Synthesis, structure, and photochemistry of exceptionally stable synthetic DNA hairpins with stilbene diether linkers

Article abstract of DOI:10.1021/ja026941o

The structure and properties of 18 hairpin-forming bis(oligonucleotide) conjugates possessing stilbene diether linkers are reported. Conjugates possessing bis(2-hydroxyethyl)stilbene 4,4′-diether linkers form the most stable DNA hairpins reported to date. Hairpins with as few as two T:A base pairs or four noncanonical G:G base pairs are stable at room temperature. Increasing the length of the hydroxyalkyl groups results in a decrease in hairpin thermal stability. On the basis of the investigation of their circular dichroism spectra, all of the hairpins investigated adopt B-DNA structures, except for a hairpin with a short poly(G:C) stem which forms a Z-DNA structure. Both the strong fluorescence of the stilbene diether linkers and their trans-cis photoisomerization are totally quenched in hairpins possessing neighboring T:A and G:C base pairs. Quenching is attributed to an electron-transfer mechanism in which the singlet stilbene serves as an electron donor and T or C serves as an electron acceptor. In contrast, in denatured hairpins and hairpins possessing neighboring G:G base pairs the stilbene diether linkers undergo efficient photoisomerization.

Full text of DOI:10.1021/ja026941o

Published on Web 09/21/2002
Synthesis, Structure, and Photochemistry of Exceptionally
Stable Synthetic DNA Hairpins with Stilbene Diether Linkers
Frederick D. Lewis,* Yansheng Wu, and Xiaoyang Liu
Contribution from the Department of Chemistry, Northwestern UniVersity,
EVanston, Illinois 60208-3113
Received May 17, 2002
Abstract: The structure and properties of 18 hairpin-forming bis(oligonucleotide) conjugates possessing
stilbene diether linkers are reported. Conjugates possessing bis(2-hydroxyethyl)stilbene 4,4′-diether linkers
form the most stable DNA hairpins reported to date. Hairpins with as few as two T:A base pairs or four
noncanonical G:G base pairs are stable at room temperature. Increasing the length of the hydroxyalkyl
groups results in a decrease in hairpin thermal stability. On the basis of the investigation of their circular
dichroism spectra, all of the hairpins investigated adopt B-DNA structures, except for a hairpin with a short
poly(G:C) stem which forms a Z-DNA structure. Both the strong fluorescence of the stilbene diether linkers
and their trans-cis photoisomerization are totally quenched in hairpins possessing neighboring T:A and
G:C base pairs. Quenching is attributed to an electron-transfer mechanism in which the singlet stilbene
serves as an electron donor and T or C serves as an electron acceptor. In contrast, in denatured hairpins
and hairpins possessing neighboring G:G base pairs the stilbene diether linkers undergo efficient
photoisomerization.
Chart 1. Structures of Stilbenedicarboxamide (Sa) and Stilbene
Diether (Sd) Diol Linkers
Introduction
Bis(oligonucleotide) conjugates with synthetic linkers con-
necting short complementary oligonucleotides are known to
form synthetic DNA or RNA hairpins which are, in some cases,
more stable than natural hairpins possessing nucleotide linkers.1
Letsinger and Wu2 reported the use of a stilbenedicarboxamide
(Sa, Chart 1) linker which formed stable hairpin structures
possessing as few as three T:A or two G:C base pairs. At the
time of their reported synthesis, the Sa-linked structures were
the most stable mini-DNA hairpins known. The ability of the
Sa linker to serve as an electron acceptor has been exploited
in our studies of hole injection and migration processes in
synthetic DNA hairpins.3
The search for a linker that would serve as an electron donor
led us to investigate synthetic hairpins with stilbene diether (Sd,
Chart 1) linkers. The linker Sd2 which possesses hydroxyethyl
substituents proved to form exceptionally stable synthetic
hairpins with poly(T:A) stems.4 The crystal structure of a Sd2-
linked hairpin possessing six base pairs has been reported,4 as
has the one-way cis-trans photoisomerization in Sd2-linked
hairpins.5 We report here the preparation of 18 stilbene diether-
linked hairpins (Chart 2) and the study of their structure, thermal
stability, fluorescence, and photoisomerization. These properties
are found to be dependent upon the length of the hydroxyalkyl
substituent as well as the number and identity of the base pairs.
The formation of stable hairpins possessing as few as two A:T,
two G:C, or four G:G base pairs is observed. All of the hairpins
have circular dichroism (CD) spectra characteristic of B-form
structures except for a G3-Sd2-C3 hairpin which has a
characteristic Z-form CD spectrum. Linker fluorescence and
photoisomerization efficiencies are strongly dependent upon the
identity of the neighboring base pair.
* To whom correspondence should be addressed. E-mail: lewis@
chem.northwestern.edu.
(1) (a) Bevers, S.; Schutte, S.; McLaughlin, L. W. J. Am. Chem. Soc. 2000,
120, 11004-11005. (b) Durand, M.; Chevrie, K.; Chassignol, M.; Thuong,
N. T.; Maurizot, J. C. Nucleic Acids Res. 1990, 18, 6353-6359. (c) Ma,
M. Y. X.; McCallum, K.; Climie, S. C.; Kuperman, R.; Lin, W. C.; Sumner-
Smith, M.; Barnett, R. W. Nucleic Acids Res. 1993, 21, 2585-2589. (d)
Salunkhe, M.; Wu, T.; Letsinger, R. L. J. Am. Chem. Soc. 1992, 114, 8768-
8772.
(2) Letsinger, R. L.; Wu, T. J. Am. Chem. Soc. 1995, 117, 7323-7328.
(3) (a) Lewis, F. D.; Letsinger, R. L.; Wasielewski, M. R. Acc. Chem. Res.
2001, 34, 159-170. (b) Lewis, F. D.; Wu, Y. J. Photochem. Photobiol., C
2001, 2, 1-16. (c) Lewis, F. D.; Liu, J.; Liu, X.; Zuo, X.; Hayes, R. T.;
Wasielewski, M. R. Angew. Chem., Int. Ed. 2002, 41, 1026-1028. (d)
Lewis, F. D.; Zuo, X.; Liu, J.; Hayes, R. T.; Wasielewski, M. R. J. Am.
Chem. Soc. 2002, 124, 4568-4569.
(4) Lewis, F. D.; Liu, X.; Wu, Y.; Miller, S. E.; Wasielewski, M. R.; Letsinger,
R. L.; Sanishvili, R.; Joachimiak, A.; Tereshko, V.; Egli, M. J. Am. Chem.
Soc. 1999, 121, 9905-9906.
