6-Azauracil or 8-aza-7-deazaadenine nucleosides and oligonucleotides: The effect of 2′-fluoro substituents and nucleobase nitrogens on conformation and base pairing

Article abstract of DOI:10.1039/b715512c

The stereoselective syntheses of 6-azauracil- and 8-aza-7-deazaadenine 2′-deoxy-2′-fluoro-β-d-arabinofuranosides 1c and 2c employing nucleobase anion glycosylation with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-α-d- arabinofuranosyl bromide 6 as the sugar component are described; the 6-azauracil 2′-deoxy-2′-fluoro-β-d-ribofuranoside 1d was prepared from 6-azauridine 8via the 2,2′-anhydro intermediate 9 and transformation of the sugar with DAST. Compounds show a preferred N-conformer population (100% N for 1c, 1d and 78% N for 2c) being rather different from nucleosides not containing the combination of a fluorine atom at the 2′-position and a nitrogen next to the glycosylation site. Oligonucleotides incorporating 1c and 2c were synthesized using the phosphoramidites 3b and 4. Although the N-conformation is favoured in the series of 6-azauracil- and 8-aza-7-deazaadenine 2′-deoxy-2′-fluoroarabinonucleosides only the pyrimidine compound 1c shows an unfavourable effect on duplex stability, while oligonucleotide duplexes containing the 8-aza-7-deazaadenine-2′-deoxy- 2′-fluoroarabinonucleoside 2c were as stable as those incorporating dA or 8-aza-7-deaza-2′-deoxyadenosine 2a. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b715512c

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
6-Azauracil or 8-aza-7-deazaadenine nucleosides and oligonucleotides: the
effect of 2ꢀ-fluoro substituents and nucleobase nitrogens on conformation and
base pairing
Frank Seela*a,b and Padmaja Chittepua,b
Received 8th October 2007, Accepted 15th November 2007
First published as an Advance Article on the web 2nd January 2008
DOI: 10.1039/b715512c
The stereoselective syntheses of 6-azauracil- and 8-aza-7-deazaadenine
2ꢀ-deoxy-2ꢀ-fluoro-b-D-arabinofuranosides 1c and 2c employing nucleobase anion glycosylation with
3,5-di-O-benzoyl-2-deoxy-2-fluoro-a-D-arabinofuranosyl bromide 6 as the sugar component are
described; the 6-azauracil 2ꢀ-deoxy-2ꢀ-fluoro-b-D-ribofuranoside 1d was prepared from 6-azauridine 8
via the 2,2ꢀ-anhydro intermediate 9 and transformation of the sugar with DAST. Compounds show a
preferred N-conformer population (100% N for 1c, 1d and 78% N for 2c) being rather different from
nucleosides not containing the combination of a fluorine atom at the 2ꢀ-position and a nitrogen next to
the glycosylation site. Oligonucleotides incorporating 1c and 2c were synthesized using the
phosphoramidites 3b and 4. Although the N-conformation is favoured in the series of 6-azauracil- and
8-aza-7-deazaadenine 2ꢀ-deoxy-2ꢀ-fluoroarabinonucleosides only the pyrimidine compound 1c shows an
unfavourable effect on duplex stability, while oligonucleotide duplexes containing the
8-aza-7-deazaadenine-2ꢀ-deoxy-2ꢀ-fluoroarabinonucleoside 2c were as stable as those incorporating dA
or 8-aza-7-deaza-2ꢀ-deoxyadenosine 2a.
reported and these compounds are accepted as substrates by
Introduction
various DNA polymerases or have found application in in vitro
selection studies of DNA aptamers.9 The 2ꢀF-araNA modification
is well suited to tune the physico-chemical and biological
properties of G-quartets.10
The introduction of a fluorine atom to an organic compound
does not result in a considerable change in the size or the shape
of the molecule. However, the fluorine substituent can alter the
physico-chemical properties and biological activity. As the C–
F bond is stronger than the C–H bond, fluorinated compounds
are chemically more stable and more resistant against metabolic
degradation.1–3 Thus fluorinated analogues of biological molecules
provide useful tools for probing or modifying the functions of
biologically active compounds.
The ability of a fluorine atom to mimic the hydroxyl group
makes it suitable for introduction into the sugar moiety of
nucleosides. The potent effect of introducing fluorine has already
led to useful therapeutic agents. Among these fluorinated nucleo-
sides are compounds like 1-(2-deoxy-2-fluoro-b-D-arabinofura-
nosyl)thymine (FMAU),4 1-(2-deoxy-2-fluoro-b-D-arabinofura-
nosyl)-5-iodouracil (FIAU) and 1-(2-deoxy-2-fluoro-b-D-
arabinofuranosyl)-5-iodocytosine,5,6 which are potent antiviral
agents, while 2ꢀ,2ꢀ-difluorocytidine (gemcitabine) shows anticancer
activity.7 Corresponding “purine” nucleosides such as 4-amino-1-
(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-1-H-pyrazolo[3,4-d]-
pyrimidine 2c display significant activity against HBV (Hepatitis B
virus).8 The polymerase catalyzed synthesis of the 5ꢀ-triphosphates
of 2ꢀ-deoxy-2ꢀ-fluoroarabinonucleosides (2ꢀF-araNTPs) has been
The higher electronegativity of the fluorine atom can alter
the conformational properties of the sugar residue by shifting
the conformational equilibrium of the pentofuranose moiety
to either the north or the south conformation as a function
of its position on the (sugar) ring, the stereochemistry, and
interactions with the neighboring atoms (substituents).11a,b Nev-
ertheless, the contribution of the electronic structure of the
heterocyclic base in conjunction with the electronegative sub-
stituent of the sugar moiety attracts attention. It was recog-
nized that 3-bromo-1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-
1-H-pyrazolo[3,4-d]pyrimidine-4,6-diamine12a having a nitrogen
atom placed next to the glycosylation site, shows rather unique
conformational properties when compared to the corresponding
purine 2ꢀ-deoxyribonucleoside12b with a 100% population of the
N-conformer.
Recently, we reported on the pH-dependent duplex stability of
oligonucleotides containing 6-aza-2ꢀ-deoxyuridine 1a.13 Because
of the pKa value of the 6-azauracil moiety (6.5), the strength of the
base pair and the resulting oligonucleotide duplex stability are pH-
dependent. At neutral pH value the duplexes are already partially
deprotonated at the modified base leading to destabilization of
the “dA–dT” base pair. The related 8-aza-7-deaza-2ꢀ-deoxy-2ꢀ-
fluoroarabinoadenosine 2c does not show such properties al-
though it favours the N-conformation. This manuscript reports on
the stereoselective synthesis of the 6-azapyrimidine 2ꢀ-deoxy-2ꢀ-
fluoronucleosides 1c, 1d as well as the related pyrazolo[3,4-d]pyri-
midine 2ꢀ-deoxy-2ꢀ-fluoroarabinonucleoside 2c (2ꢀF-ara-c7z8Ad),
aLaboratory of Bioorganic Chemistry and Chemical Biology, Center for
Nanotechnology, Heisenbergstraße 11, 48149 Mu¨nster, Germany. E-mail:
seela@uni-muenster.de; Fax: +49(0)251 53 406 857; Tel: +49(0)251 53
406 500; Web: www.seela.net
bLaboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r
Chemie, Universita¨t Osnabru¨ck, Barbarastraße 7, 49069 Osnabru¨ck,
Germany. E-mail: frank.seela@uni-osnabrueck.de
596 | Org. Biomol. Chem., 2008, 6, 596–607
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
all with a fluorine atom at the 2ꢀ-position and a nitrogen placed
next to the glycosylation site. Oligonucleotides containing 1c
and 2c were synthesized. For this the phosphoramidite building
blocks 3b and 4 were prepared and employed in solid-phase
oligonucleotide synthesis (Scheme 1). The effect of the 2ꢀ-fluoro
substituent on the conformational properties and on the duplex
stability was determined and correlated with the structurally
related purine and pyrimidine 2ꢀ-deoxyribonucleosides 2a and 1a.
2ꢀ-fluoroarabinonucleoside formation follows a similar mechanism
to that of the 2ꢀ-deoxyribonucleoside.19,20 The protected compound
7 was deprotected with 0.2 M NaOMe in MeOH to afford
nucleoside 1c (Scheme 2).
Next, the corresponding 2ꢀ-deoxy-2ꢀ-fluororibonucleoside 1d
was prepared. Generally, 2ꢀ-deoxy-2ꢀ-fluoro-b-D-ribonucleosides
can be obtained by various routes: (i) by the fluorination of
an appropriate arabinonucleoside or anhydronucleoside;21 (ii) by
condensation of a 2-fluororibosugar halide with the hetero-
cyclic base;22 and (iii) by transglycosylation of 2ꢀ-deoxy-2ꢀ-
fluororibonucleosides with another nucleobase.23 We selected
route (i) by fluorinating the 6-azauracil arabinonucleoside 10.
