Mispair-aligned N3T-alkyl-N3T interstrand cross-linked DNA: Synthesis and characterization of duplexes with interstrand cross-links of variable lengths

Article abstract of DOI:10.1021/ja0498540

Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized a cross-link that bridges the N³ positi

Full text of DOI:10.1021/ja0498540

Published on Web 07/10/2004
Mispair-Aligned N3T-alkyl-N3T Interstrand Cross-Linked DNA:
Synthesis and Characterization of Duplexes with Interstrand
Cross-Links of Variable Lengths
Christopher J. Wilds,†,‡ Anne M. Noronha,†,§ Sebastien Robidoux,| and
Paul S. Miller*,†
Contribution from the Department of Biochemistry and Molecular Biology, Bloomberg School of
Public Health, Johns Hopkins UniVersity, Baltimore, Maryland, 21205, and Department of
Chemistry and Biochemistry, Concordia UniVersity, Montreal, Quebec, H4B 1R6, Canada
Received January 9, 2004; E-mail: pmiller@jhsph.edu
Abstract: Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As
part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized
a cross-link that bridges the N3 positions of a mismatched thymidine base pair. This cross-link, which is
similar to the N3C-alkyl-N3C cross-link that has been observed between mismatched cytosine base pairs,
was introduced by first incorporating a cross-linked phosphoramidite unit at the 5′-end of an oligonucleotide
chain. Fully cross-linked duplexes were then synthesized using an orthogonal approach to selectively remove
protecting groups, thus allowing construction of the cross-linked duplex via conventional solid-phase
oligonucleotide synthesis. Short DNA duplexes with alkyl cross-links of various lengths (two, four, and
seven methylene units) were prepared, and their physical properties were studied via UV thermal
denaturation and circular dichroism spectroscopy. These linkers were found to stabilize the duplexes by
37, 31, and 16 °C for the two-, four-, and seven-carbon linkers, respectively, relative to a non-cross-linked
duplex. Circular dichroism spectra suggested that these lesions induce very little deviation in the global
structure relative to the non-cross-linked duplex DNA control. Molecular models show that the two-carbon
cross-link spans the distance between the N3 atoms of the T-T mismatch without perturbing the helix
structure, whereas the longer linkers, particularly the seven-carbon linker, tend to push the thymines apart,
creating a local distortion. This perturbation may account for the lower thermal stability of the seven-carbon
versus two-carbon cross-linked duplex.
Introduction
a number of repair pathways, including homologous recombina-
tion and nucleotide excision repair, have been implicated in
Covalent DNA lesions play important roles in the etiology
and treatment of cancer. Therapeutic alkylating agents include
monofunctional and bifunctional agents. The latter category in-
cludes platinum agents, nitrogen mustards, chloroethylnitro-
soureas, and cyclophosphamide.1-4 The major cytotoxic lesion
introduced by the latter three agents are interstrand cross-
links, which if left unrepaired, prevent DNA strand separation
and normal mitosis, interfere with transcription, and induce
apoptosis.
cross-link repair in bacteria and mammalian cells, an under-
standing of the molecular mechanisms that are involved is still
lacking.8-10 Since development of resistance is one of the
primary reasons for treatment failure, a better understanding of
the processes by which interstrand cross-links are recognized
and repaired could lead to the development of more effective
bifunctional alkylating agents and/or treatment protocols.
To carry out biological and structural investigations, it is
necessary to have access to duplexes that contain interstrand
cross-links of well-defined structure. There are two general
strategies employed in producing such duplexes. The first
A problem presented in cancer treatment is the development
of resistance to the effects of antitumor agents, which is often
due to enhanced repair of interstrand cross-links.5-7 Although
† Department of Biochemistry and Molecular Biology, Johns Hopkins
University.
(6) (a) Friedman, H. S.; Colvin, O. M.; Kaufmann, S. H.; Ludeman, S. M.;
Bullock, N.; Bigner, D. D.; Griffith, O. W. Cancer Res. 1992, 52, 5373-
5378. (b) Dong, Q.; Bullock, N.; Aliosman, F.; Colvin, O. M.; Bigner, D.
D.; Friedman, H. S. Cancer Chemother. Pharmacol. 1996, 37, 242-246.
(c) Dong, Q.; Johnson, S. P.; Colvin, O. M.; Bullock, N.; Kilborn, C.;
Runyon, G.; Sullivan, D. M.; Easton, J.; Bigner, D. D.; Nahta, R.; Marks,
J.; Modrich, P.; Friedman, H. S. Cancer Chemother. Pharmacol. 1999,
43, 73-79.
‡ Current address: Department of Chemistry and Biochemistry, Con-
cordia University.
§ Current address: Alnylam Pharmaceuticals, Cambridge, MA.
| Department of Chemistry and Biochemistry, Concordia University.
(1) Colvin, M. The Alkylating Agents; W. B. Saunders Co.: Philadelphia, 1982.
(2) Colvin, M. Alkylating Agents and Platinum Antitumor Compounds; Lea &
Febiger: Philadelphia and London, 1993.
(7) Ali-Osman, F.; Raikar, A.; Young, P. Cancer Biochem. Biophys. 1995,
14, 231-241.
(3) Pratt, W. B.; Ruddon, R. W.; Ensminger, W. D.; Maybaum, J. CoValent
(8) Friedberg, E. C.; Walker, G. C.; Siede, W. DNA Repair and Mutagenesis;
ASM Press: Washington, DC, 1995.
DNA Binding Drugs; Oxford Press: New York, 1994.
(4) Rajski, S. R.; Williams, R. M. Chem. ReV. 1998, 98, 2723-2725.
(5) O’Connor, P. M.; Kohn, K. W. Cancer Commun. 1990, 2, 387-394.
(9) Dronkert, M. L. G.; Kanaar, R. Mutat. Res. 2001, 486, 217-247.
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9
10.1021/ja0498540 CCC: $27.50 © 2004 American Chemical Society
J. AM. CHEM. SOC. 2004, 126, 9257-9265
9257

A R T I C L E S
Wilds et al.
and ChemGenes, respectively. All other chemicals and solvents were
purchased from Aldrich Chemical Co. (Milwaukee, WI). 2-Iodoethyl
phenoxyacetate was prepared from 2-iodoethanol and phenoxyacetyl
chloride. Both 4-iodobutyl phenoxyacetate and 7-iodoheptylphenoxy-
acetate were prepared by a two-step reaction: the corresponding 1,4-
and 1,7-diols (50 equiv) were reacted with phenoxyacetyl chloride (1
equiv), followed by the conversion of the free alcohol to an iodide.24
Strong anion exchange (SAX) HPLC was carried out using a Dionex
DNAPAC PA-100 column (0.4 cm × 25 cm) purchased from Dionex
Corp, Sunnyvale, CA. Reversed-phase HPLC was carried out using a
Microsorb-C-18 column (0.46 × 15 cm) purchased from Varian
Associates, Walnut Creek, CA. The SAX column was eluted with a
30 mL linear gradient of sodium chloride in a buffer that contained
100 mM Tris (pH 7.8) in 10% acetonitrile at a flow rate of 1.0 mL/
min. The C-18 column was eluted with a 20 mL linear gradient of
acetonitrile in 50 mM phosphate buffer (pH 5.8) at a flow rate of 1.0
mL/min. The columns were monitored at 260 nm for analytical runs
or 290 nm for preparative runs. Denaturing polyacrylamide gel
electrophoresis was carried out on 20 cm × 20 cm × 0.75 cm gels
containing 20% acrylamide and 7 M urea in TBE, which contained 89
mM Tris, 89 mM boric acid, and 0.2 mM ethylenediaminetetraacetate
buffered at pH 8.0. The running buffer was TBE. [32P]-Labeled
oligonucleotides were detected by autoradiography. Mass spectra were
obtained at the John Hopkins University School of Medicine Mass
Spectrometry Facility (MALDI) or from the Scripps Research Institute
Mass Spectrometry Facility (ESI).
