Nucleoside dimers analogues with a 1,2,3-triazole linkage: conjugation of floxuridine and thymidine provides novel tools for cancer treatment. Part II

Article abstract of DOI:10.1080/15257770.2019.1610891

The fluorinated nucleoside dimers with a 1,2,3-triazole linkage are novel compounds within the field of bioorganic chemistry. We report on the synthesis and properties of two groups of nucleoside dimers analogs possessing a different arrangement of the 1,

Full text of DOI:10.1080/15257770.2019.1610891

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
Nucleoside dimers analogues with a 1,2,3-triazole
linkage: conjugation of floxuridine and thymidine
provides novel tools for cancer treatment. Part II
Dagmara Baraniak, Daniel Baranowski, Piotr Ruszkowski & Jerzy Boryski
To cite this article: Dagmara Baraniak, Daniel Baranowski, Piotr Ruszkowski & Jerzy Boryski
(2019): Nucleoside dimers analogues with a 1,2,3-triazole linkage: conjugation of floxuridine and
thymidine provides novel tools for cancer treatment. Part II, Nucleosides, Nucleotides and Nucleic
Acids, DOI: 10.1080/15257770.2019.1610891
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2019.1610891
Nucleoside dimers analogues with a 1,2,3-triazole
linkage: conjugation of floxuridine and thymidine
provides novel tools for cancer treatment. Part II
Dagmara Baraniaka , Daniel Baranowskia, Piotr Ruszkowskib, and
Jerzy Boryskia
a
b
ꢀ
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; Department of
ꢀ
ꢀ
Pharmacology, Faculty of Pharmacy, Poznan University of Medical Sciences, Poznan, Poland
ABSTRACT
ARTICLE HISTORY
Received 25 February 2019
Accepted 20 April 2019
The fluorinated nucleoside dimers with a 1,2,3-triazole linkage
are novel compounds within the field of bioorganic chemistry.
We report on the synthesis and properties of two groups of
nucleoside dimers analogs possessing a different arrangement
of the 1,4-disubstituted 1,2,3-triazole linkage. Based on ana-
KEYWORDS
Floxuridine; nucleoside
dimers derivatives;
1,3-dipolar cycloaddition;
cancer therapy
3
3
3
0
0
lysis of the JHH, JH1 C2, and JH1 C6 we estimated conform-
ational preferences of sugar part and orientation around
glycosidic bond. These compounds show moderate anticancer
activity, with cytostatic studies in three different cancer
cell lines.
1. Introduction
The synthesis of biologically active substances, potential medicines, is an
important research area in the medicinal chemistry.[1–3] One of the current
concept in drug design and their development is conjugation of compounds
with a documented clinical action.[4,5] The new hybrid compounds open
the door for a modern concept—synthesis of drugs with highly active anti-
metabolic character for cancer therapy.[6–8]
Natural nucleosides, in the form of free molecules, generally do not
exhibit biological activity and are rapidly metabolized in the cytoplasm. In
contrast, modified nucleosides often present biological activity and have
found a broad use as inhibitors of tumors, viruses, or bacterial cell growth
in medicine.
Among them fluorinated nucleoside analogs showed interesting bio-
logical properties, e.g. 5-fluorouracil (FUra, 5-FU), 5-fluoro-20-deoxyuridine
(floxuridine, 5-FdU) and 50-deoxy-5-fluoro-N4-pentyloxycarbonyl-cytidine
CONTACT Dagmara Baraniak
of Sciences, Noskowskiego St 12/14, Poznan 61-704, Poland.
baraniak@ibch.poznan.pl
Institute of Bioorganic Chemistry, Polish Academy
ꢀ
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2019.1610891.
ß 2019 Taylor & Francis Group, LLC

2
D. BARANIAK ET AL.
Figure 1. 5-Fluoropyrimidine-based drugs.
(capecitabine, CAP) are anticancer drugs with broad spectrum of activity
(Figure 1).[9,10]
The best known compounds, 5-fluorouracil and 5-fluoro-20-deoxyuridine,
are effective chemotherapeutics for colorectal cancer, but they are also used
in treatment of breast, head and neck, esophageal, gastric, and several other
cancers.[11,12] In turn, capecitabine is a drug of new generation, which is
mainly used in treatment of gastrointestinal and breast cancer.[13]
Capecitabine is a specially designed prodrug of 5-FU which, contrary to the
registered drugs, can easily penetrate the cell membrane. Inside the cell, it
undergoes three enzymatic transformations (metabolized by carboxylester-
ase, cytidine deaminase, and thymidine phosphorylase, respectively), which
lead to 5-fluorouracil. Subsequently, all described drugs are converted to 5-
fluoro-20-deoxyuridine monophosphate (FdUMP)—an irreversible inhibitor
of thymidylate synthase (TS) in the cell.[13] Blocking of the TS enzyme
leads to ending of the thymidine synthesis, and consequently DNA.
FdUMP can also interfere with DNA biosynthesis and its repair. The
second anticancer mechanism of fluoropyrimidine drugs consists of their
transformation to 5-fluoro-20-deoxyuridine triphosphate (FdUTP), which
can be incorporated into cellular DNA and then inhibits the synthesis and
repair of DNA.[12,14]
However, it is never possible to avoid side effects of chemotherapeutics,
hence a natural urge to search for new nucleoside drugs with possibly large
difference between the scope of therapeutic effect and toxicity.
An interesting solution is the so-called “click” chemistry, defined in 2001
by K.B. Sharpless, which allows to couple biologically interesting com-
pounds forming covalent bonds between them.[15,16] In the concept of
“click” chemistry, the most important role plays the Huisgen 1,3-dipolar
cycloaddition reaction between organic azides and terminal alkynes leading
to 1,2,3-triazoles.[17] In 2002, Meldal[18] and Sharpless[19] have independ-
ently established that the Huisgen reaction is catalyzed by copper(I) ions
and can be performed at room temperature, leading exclusively to 1,4-
disubstituted regioisomers of 1,2,3-triazoles. This method allows to synthe-
size new hybrid drugs combining different antimetabolites in one molecule.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Scheme 1. Coupling of 30-azido-nucleosides and 50-O-propargyl-nucleosides via click chemistry.
Reagents and conditions: (a) 1 M CuSO4 (0.4 eq), NaAsc (0.8 eq), THF–H2O (3:1, v/v), rt, 24 h,
yield: 70–90%; (b) NH4F (10 eqs), abs. MeOH, reflux, 4 h, yield: 80–95%.
Among commonly applied linkers, 1,2,3-triazole ring is of special interest
since it is extremely easily to prepare by a 1,3-dipolar cycloaddition,
and biologically activity of many 1,2,3-triazole derivatives has been well
documented.[20,21]
As part of our program to develop novel nucleoside analogs, we present
a new class of synthetic fluorinated nucleoside dimers with 1,2,3-triazole as
a linkage. We use commonly known drug—5-FdU as a component to build
their architecture. The present paper describes efficient method for the syn-
thesis of two novel groups of nucleoside dimers analogs, but also presents
their biological and chemical properties in order to find the chemical fea-
ture responsible for enhancing the substance delivery system and improving
therapeutic indices.
2. Results and discussion
2.1. Chemistry
The synthesis of two series of nucleosides dimers analogs 3a–d and 4a–d
summarized in Scheme 1 was performed according to the Cu(I)-catalyzed
Huisgen reaction under classic Sharpless conditions for “click chem-
istry”.[15,19] The organic azides were represented by 30-azido-30-deoxythymi-
dine (1a, AZT) and 30-azido-20,30-dideoxy-5-fluorouridine (1b, AddFU).
AZT was commercially available, but AddFU was synthesized using a
modified method of Celewicz, as shown in Scheme 2.[14] The ethynyl deriv-
atives were used as partially protected 50-O-propargyl compounds (2a–b)
and were obtained according to the recently published procedure.[22] The

4
D. BARANIAK ET AL.
Scheme 2. Synthesis of 30-azido-20,30-dideoxy-5-fluorouridine (AddFU). Reagents and conditions:
(a) TrCl (1.2 eq), Py, 100oC, 30 min., yield: 94%; (b) MsCl (1.5 eq), Py, rt, 2 h, yield: 95%; (c) 1 M
NaOH (1 eq), 96% EtOH, reflux, 6–10 min. (product not isolated); (d) 1 M NaOH (2 eqs), reflux,
1 h, pH ¼ 6, yield: 85–95%; (e) MsCl (3 eqs), Py, rt, 2 h, yield: 86–100%; (f) LiN3 (3 eqs), DMF,
90oC, 3 h, yield: 87–97%; (g) 0.5 M HCl/MeOH, rt, 3 h, yield: 68%.
1,3-dipolar cycloaddition of 30-azido-nucleosides and 50-O-propargylated
nucleosides was used to give the dimers 3a–b, which subsequently could be
deprotected with ammonium fluoride in absolute methanol leading to the
second group of dimers 4a–d.
30-Azido-20,30-dideoxy-5-fluorouridine (1b, AddFU) was synthesized
using a method analogous to that of Horwitz[23] for preparation AZT
(Scheme 2). This nucleoside azide could be also obtained by Czernecki
method,[24] which involved two successive Mitsunobu reactions. However,
the experience showed, that the 2,30-anhydro-50-O-bezoyl-5-fluoro-20-deox-
yuridine structure was definitely more stable and less prone to nucleophilic
substitution reaction than its thymidine analog—2,30-anhydro-50-O-bezoyl-
thymidine, which was used in known synthesis of AZT.[14]
An interesting concept are dimers 13a–d and 14a–d, in which previously
discussed carbon bridges (–CH2– and –OCH2–) are constituting a linker
structure, but they are placed in a different arrangement. The other
arrangement of the linker structure could affect the biological properties of
these compounds, what would be an interesting aim of this experiment.
To synthesize the desired compounds, we took advantage of Huisgen cyclo-
addition by coupling 50-azidonucleosides: 50-azido-50-deoxythymidine (11a,
50-AZT) or 50-azido-20,50-dideoxy-5-fluorouridine (11b) with the corre-
sponding 30-O-propargyl derivatives 12a–b using again the procedure

