Gemcitabine, Pyrrologemcitabine, and 2′-Fluoro-2′-Deoxycytidines: Synthesis, Physical Properties, and Impact of Sugar Fluorination on Silver Ion Mediated Base Pairing

Article abstract of DOI:10.1002/chem.201703427

The stability of silver-mediated “dC-dC” base pairs relies not only on the structure of the nucleobase, but is also sensitive to structural modification of the sugar moiety. 2′-Fluorinated 2′-deoxycytidines with fluorine atoms in the arabino (up) and ribo (down) configuration as well as with geminal fluorine substitution (anticancer drug gemcitabine) and the novel fluorescent phenylpyrrolo-gemcitabine (^phPyrGem) have been synthesized. All the nucleosides display the recognition face of naturally occurring 2′-deoxycytidine. The nucleosides were converted into phosphoramidites and incorporated into 12-mer oligonucleotides by solid-phase synthesis. The addition of silver ions to DNA duplexes with a fluorine-modified “dC-dC” pair near the central position led to significant duplex stabilization. The increase in stability was higher for duplexes with fluorinated sugar residues than for those with an unchanged 2′-deoxyribose moiety. Similar observations were made for “dC-dT” pairs and to a minor extent for “dC-dA” pairs. The increase in silver ion mediated base-pair stability was reversed by annulation of a pyrrole ring to the cytosine moiety, as shown for 2′-fluorinated ^phPyrGem in comparison with phenylpyrrolo-dC (^phPyrdC). This phenomenon results from stereoelectronic effects induced by fluoro substitution, which are transmitted from the sugar moiety to the silver ion mediated base pairs. The extent of the effect depends on the number of fluorine substituents, their configuration, and the structure of the nucleobase.

Full text of DOI:10.1002/chem.201703427

A Journal of
Accepted Article
Title: Gemcitabine, Pyrrologemcitabine and 2'-Fluoro- 2'-
Deoxycytidines: Synthesis, Physical Properties and Impact of
Sugar Fluorination on Silver Ion Mediated Base Pairing
Authors: Frank Seela, Xiurong Guo, Peter Leonard, and Sachin
Asaram Ingale
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To be cited as: Chem. Eur. J. 10.1002/chem.201703427
Link to VoR: http://dx.doi.org/10.1002/chem.201703427
Supported by

10.1002/chem.201703427
Chemistry - A European Journal
Gemcitabine, Pyrrologemcitabine and 2’-Fluoro- 2’-Deoxycytidines: Synthesis, Physical
Properties and Impact of Sugar Fluorination on Silver Ion Mediated Base Pairing
Xiurong Guo,[a,b,+] Peter Leonard,[a,+] Sachin A. Ingale,[a,b,+] and Frank Seela*[a,b]
[a]Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstraße 11, 48149 Münster, Germany and [b]Laboratorium für Organische und
Bioorganische Chemie, Institut für Chemie neuer Materialien, Universität Osnabrück,
Barbarastraße 7, 49069 Osnabrück, Germany
[+] These authors contributed equally to this work
Corresponding author:
Prof. Dr. Frank Seela
Phone: +49 (0)251 53 406 500; Fax: +49 (0)251 53 406 857
E-mail: Frank.Seela@uni-osnabrueck.de
Homepage: www.seela.net
Supporting information for this article is available on the www under
http://dx.doi.org/10.1002/chem.2016xxxx.
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Abstract
The stability of silver mediated “dC-dC” base pairs relies not only on the structure of the
nucleobase but also is sensitive to structural modification on the sugar moiety. 2'-Fluorinated
2'-deoxycytidines with fluorine atoms in the arabino (up) and the ribo (down) configuration,
as well as with geminal fluorine substitution (anticancer drug gemcitabine) and the novel
fluorescent phenylpyrrolo-gemcitabine (phPyrGem) were synthesized. All nucleosides display
the recognition face of naturally occurring 2'-deoxycytidine. Nucleosides were converted into
phosphoramidites and incorporated in 12-mer oligonucleotides by solid-phase synthesis.
Addition of silver ions to DNA duplexes with a fluorine modified “dC-dC” pair near central
position led to significant duplex stabilization. The stability increase was higher for duplexes
with fluorinated sugar residues than those with the unchanged 2'-deoxyribose moiety. Similar
observations were made on “dC-dT” pairs and to a minor extend on the “dC-dA” pairs. The
increase of silver ion mediated base pair stability was reversed by annulation of a pyrrole ring
to the cytosine moiety as shown for 2'-fluorinated phPyrGem compared to phenylpyrrolo-dC
(phPyrdC). The phenomenon results from stereoelectronic effects induced by fluoro
substitution which are transmitted from the sugar moiety to the silver ion mediated base pairs.
This depends on the number of fluorine substituents, their configuration and the structure of
the nucleobase.
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Introduction
Metal mediated base pairs can function as an alternative (orthogonal) pairing system to
canonical Watson-Crick base pairing thereby forming double helical DNA and RNA.[1] The
introduction of metal-based functionalities provide nucleic acids unique chemical, physical
and biological properties. It allows the development of metal ion sensors, molecular wires,
DNA-based logic gates, and detection systems for single nucleotide polymorphisms.[2a-g]
Furthermore, metal mediated base pairs can be used to alter molecular properties of nucleic
acids such as electrical conductivity or charge-transfer.[2d,2h] The replacement of hydrogen
bonds within DNA base pairs by metal interactions might be used to expand the genetic
coding system.
As the persistence length of 50 nm makes DNA a rigid rod, metal DNA represents a unique
hybrid biomaterial with application in biotechnology and for the construction of nanodevices.
Recent developments on metal responsive systems demonstrated the utility of nucleic acid
metal ion assemblies.[1,2a-b] Nevertheless, applications utilizing metal DNA are still in an
initial state.
Amongst the various metal ions forming metal mediated base pairs, silver mediated base pairs
found special attention.[3] Silver ions have the capacity to interact with DNA or RNA[4] and
form extremely stable base pairs that surpass stability of canonical Watson-Crick pairs
stabilized by hydrogen bonds. An interesting aspect of the Ag+ ion is their selective binding to
nucleobases instead to the phosphodiester backbone. Often, metal mediated base pairs are
formed between identical nucleobases (homo base pairs).[5] In this regard, the dC-dC homo
pair is of particular importance. It is naturally occurring and forms stable silver ion complexes
[6] as well as stable silver mediated dC-dC homo base pairs and pairs between anomeric dC
nucleosides.[7] One or two silver ions can be captured by one base pair[8] and silver-mediated
base pairs are recognized by polymerases during DNA-amplification.[9] An X-ray structure of
a RNA duplex gave a detailed picture on the molecular structure of the C-Ag+-C interaction
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within the RNA double helix.[10a] More recently, the crystal structure of a DNA duplex was
reported which forms silver(I)-DNA hybrid nanowires consisting of silver-mediated base
pairs formed by natural nucleobases.[10b]
Earlier work on the Watson-Crick base pairing system has shown that structural modifications
on the nucleobases have a strong impact on the base pair stability. Regarding silver mediated
base pairs similar observations have been made.[11] The annulation of a pyrrole ring or an
imidazole moiety to the cytosine base led to very stable metal-mediated DNA. So far, pyrrolo-
dC and imidazolo-dC form the most stable silver mediated “dC-dC” base pairs in double
helical DNA.[12] As pyrrolo-dC and imidazolo-dC are strongly fluorescent, the silver mediated
base pairs are also fluorescent providing the system interesting new properties for detection.
