Pyrene aromatic arrays on RNA duplexes as helical templates

Article abstract of DOI:10.1039/b705933g

RNA oligomers having multiple (2 to 4) pyrenylmethyl substituents at the 2′-O-sugar residues were synthesized. UV-melting studies showed that the pyrene-modified RNAs could form duplexes with complementary RNA sequences without loss of thermal stability.

Full text of DOI:10.1039/b705933g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Pyrene aromatic arrays on RNA duplexes as helical templates
Mitsunobu Nakamura,* Yukinori Shimomura, Yukinori Ohtoshi, Kazuhiro Sasa, Haruhisa Hayashi,
Hidehiko Nakano and Kazushige Yamana*
Received 19th April 2007, Accepted 8th May 2007
First published as an Advance Article on the web 18th May 2007
DOI: 10.1039/b705933g
RNA oligomers having multiple (2 to 4) pyrenylmethyl substituents at the 2ꢀ-O-sugar residues were
synthesized. UV-melting studies showed that the pyrene-modified RNAs could form duplexes with
complementary RNA sequences without loss of thermal stability. Absorption, fluorescence, and
circular dichroism (CD) spectra revealed that the incorporated pyrenes projected toward the outside of
A-form RNA duplexes and assembled in helical aromatic arrays along the minor grooves of the RNA
duplexes. Results of computer simulations agreed with the assembled structures of the pyrenes. The
helical pyrene arrays exhibited remarkably strong excimer fluorescence, which was dependent on the
sequence contexts of RNA duplexes.
molecular dynamics (MD) calculations, and CD simulations, and
we discuss the pyrene associations along the RNA duplexes.
DNA and RNA can be used to arrange chromophores in
Introduction
defined spaces and distances. Their self-assembling properties,
into duplexes, triplexes, and quadruplexes, may be useful in
forming specifically arranged structures.1–9 Such nanoscale space
arrangements are anticipated to be applicable in materials science
research and in biotechnology.
Results
RNA synthesis
The pyrene-modified RNAs and their complements that were
used are shown in Chart 1. Introduction of pyrene moieties (Upy,
Apy, and Cpy residues) into RNA sequences was carried out by
a conventional phosphoramidite method using protected phos-
phoramidite derivatives of 2ꢀ-O-(1-pyrenylmethyl)ribonucleosides.
Upy was synthesized as previously described.21 Apy and Cpy were
prepared from unprotected ribonucleosides and pyrenylmethyl
chloride using a base promoted reaction, as shown in Scheme 1.
The protected pyrene-modified ribonucleosides were converted by
a standard method into their phosphoramidite derivatives. The
pyrene-modified RNAs Pns (n = 1–4) have a Upy domain in the
middle of the strand. The RNAs Qns (n = 1–4) have a mixed
sequence of five bases at each end of rU10 containing the Upy
domain displaced 1 to 4 bases from the edge of the rU10. The
RNAs Rns (n = 1–3) have Upy, Apy, and Cpy units in the middle of
the strand.
Pyrene has attracted particular interest among various aromatic
chromophores because of the ease of synthetic conversion and
its high quantum efficiency in both monomer and excimer
fluorescence.10–12 Attempts to incorporate multiple pyrenes into
DNA and RNA chains have been made using organic or enzymatic
synthesis methods.13–19 p-Stacked structures between pyrenes can
be formed in double-stranded or even in single-stranded DNA,
which results in characteristic excimer fluorescence due to the
interaction between pyrenes in electronically excited states and
ground states. Such fluorescence may be utilized in homogeneous
biological assays.
We have previously reported that multiple-pyrene-modified
oligo(rU20)–oligo(rA20) duplexes at consecutive 2ꢀ-positions ex-
hibit unusually strong excimer fluorescence.20 The modified du-
plexes exhibit exciton-coupled CD signals in the region of the
pyrene absorption band (300 to 380 nm), whereas the single-
stranded pyrene-modified oligo(rU20) RNAs exhibit only negative
induced CD signals. On the basis of the CD and other spectral
data, we tentatively determined the pyrene structure to be a helical
arrangement of p-aromatics along the RNA double helix. How-
ever, the detailed structural features of multiple-pyrene-modified
RNA duplexes and the effects of RNA sequences on pyrene
association have not yet been determined. This determination
is necessary to gain insight into pyrene-modified RNAs for the
development of new materials and biotechnological applications.
