Synthesis and hybridization properties of oligodeoxynucleotides with long-chain linkers

Article abstract of DOI:10.1002/hlca.200790203

New oligonucleotides with a long-chain linker (6,9-dioxa-3,12- diazatetradecane-1,14-diyl) in their backbone were synthesized, and their hybridization properties were studied by measurement of their T_m curves and fluorescence spectra. The T

Full text of DOI:10.1002/hlca.200790203

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Helvetica Chimica Acta – Vol. 90 (2007)
Synthesis and Hybridization Properties of Oligodeoxynucleotides with
Long-Chain Linkers
by Kohji Seioa)b), Takeshi Teradab)c), Masahiro Mizutab), Akihiro Ohkubob)c), Haruhiko Taguchib)c),
and Mitsuo Sekine*b)c)
a) Frontier Collaborative Research Center, Tokyo Institute of Technology
b) CREST, Japan Science and Technology Agency (JST)
c) Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku,
Yokohama, Japan (phone: þ 81-45-924-5706; fax: þ 81-45-924-5772; e-mail: msekine@bio.titech.ac.jp)
New oligonucleotides with a long-chain linker (6,9-dioxa-3,12-diazatetradecane-1,14-diyl) in their
backbone were synthesized, and their hybridization properties were studied by measurement of their Tm
curves and fluorescence spectra. The Tm analyses revealed that these oligonucleotides could bind to their
complementary strands despite the presence of the long-chain linker. We also demonstrated interesting
fluorescence properties of oligodeoxynucleotides with an anthracen-9-ylmethyl group on one of the two
N-atoms in the long-chain linker. The fluorescence intensity of these oligonucleotides increased upon
their hybridization to the complementary strands and was sensitive to the presence of the mismatch base
pairs at a specific position.
1. Introduction. – Various modifications of phosphodiester backbones have been
reported in which substituents without P-atoms have been used to develop functional
oligodeoxynucleotides (ODNs). These modifications included amides [1], hydroxyl-
amines [2][3], formacetals [4 – 8], carbonates [9][10], ureas and carbamates [11],
squaramides [12][13], and guanidines [14]. In most of these examples, the phosphate
surrogates were designed to be isosteric to the phosphodiester structure so that they
could connect the up and downstream nucleoside residues without distortion of the
original structure. On the other hand, there are some studies of replacement of the
phosphodiester backbone with long-chain linkers such as polyethylene glycols in both
DNA and RNAs [15 – 30]. Oligonucleotides with such long-chain linkers have been
developed for structural studies of hairpin loops [15 – 18], triplexes [17][19 – 21],
ribozymes [22][23], gap-DNAs [24], CpG motifs [25], circular DNAs [26][27], and
structured RNAs [28][29]. The duplex formation of oligonucleotides with long-chain
linkers has also been described [30].
In this paper, we report the synthesis of new modified 15mer ODNs – ODN1,
ODN2a, ODN2b, ODN3a, and ODN3b – that have a 6,9-dioxa-3,12-diazatetradecane-
1,14-diyl group as a long-chain linker in their backbone (see below, Fig. 1), and a study
of their hybridization affinity for natural-type 15mer targets ODN4 – 9 (see below,
Fig. 2). Because both the modified and natural-type target ODNs have the same
number of nucleotide residues, the entire structure of the long-chain linker can be
expected to drastically change its conformation upon binding to the targets. Such
ꢀ 2007 Verlag Helvetica Chimica Acta AG, Zürich

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conformation change synchronizing with the hybridization might confer interesting
properties on the modified ODNs. Keeping such possibilities in mind, we synthesized
and studied the properties of the modified ODNs. The Tm analyses revealed the
significant affinity of ODN1 for its complementary strand despite the presence of the
long-chain linker.
We also found interesting fluorescence properties of the ODNs with an anthracen-
9-ylmethyl group on their backbone (ODN2a,b, and ODN3a,b) that might be useful for
the detection of single-base mismatches by fluorescence spectroscopy. To substitute the
long-chain linker part with a fluorescent anthracene moiety, we designed a linker with
secondary amino functions as shown below in Fig. 1. The introduction of the fluorescent
group made it possible to monitor the hybridization of the modified ODNs by
fluorescence spectroscopy.
2. Results and Discussion. – 2.1. Synthesis of Dimer 1. The thymidine dimer 1 with
the long-chain linker (see below, Fig. 1) was synthesized by the coupling of the 5’ half-
unit 3 (Scheme 1) and the 3’ half-unit 8 (see Schemes 2 and 3) by reductive amination.
The 5’ half-unit 3 was synthesized from 5’-O-(4,4’-dimethoxytrityl)thymidine by
treatment with 2.2 equiv. of NaH and prop-2-en-1-yl bromide to give the 3’-O-propenyl
derivative 2 in 70% yield (Scheme 1). Compound 2 was then oxidized with a catalytic
amount of OsO4 in combination with 1.2 equiv. of NaIO4, giving the desired 3’-O-
(formylmethyl) derivative 3 in 80% yield.
Scheme 1. Synthesis of the 5’ Half-unit 3
The 3’ half-unit 8 was synthesized as shown in Schemes 2 and 3. We first synthesized
the 5’-O-(formylmethyl) derivative 5 via 4 according to a procedure similar to that

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shown in Scheme 1 by the use of 3’-O-[(tert-butyl)dimethylsilyl]thymidine in place of
5’-O-(4,4’-dimethoxytrityl)thymidine (Scheme 2). Next, we examined the reductive
amination of 5 with amine 6 [31], which proceeded most successfully when
NaBH(OAc)3 was used as a reducing agent in CHCl3 (Scheme 3). In this case,
compound 7 was obtained in 84% yield. The reducing agents BH3 · pyridine complex
and NaBH4 were not effective. Among several solvents tested, CHCl3 gave the best
results. Interestingly, the use of CH2Cl2 in place of CHCl3 resulted in a worse result,
giving the desired compound 7 in only 40% yield. Compound 7 thus obtained was
treated with 3% trichloroacetic acid to give the 3’ half-unit 8 in 88% yield. Finally,
compound 8 was coupled with the 5’ half-unit 3 under conditions similar to those of the
above-mentioned reductive amination to furnish dimer 9 in 54% yield, which was
deprotected by acid treatment to give dimer 1 in 88% yield.
Scheme 2. Synthesis of the 5’-O-(Formylmethyl) Derivative 5
2.2. Synthesis of Phosphoramidites. Next, we tried to synthesize the phosphorami-
dite derivatives 12, 18a, and 18b from the synthetic intermediates described above.
Phosphoramidites 18a and 18b were designed to monitor the hybridization of
oligodeoxynucleotides incorporating the long-chain linker by the use of the fluorescent
signal of anthracene.
The phosphoramidite 12 was synthesized starting from the protected dimer 9
(Scheme 4). The tBuMe2Si group of 9 was removed by treatment with Bu4NF to give 10
in 91% yield. The two secondary-amine moieties were both protected with a
trifluoroacetyl group by treatment with trifluoroacetic anhydride to give 11 in 83%
yield, and the remaining OH group of 11 was phosphitylated by the phosphorodia-
midite reagent to give 12 in 76% yield.
We also synthesized phosphoramidite 18a according to the procedure shown in
Scheme 5. The alkylation of compound 7 with 9-(chloromethyl)anthracene in DMF
gave 13 in 64% yield. The (MeO)Tr group of 13 was removed by treatment with 3%

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Scheme 3. Synthesis of the 3’ Half-unit 8 andDimer 9
CCl3COOH to give 14 in 93% yield. Subsequently, the primary-amine function of 14
was condensed with compound 3 by applying conditions similar to those used for the
above-mentioned reductive amination, to give dimer 15 in 55% yield. Subsequently, the
tBuMe2Si group of 15 was removed by treatment with Bu4NF (!16), and the
secondary-amino group was protected by a CF3CO group (!17). Finally, the 3’-OH
group was phosphitylated to give the desired phosphoramidite 18a. Compound 18a was
unstable under purification procedures such as gel-filtration column chromatography,
probably because of the presence of the basic amine part. This compound, however,
could be used successfully in the ensuing oligonucleotide synthesis without further
purification.
Next, we synthesized the phosphoramidite 18b (Scheme 6). In the synthesis of 18b,
we used the new protected amine 19 in place of amine 6 because the tritylthio (TrS)
group [32 – 37] of 19 could be selectively removed by a radical reduction [34] without
damaging the (MeO)2TrO group at the 5’-position of the nucleoside, as described
below. One of the amino groups of 2,2’-[ethane-1,2-diylbis(oxy)]bis[ethanamine] was
protected by the TrS group according to the procedure reported previously [32][34].
The protected amine 19 was obtained in 55% yield and coupled with the 3’-O-
(formylmethyl) derivative 3 by reductive amination. Alkylation of the secondary-
amine function of 20 with 9-(chloromethyl)anthracene gave 21 in 77% yield.

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Scheme 4. Synthesis of Phosphoramidite 12
Subsequently, the TrS group was removed by reduction with tributylstannane to give 22
in 50% yield without damaging the (MeO)2Tr group [34]. The amine 22 was condensed
with the 5’-O-(formylmethyl) derivative 5 by reductive amination to give 23 in 43%
yield. The tBuMe2Si group of 23 was removed by treatment with Bu4NF, and the amino
group was protected by a CF3CO group. Finally, the 3’-OH group was phosphitylated to
give the desired phosphoramidite 18b. Like phosphoramidite 18a, this compound was
unstable during the purification procedure and had to be used without further
purification for oligonucleotide synthesis.
2.3. Oligonucleotide Synthesis. By the use of the phosphoramidites 12, 18a, and 18b,
the oligonucleotides ODN1 (Fig. 1), ODN2a, ODN2b, ODN3a, and ODN3b (Figs. 1
and 2) were synthesized. The 3’ half of each DNA, 5’-d(Y1Y2 TCGAG)-3’, was
synthesized on CPG (controlled-pore glass) supports with a commercially available
DNA synthesizer. After the (MeO)2Tr group at the 5’-terminal nucleotide (Y1) was
removed, the CPG resin was transferred to a glass syringe. The dimer phosphoramidites
with the long-chain linker (12, 18a, and 18b) were then used for further chain
elongation by a manual coupling procedure. Subsequently, the CPG supports were
again transferred to the reaction vessel of the DNA synthesizer, and the 5’-half part of
each DNA, 5’-d(GCTCX1X2)-3’, was elongated in the synthesizer. After cleavage of the
oligonucleotide from the solid support, all protecting groups at the nucleobases and
phosphate groups and at the amino groups were removed by treatment with aqueous
ammonia. The structures of the oligonucleotides thus obtained were confirmed by
MALDI-TOF-MS analyses.