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Scheme 3. Coupling of 50-azido-nucleosides and 30-O-propargyl-nucleosides via click chemistry.
Reagents and conditions: 1 M CuSO4 (0.4 eq), NaAsc (0.8 eq), THF–H2O (3:1,v/v), rt, 24 h, yield:
70–90%; (b) NH4F (10 eqs), abs. MeOH, reflux, 4 h, yield: 80–95%.
involving copper(I) cations generated in situ from copper(II) sulfate and
sodium ascorbate (NaAsc) in THF–water medium. Next, the silylated con-
jugates 13a–d were deprotected, which led to fourth series of nucleosides
dimers analogs—1,4-disubstituted 1,2,3-triazoles (14a–d) (Scheme 3). 50-
Azido-20,50-dideoxy-5-fluorouridine (11b, 50-AddFU) was synthesized using
a method analogous to that of Chattopadhyaya[25] for preparation 50-AZT.
30-O-Propargyl nucleosides were synthesized according to the method
described in the literature.[22]
2.2. Biological activity
The synthesized nucleoside dimers were evaluated for cytotoxic activity
against three human cancer cell lines: breast (MCF-7), nasopharyngeal
(KB), and cervical (HeLa), and one non-proliferative fibroblasts cell line—
(HDF). Biological experiments were performed for 24 compounds and
compared with a reference agent, cytarabine. The resulting cytostatic activ-
ity data of the newly synthesized nucleoside dimer analogs are presented in
Table 1. Among 50-O-propargyl derivatives (2a–b), compound 2b displayed
cytostatic activity against all three cell lines with IC50 values ranging from
7.75 lM to 9.31 lM. The both 50-azidonucleosides: 11a and 11b displayed
activity in the area ca. 10 lM what means they have cytostatic action in
vitro against all human cancer cell lines. In the series of nucleoside dimers
analogs the best parameters were shown by compound 12b, with values
similar to AZT, but slightly higher all SI. The dimer 4b exhibited also rela-
tively high activity especially against HeLa cells (IC50¼7.29 lM).
Compound 11a showed the activity against HeLa cells with IC50¼10.78 lM,
against KB cells IC50 was 9.77 lM and in MCF-7 cell lines IC50 was

6
D. BARANIAK ET AL.
Table 1. In vitro cytotoxic activity of the synthesized compounds 1–4 and 11–14 in three
human cancer cell lines: cervical (HeLa), oral (KB) and breast (MCF-7) and the reference—
healthy cells: primary skin fibroblasts (HDF).
Cytotoxicity (IC50, lM)a
Compd
HeLa
SIb
KB
SI
MCF-7
SI
HDF
log Pc
1a
1b
2a
2b
3a
3b
3c
3d
4a
4b
4c
4d
11a
11b
12a
12b
13a
13b
13c
13d
14a
14b
14c
14d
7.82
7.12
20.07
7.75
1.4
1.2
1.5
2.3
6.2
1.7
2.0
1.8
1.0
2.1
1.6
1.1
1.4
1.2
1.4
2.6
1.8
1.2
3.6
1.3
6.5
1.2
2.5
1.1
1.3
6.02
5.68
19.16
8.21
18.13
40.87
39.68
34.43
9.37
1.9
1.6
1.6
2.2
6.4
1.7
1.6
2.2
1.2
2.0
1.2
1.1
1.5
1.7
1.3
2.3
1.5
1.3
3.8
1.2
6.9
1.2
2.8
1.2
1.1
7.30
6.38
19.09
9.31
18.74
34.46
33.14
42.39
10.21
8.96
49.98
21.03
7.90
9.40
20.35
7.75
10.58
8.58
24.03
8.45
1.5
1.4
1.6
1.9
6.2
2.0
1.9
1.8
1.1
1.7
1.8
1.2
1.9
1.9
1.3
2.2
1.8
1.5
3.1
1.2
5.6
1.2
3.0
1.3
1.1
11.19
8.78
–0.10
–0.38
2.67
2.38
1.15
0.86
0.86
0.58
–2.18
–2.47
–2.47
–2.75
–0.10
–0.38
2.67
2.38
1.15
0.86
0.86
30.54
17.67
116.81
67.64
62.44
74.18
10.98
15.20
88.94
24.29
14.82
17.77
26.74
16.99
18.89
12.60
73.90
10.36
493.86
17.75
204.72
16.22
4.24
18.74
40.66
30.66
42.30
10.81
7.29
55.22
22.27
10.78
14.42
19.85
6.58
10.58
10.23
20.43
7.93
75.61
15.10
81.69
14.38
3.37
7.45
70.83
22.70
9.77
10.44
20.61
7.30
12.39
9.76
19.53
8.82
71.23
14.34
73.24
14.08
3.82
0.58
87.67
15.38
67.27
12.89
3.74
–2.18
–2.47
–2.47
–2.75
–2.32[26]
Cytarabine (standard)
aIC50 is the compound concentration required to inhibit cell growth by 50%.
bSI (Selectivity Index) was calculated for each compound using formula: SI ¼ IC50 for normal cell line (HDF)/IC50
for respective cancerous cell line. A beneficial SI > 1.0 indicates a drug with efficacy against tumor cells
greater than toxicity against normal cells.
clog P (logarithm of partition coefficient) was calculated using the “miLogP” method.[27]
7.90 lM. The second derivative, 50-azido-20,50-dideoxy-5-fluorouridine
(11b), exhibited IC50 values ranging from 9.40 lM (MCF-7) to 14.42
(HeLa). The activity of compound 11b against KB cell line was 10.44 lM.
All other nucleoside derivatives displayed moderate or poor activity when
tested against three cancer cell lines in vitro. The highest calculated parti-
tion coefficient (logP) values were noted for both 50-O-propargyl derivatives
(2a–b). Hence, we predict good cell membranes permeability and positive
distribution rates only for these two latter compounds.
2.3. NMR spectroscopy and conformational analysis of 30-50 and 50-30
nucleoside dimers
Structure verification of 30–50 (3a–d, 4a–d) and 50–30 (13a–d, 14a–d)
1
nucleoside dimers were obtained by series of H, 13C, 15N, 19F and 29Si
1
NMR measurements. Unambiguous assignment of H, 13C, 15N resonances
was accomplished by careful and comprehensive analysis of 2D spectra
1
13
15
(1Hꢀ H COSY, 1Hꢀ C gHSQC and gHMBC, 1Hꢀ N gHSQC and

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
gHMBC). NMR data and spectra were provided in tables and Supporting
Information, respectively. Conformational analysis were performed based
1
on vicinal H–1H and heteronuclear 13C–1H coupling constants as well as
NOE measurements.
1
13
Thorough analysis of H and C chemical shifts of 30–50 and 50–30
nucleoside dimers revealed significant differences within sugar moieties
1
depending on position of triazole linkage compared with H and 13C NMR
data for thymidine (Tables 2 and 3).[28] Substitution of hydroxyl group in
C30 or C50 position with aromatic nitrogen of triazole caused apparent
downfield shift of resonances for H30A or H50/H500B respectively by ca.
1.1 ppm. This deshielding changes are due to magnetic anisotropy effect of
triazole current ring. In case of 30–50 nucleoside dimers less pronounced
but still noticeable deshielding effect was also observed for contiguous pro-
tons H20/H200A and H40A—chemical shifts were changed by ca.
0.6–0.7 ppm and 0.4 ppm, respectively. These downfield shifts influenced by
proximity of triazole current ring suggest rather near parallel than perpen-
dicular arrangement of triazole ring plane relative to ribose moiety.
However, for nucleoside dimers possessing 50–30 linkage this induced by
triazole current ring downfield shift of nearby proton is less perceivable,
and limited only to H40B (ca. 0.3 ppm) that may be explained by more dis-
tant location of triazole ring. On the other hand, downfield influence of
1H-1,2,3-triazol-4-yl-methylene substituent in position O50 or O30 on
chemical shift of neighboring protons is less noticeable (ca. 0.2 ppm) and
observed only for H40A and H200A of 50–30 nucleoside dimers.
In 13C NMR spectra of sugar moieties, the greatest upfield changes by of
10–11 ppm were observed for C30A and C50B upon substitution hydroxyl
group with aromatic nitrogen in 30–50 and 50–30 nucleoside dimers, respect-
ively. The opposite changes by of ca. 8 ppm were noticed for C50B and C30A
of 30–50 and 50–30 nucleoside dimers, respectively upon introduction of 1H-
1,2,3-triazol-4-yl-methylene group, and those downfield shifts are in line with
our previous observations for propargyl group at position O50 or O30.[22]
Substitution of methyl group with fluorine atom in position 5 of base
1
part triggered significant perturbation of resonance frequencies of H, 13C
and 15N of base moiety independently of the position of dimer linkage.
The largest downfield shift by of 30 ppm was observed for C5A and C5B
induced by strong withdrawing effect of fluorine atom.[29] This consider-
able decrease of electron density at carbon C5 of base caused upfield shifts
of 13C resonance frequencies of C6, C4, and C2 by ca. of 11, 6.5, and
1.5 ppm, respectively. The similar upfield changes by of 7.5 ppm were
observed for N1 resonances (Table 4). Definitely smaller alterations of reso-
nances in opposite directions were observed for H6, H3, and N3 by of
0.4–0.6, 0.5, and 2.0 ppm, respectively.

8
D. BARANIAK ET AL.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9

10
D. BARANIAK ET AL.
Table 4. 15N NMR data for compounds 3a–d, 4a–d, 13a–d and 14a–da (letters A or B corres-
pond to 30 or 50 substituted monomer of nucleoside dimer, respectively, d-scale with liquid
NH3 as reference).
N1A
N1B
N3A
N3B
N1
triazole
N2
triazole
N3
triazole
3a
3b
143.9
143.4
143.9
135.9
136.4
143.5
136.2
136.0
143.9
143.9
143.9
136.2
136.4
144.0
136.4
136.3
143.0
142.7
135.4
142.4
142.3
134.3
135.5
134.2
143.0
142.4
135.7
142.5
143.0
134.2
135.6
134.1
156.0
155.9
156.0
157.9
158.0
156.0
158.0
158.0
156.0
156.0
156.1
n.d.
253.8
253.8
253.2
253.4
253.7
253.8
253.3
253.4
244.3
244.6
244.8
244.7
244.4
244.4
244.6
244.6
359.9
359.7
359.7
359.4
359.4
359.5
359.5
359.4
362.7
363.4
363.0
363.0
363.2
363.3
362.8
363.0
354.7
354.7
354.6
354.8
354.4
354.3
354.5
354.4
352.1
353.6
353.5
353.6
353.3
353.3
353.3
353.3
3c
3d
4a
4b
4c
158.0
156.0
n.d.
4d
13a
13b
13c
13d
14a
14b
14c
14d
156.0
156.0
158.0
156.0
156.0
158.0
158.0
158.0
156.0
156.0
n.d.
156.0
157.8
157.9
157.9
Structure of 1,4-disubstituted 1H-1,2,3-triazole as a linker of 30–50 and
50–30 nucleoside dimers were confirmed by repetitive patterns of connectiv-
1
1
ities in H–13C and H–15N gHMBC spectra of all compounds as well as
13
by C NMR spectra with gated decoupling. In case of 30–50 nucleoside
dimers we observed the following H–13C long-range correlations between
1
carbon or proton of triazole and ribose moiety: C5–H30A, C30A–H5, C4/
C5–H6, C6–H50/500B, C50B–H6 (Figure 2). Additionally, H–15N gHMBC
1
connectivities observed for N1–H20/200A, N2–H30A, N3–H6 were in agree-
ment with literature data that gave us a proof of 1,4 disubstitution patterns
of 1H-1,2,3-triazole.[30] Analogously, for 50–30 nucleoside dimers possessing
reverse arrangement of 1H-1,2,3-triazol-4-yl-methylene fragment we
1
1
observed reversed patterns of H–13C and H–15N long-range correlations
i.e. C5–H50/500B, C50B–H5, C4/C5–H6, C6–H30A, C30A–H6, N1–H50/500B,
1
15
N2–H50/500B, and N3–H6 (Figure 3 and Hꢀ N gHMBC spectra in ESI).
The 15N chemical shifts (Table 4) of triazole moiety of nucleoside dimers
reveals up to 8 ppm shift compared with literature data thus leading to

