Preparation of N3-thymidine-butylene-N3-thymidine interstrand cross-linked DNA via an orthogonal deprotection strategy

Article abstract of DOI:10.1016/j.tet.2012.07.043

A DNA duplex containing an N3-thymidine-butylene-N3-thymidine interstrand cross-link (ICL) was prepared using an on-column orthogonal deprotection strategy to permit different nucleotide sequence composition around the cross-linked site. The conditions us

Full text of DOI:10.1016/j.tet.2012.07.043

Tetrahedron 68 (2012) 7787e7793
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Preparation of N3-thymidineebutyleneeN3-thymidine interstrand cross-linked
DNA via an orthogonal deprotection strategy
*
Gang Sun, Anne Noronha, Christopher Wilds
ꢀ
ꢀ
Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St. West, Montreal, Quebec, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 June 2012
Received in revised form 9 July 2012
Accepted 11 July 2012
Available online 20 July 2012
A DNA duplex containing an N3-thymidineebutyleneeN3-thymidine interstrand cross-link (ICL) was
prepared using an on-column orthogonal deprotection strategy to permit different nucleotide sequence
composition around the cross-linked site. The conditions used to remove 50-O-allyloxycarbonyl and 30-O-
tert-butyldimethylsilyl protective groups for various on-column oligonucleotide intermediates did not
affect the cross-linked lesion. Efficient removal of these groups enabled successful coupling of 20-de-
oxyphosphoramidites to produce the desired duplex with a 31% yield after deprotection and purification.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Cross-linked DNA
Chemically modified oligonucleotides
DNA damage
DNA repair
Solid-phase synthesis
1. Introduction
mammalian cells offer the potential to improve the efficacy of these
drugs in cancer therapy.
Cellular DNA, on exposure to various environmental and che-
motherapeutic agents, can undergo damage with numerous mod-
ifications identified including interstrand cross-links (ICLs). ICLs
threaten genomic and cellular integrity because they present for-
midable blocks to essential metabolic processes that require obli-
gate strand separation (replication and transcription). Even though
cells must efficiently detect and remove damage for survival,
ICL-inducing agents continue to be employed as chemotherapeutic
treatments for many cancers.1,2 However, resistance to ICL-inducing
agents due in part to efficient DNA repair, can result in recurrent
malignancies, which are unresponsive to treatment,3,4 in addition
to possibly increasing the risk of secondary cancers likely due to
their mutagenic processing in normal cells.5 Despite the substantial
progress in the fields of ICL repair in bacteria and yeast, which occur
primarily via a combination of processes including nucleotide ex-
cision repair (NER), homologous recombination (HR), and trans-
lesion synthesis (TLS)6e9 a complete understanding of such repair
in mammalian systems is still unclear.10e17 In mammalian cells, it is
thought that ICLs are repaired by the coordination of proteins from
several pathways, including NER,18,19 base excision repair (BER),20
mismatch repair (MMR),21,22 HR,23,24 TLS,25 and proteins involved
in Fanconi anemia (FA).26 Therefore, efforts to better understand
the cellular responses to and repair of ICL-inducing lesions in
One continuing approach to investigate the role that repair
pathways play in removing ICL is to use chemically synthesized
oligonucleotides, which contain adducts that represent the lesions
introduced by ICL-inducing agents. Differing strategies have been
developed to obtain ICL-containing DNA. One avenue to ICL syn-
thesis involves the incorporation of stable ICL forming precursors
into single stranded DNA using standard DNA synthesis after which
hybridization triggers ICL formation.27,28 We and others have
established an approach (mono and bidirectional) to synthesize
cross-linked nucleosides to directly introduce the ICL, which en-
ables exact placement of the desired ICL, using a combination of
solution and solid-phase synthesis.29e37 However, several of these
model ICL systems to date have contained symmetrical nucleotide
sequence composition (either complete or partial) around the
cross-linked site (Fig. 1).
We have previously reported the synthesis of N3-thym-
idineebutyleneeN3-thymidine (N3TebutyleneeN3T) ICL du-
plexes with either partial or complete symmetry around the site of
the cross-link using nucleoside dimers containing DMT and TBS
protecting groups on the 50- and 30-O functionalities using mono-
and bis-phosphoramidite approaches.33,34 In order to accomplish
the synthesis of asymmetric sequences it is imperative to design the
dimer phosphoramidite to contain three different protective groups
around the 50- and 30-O functionalities that are compatible with each
other for selective removal without compromising the ICL lesion to
introduce the cross-link into the desired ICL duplexes.38
* Corresponding author. Tel.: þ1 514 848 2424x5798; fax: þ1 514 848 2868;
e-mail address: cwilds@alcor.concordia.ca (C. Wilds).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.07.043

7788
G. Sun et al. / Tetrahedron 68 (2012) 7787e7793
50-O-dimethoxytrityl-30-O-(tert-butyldimethylsilyl)-thymidine to
produce N3-(4-iodobutyl)-50-O-dimethoxytrityl-30-O-(tert-butyl-
dimethylsilyl)-thymidine in 85% yield.33
Introduction of the levulinoyl (Lev) group at the 30-OH of 50-O-
dimethoxytrityl-thymidine with levulinic acid, in the presence of 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride(EDC)
and 4-dimethylaminopyridine (DMAP) in dioxane at rt, resulted in
compound (2) in quantitative yields. The 50-O-dimethoxytrityl group
was removed by p-toluene sulfonic acid (p-TsOH) to release a free 50-
OH followed by its conversion to the allyloxycarbonyl (Alloc) group
by reacting it with AlloceOBt and DMAP inpyridine at rt overnight to
produce compound (3) (85%).39 Dimerization was accomplished by
coupling a slight excess of compound (3) with N3-(4-iodobutyl)-50-
O-dimethoxytrityl-30-O-(tert-butyldimethylsilyl)-thymidine in ace-
tonitrile (MeCN)inthepresenceof1,8-diazabicyclo-(5.4.0)-undec-7-
ene (DBU) for 2 days at rt, whichyielded the desired compound (4) in
55% yield. The 30-O-Lev protective group was removed by treatment
with 0.5 M hydrazine in a pyridine/acetic acid buffer (v/v 9:1) for
10 min at rt to yield preamidite (5) (96%). This was then converted to
the cross-linked phosphoramidite (6) using a slight excess of N,N-
diisopropylamino cyanoethyl phosphonamidic chloride in the pres-
Fig. 1. (a) Chemical structure of the N3TebutyleneeN3T ICL and (b) sequence of ICL
oligonucleotide prepared (XL).
In this report, we describe the synthesis of 1-{N3-[50-O-(dime-
thoxytrityl)-30-O-(tert-butyldimethylsilyl)-thymidylyl]}-4-{N3-[50-
O-(allyloxycarbonyl)-thymidylyl-30-O-(
b-cyanoethyl
N,N0-diiso-
propyl)phosphoramidite]}butane (6), an asymmetric phosphor-
amidite that enables the synthesis of the N3TebutyleneeN3T ICL
and detailed procedures of the orthogonal deprotection strategy
that allows for the selective nucleotide sequence assembly around
the cross-linked site.
