agrees
2+
with Barton’s early observations with noncovalently bound Ru(phen)3
.
See Barton, J. K.; Danishefsky, A. T.; Goldberg, J. M. J. Am. Chem. Soc.
1984, 106, 2172-2176.
(41) To evaluate the influence of the absolute configuration at the RuII-donor
metal center on quenching efficiency, the bis-hetero-metalated duplex 15‚
19 has been investigated (Table 4). Duplex 15‚20 with the appropriate
stereochemistry at the donor center has served as a reference. The RuII
emission in duplex ∆-15‚19 suffers 80% quenching while the fraction
quenched in the Λ-15‚19 duplex amounts to only 72%. The duplex
incorporating a racemic RuII center exhibits 75% quenching (fraction
quenched ) 1 - IRu-Os/IRu where I represents the integrated emission curve;
error is ( 3%). In agreement with the lifetime measurements discussed
above, the ∆ complex appears to be better accommodated within the chiral
major groove. These observations indicate that quenching by the racemic
OsII center is dependent, albeit to a small extent, on the absolute
configuration at the RuII-donor metal center. The relatively small magnitude
of the effect suggests, therefore, that metal-containing oligonucleotides with
racemic metal centers can be utilized for most investigations of donor-
acceptor interactions in such assemblies.
(38) This could indicate increased electronic coupling of the metalated base
with G residues within the base pair stack, resulting in quenching of the
excited state.36
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J. AM. CHEM. SOC. VOL. 124, NO. 14, 2002 3757

A R T I C L E S
Hurley and Tor
N,N′-dimethylformamide, stored over 4-Å molecular sieves, were
obtained from Fluka. Unless otherwise specified, materials obtained
from commercial suppliers were used without further purification.
blocks are converted into their phosphoramidites and are utilized
for the efficient synthesis of modified oligonucleotides.
The metal-containing oligonucleotides form stable duplexes
that retain the B-form conformation. The relative stability of
the modified duplexes is dependent on the number and position
of the metal centers as well as on the sequence composition of
the oligonucleotides. The absolute configuration of these rigidly
linked metal complexes has little effect on the thermal stability
of the oligonucleotides, although photophysical studies suggest
the ∆ complex is slightly better accommodated within the major
groove.
The RuII center in the nucleoside, oligonucleotides, and
double-stranded DNA sequences is luminescent. Absorption and
emission spectroscopies demonstrate that the emissive charge
separated MLCT excited state is susceptible to environment
polarity and suggest that the promoted electron is localized on
the extended heterocycle-linked phenanthroline. Delocalization
of this excited-state electron density through the conjugated
acetylene linker and onto the base-stacked heterocycle is a key
feature for exploring energy and electron-transfer processes
through the DNA base stack.42
Compound 3. Nucleoside 214,15 (500 mg, 1.98 mmol), 4,4′-
dimethoxytrityl chloride (772 mg, 2.28 mmol), and 4-(dimethylamino)-
pyridine (22 mg, 0.20 mmol) were suspended in anhydrous pyridine
(7 mL) and triethylamine (250 µL). The solution was stirred for 2 h at
room temperature under argon, then evaporated under reduced pressure
to an amber foam. The crude product was purified by flash chroma-
tography on silica gel. The column was eluted first with 0.5%
triethylamine, 1% methanol in dichloromethane to remove impurities,
followed by stepwise increase to 4% methanol. The product was
obtained as a light yellow foam in 92% yield (987 mg). TLC (7%
MeOH/CH2Cl2) Rf ) 0.26; IR (KBr): 2115 cm-1 (-C≡CH); 1H NMR
(CD3CN, 400 MHz): δ 7.95 (s, 1H), 7.45 (d, J ) 7.7 Hz, 2H), 7.35
(d, J ) 8.8 Hz, 4H), 7.31 (t, J ) 8.1 Hz, 2H), 7.23 (t, J ) 7.3 Hz, 1H),
6.88 (dd, J ) 8.8, 2.6 Hz, 4H), 6.13 (t, J ) 6.6 Hz, 1H, H-1′), 4.42-
4.46 (m, 1H, H-3′), 3.96-3.98 (m, 1H, H-4′), 3.77 (s, 6H, -OCH3),
3.32 (dd, J ) 11.0, 4.8 Hz, 1H, H-5′A), 3.21 (dd, J ) 10.6, 2.6 Hz, 1H,
H-5′B), 3.16 (s, 1H, -C≡CH), 2.23-2.32 (m, 2H, H-2′); 13C NMR
(CD3CN, 101 MHz): δ 41.53, 55.96, 64.46, 72.01, 76.27, 82.66, 86.63,
87.41, 87.59, 99.23, 114.27, 114.29, 128.01, 129.09, 131.15, 131.21,
136.80, 137.05, 145.33, 146.13, 150.56, 159.89, 162.82; ESI MS calcd
for C32H30N2O7Na 577.580 [MNa]+, found 577.
Experimental Section
Compound 5a. RuII complex 4a13 (150 mg, 0.16 mmol), (dppf)-
PdCl2‚CH2Cl2 (6 mg, 0.008 mmol), and CuI (1.5 mg, 0.008 mmol)
were dissolved in a degassed solution of anhydrous DMF (1.5 mL)
and triethylamine (150 µL) under argon. A solution of nucleoside 2
(43 mg, 0.17 mmol) in a degassed solution of anhydrous DMF (1.5
mL) and triethylamine (150 µL) was added over 5 min. The solution
was stirred under argon for 3 h at room temperature, and then
evaporated under reduced pressure. The crude reaction mixture was
purified by flash chromatography (silica gel, 1% saturated aqueous
KPF6, 6% water in acetonitrile). The product fractions were concen-
trated, dissolved in cold 1:3 acetone/dichloromethane, and filtered to
remove excess KPF6. The product solution was dried, suspended in
water, and filtered. The product 5a was obtained as a mixture of
diastereomers in 81% yield (143 mg) as red solid. TLC (1% saturated
General. NMR spectra were obtained on a Varian Mercury 400 MHz
1
spectrometer. The H chemical shifts of nonexchangeable protons are
expressed relative to the residual solvent signal of acetonitrile at δ 1.94.
The 13C chemical shifts are referenced relative to CD3CN at δ 1.30.
