Synthesis and hybridization ability of oligodeoxyribonucleotides incorporating N-acyldeoxycytidine derivatives

Article abstract of DOI:10.1002/1099-0690(200112)2001:24<4583::AID-EJOC4583>3.0.CO;2-R

Oligodeoxynucleotides [11a-i: d(T₆XT₆)] incorporating various N-acyldeoxycytidine derivatives (X) have been synthesized through use of a new DBU-labile 4,5-dichlorophthaloyl linker on polymer supports. The hybridization capabilities of these oligonucleotides with the complementary d(A₆GA₆) were examined with the aid of Tm experiments. It turned out that the Tm values of DNA duplexes decreased significantly with an increase in the number of the methylene groups in the aliphatic acyl group introduced into one strand of the DNA duplex. Ab initio MO calculations and ¹H NMR analysis suggested that the acyl groups in the tested derivatives were oriented in a manner that made the formation of conventional Watson-Crick-type (W-C-type) base pairs with the guanine residue possible, with the help of an intramolecular hydrogen bond between the amide carbonyl oxygen atom and the 5-vinyl proton of the N-acylcytosine residue. Moreover, all the aromatic N-acyl groups were found markedly to decrease the thermal stability of the DNA duplexes. Ab initio calculations suggested that the base pairs formed between 4-N-acyl-1-methylcytosine derivatives and 9-methylguanine have wholly planar structures as their most stable geometries. Detailed studies of the hydrogen-bond energy of the modified base pairs also suggested that the electronic repulsion between the heteroatom of the acyl group of X and the guanine residue resulted in significant destabilization of the X-G base pair and that the hydrogen-bond network structure of water molecules around the major groove of the DNA duplex is a key factor in the stabilization of DNA duplexes. Wiley-VCH Verlag GmbH, 2001.

Full text of DOI:10.1002/1099-0690(200112)2001:24<4583::AID-EJOC4583>3.0.CO;2-R

FULL PAPER
Synthesis and Hybridization Ability of Oligodeoxyribonucleotides
Incorporating N-Acyldeoxycytidine Derivatives
Takeshi Wada,[a] Akio Kobori,[a] Shun-ichi Kawahara,[a] and Mitsuo Sekine*[a]
Keywords: Ab initio calculations / N-Acyldeoxycytidine / Bioorganic chemistry / Hydrogen bonds / Nucleotides
Oligodeoxynucleotides [11a−i: d(T6XT6)] incorporating vari-
ous N-acyldeoxycytidine derivatives (X) have been synthe-
sized through use of a new DBU-labile 4,5-dichlorophthaloyl
linker on polymer supports. The hybridization capabilities of
these oligonucleotides with the complementary d(A6GA6)
were examined with the aid of Tm experiments. It turned out
hydrogen bond between the amide carbonyl oxygen atom
and the 5-vinyl proton of the N-acylcytosine residue. More-
over, all the aromatic N-acyl groups were found markedly to
decrease the thermal stability of the DNA duplexes. Ab initio
calculations suggested that the base pairs formed between
4-N-acyl-1-methylcytosine derivatives and 9-methylguanine
that the Tm values of DNA duplexes decreased significantly have wholly planar structures as their most stable geomet-
with an increase in the number of the methylene groups in ries. Detailed studies of the hydrogen-bond energy of the
the aliphatic acyl group introduced into one strand of the modified base pairs also suggested that the electronic repul-
DNA duplex. Ab initio MO calculations and 1H NMR analysis
suggested that the acyl groups in the tested derivatives were
sion between the heteroatom of the acyl group of X and the
guanine residue resulted in significant destabilization of the
oriented in a manner that made the formation of conven- X-G base pair and that the hydrogen-bond network structure
tional Watson−Crick-type (W−C-type) base pairs with the
guanine residue possible, with the help of an intramolecular
of water molecules around the major groove of the DNA du-
plex is a key factor in the stabilization of DNA duplexes.
Introduction
Results and Discussion
Synthesis of Oligodeoxynucleotides Incorporating N-
Acylated Deoxycytidine Derivatives
4-N-Acetylcytidine and its 2Ј-O-methylated derivatives
are naturally occurring modified nucleosides, found in the
first letter of the anticodon loop and in the twelfth position
The key intermediates Ϫ 4-N-acyl-5Ј-O-(4,4Ј-dimethoxy-
trityl)deoxycytidine derivatives 3a؊c Ϫ required for incorp-
oration of these N-acyldeoxycytidine derivatives into
d(T6XT6) were obtained in high yields by treatment of de-
oxycytidine hydrochloride (1) with the corresponding acid
anhydride in the presence of triethylamine in DMF at 60
°C for 6 h,[13] followed by in situ treatment of the resulting
N-acylated products 2a؊c with 4,4Ј-dimethoxytrityl chlor-
ide in pyridine (Method A of Scheme 1).
Six 4-N-aroyldeoxycytidine derivatives 2d؊i, on the other
hand, were synthesized by trimethylsilylation followed by
N-acylation with acyl chlorides, as shown in Method B in
Scheme 1. These products could easily be extracted and
purified by silica gel column chromatography. Compounds
2d؊i were further converted into the 5Ј-O-DMTr derivat-
ives 3d؊i in the usual way. The H-phosphonate units 4a؊i
were synthesized by phosphitylation of 3a؊i with 2-chloro-
4H-1,3,2-benzodioxaphosphorin-4-one[14] followed by hy-
drolysis (Scheme 1).
of the D-stem of E. coli tRNAMet [1]
in thermophilic Ar-
,
chaebacteria tRNA,[2] and in various other tRNAs and
rRNAs.[3Ϫ10] From the well-known codonϪanticodon in-
teraction between mRNA and tRNA, this minor nucleoside
base (4-N-acetylcytosine) was expected to recognize only
the guanine base on mRNA.[11] With this background, we
studied whether oligodeoxyribonucleotides containing 4-N-
acetylcytosine bases were able, like their counterparts con-
taining N-unmodified cytosine bases, to hybridize with their
complementary DNA strands possessing guanine bases at
the opposite sites.[12] In our previous paper,[12] we reported
that the presence of 4-N-acetyl groups on cytosine bases in
oligodeoxynucleotides did not actually impair the duplex
stability relative to that of the natural type duplexes, whilst
in some cases the thermal stability of duplexes even in-
creased, to a degree of 0.7 °C per modification.
Here we report the synthesis and hybridization capabilit-
ies of oligodeoxyribonucleotides containing various N-acyl-
ated cytosine residues.
Oligodeoxynucleotides incorporating N-benzoyldeoxycy-
tosine units had been synthesized by Fraser et al.,[15] who
used a diisopropylsilanediyl linker for their solid-supported
synthesis. However, this method was not applicable to the
synthesis of d(T6XT6) 11a؊i in which X is an N-acyldeoxy-
cytidine 2a؊i; the isolated yield of the modified oligomer
11a, for example, was extremely poor. Our results appar-
[a]
Department of Life Science, Tokyo Institute of Technology,
Nagatsuta, Midoriku, Yokohama 226-8501, Japan
Fax: (internat.) ϩ 81-45/924-5772
E-mail: msekine@bio.titech.ac.jp
Supporting information for this article is available on the
WWW under http://www.eurjoc.com or from the author.
Eur. J. Org. Chem. 2001, 4583Ϫ4593
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/1224Ϫ4583 $ 17.50ϩ.50/0
4583

T. Wada, A. Kobori, S. Kawahara, M. Sekine
FULL PAPER
linker was used, 30 min were required for complete cleavage.
The thymidine-loaded HCP resin 8 was synthesized by a
two-step procedure. Firstly, treatment of 5Ј-O-(4,4Ј-dime-
thoxytrityl)thymidine (5) with 4,5-dichlorophthalic anhyd-
ride (6) in pyridine in the presence of 4-(dimethylamino)py-
ridine (DMAP) gave triethylammonium 5Ј-O-(4,4Ј-dime-
thoxytrityl)thymidine-3Ј-yl (4,5-dichloro)phthalate (7) in
96% yield. This compound was then condensed with the
amino group (35 µmol/g) of the HCP resin in CH2Cl2 in
the presence of DCC to give the desired resin after detrityl-
ation (8: 13.7 µmol/g).