5′-O-Dimethoxytrityl-3′-O-phenoxyacetyl-2′-deoxythymidine, 2.25
5′-O-Dimethoxytrityl-2′-deoxythymidine (4.0 g, 7.36 mmol) was dis-
solved in THF (20 mL) and allowed to cool to 0 °C. Triethylamine
(1.19 g, 11.72 mmol) followed by phenoxyacetyl chloride (1.89 g, 11.0
mmol) was added dropwise. After 14 h, the solvent was removed in
vacuo, the crude product was taken up in dichloromethane (50 mL),
and the solution was washed with three portions of sodium bicarbonate
(3 × 50 mL). The organic layer was dried over sodium sulfate and
concentrated to give a yellow gum. The crude product was purified by
column chromatography using dichloromethane/methanol (49:1) to
afford 3.95 g (79.3%) of product as a colorless foam. Rf (SiO2 TLC):
0.31 dichloromethane/methanol (19:1).1H NMR (400 MHz, DMSO-
d6, ppm): 1.41 (3H, C5-CH3), 2.32-2.52 (2H, H2′ and H2′′), 3.18-
3.34 (2 H, H5′ and H5′′), 3.71 (6H, -OCH3), 4.10-4.11 (1H, H4′),
4.80 (2 H, -CH2OPh), 5.38-5.40 (1H, H3′), 6.19-6.23 (1 H, H1′),
6.84-7.38 (18 H, DMT, Ph), 7.51 (1 H, H6). ESI-MS (M + Na+):
701 (calcd 701).
approach involves treatment of duplex DNA with an excess of
bifunctional alkylating agent to produce the interstrand cross-
link.11-16 This methodology suffers from poor yields because
of the production of numerous monoalkylated and other side
products that must be separated from the desired cross-linked
product and the low efficiency of the cross-linking reaction itself.
The second and more attractive approach involves de novo
synthesis of a cross-linked duplex employing a solid-phase
synthesis approach.17
We recently described the syntheses and characterization of
short DNA duplexes that contain a N4C-ethyl-N4C interstrand
cross-link using either a phosphoramidite reagent to introduce
the cross-link during automated synthesis of the cross-linked
duplex or a convertible nucleoside approach.18-21 This meth-
odology has allowed the solid-phase syntheses of a number of
novel cross-linked duplexes of well-defined structure that have
been engineered to contain an interstrand cross-link between a
mismatched C-C base pair,18 a “staggered” interstrand cross-
link at a -CG- or -GC- step,20 as well as 1,3 interstrand
-CNG- cross-links containing alkyl linkages of various
lengths.21 The latter are of particular interest because they share
similarities to the interstrand cross-links formed by reaction of
duplex DNA with bifunctional alkylators, such as mechlor-
ethamine, which forms a N7G-alkyl-N7G cross-link in sequences
containing a -GNC- sequence motif.
As part of our investigation of interstrand cross-linked DNA,
we have developed a straightforward synthesis of a novel
interstrand N3T-alkyl-N3T cross-link. This lesion is similar
to the recently discovered N3C-alkyl-N3C cross-link formed
when duplex DNA containing a C-C mismatch is treated with
mechlorethamine.22,23 In this report, we describe the syntheses
of short DNA duplexes that contain N3T-alkyl-N3T interstrand
cross-links and preliminary characterization of their physical
properties.
Experimental Section
Materials and General Methods. 5′-O-Dimethoxytrityl-2′-deoxy-
thymidine was purchased from ChemGenes Inc., Ashland, MA. 5′-O-
Dimethoxytrityl-3′-O-tert-butyldimethylsilyl-2′-deoxythymidine (1) was
prepared as previously described.18 5′-O-Dimethoxytrityldeoxyribonu-
cleoside-3′-O-(â-cyanoethyl-N,N′-diisopropyl) phosphoramidites, 3′-O-
dimethoxytrityldeoxyribonucleoside-5′-O-(â-cyanoethyl-N,N′-diisopropyl)-
phosphoramidites, and protected deoxyribonucleoside-controlled pore
glass supports were purchased from Glen Research, Inc. (Sterling, VA)
N3-[2-(Phenoxyacetyl)ethyl]-5′-O-dimethoxytrityl-3′-O-(tert-bu-
tyldimethylsilyl)-2′-deoxythymidine, 3a. 5′-O-Dimethoxytrityl-3′-O-
(tert-butyldimethylsilyl)-2′-deoxythymidine 1 (5.0 g, 7.59 mmol) was
dissolved in acetonitrile (200 mL). Then, 2-iodoethyl phenoxyacetate
(4.7 g, 15.35 mmol) was added, followed by the addition of 1,8-
diazabicyclo[5.4.0]undec-7-ene (2.36 g, 15.51 mmol). After 24 h, the
solvent was removed in vacuo, the crude product was taken up in
dichloromethane (200 mL), and the solution was washed with three
portions of sodium bicarbonate (3 × 200 mL). The organic layer was
dried over sodium sulfate, and the crude compound was purified by
silica gel column chromatography using hexanes/ethyl acetate (4:1) to
afford 5.51 g (87.5%) of the product as a colorless foam. Rf (SiO2
(11) Ojwang, J. O.; Grueneberg, D. A.; Loechler, E. L. Cancer Res. 1989, 49,
6529-6537.
(12) Rink, S. M.; Solomon, M. S.; Taylor, M. J.; Rajur, S. B.; McLaughlin, L.
W.; Hopkins, P. B. J. Am. Chem. Soc. 1993, 115, 2551-2557.
(13) Tsarouhtsis, D.; Kuchimanchi, S.; Decorte, B. L.; Harris, C. M.; Harris, T.
M. J. Am. Chem. Soc. 1995, 117, 11013-11014.
(14) Rink, S. M.; Lipman, R.; Alley, S. C.; Hopkins, P. B.; Tomasz, M. Chem.
Res. Toxicol. 1996, 9, 382-389.
(15) Warren, A. J.; Hamilton, J. W. Chem. Res. Toxicol. 1996, 9, 1063-1071.
(16) Fischhaber, P. L.; Gall, A. S.; Duncan, J. A.; Hopkins, P. B. Cancer Res.
1999, 59, 4363-4368.
1
TLC): 0.66 hexanes/ethyl acetate (1:1). H NMR (400 MHz): 0.00
(3H, SiCH3), 0.05 (3H, SiCH3), 0.83 (9H, Si-C(CH3)3), 1.61 (3H,
C5-CH3), 2.20-2.41 (2H, H2′ and H2′′), 3.22-3.38 (2 H, H5′ and
H5′′), 3.79 (6H, -OCH3), 3.81-3.92 (1H, H4′), 4.15-4.18 (2H,
CH2-N3), 4.37-4.41 (2H, CH2-OPac), 4.49-4.53 (1H, H3′), 4.72 (2
H, -CH2OPh), 6.24-6.27 (1 H, H1′), 6.94-7.46 (18 H, DMT, Ph),
7.70 (1 H, H6). ESI-MS: 837 (calcd 836.4).
