Thieme chemistry journal awardees - Where are they now? Synthesis and optical properties of nile red modified 2-deoxyuridine and 7-deaza-2- deoxyadenosine: Highly emissive solvatochromic nucleosides

Article abstract of DOI:10.1055/s-0029-1218384

A general synthetic strategy for the attachment of Nile red through a rigid acetylene linker to 2′-deoxyuridine and 7-deaza-2′-deoxyadenosine using Sonogashira coupling is demonstrated. Protection of either 5-OH or N-7 of the nucleosides increased the yie

Full text of DOI:10.1055/s-0029-1218384

3252
LETTER
Thieme Chemistry Journal Awardees – Where Are They Now?
Synthesis and Optical Properties of Nile Red Modified 2¢-Deoxyuridine and
7-Deaza-2¢-deoxyadenosine: Highly Emissive Solvatochromic Nucleosides
H
ighly Emissive
e
Solvatochr
j
omic
N
i
ucleosides Varghese,a Praveen Kumar Gajula,a,b Tushar Kanti Chakraborty,b Hans-Achim Wagenknecht*a
a
Institute for Organic Chemistry, University of Regensburg, 93040 Regensburg, Germany
Fax +49(941)9434617; E-mail: achim.wagenknecht@chemie.uni-regensburg.de
Central Drug Research Institute, CSIR, Chattar Manzil Palace, Post Box No. 173, Lucknow 226 001, UP, India
b
Received 30 July 2009
Abstract: A general synthetic strategy for the attachment of Nile
red through a rigid acetylene linker to 2¢-deoxyuridine and 7-deaza-
2¢-deoxyadenosine using Sonogashira coupling is demonstrated.
Protection of either 5¢-OH or N-7 of the nucleosides increased the
yields of the cross-couplings significantly. Both Nile red modified
2¢-deoxyuridine and 7-deaza-2¢-deoxyadenosine as well as their de-
rivatives exhibit excellent fluorescence quantum efficiencies and
positive solvatochromism. The incorporation of the Nile red modi-
fied 2¢-deoxyuridine into oligonucleotides yields bright fluorescent
probes with maintained canonical base pairing in DNA.
Key words: cross-coupling, dyes, nucleosides, solvent effects,
spectroscopy
Hans-Achim Wagenknecht, born in 1968, finished his studies of
chemistry at the University of Freiburg with a diploma thesis about
glycosidase inhibitors (Prof. Jochen Lehmann). After completion of
his Ph.D. in the group of Prof. Wolf-D. Woggon (Basel) about syn-
thetic porphyrin models of chloroperoxidase, he carried out postdoc-
toral research at the California Institute of Technology (Pasadena) in
the group of Prof. Jacqueline K. Barton. In 2000, he started to set up
an independent research group at the Technical University of Munich
in the field of bioorganic and biophysical chemistry with DNA in or-
der to obtain the habilitation in 2003. In 2005, he moved as a professor
for organic chemistry to the University of Regensburg. He obtained
several awards, including the German ORCHEM Award for Organic
Chemistry 2004, the German Bredereck 2005 Award for Bioorganic
Chemistry, and (together with Torsten Fiebig) the Swiss Grammati-
cakis-Neumann Award for Photochemistry. His current interest is the
synthesis and development of functional DNA architectures for elec-
tron transfer, photocatalysis, molecular diagnostics, imaging and
nanodevices.
The majority of covalently bound fluorescent labels for
nucleic acids apply long alkyl-chain linkers between the
chromophore and the oligonucleotides.1,2 This approach is
rationalized to be a requirement for the replication of the
corresponding modified oligonucleotides or modified nu-
cleoside triphosphates by DNA polymerases.1 The termi-
nal labeling using alkyl linkers was preferred for
molecular beacons.1,3 On the other hand, the attachment of
chromophores to DNA bases via short bridges like acety-
lene, phenylene, or a single C–C bond, etc. are more at-
tractive because such conjugation of the chromophores
with the DNA base stack can potentially help to read out
structural variations and dynamics in DNA. These modi-
fications can be considered as ‘electronically coupled
fluorescent nucleobases’ which are able to recognize the
right counterbase through Watson–Crick base pairing.
