Anthraquinones as artificial DNA building blocks

Article abstract of DOI:10.1002/ejoc.200800080

Synthesis and properties of oligodeoxynucleotides containing anthraquinone-derived building blocks with flexible linkers are described. Starting from the 1,4-, 1,5-, 1,8- and 2,6-dihydroxyanthraquinone isomers, the corresponding phosphoramidites were prepared and incorporated into oligonucleotides. The site of linker attachment was found to be of critical importance for hybrid stability. Whereas the 2,6-isomer led to a significant stabilization, all other isomers had a negative effect on the stability of the duplex. Spectroscopic studies showed that the anthraquinones behave as fluorescence quenchers. Models of anthraquinone-modified double-stranded hybrids are proposed. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200800080

FULL PAPER
DOI: 10.1002/ejoc.200800080
Anthraquinones as Artificial DNA Building Blocks
Nicolas Bouquin,[a] Vladimir L. Malinovskii,[a] and Robert Häner*[a]
Keywords: DNA / Anthraquinone / Stacking interactions / Intercalations
Synthesis and properties of oligodeoxynucleotides contain- mer led to a significant stabilization, all other isomers had a
ing anthraquinone-derived building blocks with flexible lin-
kers are described. Starting from the 1,4-, 1,5-, 1,8- and 2,6-
negative effect on the stability of the duplex. Spectroscopic
studies showed that the anthraquinones behave as fluor-
dihydroxyanthraquinone isomers, the corresponding phos- escence quenchers. Models of anthraquinone-modified
phoramidites were prepared and incorporated into oligonu-
cleotides. The site of linker attachment was found to be of
critical importance for hybrid stability. Whereas the 2,6-iso-
double-stranded hybrids are proposed.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
like structures. In particular, we were interested in the influ-
ence of the geometrical attachment of the linkers on hybrid
formation and in their properties as fluorescence quenchers.
Here, we report the synthesis of four isomeric anthraqui-
none phosphoramidites, their incorporation into oligonu-
cleotides as well as the properties of the resulting oligomers.
Introduction
The use of chemically modified nucleic acids is a rapidly
growing area. Oligonucleotides containing unnatural build-
ing blocks are commonly used in the areas of diagnostics,
supramolecular chemistry and materials research.[1–5]
Among the many modifications, building blocks lacking a
sugar or a sugar-like moiety are increasingly used as versa-
tile components. In particular, polyaromatic compounds,
which often possess interesting electronic and spectroscopic
properties, were found to integrate well into DNA without
compromising hybrid stability. Typical modifications of this
kind include stilbene,[6,7] phenanthrene,[8–12] pyrene,[13–20]
perylene,[21–25] or phenanthroline.[26,27] One of the primary
reasons for the positive effect of the modifications on sta-
bility is their tendency to develop stacking interactions
among themselves or with the nucleobases.[16,17,28,29]
Anthraquinone and its derivatives are well-known intercala-
tors.[30,31] They are a frequently found motif in DNA tar-
geting drugs.[30,32–36] Not surprisingly, conjugation of
anthraquinone to oligonucleotides has served as a common
strategy for the development of high affinity oligonucleo-
tides.[37–45] Furthermore, the low reduction potential of
anthraquinone derivatives opens possibilities for charge
transport through DNA[46–50] and electrochemical DNA
sensing.[49,51–55] In addition, anthraquinone derivatives can
act as fluorescence quenchers,[38,54,56] they are investigated
as photo-activated nucleases[57,58] and they can serve as mo-
lecular entities for supramolecular assemblies.[59] On this
background, we investigated the use of anthraquinone as
an elemental building block for the construction of DNA-
Results and Discussion
Synthesis of the Phosphoramidite Building Blocks and
Oligonucleotides
Incorporation of anthraquinone derivatives with dif-
ferent geometries into DNA should give an indication of
the effects that the attachment sites of the linkers have on
the stability of the duplex. Thus, the four dihydroxyanthra-
quinones 1–4 (the 1,4-, 1,5-, 1,8- and 2,6-isomers) were
[a] Department of Chemistry and Biochemistry, University of
Bern,
Freiestrasse 3, 3012 Bern, Switzerland
Scheme 1. Synthesis of the 1,4-substituted anthraquinone phos-
phoramidite building block 13 (DMT = 4,4Ј-dimethoxytrityl, PAM
= 2-cyanoethyl diisopropylamidochloridophosphite).