(17) Harwood, E. A.; Sigurdsson, S. T.; Edfeldt, N. B. F.; Reid, B. R.; Hopkins,
P. B. J. Am. Chem. Soc. 1999, 121, 5081-5082.
(18) Noll, D. M.; Noronha, A. M.; Miller, P. S. J. Am. Chem. Soc. 2001, 123
3405-3411.
(19) Noronha, A. M.; Noll, D. M.; Miller, P. S. Nucleosides Nucleotides Nucleic
Acids 2001, 20, 1303-1307.
(20) Noronha, A. M.; Noll, D. M.; Wilds, C. J.; Miller, P. S. Biochemistry 2002,
41, 760-771.
N3-[4-(Phenoxyacetyl)butyl]-5′-O-dimethoxytrityl-3′-O-(tert-bu-
tyldimethylsilyl)-2′-deoxythymidine, 3b. Compound 3b was synthe-
(21) Noronha, A. M.; Wilds, C. J.; Miller, P. S. Biochemistry 2002, 41, 8605-
8612.
(22) Romero, R. M.; Rojsitthisak, P.; Haworth, I. S. Arch. Biochem. Biophys.
2001, 386, 143-153.
(23) Romero, R. M.; Mitas, M.; Haworth, I. S. Biochemistry 1999, 38, 3641-
3648.
(24) Garegg, P. J.; Samuelsson, B. J. C. S. Chem. Commun. 1979, 978-980.
(25) Nikiforov, T.; Connolly, B. A. Tetrahedron Lett. 1992, 33, 2379-2382.
9
9258 J. AM. CHEM. SOC. VOL. 126, NO. 30, 2004

3
3
Mispair-Aligned N T-Alkyl-N T Cross-Linked DNA
A R T I C L E S
sized via the procedure outlined for 3a using 4-iodobutyl phenoxy-
acetate to afford the desired product in 82.2% yield. Rf (SiO2 TLC):
0.69 hexanes/ethyl acetate (1:1). 1H NMR (400 MHz, DMSO-d6,
ppm): 0.00 (3H, Si-CH3), 0.06 (3H, Si-CH3), 0.83 (9 H, Si-C(CH3)3),
1.52-1.70 (7H, -CH2-CH2- and C5-CH3), 2.19-2.41 (2 H, H2′
and H2′′), 3.20-3.33 (2 H, H5′ and H5′′), 3.78 (6H, -OCH3),
3.82-3.86 (2H, -CH2-N3-), 3.87-3.90 (1H, H4′), 4.15-4.18 (2H,
-CH2-OPac), 4.50-4.54 (1H, H3′), 4.82 (2H, OCH2OPh), 6.26 (1H,
H1′), 6.92-7.67 (18 H, DMT, Ph), 7.67 (H6). ESI-MS (M + H+):
866 (calcd 865.09).
N3-[7-(Phenoxyacetyl)heptyl]-5′-O-dimethoxytrityl-3′-O-(tert-bu-
tyldimethylsilyl)-2′-deoxythymidine, 3c. Compound 3c was synthe-
sized via the procedure outlined for 3a using 7-iodoheptyl phenoxy-
acetate to afford the desired product in 80.0% yield. Rf (SiO2 TLC):
0.52 hexanes/ethyl acetate (1:1)
1H NMR (400 MHz, DMSO-d6, ppm): 0.00 (3H, Si-CH3), 0.06
(3H, Si-CH3), 0.83 (9 H, Si-C(CH3)3), 1.29 (4H, -CH2-CH2-),
1.52-1.62 (9H, -CH2-CH2- and C5-CH3), 2.20-2.41 (2 H, H2′
and H2′′), 3.21-3.35 (2 H, H5′ and H5′′), 3.77 (6H, -OCH3),
3.81-3.85 (2H, -CH2-N3-), 3.88-3.91 (1H, H4′), 4.13-4.16 (2H,
-CH2-OPac), 4.51-4.55 (1H, H3′), 4.82 (2H, OCH2OPh), 6.25-6.28
(1H, H1′), 6.92-7.46 (18 H, DMT, Ph), 7.67 (H6). ESI-MS (M +
H+): 907 (calcd 906.4).
-CH2-N3-), 3.86-3.89 (1H, H4′), 4.49-4.53 (1H, H3′), 6.23-6.26
(1H, H1′), 6.92-7.45 (13 H, DMT), 7.65 (1H, H6). ESI-MS (M +
H+): 883 (calcd 882.3).
1-{N3-[5′-O-(Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-2′-
deoxythymidylyl]}-2-{N3-[5′-O-(dimethoxytrityl)-3′-O-(phenoxyacetyl)-
2′-deoxythymidylyl]}ethane, 5a. Compound 2 (3.68 g, 5.44 mmol)
was dissolved in acetonitrile (100 mL). Compound 4a (4.42 g, 5.44
mmol) was added, followed by the addition of 1,8-diazabicyclo[5.4.0]-
undec-7-ene (0.83 g, 5.44 mmol). After 24 h, the solvent was removed
in vacuo, the product was taken up in dichloromethane (100 mL), and
the solution was washed with three portions of sodium bicarbonate (3
× 100 mL). The organic layer was dried over sodium sulfate, and the
crude product was purified by silica gel column chromatography using
hexanes/ethyl acetate (2:3) to afford 5.82 g (78.5%) of the product as
a colorless foam. Rf (SiO2 TLC): 0.44 hexanes/ethyl acetate (1:1).
1H NMR (400 MHz): 0.00 (3H, Si-CH3), 0.05 (3H, Si-CH3), 0.84
(9H, Si-C(CH3)3), 1.49 (3H, C5-CH3), 1.56 (3H, C5-CH3),
2.16-2.56 (4H, H2′ and H2′′), 3.23-3.42 (4H, H5′ and H5′′), 3.44
(12 H, -OCH3), 3.90-3.91 (1H, H4′), 4.09-4.24 (5H, H4′ and
- N3-CH2-CH2-N3-), 4.49-4.53 (1H, H3′), 4.83 (2H, -CH2OPh),
5.48-5.50 (1H, H3′), 6.18-6.32 (2H, H1′), 6.95-7.48 (52H, DMT,
Ph), 7.60 (1H, H6), 7.64 (1H, H6). ESI-MS (M + Na+): 1386 (calcd
1386).
N3-(2-Iodoethyl-)-5′-O-dimethoxytrityl-3′-O-(tert-butyldimethyl-
silyl)-2′-deoxythymidine, 4a. Compound 3a (5.51 g, 6.58 mmol) was
dissolved in dichloromethane (50 mL), and propylamine (15 mL) was
added dropwise to the solution. After 24 h, the solvent was removed
in vacuo, the crude product was taken up in dichloromethane (250 mL),
and the solution was washed with three portions of sodium bicarbonate
(3 × 200 mL). The organic layer was dried over sodium sulfate and
evaporated to afford the crude 2-hydroxyethyl intermediate, which was
dissolved in THF (200 mL) and cooled to 0 °C. Imidazole (2.95 g,
43.47 mmol), triphenylphosphine (5.70 g, 21.74 mmol), and iodine (5.51
g, 21.74 mmol) were added sequentially. After 3 h, the solvent was
removed in vacuo, the crude product was taken up in dichloromethane
(200 mL), and the solution was washed with sodium thiosulfate (10%,
3 × 100 mL), dried over sodium sulfate, and then evaporated. The
crude product was purified via silica gel column chromatography using
hexanes/ethyl acetate (4:1) as eluent to afford 4.42 g (82.1%) of the
desired compound as a colorless foam. Rf (SiO2 TLC): 0.85 hexanes/
ethyl acetate (1:1). 1H NMR (400 MHz): 0.00 (3H, SiCH3), 0.06 (3H,
SiCH3), 0.83 (9H, Si-C(CH3)3), 1.60 (3H, C5-CH3), 2.19-2.42 (2H,
H2′ and H2′′), 3.20-3.34 (4 H, CH2-I, H5′ and H5′′), 3.79 (6H,
-OCH3), 3.88-3.90 (1H, H4′), 4.16-4.20 (2H, CH2-N3), 4.49-4.54
(1H, H3′), 6.22-6.25 (1 H, H1′), 6.93-7.45 (14 H, DMT), 7.69 (1 H,
H6). ESI-MS (M + H+): 813 (calcd 812.2).
