Syntheses and fluorescence of RNA conjugates having pyrene-modified adenosine and nitrobenzene-modified uridine base pairs

Article abstract of DOI:10.1016/j.tetlet.2010.01.081

We prepared RNA duplexes possessing 2′-O-(1-pyrenylmethyl)adenosine and 5-(4-nitrophenyl)uridine base pairs. In the duplexes, pyrene serves as a photo-excitable electron donor and 5-(4-nitrophenyl)uridine acts as an electron acceptor. The donor-acceptor-modified RNA duplexes showed very weak fluorescence originating from the pyrene monomer and excimer emissions, which occur due to electron transfer from the excited pyrene to the nitrobenzene acceptor.

Full text of DOI:10.1016/j.tetlet.2010.01.081

Tetrahedron Letters 51 (2010) 1732–1735
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Syntheses and fluorescence of RNA conjugates having pyrene-modified
adenosine and nitrobenzene-modified uridine base pairs
*
*
Minoru Fukuda, Mitsunobu Nakamura , Tadao Takada, Kazushige Yamana
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan
a r t i c l e i n f o
a b s t r a c t
We prepared RNA duplexes possessing 20-O-(1-pyrenylmethyl)adenosine and 5-(4-nitrophenyl)uridine
base pairs. In the duplexes, pyrene serves as a photo-excitable electron donor and 5-(4-nitrophenyl)uri-
dine acts as an electron acceptor. The donor–acceptor-modified RNA duplexes showed very weak fluores-
cence originating from the pyrene monomer and excimer emissions, which occur due to electron transfer
from the excited pyrene to the nitrobenzene acceptor.
Article history:
Received 6 December 2009
Revised 21 January 2010
Accepted 25 January 2010
Available online 1 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Arranging
tances has attracted much research interest. Because the spatially
arranged -aromatic molecules exhibit unique photophysical and
electroconductive properties, it should be possible to use them in
many applications, such as in molecular electronic and photonic
devices.1 Bicontinuous electron donor and acceptor arrangements
are essential for photovoltaics.2
p
-aromatic molecules in defined spaces and dis-
the Pyrs are directed into the minor groove of the duplex, whereas
the NBs are positioned at the major groove. Figure 1 shows a sche-
matic illustration of the configuration of Pyr and NB of the APyr-UNB
base pair in an A-form duplex. In this configuration, photo-induced
charge transfer from an excited Pyr to an NB across the base pair of
the duplex is expected to occur.8 In addition, consecutive incorpo-
ration of Pyrs and NBs into rAn and Un sequences, respectively, in
the same manner causes a Pyr-NB bicontinuous array along the du-
plex. Sequences of the RNA duplexes (rAn-Un) having APyr-UNB pairs
are illustrated in Chart 1.
p
DNA and RNA are attractive building blocks for self-assembled
nanostructures.3 In addition, DNA and RNA can be supramolecular
scaffolds for arranging multiple aromatics by using a wide variety
of strategies to incorporate aromatic compounds.4 We have shown
that RNA molecules having multiple pyrenylmethyl substituents
on the 20-O-sugar residues can form duplexes with complementary
RNA sequences without the loss of thermal stability. In the RNA
duplexes, covalently incorporated pyrenes (Pyrs) can be assembled
in a helical manner along the duplex. These helically assembled
pyrene arrays exhibit significantly strong excimer emissions.5
Moreover, we have demonstrated that the charge transfer occurs
with double exponential distance dependence in RNA duplexes
having continuous rAn-Un sequences as bridges, Pyr as a photo-
excitable electron donor, and 4-nitrobenzene (NB) as an electron
acceptor.6 Systems with Pyr and NB site specifically introduced
at the 20-O-position of U-strands through a one-carbon linker have
been shown to undergo photo-induced charge transfer mediated
All the RNAs employed in this work were synthesized by using a
conventional phosphoramidite method. 20-O-Pyrenylmethyl-modi-
fied adenine phosphoramidite was prepared according to a previ-
ously reported method.4b 5-(4-nitrophenyl)-20-deoxyuridine
1
was prepared in 62% yield via Suzuki–Miyaura coupling using
Pd(II) acetate in the presence of triphenylphosphine-3,30,300-trisulf-
onic acid trisodium salt (TPPTS) (Scheme 1).9,10 Nucleoside 1 was
reacted with 4,40-dimethoxytrityl chloride (DMT-Cl) to give 2 in
by
p
-stacked bases along the axis of the helix.7
In this Letter, we describe another type of donor–acceptor-mod-
ified rAn-Un duplexes using Pyr and NB. Pyr groups were incorpo-
rated at the 20-O-positions of an rA strand, and NBs were
appended at the 5-positions of a U strand in the duplex. As a result,
* Corresponding authors. Tel./fax: +81 79 267 4928 (M.N.); +81 79 267 4895
(K.Y.).
