Oligonucleotides containing 6-Aza-2′-deoxyuridine: Synthesis, nucleobase protection, pH-dependent duplex stability, and metal-DNA formation

Article abstract of DOI:10.1021/jo0702903

(Chemical Equation Presented) Oligodeoxyribonucleotides containing the nucleoside 6-aza-2′-deoxyuridine z⁶U_d (1a) were prepared by using solid-phase synthesis. As the pK_a value of this nucleoside is 6.8, unwanted side reac

Full text of DOI:10.1021/jo0702903

Oligonucleotides Containing 6-Aza-2′-deoxyuridine: Synthesis,
Nucleobase Protection, pH-Dependent Duplex Stability, and
Metal-DNA Formation
Frank Seela* and Padmaja Chittepu
Laboratory for Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology,
Heisenbergstrasse 11, 48149 Mu¨nster, Germany, and Laboratorium fu¨r Organische und Bioorganische
Chemie, Institut fu¨r Chemie, UniVersita¨t Osnabru¨ck, Barbarastrasse 7, 49069 Osnabru¨ck, Germany
Frank.Seela@uni-osnabrueck.de
ReceiVed February 16, 2007
Oligodeoxyribonucleotides containing the nucleoside 6-aza-2′-deoxyuridine z6Ud (1a) were prepared by
using solid-phase synthesis. As the pKa value of this nucleoside is 6.8, unwanted side reactions are observed.
Consequently, nitrogen-3 was protected (o-anisoyl protection). The phosphoramidite of 1a prepared on
this route was as efficient as the building blocks of canonical nucleosides in allowing multiple
incorporations into oligonucleotides. Oligonucleotide duplexes containing 1a show a pH dependence of
the Tm value. This is caused by nucleobase deprotonation occurring on compound 1a already under neutral
conditions. Metal (M)-DNA formation was studied in the presence of Zn+2 ions. It is demonstrated that
6-azauracil-modified duplexes form M-DNA already in neutral medium while alkaline conditions (above
pH 8.5) are required for natural DNA. The conformational analysis of the sugar moiety of the nucleoside
1a and its anisoyl derivative 5a shows a preferred N-conformation in solution while an S-conformation
for compound 1a was obtained in the solid state (single-crystal X-ray analysis).
Introduction
acids. In the triacetate form it is also effective as an antipsoriatic
agent.4 6-Azauridine has been incorporated in oligoribonucle-
otides to study nucleic acid folding and activity of ribozymes.5,6
The synthesis of 5-substituted-6-aza-2′-deoxyuridines was re-
ported, resulting in compounds with antiviral activity.7,8 Oli-
godeoxyribonucleotides containing 6-aza-2′-deoxyuridine are
unknown, while corresponding oligoribonucleotides have been
6-Azauridine 5′-monophosphate (6-azaUMP) is a strong
inhibitor of enzyme orotidine 5′-monophosphate decarboxylase1
as the 6-position of the pyrimidine ring can serve as an enzyme
binding site.2 The polymer-linked 6-azauridine 5′-monophos-
phate was utilized as an affinity resin for the purification of
this enzyme.3 The 6-azauracil ribonucleoside is an antineoplastic
and antimetabolite which interferes with the pyrimidine bio-
synthesis, thereby preventing the formation of cellular nucleic
(4) Raab, W.; Gmeiner, B.; Muckenhuber, P. Arch. Derm. Res. 1977,
260, 257-259.
(5) Beigelman, L.; Karpeisky, A.; Usman, N. Nucleosides Nucleotides
1995, 14, 895-899.
(6) Oyelere, A. K.; Strobel, S. A. Nucleosides Nucleotides Nucleic Acids
2001, 20, 1851-1858.
* To whom correspondence should be addressed. Phone: +49(0)251 53406
500. Fax: +49(0)251 53406 857.
(1) Miller, B. G.; Snider, M. J.; Short, S. A.; Wolfenden, R. Biochemistry
2000, 39, 8113-8118.
(7) Basnak, I.; Sun, M.; Hamor, T. A.; Focher, F.; Verri, A.; Spadari,
S.; Wroblowski, B.; Herdewijn, P.; Walker, R. T. Nucleosides Nucleotides
1998, 17, 187-206.
(8) Basnak, I.; Coe, P. L.; Walker, R. T. Nucleosides Nucleotides 1994,
13, 163-175.
(2) Ivanetich, K. M.; Santi, D. V. Prog. Nucleic Acids Res. Mol. Biol.
1992, 42, 127-156.
(3) Rosemeyer, H.; Seela, F. J. Med. Chem. 1979, 22, 1545-1547.
10.1021/jo0702903 CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/16/2007
4358
J. Org. Chem. 2007, 72, 4358-4366

Oligonucleotides Containing 6-Aza-2′-deoxyuridine
SCHEME 1. Structures of Nucleosides and
Phosphoramidites 1-3
SCHEME 2. Transformation of Nucleoside 1a to the
Phosphoramidite 3a
adenine base pairs has been suggested by Lee;20 the metal ions
are expected to replace the imino protons of the guanine or the
uracil moiety. However, other complex models have been
suggested by Alexandre21 and Lippert.22 As the formation of
M-DNA requires alkaline conditions the deprotonation of the
nucleobases becomes likely. However, neither the planar nor
the nonplanar model explains that Zn-DNA shows a metal-like
conductivity which was the result of ab initio studies.23 AFM
measurements have been performed in the presence or absence
of divalent ions such as Ni2+ or Co2+ ions showing that the
DNA shrinks under those conditions.24 Zn2+ concentrations
higher than 0.5 mM lead to the precipitation of DNA.25a As the
pKa of the deprotonation of the lactam moiety of 6-aza-2′-
deoxyuridine lies near pH 7, this modified nucleobase moiety
might serve as an efficient Zn2+ acceptor within oligonucleotide
duplexes already under neutral conditions. M-DNA is a good
conductor, and the robust nature of electron transfer in M-DNA
may have a remarkable potential for the development of
nanoelectronic devices.25b
prepared.5,6 The first chemical convergent synthesis of 6-azau-
ridine 2 (ribonucleoside) as well as its large scale preparation
using the Streptococcus faecalis was reported by Handschu-
macher.9 The improved method was reported by Vorbru¨ggen
using the Silyl-Hilbert-Johnson reaction in the presence of
Friedel-Crafts catalysts such as SnCl4 or TiCl4.10-12 The 2′-
deoxy derivative 1a was obtained via the anhydroribonucleo-
side13,14 or enzymatically.15 Later the stereochemical outcome
of the chemical glycosylation reaction was controlled by the
use of CuI as catalyst leading to the exclusive â-D-nucleoside
formation.16 5-Substituted derivatives of 6-azauridine are pre-
pared through Barton-deoxygenation of the ribonucleosides17
or by convergent synthesis.18
Earlier work has shown that the incorporation of a nitrogen
atom in the 6-position of the thymine moiety has an unfavorable
effect on the duplex stability.19 Our initial experiments on the
synthesis of 6-aza-2′-deoxyuridine phosphoramidites performed
with the unprotected nucleobase showed that the acidity of the
nucleobase causes problems. This manuscript reports on the
synthesis of the phosphoramidite building block 3b with an
anisoyl residue as N3 protecting group (Scheme 1). The
utilization of this building block allows multiple incorporations
of 1a in oligodeoxyribonucleotides with the same efficiency as
the standard phosphoramidites. As the pKa value of 6-aza-2′-
deoxyuridine lies near pH 7 it is expected the base pair stability
and the resulting oligonucleotide duplex stability is pH-
dependent. This will be studied on oligonucleotides containing
the nucleoside residue 1a in place of dT. The ease of the base-
deprotonation prompted us to investigate M-DNA (metal-DNA)
formation. M-DNA represents a complex formed by B-DNA
with certain divalent metal ions such as, Co2+, Ni2+, or Zn2+ at
high pH values (>8). The replacement of the imino protons of
guanine or uracil residues within the guanine-cytosine or uracil-
Results and Discussion
1. Synthesis and Properties of the Monomers. The first
convergent synthesis of anomeric 6-aza-2′-deoxyuridines was
performed employing the mercury salt of 3-diphenylmethyl-6-
azauracil and 2-deoxy- 3,5-di-O-(p-toluoyl)-R-D-erythro-pento-
furanosyl chloride.26 Anomeric mixtures are formed under these
nonselective glycosylation conditions. The stereoselective gly-
cosylation of silylated 6-azauracil with 2-deoxy-3,5-di-O-(p-
toluoyl)-R-D-erythro-pentofuranosyl chloride in the presence of
CuI results in the exclusive formation of the protected â-D-
nucleoside (pyrimidine numbering is used throughout the text;
systematic numbering is used in the experimental section).
