α- and β-Stilbenosides as base-pair surrogates in DNA hairpins

Article abstract of DOI:10.1039/b513694f

The synthesis, structure, and optical spectroscopy of hairpin oligonucleotide conjugates possessing synthetic stilbene C-nucleosides (stilbenosides) are reported. Synthetic methods for selective preparation of both the α- and β-stilbenosides have been dev

Full text of DOI:10.1039/b513694f

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www.rsc.org/obc | Organic & Biomolecular Chemistry
a- and b-Stilbenosides as base-pair surrogates in DNA hairpins†
Ligang Zhang,a Hai Long,a Grant E. Boldt,b Kim D. Janda,*b George C. Schatza and Frederick D. Lewis*a
Received 6th October 2005, Accepted 10th November 2005
First published as an Advance Article on the web 8th December 2005
DOI: 10.1039/b513694f
The synthesis, structure, and optical spectroscopy of hairpin oligonucleotide conjugates possessing
synthetic stilbene C-nucleosides (stilbenosides) are reported. Synthetic methods for selective
preparation of both the a- and b-stilbenosides have been developed. Both anomers are effective in
stabilizing hairpin structures when used as capping groups at the open end of the hairpin base-pair
domain. However, only the b-anomer effectively stabilizes the hairpin structure when located in the
interior of the base-pair domain opposite an abasic site. Similar results are obtained for hairpins
possessing two stilbenosides, either adjacent to each other or with one intervening base-pair. Molecular
dynamics simulations are employed to obtain averaged structures for these conjugates. The calculated
structures for the capped hairpins formed with either anomer show effective p-stacking with the
adjacent base-pair. The calculated structures for the internal stilbenosides show that the a- and
b-anomers form extrahelical and intrahelical structures, respectively. The relative orientations of the
two stilbenes in the bis-stilbenosides have been studied using a combination of exciton-coupled circular
dichroism spectroscopy and molecular modeling.
Introduction
Aromatic p–p interactions are an important contributing factor to
the stabilization of duplex DNA.1 The aromatic stacking abilities
of synthetic b-nucleosides possessing non-natural aromatic bases
have been extensively investigated by Kool and co-workers.2 They
report that the stability of self-complementary duplexes possessing
dangling synthetic b-nucleosides increases as the surface area of
the adjacent base-pair covered by the arene increases (benzene <
naphthalene < phenanthrene < pyrene).3 These workers also
investigated the thermal stability of duplexes possessing the b-
pyrenoside b-Py opposite an abasic site X (Chart 1) within the
interior of a 12-mer duplex.4,5 These duplexes proved to be slightly
more stable than a reference duplex lacking this base-pair, but less
stable than a duplex possessing an A:T base-pair in place of the
b-Py–X base-pair surrogate.
The dimensions of the conjugated aromatic trans-stilbene
Chart 1
provide an excellent match for those of the purine–pyrimidine
but not in the interior. Synthetic nucleosides can be positioned
base-pairs. Thus stilbene nucleosides (stilbenosides) might serve as
either at the end or within the interior of the base-pair domain.
effective base-pair surrogates and also serve as intrinsic fluorescent
The synthesis of the a-anomer of a stilbene C-nucleoside linked at
probes of the duplex interior. We and others have reported that the
the stilbene para position has been reported by Stra¨ssler et al.;11
stilbenedicarboxamide Sa (Chart 1) and other stilbene derivatives
however it has not been incorporated into an oligonucleotide
can serve as capping groups for duplexes and hairpins and as
conjugate. Neither has a comparative study of aromatic a- vs.
hairpin linkers in a variety of oligonucleotide conjugates.6–10 In
b-C-nucleosides been reported.
the conjugates investigated to date, the stilbene is attached to the
Molecular modeling of the isomeric stilbenosides suggested that
oligonucleotide by means of short, flexible tethers. This permits the
the meta isomers might provide more complete coverage of the
stilbene to be positioned at the end of a duplex base-pair domain,
adjacent base-pairs without deformation of the sugar–phosphate
backbone of B-DNA than would the para isomers. Thus we have
undertaken the synthesis of the anomeric stilbenosides a-St and b-
aDepartment of Chemistry, Northwestern University, Evanston, IL, 60201.
E-mail: lewis@chem.northwestern.edu
bDepartments of Chemistry and Immunology and The Skaggs Institute for
Chemical Biology, The Scripps Research Institute, 10550, N. Torrey Pines
Road, La Jolla, CA, 92037. E-mail: kdjanda@scripps.edu
† Electronic supplementary information (ESI) available: HPLC and
MALDI TOF mass spectra for conjugates. Averaged truncated structures
for 8b and 9b. Partial atomic charges and atom types for the stilbenosides
used in MD simulations. See DOI: 10.1039/b513694f
St (Chart 1) and several hairpin-forming conjugates in which one
or two stilbenosides are located either at the end or opposite an
abasic site within the interior of a short base-pair duplex domain
(Chart 2). We report here the results of our investigation of the
synthesis, structure, and optical spectroscopy of these conjugates.