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Fig. 1. Structure of the dimer unit 1 and the oligodeoxynucleotides with long-chain linkers
2.4. Hybridization Properties of Oligodeoxynucleotides with Long-Chain Linkers.
We first measured the UV melting curve of the duplex of ODN1, i.e., 5’-
d(GCTCAG[tt]GATCGAG)-3’, where [tt] represents the dithymidine unit derived
from 1 and introduced by means of 12, with the complementary strand ODN4, i.e., 5’-
d(CTCGATCAACTGAGC)-3’ (Fig. 2). We also measured the Tm value of the duplex
of the unmodified DNA strand ODN5’, i.e., 5’-d(GCTCAGTTGATCGAG)-3’, with
ODN4 to clarify the effect of the modification on the melting temperature. Although
the Tm value of the ODN1 · ODN4 duplex was lower by 98 than that of the natural-type
duplex, as shown in the Table, it was clearly revealed that ODN1 could hybridize with
the target sequence despite the presence of the long-chain linker in its backbone.
Table. Tm Values of Modified (ODN1) andNatural-Type (ODN5’) Duplexes
ODN1 · ODN4
ODN5’ · ODN4
Tm [8]
48
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Fig. 2. Sequences of the oligonucleotides used in this study. The abbreviations tt* and t*t refer to the
modified thymidine dimers. ODN4 is complementary to ODN2a and ODN2b. ODN5 is complementary
to ODN3a and ODN3b. ODN6 – 9 are mismatched targets for ODN3a. The mismatch sites are indicated
by underlines.
Next, we studied the hybridization properties of oligodeoxynucleotides ODN2a,
ODN2b, ODN3a, and ODN3b (Figs. 1 and 2) in more detail by measurement of the
fluorescence of the anthracene moiety of these oligonucleotides. ODN2a and ODN2b
have almost identical structures, except that ODN2a has an anthracene-9-ylmethyl
group at the N-atom of the long-chain linker closer to the 3’ terminus, as represented by
[tt*] (Fig. 2), and ODN2b has the same fluorescent aromatic group at the N-atom
nearer to the 5’ terminus, as represented by [t*t]. We also synthesized the
oligonucleotides ODN3a and ODN3b with ÀCT[tt*]AGÀ and ÀCT[t*t]AGÀ
sequences in place of the ÀAG[tt*]GAÀ and ÀAG[t*t]GAÀ sequences of ODN2a
and ODN2b, respectively, to explore the effect of the nearby guanine residue on the
fluorescence intensity of the anthracene moiety. ODN4 is the complementary strand of
ODN2a and ODN2b, and ODN5 is that of ODN3a and ODN3b.

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First we measured the fluorescence intensities of ODN2a, ODN2b, ODN3a, and
ODN3b in their single-strand states in various pH regions. The results are shown on the
left-hand side of Fig. 3. As the pH value of the solution increased, the fluorescence
intensity decreased in all cases. These observations agree with the fact that the
fluorescence of the anthracene moiety is quenched by the nearby lone pair on the N-
atom, and the quenching is suppressed by protonation of the N-atom [38 – 43].
Moreover, comparison between ODN2a and ODN2b revealed that ODN2a with the
anthracene moiety at the N-atom closer to the 3’ terminus showed approximately twice
as much fluorescence in all pH regions. This trend was also seen for the fluorescence
intensities of ODN3a and ODN3b.
As expected, ODN2a and ODN2b, having guanine residues at the nearest-neighbor
positions of the tt* and t*t dimer units, showed lower fluorescence intensity in
comparison with ODN3a and ODN3b, respectively. These results suggest that the
fluorescence of the anthracene moiety was quenched by the guanine residue.
Fig. 3. Fluorescence intensities of ODN2a, ODN2b, ODN3a, andODN 3b in the presence andabsence of
their complementary targets
Next, we measured the fluorescence spectra of ODN2a, ODN2b, ODN3a, and
ODN3b in the presence of their complementary strands ODN4 and ODN5.
Interestingly, upon addition of these complementary strands, the fluorescence
intensities increased about 2-fold in all pH regions. It is well known that the
fluorescence of the anthracene moiety is quenched by interaction with DNA strands

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[44 – 46]. Therefore, the observed intensity increases suggested that the anthracene
moiety on the long-chain linker was more accessible to the nucleobases in the single-
strand state than in the double-strand state. Yamana and co-workers also reported that
the fluorescence of the anthracene moiety introduced to the 2’ position of RNA
derivatives increased similarly upon hybridization to the DNA and RNA targets [46].
The observed increases in fluorescence intensities suggested to use the anthracene-
modified oligonucleotides reported in this study as oligonucleotide probes for
hybridization sensing.
Therefore, we examined the fluorescence properties of the most strongly
fluorescent ODN3a upon the addition of the target oligonucleotides with a mismatched
base (ODN6 – 9), and compared the fluorescence intensity with that of the fully
matched ODN3a · ODN5 duplex (Fig. 4). Here, ODN3a · ODN6 has a T· C mismatch
at the third position from the tt* unit, ODN3a · ODN7 has a G · T mismatch at the
second position, ODN3a · ODN9 has a T· C mismatch at the fourth position, and
ODN3a · ODN8 has an A · C mismatch at the position next to the tt* unit. Interestingly,
the mismatch-containing duplexes ODN3a · ODN9, ODN3a · ODN7, and ODN3a ·
ODN6 showed fluorescence spectra almost identical to that of the fully matched
duplex ODN3a · ODN5. In contrast, the duplex ODN3a · ODN8, containing an A · C
mismatch near tt* had significantly lower fluorescence intensity than the others. This
observation indicates that the fluorescence intensity of the anthracene moiety in
ODN3a is very sensitive to the loss of the Watson – Crick base pair only at the
neighboring position. Although the detailed mechanism of this fluorescence change
was not clear, the interesting properties of ODNs containing tt* might enable them to
be used as modified oligonucleotide probes for mismatch detection.
Fig. 4. Fluorescence intensities of duplexes of ODN3a with the target strands ODN6 – 9

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3. Conclusions. – In this work, we synthesized new oligonucleotides with a 6,9-
dioxa-3,12-diazatetradecane-1,14-diyl long-chain linker in their backbone and studied
their hybridization properties by measurement of their Tm curves and fluorescence
spectra. It should be noted that oligonucleotides with such long linkers could hybridize
with their target strands. The Tm analysis by use of ODN1 revealed the ability of this
oligonucleotide to bind its complementary strand despite the presence of the long-
chain backbone. In addition, we demonstrated the interesting fluorescence properties
of oligodeoxynucleotides with an anthracen-9-ylmethyl group at one of the two N-
atoms in the long-chain linker. The fluorescence intensity of the oligonucleotides
ODN2a, ODN2b, ODN3a, and ODN3b, increased upon their hybridization with their
complementary strands. These results indicate that the dynamic conformation change
of the long-chain linker upon hybridization changes the environment around the
anthracene residue so that their fluorescence increases.
We also examined the effect of the presence of mismatch base pairs on the
fluorescence of ODN3a. Interestingly, when ODN3a was hybridized to the target
strand ODN8 with an A · C mismatch at the nearby site of the long-chain-linked units,
the fluorescence decreased significantly. This observation suggests that the fluores-
cence of the anthracene moiety of ODN3a is very sensitive to the loss of base pairs at a
specific site.
Experimental Part
1. General. TLC: Merck silica gel 60 (F254) plates. Column chromatography (CC): silica gel C-200
(Wako Co. Ltd.); for rapid separations a minipump for a goldfish bowl was used to attain sufficient
pressure. UV Spectra: Biospec-mini spectrophotometer. Circular dichroism (CD) spectra: J-725
1
spectrometer, 0.5 cm cell. H-, 13C-, and 31P-NMR Spectra: at 500, 126, and 202 MHz, resp.; chemical
shifts d in ppm rel. to SiMe4 (¼0 ppm) or ( D)DMSO (¼2.49 ppm) for 1H, and rel. to CDCl3
6
(¼ 77.0 ppm) or (D6)DMSO (¼ 39.7 ppm) for 13C. ESI-MS: MarinerTM; in m/z. Cyclic voltammetry:
ALS electrochemical analyzer, model 600A. Elemental analyses were performed by the Microanalytical
Laboratory, Tokyo Institute of Technology at Nagatsuta.
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-(prop-2-en-1-yl)thymidine (2). To a soln. of 5’-O-
bis[(4-methoxyphenyl)phenylmethyl]thymidine (22 g, 41 mmol) in THF (200 ml), NaH (3.3 g, 89 mmol)
and 3-bromoprop-1-ene (3.5 ml, 41 mmol) were added. The soln. was stirred at r.t. for 90 min, then the
reaction was quenched by addition of MeOH (10 ml). The soln. was diluted with AcOEt (200 ml),
washed twice with brine (100 ml), dried (MgSO4), and concentrated and the residue subjected to CC
1
(silica gel, hexane/CHCl3 1 :1): 2 (17 g, 70%). H-NMR (500 MHz, CDCl5): 1.46 (s, MeÀC(5)); 2.17 –
2.51 (m, 2 HÀC(2’)); 3.30 – 3.51 (m, 2 HÀC(5’)); 3.80 (s, 2 MeO); 3.90 – 4.02 (m, CH2¼CHCH2O);
4.13 – 4.15 (m, HÀC(4’)); 4.25 – 4.27 (m, HÀC(3’)); 5.18 – 5.27 (m, CH2¼CHCH2O); 5.82 – 5.90 (m,
CH2¼CHCH2O); 6.34 – 6.37 (t, J ¼ 2.2, 1 HÀC(1’)); 6.82 – 7.41 (m, 13 H of (MeO)2Tr); 7.62 (s,
HÀC(6)); 8.23 (s, NH(3)). 13C-NMR (126 MHz, CDCl3): 11.97; 38.17; 55.41; 63.77; 70.41; 79.03; 84.31;
84.99; 87.05; 111.27; 113.41; 113.42; 117.67; 127.31; 128.15; 128.26; 130.22; 134.20; 135.50; 135.55; 135.77;
144.47; 150.24; 158.88; 163.62. ESI-MS: 604.2575 (C34H37N2NaOþ7 ; calc. 607.2420).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-(2-oxoethyl)thymidine (3). To a soln. of 2 (17 g,
28 mmol) in acetone/H2O 6 :1 (230 ml), OsO4 (7.1 ml, 570 mmol) and 4-methylmorpholine 4-oxide
(13 ml; 50% in H2O, 63 mmol) were added. The soln. was stirred in the dark at r.t. for 3 h. The solid was
removed by filtration, and the filtrate was diluted with AcOEt (250 ml). The org. layer was washed once
with sat. aq. NaHCO3 soln. (100 ml), dried (MgSO4), and concentrated. The residue was dissolved in 1,4-
dioxane/H2O 3 :1 (280 ml), and to this soln. was added NaIO4 (7.2 g, 34 mmol). The soln. was stirred in
the dark at r.t. for 13 h. The soln. was diluted with AcOEt (300 ml), washed once with sat. aq. NaHCO3