We describe the structures of the multiple-pyrene-modified
RNA duplexes that were characterized by spectroscopic analyses,
Thermal dissociation
Helical stability of pyrene-modified RNA duplexes was evaluated
by UV-melting studies in a buffer of pH 7 containing 0.1 M
NaCl, 0.01 M NaHPO4, and 1 mM EDTA. The melting profiles
for all the pyrene-modified RNA duplexes exhibited single-phase
transitions similar to those of unmodified RNA duplexes. The
melting temperatures (Tm) of the pyrene-modified RNA duplexes
are summarized in Table 1. The Tm values of Pn-X differ by 1.5–
2.5 ◦C from those of corresponding unmodified RNA duplex
P0-X. Qn-Ys (n = 1–4) melt at temperatures 2.0–3.9 ◦C higher
than Q0-Y and Rn-Zs (n = 1–3) and melt at temperatures 3.9–
7.8 ◦C lower than R0-Z. Pyrene-modified RNA duplexes are
therefore stable under conditions for structural observation at
Department of Materials Science and Chemistry, Graduate School of
Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo, 671–2201,
Japan. E-mail: mitunobu@eng.u-hyogo.ac.jp, yamana@eng.u-hyogo.ac.jp;
Fax: (+81) 79-267-4928; Tel: (+81) 79-267-4928
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Chart 1 Sequences of pyrene-modified RNAs.
Scheme 1
Table 1 Tm values for pyrene-modified RNA duplexesa
Table 2 Absorption maximums (kmax) of pyrene-modified RNAsa
Pn-X
Tm/◦C
Qn-Y
Tm/◦C
Rn-Z
Tm/◦C
RNA
k
max/nm
RNA
kmax/nm
RNA
kmax/nm
P0-X
P1-X
P2-X
P3-X
P4-X
45.0
46.5
47.5
46.6
42.7
Q0-Y
Q1-Y
Q2-Y
Q3-Y
Q4-Y
50.0
53.9
53.9
52.9
52.0
R0-Z
R1-Z
R2-Z
R3-Z
73.7
69.8
68.9
65.9
P1
P1-X
P2
P2-X
P3
348
353
348
347
349
349
349
350
348
352
Q1
Q1-Y
Q2
Q2-Y
Q3
Q3-Y
Q4
Q4-Y
349
352
349
350
350
350
349
349
R1
R1-Z
R2
R2-Z
R3
R3-Z
349
352
348
345
349
345
P3-X
P4
a Measurements were carried out at 260 nm for a 1 : 1 mixture of
oligon◦ucleotides with increase in temperature from 20 to 80 ◦C at a rate
of 0.5 C min−1. Buffer contained 0.01 M sodium phosphate, 0.1 M NaCl,
and 1 mM EDTA·2Na adjusted to pH 7.0. The concentration of RNA
was 5 × 10−5 M.
P4-X
P2ꢀ
P2ꢀ-X
a Absorption spectra were measured at room temperature in a buffer
containing 0.01 M sodium phosphate, 0.1 M NaCl, and 1 mM EDTA·2Na
adjusted to pH 7.0. The concentration of RNA was 5 × 10−5 M.
room temperature. The introduction of multiple pyrenes barely
affected the thermal stability of the RNA duplexes.
Absorption spectra
the absorption maximums of the pyrene 1La transition. The single-
stranded pyrene-modified RNAs (Pns) exhibited an absorption
maximum at a wavelength of 348 to 349 nm. Upon hybridization
with complementary RNA, the absorption maximum of P1 shifted
by about 5 nm to a lower energy. In contrast, the pyrene absorption
maximum of Pns (n = 2–4) barely shifted upon hybridization.
In the absorption spectra of pyrene-modified RNAs, a short-
wavelength (about 260 nm) absorption band attributed to the
overlapping of nucleobases and pyrenes, and a long-wavelength
(300–370 nm) absorption band attributed to the 0–0 absorption for
the 1La transition of pyrene, were observed.22,23 Table 2 summarizes
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Similarly, the absorption maximum of single-stranded Q1
appeared at 349 nm and shifted by about 3 nm to a lower energy
upon hybridization with Y. The absorption of single-stranded Qns
(n = 2–4) and double-stranded Qn-Ys (n = 2–4) appeared near
349 nm. In the cases of Rns, the absorption maximum of R1 shifted
to longer wavelengths, but the absorption maximum of R2 and R3
shifted to shorter wavelengths upon hybridization with Z.