Both stilbenoside anomers can function as capping groups but
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Scheme 2 Reagents and conditions: (a) m-bromostilbene, n-BuLi, THF
−78 ◦C, 42%; (b) TBAF, THF, 79%; (c) DMT-Cl, py, DIPEA, 88%.
by reaction with 2-cyanoethyl diisopropylchlorophosphoramidite
prior to conjugate syntheses. The preparation of trans-N,Nꢀ-bis(3-
hydroxypropyl)stilbene-4,4ꢀ-dicarboxamide (Sa) and conversion
to its monoprotected, mono-activated diol by sequential reaction
with 4,4ꢀ-dimethoxytrityl chloride and with 2-cyanoethyl diiso-
propylchlorophosphoramidite have been described previously.6
Oligonucleotide conjugates were prepared by means of con-
ventional phosphoramidite chemistry following the procedure of
Letsinger and Wu.6 Base sequences in which complementary 5ꢀ-
and 3ꢀ-ends are connected either to the base sequence 5ꢀ-ACC or
to the stilbenedicarboxamide linker Sa are known to form stable
mini-hairpins.9,16 The reference hairpins 1, 4, and 6 employed in
this investigation have short stems consisting of 3 or 5 A:T base-
pairs with a single G:C base-pair adjacent to the ACC loops
of 1 and 6. The use of hairpin rather than duplex structures
permits study of short, stable base-pair domains. The hairpins are
thermodynamically more stable than the corresponding duplexes.9
A single stilbenoside has been introduced into the hairpin struc-
tures as a dangling base (2a,b and 5a,b) and into the interior
of the hairpin stem opposite an abasic site (3b and 7a,b). Two
stilbenosides have been introduced opposite abasic sites in the
interior of the poly(dT) strand of hairpin structure 6, both in
adjacent positions (8a,b) and with an intervening dT base (9a,b).
Chart 2
only the b-anomer forms stable conjugates with an intrahelical
stilbenoside structure. Studies of the hairpin thermodynamic
stability indicate that the b-St nucleoside is comparable to b-Py in
its ability to stabilize duplex structures either as a dangling end-
capping nucleoside or opposite an abasic site within the duplex
interior. The strong electronic transition dipoles of the stilbene
chromophores permits the study of the relative orientation of
two intrahelical stilbenes using a combination of exciton-coupled
circular dichroism spectroscopy and molecular modeling.12
Results
Synthesis of the stilbenosides and oligonucleotide conjugates
The stilbene C-nucleosides a-St and b-St were prepared as
outlined in Schemes 1 and 2, respectively. The key step in
Scheme 1 is the cadmium-mediated reaction of the Grignard
derivative of m-bromostilbene with Hoffer’s chlorosugar 10.11,13,14
This reaction provides 11 as a 5 : 1 mixture of the a- and b-
anomers. The key step in Scheme 2 is coupling of lactone 13
with m-bromostilbene, which affords 14 as a 1 : 9 mixture of
a- and b-anomers.14,15 Separation of the anomers and removal
of the protecting groups provide the C-nucleosides a-St and
b-St, which are converted to their 5ꢀ-dimethoxytrityl (DMT)
derivatives 12 and 15 upon reaction with 4,4ꢀ-dimethoxytrityl
chloride in the presence of diisopropylethylamine. The stable
DMT derivatives were converted to their activated derivatives
UV and fluorescence spectra
The normalized UV absorption spectra of 15, the DMT derivative
of nucleoside b-St and the conjugates 2b and 7b are shown in
Fig. 1. The long-wavelength band is assigned to the allowed
p,p* transition of stilbene. This band is slightly red-shifted in the
conjugates vs. the stilbenoside 15. The 260 nm band is assigned
to the overlapping absorption of the nucleobases and the weaker
stilbene absorption. The UV spectral band shapes of conjugates
possessing the a- vs. b-stilbenosides are indistinguishable, as
are the spectra of conjugates possessing two stilbenosides. The
260/315 nm absorbance ratio is proportional to the ratio of the
number of base-pairs to stilbenes within the conjugate structure.
Thermal dissociation profiles for the hairpins in Chart 2 were
determined at several wavelengths using a Peltier temperature
◦
controller to provide a heating rate of 0.5 C min−1. Hyper-
chromism is observed for the 260 nm bands of all of the conjugates.
Hyperchromism is also observed for the 315 nm bands, in contrast
to the hypochromism observed for Sa-linked hairpins.17 First
derivatives of the profiles obtained at 260 nm provide melting
temperatures which are reported in Table 1. Similar values of
Scheme 1 Reagents and conditions: (a) m-bromostilbene, Mg, CdCl2, 70%;
(b) NaOMe, MeOH, 83%; (c) DMT-Cl, py, DIPEA, 88%.
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Fig. 1 UV spectra b-St (as its mono-DMT derivative 15 in acetonitrile)
and conjugates 2b and 7b (5 lM in standard buffer).
Fig. 2 Fluorescence spectra of b-St (as its mono-DMT derivative 15 in
acetonitrile) and conjugates 2b, 3b, and 7b (5 lM in standard buffer).