E-mail: robert.haener@ioc.unibe.ch
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2008, 2213–2219
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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N. Bouquin, V. L. Malinovskii, R. Häner
Table 1. The 1,4-, 1,5-, 1,8- and 2,6-substituted anthraquinone intermediates and phosphoramidites.
FULL PAPER
1,4-Isomer
1,5-Isomer
1,8-Isomer
2,6-Isomer
1
2
3
4
R1 = R2 = H
R1 = R2 = H
R1 = R2 = H
R1 = R2 = H
5
6
7
8
R1 = R2 = (CH2)2OH
R1 = R2 = (CH2)2OH
R1 = R2 = (CH2)2OH
R1 = R2 = (CH2)2OH
9
10
11
12
R1 = (CH2)2ODMT
R2 = (CH2)2OH
R1 = (CH2)2ODMT
R2 = (CH2)2OH
R1 = (CH2)2ODMT
R2 = (CH2)2OH
R1 = (CH2)2ODMT
R2 = (CH2)2OH
13
14
15
16
R1 = (CH2)2ODMT
R2 = (CH2)2OPAM
R1 = (CH2)2ODMT
R2 = (CH2)2OPAM
R1 = (CH2)2ODMT
R2 = (CH2)2OPAM
R1 = (CH2)2ODMT
R2 = (CH2)2OPAM
Table 2. Melting temperatures of hybrids containing pairs of identical anthraquinone building blocks.
[a]
[b]
Oligonucleotide
Tm [°C]
∆Tm [°C]
17
18
(5Ј) AGC TCG GTC ATC GAG AGT GCA
(3Ј) TCG AGC CAG TAG CTC TCA CGT
71.4
–
19
20
(5Ј) AGC TCG GTC AH14C GAG AGT GCA
(3Ј) TCG AGC CAG TH14G CTC TCA CGT
66.0
66.2
65.9
76.6
–5.4
–5.2
–5.5
+5.2
21
22
(5Ј) AGC TCG GTC AH15C GAG AGT GCA
(3Ј) TCG AGC CAG TH15G CTC TCA CGT
23
24
(5Ј) AGC TCG GTC AH18C GAG AGT GCA
(3Ј) TCG AGC CAG TH18G CTC TCA CGT
25
26
(5Ј) AGC TCG GTC AH26C GAG AGT GCA
(3Ј) TCG AGC CAG TH26G CTC TCA CGT
[a] Conditions: 1.0 µ oligonucleotide concentration (each strand), 10 m phosphate buffer (pH 7.4) and 100 m NaCl. [b] Difference
in Tm relative to reference duplex 17*18.
chosen for the study. Representative for all four isomers, the porated into oligonucleotides by using the phosphoramidite
synthesis of the 1,4-substituted phosphoramidite building procedure.[61,62] Deprotection (conc. NH3, 50 °C), followed
block is shown in Scheme 1. Introduction of the linkers was by HPLC purification yielded oligonucleotides 17–26
done by treatment with 2-chloroethanol in the presence of (Table 2). The correct molecular weights of all oligomers
potassium carbonate and potassium iodide following a sim- were verified by mass spectrometry (Supporting Infor-
ilar method described in the literature.[60] The obtained bis- mation).
(hydroxyethylated) compounds 5–8 were converted into the
monoprotected derivatives 9–12 by reaction with 4,4Ј-di-
Influence of Anthraquinone Building Blocks on Hybrid
methoxytrityl chloride. Phosphitylation under conventional
Stability
conditions yielded the phosphoramidite derivatives 13–16.
The structures of all anthraquinone derivatives are shown
The effect of the four non-nucleosidic building blocks on
in Table 1. The phosphoramidites were subsequently incor- the stability of the duplex was analyzed by thermal denatur-
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Eur. J. Org. Chem. 2008, 2213–2219

Anthraquinones as Artificial DNA Building Blocks
ation experiments. As shown in Table 2, incorporation of a
pair of 1,4-, 1,5- or 1,8-derivatives in each strand (19*20,
21*22 and 23*24) results in a considerable decrease in hy-
brid stability. All three regioisomeric modifications reduce
the melting temperature (Tm) by approximately 5 °C (∆Tm
= –5.4, –5.2 and –5.5 °C, respectively). In contrast, incorpo-
ration of a pair of the 2,6-isomer in opposite positions re-
sults in a significant increase in stability (∆Tm = +5.2 °C).