When two pyrenes were separated by one nucleotide in Pꢀ2, the
pyrene absorption band appeared at 348 nm and shifted to 352 nm
upon hybridization.
Fig. 2 (a) Fluorescence spectra of Rns (n = 1–3). (b) Fluorescence spectra
of Rn-Zs (n = 1–3). The measurements were carried out at a strand
concentration of 2.5 × 10−6 M at room temperature in a buffer containing
0.01 M sodium phosphate, 0.1 M NaCl, and 1 mM EDTA·2Na adjusted
to pH 7.0. The excitation wavelength was 350 nm.
Fluorescence spectra
In the fluorescence spectra of Pn-Xs reported previously,20 the
monomer intensity of P1-X was about 20 times higher than that
of P1, and the excimer intensities of Pn-Xs (n = 2–4) were about 1
to 10 times higher than that for single-stranded Pns (n = 2–4).
The fluorescence behavior of Qns and Rns was similar to that
of Pns. The mono-pyrene-modified Q1 exhibited only monomer
fluorescence in both single-stranded and double-stranded forms,
and the monomer fluorescence intensity in double-stranded Q1-
Y was about 10 times higher than that in single-stranded Q1.
The multiple-pyrene-modified Qns (n = 2–4) exhibited weak
monomer and excimer fluorescence. The double-stranded Qn-
Ys (n = 2–4) also exhibited monomer and excimer fluorescence
similar to corresponding Qns (n = 2–4) in spectral features, but
their fluorescence intensities were about 2 times higher than that
of Qns (Fig. 1). Similarly, the fluorescence intensities of Rn-Zs
were about 1 to 10 times higher than that of Rn (Fig. 2).
Fig. 3 Fluorescence spectra of P2ꢀ and P2ꢀ-X (strand concentration of
2.5 × 10−6 M) at room temperature in a buffer containing 0.01 M sodium
phosphate, 0.1 M NaCl, and 1 mM EDTA·2Na adjusted to pH 7.0. The
excitation wavelength was 350 nm.
Pn-Xs (n = 2–4) exhibited CD signals due to right-handed A-
form RNA below 300 nm and positive Cotton effects attributed to
exciton coupling between pyrenes in the region of 300 to 360 nm.24
The profiles of the exciton coupled CD signals for Pn-X (n = 2–4)
were very similar to each other, and the intensities of the signals
increased with the number of incorporated pyrenes.
Fig. 4 shows the CD spectra of Qn-Ys (n = 1–4) and Rn-Zs
(n = 1–3) at room temperature. The CD profiles below 300 nm for
all the pyrene-modified duplexes were similar to that of a typical
right-handed A-form RNA. The exciton-coupled CD signals in
Fig. 1 (a) Fluorescence spectra of Qns (n = 1–4). (b) Fluorescence spectra
of Qn-Ys (n = 1–4). The measurements were carried out at a strand
concentration of 2.5 × 10−6 M at room temperature in a buffer containing
0.01 M sodium phosphate and 0.1 M NaCl, adjusted to pH 7.0. The
excitation wavelength was 350 nm.
The fluorescence behavior of Pꢀ2 was different from that of
other multiple-pyrene-modified RNAs. Monomer and excimer
fluorescence were observed in single-stranded Pꢀ2. The monomer
fluorescence intensity was more than 10 times greater, however,
the excimer fluorescence disappeared upon hybridization with Z
(Fig. 3).
Circular dichroism spectra
CD spectroscopic analyses of the pyrene-modified RNAs were
performed to investigate structural preferences. The CD spectra of
Pns (n = 1–4) exhibited positive CD signals at wavelengths shorter
than 300 nm and negative induced CD signals in the region of 300
to 360 nm.20 On the other hand, the CD spectra of double-stranded
Fig. 4 (a) CD spectra of Qn-Ys (n = 1–4). (b) CD spectra of Rn-Zs (n =
1–3). The measurements were carried out at a strand concentration of
2.5 × 10−6 M at room temperature in a buffer containing 0.01 M sodium
phosphate, 0.1 M NaCl, and 1 mM EDTA·2Na adjusted to pH 7.0.