Table 1 Melting temperature for reference conjugates and conjugates
Circular dichroism spectra
possessing one or two stilbenosidesa
The circular dichroism (CD) spectra of conjugates 2a,b are shown
in Fig. 3a. Similar CD spectra were obtained for 3b and 7a,b (data
not shown). The bands in the 200–300 nm spectral region are
attributed to exciton coupling between the adjacent base-pairs,
and are similar to those of the mini-hairpins 1 and 6.19 Weak
negative bands are observed in the 300–340 nm spectral region of
2a,b. The CD spectra of conjugates 5a,b, 8a,b, and 9a,b are shown
in Fig. 3b,c. Their spectra in the base-pair region (200–300 nm)
are similar to those of the mini-hairpins 1 and 6. In addition, they
Hairpin
Tm/◦C
DTm/◦C
1b
36
48
59
33
64
54 (br)
64
50
47
53
46
54
46
—
12
23
−3
—
−10
0
—
−3
3
2a
2b
3b
4b
5a
5b
6
7a
7b
8a
8b
9a
9b
−4
4
−4
4
54
a Data for 5 lM solutions (0.1 M NaCl, 10 mM phosphate buffer, pH 7.2)
obtained with a heating rate of 0.5 ◦C min−1 b Data from ref. 9.
.
Tm were obtained from 280 nm profiles. In some cases, as noted
in Table 1, broad melting transitions and first derivatives were
observed.
The fluorescence spectra of the DMT derivative 15 in acetoni-
trile solution and several conjugates in aqueous buffer are shown
in Fig. 2. The vibronic structure observed for 15 is similar to that
of trans-stilbene, as is its fluorescence quantum yield (Uf = 0.10
vs. 0.05 for stilbene).18 The fluorescence of the conjugates is much
weaker than that of 15. In the case of conjugate 3b, in which the
stilbene is located adjacent to a G:C base-pair, a long-wavelength
band with a maximum near 450 nm is observed in addition to the
quenched monomer band (Fig. 2). The fluorescence intensity for
7a is approximately twice as large as that of 7b.
The stilbenoside conjugates are sensitive to ultraviolet light.
Continuous 300 nm irradiation of 7b results in rapid and complete
disappearance of its 330 nm absorption band, indicative of loss
of stilbene conjugation, rather than trans,cis photoisomerization.
HPLC analysis of the irradiated solutions showed the formation
of a complex mixture of products, discouraging attempted product
isolation and identification.
Fig. 3 CD spectra of (a) conjugates 2a and 2b, (b) conjugates 5a and 5b
(matched absorbance at 327.5 nm), and (c) conjugates 8a, 8b, 9a, and 9b
(matched absorbance at 316 nm) all in standard buffer.
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Fig. 4 Perspective view of the structures obtained from molecular dynamics simulations (loop regions omitted) viewed from the side and from the
stilbene end for 2a (a and b) and 2b (c and d). Stilbenosides are bold. The twist angles between the stilbene and base-pair long axes are −9 16◦ for 2a
and 38 6◦ for 2b.
have bands in the 300–350 nm spectral region which are more
intense than those of 2a,b and differ in both sign and intensity.
pronounced for 7a, in which the stilbene is extrahelical. The
˚
stilbene in 7b has a smaller base-stacking distance (3.4 0.4 A
vs. 4.2 0.4 A) and also a smaller twist angle (11 5◦ vs. 56
˚
6◦) in the 3ꢀT vs. 5ꢀT direction. As is the case for 7a and 7b,
the calculated structures for 8a and 9a have extrahelical stilbenes
(structures not shown), whereas the calculated structures for 8b
and 9b have intrahelical stilbenes (see ESI†). The dihedral angles
Molecular dynamics simulations
Geometries for the DNA duplex domains of 2a,b, 7a,b, 8a,b,
and 9a,b calculated using the AMBER force field by adopting
the canonical A6T6 B-form DNA structure for their base-pair
domains.20 Similar methods as described in previous work were
used to calculated the charge parameters of the stilbenoside
residues and to equilibrate the simulation systems.9 The Amber
7.0 program suite was used to run molecular dynamics simulations
in the explicit water solution.21 The total simulation time for
each conjugate structure was 4.0 ns with a time step length of
2 fs. The resulting structures represent local minima arrived at
from idealized B-DNA input structures. The twist angle and
distance between stilbenes and neighboring DNA base-pairs were
calculated by fitting the trajectories using Curves 5.2.22
between stilbenes in 8a and 8b are relatively small (−18
13◦
and 1 8◦, respectively), when compared to the dihedral angles
between stilbenes in 9aand 9b (77 10◦ and 52 8◦, respectively).
The rise and helical pitch for 8b and 9b are larger in the 5ꢀ- vs. 3ꢀ-
direction, as is the case for 7b. The p-stacking distance between
˚
stilbenes in 8b is 3.8 0.5 A.
Discussion
Capping stilbenosides
Averaged structures obtained from multiple MD snapshots
for 2a,b are shown in Fig. 4. In both structures, the stilbene is
approximately parallel to the adjacent base-pairs with p-stacking
The addition of a stilbenoside to the mini-hairpin 1 as a dangling
base results in a marked increase in hairpin stability for both
2a and 2b. The increase in Tm for 2b is approximately twice
as large as that for 2a (Table 1). Similarly, the value of Tm for
5b is the same as that for the capped hairpin 4, which has a
singly tethered Sa end-cap, whereas the Tm for 5a is 10◦ lower.
Increased thermodynamic stability has previously been observed
for hairpin structures possessing hydrophobic stilbene capping
groups.8–10 The value of DTm for a derivative of 1 possessing a Sa
˚
˚
distances of 3.2 0.8 A and 3.4 0.4 A for 2aand 2b, respectively.