In the solid-phase synthesis of d(T6XT6) 11a؊i with X ϭ
2a؊i, the chain elongation was carried out by the modified
H-phosphonate approach, with the use of 2-(benzotriazol-
1-yloxy)-1,1-dimethyl-2-(pyrrolidin-1-yl)-1,3,2-diazaphos-
pholidinium hexafluorophosphate (BOMP) as a powerful
condensing agent.[18] For oxidation of the PϪH bond of
the resulting H-phosphonate esters, 2-(phenylsulfonyl)-3-(3-
nitrophenyl)oxaziridine (PNO)[19] was used as an effective
oxidizing agent in the presence of N,O-bis(trimethylsilyl)-
acetamide (BSA), as shown in Scheme 3. At the final stage in
the solid-phase synthesis, N-acylated oligodeoxynucleotides
were released by treatment with DBU and purified by an-
ion-exchange HPLC to give sufficient amounts of oligo-
deoxyribonucleotides 11a؊i, as summarized in Table 1.
The structures of these modified oligodeoxynucleotides
were confirmed by MALDI TOF mass spectrometry. A typ-
ical example of the HPLC profile of the crude material
Scheme 1
ently indicated that serious side reactions occurred during
the synthesis,[16] and the original paper[15] did not actually
give any details of the yields of the oligodeoxynucleotides
incorporating 4-N-benzoyldeoxycytidines. To overcome this
problem, we therefore developed a new linker: 4,5-dichlor-
ophthaloyl.[12] This linker was stable during the chain
elongation and, when a highly cross-linked polystyrene
(HCP) resin[17] was employed for the solid-phase synthesis,
it could be removed within 5 min by treatment of the poly-
mer support 9 with 10% DBU in CH3CN at room temper-
ature, through an intramolecular cyclization with formation
of a phthalimide derivative 12 to release the oligodeoxynu-
cleotides (Scheme 2). When an unsubstituted phthaloyl Scheme 3
Scheme 2
4584
Eur. J. Org. Chem. 2001, 4583Ϫ4593

Oligodeoxyribonucleotides Incorporating N-Acyldeoxycytidine Derivatives
FULL PAPER
It was found that introduction of a benzoyl group into
the cytosine residue of d(A6GA6)/d(T6CT6) destabilized the
DNA duplex considerably, with a substantial decrease in
Tm, of 6.6 °C, being observed. The duplex between
d(A6GA6) and d(T6CfrT6) (11e), containing a 4-N-furoyld-
eoxycytidine unit (2e: dCfr), was also destabilized to the
same degree (by 6.8 °C), as shown in Figure 2 (B) and
Table 1. The use of dCth 2f, with a thenoyl group, resulted
in a more significant decrease (8.8 °C) in the Tm value,
down to 34.8 °C.
The thermal stabilities of the duplexes between d(A6GA6)
and oligodeoxynucleotides 11g؊i, possessing 4-N-(2-pico-
lyl)deoxycytidine (dCpc) (2g), 4-N-nicotinoyldeoxycytidine
(dCnc) (2h), and 4-N-isonicotinoyldeoxycytidine (dCin) (2i)
substituents, were also examined. The presence of the 2-
picolyl group resulted in a sharp drop of 8.1 °C in the Tm
value, while the nicotinoyl (nc) group produced a more
moderate drop (5.8 °C), as shown in Figure 2 (C) and
Table 1. The isonicotinoyl (in) group affected the duplex
stability to a degree (7.3 °C) intermediate between those of
the 2-picolyl and the nicotinoyl (ni) groups. The position of
the nitrogen atom in the pyridine ring thus had a pro-
nounced effect on the duplex stability.
Table 1. Synthesis and hybridization capability of DNA 13mers
containing 4-N-acyl-2Ј-deoxycytidine derivatives
Sequence
Compd. Isolated
Hybridization
yield (%) Tm
∆Tm
d(TTTTTTCTTTTTT)
10
Ϫ
43.6
44.0
40.9
37.8
37.0
36.8
34.8
35.5
36.3
37.8
Ϫ
0.4
d(TTTTTTCacTTTTTT) 11a
d(TTTTTTCprTTTTTT) 11b
d(TTTTTTCbuTTTTTT) 11c
d(TTTTTTCbzTTTTTT) 11d
d(TTTTTTCfrTTTTTT) 11e
d(TTTTTTCthTTTTTT) 11f
d(TTTTTTCpcTTTTTT) 11g
d(TTTTTTCncTTTTTT) 11h
d(TTTTTTCinTTTTTT) 11i
23
30
32
27
30
20
39
27
19
Ϫ2.7
Ϫ5.8
Ϫ6.6
Ϫ6.8
Ϫ8.8
Ϫ8.1
Ϫ7.3
Ϫ5.8
Structural Requirements for Preservation of Hybridization
Ability in N-Acylated Cytosine Residues
Parthasarathy reported that the X-ray crystal structure of
4-N-acetylcytidine showed intramolecular hydrogen bond-
ing between the carbonyl oxygen atom and the 5-vinyl pro-
ton.[20,21] Actually, the signal of the 5-proton of this nucle-
oside exhibited a downfield shift of δ ഠ 1.21 in the 1H
1
NMR spectrum.[20] In our hands, the H NMR spectrum
of its deoxy counterpart 2a showed a lower magnetic field
shift (∆δ ഠ 1.5) of the 5-vinylic hydrogen atom to δ ഠ 7.2
from the chemical shift δ ϭ 5.70 of deoxycytidine, as sum-
marized in Table 2. It is reasonable to postulate that this
inherent orientation of the acetyl group on the cytosine res-
idue enabled oligodeoxynucleotides containing 2a to form
the usual WϪC-type base pairs between the 4-N-acetylcyto-
sine and guanine bases, so that they exhibited better hybrid-
ization properties than the unmodified oligodeoxynucleot-
ides, as reported previously.[12] It is likely that the 4-N-pro-
pionyldeoxycytidine (2b) and 4-N-butyryldeoxycytidine (2c)
Figure 1. Anion-exchange HPLC profile of crude 11f
(dT6CthT6) (11f) obtained after deprotection is shown in
Figure 1.
Hybridization Ability of Modified Oligodeoxynucleotides
The thermal stabilities of the duplexes formed between moieties in 11b and 11c, respectively, may give rise to sim-
the N-acylated oligodeoxynucleotides [11a؊i: d(T6XT6), ilar WϪC-type hydrogen bonds at the modified position, as
X ϭ N-acylated dC 2a؊i] and the DNA 13mer d(A6GA6) evidenced by the clear-cut low magnetic field shifts of the
were examined. These experiments were carried out at 5-vinyl protons of 2b and 2c, as shown in Table 2. It is also
pH ϭ 7.0 in 10 m phosphate buffer in the presence of 1 to be expected that the same would be true for other aro-
 NaCl.
matic N-acyldeoxynucleosides, since these nucleosides ex-
To our surprise, the duplexes incorporating 4-N-propion- hibit similar low-field shifts of the resonance signals of their
yldeoxycytidine (2b) or 4-N-butyryldeoxycytidine (2c) had 5-vinyl protons.
stabilities considerably lower than that of the duplex con-
The capability of the N-acetylcytosine residue to form the
taining 4-N-acetyldeoxycytidine (2a) (Figure 2, A, and WϪC-type base pair is also supported by the theoretical ab
Table 1). On addition of a methylene group to the acetyl initio MO calculation reported in the previous paper, which
group, the Tm value dropped by approximately 3 °C. Thus, showed that the most stable geometry-optimized structure
it turned out that introduction of a butyryl group into the of 4-N-acetyl-1-methylcytosine is that with an intramolecu-
4-N position in dC resulted in sharp decrease of up to 5.8 lar hydrogen bond between the amide carbonyl oxygen
°C in the Tm value of the duplex.
atom and the 5-vinylic proton.[12] This conformer was 3.27
Eur. J. Org. Chem. 2001, 4583Ϫ4593
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T. Wada, A. Kobori, S. Kawahara, M. Sekine
FULL PAPER
Table 2. The 31P NMR chemical shifts of the 5-vinylic protons in
4-N-acylated deoxycytidine derivatives ([D5]DMSO)
Compd.
δ value
Compd.
δ value
dC
2a
2b
2c
2d
5.70
7.18
7.21
7.21
7.36
2e
2f
2g
2h
2i
7.27
7.27
7.38
7.31
7.33
kcal/mol more stable than the second most stable con-
former. This great gap between the stabilities of these con-
formers strongly suggests that the N-acetyl-1-methylcyto-
sine exists virtually entirely in this form, as attested by the
NMR analysis of 2a.
Among the oligodeoxynucleotides 11a؊i incorporating
N-acyldeoxycytidines 2a؊i, only DNA oligomer 11a, con-
taining N-acetyldeoxycytidine (2a), showed a slight stabil-
ization effect on DNA duplex formation. Any lengthening
of the alkyl chain of the acetyl group resulted in a decrease
in the thermal stability of the corresponding DNA duplex.
A similar tendency towards decreasing Tm values has been
reported in a paper by Thuong et al., concerning the ther-
mal stabilities of DNA duplexes containing 4-N-alkyldeoxy-
cytidine units.[22] These worker observed that when the N-
alkyl chain on the exo amino group of the cytosine base
became longer, the thermal stability of DNA duplexes de-
creased.