N3-(2-Iodobutyl-)-5′-O-dimethoxytrityl-3′-O-(tert-butyldimethyl-
silyl)-2′-deoxythymidine, 4b. Compound 4b was synthesized via the
procedure outlined for 4a to afford the desired product in 85.8%
yield. Rf (SiO2 TLC): 0.74 hexanes/ethyl acetate (1:1). 1H NMR
(400 MHz, DMSO-d6, ppm): -0.05 (3H, Si-CH3), 0.01 (3H, Si-CH3),
0.78 (9 H, Si-C(CH3)3), 1.55 (3H, C5-CH3), 1.57-1.78 (4H,
-CH2-CH2-), 2.14-2.36 (2 H, H2′ and H2′′), 3.14-3.31 (4 H,
-CH2-I-, H5′ and H5′′), 3.73 (6H, -OCH3), 3.80-3.84 (3H,
-CH2-N3-, H4′), 4.45-4.49 (1H, H3′), 6.18-6.21 (1H, H1′),
6.87-7.40 (13 H, DMT), 7.61 (H6). ESI-MS (M + H+): 841 (calcd
840.9).
1-{N3-[5′-O-(Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-2′-
deoxythymidylyl]}-4-{N3-[5′-O-(dimethoxytrityl)-3′-O-(phenoxyacetyl)-
2′-deoxythymidylyl]}butane, 5b. Compound 5b was synthesized via
the procedure outlined for 5a to afford the desired product in 69.6%
yield. Rf (SiO2 TLC): 0.47 hexanes/ethyl acetate (1:1). 1H NMR (400
MHz, DMSO-d6, ppm): 0.00 (3H, Si-CH3), 0.06 (3H, Si-CH3), 0.83
(9 H, Si-C(CH3)3), 1.50-1.56 (7H, C5-CH3, -CH2-CH2-), 1.59 (3
H, C5-CH3), 2.20-2.48 (4 H, H2′ and H2′′), 3.19-3.35 (4 H, H5′
and H5′′), 3.78 (12H, -OCH3), 3.80-3.90 (5H, H4′, -CH2-N3),
4.19-4.22 (1H, H4′), 4.49-4.53 (1H, H3′), 4.87 (2H, OCH2OPh),
5.46-5.49 (1H, H3′), 6.23-6.34 (2H, H1′), 6.92-7.45 (31 H, DMT,
Ph), 7.64-7.66 (2H, H6). ESI-MS (M + H+): 1392 (calcd 1391.7).
1-{N3-[5′-O-(Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-2′-
deoxythymidylyl]}-7-{N3-[5′-O-(dimethoxytrityl)-3′-O-(phenoxyacetyl)-
2′-deoxythymidylyl]}heptane, 5c. Compound 5c was synthesized via
the procedure outlined for 5a to afford the desired product in 98.2%
yield. Rf (SiO2 TLC): 0.45 hexanes/ethyl acetate (1:1). 1H NMR (400
MHz, DMSO-d6, ppm): 0.00 (3H, Si-CH3), 0.06 (3H, Si-CH3),
0.83 (9 H, Si-C(CH3)3), 1.12-1.17 (4H, -CH2-CH2-), 1.54 (3H,
C5-CH3), 1.60 (3 H, C5-CH3), 2.21-2.48 (4 H, H2′ and H2′′),
3.19-3.35 (4 H, H5′ and H5′′), 3.37-3.46 (4H, -CH2-N3), 3.78
(12H, -OCH3), 3.87-3.90 (1H, H4′), 4.49-4.53 (3H, H3′), 4.87 (2H,
OCH2OPh), 6.23-6.34 (2H, H1′), 6.92-7.45 (31 H, DMT, Ph),
7.63-7.66 (2H, H6). ESI-MS (M + H+): 1434 (calcd 1432.7).
1-{N3-[5′-O-(Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-2′-
deoxythymidylyl]}-2-{N3-[5′-O-(dimethoxytrityl)-2′-deoxythymidylyl]}-
ethane, 6a. Compound 5b (5.38 g, 3.95 mmol) was dissolved in
dichloromethane (50 mL), and propylamine (10 mL) was added. After
12 h, the solvent was removed in vacuo, the crude product was taken
up in dichloromethane (100 mL), and the solution was washed with
three portions of sodium bicarbonate (3 × 100 mL). The organic layer
was dried over sodium sulfate and evaporated to afford the crude
product, which was purified via silica gel column chromatography using
hexanes/ethyl acetate (2:3) as eluent to afford 4.64 g (82.1%) of the
desired compound as a colorless foam. Rf (SiO2 TLC): 0.15 hexanes/
N3-(2-Iodoheptyl)-5′-O-dimethoxytrityl-3′-O-(tert-butyl-dimeth-
ylsilyl)-2′-deoxythymidine, 4c. Compound 4c was synthesized via
the procedure outlined for 4a to afford the desired product in
75.2% yield. Rf (SiO2 TLC): 0.67 hexanes/ethyl acetate (1:1). 1H
NMR (400 MHz): (400 MHz, DMSO-d6, ppm): 0.00 (3H, Si-CH3),
0.06 (3H, Si-CH3), 0.83 (9 H, Si-C(CH3)3), 1.28-1.39 (6H,
-CH2-CH2-CH2-), 1.52-1.56 (2H, -CH2-), 1.60 (3H, C5-CH3),
1.74-1.80 (2H, -CH2-), 2.19-2.41 (2 H, H2′ and H2′′), 3.19-3.33
(4 H, -CH2-I-, H5′ and H5′′), 3.78 (6H, -OCH3), 3.81-3.85 (2H,
1
ethyl acetate (1:1). H NMR (400 MHz, DMSO-d6, ppm): 0.00 (3H,
Si-CH3), 0.05 (3H, Si-CH3), 0.83 (9H, Si-C(CH3)3), 1.49 (3H,
C5-CH3), 1.54 (3H, C5-CH3), 2.18-2.37 (4H, H2′ and H2′′),
3.23-3.40 (4H, H5′ and H5′′), 3.44 (12 H, -OCH3), 3.88-3.92 (1H,
H4′), 3.96-4.00 (1H, H4′), 4.49-4.53 (1H, H3′), 4.12-4.24 (4H,
-N3-CH2-CH2-N3-), 4.35-4.39 (1H, H3′), 4.49-4.54 (1H, H3′),
6.17-6.26 (2H, H1′), 6.95-7.48 (26 H, DMT), 7.58 (1H, H6), 7.64
(1H, H6). ESI-MS (M + H+): 1229.5 (calcd 1228.5).