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soln., dried (MgSO4), and concentrated and the residue subjected to CC (silica gel, CHCl3): 3 (13 g,
80%). 1H-NMR (500 MHz, CDCl3): 1.55 (s, MeÀC(5)); 2.11 – 2.16 (m, 1 HÀC(2’)); 2.40 – 2.49 (m,
1 HÀC(2’)); 3.31 – 3.56 (m, 2 HÀC(5’)); 3.80 (s, 2 MeO); 4.01 – 4.11 (m, 1 H, CH2CH¼O); 4.16 – 4.23
(m, HÀC(3’), HÀC(4’)); 6.35 – 6.37 (m, HÀC(1’)); 6.83 – 7.40 (m, 13 H of (MeO)2Tr); 7.58 (m,
HÀC(6)); 10.01 – 10.10 (m, CH2CH¼O). 13C-NMR (126 MHz, CDCl3): 12.01; 37.78; 55.40; 63.69; 74.67;
81.01; 84.05; 84.79; 87.18; 111.50; 113.43; 113.44; 127.32; 127.37; 128.15; 128.17; 128.20; 128.24; 130.17;
130.21; 135.37; 135.43; 135.54; 144.34; 150.38; 158.86; 158.91; 163.76; 199.45. ESI-MS: 609.2295
(C33H34N2NaOþ8 ; calc. 609.2213).
3’-O-[(tert-Butyl)dimethylsilyl]-5’-O-(prop-2-en-1-yl)thymidine (4). As described for 2, with 3’-O-
[(tert-butyl)dimethylsilyl]thymidine (9.8 g, 27 mmol), THF (140 ml), NaH (1.4 g, 60 mmol), and 3-
bromoprop-1-ene (2.3 ml, 27 mmol) for 4 h. Workup with MeOH (10 ml), AcOEt (150 ml), and brine
1
(100 ml) and CC (silica gel, hexane/CHCl3 3 :2), gave 4 (7.3 g, 68%). H-NMR (500 MHz, CDCl3): 0.08
(s, Me2Si); 0.89 (s, tBuSi); 1.90 (s, MeÀC(5)); 2.04 – 2.08 (m, 1 HÀC(2’)); 2.16 – 2.20 (m, 1 HÀC(2’)); 3.51
(dd, J ¼ 2.2, 10.7, 1 HÀC(5’)), 3.66 (dd, J ¼ 2.2, 10.7, 1 HÀC(5’)); 3.98 – 4.00 (m, HÀC(4’)); 4.05 – 4.07 (m,
CH2¼CHCH2O); 4.44 – 4.47 (m, HÀC(3’)); 5.14 – 5.24 (m, CH2¼CHCH2O); 5.89 – 5.96 (m,
CH2¼CHCH2O); 6.26 (t, J ¼ 6.3, HÀC(1’)); 7.66 (s, HÀC(6)); 8.07 (s, NH(3)). 13C-NMR (126 MHz,
CDCl3): À 4.84; À 4.65; 12.60; 18.00; 25.77; 41.43; 69.49; 71.97; 72.40; 85.06; 86.45; 110.75; 117.54;
134.07; 136.05; 150.66; 164.34. ESI-MS: 397.2357 (C19H33N2O16Siþ; calc. 397.2159).
3’-O-[(tert-Butyl)dimethylsilyl]-5’-O-(2-oxoethyl)thymidine (5). As described for 3, with 4 (7.3 g,
19 mmol), acetone/H2O 6 :1 (190 ml), OsO4 (3.8 g, 370 mmol), and 4-methylmorpholine-4-oxide (8.5 ml;
50% in H2O, 41 mmol) for 19 h. Then workup with AcOEt (200 ml) and sat. aq. NaHCO3 soln. (150 ml),
followed by treatment of the residue in 1,4-dioxane/H2O 3 :1 (190 ml), with NaIO4 (4.7 g, 22 mmol) for
20 h. Workup with AcOEt (200 ml) and sat. aq. NaHCO3 soln. and CC (silica gel, hexane/CHCl3 2 :3 gave
t
5 (6.4 g, 86%). 1H-NMR (500 MHz, CDCl3): 0.01 (s, Me2Si); 0.80 – 0.81 (m, BuSi); 1.81 (d, J ¼ 0.7,
MeÀC(5)); 2.12 – 2.27 (m, 2 HÀC(2’)); 3.45 – 3.89 (m, 2 HÀC(5’), CH2CH¼O); 3.97 – 4.02 (m,
HÀC(4’)); 4.42 – 4.53 (m, HÀC(3’)); 6.29 – 6.37 (m, HÀC(1’)); 7.50 – 7.58 (m, HÀC(6)); 8.12 (s,
NH(3)); 9.72 (s, CH2CH¼O). 13C-NMR (126 MHz, CDCl3): À 4.78; À 4.58; 12.62; 18.05; 25.81; 41.08;
71.15; 72.23; 85.20; 86.12; 111.12; 136.08; 150.57; 164.08; 198.02. ESI-MS: 399.1714 (C18H31N2O6Siþ; calc.
399.1951).
3’-O-[(tert-Butyl)dimethylsilyl]-5’-O-[13-(4-methoxyphenyl)-13,13-diphenyl-6,9-dioxa-3,12-diazatri-
dec-1-yl]thymidine (7). To the soln. of 6 (200 mg, 502 mmol) in CHCl3 (5 ml) was added 5 (211 mg,
502 mmol). To this soln. was added NaBH(OAc)3 (213 mg, 1.0 mmol), and the resulting mixture was
stirred at r.t. for 40 min. The soln. was diluted with AcOEt (10 ml) and washed once with sat. aq.
NaHCO3 soln. (10 ml). The org. layer was dried (MgSO4) and concentrated and the residue subjected to
1
CC (NH-silica gel, hexane/CHCl3 2 :3): 7 (338 mg, 84%). H-NMR (500 MHz, CDCl3): 0.10 (s, Me2Si);
0.91 (s, tBuSi); 1.92 (s, MeÀC(5)); 2.07 – 2.11 (m, 1 HÀC(2’)); 2.23 – 2.27 (m, 1 HÀC(2’)); 2.36 (t, J ¼ 5.6,
2 H, CH2N); 2.79 (t, J ¼ 5.3, 2 H, CH2N); 2.84 (t, J ¼ 6.1, 2 H, CH2N); 3.50 – 3.65 (m, 1 HÀC(5’),
OCH2CH2NHCH2CH2OCH2CH2OCH2CH2NH); 3.72 (dd, J ¼ 2.9, 10.8, 1 HÀC(5’)); 3.79 (s, MeO),
3.98 – 4.00 (m, HÀC(4’)); 4.42 – 4.44 (m, HÀC(3’)); 6.34 (t, J ¼ 2.8, HÀC(1’)); 6.80 – 6.83 (m, 2 arom. H);
7.15 – 7.52 (m, 12 H of (MeO)Tr); 7.53 (s, HÀC(6)). 13C-NMR (126 MHz, CDCl3): À 4.93; À 4.76; 12.61;
17.84; 25.65; 41.06; 42.95; 48.84; 49.10; 55.01; 69.94; 70.02; 70.17; 70.31; 70.40; 71.06; 71.16; 72.09; 84.84;
86.18; 110.62; 112.97; 126.03; 127.64; 128.48; 129.72; 135.56; 138.15; 146.28; 150.61; 157.71; 164.23. ESI-
MS: 803.2904 (C44H63N4O8Siþ; calc. 803.4415).
5’-O-{2-{{2-[2-(2-Aminoethoxy)ethoxy]ethyl}amino}ethyl}-3’-O-[(tert-butyl)dimethylsilyl]thymidine
(8). A soln. of 7 (590 mg, 735 mmol) in 3% CCl3COOH/CH2Cl2 (25 ml) was stirred at r.t. for 10 min. The
soln. was diluted with CHCl3 (10 ml), washed with sat. aq. NaHCO3 soln. (4 ꢀ 10 ml), dried (MgSO4),
and concentrated and the residue subjected to CC (NH-silica gel, CHCl3/MeOH/Et3N 97:3 :1): 8
1
(350 mg, 88%). H-NMR (500 MHz, CDCl3): 0.08 (s, Me2Si); 0.89 (s, tBuSi), 1.93 (s, MeÀC(5)), 2.07 –
2.18 (m, 1 HÀC(2’)); 2.21 – 2.29 (m, 1 HÀC(2’)); 2.80 – 2.88 (m, 6 H, CH2N); 3.38 (br., NH2); 3.49 – 3.74
(m, 12 H, 2 HÀC(5’), CH2O); 3.98 – 4.00 (m, HÀC(4’)); 4.39 – 4.42 (m, HÀC(3’)); 6.27 – 6.30 (t, J ¼ 6.7,
HÀC(1’)); 7.52 (s, HÀC(6)). 13C-NMR (126 MHz, CDCl3): À 4.74; À 4.56; 12.86; 18.06; 25.83; 41.36;
41.73; 49.25; 49.48; 70.27; 70.43; 70.60; 70.65; 71.39; 72.31; 73.34; 85.25; 86.45; 110.78; 135.85; 150.56;
160.11. ESI-MS: 531.1849 (C24H47N4O7Siþ; calc. 531.3214).

