Nucleosides and oligonucleotides with diynyl side chains: Base pairing and functionalization of 2′-deoxyuridine derivatives by the copper(I)-catalyzed alkyne-azide 'click' cycloaddition

Article abstract of DOI:10.1002/hlca.200790055

Oligonucleotides containing the 5-substituted 2′-deoxyuridines 1b or 1d bearing side chains with terminal C≡C bonds are described, and their duplex stability is compared with oligonucleotides containing the 5-alkynyl compounds 1a or 1c with only one nonte

Full text of DOI:10.1002/hlca.200790055

Helvetica Chimica Acta – Vol. 90 (2007)
535
Nucleosides and Oligonucleotides with Diynyl Side Chains: Base Pairing and
Functionalization of 2’-Deoxyuridine Derivatives by the Copper(I)-Catalyzed
AlkyneꢀAzide ꢀClickꢁ Cycloaddition
by Frank Seela* and Venkata Ramana Sirivolu
Laboratorium für Organische und Bioorganische Chemie, Universität Osnabrück, Barbarastrasse 7,
D-49069 Osnabrück, and
Laboratory for Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstrasse 11, D-48149Münster
(phone: þ 49(0)5419692791; mobil: þ 49(0)1737250297; fax: þ 49(0)25135406501; e-mail:
Frank.Seela@uni-osnabrueck.de, Seela@uni-muenster.de; www.seela.net)
Dedicated to Professor Wolfgang Pfleiderer
Oligonucleotides containing the 5-substituted 2’-deoxyuridines 1b or 1d bearing side chains with
terminal CꢁC bonds are described, and their duplex stability is compared with oligonucleotides
containing the 5-alkynyl compounds 1a or 1c with only one nonterminal CꢁC bond in the side chain. For
this, 5-iodo-2’-deoxyuridine (3) and diynes or alkynes were employed as starting materials in the
Sonogashira cross-coupling reaction (Scheme 1). Phosphoramidites 2b – d were prepared (Scheme 3) and
used as building blocks in solid-phase synthesis. Tm Measurements demonstrated that DNA duplexes
containing the octa-1,7-diynyl side chain or a diprop-2-ynyl ether residue, i.e., containing 1b or 1d, are
more stable than those containing only one triple bond, i.e., 1a or 1c (Table 3). The diyne-modified
nucleosides were employed in further functionalization reactions by using the protocol of the CuI-
catalyzed Huisgen – Meldal – Sharpless [2 þ 3] cycloaddition (ꢀclick chemistryꢁ) (Scheme 2). An aliphatic
azide, i. e., 3’-azido-3’-deoxythymidine (AZT; 4), as well as the aromatic azido compound 5 were linked
to the terminal alkyne group resulting in 1H-1,2,3-triazole-modified derivatives 6 and 7, respectively
(Scheme 2), of which 6 forms a stable duplex DNA (Table 3). The Husigen – Meldal – Sharpless
cycloaddition was also performed with oligonucleotides (Schemes 4 and 5).
Introduction. – Modified oligonucleotides have the potential to widen the
diagnostic application of synthetic DNA such as in primer probe interactions, DNA
chip technology, or to enlarge the repertoire of therapeutic applications (antisense
therapy) [1 – 4]. This led to the development of nucleic acid mimics containing a wide
variety of modified bases [5 – 8], sugar residues [9], or altered phosphodiester
backbones [10]. These structural modifications can influence hybridization strength,
binding affinity, mismatch discrimination, nuclease resistance, or cellular uptake.
Oligonucleotide – alkynyl conjugates are of particular importance because of their
antiviral and anticancer activity [11 – 13]. They can also be used to link any kind of
reporter groups [14] or duplex-stabilizing residues to DNA.
Among the various base modifications, the 5-position of pyrimidine nucleosides is
an ideal place for introducing structural diversity in oligonucleotides [15 – 17]. This site
has steric freedom as it lies in the major groove of the B-DNA duplex. Thus,
substituents of moderate size can be introduced without perturbing the DNA structure.
Recently, we have shown that halogen substituents present at the 5-position of
ꢂ 2007 Verlag Helvetica Chimica Acta AG, Zürich

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Helvetica Chimica Acta – Vol. 90 (2007)
pyrimidines show very little influence or even no effect on the DNA duplex stability
[18]. A significant stabilization is observed when alkynyl residues are introduced. A
series of such nucleosides containing propynyl groups (e.g., 1a) have been synthesized
and incorporated into duplex DNA [19– 24]. It was found that an increase in the chain
length from propynyl to hexynyl groups has an unfavorable influence on the base-pair
stability most likely due to the increasing hydrophobic character of the alkynyl chain
[17]. As the hydrophobic character of a side chain can dry out the grooves from H2O
molecules, an increasing hydrophilic character might have a favorable influence on the
duplex structure. As triple bonds or O-functionalities can fulfill these requirements, we
decided to use this principle for the functionalization of oligonucleotides carrying long
flexible linker arms, such as those present in the nucleosides, 1b – d.
We have already reported on the construction of polymeric networks by the
functionalization of the 2’-deoxyuridine side chain by a terminal alkyne [25]. For this
purpose the 5-octadiynyl derivative 1b was prepared containing two CꢁC bonds. This
compound was employed in the Sonogashira cross-coupling reaction resulting in the
formation of cross-linked 5,5’-bis(nucleosides). The side chains with such a terminal
unsaturation could also be used for further functionalization by means of the CuI-
catalyzed alkyne – azide coupling described independently by the Meldal [26] and
Sharpless [27] groups (ꢀclickꢁ reaction). CuI strongly catalyzes this coupling and
improves the regioselectivity by giving the 1,4-disubstituted 1H-1,2,3-triazole re-
gioisomer exclusively. So, the ꢀclickꢁ functionalization of oligonucleotides can be used
to introduce almost any reporter group in nucleic acids.
Now, we performed the Huisgen – Meldal – Sharpless cycloaddition with nucleo-
sides and oligonucleotides. In the case of oligonucleotides, the reaction was carried out
in solution as well as on solid support. Details of the synthesis of the phosphoramidite
building blocks 2b – d are given, and the stability of duplexes containing such
modifications was studied. After the appearance of a short communication [28] in
which we described the functionalization and incorporation of four octa-1,7-diynyl
nucleosides in oligonucleotides mimicking the canonical DNA constitutents, the
functionalization of alkyne-modified DNA by means of one base modification was
reported [29].

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Results and Discussion. – 1. Synthesis and Properties of Nucleosides. The synthesis
of nucleoside 1a was already reported [23]. The nucleosides 1b – d were synthesized
from 5-iodo-2’-deoxyuridine (3) [30] by the Sonogashira cross-coupling reaction [31]
(Scheme 1). The reaction was performed in dry DMF in the presence of Et3N, of
[Pd0(PPh3)4] and CuI as catalyst in a 1:2 ratio, and of an excess of the diyne or alkyne.
As a result, only one terminal CꢁC bond of the diyne was functionalized leading to the
almost exclusive formation of the mono-substituted diynyl nucleosides 1b and 1d in 72
and 84% yield, respectively. The presence of traces of a fluorescent by-product was
observed [32]. Its formation depended on the type of reactant used as well as on the
reaction time. Reactions at the second CꢁC bond occurred when compounds 1b or 1d
were isolated and employed in a second cross-coupling reaction. The structure of the
nucleosides was confirmed by the 1H-, and 13C-NMR spectra (Table 1) and by
elemental analysis.
Scheme 1
i) [Pd0(PPh3)4], CuI, Et3N, DMF, octa-1,7-diyne (!1b), oct-1-yne (!1c), or diprop-2-ynyl ether
(!1d).
The 13C-NMR chemical shifts were assigned by means of gated-decoupled spectra. The terminal
alkynyl C-atom of the side chain of 1b showed a C,H coupling (1J(C,H) ¼ 250 Hz) and the chain CH2
groups were observed in the range of d 17 – 27 (J(C,H) ¼ 125 – 129Hz). Because of the presence of an O-
atom in the dipropynyl ether moiety of 1d, the CH2 signals were shifted downfield to d(C) 56.0 and 56.7;
the d(C) of the CꢁC moieties were in agreement with literature data [31]. In the 1H-NMR spectra of 1b,
the presence of an alkyne proton at d(H) 2.74 reveals the formation of a 5-diynyl nucleoside, while for the
dipropynyl ether derivative 1d the O-atom of the side chain significantly shifts the adjacent proton signals
downfield to d(H) 4.22 – 4.37.
The introduction of the alkynyl or diynyl substituents at the 5-position of 2’-
deoxyuridines influences their physical properties, such as the pKa values, the
hydrophobic character, and the polarizability. As this may lead to changes in the
physical and biological properties of oligonucleotides, containing these nucleosides,
these properties were studied. Fig. 1,a, shows that the side chain has a significant effect
on nucleoside mobility in reversed-phase HPLC, compound 1c being the slowest
migrating nucleoside when compared with 1b and 1d. Retention times are correlated to
the hydrophobic character of the nucleosides; the presence of an additional CꢁC bond

