2,2′-bipyridine ligandoside: A novel building block for modifying DNA with intra-duplex metal complexes [2]

Full text of DOI:10.1021/ja005785n

J. Am. Chem. Soc. 2001, 123, 3375-3376
2,2′-Bipyridine Ligandoside: A Novel Building Block
3375
for Modifying DNA with Intra-Duplex Metal
Complexes
Haim Weizman and Yitzhak Tor*
Department of Chemistry and Biochemistry
UniVersity of California, San Diego
La Jolla, California 92093-0358
Figure 1. Schematic representation of an interstrand metal complex
within a DNA double helix (right) and a ligandoside (left).
ReceiVed NoVember 14, 2000
Scheme 1. Synthesis of 2,2′-bipyridine Ligandoside and Its
Phosphoramiditea
The structure, dynamics, and recognition properties of the DNA
double helix continue to fascinate chemists. While ingenious and
diverse approaches have been devised to probe the various
surfaces of DNA (e.g., groove binders, metal complexes),1 little
has been done to explore the interior of the helix. This partially
exposed domain is comprised of stacked heterocycles that project
H-bond donors and acceptors into the interior as well as the two
distinct grooves. The ability of this aromatic π-stack to mediate
charge-transfer processes has recently taken center stage2 and
prompted us to consider modification to the DNA core.3 We have
envisioned the placement of charged metal complexes at the center
of the DNA helix as illustrated in Figure 1.4,5
Nucleoside mimics, hereby coined ligandosides, where the
heterocyclic base is replaced by a strong chelator, are key to our
approach (Figure 1).6,7 A ligandoside should meet the following
requirements: (a) Be compatible with standard DNA synthesis;
(b) have higher affinity for metal ions than the heterocyclic bases;
(c) form complexes with comparable dimensions to a DNA base
pair. 2,2′-Bipyridine has been selected as the ligand due to its
high stability and affinity to numerous metal ions.8,9 Modeling
reveals that direct attachment of the bpy to the sugar results in
compression of the duplex upon metal complexation. A methylene
group has therefore been introduced between position 5 of the
ligand and the 2′-deoxy-D-ribose. The saturated methylene bridge
relaxes the ligandoside and results in better fitting of the complex
within the DNA duplex.
a Reagents and conditions: (i) THF, -78 °C, rt 1 h, 72%; (ii)
diethylazodicarboxylate, Ph3P, THF, 3 h, 87%; (iii) BCl3, THF, -78 °C,
1.5 h, 58%; (iv) DMT-Cl, DMAP, Py, 1 h, 80% (mixture of two anomers),
chromatographic resolution; (v) 2-cyanoethyl-N,N,N′,N′-tetraisopropyl-
phophordiamidite, 1H-tetrazole, CH3CN, 1.5 h, 66%.14
the lithiation of 5-methyl-2,2′-bipyridine followed by coupling
with 3,5-Di-O-benzyl-2-deoxy-D-ribofuranose (Scheme 1).12 The
resulting diol 1 is then cyclized by a Mitsunobu reaction13 to give
a 1:1 diastereomeric mixture of the protected ligandoside 2.
Removal of the benzyl groups of 2 by hydrogenolysis failed under
various conditions, but was successfully accomplished using BCl3
to give the free ligandoside 3. Selective protection of the 5′
hydroxyl with 4,4′-dimethoxytrityl facilitated the chromatographic
resolution of the two diastereoisomers. The assignment of their
absolute configuration was based on NOE experiments.14 Phos-
phitylation of the â-anomer provided the ligandoside phosphor-
amidite 5.
While numerous methods for synthesizing aromatic N- and
C-glycosides have been developed,10 synthetic approaches to
aliphatic C-nucleosides are scarce.11 Our approach is based on
(1) Erkkila, K. E.; Odom, D. T.; Barton, J. K. Chem. ReV. 1999, 99, 2777-
2795. Jamieson, E. R.; Lippard, S. J. Chem. ReV. 1999, 99, 2467-2498. Kool,
E. T. Chem. ReV. 1997, 97, 1473-1488. Nielsen, P. E. Chem. Eur. J. 1997,
3, 505-508. Waring, M. J. Bailly, C. J. Pharm. Belg. 1997, 52, 70-78.