The fluorination of arabinonucleosides undergoes SN2 displace-
ment with inversion of configuration when treated with diethy-
laminosulfur trifluoride (DAST) or with tetra-n-butylammonium
fluoride (TBAF).24,25
Results and discussion
1. Synthesis and properties of monomers
The convergent syntheses of a number of 2ꢀ-deoxy-2ꢀ-fluoro-b-
D-arabinofuranosyl nucleosides have already been reported.14,15
The preparation of the 6-aza-2ꢀ-deoxy-2ꢀ-fluoro-arabinofuranosyl
uracil 1c (6-aza-2ꢀ F-ara-U; pyrimidine numbering is used
throughout the text; the systematic numbering is used in the
experimental section) started with the known 3,5-di-O-benzoyl-
2-deoxy-2-fluoro-a-D-arabinofuranosyl bromide 6 which was ob-
tained from the commercially available 1-O-acetyl-2,3,5-tri-O-
benzoyl-b-D-ribofuranose by a three-step procedure.16–18 This
was directly coupled to the persilylated 6-azauracil 5 in the
presence of CuI following the protocol of Freskos.19 This reaction
protocol led exclusively to the formation of the protected b-D-
nucleoside 7. Apparently, the stereochemical outcome of 2ꢀ-deoxy-
The arabinonucleoside 10 was obtained from the ribonucleoside
8 via the intermediate 2,2ꢀ-anhydro-6-azauridine 9. The 3ꢀ,5ꢀ-
hydroxyl groups of 8 were protected by using the Markiewicz
clamp (1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane; TIPDS) and
further treatment with methane sulfonyl chloride followed by
TBAF yielded compound 9. Next, the 3ꢀ,5ꢀ-hydroxyl groups
were selectively protected with 3,4-dihydropyran (DHP) in DMF,
followed by the opening of the anhydro ring with the help
of mild basic hydrolysis to form the protected 6-azauridine
arabinonucleoside 10. Compound 10 was then treated with DAST
Scheme 1 Structures of nucleosides and the corresponding phosphoramidites.
Scheme 2 Reagents and conditions: (i) CuI, CHCl3, overnight, r.t.; (ii) 0.2 M NaOMe–MeOH, 2 h, r.t.
The Royal Society of Chemistry 2008 Org. Biomol. Chem., 2008, 6, 596–607 | 597
This journal is
©

View Article Online
Scheme 3 Reagents and conditions: (i) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDS), pyridine, 5 h, r.t.; (ii) CH3SO2Cl, pyridine, 16 h, r.t.;
(iii) 1 M TBAF in THF, 2 h, r.t.; (iv) 3,4-dihydropyran, DMF, p-toluenesulfonic acid, 4 h, 0 ◦C; (v) 1 N NaOH, MeOH, 2 h, r.t.; (vi) DAST, pyridine,
CH2Cl2, −60 ◦C; (vii) pyridinium p-toluenesulfonate, EtOH, 3 h, 60 ◦C.
to give the intermediate 11 in 48% yield. Finally, the removal of
the THP groups from 11 with pyridinium p-toluenesulfonate gave
the desired nucleoside 1d (Scheme 3).
of the sugar halide not involved in the glycosylation reaction. To
confirm the configuration of the fluorine atom at the 2ꢀ-position
in 1d, a H-NMR NOE spectrum was measured. Irradiation of
1
More recently, we have reported on the synthesis of 4-amino-
1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)-1-H-pyrazolo[3,4-d]-
pyrimidine 2c8 (systematic numbering is used). In this method the
glycosylation of 4-methoxypyrazolo[3,4-d]pyrimidine 12 with 3,5-
di-O-benzoyl-2-deoxy-2-fluoro-a-D-arabinofuranosyl bromide (6)
in the presence of DBU resulted in the formation of a mixture
of the a-D and b-D-anomers of N1- and N2-glycosides (94%,
overall yield) with 45% of the desired N1-b-D-anomer. Because
of the unsatisfactory stereochemical outcome of the above
method, here, we used an improved protocol. The condensation
of 4-methoxypyrazolo[3,4-d]pyrimidine 12, which was performed
with the halogenose 6 in MeCN in the presence of powdered
KOH and TDA-1 (tris[2-(2-methoxyethoxy)ethyl]amine) at r.t.,
afforded the desired N1-b-D-nucleoside 13 (50%) along with
the 3ꢀ-debenzoylated derivative 14 (9%) and a trace amount of
the N2-b-D-regioisomer 15 (7%). Deprotection of compounds
13 and 14 with 25% aqueous ammonia resulted in the removal
of the benzoyl groups and a simultaneous conversion of the
4-methoxy substituent to an amino group yielding 2c. When
the deprotection was performed in NaOMe–MeOH, it gave the
methoxy nucleoside 16 (Scheme 4).
H-1ꢀ resulted in NOE effects at H-4ꢀ (1%) and OH-3ꢀ (4.7%);
irradiation of H-3ꢀ gave NOE at H-(2ꢀ-b) (3.2%). Based on the
spatial relationships of the H-atoms the position of fluorine is
assigned as a-D (2ꢀ-F down). The assignment of the 13C NMR
chemical shifts of the base moieties in nucleosides 1c, 1d and 2c was
made according to the previous reports of related compounds.13,27
The 19F NMR spectra of 2ꢀ-deoxy-2ꢀ-fluoroarabino ortho-aza
nucleosides differ substantially from those of other 2ꢀ-deoxy-2ꢀ-
fluoroarabinonucleosides. The fluorine signal in the 19F NMR
spectra of 1c, 2c and its derivatives forms a doublet of doublets
(geminal H2ꢀ–F and vicinal H3ꢀ–F couplings) and not a doublet of
two doublets as a result of an additional vicinal H1ꢀ–F coupling.
It implies that there is only one vicinal H–F coupling observed.
Similar behaviour was also found in other ortho-aza nucleosides
such as 3-bromopyrazolo[3,4-d]pyrimidine 2ꢀ-deoxy-2ꢀ-fluoro-b-
D-arabinonucleosides.12a
3. Nucleoside conformation
It has been noticedpreviously, that the N–S pseudorotational equi-
librium of the sugar moieties of nucleosides is driven by various
gauche and anomeric effects. The anomeric effect depends on the
electronic nature of the heterocyclic base and can drive the sugar
moiety of b-D-nucleosides toward the N-type conformers.28–30
We have already studied the conformational properties of the
azapyrimidine nucleosides 1c, 1d31a,b and the azapurine nucleoside
2c in solution by applying the PSEUROT program (version 6.3).32
The use of both 3JH,H, 3JH,F coupling constants and semi-empirical
calculations using the HyperChem 7.0 program (Hypercube Inc.,
2. Spectroscopic data
The structures of all synthesized compounds were characterized by
1H, 13C and 19F NMR spectra (Table 1 and 2) as well as by elemental
analysis. In the case of 1c the configuration of the fluorine atom
was assigned as b-D, as it was proven by single crystal X-ray
analysis26 and was in accordance with the fixed stereogenic centers
598 | Org. Biomol. Chem., 2008, 6, 596–607
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
Scheme 4 Reagents and conditions: (i) MeCN, TDA-1 (= tris[2-(2-methoxyethoxy)ethyl]amine), KOH, 30 min, r.t.; (ii) 0.2 M NaOMe–MeOH, 2 h, r.t.;
(iii) NH3 in H2O–dioxane (4 : 1), 24 h, 90 ◦C.
Table 1 1H NMR data of 2ꢀ-deoxy-2ꢀ-fluoro nucleosides measured in DMSO-d6 at 298 K
Chemical shifts, dTMS/ppm
Coupling constants/Hz
3J(H,H) 3J(H,F)
1ꢀ,2ꢀ 2ꢀ,3ꢀ
Sugar
H-1ꢀ
6.58 d 5.82 dt
1c 6.40 d 5.28 dt
H-2ꢀ
H-3ꢀ
H-4ꢀ
H-5ꢀ
3ꢀ,4ꢀ
7.8
7.3
—
1ꢀ,F
3ꢀ,F Others
7
6.06 m 4.65–4.68 m 4.48–4.52 m
4.33 m 3.62–3.69 m 3.52–3.54 m
6.7 7.5
7.0 7.0
<1.0
<1.0
18.8 7.43–8.04 (s, 1 H, 5-H; m, 10 H, arom.); 12.43
(s, 1 H, NH).
18.3 4.76 (t, 1 H, 5ꢀ-OH); 5.79 (br d, 1 H, 3ꢀ-OH);
7.6 (s, 1 H, 5-H); 12.33 (s, 1 H, NH).
11 6.16 d 5.20 dd 4.27 m 4.07–4.15 m 3.69 m
<1.0
—
20.65 24.1 4.55, 4.1, 1.5 (m, 18 H, THP); 7.58 (s, 1 H,
5-H); 12.33 (s, 1 H, NH).
1d 6.07 d 5.08 dd 4.23 m 3.80–3.65 m 3.60 m
<1.0 3.9
6.3 6.97 6.8
7.8
2c 6.60 d 5.40 dt
17 6.50 d 5.28 dt
18 6.42 d 5.34 dt
4.74 m 3.80 m
4.38 m 4.01 m
4.40 m 3.74 m
3.78 m
18.0 4.84 (br t, 5ꢀ-OH); 5.86 (br d, 3ꢀ-OH); 7.80 (br
dd, NH2); 8.20 (s, H-3, H-6).
3.20 m
6.5 7.0
6.6 7.1
7.2
7.0
3.53–3.69 m
19 6.47 d 5.27 dt
20 6.76 d 5.42 dt
4.40 m 3.92 m
4.68 m 3.79 m
3.16–3.26 m
3.60–3.67 m
6.6 7.0
6.3 6.6
6.9
6.4
<1.0
<1.0
19.3 3.73 (s, 9 H, OMe); 5.89 (br d, J = 5.55,
3ꢀ-OH); 6.86–8.00 (s, 1 H, 5-H; m, 17 H,
arom.).