Helvetica Chimica Acta – Vol. 90 (2007)
1959
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl(6,9-dioxa-3,12-diazate-
tradecane-1,14-diyl)-(3’ ! 5’)-3’-O-[(tert-butyl)dimethylsilyl]thymidine 9. As described for 7, with 8
(350 mg, 677 mmol) in CHCl3 (17 ml), 3 (397 mg, 677 mmol) in CHCl3 (17 ml), and NaBH(OAc)3
(387 mg, 1.4 mmol) for 15 min. Workup with AcOEt (50 ml) and sat. aq. NaHCO 3 soln. (2 ꢀ 30 ml) and
CC (silica gel, CHCl3/MeOH (96 :4) gave 9 (403 mg, 54%). 1H-NMR (500 MHz, CDCl3): 0.08 (s, Me2Si);
0.89 (s, tBuSi); 1.45 (s, 1 MeÀC(5)); 1.95 (s, 1 MeÀC(5)); 2.07 – 2.49 (m, 4 H, HÀC(2’)); 2.81 – 2.85 (br.,
4 CH2N); 3.32 (dd, J ¼ 3.5, 10.5, 1 HÀC(5’)); 3.51 (dd, J ¼ 2.7, 10.5, 1 HÀC(5’’)); 3.54 – 3.68 (m,
2 HÀC(5’)), 6 CH2O); 3.79 (s, 2 MeO); 3.98 – 4.40 (m, 2 HÀC(3’), 2 HÀC(4’)); 6.28 (t, J ¼ 4.5,
1 HÀC(1’)); 6.37 (t, J ¼ 2.9, 1 HÀC(1’)); 6.83 (m, 2 arom. H); 7.18 – 7.40 (m, 11 arom. H); 7.50 (s,
1 HÀC(6)); 7.59 (s, 1 HÀC(6)). 13C-NMR (126 MHz, CDCl3): À 4.74; À 4.56; 11.91; 12.85; 18.05; 25.83;
37.94; 41.35; 49.12; 49.17; 49.21; 49.35; 55.36; 64.03; 68.85; 70.30; 70.36; 70.40; 70.53; 70.64; 71.21; 72.33;
80.44; 84.14; 84.85; 85.42; 86.48; 87.02; 110.71; 111.30; 113.38; 127.26; 128.10; 128.24; 130.18; 135.46;
135.50; 135.69; 135.93; 144.43; 150.56; 158.83; 164.01; 164.14. ESI-MS: 1101.6314 ([ M þ H]þ,
C57H81N6O14Siþ; calc. 1101.5580).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl(6,9-dioxa-3,12-diazate-
tradecane-1,14-diyl)-(3’ ! 5’)-thymidine (10). To a soln. of 9 (837 mg, 760 mmol) in THF (7.6 ml) was
added 1m Bu4NF in THF (836 ml), and the soln. was stirred at r.t. for 2 h. The soln. was diluted with
AcOEt (10 ml), washed once with brine (10 ml), dried (MgSO4), and concentrated and the residue
1
subjected to CC (NH-silica gel, CHCl3/MeOH 99 :1): 10 (680 mg, 91%). H-NMR (500 MHz, CDCl3):
1.44 (s, 1 MeÀC(5); 1.90 (s, 1 Me – C(5); 2.15 – 2.51 (m, 4 H, HÀC(2’)); 2.79 – 2.85 (m, 8 H, CH2N);
3.29 – 3.75 (m, 16 H, HÀC(5’), CH2O); 3.78 (s, 2 MeO); 4.01 (br., 1 HÀC(4’)); 4.15 (br., 1 HÀC(4’));
4.18 (br., 1 HÀC(3’)); 4.50 (br., 1 HÀC(3’)); 6.32 – 6.37 (m, 2 HÀC(1’)); 6.79 – 6.83 (m, 4 arom. H);
7.18 – 7.41 (m, 9 arom. H); 7.57, 7.60 (2s, 2 HÀC(6)). 13C-NMR (126 MHz, CDCl3): 11.81; 12.72; 37.77;
40.80; 48.58; 48.70; 48.88; 55.23; 63.93; 68.53; 70.01; 70.18; 70.30; 70.36; 70.66; 80.31; 83.94; 84.64; 84.92;
85.80; 86.87; 110.56; 111.22; 113.25; 127.11; 127.97; 128.10; 130.06; 135.33; 135.37; 135.56; 135.99; 144.30;
150.76; 150.80; 158.66; 164.44; 164.49. ESI-MS: 987.5160 ([M þ H]þ, C51H67N6O1þ4 ; calc. 987.4715).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl[3,12-bis(2,2,2-trifluoro-
acetyl)-6,9-dioxa-3,12-diazatetradecane-1,14-diyl]-(3’ ! 5’)-thymidine (11). To a soln. of 10 (510 mg,
507 mmol) in CH2Cl2 (5 ml) were added iPr2EtN (580 ml, 3.4 mmol) and (CF3CO)2O (233 ml, 1.7 mmol),
and the soln. was stirred at r.t. for 10 min. The soln. was diluted with AcOEt (10 ml), washed once with
brine (10 ml), dried (MgSO4), and concentrated and the residue subjected to CC (silica gel, CHCl3/
MeOH 98 :2): 11 (507 mg, 83%). 1H-NMR (500 MHz, CDCl3): 1.41 – 1.48 (m, MeÀC(5)); 1.90 – 1.92 (m,
MeÀC(5)); 2.12 – 2.47 (m, 4 HÀC(2’)); 3.29 – 3.76 (m, 4 HÀC(5’), 10 CH2 of 3’ ! 5’ linker); 3.79 (s,
2 MeO); 4.06 – 4.45 (m, 2 HÀC(3’), 2 HÀC(4’)); 6.29 – 6.36 (m, 2 HÀC(1’)); 6.84 (m, 2 arom. H); 7.21 –
7.40 (m, 11 arom. H); 7.46 (s, 1 HÀC(6)); 7.59 (s, 1 HÀC(6)); 9.99 (br., 2 NH(3)). 13C-NMR (126 MHz,
CDCl3): 0.13; 11.89; 12.97; 12.76; 37.89; 37.86; 40.20; 40.25; 47.56; 47.92; 48.25; 48.41; 48.70; 48.80; 55.41;
63.96; 64.20; 66.56; 68.27; 68.52; 68.46; 70.02; 70.14; 70.31; 70.52; 70.70; 70.74; 70.82; 70.88; 71.26; 71.62;
81.05; 84.01; 84.13; 84.76; 85.34; 85.36; 85.41; 85.48; 85.74; 87.24; 87.22; 111.02; 111.10; 111.75; 111.79;
113.45; 115.40; 117.69; 127.39; 128.18; 128.24; 130.20; 135.34; 135.49; 135.58; 135.99; 136.11; 144.33;
150.57; 150.79; 158.93; 163.85; 163.90; 163.94. ESI-MS: 1201.4032 ([ M þ Na]þ, C55H65F6N6NaO1þ6 ; calc.
1201.4180).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl[3,12-bis(2,2,2-trifluoro-
acetyl)-6,9-dioxa-3,12-diazatetradecane-1,14-diyl]-(3’ ! 5’)-thymidine 3’-(2-Cyanoethyl N,N-Diisopropyl-
phosphoramidite) (12). To a soln. of 11 (193 mg, 164 mmol) in CH2Cl2 (660 ml) were added 2-cyanoethyl
N,N,N’,N’-tetraisopropylphosphorodiamidite (62 ml, 196 mmol) and diisopropylammonium 1H-tetrazo-
lide (11 mg, 66 mmol). The mixture was stirred at r.t. for 6 h. The soln. was diluted with AcOEt (10 ml),
and washed once with sat. aq. NaHCO3 soln. (10 ml), dried (MgSO4), and concentrated and the residue
purified by gel-filtration HPLC (AcOEt): 12 (171 mg, 76%). 1H-NMR (500 MHz, CDCl3): 1.18 – 1.20 (m,
2 Me2CH); 1.44, 1.48 (2s, 1 MeÀC(5)); 1.91, 1.94 (2s, 1 MeÀC(5)); 2.14 – 2.45 (m, 4 HÀC(2’)); 2.64 (t,
J ¼ 6.1, POCH2CH2CN); 3.27 – 3.78 (m, 4 HÀC(5’), 2 Me2CH, POCH2CH2CN, 10 CH2 of 3’ ! 5’ linker);
3.80 (s, 2 MeO); 4.09 – 4.56 (m, 2 HÀC(3’), 2 HÀC(4’)); 6.24 – 6.36 (m, 2 HÀC(1’)); 6.83 – 6.85 (m, 4
arom. H); 7.18 – 7.37 (m, 9 H of (MeO)2Tr); 7.38 – 7.47 (m, 2 HÀC(6)); 7.58 (br., 2 NH(3)). 13C-NMR
(126 MHz, CDCl3): 11.89; 11.95; 12.66; 19.34; 20.48; 20.52; 21.57; 24.50; 24.55; 24.59; 24.66; 24.69; 24.71;