Circular dichroism (CD) spectral analysis of anthraquinone
hybrids 19*20, 21*22, 23*24 and 25*26 are consistent with
a DNA B-conformation (Figure 1).
Figure 2. Amber-minimized models of DNA hybrids containing
isomeric anthraquinone building blocks as indicated. Anthraqui-
none units are shown in space-filling representation.
stabilizing effects of the 2,6-isomer. In agreement with pre-
vious observations with non-nucleosidic phenanthrene
building blocks,[11] a significant destabilization was ob-
served with all regioisomeric anthraquinone moieties if
Figure 1. CD spectra of hybrids 19*20, 21*22, 23*24 and 25*26. placed opposite to a thymidine residue. Anthraquinone
Conditions: see Table 2.
groups had no stabilizing effect on DNA with an abasic site
(see Supporting Information). This is surprising, as several
Because of the well-known property of anthraquinones
to intercalate into DNA, we calculated possible models con-
taining the building blocks in an interstrand stacked ar-
reports exist in the literature describing a significant struc-
tural stabilization of abasic site-containing DNA.[26,65–67]
rangement. The minimization process was done with amber
Table 3. Melting temperatures in hybrids containing mixed pairs of
force field[63] by considering the two anthraquinone moie-
anthraquinone units.
ties and one base pair on each side. After minimization,
[a]
[b]
Oligonucleotide
Tm ∆Tm
[°C] [°C]
the remaining natural bases were attached by using B-DNA
parameters. The hybrid models thus obtained are depicted
in Figure 2. As can be seen, only the 2,6-linked anthraqui-
none units are arranged in a face-to-face stacking mode.
Stacking of the anthraquinone moieties in the hybrids con-
17 (5Ј) AGC TCG GTC ATC GAG AGT GCA
18 (3Ј) TCG AGC CAG TAG CTC TCA CGT
71.4
–
25 (5Ј) AGC TCG GTC AH26C GAG AGT GCA 68.6 –2.8
taining the other isomeric building blocks seems much less 20 (3Ј) TCG AGC CAG TH14G CTC TCA CGT
favourable. Although this would be in agreement with the
large differences observed in the stabilities of the duplex,
alternative structures can, of course, not be excluded. In
25 (5Ј) AGC TCG GTC AH26C GAG AGT GCA 70.6 –0.8
22 (3Ј) TCG AGC CAG TH15G CTC TCA CGT
fact, a recent crystal structure of a bis(alkoxy)anthraqui-
none revealed that crystal packing is stabilized by intermo-
lecular C–H···O nonclassical hydrogen bonds, whereas no
π–π stacking interactions were observed.[64]
We subsequently studied the effect of mixed pairs, in
which the most stable 2,6-isomer was placed opposite to
one of the other isomeric building blocks. The Tm values
are shown in Table 3. For all hybrids (25*20, 25*22 and
25*24) a decrease in stability (∆Tm = –2.8, –0.8, –5.3 °C,
respectively) relative to the unmodified duplex was ob-
served. The decrease in stability, however, was relatively
25 (5Ј) AGC TCG GTC AH26C GAG AGT GCA 66.3 –5.3
24 (3Ј) TCG AGC CAG TH18G CTC TCA CGT
[a] Conditions: 1.0 µ oligonucleotide concentration (each strand),
10 m phosphate buffer (pH 7.4) and 100 m NaCl. [b] Difference
in Tm relative to reference duplex 17*18.
Fluorescence Quenching by Anthraquinone Building Blocks
Anthraquinones have been described as nonfluorescent
small for hybrids 25*20 and 25*22 (cf. Table 2, ∆Tm values quenchers and are, therefore, of interest for applications in
of –5.4 and –5.2 °C, for the respective hybrids with identical diagnostic tools.[38,56] The four different anthraquinone
anthraquinone modifications), which further illustrates the building blocks were investigated for their influence on
Eur. J. Org. Chem. 2008, 2213–2219
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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N. Bouquin, V. L. Malinovskii, R. Häner
FULL PAPER
pyrene excimer fluorescence. As shown in Figure 3, all four Tm data of the different hybrids. The combination of a flu-
isomers have a significant quenching effect if placed oppo- orophore with a quencher is used in many types of molecu-
site to two pyrene building blocks. Single strand 27 contain- lar probes. Efficient quenching of excimer fluorescence is
ing two pyrene moieties next to each other shows pyrene difficult to achieve.[69,70] Excimer quenching upon hybrid
excimer fluorescence with a maximum at 505 nm.[68] Com- formation with interstrand stacking modifications opens a
plementary single strands 20, 22, 24 and 26 with the dif- new way for the design of DNA-based probes.