Moreover, the sequence-selective base-pairing properties
of such modified oligonucleotides provide the basis for
potential applications in the field of DNA-based molecu-
lar diagnostics. One suitable approach to fulfill these re-
quirements is to attach fluorophores to DNA bases either
through the C-5 position of pyrimidines or the C-7 posi-
tion of 7-deazapurines. In this context, we and others have
shown the covalent coupling of a large class of chro-
mophores to nucleobases through rigid linkers for differ-
ent applications.4–14 Very recently, we demonstrated that
the DNA polymerase catalyzed nucleotide incorporation
opposite to pyrene- and BODIPY-modified 2¢-deoxy-
uridines follows canonical base pairing.15 Moreover, the
modification sites are bypassed for further elongation.
These observations make this kind of fluorescent labels
attractive tools for chemical cell biology.
Nile red is particularly attractive as a fluorescent label be-
cause of the positive solvatochromism which has been ex-
tensively employed to probe the local polarity in
organized and constrained media.16 Hence, the incorpora-
tion of Nile red into nucleic acids is promising not only as
a new fluorescent label for DNA to probe the variation in
polarity of the microenvironment around DNA but also to
follow the interaction of DNA with other biomolecules.
Herein, we report the synthesis and optical behaviors of
Nile red-modified 2¢-deoxyuridine derivatives 9–12 and
7-deaza-2¢-deoxyadenosine derivatives 13–15.
The most convenient way of attaching chromophores to
pyrimidines and purines represents the Sonogashira cou-
pling between the alkyne functionalized chromophore and
SYNLETT 2009, No. 20, pp 3252–3257
Advanced online publication: 27.11.2009
DOI: 10.1055/s-0029-1218384; Art ID: S08609ST
© Georg Thieme Verlag Stuttgart · New York
x
x
.x
x
.2
0
0
9

LETTER
Highly Emissive Solvatochromic Nucleosides
3253
the halogenated nucleobases (Br or I) or vice versa. We adopts the syn conformation which prevents canonical
adopted this strategy for the incorporation of Nile red into base pairing in DNA. The alternative modification of the
nucleobases. For this, the alkyne-functionalized Nile red N-7 position results in a significantly increased hydrolytic
dye 1 was first synthesized by a reaction sequence accord- lability of the glycosidic bond. The only suitable alterna-
ing to a very recent literature report.17 The Sonogashira tive is via the C-7 position of 7-deazapurine. Accordingly
coupling of 5-iodo-2¢-deoxyuridine (2, 1 equiv) with 1 we synthesized 7-iodo-7-deaza-2¢-deoxyadenosine (6) ac-
(1.2 equiv) in the presence of Pd(PPh3)4 (5 mol%) and CuI cording to the methodology of Seela and co-workers.18
(10 mol%) as catalysts in a mixture of triethylamine The Sonogashira coupling of 6 with 1 under the same
(Et3N) and dimethylformamide (DMF) at 90 °C for four reaction conditions as in the case of pyrimidine 9 fur-
hours gave the corresponding Nile red functionalized nu- nished the corresponding nucleoside 13 in comparable
cleoside 9 in about 30% yield.20 The coupling efficiency poor yield (25%).24 However, better coupling efficiencies
did not improve by a higher percentage of catalysts or by were observed after the protection of the 5¢-OH group of
variation of the reaction conditions. However, the yield the sugar moiety (50% and 55% for 1425 and 1526, respec-
increased noticeably after protecting either the 5¢-OH or tively), similar to the case of pyrimidine derivatives
N-7 of 5-iodo-2¢-deoxyuridine. Thus 5¢-DMT-protected (Scheme 1).19
1021 and 5¢-(O-acyl)-protected 1122 as derivatives of 5-
The optical properties of the modified nucleosides were
investigated in solvents of different polarity.
iodo-2¢-deoxyuridine furnished coupling yields of 60%
and 55%, respectively. The N-alkylated derivative 1223
Representatively we describe here the optical behavior of
gave a yield of 55% under the same reaction conditions
9. Both absorption and emission spectra showed gradual
bathochromic shifts with increasing the solvent polarity.
