Metal-containing oligonucleotides: Solid-phase synthesis and luminescence properties [12]

Full text of DOI:10.1021/ja9739998

2194
J. Am. Chem. Soc. 1998, 120, 2194-2195
Scheme 1. Synthesis of Phosphoramidites 6a and 6ba,b
Metal-Containing Oligonucleotides: Solid-Phase
Synthesis and Luminescence Properties
Dennis J. Hurley and Yitzhak Tor*
Department of Chemistry and Biochemistry
UniVersity of California, San Diego
La Jolla, California 92093-0358
ReceiVed NoVember 24, 1997
The incorporation of photo- and redox-active transition metal
ions into oligonucleotides is a key design target for the study of
energy and electron-transfer processes through DNA,1 as well as
the development of DNA hybridization probes and sensors.2
Metal-containing oligonucleotides have been predominantly con-
structed via two major pathways: (a) the synthesis of a chelator-
containing oligonucleotide followed by metal complexation3 and
(b) the synthesis of an end-functionalized oligonucleotide to which
a metal complex can be conjugated.1,4 These approaches are
restricted primarily to modifications at the oligonucleotides’
termini and/or require the exposure of oligonucleotides to reactive
metal precursors.2-5 A direct method for the site-specific
incorporation of metal complexes during solid-phase oligonucleo-
tide synthesis has never been reported.
We now disclose a general methodology for the incorporation
of polypyridine metal complexes into oligonucleotides using
automated DNA synthesizers. We report the synthesis of novel
RuII- and OsII-containing nucleosides and their phosphoramidite
derivatives. These building blocks are sequence-specifically
incorporated into oligonucleotides in high yields using standard
solid-phase phosphoramidite chemistry. The uniquely modified
oligonucleotides form stable DNA duplexes and are useful probes
for the study of energy-transfer processes in nucleic acids.
We have previously reported that functionalized tris-chelate
complexes are excellent substrates for the powerful palladium-
mediated cross-coupling methodologies.6 This approach provides
a convenient entry into metal-containing nucleosides and is key
a Reagents: (a) 4,4′-dimethoxytrityl chloride (DMT-Cl), DMAP,
pyridine, Et3N, 92% yield; (b) 3a or 3b, (Ph3P)2PdCl2, CuI, DMF, Et3N,
sonication, 84% yield; (c) (iPr2N)2POCH2CH2CN, (1H)-tetrazole, CH3CN;
70-85% yield. b All metal-modified nucleosides were isolated as their
-
PF6 salts.8
to the successful preparation of the modified nucleosides and their
phosphoramidites. Thus, palladium-catalyzed cross-coupling
reactions between 5-ethynyldeoxyuridine7 (1) and [(bpy)2Ru(3-
bromo-1,10-phenanthroline)]2+(PF6-)2 (3a) or [(bpy)2Os(3-bromo-
-
1,10-phenanthroline)]2+(PF6
) (bpy ) bipyridyl) (3b) afford
2
nucleosides 4a and 4b, respectively (Scheme 1).8,9 The mild
conditions of this reaction allow us to apply it for the modification
of 4,4′-dimethoxytrityl-protected nucleosides. Thus, 1 is first
treated with 4,4′-dimethoxytrityl (DMT) chloride in the presence
of 4-(dimethylamino)pyridine (DMAP) to provide the DMT-
protected nucleoside 2, which is then cross-coupled to 3a or 3b
to afford the protected metal-containing nucleosides 5a and 5b,
respectively (Scheme 1). Phosphitylation of the protected nucleo-
sides 5a and 5b using (2-cyanoethoxy)bis(diisopropylamino)-
phosphine in the presence of (1H)-tetrazole provides the corre-
sponding metal-modified phosphoramidites 6a and 6b.10
(1) 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. Meggers,
E.; Kusch, D.; Giese, B. HelV. Chim. Acta 1997, 80, 640-652. See also:
Netzel, T. L. J. Chem. Educ. 1997, 74, 646-651. Diederichsen, U. Angew.
Chem., Int. Ed. Engl. 1997, 36, 2317-2319.