Detoluoylation in 0.2 M NaOMe/MeOH afforded the free
nucleoside 1a (77% yield). Initial experiments using the
unprotected nucleoside 1a for phosphoramidite synthesis failed
(Scheme 2). When the DMT-derivative 4 was phosphitylated
(9) Handschumacher, R. E. J. Biol. Chem. 1960, 235, 764-768.
(10) Niedballa, U.; Vorbru¨ggen, H. Angew. Chem. Int. Ed. Engl. 1970,
9, 461-462.
(11) Niedballa, U.; Vorbru¨ggen, H. J. Org. Chem. 1974, 39, 3654-3659.
(12) Lukevics, E.; Zablocka, A. In Nucleoside Synthesis Organosilicon
Methods; Ellis Horwood: New York, 1991; pp 97-104.
(13) Drasar, P.; Hein, L.; Beranek, J. Collect. Czech. Chem. Commun.
1976, 41, 2110-2123.
(20) (a) Lee, J. S.; Latimer, L. J. P.; Reid, R. S. Biochem. Cell Biol.
1993, 71, 162-168. (b) Aich, P.; Labiuk, S. L.; Tari, L. W.; Delbaere, L.
J. T.; Roesler, W. J.; Falk, K. J.; Steer, R. P.; Lee, J. S. J. Mol. Biol. 1999,
294, 477-485.
(14) Holy, A.; Cech, D. Collect. Czech. Chem. Commun. 1974, 39, 3157-
3167.
(21) Alexandre, S. S.; Soler, J. M.; Seijo, L.; Zamora, F. Phys. ReV. B
2006, 73, 205112-205115.
(15) Kara, J.; Sorm, F. Collect. Czech. Chem. Commun. 1963, 28, 1441-
1448.
(16) Freskos, J. N. Nucleosides Nucleotides 1989, 8, 549-555.
(17) Mitchell, W. L.; Ravenscroft, P.; Hill, M. L.; Knutsen, L. J. S.;
Judkins, B. D.; Newton, R. F.; Scopes, D. I. C. J. Med. Chem. 1986, 29,
809-816.
(18) Baker, J. J.; Mian, A. M.; Tittensor, J. R. Tetrahedron 1974, 30,
2939-2942.
(19) Sanghvi, Y. S.; Hoke, G. D.; Freier, S. M.; Zounes, M. C.; Gonzalez,
C.; Cummins, L.; Sasmor, H.; Cook, P. D. Nucleic Acids Res. 1993, 21,
3197-3203.
(22) Fusch, E.; Lippert, B. J. Am. Chem. Soc. 1994, 116, 7204-7209.
(23) Fuentes- Cabrera, M.; Sumpter, B. G.; Sponer, J. E.; Sponer, J.;
Petit, L.; Wells, J. C. J. Phys. Chem. B 2007, 111, 870-879.
(24) Moreno-Herrero, F.; Herrero, P.; Moreno, F.; Colchero, J.; Gomez-
Navarro, C.; Gomez-Herrero, J.; Baro, A. M. Nanotechnology 2003, 14,
128-133.
(25) (a) Kejnovsky, E.; Kypr, J. Nucleic Acids Res.1998, 26, 5295-
5299. (b) Wettig, S. D.; Bare, G. A.; Skinner, R. J. S.; Lee, J. S. Nano Lett.
2003, 3, 617-622.
(26) Pliml, J.; Prystas, M.; Sorm, F. Collect. Czech. Chem. Commun.
1963, 28, 2588-2597.
J. Org. Chem, Vol. 72, No. 12, 2007 4359

Seela and Chittepu
FIGURE 1. UV spectra of compound 1a measured in phosphate buffer (7.8 g of NaH2PO4‚H2O in 500 mL of H2O) (a) at 260 nm from pH 5.0
to 9.0. (b) Absorbance of compound 1a as a function of pH values measured at 245 nm from pH 1.5 to 12.5.
SCHEME 3. Synthesis of Protected Nucleosides 5a-c and
i
with Pr2NP(Cl)OCH2CH2CN/iPr2EtN in CH2Cl2 at rt, the
the Phosphoramidite 3ba
unprotected phosphoramidite 3a was formed (TLC-monitoring).
However, its chromatographical purification was impossible due
to its degradation into unresolved products (Scheme 2). We also
studied this phenomenon on the corresponding ribonucleoside
(6-azauridine) phosphoramidite and encountered the same
problem.5 Recently, it was reported that the use of the more
lipophilic 2′-O-ACE ortho ester protocol applied for HDV
ribozymes synthesis6 can overcome the difficulties appearing
in oligoribonucleotide synthesis.
As we reasoned that this behavior results from deprotonation
of the lactam moiety, the pKa value of 1a was determined by
spectrophotometric titration27 (Figure 1b) (pH 1.5-12.5) and
was found to be 6.8, while its 5-methylated derivative 1b shows
a Reagents and conditions: (for 5a) (i) Et3SiCl, pyridine, o-anisoyl
a pKa of 7.2. Both values are significantly lower than that of
chloride, 5% CF3COOH (CH2Cl2/MeOH 70:30) r.t., 82%; (5b) (ii) Et3SiCl,
pyridine, benzoyl chloride, 5% CF3COOH (CH2Cl2/MeOH 70:30) rt, 69%;
(5c) (iii) Et3SiCl, pyridine, DMAP, 2,4-dimethoxy benzoyl chloride, 5%
2′-deoxyuridine (pKa ) 9.3). Consequently, the protection of
the lactam moiety was considered employing a lipophilic
protecting group. Earlier work performed on the lactam protec-
tion of 2′-deoxyuridines or 2′-deoxythymidines has shown that
4-O-aryl,28 alkoxymethyl,29 alkoxycarbonyl,30 cyanoethyl,31
benzoyl,32,33 and anisoyl34,35 groups are useful for this purpose.