€
ence of Hunigs base. The phosphoramidite was isolated by hexane
precipitation in 71% yield and further analyzed by mass spectrome-
try, which gave the expected molecular mass (1261.5638). 31P NMR
analysis of the phosphoramidite revealed the presence of two signals
for (6) (146.71 and 146.97 ppm) in the region diagnostic for
a phosphoramidite.
2. Results and discussion
2.1. N3TebutyleneeN3T cross-linked phosphoramidite
synthesis
The cross-linked dimer phosphoramidite (6) was synthesized in
solution as illustrated in Schemes 1 and 2. The linker, 4-iodobutyl 2-
phenoxyacetate (1), was prepared in a single step in quantitative
yields by reacting 2-phenoxyacetyl chloride and potassium iodide
in tetrahydrofuran (THF). The protected alkyl linker was coupled to
2.2. Solid-phase synthesis of the N3TebutyleneeN3T ICL
The ICL duplex containing the dimer was synthesized on 1 mmol
scale employing an ABI 3400 solid-phase DNA synthesizer using
polystyrene (PS) rather than controlled-pore glass (CPG) solid-
support due to the incompatibility of triethylamine$trihydro-
fluoride (TEA$3HF) with the latter. As an added precaution, the
cyanoethyl protecting groups were removed using triethylamine
(TEA) as it has been observed that prolonged fluoride treatment
using tetrabutylammonium fluoride (TBAF) could lead to
chain cleavage.34 The assembly of the ICL (Fig. 1) utilizing
Scheme 1. Reagents and conditions: (a) KI, 2.5 equiv, THF, overnight, rt (95%).
Scheme 2. Reagents and conditions: (a) 2 equiv levulinic acid, 2 equiv EDC, 0.01 equiv DMAP, in 1,4-dioxane at rt for 16 h (quantitative yield); (b) 2.2 equiv p-TsOH, in CH2Cl2/
CH3OH (4:1) at rt for 1 h followed by 1.33 equiv AlloceOBt, 0.2 equiv DMAP, in THF/pyridine (9:1) at rt for 24 h (85%); (c) 1.0 equiv N3-(4-iodobutyl)-50-O-dimethoxytrityl-30-O-(tert-
butyldimethylsilyl)-thymidine, 2.0 equiv DBU, in CH3CN at rt for 48 h (55%); (d) 0.5 M hydrazine hydrate, in pyridine/acetic acid (1:1) at rt for 10 min (96%); (e) 1.2 equiv
ClP(OCE)(Ni-Pr2), 1.5 equiv DIPEA, in THF at rt for 1 h (71%).

G. Sun et al. / Tetrahedron 68 (2012) 7787e7793
7789
phosphoramidite (6) is illustrated in Scheme 3. The position of the
cross-link introduces asymmetry into the duplex (i.e., the nucleo-
tide composition of the oligonucleotide strands around the cross-
linked site is different). Intermediate U was assembled continu-
ously on solid support by coupling 0.15 M of (6) directly to a short
linear segment of DNA for 10 min, followed by removal of the DMT
on the ICL dimer, around which the second nucleotide chain was
grown. Chain assembly in the 30 to 50 direction proceeded smoothly
using 30-O-20-deoxyphosphoramidites (dissolved to a concentra-
tion of 0.1 M in MeCN). A higher concentration and extended
coupling time for (6) were necessary to ensure an optimal coupling
efficiency due to its larger size compared to the standard 30-O-20-
deoxyphosphoramidites. This step was followed by capping on the
synthesizer to prevent undesired chain growth in subsequent steps.
scavenger step results in oligonucleotide degradation as illustrated
by IEX HPLC analysis of an U intermediate prepared for another ICL
duplex (see Fig. 2b without scavenger-washing). The 30-O-TBS group
was then removed from the Y intermediate by treating the support
with anhydrous TEA overnight followed by TEA$3HF twice
for 30 min at rt. Reversed-phase HPLC analysis of the deprotected
intermediate revealed complete removal of the silyl group with
the shift of the major peak from 13.0 to 9.5 min in the case of
the cross-link. Continued synthesis with repetitive coupling of
50-O-20-deoxyphosphoramidites at the 30-end of intermediate Ygave
the full-length ICL duplex.
The full-length cross-linked H-duplex was cleaved from the
solid support and deprotected by treating the support with a mix-
ture of ethanolic ammonia (1:3 v/v, 0.5 mL) at 55 ꢀC, using standard
Scheme 3. Solid-phase synthesis procedure of oligonucleotide XL involving N3TebutyleneeN3T phosphoramidite (6). (i) Coupling of phosphoramidite (6); (ii) removal of 50-O-DMT
on the machine, extension with 30-O-20-deoxyphosphoramidites and capping (to afford ‘U’); (iii) removal of 50-O-Alloc protective group; (iv) extension with 30-O-20-deoxy-
phosphoramidites and capping (to afford Y); (v) removal of the 30-O-TBS protective group; (vi) extension with 50-O-20-deoxyphosphoramidites (to form H); (vii) Cleavage from the
solid support and deprotection with NH4OH/C2H5OH to give crude XL.
At each stage of the ICL duplex assembly,1e2 mg of solid support
was deprotected with ethanolic ammonia (1:3 v/v, 0.5 mL) at 55 ꢀC
for 4 h and the crude analyzed using Ion Exchange (IEX) HPLC. This
was done to monitor amidite coupling and removal of a specific
protectinggroup ateachstage of the solid-phase synthesis. TheHPLC
profiles (shown in Fig. 2) illustrate the successful coupling of dimer
phosphoramidite(6) with a major species at ca. 8 min corresponding
tothe desiredU intermediate (Fig. 2a). After subjecting the oligomer-
bound support to TEA treatment as described above, the Alloc pro-
tective group was removed with tetrakis(triphenylphosphine)pal-
ladium(0) [Pd(PPh3)4] in a butylamineeformic acid buffer solution
for 3 h at 35 ꢀC to free the 30-OH group (Scheme 3). The successful
removal of the Alloc group was determined indirectly through sub-
sequentoligonucleotide assembly to produce intermediate oligomer
Y from intermediate U (see Scheme 3). It is essential that all traces of
remnant palladium (Pd) be removed using a Pd scavenger, namely
sodium N,N-diethyldithiocarbamate to enable the successful syn-
thesis of the Y intermediate.40e42 The effect of avoiding this Pd
deprotection conditions.33 The crude oligomer was purified by IEX
HPLC, using a gradient buffer of 20e55% B in 30 min, followed by
desalting to afford the pure cross-linked duplex in 31% yield. This
ICL duplex was digested to the constituent nucleosides with
a combination of snake venom phosphodiesterase and calf in-
testinal phosphatase in a buffer containing 10 mM Tris (pH 8.1) and
2 mM magnesium chloride at 37 ꢀC for 16 h and analyzed by C-18
reversed-phase HPLC (see Supplementary data for the HPLC trace).