Most Stable Geometry-Optimized Structures of 4-N-Acyl-
1-methylcytosine Derivatives and Base Pairs with 9-
Methylguanine Evaluated by Ab Initio MO Calculation
To interpret the results described above, we calculated the
most stable geometry-optimized structures of 4-N-acyl-1-
methylcytosine derivatives 13a؊i by ab initio MO calcula-
tion at the HF/6-31G* level. These results are shown in Fig-
ure 3 (for the results for 13a؊c, see Figure 11 in the Sup-
porting Information). The most stable geometry-optimized
structures of the modified base pairs formed between 13a؊i
(meCR where R ϭ acyl groups) and 9-methylguanine (14:
meG) were also calculated at the same level base set. These
results are shown in Figure 4. In the case of the aliphatic
acyl groups, the hydrogen-bond energies are similar (Ϫ26.12
to Ϫ26.90 kcal/mol) to each other, and so these aliphatic
acyl groups did not affect the WϪC-type hydrogen bonds
essentially. However, the thermal stabilities of the duplexes
formed between d(T6XT6) 11b and 11c (X ϭ 2b, 2c) and
d(A6GA6) decreased with increasing alkyl chain length in
the acyl group. From these results, it seems likely that the
aliphatic alkyl group disturbs the hydrogen-bond network
structure around the major groove of the DNA duplexes.
Williams reported that such a water bridge structure is
known to be essential for stabilization of DNA duplexes.[23]
Among the other six 4-N-aroyl-1-methylcytosine derivat-
ives, only those with furoyl, thenoyl, and picolyl groups can
maintain flat structures suitable for base paring, as shown
in Figure 3. Benzoyl, nicotinoyl, and isonicotinoyl groups
Figure 2. Melting temperature curves of DNA duplexes incorporat-
ing 4-N-acyldeoxycytidine: panel A: square: normal duplex be-
tween d(T6CT6) 10 and d(A6GA6); diamond: duplex between
d(T6CacT6) 11a and d(A6GA6); circle: duplex between d(T6CprT6)
11b and d(A6GA6); triangle, duplex between d(T6CbuT6) 11c and
d(A6GA6); panel B: square: normal duplex between d(T6CT6) 10
and d(A6GA6); diamond: duplex between d(T6CbzT6) 11d and
d(A6GA6); circle: duplex between d(T6CfrT6) 11e and d(A6GA6);
triangle, duplex between d(T6CthT6) 11f and d(A6GA6); panel C:
square: normal duplex between d(T6CT6) 10 and d(A6GA6); dia-
mond: duplex between d(T6CpcT6) 11g and d(A6GA6); circle: du-
plex between d(T6CncT6) 11h and d(A6GA6); triangle: duplex be-
tween d(T6CinT6) 11i and d(A6GA6)
4586
Eur. J. Org. Chem. 2001, 4583Ϫ4593

Oligodeoxyribonucleotides Incorporating N-Acyldeoxycytidine Derivatives
FULL PAPER
Figure 3. Most stable geometry-optimized structures of 4-N-aroyl-1-methylcytosines determined by ab initio MO calculation at the HF/
6-31G* level of theory
Figure 4. Most stable geometry-optimized structures of modified base pairs formed between 4-N-acyl-1-methylcytosines and 9-methyl-
guanine determined by ab initio MO calculation at the HF/6-31G* level of theory, the distances between the guanine 6-oxygen atom and
one of the aromatic protons of the acyl group capable of hydrogen bonding, and the Mulliken charges of the protons (H*, H**, and
H***) marked by asterisks; the values given are the hydrogen-bond energies ∆E, defined as the difference between the energy of the most
stable energy-optimized base pair of the 4-N-acyl-1-methylcytosine derivatives 13aϪi and 9-methylguanine (14) and the total energies
of the most stable energy-optimized structures of the two components
Eur. J. Org. Chem. 2001, 4583Ϫ4593
4587

T. Wada, A. Kobori, S. Kawahara, M. Sekine
FULL PAPER
induced twisted torsion angles around X*ϪCϪCϪO and atom contributes to stabilization of the hydrogen-bond net-
OϪCϪN4ϪC4. It is interesting that the most stable geo- work around the major groove, relative to that seen with a
metry-optimized structures of the 4-N-aroyl-1-methylcyto- benzoyl substituent. In contrast, the worst result was ob-
sine derivatives are not always those used in the base-pair- tained in the case of the picolyl group. This may be ex-
ing with 9-methylguanine (14). As demonstrated in the case plained by the fact that the 2-nitrogen atom of the picolyl
of the base pairs meCfr-meG and meCnc-meG, the orienta- group cannot contribute to the outer hydrogen-bonding
tions of the aromatic rings of the furoyl and nicotinoyl network in the major groove.
groups were different, with complete rotation around X*
To understand these results, we carried out molecular
ϪCϪCϪO (Figure 3). In any event, the calculated struc- mechanics computer simulations of these DNA duplexes,
tures of the base pairs were wholly planar, as shown in Fig- using AMBER* with the GB/SA water solvation model.[28]
ure 4. Except for the meCnc-meG base pair, the other base These simulations suggested that the substituents extending
pairs showed relatively low hydrogen-bond energies com- from the exo-amino group of the cytosine ring are posi-
pared with the C-G base pair. This tendency is basically in tioned in the direction of the major groove in such a man-
agreement with the low Tm values experimentally observed ner that the hydration structure of water molecules[22] is de-
in several DNA duplexes containing 4-N-aroyldeoxycytid- stroyed (see Figure 10 in Supporting Information). In this
ine derivatives. In particular, the meCpc-meG base pair, regard, the (smallest) acetyl group is superior to the other
computed to have the lowest hydrogen-bond energy among acyl groups. This simulation model also implies that a
the calculated base pairs, did actually exhibit the lowest Tm wholly flat structure in the 4-N-aroylcytosine residues does
value, as shown in Table 1. A serious factor in this case not contribute to stabilization of the duplex, since the acyl
appears to be electronic repulsion between the nitrogen residues only extend into the major groove so that the base
atom of the pyridine ring and the 6-carbonyl oxygen atom stacking interaction is impossible.
of the guanine residue. This interaction gives rise to strained
base pairs. In the meCfr-meG base pair, the orientation of
Conclusion
the oxygen atom of the furoyl ring was the reverse of that
seen in the monomeric meCfr (13e in Figure 3), but in the
base pair a new electronic repulsion had arisen between the
oxygen and the carbonyl oxygen atom, so that the hydro-
gen-bond energy was lowered to the second worst value. In
meCbz, meCnc, and meCin, on the other hand, one of the
aromatic protons can approach the guanine carbonyl oxy-
gen atom within hydrogen-bonding distances, so that this
weak interaction can compensate for the loss of energy due
to the conformational change from the twist form of these
bases to the flat form upon base-paring. The distances be-
From these results, it appears likely that the preservation
of the hydration structure around the major groove is cru-
cial for the design of N-modified deoxycytidines that will
maintain the thermal stability. It may also be concluded
that planarity of the N-aroylcytosine moiety does not essen-
tially contribute to stabilization of DNA duplexes.
Since the major groove provides adequate space for mo-
dification of the cytosine amino group, some functional
group such as fluorescence and biotin groups can be intro-
duced through the amide linkage, but a certain destabiliza-
tion effect should be taken into account when such strat-
egies are designed in future.
This N-acyl mode for modification of cytosine may pro-
vide an alternative to the conventional use of the 5-position
of the cytosine base for substitution or introduction of
functional groups for gene diagnosis or a variety of physico-
chemical studies. In applications of work of this kind, it
should be taken into account that substituents of the acyl
type should be designed not to interfere with this hydration
structure and to avoid electronic repulsion between the
guanine residue and the substituents. Further studies in this
direction are underway.
tween the aromatic proton and the carbonyl oxygen atom
bz
˚
˚
˚
are calculated as 2.05 A, 2.02 A, and 2.01 A for meC -
meG, meCnc-meG, meCin-meG, respectively, as shown in
Figure 4 (E, H, I). Actually, electron-density analysis of the
base pairs indicated that these protons marked as H** in
Figure 4 are positively charged, with Mulliken charges of
ϩ0.32, ϩ0.34, and ϩ0.33 for meCbz-meG, meCnc-meG, and
meCin-meG, respectively. It is apparent that these protons
are more positively charged than the reverse side protons
marked as H***, which uniformly have Mulliken charges
of 0.25. On the other hand, the protons (H*) of the amide
groups each have a Mulliken charge of 0.52 each in these
three base pairs. These results strongly suggest that the de-
crease in the Tm values in the case of the aromatic acyl
groups might be attributable to poor levels of stabilization
through hydrogen-bonding energies.