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
1
13
Figure 2. Fragments of Hꢀ C gHMBC spectrum of 30–50nucleoside dimers (3a) showing
13
1Hꢀ C long range correlations for triazolemethylene linker.
1
13
Figure 3. Fragments of Hꢀ C gHMBC spectrum of 50–30nucleoside dimer (13a) showing
13
1Hꢀ C long range correlations for triazolemethylene linker.
reduction of differences between N2 and N3 resonances, but the sequence
of these 15N resonances on the 1H–15N gHMBC spectra remains
unalterable.
Resonance frequency of triazole proton H5 of 30–50 nucleoside dimers is
8.30 0.02, and it is slightly shifted downfield by of 0.2 ppm compared
with 50–30 nucleoside dimers, thus showing H5 is not reliable diagnostic
parameter for identification of 1,5 or 1,4 disubstituted 1H-1,2,3-triazole iso-
mers. Creary and coworkers demonstrated that 1,4-disubstituted-1H-1,2,3-
triazoles can easily be distinguished from the isomeric 1,5-disubstituted-
1H-1,2,3-triazoles by simple one-dimensional 13C NMR spectroscopy using

12
D. BARANIAK ET AL.
13
1
Figure 4. Expanded C NMR spectra coupled with H for carbon C5 of 30–50 (left) and 50–30
(right) nucleoside dimers.
gated decoupling.[31] Authors excellently proved that chemical shift of tri-
azole carbon C5 or C4 as well as its one or three bond C–H coupling con-
3
stants (1JCH, JCH) were diagnostic for differentiation of disubstitution type
of 1H-1,2,3-triazole moiety. The C5 signal of 1,4-disubstituted-1H-1,2,3-tri-
azoles appears at d ¼ 120 3 ppm, while the C4 signal of 1,5-disubstituted
isomer is shifted downfield and appears at d ¼ 133 3 ppm. For model
compounds, i.e. 1-benzyl-4-phenyl- and 1-benzyl-5-phenyl-1H-1,2,3-tria-
1
3
zoles, they obtained values of d ¼ 120 ppm JCH¼ 191 Hz JCH¼2.7 Hz
1
3
(doublet of triplets) and d ¼ 133 ppm JCH¼195 Hz JCH¼0 Hz (doublet),
respectively. However, taking into accounts all compounds that were inves-
1
tigated in this paper, the value range of JCH for both isomers were partially
overlapped depending on the electronegativity of substituent in position 1,4
or 1,5 of 1H-1,2,3-triazole ring.
The 13C NMR spectra with gated decoupling revealed doublet of quartets
or doublet of multiplets for carbon C5 of 30–50 and 50–30 nucleoside dimers,
respectively. The doublet of quartets results from coupling of C5 via three
3
bonds with proton H30of ribose and H6 of methylene and values of JCH
were 3.21 0.19 Hz, 3.68 0.15 Hz, and 5.49 0.34 Hz. In case of 50–30
nucleoside dimers carbon C5 is coupled with four protons of two neighbor-
ing methylene groups that results in more complex splitting pattern on 13C
3
NMR spectra, and extraction reliable values of JCH may be significantly
imprecise (Figure 4). The average one bond C5–H5 coupling constant were
3
of 196.3 0.7 Hz and fall within observed value range of JCH in Creary’s
publication. The average chemical shift of carbon C5 obtained for 30–50 and
50–30 nucleoside dimers were of 123.55 0.03 ppm and 124.69 0.03 ppm,
respectively. Our results obtained from 13C NMR spectra with gated decou-
pling measurements are in line with Creary’s conclusions and support for
structure of 1,4-disubstituted 1H-1,2,3-triazole as a linker.
In order to estimate the effect of 1H-1,2,3-triazol-4-yl-methylene linker
at position of 30–50 or 50–30 on sugar puckering and syn-anti equilibrium of
30–50 and 50–30 nucleoside dimers we subjected them to a conformational

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
analysis by means of NMR spectroscopy and PSEUROT calculations
approach.[32] According to the concept of pseudorotation the conformation
of the furanose ring can be determined in terms of the phase angle of pseu-
dorotation (P), the degree of pucker (m) and the conformational popula-
tion.[33] The computer program PSEUROT (6.0) was used to calculate the
best fit of all conformational parameters (P and m for both I and II con-
formers and corresponding mole fractions) to three experimental coupling
3
3
constants (3JH1 H2 , JH2 H3 , JH3 H4 ). The summary results of conform-
ational preferences of all compounds are presented in Table 5. In general,
conformation of 20-deoxyribose moiety in 30–50 (3a–d, 4a–d) and 50–30
(13a–d, 14a–d) nucleoside dimers showed predominant preference for S-
type sugar puckering, which are in good agreement with the general ten-
dency of 20-deoxynucleosides sugar moiety to adopt S-type conform-
ers.[34,35] The 20-deoxyribose moieties in part A and B of 30–50-dimers
prefers almost the same conformers C20-endo-C10-exo and C20-endo,
respectively, with 56–69% of total conformer population. In case of 50–30
nucleoside dimers we observed different preference for conformers of both
20-deoxyriboses. The sugar fragment A of 50–30 nucleoside dimer adopted
C20-endo and percentage share of total conformer population was 83 4%,
conformation of second sugar was located between C20-endo-C30-exo and
C30-exo. In addition, estimation of N$S equilibrium by means of simple
equations[34] (Table 5) confirmed significant predominance of S-type con-
formers, however for us, the results were less useful than those obtained
with PSEUROT approach.
0
0
0
0
0
0
We have also performed NMR experiments to determine the preferred
conformation around the glycosidic bond (C10–N1) by observing the NOE
enhancement for H–10 and H–20, upon irradiation of H-6 and by measur-
3
0
ing the heteronuclear vicinal coupling constants, i.e. JH1 C2 and
3
[36,37]
0
JH1 C6
.
For 30–50 and 50–30 nucleoside dimers, the largest NOE
enhancement was found in the H20 resonance (2–3%), which clearly indi-
cates pronounced preference to anti conformation. Furthermore, the higher
values of JH1 C6 (3.6–6.1) than JH1 C2 (1.2–2.4 Hz) confirms that anti
3
3
0
0
orientation of base plane is predominant.
3. Conclusion
The synthesis of modified (30–50)dinucleoside based on the “click” chemis-
try can deliver biologically active compounds. The 1,4-disubstituted 1,2,3-
triazole ring, formed as a result of the copper(I) ions catalyzed 1,3-dipolar
Huisgen cycloaddition, is a stable structural element, that replaces the
phosphodiester bond and constitutes an additional pharmacophore. The
3
PSEUROT calculations based on JHH for 30–50 and 50–30 nucleoside dimers

14
D. BARANIAK ET AL.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
revealed strong preference of 20-deoxyribofuranosyl moiety for the S-type
puckering between C30-exo and C-20endo-C-10exo. The interpretation of
3
3
JH1 C2 and JH1 C6 and observation of NOE enhancement for H-20 upon H-
0
0
6 irradiation, showed that anti orientation is predominant over syn. The
substitution of the negatively charged phosphoric acid subunit with a neu-
tral 1,2,3-triazole residue eliminates partially the charge in the molecule,
enabling an easier transport of the described compounds across cell mem-
branes, and leads to a lack of electrostatic repulsion while conjugated with
DNA or RNA. Furthermore, such modification of the internucleotide bond
increases the resistance to nucleases—enzymes, which are capable of hydro-
lyzing natural nucleic acids. In turn, the fluorinated nucleobases bring
active pharmacophores as well as they exhibit a natural ability to form
hydrogen bonds with the complementary bases of target nucleic acids, and
this makes possible to use them as inhibitors of enzymes involved in the
DNA synthesis. The designed dimers of nucleoside analogs with an unpro-
tected hydroxyl group in the 50-position may also be incorporated into a
newly synthesized nucleic acid strand, e.g. viral DNA chain, and because of
the lack of a hydroxyl group in the 30-position can also act as terminators
of its synthesis. It is expected that the presented synthetic nucleoside
dimers could have a potential use as nucleoside drugs, due to their chem-
ical (terminators of synthesis) or physical (disorder of helix struc-
ture) properties.
4. Experimental protocols
4.1. Chemistry
General Information
1
1D and 2D H, 13C, 15N and 29Si NMR spectra were recorded on a
Bruker Avance III 500 MHz spectrometer, equipped with a 5 mm broad-
band multinuclear (PABBO) probe in DMSO-d6 at 298K. 1D 19F NMR
spectra were recorded on a Bruker Avance II 400 MHz spectrometer.
1
Chemical shifts (d) for H, 13C and 29Si NMR were reported in ppm
relative to the tetramethylsilane (TMS) peak. For 19F and 15N NMR d
were reported in ppm, relative to trichlorofluoromethane and liquid
NH3, respectively. Mass spectra were measured with QqToF Bruker with
source of ions ESI—electrospray ionization mass spectrometer. Thin
layer chromatography (TLC) was performed on silica gel 60 F254
precoated (0.2 mm) plates and vacuum flash column chromatography on
silica gel 60 H (5–40 lm), purchased from Merck. Chemical reagents
were purchased from Acros Organics, Alfa Aesar, Carbosynth and
Sigma-Aldrich.

