Redox-active metal-containing nucleotides: Synthesis, tunability, and enzymatic incorporation into DNA

Article abstract of DOI:10.1021/ja017193q

Novel redox-active DNA labeling tags with tunable electrochemical potentials are modularly synthesized using (a) bis-substituted Ru²⁺ or Os²⁺ precursors (R₂bpy)₂ML₂, (b) substituted 2,4-pentanediones or hydroxamic acids bearing a functionalized linker, and (c) modified nucleotides. DNA polymerase efficiently incorporates the metal-containing nucleotide triphosphate into DNA oligonucleotides. Copyright

Full text of DOI:10.1021/ja017193q

Published on Web 01/30/2002
Redox-Active Metal-Containing Nucleotides: Synthesis, Tunability, and
Enzymatic Incorporation into DNA
Haim Weizman and Yitzhak Tor*
Department of Chemistry and Biochemistry, UniVersity of California, San Diego, La Jolla, California 92093-0358
Received September 28, 2001
Scheme 1. Synthesis and Electrochemical Data of Acetyacetonate
Recent developments in “lab-on-a-chip” approaches and DNA
diagnostic tools have triggered a renewed interest in the incorpora-
tion of electrochemically active modifications into oligonucle-
otides.1,2 This has been motivated by the emerging need to develop
nonoptical detection techniques that are amenable to miniaturiza-
tion.3,4 Early approaches to redox-active DNA tags utilized organic
probes (e.g., anthraquinones),5 while more recent systems have
relied on ferrocene-containing nucleosides.6,7 Two important ad-
vances remain to be addressed: (1) The development of multiple
tags with diverse and “tunable” redox potentials that maintain
similar structural and chemical features, and (2) the development
of redox-active nucleoside triphosphate that can be enzymatically
incorporated at any position by DNA polymerase.8 Here we disclose
the design, synthesis, and characterization of novel redox-active
tags with tunable electrochemical potentials, as well as the synthesis
and successful enzymatic incorporation of an electrochemically
active nucleoside triphosphate into DNA oligonucleotides.
Several criteria have been taken into consideration when design-
ing electrochemically active nucleotides and DNA-labeling agents.
The E1/2 value of the electroactive moiety should fall outside the
oxidation or reduction potentials of the natural heterocyclic bases.9
Taking into account the oxidation and reduction potentials of water
and the diminished barrier for oxidation of G residues when found
within G-tracts, useful electrochemically active DNA labels should
possess redox potentials between +0.8 and -0.7 V versus SCE.10
Within this window, a range of redox potentials should be accessible
to ensure well-resolved signals for “multicolor” detection. The
electroactive moieties should also be chemically stable and compat-
ible with polymerase reaction conditions and electrophoretic
separations.
Kinetically inert polypyridine Ru2+ and Os2+ complexes are
attractive candidates due to their chemical stability and favorable
redox properties: the Mn/n+1 couples are metal-centered and
reversible, and their E1/2 can be tuned by the selection of the
coordinated ligands.11 [Ru(bpy)3]2+ and [Ru(phen)3]2+ exhibit oxi-
dation potentials outside the proposed working range (E1/2 ≈ 1.25
V vs SCE).11 If one of the polypyridine ligands, however, is replaced
by a negatively charged ligand (e.g., an acetylacetonate or hydrox-
amate), the resulting complexes [(bpy)2Ru(L)]+ exhibit appreciably
lower oxidation potentials, well within the desired range.11,12
The synthesis of the tagged nucleotides is modular and encom-
passes the following building blocks: (a) substituted 2,4-pentanedi-
ones or hydroxamic acids bearing a functionalized linker, (b) bis-
substituted Ru2+ or Os2+ precursors (R2bpy)2ML2, and (c) modified
nucleotides. Alkylating the dianion derived from acetylacetone with
lithium 5-bromopropanoate affords a functional linker (Scheme 1).13
Treating the resulting ligand with bis-substituted bipyridine Ru2+
or Os2+ precursors gives the neutral complexes 1-3.13 Complex-
ation with metal ions activates the acetylacetonate and facilitates
Complexes Bearing a Functionalized Linker for Nucleotide Tagginga
a Reagents and Conditions: (a) THF, -78 °C to rt, 29%; (b) (R2bpy)2MCl2,
EtOH/H2O, CaCO3, reflux, 80-95%; (c) For 4: Cu(NO3)2/Ac2O, 71%; for 5
and 6: N-chlorosuccinimide, 58-60%.13
electrophilic substitution reactions (e.g., halogenation and nitration)
at the central position to give complexes 4-6 (Scheme 1).14
Cyclic voltammetry of the complexes shows a reversible wave
for the metal centered oxidation with ∆E ) 68 ( 2 mV (CH3CN,
0.1 M n-Bu4N+PF6-), close to the anticipated 1e Nerstian process.13
As expected, oxidation of the parent ruthenium complex 1 occurs
at considerably lower potentials when compared to [Ru(bpy)3]2+
,
due to the lower charge and stabilization of the higher oxidation
state by the O,O-donor ligands.15 The tunabilty of redox potentials
is demonstrated by substituting the bipyridine co-ligands, selecting
other central ions and modifying the acetonate moiety. Replacing
the bpy co-ligands with 4,4′-dimethyl-2,2′-bipyridine results in a
shift of 0.11 V to lower potentials. A more pronounce negative
shift of 0.36 V is observed for the analogous osmium complex 3
(Scheme 1). Modifying the complexes with electron-withdrawing
substituents (e.g., NO2 in 4 and Cl in 5 and 6) results in a shift to
more positive potentials.
