6-Aza-2′-deoxyisocytidine: Synthesis, properties of oligonucleotides, and base-pair stability adjustment of DNA with parallel strand orientation

Article abstract of DOI:10.1021/jo020507n

6-Aza-5-methyl-2′-deoxyisocytidine (1a) and 6-aza-2′-deoxyisocytidine (lb) have been synthesized, converted into phosphoramidite building blocks, and incorporated into oligodeoxynucleotides. The glycosylic bond stability of la,b under acidic conditions increases compared to that of 5-methyl-2′-deoxyisocytidine (2a) and 2′-deoxyisocytidine (2b). Oligonucleotides incorporating la or lb show an enhanced stability against the 3′-exonuclease snake-venom phosphodiesterase. The duplexes containing 6-azapyrimidine nucleosides la or lb have lower T_m values than duplexes containing 2a or 2b with either antiparallel or parallel chain orientation. This was used to adjust the stability of the tridentate M⁵_iC_d-dG base pair to the level of the bidentate reverse Watson-Crick dA-dT pair.

Full text of DOI:10.1021/jo020507n

6-Aza -2′-d eoxyisocytid in e: Syn th esis, P r op er ties of
Oligon u cleotid es, a n d Ba se-P a ir Sta bility Ad ju stm en t of DNA w ith
P a r a llel Str a n d Or ien ta tion
Frank Seela* and Yang He
Laboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie, Universita¨t Osnabru¨ck,
Barbarastrasse 7, D-49069 Osnabru¨ck, Germany
frank.seela@uni-osnabrueck.de
Received August 1, 2002
6-Aza-5-methyl-2′-deoxyisocytidine (1a ) and 6-aza-2′-deoxyisocytidine (1b) have been synthesized,
converted into phosphoramidite building blocks, and incorporated into oligodeoxynucleotides. The
glycosylic bond stability of 1a ,b under acidic conditions increases compared to that of 5-methyl-
2′-deoxyisocytidine (2a ) and 2′-deoxyisocytidine (2b). Oligonucleotides incorporating 1a or 1b show
an enhanced stability against the 3′-exonuclease snake-venom phosphodiesterase. The duplexes
containing 6-azapyrimidine nucleosides 1a or 1b have lower Tm values than duplexes containing
2a or 2b with either antiparallel or parallel chain orientation. This was used to adjust the stability
of the tridentate m5iCd-dG base pair to the level of the bidentate reverse Watson-Crick dA-dT
pair.
SCHEME 1
In tr od u ction
The hybridization of nucleic acids is commonly per-
formed under the formation of duplexes with antiparallel
chain orientation. The reversal of the strand orientations
from antiparallel (aps) to parallel (ps)swas originally
accomplished by the selection of particular sequences of
oligonucleotides but was limited to oligonucleotides with
dA-dT base pairs.1,2 A general way which allows the
formation of parallel-stranded hybrids incorporating the
four canonical DNA constituents in one strand requires
the structural manipulations of the monomeric nucleic
acid components of the second one. The configurational
change at the anomeric center from â-D to R-D3 or the
vantages over the naturally occurring antiparallel-
stranded hybrids: Oligonucleotides become resistant
against various enzymes, and reporter groups used for
diagnostic purposes can be located in positions which are
otherwise difficult to access.
transposition of the nucleobase substituents leading from
dG to iGd (3) (Scheme 1) or from dC to m5iCd
are
4-6
established protocols to accomplish this objective. Re-
cently, the structure of such a ps-DNA containing iGd-
dC as well as m5iCd-dG base pairs was studied by NMR
spectroscopy.7 Such a parallel hybridization offers ad-
In the case of aps-DNA the tridentate dG-dC base pair
is significantly more stable than the bidentate dA-dT
pair. Thus, duplexes being rich in dG-dC pairs show
increased stability. As a consequence oligonucleotides
containing base mismatches can be more stable than
perfectly matching duplexes containing mainly dA-dT
pairs. This problem obscures sequence data obtained
from hybridization experiments with oligonucleotide
libraries which are performed in solution or on a solid
support (biochips). Thus, it is desirable to destabilize dG-
dC-rich duplexes and/or to increase the stability of dA-
dT-rich duplexes while maintaining the sequence speci-
ficity of the hybridization. This is accomplished by
replacing one or more of the native nucleosides in the
* To whom correspondence should be addressed. Phone: +49-541-
969-2791. Fax: +49-541-969-2370.
(1) van de Sande, J . H.; Ramsing, N. B.; Germann, M. W.; Elhorst,
W.; Kalisch, B. W.; von Kitzing, E.; Pon, R. T.; Clegg, R. C.; J ovin, T.
M. Science 1988, 241, 551-227.
(2) Parvathy, V. R.; Bhaumik, S. R.; Chary, K. V. R.; Govil, G.; Liu,
K.; Howard, F. B.; Miles, H. T. Nucleic Acids Res. 2002, 30, 1500-
1511.
(3) (a) Morvan, F.; Rayner, B.; Imbach, J .-L.; Lee, M.; Hartley, J .
A.; Chang, D.-K.; Lown, J . W. Nucleic Acids Res. 1987, 15, 7027-7044.
(b) Thuong, N. T.; Asseline, U.; Roig, V.; Takasugi, M.; He´le`ne, C. Proc.
Natl. Acad. Sci. U.S.A. 1987, 84, 5129-5133. (c) Praseuth, D.;
Chassignol, M.; Takasugi, M.; Le Doan, T. L.; Thuong, N. T.; He´le`ne,
C. J . Mol. Biol. 1987, 196, 939-942.
(4) Seela, F.; Gabler, B.; Kazimierczuk, Z. Collect. Czech. Chem.
Commun. 1993, 58 (Special Issue), 170-173.
(5) Seela, F.; He, Y.; Wei, C. Tetrahedron 1999, 55, 9481-9500.
(6) Sugiyama, H.; Ikeda, S.; Saito, I. J . Am. Chem. Soc. 1996, 118,
9994-9995.
(7) Yang, X.; Sugiyama, H.; Ikeda, S.; Saito, I.; Wang, A. H.-J .
Biophys. J . 1998, 75, 1163-1171.
10.1021/jo020507n CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/06/2002
J . Org. Chem. 2003, 68, 367-377
367

Seela and He
SCHEME 2a
probe (or the target) with modified, nonstandard nucleo-
sides. The substitution of guanine residues by 7-deaza-
guanine, for example, will generally destabilize duplexes,
whereas substituting adenine residues with 7-haloge-
nated 8-aza-7-deazapurine-2,6-diamines will enhance
duplex stability.8,9 The incorporation of 6-azapyrimidine
nucleoside analogues into oligonucleotide probes gener-
ally decreases their binding affinity for complementary
nucleic acids.10
The same problem appears in parallel-stranded DNA.
For instance, the PM3 semiempirical calculation in the
gas phase suggested that the tridentate reverse Watson-
Crick base pairs of both m5iCd-dG (∆G° ) -12.0 kcal/
mol) and iGd-dC (∆G° ) -13.7 kcal/mol) are more stable
than the bidentate reverse Watson-Crick dA-dT base
pair (∆G° ) -4.6 kcal/mol) in parallel DNA. This was
underlined experimentally in various DNA duplexes
showing this parallel strand orientation.5,6 When parallel
hybridization is employed with oligonucleotide libraries
incorporating iGd-dC or m5iCd-dG base pairs, it is desir-
able to search for the stabilization of the dA-dT pair or
to destabilize the iGd-dC (or m5iCd-dG) pair to the same
level of free energy. Thus, efforts were undertaken to
harmonize the tridentate and bidentate base pair stabili-
ties in these cases.
a
Reagents and conditions: (i) TsCl, pyridine, rt, 70% for 5a ,
43% for 5b; (ii) DBU, MeCN, reflux, 47% for 6a , 50% for 6b; (iii)
NH3/MeOH, rt, 80% for 1a , 75% for 1b.
The stabilization of the reverse Watson-Crick dA-dT
base pairs in ps-DNA has already been investigated by
using base-modified analogues which are able to retain
the base pair recognition selectivity of the dA residue.
The stability of the parallel-stranded DNA duplex is
increased by incorporation of the 7-substituted 7-deaza-
2′-deoxyadenosine derivatives instead of dA.11 Studies on
the iGd-dC base pair stability have also been performed
using 7-deaza-2′-deoxyisoguanosine instead of 2′-deoxy-
isoguanosine.12 However, a significant destabilization of
the tridentate base pair was not accomplished. In this
paper we investigate the adjustment of the stability of
the tridentate m5iCd-dG base pair to that of the reverse
Watson-Crick dA-dT pair. For this purpose the pyrimi-
dine nucleoside m5iCd (2a ) or iCd (2b) is replaced by
6-aza-5-methyl-2′-deoxyisocytidine (1a ) or 6-aza-2′-deoxy-
isocytidine (1b) (Scheme 1). Likewise, the destabilization
of the iGd-m5iCd pair formed in antiparallel DNA will be
studied for comparison. The resistance of oligonucleotides
containing 1a and 1b against degradation by exonu-
cleases, e.g., snake-venom phosphodiesterase, will be
reported as well.
compounds 4a ,b, were prepared by the stereoselective
glycosylation of silylated 6-azathymine or 6-azauracil
with 2-deoxy-3,5-di-O-(p-toluoyl)-R-D-erythro-pentofura-
nosyl chloride in the presence of CuI.16,17 The toluoylated
â-D-nucleosides were isolated as major components. The
â-D-configuration was assigned according to small 1H
NMR chemical shift differences between the 4′ and 5′
proton signals.18 After detoluoylation, compounds 4a and
4b were obtained in 77% and 83% yield, respectively. For
these nucleosides, the assignment of the glycosylation
position was made according to Hall19 on the basis of UV
data. Both nucleosides show a hypsochromic shift of the
UV maxima in alkaline medium compared to a neutral
1
molecule. We have also performed H NMR NOE experi-
ments for an unambiguous assignment of the anomeric
configuration.20 Irradiation of H-C(1′) resulting in NOEs
on H-C(4′) of 3.0% (4a ) and 1.3% (4b) confirms the
configurational assignment.