Trauger, J. W.; Baird, E. E.; Dervan, P. B. Nature 1996, 382, 559-561. Kahne,
D. Chem. Biol. 1995, 2, 7-12.
(2) Netzel, T. L. J. Biol. Inorg. Chem. 1998, 3, 210-214. Grinstaff, M.
W. Angew. Chem., Int. Ed. 1999, 38, 3629-3635. Nunez, M. E.; Barton, J.
K. Curr. Opin. Chem. Biol. 2000, 4, 199-206. Stemp, E. D. A.; Holmlin, R.
E.; Barton, J. K. Inorg. Chim. Acta 2000, 297, 88-97
(3) Various strategies have been employed for modifying the periphery of
DNA with metal complexes. See, for example: 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
(4) Backbone phosphates may then be envisioned to serve as counteranions.
(5) Hydrophobic nucleoside mimics form surrogate non-hydrogen-bonded
base pairs. See: Kool, E. T.; Morales, J. C.; Guckian, K. M. Angew. Chem.,
Int. Ed. 2000, 39, 991-1009. Ogawa, A. K.; Wu, Y. Q.; McMinn, D. L.; Liu,
J. Q.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 3274-
3287.
The ligandoside (3, Xbpy) was incorporated into DNA oligo-
nucleotides using solid-phase DNA synthesis.14 A self-comple-
mentary 11-mer sequence 6 was synthesized and purified by
polyacrylamide gel electrophoresis in the presence of EDTA.
Enzymatic digestion followed by HPLC analysis confirmed the
presence of the intact ligandoside in the anticipated amount.14
A
10-mer oligonucleotide 7 that has an identical base sequence but
lacks the modified nucleoside was synthesized as a control (Figure
2).
(6) The synthesis of o-phenylenediamine furanoside, a potential metal
binder, has been reported: Tanaka, K.; Shionoya, M. J. Org. Chem. 1999,
64, 5002-5003.
(10) Postema, M. H. D. C-Glycoside Synthesis; CRC Press: Boca Raton,
Florida, 1995. See aslo: Schweitzer, B. A.; Kool E. T. J. Org. Chem. 1994,
59, 7238-7242. Chaudhuri, N. C.; Kool, E. T. Tetrahedron Lett. 1995, 36,
1795-1798.
(7) A selective, neutral, and complementary copper-mediated base pair has
been reported: Meggers, E.; Holland, P. L.; Tolman, W. B.; Romesberg, F.
E.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 10714-10715.
(8) Constable, E. C. AdV. Inorg. Chem. 1989, 34, 1-63. Kaes, C.; Katz.
A.; Hosseini, M. W. Chem. ReV. 2000, 100, 3553-3590.
(11) Boal, J. H.; Wilk, A.; Scremin, C. L.; Gray, G. N.; Phillips, L. R.;
Beaucage, S. L. J. Org. Chem. 1996, 61, 8617-8626.
(12) Wierenga, W.; Skulnick, H. I. Carbohydr. Res. 1981, 90, 41-52
(13) Harusawa, S.; Murai, Y.; Moriyama, H.; Imazu, T.; Ohishi, H.; Yoneda,
R.; Kurihara, T. J. Org. Chem. 1996, 61, 4405-4411.
(9) The low nucleophilicity of the heterocyclic bpy nitrogens precludes
the need for protecting groups during DNA synthesis.
(14) See Supporting Information for experimental details.
10.1021/ja005785n CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/04/2001

3376 J. Am. Chem. Soc., Vol. 123, No. 14, 2001
Communications to the Editor
Figure 2. Sequences of a ligandoside-containing oligonucleotide (6) and
its control sequence (7).
Figure 4. Thermal denaturation curves for ligandoside modified duplex
6 (0), control duplex 7 (O), and duplex 6 in the presence of Cu2+ (4).