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Helvetica Chimica Acta – Vol. 90 (2007)
a
Table 1. 13C-NMR Chemical Shifts of 5-Substituted Pyrimidine 2’-Deoxyribonucleosides at298 K
)
1a
1bb)
1c
1d
6
4
7b)
8
9
10
C(2)
C(4)
C(5)
C(6)
CꢁC
149.4 149.4 (6.7) 149.4 149.4
161.8 161.7 (9.4) 161.7 161.5
99.1 98.9 (< 1.9) 99.1 97.5
142.6 142.6 (184) 142.5 144.2
149.5
161.8
99
142.8
93.1
150.5 149.5 (8.2)
163.8 161.8 (9.7)
109.7 99 (< 1.9)
136.3 142.7 (184)
149.4
161.7
99.3
149.7
162
99.7
149.3
161.5
97.8
143.6
87.9,
79.4
142
142.2
89.1 92.9, 84.2
93.2 87.8,
79.5
–
–
–
–
93
93.0, 84.2 93.7
CꢁC
72
–
72.9,
72.8 79.1,
77.7
31.6, 56.7, 56 28.0, 27.6,
73
73
72.4, 71.2 72.5
78.6,
77.7
71.3 (250)
27.2, 27.1,
18.3, 17.2
–
CH2
27.8, 27.6,
24.5, 18.6
148.6,
27.1, 27.0, 31, 28.3 56.7,
18.3, 17.1
–
28.8
–
24.5, 18.6
147.5,
121.5
56
–
Triazole
–
–
–
121.1 (198)
C(1’)
C(2’)
C(3’)
C(4’)
C(5’)
MeO
C¼O
Me
84.5 84.6 (168.5) 85.4 84.8
84.6
)
70.2
87.5
61
–
83.984.6 (171.1) 84.8
85.1
)
70.7
86.2
63.9
55.3
–
85
)
c
c
c
c
c
c
c
c
c
)
)
)
)
37.1
)
)
70.2 70.2 (149.2) 71
70
59.1 70.1 (148.0) 70.4
84.5 87.5 (148.1) 85.8
70.4
85.8
63.6
55
–
87.5 87.5 (147.6) 88.4 87.6
61
–
–
60.9(141.1) 61.8 60.8
60.8 61 (141.1)
63.6
55
–
–
–
–
–
–
14.7
–
–
–
–
–
–
–
–
195.7 (6.3)
–
12.3 27.2
–
14.2
–
b
a) Measured in (D6)DMSO.
DMSO signal.
)
13C,1H-Coupling constants given in parentheses. c) Superimposed by the
Fig. 1. HPLC Profile a) of the nucleosides 1b – d, b) of the enzymatic-hydrolysis products of the
oligonucleotide 5’-d(TAG G1bC AA1b ACT)-3’, and c) of the oligonucleotide 5’-d(AGT AT1c GAC
CTA)-3’ obtained by digestion with snake-venom phosphodiesterase and alkaline phosphatase in 1m Tris ·
HCl buffer (pH 8.3) at37 8. Gradient for a): within 0 – 30 min 10 – 60% B in A; for b): 25 min 100% A,
within 25 – 60 min 0 – 50% B in A; for c): 25 min 100% A, within 25 – 55 min 0 – 60% B in A; flow rate
0.7 ml/min; A ¼ 0.1m (Et3NH)OAc (pH 7.0)/MeCN 95 :5, B ¼ MeCN.

Helvetica Chimica Acta – Vol. 90 (2007)
539
as in the octa-1,7-diynyl side chain of 1b decreases the lipophilicity when compared to
the oct-1-ynyl-substituted nucleoside 1c. In the case of nucleoside 1d, the O-atom in the
side chain makes the molecule even more hydrophilic.
To evaluate the impact of these side chains, the logP values were calculated for the
bases with the help of the ACD/logP 1.0 program. For the natural bases, the logP values
were negative which reflects the hydrophilicity of these compounds. The highest logP
value was calculated for 5-(oct-1-ynyl)uracil (base of 1c; 3.42 ꢂ 0.75); an additional
CꢁC bond as in the 5-(octa-1,7-diynyl)uracil (base of 1b) decreased the value (2.23 ꢂ
0.75), while the corresponding dipropynyl ether derivative led to the lowest value
(1.45 ꢂ 0.75). Moreover, the presence of a long hydrophobic substituent enhanced the
nucleobaseꢁs polarizability ¼ am/10ꢀ24 cm3; the calculated data (Hyperchem 7.0) were
23.23 for 1b, 24.10 for 1c, and 20.24 for 1d. These values are higher than that of the 5-
propynylated 2’-deoxyuridine 1a (14.92). The pKa-value changes were also noticeable;
they were determined UV-spectrophotometrically [33] (from pH 1.5 to 11.50) at 220 –
350 nm. The 5-alkynyl-2’-deoxyuridines showed the following pKa values: 8.7 for 1b
and 8.8 for 1c. These values are significantly lower than that of thymidine (dT) (9.5) or
of the nonfunctionalized 2’-deoxyuridine (9.34).
2. Functionalization of Nucleoside 1b and of Oligonucleotides Containing 1b by the
Huisgen – Meldal – Sharpless [2 þ 3] Cycloaddition (ꢀClickꢁ Chemistry). Recent studies
of the CuI-catalyzed [2 þ 3] dipolar cycloaddition between an organic azide and an
alkyne has attracted our attention [34][35]. Azides and alkynes are highly energetic
functional groups with a particularly narrow distribution of reactivity. The irreversible
Huisgen – Meldal – Sharpless cycloaddition of azides and alkynes is thermodynamically
favorable by ca. 30 kcal/mol. The reaction is characterized by mild and simple reaction
conditions and O2 and H2O tolerance, even in the presence of a large variety of other
functional groups. It is chemoselective affording only the desired 1,4-disubstituted 1H-
1,2,3-triazole. We report here on the use of nucleosides containing terminal CꢁC bonds
for the functionalization of nucleosides as well as of oligonucleotides employing the
protocol of such a [2 þ 3] cycloaddition. This kind of coupling chemistry was already
used to construct fluorescent oligonucleotides [36] and also to attach oligonucleotides
at a monolayer [37]. The functionalization of oligonucleotides can be employed for
primers and probes used in DNA detection, hybridization, sequencing, and nano-
technology applications.
We functionalized the octadiynylated nucleoside 1b with the antivirally active 3’-
azido-3’-dideoxythymidine (AZT; 4) having the azido group attached to the sugar
moiety and with the aromatic system 5 [38] (Scheme 2). Standard reaction conditions
were employed, and the reaction was performed in THF/H2O 3 : 1 in the presence of
copper(II) sulfate and sodium ascorbate. The ꢀclickꢁ products 6 and 7 were obtained in
excellent yields, i.e., regiospecifically, only the mono-functionalized 1,4-disubstituted
1H-1,2,3-triazole derivatives were formed. Bis-functionalization under participation of
the second CꢁC bond was not found, most likely due to steric reasons. The structures of
6 and 7 formed by the cycloaddition were confirmed by comparison of their NMR
(Table 1) and UV spectra (Fig. 2, Table 2) with those of 1b and 4.
Due to triazole-ring formation, the acetylene proton of 1b (d(H) 2.74) disappeared, and new olefinic
protons arising from the formed triazole moiety were identified at d(H) 8.0 – 9.0. In the 13C-NMR spectra