(2) (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) Bannwarth, W.;
Pfleiderer, W.; Mu¨ller, F. HelV. Chim. Acta 1991, 74, 1991-1999. (d) Telser,
J.; Cruickshank, K. A.; Schanze, K. S.; Netzel, T. L. J. Am. Chem. Soc. 1989,
111, 7221-7226. (e) Sammes, P. G.; Yahioglu, G. Nat. Prod. Rep. 1996, 13,
1-28. (f) Ihara, T.; Nakayama, M.; Murata, M.; Nakano, K.; Maeda, M. Chem.
Commun. 1997, 1609-1610.
(3) 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. Dreyer, G. R.; Dervan, P. B. Proc. Natl.
Acad. Sci. U.S.A. 1985, 82, 968-972. Chen, C.-H. B.; Sigman, D. S. J. Am.
Chem. Soc. 1988, 110, 6570-6572.
(4) 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.
Target 20-mer oligonucleotides incorporating one or two metal-
modified 2′-deoxyuridine bases at various positions were syn-
thesized on a 0.2 µmol scale using an automated DNA synthesizer
(Figure 1). When coupling times for the phosphoramidites 6a
and 6b in 0.5 M (1H)-tetrazole were extended to 5 min, reaction
efficiencies were greater than 90%.11 Removal of the finished
20-mers from the solid support using concentrated ammonium
hydroxide was followed by incubation at 55 °C for 8 h to afford
(7) Robins, M. J.; Barr, P. J. J. Org. Chem. 1983, 48, 1854-1862.
(8) See the Supporting Information for experimental details.
(9) Polypyridine complexes of RuII and OsII were selected due to their
chemical stability and favorable redox and photophysical characteristics.
See: 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. Balzani, V.; Juris, A.; Venturi, M.; Campagna, S.; Serroni, S.
Chem. ReV. 1996, 96, 759-833.
(5) For recent approaches utilizing metal-containing phosphoramidites or
H-phosphonates, see: ref 2b. Manchanda, R.; Dunham, S. U.; Lippard, S. J.
J. Am. Chem. Soc. 1996, 118, 5144-5145. Schliepe, J.; Berghoff, U.; Lippert,
B.; Cech, D. Angew. Chem., Int. Ed. Engl. 1996, 35, 646-648. Mucic, R. C.;
Herrlien, M. K.; Mirkin, C. A.; Letsinger, R. L. Chem. Commun. 1996, 555-
557. Magda, D.; Crofts, S.; Lin, A.; Miles, D.; Wright, M.; Sessler, J. L. J.
Am. Chem. Soc. 1997, 119, 2293-2294. 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.
(10) All compounds were characterized by 1H NMR, ESI-MS, UV-vis,
IR and square-wave and cyclic voltammetry. See the Supporting Information.
(11) To control the amount of reagents and reaction time, the coupling of
the modified bases was performed manually (ref 8). The decreased coupling
efficiencies relative to standard phosphoramidites are likely due to the steric
bulk of the appended metal complex and have been reported with other bulky
phosphoramidites. See: Kobertz, W. R.; Essigmann, J. M. J. Am. Chem. Soc.
1997, 119, 5960-5961.
(6) Tzalis, D.; Tor, Y. Chem. Commun. 1996, 1043-1044. Tzalis, D.; Tor,
Y. J. Am. Chem. Soc. 1997, 119, 852-853. Connors, P. J., Jr.; Tzalis, D.;
Dunnick, A. L.; Tor, Y. Inorg. Chem. In press.
S0002-7863(97)03999-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/24/1998

Communications to the Editor
J. Am. Chem. Soc., Vol. 120, No. 9, 1998 2195
Figure 1. Sequences of oligonucleotides synthesized. The RuII- and OsII-
containing deoxyuridine nucleosides (4a and 4b, respectively) are shown
in bold.