Thus, it was desirable to use such protecting groups on
nitrogen-3 of the 6-azauracil moiety (Scheme 3). Here, the
benzoyl, o-anisoyl, and the 2,4-dimethoxybenzoyl residues were
introduced using the protocol of transient protection.33 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). Among the various protecting groups, the 2,4-dimethoxy
benzoyl was not considered for further manipulations because
of the poor reaction yield. The N3-anisoyl as well as the N3-
CF3COOH (CH2Cl2/MeOH 70:30), 60 °C, 20%; (iv) (MeO)2TrCl, pyridine,
rt, 62%; (v) Pr2NP(Cl)OCH2CH2CN, Pr2EtN, CH2Cl2, rt, 61%.
i
i
benzoyl protected nucleoside 5a, 5b were converted into the
DMT derivatives. Only the anisoyl derivative 6 was isolated in
sufficient yield, while the N3-benzoyl residue was lost during
the work up procedure. The anisoyl protected compound 6 was
transformed into the phosphoramidite building block 3b
(Scheme 3), which was used for the solid-phase oligonucleotide
synthesis. The structure of all synthesized compounds was
characterized by 1H-, 13C- (Table 1), and 31P-NMR spectroscopy
1
(Supporting Information) as well as by elemental analysis. H
NMR data of compound 1a were already unambiguously
assigned.36 The 13C NMR chemical shifts of the N3-derivatives
show an up-field shift for C4 and C2 which is in agreement
with earlier work reported by Hata.37 The chemical shift
assignment of C1′, C4′ is tentative for some compounds shown
in Table 1. The position of acylation for the protected nucleoside
5a was determined by X-ray analysis.
(27) Albert, A.; Serjeant, E. P. In The Determination of Ionization
Constants; Chapman and Hall Ltd.: London, 1971; pp 44-64.
(28) Jones, S. S.; Reese, C. B.; Sibanda, S.; Ubasawa, A. Tetrahedron
Lett. 1981, 22, 4755- 4758.
2. Physical Properties of 6-Aza-2′-deoxyuridine and Its
Derivatives. The calculation of the oligonucleotide concentra-
tion requires the values of extinction coefficients of the
monomers in the medium of measurement. As deprotonation
of 1a occurs near pH 7 it was necessary to determine the UV
maxima and extinction coefficients at various pH values. The
(29) Ito, T.; Ueda, S.; Takaku, H. J. Org. Chem. 1986, 51, 931-933.
(30) Kamimura, T.; Masegi, T.; Hata, T. Chem. Lett. 1982, 965-968.
(31) Mag, M.; Engels, J. W. Nucleic Acids Res. 1988, 16, 3525-3543.
(32) Matsuzaki, J.; Hotoda, H.; Sekine, M.; Hata, T. Tetrahedron Lett.
1984, 25, 4019-4022.
(33) Sekine, M.; Fujii, M.; Nagai, H.; Hata, H. Synthesis 1987, 1119-
1121.
(34) Welch, C. J.; Chattopadhyaya, J. Acta Chem. Scand. Ser. B. 1983,
37, 147-150.
(35) Kamimura, T.; Tsuchiya, M.; Urakami, K.; Koura, K.; Sekine, M.;
Shinozaki, K.; Miura, K.; Hata, T. J. Am. Chem. Soc. 1984, 106, 4552-
4557.
(36) Seela, F.; He, Y. J. Org. Chem. 2003, 68, 367-377.
(37) Kamimura, T.; Masegi, T.; Sekine, M.; Hata, T. Tetrahedron Lett.
1984, 25, 4241-4244.
4360 J. Org. Chem., Vol. 72, No. 12, 2007

Oligonucleotides Containing 6-Aza-2′-deoxyuridine
TABLE 1. 13C NMR Chemical Shifts of the 6-Aza-2′-deoxyuridine Derivatives 1-6a
compd
C(2)b
C(4)b
C(5)b
C(1′)
C(2′)
C(3′)
C(4′)
C(5′)
MeO
56.4
1a
5a
5b
5c
4
148.2
146.7
146.8
146.7
148.1
146.6
156.5
154.5
154.9
154.5
156.5
154.4
136.0
135.9
136.1
135.8
135.7
135.7
87.2
87.5
87.5
87.3
85.2c
85.3c
36.8
36.9
37.0
36.8
37.3
37.3
70.4
70.5
70.4
70.4
70.5
70.3
84.7
85.4
85.5
85.3
85.0c
85.2c
62.1
62.2
62.0
62.1
64.4
63.9
56.5
55.0
56.4
6
a Measured in DMSO-d6 at 25 °C. b Pyrimidine numbering. c Tentative.
3
TABLE 2. Half-Life Values of the N-3 Protected Nucleosidesa
TABLE 3. JH,H Coupling Constants of the Sugar Moieties and
N/S-Conformer Populations of the Nucleosidesa
τ
λ
[nm]
compd
[min]a
3JH,H
conformation
5a
5b
5c
9.2
3.5
9.7
260
260
280
compd
1′,2′
1′,2′′
2′,3′
2′′,3′
3′,4′
%N
%S
dU43
6.70
4.82
4.60
7.20
6.55
6.60
7.28
7.29
6.50
6.70
6.50
6.10
6.18
6.50
6.45
4.30
5.80
5.98
3.30
4.00
4.10
5.93
6.05
3.20
3.70
30
58
60
28
37
70
42
40
72
63
1a42
5a
a Measured UV-spectrophotometrically in 25% aq NH3 at 25 °C.
dA44
c7z8Ad
44,b
a Measured in D2O; rms < 0.4 Hz; I∆JmaxI < 0.5 Hz. b c7z8Ad ) 8-Aza-
7-deaza 2′-deoxyadenosine.
UV maximum of 1a is shifted bathochromically under acidic
conditions (pH 5.0 ) 262 nm, pH 7.0 ) 258 nm, pH 9.0 )
255 nm) (Figure 1a). This is in agreement with data reported
for 6-azauridine.9 The extinction coefficients of 6-aza-2′-
deoxyuridine measured in sodium phosphate buffer at 260 nm
(near to the isosbestic point ) 265 nm) are almost independent
of the pH value (pH 5.0 ) 6900, pH 7.0 ) 6900, pH 9.0 )
7200) (Figure 1a). The UV spectrum of the N3-protected
nucleoside 5a in MeOH shows two maxima at 258 and
325 nm.
As the benzoyl group can be used efficiently for the protection
of dT33 it was not clear why the benzoyl group was lost during
dimethoxytritylation of 5b. Therefore, the half-life values (τ)
for the three protecting groups were determined. The reaction
was followed UV-spectrophotometrically at 260 nm (25 °C) in
25% aqueous ammonia (Table 2). From the data it is obvious
that the benzoyl group is too labile for the N-3 protection of
6-aza-2′-deoxyuridine while the anisoyl residue was suitable.
