A fluorescence-quencher pair for DNA hybridization studies based on hydrophobic base surrogates

Article abstract of DOI:10.1016/j.tet.2006.12.095

The synthesis of the novel, fluorescent 3-aminobiphenyl-C-nucleoside M as well as the corresponding building block for oligodeoxynucleotide synthesis is described. M was incorporated into oligodeoxynucleotides via standard phosphoramidite chemistry and th

Full text of DOI:10.1016/j.tet.2006.12.095

Tetrahedron 63 (2007) 3440–3449
A fluorescence-quencher pair for DNA hybridization studies
based on hydrophobic base surrogates
*
Matthias Stoop, Alain Zahn and Christian J. Leumann
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland
Received 4 October 2006; revised 18 December 2006; accepted 20 December 2006
Available online 2 February 2007
Abstract—The synthesis of the novel, fluorescent 3-aminobiphenyl-C-nucleoside M as well as the corresponding building block for oligo-
deoxynucleotide synthesis is described. M was incorporated into oligodeoxynucleotides via standard phosphoramidite chemistry and the ther-
mal stabilities of duplexes with one and three consecutive M–M, M–P, and M–O pairs, where P denotes an unmodified biphenyl C-nucleoside
and O a 3,5-dinitrobiphenyl-C-nucleoside, were determined. It was found that duplexes containing three consecutive M–O pairs were the
most stable in the series, notably more stable than a duplex with one additional natural G–C pair instead of the modified residues. Furthermore
it was found that the fluorescence of M is efficiently quenched in a duplex when placed opposite to the dinitrophenyl unit O. Thus M and O
constitute a novel fluorophore/quencher pair that is orthogonal to natural base pairs in its recognition properties enabling its use as highly
specific tags with reporting properties.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
recently found that biphenyl (P) modified oligonucleotides
(Fig. 1) readily form stable duplexes via self-recognition
of the aromatic units.10–12 Stability is most likely brought
about by interstrand intercalation of two residues in opposite
position (Fig. 1). We found that up to seven of such aromatic
pairs can be introduced into the center of a duplex with in-
creasing thermal stability per added pair. This interstrand
Fluorescence spectroscopy has lately become a method of
choice not only for studying structure and dynamics of
nucleic acids down to the single molecule level,1 but also
in various commercialized applications in biotechnology,
e.g., DNA sequencing, array hybridization, and real time
PCR applications, to name only a few of them. Since natural
nucleic acids are non-fluorescent,2 fluorescence has to be
brought about by chemical modification with fluorescent
dyes. Alternatively, oligonucleotides can be made fluores-
cent by replacing natural bases with fluorescent base ana-
logues. The most prominent analogues used in the context
outlined above are 2-aminopurine,3 ethenoadenine,4 and
pyrrolocytosine.5
Of special interest were also recent findings with
oligonucleotides containing aromatic residues instead of
the natural bases. Such constructs are of potential value as
orthogonal base pairs for the extension of the genetic alpha-
bet,6 as tools for probing the enzymatic processivity by poly-
merases,7 as tools for DNA damage detection,8 and as
variable color fluorescent tags.9
In our own investigations into oligonucleotides with
aromatic, hydrophobic residues as base replacements we
Keywords: Biphenyl DNA; Fluorescence quenching; DNA analogues;
Hydrophobic base pairs.
* Corresponding author. Tel.: +41 31 631 4355; fax: +41 31 631 3422;
e-mail: leumann@ioc.unibe.ch
Figure 1. Top: Chemical structures of the biphenyl-C-nucleotides synthe-
sized and used in this study; bottom: two possible, idealized recognition
motifs of the biphenyl units within a DNA duplex via partial interstrand
stacking interactions.
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.12.095

M. Stoop et al. / Tetrahedron 63 (2007) 3440–3449
3441
recognition motif is not restricted to biphenyl-C-nucleosides
but was also reported for non-nucleosidic phenanthrenes
and pyrenes, for which interesting excimer formation was
observed.13,14
The biphenyl scaffold is ideally suited for further chemical
modification with functional groups that change its elec-
tronic and spectroscopic properties without interfering
with its recognition properties. Biphenyl itself is fluorescent
but its emission maximum at 318 nm15 and its fluorescence
intensity is far from ideal for applications in biotechnology.
Donor-substituted biphenyls show generally red-shifted
emission wavelengths and equal to enhanced fluorescence
intensities while acceptor-substituted biphenyls, for exam-
ple, nitrobiphenyls are non-fluorescent and can therefore
act as quenchers. The goal of the present study was to
explore whether 3-aminobiphenyl-C-nucleoside N which
displays an emission maximum at 410 nm15 and 30,50-dini-
trobiphenyl-C-nucleoside O (Fig. 1) could act as such a fluo-
rescence-quencher pair.
2. Results and discussion
2.1. Synthesis of biaryl-C-nucleosides
The syntheses of the building blocks and of corresponding
modified oligodeoxynucleotides with biphenyl-C-nucleo-
sides O and P were recently published.12,16 For nucleosides
M and N, carrying an acceptor and a donor group in the prox-
imal phenyl ring, a de novo synthesis had to be devised.
Scheme 1. Reagents andconditions:(a) di-tert-butyldicarbonate (1.1 equiv),
THF, rt, 15 h; (b) 3 (1 equiv), t-BuLi (2.2 equiv), THF, ꢀ78 ꢁC to ꢀ20 ꢁC
(3 h), then 1 (0.6 equiv) in THF, ꢀ78 ꢁC, 1.5 h; (c) L-Selectride (1.5 equiv),
THF, ꢀ78 ꢁC to rt, 30 ꢁC; (d) DIAD (2.5 equiv), PPh3 (2.5 equiv), THF,
0 ꢁC, 3 h; (e) SnCl4 (4 equiv), EtOAc, rt, 3 h; (f) 9-fluorenylmethylchlorofor-
mate (2.2 equiv), i-Pr2NEt (2.2 equiv), CH2Cl2, rt, 21 h; (g) (HF3)$NEt3
(10 equiv), THF, rt, 14 h; (h) 4,40-dimethoxytrityl (DMTr) chloride
(1.2 equiv), pyridine, rt, 4 h; (i) (i-Pr)2NP(Cl)OCH2CH2CN (1.5 equiv),
i-Pr2NEt (3 equiv), THF, rt, 4 h.
Lithiation of arylhalides followed by nucleophilic addition
to lactone derivatives is a well-established pathway in C-
nucleoside synthesis.17,18 However, in the present case where
the position neighboring the site of substitution of the bi-
phenyl unit is amino-substituted, direct lithiation via ortho-
metalation seemed to be a shorter and better alternative.
Especially tert-butylcarbamate (Boc) protected aromatic
amines arewell known for directing metalation into the ortho
positon.19,20 Our synthesis therefore started with the Boc-
protected aminobiphenyl 3 that was obtained from the known
3-aminobiphenyl 221 by standard methods (Scheme 1).
Selective Boc deprotection with SnCl4 in EtOAc24 followed
by reprotection of the amine with 9-fluorenylmethylchloro-
formate (Fmoc-Cl) under basic conditions (i-Pr2NEt,
CH2Cl2) yielded Fmoc-protected nucleoside 8 in good yield.