16
D. BARANIAK ET AL.
4.1.1. 30-Azido-20,30-dideoxy-5-fluorouridine (1b)
To a solution of 10 (0.96 g, 1.36 mmol) in THF (3.9 mL) was added 0.5 M
HCl in methanol (8.2 mL) and the mixture was stirred at room temperature
for 3 h. Then the mixture was neutralized using 5% aqueous solution of
sodium bicarbonate, filtered and evaporated to dryness. The residue was
purified by silica gel column chromatography using methylene chloride—
methanol (40:1, v/v) as eluent to give pure 1b as a white powder (yield:
1
0.34 g, 68%). H NMR (DMSO-d6) d: 11.85 (brs, 1H), 8.20 (d, JHF¼7.2 Hz,
3
1H), 6.15 (td, JHH¼7.00 Hz 7.20 Hz, JHF¼1.20 Hz, 1H), 5.32 (t,
3
3JHH¼4.30 Hz 4.10 Hz, 1H), 5.22–5.20 (m, JHH¼3.40 Hz 3.10 Hz 4.80 Hz
3
5.40 Hz, 1H), 4.10 (q, JHH¼2.90 Hz 2.60 Hz 4.80 Hz, 1H), 3.64 (t,
3JHH¼3.10 Hz 3.40 Hz, 2 H), 2.28–2.26 (dd, 4.00 Hz 7.10 Hz, 2 H). 13C NMR
d: 170.04, 157.00 (d, JCF¼26.3 Hz), 149.05, 140.10 (d, JCF¼230.6 Hz), 124.45
(d, JCF¼34.5 Hz), 84.91, 84.42, 74.70, 61.27, 36.86. HRMS (ESI–) calcd for
C9H9FN5O4 [M – H]– 270.0638, found 270.0653, calcd for C9H10FN5O4Cl
[M þ Cl]– 306.0405, found 306.0422, calcd for C9H10FN5O4 [M þ H]þ
272.0795, found 272.0788, calcd for C9H9FN5O4Na [M þ Na]þ 294.0614,
found 294.0607.
4.1.2. Dimers of type 3a–d and 13a–d
In a round-bottomed flask, nucleoside azide (AZT or FAddU, 1 mmol) and
an equimolar amount of 30-O-propargyl derivative (1 mmol) were placed.
The substrates were dissolved in THF–H2O (3:1, v/v, 4 mL) and stirred
using a magnetic stirrer at room temperature until dissolved completely.
Subsequently, sodium ascorbate (80 mol%) was added. The mixture was
stirred until a homogenous solution was obtained. Finally a 1 M CuSO4
solution (40 mol%) was added and the flask was closed with glass stopper.
The reaction mixture was vigorously stirred for 24 hours at room tempera-
ture. When the reaction was completed, the solvent was removed using a
rotary evaporator, and the compound was purified on a chromatographic
column with silica gel using a methylene chloride – methanol mixture
(5!10%, v/v) as the eluent. Following the chromatographic purification
product was obtained as a white powder with 70 – 90% yield.
1
Compound 3a: H NMR (DMSO-d6) d: 11.34 (brs, 1H), 11.29 (brs, 1H),
8.31 (s, 1H), 7.81 (d, JHH¼ 1. 1H z, 1H), 7.53 (d, JHH¼1. 1H z, 1H), 6.42
3
3
(t, JHH¼6.70 Hz 6.55 Hz, 1H), 6.15 (t, JHH¼6.30 7.20 Hz, 1H), 5.38 (dt,
3JHH¼5.30 Hz 8.55 Hz 5.40 Hz, 1H), 5.29 (t, JHH¼5. 1H z 5. 1H z, 1H),
3
3
4.62 (s, 2 H), 4.39 (dt, JHH¼3.10 Hz 6.10 Hz 3.40 Hz, 1H), 4.20 (q,
3JHH¼5.40 Hz 3.55 Hz 3.55 Hz, 1H), 3.88 (q, JHH¼3.10 Hz 3.70 Hz 3.70 Hz,
3
3
1H), 3.72 – 3.68 (m, JHH¼3.55 Hz 3.70 Hz, 2JHH¼11.95 Hz 11.00 Hz, 2 H),
3
2
3.65 – 3.59 (m, JHH¼3.70 Hz 3.55 Hz, JHH¼11.00 Hz 11.95 Hz, 2 H),
3
2
2.74 – 2.68 (m, JHH¼6.70 Hz 5.30 Hz, JHH¼13.95 Hz, 1H), 2.67 – 2.61 (m,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
2
3
3JHH¼6.55 Hz 8.55 Hz, JHH¼13.95 Hz, 1H), 2.23 – 2.18 (m, JHH¼7.20 Hz
2
3
6.10 Hz, JHH¼13.30 Hz, 1H), 2.07 – 72.03 (ddd, JHH¼6.30 Hz 3.40 Hz,
2JHH¼13.30 Hz, 1H), 1.81 (d, JHH¼0.8 Hz, 3 H), 1.61 (d, JHH¼0.7 Hz, 3 H),
0.86 (s, 9 H), 0.06 (s, 6 H). 13C NMR d: 163.72, 163.66, 153.43, 150.39,
143.81, 136.23, 135.83, 123.56, 109.60, 109.56, 85.25, 84.48, 83.91, 83.75,
72.23, 69.50, 63.84, 60.70, 59.17, 39.30, 37.13, 25.65, 17.65, 12.25, 12.05,
–4.87, –4.96. 15N NMR d: 359.9, 354.7, 253.8, 156.0, 155.9, 143.9, 143.4.
29Si NMR d: 20.03. HRMS (ESI–) calcd for C29H42N7O9Si [M – H]–
660.2813, found 660.2815.
1
Compound 3b: H NMR (DMSO-d6) d: 11.82 (brs, 1H), 11.35 (brs, 1H),
8.29 (s, 1H), 7.95 (d, JHF¼7. 1H z, 1H), 7.82 (d, JHH¼1.2 Hz, 1H), 6.43 (t,
3
3JHH¼6.65 Hz 6.50 Hz, 1H), 6.10 (td, JHH¼6.30 Hz 6.75 Hz, JHF¼1.80 Hz,
3
3
1H), 5.37 (dt, JHH¼5.20 Hz 8.50 Hz 5.30 Hz, 1H), 5.30 (t, JHH¼5. 1H z
3
5.0 Hz, 1H), 4.62 (s, 2 H), 4.38 (dt, JHH¼4.20 Hz 6.10 Hz 3.75 Hz, 1H), 4.18
3
3
(dt, JHH¼5.30 Hz 3.40 Hz 3.50 Hz, 1H), 3.87 (q, JHH¼3.75 Hz 3.90 Hz
3
2
3.60 Hz, 1H), 3.74 – 3.69 (2 H, JHH¼3.40 Hz 3.90 Hz, JHH¼12.20 Hz
3
2
11.10 Hz, m), 3.65 – 3.59 (2 H, JHH¼3.50 Hz 3.60 Hz, JHH¼12.20 Hz
3
2
11.10 Hz, m), 2.72 – 2.68 (m, JHH¼6.65 Hz 5.20 Hz, JHH¼ 14.10 Hz, 1H),
3
2
2.66 – 2.62 (m, JHH¼6.50 Hz 8.50 Hz, JHH¼14.10 Hz, 1H), 2.25 – 2.21 (m,
3JHH¼6.75 Hz 6.10 Hz, JHH¼13.40 Hz, 1H), 2.11 – 2.07 (ddd, JHH¼6.30 Hz
2
3
2
4.20 Hz, JHH¼13.40 Hz, 1H), 1.81 (d, JHH¼1.0 Hz, 3 H), 0.85 (s, 9 H), 0.06
(s, 3 H), 0.04 (s, 3 H). 13C NMR d: 163.74, 157.00 (d, JCF¼26.2 Hz), 150.45,
148.96, 143.72, 139.96 (d, JCF¼230.7 Hz), 136.24, 124.59 (d, JCF¼34.3 Hz),
123.55, 109.64, 85.30, 84.52, 84.42, 83.93, 71.65, 69.12, 63.79, 60.68, 59.18,
39.00, 37.16, 25.81, 25.65, 17.64, 12.27, –4.87, –5.00. 15N NMR d: 359.7,
354.7, 253.8, 157.9, 156.0, 143.9, 135.9. 19F NMR d: –91.45. 29Si NMR d:
20.25. HRMS (ESI–) calcd for C28H39FN7O9Si [M – H]– 664.2562,
found 664.2529.
1
Compound 3c: H NMR (DMSO-d6) d: 11.89 (brs, 1H), 11.28 (brs, 1H),
8.30 (s, 1H), 8.34 (d, JHF¼7.2 Hz, 1H), 7.53 (d, JHH¼1.1 Hz, 1H), 6.38 (td,
3
3JHH¼6.50 Hz 6.00 Hz, JHF¼1.80 Hz, 1H), 6.15 (t, JHH¼6.30 Hz 7.30 Hz,
3
3
1H), 5.42 (t, JHH¼4.9 Hz 4.9 Hz, 1H), 5.37 (dt, JHH¼5.50 Hz 8.50 Hz
3
5.40 Hz, 1H), 4.62 (s, 2 H), 4.39 (dt, JHH¼3.40 Hz 6.20 Hz 3.20 Hz, 1H),
3
3
4.23 (dt, JHH¼5.40 Hz 3.40 Hz 3.40 Hz, 1H), 3.87 (q, JHH¼3.20 Hz 3.90 Hz
3
2
3.60 Hz, 1H), 3.75 – 3.69 (2 H, JHH¼3.40 Hz 3.90 Hz, JHH¼12.30 Hz
3
2
11.00 Hz, m), 3.65 – 3.59 (2 H, JHH¼3.40 Hz 3.60 Hz, JHH¼12.30 Hz
3
2
11.00 Hz, m), 2.78 – 2.73 (m, JHH¼6.50 Hz 5.50 Hz, JHH¼14.00 Hz, 1H),
3
2
2.69 – 2.63 (m, JHH¼6.00 Hz 8.50 Hz, JHH¼14.00 Hz, 1H), 2.23 – 2.18 (m,
3JHH¼7.30 Hz 6.20 Hz, JHH¼13.40 Hz, 1H), 2.07 – 2.03 (ddd, JHH¼6.30 Hz
2
3
2
3.40 Hz, JHH¼13.40 Hz, 1H), 1.65 (d, JHH¼ 0.9 Hz, 3 H), 0.86 (s, 9 H), 0.06
(s, 6 H). 13C NMR d: 163.65, 157.04 (d, JCF¼26.2 Hz), 150.39, 149.00,
143.81, 140.04 (d, JCF¼230.2 Hz), 135.82, 124.87 (d, JCF¼34.7 Hz), 123.59,