Nevertheless, also ribose modification can affect base pair stability as it is reported for DNA
vs RNA.[13] As almost nothing is known on the impact of sugar modification on silver
mediated base pairs formed by canonical bases in DNA we anticipated that structural changes
on the sugar moiety can alter the properties of silver mediated base pairing as well. This was
considered as stereoelectronic effects induced in the sugar moiety might be transmitted to the
nucleobase. To this end, we selected the fluorine atom to functionalize the 2’-position in the
dC-Ag+-dC pair. Fluorine substituents are known to stabilize the anomeric center with regard
to glycosylic bond hydrolysis due to the negative inductive effect. Furthermore, the beneficial
influence of fluorine substituents is well established in therapeutic applications of nucleosides
and oligonucleotides. The 2’-difluoro-dC nucleoside gemcitabine 3 is used for therapeutic
application as drug to treat pancreatic cancer. Antisense oligonucleotides containing fluorine
atoms at the 2'-position of the ribose moiety have shown to develop remarkable properties and
act as polymeric drugs.[14]
This work investigates the influence of 2'-fluoro substituents on the formation and stability of
silver mediated homo "dC-dC" and “pyrrolo-dC-pyrrolo-dC” base pairs. The fluorinated
nucleosides used in this study are shown in Figure 1. These nucleosides contain cytosine or
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cytosine derivatives as nucleobases that all show the recognition face of the naturally
occurring nucleoside. Some of them were known (1-4)[15] others had to be synthesized (5-6).
Phosphoramidites were prepared and used as building blocks for solid phase synthesis. The
influence of 2’-fluoro up (arabino), 2’-fluoro down (ribo) and geminal fluorine
functionalization was studied with regard to metal ion mediated base pairing. Also, an
anticipated fluorine effect was compared between dC- and phenylpyrrolo-dC residues. This
included the anticancer drug gemcitabine having two geminal fluorine atoms in the 2’-
position.
Figure 1. Structures of modified dC nucleosides.
Results and Discussion
1. Synthesis of Monomers and their Physical Properties.
For this study nucleosides 1 to 6 were synthesized. Syntheses of nucleosides 1-4[15] were
performed according to reported literature protocols whereas the syntheses of compounds 5
and 6 are described in this manuscript.
To access phPyrGem (6) two different synthetic routes (Schemes 1 and 2) were exercised. In
both cases gemcitabine (3) was used as starting material and iodinated to 5-iodo-gemcitabine
(4). Iodination of 3 has been already reported[15f] but the reaction was performed on a
milligram scale with moderate yield (58%). Moreover, glacial acetic acid was utilized as
reaction medium which complicated work-up.[15f] Now, a multigram scale protocol was
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developed using ethanol as solvent. Due to this change, the yield was increased to 90%, the
work up of the reaction mixture was simplified and gram amounts of 5-iodo-gemcitabine (4)
became easily accessible (Scheme 1).
In the first route, 5-iodo-gemcitabine (4) was converted to 5 (81% yield) by Sonogashira
cross-coupling (DMF, DIPEA, [Pd(PPh3)4], CuI), with a 4-fold excess of phenylacetylene
(Scheme 1). Protection of the 5′-OH group of 5 with a 4,4’-dimethoxytrityl chloride followed
by amino group blocking with an acetyl residue gave nucleoside 8. Formation of the pyrrole
system was achieved by intramolecular cyclization using CuI and triethylamine (→ 9). Final
cleavage of the DMT residue with trichloroacetic acid gave nucleoside 6 in 31% overall yield
(5 steps) starting from 5-iodo-gemcitabine (4).
Scheme 1. Synthesis of nucleosides 4-6. Reagents and conditions: (i) CCl4, I2, HIO3, EtOH,
45 °C, 16 h; (ii) Pd(PPh3)4, CuI, DIPEA, phenylacetylene, DMF, rt, 16 h; (iii) DMT-Cl,
pyridine, rt, 12 h; (iv) acetic anhydride, DMF, rt, 12 h; (v) CuI, triethylamine, DMF, 60 °C, 24
h; (vi) trichloroacetic acid (TCA), rt, 15 min.
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In the second route, the 5’-OH group of 5-iodo-gemcitabine (4) was protected with a DMT
residue and the amino group with an acetyl group to yield the protected iodo nucleoside 11
(Scheme 2). Then, Sonogashira cross-coupling of 11 with phenylacetylene and subsequent
cyclization gave nucleoside 9 in 58% in two steps. Cleavage of the DMT residue gave
phPyrGem (6) in 33% overall yield (4 steps). Both routes gave comparable overall yields but
the second route is one step shorter.
Scheme 2. Synthesis of nucleoside 6. Reagents and Conditions: (i) DMT-Cl, pyridine, rt, 12
h; (ii) acetic anhydride, DMF, rt, 12 h; (iii) Pd(PPh3)4, CuI, triethylamine, phenylacetylene,
DMF, rt, 16 h; (iv) trichloroacetic acid (TCA), rt, 15 min.
Recently, Hudson et al reported that 4,4’-dimethoxytritylation of 6-arylpyrrolocytidine
analogs resulted in a sluggish reaction.[16] Therefore, they added triethylamine to the reaction
mixture. However, this gave C7-DMT-subtituted arylpyrrolocytidines as main products.[16]
We found, when phPyrGem (6) was treated with 3.5 eq. of DMT-Cl in the absence of
triethylamine for 6 h the 5’-O-DMT protected nucleoside 9 was formed in 87% yield without
formation of a C7-functionalized DMT product. Under the same reaction conditions the DMT
protected nucleoside 13 was obtained from phPyrdC (12) in 70% yield. Also in this case
formation of a C-7-DMT product was not observed. Most probably, triethylamine activates
the 7-position of the nucleobase for electrophilic attack as described for the Mannich reaction
or for electrophilic halogenation.[17]
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Scheme 3. Synthesis of DMT-protected nucleosides 9 and 13.
Next, the physical properties of fluorinated nucleosides were investigated as they are
considered to be of importance in regard to formation of silver mediated base pairs. Figure 2
shows the chromatographic behavior of nucleosides on hydrophobic RP-18 silica resin and
demonstrates the influence of fluorine substituents on the lipophilicity of various cytidine
(Figure 2a) and pyrrolo-dC derivatives (Figure 2b). In the cytidine series, the ribonucleoside
is the most hydrophilic compound whereas gemcitabine the most lipophilic molecule.
However, also antipodal changes of 2’-fluoro substituents have a significant impact. The 2’-
fluoro-up nucleoside 2 is significantly more lipophilic as nucleoside 1 with the fluorine atom
in down position. Figure 2b demonstrates the increasing lipophilicity of phPyrdC (12) to
phPyrGem (6) and the even higher lipophilicity of the open chain phPyrGem precursor 5.
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Figure 2. Reversed-phase HPLC elution profiles of nucleosides: a) artificial mixture of
cytidine, 2’-deoxycytidine and 2’-deoxy-2’-fluorocytidine nucleosides 1, 2, and 3 monitored
at 260 nm using gradient system III; b) artificial mixture of nucleosides 5, 6, and 12
monitored at 260 nm using gradient system IV.