The 31P chemical shifts for metal containing species were measured
using the PF6- counterion as an internal standard. Infrared spectra were
obtained on a Perkin-Elmer 1600 FT-IR spectrometer. Absorption
spectra were recorded using an Hewlett-Packard 8452A diode array
spectrophotometer. Emission spectra were collected on a Perkin-Elmer
LS 50B luminescence spectrophotometer. Optical rotations were
determined at ambient temperature using a Perkin-Elmer 241 polarim-
eter. Electrochemical measurements were performed with a BAS CV-
50W system using platinum working and auxiliary electrodes with an
Ag/AgNO3/CH3CN reference electrode at a 100 mV/s scan rate. The
experiments were conducted using 1 mM degassed acetonitrile solutions
with 0.1 M [n-Bu4N+][PF6-] as the supporting electrolyte unless
otherwise stated. Ferrocene was used as an external reference. Mass
spectral analyses were performed by the University of California-
Riverside Mass Spectrometry Facility, and the mass value for the most
intense signal of an isotopomeric cluster is reported (see Supporting
Information of ref 8 for representative experimental and simulated
spectra). HPLC analyses were performed on a Hewlett-Packard 1050
series analytical HPLC using a Vydac reversed-phase column (0.46 ×
25 cm, 5µ C18-silica). HPLC grade solvents were obtained from Fisher
Scientific. Flash chromatography was performed using silica gel 60
(230-400 mesh, Merck) or activated basic aluminum oxide (50-200
micron, Acros). Thin-layer chromatography (TLC) was performed using
silica gel 60 F254 precoated plates (Merck). Visualization was done by
UV (254 nm) and by treatment with a solution of 4 g of cerium (IV)
sulfate, 100 g of ammonium molybdate, 100 mL of sulfuric acid, and
900 mL of water, followed by heating. 4,4′-Dimethoxytrityl chloride
and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite were ob-
tained from Aldrich. All reaction flasks used for DMT-derivatized
compounds were treated with a concentrated NaOH prior to use.
Nucleoside phosphoramidite monomers as well as ancillary reagents
for nucleic acid synthesis were obtained from Prime Synthesis, Inc.
(Aston, PA). Anhydrous acetonitrile, pyridine, dichloromethane, and
aqueous KNO3/10% H2O/CH3CN) Rf ) 0.13; [R]21 ) -38, (c ) 0.1
D
1
mg/mL in CH3CN); IR (KBr): 2216 cm-1 (-C≡C-); H NMR (CD3-
CN, 400 MHz): δ 9.34 (s, 1H), 8.65 (d, J ) 1.5 Hz, 1H), 8.62 (dd, J
) 8.3, 1.0 Hz, 1H), 8.52-8.57 (m, 3H), 8.50 (d, J ) 8.3 Hz, 1H), 8.34
(s, 1H), 8.27 (d, J ) 8.8 Hz, 1H), 8.20 (d, J ) 8.8 Hz, 1H), 8.08-8.13
(m, 4H), 8.02 (dt, J ) 8.1, 1.5 Hz, 1H), 8.01 (dt, J ) 8.1, 1.5 Hz, 1H),
7.87 (d, J ) 4.9 Hz, 1H), 7.81 (d, J ) 4.9 Hz, 1H), 7.75 (dd, J ) 8.3,
5.4 Hz, 1H), 7.65 (d, J ) 5.9 Hz, 1H), 7.53 (d, J ) 4.9 Hz, 1H), 7.44-
7.48 (m, 2H), 7.24-7.26 (m, 2H), 6.11 (dt, J ) 6.6, 1.6 Hz, 1H, H-1′),
4.33-4.36 (m, 1H, H-3′), 3.90-3.92 (m, 1H, H-4′), 3.70-3.78 (m,
2H, H-5′), 2.26-2.31 (m, 1H, H-2′A), 2.14-2.20 (m, 1H, H-2′B); 13C
NMR (CD3CN, 101 MHz): δ 41.52, 61.89, 61.91, 70.98, 71.00, 86.77,
86.81, 88.09, 88.11, 88.51, 88.53, 89.32, 98.02, 98.04, 122.85, 124.95,
125.04, 125.10, 125.12, 127.05, 128.17, 128.24, 128.37, 128.39, 128.47,
129.71, 131.10, 132.14, 137.67, 138.57, 138.59, 138.72, 138.73, 139.06,
139.09, 146.31, 147.01, 148.06, 150.01, 152.66, 152.70, 152.89, 152.93,
153.00, 153.60, 154.28, 157.63, 157.76, 157.99, 158.02, 161.76; UV
(CH3CN): λmax nm (ꢀ × 10-3) 242 (39.7), 256 (39.0), 286 (69.3), 352
(27.1), 450 (12.6); ꢀ260 ) 36,500; ESI MS calcd for C43H34F6N8O5PRu
[M]+ 988.815, found 989 [M]+, 422 [M]2+
.
Compound Λ-5a. The nucleoside Λ-5a was obtained by the same
procedure described above for 5a from nucleoside 2 and Λ-4a (see ref
31 for the resolution procedure). The product was obtained in a 71%
yield as a red foam. TLC (1% saturated aqueous KNO3/10% H2O/
CH3CN) Rf ) 0.13; [R]21 ) +451, (c ) 0.1 mg/mL in CH3CN); CD
D
(42) The effect of donor/acceptor separation, relative placement, sequence, and
degree of electronic conjugation on photophysical processes in metalated
DNAs is systematically investigated in our laboratories. These results will
be communicated in due course.
(CH3CN) 210 (0), 220 (-25.1), 244 (-14.2), 272 (-69.9), 281 (0),
292 (181.8), 332 (13.6), 377 (0), 424 (-14.2), 454 (0), 475 (11.2); 1H
NMR (CD3CN, 400 MHz): δ 8.65 (d, J ) 1.5 Hz, 1H), 8.60 (dd, J )
9
3758 J. AM. CHEM. SOC. VOL. 124, NO. 14, 2002

Ru(II) and Os(II) Nucleosides and Oligonucleotides
A R T I C L E S
8.1, 1.1 Hz, 1H), 8.47-8.55 (m, 4H), 8.40 (s, 1H), 8.25 (d, J ) 8.8
Hz, 1H), 8.19 (d, J ) 8.8 Hz, 1H), 8.06-8.12 (m, 4H), 8.00 (dt, J )
7.7, 1.5 Hz, 1H), 7.99 (dt, J ) 7.1, 1.5 Hz, 1H), 7.86 (d, J ) 5.1 Hz,
1H), 7.79 (d, J ) 4.4 Hz, 1H), 7.73 (dd, J ) 8.1, 5.1 Hz, 1H), 7.64 (d,
J ) 4.4 Hz, 1H), 7.50 (d, J ) 4.8 Hz, 1H), 7.46 (dt, J ) 6.8, 1.1 Hz,
1H), 7.44 (dt, J ) 6.0, 1.1 Hz, 1H), 7.23 (t, J ) 6.6 Hz, 2H), 6.07 (t,
J ) 5.9 Hz, 1H, H-1′), 4.32-4.36 (m, 1H, H-3′), 3.86-3.87 (m, 1H,
H-4′), 3.71-3.78 (m, 2H, H-5′), 2.26-2.32 (m, 1H, H-2′A), 2.14-
2.19 (m, 1H, H-2′B); 13C NMR (CD3CN, 101 MHz): δ 41.51, 61.69,
70.61, 86.75, 88.15, 88.63, 89.53, 98.03, 122.99, 125.05, 125.14, 125.22,
125.25, 127.14, 128.29, 128.33, 128.49, 128.51, 128.60, 129.80, 131.22,
132.24, 137.79, 138.67, 138.71, 138.83, 138.86, 139.19, 146.52, 147.10,
148.19, 150.14, 152.80, 152.81, 153.04, 153.16, 153.71, 154.42, 157.76,
157.91, 158.15, 161.88; ESI MS calcd for C43H34F6N8O5PRu [M]+
CN): λmax nm (ꢀ × 10-3) 242 (37.9), 254 (35.6), 290 (64.3), 358 (25.9),
436 (11.1), 484 (11.0), 592 (3.1); ꢀ260 ) 33 200; ESI MS calcd for
C43H34F6N8O5OsP [M]+ 1077.975, found 1078 [M]+, 466 [M]2+
.