540
Helvetica Chimica Acta – Vol. 90 (2007)
Scheme 2
of 7, the signals of the terminal CꢁC bond of 1b disappeared, and the signals of the olefinic C-atoms of
the 1,2,3-triazole moiety were identified at d(C) 121.1 and 148.6 (quaternary C-atom) (Table 1). The
unsaturated side chain of compound 1b has a strong impact on the UV spectrum as can be seen from
Fig. 2,a, the UV maximum being bathochromically shifted by 26 nm with respect to that of dT, with the
minimum at ca. 260 nm. The UV data of 1b are similar to those of 2’-deoxy-5-propynyluridine (1a)
(Table 2). The ꢀclickꢁ product 6 shows a rather different UV spectrum as this is formed by the
combination of the UVabsorbances of AZT (4), 1b, and the 1,2,3-triazole moiety (Fig. 2,b). A complete
set of data is shown in Table 2.
The mobility of the ꢀclickꢁ nucleosides 4 and 6 in reversed-phase HPLC was
determined by injecting an artificial mixture of starting materials and products onto the
column; the starting material AZT (4) and ꢀclickꢁ product 6 move faster when
compared to the 5-(octa-1,7-diynyl)-2’-deoxyuridine 1b (Fig. 3,a). The presence of a
triazole ring increases the hydrophilic character of the functionalized nucleoside 6
when compared to 1b.
To study the properties of oligonucleotides containing the modified nucleosides 1b
or the derivatives 1c,d, a series of phosphoramidites were prepared. For this purpose,

Helvetica Chimica Acta – Vol. 90 (2007)
541
Fig. 2. UV/VIS Spectra of a) dT and 1b, and b) 1b, 4, and 6 in MeOH
Table 2. Extinction Coefficients of 5-Substituted Pyrimidine 2’-Deoxyribonucleosidesa)
Wavelength
Extinction coefficient
Wavelength
Extinction coefficient
(lmax) [nm]
(e)
(lmax) [nm]
(e)
1a
1c
228
260
291
22913000
260
13000
4000
13700
1b
6
228
260
292
218
260
274
12800
3800
12400
17000
12000
14000
3800
12700
292
a) Measured in MeOH.
nucleosides 1b – d were converted to the 5’-O-(dimethoxytrityl) derivatives 8 – 10 under
standard reaction conditions. They were phosphitylated to yield the phosphoramidite
building blocks 2b – d (Scheme 3). All compounds were characterized by elemental
analyses or mass spectra, as well as by 1H- and 13C-NMR. The 13C-NMR chemical shifts
are summarized in Table 1, for other data, see Exper. Part.
3. Synthesis and Characterization of Oligonucleotides. The oligonucleotides 11 – 25
(see Schemes 4 and 5 and Table 3 for Formulae) were prepared by solid-phase synthesis
employing the phosphoramidites 2b – d as well as standard building blocks. The
coupling yields were higher than 95%. The oligonucleotides were purified by reversed-
phase HPLC (see Exper. Part). The base composition of the oligonucleotides was
determined by tandem enzymatic hydrolysis with snake-venom phosphodiesterase
followed by alkaline phosphatase in 0.1m Tris · HCl buffer (pH 8.3) at 378. The HPLC
profiles of the reaction products obtained after enzymatic digestion clearly demon-
strate that the newly incorporated nucleosides 1b or 1c migrate much slower than the
canonical DNA constituents (Fig. 1,b, and c). The oligonucleotides were also
characterized by MALDI-TOF mass spectra. The detected masses were identical with
the calculated values (see Exper. Part, Table 4).

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Helvetica Chimica Acta – Vol. 90 (2007)
Fig. 3. HPLC Profile a) of the artificial mixture of nucleosides 1b, 4, and 6 and b) of the enzymatic-
hydrolysis products of the oligonucleotide 18 obtained by digestion with snake-venom phosphodiesterase
and alkaline phosphatase in 1m Tris · HCl buffer (pH 8.3) at37 8. Gradient for a): within 0 – 40 min 0 – 40%
B in A; for b): 20 min 100% A, within 20 – 60 min 0 – 30% B in A; flow rate 0.7 ml/min; A ¼ 0.1m
(Et3NH)OAc (pH 7.0)/MeCN 95 :5, B ¼ MeCN.
Scheme 3
i
i) (MeO)2TrCl, pyridine, r.t., 5 h. ii) iPr2NP(Cl)OCH2CH2CN, Pr2EtN, CH2Cl2, r.t, 45 min.
Next, the Huisgen – Meldal – Sharpless cycloaddition was performed with oligonu-
cleotides containing 1b. For this, the self-complementary 6-mer oligonucleotide 5’-
d(GCA1bGC)-3’ (11) was synthesized in which dT was replaced by 1b. The reaction
was carried out with 86 nmol of the purified oligomer 11 and AZT (4) in tBuOH/H2O
1:9in the presence of a 1:1 complex CuSO 4 · TBTA (tris(benzyltriazoylmethyl)amine)
[39][40] and TCEP (tris(carboxyethyl)phosphine), a H2O-soluble reducing agent, to
give the functionalized oligonucleotide 12 (Scheme 4). The oligonucleotide conjugate
was purified by reversed-phase HPLC (RP-18 column) in the trityl-off modus (see
Exper. Part). The oligonucleotide conjugate was identified by MALDI-TOF-MS (see

Helvetica Chimica Acta – Vol. 90 (2007)
543
Table 3. Tm Values and Thermodynamic Data of Oligonucleotide Duplexes Containing the Nucleosides
1a – d and 6 Located Opposite dAa)
Duplex
Tm [8]
DTmb) [kcal/mol]
DGo310 [kcal/mol]
ꢀ 11.8
5’-d(TAG
3’-d(ATC
5’-d(TAG
3’-d(ATC
GTC
CAG
G1bC
CAG
AAT
TTA
AAT
TTA
ACT)-3’ (15)
TGA)-5’ (16)
ACT)-3’ (17)
TGA)-5’ (16)
50
52
51
þ 2
þ 1
ꢀ 11.9
ꢀ 11.8
5’-d(TAG
3’-d(ATC
G6C
CAG
AAT
TTA
ACT)-3’ (18)
TGA)-5’ (16)
5’-d(TAG
3’-d(ATC
5’-d(TAG
3’-d(A1bC
5’-d(TAG
3’-d(A1bC
5’-d(TAG
3’-d(ATC
GTC
CAG
GTC
CAG
G1bC
CAG
G1bC
CAG
AAT
1b1bA
AAT
TTA
AAT
TTA
ACT)-3’ (15)
TGA)-5’ (19)
ACT)-3’ (15)
1bGA)-5’ (20)
ACT)-3’ (17)
1bGA)-5’ (20)
ACT)-3’ (17)
TGA)-5’ (19)
53
53
53
55
þ 1.5
þ 1.5
þ 1.0
þ 1.6
ꢀ 12.4
ꢀ 12.0
ꢀ 12.0
ꢀ 12.3
AAT
1b1bA
5’-d(TAG
3’-d(ATC
5’-d(TAG
3’-d(A1dC
5’-d(TAG
3’-d(A1dC
G1dC
CAG
GTC
CAG
G1dC
CAG
AAT
TTA
AAT
TTA
AAT
TTA
ACT)-3’ (21)
TGA)-5’ (16)
ACT)-3’ (15)
1dGA)-5’ (22)
ACT)-3’ (21)
1dGA)-5’ (22)
52
53
53
þ 2
ꢀ 11.8
ꢀ 11.5
ꢀ 11.8
þ 1.5
þ 1.0
5’-d(TAG
3’-d(ATC
5’-d(TAG
3’-d(A1cC
5’-d(TAG
3’-d(A1cC
GTC
CAG
GTC
CAG
G1cC
CAG
AAT
1cTA
AAT
TTA
AAT
TTA
ACT)-3’ (15)
TGA)-5’ (23)
ACT)-3’ (15)
1cGA)-5’ (24)
ACT)-3’ (25)
1cGA)-5’ (24)
50.5
50.5
51
þ 0.5
þ 0.25
þ 0.3
ꢀ 11.6
ꢀ 11.7
ꢀ 11.4
5’-d(TAG
3’-d(ATC
5’-d(TAG
3’-d(ATC
5’-d(TAG
3’-d(ATC
G1aC
CAG
GTC
CAG
G1aC
CAG
AAT
TTA
AAT
1a1aA
AAT
1a1aA
ACT)-3’ (26)
TGA)-5’ (16)
ACT)-3’ (15)
TGA)-5’ (27)
ACT)-3’ (26)
TGA)-5’ (27)
52
52
53
þ 2
þ 1
þ 1
ꢀ 11.9
ꢀ 12.2
ꢀ 12.5
a) Measured at 260 nm in 1m NaCl, 100 mm MgCl2, and 60 mm Na-cacodylate (pH 7.0) with 5 mm single-
strand concentration. b) Tm increase per modification.
Exper. Part) and by enzymatic hydrolysis with snake-venom phosphodiesterase and
alkaline phosphatase (see Exper. Part and Fig. 4). Fig. 4,a, shows the HPLC profile of
the starting oligomer 11 after enzymatic hydrolysis, while Fig. 4,b, represents the HPLC
profile after enzymatic digestion of the crude reaction mixture of the ꢀclickꢁ product 12,
which clearly demonstrates the formation of the ꢀclickꢁ nucleoside 6. However, AZT (4)
exhibits the same mobility as the oligonucleotide 12 causing difficulties in the
separation during chromatographic purification.
In the second experiment, the reaction was performed with the CPG-bound
oligonucleotide 13 and AZT (4). For this purpose the CPG-bound 12-mer oligonu-