E-mail addresses: mitunobu@eng.u-hyogo.ac.jp (M. Nakamura), yamana@eng.u-
Figure 1. Schematic illustration of the configuration of pyrene (Pyr) and nitroben-
hyogo.ac.jp (K. Yamana).
zene (NB) of the A–U base pair in an A-form duplex.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.01.081

M. Fukuda et al. / Tetrahedron Letters 51 (2010) 1732–1735
P0: 5'-rAAA AAA AAA AAA AAA AAA AA-3'
1733
P1: 5'-rAAA AAA APyrAA AAA AAA AAA AA-3'
P2: 5'-rAAA AAA APyrAPyrA AAA AAA AAA AA-3'
N0: 5'-rUUU UUU UUU UUU UUU UUU UU-3'
N1: 5'-rUUU UUU UUU UUU UUNBU UUU UU-3'
N2: 5'-rUUU UUU UUU UUU UNBUNBU UUU UU-3'
NH2
N
O2N
O
N
N
N
NH
O
N
O
O
O
O
APyr
=
UNB
=
O
O
O P O-
O
O P O-
O
NH2
N
NH
Figure 2. Melting profiles of Pyr-NB-modified RNA duplexes in a buffer of pH 7
N
containing 0.1 M NaCl and 0.01 M NaH2PO4.
N
O
N
N
O
O
O
O
U =
A =
O
OCH3
O
OCH3
O P O-
O P O-
Chart 1. Sequences of Pyr donor- and NB acceptor-modified RNAs.
54% yield after purification on a silica gel column.11 Protected nucle-
oside 2 was then converted to 3 by reacting it with 2-cyanoethyl-
N,N,N0N0-tetraisopropylphosphorodiamidite in dichloromethane
containing tetrazole. Purification by using silica gel column chroma-
tography gave 3, which was used in DNA/RNA-automated synthe-
sis,12 in 81% yield.
Melting profiles of the RNA duplexes displayed a single melting
transition (Fig. 2). The melting temperatures (Tm) of the duplexes
were in the range of 42–47 °C. The introduction of APyr and UNB into
the RNA duplexes (rA20-U20) slightly destabilized the resulting Pyr-
NB-modified duplexes.
Figure 3 shows absorption spectra of the Pyr-NB-modified RNA
duplexes under identical conditions. For the Pyr-modified duplexes
(P1N0 and P2N0), pyrene 1La absorption bands were observed in
the region of 300–370 nm. A blue shift and hypochromism in the
P2N0 absorption band in comparison with that of P1N0 were ob-
served, suggesting that the two Pyrs of P2N0 interact with each
other. For the NB-modified duplexes (P0N1 and P0N2), a broad
absorption band was observed in the region of 300–370 nm. No
such absorption band was observed in the RNA possessing NB at
the 20-O-position of U.6 In addition, a broad absorption band in that
region was seen in the spectra of single-stranded N1 and single-
stranded N2, whereas in the spectrum of single-stranded N0, no
such absorption band was observed (see Supplementary data).
Therefore, the broad absorption band is possibly due to the elec-
tronic coupling between U and NB in UNB. The broad absorption
bands observed for N1 and N2 shifted to longer wavelengths and
decreased upon hybridization with P0, suggesting that the elec-
tronically coupled UNBs form base-pairs with complement bases
and stack with adjacent bases in the duplexes. In the cases of the
Pyr-NB-modified duplexes (P1N1, P2N1, and P2N2), their absorp-
Figure 3. UV–vis spectra of Pyr-NB-modified RNA duplexes measured at 22 °C in a
pH 7 buffer containing 0.1 M NaCl and 0.01 M NaH2PO4. Inset shows expanded
spectra between 300 nm and 400 nm.
tion spectra showed hypochromism in comparison with the sum
of the absorption intensities of PnN0 (n = 1 and 2) and P0Nn
(n = 1 and 2). The absorption intensities of P1N1 and P2N1 were
nearly equal to those of P1N0 and P2N0, respectively. The absorp-
tion intensity of P2N2 was slightly lower than the sum of the
intensities of the P2N0 and P0N2 absorptions bands.