Functionalized hydroxamic acids are synthesized by alkylating
benzyl-4-hydroxybenzoate with a suitable spacer followed by
activation as the pentachlorophenyl ester (Scheme 2).13 Condensa-
tion with N-methyl hydroxylamine provides the N-substituted
hydroxamic acid. Complexation with metal bipyridine carbonate
precursors gives complexes 7-9.16 The hydroxamate complexes
exhibit a reversible redox waves and their E1/2 values are negatively
shifted compared to those for the analogous acetylacetonates. The
effect of changing the co-ligands and metal ions is similar to that
observed for the acetonate complexes shown in Scheme 1. Together
with the acetylacetonate derivatives, complexes 1-9 span a potential
window of approximately 0.9 V.
A redox-active dTTP analogue 10 is synthesized by coupling
5-(3′′-aminopropynyl)-2′-deoxyuridine-5′-triphosphate to the suc-
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1568 VOL. 124, NO. 8, 2002 J. AM. CHEM. SOC.
10.1021/ja017193q CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. Synthesis and Electrochemical Data of Hydroxamate
mass and additional single positive charge at the metal center. Full-
length extension is also observed with a longer template containing
multiple dA residues.19 Taken together, these experiments demon-
strate that the modified nucleotide is very well tolerated by the
polymerase at the triphosphate level as well as part of the growing
chain.20
Complexesa
These results demonstrate that octahedral Ru2+ and Os2+ com-
plexes are viable, diverse, and tunable redox-active tags for DNA
modification. The ability of DNA polymerase to efficiently incor-
porate the metal-containing nucleotide triphosphate suggests po-
tential use in DNA diagnostics.
Acknowledgment. We thank Motorola Clinical Micro Sensors,
the UC BioSTAR Project (Grant S99-41) and the National Institutes
of Health (Grant GM 58447) for generous support.
a Reagents and Conditions: (a) Br(CH2)5CO2Et, NaH, THF, -78 °C to rt,
88%; (b) (i) H2/Pd, 92%, (ii) C6Cl5OH, DCC, 85%, (iii) NH(OH)CH3, DMAP,
59%; (c) (R2bpy)2MCO3, 30-52%.
Supporting Information Available: Additional synthetic, analyti-
cal, and electrochemical data as well as details for enzymatic synthesis
of oligonucleotides (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
Scheme 3. Synthesis of Triphosphate 10 (dRuTP)a
References
(1) Meldrum, D. R. Science 2001, 292, 515-517.
(2) For electronic detection of nucleic acids, see: Umek, R. M.; Lin, S. W.;
Vielmetter, J.; Terbrueggen, R. H.; Irvine, B.; Yu, C. J.; Kayyem, J. F.;
Yowanto, H.; Blackburn, G. F.; Farkas, D. H.; Chen, Y.-P. J. Mol. Diagn.
2001, 3, 74-84.
(3) For capillary electrophoresis chips with integrated electrochemical detec-
tion see: Woolley, A. T.; Lao, K.; Glazer, A. N.; Mathies, R. A. Anal.
Chem. 1998, 70, 684-688. For miniaturized electrochemical devices,
see: Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1997,
69, 253-258.
(4) Electrochemical methods offer both high sensitivity and adaptability to
microfabrication, see: Fan, F.-F.; Bard, A. J. Science 1995, 267, 871-
874. Gavin, P. E.; Ewing, A. G. J. Am. Chem. Soc. 1996, 118, 8932-
8936.
a Reagents and conditions: DMF/dioxane/H2O, (iPr2)NEt, quantitative.
(5) Mori, K.; Subasinghe, C.; Cohen, J. S. FEBS Lett. 1989, 249, 213-218.