ferent anthraquinone moieties opposite the two pyrene
units led to significant reduction (60 to 70%) in the fluores-
Conclusions
cence signal. The values are given in Table 4 along with the
Four isomeric anthraquinone building blocks differing in
the attachment site of the linker were synthesized and incor-
porated into oligodeoxynucleotides. The site of linker at-
tachment was found to have a strong influence on duplex
stability. Hybrids containing a pair of the 1,4-, 1,5- and 1,8-
isomers led to substantial reduction in the Tm value (∆Tm
= –5.4, –5.2 and –5.5 °C, respectively). In contrast, the 2,6-
isomer resulted in a considerable increase in stability (∆Tm
= +5.2 °C). Hybrids containing mixed pairs of isomeric
anthraquinone moieties show moderate-to-significant de-
stabilization. Molecular models suggest that the positive ef-
fect of the 2,6-isomer is a result of interstrand stacking in-
teractions between the two anthraquinone units. In the case
of the other isomers, stacking interactions seem much less
favourable. All anthraquinone building blocks act as fluo-
rescence quenchers. If placed opposite to two pyrene build-
ing blocks, excimer fluorescence is quenched by 60–70%.
The anthraquinone derivatives described here extend the set
Figure 3. Fluorescence spectra of single strand 27 containing two of artificial building blocks with potential application in di-
pyrene units and that of hybrids between 27 and complementary
strands containing the different anthraquinone building blocks.
Conditions: see Table 2.
agnostics or in DNA-based nanomaterials.
Experimental Section
General: Reactions were carried out under a nitrogen atmosphere
Table 4. Quenching of pyrene excimer fluorescence by the different
anthraquinone building blocks.
by using distilled, anhydrous solvents. Flash column chromatog-
raphy (CC) was performed by using silica gel 60 (63–32 µ, Chemie
Brunschwig AG). If compounds were sensitive to acid, the silica
was pretreated with solvent containing 1% Et3N. All NMR spectra
were measured at room temperature with a Bruker AC-300 spec-
trometer. 1H NMR chemical shifts (δ) are reported in ppm relative
to the residual undeuterated solvent (CDCl3: 7.27 ppm). Multi-
plicities are abbreviated as follows: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet. 13C NMR chemical shifts are
reported in ppm relative to the residual nondeuterated solvent
(CDCl3: 77.00 ppm). 31P NMR chemical shifts are reported in ppm
relative to 85% H3PO4 as an external standard. Electrospray ion-
ization (ESI) mass spectra were recorded with VG platform Fisons
instruments.
[a]
[b]
Oligonucleotide
Tm
[°C]
∆Tm
[°C]
Q[c]
[%]
27 (5Ј) AGC TCG GTC S S C GAG AGT GCA
20 (3Ј) TCG AGC CAG TH14G CTC TCA CGT
64.0 –7.4
64.6 –6.8
64.9 –6.5
66.3 –5.1
60
64
70
71
27 (5Ј) AGC TCG GTC S S C GAG AGT GCA
22 (3Ј) TCG AGC CAG TH15G CTC TCA CGT
27 (5Ј) AGC TCG GTC S S C GAG AGT GCA
24 (3Ј) TCG AGC CAG TH18G CTC TCA CGT
27 (5Ј) AGC TCG GTC S S C GAG AGT GCA
26 (3Ј) TCG AGC CAG TH26G CTC TCA CGT
General Method for Bis(hydroxyethylation) of Dihydroxyanthra-
quinones (5–8): A solution of dihydroxyanthraquinone (5.0 g,
20.0 mmol) was dissolved in DMF (100 mL). Potassium carbonate
(27.7 g, 200 mmol) was added to the mixture. The mixture was
stirred for 2 h at 120 °C. 2-Chloroethanol (27 mL, 400 mmol) and
potassium iodide (6.65 g, 40.0 mmol) were added to the mixture.