In addition to the UV–vis spectral studies, circular dichroism
(CD) spectroscopy was performed in order to obtain further struc-
tural information about the Pyr and NB chromophores. Figure 4
shows CD spectra of the Pyr-NB-modified RNA duplexes. In the
CD spectra of all the modified duplexes, a typical CD pattern
(<300 nm) of a right-handed A-form duplex was observed. Duplex
P1N0 exhibited no induced CD in the region between 300 and
370 nm, which is consistent with a Pyr ring directed into the minor
groove of the duplex. P2N0 exhibited negative Cotton effects in the
NO2
NO2
NO2
O
N
O
N
O
N
O
N
I
HN
O
HN
O
HN
O
HN
O
HO
DMTO
(i)
(ii)
(iii)
DMTO
HO
O
O
O
O
OH
OH
O
OH
P
CN
N
O
2
1
3
Scheme 1. Synthesis of 5-(4-nitrophenyl)-20-deoxyuridine derivatives (1–3). Reagents and conditions: (i) 4-nitrophenyl-1-boronic acid pinacol ester, Na2CO3, Pd(OAc)2,
triphenylphosphine-3,30,300-trisulfonic acid trisodium salt in CH3CN–H2O, reflux for 20 h; (ii) 4,40-dimethoxytrityl chloride in pyridine, 6 h, rt; (iii) 2-cyanoethyl-N,N,N0N0-
tetraisopropylphosphorodiamidite and tetrazole in dichloromethane, 2 h, rt.

1734
M. Fukuda et al. / Tetrahedron Letters 51 (2010) 1732–1735
intensity of P1N1 at 376 nm was smaller (35%) than that of
P1N0, where the distance between Pyr and NB is ca. 13 Å. In our
previous work,6 the fluorescence of the RNA duplex modified with
Pyr and NB at the 20-O-positions, where Pyr and NB were separated
by 8.7 Å, was quenched by 48%. These results indicate that the fluo-
rescence quenching in the present RNA system is stronger than
that in the previous system. P2N0 exhibited a Pyr excimer fluores-
cence (480 nm) in addition to Pyr monomer fluorescences (376 and
398 nm). In contrast to P2N0, P2N1 and P2N2 exhibited virtually
no excimer fluorescence and showed only very weak monomer
emission. Quenching of the P2N2 emission was more effective than
that of the P2N1 emission. Scaiano et al. have reported that nitro-
benzene can efficiently quench the pyrene intermolecular excimer
as well as the monomer fluorescence.8a The extreme decrease in
the intensity of the excimer fluorescence of P2N1 and P2N2 is
therefore due to the direct quenching of the excimer by UNB and
Figure 4. CD spectra of Pyr-NB-modified RNA duplexes measured at 22 °C in a
buffer of pH 7 containing 0.1 M NaCl and 0.01 M NaH2PO4. Inset shows expanded
spectra between 300 nm and 400 nm.
*
the decrease in the Pyr population is due to the emission quench-
ing. The electronic coupling between U and NB, as well as the base
pairing of APyr-UNB, may play important roles in the ET.8d Further
detailed studies to clarify this point are necessary.
region of 300–350 nm and a positive Cotton effect in the region of
350–370 nm due to exciton coupling between the two Pyr chro-
mophores.5a,5b On the other hand, P0N1 and P0N2 showed only
In summary, RNA duplexes having APyr-UNB base pairs showed
significant quenching of Pyr excimer and monomer fluorescences.
The present system is the first step toward bicontinuous donor–
acceptor arrays to generate charge-separated states by photo-irra-
diation of RNA duplexes.
negative induced CD signals owing to electronically coupled UNB
s
in the region of 300–370 nm, and the CD amplitude for P0N2
was about two times that for P0N1 in this region. In the CD spectra
of P1N1, P2N1, and P2N2, negative induced CD signals like those
observed in the spectra of P0N1 and P0N2 were not observed. In
the spectra of P1N1, no induced CD signals appeared in the region
of 300–370 nm. In contrast, in the spectra of P2N1 and P2N2, po-
sitive and negative Cotton effects due to exciton coupling between
Pyrs were observed. On the basis of the UV–vis and CD spectra,
there are interactions between Pyrs of the APyrAPyr domain and
those between UNBs of the UNBUNB domain in the RNA duplexes.
However, whether or not there are interactions between Pyr and
UNB in the Pyr-NB-modified duplexes is still unclear. The hypochro-
mism in their UV–vis spectra suggests some electronic interaction
between Pyrs and UNBs, whereas little information about the inter-
action could be obtained from the CD spectra.
Acknowledgment
This research was supported by a Grant-in-Aid for Scientific Re-
search from the Japan Society for the Promotion of Science (JSPS).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.01.081.