Experimental Section
In the Tm experiments, it was also observed that the loca-
tion of the nitrogen atom in the pyridinecarbonyl group is
important for stabilization of X-G base pairs. Among the
duplexes made up of d(T6XT6) 11g؊i and d(A6GA6), the
one containing the isonicotinoyl group is more stable than
the others, more stable even (as far as the Tm values are
concerned) than a duplex incorporating the benzoyl group,
1
General Remarks: H, 13C, and 31P NMR spectra were recorded at
270, 68, and 109 MHz, respectively. The chemical shifts were meas-
ured from tetramethylsilane or DSS for H NMR spectra, CDCl3
1
(δ ϭ 77) or DSS (δ ϭ 0) for 13C NMR spectra, and 85% phosphoric
acid (δ ϭ 0) for 31P NMR spectra. UV spectra were recorded with
a U-2000 spectrometer. MALDI TOF mass spectra were taken with
a MarinerTM spectrometer (PerSeptive Biosystems). Column chro-
as shown in Table 1. This result implies that the 4-nitrogen matography was performed with C-200 silica gel purchased from
4588 Eur. J. Org. Chem. 2001, 4583Ϫ4593

Oligodeoxyribonucleotides Incorporating N-Acyldeoxycytidine Derivatives
Wako Co. Ltd., and an aquarium minipump could conveniently be ine. The resulting support 8 was filtered, washed with pyridine, and
FULL PAPER
used to attain sufficient pressure for rapid chromatographic separa-
tion. Anion-exchange HPLC was done on a Gen-PakTM FAX col-
umn (Waters, 4.6 ϫ 100 mm) with an LC module 1 with a Waters
M-741 data module and a Waters column heater, with a 10Ϫ77%
linear gradient of 25 m phosphate, 1  sodium chloride buffer
(pH ϭ 6.0) in 25 m phosphate buffer (pH ϭ 6.0) at 50 °C, at a
flow rate of 1.0 mL/min for 30 min. Pyridine was distilled twice
from p-toluenesulfonyl chloride and from calcium hydride and then
dried. The degree of thymidine loading was estimated by DMTr
cation assay as 13.7 µmol/g.
Evaluation of the Stability of the DMT-T-3Ј-O-(4,5-Dichloro)-
phthaloyl Support Towards DBU: The DMT-T-3Ј-O-(4,5-dichloro)-
phthaloyl support (9 mg) was suspended in 10% DBU in CH3CN.
At intervals, the support was filtered and washed with pyridine and
CH3CN. The support was treated with 1% TFA in CH2Cl2, and the
TFA solution was filtered. The filtrate was concentrated to dryness,
and the residue was dissolved in perchloric acid/ethanol (3:2, v/v)
to estimate the amount of released DMTrT by measurement of the
UV absorbance at 498 nm (ε ϭ 71700).
˚
stored over 4-A molecular sieves. Elemental analyses were per-
formed by the Microanalytical Laboratory, Tokyo Institute of
Technology at Nagatsuta.
Computational Methods: All ab initio molecular orbital calculations
were carried out by using the Gaussian 98 program with an Ori-
gin2000/256 supercomputer. The results were analyzed using Gauss
View (version 2.0, Gaussian, Inc.) software with a Silicon Graphics
Inc. IMPACT O2 workstation. Geometry optimizations and energy
calculations were carried out at the HF/6-31G* level. The molecu-
lar mechanics simulations of d(T4C*T4)/d(A4GA4) were carried out
under the following conditions. The initial structures were con-
structed by use of the structure implemented in Macro Model (ver-
sion 6.0). The acyl moieties on cytidine derivatives were fixed in
the appropriate conformation by application of the geometry ob-
tained by ab initio calculations. The force field used was the AM-
BER* one, with modification of the distance-dependent dielectric
constant to the constant dielectric. The effect of the solvent was
included by use of the GB/SA solvent model implemented in the
software.
Typical Procedure for Solid-Phase Synthesis: Each chain-elongation
cycle consisted of detritylation [1% TFA in CH2Cl2 (1 mL), 15 s],
washing (CH2Cl2, 5 mL), coupling [0.05  H-phosphonate units,
0.2  BOMP in pyridine (200 mL); 1.5 min], and washing (pyridine,
5 mL). Generally, the average yield per cycle was estimated as
97Ϫ99% by the DMTr cation assay. After chain elongation, the
DMTr group was removed by treatment with 1% TFA in CH2Cl2
(1 mL) for 15 s, and the resin was washed with CH2Cl2. The H-
phosphonate oligomer on the HCP resin was oxidized by treatment
with a mixture of PNO (0.2 ) and BSA (0.5 ) in CH3CN (400
µL) for 10 min. After having been washed with CH2Cl2, the oligo-
mer was released from the HCP resin by treatment with 10% DBU
in CH3CN (500 µL) for 5 min. The HCP resin was removed by
filtration and washed with CH3CN. The filtrate was neutralized
with 50% CH3COOH in CH3CN (1 mL), desalted on Dowex 50 W
(NH3 form), and purified by anion-exchange HPLC. Oligomers
were analyzed by nuclease P1 digestion and MALDI TOF mass
spectrometry.
Triethylammonium 5Ј-O-(4,4Ј-Dimethoxytrityl)thymidine-3Ј-yl-(4,5-
dichloro)phthalate (7): 5Ј-O-(4,4Ј-Dimethoxytrityl)thymidine (5:
1.09 g, 2 mmol) was dried by repeated coevaporation with dry pyr-
idine and finally dissolved in dry pyridine (20 mL). 4,5-Dichloroph-
thalic anhydride (6: 1.30 g, 6 mmol) and 4-(dimethylamino)pyridine
(12.2 mg, 0.1 mmol) were added to this solution, and the mixture
was stirred at room temperature for 6 h. The mixture was concen-
trated to half its original volume, diluted with CHCl3, and washed
three times with 5% NaHCO3 (aq), and the aqueous layer was
back-extracted with CHCl3. The organic layer and washings were
combined, dried with Na2SO4, filtered, and concentrated to dry-
ness under reduced pressure. The residue was subjected on a silica
gel column. Chromatography was performed with CHCl3 con-
taining 1% triethylamine, applying a gradient of methanol (1Ϫ2%),
to give 7 (1.66 g, 96%) as a colorless foam. 1H NMR (CDCl3): δ ϭ
Tm Measurement: An appropriate oligonucleotide (2.0 µmol) and
its complementary DNA 13mer d(A6GA6) (2.0 µmol) were dis-
solved in a buffer consisting of NaCl (1.0 ⁾, sodium phosphate
(10 m⁾, and EDTA (0.1 m⁾, adjusted to pH ϭ 7.0. The solution
containing the oligonucleotides was kept at 60 °C for 10 min for
complete dissociation of the duplex into single strands, cooled at
the rate of Ϫ1.0 °C/min, and kept at 0 °C for 10 min. After that,
the melting temperatures (Tm) were determined at 260 nm, using a
Hitachi U-2000 at the rate of 1.0 °C/min.
5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-propionyl-2Ј-deoxycytidine
(3b):
Deoxycytidine hydrochloride (2.68 g, 10 mmol) was dried by re-
peated coevaporation with dry N,N-dimethylformamide and was
finally dissolved in dry N,N-dimethylformamide (100 mL). Triethyl-
amine (1.39 mL, 10 mmol) was added to the solution, and the mix-
ture was stirred at room temperature for 30 min. Propionic anhyd-
ride (1.42 mL, 11 mmol) was added to the mixture. After the mix-
ture had been stirred at 60 °C for 6 h, the solvent was removed
under reduced pressure. The residue was dried by repeated coevap-
oration with dry pyridine and finally dissolved in dry pyridine
(100 mL). 4,4Ј-Dimethoxytrityl chloride (4.07 g, 12 mmol) was ad-
ded to the solution, and the mixture was stirred at room temper-
ature for 12 h. The mixture was concentrated to half its original
volume, diluted with CHCl3, and washed three times with 5%
NaHCO3 (aq), and the aqueous layer was back-extracted with
3
1.28 (t, 9 H, J ϭ 7.3 Hz), 1.34 (s, 3 H), 2.47 (m, 1 H), 2.74 (m, 1
3
H), 3.08 (q, 6 H, J ϭ 7.2 Hz), 3.52 (m, 2 H), 3.79 (s, 6 H), 4.44
3
3
(m, 1 H), 5.66 (m, 1 H), 6.43 (dd, 1 H, J1Ј,2Ј ϭ 8.9, J1Ј,2ЈЈ
ϭ
3
5.6 Hz), 6.85 (d, J ϭ 8.9 Hz, 4 H), 7.12Ϫ7.41 (m, 9 H), 7.57 (s, 1
H, 6-H), 7.66 (s, 1 H), 7.93 (s, 1 H). 13C NMR (CDCl3): δ ϭ 8.5,
11.5, 37.5, 45.0, 55.2 63.8, 77.2, 83.3, 84.4, 87.0, 111.5, 113.2,
127.2, 128.0, 128.1, 129.2, 130.1, 130.1, 131.2, 132.5, 134.4,
135.2, 135.6, 138.7, 144.2, 150.3, 158.7, 163.7, 167.6, 170.2.