Furthermore, it has been found that fluorine substituents on the sugar moiety change the
conformation compared to ribo- or deoxyribonucleosides. Whereas a fluorine substituent in
the 2’-fluoro-up position enhances the preference for the S-type pseudorotamers, the fluorine
substituent in the down position increases the amount with N-type conformation. Geminal
fluoro substituents enhance the preference of an S-type sugar pucker in the solid-state,
whereas the N-type pucker is preferred within an oligonucleotide.[18] Changes on the pKa
value of the nucleobase are also observed going back to the fluorine modification of the sugar
residue. As already reported 2’-deoxycytidine (dC, pKa = 4.3)[12b] has a similar pKa value as
cytidine (C, pKa = 4.3).[19a] However, mono-fluoro functionalization at the 2’-position
reduces the pKa value independently from fluoro-down (1, pKa = 3.9)[15d] or fluoro-up (2, pKa
= 3.9)[19b] positioning. Gemcitabine (3, pKa = 3.6)[19c] shows the lowest pKa value due to the
two geminal electronegative fluorine substituents. The same is true for the pKa values of
phPyrGem (6, pKa = 2.3 and 11.0, see Figure S1) which are lower than those of phPyrdC (12,
pKa = 2.7 and 11.5).[12b]
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For oligonucleotides to be accessed, nucleosides 3-6 were converted into phosphoramidite
building blocks 16a-16c and 17 (Schemes 4 and 5) whereas the syntheses of
phosphoramidites of nucleosides 1 and 2 were already reported (for structures see Figure
S2).[20, 21] Also, a phosphoramidite building block of 3[22] was described with a benzoyl
protected amino group. For our study and better solubility we prepared dibutylformamidine
derivatives. First, nucleosides 3-5 were protected at the 4-amino group with a
dibutylformamidine residue affording the protected intermediates 14a (52%), 14b (78%) and
14c (77%) respectively. Then, compounds 14a-14c were converted to 5′-O-DMT derivatives
15a (71%), 15b (73%) and 15c (73%), respectively. Finally, phosphitylation of nucleosides
15a-15c under standard conditions yielded the phosphoramidites 16a (75%), 16b (61%) and
16c (80%) respectively (Scheme 4).
Scheme 4. Synthesis of phosphoramidites 16a-16c. Reagents and conditions: (i) N,N-
dibutylformamid dimethylacetal, methanol, 30 °C; (ii) DMT-Cl, pyridine, rt, 12 h; (iii)
DIPEA, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, CH2Cl2, rt, 30 min.
The 5′-O-DMT protected nucleoside 9 was converted to phosphoramidite 17 in 89% yield
(Scheme 5). All new synthesized compounds were characterized by 1H-, 13C-NMR spectra as
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well as ESI-TOF mass spectra (see Experimental section). The 1H-13C correlated (HMBC and
HSQC) NMR spectra were used to assign the 13C NMR signals. For details see the
Experimental section (for spectra, see the Supporting Information).
Scheme 5. Synthesis of phosphoramidite 17.
2. Synthesis, Characterization and Photophysical Properties of Oligonucleotides.
Oligonucleotides (ODNs) containing nucleosides 1-6 were prepared on solid-phase using the
phosphoramidites of nucleosides 1-2, phosphoramidites 16a-16c and 17 together with
standard building blocks. Nucleosides 1-6 were incorporated in central positions of the
oligonucleotides 5′-d(TAG GTC AAT ACT) (ODN-1) and 3′-d(ATC CAG TTA TGA)
(ODN-2) replacing dC and dG residues. After solid-phase synthesis, the oligonucleotides
were cleaved from the solid support and deprotected in concentrated 28% aqueous ammonia
at 55 °C for 2 h. The coupling yields of the modified building blocks were always higher than
95%. The ODNs containing nucleoside 6 (phPyrGem) are not stable in ammonia at elevated
temperature. Therefore, oligonucleotides containing nucleoside 6 were deprotected in 28%
aqueous ammonia for 16 h at room temperature. All synthesized oligonucleotides were
purified by reversed-phase HPLC (RP-18), detritylated with 2.5% dichloroacetic acid in
dichloromethane and again purified by HPLC. The contents of single peaks were isolated in
all cases (Figure S3, Supporting Information). Subsequently, the molecular masses were
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determined by MALDI-TOF mass spectra. Table 1 displays all modified oligonucleotides
used in this study together with their mass spectrometric data.
Table 1. Synthesized oligonucleotides and their molecular masses measured by MALDI-TOF
mass spectra.
M.W. calcd.[a]
M.W. found [b]
M.W. calcd.[a]
Entry
Oligonucleotide
Entry
Oligonucleotide
M.W. found [b]
3603.6
3604.4
3806.3
3806.3
ODN-1
5’-d-(TAG GTC AAT ACT)
ODN-11
5’-d-(TAG GT4 AAT ACT)
3643.6
3643.7
3766.3
3765.8
ODN-2
ODN-3
ODN-4
ODN-5
ODN-6
ODN-7
ODN-8
ODN-9
ODN-10
5’-d(AGT ATT GAC CTA)
5’-d(AGT ATT AAC CTA)
5’-d(AGT ATT TAC CTA)
5’-d-(TAG GT1 AAT ACT)
5’-d(AGT ATT 1AC CTA)
5’-d-(TAG GT2 AAT ACT)
5’-d(AGT ATT 2AC CTA)
5’-d-(TAG GT3 AAT ACT)
5’-d(AGT ATT 3AC CTA)
ODN-12
ODN-13
ODN-14
ODN-15
ODN-16
ODN-17
ODN-18
ODN-19
5’-d(AGT ATT 4AC CTA)
5’-d-(TAG GT5 AAT ACT)
5’-d(AGT ATT 5AC CTA)
5’-d-(TAG GT6 AAT ACT)
5’-d(AGT ATT 6AC CTA)
5’-d-(TAG GT12 AAT ACT)[12a]
5’-d(AGT ATT 12AC CTA)[12a]
5’-d(AGT ATT CAC CTA)[11b ]
3627.6
3629.2
3780.5
3780.2
3618.6
3619.3
3740.5
3740.4
3662.4
3662.3
3780.5
3781.5
3622.4
3622.8
3740.5
3739.9
3662.4
3664.3
3622.4
3622.8
3680.4
3680.3
3640.4
3640.6
[a] Calculated on the basis of molecular weight as [M+H]+. [b] Determined by MALDI-TOF mass spectrometry as [M+H]+ in
the linear positive mode.
Phenylpyrrolo-dC (phPyrdC) is commonly known as a strongly fluorescent nucleoside
containing a 2-oxopyrrolo[2,3-d]pyrimidine “isohypoxanthine” system.[23] Therefore, it is
widely used to explore the structure and dynamics of nucleic acids.[24] Consequently, we
investigated the fluorescence properties of the 2’-difluoro modified phPyrGem 6. Fluorescence
measurement of 6 was performed and compared to phPyrdC (12).[12b] Resulting spectra are
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displayed in Figure 3. Phenylpyrrolo nucleosides 6 and 12 show almost identical UV maxima
(359 nm for 6, and 358 nm for 12, Figure S4), fluorescence maxima (461 nm for 6, and 458
nm for 12) and Stoke’s shift (102 nm for 6, and 100 nm for 12). The fluorescence intensity of
phPyrGem (6) is about 1.4 fold lower than that of phPyrdC (12). Stereoelectronic effects
induced by geminal fluorine atoms might be responsible for these changes.
Next, the photophysical properties of phPyrGem (6) in a single-stranded (ss) oligonucleotide
and an oligonucleotide duplex (ds) were investigated. Identical concentrations in aqueous
buffer were used. A significant UV/vis bathochromic shift (11 nm) is observed when
phenylpyrrolo gemcitabine (6) is incorporated in single-stranded ODN-15 and a 7 nm UV/vis
bathochromic shift is observed when the duplex ODN-15•ODN-2 is formed (Figure S4).