The mixture was stirred at 120 °C overnight and then cooled to
room temperature and concentrated. Water (100 mL) was added to
the residue, and the mixture was extracted with CH2Cl2/2-propanol
(3:1). The organic phase was washed again with water, dried with
magnesium sulfate and evaporated under reduced pressure. The
crude product was purified by column chromatography (silica gel;
[a] Conditions: 1.0 µ oligonucleotide concentration (each strand),
10 m phosphate buffer (pH 7.4) and 100 m NaCl. [b] Difference
in Tm relative to reference duplex 17*18. [c] Reduction of excimer
fluorescence relative to single-strand 27.
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Anthraquinones as Artificial DNA Building Blocks
AcOEt + 10% MeOH). The fractions were combined and concen-
trated.
(0.225 g, 0.95 mmol) dissolved in dichloromethane (5 mL) was
added dropwise. The reaction mixture was stirred at room tempera-
ture for 2 h. The resulting mixture was directly applied on a silica-
gel column for purification (AcOEt + 1% TEA).
1,4-Bis[(hydroxyethyl)oxy]anthraquinone (5): Yield: 2.54 g (38%).
1H NMR (300 MHz, CDCl3): δ = 3.99 (t, J = 4.4 Hz, 4 H), 4.29
(t, J = 4.2 Hz, 4 H), 7.37 (s, 2 H), 7.74 (dd, J = 5.9, 3.4 Hz, 2 H),
8.18 (dd, J = 5.8, 3.4 Hz, 2 H) ppm. 13C NMR (75 MHz, CDCl3):
δ = 61.16, 73.34, 124.23, 126,90, 127.11, 134.07, 134.37, 154.79,
184.36 ppm. MS (ESI): m/z = 328 [M]+. C18H16O6 MW = 328.09.
1-[({[(2-Cyanoethoxy)(diisopropylamino)phosphanyl]oxy}ethyl)oxy]-
4-({[(4,4Ј-dimethoxytrityl)oxy]ethyl}oxy)anthraquinone (13): Yield:
0.35 g (53%). 1H NMR (300 MHz, CDCl3): δ = 1.17 (m, 12 H),
2.64 (m, 2 H), 3.58 (m, 2 H), 3.59 (m, 2 H), 3.69 (m, 2 H), 3.79 (s,
6 H), 3.86 (m, 2 H), 4.35 (m, 4 H), 6.83 (m, 4 H), 7.24 (m, 1 H),
7.28 (m, 4 H), 7.39 (m, 4 H), 7.50 (m, 2 H), 7.65 (m, 2 H), 7.87
(m, 2 H) ppm. 31P NMR (122 MHz, CDCl3): δ = 149.11 ppm.
1,5-Bis[(hydroxyethyl)oxy]anthraquinone (6): Yield: 1.82 g (27%).
1H NMR (300 MHz, CDCl3): δ = 4.02 (t, J = 4.4 Hz, 4 H), 4.31
(t, J = 4.4 Hz, 4 H), 7.30 (dd, J = 8.3, 1.1 Hz, 2 H), 7.70 (t, J =
8.1 Hz, 2 H), 7.93 (dd, J = 6.6, 1.1 Hz, 2 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 60.77, 72.11, 119.34, 120.17, 120.79, 135.26,
136.96, 159.36, 182.8 ppm. MS (ESI): m/z = 328 [M]+. C18H16O6.
MW = 328.09.
1-({[(2-Cyanoethoxy)(diisopropylamino)phosphanyl]oxy}ethyl)oxy]-
5-({[(4,4Ј-dimethoxytrityl)oxy]ethyl}oxy)anthraquinone (14): Yield:
0.39 g (60%). 1H NMR (300 MHz, CDCl3): δ = 1.18 (m, 12 H),
2.64 (m, 2 H), 3.59 (m, 4 H), 3.70 (m, 2 H), 3.79 (s, 6 H), 3.89 (m,
2 H), 4.33 (m, 4 H), 6.84 (m, 4 H), 7.24 (m, 1 H), 7.28 (m, 4 H),
7.40 (m, 4 H), 7.51 (m, 2 H), 7.66 (m, 2 H), 7.87 (m, 2 H) ppm.
31P NMR (122 MHz, CDCl3): δ = 149.10 ppm.