1960
Helvetica Chimica Acta – Vol. 90 (2007)
24.74; 37.70; 37.85; 39.42; 43.34; 43.44; 46.76; 47.44; 47.67; 47.95; 48.05; 48.18; 48.23; 48.50; 48.70; 55.36;
57.92; 58.02; 58.17; 63.84; 64.10; 66.52; 68.22; 68.30; 68.41; 69.88; 69.85; 70.17; 70.44; 70.59; 70.69; 70.78;
71.14; 71.17; 80.87; 83.94; 84.06; 84.57; 84.72; 84.76; 84.82; 87.14; 111.05; 111.12; 111.50; 111.54; 113.40;
115.38; 117.67; 117.82; 117.89; 127.32; 128.12; 128.21; 130.16; 135.35; 135.48; 135.54; 136.11; 144.32;
150.51; 150.55; 150.58; 150.61; 158.87; 163.93; 164.01. 31P-NMR (CDCl3, 203 MHz): 149.33; 149.37. ESI-
MS: 1401.5037 ([M þ H]þ, C61H78F6N7NaOP1þ6 ; calc. 1401.5439).
5’-O-[3-(Anthracen-9-ylmethyl)-13-(4-methoxyphenyl)-13,13-diphenyl-6,9-dioxa-3,12-diazatridec-1-
yl]-3’-O-[(tert-butyl)dimethylsilyl]thymidine (13). To a soln. of 7 (4.1 g, 5.0 mmol) in N,N-dimethyl-
formamide (50 ml) were added 9-(chloromethyl)anthracene (1.1 g, 5.0 mmol), Na2CO3 (11 g,
101 mmol), and KI (167 mg, 1.0 mmol). The mixture was stirred at r.t. for 2 h. The soln. was diluted
with AcOEt (200 ml), washed with brine (3 ꢀ 200 ml), dried (MgSO4), and concentrated and the residue
subjected to CC (NH-silica gel, hexane/CHCl3 1:1): 13 (3.2 g, 64%).1H-NMR (500 MHz, CDCl3): 0.04,
0.07 (2s, Me2Si); 0.87 (s, tBuSi); 1.53 (s, MeÀC(5)); 1.67 – 1.80 (m, 1 HÀC(2’)); 2.02 – 2.08 (m,
1 HÀC(2’)); 2.38 – 2.40 (m, 2 H, CH2N); 2.81 – 2.95 (m, 4 H, CH2N); 3.35 – 3.67 (m, 15 H, 2 HÀC(5’),
5 CH2O, MeO); 3.84 (s, HÀC(4’)); 4.20 (s, HÀC(3’)); 4.63 (s, NCH2-anth); 6.19 (t, J ¼ 6.3, HÀC(1’));
6.76 – 6.80 (m, 2 arom. H); 7.18 – 7.57 (m, 17 H, HÀC(6), arom. H); 8.01 (d, J ¼ 6.1, 2 arom. H); 8.40 (s, 1
arom. H); 8.53 (d, J ¼ 8.8, 2 arom. H). 13C-NMR (126 MHz, CDCl3): À 4.75; À 4.59; 12.49; 18.05; 25.83;
41.11; 43.16; 52.18; 53.54; 54.28; 55.26; 70.24; 70.30; 70.47; 70.57; 71.50; 72.63; 85.17; 86.54; 110.49;
113.17; 125.00; 125.10; 125.79; 126.26; 127.75; 127.86; 128.71; 129.18; 129.95; 130.11; 131.46; 131.54;
135.77; 138.37; 146.49; 157.93. ESI-MS: 993.3506 ([ M þ H]þ, C59H73N4O8Siþ; calc. 993.5198).
5’-O-{2-{{2-[2-(2-Aminoethoxy)ethoxy]ethyl}(anthracen-9-ylmethyl)amino}ethyl}-3’-O-[(tert-butyl)-
dimethylsilyl]thymidine (14). As described for 8, with 13 (1.0 g, 1.0 mmol) and 3% CCl3COOH/CH2Cl2
(34 ml) for 15 min. Workup with CHCl3 (30 ml) and sat. aq. NaHCO3 soln. (4 ꢀ 50 ml) and CC (NH-
silica gel, CHCl3) gave 14 (678 mg, 93%). 1H-NMR (500 MHz, CDCl3): À 0.01 to 0.01 (2s, Me2Si); 0.83
(s, tBuSi); 1.53 (s, MeÀC(5)); 1.68 – 1.79 (m, 1 HÀC(2’)); 1.98 – 2.02 (m, 1 HÀC(2’)); 2.78 – 2.90 (m, 6 H,
CH2N); 3.34 – 3.63 (m, 12 H, 2 HÀC(5’), CH2O); 3.81 – 3.82 (m, HÀC(4’), J ¼ 2.7 Hz); 4.08 (br., NH2);
4.19 – 4.21 (m, HÀC(3’)); 4.61 (s, NCH2-anth); 6.18 (t, J ¼ 6.3, HÀC(1’)); 7.21 (s, HÀC(6)); 7.40 – 7.53
(m, 4 arom. H); 7.98 (m, 2 arom. H); 8.38 (s, 1 arom. H); 8.41 – 8.49 (m, 2 arom. H). 13C-NMR (126 MHz,
CDCl3): À 4.76; À 4.59; 12.53; 18.05; 25.82; 41.12; 41.79; 52.20; 53.67; 54.25; 70.21; 70.36; 70.41; 70.46;
70.60; 72.59; 73.51; 85.19; 86.52; 110.50; 124.99; 125.08; 125.79; 127.77; 129.19; 130.07; 131.45; 131.53;
135.76; 150.33; 163.81. ESI-MS: 721.2548 ([M þ H]þ, C39H57N4O7Siþ; calc. 721.3997).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl[12-(anthracen-9-ylmeth-
yl)-6,9-dioxa-3,12-diazatetradecane-1,14-diyl]-(3’ ! 5’)-3’-O-[(tert)-butyl)dimethylsilyl]thymidine (15).
As described for 7, with 14 (579 mg, 804 mmol) and 3 (397 mg, 677 mmol) in CHCl3 (8 ml) and
NaBH(OAc)3 (431 mg, 1.6 mmol) for 5 min. Workup with AcOEt (20 ml) and sat. aq. NaHCO3 soln.
(2 ꢀ 20 ml) and CC (silica gel, CHCl3/MeOH 98 :2) gave 15 (572 mg, 55%). 1H-NMR (500 MHz,
CDCl3): 0.001, 0.025 (2s, Me2Si); 0.86 (s, tBuSi); 1.46 (s, 1 MeÀC(5)); 1.56 (s, 1 MeÀC(5)); 1.71 – 1.78 (m,
1 HÀC(2’)); 2.01 – 2.06 (m, 1 HÀC(2’)); 2.17 – 2.19 (m, 1 HÀC(2’)); 2.42 – 2.47 (m, 1 HÀC(2’)); 2.72 –
2.94 (m, 8 H, CH2N); 3.27 – 3.68 (m, 16 H, 4 HÀC(5), CH2O); 3.80 (s, 2 MeO); 3.84 – 3.85 (m,
1 HÀC(4’)); 4.12 – 4.15 (m, 1 HÀC(3’), 1 HÀC(4’)); 4.20 – 4.23 (m, 1 HÀC(3’)); 4.66 (s, NCH2-anth);
6.18 (t, J ¼ 7.5, 1 HÀC(1’)); 6.36 (dd, J ¼ 8.8, 5.0, 1 HÀC(1’)); 6.82 – 6.84 (m, 4 arom. H); 7.21 – 7.36 (m, 9
arom. H); 7.41 – 7.50 (m, 1 HÀC(6), 4 arom. H); 7.60 (s, 1 HÀC(6)); 7.98 (d, J ¼ 7.8, 2 arom. H); 8.38 (s, 1
arom. H); 8.51 (d, J ¼ 8.8, 2 arom. H). 13C-NMR (126 MHz, CDCl3): À 4.81; À 4.64; 11.86; 12.48; 17.99;
25.77; 37.85; 41.04; 49.05; 40.15; 52.10; 53.51; 54.22; 55.32; 63.95; 68.73; 70.11; 70.27; 70.36; 70.39; 70.45;
70.50; 72.50; 80.34; 84.09; 84.83; 85.20; 86.45; 86.97; 110.43; 111.28; 113.35; 124.95; 125.06; 125.74; 127.22;
127.68; 128.06; 128.20; 129.11; 130.07; 130.14; 131.38; 135.42; 135.46; 135.68; 135.79; 144.39; 150.44;
150.57; 158.79; 164.07; 164.10. ESI-MS: 1291.6973 ([ M þ H]þ, C72H91N6O14Siþ; calc. 1291.6362).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl[12-(anthracen-9-ylmeth-
yl)-6,9-dioxa-3,12-diazatetradecane-1,14-diyl]-(3’ ! 5’)-thymidine (16). As described for 10, with 15
(492 mg, 389 mmol), THF (4 ml), and Bu4NF (112 mg, 428 mmol) for 2.5 h. Workup and CC as described
1
gave 16 (371 mg, 83%). H-NMR (500 MHz, CDCl3): 1.44 (s, 1 MeÀC(5)); 1.50 (s, 1 MeÀC(5)); 1.82 –
2.46 (m, 4 HÀC(2’)); 2.71 – 2.97 (m, 8 H, CH2N); 3.25 – 3.30 (m, 1 HÀC(5’)); 3.42 – 3.74 (m, 15 H,
HÀC(5’), CH2O); 3.76 (s, 2 MeO); 3.78 – 3.81 (m, 1 HÀC(3’)); 4.11 – 4.12 (m, 1 HÀC(3’)); 4.13 – 4.28