Next, the tosylation of 4a and 4b was performed to
yield the 5′-O-p-toluoylsulfonyl derivatives 5a and 5b
selectively in 70% and 43% yield. The 2,5′-anhydro-
nucleosides 6a and 6b are then formed by refluxing 5a
and 5b with DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in
anhydrous acetonitrile. Ammonolysis of 6a and 6b fur-
nished 1a (80% yield) and 1b (75% yield). This reaction
Resu lts a n d Discu ssion
1. Mon om er s. Both 1a (m5z6iCd) and 1b (z6iCd) (py-
rimidine numbering is used throughout the text; the
systematic numbering is used in the Experimental Sec-
tion) were synthesized from 6-aza-2′-deoxythymidine (4a )
or 6-aza-2′-deoxyuridine (4b) by chemical transformation
of their nucleobases (Scheme 2). Similar work has already
been performed on 5-methyl-2′-deoxyisocytidine (2a ) and
its desmethyl derivative 2b.13-15 The starting materials,
(13) Switzer, C.; Moroney, S. E.; Benner, S. A. J . Am. Chem. Soc.
1989, 111, 8322-8323.
(14) Tor, Y.; Dervan, P. B. J . Am. Chem. Soc. 1993, 115, 4461-4467.
(15) Kowollik, G.; Langen, P. J . Prakt. Chem. 1968, 37, 311-318.
(16) 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.
(17) Freskos, J . N. Nucleosides Nucleotides 1989, 8, 549-555, 1075.
(18) Nuhn, P.; Zschunke, A.; Heller, D.; Wagner, G. Tetrahedron
1969, 25, 2139.
(19) Hall, R. H. J . Am. Chem. Soc. 1958, 80, 1145-1150.
(20) Rosemeyer, H.; Toth, G.; Golankiewicz, B.; Kazimierczuk, Z.;
Bourgeois, W.; Kretschmer, U.; Muth, H. P.; Seela, F. J . Org. Chem.
1990, 55, 5784-5790.
(8) Seela, F.; Becher, G. Nucleic Acids Res. 2001, 29, 2069-2078.
(9) He, J .; Seela, F. Tetrahedron 2002, 58, 4535-4542.
(10) Freier, S. M.; Altmann, K.-H. Nucleic Acids Res. 1997, 25,
4429-4443.
(11) Seela, F.; Wei, C.; Becher, G.; Zulauf, M.; Leonard, P. Bioorg.
Med. Chem. Lett. 2000, 10, 289-292.
(12) Seela, F.; Wei, C. Helv. Chim. Acta 1999, 82, 726-745.
368 J . Org. Chem., Vol. 68, No. 2, 2003

6-Aza-2′-deoxyisocytidine
SCHEME 3a
a
Reagents and conditions: (i) CuI, CHCl3, rt; (ii) 0.2 M NaOMe, rt; (iii) DMTCl, pyridine, rt; (iv) 2.5% DCA in CH2Cl2, rt.
TABLE 1. Ha lf-Life Va lu es of 1a a n d Der iva tives Deter m in ed in Acid ic a n d Alk a lin e Med ia
half-life (τ, min)
25% aqueous NH3
half-life (τ, min)
25% aqueous NH3
b
b
compd
0.1 M HCla
compd
12a
0.1 M HCla
1a
250
75
17
5
stable within 16 h at 25 °C
stable within 16 h at 25 °C
stable within 16 h at 60 °C
stable within 16 h at 25 °C
>440
210
16
4 min at 25 °C
3 min at 25 °C
5 min at 25 °C
7 min at 40 °C
1b
12b
23
2a 5
2b23
dmf2iCd
5
dmf2m5iCd
28
a
Hydrolysis was followed UV-spectrophotometrically (235 nm) at 40 °C for 1a ,b, at 275 nm for 12a , and at 280 nm for 12b; the half-
b
life values refer to the glycosylic bond cleavage. Hydrolysis was followed at 275 nm for 12a and 270 nm for 12b; the half-life values refer
to the cleavage of the protecting groups.
takes place much faster than in the case of the 2,5′-
anhydro derivatives of 2′-deoxy-5-methylisocytidine or its
5-desmethyl derivative.
residue on 5′-OH, the N(1) isomers 10a ,b were separated
from the N(3) isomers 11a ,b by column chromatography.
Removal of the DMT groups with 2.5% dichloroacetic acid
(in CH2Cl2) afforded compounds 1a ,b or 9a ,b. The N(1)
isomers 1a ,b were found to be identical with those
obtained from the above-mentioned nucleoside transfor-
mation. For the other isomers, glycosylation at N(6) is
unlikely, since it would lead to either a positively charged
quaternary nitrogen atom or a nonaromatic ring system
which should give rather different spectroscopic charac-
teristics. Therefore, the structures of compounds 9a and
9b were suggested to be the N(3) isomers and were
confirmed by NMR spectroscopy. The â-D-configuration
Compounds 1a and 1b can also be synthesized by a
convergent synthetic approach. The nucleobases 6-aza-
5-methylisocytosine (7a ) and 6-azaisocytosine (7b) were
prepared from pyruvic acid and chloral, respectively, via
cyclization of the corresponding guanylhydrazones.21
Silylation of 7a or 7b according to Langer22 with HMDS/
TMSCl furnished 16a or 16b, which were used for the
condensation with 2-deoxy-3,5-di-O-(p-toluoyl)-R-D-erythro-
pentofuranosyl chloride (8) in CHCl3 with CuI as a
catalyst (Scheme 3). Unlike the synthesis of 4a and 4b,
where only the N(1) regioisomer is formed, the glyco-
sylation of 16a and 16b furnished two regioisomerssthe
N(1) (1a ,b) and N(3) (9a ,b) compounds in nearly the same
yield. The ratio of isomers was determined from the
signal intensities of the anomeric protons in the 1H NMR
spectra. The formation of regioisomeric reaction products
in this CuI-catalyzed reaction was also observed earlier
upon the glycosylation of 5,6-dimethyluracil.16
1
of the anomeric centers of 9a ,b was established by H
NMR NOE experiments. Irradiation of H-C(1′) resulted
in NOEs on H-C(4′) (η ) 1.5% for 9a and η ) 1.3% for
9b).
Then, the stability of the nucleosides 1a ,b was studied
under acidic and alkaline conditions. This study was
undertaken as it was found that the parent 2′-deoxyiso-
cytidine nucleosides 2a and 2b are very labile in acidic
and alkaline media, which are used in oligonucleotide
synthesis and during the workup conditions.23-25 At first,
the hydrolysis was performed in 0.1 M HCl (40 °C). The
reaction was followed by RP18-HPLC as well as UV-
As both regioisomers show almost identical Rf values,
it was impossible to separate them chromatographically
on the stage of the toluoylated or the free nucleosides
(1a ,b or 9a ,b) (Scheme 3). However, when they were
derivatized by the DMT (4,4′-dimethoxytriphenylmethyl)
(23) Seela, F.; He, Y. Helv. Chim. Acta 2000, 83, 2527-2540.
(24) J urczyk, S.; Kodra, J . T.; Rozzell, J . D.; Benner, S. A.; Battersby,
T. R. Helv. Chim. Acta 1998, 81, 793-811.
(25) Roberts, C.; Bandaru, R.; Switzer, C. J . Am. Chem. Soc. 1997,
119, 4640-4649.
(21) Gut, J .; Hesoun, D.; Novacek, A. Collect. Czech. Chem. Commun.
1966, 31, 2014-2024.
(22) Langer, S. H.; Connell, S.; Wender, I. J . Org. Chem. 1958, 23,
50.
J . Org. Chem, Vol. 68, No. 2, 2003 369

Seela and He
F IGURE 2. Reversed-phase HPLC (250 × 4 mm RP-18
column) profiles of the hydrolysis products of 1a and 12a in
25% aqueous NH3. 1a (a) at 60 °C for 16 h (for the buffer see
Figure 1a) and (b) at room temperature for 16 h (for the buffer
see Figure 1a).
tions. However, when mild deprotection conditions (room
temperature, 16 h) were applied, no deamination took
place either on 1a or on 1b (Figure 2b).