This group was already used by Chattopadhyaya for uridine
protection.34 As it was reported that the acylation site (O-4 or
N-3) was changed for 2′-deoxyuridine depending on the reaction
conditions,38 a single-crystal X-ray analysis of compound 5a39
was performed which immediately showed that nitrogen-3 is
the only acylation site. Based on this X-ray analysis and the
13C NMR chemical shifts (Table 1) it became apparent that all
protecting groups are attached to nitrogen-3.
stants: J(H1′,H2′), J(H1′,H2′′), J(H2′,H3′), J(H2′′,H3′), J(H3′,-
H4′). During the iterations either the puckering parameters (P,
ψ
max) of the minor conformer (N) or the puckering amplitudes
of both conformers were constrained. The coupling constants
and the population of the nucleosides are shown in Table 3.
As the sugar conformation of nucleosides is controlled by
stereo electronic and anomeric effects,41 it was reasoned that
the 6-aza-2′-deoxyuridine has a particular sugar conformation.
Indeed, the nucleosides 1a, 5a show a higher N-conformation
population (60% N)42 than the corresponding 2′-deoxyuridine
(dU) (30% N).43 The presence of an additional nitrogen atom
at the position-6 of 6-aza-2′-deoxynucleoside is responsible for
a conformational bias of the sugar moiety from SfN. The same
observation has been made on other ortho-aza nucleosides such
as of pyrazolo[3,4-d]pyrimidines;44 37% N for 8-aza-7-deaza-
2′-deoxyadenosine (c7z8Ad) and 28% N for dA. The N/S
equilibrium of protected compound 5a does not show much
influence on the conformation when compared to the unpro-
tected 1a.
These results prompted us to investigate the conformation in
solid state by single-crystal X-ray analysis of 1a and 5a. In the
crystal structure of compound 1a, the conformation of the
glycosylic bond is between anti and high-anti with the O4′-
C1′-N1-C2 torsion angle of ø ) -94.0 (3)°, whereas
compound 5a has a high-anti conformation with ø ) -86.4
(3)°.39 Similar results were observed for the ribo analogue of
6-azauridine nucleoside which exhibits a high-anti orientation
with a torsion angle of -93.3°.45 In compound 1a the sugar
ring has the C(2′)-endo, C(3′)-exo (2T3) conformation (P ) 188.1
(2)°, τm ) 40.3 (2)°). Thus its pucker can be described as type-
S, it is different from the behavior of the protected nucleoside
3. Nucleoside Conformation in Solution and in Solid State.
The base moieties of the ortho-aza nucleoside 1a, 5a influence
the N/S pseudorotational equilibrium of the sugar moiety. For
this reason the conformational analysis was performed on the
3
1
basis of J(H,H) NMR coupling constants obtained from H
NMR spectra measured in D2O, with the help of PSEUROT
6.3 program.40 In this program, a minimization of the differences
between the experimental and calculated couplings is ac-
complished by a nonlinear Newton-Raphson minimization; the
quality of the fit is expressed by the root-mean-square (rms)
differences. The input contained the following coupling con-
(41) Thibaudeau, C.; Chattopadhyaya, J. Stereoelectronic effects in
nucleosides and nucleotides and their structural implications; Uppsala
University Press: Uppsala, Sweden, 1999; pp 55-135.
(42) Seela, F.; Chittepu, P.; He, Y.; He, J.; Xu, K. Nucleosides
Nucleotides Nucleic Acids 2005, 24, 847-850.
(38) Sekine, M. J. Org. Chem. 1989, 54, 2321-2326.
(39) Seela, F.; Chittepu, P.; He, Y.; Eickmeier, H.; Reuter, H. Acta
Crystallogr. 2007, C63, o173-o176.
(40) Van Wijk, L.; Haasnoot, C. A. G.; de Leeuw, F. A. A. M.;
Huckriede, B. D.; Westra Hoekzema, A. J. A.; Altona, C. PSEUROT,
Version 6.3; Leiden Institute of Chemistry, Leiden University: Leiden, The
Netherlands, 1999.
(43) Becher, G.; He, J.; Seela, F. HelV. Chim. Acta 2001, 84, 1048-
1065.
(44) Rosemeyer, H.; Zulauf, M.; Ramzaeva, N.; Becher, G.; Feiling, E.;
Mu¨hlegger, K.; Mu¨nster, I.; Lohmann, A.; Seela, F. Nucleosides Nucleotides
1997, 16, 821-828.
(45) Schwalbe, C. H.; Saenger, W. J. Mol. Biol. 1973, 75, 129-143.
J. Org. Chem, Vol. 72, No. 12, 2007 4361

Seela and Chittepu
SCHEME 4. Face to Face Base Pair Motifs of (I) dA-1a
and (II) dA-dU
motif I). According to Table 4, the Tm decrease is moderate
when two modified residues are positioned in the flanking region
(7‚10) and stronger when they are located in the center of the
duplex (7‚12) or (8‚9); similar observation was also found for
natural 2′-deoxyuridine.43 Nevertheless, the base pairing modes
of 1a and dU (Scheme 4, motif I and II) are expected to be the
same.
The strength of the base-pairing based on the pKa difference
(∆pKa) of the monomeric donor and acceptor involved in
hydrogen bonding can be used to understand the relative base
pairing strength of DNA-DNA and RNA-RNA duplexes.47
The low pKa value (pKa ) 6.8) of nucleoside 1a has a significant
influence on the base pair stability. At neutral pH the majority
of the nucleoside molecules exist as negatively charged species.
Alkali ions and water molecules can coordinate to the ring
nitrogen. As a proton is required for base pair formation its
absence destroys or weakens the base pair. At lower pH values
(below 7.0) the proton necessary for base pairing is delivered
from solution leading to the expected base pair formation thereby
increasing the Tm value of duplex melting. To prove this, the
duplex stability of the modified oligonucleotides containing 1a
was measured at pH of 6.0 (Table 4). At this pH, an increase in
the Tm by 2.5 °C per modification was observed for two
incorporations (7‚10) when compared with the Tm value at pH
7.0; two successive modifications resulted in an increase of Tm
by 7 °C. The Tm enhancement was 10 °C for four incorporations
(from 28 °C to 38 °C). The reason for this is the partial
deprotonation of the lactam moiety of 1a at pH 7.0. In the
absence of the N-3 proton WC base pairs cannot be formed at
the modified sites which normally provide the necessary stability
for duplex formation. The unmodified duplex 7‚8 does not show
a significant difference in the Tm values at different pH values.
These results clearly indicate that the destabilization of
duplexes incorporating 6-aza-2′-deoxyuridine is caused mainly
by the deprotonation of the nucleobase. In our comparison the
influence of the 5-methyl group was neglected which causes
an additional effect by 0.5-1.0 °C per modification.36 If these
two phenomena are taken into account there will be still a
destabilizing effect left which does not result from nucleobase
depotonation but which might result from the polar character
of the nucleobase and/or the high anti conformation of the
nucleoside as it was demonstrated by the single-crystal X-ray
analysis of nucleoside 1a. As we reasoned, that the ease of
compound 1a deprotonation will have a positive effect on the
M-DNA formation this phenomenon was studied next.