544
Helvetica Chimica Acta – Vol. 90 (2007)
Scheme 4
Fig. 4. HPLC Profile a) of the enzymatic-hydrolysis products of the oligonucleotide 11 and b) of
oligonucleotide 12 obtained by digestion with snake-venom phosphodiesterase and alkaline phosphatase in
1m Tris · HCl buffer (pH 8.3) at37 8. Gradient for a): 20 min 100% A, within 55 min 0 – 35% B in A; for b):
20 min 100% B, within 50 min 0 – 35% B in A; flow rate 0.7 ml/min; A ¼ 0.1m (Et3NH)OAc (pH 7.0)/
MeCN 95 :5, B ¼ MeCN.
cleotide 5’-d((5’-O-(MeO)2Tr)TAG G1bC AAT ACT)-3’ 13 was synthesized by solid-
phase synthesis by means of the protocol of phosphoramidite chemistry and the regular
phosphoramidites and modified building block 1b (Scheme 5). The (MeO)2Tr

Helvetica Chimica Acta – Vol. 90 (2007)
545
protecting group was preserved on the CPG-bound oligonucleotide, and the ꢀclickꢁ
reaction was performed with AZT (4) in H2O/DMSO/tBuOH 3 :1:1 for 12 h and the
above mentioned reagents (see Exper. Part) to give the crude modified oligomer 14. To
remove excess 4 and other starting materials present in the reaction mixture, the crude
matrix-bound 14 was washed with MeOH/H2O 1:1. Thereafter, the solid support was
removed by using standard deprotection conditions (25% aqueous NH3 solution, 608,
14 h). During this procedure, the protecting groups were also removed (Scheme 5).
Purification of the obtained 5’-O-(dimethoxytrityl)oligonucleotide conjugate was
performed by reversed-phase HPLC (RP-18 column). The (MeO)2Tr protecting group
was then removed and the formed oligonucleotide 18 purified further (see Exper. Part).
The structure of the ligation product 18 was confirmed by MALDI-TOF-MS (Table 4)
and by enzymatic hydrolysis (Fig. 3,b). From the HPLC profile it is apparent that the
compound obtained after the digest was the only modified residue and was identical to
the nucleoside 6 prepared earlier. The complete conversion of oligonucleotide 18 to the
respective triazole product 6 was observed within 12 h.
Scheme 5. Cycloaddition Performed with the Oligonucleotide 13
4. Duplex Stability of Oligonucleotides Containing 5-Substituted 2’-Deoxyuridines
1a – d and the ꢀClickꢁ-Functionalized Nucleoside 6. Earlier work performed with
oligonucleotides containing the propynylnucleoside 1a showed an increase of the
duplex stability resulting in a DTm of 1 – 28 per modification when 1a was incorporated
in the place of dT [24] (Table 3). To evaluate the influence of the base modification on
the duplex stability, hybridization experiments were performed with the oligonucleo-

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tide duplex 5’-d(TAGGTCAATACT)-3’ (15) and 3’-d(ATCCAGTTATGA)-5’ (16);
the dT residues were replaced by the modified nucleosides 1b – d. The oct-5-ynylated
nucleoside 1c shows very little influence on the duplex stability with an increase of the
Tm value of 0.25 – 0.508 per modification. According to the previous reports, the
destabilizing effect dominates when alkynyl groups higher than hexynyl were
introduced at the 5-position of 2’-deoxyuridines [17]. To study the influence of
terminal unsaturated diynes on the DNA duplex stability, the octa-1,7-diynyl residue
was chosen instead of oct-1-ynyl. The 5-(octa-1,7-diynyl)nucleoside 1b enhances the
stability of the dA · dT base pair resulting in a Tm increase of 1 – 28 per modification
depending on the position of the substitution (Table 3). This increase is similar to that
found for the propynylnucleoside 1a. In a similar way, the dipropynyl ether derivative
1d increases the stability like 1b, with a Tm increase of 1 – 28 per modification. This
study was extended to multiple incorporations of compound 1b into a series of
oligonucleotide duplexes at various positions. Replacement of two dT residues by 1b
enhances the Tm value by 38 (15 · 19, 15 · 20, Table 3). With three incorporations of 1b in
duplex 17 · 19, the Tm increases from 53 to 558 when compared with 1a in the
corresponding duplex 26 · 27. The Tm values of a series of oligonucleotides are
summarized in Table 3.
To study the influence of the modified nucleoside 6 containing the 1,2,3-triazole
moiety as well as an AZT residue on the duplex stability, the oligonucleotide 18 was
hybridized with 16. This duplex 16 · 18 (Fig. 5) shows a Tm value (518) similar to the one
of duplex 16 · 17 containing the 5-octadiynylated nucleoside 1b (Tm 528, Table 3). From
the Tm values shown above, it can be concluded that the octadiynyl and dipropynyl
ether moieties at C(5) are better stabilizers than the octynyl residue (Table 3), they
almost behave like propynyl residues, and in a few sequences, they are superior to the
propynyl group. We were surprised to see that such a bulky side chain does not interfere
with base-pair stability implying that the solvation of the side chain is of utmost
importance for the duplex stability.
Conclusions and Outlook. – Nucleosides incorporating the octa-1,7-diynyl or
dipropynyl ether moiety at the 5-position of 2’-deoxyuridine were synthesized by the
Sonogashira cross-coupling reaction resulting in a selective mono-derivatization of the
diyne starting material. The (1,7-diynyl)uracil derivative 1b bearing a long flexible
linker arm was further functionalized by the Huisgen – Meldal – Sharpless [2 þ 3]
cycloaddition (ꢀclickꢁ chemistry) at the stages of the nucleoside and oligonucleotides.
The reactions were performed in solution as well as on solid support. Oligonucleotides
incorporating the octa-1,7-diynyl and dipropynyl ether linkers at the 5-position of 2’-
deoxyuridine (base-pair motifs Ic and Id, Fig. 6) stabilize the DNA duplexes stronger
than 5-alkynyl nucleosides such as 5-propynyl-2’-deoxyuridine or 5-octynyl-2’-deoxy-
uridine (motifs Ia and Ib). Even a long linker arm formed by the cycloaddition of AZT
(4) with the (1,7-diynyl)oligonucleotide 17 (!18) yields an oligonucleotide – nucleo-
side conjugate forming a stable base pair (motif II). Therefore, side chains of this
complex structure are not disturbing the duplex structure if the environment around
the DNA helix allows the solvation by H2O molecules. In the case of motif II, the side
chain fulfills these requirements because of the polar character of the nucleoside
residues as well as of the highly polar 1,2,3-triazole moiety. Thus, for an unperturbated