(5) Lewis, F. D.; Liu, X. J. Am. Chem. Soc. 1999, 121, 11928-11929.
9
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J. AM. CHEM. SOC. 2002, 124, 12165-12173
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A R T I C L E S
Lewis et al.
Chart 2. Structures of Stilbene Diether-Linked Conjugates in
Their Hairpin Conformations, Showing Hydrogen Bonding between
Canonical (---) and Noncanonical (|||) Base Pairs
Figure 1. Absorption and fluorescence spectra of the stilbene diether Sd3
in methanol (s) and methanol-water (---) solution.
Table 1. Absorption and Fluorescence Maxima for the Stilbene
Diethers
solvent
Sd2
Sd3
Sd4
304, 326
λ
abs, nm methanol
304, 326 304, 326
methanol-water 282
278
374
0.22
0.37
386, 409
0.06
276
374
0.22
0.37
409
0.06
λfl, nm methanol
374
a
Φfl
methanol
0.21
0.35
τfl, nsb methanol
λ
Φfl
fl, nm methanol-water 386
a
methanol-water 0.02
τfl, nsc methanol-water
1.9 (79), 6.7 (21) 0.87 (70), 5.9 (30)
a Fluorescence quantum yield in deoxygenated solution determined using
quinine sulfate actinometry. b Fluorescence decay time obtained from single-
exponential fits. c Fluorescence decay times and preexponentials (values
in parentheses) obtained from dual exponential fits.
band, similar to that for 4,4′-dimethoxystilbene.8 Thus, the
hydroxyalkyl groups do not influence the spectra in methanol
solution. The absorption spectra in tetrahydrofuran are identical
to those in methanol. The stilbene diethers are insoluble in water
and sparingly soluble in methanol-water. Their absorption
spectra in methanol-water display a blue-shift in the absorption
maxima and a red-shifted onset of absorption.
The fluorescence spectra of Sd3 in methanol and methanol-
water are also shown in Figure 1, and the fluorescence maxima,
quantum yields, and decay times of the stilbene diethers are
reported in Table 1. The fluorescence parameters for the stilbene
diethers are similar to those of 4,4′-dimethoxystilbene in
methanol (Φfl ) 0.20, τfl ) 0.42 ns).9 The fluorescence decay
time for the stilbene diethers are near the time resolution of
our lifetime apparatus (ca. 0.5 ns). The fluorescence spectra are
red-shifted in methanol-water. The fluorescence quantum yields
in methanol-water are smaller than those in methanol. The
fluorescence decay in methanol-water is best fit by a dual
exponential function in which both decay components are longer
lived than the single component decay obtained in methanol
solution (Table 1). The band shapes of the fluorescence of Sd3
and Sd4 are independent of temperature below 70 °C but
resemble the spectra obtained in methanol at 90 °C.
Results
Bis(n-hydroxyalkyl)stilbene 4,4′-Diethers. The stilbene di-
ethers Sd2, Sd3, and Sd4 were prepared by the method of
Sieber.6 Their MM2-minimized structures consist of a nearly
planar rodlike stilbene with appended flexible hydroxyalkyl
groups. The hydroxyalkyl groups in Sd2 adopt the gauche
conformation favored by ethylene glycol monomethyl ether,7
whereas the hydroxyalkyl groups in Sd3 and Sd4 adopt lowest
energy fully extended trans conformations which are only
slightly more stable than the gauche conformations (Chart 1).
The distances between the two hydroxy oxygen atoms are 17.0,
21.4, and 23.7 Å for Sd2, Sd3, and Sd4, respectively.
The absorption spectra of Sd3 in methanol and 1:10 methanol/
water are shown in Figure 1, and the absorption maxima of the
stilbene diethers are reported in Table 1. The spectra in methanol
solution consist of a single allowed, long-wavelength absorption
Irradiation of Sd2 with 344 nm light in methanol or dimethyl
sulfoxide solution results in photoisomerization, yielding a
mixture of Sd2 and its cis isomer, c-Sd2 (eq 1). The photo-
(6) Sieber, R. H. Liebigs Ann. Chem. 1969, 730, 31-46.
(7) Eliel, E. L.; Wilen, S. H. Stereochemistry of Organic Compounds; Wiley:
New York, 1994.
(8) Lewis, F. D.; Bedell, A. M.; Dykstra, R. E.; Elbert, J. E.; Gould, I. R.;
Farid, S. J. Am. Chem. Soc. 1990, 112, 8055-8064.
(9) Zeglinski, D. M.; Waldeck, D. H. J. Phys. Chem. 1988, 92, 692-701.
9
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DNA Hairpins with Stilbene Diether Linkers
A R T I C L E S
Figure 2. (a) Crystal Structure of the Sd2-linked hairpin 1 and MM2-minimized structures of conjugates 4 (b), c-4 (c), and 8 (d). Hairpin linkers are at the
top of the structures.
stationary state obtained upon irradiation of either isomer is 28%
Sd2 and 82% c-Sd2. Quantum yields measured by comparison
with the secondary standard trans-stilbene are shown in eq 1.10
distance for the linker; the average distance between its outer
oxygen atoms is ca. 16.5 Å, similar to that calculated for the
Sd2 diol (Chart 1). The average interstrand distance between
phosphorus atoms bound to the Sd2 linker is 18.1 Å and thus
comparable to the average distance of 17.7 Å for phosphorus
atoms from opposite strands in all other dimer steps. Details of
the crystal structure will be reported elsewhere.
The MM2-minimized structure for conjugate 1 has a hairpin
geometry similar to that for the crystal structure. The other
conjugates in Chart 2 can also adopt minimized structures in
which the linker serves as the loop in a B-DNA hairpin structure.
Hairpin formation is consistent with the results of UV thermal
dissociation studies and circular dichroism (CD) spectra (vide
infra). The minimized structures for conjugates 4, c-4, and 8
are shown in Figure 2b-d. The minimized structure for 4
(Figure 2b), which possesses a Sd2 linker, has a loop region
similar to that of conjugate 1. The structure for c-4 (Figure 2c)
has a severely distorted loop region. The minimized structures
for conjugates 6 and 8 (Figure 2d) have wider loop regions in
which the alkyl groups extend beyond the sugar-phosphate
backbone. However, the stilbene diether chromophores remain
π-stacked with the neighboring T:A base pair.
Ultraviolet Spectra and Thermal Dissociation. The ultra-
violet absorption spectra of the stilbene diether-linked conjugates
display a long-wavelength absorption band (λmax ∼ 327 nm)
assigned to the stilbene π,π* transition and a shorter wavelength
band (λmax ∼ 260 nm) assigned to overlapping stilbene and
nucleobase absorption bands. The long wavelength bands are
slightly red-shifted with respect to that of the stilbene diether,
as shown in Figure 3 for conjugate 4.