FIGURE 2. RP-18 HPLC profile of an enzymatic hydrolysis of the
oligomer 5′-d(TAGG1aC AA1a ACT) after treatment with snake venom
phosphodiesterase and alkaline phosphatase in 0.1 M Tris. HCl buffer
(pH 7.0) at 37 °C, for (a) 4 h and for (b) 12 h.
5a which has a N-type sugar pucker with P ) 36.1 (3)°, τm
)
33.5°. From the above results it is apparent that 1a shows S-type
sugar pucker in the solid state while the preferred conformation
is N in solution. The same solution conformation is found for
5a.
4. Synthesis, Characterization, and Properties of Oligo-
nucleotides. To investigate the influence of base-modified
nucleosides on the duplex stability a series of oligonucleotides
were synthesized. The oligonucleotides 7-14 were prepared by
solid-phase synthesis using the protocol of phosphoramidite
chemistry. The coupling yields were always higher than 95%.
Deprotection of the oligomers was conducted in 25% aqueous
ammonia at 60 °C for 16 h. The oligonucleotides were
detritylated and purified by reversed-phase HPLC. The homo-
geneity of the oligonucleotides was proven by reversed-phase
HPLC as well as by MALDI-TOF mass spectrometry. The
detected masses were identical with the calculated values
(Supporting Information, see Table 1). The composition of
oligonucleotides containing 1a was determined by the complete
enzymatic hydrolysis using snake-venom phosphodiesterase
followed by alkaline phosphatase and subsequent reversed-phase
HPLC (RP-18). However, treatment under regular conditions
did not lead to complete digestion. The incubation up to 4 h at
37 °C was not sufficient for the complete digestion, which is
confirmed by the extra peak (X; nondigested oligonucleotide)
found in the HPLC profile (Figure 2). After increasing the
incubation time to 12 h at 37 °C, the additional peak disap-
peared, demonstrating the complete enzymatic hydrolysis. These
results suggest that 6-azauridine containing oligonucleotides are
partially protected from the nuclease digestion. Similar results
were observed earlier for 6-aza-2′-deoxycytidine.36
To evaluate the influence of the base modification on the
duplex stability, hybridization experiments were performed using
the oligonucleotide duplex 5′-d(TAGGTCAATACT)‚3′-d(ATC-
CAGTTATGA) (7‚8) as a reference.46 The modified residues
were introduced at various positions in one or both strands of
a DNA duplex. The replacement of the canonical thymine base
in one strand of the duplex by a 6-azauracil residue results in
a Tm decrease of 5 °C, when paired with dA (7‚11, Scheme 4,
5. Formation of M-DNA. Experiments with the aim to shift
M-DNA formation to neutral pH values have been already
performed using modified polynucleotides.48 As we want to
perform our experiments under defined conditions regarding
DNA length and composition we are now using chemically
(47) Acharya, P.; Cheruku, P.; Chatterjee, S.; Acharya, S.; Chatto-
padhyaya, J. J. Am. Chem. Soc. 2004, 126, 2862-2869.
(48) Wood, D. O.; Dinsmore, M. J.; Bare, G. A.; Lee, J. S. Nucleic Acids
Res. 2002, 30, 2244-2250.
(46) Seela, F.; Mu¨nster, I.; Lo¨chner, U.; Rosemeyer, H. HelV. Chim. Acta
1998, 81, 1139- 1155.
4362 J. Org. Chem., Vol. 72, No. 12, 2007

Oligonucleotides Containing 6-Aza-2′-deoxyuridine
TABLE 4. Tm Values and Thermodynamic Data of Oligonucleotide Duplexes Containing Regular and the Base-Modified Nucleoside at
Different pH Valuesa
pH 7.0
pH 6.0
Tm
[°C]
∆G°310
[kcal/mol]
Tm
[°C]
∆G°310
[kcal/mol]
duplex
5′-d(TAG GTC AAT ACT)-3′ (7) ‚
3′-d(ATC CAG TTA TGA)-5′ (8)
5′-d(TAG G1aC AA1a ACT)-3′ (9) ‚
3′-d(ATC C A G TTA TGA)-5′ (8)
5′-d(TAG GTC AAT ACT)-3′ (7) ‚
3′-d(A1aC CAG TTA 1aGA)-5′ (10)
5′-d(TAG GTC AAT ACT)-3′ (7) ‚
3′-d(ATC CAG 1aTA TGA)-5′ (11)
5′-d(TAG GTC AAT ACT)-3′ (7) ‚
3′-d(ATC CAG 1a1aA TGA)-5′ (12)
5′-d(TAG G1aC AA1a ACT)-3′ (9) ‚
3′-d(A1aC CAG TTA 1aGA)-5′ (10)
50
38
42
45
36
28
-11.8
-7.5
-8.6
-10.0
-7.0
-5.9
51
-
-12.1
-
47
-
-10.1
-
43
38
-8.9
-8.1
a Measured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0 and 6.0) with 5 µM single-strand concentration.
TABLE 5. Precipitation Assay Performed with 0.5 A260 Units (1.9
nmol) of the ss- (13) or ds-Oligonucleotides (7‚8) in 1.0 mL of 10
mM TRIS-HCl, 0.2 mM Sodium Phosphate at pH ) 8.5 at Various
Zn+2 Concentrations at 25 °C
TABLE 6. Recovery of ss- (13) or ds-Oligonucleotides (7‚8) from
Zn+2 Complex by Addition of 1.0 mL of 10 mM TRIS-HCl, 1 mM
EDTA at pH ) 8.5 at 25 °C
Zn2+
concn
(mM)
ss-
oligonucleotide
(%)
ds-
oligonucleotide
(%)
Zn2+
concn
(mM)
ss-
ds-
oligonucleotide
oligonucleotide
sample
sample
(13) (%)a
(7‚8) (%)b
1
2
3
4
5
0.1
0.5
1
2
5
e5
10
35
75
80
e5
-
20
70
85
1
2
3
4
5
0.1
0.5
1
2
5
100
90
70
20
0
95
90
80
15
0
a [13] ) 5′-d(TAGGTCAATACTTAGGTCAATACT). b [7‚8] ) 5′-
d(TAGGTCAATACT)‚3′-d(ATCCAGTTATGA).
leading to charge neutralization and shrinkage of the DNA.24
Nevertheless, the precipitation process is faster in the case of
ds-DNA.
synthesized oligonucleotides. This allowed us to undertake
studies on short DNA-fragments which are usually not available
by enzymatic procedures. To our best knowledge these are the
first experiments using chemically defined duplexes of 12-mers
for this purpose. In a first series of experiments the solubility
of the Zn+2 complexes was studied. This was necessary as it
was reported for high-molecular weight DNA that Zn ions
precipitate duplex DNA. This technique has the potential to
separate DNA or even oligonucleotides from other biomolecules
selectively. The precipitation experiments were preformed with
the single-stranded oligonucleotide 13 as well as with the
unmodified duplex 7‚8. At first the single-stranded (ss) oligo-
nucleotide 13 as well as the duplex 7‚8 were incubated for 20
min at various ZnCl2 concentrations (0-5 mM) at pH 8.5 (for
details, see Experimental Section). The samples were centri-
fuged, the supernatant was taken up, and the absorbance was
read at 260 nm. As the volume was always the same (1 mL),
the amount of DNA present in solution before and after Zn
treatment could be compared (Experimental Section, Table 5).