19.7 1.05–1.17 (d, 6 H, 2Me); 2.86–2.94 (m, 1 H,
CH); 4.79 (t, 1 H, J = 5.30, 5ꢀ-OH); 5.85 (d,
1 H, J = 5.72, 3ꢀ-OH); 8.57 (s, 1 H, 3-H); 8.69
(s, 1 H, 6-H); 1.29 (s, 1 H, NH).
21 6.71 d 5.46 dt
4.85 m 3.99 m
3.15 m
—
6.9
6.5
<1.0
18.0 1.06–1.17 (d, 6 H, 2Me); 2.88–2.98 (m, 1 H,
CH); 5.88 (d, 1 H, J = 5.50, 3ꢀ-OH);
6.75–7.31 (m, 13 H, arom.); 8.46 (s, 1 H, 3-H);
8.71 (s, 1 H, 6-H); 11.32 (s, 1 H, NH).
Gainesville, FL, USA, 2001) permits31a a detailed conformational
analysis of the pentofuranose ring. The input contained the fol-
coupling constants and the corresponding H–C–C–F torsion
angles. Correction terms for substituent electronegativity and for
the H–C–C (aHCC) and F–C–C (aFCC) bond angle deviations from
tetrahedral values33 are also implemented. This protocol is not
used in the present study.
In order to understand the effect of the 2ꢀ-fluoro substituent as
well as the heterocyclic base on the conformational properties, a
series of related purine and pyrimidine nucleosides were compared
3
lowing coupling constants: J(H1ꢀ,H2ꢀ), J(H2ꢀ,H3ꢀ), J(H3ꢀ,H4ꢀ),
1
J(H1ꢀ,F), J(H3ꢀ,F). They were taken from the well resolved H
NMR spectra measured in D2O. The results of semi empirical
calculations (PM3)31a are in good agreement with the X-ray
data. Recently, Chattopadhyaya and co-workers have developed
a new Karplus-type relationship between vicinal proton–fluorine
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 596–607 | 599
©

View Article Online
Table 2 13C NMR data of nucleosides in DMSO-d6
Chemical shifts, dTMS/ppm
Base
Sugar
C(2)a
C(4)a
C(5)a
C(6)a
C(7)a
(C-3)b C-1ꢀ (2JF, C
(C-6)b (C-7a)b (C-3a)b (C-4)b
)
C-2ꢀ (1JF, C
)
C-3ꢀ (2JF, C
)
C-4ꢀ (3JF, C
)
C-5ꢀ
Others
7
148.1
1c 148.3
160.0
156.0
156.2
164.5
156.3
136.8
136.2
142.2
135.5
—
—
—
—
—
—
—
—
81.4 (17.2)
92.1 (198.7)
95.1 (196.2)
89.9c
74.9 (22.2)
72.3 (20.1)
75.2
76.4 (9.7)
64.2
62.3
60.9
61.3
81.1 (17.0)
82.1 (10.1)
9
90.3c
89.3c
10 148.4
84.3
80.7
74.7
80.3
98.1, 68.5, 61.2, 30.3,
24.9, 18.9 -THP
98.7, 66.7, 62.0, 30.8,
25.7, 19.7 -THP
11 148.6
157.4
137.6
—
—
88.8 (34.6)
94.1 (184.0)
76.8 (16.2)
80.3 (10.1)
62.3
1d 148.0
18 146.7
19 146.6
2c 156.3
20 155.1
156.6
154.1
154.0
154.5
152.3
136.6
136.1
135.9
99.9
—
—
—
158.1
155.5
—
—
—
133.7
137.5
87.5 (34.6)
81.7 (17.04) 95.1 (196.5)
81.77 (17.4) 95.0 (195.9)
80.4 (18.0)
80.33 (17.7) 95.05 (196.34)
93.8 (182.2)
68.9 (16.3)
72.3 (19.81)
72.4 (20.18)
72.26 (20.06) 82.25 (10.7)
72.28 (19.87) 82.35 (10.7)
83.1
82.34 (11.0)
80.1 (11.3)
61.1
62.3
64.1
62.5
62.5
56.42 -OCH3
56.42, 55.01 -OCH3
95.1 (196.3)
103.3
34.4 -CH; 19.1, 19.07
-(CH3)2
21 155.1
152.3
103.3
155.6
137.4
80.2 (18.0)
94.84 (197.2)
72.35 (19.9)
85.23
64.3
54.9 -OCH3, 34.4 -CH;
19.1, 19.07 -(CH3)2
a Pyrimidine numbering or purine numbering. b Systematic numbering of the purine base. c Tentative.
which are compiled in Scheme 5. The top row formulas show a se-
ries of 2ꢀ-deoxyribonucleosides (dU,34 dA,351a13 and 2a35) and their
conformer populations; the lower row shows the corresponding 2ꢀ-
fluoroarabinonucleosides (2ꢀF-ara-U5Et,362b,37,38 1c and 2c31a,b).
The introduction of a fluorine atom in the 2ꢀ-arabino config-
uration of the nucleoside moiety enhances the population of the
N-conformers (dU = 30% → 2ꢀF-ara-U5Et = 44%), (dA = 28% →
2ꢀF-ara-A, (2b) = 58%). From Scheme 5, it can be concluded that
this effect is more predominant in the case of nucleosides with
an extra ring nitrogen atom placed next to the glycosylation site.
The N–S equilibrium in the case of the 2ꢀ-deoxyribonucleoside of
6-azapyrimidine 1a shows an N-conformer population of 58%,
while the 2ꢀ-fluoro derivative 1c (2ꢀF-ara “up”) shows a 100%
N-conformer population (6-aza-dU (1a) = 58% → 6-aza-2ꢀF-
ara-U (1c) = 100%). A similar situation is observed for the
azapurine nucleoside 2c (c7z8Ad, (2a) = 37% N → 2ꢀF-ara-c7z8Ad,
(2c) = 78% N; purine numbering is used). It was suggested
that these differences are connected with the stronger anomeric
effect caused by the 6-azauracil base compared to that of the
uracil moiety. The same effect is observed in the pyrazolo[3,4-
d]pyrimidine base compared to that of the purine base. The 6-
aza-2ꢀ-fluororibonucleoside 1d with the fluoro substituent in the
ribo configuration shows a 98% N-conformer population31b being
similar to that of 2ꢀF-ribo-U with 89% N-conformer (Scheme 5).
Scheme 5 Conformer population of 2ꢀ-deoxy and 2ꢀ-fluoro nucleosides.
600 | Org. Biomol. Chem., 2008, 6, 596–607 This journal is The Royal Society of Chemistry 2008
©

View Article Online
The gauche effect of the 2ꢀ-F “up” and the 3ꢀ-OH is considered to
be a contributing factor in driving the conformation towards N;
it is not the case for the ribo analogue (2ꢀ-F “down”).
The conformational properties of 1c were also studied by X-
ray analysis. In the single-crystal of the nucleoside 1c26 the sugar
moiety adopts the N conformation with a torsion angle of the
glycosylic bond in the anti range (v = −125.37 (13)◦; (3T2) with
P = 359.2◦ and sm = 31.4) similar to that found in solution.
The gauche effect between the O4ꢀ and the extra nitrogen in the
heterocyclic ring is variable and controlled by the gycosylic bond
torsional angle.
This places the O4ꢀ and N6 in a perfect gauche position in
both the N and the S conformations. However, only in the N
conformation is a clash between the 2ꢀ-F “up” and N6 avoided,
while in the S conformation the 2-oxo group of the base clashes
with the 2ꢀ-F “up” substituent.
the same as that of 1a (pKa = 6.8). These values are significantly
lower when compared to that of 2ꢀ-deoxyuridine (pKa = 9.3).
Consequently, the protection of the lactam moiety was performed
employing the o-anisoyl group by using the protocol of transient
protection.40 For that Et3SiCl was employed in the presence of
pyridine to protect the sugar hydroxyls. Then, the acylating reagent
was added in pyridine solution. The silyl groups were removed
by treatment with 5% trifluoroacetic acid in dichloromethane–
methanol (7 : 3) to yield 18. The N-3 anisoyl protected nucleoside
18 was further converted into the DMT derivative 19, later it
was transformed into the phosphoramidite building block 3b
(Scheme 6) which was used for the oligonucleotide synthesis.
Next, the phosphoramidite of the pyrazolo[3,4-d]pyrimidine
nucleoside 2c was synthesized. The amino group of 2c was
protected with an isobutyryl residue using the protocol of transient
protection.41 The protected 20 was then converted into the DMT
derivative 21 and further transformed into the phosphoramidite
4 under standard conditions (Scheme 7). The structures of all
protected compounds were characterized by 1H, 13C and 19F NMR
spectroscopy as well as by elemental analysis. The 31P NMR
spectra of 3b and 4 show a four-bond coupling between the P-
3ꢀ and F-2ꢀ.
4. Nucleobase protection and phosphoramidite synthesis
In order to study the influence of the 2ꢀ-fluoro substituent as
well as an additional ring nitrogen atom in the nucleobase,
oligonucleotides containing 1c and 2c were prepared. For that
the phosphoramidite building blocks 3b and 4 were synthesized
and employed in solid-phase oligonucleotide synthesis. Initial
experiments using the unprotected nucleoside 1c for phospho-
ramidite synthesis failed (Scheme 6). The phosphitylation of 17
with chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine in
CH2Cl2 at r.t. afforded the phosphoramidite 3a (TLC-monitoring).