18
D. BARANIAK ET AL.
109.55, 85.25, 84.80, 84.59, 83.75, 72.22, 69.49, 63.83, 60.43, 58.80, 39.30,
37.43, 25.64, 17.65, 12.04, –4.87, –4.96. 15N NMR d: 359.7, 354.6, 253.2,
158.0, 156.0, 143.5, 136.4. 19F NMR d: –91.48. 29Si NMR d: 20.03. HRMS
(ESI–) calcd for C28H39FN7O9Si [M – H]– 664.2562, found 664.2546.
1
Compound 3d: H NMR (DMSO-d6) d: 11.88 (brs, 1H), 11.82 (brs, 1H),
8.28 (s, 1H), 8.34 (d, JHF¼7. 1H z, 1H), 7.95 (d, JHF¼7.0 Hz, 1H), 6.38 (td,
3
3JHH¼6.55 Hz 6.20 Hz, JHF¼1.80 Hz, 1H), 6.10 (td, JHH¼6.30 Hz 6.70 Hz,
3
3
JHF¼1.80 Hz, 1H), 5.44 (t, JHH¼4.9 Hz 5.0 Hz, 1H), 5.37 (dt, JHH¼5.55 Hz
3
8.50 Hz 5.30 Hz, 1H), 4.61 (s, 2 H), 4.37 (dt, JHH¼4.10 Hz 6.20 Hz 4.00 Hz,
3
3
1H), 4.22 (dt, JHH¼5.30 Hz 3.00 Hz 3.00 Hz, 1H), 3.87 (q, JHH¼4.00 Hz
3.95 Hz 3.40 Hz, 1H), 3.75 – 3.71 (2 H, 3JHH¼3.00 Hz 3.95 Hz,
2JHH¼12.00 Hz 11.80 Hz, m), 3.66 – 3.60 (2 H, JHH¼3.00 Hz 3.40 Hz,
3
2JHH¼12.00 Hz 11.80 Hz, m), 2.78 – 2.72 (m, JHH¼6.65 Hz 5.55 Hz,
3
2JHH¼14.00 Hz, 1H), 2.68 – 2.62 (m, JHH¼6.20 Hz 8.50 Hz, JHH¼14.00 Hz,
3
2
3
2
1H), 2.25 – 2.20 (m, JHH¼6.70 Hz 6.20 Hz, JHH¼13.40 Hz, 1H), 2.11 – 2.07
3
2
(ddd, JHH¼6.30 Hz 4.10 Hz JHH¼13.40 Hz, 1H), 0.85 (s, 9 H), 0.06 (s,
3 H), 0.04 (s, 3 H). 13C NMR d: 157.02 (d, JCF¼26.2 Hz), 156.96 (d,
JCF¼26.1 Hz), 148.99, 148.92, 143.69, 140.02 (d, JCF¼230.2 Hz), 139.93 (d,
JCF¼230.7 Hz), 124.84 (d, JCF¼34.7 Hz), 124.55 (d, JCF¼34.2 Hz), 123.54,
85.27, 84.82, 84.60, 84.40, 71.63, 69.11, 63.77, 60.40, 58.81, 39.30, 37.43,
25.62, 17.61, –4.90, –5.02. 15N NMR d: 359.4, 354.8, 253.4, 158.0, 136.2,
136.0. 19F NMR d: –91.45, –91.49. 29Si NMR d: 20.24. HRMS (ESI–) calcd
for C27H36F2N7O9Si [M – H]– 668.2311, found 668.2286.
1
Compound 13a: H NMR (DMSO-d6) d: 11.34 (brs, 1H), 11.30 (brs,
1H), 8.10 (s, 1H), 7.48 (d, JHH¼1.1 Hz, 1H), 7.36 (d, JHH¼1.1 Hz, 1H), 6.17
3
3
(t, JHH¼7.40 Hz 6.50 Hz, 1H), 6.10 (dd, JHH¼5.70 Hz 8.55 Hz, 1H), 5.52
3
3
2
(d, JHH¼4.3 Hz, 1H), 4.73 – 4.70 (dd, JHH¼4.40 Hz JHH¼14.20 Hz, 1H),
3
2
4.64 – 4.58 (dd, JHH¼7.70 Hz JHH¼14.20 Hz, 1H), 4.58 (s, 2 H), 4.28 (m,
3JHH¼3.35 Hz 6.50 Hz 3.60 Hz, 1H), 4.15 (dt, JHH¼1.80 Hz 5.80 Hz
3
3
2.20 Hz, 1H), 4.08 (dt, JHH¼3.60 Hz 7.70 Hz 4.40 Hz, 1H), 4.00 (q,
3JHH¼2.20 Hz 3.80 Hz 3.90 Hz, 1H), 3.78 – 3.75 (dd, JHH¼3.80 Hz
3
2JHH¼11.40 Hz, 1H), 3.73 – 3.70 (dd, JHH¼ 3.90 Hz JHH¼11.40 Hz, 1H),
3
2
3
2
2.30 – 2.26 (ddd, JHH¼5.70 Hz 1.80 Hz JHH¼13.40 Hz, 1H), 2.21 – 2.15 (m,
3JHH¼6.50 Hz 6.50 Hz JHH¼14.00 Hz, 1H), 2.14 – 2.10 (m, JHH¼7.40 Hz
2
3
2
3
3.35 Hz JHH¼14.00 Hz, 1H), 2.11 – 2.08 (m, JHH¼8.55 Hz 5.80 Hz
2JHH¼13.40 Hz, 1H), 1.80 (s, JHH¼0.8 Hz, 3 H), 1.77 (s, JHH¼0.8 Hz, 3 H),
0.86 (s, 9 H), 0.06 (s, 3 H), 0.05 (s, 3 H). 13C NMR d: 163.62, 163.60,
150.36, 150.34, 143.67, 136.05, 135.35, 124.69, 109.82, 109.50, 84.05, 83.99,
83.96, 83.91, 78.13, 70.72, 63.24, 61.65, 51.19, 37.89, 36.30, 25.72, 17.91,
12.19, 12.04, –5.53, –5.56. 15N NMR d: 362.7, 352.1, 244.3, 156.0, 143.0,
142.7. 29Si NMR d: 20.16. HRMS (ESI–) calcd for C29H42N7O9Si [M – H]–
660.2813, found 660.2827.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
19
1
Compound 13b: H NMR (DMSO-d6) d: 11.86 (brs, 1H), 11.31 (brs,
1H), 8.09 (s, 1H), 7.96 (d, JHF¼6.9 Hz, 1H), 7.35 (s, 1H), 6.17 (t,
3
3JHH¼7.40 Hz 6.50 Hz, 1H), 6.08 (t, JHH¼5.80 Hz 8.10 Hz, JHF¼1.50 Hz,
1H), 5.51 (d, 3JHH¼4.3 Hz, 1H), 4.73 – 4.69 (dd, 3JHH¼4.50 Hz
3
2
2JHH¼14.20 Hz, 1H), 4.63 – 4.58 (dd, JHH¼7.50 Hz JHH¼14.20 Hz, 1H),
3
4.58 (dd, JHH¼ 4.5 Hz 2.2 Hz, 2 H), 4.28 (m, JHH¼3.30 Hz 6.50 Hz 3.60 Hz,
3
3
1H), 4.17 (dt, JHH¼2.25Hz 5.70 Hz 2.30 Hz, 1H), 4.07 (dt, JHH¼3.60 Hz
3
7.50 Hz 4.50 Hz, 1H), 4.03 (q, JHH¼2.30 Hz 3.60 Hz 3.10 Hz, 1H),
3
2
3.82 – 3.79 (dd, JHH¼3.60 Hz JHH¼11.60 Hz, 1H), 3.74 – 3.71 (dd,
3JHH¼3.10 Hz JHH¼11.60 Hz, 1H), 2.36 – 2.32 (ddd, JHH¼5.80 Hz 2.25 Hz
2
3
2JHH¼13.80 Hz, 1H), 2.21 – 2.15 (m, JHH¼6.50 Hz 6.50 Hz JHH¼14.00 Hz,
3
2
3
2
1H), 2.14 – 2.10 (m, JHH¼7.40 Hz 3.30 Hz JHH¼14.00 Hz, 1H), 2.11 – 2.08
3
2
(m, JHH¼8.10 Hz 5.70 Hz JHH¼13.80 Hz, 1H), 1.79 (s, 3 H), 0.86 (s, 9 H),
0.07 (s, 3 H), 0.06 (s, 3 H). 13C NMR d: 163.64, 156.96 (d, JCF¼ 26.1 Hz),
150.38, 148.88, 143.67, 140.00 (d, JCF¼230.8 Hz), 136.05, 124.73, 124.06 (d,
JCF¼34.1 Hz), 109.85, 84.74, 84.40, 84.01, 83.97, 78.38, 70.74, 63.13, 61.73,
51.21, 37.91, 36.86, 25.70, 17.93, 12.06, –5.68. 15N NMR d: 363.4, 353.6,
244.6, 158.0, 156.0, 142.4, 135.4. 19F NMR d: –91.37. 29Si NMR d: 21.88.
HRMS (ESI–) calcd for C28H39FN7O9Si [M – H]– 664.2562, found 664.2313;
calcd for C28H39FN7O9SiCl [M þ Cl]– 700.2329, found 700.2059.
1
Compound 13c: H NMR (DMSO-d6) d: 11.87 (brs, 1H), 11.34 (brs,
1H), 8.10 (s, 1H), 7.94 (d, JHF¼7.0 Hz, 1H), 7.48 (d, JHH¼1.0 Hz, 1H), 6.13
3
3
(t, JHH¼7.40 Hz 6.50 Hz, JHF¼1.50 Hz, 1H), 6.10 (dd, JHH¼5.80 Hz
3
3
8.45 Hz, 1H), 5.54 (d, JHH¼4.2 Hz, 1H), 4.72 – 4.69 (dd, JHH¼4.10 Hz
2JHH¼14.20 Hz, 1H), 4.66 – 4.63 (dd, JHH¼8.15 Hz JHH¼14.20 Hz, 1H),
3
2
3
4.58 (dd, JHH¼3.5 Hz 2.4 Hz, 2 H), 4.25 (m, JHH¼3.40 Hz 7.00 Hz 3.30 Hz,
3
3
1H), 4.15 (dt, JHH¼1.80 Hz 5.70 Hz 2.10 Hz, 1H), 4.07 (dt, JHH¼3.30 Hz
3
8.15 Hz 4.10 Hz, 1H), 4.00 (q, JHH¼2.10 Hz 3.60 Hz 3.95 Hz, 1H),
3
2
3.77 – 3.75 (1 H, JHH¼3.95 Hz JHH¼11.30 Hz, dd), 3.72 – 3.70 (1 H,
3JHH¼3.60 Hz JHH¼11.30 Hz, dd), 2.29 – 2.27 (ddd, JHH¼5.80 Hz 1.80 Hz
2
3
2JHH¼13.50 Hz, 1H), 2.24 – 2.20 (m, JHH¼6.50 Hz 7.00 Hz JHH¼14.00 Hz,
3
2
3
2
1H), 2.14 – 2.10 (m, JHH¼7.40 Hz 3.40 Hz JHH¼14.00 Hz, 1H), 2.10 – 2.07
3
2
(m, JHH¼ 8.45 Hz 5.70 Hz JHH¼13.50 Hz, 1H), 1.77 (d, JHH¼0.9 Hz, 3 H),
0.86 (s, 9 H), 0.06 (s, 3 H), 0.05 (s, 3 H). 13C NMR d: 163.61, 156.96 (d,
JCF¼ 26.2 Hz), 150.36, 148.98, 143.69, 140.13 (d, JCF¼ 231.2 Hz), 135.36,
125.02 (d, JCF¼ 33.9 Hz), 124.64, 109.52, 84.65, 84.28, 84.06, 83.93, 78.70,
70.64, 63.25, 61.62, 51.25, 37.94, 36.31, 25.73, 17.93, 12.20, –5.52, –5.50.
15N NMR d: 363.0, 353.5, 244.8, 158.0, 156.0, 142.3, 134.3. 19F NMR d:
–91.07. 29Si NMR d: 21.13. HRMS (ESI–) calcd for C28H39FN7O9Si
[M – H]– 664.2562, found 664.2331.
1
Compound 13d: H NMR (DMSO-d6) d: 11.85 (brs, 2 H), 8.10 (s, 1H),
7.97 (d, JHF¼ 6.9 Hz, 1H), 7.94 (d, JHF¼ 6.9 Hz, 1H), 6.14 (t, JHH¼7.45 Hz
3

