2,1,3-Benzothiadiazole-modified DNA

Article abstract of DOI:10.1002/ejoc.201200231

The use of 2,1,3-benzothiadiazole (BTD) as a structural element with advanced electronic properties for DNA hybrids is described. Bis(alkynyl)- and bis(carboxamide)-derived BTD units are shown to support duplex stability through interstrand stacking inter

Full text of DOI:10.1002/ejoc.201200231

FULL PAPER
DOI: 10.1002/ejoc.201200231
2,1,3-Benzothiadiazole-Modified DNA
Florian Garo[a] and Robert Häner*[a]
Keywords: DNA / Stacking interactions / Fluorescence / Helical structures / Electron transfer
The use of 2,1,3-benzothiadiazole (BTD) as a structural ele- bases decreases in the order G ϾϾ A Ն T Ն C and correlates
ment with advanced electronic properties for DNA hybrids is
described. Bis(alkynyl)- and bis(carboxamide)-derived BTD
units are shown to support duplex stability through in-
terstrand stacking interactions. Placement of the BTD units
opposite to a natural base, however, leads to considerable
destabilization. The bis(alkynyl)-derived BTD W is strongly
fluorescent, and quantum yields of up to 0.20 are observed.
Its fluorescence behavior is strongly dependent on the neigh-
boring nucleobases. The quenching effect of the natural
very well with the free energies for charge separation (ΔGCS)
through photoinduced electron transfer, as calculated by the
Rehm–Weller equation. Fluorescence of W is completely
quenched when it is placed against the bis(carboxamide)-
derived BTD V. The described BTD-based compounds W and
V represent valuable building blocks for the construction of
highly ordered, DNA-based materials with special optical
and electronic properties.
itself as an interesting building block for DNA-based func-
tional materials. Interactions between fluorescent labels and
DNA nucleobases have been extensively investigated, and
the effect of nucleobases on fluorescent labels is of
particular importance for the use of genetic diagnostic
probes.[52–58] The mechanism of fluorescence quenching by
DNA nucleobases is best explained by charge transfer pro-
cesses that occur between the nucleobase and the fluoro-
phore.[59–62] When the nucleobase acts as the electron-do-
nating species, the excited state of the electron-accepting la-
bel is quenched by electron transfer, and a charge-separated
state is formed. The probability of this pathway is best esti-
mated by the change in free energy for charge separation
(ΔGCS) according to the Rehm–Weller equation.[63] Here,
we report the synthesis and the electronic and structural
properties of BTD-modified DNA. BTD units were con-
nected to the DNA phosphate backbone through triple
bonds and amide groups. The influence of the BTD build-
ing blocks on the thermal stability and the fluorescence
properties are described, and the influence of the four types
of nucleobases on BTD-fluorescence is correlated with their
oxidation potentials.
Introduction
DNA represents a versatile tool for the directed assembly
of functional materials, such as chromophores, metal li-
gands, nanoparticles and proteins.[1–5] The use of function-
alized nucleic acids for applications in nanotechnology has
emerged as an independent area of research with implica-
tions in the fields of diagnostics, electronics, and material
sciences.[6–15] Modified nucleic acids containing various
types of building blocks have been described towards this
end.[16–25] As part of our work aimed at the functional ex-
pansion of DNA, we have investigated the structural and
spectroscopic effects of non-nucleosidic, polyaromatic
building blocks on nucleic acids.[26–32] 2,1,3-Benzothiadi-
azole (BTD) derivatives are widely used fluorophores with
excellent spectroscopic properties, such as high molar ab-
sorptivities, high quantum yields, and large Stoke’s
shifts.[33–35] Due to its electron-accepting properties, BTD is
a common component in “donor–acceptor” based π-conju-
gated polymers.[36–42] Derivatives find applications in pho-
tovoltaic devices, as electroluminescent materials, two-pho-
ton absorbing materials, and near-IR emitting fluoro-
phores.[43–46] Furthermore, the molecular polarization sup-
ports the formation of highly ordered structures with strong
π–π interactions.[47–49] Alkynyl-substituted BTD derivatives
were described as DNA markers, presumably acting
through an intercalative binding mode.[50,51] Because of
these structural and electronic properties, BTD presents
Results and Discussion
Preparation of the BTD-based phosphoramidite building
blocks is shown in Scheme 1. Starting from 4,7-dibromo-
2,1,3-benzothiadiazole 1, the hexynyl linker chains were in-
troduced by using the Sonogashira cross-coupling reaction
to give diol 2. 4,4Ј-Dimethoxytrityl (DMT) protection of
one hydroxy group yielded the mono-DMT-protected inter-
mediate 3. Highest yields were obtained by slowly adding
a dilute solution of DMT chloride over a period of 4 h.