For example, 9 exhibits an absorption maximum of 532
nm in diethyl ether, 551 nm in acetonitrile, and 569 nm in
(Scheme 1).
The attachment of chromophores to the C-8 position via
short linkers is unfavorable since the modified nucleobase
O
O
R2
I
N
O
N
N
O
R2
O
O
R1O
N
N
O
R1O
N
2: R1 = H, R2 = H
OH
9: R1 = H, R2 = H
O
3: R1 = DMT, R2 = H
4: R1 = H, R2 = C12H25
5: R1 = C(O)C11H23, R2 = H
10: R1 = DMT, R2 = H
11: R1 = H, R2 = C12H25
H
12: R1 = C(O)C11H23, R2 = H
OH
Pd(PPh3)4, CuI
DMF, Et3N, 90 °C, 4 h
O
N
O
N
O
O
1
N
N
NHR2
I
NHR2
N
N
N
N
R1O
N
N
O
O
R1O
13: R1 = H, R2 = H
6: R1 = H, R2 = H
OH
14: R1 = DMT, R2 = H
15: R1 = DMT, R2 = Bz
7: R1 = DMT, R2 = H
8: R1 = DMT, R2 = Bz
OH
3'-A-G-T-C-A-C-T-T- 9-T-T-C-T-G-A-C-G-5'
3'-A-G-T-C-A-C-T-T-9-T-T-C-T-G-A-C-G-5'
DNA1
X = A; DNAA
X = G; DNAG
X = C; DNAC
X = T; DNAT
DNA1A; X = A
DNA1G; X = G
DNA1C; X = C
DNA1T; X = T
3'-A-G-T-C-A-C-T-T-T-T-T-C-T-G-A-C-G-5'
5'-T-C-A-G-T-G-A-A-X-A-A-G-A-C-T-G-C-3'
5'-T-C-A-G-T-G-A-A-X-A-A-G-A-C-T-G-C-3'
3'-A-G-T-C-A-C-G-G-9-G-G-C-T-G-A-C-G-5'
DNA2
Scheme 1 Synthesis of Nile red modified nucleosides and the DNA sequences used in this study
Synlett 2009, No. 20, 3252–3257 © Thieme Stuttgart · New York

3254
R. Varghese et al.
LETTER
Table 1 Absorption and Fluorescence Properties of 9–15 in Different Solventsa
max(abs)/nm (emax·104/M–1cm–1)
MeCN DMF DMSO
l
l
max(em)/nm (f)
Et2O
EtOAc
MeOH
Et2Ob
EtOAcb MeCNc DMFc
DMSOc
MeOHc
9
532 (0.42) 537 (3.60) 551 (2.70) 560 (3.18) 568 (5.89) 569 (6.21) 574 (0.65) 599 (0.85) 622 (0.79) 629 (0.71) 640 (0.49) 644 (0.38)
10 531 (0.39) 538 (3.12) 550 (2.59) 561 (3.02) 568 (5.62) 570 (6.56) 573 (0.62) 598 (0.79) 619 (0.70) 628 (0.63) 639 (0.38) 643 (0.33)
d
11
–
529 (2.01) 539 (0.68) 553 (2.93) 559 (3.68) 559 (1.10) –d
597 (0.74) 618 (0.82) 627 (0.72) 638 (0.55) 641 (0.39)
12 522 (2.31) 533 (2.22) 547 (2.28) 556 (2.38) 563 (2.52) 564 (1.93) 575 (0.78) 598 (0.72) 621 (0.68) 626 (0.38) 639 (0.35) 643 (0.34)
13 529 (0.46) 538 (2.03) 551 (0.18) 560 (1.28) 568 (0.84) 569 (2.11) 582 (0.68) 598 (0.73) 622 (0.76) 629 (0.48) 640 (0.32) 645 (0.16)
d
14
15
–
–
533 (0.90) 546 (0.88) 554 (1.10) 562 (1.16) 564 (0.93) –d
600 (0.62) 622 (0.54) 629 (0.43) 639 (0.28) 643 (0.12)
601 (0.58) 622 (0.45) 628 (0.32) 640 (0.21) –d
d
d
532 (0.82) 547 (0.42) 554 (0.98) 563 (0.91) –d
–
a Spectra were recorded at 25 °C (c 1·10–6 M, l = 10 mm).
