Synthetic incorporation of Nile Blue into DNA using 2′-deoxyriboside substitutes: Representative comparison of (R)-and (S)-aminopropanediol as an acyclic linker

Article abstract of DOI:10.3762/bjoc.6.13

The Nile Blue chromophore was incorporated into oligonucleotides using click chemistry for the postsynthetic modification of oligonucleotides. These were synthesized using DNA building block 3 bearing an alkyne group and reacted with the azide 4. (R)-3-amino-1,2-propanediol was applied as the linker between the phosphodiester bridges. Two sets of DNA duplexes were prepared. One set carried the chromophore in an A-T environment, the second set in a G-C environment. Both were characterized by optical spectroscopy. Sequence-dependent fluorescence quenching was applied as a sensitive tool to compare the stacking interactions with respect to the chirality of the acyclic linker attachment. The results were compared to recent results from duplexes that carried the Nile Blue label in a sequentially and structurally identical context, except for the opposite chirality of the linker ((S)-3-amino-1,2-propandiol). Only minor, negligible differences were observed. Melting temperatures, UV-vis absorption spectra together with fluorescence quenching data indicate that Nile Blue stacks perfectly between the adjacent base pairs regardless of whether it has been attached via an S-or R-configured linker. This result was supported by geometrically optimized DNA models.

Full text of DOI:10.3762/bjoc.6.13

Synthetic incorporation of Nile Blue into DNA
using 2′-deoxyriboside substitutes:
Representative comparison of (R)- and (S)-
aminopropanediol as an acyclic linker
Daniel Lachmann1, Sina Berndl1, Otto S. Wolfbeis2
and Hans-Achim Wagenknecht*1
Full Research Paper
Open Access
Address:
Beilstein Journal of Organic Chemistry 2010, 6, No. 13.
1University of Regensburg, Institute for Organic Chemistry, 93040
Regensburg, Germany and 2University of Regensburg, Institute for
Analytical Chemistry, Chemo- and Biosensors, 93040 Regensburg,
Germany
doi:10.3762/bjoc.6.13
Received: 20 November 2009
Accepted: 25 January 2010
Published: 09 February 2010
Email:
Hans-Achim Wagenknecht* -
achim.wagenknecht@chemie.uni-regensburg.de
Associate Editor: S. Flitsch
© 2010 Lachmann et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
cycloaddition; fluorescence; glycol; oligonucleotide; phenoxazinium;
phosphoramidite
Abstract
The Nile Blue chromophore was incorporated into oligonucleotides using “click” chemistry for the postsynthetic modification of
oligonucleotides. These were synthesized using DNA building block 3 bearing an alkyne group and reacted with the azide 4. (R)-3-
amino-1,2-propanediol was applied as the linker between the phosphodiester bridges. Two sets of DNA duplexes were prepared.
One set carried the chromophore in an A-T environment, the second set in a G-C environment. Both were characterized by optical
spectroscopy. Sequence-dependent fluorescence quenching was applied as a sensitive tool to compare the stacking interactions with
respect to the chirality of the acyclic linker attachment. The results were compared to recent results from duplexes that carried the
Nile Blue label in a sequentially and structurally identical context, except for the opposite chirality of the linker ((S)-3-amino-1,2-
propandiol). Only minor, negligible differences were observed. Melting temperatures, UV–vis absorption spectra together with
fluorescence quenching data indicate that Nile Blue stacks perfectly between the adjacent base pairs regardless of whether it has
been attached via an S- or R-configured linker. This result was supported by geometrically optimized DNA models.
Introduction
Chemical bioanalysis and imaging of nucleic acids require can be incorporated routinely at specific positions within the
synthetic incorporation of fluorescent DNA probes for an oligonucleotide using standard phosphoramidite DNA chem-
optical readout [1-9]. A large variety of organic fluorophores istry and commercially available DNA building blocks.
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Beilstein Journal of Organic Chemistry 2010, 6, No. 13.