Synthesis and Structure of Stilbene Diether-Linked Hair-
pins. Prior to incorporation of the stilbene diethers into a
synthetic (bis)oligonucleotide conjugate, they were converted
to the monoprotected, monoactivated diol by sequential reaction
with 4,4′-dimethoxytrityl chloride and with 2-cyanoethyl diiso-
propylchlorophosphoramidite (see Experimental Section). The
conjugates 1-18 shown in Chart 2 were prepared by means of
conventional phosphoramidite chemistry using a Millipore
Expedite oligonucleotide synthesizer following the procedure
developed by Letsinger and Wu.2 The conjugates were first
isolated as trityl-on derivatives by RP HPLC, then detritylated
in 80% acetic acid for 30 min, and then repurified by RP HPLC.
A single peak was detected by both RP and IE HPLC. Molecular
weights of representative conjugates were determined by
electrospray ionization mass spectroscopy.
Irradiation of hairpin 4 under denaturing conditions in 50%
ethanol-water results in conversion to a mixture of 4 and a
single photoproduct formed in 72% yield. Formation of second-
ary photoproducts was observed after the photostationary state
was attained. Irradiation under denaturing conditions in water
at pH > 12 resulted in more complex product mixtures. The
product of irradiation in ethanol-water was isolated by HPLC
and identified as the cis isomer, c-4, by comparison of its
absorption spectrum with that of c-Sd2 (eq 1) and by its
photoisomerization in ethanol-water to yield the same mixture
of isomers as obtained from 4.
The 260 nm bands display an increase in absorbance
(hyperchromism) upon heating in aqueous buffer containing 0.1
M NaCl, whereas the 327 nm bands display a decrease in
absorbance. Thermal dissociation profiles for the Sd2-linked
hairpins 2-5 (2 × 10-6 M) in 0.1 M NaCl aqueous buffer (10
mM sodium phosphate, pH 7.2) are shown in Figure 4. The
first derivatives of these curves provide the melting temperatures
(Tm) reported in Table 2. The values of Tm for 2-5 increase
with increasing length of the polyT-polyA sequences and with
increasing salt concentration. However, they are independent
of conjugate concentration, indicative of the formation of hairpin
rather than duplex structures. The Tm of hairpin 4 is higher in
1.0 M NaCl (60.8 °C) and lower in water (50.8 °C) than in 0.1
M NaCl (54.0 °C). Lower Tm values are also observed in
ethanol-water mixtures (50.0, 37.6, and 22.2 °C , in 10, 30,
and 50% ethanol, respectively). The Tm values for c-4 are
The crystal structure of the bromine-labeled conjugate 1
obtained by crystallization from solutions containing Sr2+ ions
has previously been reported.4 The asymmetric unit consists of
four Sd2-linked hairpins in which the stilbenes form an edge-
to-face tetramer. The structure of one hairpin from the unit cell
is shown in Figure 2a. The base pairs adopt a standard B-form
DNA conformation, and the linker is π-stacked with the adjacent
G:C base pair. Both ethylenes in the linker adopt gauche
conformations, as is the case for 1,2-dimethoxyethane and poly-
(ethylene oxide).7 This results in a rather short end-to-end
(10) Lewis, F. D.; Johnson, D. E. J. Photochem. 1977, 7, 421-423.
9
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A R T I C L E S
Lewis et al.
only GC base pairs. No hyperchromism is observed upon heating
either conjugate from 10 to 60 °C in aqueous 0.1 M NaCl or
upon heating 11 in water or 50% ethanol-water. Hyper-
chromism is observed at temperatures >60 °C, as previously
observed by Letsinger and Wu2 for analogous Sa-linked
hairpins. These results are indicative of values of Tm > 80 °C.
Conjugate 13 contains two polyG arms. A freshly prepared
dilute solution of conjugate 13 (2 × 10-6 M) displays reversible
melting with a Tm value of 48.8 °C. Annealing of 13 at 85 °C
followed by slow cooling to room temperature results in a
change in the melting behavior indicative of the formation of a
more stable G-quadruplex or aggregate structures which are also
obtained at higher concentrations of 13. Conjugates 14-18
possess G:G mismatches inserted between the stilbene diether
linker and a T6:A6 duplex region. The values of TM for these
conjugates are lower than that of hairpin 5; however, their
thermal dissociation profiles are similar in appearance to that
of 5. The melting profiles for 14-18 are reversible and do not
change upon annealing. The value of TM decreases with the
number of G:G mismatch base pairs for 14-16 but increases
slightly for 17. The value of TM is lower for conjugate 18, which
possesses an Sd4 linker, than for 14, which possesses an Sd2
linker.
Figure 3. Absorption spectra of the stilbene diether Sd2 in methanol
solution and the Sd2-linked conjugate 4 in aqueous 0.1 M NaCl.
Circular Dichroism Spectra. The circular dichroism (CD)
spectra of conjugates 2-4 which possess poly(T:A) stems are
shown in Figure 5a. The spectra of 4 and 5 have a positive
band at 283 nm and a negative band at 250 nm, consistent with
formation of a B-form DNA structure in solution. The CD
spectra of 2 and 3 are similar but are broader and have red-
shifted minima. A very weak negative band is observed at longer
wavelength (300-350 nm). The CD spectrum of conjugate 9
is similar to that of 4 except that no long-wavelength band
(>300 nm) is detected.
The CD spectrum of 11 (Figure 5b), which possesses two
G:C base pairs, is similar in appearance to that of 2 (Figure
5a). However, its 254 nm minimum and 291 nm maximum are
red-shifted with respect to those of 2, and the long-wavelength
minimum at 340 nm is more pronounced. The CD spectrum of
conjugate 12 either in water or in standard buffer (Figure 5b)
is markedly different from that of the poly(T:A) hairpins,
displaying a positive peak at 260 nm and a negative peak at
281 nm characteristic of the left-hand helical conformation of
Z-form DNA. The CD spectrum of conjugate 13 (Figure 5c),
which possesses only noncanonical G:G base pairs, is similar
to that of the poly(T:A) hairpin 4. This is also the case for
conjugates 14 and 15, which possess one and two G:G base
pairs, respectively. At higher concentrations, the CD spectrum
of 13 is more complex, indicative of the formation of G-
quadruplex structures; however, the CD spectra of 14 and 15
are not concentration dependent.
Figure 4. Thermal dissociation profiles for the Sd2-linked conjugates 2-5.