However, its chromatographical purification was cumbersome due
to its degradation into unresolved products (Scheme 6). We also
observed this phenomenon for the related 6-aza-2ꢀ-deoxyuridine
phosphoramidite building block.13 As we reasoned that this
behaviour results from the lactam moiety, the pKa value of 1c was
determined by spectrophotometric titration39 and was found to be
5. Synthesis, base pairing and duplex stability of oligonucleotides
Oligonucleotide synthesis was carried out on solid phase with an
ABI 392-08 synthesizer at a 1 lmol scale employing the synthesized
phosphoramidites 3b, 4 as well as standard building blocks. The
coupling yields were always higher than 95%. The synthesis
of oligonucleotides was performed by employing the DMT-on
mode. After cleavage from the solid support, the oligomers were
deprotected in 25% aqueous ammonia solution for 14–16 h at
◦
60 C. The purification of the 5ꢀ-dimethoxytritylated oligomers
was carried out by reversed-phase HPLC (see experimental part).
i
i
Scheme 6 Reagents and conditions: (i) DMTr-Cl, pyridine, r.t.; (ii) Pr2NP(Cl)OCH2CH2CN, Pr2EtN, CH2Cl2, 30 min, r.t.; (iii) Et3SiCl, pyridine,
o-anisoyl chloride, 5% CF3COOH (CH2Cl2–MeOH 70 : 30) r.t.
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 596–607 | 601
©

View Article Online
i
Scheme 7 Reagents and conditions: (i) Me3SiCl, isobutyric anhydride, pyridine, 4 h, r.t.; (ii) DMTr-Cl, pyridine, r.t.; (iii) Pr2NP(Cl)OCH2CH2CN,
iPr2EtN, CH2Cl2, 30 min, r.t.
Table 3 Tm Values of oligonucleotide duplexes containing regular and
The molecular masses of the oligonucleotides were determined
by MALDI-TOF Biflex-III mass spectrometry (Bruker Saxonia,
Leipzig, Germany) with 3-hydroxypicolinic acid (3-HPA) as a
matrix. The detected masses were identical with the calculated
values (see experimental part, Table 5).
base-modified nucleosides at different pH valuesa
pH 7.0
pH 6.0
Duplex
Tm/◦C
DTm/◦C
Tm/◦C
DTm/◦C
To evaluate the combined effect of 2ꢀ-fluoro substitution and the
presence of a ring nitrogen placed next to the glycosylation site of
the nucleosides 1c and 2c on the duplex stability, hybridization
experiments were performed using the oligonucleotide duplex 5ꢀ-
d(TAGGTCAATACT)-3ꢀ 22 and 3ꢀ-d(ATCCAGTTATGA) 23 as
a reference. The replacement of dT in one strand of the duplex
by the 6-aza-2ꢀ-deoxy-2ꢀ-fluoroarabinouracil 1c results in a DTm
5ꢀ-d(TAG GTC AAT
ACT)-3ꢀ (22)
50
20
37
45
28
49
—
51
30
42
48
38
49
—
3ꢀ-d(ATC CAG TTA
TGA)-5ꢀ (23)
5ꢀ-d(TAG GTC AAT
ACT)-3ꢀ (22)
−30
−13
−5
−21
−9
3ꢀ-d(A1cC CAG 1c1cA
1cGA)-5ꢀ (24)
◦
of −13 C (22·25) when paired with dA (Scheme 8), while its 2ꢀ-
deoxyribonucleoside analogue 1a shows less of a decrease with a
DTm of only −5 ◦C (22·26). This effect is substantial when all the
dT residues in one strand are replaced by 1c (22·24) (Table 3).
5ꢀ-d(TAG GTC AAT
ACT)-3ꢀ (22)
3ꢀ-d(ATC CAG 1cTA
TGA)-5ꢀ (25)
5ꢀ-d(TAG GTC AAT
ACT)-3ꢀ (22)
−3
3ꢀ-d(ATC CAG 1aTA
TGA)-5ꢀ (26)
5ꢀ-d(TAG G1aC AA1a
ACT)-3ꢀ (27)
−22
−1
−13
−2
3ꢀ-d(A1aC CAG TTA
1aGA)-5ꢀ (28)
5ꢀ-d(TAG GTC 2cAT
ACT)-3ꢀ (29)
3ꢀ-d(ATC CAG TTA
TGA)-5ꢀ (23)
Scheme 8 Face to face base pair motif of dA–1c.
a Measured at 260 nm in 1M NaCl, 100 mM MgCl2 and 60 mM Na-
cacodylate (pH 7.0 and 6.0) with a 5 lM single-strand concentration.
Earlier, we anticipated that the low pKa value of 1a (pKa
=
6.8) has an unfavourable effect on the base pair stability. This
is now also suggested for the nucleoside 1c. At neutral pH the
6-azapyrimidine nucleoside molecule predominantly exists as a
negatively charged species. As this withstands base pair formation
the anion is generating a mismatch. At pH values below 7.0 a
proton is delivered from the solution leading partially or totally
to the expected base pair thereby enhancing the Tm value of
duplex melting. In order to confirm this, the duplex stability of the
modified oligonucleotides containing 1c and 1a was determined
at two different pH values (Table 3). At pH 6.0 an increase in
the Tm of 5 ◦C for one incorporation of 1c (22·25) and 10 ◦C for
four incorporations of 1c (from 20 ◦C to 30 ◦C) was observed,
when compared with Tm values measured at pH 7.0. Duplexes
containing 6-aza-2ꢀ-deoxyuridine 1a instead of the fluoro analogue
1c show higher Tm values and smaller pH-dependent changes.
Although the pKa values of lactam deprotonation are the same for
1a (6.8) and 1c (6.8) the pH-dependent duplex stability is different.
Other factors such as stacking interactions and changes in the
populations of protonated and non-protonated bases within the
duplex might be of importance.
The results obtained from the fluorinated 6-azapyrimidine
nucleoside prompted us to study the above effects on “purine”
arabinonucleoside 2c with a 2ꢀ-fluoro substituent in the “up”
position. From previous reports, it was known from oligonu-
cleotides containing 2ꢀF-ara-A; 2b and 9-(2-deoxy-2-fluoro-b-D-
arabinofuranosyl) guanine (2ꢀF-ara-G) that they do not have
much influence on the stability of DNA and RNA.37 Here,
602 | Org. Biomol. Chem., 2008, 6, 596–607
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
we studied the effect of 2ꢀ-deoxy-2ꢀ-fluoro-b-D-arabinofuranosyl
pyrazolo[3,4-d]pyrimidine (2c). For that purpose oligonucleotides
containing 2c were prepared by replacing dA in the standard
sequences (Table 4). One incorporation of 2c leads to a stable
duplex (29·23) with a Tm of 49 ◦C, and the replacement of four dA
residues by 2c (30·23) does not change this value (Table 4). These
results clearly demonstrate that 2c has no significant influence on
the duplex stability as almost similar results were found for the 2ꢀ-
deoxyribonucleoside 2a indicating that the presence of a 2ꢀ-fluorine
atom within the 8-azapurine nucleoside 2c does not change the
base pair and duplex stability (Scheme 9). Next, the effect of
such a modification on the stability of DNA–RNA hybrids was
Table 4
T
m Values of DNA–DNA and DNA–RNA duplexes containing
regular and base-modified nucleosides 2c, 2a and 1ca
DG◦
/
310
Duplex
Tm/◦C kcal mol−1 DTm/◦C
5ꢀ-d(TAG GTC AAT ACT)-3ꢀ (22)
3ꢀ-d(ATC CAG TTA TGA)-5ꢀ (23)
50
−11.8
−11.0
−11.0
−12.1
−11.6
−8.1
—
5ꢀ-d(TAG GTC 2cAT ACT)-3ꢀ (29)
3ꢀ-d(ATC CAG TTA TGA)-5ꢀ (23)
49
−1
−1
+1
0
5ꢀ-d(T2cG GTC 2c2cT 2cCT)-3ꢀ (30) 49
3ꢀ-d(ATC CAG TTA TGA)-5ꢀ (23)
5ꢀ-d(TAG GTC A2aT ACT)-3ꢀ (31)
3ꢀ-d(ATC CAG TTA TGA)-5ꢀ (23)
51
50
37
48
studied. The standard DNA–RNA hybrid (22·34) shows a Tm
=
48 ◦C, whereas four modifications with 2c result in a DTm value of
−3 ◦C (30·34) which is lower than that for the DNA–DNA duplex.
Even though nucleoside 2c has a high N-conformer population
similar to that of 1c, surprisingly we observed stable DNA–DNA
duplexes and less stable DNA–RNA hybrids for oligonucleotides
incorporating 2c. Also a dependence of the duplex stability on pH
value is not observed for 2c (29·23; Table 3) as was found for the
corresponding pyrimidine nucleosides.
3ꢀ-d(ATC C2aG TT2a TGA)-5ꢀ (32)
5ꢀ-d(T2aG GTC AAT 2aCT)-3ꢀ (33)
5ꢀ-d(TAG GTC 2cAT ACT)-3ꢀ (29)
3ꢀ-d(ATC CAG 1cTA TGA)-5ꢀ (23)
−13
—
5ꢀ-d(TAG GTC AAT ACT)-3ꢀ (22)
3ꢀ-r(AUC CAG UUA UGA)-5ꢀ (34)
−10.8
−10.4
5ꢀ-d(T2cG GTC 2c2cT 2cCT)-3ꢀ (30) 45
3ꢀ-r(AUC CAG UUA UGA)-5ꢀ (34)
−3
a Measured at 260 nm in 1M NaCl, 100 mM MgCl2 and 60 mM Na-
cacodylate at pH 7.0 with a 5 lM single-strand concentration.