b Fluorescence quantum yields were measured using rhodamine B (f = 0.69 in EtOH, lexc = 520 nm) as standard.
c Fluorescence quantum yields were measured using cresyl violet (f = 0.54 in MeOH, lexc = 530 nm) as standard.
d Spectrum did not record due to the poor solubility.
methanol (Figure 1, a). The inherent positive solvato- (fluorescence quantum yield was measured using cresyl
chromism of the dye is maintained even after the attach- violet as standard, ff = 0.54 in methanol, lexc = 580 nm;
ment of the nucleobase to the dye via an acetylene bridge Figure 1, b). However, significant quenching of fluores-
in 9. Noticeably, in diethyl ether there is a shoulder band cence (ff = ca. 10%) was observed upon duplex forma-
at around 650 nm indicating the aggregation of the nucleo- tion with perfectly matched counter strand. Interestingly,
side which is currently under investigation. Similarly, sol- in the case of duplexes having mismatch opposite to Nile
vatochromism was observed in the emission spectra of 9. red revealed higher quantum efficiency (15–20%) than
The emission maximum of 9 in diethyl ether is 574 nm, in perfectly matched duplex (Figure 2, b). This difference in
acetonitrile at 622 nm, and in methanol at 644 nm the quantum efficiency can be attributed to the solvato-
(Figure 1, b). More importantly, 9 exhibits excellent fluo- chromic behavior of the dye. It is also worth noting that a
rescent quantum yields in moderatively polar solvents large number of fluorescent labels fail due to a significant
(Table 1). The emission quantum yield of 9 in ethyl ace- quenching in the presence of DNA as result of electron
tate is 85% and decreases with increasing solvent polarity. transfer with the nucleobases, especially guanine. Nile red
It is worth noting that 9 exhibits a lower quantum yield in
diethyl ether than ethyl acetate although the former is less
polar than the latter. This observation can be attributed to
the aggregation of the dye yielding self-quenching of the
fluorescence which is in accordance with the absorption
spectrum in diethyl ether. The observed quantum yields of
the nucleoside 9 indicates that Nile red exhibits higher
quantum efficiency when attached to the nucleobase
through a rigid linker than Nile red as a C-nucleosidic
DNA base surrogate.27
The 2¢-deoxyuridine derivatives 10–12 and the 7-deaza-
2¢-deoxyadenosine derivatives 13–15 show similar ab-
sorption and fluorescence properties as described repre-
sentatively for 9. In general, the quantum yields decrease
with increasing solvent polarity (with few exceptions) ir-
respective of the protecting groups on the base. The spec-
troscopic data are summarized in Table 1.
Subsequently, we synthesized DNA128 and DNA2 using
10 as the building block after converting the 3¢-OH of 10
to the corresponding phosphoramidite derivative under
standard reaction conditions.14 Absorption spectra of
DNA1 and DNA2 exhibited the characteristic absorption
of Nile red (lmax = 615 nm; Figure 1, a). The emission
maximum of ss-DNA1 was observed at 658 nm Figure 1 Normalized absorption (a) and fluorescence (b) spectra of
9 in solvents of different polarity and DNA1 in water (c = 1·10–6 M,
l = 10 mm and lexc = 530 nm)
(lexc = 580 nm) with a quantum efficiency of ca. 20%
Synlett 2009, No. 20, 3252–3257 © Thieme Stuttgart · New York

LETTER
Highly Emissive Solvatochromic Nucleosides
3255
does not belong to those redox-active dyes since ss-DNA2 In conclusion, we have demonstrated a general and prom-
does not exhibit a significant fluorescence quenching ising synthetic strategy for the attachment of the solvato-
(ff = ca. 20%).