Compound 6 and single-stranded ODN-15 show almost identical fluorescence maxima (461
nm) with no fluorescence quenching among nucleoside 6 and ODN-15. In contrary, the
fluorescence is strongly decreased upon oligonucleotide duplex formation (ODN-15•ODN-2)
because of increased stacking interactions with neighboring bases. The duplex ODN-
15•ODN-2 containing 6 shows fluorescence at a wavelength similar to that of the monomeric
nucleoside 6 and ODN-15.
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Figure 3. Fluorescence spectra of a) monomeric nucleosides 6 and 12; b) single-stranded
ODN-15 and duplex ODN-15•ODN-2. All measurements were performed in 100 mM
NaOAc, 10 mM Mg(OAc)2 buffer (pH 7.4) at 5 µM concentration. Excitation wavelength:
368 nm. For solubility reasons 0.4% DMSO was used in case of monomeric nucleoside.
3. Influence of 2'-Fluorination on the Stability of Canonical and Silver Mediated Base
Pairs
The fluorine-carbon bond stability (109 kcal/mol)[25] makes fluorine substituents rather
resistant to metabolic transformations. The hydrophobic, but polar fluorine atom has
approximately the same atomic radius as hydrogen. Therefore, it is well accommodated in
DNA and DNA-RNA hydrids.[20,21,26] However, the fluorine van-der-Waals radius is larger
(1.47 Å) than that of hydrogen (1.20 Å) and similar to that of oxygen (1.52 Å).[27] Fluorine
has the highest electronegativity of all elements. Consequently, fluorine has been incorporated
in nucleosides, nucleotides and nucleic acids to alter chemical and medicinal properties.[28]
Gemcitabine (3) is a prominent example and one of the widely used anticancer drugs. It is
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incorporated in DNA and inhibits chain elongation.[29] The modification of the sugar by
fluorine functionalization has also a large impact on DNA stability and activity.[20,21,26]
Modification of the sugar moiety effects base pair stability by stereoelectronic effects,
increases lipophilicity and causes conformational changes.[30] Stereoelectronic effect is a term
combining steric effects and electronic properties. Both effects can be induced by fluorine
functionalization of the sugar moiety. Steric effects depend on the steric demand of
substituents whereas the electronic effects depend on the electronegativity of substituents.
Both phenomena can influence sugar conformation and base pair stability.
As nothing is known on the influence of ribose functionalization on the stability of silver
mediated base pairs formed by dC-dC mismatches and closely related derivatives we decided
to study this matter and to investigate the influence of antipodal and bis-fluorine
functionalization of dC (nucleosides 1-6 and 12). This includes functionalization of the
canonical dC-dG pair, base pair mismatches of dC-dA and dC-dT and most importantly, the
homo dC-dC pair. All investigations were performed on the standard duplex 5’-d-
(TAGGTCAATACT) • 3’-d-(ATCCAGTTATGA) ODN-1•ODN-2 containing the modified
base pairs in a near central position embedded in dA-dT pairs and replacing the canonical dC-
dG pair. The studies used Tm measurements to determine the duplex stability in the presence
and absence of silver ions.
In the first series of experiments (Table 2, left column, Table S1 and Figure S5) the dC
residues in the dC-dG pair were replaced by nucleosides 1-6 and 12 within 12-mer duplexes.
Without silver ions most duplexes showed very similar Tm values with regard to the
unmodified counterpart (around 47°C) indicating that a fluorine mono-functionalization in the
up or down position has very little influence on the base pair stability. However, geminal
fluorine functionalization (gemcitabine, 3) destabilizes the duplex, a 5-iodo substituent
(nucleoside 4) compensates the decrease, whereas a phenylacetylene residue in 5-position
(nucleoside 5) has a negative impact. Replacing dC with phPyrdC (12) or phPyrGem (6) in the
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“dC-dG” pair leads to base pair stabilization and significantly higher Tm values. When silver
ions were added the duplex stability did not change in any case. Therefore, it was concluded
that silver ions do not participate in “dC- dG” base pair formation neither in the case of
unmodified nor in 2’-fluorine modified oligonucleotides.
Next, the impact of fluorine modification was studied on duplexes with dC-dA and dC-dT
hetero base pairs (mismatches, Table 2, middle and right column, Figures S6 and S7). Also in
these cases duplexes incorporating nucleosides 1-6 and 12 showed very similar stabilities in
the absence of silver ions except those with the pyrrolo-dC skeleton that are more stable. Most
probably the increased duplex stability of pyrrolo-dC mismatches in the absence of silver ions
relies on increased stacking interactions between the phenyl residues of the phenylpyrrolo-dC
skeleton with nucleobase neighbors. Addition of silver ions leads to a Tm increase in almost
every case but not for the pyrrolo-dC derivatives 6 and 12 and the precursor 5. The stability of
fluorine modified “dC-dA” and “dC-dT” base pairs is similar to that of the unmodified base
pairs for compound 1-3 showing that fluorine modification has very little influence on
mismatch stability. Moreover, for “dC-dA” mismatches two silver ions were required to
obtain the highest duplex stability (Tm). Taken together, increase of Tm values in the presence
of silver ions points to the formation of silver mediated base pairs which is in line with
observations made on other silver mediated “dC-dA” and “dC-dT” mismatches without
fluorine modification.[9c,31] Substituents at the 5-position of the base destabilizes the silver ion
mediated base pairs.
16
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Chemistry - A European Journal
Table 2. Tm values (°C) of duplexes with modified dC residues opposite to dG, dA and dT in
the absence and presence of silver ions.
Tm [°C] with n equiv of Ag+/modified base pair[a]
5’-d-(TAG GTX AAT ACT)
3’-d-(ATC CAY TTA TGA)
Y= dG (ODN-2)
Y= dA (ODN-3)
Y= dT (ODN-4)
n=0
n=1
n=2
n=0
n=1
n=2
n=0
n=1
n=2
(ΔTm)[b]
(ΔTm)[b]
(ΔTm)[b]
(ΔTm)[b]
(ΔTm)[b]
(ΔTm)[b]
47.5
(+0.5)
47.0
(+0.0)
29.0
(+4.5)
27.5
(+4.0)
30.0
32.0
(+7.5)
30.5
(+7.0)
32.5
34.5
(+9.0)
31.0
(+5.0)
34.0
35.5
(+10.0)
32.5
(+6.5)
35.5
X = dC (ODN-1)
X= 1 (ODN-5)
X= 2 (ODN-7)
47.0
46.5
47.0
24.5
23.5
24.0
25.5
26.0
26.0
47.0
(+0.5)
47.5
(+1.0)
47.5
(+0.5)
47.5
(+0.5)
(+6.0)
(+8.5)
(+8.0)
(+9.5)
30.0
(+7.5)
32.0
(+9.5)
35.5
(+10.0)
34.0
(+8.5)
44.5
(+1.0)
44.5
(+1.0)
X= 3 (ODN-9)
22.5
25.5
43.5
25.0
(+1.5)
24.5
(+0.5)
31.0
(+3.0)
30.5
(-0.5)
29.0
(+5.5)
26.0
(+2.0)
30.0
(+2.0)
30.5
(-0.5)
29.0
(+1.0)
26.5
(0.0)
36.0
(+2.5)
35.0
(+0.5)
30.0
(+2.0)
27.5
(+1.0)
36.0
(+2.5)
36.0
(+1.5)
48.0
(+0.5)
48.0
(+0.5)
X= 4 (ODN-11)
X= 5 (ODN-13)
X= 6 (ODN-15)
X= 12 (ODN-17)
23.5
24.0
28.0
31.0
28.0
26.5
33.5
34.5
47.5
43.5
49.5
51.0
45.0
(+1.5)
45.0
(+1.5)
50.5
(+1.0)
50.0
(+0.5)
52.0
(+1.0)
52.0
(+1.0)
[a] Measured at 260 nm with 5 μM + 5 μM single-strand concentration at a heating rate of 1.0 °C/min in 100 mM NaOAc, 10 mM Mg(OAc)2, pH
= 7.4 in the presence of various concentrations of AgNO3 (0–2.0 equiv.). [b] ΔTm = Tm (after addition of 1 or 2 equiv AgNO3) - Tm (before
addition of AgNO3).