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the region of 300 to 360 nm were observed for multiple-pyrene-
modified Qn-Ys (n = 2–4) and Rn-Zs (n = 2–3).
structure, the center-to-center distances between two adjacent
pyrenes changed considerably.
The experimentally observed CD assigned to the exciton cou-
pling among the pyrenes in P4-X was theoretically evaluated using
the exciton-chirality method,27,28 where the structural parameters
for P4-X were obtained from the MD minimization. The overall
CD was estimated as a sum of exciton interactions between each
pair of pyrenes, yielding the simulated CD shown in Fig. 6.
This spectrum closely resembled the shape of the experimentally
observed CD. Therefore, the CD simulations strongly suggest that
the covalently attached pyrenes can associate helically along the
minor groove of the RNA duplex.
Discussion
For the mono-pyrene-modified RNAs, the pyrene absorption band
shifted to longer wavelengths and the pyrene-monomer fluores-
cence was enhanced upon hybridization. These results indicate
that the pyrene was free of interactions with nucleobases and
that the environment changed from hydrophobic to hydrophilic by
hybridization. Hence, the pyrene was located outside of the double
helix without intercalation.25 For the multiple-pyrene-modified
RNA, the excimer fluorescence was enhanced upon hybridization,
and the pyrene absorption band in double-stranded form appeared
at shorter wavelengths than that of the corresponding mono-
pyrene-modified RNA duplex. These spectroscopic behaviors
suggest that the pyrenes in the multiple-pyrene-modified RNA
duplex were located outside the double helix and were associated
with each other as an H-aggregate, which induces hypsochromic
shift of the absorption band.2 On the other hand, the two
pyrenes were located outside the duplex, without association in
P2ꢀ-X, because the pyrene absorption band appeared at longer
wavelengths than that of single-stranded P2ꢀ and the excimer
fluorescence observed in P2ꢀ disappeared upon hybridization.
We ran simulations of the MD of multiple-pyrene-modified
RNA duplexes to clarify the structural features of the assembled
pyrenes. The model structure for pyrene-modified RNA duplexes
was constructed based on an A-form RNA duplex using the
sequence of P4-X. The modified duplex was then optimized,
thermalized (300 K), and equilibrated using an AMBER module.26
Fig. 5 shows the energy-minimized structure of P4-X. It can be
seen that the four pyrenes of P4-X projected into the minor groove
and were associated approximately parallel with each other. The
average center-to-center distance between the two neighboring
Fig. 6 Simulated CD spectrum of P4-X obtained from the exciton-chi-
rality method using four Gaussians to simulate the absorption band of the
pyrenyl group.
Since the CD observation and the CD simulation revealed that
the exciton-coupled CD signal of the multiple-pyrene-modified
RNA duplexes depended on the number of associations and the
manner of association, the following is suggested for the pyrene
association in Qn-Y (n = 2–4) and Rn-Zs (n = 2–3) systems.
The shape of the exciton-coupled CD for Qn-Y (n = 2–4) was
similar to that for Pn-X (n = 2–4), indicating that the manner of
association of pyrenes in Qn-Y was similar to that in Pn-X. Hence,
the manner of association of pyrenes in consecutive Upy domains
was independent of the position of the domains and the context
sequence of the consecutive Upy domains. In the Rn-Z system, the
pyrene-modified duplex R2-Z containing Upy and Cpy also showed
similar exciton-coupled CDs to those of P2-X and Q2-X. This
indicates that the manner of association of pyrenes in the CpyUpy
domain was similar to that in the UpyUpy domain because Upy and
Cpy have a pyrimidine nucleobase. On the other hand, the shape
of the exciton-coupled CD signal of R3-Z containing Upy, Cpy,
and Apy differed from those of other multiple-pyrene-modified
RNA duplexes. Because the manner of association of pyrene in
purine nucleotides such as Apy is probably different from that in
pyrimidine nucleotides, such as Upy and Cpy, the addition of Apy to
the CpyUpy domain will probably induce changes in exciton-coupled
CD signals. Further research is necessary to confirm this.