[a] Department of Chemistry and Biochemistry, University of
Bern,
Freiestrasse 3, 3012 Bern, Switzerland
Fax: +41-31-631-8057
E-mail: robert.haener@ioc.unibe
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200231.
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Scheme 1. Reagents and conditions: (a) 5-hexyn-1-ol (2.2 equiv.), CuI (0.06 equiv.), bis(triphenylphosphane)palladium(II) dichloride
(0.05 equiv.), Et3N (4.4 equiv.), dioxane, 60 °C, 1 h (92%); (b) 4,4Ј-dimethoxytrityl chloride (1.0 equiv.), pyridine, DMAP (0.15 equiv.),
CH2Cl2, room temp., 4 h (45%); (c) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.1 equiv.), N,N-diisopropylethylamine
(2.0 equiv.), CH2Cl2, room temp., 1 h (4: 90%; 8: 75%); (d) CuCN (4 equiv.), pyridine, NaI (0.1 equiv.), DMF, 180 °C, 6 h (80%); (e)
25% NaOH in water, 100 °C, 2 h (85%); (f) 3-hydroxypropylamine/3-(4,4Ј-dimethoxytrityloxy)propylamine (1:1), HBTU (2.2 equiv.), N,N-
diisopropylethylamine (4.0 equiv.), DMF, room temp., 2 h (33%).
Compound 3 was phosphitylated to 4,7-dihexynyl-BTD-
Compounds 2 and 6 were characterized by UV/Vis ab-
phosphoramidite 4. For the synthesis of the second building sorbance and fluorescence spectroscopy. Figure 1 compares
block, amide linker chains were introduced by converting 1 the spectroscopic properties of the bis(alkynyl)- and the
through the Rosenmund–von Braun reaction into BTD-4,7- bis(carboxylic acid)-derived BTDs 2 and 6. The absorption
dicarbonitrile 5,[64] which was subsequently hydrolyzed to spectrum of 2 exhibited a bathochromic shift compared to
BTD-4,7-dicarboxylic acid 6.[65] Amide formation involved 6 (approximately 70 nm in the lowest energy bands). Both
treatment of 6 with a 1:1 mixture of 3-hydroxypropylamine compounds exhibit a broad unstructured band in the fluo-
and 3-(4,4Ј-dimethoxytrityloxy)propylamine[66] to provide rescence spectrum. The emission maximum of compound 2
the mono-DMT-protected intermediate 7. Phosphitylation is again redshifted by approximately 55 nm. In comparison,
finally gave the amide-derived BTD-phosphoramidite 8. the alkynyl-substituted derivative 2 showed significantly
The two building blocks 4 and 8 were incorporated into higher steady-state fluorescence intensity than the bis(carb-
DNA oligonucleotides by using phosphoramidite chemistry oxylic acid) 6 (quantum yields: 0.23 for 2 and 0.046 for 6).
(see below).[67]
Similar bathochromic shifts and increased quantum yields
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2,1,3-Benzothiadiazole-Modified DNA
Table 1. Properties of compounds 2 and 6.