Finally, the influence of fluorine modification on metal mediated “dC-dC” homo base pair
was studied in the absence and presence of one or two equivalents of silver ions.
Oligonucleotides incorporating nucleosides 1-6 and 12 were utilized and were hybridized with
complementary strands containing the same modified “dC” residues in opposite position. The
resulting duplexes contain the same fluorine modification in both strands (homo base pairs).
Base pairing was also investigated on hetero base pairs with duplexes incorporating
nucleosides 1-6 and 12 opposite to dC (Table 3, Figure S9). In this case only one strand of the
duplexes contains a fluorine modified sugar moiety. Non-modified duplexes containing dC-
dC base pairs were used as a control.
17
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Table 3. Tm values (°C) of duplexes containing modified dC residues in homo base pairs and
opposite to dC in the absence and presence of silver ions.[a]
Tm [°C] with n equiv of
Tm [°C] with n equiv of
Ag+/modified base pair
Ag+/modified base pair
Duplexes
Duplexes
n=0
n=1
n=2
n=0
n=1
n=2
(ΔTm)[b]
(ΔTm)[b]
(ΔTm)[b]
(ΔTm)[b]
5’-d-(TAG GTC AAT ACT) (ODN-1)
3’-d-(ATC CAC TTA TGA) (ODN-19)
26.5
34.0
(+7.5)
35.5
(+9.0)
5’-d-(TAG GT1 AAT ACT) (ODN-5)
3’-d-(ATC CA1 TTA TGA) (ODN-6)
23.0
22.0
20.0
21.0
33.5
34.5
5’-d-(TAG GT1 AAT ACT) (ODN-5)
3’-d-(ATC CAC TTA TGA) (ODN-19)
25.5
20.5
<20
22.0
34.5
(+9.0)
35.5
(+10.0)
(+10.5) (+11.5)
5’-d-(TAG GT2 AAT ACT) (ODN-7)
3’-d-(ATC CA2 TTA TGA) (ODN-8)
36.5
37.5
5’-d-(TAG GT2 AAT ACT) (ODN-7)
3’-d-(ATC CAC TTA TGA) (ODN-19)
36.0
(+15.5)
37.0
(+16.5)
(+14.5) (+15.5)
5’-d-(TAG GT3 AAT ACT) (ODN-9)
3’-d-(ATC CA3 TTA TGA) (ODN-10)
31.5
33.0
5’-d-(TAG GT3 AAT ACT) (ODN-9)
3’-d-(ATC CAC TTA TGA) (ODN-19)
33.0
(>+13.
0)
28.0
(+6.0)
34.5
(>+14.5)
(+11.5) (+13.0)
5’-d-(TAG GT4 AAT ACT) (ODN-11)
3’-d-(ATC CA4 TTA TGA) (ODN-12)
28.0
(+7.0)
28.5
(+7.5)
5’-d-(TAG GT4 AAT ACT) (ODN-11)
3’-d-(ATC CAC TTA TGA) (ODN-19)
29.5
(+7.5)
5’-d-(TAG GT5 AAT ACT) (ODN-13)
3’-d-(ATC CA5 TTA TGA) (ODN-14)
24.0
34.0
20.5
(-3.5)
21.5
(-2.5)
5’-d-(TAG GT5 AAT ACT) (ODN-13)
3’-d-(ATC CAC TTA TGA) (ODN-19)
29.5
31.0
33.0
(+3.5)
32.0
(+2.5)
5’-d-( TAG GT6 AAT ACT) (ODN-15)
3’-d-(ATC CA6 TTA TGA) (ODN-16)
46.5
47.0
5’-d-(TAG GT6 AAT ACT) (ODN-15)
3’-d-(ATC CAC TTA TGA) (ODN-19)
37.0
(+6.0)
37.5
(+6.5)
(+12.5) (+13.0)
5’-d-(TAG GT12 AAT ACT) (ODN-17) 35.0[12b]
3’-d-(ATC CA12 TTA TGA) (ODN-18)
30/58[c]
57.5
5’-d-(TAG GT12 AAT ACT) (ODN-17) 33.0
3’-d-(ATC CAC TTA TGA) (ODN-19)
35.5
(+2.5)
36.5
(+3.5)
(+22.5)
[a] Measured at 260 nm with 5 μM + 5 μM single-strand concentration at a heating rate of 1.0 °C/min in 100 mM NaOAc, 10 mM
Mg(OAc)2, pH = 7.4 in the presence of various concentrations of AgNO3 (0–2.0 equiv.). [b] ΔTm = Tm (after addition of 1 or 2 equiv
AgNO3) - Tm (before addition of AgNO3). [c] Biphasic melting.
At first homo base pairs (left hand side of Table 3, Figure S8) were studied. In general the Tm
values of duplexes containing homo base pairs in the absence of silver are significantly lower
than the Tm of the reference duplex. For duplexes incorporating the phenylpyrrolo nucleosides
6 and 12 the highest Tm values were observed. After addition of silver the duplex stability
increased significantly (7.5°C to 22.5°C) except for duplex ODN-13•ODN-14 containing 5-
phenylacetylene-gemcitabine (5). Duplexes with hetero base pairs with only one dC
modification in the silver mediated base pair (Table 3, right side) show low Tm values in the
18
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10.1002/chem.201703427
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absence of silver ions as it was observed for the homo base pairs. Addition of silver ions leads
to a significant increase of duplex stability pointing to silver mediated base pair formation.
The ∆Tm changes resulting from the addition of silver ions are shown in Table 3 and are also
displayed in the bar diagram of Figure 4. From this, it can be concluded that dC residues
bearing fluorine atoms in 2-position of the sugar residues either in “up”, “down” or “geminal”
positioning strengthen the stability of silver-mediated “dC-dC” homo base pairs (fluorine
substituent in both strands) or “dC-dC” hetero base pairs (fluorine substituent in one strand).
Interestingly, nucleosides 1-4 show similar Tm values no matter if incorporated in silver
mediated “dC-dC” homo or “dC-dC” hetero base pairs. Thus, fluorine modification in one
strand leads already to significant duplex stabilization whereas modification in both strands
did not lead to a substantial additional increase. Substituents in 5-position of gemcitabine
have a negative impact on these base pairs (homo and hetero pair). For phPyrGem 6 the
situation is different. Geminal difluoro substituents introduced to silver mediated phPyrdC
homo base pairs are destabilizing. Apparently, the two electronegative fluorine substituents on
the sugar moiety have a different impact on the electronic properties of the cytosine base of
gemcitabine (3) and the phenylpyrrolopyrimidine base of phPyrGem (6).
19
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Figure 4. Bar diagrams showing ∆Tm values of the DNA duplex 5’-d-(TAG GTC AAT ACT)
• 3’-d-(ATC CAC TTA TGA) ODN-1•ODN-19 modified with dC analogs 1-6 and 12. ∆Tm
corresponds to Tm after addition of 2 equiv AgNO3 - Tm before addition of AgNO3.
