Synthesis and spectroscopy of Ru(II)-bridged DNA hairpins

Article abstract of DOI:10.1039/a808491b

A difunctional Ru(II) complex has been prepared which can be incorporated into synthetic oligonucleotides by means of standard solid-phase methodology and the properties of several oligonucleotide conjugates capable of forming Ru(II)-bridged hairpins have been investigated.

Full text of DOI:10.1039/a808491b

Synthesis and spectroscopy of Ru(ii)-bridged DNA hairpins
Frederick D. Lewis,* Sara A. Helvoigt† and Robert L. Letsinger*
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL 60208, USA.
E-mail: lewis@chem.nwu.edu
Received (in Columbia, Mo, USA) 28th October 1998, Accepted 11th January 1999
A difunctional Ru(ii) complex has been prepared which can
be incorporated into synthetic oligonucleotides by means of
standard solid-phase methodology and the properties of
several oligonucleotide conjugates capable of forming
Ru(ii)-bridged hairpins have been investigated.
dimethoxytrityl cation, was > 95%. Obtained by this procedure
were the Ru-linked oligonucleotides dT4–Ru(bpy)2(dabp)2+–
dA4, dG3–Ru(bpy)2(dabp)2+–dC3, and dGCAATTGC–Ru-
(bpy)2(dabp)2+–dGCAATTGC, 3–5.10 After removal from the
solid support and deprotection the oligonucleotides were
purified by HPLC and appear as a single peak on both ion
exchange and reverse phase HPLC.6
There is continuing interest in the development of oligonucleo-
tide conjugates containing luminescent or redox-active transi-
tion metal complexes.1 These conjugates have been investigated
as alternatives to the use of radioactive isotopes for gene
sensing, as site specific photonucleases, and as luminescent
probes for the study of energy transfer and electron transfer in
duplex DNA. In most of the conjugates investigated to date the
metal complex is attached post-synthetically to the 5A-end of an
oligonucleotide by means of a flexible tether.2 Flexible tethers3
and rigid linkers4 with metal-binding ligands have also been
used to modify nucleosides which can be incorporated using
standard solid-phase methodology at a specific position in a
synthetic oligonucleotide. Hybridization of conjugates with
flexible tethers results in the formation of duplexes in which the
metal complex may be intercalated, groove bound, or only
weakly associated with the duplex. Uncertainty about the
location of the metal complex can complicate the interpretation
of electron and energy transfer measurements. Our interest in
photoinduced electron transfer in duplex DNA5 led us to
undertake the synthesis of oligonucleotide conjugates in which
a difunctional metal complex forms a linker connecting two
oligonucleotides. We report here the synthesis and spectral
properties of the first Ru(ii)-linked oligonucleotides which
possess complementary sequences capable of forming hairpin
structures.
The absorption spectrum of [Ru(bpy)2(dabp)]2+ 2a displays
maxima at 468 nm (e = 8538 dm3 mol21 cm21) and 288
Preparation of synthetic oligonucleotides with a linking metal
complex by standard solid state synthesis required the develop-
ment of a difunctional ligand in which one functional group can
be protected and the other activated. Our solution to this
problem is outlined in Scheme 1. The difunctional diamidobi-
pyridine ligand dabp (1a‡) was prepared from 2,2A-bipyridine-
4,4A-dicarboxylic acid following the method developed for the
synthesis of analogous arenedicarboxamides.6 Reaction of this
ligand with [Ru(bpy)2Cl2]·2H2O7 afforded the complex [Ru(b-
2
py)2(dabp)]2+ 2a, isolated as the PF6 salt.8 Synthesis of the
mono-protected complex [Ru(bpy)2(DMT-dabp)][PF6]2 2b was
best accomplished via reaction of the monoprotected dabp
ligand 1b with [Ru(bpy)2Cl2]·2H2O. Repeated efforts to convert
2b to 2c using standard conditions led to recovery of 2b. We
found that 2c is moderately stable when prepared in a glove box,
but is rapidly hydrolyzed to 2b upon exposure to moisture.
The instability of 2c necessitated a modified protocol for
oligonucleotide synthesis. The 3A-segment of the oligonucleo-
tide was prepared according to standard solid phase proce-
dures.9 After removal of its 5A-DMT protecting group, the
column was brought into a dry box and the oligonucleotide
coupled with 2c, using tetrazole as the activator. The column
was returned to the automatic synthesizer to complete the
synthesis of the oligonucleotide. The coupling efficiency for Ru
incorporation, as determined by monitoring release of the
Scheme 1 Reagents and conditions: i, a, SOCl2, PhCH2NEt3Cl, C2H4Cl2,
reflux; b, HOCH2CH2CH2NH2, Et3N, THF, MeOH, 50%; ii, [Ru-
(bpy)2Cl2]·2H2O, 95% ethanol, 90%; iii, a, dimethylaminopyridine,
pyridine; b, dimethoxytrityl (DMT) chloride, pyridine, 38%; iv, same as ii,
88%; v, (2-cyanoethyl)diisopropylchloride phosphoramidite, Et3N, dry
acetonitrile, glove box, not isolated; vi, see text, Expidite™ nucleic acid
synthesis system.
† Present address: Nanophase Technologies Corp., 453 Commerce St., Burr
Ridge, IL 60521, USA.
Chem. Commun., 1999, 327–328
327