However, problems can arise if labels or probes are chemically modified oligonucleotides which has been reduced from 3 (in
incompatible with the conditions of DNA synthesis, or if the normal nucleosides) to 2 (Scheme 1). However, we have shown
stepwise synthesis of the corresponding DNA building block that the ethidium dye connected to D-threoninol as a linker
fails. So-called postsynthetic modifications can overcome these bearing three carbon atoms between the phosphodiester bridges
limitations and allow the synthetic modification of a presynthe- has nearly the same optical properties as the shorter (S)-3-
sized oligonucleotide carrying a special functional group [10]. amino-1,2-propanediol linker [38]. It is important to point out,
Bioorthogonality is required for these ligation reactions in that however, that chirality of the acyclic linker system has proven
both the functional group of the oligonucleotides and the func- to be an important aspect in terms of chromophore stacking
tional group of the modifier should not be present in typical orientation [39,40], oligonucleotide function [41] and duplex
biomolecules and should react selectively with each other [11]. formation [42]. Komiyama et al. were able to show that D- and
Over the last five years the Huisgen–Meldal–Sharpless “click” L-threoninol as alternative acyclic linker systems act dif-
ligation strategy has become an important strategy for postsyn- ferently as 2′-deoxyribose substitutes by influencing the
thetic labeling of DNA [12,13]. Huisgen described first the stacking orientation of an attached azobenzene dye. The con-
[2+3]-cycloaddition between alkynes and azides yielding 1,2,3- figuration of the linker decides if the dye protrudes towards the
triazoles [14]. The utility of this reaction as a bioligation major or minor groove, which subsequently leads to an
method has grown incredibly after Meldal [15] and – almost at enhanced or diminished stability of the whole DNA duplex
the same time – Sharpless [16] had reported that the addition of [42]. Herein, we want to explore how important the chirality of
Cu(I) led to a significant increase in the reaction rate and in the 3-amino-1,2-propanediol linker is with respect to the optical
regioselectivity. This type of “click” chemistry matches the properties of an attached fluorophore. We chose Nile Blue as
requirements of bioorthogonality since both two functional the fluorescent probe for these experiments since the redox
groups, alkyne and azide, are typically not present in biopoly- properties of this phenoxazinium label exhibit a potential suffi-
mers and react selectively with each other in aqueous solutions. cient for photooxidizing guanines in the sequential neighbor-
hood [17]. As a result, fluorescence quenching is observed in
The “click” ligation can avoid the time-consuming synthesis of DNA and it is important to note that we decided to use this
phosphoramidites as DNA building blocks which is especially property as a sensitive tool to compare the electronic interac-
important for brightly emitting fluorophores that are not tions of the phenoxazinium chromophore with the DNA base
compatible with the acidic, oxidative or basic conditions of stack in order to evaluate the role of the chirality of the
automated DNA phosphoramidite chemistry and/or DNA 3-amino-1,2-propanediol linker.
workup. We recently presented the postsynthetic incorporation
of Nile Blue and a coumarin dye as representatives of base-
labile fluorophores by the “click” ligation strategy [17]. Several
other fluorophores (spanning the whole visible spectrum) for
use in “click” conjugation have been reported meanwhile [18].
The dye carrying the azide group was reacted with an alkyne
group that was incorporated into the oligonucleotides as a
nucleotide substituent. (S)-3-Amino-1,2-propanediol was used
as an acyclic linker and substitute for the 2′-deoxyriboside
Scheme 1: Chirality of C-3 of natural 2′-deoxyribofuranosides (left) in
comparison with the acyclic D-threoninol [38] and (S)-[17] and (R)-3-
amino-1,2-propanediol linker of this study (to the right).