20
D. BARANIAK ET AL.
3
6.45 Hz, JHF¼1.60 Hz, 1H), 6.08 (t, JHH¼5.80 Hz 8.00 Hz, JHF¼1.50 Hz,
1H), 5.51 (d, 3JHH¼3.6 Hz, 1H), 4.72 – 4.68 (dd, 3JHH¼3.40 Hz
3
2
2JHH¼14.30 Hz, 1H), 4.66 – 4.62 (dd, JHH¼8.20 Hz JHH¼14.30 Hz, 1H),
2
3
4.57 (dd, JHH¼12.20 Hz, 2 H), 4.27–4.24 (m, JHH¼3.30 Hz 6.90 Hz
3
3.20 Hz, 1H), 4.17 (dt, JHH¼2.40 Hz 5.70 Hz 2.25 Hz, 1H), 4.07 (dt,
3JHH¼3.20 Hz 8.20 Hz 3.40 Hz, 1H), 4.04 (q, JHH¼2.25 Hz 3.10 Hz 3.40 Hz,
3
3
2
1H), 3.82 – 3.79 (dd, JHH¼3.10 Hz JHH¼11.30 Hz, 1H), 3.74 – 3.71 (dd,
3JHH¼3.40 Hz JHH¼11.30 Hz, 1H), 2.36 – 2.32 (ddd, JHH¼5.80 Hz 2.40 Hz
2
3
2JHH¼13.70 Hz, 1H), 2.25 – 2.20 (m, JHH¼6.45 Hz 6.90 Hz JHH¼13.80 Hz,
3
2
3
2
1H), 2.16 – 2.12 (m, JHH¼7.45 Hz 3.30 Hz JHH¼13.80 Hz, 1H), 2.12 – 2.10
3
2
(m, JHH¼8.00 Hz 5.70 Hz JHH¼13.70 Hz, 1H), 0.86 (s, 9 H), 0.07 (s, 3 H),
0.06 (s, 3 H). 13C NMR d: 156.97 (d, JCF¼26.1 Hz), 149.00, 148.88, 143.68,
141.14 (d, JCF¼231.2 Hz), 140.01 (d, JCF¼230.8 Hz), 125.02 (d,
JCF¼34.0 Hz), 124.67, 124.06 (d, JCF¼34.0 Hz), 84.75, 84.65, 84.39, 84.29,
78.33, 70.65, 63.13, 61.70, 51.26, 37.95, 25.70, 17.94, –5.68. 15N NMR d:
363.0, 353.6, 244.7, 158.0, 135.5, 134.2. 19F NMR d: –91.11, –91.37. 29Si
NMR d: 21.89. HRMS (ESI–) calcd for C27H36F2N7O9Si [M – H]– 668.2311,
found 668.2066.
4.1.3. Dimers of type 4a–d and 14a–d
Ammonium fluoride (10 eqs) was added to a stirred solution of silylated
dimer 3a–d or 13a–d (1 mmol) in abs. MeOH (4 mL) and the reactants
were heated at reflux for 4 h. After that time, the silica gel was added to
the reaction mixture and it was evaporated under reduced pressure. The
residue was purified with silica gel column chromatography, using the mix-
ture methylene chloride–methanol (10!15%) as an eluent, to afford prod-
uct as a white powder with 80 – 95% yield.
1
Compound 4a: H NMR (DMSO-d6) d: 11.34 (brs, 1H), 11.26 (brs, 1H),
8.32 (s, 1H), 7.82 (d, JHH¼1.2 Hz,1H), 7.54 (d, JHH¼1.1 Hz, 1H), 6.42 (t,
3
3JHH¼6.75 Hz 6.45 Hz, 1H), 6.18 (t, JHH¼6.20 Hz 7.65 Hz, 1H), 5.38 (dt,
3
3JHH¼5.30 Hz 8.60 Hz 5.30 Hz, 1H), 5.32 (d, JHH¼4.3 Hz, 1H), 5.29 (t,
3
3JHH¼5.3 Hz 5.2 Hz 1H), 4.61 (s, 2 H), 4.24 (m, JHH¼3.50 Hz 6.10 Hz
3
3.15 Hz, 1H), 4.20 (q, JHH¼5.30 Hz 3.65 Hz 3.40 Hz, 1H), 3.88 (q,
3
3JHH¼3.15 Hz 4.10 Hz 3.40 Hz,1H), 3.72–3.68 (2 H, JHH¼4.10 Hz 3.65 Hz
3
2JHH¼12.40 Hz 10.70 Hz, m), 3.65–3.60 (2 H, JHH¼3.40 Hz 3.40 Hz
3
2JHH¼12.40 Hz 10.70 Hz, m), 2.74–2.70 (ddd, JHH¼6.75 Hz 5.30 Hz
3
2
2JHH¼14.15 Hz, 1H), 2.66–2.62 (ddd, JHH¼6.45 Hz 8.60 Hz JHH¼14.15 Hz,
3
2
1H), 2.14–2.10 (ddd, JHH¼7.65 Hz 6.10 Hz JHH¼13.20 Hz 1H), 2.08–2.05
3
2
(ddd, JHH¼6.20 Hz 3.50 Hz JHH¼13.20 Hz, 1H), 1.81 (d, 3 H), 1.64 (d,
3 H). 13C NMR d: 163.71, 163.66, 150.42, 150.40, 143.92, 136.22, 135.84,
123.54, 109.60, 109.52, 85.25, 84.46, 83.89, 83.78, 70.66, 70.11, 63.89, 60.71,
59.18, 39.30, 37.13, 12.24, 12.02. 15N NMR d: 359.4, 354.4, 253.7, 156.0,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
21
143.9. HRMS (ESIþ) calcd for C23H29N7NaO9 [M þ Na]þ 570.1924,
found 570.1988.
1
Compound 4b: H NMR (DMSO-d6) d: 11.80 (brs, 1H), 11.35 (brs, 1H),
8.30 (s, 1H), 7.95 (d, JHF¼ 7.1 Hz, 1H), 7.82 (d, JHH¼1.2 Hz, 1H), 6.43 (t,
3
3JHH¼6.80 Hz 6.45 Hz, 1H), 6.12 (td, JHH¼6.50 Hz 6.80 Hz, JHF¼1.80 Hz,
3
3
1H), 5.37 (dt, JHH¼5.30 Hz 8.65 Hz 5.30 Hz, 1H), 5.33 (d, JHH¼4.2 Hz,
3
1H), 5.29 (t, JHH¼4.2 Hz 4.7 Hz, 1H), 4.61 (brs, 2 H), 4.23–4.18 (m,
3JHH¼3.70 Hz 5.45 Hz 3.30 Hz 5.30 Hz 6.65 Hz 3.30 Hz, 2 H), 3.89 (q,
3JHH¼3.30 Hz 4.00 Hz 3.50 Hz, 1H), 3.75–3.68 (m, JHH¼3.65 Hz 4.00 Hz
3
2JHH¼12.00 Hz 10.80 Hz, 2 H), 3.67–3.60 (m, JHH¼3.30 Hz 3.50 Hz
3
2JHH¼12.00 10.80, 2 H), 2.75–2.68 (m, 3JHH¼6.80 Hz 5.30 Hz
2JHH¼14.00 Hz, 1H), 2.67–2.60 (m, JHH¼6.45 Hz 8.65 Hz JHH¼14.00 Hz,
3
2
3
2
1H), 2.17–2.13 (m, JHH¼6.80 Hz 5.45 Hz JHH¼12.90 Hz, 1H), 2.13–2.07
(m, JHH¼6.50 Hz 3.70 Hz JHH¼12.90 Hz, 1H), 1.81 (d, 3 H). 13C NMR d:
163.72, 156.99 (d, JCF¼26.1 Hz), 150.44, 148.97, 143.82, 139.92 (d,
JCF¼230.7 Hz), 136.22, 124.53 (d, JCF¼34.3 Hz), 123.49, 109.62, 85.43,
84.57, 84.48, 83.90, 70.33, 69.80, 63.86, 60.68, 59.17, 39.40, 37.17, 12.26. 15N
NMR d: 359.5, 354.5, 253.8, 156.1, 143.9, 136.2. 19F NMR d: –91.45. HRMS
(ESI–) calcd for C22H25FN7O9 [M – H]– 550.1697, found 550.1513.
3
2
1
Compound 4c: H NMR (DMSO-d6) d: 11.88 (brs, 1H), 11.26 (brs, 1H),
8.34 (d, JHF¼ 7.2 Hz, 1H), 8.30 (s, 1H), 7.53 (d, JHH¼1.1 Hz, 1H), 6.38 (td,
3
3JHH¼6.55 Hz 6.15 Hz, JHF¼1.60 Hz, 1H), 6.18 (t, JHH¼6.30 Hz 7.40 Hz,
3
3
1H), 5.42 (t, JHH¼4.9 Hz 5.0 Hz, 1H), 5.37 (dt, JHH¼5.60 Hz 8.45 Hz
3
5.30 Hz, 1H), 5.31 (d, JHH¼4.3 Hz, 1H), 4.61 (s, 2 H), 4.25–4.22 (m,
3JHH¼3.20 Hz 6.15 Hz 3.20 Hz 5.30 Hz 3.40 Hz 3.30 Hz, 2 H), 3.88 (q,
3JHH¼3.20 Hz 4.00 Hz 3.40 Hz, 1H), 3.75 – 3.70 (2 H, JHH¼3.40 Hz 4.00 Hz
3
2JHH¼12.00Hz 10.60 Hz, m), 3.65–3.60 (2 H, JHH¼3.30 Hz 3.40 Hz
3
2JHH¼12.00Hz 10.60 Hz, m), 2.79 – 2.74 (m, JHH¼6.55 Hz 5.60 Hz
3
2JHH¼13.80 Hz 1H), 2.69 – 2.63 (m, JHH¼6.15 Hz 8.45 Hz JHH¼13.80 Hz,
3
2
3
2
1H), 2.14 – 2.09 (m, JHH¼7.40 Hz 6.15 Hz JHH¼13.25 Hz, 1H), 2.08 – 2.04
(ddd, JHH¼6.30 Hz 3.20 Hz JHH¼13.25 Hz, 1H), 1.64 (d, 3 H). 13C NMR
d: 163.66, 157.04 (d, JCF¼ 26.2 Hz), 150.40, 149.00, 143.92, 140.03 (d,
JCF¼230.1 Hz), 135.84, 124.86 (d, JCF¼34.6 Hz), 123.57, 109.51, 85.25,
84.78, 84.58, 83.78, 70.66, 70.12, 63.89, 60.44, 58.80, 39.40, 37.42, 12.02. 15N
NMR d: 359.5, 354.5, 253.3, 158.0, 156.0, 144.0, 136.4. 19F NMR d: –91.47.
HRMS (ESI–) calcd for C22H25FN7O9 [M – H]– 550.1697, found 550.1672.
Compound 4d: 1H NMR (DMSO-d6) d: 11.83 (brs, 2 H), 8.35 (d,
JHF¼7.1 Hz, 1H), 8.29 (s, 1H), 7.94 (d, JHF¼7.0 Hz, 1H), 6.38 (td,
3
2
3JHH¼6.60 Hz 6.20 Hz, JHF¼1.55 Hz, 1H), 6.12 (td, JHH¼6.40 Hz 7.00 Hz,
3
3
JHF¼1.70 Hz, 1H), 5.46 (brs, 1H), 5.38 (dt, JHH¼5.80 Hz 8.55 Hz 5.40 Hz,
3
3
1H), 5.37 (d, JHH¼5.4 Hz, 1H), 4.61 (s, 2 H), 4.24–4.21 (m, JHH¼3.60 Hz
3
6.55 Hz 3.30 Hz 5.40 Hz 3.20 Hz 3.30 Hz, 2 H), 3.89 (q, JHH¼3.30 Hz