Finally, cleavage of the TIPS protection group under mild
conditions ((HF)3$NEt3 in THF) afforded the Fmoc-pro-
tected C-nucleoside 9 in acceptable yield. The b-configura-
tion at the pseudoanomeric center in 9 was again confirmed
1
Ortholithiation of 3 with tert-BuLi followed by coupling to
the TIPS-protected 20-deoxy-D-ribono-lactone 122 yielded
hydroxy ketone 4 in reasonable yield. No products arising
from the nucleophilic attack of the alternative, sterically
more hindered ortho position could be isolated. Interestingly
the ketone and not the corresponding hemiacetal was the
predominant product of this reaction, as could readily be
determined by 13C NMR. This is most likely due to conjuga-
tive stabilization of this keto-function by the Boc-protected
amino group (vinologous imide). Classical reduction to the
C-nucleoside 6 directly with Et3SiH and BF3$Et2O was
therefore expected to be difficult and indeed did not take
place. Therefore the method of Reese et al.23 was chosen
for ring closure. For this, compound 4 was reduced with
L-Selectride to the corresponding mixture of diastereomeric
diols 5 and cyclized under Mitsunobu conditions to afford
C-nucleoside 6 in a b:a anomeric mixture of ca. 5:1. The
b-configuration at the anomeric center was unambiguously
assigned by 1H NMR-NOE-spectroscopy (see Section 4).
by H NMR-NOE experiments to exclude epimerization
during protecting group manipulations. The C-nucleoside
9 was subsequently converted into the corresponding 4,40-
dimethoxytrityl (DMTr)-protected phosphoramidite build-
ing block 11 by treatment with 4,40-dimethoxytritylchloride
(DMTrCl) in pyridine (/10) followed by phosphitylation
in CH2Cl2. Phosphoramidite 11 was obtained from 2 in an
overall yield of 4%.
With intermediate 7 in hand we also prepared the corre-
sponding o-nitrobiphenyl building block 15 (Scheme 2).
For this the aromatic amino function in 7 was selectively
oxidized with dimethyldioxirane25 in acetone to give the
TIPS-protected nitro-biphenyl-C-nucleoside 12 in 62%
yield. Further transformations to the phosphoramidite build-
ing block 15 are in exact analogy to those described for the
3-aminobiphenyl-C-nucleoside-phosphoramidite 11. Proof
for b-configuration was again obtained on the level of
1
the sugar deprotected C-nucleoside 13 by H NMR-NOE

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M. Stoop et al. / Tetrahedron 63 (2007) 3440–3449
experiments (see Section 4). The overall yield for 15 starting
from 3-aminobiphenyl 2 was 3%.
Scheme 2. Reagents and conditions: (a) dimethyldioxirane (8.2 equiv),
acetone, rt, 3 h; (b) (HF3)$NEt3 (10 equiv), THF, rt, 17 h; (c) 4,40-
dimethoxytrityl (DMTr) chloride (1.2 equiv), pyridine, rt, 4 h; (d)
(i-Pr)2NP(Cl)OCH2CH2CN (1.5 equiv), i-Pr2NEt (3 equiv), THF, rt, 4 h.
Scheme 3. Mechanism of photochemical strand cleavage at o-nitrophenyl-
C-nucleotide units N.
2.2. Synthesis of modified oligodeoxynucleotides
properties of P.12 The problem of the lower affinity can
be overcome by increasing the number of biphenyl pairs.
Indeed, the Tm’s in the triply substituted series are generally
higher than that of an identical duplex with one additional
natural base pair (Table 1). This is in general agreement
with the proposed interstrand-intercalative arrangement of
the biphenyls in the duplex.12
Incorporation of 11 and 15 into oligodeoxynucleotides was
performed via standard phosphoramidite chemistry on
a DNA synthesizer. The coupling time for the modified
building blocks was extended to 6 min, and ethyl-thio-1H-
tetrazole was used as the activator.26 Typical coupling yields
for the modified building blocks, as deduced from the trityl
assay, were in the same range as for non-modified building
blocks (>98%). The oligonucleotides were detached from
the solid support and deprotected in aqueous ammonia
(25%) during 15 h at 55 ꢁC. The oligodeoxynucleotides
were purified by RP-HPLC and their structural integrity
was verified by ESIMS. (Table 2, Section 4).
There are differences in the thermal stability of the biphenyl
pairs depending on the nature of the substitutents. In the
monosubstituted duplexes, the M–M pair is less stable
than the unfunctionalized P–P pair. In the triply substituted
series, pairing of M against the other biphenyls P and O
leads to more stable duplexes than if paired against itself.
The duplex with three M–O pairs is the most stable in this
series. The sequential arrangement 50-M-P-30 versus 50-P-
M-30 has no major influence on the Tm of a corresponding
duplex, indicating stacking interactions of similar energy
at the stereochemically different junction sites.
2.3. Pairing properties of modified oligodeoxy-
nucleotides
Expectedly, oligodeoxynucleotides containing the o-nitrobi-
phenyl unit N were unstable under UV light and led to strand
scission at the site of modification, leaving behind two
distinct end-phosphorylated fragments as determined by
HPLC and ESIMS (data not shown). Strand cleavage most
likely arises via the known photochemistry of o-nitrobenzyl
units27 via oxidation of the pseudoanomeric center followed
by b-elimination of the oligonucleotide fragments (Scheme
3). This C-nucleoside is therefore not suitable for UV-
spectroscopic applications but can be used to photolytically
cleave oligodeoxynucleotides at predefined positions upon
UV irradiation. For this reason the photolytically stable dini-
tro-C-nucleoside O was used as a quencher in the following
hybridization experiments.
A reason for the diminished stability of M–M pairs com-
pared to P–P pairs could be its less hydrophobic nature,
favoring its solvation in the single stranded state. Alterna-
tively, steric interactions of the amino group with the back-
bone or neighboring bases of the same strand may have an
Table 1. Tm data of modified duplexes
50-d(GATGACXGCTAG)-30
30-d(CTACTGYCGATC)-50
50-d(GATGACX3GCTAG)-30
30-d(CTACTGY3CGATC)-50
X–Y
Tm
X–Y
Tm
M–M
R–R
C–G
T–A
40.6
M–M
P–P
M–P
P–M
M–O
O–O
46.5
49.9
48.8
48.3
54.7
50.6
42.512
51.912
47.912
Tm data were measured for the duplexes indicated in Table 1.
Inspection of the data leads to the following general observa-
tions. Compared to a natural A–Tor G–C base pair, incorpo-
ration of a single biphenyl-C-nucleoside pair destabilizes
a duplex irrespective of the presence or absence of the amino
group. This was expected on the basis of the recognition
c(duplex)¼1.2 mM in 10 mM NaH2PO4, 150 mM NaCl, pH 7.0.

M. Stoop et al. / Tetrahedron 63 (2007) 3440–3449
3443
phenyl stacking). A qualitative analysis along the same lines
may explain the higher stability of the M–O pair. Here, an
attractive dinitrophenyl/phenyl stacking arrangement is
expected in the proximal/distal model, which is not the
case in the distal/distal model.
2.4. Fluorescence properties of 3-aminobiphenyl
containing oligonucleotides
To get insight into the fluorescence properties of the modi-
fied oligomers containing the aminobiphenyl unit M and
the dinitrobiphenyl unit O in the paired and unpaired state,
emission spectra were recorded from 350 to 450 nm at dif-
ferent temperatures (Fig. 3).
Figure 2. Calculated electron densities of biphenyl (left) and 3-aminobi-
phenyl (right) in the ground state in the gas phase. Red: high electron den-
sity; blue: low electron density. All calculations were performed at the RHF
level with the 6-31G(d,p) basis set using Gaussian 03.
Single strands containing one (data not shown) or three M-
modifications showed similar spectra with an emission max-
imum at 408 nm when excited at 237 nm. The incorporated
3-aminobiphenyl units thus show fluorescence behavior sim-
ilar to that of free 3-aminobiphenyl in water (lmax¼410 nm).
From this we conclude that no excimeric coupling of
the aminobiphenyls occurs in the single strand. Compared
influence on the stacking interactions and should be consid-
ered as well. Finally, p–p stacking in the case of the M–M
pair may be less favored as a consequence of a repulsive con-
tribution of the electron rich p-systems.
to the fluorescence emission properties of aniline (lmax
¼
335 nm),28 the red-shifted emission maximum of the amino-
biphenyl chromophore indicates extended p-conjugation.
The fluorescence intensity of the single strand is reduced
with increasing temperature in a non-cooperative manner,
which excludes self-recognition as fluorescence signal mod-
ulating event. A single strand with three dinitrophenyl (O)
units as a control shows as expected no fluorescence when
irradiated at the same wavelength.