1,8-Bis[(hydroxyethyl)oxy]anthraquinone (7): Yield: 1.93 g (29%).
1H NMR (300 MHz, CDCl3): δ = 4.03 (t, J = 4.1 Hz, 4 H), 4.32
(t, J = 4.2 Hz, 4 H), 7.34 (dd, J = 8.4, 1.0 Hz, 2 H), 7.68 (t, J =
7.2 Hz, 2 H), 7.91 (dd, J = 7.7, 1.1 Hz, 2 H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 60.77, 72.53, 119.61, 120.17, 121.20, 134.47,
135.02, 159.00, 182.82, 184.15 ppm. MS (ESI): m/z = 328 [M]+.
C18H16O6. MW = 328.09.
1-({[(2-Cyanoethoxy)(diisopropylamino)phosphanyl]oxy}ethyl)oxy]-
8-({[(4,4Ј-dimethoxytrityl)oxy]ethyl}oxy)anthraquinone (15): Yield:
0.35 g (53%). 1H NMR (300 MHz, CDCl3): δ = 1.18 (m, 12 H),
2.64 (m, 2 H), 3.59 (m, 4 H), 3.70 (m, 2 H), 3.79 (s, 6 H), 3.89 (m,
2 H), 4.33 (m, 4 H), 6.84 (m, 4 H), 7.24 (m, 1 H), 7.28 (m, 4 H),
7.40 (m, 4 H), 7.51 (m, 2 H), 7.66 (m, 2 H), 7.87 (m, 2 H) ppm.
31P NMR (122 MHz, CDCl3): δ = 148.86 ppm.
2,6-Bis[(hydroxyethyl)oxy]anthraquinone (8): Yield: 1.98 g (30%).
1H NMR (300 MHz, CDCl3): δ = 4.04 (t, J = 3.8 Hz, 4 H), 4.29
(t, J = 4.3 Hz, 4 H), 7.29 (m, 2 H), 7.74 (m, 2 H), 8.26 (m, 2 H)
ppm. MS (ESI): m/z = 328 [M]+. C18H16O6. MW = 328.09.
2-({[(2-Cyanoethoxy)(diisopropylamino)phosphanyl]oxy}ethyl)oxy]-
6-({[(4,4Ј-dimethoxytrityl)oxy]ethyl}oxy)anthraquinone (16): Yield:
0.25 g (38%). 1H NMR (300 MHz, CDCl3): δ = 1.19 (m, 12 H),
2.65 (m, 2 H), 3.55 (m, 2 H), 3.61 (m, 2 H), 3.78 (s, 6 H), 3.79 (m,
2 H), 4.01 (m, 2 H), 4.33 (m, 4 H), 6.84 (m, 4 H), 7.22 (m, 1 H),
7.28 (m, 4 H), 7.34 (m, 4 H), 7.46 (m, 2 H), 7.73 (m, 2 H), 8.25
(m, 2 H) ppm. 31P NMR (122 MHz, CDCl3): δ = 148.86 ppm.
General Method for 4,4Ј-Dimethoxytrityl (DMT) Protection of
Bis[(hydroxyethyl)oxy]anthraquinones (9–12): The diol (1.0 g,
3.0 mmol) was dissolved in absolute pyridine (8 mL). 4,4Ј-Dimeth-
oxytrityl chloride (1 g, 3.0 mmol) dissolved in absolute pyridine
(8 mL) was added dropwise. After stirring at room temperature for
6 h, saturated aqueous sodium hydrogen carbonate solution was
added. After extraction with dichloromethane and concentration
under reduced pressure, the product was purified by column
chromatography (silica gel; AcOEt + 1% TEA). The fractions were
combined and concentrated.
Synthesis and Analysis of Oligonucleotides: Cyanoethyl phos-
phoramidites from Transgenomic (Glasgow, UK) were used for oli-
gonucleotide synthesis. Oligonucleotides 17–27 were prepared by
automated oligonucleotide synthesis by a standard synthetic pro-
cedure (“trityl-off” mode) with a 394-DNA/RNA synthesizer (Ap-
plied Biosystems). Cleavage from the solid support and final depro-
tection was done by a treatment with 33% aqueous NH3 at 55 °C
overnight. All oligonucleotides were purified by ion exchange
HPLC (Tricorn column SOURCE 15Q 4.6/100 PE 100 15 µm,
Merck, L-6250 Intelligent Pump); eluent A = Na2HPO4 (20 m),
pH 11.5; eluent B = Na2HPO4 (20 m) + NaCl (2 ), pH 11.5;
gradient 0–60% B over 30 min at 25 °C. MS (ESI, negative mode,
CH3CN/H2O/TEA) of oligonucleotides was performed with a Sciex
QSTAR pulsar, (hybrid quadrupole time-of-flight mass spectrome-
ter, Applied Biosystems); data of oligomers 17–27 are given in the
Supporting Information.