Measurements were performed in 400 mM Tris-acetate pH 7.8, in the
presence of 2 × 10-6 M oligonucleotide for 6 and 7, and 2 × 10-6
M
oligonucleotide + 1 × 10-6 M Cu(OAc)2 for 6+Cu2+
.
The observed melting temperature of the ligandoside-containing
DNA 6 was the same as the 10-mer control (Tm ) 56.5 °C), yet
the slope of the melting curve was less steep, suggesting lower
cooperativity of the melting process (Figure 4). A priori, the bpy
ligands can be either stacked inside the DNA duplex or exposed
to the solvent. At neutral and basic pHs, the bpy ligands are
uncharged and are therefore expected to minimize interactions
with water. The shape of the melting curve suggests that the free
ligands are likely to be buried within the helix, causing disruption
of the melting process. Upon addition of 1.0 equiv of Cu2+ to
the ligandoside-containing duplex 6, a significant increase in the
Tm value is observed (Tm ) 64 °C), and a cooperative melting
curve is restored (Figure 4). This stabilization is in agreement
with an interstrand, intraduplex tetracoordinated complex forma-
tion. It is likely that, within the duplex, solvent molecules are
excluded and the backbone phosphate groups serve as the
counterions. It is important to note that the melting curve of the
control duplex 7 dose not change upon addition of 1 equiv of
Figure 3. Absorption spectra of ligandoside 3 and the modified DNA 6
upon addition of Cu2+. (a) Unattached ligandoside (s 3, - - - 3+Cu2+);
(b) Modified DNA and a control sequence (s 6, - - - 6+Cu2+
‚ - ‚ - 7); (c) Difference spectra (s 6-7, - - - 6+Cu2+-7).
,
The UV spectrum of the free ligandoside 3 displays two bands
at 240 and 284 nm, characteristic to the bpy π-π* transitions
(Figure 3a).15 Upon addition of 0.5 equiv of Cu(OAc)2 the
absorptions bands are shifted to 248 and 304 nm, respectively.
The UV spectrum of the ligandoside-containing sequence 6, is
predominated by the 260 nm absorption of the DNA heterocycles
(Figure 3b). Subtracting the spectrum of the control DNA
sequence 7 (Figure 3b) from that of the ligandoside-containing
DNA 6 reveals the characteristics transitions of the ligandoside
(Figure 3c). Upon addition of 1.0 equiv of Cu2+ to the modified
DNA duplex 6, a new band appears as a shoulder around 305
nm (Figure 3b).16 Subtracting the spectrum of the control DNA
sequence 7 from that of the metalated ligandoside-containing
DNA 6-Cu2+ unveils a shift in the transitions associated with
the chelator, indicative of metal complex formation (Figure 3c).
Addition of Cu2+ to the control sequence 7 results in no spectral
changes.
Cu2+
.
These results demonstrate the utilization of a ligandoside, an
unnatural nucleoside mimic, for the incorporation of a charged
homoleptic metal complex inside a DNA duplex. This strategy
represents a unique approach for metal-mediated interstrand
binding. Such noncovalent interactions can be viewed as tunable
cross-linking. Metal coordination is typically stronger than
hydrogen bonds and, unlike covalent bonds, can be easily
“denatured” in the presence of competing ligands.
Acknowledgment. We thank the National Institutes of Health (Grant
number GM 58447) for generous support.
Supporting Information Available: Additional data regarding
synthesis and structural assignment of the ligandoside, thermal de-
naturation measurements, HPLC, and enzymatic digestions of the
oligonucleotides (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org
Thermal denaturation experiments have been utilized to
compare the relative stability of the various duplexes (Figure 4).14
(15) Due to the high affinity of bpy to numerous metal ions, all solvents
and reagents were purified over Chelex 100 resin prior to use.
(16) Note that since oligonucleotides 6 and 7 are self-complementary, the
amount of Cu2+ added is given per duplex and not per strand.
JA005785N