Compound 2
Compound 6
Abs. λmax [nm] (ε [Lmol–1 cm–1])[a]
Emission λmax [nm] (quantum yield)[b]
Reduction potential [V][c]
240 (16400), 260 (22900), 270 (26100), 310 (8700), 320 (12700), 390 (8000)
230 (12400), 315 (11300)
460 (0.05)
515 (0.23)
–1.12
–0.67
[a] 10 μm in water. [b] Quinine sulfate as fluorescence standard. [c] Versus Ag/AgCl; concentration 1.0 mm (2) and 0.5 mm (6); 0.1 m
tetrabutylammonium hexafluorophosphate in acetonitrile.
were observed with pyrene.[28] Moreover, replacement of the Table 2. Change in Gibbs free enthalpies of photoinduced charge
separation (ΔGCS) between 2 or 6 and the four DNA nucleobases.
amide linkers by triple bonds in non-nucleosidic aromatic
Eox [V vs. SCE][a]
ΔGCS(2) [eV][b]
ΔGCS(6) [eV][b]
DNA building blocks increased the hydrophobicity of the
units, which led to a substantial increase in the stacking
interactions.[28,68] In combination with the described intra-
molecular polarization of BTD, the alkynyl linkers should
lead to a preferential hybrid stability in complementary
DNA strands containing building block 2. In addition, the
advanced fluorescence properties of this compound are
worth further investigation in the context of the DNA envi-
ronment, especially with regard to their electronic interac-
tions with nucleobases. In general, BTD derivatives are
known as strong electron acceptors. The reduction poten-
tials of 2 and 6 were determined by cyclic voltammetry to
be –1.12 and –0.67 V, respectively (vs. Ag/AgCl, see the
Supporting Information). This and other relevant electronic
properties of the two monomeric building blocks are sum-
marized in Table 1.
Base
G
A
T
1.25
1.72
1.87
1.90
–0.40
0.07
0.22
0.25
–1.16
–0.69
–0.54
–0.51
C
[a] Values taken from the literature.[53] [b] Calculated according to
Rehm and Weller.[63] The values for the excitation energies were
2.80 eV (443 nm; 2) and 3.10 eV (400 nm; 6). For more details see
Figure 1 and the Supporting Information.
The two building blocks 4 and 8 were incorporated into
DNA oligonucleotides by phosphoramidite chemistry.[67]
The studied oligonucleotides 9–20 are shown in Table 3.
Oligonucleotides 9 and 20 contain a 4,7-dihexynyl-derived
BTD unit (abbreviated as W) in the middle of the sequence.
Based on the calculations described above, guanine was ex-
pected to have a strong quenching effect. Therefore, the
flanking DNA parts were composed exclusively of AT base
pairs. Duplex formation with any of the oligonucleotides
10–19 leads to a hybrid with one of the natural nucleobases
or a 4,7-dicarboxamide-derived BTD unit (abbreviated as
V) opposite to W. This set allows the effect of W on hybrid
stability as well as the quenching effect of the individual
nucleobases on BTD fluorescence to be established.
Table 3. Oligonucleotides 9–20 and the structures of BTD-based
building blocks W and V.
Figure 1. UV/Vis absorbance and fluorescence spectra of com-
pounds 2 (10 μm; orange/red; λex = 350 nm) and 6 (10 μm: light-
blue/blue; λex = 350 nm) in water.
The feasibility of photoinduced electron transfer (PET)
between the chromophores and the four canonical DNA
nucleobases adenine (A), guanine (G), cytosine (C), and
thymine (T) was calculated by using the Rehm–Weller equa-
tion[52,63] (see the Supporting Information) and are summa-
rized in Table 2. Based on these calculations, the change in
Gibbs free energy of the photoinduced charge separation
(ΔGCS) between 2 and the nucleobases is exergonic only for
guanine; for the three other bases, ΔGCS is slightly or
strongly endergonic. All ΔGCS values for PET between 6
and the four DNA nucleobases were strongly exergonic.
Melting temperature (Tm) values of the hybrids were de-
These values of ΔGCS helped to delineate the quenching ef- termined by conventional thermal denaturation (see the
fects of the nucleobases on the BTD building blocks (see Supporting Information). Introduction of W opposite one
below).
of the natural bases led to a considerable decrease in the
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Tm of the hybrid (10 or 14 °C compared to the hybrid with W and V into DNA strands caused a slight bathochromic
an AT or a GC; see Table 4 and the Supporting Infor- shift of the absorbance bands compared to the spectra of 2
mation). Replacement of an entire base pair with a W*V and 6 (8 and 9 nm). A hypochromic effect was observed in
or W*W pair, however, resulted in a significantly smaller hybrid 19*20 (W and V) at 400 nm compared to hybrid
destabilization. A W*W pair (9*20) has approximately the 17*20 (W next to C), which serves as an indication for aro-
same stabilizing effect as an AT base pair (cf. 10*16). Tm matic stacking interactions between W and V. For steady-
values of hybrids with any of the four DNA bases in the state fluorescence measurements, W was excited at 400 nm.