In light of this instability problem the N,N-dimethyl-
formamidine (dmf) protecting group was chosen for the
amino group protection of 1a or 1b.26a,b This protecting
group has already been used for the protection of other
isocytidine derivatives27 and 2′-deoxyisocytidine deriva-
tives.23 Treatment of 1a and 1b with dimethylformamide
dimethyl acetal in methanol furnished the amidines 12a
and 12b in almost quantitative yield (Scheme 4). The
applicability of the amidines 12a and 12b was also
investigated with regard to their stability under acidic
and basic conditions. As shown in Figure 1c,d, the free
base 7a is the final product of the acidic hydrolysis of
12a , and a small amount of dmf-protected 6-aza-5-
methylisocytosine (13a ) can be detected as an intermedi-
ate. Compound 12b underwent the same reaction but
with a faster rate. The acidic removal of the dmf group
has been described previously.5 According to Table 1, the
dmf group increases the stability of the glycosylic bond
of 1a ,b as described for other 2′-deoxyisocytidine deriva-
tives, such as the dmf-protected 5-methyl-2′-deoxyisocy-
tidine (dmf2m5iCd) or dmf-protected 2′-deoxyisocytidine
(dmf2iCd). Under the mild deprotection conditions the dmf
group of 12a or 12b was removed to give 1a or 1b via an
intermediate, which is expected to be the formyl deriva-
tive.28 The half-lives of 12a and 12b were determined UV-
spectrophotometrically to be 4 and 3 min (Table 1).
Compounds 12a and 12b were then converted into the
DMT derivatives 14a and 14b (68%, 70%) by means of
the standard conditions29 (Scheme 4). The phosphor-
F IGURE 1. Reversed-phase HPLC (250 × 4 mm RP-18
column) profiles of the hydrolysis products of 1a and 12a in
0.1 M HCl at 40 °C. Samples were taken at the times indicated.
Hydrolysis of 1a for (a) 90 min and (b) for 4 h. Buffer A: 5%
MeCN in 0.1 M (Et3NH)OAc, pH 7.0, 0.6 mL/min. Hydrolysis
of 12a (c) for 4 h and (d) for 20 h. Buffer: 10% MeCN in buffer
A, 0.6 mL/min.
spectrophotometrically. According to Figure 1a, com-
pound 1a is not significantly hydrolyzed within 90 min.
After 4 h (Figure 1b), half of the educt is degraded by
liberation of the base 7a . Compound 1b gave 7b with a
faster rate. According to the half-life values (Table 1),
the presence of a nitrogen in position 6 of the pyrimidine
ring increases the glycosylic bond stability toward acid
by a factor of 15 compared to that of the parent com-
pounds and the methyl group increases the stability 3
times further.
Next, the stability of 1a and 1b under alkaline condi-
tions was studied. As shown in Figure 2a compound 1a
was deaminated to give 4a under standard oligonucleo-
tide deprotection conditions (25% aqueous NH3, 60 °C,
16 h); the same reaction gave 4b from 1b with a faster
rate. This shows that the 6-pyrimidine nitrogen (1a ,b)
increases the deamination velocity under alkaline condi-
(26) (a) McBride, L. J .; Kierzek, R.; Beaucage, S. L.; Caruthers, M.
H. J . Am. Chem. Soc. 1986, 108, 2040-2048. (b) Seela, F.; Kro¨schel,
R.; He, Y. Nucleosides, Nucleotides Nucleic Acids 2001, 20, 1283-1286.
(27) Wang, D.; Ts’o, P. O. P Nucleosides Nucleotides 1996, 15, 387-
397.
(28) Zemlicka, J . Collect. Czech. Chem. Commun. 1970, 35, 3572-
3578.
(29) Alul, R. In DNA Probes; Keller, G. H., Manak, M. M., Eds.;
Stockton Press: New York, 1993; p 73.
370 J . Org. Chem., Vol. 68, No. 2, 2003

6-Aza-2′-deoxyisocytidine
SCHEME 4a
a
Reagents and conditions: (i) Me2NCH(OMe)2, MeOH, rt, 98% for 12a , 97% for 12b; (ii) DMTCl, pyridine, rt, 68% for 14a , 70% for
14b; (iii) CN(CH2)2OP(Cl)N(i-Pr)2, (i-Pr)2EtN, CH2Cl2, rt, 87% for 15a , 85% for 15b.
TABLE 2. MALDI-TOF Ma ss Sp ectr om etr ic Da ta
amidites 15a ,b were obtained after chromatographic
(P ositive Mod e) of a n Oligon u cleotid e Digest Clea ved by
Sn a k e-Ven om P h osp h od iester a sea
workup. All monomeric compounds were characterized
1
by H, 13C, and 31P NMR spectroscopy as well as by UV
spectroscopy and elemental analyses (see the Experi-
mental Section). The 13C NMR chemical shifts were
assigned form the gated-decoupled 13C NMR spectra.
2. Oligon u cleotid es. 2.1. Syn th esis a n d An a lysis
by MALDI-TOF Ma ss Sp ectr om etr y. The oligonucleo-
tide synthesis was performed on a solid phase employing
standard phosphoramidite chemistry. The phosphor-
amidites 15a and 15b (from 1a or 1b) and also the others
gave coupling yields higher than 95%. After the synthe-
sis, the oligonucleotides 17, 18, 22, 23, 24, and 31-37
(Tables 3 and 4) were deprotected in concd aqueous
ammonia at 60 °C for 16 h. The oligonucleotides incor-
porating 1a , 1b, or 2b (Tables 3 and 4) were deprotected
in concd aqueous ammonia solution at room temperature
(16 h). In these cases, the dA and dG phosphoramidites
with labile 4-(tert-butylphenoxy)acetyl (tac) protecting
groups were used. The 5′-DMT oligonucleotides were
purified by RP18-HPLC (see the Experimental Section),
and the DMT groups were removed with 2.5% CHCl2-
COOH in CH2Cl2 (5 min, room temperature). The detri-
tylated oligomers were purified again, desalted by RP18-
HPLC, and lyophilized. The homogeneity of the oligo-
nucleotides was proven by both ion-exchange chroma-
tography and reversed-phase HPLC, as well as by
MALDI-TOF mass spectrometry (data see the Supporting
Information). According to the increased stability of 1a ,b
compared to 2a ,b toward acid, only the oligonucleotides
containing 1a ,b gave the expected MH+ peaks without
forming degradation products. Oligonucleotides incorpo-
rating either compound 2a or 2b were partially degraded
by the acidic matrix. Apart from the MH+ peaks ad-
ditional peaks which resulted from the stepwise “depy-
rimidination” were present as the result of the low
N-glycosylic bond stability.
a
For details see Figure 3.
residue is partially resistant against hydrolysis by the
exonuclease. Also other cases were reported where other
base-modified nucleosides retard the phosphodiester hy-
drolysis of oligonucleotides by exonucleases.30 Therefore,
the partial enzymatic hydrolysis of oligonucleotides
containing 1a or 1b by 3′ f 5′ snake-venom phosphodi-
esterase was investigated by MALDI-TOF spectrometry.
The digestion pattern of 5′-d(T 1a ATAA1a T1a 1a TA)
(20) (1.4 A260 units, SVP, 0.1 unit, in H2O (25 µL) at 37
°C) is shown in Figure 3. From the reaction mixtures
aliquots (1 µL) were taken after different time intervals
for MALDI-TOF mass spectrometric analysis. Within 60
min the hydrolysis of 20 gave only three peaks corre-
sponding to the full-length oligonucleotide (3626.7 Da),
as well as the n - 1 and n - 2 oligomers (Figure 3a and
Table 2). Upon excessive treatment (20 h), the first two
peaks disappeared, and only the peak corresponding to
5′-d(T 1a ATAA1a T1a 1a ) (3006.6 Da) was detected (Fig-
ure 3b). After 25 h, the modified nucleotides were split
off and a ladder pattern was obtained with peaks being
in accordance with the sequence (Table 2). As the
truncated oligonucleotides containing 1a at the 3′-
terminus (3006.6, 2703.0, and 2094.8 Da) have much
higher intensities than the oligonucleotides with regular
residues at the 3′-terminus, the resistance toward phos-
phodiester cleavage induced by the modified base is
apparent (Figure 3c). It is not clear whether the change
of the electronic properties of the base or their high-anti
conformation affects the binding and the processing of
the oligonucleotide. The phenomenon might be exploited
The base composition of oligonucleotides containing 1a
or 1b was determined by complete enzymatic hydrolysis
using snake-venom phosphodiesterase followed by alka-
line phosphatase and subsequent reversed-phase HPLC
chromatography. No deamination to 6-aza-2′-deoxythy-
midine or 6-aza-2′-deoxyuridine was observed. However,
treatment under the regular conditions did not lead to a
complete cleavage. Only an extended incubation (48 h
instead of 45 min) led to complete digestion. Thus, the
phosphodiester bond of the base-modified nucleotide
(30) Rosemeyer, H.; Ramzaeva, N.; Becker, E. M.; Feiling, E.; Seela,
F. Bioconjugate Chem. 2002, 13, 1274-1285.
J . Org. Chem, Vol. 68, No. 2, 2003 371

Seela and He
rimidine nucleosides in aps-DNA16 prompted us to in-
vestigate this modification in ps-DNA with the final aim
to harmonize the m5iCd-dG base pair (motif IIa) stability
to the level of a reverse Watson-Crick dA-dT pair (motif
I). For this purpose, oligonucleotides were studied car-
rying the 6-azapyrimidine nucleoside analogue 1a or 1b
instead of m5iCd or iCd. They were hybridized in a parallel
way with those containing dG opposite 6-azapyrimidine
residues. The base pair of dG-m5z6iCd is shown in motif
IIc.