22
D. BARANIAK ET AL.
3
2
4.10 Hz 3.40 Hz, 1H), 3.74 – 3.72 (2 H, JHH¼3.20 Hz 4.10 Hz JHH
¼
3
2
12.00 Hz 10.70 Hz, m), 3.66 – 3.61 (2 H, JHH¼3.30 Hz 3.40 Hz JHH
¼
12.00 Hz 10.70 Hz, m), 2.79 – 2.74 (m, 3JHH¼6.60 Hz 5.80 Hz
2JHH¼13.80 Hz, 1H), 2.68 – 2.62 (m, JHH¼6.20 Hz 8.55 Hz JHH¼13.80 Hz
3
2
3
2
1H), 2.16 – 2.11 (m, JHH¼7.00 Hz 6.55 Hz JHH¼13.30 Hz, 1H), 2.10 – 2.08
(m, JHH¼6.40 Hz 3.60 Hz JHH¼13.30 Hz, 1H). 13C NMR d: 157.08 (d,
JCF¼26.1 Hz), 157.01 (d, JCF¼26.1 Hz), 149.06, 148.97, 143.81, 140.04 (d,
JCF¼ 230.2 Hz), 139.92 (d, JCF¼230.8 Hz), 123.55, 124.84 (d, JCF¼34.7 Hz),
124.52 (d, JCF¼34.4 Hz), 85.43, 84.85, 84.61, 84.57, 70.31, 69.80, 63.85,
60.41, 58.83, 39.40, 37.44. 15N NMR d: 359.4, 354.4, 253.4, 136.4, 136.3. 19F
NMR d: –91.42, –91.48. HRMS (ESI–) calcd for C21H22F2N7O9 [M – H]–
554.1447, found 554.1417.
3
2
1
Compound 14a: H NMR (DMSO-d6) d: 11.30 (brs, 2 H), 8.10 (s, 1H),
3
7.70 (d, JHH¼1.1 Hz, 1H), 7.35 (d, JHH¼1.1 Hz, 1H), 6.16 (t, JHH¼7.50 Hz
3
3
6.40 Hz, 1H), 6.11 (dd, JHH¼5.75 Hz 8.50 Hz, 1H), 5.51 (d, JHH¼4.4 Hz,
3
3
1H), 5.12 (t, JHH¼5.2 Hz 5.3 Hz, 1H), 4.73 – 4.70 (dd, JHH¼4.40 Hz
2JHH¼14.20 Hz, 1H), 4.64 – 4.59 (dd, JHH¼7.70 JHH¼14.20 1H), 4.57 (s,
3
2
3
2 H), 4.26–4.30 (m, JHH¼3.80 Hz 6.45 Hz 3.40 Hz, 1H), 4.21 (dt,
3JHH¼2.05 Hz 5.70 Hz 2.30 Hz, 1H), 4.07 (dt, JHH¼3.40 Hz 7.70 Hz
3
3
4.40 Hz, 1H), 3.95 (q, JHH¼2.30 Hz 3.90 Hz 3.90 Hz, 1H), 3.61 – 3.57 (1 H,
3JHH¼3.90 Hz 2JHH¼11.80 Hz, dd), 3.57 – 3.54 (1 H, 3JHH¼3.90 Hz
2JHH¼11.80 Hz, dd), 2.27 – 2.22 (ddd, JHH¼5.75 Hz 2.05 Hz JHH¼13.55 Hz,
3
2
3
3
1H), 2.21 – 2.15 (m, JHH¼6.40 Hz 6.45 Hz JHH¼13.90 Hz, 1H), 2.15 – 2.11
(m, 3JHH¼7.50 Hz 3.80 Hz 2JHH¼13.90 Hz, 1H), 2.11 – 2.07 (m,
3JHH¼8.50 Hz 5.70 Hz JHH¼13.55 Hz 1H), 1.80 (s, 3 H), 1.77 (s, 3 H). 13C
2
NMR d: 163.69, 163.65, 150.46, 150.40, 143.85, 136.05, 135.97, 124.70,
109.86, 109.53, 84.59, 84.03, 83.96, 83.80, 79.09, 70.74, 61.76, 61.50, 51.16,
37.87, 36.27, 12.25, 12.06. 15N NMR d: 363.2, 353.3, 244.4, 156.0, 143.0,
142.4. HRMS (ESI–) calcd for C23H28N7O9 [M – H]– 546.1948,
found 546.1775.
1
Compound 14b: H NMR (DMSO-d6) d: 11.79 (brs, 1H), 11.32 (brs,
1H), 8.20 (d, JHF¼7.2 Hz, 1H), 8.10 (s, 1H), 7.34 (d, JHH¼1.2 Hz, 1H), 6.16
3
3
(t, JHH¼7.40 Hz 6.65 Hz, 1H), 6.08 (td, JHH¼5.90Hz 8.00 Hz, JHF¼1.80 Hz,
3
3
1H), 5.52 (d, JHH¼4.0 Hz, 1H), 5.24 (t, JHH¼4.8 Hz 4.9 Hz, 1H),
3
2
4.73 – 4.69 (dd, JHH¼4.60 Hz JHH¼14.30 Hz 1H), 4.63 – 4.59 (dd,
3JHH¼7.60 Hz JHH¼14.30 Hz, 1H), 4.57 (s, 2 H), 4.26–4.29 (m,
2
3JHH¼3.10 Hz 6.30 Hz 3.65 Hz, 1H), 4.22 (dt, JHH¼2.50 Hz 5.75 Hz
3
3
2.35 Hz, 1H), 4.08 (dt, JHH¼3.65 Hz 7.60 Hz 4.60 Hz, 1H), 3.98 (q,
3JHH¼2.35 Hz 3.45 Hz 3.55 Hz, 1H), 3.63 – 3.59 (dt, JHH¼3.45 Hz
3
2JHH¼11.60 Hz 1H), 3.59 – 3.55 (dt, JHH¼3.55 Hz JHH¼11.60 Hz 1H),
3
2
3
2
2.31 – 2.27 (ddd, JHH¼5.90 Hz 2.50 Hz JHH¼13.60 Hz, 1H), 2.20 – 2.15 (m,
3JHH¼6.65 Hz 6.30 Hz JHH¼13.90 Hz, 1H), 2.15 – 2.11 (m, JHH¼7.40 Hz
2
3

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
23
2
3
3.10 Hz JHH¼13.90 Hz 1H), 2.11 – 2.07 (ddd, JHH¼8.00 Hz 5.75 Hz
2JHH¼13.60 Hz, 1H), 1.79 (s, 3 H). 13C NMR d: 163.66, 157.03 (d,
JCF¼26.1 Hz), 150.40, 149.04, 143.83, 140.02 (d, JCF¼230.3 Hz), 136.06,
124.72, 124.62 (d, JCF¼34.4 Hz), 109.87, 84.88, 84.57, 84.03, 83.96, 78.85,
70.75, 61.80, 61.33, 51.17, 37.87, 36.71, 12.07. 15N NMR d: 363.3, 353.3,
244.4, 142.5, 135.7. 19F NMR d: –91.23. HRMS (ESI–) calcd for
C22H25FN7O9 [M – H]– 550.1697, found 550.1707.
1
Compound 14c: H NMR (DMSO-d6) d: 11.86 (brs, 1H), 11.30 (brs,
1H), 8.11 (s, 1H), 7.92 (d, JHF¼7.0 Hz, 1H), 7.70 (d, JHH¼1.1 Hz, 1H), 6.13
3
3
(t, JHH¼7.45 Hz 6.55 Hz, JHF¼1.60 Hz, 1H), 6.11 (dd, JHH¼5.75 Hz
3
3
8.40 Hz, 1H), 5.53 (d, JHH¼4.5 Hz, 1H), 5.12 (t, JHH¼4.6 Hz 4.3 Hz, 1H),
3
2
4.73 – 4.69 (dd, JHH¼4.25 Hz JHH¼14.20 Hz, 1H), 4.66 – 4.62 (dd,
3JHH¼8.10 Hz 2JHH¼14.25 Hz 1H), 4.57 (s, 2 H), 4.27–4.23 (m,
3JHH¼3.65 Hz 6.45 Hz 3.60 Hz, 1H), 4.21 (dt, JHH¼2.15 Hz 5.90 Hz
3
3
2.25 Hz, 1H), 4.07 (dt, JHH¼3.60 Hz 8.10 Hz 4.25 Hz, 1H), 3.95 (q,
3JHH¼2.25 Hz 3.85 Hz 3.85 Hz, 1H), 3.61 – 3.57 (1 H, JHH¼3.85 Hz
3
2JHH¼13.80 Hz, dt), 3.57 – 3.53 (1 H, JHH¼3.85 Hz JHH¼13.80 Hz, dt),
3
2
3
2
2.27 – 2.19 (m, JHH¼5.75 Hz 2.15 Hz 6.55 Hz 6.45 Hz JHH¼13.80 Hz
3
13.90 Hz, 2 H), 2.17 – 2.10 (m, JHH¼7.45 Hz 3.65 Hz 8.40 Hz 5.90 Hz
2JHH¼13.90 Hz 13.80 Hz, 2 H), 1.77 (s, 3 H). 13C NMR d: 163.69, 156.98 (d,
JCF¼26.1 Hz), 150.46, 149.01, 143.87, 140.13 (d, JCF¼231.2 Hz), 135.98,
125.03 (d, JCF¼33.8 Hz), 124.64, 109.54, 84.69, 84.59, 84.27, 83.80, 79.06,
70.65, 61.73, 61.50, 51.21, 37.92, 36.27, 12.26. 15N NMR d: 362.8, 353.3,
244.6, 157.8, 156.0, 143.0, 134.2. 19F NMR d: –91.10. HRMS (ESI–) calcd
for C22H25FN7O9 [M – H]– 550.1697, found 550.1510.
1
Compound 14d: H NMR (DMSO-d6) d: 11.84 (brs, 2 H), 8.21 (d,
JHF¼7.2 Hz, 1H), 8.11 (s, 1H), 7.93 (d, JHF¼7.0 Hz, 1H), 6.13 (td,
3
3JHH¼7.40 Hz 6.40 Hz, JHF¼1.70 Hz, 1H), 6.08 (td, JHH¼6.00 Hz 7.90 Hz,
3
3
JHF¼1.80 Hz, 1H), 5.52 (d, JHH¼4.3Hz, 1H), 5.22 (t, JHH¼4.9 Hz 4.9 Hz,
3
2
1H), 4.73 – 4.69 (dd, JHH¼4.30 Hz JHH¼14.30 Hz, 1H), 4.66 – 4.62 (dd,
3JHH¼8.00 Hz JHH¼14.30 Hz, 1H), 4.57 (s, 2 H), 4.27–4.23 (m,
2
3JHH¼3.70 Hz 6.45 Hz 3.50 Hz, 1H), 4.22 (dt, JHH¼2.50 Hz 5.80 Hz
3
3
2.20 Hz, 1H), 4.08 (dt, JHH¼3.50 Hz 8.00 Hz 4.30 Hz 1H), 3.98 (q,
3JHH¼2.20 Hz 3.50 Hz 3.50 Hz, 1H), 3.64 – 3.60 (dt, JHH¼3.50 Hz
3
2JHH¼11.70 Hz, 1H), 3.59 – 3.55 (dt, JHH¼3.50 Hz JHH¼11.70 Hz, 1H),
3
2
3
2
2.31 – 2.27 (ddd, JHH¼6.00 Hz 2.50 Hz JHH¼13.80 1H), 2.25 – 2.18 (m,
3JHH¼6.40 Hz 6.45 Hz JHH¼14.00 Hz 1H), 2.17–2.13 (m, JHH¼7.90 Hz
2
3
2
3
5.80 Hz JHH¼13.80 Hz, 1H), 2.13 – 2.10 (ddd, JHH¼7.40 Hz 3.70 Hz
2JHH¼14.00 Hz, 1H). 13C NMR d: 157.02 (d, JCF¼26.2 Hz), 157.01 (d,
JCF¼26.1 Hz), 149.03, 143.85, 140.15 (d, JCF¼231.2 Hz), 140.01 (d,
JCF¼230.4 Hz), 125.02 (d, JCF¼34.0 Hz), 124.66, 124.64 (d, JCF¼34.4 Hz),
84.88, 84.69, 84.58, 84.27, 78.83, 70.66, 61.78, 61.33, 51.22, 37.92, 36.71. 15N