References and notes
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Fluorescence spectra of the Pyr-NB-modified RNA duplexes are
shown in Figure 5. In the spectrum of P1N1, Pyr monomer fluores-
cence (376 and 398 nm) was observed, and its fluorescence inten-
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the excited Pyr (Pyr ) to the UNB acceptor.13 The fluorescence
*
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Figure 5. Fluorescence spectra of Pyr-NB-modified RNA duplexes measured at
22 °C in a buffer of pH 7 containing 0.1 M NaCl and 0.01 M NaH2PO4. Inset shows
expanded spectra between 440 and 550 nm. The excitation wavelength was
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11. 4,40-Dimethoxytrityl chloride (0.53 g, 1.6 mmol) was added to a solution of 1
(0.50 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 the solution was extracted with water.
The organic phase was dried over Na2SO4, filtered, and evaporated to near
8. (a) Focsaneanu, K.-S.; Scaiano, J. C. Photochem. Photobiol. Sci. 2005, 4, 817–821;
(b) Lewis, F. D.; Daublain, P.; Deles Santos, G. B.; Liu, W.; Asatryan, A. M.;
Markarian, S. A.; Fiebig, T.; Raytchev, M.; Wang, Q. J. Am. Chem. Soc. 2006, 128,
4792–4801; (c) Meaney, M. S.; McGuffin, V. L. Anal. Chim. Acta 2008, 610, 57–
67; (d) Lin, Z.; Lawrence, C. M.; Xiao, D.; Kireev, V. V.; Skourtis, S. S.; Sessler, J.
L.; Beratan, D. N.; Rubtsov, I. V. J. Am. Chem. Soc. 2009, 131, 18060–18062.
9. (a) Amann, N.; Wagenknecht, H.-A. Synlett 2002, 687–691; (b) Mayer, E.;
Valis, L.; Huber, R.; Amann, N.; Wagenknecht, H.-A. Synthesis 2003, 15, 2335–
2340.
dryness. The concentrated solution was added to
a silica gel column
chromatograph and eluted with 9:1 (v/v) CH2Cl2/CH3OH. The appropriate
fractions were pooled and then evaporated to near dryness. The residual
solution was poured into cooled hexane, and pure 2 was obtained by filtering.
Yield 54% (0.50 g). TLC (9:1, CH2Cl2/CH3OH, v/v), Rf 0.42. 1H NMR (DMSO-d6): d
2.25 (2H, 20-CH2), 3.66 (2H, 50-CH2), 3.67 (6H, OCH3), 3.95 (1H, 30-CH), 4.29 (1H,
40-CH), 5.36 (1H, 30-OH), 6.22 (1H, 10-CH), 6.73–7.31 (total 13H, Ar of DMT),
7.58 (2H, Ar of nitrophenyl), 7.89 (2H, Ar of nitrophenyl), 7.91 (1H, 6-CH of
uracil), 11.78 (1H, 3-NH of uracil).
10. To a solution of 5-iodo-20-deoxyuridine (1.0 g, 2.8 mmol), 4-nitrophenyl-1-
boronic acid pinacol ester (0.77 g, 3.1 mmol), and Na2CO3 (0.90 g, 8.5 mmol) in
CH3CN–H2O (20 mL, 10:1), palladium acetate (0.032 g, 0.14 mmol) and TPPTS
(0.24 g, 0.42 mmol) were added. The solution was refluxed for 20 h under a N2
atmosphere, diluted with 30 mL of water, and extracted with ethyl acetate
(30 mL ꢀ 3). The organic phase was dried over Na2SO4, filtered, and
evaporated. The crude product was separated on a silica gel column and
eluted with 9:1 (v/v) CH2Cl2/CH3OH to afford 1. Yield, 62% (0.61 g); TLC (9:1,
CH2Cl2- CH3OH, v/v), Rf 0.28. 1H NMR (DMSO-d6): d 2.26 (2H, 20-CH2), 3.66 (2H,
50-CH2), 3.84 (1H, 30-CH), 4.31 (1H, 40-CH), 5.25 (1H, 50-OH), 5.29 (1H, 30-OH),
6.21 (1H, 10-CH), 7.88 (2H, Ar), 8.21 (2H, Ar), 8.54 (1H, 6-CH of uracil), 11.69
(1H, 3-NH of uracil).
12. 2-Cyanoethyl-N,N,N0N0-tetraisopropylphosphorodiamidite (0.294 mL, 0.93 mmol)
was added to
a solution of 2 (0.50 g, 0.78 mmol) and tetrazole (0.055 g,
0.78 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, filtered, and then evaporated to near dryness. The product 3 was
purified by silica gel column chromatography with 45/45/10 (v/v)CH2Cl2/
AcOEt/Et3N as the eluent. Yield, 81% (0.54 g); TLC (45/45/10, CH2Cl2–AcOEt–
Et3N, v/v), Rf 0.64.
13. The Pyr fluorescence of P1 in single stranded state was quenched upon
hybridization with N0 (see Supplementary data).