9
J. AM. CHEM. SOC. VOL. 126, NO. 30, 2004 9259

A R T I C L E S
Wilds et al.
1-{N3-[5′-O-(Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-2′-
deoxythymidylyl]}-4-{N3-[5′-O-(dimethoxytrityl)-2′-deoxythymidylyl]}-
butane, 6b. Compound 6b was synthesized via the procedure outlined
for 6a to afford the desired product in 98.1% yield. Rf (SiO2 TLC):
0.13 hexanes/ethyl acetate (1:1). 1H NMR (400 MHz, DMSO-d6,
ppm): 0.00 (3H, Si-CH3), 0.06 (3H, Si-CH3), 0.832 (9 H,
Si-C(CH3)3), 1.48-1.53 (7H, -CH2-CH2- and C5-CH3), 1.59 (3H,
C5-CH3), 2.19-2.41 (4 H, H2′ and H2′′), 3.20-3.34 (4H, H5′ and
H5′′), 3.78 (12H, -OCH3), 3.80-3.97 (6H, H4′, -CH2-N3-),
4.37-4.40 (1H, H3′), 4.49-4.53 (1H, H3′), 6.23-6.31 (2H, H1′), 6
.93-7.45 (26 H, DMT), 7.62 (H6), 7.67 (H6). ESI-MS (M + Na+):
1280 (calcd 1280).
1-{N3-[5′-O-(Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-2′-
deoxythymidylyl]}-7-{N3-[5′-O-(dimethoxytrityl)-2′-deoxythymidylyl]}-
heptane, 6c. Compound 6c was synthesized via the procedure outlined
for 6a to afford the desired product in 94.2% yield. Rf (SiO2 TLC):
0.28 hexanes/ethyl acetate (1:1). 1H NMR (400 MHz, DMSO-d6,
ppm): 0.00 (3H, Si-CH3), 0.06 (3H, Si-CH3), 0.83 (9 H, Si-C(CH3)3),
1.12-1.17 (4H, -CH2-CH2-), 1.54 (3H, C5-CH3), 1.60 (3 H,
C5-CH3), 2.21-2.48 (4 H, H2′ and H2′′), 3.19-3.35 (4 H, H5′ and
H5′′), 3.37-3.46 (4H, -CH2-N3), 3.78 (12H, -OCH3), 3.87-3.90
(1H, H4′), 4.49-4.53 (3H, H3′), 6.23-6.34 (2H, H1′), 6.92-7.45 (26
H, DMT, Ph), 7.63-7.66 (2H, H6). ESI-MS (M + H+): 1300 (calcd
1298.6).
1-{N3-[5′-O-(Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-2′-
deoxythymidylyl]}-2-{N3-[5′-O-(dimethoxytrityl)-2′-deoxythymidy-
lyl-3′-O-(â-cyanoethyl N,N′-diisopropyl)phosphoramidite]}ethane,
7a. Compound 6a (0.500 g, 0.407 mmol) was dissolved in dichlo-
romethane (2.5 mL) and diisopropylamine tetrazolide (0.243 g 1.42
mmol) added. N,N,N′,N′-2-cyanotetraisopropylphosphorane (0.441 g,
1.46 mmol) was added. After 4 h, the reaction was quenched by the
addition of dichloromethane (50 mL), and the solution was extracted
with sodium bicarbonate (5%, 3 × 50 mL). The organic layer was
dried over sodium sulfate and evaporated to afford the crude product,
which was precipitated from hexanes to yield 0.41 g (70.6%) of product
as a colorless powder. Rf (SiO2 TLC): 0.47 hexanes/ethyl acetate (1:
1). 31P NMR (161.8 MHz, acetone-d6, ppm): 149.2, 149.5. ESI-MS
(M + K+): 1453 (calcd 1453.6).
1-{N3-[5′-O-(Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-2′-
deoxythymidylyl]}-4-{N3-[5′-O-(dimethoxytrityl)-2′-deoxythymidy-
lyl-3′-O-(â-cyanoethyl N,N′-diisopropyl)phosphoramidite]}butane,
7b. Compound 7b was synthesized via the procedure outlined for 7a
to afford the desired product in 85.6% yield. Rf (SiO2): 0.50 hexanes/
ethyl acetate (1:1) 31P NMR (161.8 MHz, acetone-d6, ppm): 149.2,
149.3. ESI-MS (M + K+): 1481 (calcd 1481.6).
1-{N3-[5′-O-(Dimethoxytrityl)-3′-O-(tert-butyldimethylsilyl)-2′-
deoxythymidylyl]}-7-{N3-[5′-O-(dimethoxytrityl)-2′-deoxythymidy-
lyl-3′-O-(â-cyanoethyl N,N′-diisopropyl)phosphoramidite]}heptane,
7c. Compound 7c was synthesized via the procedure outlined for 7a to
afford the desired product in 72.0% yield. Rf (SiO2 TLC): 0.52 hexanes/
ethyl acetate (1:1). 31P NMR (161.8 MHz, acetone-d6, ppm): 149.0,
149.1. ESI-MS (M + H+): 1499 (calcd 1498.8).
Syntheses and Purification of Cross-Linked Duplexes. The cross-
linked duplexes, whose sequences are shown in Figure 1, were
assembled on an Applied Biosystems model 392A synthesizer using
standard cyanoethylphosphoramidite chemistry.26 Long-chain alkyl-
amine-controlled pore glass (CPG) was used as the solid support. The
nucleoside phosphoramidites were prepared in anhydrous acetonitrile
at a concentration of 0.15 M for the 3′-phosphoramidites or 0.3 M for
the 5′-phosphoramidites. Assembly of sequences was carried out as
follows: (a) detritylation: 2% dichloroacetic acid in dichloromethane;
(b) nucleoside phosphoramidite coupling time of 2 min for commercial
phosphoramidites and 10 min for the T-T cross-linked phosphora-
midite; (c) capping: 1:1 (v/v) of acetic anhydride/collidine/tetrahydro-
Figure 1. Sequences of the cross-linked duplexes. The 5′ end of each strand
of the duplex is denoted by “d-”. The structure of the N3T-ethyl-N3T cross-
link is shown at the right.
Table 1. Amounts, Retention Times, Nucleoside Ratios, and Mass
Spectral Data for Cross-Linked Duplexes
Nucleoside Ratios
timeb composition expected observed expected observed
Mass
cross-linked
duplexes
A
retention nucleoside
260
unitsa
XL-2
XL-4
XL-7
272 (22) 26.3
153 (24) 26.4
168 (21) 26.7
dC
dG
dT
dA
dT-dT
dC
dG
dT
dA
dT-dT
dC
dG
dT
dA
1.00
1.00
1.50
1.50
0.25
1.00
1.00
1.50
1.50
0.25
1.00
1.00
1.50
1.50
0.25
1.00 6687.21 6691.05
1.19
1.81
1.53
0.22
1.0
6717.08 6718.78
6759.03 6759.82
1.19
1.82
1.51
0.22
1.0
1.07
1.64
1.58
0.20
dT-dT
a
Amount of crude cross-linked duplex purified by SAX HPLC. The
b
numbers in parentheses indicate the amount of pure duplex obtained.