Helvetica Chimica Acta – Vol. 90 (2007)
547
Fig. 5. a) DNA Duplex 16 · 17 containing 2’-deoxy-5-(octa-1,7-diynyl)uridine (1b). b) DNA Duplex 18 ·
16 containing post-modified nucleoside 6.
DNA structure, it is of utmost importance to select reporter groups – dyes or other
functionalizing residues which do not change the water shells of a DNA molecule.
We thank Dr. H. Rosemeyer and Dr. Y. He for measuring the NMR spectra. We also thank Mr. K. I.
Shaikh for the MALDI-TOF mass spectra, Mrs. E. Michalek for the oligonucleotide syntheses, Mrs. P.
Chittepu for measurement of the pKa values, and Dr. P. Leonard and Mrs. S. Budow for reading the
manuscript. Financial support from ChemBiotech, Münster, is gratefully acknowledged.
Experimental Part
General. All chemicals were purchased from Aldrich, Sigma, or Fluka (Sigma-Aldrich Chemie
GmbH, Deisenhofen, Germany). Solvents were of technical grade and freshly distilled before use. Thin
layer chromatography (TLC): aluminium sheets, silica gel 60 F254, 0.2 mm layer (VWR, Germany).
Column flash chromatography (FC): silica gel 60 (VWR, Germany), at 0.4 bar; sample collection with an

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Helvetica Chimica Acta – Vol. 90 (2007)
Fig. 6. Base-pair motifs formed by 1a – d and 6
UltroRac-II fraction collector (LKB Instruments, Sweden). UV Spectra: U-3200 spectrometer (Hitachi,
Tokyo, Japan); lmax (e) in nm. NMR Spectra: Avance-DPX-250 or AMX-500 spectrometers (Bruker,
1
Karlsruhe, Germany); at 250.13 MHz for H and 13C; d in ppm rel. to Me4Si as internal standard or
external 85% H3PO4 for 31P; J values in Hz. Elemental analyses were performed by the Mikroanaly-
tisches Laboratorium Beller (Gçttingen, Germany). Electron-spray-ionization (ESI) MS for the
nucleosides: Bruker-Daltonics-MicroTOF spectrometer with loop injection (Bremen, Germany).
MALDI-TOF-MS for the oligonucleotides: Biflex-III mass spectrometer (Bruker Saxonia, Leipzig,
Germany), with 3-hydroxypicolinic acid (3-HPA) as a matrix; detected masses were identical with the
calculated values (Table 4).
Synthesis, Purification, and Characterization of the Oligonucleotides 11 – 25. The oligonucleotide
synthesis was performed on a DNA synthesizer, model 392-08 (Applied Biosystems, Weiterstadt,
Table 4. Molecular Masses [M þ H]þ of the Oligonucleotides Measured by MALDI-TOF Mass
Spectrometry
[M þ H]þ (calc.)
[M þ H]þ (found)
5’-d(GCA 1bGC)-3’ (11)
5’-d(GCA 6GC)-3’ (12)
1882.4
2148.1
3645.4
3645.4
3735.5
4002.4
3825.7
3825.7
3721.6
3801.6
3739.6
3831.8
3739.6
1882.9
2147.2
3645.0
3645.1
3735.4
4002.4
3825.4
3826.1
3721.8
3801.6
3739.4
3832.5
3739.0
5’-d(TAG GTC AAT ACT)-3’ (15)
5’-d(AGT ATT GAC CTA)-3’ (16)
5’-d(TAG G1bC AAT ACT)-3’ (17)
5’-d(TAG G6C AAT ACT)-3’ (18)
5’-d(AGT A1b1b GAC CTA)-3’ (19)
5’-d(AG1b ATT GAC C1bA)-3’ (20)
5’-d(TAG G1dC AAT ACT)-3’ (21)
5’-d(AG1d ATT GAC C1dA)-3’ (22)
5’-d(AGT AT1c GAC CTA)-3’ (23)
5’-d(AG1c ATT GAC C1cA)-3’ (24)
5’-d(TAG G1cC AAT ACT)-3’ (25)

Helvetica Chimica Acta – Vol. 90 (2007)
549
Germany) at a 1-mmol scale, with the phosphoramidites 2b – d and following the synthesis protocol for 3’-
(2-cyanoethyl phosphoramidites) (userꢁs manual for the 392 DNA synthesizer, Applied Biosystems,
Weiterstadt, Germany). The coupling efficiency was always higher than 95%. After cleavage from the
solid support with 25% aq. NH3 soln. for 14 – 16 h at 608, the oligonucleotides were deprotected in the
same soln. for 14 – 16 h at 608. The purification of the 5’-O-(dimethoxytrityl)oligomers was carried out on
reversed-phase HPLC (Merck-Hitachi-HPLC; RP-18 column; gradient system (A ¼ 0.1m (Et3NH)OAc
(pH 7.0)/MeCN 95 :5, B ¼ MeCN): 3 min 20% B in A, 12 min 20 – 50% B in A, and 25 min 20% B in A;
flow rate 1.0 ml/min. The purified ꢀtrityl-onꢁ oligonucleotides were treated with 2.5% CHCl2COOH/
CH2Cl2 for 5 min at 08 to remove the 4,4’-dimethoxytrityl residues. The detritylated oligomers were
purified again by reversed-phase HPLC (gradient: 0 – 20 min 0 – 20% B in A; flow rate 1 ml/min). The
oligomers were desalted on a short column (RP-18, silica gel) and lyophilized on a Speed-Vac evaporator
to yield colorless solids which were frozen at ꢀ 248.
Melting curves were measured with a Cary-1/3 UV/VIS spectrophotometer (Varian, Australia)
equipped with a Cary thermoelectrical controller. The temp. was measured continuously in the reference
cell with a Pt-100 resistor, and the thermodynamic data of duplex formation were calculated by the
Meltwin 3.0 program [41].
The enzymatic hydrolysis of the oligonucleotides containing 1b, 1c, and 6 was performed as described
[5] with snake-venom phosphodiesterase (EC 3.1.15.1, Crotallus adamanteus) and alkaline phosphatase
(EC 3.1.3.1, Escherichia coli from Roche Diagnostics GmbH, Germany) in 0.1m Tris · HCl buffer (pH 8.3)
at 378, which was analyzed by reversed-phase HPLC (RP-18, at 260 nm) showing the peaks of the
modified and unmodified nucleosides (Fig. 1,b and c; Fig. 3,b; Fig. 4,b). Quantification of the
constituents were made on the basis of the peak areas, which were divided by the extinction coefficients
e260 of the nucleosides: dA 15400 (H2O), dC 7300 (H2O), dG 11700 (H2O), dT 8800 (H2O), 1b 3800
(MeOH), 1c 3800 (MeOH), and 6 12000 (MeOH).
Huisgen – Meldal – Sharpless [2 þ 3] Cycloaddition of 11 with 4 Performed in Aqueous Solution
Yielding 12. To the single-stranded oligonucleotide 11 (5 A260 units, 87 nmol), CuSO4 · TBTA ligand
complex (20 ml of a 10 mm stock soln. in tBuOH/H2O 1:9), tris(carboxyethyl)phosphine (TCEP; 20 ml of
a 10 mm stock soln. in H2O), AZT (4; 25 ml of a 10 mm stock soln. in dioxane/H2O 1:1) and 35 ml of 10%
tBuOH were added, and the reaction was run at r.t. for 12 h. The product 5’-d(GCA6GC)-3’ (12) was
purified by reversed-phase HPLC (see above). Composition by enzymatic hydrolysis: Fig. 4,b. MALDI-
TOF-MS: Table 4.
Huisgen – Meldal – Sharpless [2 þ 3 ] Cycloaddition of 13 with 4 Performed on Solid SupportYielding
18. To the single-stranded oligonucleotide 13 attached to a solid support (40 mg, 36 mmol/g loading
500 Š) bearing the (MeO)2Tr protected residue as well as protected nucleobases adenine and cytosine
with tBPA (¼ (4-tert-butylphenoxy)acetyl) protecting group and guanine with iB (¼ isobutyryl)
protecting group, an aq. soln. of 70 ml of H2O/DMSO/tBuOH 3 :1:1, CuSO4 · TBTA ligand complex
(40 ml of a 10 mm stock soln. in tBuOH/H2O 1:9), tris(carboxyethyl)phosphine (TCEP; 40 ml of a 10 mm
stock soln. in H2O), AZT (4; 50 ml of a 10 mm stock soln. in dioxane/H2O 1:1) were added, and the
mixture was stirred at r.t. for 12 h and then concentrated. The crude modified CPG-bound 14 was shaken
with 4 ml of H2O/MeOH 1:1. After cleavage from the CPG by 25% aq. NH3 soln. for 14 h at 608 the
ꢀtrityl-onꢁ oligonucleotide was further purified by reversed-phase HPLC (see above). The detritylated
oligonucleotide 18 was also purified by reversed-phase HPLC (see above). MALDI-TOF: Table 4.
Enzymatic hydrolysis: Fig. 3,b.
1-(2-Deoxy-b-d-erythro-pentofuranosyl)-5-(octa-1,7-diynyl)uracil (¼ a-(Hept-6-ynylidyne)thymi-
dine; 1b). To a suspension of 5-iodo-2’-deoxyuridine (3; 1.0 g, 2.82 mmol) and CuI (108 mg, 0.57 mmol)
in anh. DMF (15 ml) was added successively [Pd(PPh3)4] (323 mg, 0.28 mmol), anh. Et3N (570 mg,
5.64 mmol), and octa-1,7-diyne (3.0 g, 28.3 mmol). The mixture was stirred at r.t. under Ar until the
starting material was consumed (TLC monitoring). The mixture was diluted with MeOH/CH2Cl2 1:1
(30 ml), and Dowex 1X8 (100 – 200 mesh; 1.0 g, hydrogen carbonate form) was introduced. After
additional stirring for 1 h, the mixture was filtered and the resin washed with MeOH/CH2Cl2 1:1 (50 ml).
The combined filtrate was concentrated and the residue purified by FC (silica gel, column 15 ꢃ 3 cm,
CH2Cl2/MeOH 95 :5): 1b (0.68 g, 72%). Colorless amorphous solid. TLC (CH2Cl2/MeOH 9:1): Rf 0.42.
UV (MeOH): 228 (12800), 292 (12400). 1H-NMR ((D6)DMSO): 1.55 – 1.57 (m, 2 CH2); 2.11 (m,