(43 550) which are red-shifted with respect to those of
[Ru(bpy)3]2+, as previously observed for Ru(ii) complexes with
a single bipyridine-4,4A-dicarboxamide ligand.11 The spectra of
3–5 have maxima below 300 nm for the nucleobases which
overlap the shorter wavelength Ru2+ band. The 260 nm bands of
3–5, but not the 468 nm bands exhibit hypochromism. The
complex 2a and conjugates 3–5 display a single broad emission
band with a maximum at 665 nm, similar to that reported for
other Ru(ii) complexes possessing a single bipyridine-4,4A-
dicarboxamide ligand.11 The luminescence quantum yields and
decay times are summarized in Table 1. The neighboring
nucleobases have little effect on the photophysical behavior of
the Ru(ii) complex, in accord with previous observations for
[Ru(bpy)3]2+ covalently attached to oligonucleotides.3
bridging metal complex. Whereas the precise structures of these
conjugates remain to be established, the metal complex is likely
located at the end of a duplex region of a hairpin structure. The
excited Ru(ii) complex selected for this study is not quenched
by nucleobases, however excited state redox potentials can be
tuned over a wide range by variation of the nonbridging ligands
or metal.13 Similarly, the use of nucleobase analogs such as
6-oxoguanine or 7-deazaguanine which have low oxidation
potentials should increase the driving force for photoinduced
electron transfer.14 In addition, hybridization of metal linked
conjugates possessing noncomplementary arms with unlabelled
oligonucleotides should position the metal center near a specific
location in the unlabelled strand. Thus the availability of
difunctional metal complexes which can be introduced into
oligonucleotides via automated phosphoramidite chemistry
serves to extend structural diversity currently available with
monofunctional metal complexes.2–4 The long lifetimes and
moderately large fluorescence quantum yields make these
Ru(ii) complexes particularly well-suited for studies of long
range energy and electron transfer.
Table 1 Emission lifetimes and quantum yields for complexes 2a, 3, 4
and 5
a
Complex
t/ns
Fe
2a
3
4
850
815
790
608
0.013
0.018
0.016
0.019
This work has been supported by the Division of Chemical
Sciences, Offices of Basic Energy Sciences, US Department of
Energy.
5
a
Values are reported relative to [Ru(bpy)3][PF6]2 in water (Fe = 0.042)
and calculated according to published procedures (J. N. Demas and G. A.
Crosby, J. Phys. Chem., 1971, 75, 991).
Notes and references
Molecular modeling indicates that the conjugates 3–5 can
adopt low energy hairpin conformations as shown schemat-
ically in Fig. 1; however, these conjugates also might form
duplexes in which the Ru(ii) complexes occupy bulges on
opposite strands. In the case of 5 a value of TM = 50 °C is
obtained from the thermal dissociation profile in 0.1 M NaCl.
This value is independent of concentration (1.0–5.0 mM). In the
case of 4 the observed value of TM = 50 °C in 1.0 M NaCl is
higher than that calculated for either a GGG/CCC duplex
[TM(calc.) = 242 °C] or for two such duplex segments
[TM(calc.) = 20 °C] with no contribution from the Ru(ii) linkers
and no cooperativity in melting of the two segments.12 This
evidence supports the tentative assignment of a hairpin vs.
duplex structures for 4 and 5. The conjugates 3 and 4 have broad
thermal dissociation profiles (not shown) and values of TM
( < 20 and 50 °C, respectively, in 1.0 M NaCl) lower than those
of the analogous stilbene dicarboxamide-bridged hairpins (TM
= 49 and > 80 °C, respectively, in 1.0 M NaCl).7 This may
reflect a better fit for the stilbene vs. Ru(ii) linker across the
double helix. The broad thermal dissociation profiles for 3–5
may reflect multiple conformations for the hairpin loop region
as well as the presence of two diastereomeric octahedral
complexes.
‡ Selected data for 1a: ES/MS (DMSO–H2O): m/z 359 NMR (DMSO-d6,
1H): d 8.95 (t, 2H), 8.85 (d, 2H), 8.75 (s, 2H), 7.8 (d, 2H), 4.5 (t, 2H), 3.5
(t, 4H), 3.3 (t, 4H), 1.7 (q, 4H). For 1b: NMR (DMSO-d6, 1H): d 9.9 (t, 2H),
9.85 (d, 2H), 9.75 (d, 2H); 7.8 (dd, 2H), 7.35 (d, 2H), 7.2 (m, 5H), 6.8 (d,
4H), 3.65 (s, 6H), 3.4 (m, 8H), 1.85 (q, 2H), 1.7 (q, 2H). For 2a: ES/MS
(MeCN): m/z 917 ([Ru(bpy)2dabp][PF6]+), 386 ([Ru(bpy)2dabp]2+). UV–
VIS (MeCN): 468 nm (e = 8538 dm3 mol21 cm21); 350 (6847), 288
(43550), 236 (37107). For 2b: ES/MS (MeCN): m/z 1219 ([Ru(bpy)2(DMT-
dabp)][PF6]+); 537 ([Ru(bpy)2(DMT-dabp)]2+). UV–VIS (MeCN): 468,
350, 288, 236 nm. For 2c: NMR (MeCN, 31P): d +140 (two singlets).
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The preliminary results reported here provide a potentially
versatile method for the introduction of a bipyridyl-complexed
metal ion at a specific location in a synthetic conjugate. The
three conjugates prepared in this study have complementary
arms and thus are capable of forming hairpin structures with a
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10 tert-Butyl hydroperoxide (1.0 M dichloromethane) was used as the
oxidizer instead of I2 for the completion of the synthesis. Overall yields
ranged from 3–5% after purification.
11 A. Juris, V. Balzani, F. Barigelletti, S. Campanga, P. Belser and A. Von
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Fig. 1 Schematic structure for the hairpin conformation of 5.
Communication 8/08491B
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Chem. Commun., 1999, 327–328