The calculated structure for 2b has a normal helical pitch of 38
6◦ between the long axes of stilbene and the adjacent base-pair.
However, the twist angle for 2a is −9 16◦, a negative twist being
necessary to provide an extent of p-stacking similar to that for 2b.
Averaged structures for 7a,bare shown in Fig. 5. Both structures
display significant perturbation of B-DNA geometry. This is most
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Fig. 5 Averaged truncated structures (loop regions omitted) for (a) 7a and (b) 7b (dashed line shows center of base-pairs) obtained from molecular
dynamics simulations. Stilbenosides are bold.
linker attached to the 5ꢀ-terminus of 1 via a flexible linker is 18 ◦C,
intermediate between the values for 2a and 2b.8,9 The presence
of an Sa capping group in hairpin 4 also results in a significant
increase in Tm. Kool and co-workers have investigated the ther-
modynamics of melting for self-complementary duplexes having a
dangling synthetic b-nucleosides possessing aromatic hydrocarbon
hydrophobic◦groups, the most effective of which is b-Py (Chart 1,
DTm = 23.1 C for 5 lM duplex in 1.0 M NaCl).3 Dogan et al.
report a similar value of DTm for a self-complementary duplex
with a 5ꢀ-tethered stilbene.10 Both the pyrenoside and the tethered
stilbene duplexes have two hydrophobic aromatic capping groups,
whereas 2b has a single capping group. Thus the unsubstituted
stilbene ring of 2b appears to have a hydrophobic capping effect
on duplex stability comparable to or greater than those of Kool’s
b-Py or Dogan’s stilbene.
The effect of an abasic site on the structure and stability of
duplex DNA has been extensively investigated.23,24 Duplexes in
which a purine nucleobase is located opposite the abasic site
are generally more stable than those with a pyrimidine opposite
the abasic site; however both apurinic and apyrimidinic duplexes
adopt B-DNA geometries with intrahelical bases.24 The presence
of a single a-dA nucleoside opposite dT in a 9-mer duplex causes
only a small decrease in duplex stability (DDG◦ = 0.2 kcal mol−1)
when compared to a normal b-dA–dT base-pair.25 Molecular
modeling indicates that the a-dA–dT base-pair has an intrahelical
base-paired structure. The inability of the a-St–X base-pair to form
a hydrogen-bonded structure may account for the extrahelical
location of the stilbene in 7a predicted by molecular modeling
(Fig. 5a). In the case of 7b, the increase in base-stacking of the
intrahelical stilbene may be limited by the poor overlap with the
base-pair in the 5ꢀT direction, as predicted by molecular modeling
(Fig. 5b).
The CD spectra of 2a,b and 5a,b in the base-pair region (200–
300 nm, Fig. 3a,b) are similar to those of 1 and 4, and presumably
are dominated by the short A3:T3 base-pair domains (A-tracts).
Molecular modeling (Fig. 4) suggests that 2b adopts a more
Two internal stilbenosides
˚
“normal” B-DNA capping geometry with a rise of 3.4 A and a
helical pitch of 38◦ between the stilbene long axis and the adjacent
A:T base-pair. In comparison, 2a adopts a smaller helical pitch
of −9◦, presumably in order to achieve optimal stilbene–base-pair
overlap.
Incorporation of a second stilbenoside into the hairpin structure 6
causes little additional change in the Tm values of 8a,bor 9a,bwhen
compared to 7a,b (Table 1). Their base-pair CD spectra (Fig. 3c)
are similar to those of 2a,b (Fig. 3a), indicating that the short A-
tract base-pair domains separating the hairpin loop and proximal
stilbene remain largely intact. Matray and Kool prepared a duplex
having two b-Py–X base-pairs separated by two intervening G:C
base-pairs.4 They report an abnormal thermal dissociation profile
with a high apparent Tm, but a CD spectrum characteristic of
B-DNA.
The calculated structures of 8a and 9a (not shown) are highly
disordered with extrahelical stilbenes. The calculated structures of
8b and 9b both have intrahelical stilbenes with distorted base-
paired geometries in the region of the two stilbenosides (see
ESI†). The two stilbenes in 8b have a slipped parallel relationship
in which the stilbene long axes are aligned with a p-stacking
Internal stilbenosides
The presence of a stilbenoside opposite an abasic site in the
interior of the base-pair stem of the mini-hairpin 3b results in a
small decrease in thermal stability when compared to 1 (Table 1).
Comparison of the Tm values for 7a,b with those for 6 indicates
that 7b is slightly more stable and 7a is slightly less stable. Kool
and co-workers observed a similar increase in Tm for duplexes
possessing b-Py opposite an abasic site.4,5 The CD spectra of 3b
and 7a,b in the base-pair region is similar to those of 1 or 6, and
presumably is dominated by the short A-tracts.