Helvetica Chimica Acta – Vol. 90 (2007)
1961
(m, 2 HÀC(4’)); 4.52 (d, J ¼ 13, 1 H, CH2N-anth); 4.62 (d, J ¼ 13, 1 H, CH2N-anth); 6.25 (t, J ¼ 7.8,
1 HÀC(1’)); 6.37 (dd, J ¼ 5.9, 8.3, 1 HÀC(1’)); 6.81 – 6.85 (m, 4 arom. H); 7.23 – 7.50 (m, 14 arom. H); 7.61
(s, 1 arom. H); 7.97 (d, J ¼ 8.1, 2 arom. H); 8.38 (s, 1 arom. H); 8.51 (d, J ¼ 8.8, 2 HÀC(6)). 13C-NMR
(126 MHz, CDCl3): 11.89; 12.45; 37.86; 40.95; 49.01; 49.07; 51.98; 53.66; 54.06; 55.31; 63.99; 68.45; 69.75;
69.89; 70.32; 70.36; 70.56; 70.74; 71.45; 80.41; 84.07; 84.80; 85.21; 86.16; 86.96; 110.30; 111.32; 113.34;
124.93; 125.07; 127.22; 127.75; 128.06; 128.20; 129.13; 129.95; 130.14; 131.37; 131.42; 135.41; 135.45;
135.68; 135.90; 144.39; 150.56; 150.69; 158.77; 164.20; 164.23. ESI-MS: 1177.6360 ([ M þ H]þ,
C66H77N6Oþ14 ; calc. 1177.5497).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl[12-(anthracen-9-ylmeth-
yl)-3-(2,2,2-trifluoroacetyl)-6,9-dioxa-3,12-diazatetradecane-1,14-diyl]-(3’ ! 5’)-thymidine (17). As de-
scribed for 11, with 16 (365 mg, 310 mmol), CH2Cl2 ( 3 ml),iPr2EtN (160 ml, 930 mmol), and (CF3CO)2O
(65 ml, 470 mmol) for 20 min. Workup AcOEt (15 ml) and brine (10 ml) and CC (silica gel, CHCl3/MeOH
1
99 :1) gave 17 (326 mg, 83%). H-NMR (500 MHz, CDCl3): 1.41 – 1.59 (m, 6 H, 2 MeÀC(5)); 1.79 – 1.83
(m, 1 HÀC(2’)); 2.12 – 2.17 (m, 2 HÀC(2’)); 2.32 – 2.34 (m, 1 HÀC(2’)); 2.77 – 3.73 (m, 24 H, CH2N,
CH2O, HÀC(5’)); 3.76 (s, 2 MeO); 3.91 – 4.02 (m, 2 HÀC(3’)); 4.03 – 4.21 (m, 2 HÀC(4’)); 4.56 (d, J ¼
12.9, 1 H, CH2N-anth); 4.64 (d, J ¼ 13.4, 1 H, CH2N-anth); 6.24 – 6.34 (m, 2 HÀC(1’)); 6.83 (m, 4 arom.
H); 7.21 – 7.48 (m, 13 H, HÀC(6’), arom. H); 7.59 (s, 1 HÀC(6’)); 7.96 (d, J ¼ 8.3, 2 arom. H); 8.36 (s, 2
arom. H); 8.57 (d, J ¼ 9.0, 2 arom. H); 9.94 (br., 2 NH(3)). 13C-NMR (126 MHz, CDCl3): 11.76; 11.82;
12.34; 37.43; 37.62; 40.44; 47.92; 48.08; 48.29; 48.42; 51.81; 53.50; 53.77; 55.17; 63.59; 63.85; 66.16; 67.91;
68.14; 69.63; 69.90; 70.02; 70.30; 70.56; 70.67; 71.85; 80.43; 80.48; 83.70; 83.83; 84.51; 84.99; 85.90; 86.87;
86.89; 110.37; 111.34; 111.37; 113.21; 124.84; 124. 92; 125.65; 127.13; 127.58; 127.96; 128.02; 128.97; 129.89;
129.99; 131.20; 135.15; 135.47; 135.76; 144.18; 150.59; 150.68; 150.71; 158.63; 164.17; 164.23. ESI-MS:
1273.5637 ([M þ H]þ; calc. 1273.5320).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl[12-(anthracen-9-ylmeth-
yl)-3-(2,2,2-trifluoroacetyl)-6,9-dioxa-3,12-diazatetradecane-1,14-diyl]-(3’ ! 5’)-thymidine 3’-(2-Cya-
noethyl N,N-Diisopropylphosophoramidite) (18a). As described for 12, with 17 (150 mg, 118 mmol),
CH2Cl2 (470 ml), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (107 mg, 354 mmol), and
diisopropylammonium 1H-tetrazolide (3.3 mg, 47 mmol) for 3 h. Workup with AcOEt (10 ml) and sat.
aq. NaHCO3 soln. (10 ml) yielded crude 18a, which was used without further purification for the next
1
oligonucleotide synthesis because of the instability on CC purification. H-NMR (500 MHz, CDCl3):
1.14 – 1.31 (m, 12 H, Me2CH); 1.45 – 1.53 (m, 2 MeÀC(5)); 1.73 – 2.30 (m, 4 HÀC(2’); 2.52 – 2.58 (m, 2 H,
CH2N); 2.73 – 2.90 (m, 4 HÀC(5’)); 3.19 – 3.75 (m, 22 H, CH2N, CH2O); 3.77 (s, 2 MeO); 3.95 – 4.37 (m,
4 H, HÀC(3’), HÀC(4’)); 4.64 – 4.65 (m, CH2N-anth); 6.20 – 6.31 (m, 2 H – C(1’)); 6.83 (m, 4 arom. H);
7.22 – 7.58 (m, 13 arom. H); 7.98 (m, 2 arom. H); 8.19 (s, 1 arom. H); 8.50 – 8.53 (m, 2 arom. H); 9.41 (s,
1 H, NH(3)). 13C-NMR (126 MHz, CDCl3): 11.61; 11.80; 11.86; 12.31; 12.37; 14.18; 15.26; 19.83; 19.89;
20.29; 20.35; 21.02; 22.21; 22.83; 23.12; 23.46; 24.37; 24.43; 24.49; 24.51; 24.55; 24.61; 37.52; 37.71; 39.63;
39.66; 39.70; 39.74; 43.15; 43.18; 43.25; 43.28; 45.53; 45.57; 45.61; 45.65; 47.98; 48.14; 48.31; 48.51; 52.02;
53.47; 53.83; 53.90; 55.22; 57.90; 58.05; 58.09; 58.24; 58.60; 58.62; 60.37; 63.65; 63.91; 65.82; 66.26; 67.97;
68.29; 69.98; 70.02; 70.17; 70.40; 70.66; 70.71; 70.80; 74.17; 74.30; 74.43; 74.56; 80.45; 80.50; 83.74; 83.90;
84.58; 84.96; 85.03; 85.13; 85.41; 85.44; 86.95; 86.97; 110.47; 110.59; 111.29; 111.31; 113.28; 117.54; 117.69;
124.90; 125.00; 125.71; 127.18; 127.20; 127.60; 127.62; 128.00; 128.11; 129.04; 130.06; 131.31; 131.38;
135.27; 135.37; 135.43; 135.58; 144.28; 150.38; 150.45; 150.51; 150.53; 158.75; 163.91; 163.94; 163.96;
171.13. 31P-NMR (CDCl3, 203 MHz): 149.24; 149.57. ESI-MS: 1473.5786 ([M þ H]þ, C77H93F3N8O16Pþ;
calc. 1473.6106).
N-{2-[2-(2-Aminoethoxy)ethoxy]ethyl}-a,a-diphenylbenzenemethanesulfenamide (19). To the soln.
of 2,2’-[(ethane-1,2-diylbis(oxy)]bis[ethanamine] (2.0 ml, 14 mmol) in THF/H2O 19 :1 (270 ml) were
added Na2CO3 (1.4 g, 14 mmol) and a,a-diphenylbenzenemethanesulfenyl chloride (2.1 g, 6.8 mmol).
The soln. was stirred at r.t. for 1 h and then diluted with AcOEt (500 ml). The org. layer was washed once
with sat. aq. NaHCO3 soln. (500 ml), dried (MgSO4), and concentrated and the residue subjected to CC
(silica gel, CHCl3/MeOH 96 :4): 19 (1.6 g, 55%). 1H-NMR (500 MHz, CDCl3): 2.66 – 2.70 (m, 2 H,
CH2N); 2.81 – 2.85 (m, 2 H, CH2N); 3.29 – 3.13 (t, J ¼ 5.4, 2 H, CH2O); 3.46 – 3.49 (m, 4 H, CH2O); 3.53 –
3.55 (m, 2 H, CH2O); 7.21 – 7.48 (m, 15 arom. H). 13C-NMR (126 MHz, CDCl3): 41.58; 51.93; 69.89;