chromic and highly emissive Nile red dye to the 5-
position of 2¢-deoxyuridine and the 7-position of 7-deaza-
2¢-deoxyadenosine through a rigid acetylene spacer. The
correspondingly modified nucleosides 9 and 13 and their
derivatives 10–1223 and 14, 15,25,26 respectively, exhibit
high emission quantum yields and positive solvato-
chromism. Moreover, the Nile red modified uridine base
was successfully incorporated into DNA. The quantum
yields of the Nile red modified duplexes are suitable for
bioanalytical applications. The canonical base pairing in
DNA is maintained. Furthermore, the solvatochromic be-
havior of the dye is suitable for probing the structure and
dynamics of DNA and for following the interaction of
DNA with other biologically important molecules such as
proteins. The applications of Nile red modified oligonu-
cleotides in DNA-hybridization assays are in progress in
our laboratory.29
Thermal denaturation experiments with DNA1A revealed
that the presence of the Nile red modified uridine destabi-
lizes the duplex when compared with the unmodified
DNAA (Table 2). However, the Tm value for DNA1A
supports the canonical A:dU base pairing even though the
Tm differences between the matched and mismatched
modified duplexes are less compared to the corresponding
modified duplexes. Especially the stabilization of the mis-
matched modified duplexes DNA1C and DNA1T could
result from a better ground state p-stacking interactions of
the Nile red chromophore with the adjacent bases. The lat-
ter interpretation is supported by the absorption spectra
that showed small bathchromic shifts (ca. 2 nm) with pu-
rine counterbases (DNA1A, DNA1G) and ca. 12 nm
shifts in the presence of pyrimidines (DNA1C, DNA1T;
Figure 2, a).
Table 2 Melting temperature of the Modified and Natural Duplexesa
Acknowledgment
Modified duplexes Tm (°C)
Natural duplexes Tm (°C)
We acknowledge the financial support from Deutsche Forschungs-
gemeinschaft (GK 640, RV), the University of Regensburg, and
the German Academic Exchange Service (DAAD) together with
the BASF company within the Indian–German Graduate School
INDIGO (PG).
DNA1A
DNA1G
DNA1C
DNA1T
57.4
55.1
56.3
55.2
DNAA
DNAG
DNAC
DNAT
62.5
55.0
52.8
53.2
References and Notes
a Conditions: DNA (2.5 mM), Na-Pi buffer (10 mM), pH 7, NaCl (250
mM).
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Figure 2 Absorption (a) and fluorescence (b) spectra of ss-DNA1
and duplexes DNA1A-T, 2.5 mM duplex, 10 mM Na-Pi buffer, pH 7,
250 mM NaCl, 20 °C, lexc = 580 nm. Insets show normalized spectra.
Synlett 2009, No. 20, 3252–3257 © Thieme Stuttgart · New York

3256
R. Varghese et al.
LETTER
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4 H), 6.64 (dd, J = 9.1, 2.7 Hz, 1 H), 6.42 (d, J = 2.5 Hz, 1
H), 6.34 (m, 1 H), 6.32 (s, 1 H), 4.58 (m, 1 H), 4.07 (m, 1 H),
3.62 (s, 3 H), 3.60 (s, 3 H), 3.49–3.37 (m, 6 H), 3.33 (m, 1
H), 2.53 (m, 1 H), 2.48 (m, 1 H), 1.21 (t, J = 4.7 Hz, 6 H)
ppm. 13C NMR (75 MHz, CDCl3): d = 183.1, 161.1, 158.5,
152.2, 150.9, 149.1, 146.8, 144.4, 142.9, 138.87, 135.5,
135.4, 133.0, 132.5, 132.1, 132.0, 131.7, 131.6, 129.9,
128.6, 128.4, 128.1, 127.9, 127.0, 125.5, 125.2, 125.1,
113.3, 113.1, 110.0, 100.1, 96.1, 93.3, 87.1, 86.8, 72.2, 63.5,
55.1, 45.1, 12.6 ppm. ESI-MS: m/z (%) = 871.4 (100) [M +
H]+.