Compound 6a. A mixture of nucleoside 3 (484 mg, 0.87 mmol),
4a (800 mg, 0.83 mmol), Pd(PPh3)2Cl2 (29 mg, 0.042 mmol), and CuI
(8 mg, 0.042 mmol) was dissolved in a degassed solution of anhydrous
DMF (15 mL) and triethylamine (2 mL) under argon. The mixture was
sonicated for 8 h at 50 °C under an argon atmosphere and the solvent
was removed under reduced pressure. The crude reaction mixture was
purified by flash chromatography (basic alumina, 0.5% saturated
aqueous KPF6, 2.5% water in acetonitrile). The product fractions were
concentrated, dissolved in dichloromethane, and filtered to remove
excess KPF6. The product 6a was obtained as a mixture of diastereomers
in 83% yield (600 mg) as a red foam. TLC (1% saturated aqueous
KNO3/10% H2O/CH3CN) Rf ) 0.28; IR (KBr): 2207 cm-1 (-C≡C-);
1H NMR (CD3CN, 400 MHz): δ 8.61 (dd, J ) 2.9, 1.2 Hz, 0.5H),
8.59 (dd, J ) 2.9, 1.2 Hz, 0.5H), 8.45-8.52 (m, 3H), 8.39 (t, J ) 7.1
Hz, 1H), 8.38 (s, 0.5H), 8.27 (s, 0.5H), 8.23 (d, J ) 8.8 Hz, 0.5H),
8.21 (d, J ) 8.8 Hz, 0.5H), 7.93-8.11 (m, 6.5H), 7.84 (d, J ) 5.6 Hz,
0.5H), 7.83 (d, J ) 8.8 Hz, 0.5H), 7.77 (d, J ) 5.6 Hz, 0.5H), 7.68-
7.75 (m, 2H), 7.55 (d, J ) 5.6 Hz, 0.5H), 7.49-7.53 (m, 1.5H), 7.35-
7.45 (m, 5.5H), 7.30-7.34 (m, 2H), 7.22-7.27 (m, 2.5H), 7.18 (t, J )
5.9 Hz, 0.5H), 7.09 (app quin, J ) 7.4 Hz, 2H), 6.97 (t, J ) 7.4, 0.5
Hz), 6.84 (t, J ) 7.4 Hz, 0.5H), 6.78 (d, J ) 9.3 Hz, 1H), 6.74 (d, J
) 9.3 Hz, 1H), 6.71 (d, J ) 8.8 Hz, 1H), 6.57 (d, J ) 9.3 Hz, 1H),
6.14 (dt, J ) 6.3, 1.0 Hz, 1H, H-1′), 4.58 and 4.49 (quin, J ) 3.9 Hz,
1H, H-3′), 4.01-4.04 and 3.95-3.97 (m, 1H, H-4′), 3.58, 3.57, 3.56,
and 3.47 (s, 6H, -OCH3), 3.40 and 3.37 (d, J ) 4.2 Hz, 1H, 3′-OH),
3.34 and 3.14 (dd, J ) 10.8, 2.0 Hz, 1H, H-5′A), 3.31 and 3.21 (dd, J
) 10.8, 2.0 Hz, 1H, H-5′B), 2.30-2.40 (m, 4H, H-2′); 13C NMR (CD3-
CN, 101 MHz): δ 42.05, 42.08, 55.62, 55.71, 55.76, 63.49, 63.71,
71.36, 71.41, 86.56, 87.14, 87.34, 87.40, 87.44, 87.49, 88.11, 88.20,
89.00, 89.23, 98.20, 98.32, 113.95, 113.98, 114.02, 114.08, 122.53,
122.61, 124.79, 124.95, 125.00, 125.03, 125.08, 127.01, 127.48, 127.76,
128.12, 128.17, 128.22, 128.26, 128.28, 128.30, 128.39, 128.45, 128.61,
18.69, 128.76, 129.53, 129.58, 130.39, 130.54, 130.71, 130.75, 130.81,
132.11, 132.18, 136.23, 136.38, 136.57, 136.80, 137.63, 137.67, 138.58,
138.62, 138.64, 138.69, 139.62, 140.00, 145.27, 145.40, 145.61, 146.03,
146.74, 146.80, 147.93, 149.95, 152.48, 152.56, 152.61, 152.71, 152.76,
152.79, 152.82, 153.54, 153.70, 157.55, 157.58, 157.67, 157.70, 157.83,
157.88, 159.12, 159.29, 159.32, 159.35, 161.91, 161.94; ESI MS calcd
988.815, found 989 [M]+, 422 [M]2+
.
Compound ∆-5a. The nucleoside ∆-5a was obtained by the same
procedure described above for 5a from nucleoside 2 and ∆-4a (see ref
31 for the resolution procedure). The product was obtained in a 64%
yield as a red foam. TLC (1% saturated aqueous KNO3/10% H2O/
CH3CN) Rf ) 0.13; [R]21 ) -594, (c ) 0.1 mg/mL in CH3CN); CD
D
(CH3CN) 209(0), 219 (31.4), 244 (18.7), 272 (76.7), 281 (0), 291
1
(-190.8), 332 (-13.7), 376 (0), 421 (15.7), 453 (0), 475 (12.6); H
NMR (CD3CN, 400 MHz): δ 9.34 (s, 1H), 8.65 (d, J ) 1.5 Hz, 1H),
8.61 (dd, J ) 8.2, 1.1 Hz, 1H), 8.48-8.56 (m, 4H), 8.35 (s, 1H), 8.26
(d, J ) 8.8 Hz, 1H), 8.20 (d, J ) 8.8 Hz, 1H), 8.07-8.13 (m, 4H),
8.02 (dt, J ) 7.7, 1.5 Hz, 1H), 8.00 (dt, J ) 7.9, 1.5 Hz, 1H), 7.86 (d,
J ) 4.8 Hz, 1H), 7.79 (d, J ) 5.1 Hz, 1H), 7.74 (dd, J ) 8.4, 5.5 Hz,
1H), 7.64 (d, J ) 5.1 Hz, 1H), 7.52 (d, J ) 5.1 Hz, 1H), 7.46 (dt, J )
6.2, 1.5 Hz, 1H), 7.45 (dt, J ) 6.0, 1.1 Hz, 1H), 7.24 (t, J ) 6.2 Hz,
2H), 6.10 (t, J ) 6.2 Hz, 1H, H-1′), 4.32-4.36 (m, 1H, H-3′), 3.89-
3.91 (m, 1H, H-4′), 3.69-3.78 (m, 2H, H-5′), 2.26-2.32 (m, 1H, H-2′A),
2.14-2.19 (m, 1H, H-2′B); 13C NMR (CD3CN, 101 MHz): δ 41.56,
61.97, 71.07, 86.91, 88.21, 88.65, 89.40, 98.16, 122.99, 125.11, 125.20,
125.25, 125.28, 127.21, 128.33, 128.41, 128.52, 128.55, 128.63, 129.87,
131.26, 132.31, 137.84, 138.74, 138.76, 138.89, 138.90, 139.27, 146.51,
147.20, 148.24, 150.20, 152.84, 152.89, 153.12, 153.18, 153.79, 154.47,
157.82, 157.94, 158.18, 158.20, 162.00; ESI MS calcd for C43H34F6N8O5-
PRu [M]+ 988.815, found 989 [M]+, 422 [M]2+
.