C45H49Cl2N3O10·H2O (880.8): C 61.36, H 5.84, Cl 8.05, N 4.77;
found C 61.02, H 5.98, Cl 8.53, N 5.09.
Preparation of Thymidine-Loaded Highly Cross-Linked Polystyrene
Support 8: Triethylammonium 5Ј-O-(4,4Ј-dimethoxytrityl)thymid-
ine-3Ј-yl-(4,5-dichloro)phthalate (7: 70 mg, 87.4 mmol) was dried CHCl3. The organic layer and the washings were combined and
by repeated coevaporation with dry pyridine and finally dissolved dried with Na2SO4, filtered, and concentrated to dryness under re-
in dry CH2Cl2 (2 mL). Dicyclohexylcarbodiimide (90 mg, duced pressure. The residue was subject on a silica gel column.
437.5 mmol) and HCP (500 mg 17.5 µmol) were added to the solu- Chromatography was performed with CHCl3 containing 1% pyrid-
tion. The mixture was kept at room temperature for 3 h. The solid ine, applying a gradient of methanol (1Ϫ2%) to give 3b (5.19 g,
support was filtered and washed with pyridine. Unreacted amino 89%) as a colorless foam. 1H NMR (CDCl3): δ ϭ 1.18 (t, 3 H,
groups were blocked by treatment with 10% (CH3CO)2O in pyrid-
3J ϭ 7.6), 2.24 (m, 1 H, 2Ј-H), 2.24 (q, 3J ϭ 7.6 Hz, 2 H), 2.76 (m,
Eur. J. Org. Chem. 2001, 4583Ϫ4593
4589

T. Wada, A. Kobori, S. Kawahara, M. Sekine
FULL PAPER
1 H), 3.45 (m, 2 H), 3.81 (s, 6 H), 4.14 (m, 1 H), 4.50 (m, 1 H), maintained at room temperature for 2 h. The mixture was then co-
3
3
6.29 (dd, 1 H, J1Ј,2Ј ϭ 5.9, J1Ј,2ЈЈ ϭ 5.9 Hz), 6.85 (d, 3J ϭ 7.9 Hz, oled in an ice bath and water (3 mL) was added. After 5 min, aque-
4 H), 7.20Ϫ7.42 (m, 10 H, 5-H), 8.23 (d, 3J ϭ 7.6 Hz, 1 H). 13C ous ammonia (29%, 10 mL) was added and the mixture was stirred
NMR (CDCl3): δ ϭ 8.5, 30.2, 41.9, 55.0, 62.7, 70.8, 77.2, 86.5, at room temperature for 15 min. The reaction mixture was then
86.6, 87.3, 96.6, 113.1, 126.9, 127.8, 127.9, 129.8, 129.9, 135.1,
135.3, 144.0, 144.4, 155.5, 158.4, 162.5, 174.2. MS: m/z calcd. for
(C33H35N3O7 ϩ H) [M ϩ Hϩ] 586.2553, found 586.2553.
concentrated to near dryness and the residue was dissolved in aque-
ous NaHCO3 (5%, 45 mL) and extracted five times with CHCl3/
pyridine (1:1, v/v). The organic layers were combined and dried
with Na2SO4, filtered, and concentrated to dryness under reduced
pressure. The residue was loaded onto a silica gel column. Chroma-
tography was performed with CHCl3 containing pyridine, applying
a gradient of methanol (10Ϫ15%) to give 2g (552 mg, 81%) as
4-N-Butyryl-5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-deoxycytidine (3c):
A
procedure similar to that described above was performed using de-
oxycytidine hydrochloride (2.68 g, 10.0 mmol) and butyric anhyd-
ride (1.80 mL, 11 mmol), to give 3c (5.02 g, 84%) as a colorless
1
foam. H NMR ([D6]DMSO): δ ϭ 2.05 (m, 1 H), 2.33 (m, 1 H),
1
3
3
foam. H NMR (CDCl3): δ ϭ 0.87 (t, J ϭ 7.3 Hz, 3 H), 1.58 (m,
3.60 (m, 2 H), 3.88 (m, 1 H), 4.23 (m, 1 H), 5.08 (t, J ϭ 5.0 Hz,
3
3
1 H), 5.28 (d, 3J ϭ 4.0 Hz, 1 H), 6.11 (dd, 1 H, J1Ј,2Ј ϭ 6.3,
2 H, N-butyryl), 2.14 (m, 1 H), 2.28 (t, 2 H, J ϭ 7.3), 2.76 (m, 1
3J1Ј,2ЈЈ ϭ 6.3 Hz), 7.38 (d, 3J ϭ 7.6 Hz, 1 H), 7.74 (m, 1 H),
8.07Ϫ8.19 (m, 2 H), 8.47 (d, 3J ϭ 7.3 Hz, 1 H) 8.74 (d, 3J ϭ 0.5 Hz,
1 H). 13C NMR ([D6]DMSO): δ ϭ 41.2, 61.1, 70.1, 86.7, 88.2,
95.0, 122.9, 128.4, 138.8, 146.1, 147.9, 149.1, 154.5, 161.2, 163.4.
C15H16N4O5 (332.3): C 54.21, H 4.85, N 16.86; found C 53.89, H
4.68, N 16.81.
H), 3.35 (m, 2 H), 3.71 (s, 6 H), 4.10 (m, 1 H), 4.43 (m, 1 H), 6.23
(dd, 1 H, J1Ј,2Ј ϭ 5.6, J1Ј,2ЈЈ ϭ 5.6 Hz), 6.77 (d, 3J ϭ 8.6 Hz, 4
H), 7.14Ϫ7.34 (m, 10 H, 5-H), 8.15 (d, 3J ϭ 7.3 Hz, 1 H). 13C
NMR (CDCl3): δ ϭ 13.4, 18.1, 39.1, 42.0, 55.1, 62.8, 70.8, 77.2,
86.5, 86.7, 87.4, 96.6, 113.2, 127.0, 127.9, 128.0, 129.9, 129.9, 135.3,
135.4, 144.1, 144.5, 155.5, 158.5, 162.4, 173.4. C34H37N3O7 (599.7):
calcd. C 68.10, H 6.22, N 7.01; found C 68.10, H 6.38, N 6.81.
3
3
4-N-Nicotinoyl-2Ј-deoxycytidine (2h): Deoxycytidine hydrochloride
(1: 804 mg, 3 mmol) was dried by repeated coevaporation with dry
pyridine and finally suspended in dry pyridine (25 mL). Chlorotri-
methylsilane (1.90 mL, 15.0 mmol) was added to the solution. After
4-N-Furoyl-2Ј-deoxycytidine (2e): Deoxycytidine hydrochloride
(804 mg, 3 mmol) was dried by repeated coevaporation with dry
pyridine and finally suspended in dry pyridine (25 mL). Chlorotri-
methylsilane (1.90 mL, 15.0 mmol) was added to the solution. After the solution had been stirred for 15 min, nicotinoyl chloride hydro-
the solution had been stirred for 15 min, 2-furoyl chloride
(1.48 mL, 15 mmol) was added, and the reaction was maintained
chloride (2.67 g, 15 mmol) was added, and the reaction mixture
was maintained at room temperature for 2 h. The mixture was then
at room temperature for 2 h. The mixture was then cooled in an cooled in an ice bath, and water (3 mL) was added. After 5 min,
ice bath, and water (3 mL) was added. After 5 min, aqueous ammo- aqueous ammonia (29%, 10 mL) was added and the mixture was
nia (29%, 10 mL) was added, and the mixture was stirred at room stirred at room temperature for 15 min. The reaction mixture was
temperature for 15 min. The reaction mixture was then concen- then concentrated to dryness, and the residue was dissolved in
trated to dryness, and the residue was dissolved in water (45 mL).
water (45 mL). The solution was washed with a 15-mL portion of
The solution was washed with a 15-mL portion of ethyl acetate. ethyl acetate. Cooling of the solution and filtration gave 4-N-nicoti-
Crystallization began immediately after separation of the layers.