Our studies were focused toward oligonucleotides
containing m5iCd-dG base pairs apart from dA-dT pairs
but not incorporating iGd-dC pairs (Table 3). In one set
of experiments only one strand was modified. As refer-
ence, the parallel duplex 36‚37 was chosen incorporating
only dA-dT pairs. The Tm difference between 36‚37 and
22‚18 is 24 °C, which corresponds to 6 °C for one 2a -dG/
dA-dT base pair replacement. The usage of the des-
methylated residue 2b, which is rather sensitive to acidic
and alkaline conditions, reduces this difference to 20 °C
(5 °C for one 2b-dG/dA-dT pair substitution). A strong
destabilization of the tridentate base pair is observed
when 6-aza-5-methyl-2′-deoxyisocytidine (see 22‚20) re-
places 5-methyl-2′-deoxyisocytidine (22‚18). The Tm dif-
ference between duplex 22‚20 and duplex 36‚37 is now
only 15 °C corresponding to about 3.5 °C for one 1a -dG/
dA-dT substitution and 10 °C in the case of the desmeth-
ylated duplexes (22‚21 vs 36‚37), which equals 2.5 °C per
1b-dG/dA-dT base pair replacement. For comparison, the
modified base residues were also incorporated into both
strands. In such cases, the parallel duplex 31‚33 was
used as a reference. The ∆Tm is 19 °C between 23‚24 and
31‚33, corresponding to 4.8 °C for the presence of one
2a -dG base pair. In such cases, the replacement of 2a by
1a led to a ∆Tm ) 10 °C between duplexes 25‚26 and
31‚33, which corresponds to 2.5 °C for one 1a -dG/dA-dT
base pair replacement. By removing the methyl groups,
a further destabilization is observed (∆Tm ) 5 °C between
duplexes 27‚28 and 31‚33), which equals only around 1
°C per 1b-dG/dA-dT base pair replacement. The methyl
effect is also shown by comparing the Tm values of the
duplexes 25‚26 with those of 25‚28 or 27‚26.
F IGURE 3. MALDI-TOF mass spectra (positive mode) taken
from a partial enzymatic digest of the oligonucleotide 20 by
snake-venom phosphodiesterase: (a) 60 min treatment, (b) 20
h treatment, and (c) 25 h treatment. 3-Hydroxypicolinic acid
was used as matrix. For details see the Experimental Section.
to increase the stability of ps-DNA or aps-DNA in
antisense technology.
2.2. Sta bility of th e Du p lexes. Very little has been
reported on the influence of base-modified nucleosides
on the base pair stability of parallel-stranded DNA.31 As
we have accomplished the harmonization of stability of
a bidentate dA-dT base pair and the tridentate dG-dC
pair,8,9 we have now focused our interest to adjust the
base pair stability of the reverse Watson-Crick dA-dT
base pairs (motif I) to the m5iCd-dG pair (motif IIa) in
parallel DNA. The stabilization of motif I was already
studied with modified dA analogues incorporated in ps-
duplexes. On the other hand, the destabilization of the
tridentate iGd-dC base pairs failed when iGd analogues
were used.12 The observed destabilization of 6-azapy-
The influence of the 6-azapyrimidine nucleosides was
also studied in aps-DNA. Earlier, it was shown that the
base pairs of iGd-m5iCd or iGd-iCd (motif IVa,b) are more
stable than the dG-dC pair (motif III).5,25,32 Moreover,
enzymatic incorporation of the base pair was accom-
plished.33,34 The stability of the corresponding isoguanine-
(31) Seela, F.; He, Y. Parallel DNA: The application of base-modified
nucleosides to control chain orientation. In Modified Nucleosides,
Synthesis and Applications; Loakes, D., Ed.; Transworld Research
Network: Trivandrum, Kerala, India, 2002; pp 57-85.
(32) Horn, T.; Chang, C.; Collins, M. L. Tetrahedron Lett. 1995, 36,
2033-2036.
(33) Switzer, C. Y.; Moroney, S. E.; Benner, S. A. Biochemistry 1993,
32, 10489-10496.
372 J . Org. Chem., Vol. 68, No. 2, 2003

6-Aza-2′-deoxyisocytidine
TABLE 3. Tm Va lu es a n d Th er m od yn a m ic Da ta of
TABLE 4. Tm Va lu es a n d Th er m od yn a m ic Da ta of
P a r a llel Du p lexesa
An tip a r a llel Du p lexesa
a,b
See Table 3.
the Tm difference between tridentate- and bidentate-pair-
rich duplexes can be reduced to 12 °C (motif IVa f motif
IVc) and 7 °C (motif IVa f motif IVd), respectively. Thus,
the 6-azapyrimidine nucleosides 1a and 1b reduce the
stability of base pairs in ps- and aps-DNA. While an
adjustment of the tridentate vs bidentate base pair
stability was achieved by the 6-azapyrimidine nucleo-
sides, a complete harmonization of the stability of the
m5iCd-dG base pair to that of the reverse Watson-Crick
base pair of dA-dT, however, was not fully accomplished.
Further investigations with other modified nucleoside
residues are in progress.
a
Measured UV-spectrophotometrically at 260 nm in 1 M NaCl,
100 mM MgCl2, and 60 mM sodium cacodylate (pH 7.0). The
oligonucleotide concentration is 5 µM + 5 µM. Compared with
corresponding duplex 31‚33 or 36‚37.
b
isocytosine base pair incorporated in RNA fragments was
investigated recently.35 The pairing properties of pyra-
nosyl RNA containing isoguanine or 5-methylisocytosine
residues was investigated.36
Con clu sion
A nitrogen present in the 6-position of pyrimidine
nucleosides such as in 1b and its methyl derivative 1a
stabilizes the glycosylic bond against acidic degradation
but leads to a faster deamination rate in alkaline
medium. Oligonucleotides containing multiple incorpora-
tions of 1a and 1b residues can be synthesized when the
2-amino group is blocked with a (dimethylamino)meth-
ylidene residue and mild deprotection conditions are
chosen. These oligonucleotides show an enhanced stabil-
ity toward exonuclease, e.g., snake-venom phosphodi-
esterase. The 6-azapyrimidine nucleosides destabilize
both aps- and ps-DNA. This allows the adjustment of the
stability of oligonucleotides incorporating m5iCd-dG base
pairs to that of oligonucleotides with reverse Watson-
Crick dA-dT pairs. The adjustment is of particular
importance for the parallel hybridization performed with
oligonucleotide libraries in solution or on polymeric
surfaces (biochips). Although the harmonization of the
stability of the two base pairs formed in ps-DNA was not
completely achieved, the use of the 6-azapyrimidine
nucleosides 1a and 1b reduces the stability between the
tridentate and bidentate pairs significantly.
According to Table 4, a ∆Tm of -16 °C was observed
between a duplex containing 4 dG-dC and 8 dA-dT base
pairs (29‚30) and that incorporating 12 dA-dT pairs (31‚
32). This corresponds to a 4 °C stability increase of the
tridentate vs bidentate base pair. The related duplexes
containing four iGd-m5iCd pairs (motif IVa) instead of four
dA-dT pairs (17‚18) amounts to a ∆Tm ) -23 °C in
relation to the duplex 34‚35. For the related substitution
of m5iCd with the 6-azapyrimidine nucleoside 1a or 1b,
(34) Piccirilli, J . A.; Krauch, T.; Moroney, S. E.; Benner, S. A. Nature
1990, 343, 33-37.
(35) Chen, X.; Kierzek, R.; Turner, D. H. J . Am. Chem. Soc. 2001,
123, 1267.
(36) Krishnamurthy, R.; Pitsch, S.; Minton, M.; Miculka, C.; Windhab,
N.; Eschenmoser, A. Angew. Chem. 1996, 108, 1619-1623.