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˚
distance of 3.8 0.5 A. The slipped geometry reduces electron–
by eqn (1),
electron repulsion and is commonly observed for pairs of aromatic
p
De ≈
laldR−2 sin(2h)
(1)
molecules.26 The two stilbenes in 9b have a parallel geometry with
da
4k
a dihedral angle of 52
8◦, somewhat smaller than the value
where ld and la are the electronic transition dipole moments of
the two chromophores, Rda is their center-to-center distance, and
h is the angle between their transition dipoles.8,9,29,31 The sin(2h)
dependence results in zero intensity when h = 0, 90, or 180◦,
and maximum intensity when h = 45 or 135◦. The low EC-CD
intensity for 8b (Fig. 3c) is consistent with the small dihedral
angle between the stilbene long-axes obtained from molecular
for two 36◦ steps in B-DNA. The calculated stilbene–stilbene
dihedral angles are consistent with the EC-CD spectra (vide infra).
However, it should be borne in mind that the calculated structures
represent local minima and may not accurately reflect the solution
structure of these conjugates.
modeling (1
8◦). Significantly higher intensity EC-CD bands
Electronic interactions
are observed for 9b (Fig. 3c) even though the value of Rda is
approximately twice as large as that for 8b. The larger EC-CD
intensity is consistent with the calculated dihedral angle (52
8◦) which is close to the optimum angle of 45◦. The weaker EC-
CD signals for the a-stilbenosides 8a and 9a may reflect their
highly disordered, fluctional structures. We note that the intensity
of the EC-CD spectrum is dependent upon the magnitude of the
transition dipoles as well as the geometry of the two coupled
chromophores. The larger dipole for the stilbene vs. pyrene long-
wavelength transition makes the stilbenosides better suited for this
application.
The CD spectra of conjugates 5a and 5b (Fig. 3b) display
moderately intense negative bands in the 300–350 nm spectral
region attributed to exciton coupling between the Sa and St
chromophores. The irregular band shapes are a consequence
of coupling between chromophores with different absorption
maxima.31 The EC-CD intensity for 5b is stronger than that for
5a, indicative of a difference in capping structure similar to that
calculated for 2a vs. 2b (Fig. 4). The Sa-capped hairpin 4 (Chart
2), which has three intervening A:T base-pairs also has a negative
long-wavelength EC-CD band.9
Small red-shifts are observed in the long wavelength region of
the absorption spectra for conjugates 2a,b, 3b, and 7a,b when
compared to the spectra of the mono-DMT derivative of b-St
(Fig. 1). We have attributed similar UV spectral shifts for Sa-linked
hairpins and capped hairpins to weak electronic interactions
between the stilbene chromophore and adjacent base-pair.8,9,17
The weak long-wavelength CD bands for 2a,b (Fig. 3a), 3b, and
7a,b are also similar to those observed for Sa-linked hairpins and
capped hairpins. These bands are attributed to induced circular
dichroism, by analogy to the CD spectra of intercalating dyes
which have their long axes parallel to those of the adjacent base-
pairs.27
Efficient quenching of stilbene fluorescence in 2a,b, 3b, and
7a,b by neighboring nucleobases (Fig. 2) prevents the use of
the stilbenosides as intrinsic fluorescence probes of the base-pair
dynamics. Quenching might occur via either electron transfer or a
photochemical reaction involving the stilbene and a neighboring
base. Quenching of the fluorescence of stilbene hairpin linkers
having either electron-accepting or electron-donating substituents
by neighboring base-pairs has been attributed to electron transfer
in which the excited stilbene singlet can served as either an electron
acceptor or donor.7,17 An alternative explanation for the efficient
fluorescence quenching of the stilbenoside conjugates is the occur-
rence of photochemical addition reactions between the stilbene
and adjacent nucleobases. Both stilbene and thymine are known
to undergo efficient [2 + 2] duplex-templated photodimerization
reactions.28 Stilbene photodimerization is possible for 8a,b, but
not for the other conjugates. Cross-addition between stilbene and
thymine could account for the occurrence of efficient fluorescence
quenching, bleaching of the stilbene 315 nm absorption band, and
the complex appearance of HPLC traces for irradiated solutions.
Conjugates 5a,b, 8a,b, and 9a,b, which possess two stilbene
chromophores, have stronger long-wavelength CD bands than
do conjugates possessing a single stilbene chromophore (Fig. 3).
These CD bands are attributed to exciton coupling (EC-CD)
between the two stilbene chromophores.29,30 As previously reported
for capped hairpin structures possessing two Sa chromophores,
exciton coupling results in the observation of bisignate CD spectra,
the sign and intensity of which are dependent upon both the
distance between the stilbene chromophores and the dihedral
angle between their electronic transition dipoles.8,9 A second,
shorter wavelength band of the EC-CD spectrum should appear
below 300 nm but is largely obscured by the strong positive 280 nm
band of the base-pair CD spectrum.
Concluding remarks
The structure and properties of oligonucleotides possessing syn-
thetic stilbene C-nucleosides have been investigated for the first
time. Both the a- and b-stilbenosides stabilize duplex structures
when employed as dangling end-capping groups in conjugates
2a,b and 5a,b. When incorporated into duplex interior opposite
an abasic site, the b-anomer results in a modest increase in duplex
stability, whereas the presence of the a-anomer destabilizes the
duplex. Similar results were obtained for conjugates possessing
two stilbenosides either adjacent to each other or separated by
one base-pair. The ability of the b-stilbenoside to stabilize duplex
structures either as a capping group or opposite an abasic site
within the duplex interior is similar to that of Kool’s b-pyrenoside.