noyl-2Ј-deoxycytidine 2h (684 mg, 71%). M.p. 161Ϫ163 °C. 1H
Cooling of the solution gave 4-N-furoyl-2Ј-deoxycytidine (684 mg,
NMR ([D6]DMSO): δ ϭ 2.05 (m, 1 H), 2.31 (m, 1 H), 3.61 (m, 2
3
71%). M.p. 153Ϫ155 °C. 1H NMR ([D6]DMSO): δ ϭ 2.03 (m, 1 H), 3.87 (m, 1 H), 4.23 (m, 1 H), 5.07 (t, J ϭ 5.3 Hz, 1 H), 5.28
H), 2.29 (m, 1 H), 3.59 (m, 2 H), 3.85 (m, 1 H), 4.14 (m, 1 H), 5.05 (d, 3J ϭ 4.0 Hz, 1 H), 6.13 (dd, 1 H, 3J1Ј,2Ј ϭ 6.3, 3J1Ј,2ЈЈ ϭ 6.3 Hz),
3
3
3
3
(m, 1 H), 5.26 (m, 1 H), 6.11 (dd, 1 H, J1Ј,2Ј ϭ 6.3, J1Ј,2ЈЈ
ϭ
7.31 (d, 1 H, J ϭ 7.3), 7.54 (m, 1 H), 8.31 (d, J ϭ 7.9 Hz, 1 H),
3
3
3
3
6.3 Hz), 6.70 (m, 1 H), 7.27 (d, J ϭ 7.3 Hz, 1 H), 7.71 (m, 1 H), 8.41(d, J ϭ 7.6 Hz, 1 H), 8.76 (d, J ϭ 4.6 Hz, 1 H), 9.09(t, J ϭ
7.98 (m, 1 H), 8.36 (d, 3J ϭ 7.3 Hz, 1 H). 13C NMR ([D6]DMSO): 1.0 Hz, 1 H). 13C NMR ([D6]DMSO): δ ϭ 41.1, 61.1, 70.1, 86.5,
δ ϭ 41.1, 61.1, 70.1, 86.4, 88.1, 96.1, 112.5, 117.4, 145.2, 146.3, 88.2, 96.3, 123.6, 129.4, 136.4, 145.3, 149.6, 153.1, 154.4, 162.9,
147.8, 154.4, 157.7, 162.7. C14H15N3O6·1/2H2O (330.3): C 50.91, H
166.7. C15H16N4O5·H2O (350.3): C 51.43, H 5.18, N 15.99; found
4.88, N 12.72; found C 50.62, H 4.66, N 12.70.
C 51.18, H 5.09, N 16.38.
4-N-Isonicotinoyl-2Ј-deoxycytidine (2i): Deoxycytidine hydro-
chloride (1: 804 mg, 3 mmol) was dried by repeated coevaporation
with dry pyridine and finally suspended in dry pyridine (25 mL).
Chlorotrimethylsilane (1.90 mL, 15.0 mmol) was added to the solu-
tion. After the solution had been stirred for 15 min, isonicotinoyl
chloride hydrochloride (2.67 g, 15 mmol) was added, and the reac-
tion mixture was maintained at room temperature for 2 h. The mix-
ture was then cooled in an ice bath, and water (3 mL) was added.
After 5 min, aqueous ammonia (29%, 10 mL) was added, and the
mixture was stirred at room temperature for 15 min. The reaction
mixture was then concentrated to dryness, and the residue was dis-
solved in water (45 mL). The solution was washed with a 15-mL
portion of ethyl acetate. Crystallization began immediately after
separation of the layers. Cooling of the solution gave 4-N-nicoti-
4-N-Thenoyl-2Ј-deoxycytidine (2f): A procedure similar to that de-
scribed above was performed with deoxycytidine hydrochloride
(804 mg, 3 mmol) and 2-thenoyl chloride (1.59 mL, 15 mmol), to
give 2e (556 mg, 55%). M.p. 205Ϫ208 °C. H NMR ([D6]DMSO):
δ ϭ 2.04 (m, 1 H), 2.30 (m, 1 H), 3.59 (m, 2 H), 3.86 (m, 1 H),
1
4.22 (m, 1 H), 5.07 (m, 1 H), 5.27 (m, 1 H), 6.12 (dd, 1 H, J1Ј,2Ј
ϭ
6.3, J1Ј,2ЈЈ ϭ 6.3 Hz), 7.21 (m, 1 H), 7.27 (d, 3J ϭ 7.3 Hz, 1 H),
7.96 (m, 1 H), 8.25 (m, 1 H), 8.36 (d, 3J ϭ 7.3 Hz, 1 H). 13C NMR
([D6]DMSO): δ ϭ 41.1, 61.1, 70.1, 86.4, 88.1, 96.2, 128.8, 131.8,
134.4, 138.8, 145.1, 154.3, 162.1, 162.8. C14H15N3O5S·1/2H2O
(346.4): C 48.55, H 4.66, N 12.13, S 9.26; found C 48.81, H 4.36,
N 12.53, S 9.09.
3
4-N-Picolyl-2Ј-deoxycytidine (2g): Deoxycytidine hydrochloride (1:
804 mg, 3 mmol) was dried by repeated coevaporation with dry noyl-2Ј-deoxycytidine 2i (684 mg, 71%). M.p. 198Ϫ200 °C. 1H
pyridine and finally suspended in dry pyridine (25 mL). Chlorotri-
methylsilane (1.90 mL, 15.0 mmol) was added to the solution. After H), 3.87 (m, 1 H), 4.22 (m, 1 H), 5.08 (t, J ϭ 5.3 Hz, 1 H), 5.28
the solution had been stirred for 15 min, 2-picolyl chloride hydro-
(d, 3J ϭ 4.3 Hz, 1 H), 6.12 (dd, 1 H, 3J1Ј,2Ј ϭ 6.3, 3J1Ј,2ЈЈ ϭ 6.3 Hz),
chloride (2.67 g, 15 mmol) was added and the reaction mixture was 7.33 (d, 3J ϭ 7.3 Hz, 1 H), 7.87 (d, 3J ϭ 5.3 Hz, 2 H), 8.42 (d, 3J ϭ
NMR ([D6]DMSO): δ ϭ 2.06 (m, 1 H), 2.28 (m, 1 H), 3.60 (m, 2
3
4590
Eur. J. Org. Chem. 2001, 4583Ϫ4593

Oligodeoxyribonucleotides Incorporating N-Acyldeoxycytidine Derivatives
FULL PAPER
3
7.3 Hz, 1 H) 8.76 (d, J ϭ 5.6 Hz, 2 H). 13C NMR ([D6]DMSO): (880 mg, 92%) as a colorless foam. 1H NMR (CDCl3): δ ϭ 2.30
δ ϭ 41.1, 61.1, 70.1, 86.5, 88.2, 96.3, 122.2, 140.6, 145.5, 150.5, (m, 1 H), 2.76 (m, 1 H), 3.46 (m, 2 H), 3.79 (s, 6 H), 4.18 (m, 1
3
3
154.4, 162.8, 166.5. C15H16N4O5 (332.3): C 54.21, H 4.85, N 16.86;
found C 54.22, H 4.80, N 17.03.
H), 4.56 (m, 1 H), 6.31 (dd, 1 H, J1Ј,2Ј ϭ 5.6, J1Ј,2ЈЈ ϭ 5.6 Hz),
6.85 (d, 3J ϭ 7.9 Hz, 4 H), 7.10Ϫ7.68 (m, 13 H, 5-H), 8.27 (d, 3J ϭ
7.6 Hz, 1 H). 13C NMR (CDCl3): δ ϭ 41.9, 55.1, 62.7, 70.7, 77.2,
86.5, 86.8, 87.2, 97.2, 113.2, 127.0, 128.0, 129.9, 130.0, 130.5, 133.0,
135.2, 135.4, 138.2, 144.1, 144.4, 158.5, 161.9. C36H34N4O7 (634.7):
calcd. C 68.13, H 5.40, N 8.83; found C 67.84, H 5.66, N 8.59.