Exp er im en ta l Section
Gen er a l P r oced u r es. See ref 5. The solid-phase synthesis
of oligonucleotides was carried out on an automated DNA
J . Org. Chem, Vol. 68, No. 2, 2003 373

Seela and He
TABLE 5. 13C NMR Ch em ica l Sh ifts of Nu cleosid es a n d Der iva tivesa
C(2)
C(4)
C(5)
CdN
Me
Me2N
C(1′)
C(2′)
C(3′)
C(4′)
C(5′)
MeO
1a
1b
9a
9b
7a
155.6
155.2
155.4
155.1
156.5
156.1
160.3
160.2
159.0
159.0
158.9
159.0
148.8
148.2
148.4c
147.8
157.8
157.1
158.0
157.9
158.0
157.9
162.5
162.3
162.5
162.3
163.6
163.4
164.1
164.1
162.9
162.8
162.8
162.8
156.6
156.5
156.5c
156.4
163.0
163.0
162.5
162.5
162.3
162.3
146.9
139.4
147.0
139.5
146.1
139.0
148.1
140.5
148.5
140.7
148.3
140.5
143.4
136.0
144.9
136.0
148.2
140.3
144.8
144.9
139.7
139.3
16.9
88.2
88.3
87.4
87.6
37.2
37.1
37.0
37.0
70.5
70.3
69.7
69.7
87.6
87.6
86.1
86.1
62.0
61.8
60.9
60.8
17.0
16.7
16.9
17.2
16.8
16.3
16.1
16.7
7b
13a
13b
12a
12b
14a
14b
4a
4b
5a
5b
6a
158.9
159.1
158.8
158.9
157.9
157.9
34.7/40.8
34.8/40.9
35.1/41.0
35.1/41.0
35.1/41.0
35.2/41.1
86.3
86.3
86.8
86.6
87.2
87.1
85.0
85.1
87.6
87.6
87.3
87.4
87.5
87.4
37.6
37.4
b
70.9
70.7
71.4
70.6
70.6
70.4
70.7
70.5
70.3
70.1
70.4
70.8
70.4
70.0
87.4
87.3
85.7
85.2
84.5
84.6
83.3
83.2
86.6
87.0
84.2
85.7
84.2
85.4
62.5
62.3
65.0
64.3
62.2
62.0
70.3
70.0
62.0
61.8
63.5
64.6
63.5
64.0
54.8
54.9
b
36.9
36.8
37.4
37.1
37.0
36.9
37.1
37.7
37.3
37.5
6b
11a
10a
11b
10b
17.0
16.7
54.9
54.9
55.0
54.9
a
b
Measured in d6-DMSO at 25 °C. Superimposed by d6-DMSO. c Tentative.
synthesizer for phosphoramidite chemistry. All reagents are
commercially available and were used as received. The sol-
vents were purified and dried according to the standard
procedures. Thin-layer chromatography (TLC) was performed
on aluminum sheets of silica gel 60 F254 (0.2 mm). Flash
chromatography (FC) was performed at 0.4 bar on silica gel
60 H. Reversed-phase HPLC was performed on a 4 × 250 mm
RP-18 (10 µm) column with an HPLC pump, a variable-
wavelength monitor, a controller, and an integrator. For UV
spectra, λmax is in nanometers and ꢀ is in M-1 cm-1; half-life
values (τ) were measured with a UV spectrophotometer,
connected with a temperature controller. Melting curves were
determined using a UV/vis spectrophotometer equipped with
a thermoelectrical controller. The calculation of thermody-
namic data was performed with the program MeltWin (version
3.1)37 using the curve fitting of the melting profiles according
to a two-state model. The standard errors of thermodynamic
data for ∆H° and ∆S° obtained from the curve fitting are
within an error of (15%. For NMR spectra, δ values are in
parts per million downfield from the chemical shift of internal
SiMe4 (1H, 13C (Table 5)) or external 85% H3PO4 (31P). MALDI-
TOF mass spectra were recorded in the reflector mode. For
details see ref 30.
Com p osition An a lysis of Oligon u cleotid es. The oligo-
nucleotides (0.2 A260 unit) were dissolved in 0.1 M Tris-HCl
buffer (pH 8.3, 200 µL) and treated with snake-venom phos-
phodiesterase (EC 3.1.4.1, Crotalus adamanteus, 3 µL) at 37
°C for 45 min and then with alkaline phosphatase (EC 3.1.3.1,
E. coli, 3 µL) at 37 °C for 30 min for the unmodified oligonu-
cleotides. For oligonucleotides containing 1a ,b, the incubation
time was extended to 48 h. The mixture was analyzed on
reversed-phase HPLC (RP-18, gradient III, at 260 nm).
Quantification of the constituents was made on the basis of
the peak areas, which were divided by the extinction coef-
Silyla tion of 6-Aza u r a cil, 6-Aza th ym in e, 6-Aza -5-m eth -
ylisocytosin e (7a ), a n d 6-Aza isocytosin e (7b). This general
method described for the silylation of 6-azauracil was used for
all the other heterocycles. A suspension of 6-azauracil (5.0 g,
44 mmol), HMDS (90 mL), and TMSCl (10 mL) was heated to
reflux (170 °C) under anhydrous conditions for 3 h. The NH4-
Cl sublimed nearly quantitatively into the reflux condenser
and was thus removed from the reaction mixture. The excess
of HMDS/TMSCl was removed by distillation in vacuo. The
residual oil solidified upon storage in vacuo to furnish a powder
(11.0 g). The product was used directly without further
purification in the following glycosylation reactions.
Glycosyla tion of Silyla ted 6-Aza -5-m eth ylisocytosin e
(16a ) w ith th e Ha logen ose 8. To a stirred solution of
compound 8 (13.1 g, 33.7 mmol) in dry CHCl3 (400 mL) was
added CuI (6.8 g, 35.7 mmol), and stirring was continued for
5 min at rt under exclusion of moisture. The silylated base
16a (7.5 g, 27.7 mmol) was added to the solution, and the
mixture was stirred for 3 h at rt. The reaction was quenched
with saturated aqueous NaHCO3 (400 mL). Stirring was
continued for 15 min, and the two-phase mixture was filtered
through a pad of Celite. The CHCl3 layer was separated,
washed with saturated aqueous NaCl (200 mL), dried (Na2-
SO4), concentrated, and applied to an FC column (silica gel;
column 5.5 × 10 cm; CH2Cl2/MeOH, 95:5). Isolation of the main
zone afforded a colorless solid, which was a mixture of the N(1)/
N(3)-isomers in a 1:1 ratio (determined from the anomeric
proton signal). This regioisomeric mixture was deprotected
with 0.2 M NaOMe (100 mL) at rt for 1 h. The solution was
neutralized (Dowex-50, H+-form) and filtered. The resin was
washed with MeOH, and the combined filtrate was concen-
trated and applied to an FC column (silica gel; column 4.5 ×
11 cm; CH2Cl2/ MeOH, 95:5 f 3:1). A colorless powder (2.9 g,
43%) of a 1a /9a mixture was isolated which proved to be
nonseparable.
ficients of the nucleosides (ꢀ260
15400; 1a , 6100; 1b, 5800).
: dG, 11700; dT, 8800; dA,
3-Am in o-4-[2-deoxy-5-O-(4,4′-dim eth oxytr ityl)-â-D-er yth -
r o-p en tofu r a n osyl]-6-m eth yl-1,2,4-tr ia zin -5(4H)-on e (11a )
a n d 3-Am in o-2-[2-d eoxy-5-O-(4,4′-d im et h oxyt r it yl)-â-D-
er yt h r o-p e n t ofu r a n osyl]-6-m e t h yl-1,2,4-t r ia zin -5(2H )-
on e (10a ). The mixture of 1a /9a (1.7 g, 7.0 mmol) was dried
by coevaporation with anhydrous pyridine (3 × 10 mL) and
dissolved in anhydrous pyridine (10 mL). Then, 4,4′-dimeth-
oxytrityl chloride (4.1 g, 12.1 mmol) was added in two portions
(every 10 min) at rt under stirring, and stirring was continued
for 2 h. MeOH (5 mL) was added, and after 10 min the solution
MALDI-TOF An a lysis of th e Oligon u cleotid e Digests.
The oligonucleotides (1.4 A260 units) were dissolved in H2O to
a
total volume of 25 µL and treated with snake-venom
phosphodiesterase (EC 3.1.4.1, C. adamanteus, 1 µL, 0.1 unit)
at 37 °C. Aliquots of 1 µL of the enzymatic digests were
analyzed by MALDI-TOF mass spectrometry.
(37) McDowell, J . A.; Turner, D. H. Biochemistry 1996, 35, 14077-
14089.
374 J . Org. Chem., Vol. 68, No. 2, 2003

6-Aza-2′-deoxyisocytidine
was evaporated to dryness. The residue was dissolved in CH2-
Cl2 (35 mL), washed with 5% aqueous NaHCO3 (2 × 15 mL)
and H2O (20 mL), and dried (Na2SO4). After evaporation a
foam was formed which was redissolved in a small volume of
CH2Cl2 and applied to an FC column (silica gel; column 4 ×
12 cm; CH2Cl2/MeOH, 95:5).
5.98 (t, 1H); 7.40 (s, 1H); 7.43 (s, 2H). Anal. Calcd for
C8H12N4O4 (228.2): C, 42.10; H, 5.30; N, 24.55. Found: C,
42.11; H, 5.35; N, 24.36.