Table 2. Melting Temperatures for Stilbene Diether-Linked
Hairpinsa
hairpin
Tm, °C
78.4
30 (33)
46.7 (48.9)
54.0 (60.8)
30 (33)
64.2 (73.1)
53
hairpin
Tm, °C
1
2
3
4
c-4
5
6
7
8
10
11
12
13
14
15
16
17
18
72.4
>80
>80
48.8
53.4
49.8
46.5
47.7
50.2
66.3
48.3
9
56.6
a Values for 2 × 10-6 M conjugates in standard buffer (10 mM sodium
phosphate, pH 7.2) containing 0.1 M NaCl or 1.0 M NaCl (values in
parentheses).
Conjugate Fluorescence and Photoisomerization. The
hairpins 1-12 are very weakly fluorescent (Φf < 10-3). The
weak fluorescence is attributed to quenching of the strong
stilbene diether fluorescence (Table 1) by the neighboring A:T
or G:C base pair.4 In contrast, relatively strong fluorescence is
observed for the hairpins 13-17, which possess one or more
G:G base pairs adjacent to the stilbene diether linker. The
fluorescence excitation and emission spectra of 13 (Figure 6)
are similar to those of the stilbene diether linkers in methanol
solution (Figure 1). Fluorescence data for 13-17 are sum-
significantly lower than those of 4, in 1.0 M NaCl (33 °C), 0.1
M NaCl (30 °C), and 30% ethanol-water (26 °C).
Conjugates 1 and 10 possess a combination of G:C and A:T
base pairs. Their melting transitions are above 70 °C in 0.1 M
aqueous NaCl. Since these conjugates are related to conjugate
5 by the replacement of two A:T base pairs with G:C base pairs
in the case of 1 and by the addition of one G:C base pair in the
case of 10, it is not surprising that their Tm values are appreciably
higher than that of 5 (Table 2). Conjugates 11 and 12 possess
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DNA Hairpins with Stilbene Diether Linkers
A R T I C L E S
Figure 6. Fluorescence excitation and emission spectra of conjugate 13 in
standard buffer.
Table 3. Fluorescence Data for Hairpins with G:G Base Pairsa
hairpin
Φflb
τs, nsc
13
14
15
16
17
0.17 ( 0.01
0.12 ( 0.02
0.19 ( 0.01
0.24 ( 0.01
0.24 ( 0.01
0.92 (96%), 4.1 (4%)
0.32 ( 0.1
0.53 ( 0.03
0.69 ( 0.05
0.69 ( 0.05
a Data for deoxygenated 2 × 10-6 M solutions in standard buffer.
b Fluorescence quantum yields determined using quinine sulfate actinometry.
c Single- or double-exponential decay times (preexponentials in parentheses).
Quantum yields for reversible isomerization of 4 and c-4 are
Φtc ) 0.19 and Φct ) 0.10. No isomerization is observed when
4 is irradiated in standard buffer (0.1 M NaCl, 10 mM sodium
phosphate, pH 7.2). In contrast, irradiation of c-4 results in
quantitative (>95%) conversion to 4 under conditions where it
is either partially denatured (30% ethanol-water, 25 °C) or fully
base-paired (0.1 M NaCl, 25 °C, Φct ) 0.17). Trans-cis
isomerization results in bleaching of the 327 nm band assigned
to the trans-stilbene diether linker. Thus, the occurrence of
isomerization can be qualitatively evaluated by monitoring the
327 nm absorption during irradiation. During the time required
for quantitative conversion of c-4 to 4 in 0.1 M NaCl, no
bleaching of the 327 band is observed for hairpins with A:T or
G:C base pairs adjacent to the Sd linkers. However, rapid
bleaching of the 327 band is observed for hairpins 13 and 17,
both of which have adjacent G:G base pairs.
Discussion
Figure 5. Circular dichroism spectra of 2 × 10-6 M conjugates in aqueous
buffer (0.1 M NaCl, 10 mM sodium phosphate): (a) conjugates 2-4; (b)
conjugates 11 and 12; (c) conjugates 13-15.
Stilbene Diethers. The absorption and fluorescence spectra
(Figure 1), fluorescence quantum yields, and fluorescence decay
times of the stilbene diethers Sd2-4 (Table 1) are similar to
marized in Table 3. Fluorescence quantum yields and decay
times for hairpins 16 and 17 are similar to the values for the
diol Sd2 in methanol solution. Both quantum yields and decay
times decrease for 15 and 14, which have fewer G:G base pairs.
Dual exponential fluorescence decay is observed for hairpin 13;
however, the short-lived component accounts for 96% of the
fluorescence intensity and has a decay time similar to those of
16 and 17.
those of 4,4′-dimethoxystilbene in methanol solution (Φfl
)
0.20, τfl ) 0.42 ns).9 Thus, the hydroxyalkyl groups have little
effect on the photophysical properties of the stilbene diethers
in methanol solution. The stilbene diethers undergo reversible
photoisomerization in methanol solution. The sum of the
isomerization (eq 1) and fluorescence quantum yields is 0.93
( 0.1, in accord with the usual mechanism for stilbene
isomerization via a twisted singlet intermediate.11 The absorption
spectra of the stilbene diethers is broadened and its maximum
As described above, irradiation of denatured 4 (50% ethanol-
water, 25 °C) results in formation of a mixture of 4 and c-4.
9
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A R T I C L E S
Lewis et al.
Table 4. Thermodynamic Parameters for Hairpin Formationa
blue-shifted in aqueous solution, whereas the fluorescence
spectrum is red-shifted (Figure 1). These changes are similar
to those observed by Whitten and co-workers for stilbene
aggregates in Langmuir-Blodgett (LB) films and attributed to
exciton coupling in the H-aggregates.12
We assume that the stilbene diethers form laminar aggregates,
similar to those of known bolaamphiphiles possessing aromatic
cores and pendant hydroxyalkyl groups.13 The fluorescence
quantum yields of the stilbene aggregates in aqueous solution
are lower than those of the isolated molecules in methanol
solution; however, the fluorescence decay times are longer in
aqueous solution (Table 1). The increased lifetimes can be
attributed to an increased barrier for torsion about the stilbene
double bond in the stilbene diether aggregates. The stilbene
aggregates display remarkable thermal stability. Fluorescence
melting studies indicate that the transition from aggregate to
monomer occurs above 75 °C.