counter strand facing W (hybrids with 9 or 20) were equal The emission spectra of the same three hybrids discussed
within the experimental error. Overall, the Tm values were above are shown in Figure 2b. Emission curves are identical
constant within 1 °C for all combinations with a natural in shape and position. Considerable differences, however,
DNA base opposite W. This is in agreement with previous were observed in their intensities. The fluorescence of the
studies involving other non-nucleosidic aromatic building bis(carboxamide)-derived BTD V was nearly completely
blocks and their effects on DNA hybrids,[69] and suggests quenched after incorporation into DNA strands, which is
that there is little interaction between W and the nucleo- not unexpected, because the corresponding monomer 6 also
bases. Interaction between pairs of W or between W and V, showed a relatively weak fluorescence (Figure 1). Addition-
restore the duplex stability nearly completely. The stability ally, the ΔGCS values for this building block with all four
of hybrid 9*20 (W*W) is an indication that the high polar- DNA bases are strongly exergonic (see the Supporting In-
ization in BTD leads to favorable π–π stacking interac- formation), which explains why the two DNA strands con-
tions.[49] The combination of W and V (9*14 and 19*20) taining V were nonfluorescent (see the Supporting Infor-
are also favorable, albeit to a lesser extent. Considering that mation). The quenching effect exerted by the natural bases
the aromatic surfaces of W and V are smaller than those on the fluorescence of W follows the order G ϾϾ A Ն T
of other non-nucleosidic polyaromatic DNA building Ն C and, thus, closely follows the calculated values of ΔGCS
blocks, such as pyrenes, phenanthrenes, or perylenebis- obtained for this series (Table 2). Hybrid 17*20 (W against
(imides),[70,71] W and V efficiently stabilize DNA hybrids C) has a quantum yield of almost 0.2, which is close to the
by interstrand stacking interactions. The smaller aromatic quantum yield of the monomeric building block 2 in water.
surface is largely compensated for by the polarity of the This shows that A, T, and C residues in the immediate
BTD core.
neighborhood of the BTD fluorophore have very little or
no quenching effect.
Table 4. Quantum yields (Φ) and Tm values of hybrids.
Hybrid
Pair
Tm [°C][a]
ΔTm [°C][b]
Φ[c]
16*10
17*13
9*10
9*11
9*12
9*13
9*14
T*A
C*G
W*A
W*T
W*C
W*G
W*V
41.5
44.5
32.0
32.0
31.5
32.5
39.5
–
–
–
–
–9.5/–12.5
–9.5/–12.5
–10.0/–13.0
–9.0/–12.0
–2.0/–5.0
0.12
0.12
0.12
0.02
Ͻ0.01
15*20
16*20
17*20
18*20
19*20
9*20
A*W
T*W
C*W
G*W
V*W
W*W
32.0
32.5
32.0
32.0
40.0
41.5
–9.5/–12.5
–9.0/–12.0
–9.5/–12.5
–9.5/–12.5
–1.5/–4.5
0.0/–3.0
0.13
0.16
0.20
0.03
Ͻ0.01
0.11
[a] 1.0 μm single-strand concentration, 100 mm NaCl, 10 mm so-
dium phosphate buffer, pH = 7.0; experimental error Ϯ1.0 °C (see
also the Supporting Information). [b] First value against 16*10,
second against 17*13. [c] Coumarin 343 was used as fluorescence
standard (see the Supporting Information).
Figure 2. UV/Vis absorbance (a) and fluorescence spectra (b) of
hybrids 9*20 (blue), 17*20 (black) and 19*20 (red). Temperature:
15 °C; λex = 400 nm.
UV/Vis absorbance spectra of all hybrids were recorded.