between the phosphodiester bridges. Similar propanediol de- Results and Discussion
rivatives have been used extensively as alternative and simpli- The DNA building block ethynyl (2R)-3-(4,4′-dimethoxytrityl)-
fied phosphodiester linkers in the 1990s [19-23], and have been 2-[(2-cyanoethoxy)(diisopropylamino)phosphinooxy]propylcar-
further explored for glycol nucleic acid (GNA) [24-26] twisted bamate (3) contains the propargyl group attached to the glycol
intercalating nucleic acids (TINA) [27,28], and by our group for part by a carbamate function (Scheme 2). In comparison to our
fluorescent DNA base substitutions by ethidium [29,30], indole earlier synthetic protocols for incorporation of ethidium [29,30],
[31,32], thiazole orange [33,34], perylene bisimide [35,36] and indole [31] and phenothiazine[37] the NH group of the
phenothiazine [37]. This 2′-deoxyriboside substitution provides carbamate is less nucleophilic and need not be protected during
high chemical stability and conformational flexibility for the phosphoramidite synthesis. This facilitates the preparation of
chromophore to intercalate.
DNA building blocks as we have shown with indole [32] and
thiazole orange [33,34]. DMT-protected (R)-3-amino-1,2-
The major difference between the 3-amino-1,2-propanediol propanediol 1 as a precursor was synthesized according to lit-
linker and the 2′-deoxyribofuranoside is the number of carbon erature [29,43]. The hydroxy function of commercially avail-
atoms between the phosphodiester bridges in the corresponding able 2-propyn-1-ol was converted into an activated ester by
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Beilstein Journal of Organic Chemistry 2010, 6, No. 13.
1,1′-carbonyldiimidazole and subsequent acylation gave
conjugate 2 in 44% yield. The synthesis of phosphoramidite 3
was finished by standard procedures, and 3 was applied directly
for automated DNA synthesis. Presynthesized oligonucleotides
for DNA1 and DNA2 were reacted with the Nile Blue azide 4
according to our “click”-type ligation protocol, worked up and
purified according to literature [17]. The two Nile Blue-modi-
fied oligonucleotides DNA1 and DNA2 were identified by ESI
mass spectrometry, quantified by UV–vis absorption and char-
acterized by fluorescence spectroscopy (including quantum
yields) and measurement of melting temperatures (Tm). DNA
duplexes are formed by heating in the presence of 1.2 equiv of
unmodified counterstrands at 90 °C for 10 min followed by
slow cooling to r.t. The two representative oligonucleotides,
DNA1 and DNA2, expose the blue phenoxazinium chromo-
phore to two different variations in the neighborhood: (i) the
dye was placed either in an A-T environment (DNA1Y) or next
to G-C base pairs (DNA2Y), and (ii) within each duplex set,
DNA1Y and DNA2Y, the base opposite to the chromophore
site was varied (Y = A, T, C or G).
Representatively, we compared melting temperatures of the
Nile Blue-modified duplexes DNA1A and DNA2A with the
corresponding unmodified DNA sequences that bear a standard
T instead of the Nile Blue chromophore (Table 1). The replace-
ment of the natural 2′-deoxyribofuranoside by the R-configured
acyclic glycol linker results in a decrease of thermal stability by
3.4 °C in DNA1A and 1.3 °C in DNA2A. Destabilization of the
corresponding duplexes with the S-configured linker was
similar [16].That means that there is no significant difference
(ΔTm ≤ 2.5 °C) between the S- and R-configured acyclic linker
that attaches Nile Blue to DNA. Obviously, both configurations
Scheme 2: Synthesis of the R-configured DNA building block 3 and
postsynthetic click ligation of the Nile Blue-modified single strands (ss)
DNA1 and DNA2 that form the double strands (ds) DNA1Y and
DNA2Y with the counterparts Y = A, T, C, G; a) 2-propyn-1-ol, 1,1′-
carbonyldiimidazole, DMF, r.t., 27 h; 44%; b) 2-cyanoethyl-N,N-diisop-
ropylchlorophosphoramidite, EtN(iPr)2, CH2Cl2, r.t., 3 h.
Table 1: Melting temperatures (Tm) and quantum yields (ΦF) of duplex sets DNA1Y and DNA2Y bearing the R-configured linker in comparison to the
corresponding values with the S-configured linker [17].