In addition to the four standard 20-deoxynucleosides, one addi-
tional peak was observed with a retention time of 14 min identical
to the completely deprotected dimer (6). The ratios of the compo-
nent 20-deoxynucleosides and cross-linked nucleosides were con-
sistent with the theoretical composition of ICL duplex (see
Supplementary data). The molecular weight of the cross-link du-
plex as determined by ESI-TOF mass spectrometry was in close
agreement with the expected theoretical value (6110.0 Da).
The overall yield of the cross-linked oligonucleotide was
0.31 mmol (31% from a 1 mmol scale synthesis) after purification and

7790
G. Sun et al. / Tetrahedron 68 (2012) 7787e7793
Fig. 2. (a) IEX HPLC chromatographs for intermediates in the synthesis of XL prepared with phosphoramidite (6). (i) ‘U’ intermediate; (ii) ‘Y’ intermediate; (iii) Crude XL; (b): IEX
HPLC chromatograph of an ‘U’ intermediate without residual Pd scavenging treatment. The column, which was monitored at 260 nm, was eluted at rt with a gradient of 20e55%
buffer B in 30 min. Buffer A: 10% acetonitrile, 100 mM TriseHCl (pH 7.8); buffer B: 10% acetonitrile, 100 mM TriseHCl (pH 7.8), 1 M NaCl.
desalting. Cross-linked duplexes prepared by methods described by
Kishi and co-workers were reported to afford 0.15 mol of material
based on a 1
mol after HPLC purification.43 Among the dimers they
cumulative base stacking to the stability of the ApTcompared to the
TpT step suggesting that the latter is more deformable within a DNA
stem. The constraints of the butyl tether perturb the structure very
slightly, resulting in accommodation in a region between major and
minor grooves that is in a staggered fashion, which deviates from
being perpendicular to the stem axis. However, the canonical B-DNA
local structure throughout the stem is observed.
m
m
investigated, one contained a 50-O-tert-butyldiphenylsilyl (TBDPS)
group, reported to couple with similar efficiency to other phos-
phoramidites studied. One explanation for the higher yield of final
product we observed may be attributed the use of the 30-O-tert-
butyldimethylsilyl (TBS) protective group, which is easier to
remove, enabling more efficient extension during phosphoramidite
coupling to produce the desired ICL.
3. Conclusion
Preliminary attempts to prepare the ICL with an orthogonal
deprotection strategy involving the levulinoyl (Lev) and phenoxy-
acetyl (Pac) groups present in 30-O-phenoxyacetyl-50-O-levulinoyl-
thymidine, were unsuccessful (see Supplementary data for the
synthesis and characterization of these compounds).33,44e47 We
believe the 30-O-Pac group was partially removed from the dimer
under conditions to remove the tert-butyldimethylsilyl (TBS) group
during one of the assembly steps in duplex construction. The Pac
group has been shown to be extremely labile under mild alkaline
conditions (0.001 mol/L Na2CO3 in CH3OH/CH2Cl2).44,48,49 In our
hands, attempts to remove the 50-O-Lev group on the oligomer-
bound solid support with hydrazine resulted in several undesired
products on sequential extension, which could be due to the re-
moval of benzoyl protected cytidine residues as reported
previously.50
The stability of the XL duplex was assessed by ultraviolet ther-
mal denaturation experiments (see Supplementary data). The
profiles for the ICL and control duplex were sigmoidal indicating
normal cooperativity and they displayed thermal melts of about 66
and 25 ꢀC, respectively.
The CD spectra of the XL duplex as well as the non-cross-linked
control were recorded at 10 ꢀC. The CD spectra of the duplex
exhibited signatures characteristic of B-form DNA with a positive
maximum peak centered around 280 nm, a negative peak at ap-
proximately 250 nm, and a cross over around 270 nm.51,52 In general
the CD spectra were red shifted relative to the non-cross-linked
controls (see Supplementary data). Previously, Webba da Silva and
co-workers53 have shown that duplexes containing the
N3TebutyleneeN3T ICL display dramatic widening of the major
groove of the B-DNA stem without disruption of WatsoneCrick base
pairing due to the dominant contribution of cooperative and
An ICL duplex containing an N3TebutyleneeN3T ICL has been
successfully synthesized using a novel phosphoramidite (6) and
solid-phase synthesis to afford the duplex in a 31% yield from
a 1 mmol scale synthesis. The orthogonal deprotection strategy may
be valuable for the synthesis of other ICL duplexes to produce
substrates for various DNA repair studies.
4. Experimental section
4.1. General
50-O-Dimethoxytrityl-thymidine, N,N-diisopropylamino cya-
noethyl phosphonamidic chloride, and 30-O-dimethoxytrityl-20-
deoxyribonucleoside-50-O-( -cyanoethyl-N,N0-diisopropyl)phos-
b
phoramidites were purchased from ChemGenes Inc. (Wilmington,
MA). 50-O-Dimethoxytrityl-20-deoxyribonucleoside-30-O-(
b-cya-
noethyl-N,N0-diisopropyl)phosphoramidites, protected 20-deoxy-
ribonucleosideepolystyrene supports, and ancillary reagents for
solid-phase synthesis were purchased from Glen Research (Ster-
ling, Virginia). Allyl chloroformate, n-butylamine, 1,8-diazabicyclo
[5.4.0]undec-7-ene (DBU), formic acid, 1-hydroxybenzotriazole
hydrate, hydrazine hydrate, levulinic acid, phenoxyacetyl
chloride, tert-butyldimethylsilyl chloride (TBSeCl), p-toluene sul-
fonic acid (p-TsOH), diisopropylethylamine (DIPEA), 4-dimethy-
laminopyridine (DMAP), triphenylphosphine (PPh3), sodium N,N-
diethyldithiocarbamate,
tetrakis(triphenyl
phosphine)palla-
dium(0), and all other chemicals and solvents for synthesis were
purchased from the Aldrich Chemical Company (Milwaukee, WI).
50-O-Dimethoxytrityl-30-O-(tert-butyldimethylsilyl)-thymidine,
N3-[4-(phenoxyacetyl)butyl]-50-O-dimethoxytrityl-30-O-(tert-butyl-
dimethylsilyl)-thymidine, N3-(4-iodobutyl)-50-O-dimethoxytrityl-

G. Sun et al. / Tetrahedron 68 (2012) 7787e7793
7791
30-O-(tert-butyldimethylsilyl)-thymidine, 50-O-dimethoxytrityl-30-
O-phenoxyacetyl-thymidine were all prepared with minor modi-
fications to published procedures.33
was washed with distilled water (150 mL), dried over sodium sul-
fate, and concentrated to give a colorless gum. The residue was
purified by FCC using CH3OH/CH2Cl2 (1:9) to afford the in-
termediate. This intermediate (0.620 g, 1.822 mmol) was then dis-
Flash column chromatography (FCC) was performed using silica
gel 60 (230e400 mesh) obtained from Silicycle (Quebec City, QC).