The combination of V and W (hybrid 19*20) results in
The spectra of three different types of hybrids are shown in almost complete quenching of W (Φ Ͻ 0.01, Table 4) lead-
Figure 2a and are representative for the whole study (see ing to an on/off behavior of the W fluorescence depending
also the Supporting Information). The region below 300 nm on the presence or absence of V. The emission curves of
is dominated by the absorption bands of the nucleobases, hybrids 17*20 (W opposing C) and 9*20 (W opposing W)
whereas the area between 300 and 470 nm shows only elec- differ only slightly in their intensities but are identical in
tronic transitions from the BTD derivatives W and/or V. shape and position. Association of two bis(alkynyl)-derived
The band with a maximum at 400 nm is due to the bis(alk- BTD molecules does not lead to excimer fluorescence. In-
ynyl)-derived BTD W, whereas the 325 nm band represents stead, a certain degree of self-quenching is observed. Self-
an overlay of W and V absorbance. The incorporation of quenching was reported for syn- and anti-[2.2](4,7)benzo-
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2,1,3-Benzothiadiazole-Modified DNA
thiadiazolophanes. The quenching effect was much stronger of the experimental fluorescence quantum yields of W in
when the thiadiazole units were arranged syn to each hybrids containing 20 followed exactly the order of the oxi-
other.[72] An anti arrangement resulted in only partial dation potentials of the DNA bases (Table 2). The same
quenching. Partial quenching was observed in hybrids, quenching effects were also observed when G was placed
which suggests an anti arrangement of the two BTD units. directly next to W in the same strand (see the Supporting
An energy-minimized structure of hybrid 9*20, in which the Information).
two bis(alkynyl)-derived BTD units W are π-stacked in an
interstrand fashion, is shown in Figure 3.
Conclusions
Two types of 2,1,3-benzothiadiazole-based phosphoram-
idite building blocks were synthesized. The building blocks
were integrated into oligodeoxynucleotides through alkynyl
or carboxamide linkers (W and V). Both building blocks
were found to support duplex stability in hybrids containing
a BTD unit in each of the strands in opposite positions
through interstrand stacking interactions. This stabilization
is attributed to the high polarization in the BTD molecules
caused by the electron-withdrawing thiadiazole and the fa-
vorable π–π stacking interactions resulting therefrom.
Placement opposite to a natural base, however, resulted in
Figure 3. An Amber-minimized HyperChem model of hybrid 9*20 considerable destabilization. The bis(alkynyl)-derived BTD
with the two W units in orange (top). The anti arrangement of π–
π-stacked thiadiazole units is illustrated below.
W exhibited a fluorescence behavior that was strongly de-
pendent on the neighboring nucleobases. The quenching ef-
fect decreased in the order G ϾϾ A Ն T Ն C and corre-
lated very well with the calculated free energies for charge
separation (ΔGCS) through photoinduced electron transfer.
Fluorescence of W is essentially completely quenched when
it is placed against the bis(carboxamide)-derived BTD V.
The described BTD-based compounds W and V represent
valuable building blocks for the construction of highly or-
dered, DNA-based materials with special optical and elec-
tronic properties.
The results show that the fluorescence efficiency of W
critically depends on the neighboring groups in the direct
vicinity. The quantum yields of the hybrids are displayed
graphically in Figure 4. White and black bars represent hy-
brids in which W is sandwiched between two adenine or
thymidine units, respectively. Generally, the highest quan-
tum yield resulted from combinations with C (17*20),
whereas the fluorescence was quenched in all combinations
with G. The quenching effect of G can be explained by the
exergonic ΔGCS value for the W*G combination. The values
Experimental Section
General: Compounds 5 and 6 were synthesized according to proto-
cols described in the literature.[64,65] All other compounds were syn-
thesized as described below and analyzed by NMR spectroscopy
(Bruker, 300 MHz) and mass spectrometry [LTQ Orbitrap XL; ac-
curate mass determination with nanospray ionization (NSI) in the
positive mode; acetonitrile as solvent]. The natural nucleotide phos-
phoramidite building blocks were purchased from SAFC Proligo
(Hamburg, Germany). Oligonucleotides 9, 14, 19, 20, 21, 26, 31,
and 32 were synthesized with an Applied Biosystems 394-DNA/
RNA synthesizer by using standard synthetic procedures (“trityl-
off” mode). The coupling time for the artificial building blocks was
prolonged to 2 min. The coupling yields for all building blocks per
single step were between 95 and 99% (“trityl assay”). Cleavage
from the solid support and final deprotection were achieved by
treatment with 30% NH4OH solution (55 °C, 2 h). All oligonucleo-
tides were purified by reverse-phase HPLC (LiChrospher 100 RP-
18 5 μm column; 0.1 m triethylammonium acetate at pH = 7.0 and
acetonitrile). The fully natural oligonucleotides (10–13, 15–18, 22–
25, and 27–30) were purchased from Microsynth AG (Balgach,
Switzerland). All oligonucleotides were characterized with a Shim-
adzu LCMS-2010EV (Waters XTerra MS C-18 3.5 μm column;
50 mm ammonium formate and acetonitrile) and quantified by UV
absorption measurements at 260 nm (molar extinction coefficient
Figure 4. Quantum yields of the hybrids listed in Table 4. White
bars: hybrids containing strand 9; black bars: hybrids containing
strand 20; B indicates the partner in the counter strand.