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Helvetica Chimica Acta – Vol. 90 (2007)
69.98; 70.05; 70.36; 70.75; 73.21; 73.24; 126.05; 126.44; 127.59; 128.49; 129.74; 144.06. ESI-MS: 423.6831
([M þ H]þ, C25H31N2O2Sþ; calc. 423.2106).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-(14,14,14-triphenyl-6,9-dioxa-13-thia-3,12-diazate-
tradec-1-yl)thymidine (20). As described for 7, with 3 (1.8 g, 3.0 mmol), CHCl3 (30 ml), 19 (1.3 g,
3.0 mol), and NaBH(OAc)3 (1.3 mg, 6.0 mmol) for 15 min. Workup with CHCl3 (20 ml) and (1 ꢀ 20 ml)
1
and CC (NH-silica gel, hexane/CHCl3 1:4) gave 20 (2.0 g, 66%). H-NMR (500 MHz, CDCl3): 1.46 (s,
MeÀC(5)); 2.16 – 2.22 (m, 1 HÀC(2’)); 2.49 – 2.53 (m, 1 HÀC(2’)); 2.61 – 2.81 (m, 6 H, CH2N); 3.07 –
3.61 (m, 12 H, 2 HÀC(5’), CH2O); 3.75 (s, 2 MeO); 4.12 – 4.13 (m, HÀC(4’)); 4.18 – 4.19 (m,
HÀC(3’)); 6.35 (dd, J ¼ 5.6, 7.8, HÀC(1’)); 6.83 – 7.48 (m, 28 H of (MeO)2Tr and TrS); 7.61 (s,
HÀC(6)). 13C-NMR (126 MHz, CDCl3): 12.12; 38.14; 49.36; 49.43; 52.39; 55.52; 64.17; 69.22; 70.42;
70.51; 70.79; 70.84; 71.22; 80.49; 84.30; 85.04; 87.17; 111.43; 113.56; 126.89; 127.42; 128.04; 128.27; 128.42;
128.96; 130.21; 130.37; 135.66; 135.71; 135.86; 144.53; 144.65; 150.85; 159.00; 164.42. ESI-MS: 993.2842
([M þ H]þ, C58H65N4O9Sþ; calc. 993.4472).
3’-O-[3-(Anthracen-9-ylmethyl)-14,14,14-triphenyl-6,9-dioxa-13-thia-3,12-diazatetradec-1-yl]-5’-O-
[bis(4-methoxyphenyl)phenylmethyl]thymidine (21). As described for 13, with 20 (2.0 g, 2.0 mmol), of
N,N-dimethylformamide (20 ml), 9-(chloromethyl)anthracene (450 g, 2.0 mmol), Na2CO3 (4.2 g,
40 mmol), and KI (33 mg, 594 mmol) for 2 h. Workup with AcOEt (100 ml) and brine (3 ꢀ 100 ml)
1
and CC as described gave 21 (1.8 g, 77%). H-NMR (500 MHz, CDCl3): 1.44 (s, MeÀC(5)); 1.95 – 2.03
(m, 1 HÀC(2’)); 2.22 – 2.37 (m, 1 HÀC(2’)); 2.59 – 2.84 (m, 6 H, CH2N); 2.95 – 3.54 (m, 12 H, 2 HÀC(5’),
CH2O); 3.75 (s, 2 MeO); 3.88 – 3.91 (m, HÀC(4’), HÀC(3’)); 4.62 (s, NCH2-anth); 6.22 – 6.26 (dd, J ¼ 5.6,
8.3, HÀC(1’)); 6.77 – 7.46 (m, 32 H of (MeO)2Tr, TrS, and anth); 7.54 (s, HÀC(6)); 7.94 (m, 2 arom. H);
8.02 (br., 1 NH(3)); 8.38 (s, 1 arom. H); 8.57 (m, 2 arom. H). 13C-NMR (126 MHz, CDCl3): 11.90; 11.92;
37.89; 52.10; 52.15; 53.84; 54.23; 55.27; 55.29; 63.89; 68.52; 70.11; 70.14; 70.20; 70.61; 70.98; 79.93; 84.14;
84.83; 86.92; 111.15; 113.31; 113.32; 124.94; 125.26; 125.68; 126.26; 126.66; 127.19; 127.55; 127.82; 128.04;
128.19; 128.75; 129.01; 129.99; 130.14; 130.49; 131.39; 131.46; 135.46; 144.30; 144.43; 150.41; 158.76;
163.98. ESI-MS: 1183.5185 ([M þ H]þ, C73H75N4O9Sþ; calc. 1183.5254).
3’-O-{2-{{2-[2-(2-Aminoethoxy)ethoxy]ethyl}(anthracen-9-ylmethyl)amino}ethyl}-5’-O-[bis(4-meth-
oxyphenyl)phenylmethyl]thymidine (22). To a soln. of 21 (1.7 g, 1.4 mmol) in toluene (14 ml) was added
tributylstannane (2.9 ml, 11 mmol), and the mixture was stirred at 1308 for 3 h. The mixture was
subjected to CC (NH-silica gel, CHCl3): 22 (647 mg, 50%). 1H-NMR (500 MHz, CDCl3): 1.49 (s,
MeÀC(5)); 1.97 – 2.29 (m, 2 HÀC(2’)); 2.77 – 2.86 (m, 6 H, CH2N); 3.10 – 3.54 (m, 12 H, 2 HÀC(5’),
CH2O); 3.72 (s, 2 MeO); 3.91 – 3.93 (m, HÀC(4’), HÀC(3’)); 4.61 (s, CH2N-anth); 6.29 – 6.31 (t, J ¼ 6.3,
HÀC(1’)); 6.77 – 7.46 (m, 17 H of (MeO)2Tr and anth); 7.51 (s, HÀC(6)); 7.91 (m, 2 arom. H); 8.38 (s, 1
arom. H); 8.52 (m, 2 arom. H). 13C-NMR (126 MHz, CDCl3): 13.70; 16.22; 27.03; 28.20; 37.71; 41.56;
52.02; 53.76; 54.06; 55.14; 63.80; 68.35; 70.00; 70.10; 70.18; 73.25; 79.84; 83.88; 84.70; 86.74; 113.16;
113.18; 124.80; 125.13; 125.54; 127.02; 127.41; 127.90; 128.07; 128.87; 128.97; 130.01; 130.37; 131.26;
131.32; 135.39; 144.38; 158.61. ESI-MS: 909.2227 ([M þ H]þ, C54H61N4O9þ ; calc. 909.4439).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl[3-(anthracen-9-ylmeth-
yl)-6,9-dioxa-3,12-diazatetradecane-1,14-diyl]-(3’ ! 5’)-3’-O-[(tert-butyl)dimethylsilyl]thymidine (23).
As described for 7, with 22 (647 mg, 713 mmol) and 5 (284 mg, 713 mmol) in CHCl3 (7 ml) and
NaBH(OAc)2 (302 mg, 1.4 mmol) for 5 min. Workup with CHCl3 (10 ml) and brine (1 ꢀ 20 ml) and CC
(silica gel, CHCl3/MeOH 96 :4) gave 23 (393 mg, 43%). 1H-NMR (500 MHz, CDCl3): 0.10 (s, Me2Si);
0.82 (s, tBuSi); 1.41 (s, 1 MeÀC(5)); 1.84 (s, 1 MeÀC(5)); 1.91 – 2.23 (m, 4 HÀC(2’)); 2.70 – 2.81 (m, 8 H,
CH2N); 3.02 – 3.61 (m, 16 H, 4 HÀC(5’), CH2O); 3.69 (s, 2 MeO); 3.82 – 3.99 (m, 2 HÀC(4’), 1 HÀ(3’));
4.32 (m, 1 HÀC(3’)); 4.56 (s, CH2N-anth); 6.29 – 6.31 (m, 2 HÀC(1’)); 6.71 – 7.41 (m, 17 H of (MeO)2Tr
and anth); 7.48 (s, 1 HÀC(6)); 7.87 (m, 2 arom. H); 8.38 (s, 1 arom. H); 8.47 (m, 2 arom. H). 13C-NMR
(126 MHz, CDCl3): À 4.83; À 4.65; 11.88; 12.69; 17.94; 25.74; 37.81; 40.99; 48.65; 48.89; 52.06; 53.74;
54.22; 63.81; 68.34; 69.66; 70.01; 70.07; 70.29; 70.42; 70.55; 72.09; 79.89; 84.03; 84.70; 85.25; 86.17; 86.83;
110.76; 111.17; 113.24; 124.88; 125.18; 125.63; 127.10; 127.48; 127.97; 128.10; 128.93; 130.00; 130.06;
130.39; 131.30; 131.37; 135.38; 135.59; 135.97; 144.35; 150.55; 150.62; 158.67; 164.18. ESI-MS: 1291.6952
([M þ H]þ, C72H91N6O14Siþ; calc. 1291.6362).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl[3-(anthracen-9-ylmeth-
yl)-6,9-dioxa-3,12-diazatetradecane-1,14-diyl]-(3’ ! 5’)-thymidine (24). As described for 10, with 23