Experimental
General
All chemicals were purchased from Acros, Aldrich, Sigma, or
Fluka (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany).
Solvents were of laboratory grade. Thin layer chromatography
(TLC): aluminium sheets, silica gel 60 F254, 0.2 mm layer (VWR
International, Darmstadt, Germany). Column flash chromatogra-
phy (FC): silica gel 60 (VWR International, Darmstadt, Germany)
at 0.4 bar; sample collection with an UltroRac II fraction collector
(LKB Instruments, Sweden). UV spectra: U-3200 spectrometer
(Hitachi, Tokyo, Japan); kmax (e) in nm. NMR spectra: Avance-
250 or AMX-500 spectrometers (Bruker, Karlsruhe, Germany), at
250.13 MHz for 1H and 13C; d in ppm relative to Me4Si as internal
standard, or external standard CFCl3 for 19F, or external standard
85% H3PO4 for 31P. The J values are in Hz. Elemental analyses were
performed by Mikroanalytisches Laboratorium Beller (Go¨ttingen,
Germany). The melting curves were measured with a Cary-1/3
UV–vis spectrophotometer (Varian, Australia) equipped with a
Cary thermoelectrical controller. The temperature was measured
continuously in the reference cell with a Pt-100 resistor, and the
thermodynamic data of duplex formation were calculated by the
Meltwin 3.0 program.42a,b
Scheme 9 Face to face base pair motif of 2c–dT.
Conclusions
Oligonucleotides containing 6-azapyrimidine- or 8-aza-7-deaza-
purine 2ꢀ-deoxy-2ꢀ-fluoroarabinonucleosides 1c, 2c were synthe-
sized using the phosphoramidite building blocks 3b and 4 by
employing solid-phase synthesis. The nucleosides 1c, 2c as well
as the 2ꢀ-deoxy-2ꢀ-fluoro ribofuranosyl nucleoside 1d were either
prepared by convergent stereoselective synthesis or by nucleoside
transformation. A fluoro substituent in the 2ꢀ-’up” position drives
the sugar conformation towards a higher N-population (dU =
30% → 2ꢀF-ara-U5Et = 44%; dA = 28% → 2ꢀF-ara-A, 2b = 58%).
A combination of a 2ꢀ-fluoroarabino substituent and a nitrogen
placed next to the glycosylation site drives the N-conformation
near to 100% (1c = 100% and 2c = 78%). The destabilization
found for duplexes incorporating 1c is caused by the deprotonation
of the nucleobase as well as the fluoro substitution, which is
clearly evidenced from the higher Tm values of the 2ꢀ-deoxyribo
counterpart 1a. In the case of purine nucleoside 2c we observed
no negative influence of the 2ꢀ-F substituent and the 8-aza-
modification on the duplex stability of oligonucleotides. Even
though nucleoside 2c has a high N-conformer population similar
to that of 1c, surprisingly more stable DNA–DNA duplexes were
formed than DNA–RNA hybrids.
Synthesis, purification and characterization of oligonucleotides
The oligonucleotide synthesis was performed on a DNA syn-
thesizer, model ABI 392–08 (Applied Biosystems, Weiterstadt,
Germany) at a 1lmol scale using the phosphoramidites and fol-
lowing the synthesis protocol for 3ꢀ-cyanoethyl phosphoramidites
(users’ manual for the 392 DNA sythesizer, Applied Biosystems,
Weiterstadt, Germany). The coupling efficiency was always higher
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 596–607 | 603
©

View Article Online
Table 5 Molecular masses [M + H]+ of oligonucleotides measured by
by addition of saturated aqueous NaHCO3 (15 ml), stirring was
continued for 15 min and the reaction mixture was filtered through
a pad of celite. The CHCl3 layer was separated, washed with
saturated aqueous NaCl (50 ml), dried (Na2SO4), concentrated,
and then applied to FC (silica gel, column 10 × 3 cm, eluted with
petroleum ether–EtOAc, 2 : 1) to yield the title compound 7 as a
colorless foam (0.72 g, 67%) (found: C, 58.22; H, 3.84; N, 9.21%.
C22H18FN3O7 requires C, 58.02; H, 3.98; N, 9.23%); TLC (silica
gel, petroleum ether–EtOAc, 2 : 1): Rf 0.33; kmax(MeOH)/nm 263
(e/dm3 mol−1 cm−1 7 000).
MALDI-TOF mass spectrometry
Oligonucleotide
[M + H]+ (calc.) [M + H]+ (found)
5ꢀ-d(TAG GTC AAT ACT)-3ꢀ (22)
5ꢀ-d(AGT ATT GAC CTA)-3ꢀ (23)
3645.4
3645.4
3645
3645
3665
3631
3619
3619
3662
3716
5ꢀ-d(AG1c A1c1c GAC C1cA)-3ꢀ (24) 3665.3
5ꢀ-d(AGT AT1a GAC CTA)-3ꢀ (26) 3631.7
5ꢀ-d(TAG G1aC AA1a ACT)-3ꢀ (27) 3619.3
5ꢀ-d(AG1a ATT GAC C1aA)-3ꢀ (28) 3619.3
5ꢀ-d(TAG GTC 2cAT ACT)-3ꢀ (29)
3661.4
5ꢀ-d(T2cG GTC 2c2cT 2cCT)-3ꢀ (30) 3716.4
2-(2-Deoxy-2-fluoro-b-D-arabinofuranosyl)-1,2,4-triazin-
3,5(2H,4H)-dione (1c)
than 95%. After cleavage from the solid support the oligonu-
cleotides were deprotected with 25% aqueous NH3 for 14–16 h
at 60 ◦C. The purification of the 5ꢀ-O-(dimethoxytrityl)oligomers
was carried out on reversed-phase HPLC (Merck–Hitachi-HPLC:
250 × 4 mm RP-18 column) with the following gradient system
[A: 0.1M (Et3NH)OAc (pH 7.0)–MeCN 95 : 5 and B: MeCN];
gradient: 3 min 20% B in A, 12 min 20–50% B in A and 25 min
20% B in A with a flow rate of 1 ml min−1. The purified ‘trityl-
on’ oligonucleotides were ◦treated with 2.5% dichloroacetic acid
in CH2Cl2 for 5 min at 0 C to remove the 4,4ꢀ-dimethoxytrityl
residues. The detritylated oligomers were purified again by
reversed-phase HPLC (gradient: 0–20 min 0–20% B in A, flow
rate 1 ml min−1). The oligomers were desalted on a short column
(RP-18, silica gel) and lyophilized on a Speed-Vac evaporator to
yield colorless solids which were frozen at −24 ◦C.
Compound 7 (0.21 g, 0.46 mmol) was suspended in NaOMe–
MeOH (0.2 N, 20 ml), and the solution was stirred at r.t. for 2 h.
The solution was neutralized (Dowex-50 H+-form) and filtered.
The resin was washed with MeOH, and the combined filtrate was
concentrated and applied to FC (silica gel, column 10 × 3 cm,
eluted with CH2Cl2–MeOH, 9 : 1) furnishing 1c as a colorless solid
(0.10 g, 88%) (found: C, 38.68; H, 4.06; N, 16.76%. C8H10FN3O5
requires C, 38.87; H, 4.08; N, 17.00%); TLC (silica gel, CH2Cl2–
MeOH, 9 : 1): Rf 0.42; kmax(MeOH)/nm 263 (e/dm3 mol−1 cm−1
2
ꢀ
5 900); dF (250 MHz; [d6]DMSO; Me4Si) −205.18 (dd, JF, H2
=
53, 3JF, H3 = 20 Hz).
ꢀ
The molecular masses of the oligonucleotides were determined
by MALDI-TOF Biflex-III mass spectrometry (Bruker Saxonia,
Leipzig, Germany) with 3-hydroxypicolinic acid (3-HPA) as a
matrix. The detected masses were identical with the calculated
values (Table 5).
2,2ꢀ-O-Anhydro(b-D-arabinofuranosyl)-1,2,4-triazin-3,5(2H,4H)-
dione (9)
To a solution of compound 8 (2.02 g, 8.24 mmol) in pyridine
(36.8 ml) was added 1,3-dichloro-1,1,3,3-tetraisopropyl-disiloxane
(2.5 ml) and the mixture stirred at r.t. for 5 h. The solution
was then evaporated and the residue dissolved in EtOAc. This
was successively washed with cold aqueous NaHCO3, saturated
aqueous NaCl, then dried over Na2SO4, and evaporated to dryness.
The resulting foam was dissolved in pyridine (34 ml), and to this
solution methane sulfonyl chloride was added (0.8 ml) and the
mixture stirred for 16 h at r.t. After that H2O (2.6 ml) was added
to the reaction mixture, the solvents were removed by evaporation,
and the residue was dissolved in toluene. The organic phase was
extracted twice with 5% aqueous NaHCO3, dried over Na2SO4
and evaporated to dryness. Residual pyridine was co-evaporated
with toluene. The above residue was dissolved in THF (35 ml),
and to this solution 1M tetra-n-butylammonium fluoride in THF
(2.2 ml) was added and stirred at r.t. for about 2 h. The solvent
was removed by evaporation and the residue dissolved in H2O and
extracted with EtOAc. The aqueous phase was evaporated and
residual water was removed by co-evaporation with ethanol. The
residue was applied to FC (silica gel, column 15 × 3 cm, eluted
with CH2Cl2–MeOH, 4 : 1) furnishing 9 as a colorless solid (1.3 g,
70%) (found: C, 42.48; H, 3.90; N, 18.39%. C8H9N3O5 requires C,
42.30; H, 3.99; N, 18.50%); TLC (silica gel, CH2Cl2–MeOH, 8 : 2):
Rf 0.7; kmax (MeOH)/nm 256 (e/dm3 mol−1 cm−1 6 300) and 221
(7 100). dH (250.13 MHz; [d6]DMSO; Me4Si) 3.22–3.42 (2 H, m,
5ꢀ-H), 4.14 (1 H, m, 4ꢀ-H), 4.44 (1 H, m, 3ꢀ-H), 4.98 (1 H, ‘t’, J 4.9,
5ꢀ-OH), 5.28 (1 H, d, J 5.9, 2ꢀ-H), 5.92 (1 H, ‘t’, J 4.1, 3ꢀ-OH), 6.33
(1 H, d, J 5.9, 1ꢀ-H), 7.63 (1 H, s, 5-H).