Figure 3. Steady-state emission spectra of modified oligonucleotides in
degassed 0.01 M sodium phosphate buffer pH 7.0, 0.1 M NaCl. Top:
duplex 8‚12 (solid line), a 1:1 mixture of 8 and 9 (-‚-), and a duplex
containing proximal Ru and Os 8‚13 (dashed line). Bottom: duplex 10‚12
(solid line) and duplex 10‚13 (dashed line).
center.9,12 Intermolecular quenching can be excluded since a 1:1
mixture of the noncomplementary oligonucleotides 8 and 9 shows
essentially the same emission intensity as duplex 8‚12 (Figure
3a, top). This behavior is distance-dependent as demonstrated
by comparing duplex 8‚13 to duplex 10‚13. In this case, where
the OsII center is more remote, only 40% quenching of the RuII-
based emission is observed (Figure 3b, bottom). To the best of
our knowledge, this is the first example of energy-transfer
processes in DNA oligonucleotides that are sequence-specifically
modified with polypyridine metal complexes.
The data presented here establish a novel and powerful
approach for the site-specific incorporation of polypyridine-metal
complexes into synthetic oligonucleotides using automated phos-
phoramidite chemistry. The versatile phosphoramidite synthesis
and the compatibility with existing automated DNA synthesizers
provides enormous flexibility for rapid construction of oligo-
nucleotide-metal conjugates. The presence of photo- and redox-
active metal centers in these oligonucleotides makes them
extremely important probes for the study of photophysical
processes in nucleic acids.
Figure 2. Thermal denaturation curves for control duplex 7‚12 (b), OsII-
containing duplex 9‚12 (4), and a single-mismatch duplex 7‚14 (0)
determined in 0.01 M sodium phosphate buffer pH 7, 0.1 M NaCl.8
the deprotected oligomers 8-11 and 13 that were purified by gel
electrophoresis.8 Analytical denaturing polyacrylamide gel elec-
trophoresis confirmed the purity of the modified oligonucleotides,
and enzymatic digestion followed by HPLC analysis verified the
presence of the intact metal-containing nucleosides.8
The presence of the a metal-containing nucleoside has a
relatively small effect on duplex stability as determined by thermal
denaturation curves (Figure 2). The melting temperature (Tm) of
the unmodified duplex derived from oligonucleotide 7 and its
complementary sequence 12 is 78 °C. When the metal-containing
nucleoside is located at the 5′-end, as in duplex 10‚12, the Tm is
essentially the same. Duplexes 8‚12 and 9‚12, in which the metal-
containing nucleoside is in the middle of the duplex, are slightly
less stable with a Tm at 75 °C. Yet, this destabilization is far
smaller than the effect of a single mismatch on duplex stability
as demonstrated for duplex 7‚14 containing a T-T “pair” at the
same position (Tm ) 69 °C, Figure 2).
Steady-state emission profiles of iso-absorptive oligonucleotide
solutions in degassed phosphate buffer are shown in Figure 3.
The RuII-containing duplex 8‚12 shows a typical metal-centered
emission at 630 nm upon excitation of the visible metal-to-ligand
charge-transfer (MLCT) band at 456 nm. Upon hybridization of
the RuII-containing oligonucleotide 8 to a complementary OsII-
containing oligonucleotide 13, a substantial drop in the emission
intensity (ca. 70-85%) is observed. This suggests an “intra-
duplex” quenching of the excited RuII center by the proximal OsII
Acknowledgment. We thank Clinical Micro Sensors, Inc., and the
UC Biotechnology STAR Project for generous support of this research
and Professor David R. Kearns for the use of his temperature-controlled
UV spectrophotometer.
Supporting Information Available: Synthetic procedures and ana-
lytical data for all new derivatives as well as procedures and data for
oligonucleotide synthesis, purification, digestion, melting, and fluores-
cence studies (13 pages). See any current masthead page for ordering
information and Web access instructions.
JA9739998
(12) De Cola, L.; Barigelletti, F.; Balzani, V.; Belser, P.; Von Zelewsky,
A.; Seel, C.; Frank, M.; Vo¨gtle, F. in Supramolecular Chemistry; Balzani,
V., De Cola, L., Eds, Kluwer: Dordrecht, The Netherlands, 1992; pp 157-
180.