In order to verify the formation of silver-mediated “dC-dC” pairs, and to determine the
amount of silver ions bound to the homo base pairs and hetero base pairs, stoichiometric
titration experiments were performed using UV absorbance or fluorescence changes (Figure 5
a-f). From the titration curves it can be concluded that homo base pairs of 1 (fluoro-down dC)
and 2 (fluoro-up dC) bind one silver ion per base pair. In case of phPyrGem (6) two silver ions
are bound as reported for phPyrdC (12).[12b] For gemcitabine (3) the situation is more complex.
Contrary to oligonucleotide duplexes incorporating the mono fluoro nucleosides 1 or 2 the
duplex with gemcitabine 3 binds two silver ions. However, a break is observed during the
titration. After addition of one silver ion the second silver ion is bound less strongly (Figure
5d). Apparently, two electronegative fluorine substituents alter the electronic surface potential
of the duplex. This electronic perturbation has been already discussed in other cases[18b] and is
20
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10.1002/chem.201703427
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the source of the one electron oxidation of gemcitabine and the formation of C-2' and C-3'
sugar radicals.[32] Furthermore, the sugar moiety of gemcitabine adopts the N-conformation in
oligonucleotide duplexes whereas dC displays an S-sugar pucker.[18b] The high
electronegativity of two fluorine substituents might be the reason for the special behavior of
gemcitabine. Additional factors such as the enhanced lipophilicity of fluorinated nucleoside
residues have to be considered. Nevertheless, a base pair stability increase is not observed
when the second silver ion is bound. Thus, the second silver ion is not participating in base
pairing or is necessary to compensate the negative impact of geminal fluorine modification.
Figure 5. Titration graphs displaying consumption of equivalents of silver/duplex vs changes
in UV absorbance (measured at 260 nm) or fluorescence emission (measured at 460 nm): a)
ODN-1•ODN-19 (dC-dC); b) ODN-5•ODN-6 (1-1); c) ODN-7•ODN-8 (2-2); d) ODN-
9•ODN-10 (3-3); e) ODN-15•ODN-16 (6-6); f) fluorescence titration for duplex ODN-15•
ODN-16 (6-6). All experiments were performed in aqueous 100 mM NaOAc, 10 mM
Mg(OAc)2, pH = 7.4.
21
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6. Conclusion
DNA functionalization at the 2'-position of the sugar moiety has a strong impact on the
formation and stability of silver mediated "dC-dC" base pairs. In this study, dC analogs with
fluorine in the arabino (up) and ribo (down) configuration, as well as the anticancer drug
gemcitabine 3 and the new fluorescent phPyrGem 6 analog both with two geminal fluorine
substituents were incorporated in 12-mer DNA fragments near central positions utilizing
phosphoramidites (16a-c, 17) and solid-phase synthesis. All modified nucleosides display the
same recognition face as the naturally occurring 2'-deoxycytidine. To this end, a series of new
2'-deoxycytidine analogs were synthesized; one with an annulated pyrrole ring (6) and two 5-
substituted derivatives (4, 5). Starting material was 5-iodogemcitabine (4) which was
converted to the 5-phenylacetylene derivative 5 by Sonagashira cross coupling. After
cyclization phPyrGem (6) was obtained. An alternative route utilizing a different sequence of
reaction steps was established. Photophysical data of nucleosides, oligonucleotides and
duplexes demonstrate reduced fluorescence of phPyrGem (6) with two geminal fluorine atoms
in the sugar moiety compared to the unmodified phPyrdC (12).
The impact of antipodal (fluoro-up/fluoro-down) and geminal 2’-fluorine modification
(gemcitabine and phenylpyrrolo-gemcitabine) was studied by hybridization of complementary
strands (12-mers) with mismatches in the absence and presence of silver ions. Addition of
silver ions to fluorine modified “dC-dC” pairs led to significant duplex stabilization. The
increase measured by Tm changes was significantly higher for duplexes with fluorinated sugar
residues than for those with 2'-deoxycytidine. Similar observations found for "dC-dC" pairs
were made on “dC-dT” pairs and to a minor extend on the “dC-dA” pair. The increase of
silver ion mediated base pair stability was reversed by annulation of a pyrrole ring to the
cytosine moiety as shown for 2'-fluorinated phPyrGem (6) compared to phPyrdC (12).
Apparently, the nucleobase structure is of utmost importance for the transmission of
electronic effects from the sugar moiety to the metal mediated base pair.
22
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Herein, we report for the first time on stereoelectronic effects induced by 2’-fluorination of
the sugar moiety that are transmitted to the silver-mediated base pair. The impact depends on
the number of substitutions, the configuration and the structure of the nucleobase.
Consequently 2’ fluorine modification on the sugar moiety can be used to modulate silver
mediated base pair stability depending on the configuration of the fluorine atom and the
number of fluorine replacements. All structural modifications did not change the recognition
face of 2'-deoxycytidine. One or two silver ions were bound by the particular base pair. Figure
6 summarizes possible silver mediated base pair structures.
Figure 6. Proposed structures of silver mediated base pairs demonstrating the fluorine effect
on the base pair stability.
23
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Experimental Section
General methods and materials
All chemicals and solvents were of laboratory grade as obtained from commercial suppliers
and were used without further purification. Reversed-phase HPLC was carried out on a 4 x
250 mm RP-18 (10 μm) LiChrospher 100 column with a HPLC pump connected with a
variable wavelength monitor, a controller and an integrator. The molecular masses of the
oligonucleotides were determined by MALDI-TOF mass spectrometry in the linear positive
mode with 3-hydroxypicolinic acid (3-HPA) as a matrix. Fluorescence spectra were measured
with a fluorescence spectrophotometer in the wavelength range between 220 to 600 nm at
room temperature using standard quartz cuvettes with a path length of 1 cm. The thermal
melting curves were measured with a UV-vis spectrophotometer equipped with a
thermoelectrical controller. The temperature was measured continuously in the reference cell
with a Pt-100 resistor with a heating rate of 1 °C min-1. Tm values were determined from the
melting curves using the software MELTWIN, version 3.0.[33]
Oligonucleotide syntheses and characterization
Solid-phase oligonucleotide syntheses were performed on an ABI 392-08 synthesizer at 1
µmol scale (trityl-on mode) employing the phosphoramidites of nucleosides 1, 2[20,21]
,
phosphoramidites 16a-16c, 17 as well as the standard building blocks with an average
coupling yield over 95%. After cleavage from the solid support, oligonucleotides containing
nucleosides 1-5 were deprotected in 28% aqueous ammonia at 55 °C for 2 h. While
oligonucleotides containing nucleoside 6 were deprotected in 28% aqueous ammonia for
overnight at room temperature. The DMT-containing oligonucleotides were purified by
reversed-phase HPLC (RP-18) with the gradient system at 260 nm: (A) MeCN, (B) 0.1 M
(Et3NH)OAc (pH 7.0)/MeCN, 95:5; gradient I: 0-3 min 10-15% A in B, 3-15 min 15-50% A
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Chemistry - A European Journal
in B; flow rate 0.8 mL/min. The purified “trityl-on” oligonucleotides were treated with 2.5%
CHCl2COOH/CH2Cl2 for 2 min at 0 °C to remove the 4,4′-dimethoxytrityl residues. The
detritylated oligomers were purified again by reversed-phase HPLC with gradient II: 0-20 min
0-20% A in B; 20-25 min, 20% A in B; flow rate 0.8 mL/min. For Figure 2 following gradient
system was used, gradient III: (B) 0.1 M (Et3NH)OAc (pH 7.0)/MeCN, 98:2; 0-25 min 100%
B; flow rate 0.8 mL/min; gradient IV: (A) MeCN, (B) 0.1 M (Et3NH)OAc (pH 7.0)/MeCN,
95:5; 0-25 min 20% A in B; flow rate 0.8 mL/min. The oligonucleotides were desalted on a
short column (RP-18) using water for elution of salt, while the oligonucleotides were eluted
with H2O/MeOH (2:3). The oligonucleotides were lyophilized on a Speed-Vac evaporator to
yield colorless solids which were frozen at -24 °C. The purity of all oligonucleotides were
confirmed by RP-18 HPLC (Figure S3, Supporting Information) and MALDI-TOF mass
spectrometry (see Tables 1). The extinction coefficients ε260 (H2O) of the nucleosides are: dA
15400, dG 11700, dT 8800, dC 7300, 3 11900 (MeOH), 4 4400 (MeOH), 5 15100 (MeOH)
and 6 27700 (MeOH). The extinction coefficients of the oligonucleotides were calculated
from the sum of the extinction coefficients of nucleoside constituents.