˚
pyrenes was 6.5 A. The MD simulations were then performed
with an appropriate force field in which the equilibrated structures
were subjected to 1 ns (1 000 000 steps) in the simulations at
constant temperature (300 K) and pressure (1 atm) with standard
relaxation times of 1 fs. We found that whereas the multiple-
pyrene-modified RNA duplex rigidly retained its original A-form
Conclusions
We demonstrated that multiple-pyrene-modified RNA duplexes
had pyrene structures in helical association along the minor
groove of the RNA duplexes. The pyrene association showed that
the exciton-coupled CD signals and pyrene excimer fluorescence
had intensities depending on RNA sequences and contexts.
These findings will improve insight into sequence design of
Fig. 5 MD minimized structure of P4-X.
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pyrene-modified RNAs for the development of new materials and
biotechnological applications.
5ꢀ -Dimethoxytrityl-2ꢀ -O-(pyren-1-ylmethyl)-N-benzoyl-adeno-
sine (3). 4,4ꢀ-Dimethoxytrityl chloride (0.43 g, 1.28 mmol) was
added to a solution of 2 (0.43 g, 0.73 mmol), which was dried
by coevaporation with pyridine three times, in pyridine (4.3 ml).
After stirring for 6 h at room temperature, the solution was
concentrated to near dryness. The residual material was dissolved
in dichloromethane (150 ml) and then washed with water. The
organic phase was dried over Na2SO4 and evaporated to near
dryness. The product 3 was purified with silica gel column
chromatography with dichloromethane containing methanol (9 :
1, CH2Cl2–MeOH, v/v). The appropriate fractions were pooled
and then evaporated to near dryness. The residual solution was
poured into cooled hexane and the precipitate was collected. Yield,
56% (0.29 g); TLC (9 : 1, CH2Cl2–MeOH, v/v), Rf 0.78; 1H-NMR
(DMSO-d6): 3.68 (s, 6H, OCH3), 4.19 (m, 1H, 4ꢀ-CH), 4.60 (m, 1H,
3ꢀ-CH), 4.96 (dd, 1H, 2ꢀ-CH), 5.26 (d, 1H, CH2-pyrene), 5.49 (d,
1H, CH2-pyrene), 5.60 (d, 1H, 3ꢀ-OH), 6.25 (d, 1H, 1ꢀ-CH), 6.79
(d, 4H, aromatic of DMT), 7.15–7.23 (m, total 7H, aromatic of
DMT), 7.33 (d, 2H, aromatic of DMT), 7.56 (dd, 2H, aromatic of
benzoyl), 7.62 (dd, 1H, aromatic of benzoyl), 7.99–8.26 (m, total
11H, aromatic of benzoyl and pyrene), 8.43 (s, 1H, adenine), 8.47
(s, 1H, adenine) and 11.08 (s, 1H, amide).
Experimental
General methods
1H NMR spectra were recorded on a Bruker DRX-500 spec-
trometer, in which chemical shifts (d ppm) were determined on
the basis of a residual peak of solvent (2.49 for DMSO-d6).
Absorption spectra in solution were recorded on a Hitachi U-3500
spectrophotometer, steady-state fluorescence spectra in solution
were measured on a Hitachi F-2500 spectrofluorometer using
excitation and emission slits of 5 nm and corrected. Thermal
denaturations of RNA duplexes were carried out using a Bechman
Coulter Du800. CD spectra were recorded on a JASCO 715
spectropolarimeter.
Syntheses of pyrene modified phosphoramidite monomer according
to Scheme 1
2ꢀ-O-(Pyren-1-ylmethyl)-adenosine (1). 1-Pyrenyl methyl chlo-
ride (1.3 g, 5.16 mmol) was added to a solution of adenosine
(2.1 g, 7.74 mmol) and NaH (0.28 g) in DMF (40 ml). The reaction
mixture was stirred overnight at room temperature and then 10 ml
of water were added to the solution. Dichloromethane (150 ml) was
then added to the solution and the solution was washed with water.