Thin layer chromatography (TLC) was performed using precoated
TLC plates (Merck, Kieselgel 60 F254, 0.25 mm) purchased from EMD
Chemicals Inc. (Gibbstown, NJ). All solvents for column chroma-
tography were obtained from Mallinckrodt Baker, Inc. (Phillipsburg,
NJ). 1H and 13C NMR spectra were recorded on a Varian 500 MHz
NMR spectrometer at rt at frequencies of 500 and 125.7 MHz for 1H
and 13C, respectively. Chemical shifts were reported in parts per
million downfield from tetramethylsilane. 31P NMR spectra (1H
decoupled) were recorded at a frequency of 202.4 MHz with H3PO4
used as an external standard.
solved in
a mixture of pyridine/THF (9:1, 70 ml) to which
were added allyl 1-hydroxybenzotriazole carbonate (0.531 g,
2.423 mmol) and DMAP (0.045 g, 0.364 mmol) at rt. After 24 h, the
solvent was removed in vacuo, the residue was taken up in CH2Cl2
(100 mL), and the solution was washed with aqueous NaHCO3
(3%, 100 mL). The organic layer then was dried over sodium sulfate
and purified by FCC using hexane/ethyl acetate (7:3) as eluent to
afford compound (3) as a colorless foam (1.285 g, 85%). Rf (SiO2
TLC): 0.72 in hexane/ethyl acetate (7:3). 1H NMR (CDCl3, ppm):
d
1.93 (s, 3H, CH3eC5), 2.21 (s, 3H, CH3 of Lev), 2.22e2.27 (ddd,
J¼14.0, 8.5, 6.5 Hz, 1H, H20), 2.41e2.45 (ddd, J¼14.0, 5.5, 1.5 Hz, 1H,
H200), 2.58e2.61 (t, J¼6.5 Hz, 2H, CH2 of Lev), 2.77e2.80 (t, J¼6.5 Hz,
2H, CH2 of Lev), 3.76e3.81 (dd, J¼11.5, 2.5 Hz, 1H, H50), 4.24e4.26
(ddd, J¼5.0, 2.5, 2.5 Hz, 1H, H40), 4.41e4.44 (dd, J¼11.5, 2.5 Hz, 1H,
H500), 4.66e4.68 (d, J¼6.0 Hz, 2H, CH2 of Alloc), 5.26e5.30 (ddd,
J¼6.5, 5.0, 1.5 Hz, 1H, H30), 5.30e5.33 (dd, J¼10.5, 1.0 Hz, 1H, CH of
Alloc), 5.37e5.40 (dd, J¼17.0, 1.0 Hz, 1H, CH of Alloc), 5.90e5.96
(ddt, J¼17.0, 10.5, 6.0 Hz, 1H, CH of Alloc), 6.40e6.43 (dd, J¼8.5,
5.5 Hz, 1H, H10), 7.42 (s, 1H, H6). 13C NMR (CDCl3, ppm): 12.6, 27.9,
29.7, 37.2, 37.8, 67.3, 69.0, 74.7, 82.1, 84.4, 111.7, 119.7, 131.0, 135.0,
150.6, 154.4, 163.8, 172.4, 206.4. ESI-MS (MþNaþ): 447.1375 (calcd
447.1380).
4.2. Synthesis
4.2.1. 4-Iodobutyl-1-phenoxyacetate (1). Phenoxyacetyl chloride
(3.412 g, 20.00 mmol) and potassium iodide (8.300 g, 50.00 mmol)
were dissolved in THF (50 mL) at room temperature (rt). After 24 h,
NaHCO3 (1.681 g, 20.00 mmol) was added slowly to the solution.
This was filtered and the solvent was removed in vacuo. The crude
product was taken up in CH2Cl2 (100 mL) and the reaction
quenched with aqueous NaHCO3 (3%, 100 mL). The organic layer
was washed with distilled water (100 mLꢁ2), dried over sodium
sulfate, and concentrated to give a viscous liquid. The crude product
was purified by flash column chromatography (FCC) using a hex-
ane/ethyl acetate (1:1) mixture to afford compound (1) as a yel-
lowish oil (4.26 g, 95%). Rf (SiO2 TLC): 0.76 in hexane/ethyl acetate
4.2.4. 1-{N3-[50-O-(Dimethoxytrityl)-30-O-(tert-butyldimethylsilyl)-
thymidylyl]}-4-{N3-[50-O-(allyloxycarbonyl)-30-O-(levulinoyl)-thy-
midylyl]}butane (4). N3-(4-Iodobutyl)-50-O-dimethoxytrityl-30-O-
(tert-butyldimethylsilyl)-thymidine (0.452 g, 0.537 mmol) and
a slight excess of compound (3) (0.251 g, 0.592 mmol) were dis-
solved in MeCN (20 mL) to which was added DBU (0.164 g,
1.074 mmol) at rt. After 48 h, the solvent was removed in vacuo, the
residue was taken up with CH2Cl2 (100 mL), and the solution was
washed with aqueous NaHCO3 (3%, 100 mL). The organic layer was
dried over sodium sulfate and purified by FCC with a gradient of
hexane/ethyl acetate (5:5 to 1:9) as eluent, to produce compound
(4) as a colorless foam (0.336 g, 55 %). Rf (SiO2 TLC): 0.76 in hexane/
(1:1). 1H NMR (CDCl3, ppm):
d
1.63e1.71 (tt, J¼8.0, 6.5 Hz, 2H, CH2),
1.72e1.79 (tt, J¼8.0, 6.5 Hz, 2H, CH2), 3.08e3.10 (t, J¼6.5 Hz, 2H,
CH2), 4.15e4.17 (t, J¼6.5 Hz, 2H, CH2), 4.56 (s, 2H, OCH2 of Pac),
6.83e6.85 (dd, J¼8.5, 0.5 Hz, 2H, Ph of Pac), 6.93e6.94 (dd, J¼7.0,
0.5 Hz,1H, Ph of Pac), 7.19e7.23 (dd, J¼8.5, 7.0 Hz, 2H, Ph of Pac). 13C
NMR (CDCl3, ppm): 5.9, 29.4, 29.8, 64.0, 65.3, 114.6, 114.6, 121.8,
129.6, 129.6, 157.8, 169.0. ESI-MS (MþKþ): 372.9655 (calcd
372.9703).
4.2.2. 50-O-Dimethoxytrityl-30-O-levulinoyl-thymidine (2). To a so-
lution of 50-O-dimethoxytrityl-thymidine (5.446 g, 10.00 mmol) in
1,4-dioxane (60 mL) at rt were added EDC (3.820 g, 20.00 mmol),
DMAP (0.012 g, 0.10 mmol), and levulinicacid (2.322 g, 20.00 mmol).