Retention times (in minutes) of cross-linked duplexes on SAX HPLC using
a 0.0-0.5 M linear gradient of sodium chloride.
furan 1:1:8 (v/v/v/; solution A) and 1-methyl-1 H-imidazole/tetrahy-
drofuran 16:84 (w/v; solution B); (d) oxidation: 0.1 M iodine in
tetrahydrofuran/water/pyridine 2.5:2:1. The cyanoethyl groups were
removed from the CPG-linked oligomers by treating the support with
1 mL of anhydrous triethylamine (TEA) for at least 12 h. The support
was washed with 30 mL of anhydrous acetonitrile followed by
anhydrous tetrahydrofuran. The tert-butyldimethylsilyl group was
removed from the partial duplex by treating the support with 1 mL of
1.0 M solution of tetra-n-butylammonium fluoride in tetrahydrofuran
(stored over molecular sieves) for 20 min. The support was then washed
with 30 mL of anhydrous tetrahydrofuran and 30 mL of acetonitrile
and then dried via high vacuum (20 min). The last segment of the duplex
was then synthesized using 5′ phosphoramidites with a total detritylation
exposure of 130 s. The 3′-terminal trityl group was removed by the
synthesizer.
The oligomer-derivatized CPG was transferred from the reaction
column to autosampler vials fitted with Teflon-lined caps, and the
protecting groups were removed by treatment with a mixture of
concentrated ammonium hydroxide: ethanol (0.3 mL:0.1 mL) for 4 h
at 55 °C. The cross-linked duplexes were purified by SAX HPLC. The
retention times of the duplexes are given in Table 1. The purified
oligomers were desalted using C-18 SEP PAK cartridges (Waters, Inc.).
The amounts of purified oligomers obtained are shown in Table 1.
The cross-linked oligomers (0.2 A260 unit) were characterized by
digestion with a combination of snake venom phosphodiesterase (0.28
unit) and calf intestinal phosphatase (5 units) in a buffer containing 10
mM Tris (pH 8.1) and 2 mM magnesium chloride as previously
described.18 The resulting mixture of nucleosides was analyzed by
reversed-phase HPLC, and the ratio of nucleosides was determined.
The results are given in Table 1. The molecular weights of each cross-
(26) Beaucage, S.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859-1862.
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Mispair-Aligned N T-Alkyl-N T Cross-Linked DNA
A R T I C L E S
Scheme 1. Synthesis of the N3T-alkyl-N3T Cross-Linka
a
(A) Compound 1, I-CH2-(CH2)n-OPac (where n ) 1, 3, or 6; 2 equiv), DBU (2.05 equiv), acetonitrile, 24 h. (B) Propylamine/CH2Cl2 (4:1), 12 h.
(C) Ph3P (3.3 equiv), imidazole (6.6 equiv), I2 (3.3 equiv), THF, 3 h. (D) Compound (2) (1 equiv), DBU (2 equiv), acetonitrile, 24 h. (E) Propylamine/
CH2Cl2 (5:1), 12 h. (F) P(OCE)(NiPr2)2 (3.6 equiv), diisopropyltetrazolide (3.5 equiv), CH2Cl2, 2 h.
linked oligomer were determined by MALDI-TOF mass spectrometry,
and the results are shown in Table 1.
molar ellipticity was calculated from the equation [θ] ) θ/Cl, where θ
is the relative ellipticity (mdeg), C is the molar concentration of
oligonucleotides (mol/L), and l is the path length of the cell (cm). The
data were processed on a PC computer using Windows-based software
supplied by the manufacturer (JASCO, Inc.) and transferred into
Microsoft Excel for presentation.
UV Thermal Denaturation Studies. Molar extinction coefficients
for the oligonucleotides and cross-linked duplexes were calculated from
those of the mononucleotides and dinucleotides according to nearest-
neighbor approximations (units ) 104 M-1 cm-1).27 Non-cross-linked
duplexes were prepared by mixing equimolar amounts of the interacting
strands and lyophilizing the resulting mixture to dryness. The resulting
pellet was then redissolved in 90 mM sodium chloride, 10 mM sodium
phosphate, and 1 mM EDTA buffer (pH 7.0) to give a final
concentration of 2 µM in each strand. The cross-linked duplexes were
dissolved in the same buffer to give a final concentration of 2 µM.
The solutions were then heated to 65 °C for 15 min, cooled slowly to
room temperature, and stored at 4 °C overnight before measurements.
Prior to the thermal run, samples were degassed by placing them in a
speed-vac concentrator for 2 min. Denaturation curves were acquired
at 260 nm at a rate of heating of 0.5 °C/min., using a Varian CARY
model 3E spectrophotometer fitted with a six-sample thermostated cell
block and a temperature controller. The data were analyzed in
accordance with the convention of Puglisi and Tinoco27 and transferred
to Microsoft Excel.
Circular Dichroism (CD) Spectra Spectroscopy. Circular dichro-
ism spectra were obtained on a Jasco J-700 spectropolarimeter equipped
with a NESLAB RTE-111 circulating bath. Samples were allowed to
equilibrate for 5-10 min at 5 °C in 90 mM sodium chloride, 10 mM
sodium phosphate, and 1 mM EDTA (pH 7.0), at a final concentration
of 2 µM in each strand for the cross-linked duplexes and ca. 4 µM for
control duplexes. Each spectrum is an average of 5 scans. Spectra were
collected at a rate of 100 nm/min with a bandwidth of 1 nm and
sampling wavelength of 0.2 nm using fused quartz cells (Hellma, 165-
QS). The CD spectra were recorded from 350 to 200 nm at 5 °C. The
Results and Discussion
Syntheses and Characterization of Cross-Linked Duplexes.
The structures of the N3T-alkyl-N3T cross-links and the cross-
linked duplexes are shown in Figure 1. The synthetic approach
used to construct the cross-link is shown in Scheme 1. Starting
from 5′-O-dimethoxytrityl-3′-O-tert-butyldimethylsilyl-2′-deoxy-
thymidine 1, the N3 position of the heterocycle was alkylated
using DBU and iodoalkyl phenoxyacetate to form adducts 3a-c
in a fashion similar to the synthesis of thymine or uracil
nucleotide analogues that require protection of the N3 posi-
tion.28,29 Depending on the iodoalkane used, it is possible to
synthesize adducts having linkers of various lengths. The
phenoxyacetyl group was removed from 3a-c using propyl-
amine to the give the free alcohol, which was subsequently
converted to iodide 4a-c in good yield using triphenylphos-
phine, imidazole, and iodine.24 The protected cross-link 5a-c
was then formed by deprotonating the N3-H of 5′-O-dimethoxy-
trityl-3′-O-phenoxyacetyl-deoxythymidine, 2, with DBU, fol-
lowed by the addition of iodo adduct 4a-c. In the case of the
four- and seven-carbon linker adducts the reaction proceeded
(28) Altmann, K. H.; Kesselring, R.; Francotte, E.; Rihs, G. Tetrahedron Lett.
1994, 35, 2331-2334.
(29) Krecmerova, M.; Hrebacebecky, J.; Holy, A. Collect. Czech. Chem.
Commun. 1990, 55, 2521-2536.
(27) Puglisi, J. D.; Tinoco, I., Jr. Methods Enzymol. 1989, 180, 304-325.
9
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A R T I C L E S
Wilds et al.