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Chemistry - A European Journal
Table 4. 13C-NMR data of gemcitabin derivatives[a]
C2[b]
C2[c]
C5[b]
C6[b]
C4[c]
C7[b]
C5[c]
C8[b]
C6[c]
C4[b]
C1’
C2’
C3’
C4’
C5’ N=CH C≡C
C4a[c]
C7a[c]
83.5
(32.9)
123.1
(258)
68.7
(22.6)
80.4
(4.5)
3
4
154.7
153.4
165.7
163.8
94.6
57.7
140.8
146.4
-
-
-
-
58.9
58.4
-
-
-
-
83.5
(32.3)
122.9
(258)
67.9
(22.4)
80.5
(4.5)
83.8
(30.0)
122.9
(256)
68.2
(23.0)
80.7
(4.0)
80.9
90.6
5
6
153.0
153.6
154.7
163.8
160.5
171.5
94.0
109.9
102.2
144.3
140.6
141.6
-
96.8
-
-
136.0
-
58.6
58.7
58.7
-
-
84.8
(34.2)
123.1
(259)
68.3
(23.1)
80.8
(5.0)
-
-
83.9
(38.8)
123.0
(258)
68.4
(22.1)
80.5
(4.2.)
14a
157.9
84.1
(32.0)
122.7
(258)
69.2
(22.5)
78.7
(4.0)
15a
14b
15b
14c
15c
7
154.5
153.8
153.7
153.1
153.4
152.9
152.1
153.3
152.6
153.6
171.4
168.3
168.4
169.8
170.4
163.8
161.5
163.9
162.0
160.5
102.3
70.0
70.3
98.4
99.2
94.2
94.3
58.0
61.8
109.8
141.4
146.3
146.2
144.6
144.5
144.5
144.4
146.4
149.5
140.3
-
-
61.6
58.3
61.9
58.5
62.4
62.0
61.9
62.0
62.0
60.9
158.0
-
-
-
83.9
(33.1)
122.9
(258)
67.9
(21.1)
80.6
(4.5)
-
-
158.3
84.4
(n.d.)
122.6
(258)
69.8
(23.1)
78.8
(n.d.)
-
-
158.3
84.1
(29.2)
122.9
(260)
68.1
(22.1)
80.7
(n.d.)
83.8
91.9
-
-
157.9
83.8
(n.d.)
123.1
(257)
70.2
(22.1)
79.6
(4.0)
83.8
92.5
-
-
158.5
84.2
(n.d.)
122.7
(257)
69.7
(22.0)
80.4
(n.d.)
80.4
90.9
-
-
-
-
-
-
-
85.1
(n.d.)
122.7
(258)
69.5
(23.1)
79.5
(2.0)
79.7
95.3
8
-
-
84.0
(n.d.)
122.6
(258)
69.9
(23.1)
78.9
(n.d.)
10
-
-
-
-
-
-
-
85.6
(n.d.)
122.5
(258)
69.7
(22.0)
79.5
(n.d.)
11
84.8
(n.d.)
122.8
(258)
68.4
(21.1)
78.8
(n.d.)
9
96.2
135.5
[a] Measured in DMSO-d6 at 298 K. [b] Pyrimidine numbering. [c] Systematic numbering. n.d.: not detected.
1-(2-Deoxy-2,2’-difluoro-β-D-erythro-pentofuranosyl)-5-iodopyrimidin-2H-2-one (4)
A mixture of ethanol (40 mL), carbon tetrachloride (40 mL) and iodine (1.730 g, 6.82 mmol)
was stirred for 10 min at room temperature. 2'-Deoxy-2',2'-difluorocytidine (4.600 g, 17.48
mmol) was added. Then reaction mixture was warmed at 45°C and treated with a solution of
iodic acid (1.73 g, 9.83 mmol) in water (4.6 mL). Afterward reaction mixture was stir at 45°C
for 16 h (TLC monitoring). After completion of reaction, the mixture was allowed to cool.
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10.1002/chem.201703427
Chemistry - A European Journal
Then solvent was evaporated and the remaining residue was adsorbed on silica gel and
applied to FC (silica gel, column: 15 x 5 cm, CH2Cl2/MeOH 90:10). Evaporation of the main
zone afforded compound 4 as colorless solid (6.100 g, 90%). Analytical data were in
accordance to the literature.[15f]
4-Amino-1-(2-deoxy-2,2’-difluoro-β-D-erythro-pentofuranosyl)-5-
(phenylethynyl)pyrimidin-2H-2-one (5)
5-Iodo-2,2’-difluoro-2’-deoxycytidine (4) (963 mg, 2.47 mmol) was dissolved in dry DMF
(10 mL), then CuI (96 mg, 0.50 mmol), tetrakis(triphenylphosphine) Pd(0) (288 mg, 0.25
mmol), N,N-diisopropylethylamine (732 mg, 963 µL, 5.66 mmol) and phenylacetylene (1.08
mL, 1.0 g, 9.8 mmol) were introduced. The solution was stirred at rt for overnight. The
solvent was removed under vacuo, and the resulting oily residue was co-evaporated with
toluene twice (2 x 20 mL), adsorbed on silica gel and applied to FC (silica gel, column: 15 x 4
cm, CH2Cl2/MeOH, 85:15). Evaporation of the solvent from the main zone afforded 5 as
yellowish foam (728 mg, 81%). Rf = 0.6 (CH2Cl2/MeOH, 85:15); 1H NMR (300 MHz,
[D6]DMSO, 25 °C): δ = 3.63-3.68 (m, 2H, 2 x H-5’), 3.81-3.93 (m, 1H, H-4’), 4.16-4.29 (m,
1H, H-3’), 5.40 (t , J = 4.8 Hz, 1H, HO-5’), 5.68 (t, J = 6.9 Hz, 1H, H-1’), 6.27 (d, J = 6.3 Hz,
HO-3’), 7.29 (br, 1H, NH), 7.39-7.41 (m, 3H, Ar-H), 7.60-7.63 (m, 2H, Ar-H), 8.04 (br, 1H,
NH), 8.26 (s, 1H, H-6); 19F NMR (376 MHz, [D6]DMSO, 25 °C): δ = -116.14 to -117.56;
UV/Vis (MeOH): λmax (ε) = 305 (13300), 283 (16300), 270 nm (17100 dm3 mol-1 cm-1);
HRMS (ESI-TOF) m/z: calcd. For C17H15F2N3O4Na: 386.0923 [M+Na]+; found 386.0924.