Next, the precipitate of each experiment was dissolved in 1 mL
of buffer containing EDTA to recover the free DNA. EDTA
complexes Zn+2 ions and leads to the release of B-DNA. The
results of these experiments are shown in the Experimental
Section (Table 6). From the data of the two tables (Experimental
Section), it can be concluded that short oligonucleotides can
be precipitated almost quantitatively with Zn+2 ions with a
concentration higher than 5 mM; the DNA is almost completely
recovered by the addition of EDTA. We noticed that ss-DNA
start to precipitate at about the same Zn+2 concentrations as
duplex DNA. This implies that DNA precipitation results from
complex formation of Zn ions with the phosphodiester backbone
M-DNA does not bind ethidium bromide, thus a ‘fluorescence
assay’ in the presence of ethidium bromide (EB) can be used
to monitor the M-DNA formation.20 The qualitative and
quantitative elements of this assay have been reviewed by
Boger.49 However, it is also possible to monitor EB binding by
UV measurements. There are two possible binding sites for the
Zn+2-DNA interactions, the negatively charged phosphates of
the backbone and/or the nucleobase binding sites.50 To avoid
precipitation of the complex, the Zn+2 concentration was always
kept below 0.5 mM, preferentially in a concentration range of
0.2 to 0.25 mM Zn+2. The UV absorption maximum of free
EB is located at 480 nm and is not altered when Zn+2 ions and/
or EDTA are added. However, it is shifted bathochromically to
525 nm when EB is bound to duplex DNA. When 0.25 mM
ZnCl2 was added to this complex, EB is released from DNA
and the spectrum of the free EB is formed back with the UV
maximum at 480 nm.51 For comparison we performed this
experiment on single-stranded DNA at pH 8.5 and 7.0; after
addition of EB there is no shift in the absorbance due to non-
intercalation of EB. Next, we used two different unmodified
duplexes, the duplex of the 12-mers 7‚8 and of the 24-mers
13‚14 as well as the duplex of 12-mers 9‚12 containing four
6-aza-2′-deoxyuridine residues. According to the Figures 3a,b,
4a,b, and 5a,b, the bathochromic shift of the EB maximum
(49) Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C.; Hedrick, M.
P. J. Am. Chem. Soc. 2001, 123, 5878-5891.
(50) Wang, Q.; Lo¨nnberg, H. J. Am. Chem. Soc. 2006, 128, 10716-
10728.
(51) Wettig, S. D.; Li, C.-Z.; Long, Y.-T.; Kraatz, H.-B.; Lee, J. S. Anal.
Sci. 2003, 19, 23-26.
J. Org. Chem, Vol. 72, No. 12, 2007 4363

Seela and Chittepu
FIGURE 3. Absorption spectra of 5 µM ethidium bromide at (a) pH 8.5, (b) pH 7.0. Solid line, EB; dashed line, EB with 4 µM DNA (7‚8)
5′-d(TAGGTCAATACT)‚3′-d(ATCCAGTTATGA); dotted line, EB with DNA (7‚8) and 0.25 mM ZnCl2.
FIGURE 4. Absorption spectra of 5 µM ethidium bromide at (a) pH 8.5, (b) pH 7.0. Solid line, EB; dashed line, EB with 4 µM DNA (13‚14)
5′-d(TAGGTCAATACTTAGGTCAATACT)‚(ATCCAGTTATGA ATCCAGTTATGA); dotted line, EB with DNA (13‚14) and 0.25 mM ZnCl2.
induced by the addition of duplex DNA is apparent in all cases.
From that it is concluded that the base modified duplex binds
EB in a similar manner as the duplexes formed by the 12-mers
or the 24-mers at pH 8.5 and 7.0. However, significant changes
occur when Zn ions are added. At pH 8.5 the maxima of EB of
the unmodified duplexes are shifted fully or partially to the
maximum of free EB, indicating the release of the dye by the
addition of Zn+2 ions. Nevertheless, the EB spectra are
broadened due to a bathochromic shift of the second shorter
EB band into the range of the longer wave length absorption.
This shift of the EB spectrum is only observed in the presence
of DNA at pH 8.5 but not at pH 7.0 (Figure 3b, 4b). This was
explained earlier with the formation of a Zn-DNA complex at
pH 8.5 in which Zn occupies positions in the core of the DNA
helix thereby replacing the imino protons of the dG-dC and dA-
dT base pairs.20 The exact structure of this complex is unknown
and other binding positions have been discussed.23 However,
all models involve the nucleobases and explain the release of
EB by the interaction with Zn+2 ions which does not allow EB
intercalation.
Next the spectral changes for the unmodified duplex 7‚8 were
compared with that containing 6-aza-2′-deoxyuridine. From
Figure 3b it is obvious that M-DNA formation does not take
place at pH 7.0 in the case of the unmodified duplex. This does
not mean that Zn ion cannot be bound to duplex DNA as
interactions with the negatively charged sugar phosphate
backbone are still possible. However, according to Figure 3b
EB is not released from duplex DNA. This is supported by the
fact that free EB is not released by the addition of Zn+2 ions
when the pH is 7.0 and the canonical nucleobases are not
deprotonated, a phenomenon which seems to be required for
the binding of Zn+2 ions (Figures 3b, 4b). This situation is
different in the case of the base modified duplex 9‚12. At both
pH 8.5 and 7.0 the maxima of the EB are restored after Zn+2
addition (Figure 5a,b). This implies that deprotonation of the
nucleobases is required when Zn+2 will bind to the nucleobases.
According to the melting temperatures of the duplexes at pH
7.0 and 6.0 (see Table 4), the 6-azauracil base is partially
deprotonated at neutral pH. However, duplex formation is still
observed, as the unmodified base pairs are formed even under
the low salt condition used for the EB experiments. The
conversion of M-DNA to B-DNA is achieved by adding EDTA
which chelates the metal ion and restoring the UV maxima to
525 nm (data not shown). Thus, the M-DNA formation at neural
pH can be related to the lower pKa values of the imino proton
in the respective nucleobase; it is hypothesized that the lower
pKa of the base allows the imino proton to be replaced by the
metal ion at lower pH. On the basis of these observations,
compound 1a shows favorable properties regarding M-DNA
formation which is illustrated by the lower pKa value (pKa )
6.8) of 1a compared to dU (pKa )9.5).
Conclusions and Outlook
The N-3 protection of 6-aza-2′-deoxyuridine (1a) leads to
phosphoramidite building block 3b which gives the same high
coupling yield as the phosphoramidites of the canonical bases.