1-Bromo-2-deoxy-2-fluoro-3,5-di-O-benzoyl-a-D-arabinofuranose
(6)16–18
To a solution of 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-a-D-arabino-
furanose (1.0 g, 2.15 mmol) in CH2Cl2 (6.0 ml), 30% solution of
HBr in acetic acid (1.2 ml) was added, and the reaction mixture
was stirred at r.t. for 16 h, and evaporated to dryness. The oily
residue was redissolved in CH2Cl2 (20 ml), then washed with
water (10 ml) and then with aqueous saturated NaHCO3 solution
(10 ml), dried over Na2SO4, and concentrated to a viscous syrup
which was further dried under high vacuum for 18 h at r.t. The
colourless syrup 6 (0.86 g, 95%) was used in the next step without
purification.
2-(2-Deoxy-2-fluoro-3,5-di-O-benzoyl-b-D-arabinofuranosyl)-
1,2,4-triazin-3,5(2H,4H)-dione (7)
A suspension of 6-azauracil (0.35 g, 3.09 mmol), hexamethyld-
isilazane (HMDS) (1.2 ml) and chlorotrimethylsilane (TMSCl)
(0.11 ml) was heated to reflux (170 ◦C) under anhydrous conditions
for 3 h. The excess amount of HMDS–TMSCl was removed
by distillation in vacuo. The residual oil solidified upon storage
in vacuo to furnish 5 (0.77 g, 2.98 mmol). A mixture of 6 (0.93 g,
2.01 mmol) and 5 (0.61 g, 2.4 mmol) in dry chloroform (9 ml) was
stirred at r.t. under argon, CuI (0.5 g, 2.6 mmol) was added to
the solution and stirred for 24 h at r.t. The reaction was quenched
604 | Org. Biomol. Chem., 2008, 6, 596–607
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
1-(3,5-Di-O-tetrahydropyran-2-yl-b-D-arabinofuranosyl)-1,2,4-
triazin-3,5(2H,4H)-dione (10)
Glycosylation of 4-methoxy-1H-pyrazolo-[3,4-d]pyrimidine (12)
with the sugar bromide 6
To a suspension of 9 (0.25 g, 1.10 mmol) in DMF (4.4 ml) and 3,4-
dihydropyran (2.64 ml) was added p-toluenesulfonic acid (0.2 g)
at 0 ◦C for 4 h, after which time a clear solution was obtained.
This was neutralized with Et3N and then evaporated to dryness.
The residue was redissolved in EtOAc, washed with saturated
NaHCO3 and the organic phase was dried over Na2SO4. Removal
of the solvent gave a residue which was triturated with hexanes and
filtered. The filter cake was washed with hexanes and dried to give
the protected compound as a white solid, which was suspended
in MeOH (4.5 ml) and 1 N NaOH (1.5 ml) and stirred at r.t.
for 2 h, then neutralized with dilute acetic acid. The mixture
was evaporated to dryness and the residue was applied to FC
(silica gel, 15 × 3 cm, eluted with CH2Cl2–MeOH, 98 : 2) to give
the title compound 10 as a colorless foam (0.32 g, 70%) (found:
C, 52.72; H, 6.59; N, 10.30%. C18H27N3O8 requires C, 52.29; H,
6.58; N, 10.16%); TLC (silica gel, CH2Cl2–MeOH, 95 : 5): Rf 0.6;
kmax(MeOH)/nm 266 (e/dm3 mol−1 cm−1 5 600); dH (250.13 MHz;
[d6]DMSO; Me4Si) 1.45–1.61 (12 H, m, H of THP), 3.36–3.92
(7 H, m, 5ꢀ-H, H of THP and 4ꢀ-H), 4.19 (1 H, m, 3ꢀ-H), 4.44 (m,
1 H, 2-ꢀH), 4.57–4.74 (2 H, m, H of THP), 5.49 (1 H, d, J 5.9,
2ꢀ-OH), 6.18 (1 H, d, J 7.0, 1ꢀ-H), 7.59 (1 H, s, 5-H), 12.21 (1 H, s,
NH).
4-Methoxy-1H-pyrazolo-[3,4-d]pyrimidine
(12)43
(0.2
g,
1.3 mmol) and KOH (0.36 g, 6.4 mmol) were suspended in
MeCN (20 ml) and stirred for 10 min at r.t. Then, the phase-
transfer catalyst TDA-1 (0.1 ml) was added, and stirring was
continued for another 10 min. A solution of bromide 6 (0.6 g,
1.42 mmol) in MeCN was added in 3 portions to the reaction
mixture during 10 min, and it was stirred for another 10 min and
filtered. The filtrate was evaporated to dryness, and the residue
was applied to FC (silica gel, column 15 × 3 cm, eluted with
petroleum ether–EtOAc). The fractions containing individual
compounds were combined to give the three compounds below in
the order of their elution.
1-(2-Deoxy-2-fluoro-3,5-di-O-benzoyl-b-D-arabinofuranosyl)-4-
methoxy-1H-pyrazolo[3,4-d]pyrimidine (13)
Colorless solid (0.33 g, 50.3%); TLC (silica gel, petroleum
ether–ethyl acetate, 2 : 1): Rf 0.63; kmax(MeOH)/nm 231
(e/dm3 mol−1 cm−1 31 000), which is identical to the compound
previously synthesized.8
2-(2-Deoxy-2-fluoro-3,5-di-O-benzoyl-b-D-arabinofuranosyl)-4-
methoxy-1H-pyrazolo[3,4-d]pyrimidine (15)
Colorless solid (45 mg, 6.8%); TLC (silica gel, petroleum
ether–ethyl acetate, 2 : 1): Rf 0.52; kmax(MeOH)/nm 222
(e/dm3 mol−1 cm−1 32 800), which is identical to that of a previous
report.8
1-(2-Deoxy-2-fluoro-3,5-di-O-tetrahydropyran-2-yl-b-D-
ribofuranosyl)-1,2,4-triazin-3,5(2H,4H)-dione (11)
To a stirred mixture of 10 (0.25 g, 0.60 mmol) in CH2Cl2 (3.6 ml)
and pyridine (0.6 ml) was added diethylaminosulfur trifluoride
(DAST) (0.30 g, 1.86 mmol) at −60 C under N2. The resulting
1-(5-O-Benzoyl-2-deoxy-2-fluoro-b-D-arabinofuranosyl)-4-
methoxy-1H-pyrazolo[3,4-d]pyrimidine (14)
◦
mixture was slowly warmed to room temperature and then refluxed
for 4 h. The reaction was quenched with saturated NaHCO3 and
ice-water, then extracted with CH2Cl2, washed with saturated
NaHCO3 and dried over Na2SO4. Removal of the solvent gave
a dark-brown syrup. The residue was applied to FC (silica gel
column, 15 × 3 cm, eluted with CH2Cl2–MeOH, 99 : 1) to give
11 as a colorless foam (0.12 g, 48%) (found: C, 52.20; H, 6.24;
N, 10.06%. C18H26FN3O7 requires C, 52.04; H, 6.31; N, 10.12%);
TLC (silica gel, CH2Cl2–MeOH, 9 : 1): Rf 0.77; kmax (MeOH)/nm
261 (e/dm3 mol−1 cm−1 5 700); dF (250 MHz; [d6]DMSO; Me4Si)
Colorless solid (45 mg, 8.7%) (found: C, 56.01; H, 4.69; N, 14.64%.
C18H17FN4O5 requires C, 55.67; H, 4.41; N, 14.43%); TLC (silica
gel, petroleum ether–ethyl acetate, 2 : 1): Rf 0.36; kmax(MeOH)/nm
230 (e/dm3 mol−1 cm−1 30 800); dF (250 MHz; [d6]DMSO; Me4Si)
−205.69 (dd, 2JF, H2 = 53, 3JF, H3 = 18).
ꢀ
ꢀ
2-[2-Deoxy-5-O-(4,4ꢀ-dimethoxytrityl)-2-fluoro-b-D-
arabinofuranosyl]-1,2,4-triazin-3,5(2H,4H)-dione (17)
Compound 1c (0.37 g, 1.50 mmol) was suspended in anhydrous
pyridine (5 ml), 4,4ꢀ-dimethoxytrityl chloride (0.59 g, 1.78 mmol)
was added in portions and the mixture was stirred at r.t. for 3 h.
The reaction was quenched by the addition of MeOH and the
mixture was evaporated to dryness. The residue was dissolved in
CH2Cl2, washed with H2O and dried over Na2SO4. The organic
layer was co-evaporated with toluene and the residue was applied
to FC (silica gel, column 12 × 3 cm, eluted with CH2Cl2–MeOH,
9 : 1) furnishing 17 as a colorless foam (0.63 g, 76.5%). TLC
(silica gel, CH2Cl2–MeOH, 9 : 1): Rf 0.74; kmax (MeOH)/nm 233
(e/dm3 mol−1 cm−1 28 700), 267 (8 900).