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2 HꢀC(2’)); 2.19( m, CH2); 2.38 (m, CH2); 2.74 (s, CꢁCH); 3.55 – 3.60 (m, 2 HꢀC(5’)); 3.77 (m,
HꢀC(4’)); 4.22 (m, HꢀC(3’)); 5.06 (t, J ¼ 4.8, OHꢀC(5’)); 5.21 (d, J ¼ 4.2, OHꢀC(3’)); 6.10 (t, J ¼ 6.6,
HꢀC(1’)); 8.10 (s, HꢀC(6)); 11.53 (s, NH). Anal. calc. for C17H20N2O5 (332.14): C 61.44, H 6.07, N 8.43;
found: C 61.42, H 6.03, N 8.38.
1-(2-Deoxy-b-d-erythro-pentofuranosyl)-5-(oct-1-ynyl)uracil ((¼a-Heptylidynethymidine; 1c) [42].
As described for 1b, with 3 (1.0 g, 2.82 mmol) and oct-1-yne (1.86 g, 16.9mmol). FC (silica gel, column
15 ꢃ 3 cm, CH2Cl2/MeOH 94 :6) gave 1c (0.74 g, 78%). Colorless amorphous solid. TLC (CH2Cl2/MeOH
9 5 :5):Rf 0.23. UV (MeOH): 229(13000), 292 (12700). 1H-NMR ((D6)DMSO): 0.84 (t, Me); 1.34 – 1.48
(m, 4 CH2); 2.10 (m, 2 HꢀC(2’)); 2.35 (t, CH2); 3.57 (m, 2 HꢀC(5’)); 3.78 (m, HꢀC(4’)); 4.22 (m,
HꢀC(3’)); 5.08 (t, OHꢀC(5’)); 5.24 (d, J ¼ 3.7, OHꢀC(3’)); 6.12 (t, J ¼ 6.6, HꢀC(1’)); 8.10 (s, HꢀC(6));
11.60 (s, NH).
1-(2-Deoxy-b-d-erythro-pentofuranosyl)-5-[3-(prop-2-ynyloxy)prop-1-ynyl]uracil ((¼a-[2-(Prop-
2-ynyloxy)ethylidyne]thymidine; 1d). As described for 1b, with 3 (700 mg, 1.98 mmol), CuI (76 mg,
0.40 mmol), anh. DMF (10 ml), [Pd(PPh3)4] (230 mg, 0.20 mmol), anh. Et3N (398 mg, 3.94 mmol), and
diprop-2-ynyl ether (1.5 g, 16.0 mmol). Purification by FC (silica gel, column 15 ꢃ 3 cm, CH2Cl2/MeOH
96 :4) yielded 1d (0.53 g, 84%). Yellowish amorphous solid. TLC (CH2Cl2/MeOH 9: 1): Rf 0.46. UV
1
(MeOH): 230 (12000), 291 (12500). H-NMR ((D6)DMSO): 2.08 – 2.15 (m, 2 HꢀC(2’)); 3.47 (s, C ꢁ
CH); 3.53 – 3.64 (m, 2 HꢀC(5’)); 3.79( m, HꢀC(4’)); 4.22 (m, 3 H, CH2, HꢀC(3’)); 4.37 (m, CH2);
5.10 (t, OHꢀC(5’)); 5.23 (d, J ¼ 3.3, OHꢀC(3’)); 6.10 (t, J ¼ 6.3, HꢀC(1’)); 8.26 (s, HꢀC(6)); 11.63 (s,
NH). Anal. calc. for C15H16N2O6 (320.10): C 56.25, H 5.04, N 8.75; found: C 56.02, H 5.10, N 8.56.
Copper(I)-Catalyzed Cycloaddition: General Procedure. To a mixture of 1b (100 mg, 0.30 mmol)
and 1-(5-azido-3-nitrophenyl)ethan-1-one (5; 86.5 mg, 0.42 mmol) in THF/H2O 3 :1 (8 ml), was added
1m aq. sodium ascorbate (90 ml, 0.09mmol; freshly prepared, followed by 7.5% aq. copper(II) sulfate
pentahydrate soln. (70 ml, 0.021 mmol). The emulsion was stirred for 20 h at r.t., concentrated and
applied to FC (silica gel, column 10 ꢃ 3 cm, CH2Cl2/MeOH 94 :6).
a-{5-[1-(3-Acetyl-5-nitrophenyl)-1H-1,2,3-triazol-4-yl]pentylidyne}thymidine (7). As described in
the General Procedure. From the main zone of the FC 7 (130 mg, 80%) was isolated. Yellowish solid. TLC
1
(CH2Cl2/MeOH 9:1): Rf 0.49. UV (MeOH): 227 (30000), 288 (12000). H-NMR ((D6)DMSO): 1.57 –
1.85 (m, 2 CH2); 2.08 – 2.12 (m, 2 HꢀC(2’)); 2.44 (m, CH2); 2.76 (s, COMe); 2.81 (m, CH2); 3.56 (m,
2 HꢀC(5’)); 3.77 (m, HꢀC(4’)); 4.23 (m, HꢀC(3’)); 5.09( t, J ¼ 4.8, OHꢀC(5’)); 5.25 (d, J ¼ 4.2,
OHꢀC(3’)); 6.10 (t, J ¼ 6.6, HꢀC(1’)); 8.12 (s, HꢀC(6)); 8.65 – 8.91 (m, arom. H); 8.99 (s, C¼CH); 11.54
(s, NH). ESI-HR-MS: 561.17 ([M þ Na])þ, C25H26N6O8þ ; calc. 538.18).
a-{5-{1-[(2S,3S,5R)-Tetrahydro-2-(hydroxymethyl)-5-(1,2,3,4-tetrahydro-5-methyl-2,4-dioxopyrimi-
din-1-yl)furan-3-yl]-1H-1,2,3-triazol-4-yl}pentylidyne}thymidine (6). As described in the General Proce-
dure, with 1b (50 mg, 0.15 mmol), AZT (4; 40 mg, 0.15 mmol), 1m of sodium ascrobate (120 ml,
0.12 mmol), and copper(II) sulfate pentahydrate soln. (100 ml, 0.03 mmol of a 7.5% stock soln. in H2O)
dissolved in THF/H2O 3 :1 (4 ml) for 30 h. FC (silica gel, column 10 ꢃ 3 cm, CH2Cl2/MeOH 9:1) gave 6
(62 mg, 69%) from the main zone. Colorless foam. TLC (CH2Cl2/MeOH 9:1): Rf 0.24. UV (MeOH): 218
(17000), 274 (14000). 1H-NMR ((D6)DMSO; nucleoside atom numbering for the AZT-derived moiety,
i.e., doubly (’’) and triply (’’’) primed locants): 1.54 – 1.72 (m, 2 CH2); 1.80 (s, MeꢀC(5’’)); 2.08 – 2.68 (m,
8 H, HꢀC(2’), HꢀC(2’’’), CH2); 3.55 – 3.62 (m, 4 H, HꢀC(5’), HꢀC(5’’’)); 3.77 (m, 2 H, HꢀC(4’),
HꢀC(4’’’)); 4.17 – 4.22 (m, 2 H, HꢀC(3’), OHꢀC(5’’’)); 5.14 (t, J ¼ 4.9, OHꢀC(5’)); 5.25 (d, J ¼ 4.17,
OHꢀC(3’)); 5.30 (m, HꢀC(3’’’)); 6.10 (t, J ¼ 6.55, HꢀC(1’)); 6.40 (t, J ¼ 6.55, HꢀC(1’’’)); 7.81 (s,
HꢀC(6’’)); 8.06 (s, C¼CH); 8.13 (s, HꢀC(6)); 11.36 (s, HN(3’’)); 11.56 (s, HN(3)). ESI-HR-MS: 622.22
([M þ Na]þ, C27H33N7Oþ9 ; calc. 599.23).
1-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-5-(octa-1,7-diynyl)uracil (¼ 5’-
O-[Bis(4-methoxyphenyl)phenylmethyl]-a-(hept-6-ynylidyne)thymidine; 8). Compound 1b (0.5 g,
1.5 mmol) was dried by repeated co-evaporation with anh. pyridine (2 ꢃ 5 ml) before dissolving in
anh. pyridine (10 ml). Then 4,4’-dimethoxytrityl chloride (0.61 g, 1.80 mmol) was added in three portions
to the remaining soln. at r.t. under stirring for 5 h. Thereupon, MeOH (2 ml) was added, and the mixture
was stirred for another 30 min. The mixture was dissolved in CH2Cl2 (2 ꢃ 50 ml) and extracted with 5%
aq. NaHCO3 soln. (100 ml) followed by H2O (80 ml), dried (Na2SO4), and then concentrated.
Purification by FC (silica gel, column 15 ꢃ 3 cm, CH2Cl2/acetone 80 :20) gave 8 (0.83 g, 87%). Colorless