A comparison of the structural and electronic properties of
the unmodified biphenyl and 3-aminobiphenyl by ab initio
calculation in the ground state (Fig. 2) shows that the biarylic
axis rotates the two phenyl rings out of plane to approxi-
mately the same extent and behaves as a conjugation barrier,
leaving the electron densities of the non-substituted phenyl
ring in 3-aminobiphenyl on the same level as that in the
biphenyl itself. Excluding solvation effects, the difference
in stability between M and P pairs seems thus to be rather
of electronic than of structural origin. Given the two most
likely, idealized stacking recognition patterns outlined in
Figure 1, the observed stability order favors a structural rec-
ognition pattern of the proximal/distal type (aminophenyl/
phenyl stacking) rather than the distal/distal type (phenyl/
A mixture of complementary strands containing three
opposing fluorescent M-units shows enhanced fluorescence
intensity, irrespective of their structural state (duplex vs
single strand). Thus, the fluorescence intensity of the amino-
biphenyl residues is additive and fairly independent of
their structural environment. Also here, slightly reduced
Figure 3. Fluorescence emission spectra at different temperatures of d(GATGACM3GCTAG), top left; d(CTAGCO3GTCATC), top right; d(GATGACM3GC-
TAG)$d(CTAGCM3GTCATC), bottom left; d(GATGACM3GCTAG)$d(CTAGCO3GTCATC), bottom right. c(duplex)¼1.2 mM in 3.5 mM MgCl2, 50 mM
KCl, 10 mM Tris, pH 8.0. Excitation wavelength: 237 nm.

3444
M. Stoop et al. / Tetrahedron 63 (2007) 3440–3449
are typical for B-DNA. The duplex with three M–M pairs
shows a decreased minimum with a shoulder around
240 nm. The positive maximum around 280 nm is slightly
increased in intensity. In this case the deviations from the
typical B-DNA pattern most likely arise from the increased
weight of the spectroscopic properties of the aminobiphenyl
chromophore.
3. Conclusions
We presented here the synthesis of the novel fluorescent 3-
aminobiphenyl-C-nucleoside M. Incorporation of M into
DNA duplexes was efficiently performed by classical phos-
phoramidite chemistry. While single incorporations in both
strands generally destabilized a duplex, triple incorporations
increased its stability to the level of an additional natural base
pair. Efficient fluorescence quenching of the fluorescence
of M is observed in a duplex with the dinitrobiphenyl-
C-nucleoside O in opposite position. Thus the M–O system
constitutes a novel fluorescence-quencher pair that stabilizes
a DNA duplex via interstrand-intercalative stacking interac-
tions and that report presence or absence of a corresponding
target by modulation of the fluorescence signal of M. This
fluorophore/quencher system can be inserted into the center
of DNA duplex without destabilizing it. The signal sensitiv-
ity can be enhanced by adding further M–O pairs to the
duplex. Furthermore, the M–O pair is orthogonal to natural
base pairs in their recognition properties, enabling its use as
highly specific tags with reporting properties.
Figure 4. Fluorescence melting curve of the duplex d(GATGACM3GC-
TAG)$d(CTAGCO3GTCATC). c(duplex)¼1.2 mM in 3.5 mM MgCl2,
50 mM KCl, 10 mM Tris, pH 8.0. Excitation wavelength: 237 nm; emission
wavelength: 408 nm.
fluorescence intensity at higher temperature was observed.
The emission maximum does not change during melting,
excluding the formation of excimers also in the duplex.
Excimer formation of free aminobiphenyl is not described
in the literature but is known for the unsubstituted
biphenyl.29,30
A duplex with three M–O pairs shows low fluorescence in-
tensity in the paired state and high intensity in the denatured
state. Thus efficient fluorescence quenching of M by O oc-
curs in the duplexed but not in the single stranded state.
The cooperative nature of this change in fluorescence inten-
sity can be seen easily in a corresponding fluorescence melt-
ing curve (Fig. 4). The Tm observed (53 ꢁC) is of the same
order as that determined from UV-melting curves (Table 1).
4. Experimental
4.1. General
All reactions were carried out under argon in glassware,
which had been oven dried prior to use. Solvents for reactions
(CH2Cl2, CH3CN, Et2O, THF, toluene) were dried and puri-
fied by filtration over alumina or purchased as crown cap bot-
tles from Fluka (pyridine). Solvents for extractions and flash
chromatography (FC) were distilled before use. 2-Cyano-
ethyldiisopropyl chlorophosphoramidite,31 dimethyldioxir-
2.5. CD spectroscopy
A preliminary investigation on the influence of M-residues
on the structural properties of the oligonucleotide was per-
formed by CD spectroscopy (Fig. 5). Duplexes with one or
three M–M pairs were measured. The spectra of the duplex
with one M-residue in each strand show cotton effects that
ane,32 and TIPDSCl2 were prepared as described. Silica
33
gel thin layer chromatography (TLC) was performed using
SIL G-25 UV254 from Macherey-Nagel. Visualization was
done either by UV light (254 nm) or by staining solutions
(2.1 g cerium(IV) sulfate, 4.2 g phosphomolybdic acid,
12 ml H2SO4, 180 ml H2O or 10 ml p-anisaldehyde, 2 ml
acetic acid, 10 ml H2SO4, 180 ml EtOH). FC was performed
1
with Silica gel 60 (230–400 mesh) from Fluka. H NMR
spectra were recorded at 300 MHz on a Bruker AC-300 or
on a Bruker Avance spectrometer or at 400 MHz on a Bruker
DRX-400 spectrometer. Chemical shifts are reported in parts
per million using TMS as an internal standard. Coupling
constants J are in hertz. Multiplicities are abbreviated as
follows: s¼singlet, d¼doublet, t¼triplet, m¼multiplet. 13C
NMR spectra were recorded at 75 MHz on a Bruker AC-300
or on a Bruker Avance spectrometer or at 100 MHz on a
Bruker DRX-400 spectrometer. Chemical shifts are reported
in parts per million using TMS as an internal standard.
Multiplicities are established by DEPT experiments and are
abbreviated as follows: s¼singlet, d¼doublet, t¼triplet,
Figure 5. CD spectra of duplex with one M–M pair (red line) and duplex
with three M–M pairs (black line). c(duplex)¼3.6 mM in 10 mM NaH2PO4,
150 mM NaCl, pH 7.0, T¼20 ꢁC.

M. Stoop et al. / Tetrahedron 63 (2007) 3440–3449
3445
1
1
q¼quadruplet. H/1H and H/13C correlation experiments
(COSY, HMSC) were used for signal assignments. 31P
NMR spectra were recorded at 121 MHz on a Bruker Avance
spectrometer using 85% H3PO4 as an external standard. Dif-
(2d); 120.68 (s); 119.55 (d); 117.58 (d); 80.41 (s); 75.30
(d); 68.23 (d); 62.05, 46.03 (2t); 28.34 (q); 17.54, 17.39,
17.34, 17.31, 17.23, 17.13 (6q); 13.38, 12.89, 12.69, 12.60
(4d). HR-ESI+-MS (C34H53NO7Si2Na, [M+Na]+): calcd:
666.3258; found: 666.3243. IR (neat, cmꢀ1): 2944m,
2867m, 1730m, 1651m, 1612m, 1561m, 1524m, 1495m,
1454m, 1414m, 1367m, 1231m, 1150s, 1084m, 1047s,
1026s, 991m, 958m, 884m, 862m, 759m, 694s.
1
ference H-NOE experiments were recorded on a Bruker
DRX-400 spectrometer. Electron impact (EI) mass spectra
were recorded on a AutoSpeq Q VG with an ionization energy
of 70 eV. Electrospray ionization mass spectra (ESI) were
recorded either on a Fisons Instrument VG Platform (low
resolution) or on an Applied Biosystems, Sciex QSTAR Pulsar
(high resolution). IR-spectra were recorded either on a Jasco
FT/IR-460plus or on a Perkin–Elmer Spectrum One. The
absorption bands n are indicated in cmꢀ1. The intensities
are abbreviated as follows: s (strong), m (medium), w (weak).