2-(2-Deoxy-5-O-t osyl-â-D-er yth r o-p en t ofu r a n osyl)-6-
m eth yl-1,2,4-tr ia zin -3,5(2H,4H)-d ion e (5a ). 6-Azathymidine
(4a ) (2.64 g, 10.9 mmol) was coevaporated with anhydrous
pyridine (3 × 10 mL) and then dissolved in anhydrous pyridine
(30 mL). To the solution was added p-toluenesulfonyl chloride
(2.3 g, 12.1 mmol) at 0 °C, and the resulting solution was
stirred at rt overnight. After being quenched with H2O (25
mL), the solution was extracted with CHCl3 (3 × 50 mL). The
combined organic layer was washed with saturated aqueous
NaHCO3 (2 × 60 mL) and H2O (2 × 60 mL), dried (Na2SO4),
concentrated, and then applied to an FC column (silica gel;
column 4.5 × 11 cm; CH2Cl2/MeOH, 9:1). Collection of the main
fraction and evaporation gave compound 5a as a colorless foam
(3.0 g, 70%). TLC (CH2Cl2/MeOH, 9:1): Rf 0.42. UV (MeOH):
From the faster migrating main zone compound 11a was
isolated. Colorless foam (880 mg, 23%). TLC (CH2Cl2/MeOH/
1
Et3N, 95:5:0.2): Rf 0.25. UV (MeOH): 234 (27300). H NMR
(d6-DMSO): δ 2.08 (s, 3H); 2.34-2.51 (m, 2H); 3.00-3.19 (m,
2H); 3.74 (s, 6H); 4.07 (m, 2H); 5.33 (d, 1H); 6.05 (t, 1H); 6.88-
7.40 (m, 15H). Anal. Calcd for C30H32N4O6 (544.6): C, 66.16;
H, 5.92; N, 10.29. Found: C, 66.21; H, 5.80; N, 9.93.
The slower migrating zone furnished compound 10a . Color-
less foam (1.3 g, 34%). TLC (CH2Cl2/MeOH/Et3N, 95:5:0.2): Rf
1
0.18. UV (MeOH): 234 (27900). H NMR (d6-DMSO): δ 1.79
(s, 3H); 2.11-2.57 (m, 2H); 3.02 (m, 2H); 3.72 (s, 6H); 3.94 (m,
1H); 4.32 (m, 1H); 5.28 (d, 1H); 6.14 (dd, 1H); 6.80-7.33 (m,
13H). 7.37(s, 2H). Anal. Calcd for C30H32N4O6 (544.6): C, 66.16;
H, 5.92; N, 10.29. Found: C, 66.09; H, 5.86; N, 10.25.
Glycosyla tion of Silyla ted 6-Aza isocytosin e (16b) w ith
th e Ha logen ose 8. Compound 16b (10.6 g, 41.3 mmol) was
treated as described for 16a with 8 (13.0 g, 33.4 mmol) and
CuI (7.6 g, 39.9 mmol). FC (silica gel; column 5.5 × 10 cm;
CH2Cl2/MeOH, 95:5) furnished the toluoylated N(1)/N(3) mix-
ture. It was detoluoylated as described above for 16a . FC (silica
gel; column 4.5 × 11 cm; CH2Cl2/MeOH, 9:1 f 3:1) furnished
a mixture of 1b/9b (4.3 g, 56%).
1
223 (14100), 264 (6000). H NMR (d6-DMSO): δ 1.98 (s, 3H);
2.09-2.30 (m, 2H); 2.38 (s, 3H); 3.88 (m, 2H); 4.16 (m, 1H);
4.28 (m, 1H); 5.38 (d, 1H); 6.27 (dd, 1H); 7.38-7.77(dd, 4H);
12.09 (s, 1H). Anal. Calcd for C16H19N3O7S (397.4): C, 48.36;
H, 4.82; N, 10.57. Found: C, 48.42; H, 4.62; N, 10.47.
2-(2-Deoxy-5-O-tosyl-â-D-er yth r o-p en tofu r a n osyl)-1,2,4-
tr ia zin -3,5(2H,4H)-d ion e (5b). As described for 5a , 6-aza-
2′-deoxyuridine (4b) (2.8 g, 12.2 mmol) in anhydrous pyridine
(35 mL) was treated with p-toluenesulfonyl chloride (3.1 g, 16.3
mmol). FC (silica gel; column 4.5 × 7 cm; CH2Cl2/MeOH, 8:1)
yielded compound 5b as a colorless foam (2.0 g, 43%). TLC
(CH2Cl2/MeOH, 8:1): Rf 0.56. UV (MeOH): 224 (14100), 262
(6000). 1H NMR (d6-DMSO): δ 2.07-2.28 (m, 2H); 2.40 (s, 3H);
3.86 (m, 2H); 4.16 (m, 1H); 4.20 (m, 1H); 5.39 (d, 1H); 6.26
(dd, 1H); 7.38-7.77(dd, 5H); 12.20 (s, 1H). Anal. Calcd for
3-Am in o-4-[2-deoxy-5-O-(4,4′-dim eth oxytr ityl)-â-D-er yth -
r o-pen tofu r an osyl]-1,2,4-tr iazin -5(4H)-on e (11b) an d 3-am -
in o-2-[2-deoxy-5-O-(4,4′-d im eth oxytr ityl)-â-D-er yth r o-p en -
tofu r a n osyl]-1,2,4-tr ia zin -5(2H)-on e (10b). As described for
1a /9a , the mixture of 1b/9b (1.0 g, 4.2 mmol) was treated with
4,4′-dimethoxytrityl chloride (2.8 g, 8.3 mmol) in anhydrous
pyridine (55 mL). It was worked up by FC (silica gel; column
4 × 12 cm; CH2Cl2/MeOH/Et3N, 9:1:0.2).
C
15H17N3O7S (383.4): C, 46.99; H, 4.47; N, 10.96. Found: C,
47.00; H, 4.59; N, 11.02.
2,5′-An h yd r o-6-a za th ym id in e (6a ). Compound 5a (2.8 g,
7.0 mmol) was dissolved in dry MeCN (dried over 4 Å
molecular sieves). DBU (1.9 mL, 12.9 mmol) was added, and
the reaction mixture was refluxed at 120 °C for 1.5 h.
Evaporation of the mixture gave an oil which was applied to
an FC column (silica gel; column 2.5 × 12 cm; CH2Cl2/MeOH,
9:1). Trituration with acetone yielded 6a as a colorless powder
(760 mg, 47%). TLC (CH2Cl2/MeOH, 9:1): Rf 0.69. UV
(MeOH): 228 (6700), 255 (6800). 1H NMR (d6-DMSO): δ 2.10-
2.56 (m, 2H); 2.12 (s, 3H); 3.47-3.71(m, 2H); 4.28-4.67 (m,
2H); 5.25 (d, 1H); 6.25 (dd, 1H).
2,5′-An h yd r o-6-a za -2′-d eoxyu r id in e (6b). As described
for 6a , compound 5b (1.8 g, 4.7 mmol) dissolved in dry MeCN
was treated with DBU (1.3 mL, 8.5 mmol) for 1 h. FC (silica
gel; column 4.3 × 9.5 cm; CH2Cl2/MeOH, 8:1) followed by
precipitation from acetone afforded 6b as a colorless powder
(496 mg, 50%). TLC (CH2Cl2/MeOH, 8:1): Rf 0.33. UV
(MeOH): 223 (8200), 257 (5600). 1H NMR (d6-DMSO): δ 2.10-
2.52 (m, 2H); 3.32-3.45(m, 2H); 3.72 (m, 1H); 4.27 (m, 1H);
5.25 (d, 1H); 6.25 (dd, 1H); 7.66 (s, 1H).
3-Am in o-2-(2-deoxy-â-D-er yth r o-pen tofu r an osyl)-6-m eth -
yl-1,2,4-tr ia zin -5(2H)-on e (1a ). Meth od A. 6a (700 mg, 3.1
mmol) was dissolved in MeOH (100 mL) which was presatu-
rated with NH3 at 0°C. The solution was stirred in a sealed
bottle at rt until the starting material had disappeared (20 h;
TLC monitoring with CH2Cl2/MeOH, 3:1). The solvent was
evaporated to dryness, and the residue was applied to an FC
column (silica gel; column 2.5 × 12 cm; CH2Cl2/MeOH, 3:1).
Evaporation of the main fraction gave compound 1a as a
colorless powder (600 mg, 80%). TLC (CH2Cl2/MeOH, 3:1): Rf
0.55. UV (MeOH): 247 (7300). 1H NMR (d6-DMSO): δ 2.03
(s, 3H); 2.04-2.56 (m, 2H); 3.48 (m, 2H); 3.74 (m, 1H); 4.29
(m, 1H); 4.85 (t, 1H); 5.24 (d, 1H); 6.05 (t, 1H); 7.29 (s, 2H).
Anal. Calcd for C9H14N4O4 (242.2): C, 44.63; H, 5.83; N, 23.13.
Found: C, 44.82; H, 5.99; N, 22.89.
From the faster migrating main zone compound 11b was
isolated. Colorless foam (500 mg, 22%). TLC (CH2Cl2/MeOH/
Et3N, 9:1:0.2): Rf 0.39. UV (MeOH): 234 (26500). 1H NMR
(d6-DMSO): δ 2.34-2.58 (m, 2H); 2.94-3.19 (m, 2H); 3.74 (s,
6H); 4.04 (m, 2H); 5.33 (br s, 1H); 6.09 (t, 1H); 6.88-7.40 (m,
14H); 7.48 (s, 2H). Anal. Calcd for C29H30N4O6 (530.6): C,
65.65; H, 5.70; N, 10.56. Found: C, 65.41; H, 5.82; N, 10.55.