(22) Compound 11: 1H NMR (300 MHz, CDCl3): d = 8.41 (s, 1
H), 8.37 (s, 1 H), 7.94 (d, J = 8.2 Hz, 1 H), 7.64–7.35 (m, 2
H), 6.55 (m, 1 H), 6.34–6.25 (m, 3 H), 4.67 (m, 1 H), 4.08–
3.85 (m, 4 H), 3.39 (q, J = 7.1 Hz, 4 H), 2.50–2.27 (m, 3 H),
1.63–1.53 (m, 2 H), 1.26–1.16 (m, 24 H), 0.83 (t, J = 7.1 Hz,
3 H) ppm. 13C NMR (75 MHz, CDCl3): d = 181.2, 161.2,
151.2, 151.0, 150.1, 146.6, 144.4, 138.5, 135.0, 132.9,
132.1, 132.0, 131.6, 128.6, 128.4, 125.4, 109.0, 104.4,
101.1, 99.5, 96.1, 91.3, 89.5, 87.5, 74.2, 67.6, 46.0, 45.1,
31.9, 29.3, 29.6, 29.5, 27.0, 22.7, 14.1, 12.6 ppm. ESI-
HRMS: m/z calcd for C43H52N4O7 [M+]: 736.3836; found:
736.3845.
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Ramamurthy, V. Langmuir 2000, 16, 265.
(23) Compound 12: 1H NMR (300 MHz, CDCl3): d = 8.7 (d,
J = 1.3 Hz, 1 H), 8.27–8.19 (m, 2 H), 7.97 (s, 1 H), 7.69–7.38
(m, 2 H), 6.65 (dd, J = 9.0, 2.7 Hz, 1 H), 6.43 (d, J = 2.7 Hz,
1 H), 6.35 (s, 1 H), 6.24 (t, J = 6.3 Hz, 1 H), 4.52–4.12 (m, 4
H), 3.44 (q, J = 6.8 Hz, 4 H), 2.56–2.34 (m, 4 H), 2.21 (m, 1
H), 1.59–1.49 (m, 1 H), 1.26–1.04 (m, 22 H), 0.78 (t, J = 6.8
Hz, 3 H) ppm. 13C NMR (75 MHz, CDCl3): d = 181.7, 176.5,
161.1, 151.4, 150.5, 149.6, 146.5, 143.6, 138.0, 135.4,
132.9, 132.2, 131.6, 131.0, 128.4, 127.2, 125.1, 108.0,
105.2, 102.1, 100.3, 96.4, 91.3, 86.0, 85.8, 72.7, 67.5, 60.4,
45.2, 35.8, 31.9, 30.9, 29.7, 29.5, 22.6, 21.0, 14.2, 12.6 ppm.
ESI-HRMS: m/z calcd for C43H50N4O8 [M+]: 750.3629;
found: 750.3604.
(b) Meinershagen, J. L.; Bein, T. J. Am. Chem. Soc. 1999,
121, 448. (c) Datta, A.; Mandal, D.; Pal, S. K.;
Bhattacharyya, K. J. Phys. Chem. B 1997, 101, 10221.
(17) Han, J.; Jose, J.; Mei, E.; Burgess, K. Angew. Chem. Int. Ed.
2007, 46, 1684.
(18) Seela, F.; Zulauf, M. Synthesis 1996, 726.