After 12 h the solvent was removed in vacuo, the residue was taken
up in CH2Cl2 (100 mL) and the solution washed with aqueous
NaHCO3 (3%, 100 mL). The organic layer was dried over sodium sul-
fate, purifiedbyFCCtoyield compound (2)asacolorless foam(6.42 g,
99%). Rf (SiO2 TLC):0.40 inhexane/ethyl acetate(1:9).1HNMR(CDCl3,
ethyl acetate (1:9). 1H NMR (CDCl3, ppm):
d 0.00 (s, 3H, SiCH3), 0.06
(s, 3H, SiCH3), 0.86 (s, 9H, SiC(CH3)3), 1.53 (s, 3H, C5eCH3), 1.71e1.74
(m, 4H, CH2 of butylene), 1.95 (s, 3H, C5eCH3), 2.24 (s, 3H, CH3 of
Lev), 2.23e2.25 (m, 1H, H20), 2.23e2.27 (m, 1H, H200), 2.36e2.39 (m,
1H, H20), 2.46e2.50 (m, 1H, H200), 2.62e2.64 (t, J¼6 Hz, 2H, CH2 of
Lev), 2.80e2.83 (t, J¼6 Hz, 2H, CH2 of Lev), 3.28e3.30 (dd, J¼10.5,
1.5 Hz, 1H, H50), 3.50e3.52 (dd, J¼10.5, 1.5 Hz, 1H, H500), 3.83 (s, 6H,
OCH3 of DMT), 3.99e4.02 (m, 1H, H40), 3.99e4.02 (t, J¼5.5 Hz, 4H,
CH2 of butylene), 4.21e4.24 (m, 1H, H40), 4.43e4.48 (m, 2H, H50 and
H500), 4.54e4.57 (m, 1H, H30), 4.70e4.72 (d, J¼6.0 Hz, 2H, OCH2 of
Alloc), 5.31e5.34 (m, 1H, H30), 5.33e5.36 (dd, J¼10.5, 1.0 Hz, 1H, CH
of Alloc), 5.40e5.44 (dd, J¼17.0, 1.0 Hz, 1H, CH of Alloc), 5.95e6.02
(ddt, J¼17.0,10.5, 6.0 Hz,1H, CH of Alloc), 6.38e6.42 (m, 2H, 2ꢁH10),
6.87e6.89 (m, 4H, Ph of DMT), 7.28e7.44 (m, 9H, Ph of DMT), 7.46
(s, 1H, H6), 7.67 (s, 1H, H6). 13C NMR (CDCl3, ppm): ꢂ4.9, ꢂ4.7, 12.7.
13.3, 17.9, 25.3, 25.7, 27.9, 29.7, 37.2, 37.8, 41.0, 41.2, 41.6, 55.3, 62.9,
67.3, 69.0, 72.0, 74.8, 82.0, 85.2, 85.5, 86.6, 86.8, 110.2, 110.8, 113.3,
113.3, 119.6, 127.1, 128.0, 128.2, 130.1, 130.1, 131.1, 132.9, 133.5, 135.5,
135.6, 144.4, 150.9, 151.0, 154.5, 158.7, 163.2, 163.5, 172.3, 206.1. ESI-
MS (MþNaþ): 1159.4929 (calcd 1159.4923).
ppm): d 1.29 (s, 3H, C5eCH3), 2.12 (s, 3H, CH3 of Lev), 2.32e2.38 (ddd,
J¼13.5, 7.0, 6.0 Hz, 1H, H20), 2.58e2.62 (ddd, J¼13.5, 5.5, 4.5 Hz, 1H,
H200), 2.63e2.66 (t, J¼6.5 Hz, 2H, CH2 of Lev), 2.67e2.70 (J¼6.5 Hz,
2H, CH2 of Lev), 3.35e3.38 (dd, J¼13.0, 2.5 Hz, 1H, H50), 3.39e3.42
(dd, J¼13.0, 2.5 Hz, 1H, H500), 3.72 (s, 6H, OCH3 of DMT), 4.06 (ddd,
J¼5.0, 2.5, 2.5 Hz, 1H, H40), 5.38e5.40 (ddd, J¼6.0, 5.0, 4.5 Hz, 1H,
H30), 6.36e6.38 (dd, J¼7.0, 5.5 Hz, 1H, H10), 6.75e6.77 (m, 4H, Ph of
DMT), 7.17e7.31 (m, 9H, Ph of DMT), 7.53 (s,1H, H6). 13C NMR (CDCl3,
ppm): 11.6, 28.0, 29.8, 37.8, 37.9, 55.3, 63.7, 75.7, 84.0, 84.3, 87.2,111.6,
113.3, 127.2, 128.0, 128.1, 130.1, 130.1, 135.1, 135.2, 135.5, 144.2, 150.4,
158.8, 158.8, 163.6, 172.2, 206.3. ESI-MS (MþNaþ): 665.2483 (calcd
665.2475).
4.2.5. 1-{N3-[50-O-(Dimethoxytrityl)-30-O-(tert-butyldimethylsilyl)-
thymidylyl]}-4-{N3-[50-O-(allyloxycarbonyl)-thymidylyl]}butane
(5). Compound (4) (0.441 g, 0.388 mmol) was treated with 10 mL
of HPAA (0.5 M hydrazine in a buffer composed of 9 mL pyridine
and 1 mL glacial acetic acid) at rt. After 10 min, the solution was
diluted with CH2Cl2 (100 mL) and washed with aqueous NaHCO3
4.2.3. 50-O-Allyloxycarbonyl-30-O-levulinoyl-thymidine (3). To com-
pound (2) (2.310 g, 3.594 mmol), dissolved in a mixture of CH2Cl2/
CH3OH (4:1, 75 mL), was added p-TsOH (1.505 g, 7.910 mmol) at rt.
After 30 min, the solution was diluted with CH2Cl2 (300 mL) and
then washed with aqueous NaHCO3 (3%, 150 mL). The organic layer

7792
G. Sun et al. / Tetrahedron 68 (2012) 7787e7793
(3%, 100 mL). The water phase was extracted twice with CH2Cl2
(100 mL). The combined organic layer was dried over sodium sul-
fate and purified by FCC using a solvent system of hexane/ethyl
acetate (1:9) to afford compound (5) as a colorless foam (0.387 g,
96%). Rf (SiO2 TLC): 0.56 in hexane/ethyl acetate (1:9). 1H NMR
oligonucleotide) in a buffer solution of butylamine/formic acid (1:1,
100 equiv to the support bound oligonucleotide) for 3 h at 35 ꢀC.