4.1.3. 1-[30-N-(Butyloxycarbonyl)aminobiphen-40-yl]-
3,5-O-tetraisopropyldisilyloxy-1,4-dihydroxypentan (5).
L-Selectride (1 M in THF, 2.76 ml, 2.76 mmol, 1.5 equiv)
was added dropwise to a stirred solution of 4 (1.18 g,
1.84 mmol) in dry THF (12 ml) at ꢀ78 ꢁC over 10 min.
The solution was stirred for 5 min at this temperature and
then 30 min at rt. The mixture was quenched with satd aq
NaHCO3 (40 ml) and then extracted with CH2Cl2 (3ꢂ
80 ml). The combined organic phases were washed with
satd aq NaHCO3 (50 ml), H2O (50 ml), and brine (50 ml),
dried (MgSO4), and concentrated in vacuo. Purification by
FC (hexane/EtOAc, 10:2) yielded the title compound 5
(906.6 mg, 1.40 mmol, 76%) as a white foam. TLC (hex-
ane/EtOAc, 10:2): Rf¼0.2. 1H NMR (300 MHz, CDCl3,
d (ppm)): 8.59 (s, 1H); 8.33 (s, 1H); 7.63–7.60 (m, 2H);
7.43–7.38 (m, 2H); 7.34–7.31 (m, 1H); 7.23–7.20 (m, 2H);
5.27–5.23 (m, 1H); 4.24 (dd, J1¼11.7, J2¼0.9, 1H); 4.12–
4.07 (m, 1H); 3.91 (dd, J1¼11.8, J2¼2.0, 1H); 3.79 (d,
J¼9.61, 1H); 2.49–2.39 (m, 1H); 2.22–2.15 (m, 1H); 1.53
(s, 9H); 1.18–1.03 (m, 28H). 13C NMR (75 MHz, CDCl3,
d (ppm)): 153.23 (s); 141.24, 140.85, 137.77, 129.59 (4s);
128.61, 127.59, 127.24, 121.43, 119.45 (5d); 79.92 (s);
72.49, 72.05, 70.42 (3d); 62.47, 39.03 (2t); 28.43 (q); 17.69,
17.55, 17.32, 17.27, 17.26, 17.21 (6q); 13.35, 13.31, 12.65,
12.55 (4d). HR-ESI+-MS (C34H55NO7Si2Na, [M+Na]+):
calcd: 668.3414; found: 668.3421. IR (neat, cmꢀ1):
3336w, 2944w, 2867w, 2359w, 1727m, 1587w, 1570w,
1532w, 1494w, 1464m, 1415m, 1389w, 1366m, 1241m,
1159m, 1048s, 1026s, 918w, 885m, 826m, 761m, 695s.
4.1.1. 3-N-(tert-Butyloxycarbonyl)aminobiphenyl (3). A
solution of 3-aminobiphenyl (2)21 (3.38 g, 20.0 mmol) and
di-tert-butyldicarbonate (4.80 g, 22.0 mmol, 1.1 equiv) in
dry THF (50 ml) was stirred at rt for 15 h. The mixture
was adsorbed on silica gel and purified by FC (hexane/
EtOAc, 9:1). The resulting oil was crystallized from hexane
to yield 3 as white needles (3.75 g, 13.9 mmol, 70%). TLC
(hexane/EtOAc, 9:1): Rf¼0.3. 1H NMR (300 MHz,
DMSO-d6, d (ppm)): 9.43 (s); 7.79 (s, 1H); 7.60–7.57 (m,
2H); 7.49–7.42 (m, 3H); 7.39–7.31 (m, 2H); 7.24 (m, 1H);
1.49 (s, 9H). 13C NMR (75 MHz, DMSO-d6, d (ppm)):
152.83 (s); 140.72 (s); 140.33 (s); 140.08 (s); 129.20 (d);
128.91 (2d); 127.46 (d); 126.60 (2d); 120.47 (d); 117.19
(d); 116.38 (d); 79.12 (s); 28.13 (q). EIMS: 269 (19, M+),
213 (63), 169 (100), 57 (87). HR-EI-MS (C17H19NO2):
calcd: 269.141579; found: 269.141480. IR (KBr, cmꢀ1):
3348s, 3055w, 2993m, 2980m, 2934w, 1690s, 1610m,
1600m, 1578w, 1539s, 1500m, 1481s, 1450m, 1405s,
1393m, 1375m, 1366m, 1314m, 1291m, 1278m, 1243s,
1183m, 1152s, 1091m, 1072m, 1047m, 1020m, 996w, 885m,
851m, 800w, 789m, 779w, 761s, 713m, 697m, 662m, 621w,
605w, 521w, 469w.
4.1.2. 1-[30-N-(Butyloxycarbonyl)aminobiphen-40-yl]-
3,5-O-tetraisopropyldisilyloxy-4-hydroxypentan-1-one
(4). t-BuLi (1.7 M in pentane, 5.18 ml, 8.8 mmol, 2.2 equiv)
was added to a stirred solution of 3 (1.08 g, 4.00 mmol) in
dry THF (10 ml) at ꢀ78 ꢁC. The solution was stirred for 1 h
before it was allowed to react at ꢀ20 ꢁC for another 2 h. The
reaction mixture was cooled again to ꢀ78 ꢁC and a solution
of lactone 122 (884.1 mg, 2.36 mmol, 0.59 equiv) in dry THF
(10 ml) was added. After stirring for 1.5 h at this temperature
the solution was quenched with satd aq NH4Cl (50 ml) and
then extracted with TBME (3ꢂ80 ml). The combined or-
ganic phases were washed with satd aq NH4Cl (40 ml),
H2O (40 ml), and brine (40 ml), dried (MgSO4), and concen-
trated in vacuo. Purification by FC (hexane/EtOAc, 20:1/
12:1/10:1) yielded the title compound 4 (520.8 mg, 34%)
4.1.4. 3-N-(Butyloxycarbonyl)amino-4-(20-deoxy-30,50-O-
tetraisopropyldisilyloxy-b-D-ribofuranosyl)biphenyl (6).
DIAD (678 ml, 3.50 mmol, 2.5 equiv) was added dropwise
to a stirred solution of 5 (903 mg, 1.40 mmol) and PPh3
(918 mg, 3.50 mmol, 2.5 equiv) in dry THF (25 ml) at
0 ꢁC. This solution was stirred for 3 h at this temperature.