Hairpins with T:A Base Pairs. The thermal dissociation
profiles and CD spectra of conjugates 2-9 indicate that they
form hairpin structures with T:A base pairing between their
polyT and polyA arms. Their CD spectra (Figure 5a) show
negative and positive peaks near 250 and 283 nm, characteristic
of B-DNA.14 Even conjugate 2, which can form only two T:A
base pairs, displays significant hyperchromism with a derivative
at 33 °C (Figure 4) and a room-temperature CD spectrum
(Figure 5a) similar to those of its homologues 3-5. Increasing
the number of T:A base pairs in the series 2-5 results in an
increase in the value of Tm (Table 2) and sharpening of the CD
spectra (Figure 5a).
The Tm values of the hairpins 2-5 (Table 2) are higher than
those of the analogous stilbenediamide-linked hairpins (Chart
1) reported by Letsinger and Wu.2 Thus, the stilbene diether
linkers form the most stable synthetic polyA-linker-polyT
hairpins reported to date. To our knowledge, no DNA or RNA
oligonucleotides have been reported to form hairpin structures
with short poly(T:A) stems.15 The exceptional stability of the
Sd2-linked hairpins is, no doubt, related to the compact structure
of the linker, as observed in the crystal structure of hairpin 1 in
which the average plane-to-plane separation between the stilbene
linker chromophore and adjacent G:C base pair is 3.25 Å,
somewhat shorter than the 3.4 Å average base stacking distance
in B-form DNA.4 The π-stacking interaction can account for
the red-shifted absorption of the linker chromophore (Figure
3) and the weak negative circular dichroism observed between
300 and 350 nm (Figure 5a). In view of the crystal structure of
hairpin 1 it is somewhat surprising that the Sd2-linked hairpins
display neither a strong induced CD spectrum nor a Cotton
effect.16 One possible explanation for the absence of strong
induced circular dichroism is that the dihedral angle between
−∆G°298
kcal/mol
,
−∆H°,
kcal/mol
−∆S°,
cal/(mol‚K)
hairpin
solvent
Tm,b °C
4
0.1 M NaCl
water
54.0
50.8
37.6
26.1
48.3
48.8
53.4
2.5
1.5
1.2
0.08
1.5
2.8
2.5
23.2
18.4
29.2
22.9
21.1
37.4
28.5
69.5
56.9
93.8
76.3
65.7
116
30% EtOH-water
30% EtOH-water
0.1 M NaCl
0.1 M NaCl
0.1 M NaCl
c-4
8
13
14
87.1
a Determined from absorption data using the method of ref 19. b Data
from Table 2.
the transition moments of the stilbene and adjacent base
chromophores is near 0 or 180°, in which case the amplitude
of the Cotton effect will vanish.17
The Tm values for hairpins 4 and 6, which possess four A:T
base pairs, and 5 and 7, which possess six A:T base pairs, are
similar (Table 2). Thus, increasing the length of the alkane
chains in the stilbene diether linkers from two to three
methylenes has little effect on the thermal stability of the hairpin.
In contrast the Tm value for hairpin 8, which possesses a Sd4
linker, is lower than that of either 4 or 6; however, its CD
spectrum is similar to that of 4. The minimized structure of 8
(Figure 2d) possesses a gauche-gauche turn in one of the
tetramethylene linkers. The Tm value for hairpin 9, which
possesses a Sd4 linker, is also significantly lower than that of
either 5 or 7, which possess Sd2 or Sd3 linkers, respectively.
The Tm values of 8 and 9 are remarkably similar to those of the
corresponding Sa-linked hairpins, which have the same number
of atoms separating the two hydroxyl groups (Chart 1).2 Thus,
it appears that the geometry of the linker rather than its donor
or acceptor properties determines the thermal stability of these
synthetic hairpins. It is interesting to note that the melting
temperatures of DNA hairpins with polyT loops decrease with
the length of the loop.18
Thermodynamic parameters for formation of DNA hairpins
can be readily obtained from analysis of the absorption spectral
data using the method of Marky and Breslauer19 in cases where
the low- and high-temperature plateaus of the melting curve
can be determined. This is not possible for conjugates 2 and 3
for which the melting transition occurs over a broad temperature
range (Figure 4) or for conjugates with melting temperatures
over 60 °C for which the high-temperature plateau is not
resolved. Thermodynamic parameters for conjugates 4, c-4, and
8 are reported in Table 4. The values of Tm for 4 decrease, and
∆G becomes less favorable, as expected, as the solvent is
changed from 0.1 M NaCl to water or ethanol-water.15
Reducing the salt concentration results in a less favorable ∆H°,
which is partially compensated by a more favorable ∆S°.
Addition of ethanol makes ∆H° more favorable and ∆S° less
favorable, as previously reported by Hickey and Turner20 for
RNA duplex formation. The lower melting temperature of c-4
versus 4 in 30% ethanol-water is largely a consequence of less
favorable entropy, in accord with the distorted hairpin structure
of c-4 (Figure 2c). The lower melting temperature and less
(11) (a) Saltiel, J.; Sun, Y.-P. In Photochromism, Molecules and Systems; Duerr,
H., Boaus-Laurent, H., Eds.; Elsevier: Amsterdam, 1990; pp 64-164. (b)
Waldeck, D. H. Chem. ReV. 1991, 91, 415-436.
(12) (a) Whitten, D. G. Acc. Chem. Res. 1993, 26, 502-509. (b) Song, X.;
Geiger, C.; Vaday, S.; Perlstein, J.; Whitten, D. G. J. Photochem. Photobiol.,
A 1996, 102, 39-45.
(13) (a) Hentrich, F.; Tschierske, C.; Diele, S.; Sauere, C. J. Mater. Chem. 1994,
4, 1547-1558. (b) Ko¨lbel, M.; Beyersdorff, T.; Cheng, X. H.; Tschierske,
C.; Kain, J.; Diele, S. J. Am. Chem. Soc. 2001, 123, 6809-6818.
(14) Bloomfield, V. A.; Crothers, D. M.; Tinoco, I. Nucleic Acids. Structures,
Properties, and Functions; University Science Books: Sausalito, CA, 2000.
(15) Turner, D. H. In Nucleic Acids. Structures, Properties, and Functions;
Bloomfield, V. A., Crothers, D. M., Tinoco, I., Eds.; University Science
Books: Sausalito, CA, 2000.
(17) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy; University
Science Books: Mill Valley, CA, 1983.
(18) Hilbers, C. W.; Haasnoot, C. A. G.; de Bruin, S. H.; Joordens, J. J. M.;
Van Der Marel, G. A.; Van Boom, H. H. Biochemie 1985, 67, 685-695.
(19) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601-1620.
(20) Hickey, D. R.; Turner, D. H. Biochemistry 1985, 24, 2086-2094.