24
D. BARANIAK ET AL.
NMR d: 363.0, 353.3, 244.6, 157.9, 135.6, 134.1. 19F NMR d: –91.11, 91.26
HRMS (ESIþ) calcd for C21H23F2N7NaO9 [M þ Na]þ 578.1423,
found 578.1427.
4.1.4. 30-O-Mesyl-50-O-trityl-5-fluoro-20-deoxyuridine (6)
In a round-bottomed flask equipped with a reflux condenser and CaCl2
tube, 5-fluoro-20-deoxyuridine (0.90 g, 0.36 mmol) was coevaporated two
times with anhydrous pyridine and next dissolved in pyridine (18 mL).
To the solution trityl chloride (1.25 g, 1.2 eq) was added and the mixture
was being stirred at 100 ꢁC for 30 min.[38] After that the mixture was
cooled to room temperature, followed by addition of the methanesulfonyl
chloride (0.44 mL, 1.5 eq) and the mixture was stirred at room tempera-
ture for 2 h (it was possible to carry out the reaction in the refrigerator
overnight). Then chloroform was added to the reaction mixture and it
was extracted with 5% aqueous solution of sodium bicarbonate. The
organic layer was dried over anhydrous magnesium sulfate, filtered and
evaporated to dryness. To the residue was added toluene and the mixture
was evaporated to remove traces of pyridine. The crude compound 6 was
dried in a vacuum exsiccator over P2O5 (a white powder, yield:
1.96 g, 95%).
4.1.5. 2,30-Anhydro-50-O-trityl-5-fluoro-20-deoxyuridine (7)
30-O-Mesyl-50-O-trityl-5-fluoro-20-deoxyuridine (6) (1.86 g, 3.28 mmol) was
dissolved in 96% ethanol (62 mL).[38] To a stirred solution sodium hydrox-
ide (1 M NaOH, 3.13 mL, 1 eq) was added and the mixture was heated in a
oil bath at 90 ꢁC for 10 min. It was observed the appearance of the product
7 - a white solid, which was not isolated. (To obtain the product 7, solvents
should be evaporated to dryness and add water to dissolve the salts –
sodium mesylate. The water-insoluble anhydro-derivative, can be filtered
off and recrystallized from ethanol.)
4.1.6. 1-(5-O-Trityl-2-deoxy-b-D-lyxofuranosyl)-5-fluorouracil (8)
To a stirred solution of 7 an additional portion of 1 M NaOH was added
and the solution allowed to remain at 85 – 90 ꢁC for 1 hour with stirring.
The pH of the solution was brought to 6 using 0.5% HCl and the volume
reduced in vacuum. Gradually the white product 8 was precipitated, filtered
off, washed with water and dried in a vacuum exsiccator over P2O5
(a white powder, yield: 85 – 95%).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
25
4.1.7. 1-(2-Deoxy-3-O-mesyl-5-O-trityl-b-D-threo-pentofuranosyl)-
5-fluorouracil (9)
To a stirred solution of compound 8 (0.48g, 0.98mmol) in pyridine (10mL)
was added methanesulfonyl chloride (0.23 mL, 2.93 mmol, 3.0 eqs) and the
stirring was continued at room temperature for 2 h. Then 5% aqueous solution
of sodium bicarbonate (20 mL) was added to the reaction mixture and it was
extracted with chloroform (3 x 30 mL). The organic layer was washed with
water (20 mL), dried over anhydrous magnesium sulfate, filtered and evapo-
rated to dryness. To the residue was added toluene (20 mL) and the mixture
was evaporated to remove traces of pyridine. The crude compound 9 was
dried in a vacuum exsiccator over P2O5 (a white powder, yield: 86 – 100%).
4.1.8. 30-Azido-50-trityl-5-fluoro-20,30-dideoxyuridine (10)
To a solution of 9 (1 g, 1.95 mmol) in DMF was added lithium azide
(0.14 g, 2.93 mmol) and the mixture was stirred at 90 ꢁC for 3 h. Then the
mixture was evaporated to dryness and the residue was purified by silica
gel column chromatography using methylene chloride – methanol
(10!15%, v/v) as eluent to give pure 10 (a white powder, yield: 87 – 97%).
4.1.9. 50-Azido-5-fluoro-20,50-dideoxyuridine (11b)
A solution of 50-O-tosyl-5-fluoro-20-deoxyuridine (300 mg, 1 mmol), sodium
azide (87 mg, 1.8 mmol) in N,N-dimethylformamide (DMF) (10 mL) was
stirred at 100 ꢁC for 3 h under an atmosphere of argon.[39,40] After cooling,
the solution was filtered, and the filtrate was evaporated under reduced
pressure. The residue was purified by silica gel column chromatography
using methylene chloride–methanol (5!15%, v/v) as eluent to give pure
1
11b (yield: 203 mg, 80 – 86%). H NMR (DMSO-d6) d: 11.87 (brs, 1H), 7.97
3
(d, JHF¼7.0 Hz, 1H), 6.16 (td, JHH¼7.56 Hz 6.18 Hz, JHF¼1.40 Hz, 1H),
3
3
5.42 (d, JHH¼4.30 Hz), 4.19–4.16 (m, JHH¼4.3 Hz 3.70 Hz 7.40 Hz 6.50 Hz,
3
1H), 3.86–3.83 (dt, JHH¼4.00 Hz 3.90 Hz 6.40 Hz), 3.63–3.61 (dd,
3JHH¼6.60 Hz JHH¼13.20 Hz, 1H), 3.57–3.53 (dd, JHH¼4.20 Hz JHH
¼
2
3
2
3
2
13.20 Hz, 1H), 2.29–2.24 (dd, JHH¼7.20 Hz 6.80 Hz JHH¼14.10 Hz, 1H),
2.12–2.08 (ddd, JHH¼3.8 Hz 6.40 Hz JHH¼13.60 Hz) 13C NMR d: 156.96
(d, JCF¼26.2 Hz) 149.06, 140.08 (d, JCF¼231.1 Hz), 124.92 (d, JCF¼33.9 Hz),
84.70, 84.50, 70.55, 51.57, 38.05 19F NMR d: –91.22
3
2
HRMS (ESI–) calcd for C9H9FN5O4 [M – H]– 270.0638, found 270.0633.
4.2. Biological evaluation
4.2.1. Cell cultures
Human KB (carcinoma nasopharynx), HeLa (cervical cancer cell line) and
MCF-7 (breast cancer cell line) were obtained from European Collection of

26
D. BARANIAK ET AL.
Cell Cultures (ECACC) supplied by Sigma-Aldrich. HDF (human dermal
fibroblasts) cell lines were purchased from American Type Culture
Collection (ATCC, Manassas, VA, USA). MCF-7 were cultured in DMEM
medium. HeLa cell line was cultured in RPMI-1640 medium, and KB cell
lines were grown in EMEM medium. Human dermal fibroblasts were
maintained in Fibroblasts Growth Medium. Each medium was supple-
mented with 10% fetal bovine serum, and 1% penicillin/streptomycin solu-
tions. All cells were maintained in a humidified atmosphere of 5% CO2
at 37 ꢁC.
4.2.2. In vitro cytotoxicity assay
The cytostatic effects of the investigated compounds were evaluated using
rapid colorimetrcic assay MTT. This method is based on the reaction
between mitochondria enzymes (dehydrogenases) of the cell lines: HeLa,
KB, MCF-7 and HDF and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT). The final product of this reaction gives violet forma-
zan crystals. The number of live cells is proportional to the amount of
formazan and is represented by the violet dye reagent. 180 mL of suspended
cells (2 x 104 cells/1 mL) was seeded in 96-well microplates. In the first row
which was considered as the blank probe, only medium was added. After
24 hours of incubation 20 mL of specific concentrations of the tested com-
pounds were added (0.1–20 micromoles) and incubated for the next
48 hours. Then 10 mL of MTT reagent was added to each well and the
plates were incubated for an additional 3 hours. The formazan crystals were
dissolved in 100 mL 10% natrium dodecylsulphate (NaDS) solution per well
and the absorbances were determined at 545 nm using Thermo
Spectrophotometer. The IC50 concentrations were calculated using
GraphPad Prism software. Statistical analyses for the cytotoxic activity were
carried out using the nonparametric Kruskal-Wallis H test for unpaired
data and ANOVA Friedman two-way analysis of variance test for paired
data. Statistical significance was tested using Dunn’s post-hoc test.
Acknowledgments
We are grateful to Prof. Maciej Stobiecki for ME ES analysis of novel compounds.
Financial support from the Polish Ministry of Science and Higher Education (statutory
financing and the KNOW program), is greatfully acknowledged.
ORCID
Dagmara Baraniak
http://orcid.org/0000-0002-0970-8297

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
27
Funding
Financial support from the Polish Ministry of Science and Higher Education (statutory
financing and the KNOW program), is greatfully acknowledged.
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