Duplex
R configuration
S configuration [17]
Differences R–S
Tm (°C)
ΦF
Tm (°C)
ΦF
ΔTm (°C)
ΔΦF
DNA1 (ss)
DNA1A
DNA1T
–
0.15
–
0.16
–
−0.01
+0.02
+0.03
−0.01
−0.01
−0.01
<0.01
<0.01
<0.01
<0.01
59.1 (−3.4)a
0.17
0.22
0.21
0.05
0.04
0.02
0.02
0.02
0.01
56.6 (−5.9)a
60.4
0.15
0.19
0.22
0.06
0.05
0.02
0.02
0.01
0.01
+2.5
−1.9
+2.2
−0.7
–
58.5
60.7
59.1
–
DNA1C
DNA1G
DNA2 (ss)
DNA2A
DNA2T
58.5
59.8
–
66.7 (−1.3)b
64.2
67
65.5 (−2.5)b
66.5
+1.2
−2.3
−0.5
−0.9
DNA2C
DNA2G
67.5
66.8
67.7
aIn comparison to an unmodified duplex with T instead of the chromophore: Tm = 62.5 °C [17].
bIn comparison to an unmodified duplex with T instead of the chromophore: Tm = 68.0 °C [17].
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Beilstein Journal of Organic Chemistry 2010, 6, No. 13.
induce a rather small destabilization, but do not exhibit any strands or even more pronounced with adjacent base pairs in the
clear dependence of the thermal stability of the DNA duplex on double strands. The base that is placed opposite to the Nile Blue
chirality. In comparison with the literature our results are chromophore has no significant influence on the optical proper-
remarkable with respect to three different aspects. (i) Our ties since the absorption spectra are remarkably similar. The
results stand in contrast to experiments with D- or L-threoninol latter result is similar to the S-configured linker [16]. All results
[42], the alternative acyclic linker mentioned above, where chir- together indicate once more that it does not matter if an S- or
ality decided about duplex stability. (ii) Single glycol modifica- R-configured acyclic linker system is used to attach this chro-
tions typically yield a strong destabilization [19-23]. For mophore to DNA.
instance, incorporation of indole as a single base surrogate into
oligonucleotides resulted in strong destabilization of the DNA We interpret the similarity of the absorption properties together
duplexes (ca. −12 °C) [32]. Our results with Nile Blue indicate with the results from the thermal dehybridization studies, as
that the interactions of the hydrophobic chromophore with the discussed above, as a result of the intercalation of the Nile Blue
adjacent base pairs regain some of the lost thermal stability that dye in duplex DNA. To further explore the optical properties
is caused by the glycol linker. (iii) The melting temperatures of we recorded steady state fluorescence spectra of the Nile Blue-
Nile Blue-modified duplexes are similar within each duplex set, modified single and double strands using an excitation
DNA1Y or DNA2Y. This is typical for chromophores as base wavelength of 610 nm (Figure 2). The emission maximum of
surrogates [26-36] since they do not exhibit any preferential the chromophore is shifted from 665 nm (in case of the isolated
base pairing properties.
dye in ethanol) to 677–679 nm for the modified DNA single
and double strands. In contrast to the absorption properties that
The UV–vis absorption properties of all Nile Blue-modified were pretty similar for all Nile Blue-modified duplexes, fluores-
duplexes are remarkably different from the Nile Blue dye in cence spectra show the influence of the sequential neighbor-
ethanol but remarkably similar within the duplex sets DNA1Y hood. The quantum yields of the Nile Blue-modified single
and DNA2Y. The Nile Blue base surrogate of the modified strands are reduced from 33% for the isolated dye in ethanol to
single strands is displayed by the characteristic absorption in the 15% in the single strand DNA1 and 4% in DNA2. Duplexes of
range between 600 and 550 nm (Figure 1). The observed differ- the sets DNA1Y and DNA2Y range from 17 to 22% provided
ences between the Nile Blue dye and the corresponding modi- guanines are not placed in the vicinity of the phenoxazinium
fied oligonucleotides in the visible range are the loss of one dye.