A 0.5 M solution of I2 in CH2Cl2 was added dropwise until
the iodine coloration just persisted. After concentration un-
der reduced pressure, the residue was taken up in satd aq
NaHCO3 (40 ml). The mixture was extracted with CH2Cl2
(3ꢂ100 ml). The combined organic phases were washed
with satd aq NaHCO3 (50 ml), dried (MgSO4), and concen-
trated in vacuo. Purification by FC (hexane/EtOAc, 100:1/
9:1) yielded the title compound 6 (585.5 mg, 0.93 mmol,
67%, b/aw5:1) as a white foam. TLC (2% EtOAc in hexane):
Rf¼0.1. 1H NMR (300 MHz, CDCl3, d (ppm)): 8.25 (s, 1H);
8.01 (s, 1H); 7.63–7.60 (m, 2H); 7.43–7.38 (m, 2H); 7.35–
7.29 (m, 1H); 7.24–7.22 (m, 2H); 5.16 (dd, J1¼9.8,
J2¼6.1, 1H); 4.59–4.54 (m, 1H); 4.19 (dd, J1¼11.1, J2¼3.6,
1H); 4.04–3.98 (m, 1H); 3.84 (dd, J1¼10.9, J2¼9.6, 1H);
2.46–2.35 (m, 1H); 2.28 (ddd, J1¼13.4, J2¼6.1, J3¼2.6,
1H); 1.53 (s, 9H); 1.12–1.02 (m, 28H). 1H NMR-
NOE (400 MHz, CDCl3, d (ppm)): 5.16 (H-C(10))/4.01
(H-C(40), 6%), 2.28 (Ha-C(20), 4%); 4.57 (H-C(30))/4.01
1
as a yellow foam. TLC (hexane/EtOAc, 9:1): Rf¼0.2. H
NMR (300 MHz, CDCl3, d (ppm)): 10.95 (s); 8.76 (d,
J¼1.9, 1H); 8.00 (d, J¼8.3, 1H); 7.69–7.66 (m, 2H); 7.48–
7.36 (m, 3H); 7.26 (dd, J1¼8.3, J2¼1.9, 1H); 4.46–4.39 (m,
1H); 4.20 (dd, J1¼11.7, J2¼1.1, 1H); 3.84 (dd, J1¼11.7,
J2¼2.1, 1H); 3.56 (dd, J1¼15.4, J2¼3.6, 1H); 3.46–3.39
(m, 1H); 3.23 (dd, J1¼15.5, J2¼7.1, 1H); 2.46 (d, J¼9.21,
1H); 1.55 (s); 1.09–0.96 (m, 28H). 13C NMR (75 MHz,
CDCl3, d (ppm)): 202.58 (s); 153.14 (s); 147.25 (s); 142.19
(s); 139.76 (s); 131.96 (d); 128.82 (2d); 128.39 (d); 127.42

3446
M. Stoop et al. / Tetrahedron 63 (2007) 3440–3449
(H-C(40), 3%), 2.40 (Hb-C(20), 6%). 13C NMR (75 MHz,
CDCl3, d (ppm)): 152.94 (s); 141.82, 140.67, 137.69 (3s);
128.64, 127.85, 127.37, 127.24, 121.46 (5d); 87.43 (d);
80.13 (s); 79.79, 74.87 (2d); 64.60, 41.03 (2t); 28.39 (q);
17.59, 17.48, 17.43, 17.40, 17.30, 17.12, 17.06, 17.00 (8q);
13.50, 13.44, 13.07, 12.62 (4d). HR-ESI+-MS (C34H53NO6-
Si2Na, [M+Na]+): calcd: 650.3309; found: 650.3300. IR
(neat, cmꢀ1): 3357w, 2944m, 2867m, 2360m, 2341w,
1731m, 1587w, 1570m, 1532w, 1494w, 1464m, 1417m,
1389w, 1366m, 1242m, 1156s, 1085m, 1032s, 955m, 919w,
884m, 824m, 759m, 693s, 637m.
125.14, 124.99, 120.08, 120.05, 120.01 (12d); 87.82, 83.70,
74.42 (3d); 66.84, 64.34 (2t); 47.21 (d); 40.96 (t); 17.63,
17.48, 17.45, 17.40, 17.32, 17.13, 17.09, 17.00 (8q); 13.57,
13.51, 13.15, 12.57 (4d). HR-ESI+-MS (C44H56NO6Si2,
[M+H]+): calcd: 750.3646; found: 750.3656. IR (neat,
cmꢀ1): 3346w, 2943m, 2866m, 2359w, 1736m, 1587w,
1570m, 1536m, 1496w, 1450m, 1417m, 1243m, 1206s,
1135m, 1084m, 1033s, 884m, 824w, 758s, 737s, 693s,
662m, 651m, 642m, 610m.
4.1.7. 3-N-(9-Fluorenylmethoxycarbonyl)amino-4-(20-
deoxy-b-D-ribofuranosyl)biphenyl (9). To a mixture of 8
(129 mg, 0.17 mmol) in dry THF (8.5 ml) was added drop-
wise (HF)3$NEt3 (277 ml, 1.72 mmol, 10 equiv). After stir-
ring for 14 h at rt, the solution was concentrated in vacuo.
Purification by FC (2% MeOH in CH2Cl2) yielded the title
compound 9 (53.9 mg, 0.11 mmol, 63%, b only) as a color-
4.1.5. 3-Amino-4-(20-deoxy-30,50-O-tetraisopropyldisilyl-
oxy-b-D-ribofuranosyl)biphenyl (7). SnCl4 (435 ml,
3.72 mmol, 4 equiv) was added dropwise to a stirred solu-
tion of 6 (585 mg, 0.93 mmol) in dry EtOAc (14 ml) at rt.
This solution was stirred for 3 h at this temperature. The
mixture was quenched with satd aq NaHCO3 (40 ml) and
then extracted with TBME (3ꢂ100 ml). The combined or-
ganic phases were washed with satd aq NaHCO3 (50 ml),
H2O (50 ml), and brine (50 ml), dried over MgSO4, and
concentrated in vacuo. Purification by FC (hexane/EtOAc,
10:1, 1% Et3N) yielded the title compound 7 (483.2 mg,
0.92 mmol, 98%, b/aw4:1) as a slightly yellow oil. TLC
(hexane/EtOAc, 10:1): Rf¼0.2. 1H NMR (300 MHz,
CDCl3, d (ppm)): 7.56–7.53 (m, 2H); 7.42–7.38 (m, 2H);
7.34–7.29 (m, 1H); 7.16 (d, J¼7.92, 1H); 6.98–6.90 (m,
2H); 5.15 (dd, J1¼8.7, J2¼7.0, 1H); 4.64–4.58 (m, 1H);
4.40 (s, 2H, amine); 4.12 (dd, J1¼10.5, J2¼2.4,1H); 3.93–
3.83 (m, 2H); 2.57–2.41 (m, 1H); 2.29 (ddd, J1¼12.8,
J2¼7.0, J3¼4.1, 1H); 1.13–0.96 (m, 28H). 13C NMR
(75 MHz, CDCl3, d (ppm)): 141.87, 141.07 (2s); 128.62,
127.96, 127.21, 127.03 (4d); 123.71 (s); 117.23, 115.31
(2d); 86.54, 78.86, 73.32 (3d); 63.45, 39.40 (2t); 17.61,
17.49, 17.46, 17.40, 17.27, 17.12, 17.10, 17.00 (8q); 13.53,
13.37, 13.09, 12.58 (4d). HR-ESI+-MS (C29H46NO4Si2,
[M+H]+): calcd: 528.2965; found: 528.2965. IR (neat,
cmꢀ1): 3453w, 3361w, 2943m, 2866m, 2361w, 1620w,
1565w, 1487w, 1463m, 1419w, 1385w, 1317w, 1285w,
1246w, 1180w, 1117m, 1082m, 1033s, 957m, 918m, 883m,
758s, 693s, 637m.
1
less foam. TLC (5% MeOH in CH2Cl2): Rf¼0.5. H NMR
(300 MHz, CDCl3, d (ppm)): 8.62 (s, 1H); 8.28 (s, 1H);
7.77 (d, J¼7.3, 2H); 7.62–7.57 (m, 4H); 7.42–7.29 (m,
7H); 7.20 (dd, J1¼7.9, J2¼1.7, 1H); 7.11 (d, J¼8.1, 1H);
5.13 (dd, J1¼11.1, J2¼5.5, 1H); 4.72 (d, J¼5.1, 2H); 4.29
(s, 1H); 4.25 (t, J¼5.1, 1H); 3.91 (d, J¼2.6, 1H); 3.49 (s,
1
2H); 2.12 (m, 1H); 1.97 (dd, J1¼13.1, J2¼5.4, 1H). H
NMR-NOE (400 MHz, CDCl3, d (ppm)): 5.13 (H-C(10))/
3.91 (H-C(40), 4%), 1.97 (Ha-C(20), 5%); 4.29 (H-C(30))/
3.91 (H-C(40), 2%), 2.12 (Hb-C(20), 5%). 13C NMR
(100 MHz, CDCl3, d (ppm)): 143.86, 143.77, 141.87,
141.43, 141.25, 140.48, 137.39 (7s); 128.84, 128.69,
127.84, 127.76, 127.46, 127.42, 127.36, 127.18, 124.74,
124.71, 119.96, 119.91 (12d); 87.51, 81.42, 73.37 (3d);
65.28, 62.41 (2t); 47.36 (d); 41.92 (t). HR-ESI+-MS
(C32H29NO5Na, [M+Na]+): calcd: 530.1943; found:
530.1948. IR (neat, cmꢀ1): 3322w, 2360w, 1701w, 1568w,
1536w, 1449w, 1415m, 1208m, 1044m, 945w, 907m,
759m, 731s, 696m.