The additional ring nitrogen atom of 1a causes a special
glycosylic bond conformation (high-anti) which was studied in
solution and in solid state.39 DNA duplexes containing 1a-dA
4364 J. Org. Chem., Vol. 72, No. 12, 2007

Oligonucleotides Containing 6-Aza-2′-deoxyuridine
FIGURE 5. Absorption spectra of 5 µM ethidium bromide at (a) pH 8.5, (b) pH 7.0. Solid line, EB; dashed line, EB with 4 µM DNA (9‚12)
5′-d(TAGG1aCAA1aACT)‚3′-d(ATCCAG1a1aATGA); dotted line, EB with DNA (9‚12) and 0.25 mM ZnCl2.
OH (7:3, 20 mL), stirred at room temperature for 1 h, and
evaporated. To this residue was added 5% aq NaHCO3. The mixture
was extracted with CH2Cl2, the extract was dried over Na2SO4
and evaporated, and the residue was applied to FC (silica gel,
column 15 × 3 cm, CH2Cl2/MeOH, 98:2). Evaporation of the main
zone afforded a colorless foam, which was recrystallized from
MeOH to give 5a as colorless crystals (0.65 g, 82%). mp ) 141
°C, UV (MeOH); 258 (15200), 260 (15000), 325 (4300). TLC (silica
gel, CH2Cl2/MeOH, 95:5): Rf 0.26. 1H NMR (DMSO-d6,
250 MHz): δ 2.15 [m, 1H, HR-C(2′)], 2.50 [m, 1H, Hâ-C(2′)],
3.48 [m, 2H, H-C(5′)], 3.74 [m, 1H, H-C(4′)], 3.78 [s, 3H, MeO],
4.30 [m, J ) 4.78, 1H, H-C(3′)], 4.70 [t, J ) 5.37, 1H, OH-
C(5′)], 5.25 (d, J ) 4.50, 1H, OH-C(3′)], 6.33 [t, J ) 5.87, 1H,
H-C(1′)], 7.18 [m, 2H, aromatic], 7.60 [m, 1H, aromatic], 7.84
[s, 1H, H-C(5)], 8.01 [m, 1H, aromatic]. Anal. calcd for C16H17N3O7
(363.32): C, 52.89; H, 4.72; N, 11.57. Found: C, 52.81; H, 4.75;
N, 11.77.
base pairs are less stable than those of dT-dA. We found that
this destabilization is largely caused by the deprotonation of
the 6-aza-2′-deoxyuridine residue which takes place already
under neutral condition. A pH decrease of the buffer solution
increases the duplex stability. Oligonucleotides containing 1a
show an enhanced stability against the 3′-exonucleases (snake-
venom phosphodiesterase). The ease of deprotonation which
causes an unfavorable effect on the duplex stability results in a
very favorable behavior regarding M-DNA formation. This
metal DNA is now formed under physiological as well as
alkaline conditions unlike canonical DNA which is formed only
at alkaline pH. Because of its conductive properties M-DNA
has the capacity to act as a nanowire,25b and the use of the 6-aza-
2′-deoxyuridine in place of dT can lead its application in the
preparation of nano electronic devices being formed already
under physiological conditions.
2-(2-Deoxy-â-D-erythro-pentofuranosyl)-N4-benzoyl-1,2,4-tri-
azin-3,5(2H,4H)-dione (5b). Compound (1a) (0.2 g, 0.87 mmol)
was treated with triethylsilyl chloride (0.4 mL, 2.36 mmol) in
pyridine (2 mL) and stirred at room temperature for 1 h and then
benzoyl chloride (0.29 mL, 2.5 mmol) was added. After being
stirred for 3 h, the mixture was evaporated and coevaporated with
toluene to remove pyridine completely. The residue was further
treated with 5% CF3COOH in CH2Cl2-CH3OH (7:3, 8 mL) stirred
at room temperature for 1 h and evaporated. To this residue was
added 5% aq NaHCO3, and the mixture was extracted with CH2-
Cl2, the extract was dried over Na2SO4 and evaporated, and the
residue was applied to FC (silica gel, column 12 × 3 cm, CH2-
Cl2/MeOH, 95:5). Evaporation of the main fractions afforded 5b
as colorless foam (0.2 g, 69%). UV (MeOH); 254 (16000), 260
(14100). TLC (silica gel, CH2Cl2/MeOH 95:5): Rf 0.27. 1H NMR
(DMSO-d6, 250 MHz): δ 2.08 [m, HR-C(2′)], 2.49 [m, 1H, Hâ-
C(2′)], 3.73 [m, 2H, H-C(5′)], 4.12 [m, 1H, H-C(4′)], 4.29 [t, J
) 4.9, 1H, H-C(3′)], 4.67 [t, J ) 5.75, 1H, OH-C(5′)], 5.23 [t,
J ) 4.60, 1H, OH-C(3′)], 6.30 [t, J ) 5.70, 1H, H-C(1′)], 7.59
[m, 2H, aromatic], 7.83 [s, 1H, H-C(5)], 8.12 [m, 2H, aromatic].
Anal. Calcd for C15H15N3O6 (333.30): C, 54.05; H, 4.54; N, 12.61
Found: C, 53.99; H, 4.50; N, 12.52.
2-(2-Deoxy-â-D-erythro-pentofuranosyl)-N4-(2,4-dimethoxy-
benzoyl)-1,2,4-triazin-3,5(2H,4H)-dione (5c). Compound (1a)
(0.15 g, 0.65 mmol) was treated with triethylsilyl chloride
(0.70 mL, 4.19 mmol) in pyridine (3.0 mL) and stirred at room
temperature for 1 h, and then 2,4-dimethoxybenzoyl chloride (36
mg, 0.18 mmol) and DMAP were added. The reaction mixture was
stirred at 60 °C for overnight. The mixture was evaporated and
coevaporated with toluene to remove pyridine completely. The
residue was further treated with 5% CF3COOH in CH2Cl2-CH3-
OH (7:3, 6 mL), stirred at room temperature for 1 h, and evaporated.
To this residue was added 5% aq NaHCO3, and the mixture was
Experimental Section
M-DNA Sample Preparation. Ethidium bromide (5 µM) was
dissolved in 1 mL of buffer solution (10 mM NaCl and 10 mM
Tris-HCl, pH 8.5 or 7.0), and the UV absorbance was measured.
To this 4 µM of DNA duplex (bases) (7‚8 or 13‚14 or 9‚12) was
added, and the absorbance was read. To the same solution was
added 0.25 mM ZnCl2, and the mixture was incubated for 20 min
at rt for the formation of M-DNA followed by UV measurement.
To convert back to B-DNA, 2 mM EDTA (20 µL of 100 mM stock
solution in H2O) was added to each sample and the absorbance
was measured.
Precipitation of DNA Using Zn+2. The oligonucleotides 13 or
7‚8 (1.9 nmol) were dissolved in 1.0 mL of buffer (10 mM Tris-
HCl, 0.2 mM sodium phosphate, pH 8.5). To this solution were
added various concentrations of ZnCl2 (0-5 mM, from 50 mM
stock solution), the mixture was incubated for 20 min at room
temperature, then the samples were centrifuged at 5000 rpm for
5 min, the supernatant was carefully taken out, and the absorbance
was measured at 260 nm. Based on that, the amount of precipitated
DNA was calculated (Table 5). To recover the DNA, the above
precipitated samples were suspended in buffer (1.0 mL of 10 mM
TRIS-HCl, 1 mM EDTA, pH 8.5), and UV absorbance was
measured at 260 nm (Table 6).