absorption band and a red-shift from 633 nm to 653 nm for the
second absorption signal. A further small bathochromic shift of The fluorescence is quenched significantly if guanine is present
ca. 5 nm is observed upon hybridization of the modified single as the counterbase (DNA1G), or as part of the adjacent base
strands DNA1 and DNA2 to the corresponding DNA duplexes. pairs (DNA2A, DNA2T, DNA2C), or both (DNA2G). It
These observations can be attributed to strong excitonic interac- becomes clear from a rough estimation of the excited state
tions of the chromophore with adjacent DNA bases in the single potential for the Nile Blue dye that guanine oxidation by an
Figure 1: UV–vis absorption spectra of single-stranded DNA1 and
DNA2, and the corresponding duplexes DNA1Y and DNA2Y (2.5 μM)
in sodium phosphate buffer (10 mM) of pH 7.0, NaCl (250 mM),
λexc = 610 nm.
Figure 2: Fluorescence spectra of single-stranded DNA1 and DNA2,
and corresponding duplexes DNA1Y and DNA2Y (2.5 μM) in sodium
phosphate buffer (10mM) of pH 7.0, NaCl (250 mM),
λexc = 610 nm.
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Beilstein Journal of Organic Chemistry 2010, 6, No. 13.
photoinduced electron transfer causes the fluorescence
quenching. If the singlet–singlet transition energy E00 = 1.9 eV
is added to the reduction potential Ered −0.3 V (vs. NHE), the
excited state potential is estimated to be E*red = 2.2 eV [17].
Hence, a photooxidation of guanine is highly favorable because
the oxidation potential of guanine is only ~1.3–1.4 V [44].
Hence sequence-dependent fluorescence quenching of the Nile
Blue dye in DNA was expected and was used as a sensitive tool
to compare the electronic interactions of the chromophore with
the DNA base stack in order to evaluate the role of chirality of
the glycol linker (R- vs. S-configuration). It is important to point
out that a similar emission profile is observed regardless of
whether the chromophore has been attached via the
R-configured linker (this study) or the S-configured one [17].
The comparison of quantum yields (Table 1) reflects only very
minor differences that are within the experimental error.
It was quite surprising to observe that in contrast to experi-
ments with D- and L-threoninol [39-42] the chirality of the
3-amino-1,2-glycol linker as a substitute for the 2′-deoxyri-
boside in our studies had no influence on the optical properties
of the attached fluorophore. In order to image this result we
worked out molecular models for the duplex DNA1A (R-con-
figuration) and the corresponding duplex bearing the linker in
Figure 3: Models for DNA1A bearing the (R)-3-amino-1,2-propanediol
linker (left) and the corresponding duplex with identical sequence but
with the (S)-3-amino-1,2-propanediol linker (right); AMBER force field,
HyperChem 7.5.
the inverted configuration (S). We assumed a base inserted posi- UV–vis absorption properties together with fluorescence
tion of the Nile Blue chromophore in both cases (Figure 3). quenching properties indicate that the chromophore Nile Blue
Hence the tether between the 3-amino-1,2-propanediol moiety stacks perfectly between the adjacent base pairs regardless of
and the Nile Blue chromophore which consists of two short whether it has been attached via an S- or R-configured linker.
alkyl chains and the triazolyl group represents the most critical This experimental result can be imaged by geometrically opti-
issue for intercalation. From both molecular models it became mized DNA models. Our result is remarkable regarding
obvious that this tether is long and flexible enough allowing the opposite results with the L-/D-threoninol linkers [37-40] that
Nile Blue chromophore to intercalate in a nearly perfectly revealed strong differences in stacking and also in function of
stacked position between the adjacent base pairs. This modeling attached chromophores depending on the chirality of the linker.
helps to rationalize the experimental result which is the simil- We were able to show, however, that it does not matter whether
arity of the optical properties between the two duplexes.