The slower migrating zone gave compound 10b. Colorless
foam (800 mg, 34%). TLC (CH2Cl2/MeOH/Et3N, 95:5:0.2): Rf
0.35. UV (MeOH): 234 (25000). 1H NMR (d6-DMSO): δ 2.11-
2.61 (m, 2H); 3.01 (m, 2H); 3.72 (s, 6H); 3.89 (m, 1H); 4.32 (m,
1H); 5.30 (d, 1H); 6.14 (dd, 1H); 6.80-7.37 (m, 13H); 7.56 (2H).
Anal. Calcd for C29H30N4O6 (530.6): C, 65.65; H, 5.70; N, 10.56.
Found: C, 65.25; H, 5.86; N, 10.25.
3-Am in o-2-(2-deoxy-â-D-er yth r o-pen tofu r an osyl)-6-m eth -
yl-1,2,4-tr ia zin -5(4H)-on e (9a ). A solution of 11a (450 mg,
0.8 mmol) in CH2Cl2 (15 mL) was treated with 2.5% DCA in
CH2Cl2 (5 mL) for 5 min at room temperature. When the DMT
group was removed (TLC monitoring), it was neutralized with
Et3N. The solvent was evaporated to give an oily residue which
was applied to an FC column (silica gel; column 4 × 7.5 cm;
CH2Cl2/MeOH, 95:5 f 3:1). Compound 9a was obtained as a
colorless solid (200 mg, nearly quantative yield). TLC (CH2-
Cl2/MeOH, 3:1): Rf 0.55. UV (MeOH): 247 (7700). 1H NMR
(d6-DMSO): δ 2.06 (s, 3H); 2.46 (m, 2H); 3.55 (m, 2H); 3.90
(m, 1H); 4.09 (m, 1H); 4.74 (t, 1H); 5.32 (d, 1H); 5.94 (t, 1H-
C); 7.28 (s, 2H). Anal. Calcd for C9H14N4O4 (242.2): C, 44.63;
H, 5.83; N, 23.13. Found: C, 44.56; H, 5.75; N, 23.20.
3-Am in o-2-(2-d eoxy-â-D-er yth r o-p en tofu r a n osyl)-1,2,4-
tr ia zin -5(4H)-on e (9b). Compound 11b (500 mg, 0.9 mmol)
in CH2Cl2 (15 mL) was treated with 2.5% DCA in CH2Cl2 as
described for 11a . FC (silica gel; column 2.2 × 10.5 cm; CH2-
Cl2/MeOH, 95:5 f 3:1) furnished 9b as a colorless solid (140
mg, 65%). TLC (CH2Cl2/MeOH, 8:1): Rf 0.15. UV (MeOH): 254
Meth od B. Compound 10a (600 mg, 1.1 mmol) was treated
as described for 11a . After purification by FC (silica gel;
column 2.5 × 12 cm; CH2Cl2/MeOH, 9:1 f 3:1) compound 1a
1
(6200). H NMR (d6-DMSO): δ 2.04-2.56 (m, 2H); 3.34-3.43
(m, 2H); 3.86 (m, 1H); 4.09 (m, 1H); 4.74 (t, 1H); 5.30 (d, 1H);
J . Org. Chem, Vol. 68, No. 2, 2003 375

Seela and He
was obtained as a colorless powder (250 mg, 94%) showing the
same analytical data as described above.
mg, 1.42 mmol) was dried by coevaporation with anhydrous
pyridine (3 × 10 mL) and then dissolved in anhydrous pyridine
(10 mL). 4,4′-Dimethoxytrityl chloride (673 mg, 2.0 mmol) was
added in two portions (every 10 min) at rt under stirring. The
mixture was stirred for a further 2 h. After addition of MeOH
(5 mL), the reaction mixture was evaporated and the residue
dissolved in CH2Cl2 (35 mL). The solution was washed with
5% aqueous NaHCO3 (2 × 15 mL) and H2O (20 mL) and dried
(Na2SO4), and the organic layer was evaporated to give a
colorless foam. This was applied to an FC column (silica gel,
column 4 × 12 cm). After the column was washed with CH2-
Cl2/acetone/Et3N (70:30:0.5), compound 14a was eluted with
CH2Cl2/MeOH/Et3N (90:10:0.1) to give a colorless foam (580
mg, 68%). TLC (CH2Cl2/MeOH/Et3N, 90:10:0.1): Rf 0.46. UV
(MeOH): 236 (33600), 275 (36300). 1H NMR (d6-DMSO): δ
1.78 (s, 3H); 2.12-2.40 (m, 2H); 3.11 (s, 3H); 3.21 (s, 3H); 3.02
(m, 2H); 3.72 (s, 6H); 3.94 (m, 1H); 4.32 (m, 1H); 5.25 (d, 1H);
6.94 (t, 1H); 6.82-7.59 (m, 14H); 8.68 (s, 1H). Anal. Calcd for
3-Am in o-2-(2-d eoxy-â-D-er yth r o-p en tofu r a n osyl)-1,2,4-
tr ia zin -5(2H)-on e (1b). Meth od A. Compound 1b was pre-
pared as described for 1a from 6b (900 mg, 4.2 mmol) in MeOH
(100 mL) which was presaturated with NH3 at 0 °C. Colorless
powder (719 mg, 75%). TLC (CH2Cl2/MeOH, 8:1): Rf 0.15. UV
(MeOH): 253 (6300). 1H NMR (d6-DMSO): δ 2.04-2.56 (m,
2H); 3.45 (m, 2H); 3.74 (m, 1H); 4.26 (m, 1H); 4.86 (t, 1H);
5.26 (d, 1H); 6.06 (t, 1H); 7.34 (s, 1H); 7.46 (s, 2H). Anal. Calcd
for C8H12N4O4 (228.2): C, 42.11; H, 5.30; N, 24.55. Found: C,
42.21; H, 5.40; N, 24.25.
Meth od B. Compound 10b (500 mg, 0.9 mmol) was treated
as described for 11b. Purification by FC (silica gel; column
2.5 × 12 cm; CH2Cl2/MeOH, 9:1 f 3:1) afforded 1b as a
colorless powder (140 mg, 65%) showing analytical data
identical to that described above.
3-[(N,N-Dim eth yla m in o)m eth ylid en e]a m in o-6-m eth yl-
1,2,4-t r ia zin -5(2H)-on e (13a ). To a suspension of 6-aza-5-
methylisocytosine21 (126 mg, 1.0 mmol) in MeOH (20 mL) was
added N,N-dimethylformamide dimethyl acetal (0.3 mL, 2.3
mmol). The reaction mixture was stirred at 50 °C. After the
starting material had disappeared (∼2 h; TLC monitoring with
CH2Cl2/MeOH, 9:1), the mixture was evaporated to dryness.
The resulting white powder was dissolved in CH2Cl2 and
applied to an FC column (silica gel; column 2.5 × 12 cm; CH2-
Cl2/MeOH, 4:1). Compound 13a was obtained as a colorless
powder (180 mg, nearly quantitative yield). TLC (CH2Cl2/
C
33H37N5O6 (599.7): C, 66.09; H, 6.22; N, 11.68. Found: C,
66.36; H, 6.31; N, 11.64.
2-[2-Deoxy-5-O-(4,4′-dim eth oxytr ityl)-â-D-er yth r o-pen to-
fu r a n osyl]-3-[(N,N-d im eth yla m in o)m eth ylid en e]a m in o-
1,2,4-tr ia zin -5(2H)-on e (14b). Compound 14b was prepared
as described for 14a with compound 12b (330 mg, 1.2 mmol)
and 4,4′-dimethoxytrityl chloride (673 mg, 2.0 mmol). This
compound was obtained as a colorless foam after FC (silica
gel; column 4 × 12 cm; CH2Cl2/acetone/Et3N, 70:30:0.5 f CH2-
Cl2/MeOH/Et3N, 90:10:0.1) (480 mg, 70%). TLC (CH2Cl2/
MeOH/Et3N, 90:10:0.1): Rf 0.46. UV (MeOH): 235 (34900),
1
MeOH, 9:1): Rf 0.33. UV (MeOH): 272 (32000). H NMR (d6-
1
276 (34900). H NMR (d6-DMSO): δ 2.14-2.42 (m, 2H); 3.13
DMSO): δ 2.05 (s, 3H), 3.04 (s, 3H); 3.17 (s, 3H); 8.65 (s, 1H);
12.61 (s, 1H). Anal. Calcd for C7H11N5O (181.2): C, 46.40; H,
6.12; N, 38.65. Found: C, 46.67; H, 6.25; N, 38.26.
(s, 3H); 3.23 (s, 3H); 3.02 (m, 2H); 3.72 (s, 6H); 3.85 (m, 1H);
4.33 (m, 1H); 5.25 (d, 1H); 6.91 (t, 1H); 6.82-7.37 (m, 13H);
8.68 (s, 1H). Anal. Calcd for C32H35N5O6 (585.7): C, 65.63; H,
6.02; N, 11.96. Found: C, 65.56; H, 6.10; N, 11.89.