The support was then washed with 30 mL each of anhydrous THF
and MeCN followed by drying via high vacuum (30 min). The
support bound oligomers was then treated with the Pd scavenger
(CDCl3, ppm):
d
0.00 (s, 3H, SiCH3), 0.06 (s, 3H, SiCH3), 0.86 (s, 9H,
N,N-diethyldithiocarbamate as a saturated solution in MeCN
SiC(CH3)3), 1.53 (s, 3H, C5eCH3), 1.71e1.73 (m, 4H, CH2 of butylene),
1.95 (s, 3H, C5eCH3), 2.23e2.29 (m, 2H, H20 and H200), 2.35e2.38
(m, 1H, H20), 2.41e2.45 (m, 1H, H200), 3.27e3.31 (dd, J¼14.0, 3.0 Hz,
1H, H50), 3.50e3.54 (dd, J¼14.0, 3.5 Hz, 1H, H500), 3.83 (s, 6H, OCH3
of DMT), 3.97e4.00 (m, 1H, H40), 3.98e4.00 (t, J¼6.0 Hz, 4H, CH2 of
butylene), 4.15e4.18 (m, 1H, H40), 4.45e4.48 (m, 2H, H50 and H500),
4.50e4.53 (m, 2H, 2ꢁH30), 4.69e4.71 (d, J¼6.0 Hz, 2H, OCH2 of
Alloc), 5.52e5.55 (dd, J¼10.5, 1.0 Hz, 1H, CH of Alloc), 5.58e5.62
(dd, J¼17.0, 1.0 Hz, 1H, CH of Alloc), 5.94e6.01 (ddt, J¼17.0, 10.5,
6.0 Hz, 1H, CH of Alloc), 6.39e6.42 (m, 2H, 2ꢁH10), 6.87e6.89 (m,
4H, Ph of DMT), 7.29e7.44 (m, 9H, Ph of DMT), 7.46 (s, 1H, H6), 7.68
(s, 1H, H6). 13C NMR (CDCl3, ppm): ꢂ4.9, ꢂ4.7, 12.7, 13.3, 15.3, 18.0,
25.3, 25.7, 40.6, 41.0, 41.1, 41.7, 55.3, 62.8, 66.7, 69.0, 71.2, 83.8, 85.3,
85.4, 86.6, 86.8, 110.2, 110.4, 113.2, 113.3, 119.7, 127.1, 128.0, 128.2,
130.0, 130.1, 131.1, 133.2, 133.6, 135.5, 135.5, 144.3, 150.8, 150.9,
154.8, 158.7, 163.4, 163.5. ESI-MS (MþNaþ): 1061.4567 (calcd
1061.4555).
(3 mLꢁ2) for a total of 1 h at rt. This was followed by washing with
30 mL each of CH2Cl2 and MeCN followed by drying via high vac-
uum (30 min). Chain assembly was continued using 30-O-20-deox-
yphosphoramidites followed by detritylation and an additional
capping step for the 50-OH group. The polystyrene-linked oligomers
were treated with 1 mL of anhydrous TEA for 12 h. Then, the sup-
port was washed with 30 mL of anhydrous MeCN followed by an-
hydrous THF. Then, the TBS group was removed from the partial
duplex by treating the support with 2ꢁ1 mL TEA$3HF for a total of
1 h. The support was then washed with 30 mL each of anhydrous
THF and MeCN followed by drying via high vacuum (20 min). The
final extension of the cross-linked duplex was then achieved using
50-O-20-deoxyphosphoramidites with a total detritylation time of
130 s with removal of the 30-terminal trityl group by the synthe-
sizer to yield duplexes on the solid support.
The oligomer-derivatized polystyrene was transferred from the
reaction column to screw cap microfuge tubes fitted with Teflon
lined caps and the oligomer released from the support and pro-
tecting groups removed by treatment with a mixture of concen-
trated ammonium hydroxide/ethanol (0.3 mL:0.1 mL) for 4 h at
55 ꢀC. The cross-linked final product was separated from pre-
terminated products by IEX HPLC using a Dionex DNAPAC PA-100
column (0.4 cmꢁ25 cm) purchased from Dionex Corp, (Sunny-
vale, CA) with a linear gradient of 20e55% buffer B over 30 min
(buffer A: 100 mM TriseHCl, pH 7.5, 10% MeCN and buffer B:
100 mM TriseHCl, pH 7.5, 10% MeCN, 1 M NaCl) at 40 ꢀC. The col-
umns were monitored at 260 nm for analytical runs or 280 nm for
preparative runs. The purified oligomer was desalted using C-18
SEP PAK cartridges (Waters Inc.) as previously described.34
4.2.6. 1-{N3-[50-O-(Dimethoxytrityl)-30-O-(tert-butyldimethylsilyl)-
thymidylyl]}-4-{N3-[50-O-(allyloxycarbonyl)-thymidylyl-30-O-(
b-cya-
noethyl N,N0-diisopropyl)phosphoramidite]}butane (6). To com-
pound (5) (0.317 g, 0.305 mmol) in THF (1.25 mL) was added DIPEA
(0.059 g, 0.458 mmol) followed by N,N-diisopropylamino cya-
noethyl phosphonamidic chloride (0.087 g, 0.366 mmol). After
30 min, the reaction mixture was diluted with ethyl acetate
(50 mL). The organic layer was washed with aqueous NaHCO3 (3%,
100 mLꢁ2) followed by saturated NaCl (100 mL), then dried over
sodium sulfate. After concentration in vacuo, the gum was pre-
cipitated from hexane to yield compound 6 as a colorless foam
(0.269 g 71%). Rf (SiO2 TLC): 0.81 in hexane/ethyl acetate (2:8). 31P
NMR (acetone-d6, ppm): 146.71, 146.97. ESI-MS (MþNaþ):
1261.5638 (calcd 1261.5634).
4.3.2. Enzymatic digests. The cross-linked oligomer (0.1 A260 units)
was characterized by enzymatic digestion (0.28 units of snake
venom phosphodiesterase and 5 units of calf intestinal phosphatase
in a buffer containing 2 mM magnesium chloride and 10 mM Tris at
pH 8.1) for a minimum of 36 h at 37 ꢀC.34 The resulting mixture of
nucleosides was analyzed by reversed-phase HPLC carried out using
4.3. Assembly of cross-linked DNA duplexes, purification, and
characterization
4.3.1. Assembly of cross-linked DNA duplexes. The cross-linked du-
plex (shown in Fig. 1) was assembled using an Applied Biosystems
a SymmetryÒ C-18 5
mm column (0.46ꢁ15 cm) purchased from
Waters Inc., Milford, MA. The C-18 column was eluted with a linear
gradient of 0e60% buffer B over 30 min (buffer A, 50 mM sodium
phosphate, pH 5.8, 2% MeCN and buffer B, 50 mM sodium phos-
phate, pH 5.8, 50% MeCN). The resulting peaks were identified byco-
injection with the corresponding standards and eluted at the fol-
lowing times: dC (4.6 min), dG (6.8 min), dT (7.4 min), dA (7.9 min),
and cross-linked dimer (14.2 min) and the ratio of nucleosides was
determined. The molar extinction coefficient of N3-thymidinee
butyleneeN3-thymidine was determined previously.33
Model 3400 synthesizer on a 1
mmol scale employing standard
b-cyanoethyl phosphoramidite cycles supplied by the manufac-
turer with slight modifications to coupling times described below.