Helvetica Chimica Acta – Vol. 90 (2007)
551
foam. TLC (CH2Cl2/MeOH 95 :5): Rf 0.31. UV (MeOH): 233 (28000), 284 (10300). 1H-NMR
((D6)DMSO)): 1.39( m, 2 CH2); 2.06 – 2.26 (m, 6 H, HꢀC(2’), CH2); 2.72 (s, CꢁCH); 3.10 – 3.23 (m,
2 HꢀC(5’)); 3.73 (s, 2 MeO); 3.91 (m, HꢀC(4’)); 4.28 (m, HꢀC(3’)); 5.33 (d, J ¼ 3.63, OHꢀC(3’)); 6.12
(t, J ¼ 6.6, HꢀC(1’)); 6.86 – 7.42 (m, arom. H); 7.87 (s, HꢀC(6)); 11.60 (s, NH). Anal. calc. for C38H38N2O7
(634.27): C 71.91, H 6.03, N 4.41; found: C 71.75, H 6.05, N 4.37.
1-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-5-(octa-1-ynyl)uracil (¼ 5’-O-
[Bis(4-methoxyphenyl)phenylmethyl]-a-heptylidynethymidine; 9). As described for 8, with 1c (0.4 g,
1.19mmol) and 4,4 ’-dimethoxytrityl chloride (0.48 g, 1.42 mmol). Purification by FC (silica gel, column
15 ꢃ 3 cm, CH2Cl2/MeOH 95 :5) furnished 9 (0.64 g, 84%). Colorless foam. TLC (CH2Cl2/MeOH 95 :5):
Rf 0.31. UV (MeOH): 233 (26000), 284 (9600). 1H-NMR ((D6)DMSO): 0.83 (t, Me); 1.18 – 1.23 (m,
4 CH2); 2.11 (m, 2 HꢀC(2’)); 2.20 (t, CH2); 3.10 – 3.20 (m, 2 HꢀC(5’)); 3.73 (s, 2 MeO); 3.91 (m,
HꢀC(4’)); 4.28 (m, HꢀC(3’)); 5.33 (d, J ¼ 3.63, OHꢀC(3’)); 6.12 (t, J ¼ 6.62, HꢀC(1’)); 6.86 – 7.42 (m,
arom. H); 7.87 (s, HꢀC(6)); 11.60 (s, NH). Anal. calc. for C38H42N2O7 (638.30): C 71.45, H 6.63, N 4.39;
found: C 71.29, H 6.70, N 4.40.
1-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-5-[3-(prop-2-ynyloxy)prop-1-
ynyl]uracil (¼ 5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-a-[2-(prop-2-ynyloxy)ethylidyne]thymidine;
10). As described for 8, with 1d (0.35 g, 1.09mmol) anh. pyridine (8 ml), and 4,4 ’-dimethoxytrityl
chloride (0.44 g, 1.3 mmol). Purification by FC (silica gel, column 15 ꢃ 3 cm, CH2Cl2/acetone 80 :20) gave
10 (0.58 g, 85%). Colorless foam. TLC (CH2Cl2/MeOH 95 :5): Rf 0.34. UV (MeOH): 232 (29000), 283
(11000). 1H-NMR ((D6)DMSO): 2.08 – 2.30 (m, 2 HꢀC(2’)); 3.10 – 3.28 (m, 2 HꢀC(5’)); 3.47 (s,
CꢁCH); 3.73 (s, 2 MeO); 3.92 (m, HꢀC(4’)); 4.07 (m, CH2); 4.17 (m, CH2); 4.28 (m, HꢀC(3’)); 5.33 (d,
J ¼ 4.4, OHꢀC(3’)); 6.12 (t, J ¼ 6.52, HꢀC(1’)); 6.86 – 7.42 (m, arom. H); 7.97 (s, HꢀC(6)); 11.69( s, NH).
Anal. calc. for C36H34N2O8 (622.23): C 69.44, H 5.50, N 4.50; found: C 69.34, H 5.70, N 4.66.
1-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-5-(octa-1,7-diynyl)uracil 3’-(2-
Cyanoethyl Diisopropylphosphoramidite) (¼ 5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-a-(hept-6-yny-
lidyne)thymidine 3’-(2-Cyanoethyl Diisopropylphosphoramidite); 2b). A stirred soln. of 8 (110 mg,
0.17 mmol) in anh. CH2Cl2 (5 ml) was pre-flushed with Ar and treated with iPr2EtN (55 ml, 0.32 mmol) at
r.t. followed by 2-cyanoethyl diisopropylphosphoramidochloridite (59 ml, 0.27 mmol). After stirring for
45 min at r.t., the soln. was diluted with CH2Cl2 (30 ml) and extracted with 5% aq. NaHCO3 soln. (20 ml).
The org. layer was dried (Na2SO4) and concentrated. FC (silica gel, column 10 ꢃ 2 cm, CH2Cl2/acetone
95 :5) gave 2b (110 mg, 76%). Colorless foam. TLC (CH2Cl2/acetone 95 :5): Rf 0.70. 31P-NMR (CDCl3):
150.25; 149.76.
1-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-5-(oct-1-ynyl)uracil 3’-(2-Cya-
noethyl Diisopropylphosphoramidite) (¼ 5’-O-[Bis(4-methoxyphenyl)phenylmethyl]-a-heptylidynethy-
midine 3’-(2-Cynoethyl Diisopropylphosphoramidite); 2c). As described for 2b, with 9 (110 mg,
i
0.17 mmol), Pr2EtN (55 ml, 0.32 mmol), and 2-cyanoethyl diisopropylphosphoramidochloridite (59 ml,
0.27 mmol). FC (silica gel, column 10 ꢃ 2 cm, CH2Cl2/acetone 95 :5) gave 2c (113 mg, 78%). Colorless
foam. TLC (CH2Cl2/acetone 95 :5): Rf 0.66. 31P-NMR (CDCl3): 150.21; 149.74.
1-[2-Deoxy-5-O-(4,4’-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-5-[3-(prop-2-ynyloxy)prop-1-
ynyl)uracil 3’-(2-Cyanoethyl Diisopropylphosphoramidite) (¼ 5’-O-[Bis(4-methoxyphenyl)phenylmeth-
yl]-a-[2-(prop-2-ynyloxy)ethylidyne]thymidine 3’-(2-Cyanoethyl Diisopropylphosphoramidite); 2d). As
described for 2b, with 10 (250 mg, 0.40 mmol), iPr2EtN (130 ml, 0.75 mmol) and 2-cyanoethyl
diisopropylphosphoramidochloridite (122 ml, 0.56 mmol). FC (silica gel, column 10 ꢃ 2 cm, CH2Cl2/
acetone 95 :5) gave 2d (248 mg, 75%). Colorless foam. TLC (CH2Cl2/acetone 95 :5): Rf 0.71. 31P-NMR
(CDCl3): 150.19; 149.65.
REFERENCES
[1] E. Uhlmann, A. Peyman, Chem. Rev. 1990, 90, 543.
´ `
[2] D. Praseuth, A. L. Guieysse, C. Helene, Biochim. Biophys. Acta 1999, 1489, 181.
[3] S. L. Beaucage, R. P. Iyer, Tetrahedron 1993, 49, 6123.
[4] J. Goodchild, Bioconjugate Chem. 1990, 1, 165.