Helvetica Chimica Acta – Vol. 90 (2007)
1963
(609 mg, 472 mmol), THF (5 ml), and Bu4NF (197 mg, 755 mmol) for 8 h. Workup and CC as described
gave 24 (419 mg, 75%). 1H-NMR (500 MHz, CDCl3): 1.45 (s, 1 MeÀC(5)); 1.88 (s, 1 MeÀC(5)); 1.96 –
2.32 (m, 4 HÀC(2’)); 2.72 – 2.85 (m, 8 H, CH2N); 3.07 – 3.66 (m, 16 H, 4 HÀC(5’), CH2O); 3.74 (s,
2 MeO); 3.86 – 3.98 (m, 2 HÀC(4’), 1 HÀC(3’)); 4.44 (m, 1 HÀC(3’)); 4.61 (s, CH2N anth); 6.26 – 6.30
(m, 2 HÀC(1’)); 6.77 – 7.46 (m, 17 H of (MeO)2Tr and anth); 7.54 (s, HÀC(6)); 7.92 (d, J ¼ 8.3, 2 arom.
H); 8.36 (s, 1 arom. H); 8.48 (d, J ¼ 8.8, 2 arom. H). 13C-NMR (126 MHz, CDCl3): 11.93; 12.77; 37.84;
40.52; 48.67; 48.88; 52.11; 53.81; 54.29; 55.29; 63.88; 68.42; 69.98; 70.08; 70.12; 70.24; 70.40; 70.53; 71.20;
79.98; 84.09; 84.76; 84.94; 85.39; 86.90; 110.78; 111.26; 113.28; 113.29; 1224.95; 125.23; 125.70; 127.16;
127.54; 128.03; 128.15; 128.98; 130.11; 130.43; 131.34; 131.42; 135.43; 135.67; 135.97; 144.40; 150.68;
150.74; 158.71; 164.28; 164.34. ESI-MS: 1177.4326 ([ M þ H]þ, C66H77N6O1þ4 ; calc. 1177.5497).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl[3-(anthracen-9-ylmeth-
yl)-12-(trifluoroacetyl)-6,9-dioxa-3,12-diazatetradecane-1,14-diyl]-(3’ ! 5’)-thymidine (25). As described
for 11, with 24 (406 mg, 345 mmol), CH2Cl2 ( 4 ml),iPr2EtN (177 ml, 1.0 mmol), and (CF3CO)2O ( 72ml,
517 mmol) for 30 min. Workup with AcOEt (20 ml) and brine (20 ml) and CC (silica gel, CHCl3/MeOH
1
99 :1) gave 25 (350 mg, 80%). H-NMR (500 MHz, CDCl3): 1.46 (s, 1 MeÀC(5)); 1.88 (s, 1 MeÀC(5));
2.00 – 2.31 (m, 4 HÀC(2’)); 2.77 – 2.84 (m, 4 H, CH2N); 3.08 – 3.72 (m, 20 H, CH2N, CH2O); 3.74 (s,
2 MeO); 3.77 – 3.98 (m, 2 HÀC(4’), 1 HÀC(3’)); 4.29 – 4.34 (m, 1 HÀC(3’)); 4.61 (s, CH2N-anth); 6.23 –
6.30 (m, 2 HÀC(1’)); 6.77 – 7.47 (m, 17 H of (MeO)2Tr and anth); 7.56 (s, HÀC(6)); 7.93 (d, J ¼ 8.8, 2
arom. H); 8.38 (s, 1 arom. H); 8.57 (d, J ¼ 8.3, 2 arom. H); 9.60 (s, 2 NH(3)). 13C-NMR (126 MHz,
CDCl3): 11.91; 12.69; 12.73; 37.86; 40.17; 40.33; 47.31; 47.70; 48.14; 52.06; 53.90; 54.14; 54.22; 55.30; 63.91;
67.96; 68.36; 69.83; 70.01; 70.09; 70.28; 70.49; 70.67; 70.95; 71.22; 71.42; 80.12; 84.16; 84.81; 85.00; 85.15;
85.25; 85.47; 86.95; 110.96; 111.35; 111.37; 113.30; 113.31; 125.00; 125.18; 125.37; 125.75; 127.20; 127.61;
128.05; 128.16; 128.29; 129.04; 129.10; 130.13; 130.33; 131.36; 131.43; 135.40; 135.74; 136.00; 144.38;
150.64; 150.67; 150.70; 150.72; 158.74; 164.05; 164.12; 164.17; 164.23; 164.28. ESI-MS: 1273.5462 ([ M þ
H]þ, C68H76F3N6O1þ5 ; calc. 1273.5320).
5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-3’-O-dephosphinicothymidylyl[3-(anthracen-9-ylmeth-
yl)-12-(trifluoroacetyl)-6,9-dioxa-3,12-diazatetradecane-1,14-diyl]-(3’ ! 5’)-thymidine 3’-(2-Cyanoethyl
N,N-Diisopropylphosphoramidite) (18b). As decribed for 12, with 25 (150 mg, 118 mmol), CH2Cl2
(470 ml), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (85 mg, 282 mmol), and diisopropy-
lammonium 1H-tetrazolide (3.3 mg, 47 mmol) for 4 h. Workup with AcOEt (10 ml) and sat. aq. NaHCO3
soln. (10 ml) yielded crude 18b, which was used without further purification for the next oligonucleotide
synthesis because of the instability on CC purification. 1H-NMR (500 MHz, CDCl3): 1.15 – 1.31 (m, 12 H,
Me2CH); 1.46 – 1.91 (m, 2 MeÀC(5)); 1.97 – 2.42 (m, 4 HÀC(2’)); 2.58 – 2.62 (m, 2 H, CH2N); 2.74 – 2.86
(m, 4 HÀC(5’)); 3.08 – 3.84 (m, 22 H, CH2N, CH2O); 3.75 (s, 2 MeO); 3.88 – 4.49 (m, 4 H, HÀC(3’),
HÀC(4’)); 4.63 (s, CH2N-anth); 6.23 – 6.33 (m, 2 HÀC(1’)); 6.77 – 8.53 (m, 24 H, 2 HÀC(6), 22 arom.
H); 9.13 (s, NH(3)). 13C-NMR (126 MHz, CDCl3): 11.98; 12.73; 2048; 20.53; 21.14; 22.32; 22.93; 22.96;
23.06; 23.22; 23.56; 24.48; 24.53; 24.57; 24.64; 24.67; 24.70; 24.73; 37.86; 39.38; 43.31; 43.29; 43.39;
45.62;45.66; 47.34; 47.56; 47.80; 47.92; 47.99; 52.16; 53.91; 54.30; 55.32; 57.97; 58.03; 58.13; 58.18; 63.91;
67.81; 68.04; 68.35; 68.48; 69.41; 69.59; 70.10; 70.21; 70.26; 70.51; 70.72; 70.91; 71.04; 73.28; 73.41; 73.82;
73.98; 80.03; 80.06; 84.15; 84.81; 85.08; 85.11; 85.19; 85.24; 85.27; 86.94; 111.05; 111.13; 111.16; 111.22;
111.25; 113.31; 113.32; 116.03; 117.77; 117.83; 124.99; 125.23; 124.72; 127.20; 127.59; 128.05; 128.19;
129.04; 130.14; 130.41; 130.44; 131.38; 131.46; 135.45; 135.62; 135.70; 135.72; 135.99; 136.03; 144.42;
150.42; 150.49; 150.54; 150.57; 158.77; 163.85; 163.94; 163.96. 31P-NMR (CDCl3, 203 MHz): 149.60;
149.78. ESI-MS: 1473.5905 ([M þ H]þ, C77H92F3N8O16Pþ; calc. 1473.6399).
Oligonucleotide Synthesis. Phosphoramidites 12, 18a, and 18b and the commercially available
deoxynucleoside phosphoroamidites were used. The 3’-half of each DNA, d(Y1Y2 TCGAG), was
synthesized on a CPG support by use of an ABI-392 DNA synthesizer. After the (MeO)2Tr group at the
5’-terminal nucleotide (Y1) was removed, the CPG was transferred to a glass syringe. The appropriate
phosphoramidite, 12, 18a, or 18b, was coupled to the free 5’-OH group according to the following manual
coupling procedure: 1) Coupling: 20 equiv. of phosphoramidite, 80 equiv. of 1H-tetrazole, MeCN
(250 ml), 5 min. 2) Capping: 1.1m Ac2O, 133 mm N,N-dimethylpyridin-4-amine in pyridine, 2 min. 3)
Oxidation: 0.1m I2 in pyridine/H2O 9 :1. Subsequently, the CPG supports were transferred to the column
for the DNA synthesizer, and the 5’-half parts of each DNA, d(GCTCX1X2), were elongated on the

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Helvetica Chimica Acta – Vol. 90 (2007)
synthesizer. After the cleavage from the solid supports, the protecting groups at the nucleobases and
phosphates and the CF3CO groups at the amino groups were removed by treatment with aq. ammonia
overnight. The desired oligonucleotides were purified by anion-exchange HPLC (Genpak Fax, 10 mm
Na3PO4 applying of the gradient 1.0m NaCl ! 10 mm Na3PO4 in 1%/min). The structures of the
oligonucleotides were confirmed by the MALDI-TOF-MS.
5’-d(GCTCAG[tt]GATCGAG)-3’:(ODN1) yield 16%. MALDI-TOF-MS: 4750.6 (calc. 4745.0).
5’-d(GCTCAG[tt*]GATCGAG)-3’:(ODN2a) yield 12%. MALDI-TOF-MS: 4932.0 (calc. 4933.1).
5’-d(GCTCAG [t*t] GATCGAG)-3’:(ODN2b) yield 11%. MALDI-TOF-MS: 4931.6 (calc. 4933.1).
5’-d(GCTCCT[tt*]AGTCGAG)-3’:(ODN3a) yield 9%, MALDI-TOF-MS: 4882.8 (calc. 4884.1).
5’-d(GCTCCT[t*t]AGTCGAG)-3’:(ODN3b) yield 19%. MALDI-TOF-MS: 4883.0 (calc. 4884.1).
Fluorescence Spectra. For the natural-type ODNs, the e260 were calculated according to [47]. The e260
of an ODN incorporating a long-chain linker without the anthracene moiety was assumed to be identical
to that of the natural-type ODN having the same nucleotide sequences. In the case of an ODN having an
anthracene moiety, the e260 was estimated as the sum of e260 of the ODN and 5500. ODN1: 145700;
ODN2a: 151200; ODN2b: 151200; ODN3a: 143800; ODN3b: 143800. The sample ODNs were dissolved
in 0.1m Tris · HCl, 0.1m NaCl. ODN concentrations are described below. The directly observed fluorescent
intensities were divided by these concentrations to give the fluorescent intensities of the hypothetical 1m
solns. and used to draw Figs. 3 and 4.
Experiment shown in Fig. 3: Single strand ODN2a, 104 nm; ODN2b, 102 nm; ODN3a, 140 nm;
ODN3b, 140 nm. Double strand ODN2a · ODN4, 125 nm; ODN2b · ODN4: 120 nm; ODN3a · ODN5,
121 nm; ODN3b · ODN5, 120 nm. The measurements were carried out at 108.
Experiment shown in Fig. 4: ODN3a · ODN5, 120 nm; ODN3a · ODN6, 130 nm; ODN3a · ODN7,
130 nm; ODN3a · ODN8, 130 nm; ODN3a · ODN9, 80 nm. The measurements were carried out at
pH 8.0.
REFERENCES
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ReceivedJune 14, 2007