Solutions of the nucleoside phosphoramidites containing standard
protecting groups were prepared in anhydrous MeCN at a concen-
tration of 0.1 M for the 30-O-20-deoxyphosphoramidites, 0.15 M for
the cross-linked phosphoramidite (6), and 0.2 M for the 50-O-20-
deoxyphosphoramidites. Assembly of sequences first involved
detritylation (3% trichloroacetic acid [TCA] in CH2Cl2), followed by
nucleoside phosphoramidite coupling with commercial 30-O-20-
deoxyphosphoramidites (2 min), 50-O-20-deoxyphosphoramidites
(3 min) or cross-linked phosphoramidite (6) (10 min); Subsequent
capping with acetic anhydride/pyridine/tetrahydrofuran (1:1:8,
v/v/v) and N-methyl-imidazole/tetrahydrofuran (16:84 w/v)
and oxidation (0.02 M iodine in tetrahydrofuran/water/pyridine
2.5:2:1) followed every coupling. To form the Y intermediate, the
cyanoethyl groups were removed from the polystyrene-linked
oligomers by treating the support with 1 mL of anhydrous trie-
thylamine (TEA) for at least 12 h. The support was then washed
with 30 mL of anhydrous MeCN followed by anhydrous THF. The 50-
O-alloc group was removed from the partial duplex by treating the
support with Pd(PPh3)4/PPh3 (10:20 equiv to the support bound
4.3.3. Mass spectrometry. ESI mass spectra for small molecules
were recorded at the McGill University Department of Chemistry
Mass Spectrometry Facility with a Finnigan LCQ DUO mass spec-
trometer in methanol or acetone. ESI mass spectra for oligonucle-
otides were obtained at the Concordia University Centre for
Biological Applications of Mass Spectrometry (CBAMS) using
a Micromass Qtof2 mass spectrometer (Waters) equipped with
a nanospray ion source. The mass spectrometer was operated in full
scan, negative ion detection mode.
4.3.4. UV thermal denaturation studies. Molar extinction co-
efficients for the oligonucleotides were calculated from those of the
mononucleotides and dinucleotides according to nearest neighbor

G. Sun et al. / Tetrahedron 68 (2012) 7787e7793
7793
approximations.54,55 Non-cross-linked duplexes were prepared by
mixing equimolar amounts of the interacting strands and lyophi-
lizing the mixture to dryness. The resulting pellets (both controls
and cross-linked duplex) were then re-dissolved in 90 mM sodium
chloride, 10 mM sodium phosphate, 1 mM EDTA buffer (pH 7.0) to
9. Dronkert, M. L. G.; Kanaar, R. Mutat. Res. 2001, 486, 217e247.
10. Helleday, T.; Petermann, E.; Lundin, C.; Hodgson, B.; Sharma, R. A. Nat. Rev.
Cancer 2008, 8, 193e204.
€
11. Raschle, M.; Knipscheer, P.; Enoiu, M.; Angelov, T.; Sun, J.; Griffith, J. D.; Ellenberger,
€
T. E.; Scharer, O. D.; Walte, J. C. Cell 2008, 134, 969e980.
12. Hanada, K.; Budzowska, M.; Modesti, M.; Maas, A.; Wyman, C.; Essers, J.; Kanaar, R.
EMBO J. 2006, 25, 4921e4932.
give a final concentration of 2.8 mM for control and cross-linked
13. Niedernhofer, L. J.; Odijk, H.; Budzowska, M.; van Drunen, E.; Maas, A.; Theil, A.
F.; de Wit, J.; Jaspers, N. G. J.; Beverloo, H. B.; Hoeijmakers, J. H. J.; Kanaar, R. Mol.
Cell. Biol. 2004, 24, 5776e5787.
duplex. The solutions were then heated to 90 ꢀC for 10 min,
cooled slowly to rt, and stored at 4 ꢀC overnight before measure-
ments. 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 6-sample
thermostated cell block and a temperature controller. The data
were analyzed in accordance with the convention of Puglisi and
Tinoco54 and transferred to Microsoft ExcelÔ.
14. Li, L.; Peterson, C. A.; Lu, X.; Wei, P.; Legerski, R. J. Mol. Cell. Biol. 1999, 19,
5619e5630.
€
15. Scharer, O. D. ChemBioChem 2005, 6, 27e32.
16. Smeaton, M. B.; Hlavin, E. M.; Noronha, A. M.; Murphy, S. P.; Wilds, C. J.; Miller,
P. S. Chem. Res. Toxicol. 2009, 22, 1285e1297.
17. Zheng, H.; Wang, X.; Legerski, R. J.; Glazer, P. M.; Li, L. DNA Repair 2006,
5, 566e574.
18. Bessho, T.; Mu, D.; Sancar, A. Mol. Cell. Biol. 1997, 17, 6822e6830.
19. Batty, D. P.; Wood, R. D. Gene 2000, 241, 193e204.
20. Couve, S.; Mace-Aime, G.; Rosselli, F.; Saparbaev, M. K. J. Biol. Chem. 2009, 284,
11963e11970.
21. Wu, Q.; Christensen, L. A.; Legerski, R. J.; Vasquez, K. M. EMBO Rep. 2005,
6, 551e557.
22. Zhao, J.; Jain, A.; Iyer, R. R.; Modrich, P. L.; Vasquez, K. M. Nucleic Acids Res. 2009,
37, 4420e4429.
23. Clingen, P. H.; De Silva, I. U.; McHugh, P. J.; Ghadessy, F. J.; Tilby, J.; Thurston, D.
E.; Hartley, J. A. Nucleic Acids Res. 2005, 33, 3283e3291.
24. De Silva, I. U.; McHugh, P. J.; Clingen, P. H.; Hartley, J. A. Mol. Cell. Biol. 2000, 20,
7980e7990.
4.3.5. Circular dichroism (CD) spectroscopy. Circular dichroism
spectra were acquired on a Jasco J-815 spectropolarimeter equip-
ped with a Julaba F25 circulating bath. Samples were allowed to
equilibrate for 5e10 min at 10 ꢀC in 90 mM sodium chloride, 10 mM
sodium phosphate, 1 mM EDTA (pH 7.0), at a final concentration of
2.8 mM for the cross-linked duplex and ca. 2.8 mM for the control
25. Wang, X.; Peterson, C. A.; Zheng, H.; Nairn, R. S.; Legerski, R. J.; Li, L. Mol. Cell.
duplexes. Each spectrum was an average of five 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 (Starna 29-
Q-10). The CD spectra were recorded from 350 to 200 nm at 10 ꢀC.
Biol. 2001, 21, 713e720.
26. Wang, W. Nat. Rev. Genet. 2007, 8, 735e748.
€
27. Alzeer, J.; Scharer, O. D. Nucleic Acids Res. 2006, 34, 4458e4466.
€
28. Hentschel, S.; Alzeer, J.; Angelov, T.; Scharer, O. D.; Luedtke, N. W. Angew. Chem.,
Int. Ed. 2012, 51, 3466e3469.