(16) Ardhammar, M.; Kurucsev, T.; Norde´n, B. In Circular Dichroism; Berova,
N., Nakanishi, K., Woody, R. W., Eds.; Wiley: New York, 2000.
9
12170 J. AM. CHEM. SOC. VOL. 124, NO. 41, 2002

DNA Hairpins with Stilbene Diether Linkers
A R T I C L E S
favorable free energy for hairpin formation of 8 versus 4 is seen
to be a consequence of less favorable enthalpy rather than an
increase in entropy. The presence of a gauche-gauche turn in
the minimized structure for 8 (Figure 2d) could account for its
smaller (less negative) value of ∆H°.
Freshly deprotected dilute solutions of conjugate 13 form a
monomeric hairpin structure in 0.1 M NaCl solution. The hairpin
displays reversible melting, with a Tm (48.8 °C) similar to that
reported for d(GGGA)n oligomers.26 The CD spectrum of 13
(Figure 5c) is similar to that of the poly(T:A) hairpins (Figure
5a), which adopt B-DNA structures. Upon being annealed at
80 °C followed by slow cooling to room temperature, conjugate
13 forms an aggregate structure assigned to a parallel quadruplex
on the basis of its CD spectrum.27 The quadruplex does not
display a melting transition below 85 °C. On the basis of this
preliminary evidence, it seems likely that the monomeric hairpin
is the kinetic product and the parallel quadruplex the thermo-
dynamic product of base-pairing in 13. The thermodynamic data
for 13 (Table 4) indicate that it forms a slightly more stable
hairpin than the poly(T:A) hairpin 4 as a consequence of a more
favorable enthalpy of formation which is largely compensated
by a less favorable entropy of formation. The more favorable
enthalpy may reflect more extensive π-stacking overlap for the
G:G vs T:A steps.
Introduction of a single G:G mismatch in hairpin 14 results
in a lowering of Tm compared to that of hairpin 5 (53.4 vs 64.2
°C, Table 2). This decrease is similar to that observed for RNA
duplex structures upon substitution of a G:G for a G:C base
pair.25 Thermodynamic parameters for 14 are reported in Table
4. The values of Tm and ∆G° are similar to those for the poly-
(T:A) hairpin 4, which has two fewer T:A base pairs and no
G:G base pair. The more favorable ∆H° for 14 vs 4 is
compensated for by the less favorable ∆S°. Substitution of a
G:G base pair for a G:C base pair in RNA duplexes also results
in compensating changes in ∆H° and ∆S°.25 Smaller decreases
in Tm are observed upon introduction of a second or third G:G
tandem mismatch in hairpins 15 and 16. The Tm of 17 which
has four tandem mismatches is slightly higher than that of 16
and is similar to that of 13 which also has four G:G base pairs
but lacks a poly(T:A) stem region. Thus, the stability of the
G:G containing hairpins appears to be determined primarily by
the structure of the hairpin loop region.
Hairpins with G:C Base Pairs. Hairpins 1 and 10-12 have
melting temperatures >70 °C in 0.1 M NaCl (Table 2). The
presence of G:C base pairs in 1 and 10 results in a more stable
hairpin than the poly(T:A) hairpin 5, which has a Tm ) 64.2
°C. Hairpins 11 and 12 can form two or three G:C base pairs,
respectively. An analogue of 11 possessing a stilbenediamide
linker (Chart 1) connecting two G:C base pairs has a broad
thermal dissociation profile with a Tm ∼ 60 °C in aqueous 0.1
M NaCl.2 Thus, the Sd2-linked hairpin 11 is more stable than
the stilbenediamide-linked hairpin, as is the case for the poly-
(T:A) hairpins. Hairpin 11 is also more stable than DNA
minihairpins with 5′GCGAAGC and related sequences.21
The CD spectrum of 11 (Figure 5b) is similar to those of the
poly(A:T) hairpins which adopt B-form structures (Figures 5a).
The induced circular dichroism of the Sd2 linker in 11 is much
stronger than that in 2-5. This may reflect a stronger interaction
of the stilbene chromophore with an adjacent G:C than with an
adjacent A:T or better structural definition of the loop region
with an adjacent G:C. The stilbene-G:C π-stacking distance
observed in the crystal structure of hairpin 1 (Figure 2a) is, in
fact, remarkably short (3.25 Å).4 The CD spectrum of 12 (Figure
5b) is markedly different from those of 11 or the poly(A:T)
hairpins and is similar to that of the Z-DNA duplex formed by
the self-complementary hexanucleotide d(CGCGCG).14 Alter-
nating d(CG) sequences are commonly found to adopt Z-form
duplex and hairpin structures.22,23 While out-of-alternation
Z-DNA structures are less common, the sequence d(CCCGGG)
has been reported to adopt a Z-DNA structure.22 Hairpin 12
presumably can adopt a base-paired geometry similar to that of
d(CCCGGG) and thus provides the simplest model for a Z-DNA
structure reported to date.
Hairpins with G:G Base Pairs. G:G forms the most stable
mismatched base pair and is commonly encountered both in
DNA and RNA.24,25 The G:G base pair can adopt a number of
structures, the syn-anti (Hoogsteen) being the most general for
single G:G mismatches in B-DNA duplexes. While single G:G
mismatches do not distort the π-stacking of B-DNA, they do
distort the sugar geometry, leading to a decrease in duplex
stability. Tandem G:G mismatches in duplex DNA have not
been widely investigated due to the propensity of G-rich regions
to form G-quadruplex structures.25 An exception is provided
by d(GGGA)n oligomers studied by Huertas and Azor´ın,26 which
form stable intramolecular hairpin structures stabilized by
tandem G:G base pairs. These hairpins are moderately stable,
having Tm values between 40 and 50 °C in 50 mM NaCl. The
G:G base pair structure in these hairpins is not known; however,
the N7 group of one of the two guanines is most likely involved.
The CD spectra of 14 and 15 (Figure 5c) are similar to those
of the poly(T:A) hairpins and thus are assumed to adopt B-form
structures. No negative band for the stilbene diether chro-
mophore is observed around 330 nm. This may indicate that
the interaction between the stilbene diether and the adjacent
base pair is weaker for G:G than it is for G:C or T:A.
Introduction of a single G:G mismatch in B-DNA is known
to result in a significant increase in the helical width.24 This
suggests the possibility that the Sd2 linker may be too short to
optimally span the G:G mismatch in 14. Conjugate 18 possesses
an Sd4 linker which should be capable of spanning a wider
helix (Chart 1). However, the lower Tm value for hairpin 18 vs
14 (Table 2) suggests that the Sd4 linker may be longer than
needed for this purpose. It is interesting to note that the
difference in Tm values for 18 vs 14 (50.2 vs 53.4 °C) is smaller
than that for 9 vs 5 (56.6 vs 64.2 °C), suggesting that the
difference in stability for the shorter vs longer linker is smaller
in the case of the wider G:G-containing helix. It would be
interesting to determine if more stable hairpins possessing G:G
mismatches can be obtained using the Sd3 linker.