Compound 5b. A mixture of nucleoside 2 (16 mg, 0.062 mmol),
OsII complex 4b (50 mg, 0.048 mmol), Pd(dppf)Cl2‚CH2Cl2 (3 mg,
0.004 mmol), and CuI (0.5 mg, 0.003 mmol) was dissolved in a
degassed solution of anhydrous DMF (1.5 mL) and triethylamine (100
µL) under argon. The solution was sonicated for 8 h at 50 °C, then the
solvent removed under reduced pressure. The crude reaction mixture
was purified by flash chromatography (silica gel, 1% saturated aqueous
KPF6, 8% water in acetonitrile). The product fractions were concen-
trated, dissolved in cold 1:3 acetone/dichloromethane, and filtered to
remove excess KPF6. The product solution was dried, suspended in
water, and filtered. The product 5b was obtained as a mixture of
diastereomers in 86% yield (50 mg) as a dark brown foam. TLC (1%
saturated aqueous KNO3/10% H2O/CH3CN) Rf ) 0.13; IR (KBr): 2216
for C64H52F6N8O7PRu [M]+ 1291.182, found 1291 [M]+, 573 [M]2+
.
Compound Λ-6a. Product Λ-6a was obtained by the same procedure
described above for 6a from compounds 3 and Λ-4a (see ref 31 for
the resolution procedure). The product was obtained as a red foam in
74% yield. TLC (1% saturated aqueous KNO3/10% H2O/CH3CN) Rf
1
) 0.28; H NMR (CD3CN, 400 MHz): δ 8.61 (dd, J ) 7.8, 1.0 Hz,
1H), 8.53 (d, J ) 8.3 Hz, 1H), 8.47-8.50 (m, 3H), 8.40 (s, 1H), 8.23
(d, J ) 8.8 Hz, 1H), 8.10 (dt, J ) 7.8, 1.0 Hz, 1H), 7.99-8.07 (m,
4H), 7.97 (d, J ) 1.5 Hz, 1H), 7.85-7.86 (m, 2H), 7.77 (d, J ) 4.9
Hz, 1H), 7.74 (dd, J ) 7.8, 4.9 Hz, 1H), 7.52 (t, J ) 4.4 Hz, 2H),
7.42-7.46 (m, 5H), 7.39 (d, J ) 9.3 Hz, 2H), 7.33 (d, J ) 8.8 Hz,
2H), 7.26 (dt, J ) 5.4, 1.0 Hz, 1H), 7.19 (dt, J ) 5.8, 1.5 Hz, 1H),
7.08 (t, J ) 7.3 Hz, 2H), 6.84 (t, J ) 7.3 Hz, 1H), 6.79 (d, J ) 8.8 Hz,
2H), 6.74 (d, J ) 8.8 Hz, 2H), 6.14 (t, J ) 6.4 Hz, 1H, H-1′), 4.50-
4.52 (m, 1H, H-3′), 4.02-4.04 (m, 1H, H-4′), 3.59 and 3.57 (s, 6H,
-OCH3), 3.40 (d, J ) 4.4 Hz, 1H, 3′-OH), 3.34 (dd, J ) 10.8, 2.4 Hz,
1H, H-5′A), 3.15 (dd, J ) 10.8, 3.4 Hz, 1H, H-5′B), 2.37-2.42 (m, 1H,
H-2′A), 2.32-2.36 (m, 1H, H-2′B); 13C NMR (CD3CN, 101 MHz): δ
42.08, 55.71, 55.76, 63.70, 71.42, 87.13, 87.38, 87.50, 88.07, 89.21,
98.19, 113.97, 114.07, 122.52, 124.95, 124.99, 125.02, 125.07, 126.99,
127.47, 128.11, 128.25, 128.29, 128.38, 128.68, 129.51, 130.54, 130.70,
130.81, 132.10, 136.21, 136.56, 137.62, 138.55, 138.60, 138.67, 138.70,
139.64, 145.59, 146.02, 146.73, 147.92, 149.84, 152.55, 152.61, 152.71,
1
cm-1 (-C≡C-); H NMR (CD3CN, 400 MHz): δ 9.31 (s, 1H), 8.49-
8.54 (m, 3H), 8.47 (d, J ) 8.3 Hz, 1H), 8.42 (d, J ) 1.5 Hz, 1H), 8.40
(d, J ) 8.3 Hz, 1H), 8.35 (s, 1H), 8.26 (d, J ) 8.8 Hz, 1H), 8.19 (d,
J ) 8.8 Hz, 1H), 8.03 (s, 1H), 8.00 (d, J ) 4.9 Hz, 1H), 7.90-7.93
(m, 2H), 7.80-7.85 (m, 2H), 7.78 (d, J ) 5.9 Hz, 1H), 7.71 (d, J )
5.4 Hz, 1H), 7.67 (dd, J ) 8.3, 5.4 Hz, 1H), 7.53 (d, J ) 5.9 Hz, 1H),
7.41 (d, J ) 5.4 Hz, 1H), 7.35-7.39 (m, 2H), 7.13-7.17 (m, 2H),
6.11 (t, J ) 6.3 Hz, 1H, H-1′), 4.32-4.35 (m, 1H, H-3′), 3.89-3.91
(m, 1H, H-4′), 3.70-3.77 (m, 2H, H-5′), 2.26-2.30 (m, 1H, H-2′A),
2.15-2.20 (m, 1H, H-2′B); 13C NMR (CD3CN, 101 MHz): δ 41.54,
61.90, 70.95, 86.82, 86.85, 88.02, 88.04, 88.59, 88.61, 89.49, 98.08,
123.19, 125.24, 125.29, 125.38, 125.40, 127.26, 128.71, 128.76, 128.82,
128.91, 129.88, 131.42, 132.54, 137.45, 138.04, 138.10, 138.22, 138.93,
146.45, 149.59, 150.11, 150.38, 151.84, 152.04, 152.13, 152.17, 152.31,
153.08, 153.86, 159.63, 159.79, 159.96, 159.98, 161.97; UV (CH3-
9
J. AM. CHEM. SOC. VOL. 124, NO. 14, 2002 3759

A R T I C L E S
Hurley and Tor
152.82, 153.52, 157.57, 157.70, 157.82, 157.88, 159.33, 161.71; ESI
MS calcd for C64H52F6N8O7PRu [M]+ 1291.182, found 1291 [M]+, 573
dried over Na2SO4 and concentrated. The crude product was purified
by flash chromatography (basic alumina, 0.5% saturated aqueous KPF6,
2.5% water in acetonitrile). The product fractions were concentrated,
dissolved in dichloromethane, and filtered to remove excess KPF6. The
product 7a was obtained in 80% yield (136 mg) as a red foam. TLC
[M]2+
.