4-Amino-1-(2-deoxy-2,2’-difluoro-5-O-(4,4’-dimethoxytrityl)-β-D-erythro-pentofuranosyl)-
5-(phenylethynyl)pyrimidin-2H-2-one (7)
Compound 5 (1.0 g, 2.75 mmol) was dried by repeated co-evaporation with dry pyridine (3 x
10 mL) and dissolved in dry pyridine (15 mL). To the solution was added 4,4’-
dimethoxytrityl chloride (1.5 g, 4.43 mmol) and the reaction mixture was stirred at rt for 12 h.
27
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10.1002/chem.201703427
Chemistry - A European Journal
The solution was diluted with CH2Cl2 (100 mL) and poured into 5% NaHCO3 solution. The
organic layer was dried over Na2SO4, evaporated and applied to FC (silica gel, column: 15 x 5
cm, CH2Cl2/acetone 4:1). Evaporation of the main zone afforded compound 7 as colorless
foam (1.6 g, 87%). Rf = 0.30(CH2Cl2/acetone, 85:15); 1H NMR (300 MHz, [D6]DMSO, 25
°C): δ = 3.40-3.44 (m, 2H, H-5’), 3.66 (s, 6H, OCH3), 4.03-4.07 (m, 1H, H-4’), 4.31-4.42 (m,
1H, H-3’), 6.18 (t, J = 8.0 Hz, 1H, H-1’), 6.33 (d, J = 8.0 Hz, 1H, HO-3’), 6.86-6.88 (m, 6H,
Ar-H), 7.14-7.45 (m, 13H, Ar-H), 7.97 (s, 1H, H-6), 8.08 (s, 1H, NH); 19F NMR (376 MHz,
[D6]DMSO, 25 °C): δ = -114.77 to 116.77 (m, CF2); UV/Vis (MeOH): λmax (ε) = 307 (12700),
282 (18400), 270 (20300), 234 nm (32300 dm3 mol-1 cm-1); HRMS (ESI-TOF) m/z: calcd. for
C38H33F2N3O6Na: 688.2229 [M+Na]+; found 688.2225.
4-(Acetyl)amino-1-(2-deoxy-2,2’-difluoro-5-O-(4,4’-dimethoxytrityl)-β-D-erythro-
pentofuranosyl)-5-(phenylethynyl)pyrimidin-2H-2-one (8)
Compound 7 (710 mg, 1.07 mmol) was dissolved in DMF (5 mL), then acetic anhydride
(1.501 g, 1.39 mL, 14.70 mmol) was added and the solution was stirred for 12 h at rt. The
solvent was evaporated and the remaining residue was co-evaporated with MeOH (3 x 3 mL)
and applied to FC (silica gel, column: 8 x 3 cm, CH2Cl2/acetone 4:1). Evaporation of the main
zone afforded compound 8 as yellowish foam (552 mg, 73%). Rf = 0.53(CH2Cl2/acetone, 8:2);
1H NMR (300 MHz, [D6]DMSO, 25 °C): δ = 2.35 (s, 3H, CH3), 3.27-3.31 (m, 1H, H-5’),
3.45-3.51 (m, 1H, H-5’), 3.66 (s, 3H, OCH3), 3.67 (s, 3H, OCH3), 4.11-4.15 (m, 1H, H-4’),
4.34-4.36 (m, 1H, H-3’), 6.20 (t, J = 7.2 Hz, 1H, H-1’), 6.38 (d, J = 6.6 Hz, 1H, HO-3’), 6.86-
6.88 (m, 4H, Ar-H), 7.14-7.45 (m, 14H, Ar-H), 8.29 (s, 1H, H-6), 9.87 (s, 1H, NH); 19F NMR
(376 MHz, [D6]DMSO, 25 °C): δ = -114.94 to -116.41 (m, CF2); UV/Vis (MeOH): λmax (ε) =
298 (15600), 282 (20600), 275 (19300), 232 nm (33200 dm3 mol-1 cm-1); HRMS (ESI-TOF)
m/z: calcd. for C40H35F2N3O7Na: 730.2335 [M+Na]+; found 730.2335.
28
This article is protected by copyright. All rights reserved.

10.1002/chem.201703427
Chemistry - A European Journal
6-(3-Phenyl)-3-[2-deoxy-2,2’-difluoro -5-O-(4,4’-dimethoxytrityl)-β-D-erythro-
pentofuranosyl]pyrrolo-[2,3-d]pyrimidin-2H-2-one (9)
From compound 8: A mixture of 8 (420 mg, 0.59 mmol), CuI (154 mg, 0.81 mmol) in
triethylamine (10 mL) and DMF (10 mL) was heated at 60 °C for 24 h. The solvent was
evaporated and the remaining residue was adsorbed on silica gel (20 g) and applied to FC
(silica gel, column: 8 x 3 cm, CH2Cl2/MeOH 95:5). Evaporation of the main zone afforded
compound 9 as yellowish foam (267 mg, 68%). Rf = 0.33 (CH2Cl2/MeOH, 95:5); 1H NMR
(400 MHz, [D6]DMSO, 25 °C): δ = 3.41-3.43 (m, 1H, H-5’), 3.54-3.58 (m, 1H, H-5’), 3.73
and 3.74 (2s, 6H, 2 x OCH3), 4.09-4.13 (m, 1H, H-4’), 4.52-4.62 (m, 1H, H-3’), 5.71 (s, 1H,
H-5), 6.38 (t, J = 6.8 Hz, 1H, H-1’), 6.48 (d, J = 6.4 Hz, 1H, HO-3’), 6.94-6.96 (m, 4H, Ar-
H), 7.28-7.48 (m, 12H, Ar-H), 7.69 (d, J = 7.6 Hz, 2H, Ar-H), 8.58 (s, 1H, H-6), 11.93 (s, 1H,
NH); 19F NMR (376 MHz, [D6]DMSO, 25 °C): δ = -117.8; UV/Vis (MeOH): λmax (ε) = 370
(5200), 268 (27000), 261 (27200), 235 nm (31000 dm3 mol-1 cm-1); HRMS (ESI-TOF) m/z:
calcd. for C38H33F2N3O6Na: 688.2230 [M+Na]+; found 688.2247.
From compound 11: Compound 11 (500 mg, 0.68 mmol) was dissolved in dry DMF (5 mL)
and triethylamine (4 mL). Then, CuI (30 mg, 0.16 mmol), tetrakis(triphenylphosphine) Pd(0)
(90 mg, 0.08 mmol), and phenylacetylene (300 µL, 2.7 mmol) were introduced. The solution
was stirred at rt overnight. The solvent was removed under vacuo, and the resulting oily
residue was co-evaporated with toluene twice (2 x 20 mL), adsorbed on silica gel and applied
to FC (silica gel, column: 15 x 4 cm, CH2Cl2/MeOH, 95:5). Evaporation of the solvent from
the main zone afforded 9 (262 mg, 58%) as yellowish foam.
From compound 6: Compound 6 (50 mg, 0.14 mmol) was dried by repeated co-evaporation
with dry pyridine (3 x 2 mL) and dissolved in dry pyridine (1 mL). To the solution was added
4,4’-dimethoxytrityl chloride (167 mg, 0.49 mmol) in four portions and the reaction mixture
was stirred at rt for 6 h (TLC-control). The solution was diluted with CH2Cl2 (20 ml) and
29
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