4.1.8. 3-N-(9-Fluorenylmethoxycarbonyl)amino-4-[(50-
O-4,40-dimethoxytriphenyl)methyl-20-deoxy-b-D-ribo-
furanosyl]biphenyl (10). Diol 9 (144 mg, 0.28 mmol) was
coevaporated with pyridine (3ꢂ2 ml) and then dissolved in
pyridine (1.2 ml). DMTrCl was added in portions over 2 h
(4ꢂ28.8 mg; totally 0.34 mmol, 1.2 equiv). After stirring
for another 90 min at rt, the mixture was diluted with EtOAc
(100 ml) and washed with satd aq NaHCO3 solution (3ꢂ
25 ml). The combined aqueous phases were extracted with
EtOAc (3ꢂ25 ml). The combined organic phases were dried
(MgSO4) and concentrated in vacuo. The residue was co-
evaporated with toluene (3ꢂ4 ml). Purification by FC
(equilibration with hexane+1% Et3N, then hexane; elution
with hexane/EtOAc, 7:3) yielded the title compound 10
(168.0 mg, 0.21 mmol, 74%) as a colorless foam. TLC (hex-
ane/EtOAc, 7:3): Rf¼0.2. 1H NMR (300 MHz, CDCl3,
d (ppm)): 8.51 (s, 1H); 8.31 (s, 1H); 7.76 (d, J¼7.5, 2H);
7.64–7.61 (m, 2H); 7.58–7.53 (m, 2H); 7.44–7.27 (m,
11H); 7.25–7.12 (m, 7H); 6.72–6.68 (m, 4H); 5.34 (dd,
J1¼10.4, J2¼5.7, 1H); 4.46–4.42 (m, 1H); 4.40–4.29 (m,
2H); 4.20–4.16 (m, 2H); 3.68 (d, J¼2.8, 6H); 3.42 (dd,
J1¼9.8, J2¼5.1, 1H); 3.27 (dd, J1¼10.0, J2¼5.7, 1H);
2.45–2.35 (m, 1H); 2.32–2.25 (m, 1H); 1.87 (s, 1H). 13C
NMR (75 MHz, CDCl3, d (ppm)): 158.49, 153.68, 144.73,
143.93, 143.84, 141.83, 141.30, 140.45, 137.55, 135.80,
135.78 (11s); 130.05, 128.72, 128.14, 127.82, 127.73,
4.1.6. 3-N-(9-Fluorenylmethoxycarbonyl)amino-4-
(20-deoxy-30,50-O-tetraisopropyldisilyloxy-b-D-ribofura-
nosyl)biphenyl (8). A mixture of 7 (206 mg, 0.39 mmol),
9-fluorenylmethylchloroformate (222 mg, 0.86 mmol,
2.2 equiv), and N-ethyldiisopropylamine (0.147 ml,
0.86 mmol, 2.2 equiv) in dry CH2Cl2 (2 ml) was stirred for
21 h at rt. The mixture was quenched with H2O (20 ml) and
then extracted with CH2Cl2 (3ꢂ80 ml). The combined or-
ganic phases were washed with H2O (30 ml) and brine
(30 ml), dried (MgSO4), and concentrated in vacuo. Purifica-
tion by FC (2% EtOAc in hexane) yielded the title compound
8 (236.3 mg, 0.31 mmol, 81%, b/aw4:1) as a white foam.
1
TLC (hexane/EtOAc, 10:1): Rf¼0.6. H NMR (300 MHz,
CDCl3, d (ppm)): 8.30 (s, 1H); 7.80–7.77 (m, 2H); 7.62–
7.60 (m, 4H); 7.44–7.22 (m, 10H); 5.21–5.13 (m, 1H);
4.58–4.54 (m, 1H); 4.52–4.45 (m, 2H); 4.31 (t, J¼7.0, 1H);
4.15 (dd, J1¼11.0, J2¼3.9, 1H); 4.08–4.02 (m, 1H); 3.75
(dd, J1¼10.8, J2¼9.9, 1H); 2.38–2.26 (m, 2H); 1.15–0.98
(m, 28H). 13C NMR (100 MHz, CDCl3, d (ppm)): 153.50,
143.87, 143.77, 141.88, 141.39, 141.36, 140.43, 137.27
(8s); 128.71, 127.90, 127.74, 127.50, 127.20, 127.19, 127.10,

M. Stoop et al. / Tetrahedron 63 (2007) 3440–3449
3447
127.51, 127.30, 127.21, 127.13, 126.82, 125.18, 119.98,
113.10, 86.67 (14d); 86.38 (s); 79.00, 74.05 (2d); 67.07,
64.43 (2t); 55.12 (q); 47.06 (d); 40.36 (t). HR-ESI+-MS
(C53H47NO7, [M+Na]+): calcd: 832.3250; found:
832.3228. IR (neat, cmꢀ1): 3328w, 2929w, 2360w, 1732m,
1606m, 1569m, 1535m, 1507m, 1448m, 1415m, 1301m,
1247s, 1212m, 1175m, 1034s, 949m, 910m, 826m, 759m,
738s, 698s, 647m, 635m, 614m.
(d, J1¼3.39, 2H); 3.87–3.82 (m, 1H); 2.80–2.70 (m, 1H);
2.12 (ddd, J1¼12.8, J2¼7.7, J3¼5.1, 1H); 1.12–0.99 (m,
28H). 13C NMR (75 MHz, CDCl3, d (ppm)): 147.32,
141.44, 138.63, 138.56 (4s); 132.13, 129.27, 128.57,
128.41, 127.18, 123.04 (6d); 85.01, 74.63, 70.40 (3d);
61.91, 42.07 (2t); 17.70, 17.61, 17.54, 17.35, 17.22, 17.13,
16.98 (7q); 13.62, 13.37, 13.07, 12.73 (4d). HR-ESI+-MS
(C29H44NO6Si2, [M+H]+): calcd: 558.2707; found:
558.2723. IR (neat, cmꢀ1): 2943m, 2866m, 2361w, 1530m,
1508w, 1464m, 1386w, 1348m, 1245w, 1122m, 1091m,
1065m, 1034s, 957w, 919w, 883s, 851m, 760m, 693s, 607m.