552
Helvetica Chimica Acta – Vol. 90 (2007)
[5] F. Seela, G. Becher, Nucleic Acids Res. 2001, 29, 2069.
[6] F. Seela, G. Becher, Synthesis 1998, 207.
[7] F. Seela, M. Zulauf, J. Chem. Soc., Perkin Trans. 1 1998, 3233.
[8] F. Seela, H. Steker, Helv. Chim. Acta 1985, 68, 563.
[9] J. He, I. Mikhailopulo, F. Seela, J. Org. Chem. 2003, 68, 5519.
[10] G. Haaima, H. F. Hansen, L. Christensen, O. Dahl, P. E. Nielsen, Nucleic Acids Res. 1997, 25, 4639.
[11] P. Vincent, J.-P. Beaucourt, L. Pichat, J. Balzarini, E. De Clercq, Nucleosides Nucleotides 1985, 5, 429.
[12] E. De Clercq, J. Descamps, J. Balzarini, J. Giziewicz, P. J. Barr, M. J. Robins, J. Med. Chem. 1983, 26,
661.
[13] R. A. Sharma, I. Kavai, R. G. Hughes Jr., M. Bobek, J. Med. Chem. 1984, 27, 410.
[14] F. Seela, N. Ramzaeva, P. Leonard, Y. Chen, H. Debelak, E. Feiling, R. Krçschel, M. Zulauf, T.
Wenzel, T. Frçhlich, M. Kostrzewa, Nucleosides Nucleotides Nucleic Acids 2001, 20, 1421.
[15] B. C. Froehler, S. Wadwani, T. J. Terhorst, S. R. Gerrard, Tetrahedron Lett. 1992, 33, 5307.
[16] L. E. Xodo, G. Manzini, F. Quadrifoglio, G. van der Marel, J. van Boom, Nucleic Acids Res. 1991, 19,
1505.
´
´
´
´
[17] J. Sagi, A. Szemzç, K. Ebinger, A. Szabolcs, G. Sagi, E. Ruff, L. Ötvçs, Tetrahedron Lett. 1993, 34,
2191; T. Kottysch, C. Ahlborn, F. Brotzel, C. Richert, Chem.-Eur. J. 2004, 10, 4017.
[18] G. Becher, J. He, F. Seela, Helv. Chim. Acta 2001, 84, 1048.
[19] T. W. Barnes III, D. H. Turner, J. Am. Chem. Soc. 2001, 123, 4107.
[20] T. W. Barnes III, D. H. Turner, Biochemistry 2001, 40, 12738.
[21] R. W. Wagner, M. D. Matteucci, J. G. Lewis, A. J. Gutierrez, C. Moulds, B. C. Froehler, Science
(Washington, D.C.) 1993, 260, 1510.
[22] A. J. Gutierrez, M. D. Matteucci, D. Grant, S. Matsumura, R. W. Wagner, B. C. Froehler,
Biochemistry 1997, 36, 743.
[23] M. Ahmadian, P. Zhang, D. E. Bergstrom, Nucleic Acids Res. 1998, 26, 3127.
[24] J. He, F. Seela, Nucleic Acids Res. 2002, 30, 5485.
[25] F. Seela, A. M. Jawalekar, V. R. Sirivolu, H. Rosemeyer, Y. He, P. Leonard, Nucleosides Nucleotides
Nucleic Acids 2005, 24, 855.
[26] C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057.
[27] V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem., Int. Ed. 2002, 41, 2596.
[28] F. Seela, V. R. Sirivolu, Chem. Biodiv. 2006, 3, 509.
[29] J. Gierlich, G. A. Burley, P. M. E. Gramlich, D. M. Hammond, T. Carell, Org. Lett. 2006, 8, 3639.
[30] J. Asakura, M. J. Robins, J. Org. Chem. 1990, 55, 4928.
[31] F. Seela, M. Zulauf, Synthesis 1996, 726.
[32] M. J. Robins, P. J. Barr, J. Org. Chem. 1983, 48, 1854.
[33] A. Albert, E. P. Serjeant, in ꢀThe Determination of Ionization Constantsꢁ, Chapman and Hall,
London, 1971, p. 44 – 64.
[34] V. D. Bock, H. Hiemstra, J. H. van Maarseveen, Eur. J. Org. Chem. 2006, 51.
[35] W. H. Binder, C. Kluger, Curr. Org. Chem. 2006, 10, 1791.
[36] T. S. Seo, Z. Li, H. Ruparel, J. Ju, J. Org. Chem. 2003, 68, 609.
[37] N. K. Devaraj, G. P. Miller, W. Ebina, B. Kakaradov, J. P. Collman, E. T. Kool, C. E. D. Chidsey, J.
Am. Chem. Soc. 2005, 127, 8600.
[38] F. Seela, Z. Naturforsch., C: Biosci. 1976, 31, 389.
[39] R. L. Weller, S. R. Rajski, Org. Lett. 2005, 7, 2141.
[40] T. R. Chan, R. Hilgraf, K. B. Sharpless, V. V. Fokin, Org. Lett. 2004, 6, 2853.
[41] M. Petersheim, D. H. Turner, Biochemistry 1983, 22, 256.
[42] A. Carangio, C. McGuigan, G. Andrei, R. Snoeck, E. De Clercq, J. Balzarini, Antivir. Chem.
Chemother. 2001, 12, 187.
Received November 23, 2006