Molecular modeling indicates that the difference in stability of
duplexes possessing a- vs. b-stilbenosides can be related to their
structures. As capping groups, both anomers adopt structures
in which the stilbene effectively covers the hydrophobic face
of the terminal base-pair. However, when located within the
base-pair domain, the a-anomer adopts an extrahelical geometry
whereas the b-anomer adopts an intrahelical geometry. The strong
electronic transition dipole moment of the stilbene chromophore
permits observation of exciton-coupled circular dichroism for
conjugates possessing two stilbene chromophores. The combina-
tion of EC-CD spectroscopy with molecular modeling permits
assignment of the relative geometry of the two chromophores.
The intensity of the positive and negative bands of the EC-CD
spectrum for two identical parallel chromophores can be described
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1,2-Dideoxy-a-1-(3-trans-stilbene)-D-ribofuranose (a-St).
Freshly prepared 0.5 M NaOMe in MeOH was added to a
solution of protected nucleoside 11 (500 mg, 0.835 mmol) in
1 : 1 MeOH–CH2Cl2 (10 mL). After stirring for 4 h at room
temperature, saturated NH4Cl (20 mL) was added, and the
solvent was evaporated. The crude material was purified using
flash chromatography (CH2Cl2–MeOH, 19 : 1) to yield the desired
compound as a white solid (205 mg, 83% yield). 1H NMR (CDCl3,
300 MHz) d 7.46–7.16 (m, 9H), 7.05 (s, 2H), 5.05 (dd, 1H, J =
7.3 Hz), 4.40 (dd, 1H, J = 6.5, 6.5 Hz), 4.07–4.04 (m, 1H), 3.72
(m, 2H), 2.63 (ddd, 1H, J = 13.2, 6.5, 6.5 Hz), 2.07–2.01 (m, 1H).
Experimental
Synthetic procedures
All reactions were carried out under an argon atmosphere
with dry solvents under anhydrous conditions, unless other-
wise noted. Yields refer to chromatographically and spectro-
scopically (1H NMR) homogeneous materials, unless otherwise
stated. Reagents purchased were of the highest commercial
quality and used without further purification, unless otherwise
stated. Methylene chloride and chloroform were distilled from
calcium hydride. Tetrahydrofuran (THF) was distilled from
sodium/benzophenone. Methanol was distilled from magnesium.
Analytical thin-layer chromatography (TLC) was performed using
0.25 mm pre-coated silica gel Kieselgel 60 F254 plates. Visual-
ization of the chromatogram was by UV absorbance, iodine,
dinitrophenylhydrazine, ceric ammonium molybdate, ninhydrin or
potassium permanganate, as appropriate. Preparative and semi-
preparative TLC was performed using Merck 1 mm or 0.5 mm
coated silica gel Kieselgel 60 F254 plates respectively. Merck
silica gel (60, particle size 0.040–0.063 mm) was used for flash
column chromatography. NMR spectra were recorded on a Varian
300 MHz instrument and calibrated using residual undeuterated
solvent as an internal reference. The following abbreviations were
used to explain the multiplicities: s = singlet, d = doublet, ddd =
doublet of doublets, t = triplet, m = multiplet. Electrospray
ionization (ESI) time-of-flight reflectron experiments were per-
formed on an Agilent ESI-TOF mass spectrometer. Samples are
electrosprayed into the TOF reflectron analyzer at an ESI voltage
of 4000 V and a flow rate of 200 lL min−1). Compounds 10,13 13,15
and 3-bromostilbene32 were synthesized according to literature
procedures.
1
13C{ H} NMR (CDCl3, 75 MHz) d 142.9, 137.9, 137.4, 129.3,
129.1, 128.9, 128.6, 127.9, 126.7, 126.1, 125.1, 123.9, 85.4, 79.8,
73.2, 62.6, 43.6. ESI-TOF calc. C19H20O3 [M + Na+]: 319.1305,
found: 319.1305.
1,2-Dideoxy-a-1-(3-trans-stilbene)-5-O-p-dimethoxytrityl-D-
ribofuranose (12). The C-nucleoside b-St (430 mg, 1.45 mmol)
was dissolved in a 1 : 1 mixture of pyridine and methylene
chloride (18 mL). Diisopropylethylamine (280 mg, 2.17 mmol)
and 4,4ꢀ-dimethoxytrityl (DMT) chloride (982 mg, 2.90 mmol)
were added to the mixture and stirred for 4 h at room temperature
and then quenched with methanol (10 mL). The resulting mixture
was concentrated in vacuo and purified by flash chromatography
(EtOAc–hexanes, from 1 : 5 to 1 : 1) to yield the desired compound
as a yellow foam (764 mg, 88% yield). 1H NMR (C6D6, 300 MHz)
d 7.69–7.65 (m, 3H), 7.51 (dd, 4H, J = 9.1, 1.8 Hz), 7.29–6.97 (m,
12H), 6.73 (d, 4H, J = 9.1 Hz), 5.05 (dd, 1H, J = 7.3, 7.3 Hz),
4.31 (ddd, 1H, J = 5.0, 5.0, 5.0 Hz), 4.23 (ddd, 1H, J = 5.0,
5.0, 5.0 Hz), 3.54 (dddd, 2H, J = 5.0, 5.0, 5.0, 5.0 Hz) 3.25 (s,
3H), 2.24 (s, 1H), (dd, 1H, J = 12.6, 6.8, 6.8 Hz), 1.87 (ddd, 1H,
1
J = 12.6, 6.8, 6.8 Hz). 13C{ H} NMR (C6D6, 75 MHz) d 159.0,
145.7, 144.3, 137.7, 137.7, 136.4, 136.5, 129.1, 129.0, 128.8, 128.7,
128.6, 128.0, 127.6, 126.9, 126.8, 125.6, 125.3, 124.3, 86.6, 85.2,
79.7, 74.7, 65.1, 54.6, 43.9. ESI-TOF calc. C40H38O5 [M + Na+]:
621.2611 found: 621.2612.