Scheme 2. Construction of Cross-Linked Duplexes via Solid
Phase Synthesis
Figure 2. Strong anion exchange HPLC profiles of crude XL-2 (blue),
XL-4 (red), and XL-7 (green). The column, which was monitored at 260
nm, was eluted with a gradient of 0-0.5 M sodium chloride in 100 mM
Tris-HCl, pH 7.8, 10% acetonitrile, at a flow rate of 1.0 mL/min.
with TBAF was limited to a reaction time of 20 min as extended
treatment results in significant cleavage of the oligonucleo-
tide from the solid support, a result shown previously by Damha
et al. in model studies using DMT-dT-(LCAA-CPG).30 To
determine the extent of removal of the silyl group, a small
amount (approximately 1 mg) of CPG was treated with a mixture
of concentrated ammonium hydroxide/ethanol (3:1) at 55 °C
for 4 h. These conditions deprotect RNA without loss of the
silyl group. The hydrolyzate was analyzed by C-18 reversed-
phase HPLC. The desilyated oligomers, XL-d, have a shorter
retention time on the reversed-phase column and are easily
distinguished from the slower moving silyated oligomers,
XL-c. Removal of the TBDMS group by treatment of the
Y-intermediate with TBAF resulted in variable yields of
desilylation with the different oligomers. For example, in the
case of the four-carbon linker, the removal of the TBDMS group
was quantitative; however, in the case of the two- and seven-
carbon linkers, the TBDMS group was removed 50 and 61%,
respectively. Variable amounts of desilation have also been
observed previously in the synthesis of N4C-alkyl-N4C cross-
linked duplexes.18-21
smoothly. However, initial attempts to synthesize the two-carbon
linker adduct were not successful because of the conversion of
iodo adduct 4a to alcohol precursor 3a when using two or more
equivalents of DBU. This undesired side reaction was minimized
by using 1 equiv of DBU when coupling 2 to 4a. The 3′-O-
phenoxyacetyl group of dimer 5a-c was removed with propyl-
amine to afford 6a-c, which was then phosphitylated to give
the protected cross-link phosphoramidites 7a-c for solid-phase
synthesis.
The solid-phase synthetic strategy used to prepare the cross-
linked duplexes, which is similar to that used previously to
prepare N4C-alkyl-N4C cross-linked duplexes,18-21 is shown in
Scheme 2. Syntheses were carried out on a 2 µmol scale. The
first arm of the duplex, XL-a, was synthesized on a controlled
pore glass support. The N3T-alkyl-N3T cross-link phosphora-
midite 7a-c was used to introduce the cross-link at the 5′ end
of the oligomer to give XL-b. The dimethoxytrityl groups were
removed by brief treatment with 2% dichloroacetic acid in
methylene chloride. The second and third arms of the duplex
were synthesized simultaneously by repetitive coupling with
protected nucleoside-3′-phosphoramidites to give, after detri-
tylation and acetylation, the branched Y-intermediate, XL-c. The
tert-butyldimethylsilyl (TBDMS) group was then removed from
XL-c by treating the support with anhydrous tetra-n-butylam-
monium fluoride (TBAF) in tetrahydrofuran. Because fluoride-
mediated desilylation can result in cleavage of adjacent â-cy-
anoethylphosphotriester linkages,30 XL-c was first treated with
anhydrous triethylamine to convert the triester groups to
phosphodiester linkages. A similar strategy was used previously
in the synthesis of branched oligoribonucleotides and in the
syntheses of DNA duplexes containing N4C-alkyl-N4C inter-
strand cross-links.18-21,30 In addition, treatment of the support
The full-length cross-linked duplexes, XL-2, 4, and 7, were
prepared by repetitive coupling of the 3′-end of the Y-inter-
mediate XL-d with protected nucleoside 5′-phosphoramidites.
To maximize the coupling yields, the phosphoramidites were
used at a concentration of 0.3 M instead of the usual 0.15 M
concentration used for nucleoside 3′-phosphoramidites. In addi-
tion, it was found necessary to increase the total detritylation
time to 130 s to effect complete removal of the 3′-O-di-
methoxytrityl groups.
The cross-linked oligomers were deprotected by treating the
support with a mixture of concentrated ammonium hydroxide/
ethanol (3:1) at 55 °C for 4 h. The duplexes were purified by
strong anion exchange HPLC using a sodium chloride gradient
in buffer that contained 100 mM Tris-HCl and 10% acetonitrile
as shown in Figure 2. The major side product in these syntheses
was the deprotected form of XL-c, which resulted from
incomplete desilylation of the TBS-protected Y-intermediate.
The duplexes, whose retention times are shown in Table 1,
eluted as single peaks and were obtained in approximately
8-16% isolated and 5-6% overall yield. Despite the low overall
yield, this procedure provides sufficient quantities of cross-linked
(30) Braich, R. S.; Damha, M. J. Bioconjugate Chem. 1997, 8, 370-377.
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Mispair-Aligned N T-Alkyl-N T Cross-Linked DNA
A R T I C L E S
increases as the length of the linker increases from two (3.6 Å)
to four (6.1 Å) to seven (8.4 Å) methylene groups.
The prediction that the cross-links do not greatly affect the
structure of B-form DNA was confirmed by CD spectra. The
CD spectra of cross-linked duplexes XL-2, XL-4, and XL-7
and the non-cross-linked control were recorded at 5 °C (Figure
5). Under these conditions, both the cross-linked and non-cross-
linked oligomers should be in their base-paired, duplex form.
In all cases, the CD spectra of the duplexes exhibit signatures
characteristic of B-form DNA. In the case of XL-4 and XL-7,
the peak maxima are slightly blue-shifted relative to the non-
cross-linked duplex and XL-2.
Ultraviolet thermal denaturation experiments were carried out
to assess the effects of the various cross-links on duplex stability.
Thermal denaturation curves for cross-linked duplexes XL-2,
XL-4, XL-7, along with a non-cross-linked control duplex,
which contains an A-T base pair in place of the T-T cross-
link, are shown in Figure 6. The denaturation temperature, which
is defined as the midpoint of the thermal denaturation curve, of
each duplex is listed in Table 2. Each cross-linked oligomer
displayed a sigmoidal denaturation profile. Although the overall
hyperchromicities of the transition curves of the cross-linked
duplexes and the non-cross-linked control are approximately
the same, there are significant differences in the breadths of
the denaturation curves. The breadth of the denaturation curve
of XL-2, with a denaturation temperature of approximately
77 °C, is similar to that of the control duplex, suggesting that
these duplexes denature in a highly cooperative manner. The
much broader transitions seen for XL-4 and XL-7 suggest that
these duplexes denature in a less cooperative manner than does
XL-2.
As was seen previously with N4C-alkyl-N4C cross-linked
duplexes,18,20,21 the denaturation temperatures of the N3T-alkyl-
N3T cross-linked duplexes are 16-37 °C higher than the melting
temperature of the non-cross-linked control. This increased
thermal stability is most likely a consequence of covalently
linking the two strands of the duplex. Thus, denaturation/
renaturation of the cross-linked duplex is a unimolecular process,
which is independent of the strand concentration, whereas
denaturation/renaturation of the non-cross-linked control is a
unimolecular process for denaturation and bimolecular for
renaturation.
Figure 3. Polyacrylamide gel electrophoresis of cross-linked duplexes.
Cross-linked duplexes XL-2 (lane 1), XL-4 (lane 2), XL-7 (lane 3), and a
control oligomer, dT22 (lane 4) were each 5′ end-labeled with γ[32P]-ATP
and subjected to electrophoresis on a 20% polyacrylamide gel run under
denaturing conditions. XC and BPB are xylene cyanol and bromophenol
blue dyes, respectively.
oligomers for biological and physical studies, including struc-
tural studies by high-resolution NMR spectroscopy.