Compound ∆-6a. Product ∆-6a was obtained by the same procedure
described above for 6a from compounds 3 and ∆-4a (see ref 31 for
the resolution procedure). The product was obtained as a red foam in
68% yield. TLC (1% saturated aqueous KNO3/10% H2O/CH3CN) Rf
1
(1% saturated aqueous KNO3/10% HO/CH3CN) Rf ) 0.34; H NMR
(CD3CN, 400 MHz): δ 8.60-8.63 (m, 1H), 8.49-8.54 (m, 3H), 8.45
(d, J ) 6.8 Hz, 0.5H), 8.41 (t, J ) 7.3 Hz, 1H), 8.33 (d, J ) 6.8 Hz,
0.5H), 8.24 (d, J ) 9.4 Hz, 0.5H), 8.21 (d, J ) 9.4 Hz, 0.5H), 7.96-
8.12 (m, 6H), 7.90 (d, J ) 9.3 Hz. 0.5H), 7.86 (d, J ) 5.4 Hz), 7.72-
7.82 (m, 3H), 7.50-7.57 (m, 2H), 7.25-7.46 (m, 10H), 7.20 (t, J )
6.8 Hz, 0.5H), 7.13 (t, J ) 7.8 Hz, 1H), 7.07 (t, J ) 7.3 Hz, 1H), 6.98
(t, J ) 6.8 Hz, 0.5H), 6.82 (d, J ) 8.8 Hz, 1H), 6.72-6.80 (m, 2H),
6.61 (d, J ) 8.3 Hz, 1H), 6.14-6.19 (m, 1H, H-1′), 4.72-4.76 and
4.67-4.71 (m, 1H, H-3′), 4.09-4.20 (m, 1H, H-4′), 3.18-3.83 (m,
12H, -NCH(CH3)2, -OCH2CH2CN, OCH3, H-5′), 2.63-2.66 and 2.50-
2.54 (m, 2H, -OCH2CH2CN), 2.47-2.58 (m, 2H, H-2′), 1.13-1.19 (m,
1
) 0.28; H NMR (CD3CN, 400 MHz) δ 8.62 (d, J ) 8.3 Hz, 1H),
8.51 (d, J ) 8.3 Hz, 1H), 8.49 (d, J ) 7.8 Hz, 1H), 8.40 (t, J ) 8.3
Hz, 2H), 8.29 (d, J ) 1.5 Hz, 1H), 8.25 (dd, J ) 8.9, 1.5 Hz, 1H),
8.10 (t, J ) 7.8 Hz, 1H), 8.05 (d, J ) 5.4 Hz, 1H), 7.97-8.03 (m,
4H), 7.96 (d, J ) 8.8 Hz, 1H), 7.80 (d, J ) 5.4 Hz, 1H), 7.72-7.75
(m, 3H), 7.58 (d, J ) 5.8 Hz, 1H), 7.55 (d, J ) 5.4 Hz, 1H), 7.41-
7.45 (m, 3H), 7.37 (t, J ) 7.8, 1H), 7.34 (dd, J ) 8.8, 1.5 Hz, 2H),
7.24-7.28 (m, 4H), 7.12 (t, J ) 7.3 Hz, 2H), 6.98 (dt, J ) 7.3, 1.0
Hz, 1H), 6.72 (dd, J ) 8.8, 1.0 Hz, 2H), 6.57 (dd, J ) 8.8,1.0 Hz,
2H), 6.15 (t, J ) 6.3 Hz, 1H, H-1′), 4.59-4.61 (m, 1H, H-3′), 3.96-
3.98 (m, 1H, H-4′), 3.57 and 3.48 (s, 6H, -OCH3), 3.32 (dd, J ) 10.7,
2.0 Hz, 1H, H-5′A), 3.22 (dd, J ) 10.7, 2.0 Hz, 1H, H-5′B), 2.36-2.39
(t, J ) 4.9 Hz, 2H, H-2′); 13C NMR (CD3CN, 101 MHz) δ 42.09,
55.67, 55.75, 63.52, 71.43, 86.61, 87.40, 87.51, 88.24, 89.03, 98.40,
114.03, 114.11, 122.69, 124.88, 125.04, 125.08, 127.11, 127.84, 128.25,
128.31, 128.38, 128.50, 128.54, 128.70, 128.85, 129.67, 129.90, 130.48,
130.80, 130.84, 132.29, 136.47, 136.89, 137.77, 138.68, 138.74, 138.79,
138.83, 140.12, 145.36, 145.49, 146.37, 146.91, 148.02, 149.97, 152.59,
152.88, 152.91, 152.95, 153.66, 153.79, 157.66, 157.69, 157.81, 157.99,
159.25, 159.42, 161.90; ESI MS calcd for C64H52F6N8O7PRu [M]+
9H, -NCH(CH3)2), 1.08 and 1.04 (d, J ) 6.8 Hz, 3H, -NCH(CH3)2; 31
NMR (CD3CN, 269 MHz) δ 292.69, 292.62, 292.54 (m); ESI MS calcd
for C73H69F6N10O8P2Ru [M]+ 1491.339, found 1492 [M]+, 673 [M]2+
P
.
Phosphoramidite Λ-7a. The red foam Λ-7a was obtained by the
same procedure described above for phosphoramidite 7a from com-
pound Λ-6a in 90% yield. TLC (1% saturated aqueous KNO3/10%
1
H2O/CH3CN) Rf ) 0.34; H NMR (CD3CN, 400 MHz): δ 8.59 (d, J
) 7.8 Hz, 1H), 8.43-8.52 (m, 5H), 8.19 (d, J ) 8.8 Hz, 1H), 7.93-
8.10 (m, 6H), 7.84 (d, J ) 5.9 Hz, 1H), 7.70-7.77 (m, 3H), 7.49 (d,
J ) 5.9 Hz, 2H), 7.16-7.45 (m, 11H), 7.04 (t, J ) 7.8 Hz, 2H), 6.80
(d, J ) 8.8 Hz, 2H), 6.71-6.76 (m, 3H), 6.14 and 6.16 (t, J ) 5.9 Hz,
1H, H-1′), 4.62-4.68 (m, 1H, H-3′), 4.12-4.18 (m, 1H, H-4′), 3.10-
3.80 (m, 12H, -NCH(CH3)2, -OCH2CH2CN, -OCH3, H-5′), 2.63 and
2.50 (t, J ) 5.9 Hz, 2H, -OCH2CH2CN), 2.43-2.55 (m, 2H, H-2′),
1.12-1.16 (m, 9H, -NCH(CH3)2), 1.03 (d, J ) 6.8 Hz, 3H, -NCH-
(CH3)2); 31P NMR (CD3CN, 269 MHz) δ 292.57, 292.46; ESI MS calcd
1291.182, found 1291 [M]+, 573 [M]2+
.