2-(2-Deoxy-â-D-erythro-pentofuranosyl)-N4-(2-methoxybenzoyl)-
1,2,4-triazin-3,5(2H,4H)-dione (5a). Compound 1a16 (0.5 g,
2.18 mmol) was treated with triethylsilyl chloride (0.94 mL,
5.61 mmol) in pyridine (3.5 mL) and stirred at room temperature
for 1 h, and then o-anisoyl chloride (0.49 mL, 3.29 mmol) was
added. After being stirred for 3 h, the mixture was evaporated and
coevaporated with toluene to remove pyridine completely. The oily
residue was further treated with 5% CF3COOH in CH2Cl2-CH3-
J. Org. Chem, Vol. 72, No. 12, 2007 4365

Seela and Chittepu
extracted with CH2Cl2. The extract was dried over Na2SO4, and
the evaporated residue was applied to FC (silica gel, column 12 ×
3 cm, CH2Cl2/MeOH, 98:2). Evaporation of the main fraction
afforded 5c; colorless solid (51 mg, 20%). UV (MeOH); 317 (8400),
277 (15800). 1H NMR (DMSO-d6, 250 MHz): δ 2.16 [m, 1H, HR-
C(2′)], 2.45 [m, 1H, Hâ-C(2′)], 3.46 [m, 2H, H-C(5′)], 3.72 [m,
1H, H-C(4′)], 3.89 [s, 3H, MeO], 4.28 [m, 1H, H-C(3′)], 4.74 [t,
1H, OH-C(5′)], 5.27 [d, J ) 4.5, 1H, OH-C(3′)], 6.32 [t, J )
5.50, 1H, H-C(1′)], 6.66 [s, 1H, aromatic], 6.74 [d, 1H, aromatic],
7.81 [s, 1H, H-C(5)], 7.97 [d, 2H, aromatic].
3MeO], 3.90 [m, 1H, H-C(4′)], 4.31 [m, 1H, H-C(3′)], 5.29 [d,
J ) 5.25, 1H, OH-C(3′)], 6.37 [t, J ) 3.62, 1H, H-C(1′)], 6.86
[m, 4H, aromatic], 7.24-8.00 [m, 13H, aromatic, H-C(5)]. Anal.
Calcd for C37H35N3O9 (665.69): C, 66.76; H, 5.30; N, 6.31.
Found: C, 66.54; H, 5.15; N, 6.35.
2-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-â-D-erythro-pentofura-
nosyl]-N4-(2-methoxybenzoyl)-1,2,4-triazin-3,5(2H,4H)-dione 3′-
(2-Cyanoethyl Diisopropylphosphoramidite) (3b). A stirred
solution of 6 (0.20 g, 0.30 mmol) in anhyd CH2Cl2 (5 mL) was
pre-flushed with Ar and treated with (i-Pr)2EtN (0.08 mL,
0.51 mmol) followed by 2-cyanoethyl diisopropyl phosphorami-
dochloridite (0.08 mL, 0.35 mmol) at room temperature. After 30
min, the mixture was diluted with CH2Cl2 (20 mL), washed with
aq 5% NaHCO3 (10 mL), dried over Na2SO4, and evaporated to
an oil. This residue was applied to FC (silica gel, column 10 × 3
cm, CH2Cl2/acetone/Et3N, 95:5:0.2) to give compound 3b as a
colorless foam (0.16 g, 61%). TLC (silica gel, CH2Cl2/acetone, 95:
5): Rf 0.87. 31P NMR (CDCl3): 149.85, 149.69.
2-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-â-D-erythro-pentofura-
nosyl]-1,2,4-triazin-3,5(2H,4H)-dione (4). Compound 1a (0.08 g,
0.35 mmol) was suspended in anhyd pyridine (1.5 mL), 4,4′-
dimethoxytrityl chloride (0.14 g, 0.42 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
coevaporated with toluene and applied to FC (silica gel, column
12 × 3 cm, CH2Cl2/MeOH, 90:10), furnishing a colorless foam
(0.17 g, 92%). UV (MeOH): 233 (20300), 260 (6400), 267 (6800).
Acknowledgment. We thank Mr. Ping Ding for the Zn-
precipitation experiments. We also thank Dr. Helmut Rosemeyer
and Dr. Xiaomei Zhang for measuring the NMR spectra, Mrs.
E. Michalek for oligonucleotide synthesis, and Dr. K. I. Shaikh
for the MALDI-TOF mass spectra. We are grateful to Dr. Yang
He for providing us with the protected nucleoside 5c and V. R.
Sirivolu, Dr. P. Leonard, S. Budow, and K. Xu for reading the
manuscript. Financial support by ChemBiotech (Mu¨nster,
Germany) and the BMBF (Germany) is gratefully acknowl-
edged.
1
TLC (silica gel, CH2Cl2/MeOH, 9:1): Rf 0.8. H NMR (DMSO-
d6, 250 MHz): δ 2.11 [m, 1H, HR-C(2′)], 2.35 [m, 1H, Hâ-C(2′)],
3.01 [m, 2H, H-C(5′)], 3.72 [s, 3H, 2MeO], 3.86 [m, 1H,
H-C(4′)], 4.24 [m, 1H, H-C(3′)], 5.27 [d, J ) 4.75, 1H, OH-
C(3′)], 6.34 [t, 1H, H-C(1′)], 6.83 [m, 4H, aromatic], 7.21-7.40
[m, 10H, aromatic, H-C(5)], 12.28 [s, NH]. Anal. Calcd for
C29H29N3O7 (531.56): C, 65.53; H, 5.50; N, 7.91. Found: C, 65.41;
H, 5.21; N, 8.00.
2-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-â-D-erythro-pentofura-
nosyl]-N4-(2-methoxybenzoyl)-1,2,4-triazin-3,5(2H,4H)-dione (6).
As described for 4, compound 5a (0.3 g, 0.83 mmol) in anhyd
pyridine (4 mL) was treated with 4,4′-dimethoxytrityl chloride
(0.33 g, 0.99 mmol). The organic layer was evaporated and applied
to FC (silica gel, column 10 × 3 cm, CH2Cl2/acetone 98:2) to afford
6 as colorless foam (0.34 g, 62%). UV (MeOH): 235 (25300),
260 (14800), 324 (3800). TLC (silica gel, CH2Cl2/acetone, 95:5):
Supporting Information Available: Synthesis, purification, and
characterization of oligonucleotides 7-14; molecular masses ([M
+ H]+) of oligonucleotides 7-14 measured by MALDI-TOF mass
spectrometry (Table 1); 1H NMR and 13C NMR of new compounds
4-6; and 31P NMR data of phosphoramidite 3b. This material is
available free of charge via the Internet at http://pubs.acs.org.
1
Rf 0.64. H NMR (DMSO-d6, 250 MHz): δ 2.18 [m, 1H, HR-
C(2′)], 2.45 [m, 1H, Hâ-C(2′)], 3.10 [m, 2H, H-C(5′)], 3.72 [s,
JO0702903
4366 J. Org. Chem., Vol. 72, No. 12, 2007