Samples of each duplex were digested with a combination
of snake venom phosphodiesterase and calf intestinal phos-
phatase, and the digests were analyzed by C-18 reversed-phase
HPLC. As shown in Table 1, each duplex was hydrolyzed to
its component nucleosides and the N3T-alkyl-N3T cross-link in
ratios consistent with the structure of the particular duplex. In
addition to this characterization, each duplex was analyzed by
MALDI TOF mass spectrometry. As shown in Table 1, the
resulting mass-to-charge ratios were consistent with the structure
of the duplex. Analysis of the final products via electrophoresis
on denaturing polyacrylamide gels, as shown in Figure 3, reveal
that the cross-linked duplexes are of sufficient purity for
structural and biological investigations.
Physical Studies. To evaluate the effect of the N3-alkyl-N3
cross-link on B-form DNA, molecular models of a T-T
mismatch as well as those of the cross-linked duplexes contain-
ing the two-, four-, and seven-carbon linkers were generated
using the program Hyperchem. These models were geometry-
optimized using the Amber force field, with the final models
shown in Figure 4. In all cases, the models predict that the cross-
links should have little effect on the global structure of the
duplexes. Thus, the overall geometry of the cross-linked
duplexes appears to be that of B-form DNA. The base pairs
flanking the cross-link maintain Watson-Crick hydrogen bond-
ing, although in the case of the four- and seven-carbon cross-
links there appears to be some buckling of the neighboring base
pairs (Figure 4, models D and E). The N3-N3 distance of the
T-T mismatch was found to be 3.8 Å. As expected, this distance
The denaturation temperature of the cross-linked duplex is
dependent upon the length of the N3T-alkyl-N3T cross-link.
As seen in Table 2, the order of stability is XL-2 > XL-4 >
XL-7. The denaturation temperatures of XL-2 and XL-4 differ
by only 6 °C, whereas that of XL-7 is 21 °C less than that of
XL-2. A similar effect was seen for duplexes that contained a
directly opposed N4C-alkyl-N4C-type interstrand cross-link.18
Here the duplex with a two-carbon linker has the highest thermal
denaturation temperature (25 °C higher than the control),18
whereas duplexes with a three- or four-carbon linker have
increased denaturation temperatures of 20 and 17 °C, respec-
tively (unpublished results). For both two- and four-carbon
linkers, the denaturation temperatures of the directly opposed
N3T-alkyl-N3T cross-link is greater than those of the N4C-alkyl-
N4C type interstrand cross-link.
The relative thermal stabilities of the duplexes are reflected
in their electrophoretic mobilities on a denaturing polyacryl-
amide gel, as shown in Figure 3. Cross-linked duplexes
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J. AM. CHEM. SOC. VOL. 126, NO. 30, 2004 9263

A R T I C L E S
Wilds et al.
Figure 4. Molecular models of a (A) complementary duplex, d(CGAAATTTTCG)/d(GCTTTAAAAGC), (B) self-complementary duplex d(CGAAATTTTCG)2
that contains a mismatched T-T, and self-complementary duplexes d(CGAAAT*TTTCG)2 where T* is N3T-alkyl-N3T (shown in green) with alkyl linkers
that are (C) two, (D) four, and (E) seven carbon atoms long. The models were built using Hyper Chem molecular modeling software and energy minimized
using the Amber force-field.
Figure 5. Circular dichroism spectra of cross-linked duplex XL-2 (‚‚‚),
Figure 6. Absorbance (A260) versus temperature profiles of cross-linked
duplex XL-2 (‚‚‚), XL-4 (- - - ), XL-7 (-‚-), and non-cross-linked
control (s). Solutions containing 2 µM duplex in 90 mM sodium chloride,
10 mM sodium phosphate, and 1 mM ethylenediaminetetraacetate buffer,
pH 7.0, were heated at 0.5 °C/min.
XL-4 (- - - ), XL-7 (-‚-), and non-cross-linked control (s). Solutions
containing 1 µM duplex in 90 mM sodium chloride, 10 mM sodium
phosphate, and 1 mM ethylenediaminetetraacetate buffer, pH 7.0, were
heated at 0.5 °C/min. Spectra were recorded at 5 °C.
XL-2 and XL-4 each have a higher electrophoretic mobility
than XL-7, whose mobility is similar to that of dT22. It is
unlikely that the difference in mobility between XL-2 or XL-4
and XL-7 is due solely to the increased molecular weight of
the cross-link. Rather, it appears that XL-7, which consists of
22 nucleotides, is denatured under the conditions of the
experiment, whereas XL-2 and XL-4, which have higher thermal
stabilities, migrate as base-paired duplexes, even in the presence
of 7 M urea. Similar behavior was seen when N4C-alkyl-N4C,
-CNG--type interstrand cross-linked duplexes were electro-
phoresed under denaturing conditions.21 The thermally more
stable four- and seven-carbon linked duplexes migrated with a
higher mobility than the less stable two-carbon linked duplex.
Previous modeling and NMR studies of a C-C ethyl cross-
linked duplex revealed that the ethyl linker protrudes into the
major groove of the duplex.19,31 This lesion induces an overall
axis bend toward the major groove, narrowing of the major and
Table 2. Thermal Denaturation of Cross-Linked Duplexes and
Their Non-Cross-Linked Controls
duplex
Tm (°C)a
XL-2
XL-4
XL-7
control
77
71
56
40
a
Experiments were carried out in buffer containing 90 mM sodium
chloride, 10 mM sodium phosphate, and 1 mM ethylenediaminetetraacetate
buffer (pH 7.0). The concentration of each strand was 2 µM.
minor grooves around the cross-link, and buckling of base pairs
around the cross-linked site. In the case of the T-T ethyl cross-
link, the lesion lies in neither the major nor minor groove but
rather appears to pass through the helical axis. Models suggest
that the length of the two-carbon cross-link is sufficient to span
the distance between the N3 atoms of the T-T mismatch without
perturbing the helix structure and surrounding base pairs.
Consequently, the two-carbon linked duplex has a high thermal
stability relative to the non-cross-linked control. The longer
(31) da Silva, M. W.; Noronha, A. M.; Noll, D. M.; Miller, P. S.; Colvin, O.
M.; Gamcsik, M. P. Biochemistry 2002, 41, 15181-15188.
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9264 J. AM. CHEM. SOC. VOL. 126, NO. 30, 2004

3
3
Mispair-Aligned N T-Alkyl-N T Cross-Linked DNA
A R T I C L E S
linkers, particularly the seven-carbon linker, however, tend to
push the thymines apart, creating a local distortion at the site
of the cross-link and affecting base pairs on either side of the
cross-link. The relatively lower thermal stability of the seven-
carbon cross-linked duplex reflects this perturbation. Further
insights into the structures of these duplexes await high
resolution NMR analyses, which are currently in progress.
The T-T cross-link mimics to some extent C-C and G-C-
type cross-links in that linkage occurs at the N3 position of the
pyrimidine base. Because alkylation at this site can potentially
interfere with base pairing, this type of lesion would be expected
to present a serious obstacle to faithful repair of the lesion. We
are currently testing this hypothesis by studying repair of T-T
cross-linked DNA in cell extracts and whole cells.
Acknowledgment. This research was supported by a grant
from the National Cancer Institute (CA082785). A.M.N. and
C.J.W. were each supported in part by postdoctoral fellowships
from the Natural Sciences and Engineering Research Council
of Canada (NSERC).
JA0498540
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