(21) (a) Varani, G. Annu. ReV. Biophys. Biomol. Struct. 1995, 24, 379-404.
(b) Yoshizawa, S.; Kawai, G.; Watanabe, K.; Miura, K.; Hirao, I.
Biochemistry 1997, 36, 4761-4767.
(22) Basham, B.; Eichman, B. F.; Ho, P. S. In Oxford Handbook of Nucleic
Acid Structure; Neidle, S., Ed.; Oxford: Oxford, UK, 1999.
(23) Chattophdhyaya, R.; Grzeskowiak, K.; Dickerson, R. E. J. Mol. Biol. 1990,
211, 189-197.
Hairpin Fluorescence and Photoisomerization. Unlike the
unmodified stilbene diether linkers which are strongly fluores-
(24) Skelly, J. V.; Edwards, K. J.; Jenkins, T. C.; Neidle, S. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 804-808.
(25) Burkard, M. E.; Turner, D. H. Biochemistry 2000, 39, 11748-11762.
(26) Huertas, D.; Azor´ın, F. Biochemistry 1996, 35, 13125-13135.
(27) Lewis, F. D.; Wu, Y.; Liu, X.; Zhang, L. Unpublished results.
9
J. AM. CHEM. SOC. VOL. 124, NO. 41, 2002 12171

A R T I C L E S
Lewis et al.
cent (Table 1), hairpins 1-12 are very weakly fluorescent (Φfl
< 10-2). For example, the fluorescence quantum yields for
hairpins 5 and 12 are 7 × 10-3 and 5 × 10-3, respectively.
Their fluorescence excitation and emission spectra are similar
to those for hairpin 13 (Figure 6). The weak fluorescence of
hairpins with adjacent T:A or G:C base pairs has been attributed
to electron-transfer quenching in which the stilbene diether
serves as an electron donor and either T or C as an electron
acceptor.4 The occurrence of fluorescence quenching via rapid
electron transfer is supported by picosecond time-resolved
transient absorption spectroscopy, which shows the rapid
conversion of the stilbene diether singlet to its cation radical.
The free energy of the photochemical electron-transfer process
can be estimated using Weller’s equation (eq 2), using the Sd2
singlet excitation energy (Es ) 3.45 V) and ground-state
oxidation potential (Eox ) 0.92 eV vs SCE in DMF solution)
and the nucleobase oxidation potentials (Erdn ) -2.26 and
-2.36 eV for dT and dC, respectively). In contrast to the
exergonic reduction of T or C, reduction of G (Erdn < -3.0
eV) is calculated to be endergonic using the single nucleotide
reduction potentials in nonaqueous solution. In accord with this
calculation, hairpins 13, 16, and 17, which have three or four
tandem G:G base pairs, have fluorescence quantum yields
similar to those of stilbene diether linkers and somewhat longer
singlet decay times (Tables 1 and 3). The lower values of Φfl
and τs for hairpins 14 and 15 may reflect quenching by the
nearest T:A base pair.
entropy of hairpin formation; (b) the preferred gauche confor-
mation of the flexible 1,2-dioxoethanes which connect the
stilbene to the sugar-phosphate backbone; (c) its total length
which matches the B-form helix diameter; (d) the π-stacking
interaction between the electron-rich stilbene and the electron-
poor adjacent base pair. The hairpin c-4, which possesses a
c-Sd2 linker (Figure 2c), is much less stable than hairpin 4, as
a consequence of the nonplanar stilbene which cannot form a
π-stacked complex with the adjacent A:T base pair and an end-
to-end distance that is shorter than that for the Sd2 linker. The
thermal stability of hairpins with poly(T:A) stems and Sd3
linkers are similar to those with Sd2 linkers. Hairpins possessing
Sd4 linkers are less stable, plausibly due to the less favorable
entropy for folding the longer polymethylene chains (Figure 2d).
All of the conjugates have CD spectra indicative of B-DNA
structures, except for 12, in which the G3-C3 stem evidently
adopts a left-handed Z-DNA structure.
The stilbene diether linkers also form stable hairpin structures
containing noncanonical G:G base pairs. To our knowledge, 13
is the first hairpin-forming conjugate reported to have a poly-
(G:G) stem. Annealing of 13 results in the formation of more
stable quadruplex structures. The formation of stable hairpins
is also observed for conjugates 14-18, which contain one or
more G:G base pairs between the linker and a poly(T:A) stem.
The fluorescence and photoisomerization of the stilbene diether
linker is strongly quenched by adjacent T:A or G:C base pairs
but not by G:G base pairs. Quenching is proposed to occur via
an electron-transfer mechanism in which the singlet stilbene
serves as an electron donor and T or C as an electron acceptor.
The dynamics of the charge injection process are currently under
investigation.
∆Get ) -(ES + Erdn) + Eox
(2)
Hairpin 4 undergoes moderately efficient photoisomerization
under denaturing conditions (50% ethanol-water) but not under
conditions where it forms a stable hairpin structure. The
photostability of 4 and other hairpins in which the Sd linker is
adjacent to an A:T or G:C base pair may reflect either very
rapid electron-transfer quenching or a well-ordered loop geom-
etry (Figure 2b) which inhibits photoisomerization. Irradiation
of c-4 under conditions where it forms a stable hairpin structure
results in one-way isomerization to yield 4 with a quantum yield
of 0.17. Incomplete quenching of the photoisomerization of
singlet c-4 might reflect a lower inherent barrier for isomer-
ization of cis- versus trans-stilbenes or the “looser” geometry
of the hairpin loop region of c-4. A looser loop geometry with
poor π-stacking between the cis-linker and adjacent base pair
should result both in slower electron-transfer quenching and in
a lower barrier to double bond torsion. Photoisomerization of
the Sd2 linker is also observed for hairpins possessing G:G base
pairs adjacent to the linker. Both inefficient electron-transfer
quenching and a distorted hairpin loop geometry may facilitate
the isomerization process.
Experimental Section
Bis(2-hydroxyalkyl)stilbene 4,4′-Diethers. The stilbene di-
ether Sd2 was prepared by the method of Sieber6 via the reaction
of 2-phenoxyethanol with chloroacetaldehyde dimethyl acetal
in 1:1 acetic acid-sulfuric acid followed by isolation of the
organic product and heating to 200-250 °C on a sand bath.