(19) General Procedure of Sonogashira Coupling
A mixture of 1 (125 mg, 0.364 mmol), 5-iodo-2¢-
deoxyuridine, 2 (100 mg, 0.28 mmol), Et3N (0.6 mL, 4
mmol), Pd(PPh3)4 (42 mg, 0.036 mmol), and CuI (15 mg,
0.072 mmol) were dissolved in DMF (5 mL). After the
solution was degassed three times via the freeze–pump–
thaw method, the mixture was heated at 90 °C for 4 h. The
reaction solvent was removed under reduced pressure, and
the crude product was purified by flash column
(24) Compound 13: 1H NMR (300 MHz, CDCl3): d = 8.69 (s, 1
H), 8.24 (br s, 1 H), 8.21 (s, 1 H), 7.62 (s, 1 H), 7.45–7.20
(m, 2 H), 6.79–6.73 (m, 2 H), 6.43 (d, J = 2.4 Hz, 1 H), 6.34
(s, 1 H), 4.56 (m, 1 H), 4.01 (m, 1 H), 3.71–3.61 (m, 2 H),
3.42 (q, J = 7.1 Hz, 4 H), 2.57–2.25 (m, 2 H), 1.23–1.14 (m,
6 H) ppm. 13C NMR (75 MHz, CDCl3): d = 180.6, 165.2,
156.8, 151.7, 150.4, 148.8, 146.9, 139.2, 135.9, 135.0,
132.0, 131.9, 130.5, 127.7, 126.5, 124.4, 113.9, 110.7,
103.0, 110.7, 104.6, 103.0, 93.2, 90.3, 87.3, 84.2, 83.4, 69.8,
63.7, 48.3, 45.6, 13.0 ppm. ESI-MS: m/z (%) = 591.2 (100)
[M + H]+.
chromatography (SiO2, 2–3% MeOH in EtOAc eluent)
furnishing the desired product 9 (48 mg, 30%) as a dark
green solid. Rf = 0.4 (SiO2, 5% MeOH in EtOAc); mp
>200 °C.
(20) Analytical Data
(25) Compound 14: 1H NMR (300 MHz, CDCl3): d = 8.70 (s, 1
H),8.29–8.21 (m, 2 H), 7.66–7.11 (m, 14 H), 6.82–6.62 (m,
4 H), 6.46 (d, J = 2.4 Hz, 1 H), 6.36 (s, 1 H), 4.57 (m, 1 H),
4.05 (m, 1 H), 3.77–3.63 (m, 8 H), 3.45 (q, J = 7.1 Hz, 4 H),
2.53–2.27 (m, 2 H), 1.26–1.16 (m, 6 H) ppm. 13C NMR (75
MHz, CDCl3): d = 182.2, 158.5, 157.4, 155.0, 152.1, 150.8,
149.4, 146.9, 144.6, 142.6, 139.2, 136.5, 135.7, 135.6,
133.7, 132.1, 132.0, 130.2, 130.1, 128.4, 128.2, 127.9,
126.3, 125.1, 113.2, 113.1, 111.7, 109.7, 101.9, 100.3, 96.2,
92.8, 86.6, 85.5, 81.1, 71.6, 69.5, 66.9, 55.2, 53.7, 45.1, 12.6
ppm. ESI-MS: m/z (%) = 893.3 (100) [M + H]+.
Compound 9: 1H NMR (300 MHz, CDCl3): d = 11.79 (s, 1
H), 8.56 (d, J = 1.7 Hz, 1 H), 8.51 (s, 1 H), 8.13 (d, J = 8 Hz,
1 H), 7.75–7.69 (m, 2 H), 6.87 (dd, J = 9.0, 2.5 Hz, 1 H), 6.70
(d, J = 2.5 Hz, 1 H), 6.31 (s, 1 H), 6.15 (t, J = 6.5 Hz, 1 H),
5.25 (d, J = 4.0 Hz, 1 H), 5.24 (t, J = 5.0 Hz, 1 H), 4.32–4.24
(m, 1 H), 3.83 (q, J = 3.3 Hz, 1 H), 3.73–3.58 (m, 2 H), 3.52
(q, J = 6.7 Hz, 4 H), 2.27–2.15 (m, 2 H), 1.17 (t, J = 7.0 Hz,
6 H) ppm. 13C NMR (75 MHz, DMSO-d6): d = 181.0, 161.3,
151.9, 151.0, 149.5, 146.4, 144.5, 137.0, 134.4, 131.69,
131.53, 131.1, 130.0, 125.5, 125.2, 124.3, 114.2, 110.4,
104.5, 97.6, 95.8, 91.2, 87.5, 85.2, 84.8, 69.8, 60.7, 44.4,
12.3 ppm. ESI-MS: m/z (%) = 569.2 (100) [M + H]+. ESI-
HRMS: m/z calcd for C31H28N4O7 [M + H]+: 569.2036;
found: 569.2026.