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F. Garo, R. Häner
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at 260 nm: 20000 m–1 cm–1 for W and 9100 m–1 cm–1 for V). The
solutions for all spectroscopic measurements were 1 μm single-
strand concentration (2 μm total oligonucleotide concentration),
100 mm sodium chloride, and 10 mm sodium phosphate buffer at
pH = 7.0. UV/Vis absorbance and thermal denaturation experi-
ments were carried out with a Varian Cary-100 Bio-UV/Vis spec-
trophotometer. Melting-temperature values (Tm) were determined
from the 1st derivative of the second cooling ramp of a cooling-
heating-cooling cycle (temperature range 10–80 °C, temperature
gradient 0.5 °C/min, optic path length 10 mm). Fluorescence data
were collected with a Varian Cary Eclipse fluorescence spectropho-
tometer. The instrumental setup was 5 nm slit width (excitation and
emission), 800 V detector sensitivity and 15 °C probe temperature
(if not mentioned differently). Cyclic voltammetry measurements
were carried out with a Metrohm 663 VA Stand with a PGStat 20.
The scan speed was 100 mV/s, sample concentration was 1.0 mm in
acetonitrile with 0.1 m tetrabutylammonium hexafluorophosphate.
Molecular modeling by using HyperChem (HyperCube, version
8.5) was performed as follows: the ideal DNA double strand (tem-
plate) was disconnected at the corresponding phosphate units, and
the W building blocks were introduced. The geometry of the whole
resulting duplex was optimized by applying the Amber force field.
4,7-Dihexynyl-Derived BTD Phosphoramidite (4): To a solution of
3 (500 mg, 0.79 mmol) and N,N-diisopropylethylamine (270 μL,
1.59 mmol) in anhydrous dichloromethane (16 mL), 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (130 μL, 0.87 mmol) was
added dropwise. The reaction mixture was stirred at room tempera-
ture for 1 h, then concentrated under reduced pressure and the
crude product purified by column chromatography [ethyl acetate/
hexane, 1:1 with 2% triethylamine]. Compound 4 was isolated as a
1
yellow gel (598 mg, 90%). H NMR (300 MHz, CDCl3): δ = 7.66–
7.52 (m, 2 H), 7.49–7.40 (m, 2 H), 7.38–7.14 (m, 7 H), 6.87–6.74
(m, 4 H), 3.96–3.50 (m, 12 H), 3.13 (t, J = 5.63 Hz, 2 H), 2.72–
2.50 (m, 6 H), 1.96–1.68 (m, 8 H), 1.20 (s, 3 H), 1.19 (s, 3 H), 1.18
(s, 3 H), 1.17 (s, 3 H) ppm. 13C (300 MHz, CDCl3) δ = 158.5, 154.8,
145.5, 136.8, 132.5, 132.4, 130.2, 128.3, 127.8, 126.7, 117.7, 117.5,
117.3, 113.1, 98.9, 98.5, 85.9, 77.4, 77.0, 63.4, 63.2, 63.0, 58.6, 58.3,
55.3, 43.3, 43.1, 30.6, 30.5, 29.6, 25.8, 25.3, 24.83, 24.78, 24.74,
24.68, 20.6, 20.5, 20.0, 19.8 ppm. 31P NMR (300 MHz, CDCl3): δ
= 147.6 ppm. HRMS (NSI): calcd. for C48H55N4O5PS [M]+ 830.36;
found 831.3714 [M + H]+.