The organic layer was dried over Na2SO4 and then evaporated
to near dryness. The residual material was purified by silica
gel column chromatography with dichloromethane containing
methanol (9 : 1, CH2Cl2–MeOH, v/v). Yield, 23% (0.57 g); TLC
(9 : 1, CH2Cl2–MeOH, v/v), Rf 0.29; 1H-NMR (DMSO-d6): 3.59
(m, 1H, 5ꢀ-CH2), 3.78 (m, 1H, 5ꢀ-CH2), 4.07 (m, 1H, 4ꢀ-CH), 4.50
(m, 1H, 3ꢀ-CH), 4.79 (dd, 1H, 2ꢀ-CH), 5.16 (d, 1H, CH2-pyrene),
5.43 (d, 1H, CH2-pyrene), 5.52 (d, 1H, 3ꢀ-OH), 6.11 (d, 1H, 1ꢀ-
CH), 7.30 (s, 2H, NH2), 7.95–8.24 (m, total 9H, pyrene), 8.32 (s,
1H, adenine) and 8.51 (s, 1H, adenine).
5ꢀ -Dimethoxytrityl-2ꢀ -O-(pyren-1-ylmethyl)-N-benzoyl-adeno-
sine, 3ꢀ-{(2-cyanoethyl)-(N,N -diisopropyl)}-phosphoramidite (4).
2-Cyanoethyl-N,N,N ꢀ,Nꢀ-tetraisopropylphosphateamidite (0.07 ml,
0.2 mmol) was added to a solution of 3 (0.13 g, 0.15 mmol) and
tetrazole (0.01 g, 0.15 mmol) in dry dichloromethane (1 mL).
The solution was stirred for 2 h at room temperature and
then dichloromethane (5 ml) was added to the solution. The
solution was washed with 10% NaHCO3 aqueous solution,
dried over Na2SO4 and then evaporated to near dryness. The
product 4 was purified by silica gel column chromatography with
dichloromethane containing ethyl acetate and triethylamine (45 :
45 : 10, CH2Cl2–AcOEt–Et3N, v/v). Yield, 89% (0.31 g); TLC (45 :
45 : 10, CH2Cl2–AcOEt–Et3N, v/v), Rf 0.75.
2ꢀ-O-(Pyren-1-ylmethyl)-cytidine (5). 1-Pyrenyl methyl chlo-
ride (2.0 g, 8.0 mmol) was added to a solution of cytidine (2.9 g,
12.0 mmol) and NaH (0.43 g) in DMF (60 ml) at 0 ◦C. The reaction
mixture was stirred overnight at room temperature and then 10 ml
of water were added to the solution. Dichloromethane (150 ml) was
then added to the solution and the solution was washed with water.
The organic layer was dried over Na2SO4 and then evaporated
to near dryness. The residual material was purified by silica
gel column chromatography with dichloromethane containing
methanol (9 : 1, CH2Cl2–MeOH, v/v). Yield, 44% (1.62 g); TLC
(9 : 1, CH2Cl2–MeOH, v/v), Rf 0.33; 1H-NMR (DMSO-d6): 3.60
(m, 1H, 5ꢀ-CH2), 3.69 (m, 1H, 5ꢀ-CH2), 3.93 (m, 1H, 4ꢀ-CH), 4.07
(dd, 1H, 2ꢀ-CH), 4.19 (m, 1H, 3ꢀ-CH), 5.11 (m, 1H, 5ꢀ-OH), 5.22 (d,
1H, 3ꢀ-OH), 5.24 (d, 1H, CH2-pyrene), 5.39 (d, 1H, CH2-pyrene),
5.67 (d, 1H, cytosine), 6.06 (d, 1H, 1ꢀ-CH), 7.16 (s, 2H, NH2), 7.90
(d, 1H, cytosine) and 8.08–8.31 (m, total 9H, aromatic of pyrene).
2ꢀ-O-(Pyren-1-ylmethyl)-N-benzoyl-adenosine (2). Trimethyl-
silyl chloride (0.66 ml, 5.19 mmol) was added to a solution
of 1 (0.5 g, 1.04 mmol), which was dried by coevaporation
with pyridine three times, in pyridine (10 ml). After stirring for
30 min, benzoyl chloride (0.36 ml, 3.1 mmol) was added to the
solution. The solution was stirred for 7 h at room temperature, and
10 ml of 28% NH3 aqueous solution was added to the solution.