Kertesz, V.; Whittemore, N. A.; Inamati, G. B.; Manoharan, M.; Cook,
P. D.; Baker, D. C.; Chambers, J. Q. Electroanalysis 2000, 12, 889-894.
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Tao, C.; Strong, M.; Choing, Y. J. Org. Chem. 2001, 66, 2937-2942.
Yu, C. J.; Yowanto, H.; Wan, Y.; Meade, T. J.; Chong, Y.; Strong, M.;
Donilon, L. H.; Kayyem, J. F.; Gozin, M.; Blackburn, G. F. J. Am. Chem.
Soc. 2000, 122, 6767-6768.
(7) For a review on metallorganic labels for DNA, see: Sloop, F. V.; Brown,
G. M.; Sachleben, R. A.; Garrity, M. L.; Elbert, J. E.; Jacobson, K. B.
New. J. Chem. 1994, 18, 317-326. For related DNA modifications:
Hurley, D. J.; Tor, Y. J. Am. Chem. Soc. 1998, 120, 2194-2195. Lewis,
F. D.; Helvoigt, S. A.; Letsinger, R. L. Chem. Commun. 1999, 327-328.
Khan, S. I.; Beilstein, A. E.; Grinstaff, M. W. Inorg. Chem. 1999, 38,
418-419. Rack, J. J.; Krider, E. S.; Meade, T. J. J. Am. Chem. Soc. 2000,
122, 6287-6288 and references therein.
(8) For end labeling, see: Anne, A.; Blanc, B.; Moiroux, J. Bioconjugate
Chem. 2001, 12, 396-405.
(9) Steenken, S.; Telo, J. P.; Novais, H. M.; Candeias, L. P. J. Am. Chem.
Soc. 1992, 114, 4701-4709; Seidel, C. A. M.; Schultz, A.; Sauer, M. H.
M. J. Phys. Chem. 1996, 100, 5541-5553; Steenken, S.; Jovanovic, S.
V. J. Am. Chem. Soc. 1997, 119, 617-618.
(10) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and
Applications, 2nd ed.; John Wiley & Sons, Inc.: New York, 2001.
(11) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.; von
Zelewsky, A. Coord. Chem. ReV. 1988, 84, 85-277.
(12) Ghosh, P.; Chakravorty, A. Inorg. Chem. 1984, 23, 2242-2248
(13) See Supporting Information for additional experimental details.
(14) Collman, J. P. Angew. Chem., Int. Ed. Engl. 1965, 4, 132-138.
(15) Converting the terminal carboxylate to benzylamide does not affect the
E1/2 value of the complex.13
Figure 1. Enzymatic incorporation of a Ru2+ modified nucleotide dRuTP.
(Top): Template and 32P-labeled primer oligonucleotides. (Bottom): Primer
extension experiments using the Klenow fragment of DNA polymerase
resolved on a 20% PAGE. (Lanes 1 and 2): C and T sequencing lanes,
respectively. (Lane 3): Full-length product is generated in the presence of
all triphosphates. (Lane 4): No deleterious effect is observed in the presence
of all triphosphate and dRuTP. (Lane 5): Full-length product containing a
single Ru-containing base is observed. (Lane 6): Truncated product is
generated in the absence of dTTP and dRuTP.
cinimide active ester of the metal complex (Scheme 3).13 The
enzymatic incorporation of the modified base 10 was tested using
a 5′-32P-labeled 13-mer primer and a 22-mer template (Figure 1).17
Primer extension in the presence of all four dNTPs gives the expec-
ted full-length product (lane 3). Addition of dRuTP to the four dN-
TPs does not interfere with the enzymatic elongation process (lane
4). If dTTP is eliminated, premature termination occurs right after
the CGGC site, yielding an 18-mer product (lane 6). When dTTP
is replaced by dRuTP, only one band appears, and premature term-
ination is not observed (lane 5).18 The full-length 22-mer metal-
containing DNA displays a slower electrophoretic mobility when
compared to the corresponding native fragments due to the increased
(16) An analogous complex to 7, in which the hydroxamic acid is not substituted
[i.e., R-CON(OH)H], has been found to be unstable.13
(17) The template contains a single adenosine residue to unequivocally
determine the incorporation of the modified nucleotide and the generation
of a full-length product.
(18) A similar faint band also appears when dRuTP is mixed with dTTP in a
1:1 ratio (compare lanes 4 and 5), suggesting that the modified nucleotide
can compete to an extent with the native nucleotide for incorporation
opposite dA.
(19) Template: GCT-GAG-TGA-TAT-CGC-AGC-ATC-AGT-ACC.
(20) The ability of a polymerase to accept a modified dNTP as a substrate and
extend the growing chain beyond the modification site is significantly
more demanding than end labeling or chain termination.
JA017193Q
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