The resulting crystalline solid was recrystallized from pyridine
and sublimed prior to use. Sd3 and Sd4 were prepared by the
analogous reactions of 3-phenoxypropanol and 4-phenoxy-
butanol, respectively. The former was prepared from the reaction
of sodium phenoxide with 3-bromo-1-propanol, and the latter,
via the reduction of ethyl 4-phenoxyl-n-butyrate with lithium
aluminum hydride. Obtained in this fashion were the following:
Bis(2-hydroxyethyl)stilbene 4,4′-Diether (Sd2). Mp: 259
1
°C (lit.6 mp 254 °C). H NMR (DMSO-d6): δ 3.55 (m, 4H),
4.04 (t, 4H), 4.56 (t, 2H), 6.91 (d, 4H), 7.02 (s, 2H), 7.48 (d,
4H).
Bis(3-hydroxypropyl)stilbene 4,4′-Diether (Sd3). Mp: 236
1
°C (lit.6 mp 221 °C). H NMR (DMSO-d6): δ 1.86 (m, 4H),
Concluding Remarks
3.55 (t, 4H), 4.05 (t, 4H), 4.57 (t, <1H), 6.92 (d, 4H), 7.02 (s,
Bis(oligonucleotide) conjugates possessing a Sd2 stilbene
diether linker are capable of forming remarkably stable hairpins
with a variety of base-paired stems. Conjugates possessing only
two T:A or G:C base pairs have melting temperatures of 30
and >80 °C, respectively, in 0.1 M NaCl. On the basis of these
results, the Sd2 linker forms more stable hairpins than any
synthetic or natural hairpin loop reported to date. The thermal
stability of hairpins possessing a Sd2 linker is attributed to
several factors: (a) the rodlike stilbene which reduces the
2H), 7.48 (d, 4H).
Bis(4-hydroxybutyl)stilbene 4,4′-Diether (Sd4). Mp: 203
°C. 1H NMR (DMSO-d6): δ 1.56 (m, 4H), 1.75 (m, 4H), 3.47
(>4H overlapped with 1H in H2O), 3.98 (t, 4H), 4.46 (t, ∼2H),
6.91 (d, 4H), 7.02 (s, 2H), 7.48 (d, 4H).
Preparation of 4,4′-Dimethoxytrityl Derivatives. The stil-
bene diethers were converted to their mono(dimethoxytrityl)
(DMT) derivatives using the method of Letsinger and Wu.2
Obtained by this method in 25-40% yield were the following:
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12172 J. AM. CHEM. SOC. VOL. 124, NO. 41, 2002

DNA Hairpins with Stilbene Diether Linkers
A R T I C L E S
Sd2-Mono-DMT. 1H NMR (CD3Cl): δ 3.43 (t, 2H), 3.8 (s,
6H), 3.95 (t, 2H), 4.15 (m, 4H), 6.82 (d, 4H), 6.92 (m, 6H),
7.2-7.5 (m 13H).
spectroscopy using a Micromass Quattro II atomospheric
pressure ionizaton system. Conjugates were purified using a
Nensorb 20 nucleic acid purification cartridge prior to direct
loop injection.
1
Sd3-Mono-DMT. H NMR (CD3Cl): δ 2.1 (p, 4H), 3.3 (t,
Conjugate 1. MS (m/z): calcd for C135BrH165N44O76P12,
4071.65; found, 4071.1 ( 0.15.
Conjugate 5. MS (m/z): calcd for C138H158N42O76P12,
3992.70; found, 3992.61 ( 0.15.
Conjugate 13. MS (m/z): calcd for C98H108N40O52P8, 2933.68;
found, 2933.62 ( 0.12.
Methods. Methods employed for the recording of electronic
absorption and fluorescence spectra, determination of fluores-
cence quantum yields and lifetimes, recording of circular
dichroism spectra, and determination of molecular mechanics
calculations have been previously described.28 All spectral data
was obtained for solutions of 2 × 10-6 M hairpins in aqueous
sodium phosphate buffer (10 mM, pH 7.2) containing 0.1 M
NaCl, unless otherwise noted.
2H), 3.8 (s, 6H), 3.9 (t, 2H), 4.15, 7.45-6.88.
Sd4-Mono-DMT. 1H (CD3Cl): δ 1.8 (m, 4H), 1.88 (m, 4H),
3.15 (2H), 3.75 + 3.8 (8H), 3.96 (t, 2H), 4.05 (t, 2H), 6.94-
6.83 (m, 8H), 7.3-7.5.
Preparation of Phosphoramidite Derivatives. The DMT
derivatives were converted to their cyanoethyl-N,N-diisopropyl
phosphoramidite derivatives by the method of Letsinger and
Wu.2 Obtained by this method in 50-70% yield were the
following:
1
Sd2-Mono-DMT-Phosphoramidite. H NMR (CD3Cl): δ
1.2 (d, 12H), 2.6 (t, 2H), 3.45 (t, 2H), 3.65 (m, 2H), 3,80 (s,
6H), 4.18 (m, 4H), 6.9 (m, 10H), 7.2-7.5 (m, 13H). 31P NMR
(CD3Cl, H3PO4 as external standard): δ 149.87.
1
Sd3-Mono-DMT-Phosphoramidite. H NMR (CD3Cl): δ
1.2 (m, 12H), 2.1 (m, 4H), 2.65 (t, 2H), 3.30 (t, 2H), 3.60 (m,
2H), 3,80 (s, 6H), 3.85 (m, 2H), 4.16 (m, 4H), 6.8-6.95 (m,
10H), 7.35 (d, ∼6H), 7.45 (m, 7H). 31P NMR (CD3Cl, H3PO4
as external standard): δ 148.9 (s).
Bis(oligonucleotide) Conjugates. The preparation, purifica-
tion, and characterization of the bis(oligonucleotide) conjugates
1-18 followed the method of Letsinger and Wu2 as imple-
mented by Lewis et al.28 Molecular weights of selected
conjugates were determined by electrospray ionization mass
Acknowledgment. This research is supported by a grant from
the Division of Chemical Sciences, Office of Basic Energy
Sciences, U.S. Department of Energy, under Contract DE-FG02-
96ER14604. We thank Robert L. Letsinger for stimulating our
interest in hairpin-forming DNA conjugates.
JA026941O
(28) Lewis, F. D.; Wu, T.; Liu, X.; Letsinger, R. L.; Greenfield, S. R.; Miller,
S. E.; Wasielewski, M. R. J. Am. Chem. Soc. 2000, 122, 2889-2902.
9
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