DMT-Protected 4,7-Diamido-Derived BTD (7): To a solution of 6
(250 mg, 1.12 mmol) and N,N-diisopropylethylamine (765 μL,
4.46 mmol) in anhydrous DMF (4 mL) under argon, 3-(4,4Ј-di-
methoxytrityloxy)propylamine[66] (421 mg, 1.12 mmol) and 3-hy-
droxypropylamine (85 μL, 1.12 mmol) in anhydrous DMF (4 mL)
was added. O-(Benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium
hexafluorophosphate (HBTU; 931 mg, 2.46 mmol) was added at
once to the reaction mixture. The mixture was stirred at room tem-
perature for 2 h, then diluted with ethyl acetate and washed once
with saturated aqueous ammonium chloride, saturated aqueous so-
dium hydrogen carbonate, and saturated aqueous sodium chloride.
The organic phase was dried with magnesium sulfate, filtered, and
concentrated under reduced pressure. The crude product was puri-
fied by column chromatography [ethyl acetate with 2% triethyl-
amine, changed to ethyl acetate/methanol (95:5) with 2% triethyl-
amine after the bis(DMT)-protected product was eluted]. Com-
pound 7 was isolated as a brownish foam (240 mg, 33%). 1H NMR
(300 MHz, CDCl3): δ = 9.23 (t, J = 6.22 Hz, 1 H), 9.02 (t, J =
5.37 Hz, 1 H), 8.79–8.66 (m, 2 H), 7.48–7.11 (m, 9 H), 6.78–6.67
(m, 4 H), 3.91–3.60 (m, 12 H), 3.29 (t, J = 5.84 Hz, 2 H), 3.10 (t,
J = 6.50 Hz, 1 H), 2.09–1.83 (m, 4 H) ppm. 13C NMR (300 MHz,
CDCl3): δ = 163.8, 162.4, 158.5, 152.2, 152.2, 145.1, 136.4, 133.3,
132.9, 130.2, 128.4, 127.9, 127.2, 126.9, 113.1, 86.3, 77.4, 61.6, 59.4,
55.3, 38.3, 36.8, 32.8, 29.8 ppm. HRMS (NSI): calcd. for
C35H36N4O6S [M]+ 640.24; found 663.2247 [M + Na]+.
4,7-Dihexynyl-Derived BTD Diol (2): A solution of 4,7-dibromo-
2,1,3-benzothiazole (700 mg, 2.38 mmol), 5-hexyn-1-ol (580 μL,
5.24 mmol), and Et3N (1.46 mL, 10.48 mmol) in 1,4-dioxane
(15 mL) was degassed for 5 min with a stream of argon directly
into the solution. Bis(triphenylphosphane)palladium(II) dichloride
(84 mg, 0.12 mmol) and copper(I) iodide (27 mg, 0.14 mmol) were
added at once. The suspension was stirred at 60 °C for 1 h. The
reaction mixture was diluted with ethyl acetate and washed three
times with saturated aqueous ammonium chloride and twice with
saturated aqueous sodium chloride. The organic layer was dried
with magnesium sulfate, filtered, and concentrated under reduced
pressure. The crude product was purified by column chromatog-
raphy (ethyl acetate/dichloromethane, 1:1). Compound 2 was iso-
lated as an orange solid (721 mg, 92%); Rf = 0.22 (ethyl acetate/
dichloromethane, 1:1). 1H NMR (300 MHz, CDCl3): δ = 7.59 (s, 2
H), 3.81–3.71 (m, 4 H), 2.69–2.60 (m, 4 H), 1.85–1.76 (m, 8 H),
1.68–1.55 (m, 2 H) ppm. 13C NMR (300 MHz, CDCl3): δ = 154.8,
132.4, 117.3, 98.7, 77.4, 62.4, 32.1, 25.0, 19.9 ppm. HRMS (NSI):
calcd. for C18H20N2O2S [M]+ 328.12; found 329.1320 [M + H]+.