1,2-Dideoxy-a-1-[3-trans-stilbene]-3,5-di-O-(p-toluoyl)-D-erythro-
pentitol (11).
A
solution of m-bromostilbene32 (700 mg,
2.70 mmol) in THF (15 mL) was slowly added to Mg turnings
(139 mg, 3.38 mmol). After the addition of ∼2 mL of the m-
bromostilbene solution, I2 crystals and two drops of dibromoe-
thane were added to start the reaction. The rest of the m-
bromostilbene was then added slowly, and the solution heated
under reflux for 2 h. Subsequently, CdCl2 (576 mg, 3.14 mmol) was
added, and the mixture was heated for another 2 h under reflux.
After cooling to room temperature, a solution of chlorosugar 1013
(1.22 g, 3.14 mmol) was added slowly via a dropping funnel and
stirred for 16 h. The solution was dried in vacuo to remove the
THF and the residue was dissolved in CH2Cl2. The resulting
organic solution was washed twice with saturated NH4Cl and
dried over Na2SO4. The mixture was concentrated in vacuo and
purified by flash chromatography (EtOAc–hexanes, from 1 : 7 to
1 : 5) to yield the desired compound as a white foam (1.0 g, 70%
3,5-O-((1,1,3,3-Tetraisopropyl)disiloxanediyl)-1,2-dideoxy-b-1-
(3-trans-stilbene)-D-ribofuranoses (14). To a solution of the
3-bromo-trans-stilbene32 (2.1 g, 8.32 mmol) in anhydrous THF
◦
(20 mL) under N2 and at −78 C was added n-BuLi (5.2 mL of
1.8 M in hexanes). The mixture was stirred at −78 ◦C for 30 min
and was then added via cannula to a solution of 1315 (2.0 g,
5.34 mmol) in anhydrous THF (20 mL) at −78 ◦C. After 1 h, the
reaction mixture was quenched at −78 ◦C with sat. aqueous NH4Cl
(80 mL) and then extracted with diethyl ether (2 × 200 mL). The
combined ether phases were washed with sat. aqueous NH4Cl,
water, and brine, and were dried over anhydrous Na2SO4. Con-
centration in vacuo produced an oil that was used without further
purification. A solution of the crude oil in CH2Cl2 (20 mL) under
◦
1
yield). a : b 5 : 1. H NMR (CDCl3, 300 MHz) d 7.56–7.51 (m,
N2 and at −78 C was treated with Et3SiH (1.86 g, 16.00 mmol)
3H), 7.48–7.42 (m, 3H), 7.38–7.16 (m, 13H), 7.13 (s, 2H), 6.85
(d, 4H, J = 8.8 Hz), 5.16 (dd, 1H, J = 7.4 Hz), 4.46 (m, 1H),
4.22 (ddd, 1H, J = 6.0, 4.6, 4.6), 3.80 (s, 6H), 3.41 (dd, 1H, J =
9.4, 4.6 Hz), 3.25 (dd, 1H, J = 9.4, 6.0 Hz), 2.73 (m, 1H), 2.07
and BF3·OEt2 (2.27 g, 16.00 mmol). The resulting solution was
◦
◦
stirred at −78 C for 6 h, and then quenched at −78 C by the
addition of sat. NaHCO3 (100 mL). The resulting mixture was
extracted with CH2Cl2 (2 × 50 mL). The combined organic phases
were dried over anhydrous Na2SO4 and concentrated in vacuo.
Flash chromatography of the crude product using toluene as the
eluent yielded the desired compound as a colorless oil (1.2 g,
1
(m, 1H). 13C{ H} NMR (CDCl3, 75 MHz) d 166.4, 166.1, 144.0,
143.8, 142.9, 137.5, 137.2, 129.8, 129.7, 129.6, 129.1, 129.1, 129.1,
128.9, 128.7, 128.7, 128.6, 128.5, 127.6, 127.0, 126.8, 126.5, 125.6,
125.0, 123.7, 82.4, 80.2, 64.6, 40.2, 21.7. ESI-TOF calc. C35H32O5
[M + H+]: 533.2322, found: 533.2313.
1
42% yield) a : b 1 : 9. H NMR (CDCl3, 300 Hz) d 7.52–7.46
(m, 4H), 7.42–7.21 (m, 8H), 7.09 (s, 2H), 5.10 (dd, 1H, J = 7.2,
320 | Org. Biomol. Chem., 2006, 4, 314–322
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7.2 Hz), 4.53 (m, 1H), 4.15 (m, 1H), 3.91 (m, 2H), 2.38 (ddd,
lowing synthesis, the conjugates were first isolated as trityl-on
derivatives by reverse phase (RP) HPLC, then detritylated in 80%
acetic acid for 30 min, and repurified by RP-HPLC as needed.