5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-furoyl-2Ј-deoxycytidine (3e): 4-N-
Furoyl-2Ј-deoxycytidine (2e: 481 mg, 1.5 mmol) was dried by re-
peated coevaporation with dry pyridine and finally dissolved in dry
pyridine (15 mL). 4,4Ј-Dimethoxytrityl chloride (610 mg,
1.8 mmol) was added to the solution, and the mixture was stirred
at room temperature for 12 h. The mixture was concentrated to
half its volume, diluted with CHCl3, and washed three times with
5% aqueous NaHCO3, and the aqueous layer was back-extracted
with CHCl3. The organic layer and washings were combined, dried
with Na2SO4, filtered, and concentrated to dryness under reduced
pressure. The residue was loaded onto a silica gel column. Chroma-
tography was performed with CHCl3 containing 1% pyridine, ap-
plying a gradient of methanol (0.5Ϫ1%), to give 3e (804 mg, 86%)
as a colorless foam. 1H NMR (CDCl3): δ ϭ 2.29 (m, 1 H), 2.76
(m, 1 H), 3.47 (m, 2 H), 3.79 (s, 6 H), 4.17 (m, 1 H), 4.54 (m, 1
5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-isonicotinoyl-2Ј-deoxycytidine (3i):
A procedure similar to that described above was performed with 4-
N-isonicotinoyl-2Ј-deoxycytidine (2i: 505 mg, 1.5 mmol), to give 3i
(880 mg, 92%) as a colorless foam. 1H NMR (CDCl3): δ ϭ 2.31
(m, 1 H), 2.78 (m, 1 H), 3.48 (m, 2 H), 3.77 (s, 6 H), 4.21 (m, 1
3
3
H), 4.62 (m, 1 H), 6.27 (dd, 1 H, J1Ј,2Ј ϭ 5.3, J1Ј,2ЈЈ ϭ 5.3 Hz),
6.84 (d, 3J ϭ 6.3 Hz, 4 H), 7.12Ϫ7.44 (m, 10 H, 5-H), 7.69 (d, 3J ϭ
5.6 Hz, 2 H), 8.38 (d, 3J ϭ 7.3 Hz, 1 H), 8.69 (d, 3J ϭ 5.3 Hz, 2
H). 13C NMR (CDCl3): δ ϭ 41.9, 55.1, 62.4, 70.2, 77.2, 86.5, 86.8,
87.3, 97.4, 113.2, 121.4, 127.0, 128.0, 128.0, 129.9, 130.0, 135.2,
135.5, 140.6, 144.2, 144.9, 150.5, 154.4, 158.5, 162.1, 166.3.
C36H34N4O7 (634.7): C 68.13, H 5.40, N 8.83; found C 67.98, H
5.70, N 8.59.
3
3
H), 6.30 (dd, 1 H, J1Ј,2Ј ϭ 5.9, J1Ј,2ЈЈ ϭ 5.9 Hz), 6.58 (d, 3J ϭ
7.9 Hz, 4 H), 6.58 (m, 1 H), 7.13Ϫ7.42 (m, 11 H, 5-H), 7.54 (m, 1
3
H, N-furoyl), 8.27 (d, J ϭ 7.6 Hz, 1 H). 13C NMR (CDCl3): δ ϭ
Typical Procedure for the Synthesis of Triethylammonium 4-N-Acyl-
5Ј-O-(4,4Ј-O-dimethoxytrityl)-2Ј-deoxycytidin-3Ј-yl
42.1, 55.2, 62.8, 70.9, 77.3, 86.7, 86.9, 87.5, 96.5, 113.0, 113.3,
127.1, 127.8, 128.1, 129.2, 130.0, 130.1, 135.3, 135.5, 144.3, 144.9,
145.9, 146.0, 155.3, 158.6, 161.7. C35H33N3O8·1/2H2O (632.7): C
66.45, H 5.42, N 6.64; found C 66.59, H 5.55, N 6.62.
Phosphonate
(4a؊i): 4-N-Acetyl-5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-deoxycytidine[24]
(3a: 572 mg, 1 mmol) was dried by repeated coevaporation with dry
pyridine and finally dissolved in dry pyridine (1 mL) and dry diox-
ane (3 mL). 2-Chloro-5,6-benzo-1,3,2-dioxaphosphorin-4-one[18]
(1.25  stock solution in dioxane) was added to the solution. After
having been stirred at room temperature for 15 min, the mixture
was treated with H2O/pyridine (1:1, v/v) and stirred at room tem-
perature for 30 min. The mixture was concentrated to dryness un-
der reduced pressure. The residue was dissolved in CHCl3 and
washed three times with 5% aqueous NaHCO3, and the aqueous
layer was back-extracted with CHCl3. The organic layer and the
washings were combined and dried with Na2SO4, filtered, and con-
centrated to dryness under reduced pressure. The residue was
loaded onto a silica gel column. Chromatography was performed
with CHCl3 containing 1% triethylamine, applying a gradient of
methanol (4Ϫ5%) to give 4a (588 mg, 80%) as a colorless foam. 1H
5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-thenoyl-2Ј-deoxycytidine (3f):
A
procedure similar to that described above was performed with 4-N-
thenoyl-2Ј-deoxycytidine (2f: 505 mg, 1.5 mmol) to give 3f (880 mg,
1
92%) as a colorless foam. H NMR (CDCl3): δ ϭ 2.30 (m, 1 H),
2.76 (m, 1 H), 3.46 (m, 2 H), 3.79 (s, 6 H), 4.18 (m, 1 H), 4.56 (m,
3
3
3
1 H), 6.31 (dd, 1 H, J1Ј,2Ј ϭ 5.6, J1Ј,2ЈЈ ϭ 5.6 Hz), 6.85 (d, J ϭ
7.9 Hz, 4 H), 7.10Ϫ7.68 (m, 13 H), 8.27 (d, 3J ϭ 7.6 Hz, 1 H). 13C
NMR (CDCl3): δ ϭ 41.9, 55.1, 62.7, 70.7, 77.2, 86.5, 86.8, 87.2,
97.2, 113.2, 127.0, 128.0, 129.9, 130.0, 130.5, 133.0, 135.2, 135.4,
138.2, 144.1, 144.4, 158.5, 161.9. MS: m/z calcd. for (C35H33N3O7S
ϩ H) [M ϩ Hϩ] 640.2117, found 640.2120.
5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-picolyl-2Ј-deoxycytidine (3g): 4-N-
Picolyl-2Ј-deoxycytidine (2g: 505 mg, 1.5 mmol) was dried by re-
peated coevaporation with dry pyridine and finally dissolved in dry
pyridine (15 mL). 4,4Ј-Dimethoxytrityl chloride (610 mg,
1.8 mmol) was added to the solution, and the mixture was stirred
at room temperature for 12 h. The mixture was concentrated to
half its volume, diluted with CHCl3, and washed three times with
5% aqueous NaHCO3, and the aqueous layer was back-extracted
with CHCl3. The organic layer and washings were combined, dried
with Na2SO4, filtered, and concentrated to dryness under reduced
pressure. The residue was subject on a silica gel column. Chromato-
graphy was performed with CHCl3 containing 1% pyridine, apply-
ing a gradient of methanol (0.5Ϫ1%) to give 3g (880 mg, 92%) as
3
NMR (CDCl3): δ ϭ 1.29 (t, 9 H, J ϭ 7.6 Hz), 2.20 (s, 3 H), 2.51
(m, 1 H), 2.75 (m, 1 H), 3.06 (q, 6 H, J ϭ 7.2 Hz), 3.45 (m, 2 H),
3.79 (s, 6 H), 4.25 (m, 1 H), 4.98 (m, 1 H), 6.25 (dd, 1 H, J1Ј,2Ј
3
3
ϭ
5.6, 3J1Ј,2ЈЈ ϭ 5.6 Hz), 6.84 (d, 3J ϭ 8.9 Hz, 4 H), 6.89 (d, 1 H, 1J ϭ
618.9 Hz), 7.12Ϫ7.41 (m, 10 H), 8.22 (d, 3J ϭ 7.6 Hz, 1 H). 13C
NMR (CDCl3): δ ϭ 8.38, 24.7, 40.6, 45.3, 55.1, 62.0, 71.6, 84.9,
86.7, 96.3, 113.1, 126.8, 127.8, 128.8, 129.9, 135.1, 135.3, 137.7,
144.1, 154.9, 158.5, 162.7, 171.1. 31P NMR (CDCl3): δ ϭ3.56 (dd,
1J ϭ 618.9, J ϭ 8.5 Hz). MS: m/z calcd. for C38H49N4O9P (M ϩ
4
Hϩ Ϫ Et3N) 636.2110, found 636.2200.