Nile Blue has been attached to a DNA duplex via an R- or
S-configured acyclic glycol linker.
Conclusion
“Click” chemistry allows the postsynthetic modification of Experimental
oligonucleotides that were presynthesized using the DNA Materials and Methods. Chemicals were purchased from
building block 3 bearing an alkyne group and the azide 4 of the Aldrich, Alfa Aesar and Merck. Unmodified oligonucleotides
base-labile phenoxazinium dye Nile Blue. The chromophore were purchased from Metabion. T.l.c. was performed on Fluka
was incorporated as a DNA base surrogate using (R)-3-amino- silica gel 60 F254 coated aluminium foil. Flash chromatography
1,2-propanediol as the linker between the phosphodiester was carried out with silica gel 60 from Aldrich (60–43 µm).
bridges. Two DNA duplex sets were prepared with the phen- Spectroscopic measurements were recorded in sodium phos-
oxazinium azide 4 (one set carried the chromophore in an A-T phate buffer solution (10 mM) with NaCl (250 mM) at pH 7.0
environment, the second set in a G-C environment) and charac- using quartz glass cuvettes (10 mm). ESI mass spectra were
terized by optical spectroscopy. A sequence-dependent fluores- acquired in the central analytical facility of the faculty on a
cence quenching of the chromophore was applied as a sensitive ThermoQuest Finnigan TSQ 7000 in negative and positive
tool to compare stacking interactions with respect to the chir- ionisation mode. NMR spectra were recorded on a Bruker
ality of the acyclic linker attachment. In fact, only minor and Avance 300 spectrometer in deuterated solvents that had been
negligible differences were observed. Melting temperatures, dried over basic alumina. Chemical shifts are given in ppm
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Beilstein Journal of Organic Chemistry 2010, 6, No. 13.
relative to TMS. Absorption spectra and the melting tempera- performed using a modified protocol. Activator solution
tures (2.5 µM DNA, 250 mM NaCl, 10–90 °C, 0.7 °C/min, step (0.45 M tetrazole in acetonitrile) was pumped together with the
width 1.0 °C) were recorded on a Varian Cary 100 spectro- building block (0.15 M in acetonitrile) through the CPG vial.
meter equipped with a 6 × 6 cell changer unit. Fluorescence The coupling time was extended to 61 min with an intervening
spectra were acquired on a Jobin-Yvon Fluoromax 3 fluori- step after 30.8 min for washing and refreshing the activator/
meter in spectral steps of 1 nm and an integration time of 0.2 s. phosphoramidite solution in the CPG vial. The CPG vial was
All spectra were recorded with an excitation and emission band- flushed with dry acetonitrile after coupling. After preparation,
pass of 2 nm and are corrected for Raman emission from the the trityl-off oligonucleotides were cleaved from the resin and
buffer solution.
deprotected by treatment with concd. NH4OH at r.t. for 24 h.
Synthesis of 2. 2-Propyn-1-ol (40 µL, 700 µmol) was “Click” ligation. The azide 4 [18] (114 µL, 10 mM), Cu(I)
dissolved in DMF (5 mL). 1,1′-Carbonyldiimidazole (114 mg, (17 µL, 100 mM), TBTA (34 µL, 100 mM), each in DMSO :
700 µmol) was added and the solution was stirred at r.t. for 3 h. tBuOH = 3 : 1, and sodium ascorbate (25 µL, 400 mM) in H2O
3-(4,4′-Dimethoxytrityl)-2-hydroxypropylamine (1) (268 mg, were added to the oligonucleotide (1 µmol). The reaction mix-
700 µmol) was added and the solution was stirred for another ture was vortexed, shaken overnight at r.t. and then evaporated
24 h at r.t. and then evaporated to dryness. The crude product to dryness using a speedvac. Sodium acetate (100 µL,
was purified by flash chromatography (CH2Cl2, 0.1% NEt3) 0.15 mmol) was added and the mixture stored for 1 h at r.t.