Compound 6b. The dark brown 6b was obtained by the same
procedure described above for 6a from compounds 3 and OsII complex
4b as a mixture of diastereomers in 83% yield. TLC (1% saturated
aqueous KNO3/10% H2O/CH3CN) Rf ) 0.29; IR (KBr): 2207 cm-1
1
(-C≡C-); H NMR (CD3CN, 400 MHz): δ 9.28 (s, 1H), 8.50 (t, J )
for C73H69F6N10O8P2Ru [M]+ 1491.339, found 1492 [M]+, 673 [M]2+
.
7.3 Hz, 1H), 8.44-8.47 (m, 1.5H), 8.36-8.41(m, 2H), 8.38 (s, 0.5H),
8.27 (s, 0.5H), 8.23 (d, J ) 9.3 Hz, 0.5H), 8.21 (d, J ) 8.8 Hz, 1H),
7.97 (dt, J ) 5.4, 1.0 Hz, 1H), 7.94 (d, J ) 8.8 Hz, 0.5H), 7.88-7.92
(m, 2H), 7.80-7.86 (m, 3.5H), 7.76 (d, J ) 5.4 Hz, 0.5H), 7.70 (d, J
) 5.4 Hz, 0.5H), 7.65-7.67 (m, 1.5H), 7.61 (d, J ) 5.4 Hz, 0.5H),
7.48 (d, J ) 1.5 Hz, 0.5H), 7.29-7.45 (m, 9H), 7.25-7.28 (m, 1.5H),
7.21 (d, J ) 1.5 Hz, 0.5H), 7.06-7.17 (m, 4H), 6.98 (t, J ) 7.3 Hz,
0.5H), 6.85 (t, J ) 7.3 Hz, 1H), 6.79 (d, J ) 8.8 Hz, 1H), 6.74 (d, J
) 9.3 Hz, 1H), 6.71 (d, J ) 8.8 Hz, 1H), 6.57 (d, J ) 9.3 Hz, 1H),
6.14 (dt, J ) 6.4, 2.0 Hz, 1H, H-1′), 4.57-4.61 and 4.48-5.52 (quin,
J ) 3.9 Hz, 1H, H-3′), 4.02-4.04 and 3.95-3.97 (m, 1H, H-4′), 3.59,
3.58, 3.57, and 3.48 (s, 6H, -OCH3), 3.43 and 3.49 (d, J ) 4.2 Hz, 1H,
3′-OH), 3.33 and 3.14 (dd, J ) 11.2, 3.0 Hz, 1H, H-5′A), 3.31 and
Phosphoramidite ∆-7a. Product ∆-7a was obtained in 68% yield
as described above for phosphoramidite 7a. TLC (1% saturated aqueous
KNO3/10% H2O/CH3CN) Rf ) 0.34; 1H NMR (CD3CN, 400 MHz): δ
8.61 (d, J ) 7.3 Hz, 1H), 8.50 (d, J ) 7.8 Hz, 1H), 8.47 (d, J ) 7.8
Hz, 1H), 8.39 (t, J ) 7.8 Hz, 2H), 8.32 (d, J ) 6.4 Hz, 1H), 8.23 (d,
J ) 9.3 Hz, 1H), 8.08 (t, J ) 8.3 Hz, 1H), 7.94-8.04 (m, 5H), 7.90
(d, J ) 8.8 Hz, 1H), 7.79 (d, J ) 5.4 Hz, 1H), 7.70-7.74 (m, 2H),
7.56 (d, J ) 5.9 Hz, 1H), 7.52 (d, J ) 5.4 Hz, 1H), 7.41-7.45 (m,
3H), 7.33-7.39 (m, 4H), 7.23-7.30 (m, 4H), 7.11 (t, J ) 7.8 Hz, 2H),
6.95-6.98 (m, 1H), 6.71 (d, J ) 7.8 Hz, 2H), 6.58 (d, J ) 8.8 Hz,
2H), 6.13 and 6.15 (t, J ) 6.4 Hz, 1H, H-1′), 4.70-4.76 (m, 1H, H-3′),
4.07-4.13 (m, 1H, H-4′), 3.31-3.83 (m, 12H, -NCH(CH3)2, -OCH2-
CH2CN, -OCH3, H-5′), 2.64-2.67 and 2.49-2.51 (t, J ) 5.9 Hz, 2H,
-OCH2CH2CN), 2.54 (t, J ) 5.9 Hz, 2H, H-2′), 1.15-1.21 (m, 9H,
-NCH(CH3)2), 1.08 (d, J ) 6.8 Hz, 3H, -NCH(CH3)2); 31P NMR (CD3-
CN, 269 MHz) δ 292.50, 292.45, 292.39; ESI MS calcd for
3.20 (dd, J ) 10.7, 2.5 Hz, 1H, H-5′B), 2.30-2.40 (m, 2H, H-2′); 13
C
NMR (CD3CN, 101 MHz): δ 42.07, 42.10, 55.67, 55.74, 55.79, 63.49,
63.71, 71.39, 71.44, 86.57, 87.16, 87.35, 87.39, 87.46, 87.52, 87.95,
88.03, 89.06, 89.32, 98.20, 98.32, 113.97, 114.00, 114.04, 114.09,
122.78, 122.83, 124.99, 125.13, 125.18, 125.29, 127.17, 127.51, 127.79,
128.40, 128.60, 128.63, 128.71, 128.77, 128.84, 129.64, 129.70, 129.80,
130.41, 130.72, 130.77, 130.84, 130.93, 132.47, 132.55, 136.24, 136.40,
136.59, 136.80, 137.35, 137.34, 137.97, 138.02, 138.09, 138.17, 138.21,
139.50, 139.88, 145.27, 145.42, 145.63, 146.03, 149.26, 149.34, 149.90,
150.14, 150.16, 151.60, 151.68, 151.86, 151.97, 151.99, 152.08, 152.99,
153.01, 153.08, 153.24, 159.16, 159.33, 159.37, 159.45, 159.50, 159.62,
159.67, 159.74, 159.77, 161.82, 161.85; ESI MS calcd for C64H52F6N8O7-
C73H69F6N10O8P2Ru [M]+ 1491.339, found 1492 [M]+, 673 [M]2+
.
Phosphoramidite 7b. The dark brown foam 7b was obtained in
84% yield from 6b by the same procedure described above for
phosphoramidite 7a. TLC (1% saturated aqueous KNO3/10% H2O/CH3-
CN) Rf ) 0.35; 1H NMR (CD3CN, 400 MHz): δ 8.42-8.50 (m, 3.5H),
8.35-8.40 (m, 2H), 8.30 (d, J ) 5.1 Hz, 0.5H), 8.21 (d, J ) 9.2 Hz,
0.5H), 8.19 (d, J ) 8.8 Hz, 0.5H), 7.95-7.97 (m, 1H), 7.85-7.92 (m,
3H), 7.75-7.73 (m, 3.5H), 7.70 (d, J ) 5.9 Hz, 0.5H), 7.63-7.67 (m,
1.5H), 7.60 (d, J ) 5.5 Hz, 0.5H), 7.26-7.45 (m, 10.5H), 7.00-7.17
(m, 5H), 6.80 (d, J ) 8.8 Hz, 1H), 6.70-6.78 (m, 2.5H), 6.59 (d, J )
8.8 Hz, 1H), 6.11-6.17 (m, 1H, H-1′), 4.65-4.76 (m, 1H, H-3′), 4.07-
4.19 (m, 1H, H-4′), 3.16-3.83 (m, 4H, -NCH(CH3)2, -OCH2CH2CN),
3.59 (d, J ) 1.1 Hz, 1.5H, -OCH3), 3.57 (d, J ) 1.8 Hz, 1.5H, -OCH3),
3.55 (d, J ) 4.0 Hz, 1.5H, -OCH3), 3.48 (s, 1.5H, -OCH3), 3.31-
3.34 (m, 1H, H-5′A), 3.18 (dt, J ) 11.0, 2.9 Hz, 1H, H-5′B), 2.62-
2.67 and 2.52-2.55(m, 2H, -OCH2CH2CN), 2.42-2.58 (m, 2H, H-2′),
OsP [M]+ 1380.342, found 1382 [M]+, 618 [M]2+
.