RP-HPLC analysis was carried out on a chromatograph with a
Phenomenex Hyperclone ODS-5 (C18) column (4.6 × 250 mm)
and a 1% gradient of acetonitrile in 0.03 M triethylammonium
acetate buffer (pH 7.0) with a flow rate of 1.0 mL min−1.
Molecular weights were determined by means of MALDI TOF
mass spectroscopy following desalting using a NAP 5 column.
1H, J = 12.7, 6.8, 4.3), 2.09 (ddd, 1H, J = 12.7, 6.8, 6.8 Hz),
1
2.13–0.93 (m, 24H). 13C{ H} NMR (CDCl3, 300 Hz) d 139.1,
135.2, 135.1, 128.8, 128.7, 128.5, 128.0, 127.8, 127.7, 127.7, 126.5,
126.4, 125.8, 123.5, 94.2, 82.3, 70.7, 62.9, 13.2, 12.5. ESI-TOF
calc. C31H46O4Si2 [M + H+]: 538.2935, found: 538.2933.
1,2-Dideoxy-b-1-(3-trans-stilbene)-D-ribofuranose (b-St). To a
solution of 14 (1.2 g, 2.22 mmol) in THF (44 mL) was added
tetrabutylammonium fluoride (6.66 mL of 1 M solution in THF).
The resulting mixture was stirred at room temperature for 3 h, then
5% aqueous NH4HCO3 (100 mL) was added to quench the reac-
tion. The mixture was extracted with diethyl ether (3 × 100 mL),
and the combined organic extracts were washed with 5% aqueous
NH4HCO3 (100 mL), water (100 mL), and brine (100 mL).
The organic layer was then dried over anhydrous Na2SO4 and
concentrated in vacuo to give the crude product as a white solid.
The crude material was purified using flash chromatography
(CH2Cl2–MeOH, 19 : 1) to yield the desired compound as a white
Electronic spectroscopy
UV spectra and thermal dissociation profiles were determined
using a Perkin–Elmer lambda 2 UV spectrophotometer equipped
with a Peltier temperature programmer for automatically increas-
ing the temperature at the rate of 0.5 ◦C min−1. Circular dichroism
spectra were obtained using a JASCO J-715 spectrometer at
the indicated concentrations. Fluorescence studies were obtained
using a Spex FluoroMax spectrometer. Photo-irradiation was
carried out in a Rayonet reactor equipped with 300 nm lamps.
Unless otherwise noted, all the spectroscopic studies were done in
0.1 M NaCl, 10 mM Phosphate buffer (pH 7.2, standard buffer)
using freshly prepared sample solutions.
1
solid (520 mg, 79% yield). H NMR (CDCl3, 300 MHz) d 7.44–
7.13 (m, 9H), 5.11 (dd, 1H, J = 10.3, 5.9 Hz), 4.36–4.34 (m, 1H),
3.99–3.95 (m, 1H), 3.79–3.67 (m, 2H), 2.22 (ddd, 1H, J = 13.2,
1
5.6, 1.5 Hz), 1.97 (m, 1H). 13C{ H} NMR (CDCl3, 75 MHz) 141.6,
137.8, 137.3, 129.3, 129.1, 128.9, 128.5, 127.9, 126.7, 126.1, 125.4,
125.4, 87.3, 80.3, 73.9, 63.5, 43.9. ESI-TOF calc. C19H20O3 [M +
Na+]: 319.1305, found: 319.1306.
Acknowledgements
1,2-Dideoxy-b-1-(3-trans-stilbene)-5-O-p-dimethoxytrityl-D-ribo-
furanose (15). The C-nucleoside b-St (430 mg, 1.45 mmol)
was dissolved in a 1 : 1 mixture of pyridine and methylene
chloride (18 mL). Diisopropylethylamine (280 mg, 2.17 mmol)
and 4,4ꢀ-dimethoxytrityl (DMT) chloride (982 mg, 2.90 mmol)
were added to the mixture and the reaction was stirred for 4 h at
room temperature and then quenched with methanol (10 mL).
The resulting mixture was concentrated in vacuo and purified by
flash chromatography (EtOAc–hexanes, from 1 : 5 to 1 : 1) to yield
the desired compound as a yellow foam (764 mg, 88% yield). 1H
NMR (C6D6, 300 MHz) d 7.84 (s, 1H), 7.70 (d, 2H, J = 7.3 Hz),
7.53 (dd, 4H, J = 8.8, 3.0 Hz), 7.29–7.22 (m, 3H), 7.18–6.96 (m,
12H), 6.72 (d, 4H, J = 8.8 Hz), 5.26 (dd, 1H, J = 8.5, 8.5 Hz),
4.15 (t, 2H, J = 3.5 Hz), 3.54 (dd, 1H, J = 9.7, 4.1 Hz), 3.39 (dd,
1H, J = 9.7, 4.1 Hz), 3.21 (s, 3H), 3.20 (s, 3H), 2.04–2.00 (m,
Financial support for this research was provided by grants from
the National Science Foundation (CHE-0400663 to FDL) and
The Skaggs Institute for Chemical Biology (KDJ).
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