DMT-Protected 4,7-Dihexynyl-Derived BTD (3): To a solution of 2
(600 mg, 1.82 mmol), pyridine (180 μL, 2.19 mmol), and 4-(dimeth-
ylamino)pyridine (33 mg, 0.27 mmol) in dichloromethane (13 mL),
4,4Ј-dimethoxytrityl chloride (618 mg, 1.82 mmol) in anhydrous
dichloromethane (13 mL) was added dropwise at room temperature
over 3 h. The reaction mixture was stirred at room temperature for
1 h, then diluted with ethyl acetate and washed twice with saturated
aqueous ammonium chloride, once with saturated aqueous sodium
hydrogen carbonate and once with saturated aqueous sodium
chloride. The organic layer was dried with magnesium sulfate, fil-
tered, and concentrated under reduced pressure. The crude product
was purified by column chromatography [ethyl acetate/hexane, 4:3
with 2% triethylamine]. Compound 3 was isolated as a yellow gel
4,7-Diamido-Derived BTD Phosphoramidite (8): To a solution of 7
(200 mg, 0.31 mmol) and N,N-diisopropylethylamine (110 μL,
0.63 mmol) in anhydrous dichloromethane (6 mL), 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (51 μL, 0.34 mmol) was
added dropwise. The reaction mixture was stirred at room tempera-
ture for 1 h, then concentrated under reduced pressure and the
crude product purified by column chromatography (ethyl acetate
with 2% triethylamine). Compound 8 was isolated as a brownish
1
foam (200 mg, 75%). H NMR (300 MHz, CDCl3): δ = 9.16 (t, J
= 5.56 Hz, 1 H), 9.06 (t, J = 5.37 Hz, 1 H), 8.79–8.63 (m, 2 H),
7.46–7.05 (m, 9 H), 6.78–6.70 (m, 4 H), 3.96–3.53 (m, 16 H), 3.32
(525 mg, 45%). 1H NMR (300 MHz, CDCl3): δ = 7.58 (m, 2 H), (t, J = 5.84 Hz, 2 H), 2.68 (t, J = 6.31 Hz, 2 H), 2.14–1.90 (m, 4
7.47–7.40 (m, 2 H), 7.37–7.15 (m, 7 H), 6.85–6.77 (m, 4 H), 3.81–
3.71 (m, 8 H), 3.13 (t, J = 5.65 Hz, 2 H), 2.69–2.53 (m, 4 H), 1.90–
1.72 (m, 8 H), 1.49 (br. s, 1 H) ppm. 13C NMR (300 MHz, CDCl3):
δ = 158.5, 154.8, 145.5, 136.8, 132.4, 130.2, 128.4, 127.8, 126.7,
117.5, 117.3, 113.1, 99.0, 98.6, 85.9, 77.4, 77.0, 63.0, 62.5, 55.3,
H), 1.21 (s, 3 H), 1.20 (s, 3 H), 1.19 (s, 3 H), 1.17 (s, 3 H) ppm.
13C NMR (300 MHz, CDCl3): δ = 162.7, 162.5, 158.5, 152.2, 145.1,
136.4, 133.0, 132.9, 130.2, 128.4, 128.1, 127.8, 127.7, 126.9, 117.7,
113.1, 86.3, 77.4, 61.5, 61.3, 58.7, 58.4, 55.3, 43.3, 43.2, 38.2, 37.5,
31.2, 31.1, 29.8, 24.8, 24.7, 20.6, 20.5 ppm. 31P NMR (300 MHz,
32.1, 29.6, 25.8, 25.0, 20.0, 19.9 ppm. HRMS (NSI): calcd. for CDCl3): δ = 147.9 ppm. HRMS (NSI): calcd. for C44H53N6O7PS
C39H38N2O4S [M]+ 630.26; found 653.2447 [M + Na]+. [M]+ 840.34; found 863.3331 [M + Na]+.
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Eur. J. Org. Chem. 2012, 2801–2808

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F. Garo, R. Häner
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Received: February 27, 2012
Published Online: March 30, 2012
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Eur. J. Org. Chem. 2012, 2801–2808