Phosphoramidite 7a. To a solution of 6a (150 mg, 0.104 mmol)
and (1H)-tetrazole (7 mg, 0.099 mmol) in 2 mL anhydrous acetonitrile
was added 2-cyanoethyl-N,N,N′,N′-tetraisopropyl-diphosphoramidite (63
mg, 0.209 mmol). The reaction was stirred for 4 h at room temperature
under argon atmosphere. The reaction mixture was diluted with cold
1% triethylamine/dichloromethane (300 mL), and washed with 1 M
NaHCO3 (2 × 20 mL) and brine (2 × 20 mL). The organic phase was
9
3760 J. AM. CHEM. SOC. VOL. 124, NO. 14, 2002

Ru(II) and Os(II) Nucleosides and Oligonucleotides
A R T I C L E S
1.12-1.20 (m, 9H, -NCH(CH3)2), 1.08 (d, J ) 6.7 Hz, 1.5H, -NCH-
(CH3)2), 1.04 (d, J ) 6.8 Hz, 1.5H, -NCH(CH3)2); 31P NMR (CD3CN,
202 MHz) δ 292.34 (m); ESI MS calcd for C73H69F6N10O8OsP2 [M]+
corporation into single stranded DNA. 8-mer oligonucleotides (10-
13) were purified by reverse-phase HPLC following gel purification
using the following conditions: Vydac C-8 analytical column, 4.6 ×
250 mm, 5µ, 1-12% CH3CN in 50 mM triethylammonium acetate,
pH 7.0 over 12 min, with a gradient to 50% CH3CN over the next 30
min. The retention time of the metal-modified 8-mer oligonucleotides
was approximately 12 min.
1580.559, found 1581 [M]+, 717 [M]2+
.
[(bpy)2Ru(3-phenylethynyl-1,10-phenanthroline)]2+ 8. RuII com-
plex 4a13 (50 mg, 0.052 mmol), Pd(dppf)Cl2‚CH2Cl2 (2.4 mg, 0.003
mmol), and CuI (1.0 mg, 0.003 mmol) were dissolved in a degassed
solution of anhydrous DMF (3.0 mL), triethylamine (500 µL), and
phenylacetylene (53 mg, 0.52) under argon. The solution was stirred
under argon for 20 min at room temperature, then evaporated under
reduced pressure. The crude reaction mixture was purified by flash
chromatography (silica gel, 0.2% saturated aqueous KNO3, 5% water
in acetonitrile). The product fractions were concentrated, dissolved in
Milli-Q water, and extracted into dichloromethane after the addition
of excess KPF6. The product solution was dried over Na2SO4, filtered,
and evaporated to dryness to yield product 8 in 94% yield (48 mg).
Crude metal-containing oligonucleotides were alternatively purified
by trityl-on reverse phase HPLC. The finished oligonucleotide was
cleaved from the solid support, and the exocyclic amines were
deprotected by treatment with 1.0 mL of 30% aqueous ammonium
hydroxide for 24 h at room temperature. The solution was lyophilized,
and the crude oligonucleotide was purified using semi-prep HPLC
(Vydac C-8 column, 10 × 250 mm, 5µ, 300 Å pore size). HPLC mobile
phase: 20-25% acetonitrile gradient in 50 mM triethylammonium
acetate pH 7.0 over 30 min. Trityl-on metalated oligonucleotides
typically eluted between 12 and 20 min depending on length, and were
lyophilized immediately after collection. The purified oligonucleotide
was then treated with 0.5 mL of 80% acetic acid at room temperature
for 20 min. The solution was lyophilized, dissolved in 1× TBE buffer
pH 8.3, and applied to a Sep-pak cartridge. The oligonucleotide was
desalted and eluted using 20% CH3CN/H2O. Quantification of the
HPLC purified oligonucleotides was performed as above.
1
TLC (1% saturated aqueous KNO3/10% H2O/CH3CN) Rf ) 0.25; H
NMR (CD3CN, 400 MHz): δ 8.74 (d, J ) 1.5 Hz, 1H), 8.62 (dd, J )
8.4, 1.1 Hz, 1H), 8.49-8.56 (m, 3H), 8.28 (d, J ) 8.8 Hz, 1H), 8.23
(d, J ) 8.8 Hz, 1H), 8.22 (d, J ) 1.8 Hz, 1H), 8.08-8.12 (m, 3H),
7.99-8.04 (m, 2H), 7.87 (d, J ) 5.1 Hz, 1H), 7.82 (d, J ) 5.5 Hz,
1H), 7.75 (dd, J ) 8.1, 5.1 Hz, 1H), 7.66 (d, J ) 5.1 Hz, 1H), 7.52-
7.56 (m, 3H), 7.42-7.48 (m, 5H), 7.22-7.27 (m, 2H); 13C NMR (CD3-
CN, 101 MHz): δ 85.32, 96.14, 122.19, 123.05, 125.03, 125.11, 125.16,
125.21, 127.11, 128.24, 128.28, 128.45, 128.59, 129.73, 130.72, 131.20,
132.23, 132.53, 137.73, 138.65, 138.78, 139.50, 147.17, 148.16, 152.73,
152.77, 152.94, 153.01, 153.67, 154.68, 157.73, 157.87, 158.06, 158.08;
UV (CH3CN): λmax nm (ꢀ × 10-4) 242 (4.0), 286 (7.5), 332 (2.7), 450
(1.3); ESI MS calcd for C40H28F6N6PRu [M]+ 838.72, found 838.8
[M]+.
Enzymatic Degradation of Oligonucleotides. The general proce-
dure is as follows: To a 2-nmol pellet of DNA were added 10 µL of
1 M MgCl2, 10 µL of 10× AP buffer (New England Biolabs), 2 µL
(0.3 U/µL) of nuclease P1 (Boehringer Mannheim), 1.5 µL (0.003 U/µL)
of snake venom phosphodiesterase (Boehringer Mannheim), 1.5 µL
(10 U/µL) of alkaline phosphatase (New England Biolabs), and 75 µL
of water. The reaction mixture was incubated at 37 °C for 6 h, passed
through a 0.45 µm Nylon-66 micro-centrifuge filter (Rainin), and
analyzed by HPLC (Vydac C18 column, 4.6 × 250 mm, 5µ RP-silica)
using a photodiode array detector with detection at 260, 358, and 450
nm. HPLC mobile phase: 1-12% acetonitrile gradient in 100 mM
triethylammonium acetate pH 5.5 over 12 min, followed by 12-99%
acetonitrile gradient over 30 min. Peaks were identified by retention
time and UV spectra, as well as by comparison with authentic standards.