The molar ellipticity was calculated from the equation [
q
]¼
3
/Cl,
29. Huang, H.; Hopkins, P. B. J. Am. Chem. Soc. 1993, 115, 9402e9408.
30. Harwood, E. A.; Hopkins, P. B.; Sigurdsson, S. T. J. Am. Chem. Soc. 1999, 121,
5081e5082.
31. Harwood, E. A.; Hopkins, P. B.; Sigurdsson, S. T. J. Org. Chem. 2000, 65,
2959e2964.
32. Wilds, C. J.; Booth, J. D.; Noronha, A. M. Tetrahedron Lett. 2006, 47, 9125e9128.
33. Wilds, C. J.; Noronha, A. M.; Robidoux, S.; Miller, P. S. J. Am. Chem. Soc. 2004, 126,
9257e9265.
where is the relative ellipticity (mdeg), C is the molar concen-
3
tration of oligonucleotides (mol/L), and l is the path length of the
cell (cm). The data were processed on a PC computer using Win-
dowsÔ based software supplied by the manufacturer (JASCO, Inc.)
and transferred into Microsoft ExcelÔ for presentation.
34. Wilds, C. J.; Palus, E.; Noronha, A. M. Can. J. Chem. 2007, 85, 249e256.
35. Wilds, C. J.; Xu, F.; Noronha, A. M. Chem. Res. Toxicol. 2008, 21, 686e695.
36. Webba da Silva, M.; Noronha, A. M.; Noll, D. M.; Miller, P. S.; Colvin, O. M.;
Gamcsik, M. P. Biochemistry 2002, 41, 15181e15188.
37. Noll, D. M.; Noronha, A. M.; Miller, P. S. J. Am. Chem. Soc. 2001, 123, 3405e3411.
38. Schelhaas, D.-C. M.; Waldmann, H. Angew. Chem., Int. Ed. 1996, 35, 2056e2083.
39. McManus, F. P.; Fang, Q.; Booth, J. D.; Noronha, A. M.; Pegg, A. E.; Wilds, C. J. Org.
Biomol. Chem. 2010, 8, 4414e4426.
40. Asanuma, H.; Hara, Y.; Noguchi, A.; Sano, K.; Kashida, H. Tetrahedron Lett. 2008,
49, 5144e5146.
41. Spinelli, N.; Meyer, A.; Hayakawa, Y.; Imbach, J.-L.; Vasseur, J.-J. Eur. J. Org. Chem.
2002, 49e56.
Acknowledgements
The authors are grateful for financial support from the Natural
Sciences and Engineering Research Council of Canada (NSERC), the
Canada Foundation for Innovation (CFI) and the Canada Research
Chair (CRC) program. G.S. is the recipient of a doctoral Bourse
ꢀ
ꢀ
d’Excellence pour Etudiants Etrangers (MELS) from Le Fonds
ꢀ ꢀ
Quebecois de la Recherche sur la Nature et les Technologies
(FQRNT). We are also indebted Mr. Nadim Saade and Dr. Bruce
Lennox at McGill University for ESI-MS analysis of nucleosides.
42. Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J. Am. Chem. Soc. 1990, 112,
1691e1696.
43. Li, H.-Y.; Qiu, Y.-L.; Moyroud, E.; Kishi, Y. Angew. Chem., Int. Ed. 2001, 40,
1471e1475.
44. Wuts, P. G. M.; Greene, T. W. Greene’s Protective Groups in Organic Synthesis;
John Wiley & Sons: Hoboken, NJ, 2006; 1e1082.
Supplementary data
These data include 1H, 13C, and COSY NMR spectra, HPLC
traces, mass spectra, UV thermal denaturation profiles, CD
spectra, and additional synthetic procedures. Supplementary data
associated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.tet.2012.07.043.
45. Sergueeva, Z. A.; Sergueev, D. S.; Ribeiro, A. A.; Summers, J. S.; Ramsay
€
Shaw, B. In Perspectives in Nucleoside and Nucleic Acid Chemistry; Kisakurek,
M. V., Rosemeyer, H., Eds.; Wiley-VCH: Weinheim: Germany, 2000;
pp 109e123.
46. Townsend, L. B. In Chemistry of Nucleosides and Nucleotides; Townsend, L. B.,
Ed.; Plenum: New York, NY, 1994; Vol. 3, pp 121e140.
47. Schulhof, J. C.; Molko, D.; Teoule, R. Tetrahedron Lett. 1987, 28, 51e54.
48. Duynstee, H. I.; De Koning, M. C.; Ovaa, H.; Van Der Marel, G. A.; Van Boom, J. H.
Eur. J. Org. Chem. 1999, 2623e2632.
References and notes
49. Virta, P. ARKIVOC 2009, 54e83.
50. Kochetkov, N. K.; Budovskii, E. I. In Organic Chemistry of Nucleic Acids; Plenum:
New York, NY, 1972; Vol. B, pp 269e348.
51. Johnson, W. C., Jr. In Methods of Biochemical Analysis; Glick, D., Ed.; Wiley-VCH:
New York, NY, 1985; Vol. 31, p 61.
52. Johnson, W. C., Jr. In Circular Dichroism and the Conformational Analysis
of Biomolecules; Fasman, G. D., Ed.; Plenum: New York, NY, 1996; pp 433e468.
53. Webba da Silva, M.; Bierbryer, R. G.; Wilds, C. J.; Noronha, A. M.; Colvin, O. M.;
Miller, P. S.; Gamcsik, M. P. Bioorg. Med. Chem. 2005, 13, 4580e4587.
54. Puglisi, J. D.; Tinoco, I. In Methods in Enzymology; Dahlberg, J. E., Abelson, J. N.,
Eds.; Academic: New York, NY, 1989; Vol. 180, pp 304e325.
55. Tataurov, A. V.; You, Y.; Owczarzy, R. Biophys. Chem. 2008, 133, 66e70.
1. Noll, D. M.; Noronha, A. M.; Wilds, C. J.; Miller, P. S. Front. Biosci. 2004, 9, 421e437.
2. Noll, D. M.; Mason, T. M.; Miller, P. S. Chem. Rev. 2005, 106, 277e301.
3. Boulikas, T.; Vougiouka, M. Oncol. Rep. 2003, 10, 1663e1682.
4. Sampath, D.; Plunkett, W. Curr. Oncol. Rep. 2007, 9, 361e367.
5. Wang, S. Q.; Setlow, R.; Berwick, M.; Polsky, D.; Marghoob, A. A.; Kopf, A. W.;
Bart, R. S. J. Am. Acad. Dermatol. 2001, 44, 837e846.
6. Cole, R. S. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 1064e1068.
7. Magana-Schwencke, N.; Henriques, J. A.; Chanet, R.; Moustacchi, E. Proc. Natl.
Acad. Sci. U.S.A. 1982, 79, 1722e1726.
8. Van Houten, B.; Gamper, H.; Holbrook, S. R.; Hearst, J. E.; Sancar, A. Proc. Natl.
Acad. Sci. U.S.A. 1986, 83, 8077e8081.