After stirring for 1 h, the solution was concentrated to near
dryness. The residual material was dissolved in dichloromethane
(150 ml) and was then washed with water. The organic phase
was dried over Na2SO4 and then evaporated to near dryness. The
product 2 was purified by silica gel column chromatography with
dichloromethane containing methanol (9 : 1, CH2Cl2–MeOH,
v/v). Yield, 93% (0.90 g); TLC (9 : 1, CH2Cl2–MeOH, v/v), Rf
1
0.56; H-NMR (DMSO-d6): 3.61 (m, 1H, 5ꢀ-CH2), 3.69 (m, 1H,
5ꢀ-CH2), 4.09 (m, 1H, 4ꢀ-CH), 4.53 (m, 1H, 3ꢀ-CH), 4.83 (dd, 1H,
2ꢀ-CH), 5.19–5.21 (m, total 2H, 5ꢀ-OH and CH2-pyrene), 5.50 (d,
1H, CH2-pyrene), 5.60 (d, 1H, 3ꢀ-OH), 6.23 (d, 1H, 1ꢀ-CH), 7.56
(dd, 2H, aromatic of benzoyl), 7.62 (dd, 1H, aromatic of benzoyl),
7.96–8.26 (m, total 11H, aromatic of benzoyl and pyrene), 8.54 (s,
1H, adenine), 8.59 (s, 1H, adenine) and 11.09 (s, 1H, amide).
2ꢀ-O-(Pyren-1-ylmethyl)-N-benzoyl-cytidine (6). Trimethylsi-
lyl chloride (0.69 ml, 5.45 mmol) was added to a solution of 5
(0.5 g, 1.1 mmol), which was dried by coevaporation with pyridine
three times, in pyridine (10 ml). After stirring for 30 min, benzoyl
chloride (0.38 ml, 3.3 mmol) was added to the solution. The
solution was stirred for 7 h at room temperature, and then 10 ml of
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28% NH3 aqueous solution was added to the solution at 0 ◦C. After
stirring for 1 h, the solution was concentrated to near dryness.
The residual material was dissolved in dichloromethane and then
washed with water. The organic phase was dried over Na2SO4 and
evaporated to near dryness. Silica gel column chromatography of
the residual material gave pure 6 with dichloromethane containing
methanol (9 : 1, CH2Cl2–MeOH, v/v) as eluent. Yield, 47%
MD simulation
The modified nucleoside, 2ꢀ-O-(1-pyrenylmethyl)uridine, was con-
structed using a support program for AMBER on the basis of our
originally developed molecular graphics program. Formal charges
for the modified nucleoside were calculated by MOPAC, and each
atom in the pyrenyl residue was defined for molecular model
building using “parm94” for AMBER.26 The starting model for
P4-X was optimized, thermalized (300 K) and equilibrated before
MD simulation. The equilibrated structures were neutralized by
adding a suitable number of Na+ ions and solvated in rectangular
boxes containing about 5400 of TIP3P water molecules such that
the minimum distance from the duplex to the periodic boundary
1
(0.29 g); TLC (9 : 1, CH2Cl2–MeOH, v/v), Rf 0.49; H-NMR
(DMSO-d6): 3.63 (m, 1H, 5ꢀ-CH2), 3.75 (m, 1H, 5ꢀ-CH2), 4.01 (m,
1H, 4ꢀ-CH), 4.16 (dd, 1H, 2ꢀ-CH), 4.24 (m, 1H, 3ꢀ-CH), 5.21 (m,
1H, 5ꢀ-OH), 5.32 (d, 1H, 3ꢀ-OH), 5.43 (d, 1H, CH2-pyrene), 5.52
(d, 1H, CH2-pyrene), 6.12 (d, 1H, 1ꢀ-CH), 7.44 (d, 1H, cytosine),
7.52 (dd, 2H, aromatic of benzoyl), 7.62 (dd, 1H, aromatic of
benzoyl), 7.85 (d, 1H, cytosine), 8.00–8.30 (m, total 11H, aromatic
of benzoyl and pyrene) and 11.18 (s, 1H, amide).
˚
was 10 A. The MD simulation of P4-X was performed using the
“sander” module of the AMBER 7.0 software package with an
appropriate force field.