−199.16 (dt, 2JF, H2 = 52, 3JF, H3 = 24, 3JF, H1 = 21).
ꢀ
ꢀ
ꢀ
1-(2-Deoxy-2-fluoro-b-D-ribofuranosyl)-1,2,4-triazin-3,5(2H,4H)-
dione (1d)
A solution of compound 11 (0.14 g, 0.34 mmol) in EtOH
(8.5 ml) was stirred with pyridinium p-toluene sulfonate (0.17 g,
0.67 mmol) at 60 ◦C for 3 h. The mixture was then evaporated and
the residue was applied to FC (silica gel column, 15 × 3 cm, eluted
with CH2Cl2–MeOH, 9 : 1) to give 1d as a colorless foam (72 mg,
87%) (found: C, 38.62; H, 4.24; N, 16.64%. C8H10FN3O5 requires
C, 38.87; H, 4.08; N, 17.00%); TLC (silica gel, CH2Cl2–MeOH,
9 : 1): Rf 0.37; kmax(MeOH)/nm 263 (e/dm3 mol−1 cm−1 5 300); dF
2-(2-Deoxy-2-fluoro-b-D-arabinofuranosyl)-N4-(2-
methoxybenzoyl)-1,2,4-triazin-3,5(2H,4H)-dione (18)
(250 MHz; [d6]DMSO; Me4Si) −202.50 (dt, 2JF, H2 = 52, 3JF, H3
=
Compound 1c (0.25 g, 1.01 mmol) was treated with triethylsilyl
chloride (0.44 ml, 2.62 mmol) in pyridine (1.6 ml) and stirred at
ꢀ
ꢀ
24, 3JF, H1 = 21).
ꢀ
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 596–607 | 605
©

View Article Online
r.t. for 30 min and then o-anisoyl chloride (0.3 ml, 2.01 mmol) was
added. After being stirred for 5 h, the mixture was evaporated then
co-evaporated with toluene to remove pyridine completely. The
oily residue was further treated with 5% CF3COOH in CH2Cl2–
CH3OH (7 : 3, 10 ml), stirred at r.t. for 1 h and evaporated. To this
residue 5% aqueous NaHCO3 was added and the mixture extracted
with CH2Cl2, the extract was dried over Na2SO4, evaporated and
the residue was applied to FC (silica gel, column 15 × 3 cm,
eluted with CH2Cl2–MeOH, 98 : 2). Evaporation of the main zone
afforded a colorless foam, which was recrystallized from MeOH
to give 18 as colorless crystals (0.29 g, 75%) (found: C, 50.10;
H, 4.20; N, 10.85; F, 4.60%. C16H16FN3O7 requires C, 50.40; H,
4.23; N, 11.02; F, 4.98%); TLC (silica gel, CH2Cl2–MeOH, 95 : 5):
Rf 0.29; kmax(MeOH)/nm 259 (e/dm3 mol−1 cm−115 200) and 324
added, and the solution stirred for another 15 min. The solution
was evaporated to dryness, dissolved in 10 ml H2O, and extracted
with CH2Cl2 (3 × 15 ml). The organic layer was dried over Na2SO4,
evaporated and the residue was applied to FC (silica gel, column
15 × 3 cm, eluted with CH2Cl2–MeOH, 95 : 5). Compound 20
was obtained as a colorless foam (0.17 g, 90%) (found: C, 49.69;
H, 5.51; N, 20.7%. C14H18FN5O4 requires C, 49.55; H, 5.35; N,
20.64%); TLC (CH2Cl2–MeOH 90 : 10): Rf 0.65; kmax(MeOH)/nm
267 (e/dm3 mol−1 cm−1 10 400) and 232 (9 400); dF (250 MHz;
[d6]DMSO; Me4Si) −205.14 (dd, 2JF, H2 = 54, 3JF, H3 = 19).
ꢀ
ꢀ
2-[2-Deoxy-5-O-(4,4ꢀ-dimethoxytrityl)-2-fluoro-b-D-
arabinofuranosyl]-4-(isobutyrylamino)-2H-pyrazolo[3,4-
d]pyrimidine (21)
2
ꢀ
(4 200); dF (250 MHz; [d6]DMSO; Me4Si) −205.18 (dd, JF, H2
=
Compound 20 (0.3 g, 0.88 mmol) was suspended in anhydrous
pyridine (3.5 ml), 4,4ꢀ-dimethoxytrityl chloride (0.35 g, 1.03 mmol)
was added in portions and the mixture was stirred at r.t. for 3 h. The
reaction was quenched by the addition of MeOH and the mixture
was evaporated to dryness. The residue was dissolved in CH2Cl2,
washed with H2O and dried over Na2SO4. The organic layer was
co-evaporated with toluene and the residue was applied to FC
(silica gel, column 12 × 3 cm, eluted with CH2Cl2–MeOH, 90 : 10)
furnishing 21 as a colorless foam (0.49 g, 86%) (found: C, 65.45;
H, 5.80; N, 10.80%. C35H36FN5O6 requires C, 65.51; H, 5.65; N,
10.91%). TLC (CH2Cl2–acetone 9 : 1): Rf: 0.83; kmax (MeOH)/nm
267 (e/dm3 mol−1 cm−1 12 400), 233 (30 200); dF (250 MHz;
53, 3JF, H3 = 20).
ꢀ
2-[2-Deoxy-5-O-(4,4ꢀ-dimethoxytrityl)-2-fluoro-b-D-
arabinofuranosyl]-N4-(2-methoxybenzoyl)-1,2,4-triazin-
3,5(2H,4H)-dione (19)
Compound 18 (0.25 g, 0.65 mmol) was suspended in anhydrous
pyridine (3.1 ml), 4,4ꢀ-dimethoxytrityl chloride (0.26 g, 0.78 mmol)
was added in portions and the mixture was stirred at room
temperature for 3 h. The reaction was quenched by the addition
of MeOH and the mixture was evaporated to dryness. The residue
was dissolved in CH2Cl2, washed with H2O and dried over
Na2SO4. The organic layer was co-evaporated with toluene and
the residue was applied to FC (silica gel, column 12 × 3 cm,
eluted with CH2Cl2–acetone, 9 : 1) furnishing 19 as a colorless
foam (0.27 g, 60%) (found: C, 65.32; H, 5.21; N, 5.97; F, 2.60%.
C37H34FN3O9 requires C, 65.00; H, 5.01; N, 6.15; F, 2.78%); TLC
(silica gel, CH2Cl2–acetone, 95 : 5): Rf 0.78; kmax(MeOH)/nm
260 (e/dm3 mol−1 cm−1 17 800) and 323 (3 400); dF (250 MHz;
[d6]DMSO; Me4Si) −205.77 (dd, 2JF, H2 = 54, 3JF, H3 = 19).
ꢀ
ꢀ
2-[2-Deoxy-5-O-(4,4ꢀ-dimethoxytrityl)-2-fluoro-b-D-
arabinofuranosyl]-4-(isobutyrylamino)-2H-pyrazolo[3,4-d]-
pyrimidine 3ꢀ-(2-cyanoethyl N,N-diisopropylphosphoramidite) (4)
A stirred solution of 21 (0.31 g, 0.48 mmol) in anhydrous CH2Cl2
(8 ml) was pre-flushed with argon and treated with (i-Pr)2EtN
(0.14 ml, 0.80 mmol) followed by 2-cyanoethyl diisopropyl phos-
phoramidochloridite (0.12 ml, 0.53 mmol) at r.t. After 30 min, the
mixture was diluted with CH2Cl2 (20 ml), and quenched by adding
a 5% aqueous NaHCO3 solution (20 ml). Then, the aqueous layer
was extracted with CH2Cl2 (3 × 15 ml), the combined organic
layers dried over Na2SO4 and evaporated to an oil. The residue was
applied to FC (silica gel, column 10 × 3 cm, eluted with CH2Cl2–
acetone–Et3N, 95 : 5: 0.2) to give compound 4 as a colorless foam
(0.31 g, 76%). TLC (silica gel, CH2Cl2–acetone, 95 : 5): Rf 0.87. dP
(CDCl3) 152.55, 152.27.
[d6]DMSO; Me4Si) −205.53 (dd, 2JF, H2 = 54, 3JF, H3 = 19).
ꢀ
ꢀ
2-(2-Deoxy-2-fluoro-b-D-arabinofuranosyl)-N4-(2-
methoxybenzoyl)-1,2,4-triazin-3,5(2H,4H)-dione)
3ꢀ-(2-cyanoethyl diisopropylphosphoramidite) (3b)
A stirred solution of 19 (0.24 g, 0.35 mmol) in anhydrous CH2Cl2
(6 ml) was pre-flushed with argon and treated with (i-Pr)2EtN
(0.11 ml, 0.63 mmol) followed by 2-cyanoethyl diisopropyl phos-
phoramidochloridite (0.09 ml, 0.40 mmol) at r.t.. After 30 min, the
mixture was diluted with CH2Cl2 (20 ml), washed with 5% aqueous
NaHCO3 (10 ml), dried over Na2SO4, and evaporated to an oil.
The residue was applied to FC (silica gel, column 10 × 3 cm, eluted
with CH2Cl2–acetone–Et3N, 95 : 5: 0.2) to give compound 3b as
a colorless foam (0.19 g, 61%). TLC (silica gel, CH2Cl2–acetone,
95 : 5): Rf 0.85. dP (CDCl3) 152.59, 152.27.