4.1.9. 3-N-(9-Fluorenylmethoxycarbonyl)amino-4-[(50-
O-4,40-dimethoxytriphenyl)methyl-30-(N,N-diisopropyl-
amino-2-cyanoethyl-phosphino)-20-deoxy-b-D-ribofuran-
osyl]biphenyl (11). To a solution of 10 (121 mg, 0.15 mmol)
in dry THF (4 ml), N,N-diisopropylethylamine (76.8 ml,
0.45 mmol, 3 equiv) followed by 2-cyanoethyl-
diisopropylchlorophosphoramidite (50.3 ml, 0.23 mmol,
1.5 equiv) was added at rt. After stirring for 4 h (TLC con-
trol), EtOAc (100 ml) was added and the mixture was
washed with satd aq NaHCO3 solution (3ꢂ25 ml). The com-
bined aqueous phases were extracted with EtOAc
(3ꢂ25 ml). The combined organic phases were dried
(MgSO4) and concentrated in vacuo. Purification by FC
(equilibration with hexane+1% Et3N, then hexane; elution
with hexane/EtOAc, 7:3) yielded the title compound 11
(123.1 mg, 0.12 mmol, 81%) as a white foam. TLC (silica
gel; hexane/EtOAc, 7:3): Rf¼0.5. 1H NMR (300 MHz,
CDCl3, d (ppm)): 8.68 (s, 1H); 8.36 (s, 1H); 7.77 (d,
J¼7.5, 2H); 7.65–7.53 (m, 4H); 7.45–7.29 (m, 12H); 7.23–
7.16 (m, 6H); 6.72–6.67 (m, 4H); 5.39–5.32 (m, 1H); 4.59–
4.41 (m, 1H); 4.35–4.26 (m, 3H); 4.16 (t, J¼7.8, 1H);
3.91–3.63 (m, 10H); 3.35–3.27 (m, 2H); 2.67–2.41 (m,
4H); 1.32–1.12 (m, 12H). 31P NMR (121 MHz, CDCl3,
d (ppm)): 149.15, 148.57. 13C NMR (100 MHz, CDCl3,
d (ppm)): 158.44, 158.43, 153.74, 153.70, 144.75, 143.98,
143.85, 141.83, 141.76, 141.28, 140.50, 140.47, 137.72
(13s); 130.12, 130.08, 128.72, 128.23, 128.19, 127.76,
127.71, 127.50, 127.28, 127.22, 127.11, 126.78, 126.73,
125.21, 121.91, 119.99 (16d); 117.53, 117.45 (2s); 113.06,
113.04, 86.42 (3d); 86.38, 86.26, 86.21 (3s); 86.14, 86.07,
79.22, 78.97, 75.58, 75.40, 75.34, 75.16 (8d); 67.17, 64.30,
64.19, 58.49, 58.38, 58.30, 58.19 (7t); 55.12 (q); 47.03,
43.41, 43.35, 43.29, 43.23 (5d); 39.53 (t); 24.68, 24.60,
24.57, 24.49 (4q); 20.45, 20.38, 20.26, 20.19 (4t). ESI+-
MS: 1010.4 ([M+H]+). IR (KBr, cmꢀ1): 3435m, 3066w,
2968m, 2932m, 2838w, 2254w, 1735m, 1609m, 1588m,
1571m, 1539m, 1510s, 1479m, 1464m, 1452m, 1416m,
1397m, 1365m, 1303m, 1251s, 1212s, 1180s, 1157m,
1127m, 1077s, 1036s, 1002m, 979m, 891m, 829m, 791w,
761m, 742m, 727m, 700m, 585m, 544w.
4.1.11. 3-Nitro-4-(20-deoxy-b-D-ribofuranosyl)biphenyl
(13). To a mixture of 12 (393 mg, 0.70 mmol) in dry THF
(35.5 ml) was added dropwise (HF)3$NEt3 (1.16 ml,
7.04 mmol, 10 equiv). After stirring for 17 h at rt, the solu-
tion was concentrated in vacuo. Purification by FC (silica
gel; 2% MeOH in CH2Cl2) and recrystallization from
CH2Cl2/hexane yielded the title compound 13 (142.6 mg,
0.45 mmol, 64%, b only) as a white solid. TLC (5%
1
MeOH in CH2Cl2): Rf¼0.3. H NMR (300 MHz, CDCl3,
d (ppm)): 8.19 (s, 1H); 7.89–7.83 (m, 2H); 7.62–7.60 (m,
2H); 7.52–7.42 (m, 3H); 5.70 (dd, J1¼9.4, J2¼6.2, 1H);
4.48–4.44 (m, 1H); 4.07–4.04 (m, 1H); 3.97–3.84 (m, 2H);
2.64 (ddd, J1¼13.3, J2¼6.0, J3¼2.5); 2.08–1.98 (m, 1H).
1H NMR-NOE (400 MHz, MeOD, d (ppm)): 5.59 (H-
C(10))/3.97 (H-C(40), 3%), 2.54 (Ha-C(20), 5%); 4.33 (H-
C(30))/3.97 (H-C(40), 3%), 1.93 (Hb-C(20), 6%). 13C
NMR (75 MHz, CDCl3, d (ppm)): 148.02, 41.62, 138.29,
136.29 (4s, C(4)); 131.88, 129.18 (2d); 128.53, 127.90,
127.04, 122.83, 86.79, 76.10, 73.35 (7d, C(300)); 63.35,
43.90 (2t). HR-ESI+-MS (C17H17NO5, [M+Na]+): calcd:
338.1004; found: 338.1005. IR (neat, cmꢀ1): 3222w,
2360w, 1531m, 1340s, 1090m, 1052s, 998m, 954m, 893w,
850w, 753s, 697m, 680m, 620w.
4.1.12. 3-Nitro-4-[(50-O-4,40-dimethoxytriphenyl)methyl-
20-deoxy-b-D-ribofuranosyl]biphenyl (14). Diol 13
(162 mg, 0.51 mmol) was coevaporated with pyridine
(3ꢂ4 ml) and then dissolved in pyridine (2.2 ml). DMTrCl
was added in portions over 2 h (4ꢂ52.6 mg; totally
0.62 mmol, 1.2 equiv). After stirring another 90 min at rt,
the mixture was diluted with EtOAc (100 ml) and washed
with satd aq NaHCO3 solution (3ꢂ25 ml). The combined
aqueous phases were extracted with EtOAc (3ꢂ25 ml).
The combined organic phases were dried (MgSO4) and
concentrated in vacuo. The residue was coevaporated with
toluene (3ꢂ4 ml). Purification by FC (hexane/EtOAc, 8:2,
1% Et3N) yielded the title compound 14 (284.7 mg,
0.46 mmol, 90%) as a yellow foam. TLC (silica gel; hex-
ane/EtOAc, 7:3): Rf¼0.4. 1H NMR (300 MHz, CDCl3,
d (ppm)): 8.21 (d, J¼1.9, 1H); 8.03 (d, J¼8.3, 1H); 7.75
(dd, J1¼8.1, J2¼1.9, 1H); 7.62–7.58 (m, 2H); 7.51–7.46
(m, 4H); 7.44–7.40 (m, 1H); 7.39–7.35 (m, 4H); 7.33–7.27
(m, 2H); 7.25–7.19 (m, 1H); 6.87–6.82 (m, 4H); 5.71 (dd,
J1¼8.8, J2¼6.3, 1H); 4.46–4.41 (m, 1H); 4.10–4.07 (m,
1H); 3.79 (s, 6H); 3.43 (d, J¼4.5, 2H); 2.64 (ddd,
J1¼13.2, J2¼6.4, J3¼3.4, 1H); 2.11–2.01 (m, 1H); 1.78 (s,
1H). 13C NMR (75 MHz, CDCl3, d (ppm)): 158.58, 147.81,
144.79, 141.36, 138.39, 137.37, 135.97 (7s); 131.91, 130.15,
130.13, 129.14, 128.45, 128.42, 128.23, 127.90, 127.03,
126.90, 122.71, 113.21 (12d); 86.43 (s); 85.72, 75.89,
73.90 (3d); 63.96 (t); 55.23 (q); 43.49 (t). HR-ESI+-MS
4.1.10. 3-Nitro-4-(20-deoxy-30,50-O-tetraisopropyldisilyl-
oxy-b-D-ribofuranosyl)biphenyl (12). To a solution of 7
(240 mg, 0.45 mmol) in acetone (9.5 ml) was added dime-
thyldioxirane (0.085 M in acetone, 44 ml, 3.7 mmol,
8.2 equiv). After stirring for 3 h at rt in the dark, the solution
was concentrated in vacuo and then coevaporated with tolu-
ene (3ꢂ4 ml). Purification by FC (2% EtOAc in hexane)
yielded the title compound 12 (156.6 mg, 0.28 mmol, 62%
a:bw10:1) as a yellow oil. TLC (2% EtOAc in hexane):
1
Rf¼0.3. H NMR (300 MHz, CDCl3, d (ppm)): 8.26 (d,
J¼1.9, 1H); 8.05 (d, J¼8.3, 1H); 7.83 (dd, J1¼8.1, J2¼1.9,
1H); 7.60–7.59 (m, 2H); 7.51–7.41 (m, 3H); 5.66 (dd,
J1¼8.6, J2¼5.0, 1H); 4.47 (dd, J1¼15.3, J2¼7.7, 1H); 4.12

3448
M. Stoop et al. / Tetrahedron 63 (2007) 3440–3449
(C38H35NO7, [M+Na]+): calcd: 640.2311; found: 640.2315.