Nucleoside ratios were determined by integration of peak areas at 260
nm and normalization using the extinction coefficients listed above.
Enzymatic digestion performed with snake venom phosphodiesterase
and alkaline phosphatase resulted in incomplete digestion and termina-
tion at the nonnatural base. The endonuclease nuclease P1 is required
for complete degradation of these metal modified oligonucleotides to
their constituent nucleosides.23
Oligonucleotide Synthesis. All oligonucleotides were prepared on
a 0.2 µmol scale on 500-Å CPG solid support using a Milligen Cyclone
Plus DNA synthesizer. Metal modified residues were introduced site-
specifically by trityl-off synthesis of the base oligonucleotide, followed
by the manual coupling of the desired metal-modified phosphoramidite.
In a typical procedure, the modified nucleoside (0.02 mmol) is dissolved
in 200 µL of 0.5 M (1H)-tetrazole in acetonitrile. This activated
phosphoramidite solution is immediately applied via syringe to the
synthesis column containing the CPG-bound base oligonucleotide.
Coupling times for the modified phosphoramidites were extended to 5
min to ensure maximum coupling efficiency. The column was rinsed
with dry acetonitrile (5 mL), and returned to the automated synthesizer
for the standard oxidation and capping steps. Coupling efficiencies for
the base-modified phosphoramidites as monitored by trityl cation assay
were consistently above 90%. Shorter coupling times and/or less
concentrated (1H)-tetrazole yielded significantly lower coupling ef-
ficiency, ranging from 25 to 40%. The remainder of the desired
oligonucleotide was synthesized normally using the trityl-off procedure.
The oligonucleotide was cleaved from the solid support by treatment
with 1.0 mL of 30% aqueous ammonium hydroxide for 3 h at room
temperature. The resulting solution was incubated at 55 °C for 8 h.
Oligonucleotides containing no modified bases were synthesized using
standard Milligen/Biosearch Cyclone Plus protocols. The crude product
from the automated oligonucleotide synthesis was lyophilized and
purified by preparative 20% denaturing polyacrylamide gel electro-
phoresis. The product was visualized by UV shadowing, and the desired
band excised from the gel. The band was crushed, then extracted with
1× TBE buffer pH 8.3 (90 mM Tris, 90 mM boric acid, 1 mM EDTA)
for 24-36 h. The extract was filtered (0.45 µm nylon), and desalted
using a Sep-pak cartridge (Waters Corporation, MA). Oligonucleotides
were eluted from the Sep-pak cartridge using 40% acetonitrile/water.
The concentration of the single-stranded oligonucleotides was deter-
mined at 260 nm using the following molar extinction coefficients:
15400 (A), 11700 (G), 8800 (T), 7300 (C), 36500 (URu), 33200 (UOs).
This procedure assumes that the absorption properties of the free
5-modified 2′-deoxyuridine nucleosides remain unchanged upon in-
Circular Dichroism. CD spectra were collected on an Aviv model
202 spectropolarimeter at 25 °C in a 1-cm path length cell at 1-nm
intervals using a 4 s averaging time. The concentrations of metal-
modified nucleosides were 2 × 10-5 M in spectral grade acetonitrile.
The concentrations of DNA duplexes were 1 µM in 100 mM NaCl, 10
mM phosphate, pH 7.0.
Thermal Denaturation Studies. All hybridizations and UV melting
experiments were carried out in 100 mM NaCl, 10 mM phosphate
buffer, pH 7.0 containing a one-to-one ratio of complementary
oligonucleotides at 2 µM duplex concentration. After samples were
prepared, they were heated to 90 °C for 5 min, cooled to room
temperature for 2-3 h, then cooled to 4 °C prior to melting temperature
(Tm) measurement. Thermal denaturation studies were carried out in a
Teflon-stoppered 1.0-cm path length quartz cell on a Varian-Cary 1E
spectrophotometer with a temperature-controlled cell compartment.
Samples were equilibrated at the starting temperature for 20 min.
Heating runs were performed between 50 and 90 °C at a scan rate of
0.5 °C min-1 with optical monitoring at 260 nm. All duplexes displayed
sharp, two-state transition curves from duplex to single-stranded DNA.
Similar results were seen in cooling curves. Tm values were determined
by computer fit of the melting data, followed by calculation of the
9
J. AM. CHEM. SOC. VOL. 124, NO. 14, 2002 3761

A R T I C L E S
Hurley and Tor
first derivative of the resulting melting curve. Uncertainty in Tm values
is estimated to be ( 0.5 °C.
was measured before and after each set of data acquisition to ensure
the stability and accuracy of the setup.
Steady-State Luminescence Experiments. Steady-state lumines-
cence experiments were conducted at 20 °C with excitation at 450 nm
on a Perkin-Elmer LS 50B luminescence spectrophotometer. Argon-
degassed samples were measured at a duplex concentration of 2 µM
in 100 mM NaCl, 10 mM sodium phosphate buffer at pH 7.0.
Quenching efficiencies were determined by integration of the emission
curves obtained from duplicated experiments on two separately prepared
samples.
Time-Resolved Emission Spectroscopy. The excited-state lifetimes
of the emissive complexes were measured using the same samples used
for the steady-state luminescence studies. The degassed solutions were
excited by a 4-ns pulsed dye-laser. The laser was tuned to 450 nm for
measurement of free nucleosides and singly metal-modified strands and
duplexes. For duplexes containing both OsII- and RuII-modified
nucleotides, the laser was tuned to 467 nm. At this wavelength, the
ratio of the extinction coefficients for the RuII nucleoside 5a and the
OsII nucleoside 5b is 1.0. The emitted light was collected at 90° and
focused into a monochromator. Detection was achieved by a photo-
multiplier tube connected to a LeCroy Digitizing oscilloscope. The data
from the scope was transferred to a PC and processed by using routine
software of local origin. A sample of [Ru(bpy)3](PF6)2 in acetonitrile
Acknowledgment. We thank the National Institutes of Health
(GM 58447) for supporting this research and Clinical Micro
Sensors, Inc., and the UC Biotechnology STAR Project for
supporting the early stages of the project. We are grateful to
Professor Doug Magde (UCSD) for his help and insightful
comments regarding the time-resolved luminescence measure-
ments. We thank Susan Seaman and Edith (Phoebe) Glazer for
assistance with electrochemical measurements and Dr. Natia
Frank for her help with measuring transient absorption spectra.
Supporting Information Available: Additional data regarding
solvatochromism studies, determination of the excited-state
localization and redox potential, as well as thermal denaturation
measurements, HPLC and enzymatic digestions of metalated
oligonucleotides, and synthesis and NMR spectroscopy of
diastereomerically pure metalated nucleosides (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA0123103
9
3762 J. AM. CHEM. SOC. VOL. 124, NO. 14, 2002