1-({[(4,4Ј-Dimethoxytrityl)oxy]ethyl}oxy)-4-[(hydroxyethyl)oxy]-
anthraquinone (9): Yield: 0.66 g (35 %). 1H NMR (300 MHz,
CDCl3): δ = 3.78 (s, 6 H), 3.98 (m, 4 H), 4.28 (m, 4 H), 6.83 (m, 4
H), 7.18 (m, 2 H), 7.28 (m, 3 H), 7.37 (m, 4 H), 7.48 (m, 2 H), 7.73
(m, 2 H), 8.18 (m, 2 H) ppm.
1-({[(4,4Ј-Dimethoxytrityl)oxy]ethyl}oxy)-5-[(hydroxyethyl)oxy]-
anthraquinone (10): Yield: 0.59 g (31 %). 1H NMR (300 MHz,
CDCl3): δ = 3.79 (s, 6 H), 4.01 (m, 4 H), 4.31 (m, 4 H), 6.84 (m, 4
H), 7.15 (m, 1 H), 7.30 (m, 4 H), 7.42 (m, 4 H), 7.50 (m, 2 H), 7.70
(m, 2 H), 7.95 (m, 2 H) ppm.
1-({[(4,4Ј-Dimethoxytrityl)oxy]ethyl}oxy)-8-[(hydroxyethyl)oxy]-
anthraquinone (11): Yield: 0.70 g (37 %). 1H NMR (300 MHz,
CDCl3): δ = 3.78 (s, 6 H), 3.82 (m, 4 H), 4.26 (m, 4 H), 6.85 (m, 4
H), 7.15 (m, 1 H), 7.30 (m, 4 H), 7.38 (m, 4 H), 7.48 (m, 2 H), 7.62
(m, 2 H), 7.88 (m, 2 H) ppm.
Thermal Denaturation Experiments: Carried out with a Varian
Cary-100 Bio-UV/Vis spectrometer equipped with a Varian Cary-
block temperature controller. Data were collected with Varian
WinUV software at 260 nm (cooling–heating–cooling cycles in
the temperature range of 10–90 °C, temperature gradient of
0.5 °Cmin–1). Experiments were carried out for 1.0-µ oligonucleo-
tide concentration (each strand), 10-m phosphate buffer and 100-
m NaCl at pH 7.4. Data were analyzed with Kaleidagraph soft-
ware from Synergy software. Melting temperature (Tm) values were
determined as the maximum of the first derivative of the smoothed
melting curve.
2-({[(4,4Ј-Dimethoxytrityl)oxy]ethyl}oxy)-6-[(hydroxyethyl)oxy]-
anthraquinone (12): Yield: 0.49 g (26 %). 1H NMR (300 MHz,
CDCl3): δ = 3.78 (s, 6 H), 4.05 (m, 4 H), 4.28 (m, 4 H), 6.82 (m, 4
H), 7.20 (m, 1 H), 7.28 (m, 4 H), 7.35 (m, 4 H), 7.46 (m, 2 H), 7.74
(m, 2 H), 8.25 (m, 2 H) ppm.
General Method for the Phosphitylation of Monoprotected Anthra-
quinones (13–16): The alcohol (0.50 g, 0.79 mmol) and ethyldi-
isopropylamine (0.25 g, 2.0 mmol) were dissolved in dichlorometh-
ane (10 mL). 2-Cyanoethyl diisopropylamidochloridophosphite
Fluorescence Data: Collected for 1.0-µ oligonucleotide solutions
(1.0 µ of each strand in case of double strands) in phosphate
Eur. J. Org. Chem. 2008, 2213–2219
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
2217

N. Bouquin, V. L. Malinovskii, R. Häner
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Received: January 23, 2008
Published Online: March 25, 2008
Eur. J. Org. Chem. 2008, 2213–2219
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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