5ꢀ-Dimethoxytrityl-2ꢀ-O-(pyren-1-ylmethyl)-N-benzoyl-cytidine
(7). 4,4ꢀ-Dimethoxytrityl chloride (0.36 g, 0.82 mmol) was added
to a solution of 6 (0.46 g, 0.82 mmol), which was dried by
coevaporation with pyridine three times, in pyridine (4.1 ml).
The solution was stirred for 6 h at room temperature and then
concentrated to near dryness. The residual material was dissolved
in dichloromethane and then washed with water. The organic
phase was dried over Na2SO4 and evaporated to near dryness. The
product 7 was purified with silica gel column chromatography with
dichloromethane containing methanol (9 : 1, CH2Cl2–MeOH,
v/v). The appropriate fractions were pooled and then evaporated
to near dryness. The residual solution was poured into cooled
hexane and then the precipitate was collected. Yield, 50% (0.35 g);
TLC (9 : 1, CH2Cl2–MeOH, v/v), Rf 0.88; 1H-NMR (DMSO-d6):
3.70 (s, 6H, OCH3), 4.11–4.18 (m, total 2H, 2ꢀ-CH and 4ꢀ-CH), 4.43
(m, 1H, 3ꢀ-CH), 5.44 (m, 1H, 3ꢀ-OH), 5.55 (s, 2H, CH2-pyrene),
6.13 (d, 1H, 1ꢀ-CH), 6.86 (d, 4H, aromatic of DMT), 7.21–7.30 (m,
total 9H, aromatic of DMT), 7.36 (d, 1H, cytosine), 7.53 (dd, 2H,
aromatic of benzoyl), 7.63 (dd, 1H, aromatic of benzoyl), 7.97 (d,
1H, cytosine), 8.02–8.32 (m, total 11H, aromatic of benzoyl and
pyrene) and 11.21 (s, 1H, amide).
Theoretical CD calculation
In this work, the absorption bands of pyrene were deconvoluted
into four vibronic peaks. Each vibronic peak was regarded as
a separate transition and fitted with a Gaussian function.29 The
transition dipole moment for the La band was assumed to be
polarized in the direction of the long axis as an La band and
1
1
1
to be common among all the vibronic peaks in the La band.
The magnitude of the transition dipole moment for each vibronic
peak was determined from UV absorption. Each transition dipole
moment vector was placed in the center of the pyrene ring.
The mutual orientation of the transition dipole moments of the
neighboring pyrene groups and their positions were determined
from the geometries obtained by the MD minimized structure.
Only the interactions between the like transitions were taken into
consideration and the overall CD was estimated as a sum of exciton
interactions between various pairs of pyrenes.
Acknowledgements
The authors thank Prof. Yoshihisa Inoue and Dr Hideo Nishino
for measurements of CD spectra. This research was supported by
a Grant-in-Aid for Scientific Research from JSPS.
5ꢀ-Dimethoxytrityl-2ꢀ-O-(pyren-1-ylmethyl)-N-benzoyl-cytidine,
3ꢀ-{(2-cyanoethyl)-(N,N -diisopropyl)}-phosphoramidite (8). 2-
Cyanoethyl-N,N,N ꢀ,Nꢀ-tetraisopropylphosphateamidite (0.17 ml,
0.53 mmol) was added to a solution of 7 (0.35 g, 0.41 mmol)
and tetrazole (0.03 g, 0.41 mmol) in dry dichloromethane
(1 mL). The solution was stirred for 2 h at room temperature
and then dichloromethane (5 ml) was added to the solution.
The solution was washed with 10% NaHCO3 aqueous solution,
dried over Na2SO4 and then evaporated to near dryness. The
product 8 was purified by silica gel column chromatography with
dichloromethane containing ethyl acetate and triethylamine (45 :
45 : 10, CH2Cl2–AcOEt–Et3N, v/v). Yield, 88% (0.39 g); TLC
(45 : 45 : 10, CH2Cl2–AcOEt–Et3N, v/v), Rf 0.67.
Notes and references
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RNA syntheses
All the RNAs were prepared by conventional phosphoramidite
chemistry using an automated DNA synthesizer. After the
recommended workup, they were purified by reversed-phase
HPLC and characterized by UV–vis absorption and fluorescence
spectroscopy.
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