yielding a yellow solid (44%). T.l.c. (CH2Cl2 : MeOH = 100 : Ethanol (1 mL) was added and the mixture vortexed and frozen
2) Rf = 0.3. 1H NMR (300 MHz, [d6]-acetone): δ = 7.51–7.48 (−20 °C) overnight. The suspension was centrifuged (13
ppm (m, 2H, arom. DMT), 7.37–7.28 (m, 7H, arom. DMT), 000 rpm, 15 min) and the supernatant removed. The pellet was
6.89–6.86 (m, 4H, arom. DMT), 6.26 (m, 1H, NH), 4.64 (d, J = washed twice with ethanol (500 µL, 70%) and then dissolved in
2.47, 2H, CH2CCH), 4.17 (d, J = 5.21, 1H, OH), 3.92–3.86 (m, water (500 µL). Prior to purification by HPLC the DNA was
1H, CH2CHCH2), 3.79 (s, 6H, OMe), 3.45–3.37 (m, 1H, desalted by NAP-5 column (GE Healthcare).
CH2CHCH2), 3.22–3.15 (m, 1H, CH2CHCH2), 3.13–3.05 (m,
2H, CH2CHCH2), 2.98 (t, J = 2.47, 1H, CCH). MS (FAB): m/z DNA purification. The modified oligonucleotides were puri-
(%) 303.1 (100) [DMT]+, 475,5 [MH+]. HRMS (FAB): M+ fied by HPLC on a semipreparative RP-C18 column (300 Å,
calc. for C28H30NO6 [MH+]: 476.2073; found: 476.2085.
Supelco) using the following conditions: A = NH4OAc buffer
(50 mM), pH = 6.5; B = acetonitrile; gradient 0–30% B over
Synthesis of 3. Compound 2 (350 mg, 0.74 mmol) was 50 min, flow rate 2.5 mL/min, UV–vis detection at 260 and
dissolved under argon in dry CH2Cl2 (5 mL). Dry ethyldiisop- 641 nm. The oligonucleotides were lyophilized and quantified
ropylamine (380 µL, 2.21 mmol) and 2-cyanoethyl-N,N-diisop- by their absorbance in water at 260 nm on a Varian Cary 100
ropylchlorophosphoramidite (181 µL, 0.81 mmol) were added. spectrometer. Duplexes were formed by heating to 90 °C
The solution was stirred for 3 h at r.t. Methanol (20 µL) was (15 min) followed by slow cooling. MS (ESI): DNA1: calc.
added to the mixture and stirred for 0.5 h to stop the reaction. 5455, found m/z = 1362.9 [M/4]4−, 1817.7 [M/3]3−; ε (260 nm)
The solution was evaporated to dryness and dissolved in dry = 159 090 [mol L−1 cm−1]; DNA2: calc. 5503, found m/z
MeCN (6.1 mL) and applied directly for automated DNA syn- =1375.1 [M/4]4−, 1833.8 [M/3]3−; ε (260 nm) = 164 290 [mol
thesis. T.l.c. (CH2Cl2 : MeOH = 100 : 2): Rf = 0.6. 1H NMR L−1 cm−1].
(300 MHz, [d6]-C3D6O): δ = 1.06–1.19 (m, 12H, 4*Me (iProp)),
2.58 (t, J = 6.04, 1H, ≡H), 2.76 (m, 2H), 2.95 (m, 1H), 3.07 (m, Acknowledgements
1H), 3.22 (m, 1H), 3.36 (m, 1H), 3.54 (m, 1H), 3.61 (m, 2H), This work was supported by the Deutsche Forschungsge-
3.85 (m, 1H), 3.75 (s, 6H, 2*OMe), 4.10 (m, 1H), 4.60 (dd, Jz = meinschaft (Wa 1386/9) and the University of Regensburg.
2.47, CH2≡), 6.20 (m, 1H, NH), 6.83–6.88 (m, 4H, arom.),
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