(26) Compound 15: 1H NMR (300 MHz, CD3CN): d = 8.5 (s, 1
H), 8.01–7.98 (m, 2 H), 7.79–7.52 (m, 7 H), 7.46–7.11 (m,
10 H), 6.85–6.74 (m, 6 H), 6.60–6.52 (m, 2 H), 6.45 (m, 1
H), 5.32 (br, 1 H), 4.48 (m, 1 H), 3.93 (m, 1 H), 3.74 (d,
J = 2.7 Hz, 6 H), 3.69 (m, 2 H), 3.48 (q, J = 7.1 Hz, 4 H),
(21) Compound 10: 1H NMR (300 MHz, CDCl3): d = 8.58 (d,
J = 1.4 Hz, 1 H), 8.26 (s, 1 H), 8.17 (br s, 1 H), 7.96 (d,
J = 8.0 Hz, 1 H), 7.52 (d, J = 7.4 Hz, 1 H), 7.42 (d, J = 7.4
Hz, 2 H), 7.31 (m, 4 H), 7.25 (s, 1 H), 7.20 (s, 1 H), 7.14–
7.06 (m, 1 H), 6.88 (dd, J = 8.2, 1.7 Hz, 1 H), 6.78–6.71 (m,
2.52 (m, 1 H), 2.28 (m, 1 H), 1.18–1.14 (m, 6 H) ppm. 13
C
NMR (75 MHz, CD3CN): d = 182.2, 169.0, 160.7, 157.0,
155.2, 152.9, 150.8, 146.2, 144.0, 141.0, 139.1, 136.9,
135.9, 135.6, 134.3, 133.9, 133.1, 131.6, 131.1, 130.6,
Synlett 2009, No. 20, 3252–3257 © Thieme Stuttgart · New York

LETTER
129.6, 129.3, 128.6, 128.4, 127.7, 127.3, 126.6, 124.7,
Highly Emissive Solvatochromic Nucleosides
3257
The oligonucleotides were dried and purified by reverse-
phase HPLC using the following conditions: A = NH4OAc
buffer (50 mM), pH = 6.5; B = MeCN; gradient = 0–20% B
over 50 min. The oligonucleotides were lyophilized and
quantified by their absorbance at 260 nm on a Varian Cary
Bio 100 spectrometer. Duplexes were prepared by heating of
Nile red modified oligonucleotides in the presence of 1 equiv
unmodified complementary strand to 90 °C (10 min),
followed by slow cooling to r.t. ESI-MS: DNA1: m/z calcd
5475.4; found: 5475.1 (100%); DNA2: m/z calcd 5575.4;
found = 5575.1 (100%).
114.0, 108.9, 106.5, 110.2, 102.5, 101.4, 97.8, 91.2, 87.6,
85.3, 82.6, 73.2, 64.8, 52.6, 45.7, 38.4, 13.5 ppm. ESI-MS:
m/z (%) = 997.4 (100) [M + H]+.
(27) Okamoto, A.; Tainaka, K.; Fujiwara, Y. J. Org. Chem. 2006,
71, 3592.
(28) The oligonucleotides were prepared on an Expedite 8909
DNA synthesizer (Applied Biosystems) via standard
phosphoramidite protocols using CPGs (1 mmol) with a
longer coupling time of 15 min and a higher concentration of
the phosphoramidite (0.1 M). After preparation, the trityl-
off oligonucleotide was cleaved off the resin and was
deprotected by treatment with concd NH4OH at r.t. for 10 h.
(29) Varghese, R.; Wagenknecht, H.-A. Chem. Eur. J. 2009, 15,
9307.
Synlett 2009, No. 20, 3252–3257 © Thieme Stuttgart · New York

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