IR (neat, cmꢀ1): 2931w, 2360m, 1733w, 1606w, 1528m,
1506s, 1443m, 1349m, 1299m, 1246s, 1174m, 1031s, 826s,
754s, 724m, 697s, 680s, 647m, 624m, 612s.
Table 2. Sequence and MS data for the oligodeoxynucleotides carrying the
biphenyl-C-nucleotides M and N
X,Y n m/z calcd. m/z found Yield (O.D.260
)
d(GATGACXnGCTAG) M 1 3727.4
3727.6
4422.2
3757.6
13.6
17.8
3.5
M
N
3 4421.9
1 3757.5
4.1.13. 3-Nitro-4-[(50-O-4,40-dimethoxytriphenyl)methyl-
30-(N,N-diisopropylamino-2-cyanoethyl-phosphino)-20-
deoxy-b-D-ribofuranosyl]biphenyl (15). To a solution of
14 (285 mg, 0.46 mmol) in dry THF (12 ml) was added at
rt N,N-diisopropylethylamine (236 ml, 1.38 mmol, 3 equiv)
followed by 2-cyanoethyldiisopropyl chlorophosphorami-
dite (151 ml, 0.69 mmol, 1.5 equiv). After stirring for 4 h
(TLC control), EtOAc (100 ml) was added and the mixture
was washed with satd aq NaHCO3 solution (3ꢂ25 ml).
The combined aqueous phases were extracted with EtOAc
(3ꢂ25 ml). The combined organic phases were dried
(MgSO4) and concentrated in vacuo. Purification by FC
(hexane/EtOAc, 8:2, 1% Et3N) yielded the title compound
15 (300.0 mg, 0.37 mmol, 80%) as a white foam. TLC (hex-
ane/EtOAc, 8:2): Rf¼0.3. 1H NMR (300 MHz, CDCl3,
d (ppm)): 8.22–8.21 (m, 1H); 8.08 (dd, J1¼12.4, J2¼8.2,
1H); 7.75 (dt, J1¼8.2, J2¼2.2, 1H); 7.61–7.59 (m, 2H);
7.51–7.46 (m, 4H); 7.43–7.35 (m, 5H); 7.32–7.28 (m, 2H);
7.24–7.21 (m, 1H); 6.86–6.81 (m, 4H); 5.70 (dd, J1¼9.3,
J2¼5.7, 1H); 4.59–4.49 (m, 1H); 4.24–4.23 (m, 1H); 3.90–
3.83 (m, 1H); 3.79, 3.78 (2s, 6H); 3.68–3.55 (m, 3H);
3.50–3.34 (m, 2H); 2.83–2.71 (m, 1H); 2.66 (t, J¼6.3, 1H);
2.44 (t, J¼6.5, 1H); 2.08–1.97 (m, 1H); 1.19–1.07 (m,
12H). 31P NMR (121 MHz, CDCl3, d (ppm)): 148.90,
147.98. 13C NMR (100 MHz, CDCl3, d (ppm)): 158.54,
147.92, 147.87, 144.83, 144.78, 141.35, 141.34, 138.39,
137.32, 137.12, 136.03, 135.98 (12s); 131.98, 131.95,
130.24, 130.21, 130.17, 130.15, 129.14, 128.52, 128.44,
128.35, 128.27, 127.84, 127.03, 126.87, 126.83, 122.73,
122.67 (17d); 117.68, 117.47 (2s); 113.13 (d); 86.27 (s);
85.47, 85.44 (2d); 85.42 (s); 76.25, 76.16, 75.43, 75.27,
75.12, 74.95 (6d); 63.77, 63.45, 58.56, 58.38, 58.34, 58.15
(6t); 55.24, 55.22 (2q); 43.42, 43.29, 43.16, 42.88, 42.84
(5d); 42.69, 42.65 (2t); 24.67, 24.63, 24.60, 24.57, 24.53,
24.50, 24.45 (7q); 20.47, 20.41, 20.21, 20.14 (4t). ESI+-
MS: 818.4 ([M+H]+). IR (KBr, cmꢀ1): 3439m, 3060w,
3036w, 2968m, 2932m, 2874m, 2838w, 2254w, 2051w,
1609m, 1584w, 1532s, 1510s, 1464m, 1447m, 1397w,
1365m, 1302m, 1252s, 1202m, 1180s, 1157m, 1084m,
1035s, 979m, 899m, 880m, 829m, 791m, 758m, 727m,
700m, 641w, 585m, 523w.
d(CTACTGYnCGATC)
M
M
N
1 3638.4
3 4333.1
1 3668.6
3638.5
4333.2
3668.5
12.0
11.4
2.3
a 15RPC ST 4.6/100 column from Pharmacia Biotech with
a gradient of B: 0.1 M triethylammonium acetate in H2O/
CH3CN 1:8, pH 7 in A: 0.1 M triethylammonium acetate in
H2O, pH 7. The flow rate was set to 1 ml/min. The detection
wavelength was 260 and 280 nm, respectively. All oligo-
deoxynucleotides containing the novel modifications M
and N were analyzed by ESIMS spectrometry (Table 2).
4.3. CD spectroscopy
Circular dichroism spectra were recorded on a Jasco J-715
spectropolarimeter equipped with a Jasco PFO-350S tem-
perature controller.
4.4. Tm Measurements
UV-melting curves were measured on a Varian Cary 100 bio
UV–vis spectrophotometer. Absorbances of duplexes in a
1:1 stoichiometric ratio were monitored at 260 nm and the
heating rate was set to 0.5 ꢁC/min. Extinction coefficients
for the novel C-nucleosides at 260 nm (3260 nm) were deter-
mined to be 11,800 Mꢀ1 cmꢀ1 for M and 21,900 Mꢀ1 cmꢀ1
for O. A heating–cooling–heating cycle in the temperature
range 10–90 ꢁC was applied. The first derivative of the melt-
ing curves was obtained using MicroCal Origin 5.0 soft-
ware. Deviations of Tm values from repeated runs were
found to be less than 0.5 ꢁC. To avoid evaporation of the
solution, dimethylpolysiloxane was layered over the sample
solution in the UV cuvette.
4.5. Fluorescence measurements
Fluorescence spectra were measured on a Varian Cary
Eclipse fluorescence spectrometer. The emission spectra
were recorded at a scan speed of 600 nm per minute with
a photomultiplier voltage of 700 V. The excitation and emis-
sion slits were set to 5 nm. The temperature-dependent mea-
surements were recorded from 20 to 80 ꢁC with a heating rate
of 0.5 ꢁC/min. After reaching the maximum temperature, the
samples were cooled and another heating cycle was started
(totally three cycles). Toavoid evaporation of the solution, di-
methylpolysiloxane was layered over the samples in the cell.
4.2. Oligodeoxyonucleotide synthesis
Oligonucleotides were synthesized on the 1.0 mmol scale on
a Expedite Nucleic Acid Synthesizer (8909) from Applied
Biosystems using standard phosphoramidite chemistry. The
phosphoramidites of the natural nucleosides were from
Glen Research. The solvents and reagents used for the syn-
thesis were prepared according to the manufacturer’s proto-
cols. 5-(Ethylthio)-1H-tetrazole (0.25 M in CH3CN) was
used as a coupling reagent and 3% dichloroacetic acid in
CH3CN was used for the detritylation step. After synthesis,
the oligonucleotides were detached and deprotected in concd
aq NH3 (55 ꢁC, 15 h) and filtered through Titan HPLC-filters
(Teflon, 0.45 mm) from Infochroma AG. All oligodeoxy-
Acknowledgements
Financial support from the Swiss National Science Founda-
tion (Grant no. 200020-107692) is gratefully acknowledged.
References and notes
¨
nucleotides were purified by RP-HPLC on an Akta Basic
10/100 system from Amersham Pharmacia Biotech using
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