3-[(N,N-Dim eth yla m in o)m eth ylid en e]a m in o-1,2,4-tr i-
a zin -5(2H)-on e (13b). Compound 13b was prepared as
described for 13a from 6-azaisocytosine (112 mg, 1.0 mmol)
with N,N-dimethylformamide dimethyl acetal (0.3 mL, 2.3
mmol). Evaporation of the main fraction gave 13b as a
colorless powder (160 mg, 96%). TLC (CH2Cl2/MeOH, 9:1): Rf
2-[2-Deoxy-5-O-(4,4′-dim eth oxytr ityl)-â-D-er yth r o-pen to-
fu r a n osyl]-3-[(N,N-d im eth yla m in o)m eth ylid en e]a m in o-
6-m eth yl-1,2,4-tr ia zin -5(2H)-on e 3′-O-(2-Cya n oeth yl d i-
isopr opylph osph or am idite) (15a). To a solution of compound
14a (590 mg, 1.0 mmol) in dry CH2Cl2 (15 mL) was added
2-cyanoethyl diisopropylphosphoramidochloridite (0.3 mL 1.3
mmol) together with N-ethyldiisopropylamine (0.3 mL, 1.7
mmol) under argon at rt. The mixture was stirred for 20 min
at room temperature. Then, 5% aqueous NaHCO3 (15 mL) was
added, and the mixture was extracted with CH2Cl2 (3 × 15
mL). The extract was dried (Na2SO4) and evaporated to an
oil. This residue was applied to an FC column (silica gel;
column 4 × 7.5 cm; CH2Cl2/MeOH/Et3N, 95:5:0.2), giving
compound 15a as a colorless foam (680 mg, 87%). TLC (CH2-
Cl2/MeOH/Et3N, 95:5:0.2): Rf 0.54. UV (MeOH): 235 (30800),
276 (30700). 31P NMR (CDCl3): δ 149.3, 149.7.
2-[2-Deoxy-5-O-(4,4′-dim eth oxytr ityl)-â-D-er yth r o-pen to-
fu r a n osyl]-3-[(N,N-d im eth yla m in o)m eth ylid en e]a m in o-
1,2,4-tr ia zin -5(2H)-on e 3′-O-(2-Cya n oeth yl d iisop r op yl-
p h osp h or a m id ite) (15b). Compound 14b (350 mg, 0.6 mmol)
in dry CH2Cl2 (15 mL) was treated with 2-cyanoethyl diiso-
propylphosphoramidochloridite (0.15 mL, 0.7 mmol) together
with N-ethyldiisopropylamine (0.15 mL, 0.86 mmol) as de-
scribed for 14a . FC furnished the title compound as a foam
(400 mg, 85%). TLC (CH2Cl2/MeOH/Et3N, 95:5:0.2): Rf 0.34.
UV (MeOH): 236 (29800), 275 (32600). 31P NMR (CDCl3): δ
149.6, 149.9.
Oligon u cleotid es. The oligonucleotide synthesis was car-
ried out on a 1 µmol scale using the phosphoramidites of 1a ,b
and the phosphoramidite of iGd (3) on a synthesizer. After the
synthesis the oligonucleotides 17, 18, 22, 23, 24, and 31-37
were deprotected in 25% aqueous NH3 at 60 °C for 16 h. The
oligonucleotides containing 1a ,b and 2b were deprotected in
25% aqueous NH3 at room temperature for 16 h. Accordingly,
the dA and dG residues were protected with the labile 4-(tert-
butylphenoxy)acetyl (tac) group which can be removed within
2 h at room temperature with 25% aqueous NH3. Oligonucleo-
tides were purified by reversed-phase HPLC using the follow-
1
0.21. UV (MeOH): 272 (29500). H NMR (d6-DMSO): δ 3.05
(s, 3H); 3.18 (s, 3H); 7.36 (s, 1H); 8.66 (s, 1H); 12.86 (s, 1H).
Anal. Calcd for C6H9N5O (167.2): C, 43.11; H, 5.43; N, 41.89.
Found: C, 43.25; H, 5.33; N, 41.72.
2-(2-Deoxy-â-D-er yth r o-pen tofu r an osyl)-3-[(N,N-dim eth -
ylam in o)m eth yliden e]am in o-6-m eth yl-1,2,4-tr iazin -5(2H)-
on e (12a ). To a suspension of compound 1a (400 mg, 1.7 mmol)
in MeOH (35 mL) was added N,N-dimethylformamide dimeth-
yl acetal (0.6 mL, 4.5 mmol). The reaction mixture was stirred
at rt for ∼2 h (TLC monitoring with CH2Cl2/MeOH, 9:1). The
reaction mixture was evaporated to dryness and the residue
applied to an FC column (silica gel; column 4.5 × 5 cm; CH2-
Cl2/MeOH, 4:1). Evaporation of the main fraction gave com-
pound 12a as a colorless powder (483 mg, 98%). TLC (CH2Cl2/
1
MeOH, 9:1): Rf 0.33. UV (MeOH): 276 (31200). H NMR (d6-
DMSO): δ 2.05-2.43 (m, 2H); 2.07 (s, 3H); 3.08 (s, 3H); 3.20
(s, 3H); 3.50 (m, 2H); 3.71 (m, 1H); 4.31 (m, 1H); 4.68 (t, 1H);
5.19 (d, 1H); 6.86 (t, 1H); 8.61 (s, 1H). Anal. Calcd for
C
12H19N5O4 (297.3): C, 48.48; H, 6.44; N, 23.56. Found: C,
48.31; H, 6.39; N, 23.37.
2-(2-Deoxy-â-D-er yth r o-pen tofu r an osyl)-3-[(N,N-dim eth -
ylam in o)m eth yliden e]am in o-1,2,4- tr iazin -5(2H)-on e (12b).
As described for 12a , compound 12b was obtained from 1b
(100 mg, 0.4 mmol) by reaction with N,N-dimethylformamide
dimethyl acetal (0.16 mL, 1.2 mmol). Colorless powder (120
mg, 97%). TLC (CH2Cl2/MeOH, 9:1): Rf 0.23. UV (MeOH): 276
(27000). 1H NMR (d6-DMSO): δ 2.08-2.47 (m, 2H); 3.09 (s,
3H); 3.22 (s, 3H); 3.47 (m, 2H); 3.70 (m, 1H); 4.27 (m, 1H);
4.69 (t, 1H); 5.21 (d, 1H); 6.86 (t, 1H); 7.51 (s, 1H); 8.62 (s,
1H). Anal. Calcd for C11H17N5O4 (283.3): C, 46.64; H, 6.05; N,
24.72. Found: C, 46.65; H, 6.04; N, 24.64.
2-[2-Deoxy-5-O-(4,4′-dim eth oxytr ityl)-â-D-er yth r o-pen to-
fu r a n osyl]-3-[(N,N-d im eth yla m in o)m eth ylid en e]a m in o-
6-m eth yl-1,2,4-tr ia zin -5(2H)-on e (14a ). Compound 12a (422
376 J . Org. Chem., Vol. 68, No. 2, 2003

6-Aza-2′-deoxyisocytidine
ing solvent systems: 0.1 M aqueous (Et3NH)OAc (pH 7.0) (A),
MeCN (B), and 0.1 M (Et3NH)OAc (pH 7.0)/MeCN, 95:5 (C).
They were used in the following order: gradient I, 3 min 15%
B in C, 12 min 15-40% B in C, 5 min 40-15% B in C with a
flow rate of 1.0 mL/min; gradient II, 20 min 0-20% B in C
with a flow rate of 1.0 mL/min; gradient III, 30 min 100% C
with a flow rate of 0.6 mL/min. The oligomers carrying 5′-DMT
residues were purified by HPLC (250 × 4 mm, RP-18 column)
using gradient I. The DMT residues were removed by treating
the oligomers with 2.5% Cl2CHCOOH/CH2Cl2 for 5 min at
room temperature. The detritylated oligomers were purified
by HPLC (250 × 4 mm RP-18 column) using gradient II. The
oligomers separated by RP18-HPLC were desalted on a 4 cm
column (RP-18, silica gel) with H2O to elute the salt, while
the oligomers were eluted by MeOH/H2O (3:2). The purified
oligonucleotides were lyophilized on an evaporator to yield
colorless solids which were dissolved in 100 µL of H2O and
stored frozen at -18 °C. The synthesized oligonucleotides were
characterized by enzymatic hydrolysis as well as by MALDI-
TOF mass spectrometry.
Ack n ow led gm en t. We gratefully acknowledge fi-
nancial support by the Roche Diagnostics GmbH. We
thank Mrs. E. Feiling for the synthesis of the oligo-
nucleotides, Mr. K. Shaikh for the measurement of the
MALDI-TOF mass spectra, and Dr. H. Rosemeyer for
helpful discussions.
Su p p or t in g In for m a t ion Ava ila b le: 13C and 1H NMR
coupling constants (Hz) of 1a ,b and 9a ,b and MALDI-TOF
mass spectral data (MH+) of selected oligonucleotides. This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O020507N
J . Org. Chem, Vol. 68, No. 2, 2003 377