Acknowledgements
We especially thank Dr Igor A. Mikhailopulo for PSEUROT
calculations, Dr J. He for helpful discussions, Mrs S. Budow and
K. Xu for measuring the NMR spectra, Dr K. I. Shaikh for
the MALDI-TOF spectra and Mrs E. Michalek and Mr Tran
for the oligonucleotide syntheses. Financial support by Roche
Diagnostics GmbH, Germany and ChemBiotech, Mu¨nster is
gratefully acknowledged.
2-(2-Deoxy-2-fluoro-b-D-arabinofuranosyl)-4-(isobutyrylamino)-
2H-pyrazolo[3,4-d]pyrimidine (20)
Compound 2c (0.15 g, 0.56 mmol) was repeatedly evaporated with
pyridine and suspended in pyridine (2.5 ml). Me3SiCl (0.34 ml)
was added and stirred for 15 min, followed by the addition of
isobutyric anhydride (1.45 ml) and stirring was continued for 4 h at
r.t. The reaction mixture was cooled in an ice-bath, and 0.5 ml H2O
was added. After 15 min, 0.5 ml 25% aqueous NH3 solution was
References
1 V. E. Marquez, C. K.-H. Tseng, H. Mitsuya, S. Aoki, J. A. Kelley, H.
Ford, Jr., J. S. Roth, S. Broder, D. G. Johns and J. S. Driscoll, J. Med.
Chem., 1990, 33, 978–985.
606 | Org. Biomol. Chem., 2008, 6, 596–607
This journal is
The Royal Society of Chemistry 2008
©

View Article Online
2 R. Masood, G. S. Ahluwalia, D. A. Cooney, A. Fridland, V. E.
Marquez, J. S. Driscoll, Z. Hao, H. Mitsuya, C.-F. Perno, S. Broder
and D. G. Johns, Mol. Pharmacol., 1990, 37, 590–596.
3 R. Filler and S. M. Naqvi, in Organofluorine Chemicals and Their
Industrial Applications, ed. R. E. Banks and E. Horwood, Holsted
Press, New York, 1979, p. 123; D. E. Bergstrom and D. J. Swartling,
in Fluorine-Containing Molecules. Structure, Reactivity, Synthesis and
Applications, ed. J. F. Liebman, A. Greenberg and W. R. Dolbier, Jr.,
VCH, New York, 1988, p. 259.
23 J. V. Tuttle, M. Tisdale and T. A. Krenitsky, J. Med. Chem., 1993, 36,
119–125.
24 M. Ikehara and J. Imura, Chem. Pharm. Bull., 1981, 29, 1034–1038.
25 H. Hayakawa, F. Takai, H. Tanaka, T. Miyasaka and K. Yamaguchi,
Chem. Pharm. Bull., 1990, 38, 1136–1139.
26 F. Seela, P. Chittepu, J. He and H. Eickmeier, Acta Crystallogr., Sect.
C: Cryst. Struct. Commun., 2004, C60, o884–o886.
27 F. Seela and H. Steker, Helv. Chim. Acta, 1985, 68, 563–570.
28 C. B. Anderson and D. T. Sepp, J. Org. Chem., 1967, 32, 607–611.
29 D. Barbry, D. Couturier and G. Ricart, J. Chem. Soc., Perkin Trans. 2,
1982, 249–254.
4 K. A. Watanabe, U. Reichman, K. Hirota, C. Lopez and J. J. Fox,
J. Med. Chem., 1979, 22, 21–24.
5 K. A. Watanabe, T.-L. Su, R. S. Klein, C. K. Chu, A. Matsuda, M.
Woo Chun, C. Lopez and J. J. Fox, J. Med. Chem., 1983, 26, 152–156.
6 K. A. Watanabe, T.-L. Su, U. Reichman, N. Greenberg, C. Lopez and
J. J. Fox, J. Med. Chem., 1984, 27, 91–94.
7 L. W. Hertel, J. S. Kroin, J. W. Misner and J. M. Tustin, J. Org. Chem.,
1988, 53, 2406–2409.
8 A. T. Shortnacy-Fowler, K. N. Tiwari, J. A. Montgomery, R. W.
Buckheit, Jr., J. A. Secrist, III and F. Seela, Helv. Chim. Acta, 1999,
82, 2240–2245.
9 C. G. Peng and M. J. Damha, J. Am. Chem. Soc., 2007, 129, 5310–5311.
10 C. G. Peng and M. J. Damha, Nucleic Acids Res., 2007, 35, 4977–4988.
11 (a) Y. C. Liaw, Y. G. Gao, V. E. Marquez and A. H.-J. Wang, Nucleic
Acids Res., 1992, 3, 459–465; (b) J. J. Barchi, Jr., L.-S. Jeong, M. A.
Siddiqui and V. E. Marquez, J. Biochem. Biophys. Methods, 1997, 34,
11–29.
12 (a) J. He, I. Mikhailopulo and F. Seela, J. Org. Chem., 2003, 68,
5519–5524; (b) F. Seela, V. R. Sirivolu, J. He and H. Eickmeier, Acta
Crystallogr., Sect. C: Cryst. Struct. Commun., 2005, C61, o67–o69.
13 F. Seela and P. Chittepu, J. Org. Chem., 2007, 72, 4358–4366.
14 K. W. Pankiewicz, Carbohydr. Res., 2000, 327, 87–105.
15 K. A. Watanabe, Collection Symposium Series, 2002, 5, 48.
16 H. G. Howell, P. R. Brodfuehrer, S. P. Brundidge, D. A. Benigni and C.
Sapino, Jr., J. Org. Chem., 1988, 53, 85–88.
30 C. Thibaudeau and J. Chattopadhyaya, in Stereoelectronic effects in
nucleosides and nucleotides and their structural implications, Uppsala
University Press, Sweden, 1999, pp. 55–135.
31 (a) I. A. Mikhailopulo, Y. A. Sokolov, J. He, P. Chittepu, H. Rosemeyer
and F. Seela, Nucleosides, Nucleotides Nucleic Acids, 2005, 24, 701–705;
(b) F. Seela, X. Peng, H. Li, P. Chittepu, K. I. Shaikh, J. He, Y. He and
I. Mikhailopulo, Collection Symposium Series, 2005, 7, 1–20.
32 L. van Wijk, C. A. G. Haasnoot, F. A. A. M. de Leeuw, B. D. Huckriede,
A. J. A. Westra Hoekzema and C. Altona, PSEUROT 6.3, Leiden
Institute of Chemistry, Leiden University, The Netherlands, 1999.
33 C. Thibaudeau, J. Plavec and J. Chattopadhyaya, J. Org. Chem., 1998,
63, 4967–4984.
34 G. Becher, J. He and F. Seela, Helv. Chim. Acta, 2001, 84, 1048–1065.
35 H. Rosemeyer, M. Zulauf, N. Ramzaeva, G. Becher, E. Feiling,
K. Mu¨hlegger, I. Mu¨nster, A. Lohmann and F. Seela, Nucleosides
Nucleotides, 1997, 16, 821–828.
36 T.-L. Su, K. A. Watanabe, R. F. Schinazi and J. J. Fox, J. Med. Chem.,
1986, 29, 151–154.
37 T. Tennila¨, E. Azhayeva, J. Vepsa¨la¨inen, R. Laatikainen, A. Azhayev
and I. A. Mikhailopulo, Nucleosides, Nucleotides Nucleic Acids, 2000,
19, 1861–1884.
38 I. A. Mikhailopulo, T. I. Pricota, G. G. Sivets and C. Altona, J. Org.
Chem., 2003, 68, 5897–5908.
17 K. S. Gudmundsson, G. A. Freeman, J. C. Drach and L. B. Townsend,
J. Med. Chem., 2000, 43, 2473–2478.
18 C. H. Tann, P. R. Brodfuehrer, S. P. Brundidge, C. Sapino, Jr. and H. G.
Howell, J. Org. Chem., 1985, 50, 3644–3647.
19 J. N. Freskos, Nucleosides Nucleotides, 1989, 8, 549–555.
20 F. Seela and Y. He, J. Org. Chem., 2003, 68, 367–377.
21 Y. Choi, L. Li, S. Grill, E. Gullen, C. Soo Lee, G. Gumina, E. Tsujii,
Y.-C. Cheng and C. K. Chu, J. Med. Chem., 2000, 43, 2538–2546.
22 I. A. Mikhailopulo, G. G. Sivets, N. E. Poopeiko and N. B. Khripach,
Carbohydr. Res., 1995, 278, 71–89.
39 A. Albert and E. P. Serjeant, in The Determination of Ionization
Constants, Chapman and Hall Ltd, London, 1971, pp. 44–64.
40 M. Sekine, M. Fujii, H. Nagai and T. Hata, Synthesis, 1987, 1119–1121.
41 G. S. Ti, B. L. Gaffney and R. A. Jones, J. Am. Chem. Soc., 1982, 104,
1316–1319.
42 (a) M. Petersheim and D. H. Turner, Biochemistry, 1983, 22, 256–263;
(b) K. J. Breslauer, in Protocols for Oligonucleotide Conjugates Synthesis
and Analytical Techniques, ed. S. Agrawal, Humana Press, Totowa, New
Jersey, 1994, pp. 347–372.
43 R. K. Robins, J. Am. Chem. Soc., 1956, 78, 784–790.
This journal is
The Royal Society of Chemistry 2008
Org. Biomol. Chem., 2008, 6, 596–607 | 607
©