Triethylammonium 5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-propionyl-2Ј-
deoxycytidin-3Ј-yl Phosphonate (4b): A procedure similar to that
described above was performed with 5Ј-O-(4,4Ј-dimethoxytrityl)-
4-N-propionyl-2Ј-deoxycytidine (3b: 586 mg, 1 mmol), to give 4b
(518 mg, 69%) as a colorless foam. 1H NMR (CDCl3): δ ϭ 1.18 (t,
1
a colorless foam. H NMR (CDCl3): δ ϭ 2.30 (m, 1 H), 2.76 (m,
1 H), 3.46 (m, 2 H), 3.79 (s, 6 H), 4.18 (m, 1 H), 4.56 (m, 1 H),
3
3
6.31 (dd, 1 H, J1Ј,2Ј ϭ 5.6, J1Ј,2ЈЈ ϭ 5.6 Hz), 6.85 (d, 3J ϭ 7.9 Hz,
4 H), 7.10Ϫ7.68 (m, 13 H), 8.27 (d, J ϭ 7.6 Hz, 1 H). 13C NMR
3
3J ϭ 7.6 Hz, 3 H), 1.33 (t, 9 H, J ϭ 7.3 Hz), 2.33Ϫ2.47 (m, 3 H,
2Ј-H), 2.82 (m, 1 H, 2ЈЈ-H), 3.06 (q, 6 H, J ϭ 7.6 Hz), 3.43 (m, 2
3
(CDCl3): δ ϭ 41.9, 55.1, 62.7, 70.7, 77.2, 86.5, 86.8, 87.2, 97.2,
113.2, 127.0, 128.0, 129.9, 130.0, 130.5, 133.0, 135.2, 135.4, 138.2,
144.1, 144.4, 158.5, 161.9. C36H34N4O7 (634.7): C 68.13, H 5.40, N
8.83; found C 68.10, H 5.19, N 8.49.
3
H), 3.80 (s, 6 H), 4.30 (m, 1 H), 4.95 (m, 1 H), 6.26 (dd, 1 H,
3J1Ј,2Ј ϭ 6.3, J1Ј,2ЈЈ ϭ 6.3 Hz), 6.84 (d, J ϭ 7.3 Hz, 4 H), 6.89 (d,
3
3
1
3
1 H, J ϭ 619.9 Hz), 7.09 (d, J ϭ 9.7 Hz, 1 H), 7.19Ϫ7.40 (m, 9
5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-nicotinoyl-2Ј-deoxycytidine (3h): A H), 8.21 (d, J ϭ 7.6 Hz, 1 H, 6-H). 13C NMR (CDCl3): δ ϭ 41.9,
procedure similar to that described above was performed with 4- 55.0, 62.7, 70.7, 77.2, 86.5, 86.8, 87.2, 97.2, 113.2, 127.0, 128.0,
N-nicotinoyl-2Ј-deoxycytidine (2h: 505 mg, 1.5 mmol), to give 3h 129.9, 130.0, 130.5, 133.0, 135.2, 135.4, 138.2, 144.1, 144.4, 158.5,
3
Eur. J. Org. Chem. 2001, 4583Ϫ4593
4591

T. Wada, A. Kobori, S. Kawahara, M. Sekine
161.9. 31P NMR (CDCl3): δ ϭ 3.7. C39H51N4O9P·H2O (768.8): C (t, 9 H, J ϭ 7.3 Hz), 2.33 (m, 1 H), 2.70 (m, 1 H), 2.96 (q, 6 H,
FULL PAPER
3
60.93, H 6.95, N 7.24; found C 60.97, H 6.90, N 6.97.
3J ϭ 7.3 Hz), 3.39 (m, 2 H), 3.71 (s, 6 H), 4.21 (m, 1 H), 4.91 (m,
3
3
3
1 H), 6.19 (dd, 1 H, J1Ј,2Ј ϭ 5.6, J1Ј,2ЈЈ ϭ 5.6 Hz), 6.77 (d, J ϭ
Triethylammonium 4-N-Butyryl-5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-de-
oxycytidin-3Ј-yl Phosphonate (4c): A procedure similar to that de-
scribed in the case of 4a was performed with 4-N-butyryl-5Ј-O-
(4,4Ј-dimethoxytrityl)-2Ј-deoxycytidine (3c: 600 mg, 1 mmol), to
give 4c (566 mg, 74%) as a colorless foam. 1H NMR (CDCl3): δ ϭ
1
15.4 Hz, 4 H), 6.79 (d, 1 H, JPϪH ϭ 619.6 Hz), 7.13Ϫ7.34 (m, 11
3
3
H), 8.15Ϫ8.22 (m, 2 H), 8.67 (d, J ϭ 5.0 Hz, 1 H), 9.13 (d, J ϭ
2.0 Hz, 1 H). 13C NMR (CDCl3): δ ϭ 40.7, 45.4, 55.0, 62.2, 72.0,
77.2, 85.2, 85.3, 86.6, 86.7, 97.7, 113.1, 123.2, 126.9, 127.8, 128.0,
129.7, 129.9, 129.9, 135.1, 135.3, 135.7, 144.0, 144.1, 149.5, 152.9,
158.4, 161.9. 31P NMR (CDCl3): δ ϭ 3.7. C42H50N5O9P·2H2O
(835.9): C 60.35, H 6.51, N 8.38; found C 59.99, H 6.55, N 8.62.
3
3
0.93 (t, J ϭ 7.3 Hz, 3 H), 1.29 (t, 9 H, J ϭ 6.9 Hz), 1.79 (m, 2
3
H), 2.34Ϫ2.48 (m, 3 H), 2.80 (m, 1 H), 3.06 (q, 6 H, J ϭ 6.6 Hz),
3.48 (m, 2 H), 3.79 (s, 3 H), 3.79 (s, 3 H), 4.30 (m, 1 H), 4.98 (m,
3
3
3
1 H), 6.26 (dd, 1 H, J1Ј,2Ј ϭ 5.6, J1Ј,2ЈЈ ϭ 5.6 Hz), 6.84 (d, J ϭ
7.9 Hz, 4 H), 6.89 (d, 1 H, 1J ϭ 619.9 Hz), 7.15Ϫ7.42 (m, 9 H),
8.20 (d, 3J ϭ 7.6 Hz, 1 H). 13C NMR (CDCl3): δ ϭ 8.5, 13.5, 18.3,
39.5, 41.0, 45.5, 55.2, 62.4, 72.2, 77.2, 86.5, 86.9, 86.9, 90.1, 113.1,
127.7, 129.0, 129.1, 135.3, 135.5, 155.1, 158.6, 162.0, 172.9. 31P
NMR (CDCl3): δ ϭ 3.9. C40H53N4O9P·H2O (782.9): C 61.37, H
7.08, N 7.16; found C 61.22, H 5.38, N 6.98.
Triethylammonium 5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-isonicotinoyl-
2Ј-deoxycytidin-3Ј-yl phosphonate (4i): 1H NMR (CDCl3): δ ϭ 1.33
3
(t, 9 H, J ϭ 7.3 Hz), 2.43 (m, 1 H), 2.82 (m, 1 H), 3.06 (q, 6 H,
3J ϭ 7.3 Hz), 3.48 (m, 2 H), 3.80 (s, 6 H), 4.31 (m, 1 H), 4.99 (m,
3
3
1 H), 6.29 (dd, 1 H, J1Ј,2Ј ϭ 5.6, J1Ј,2ЈЈ ϭ 5.6 Hz), 6.85 (4 H, d),
1
6.90 (d, 1 H, JPϪH ϭ 619.6 Hz), 7.21Ϫ7.42 (m, 11 H), 7.79 (d,
3J ϭ 5.6 Hz, 1 H), 8.29 (d, 3J ϭ 7.6 Hz, 1 H, 6-H), 8.81(d, J ϭ
6.3 Hz, 2 H). 13C NMR (CDCl3): δ ϭ 8.3, 40.6, 45.2, 54.9, 62.0,
71.7, 77.2, 85.1, 85.2, 86.5, 86.6, 97.4, 113.0, 121.5, 126.8, 127.7,
127.9, 129.6, 129.8, 135.0, 135.1, 140.8, 143.9, 144.2, 150.3, 153.4;
31P NMR (CDCl3): δ ϭ 4.0. C42H50N5O9P·2H2O (835.9): C 60.35,
H 6.51, N 8.38; found C 60.31, H 6.60, N 7.99.
Triethylammonium
5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-furoyl-2Ј-de-
oxycytidin-3Ј-yl Phosphonate (4e): A procedure similar to that de-
scribed in the case of 4a was performed with 5Ј-O-(4,4Ј-
dimethoxytrityl)-4-N-furoyl-2Ј-deoxycytidine (3e: 624 mg, 1 mmol)
1
to give 4e (584 mg, 74%) as a colorless foam. H NMR (CDCl3):
3
δ ϭ 1.32 (t, 9 H, J ϭ 7.3 Hz), 2.39 (m, 1 H), 2.86 (m, 1 H), 3.05
3
(q, 6 H, J ϭ 7.3 Hz), 3.79 (s, 6 H), 4.32 (m, 1 H), 4.96 (m, 1 H),
3
3
6.28 (dd, 1 H, J1Ј,2Ј ϭ 5.9, J1Ј,2ЈЈ ϭ 5.9 Hz), 6.84 (d, 3J ϭ 8.9 Hz,
1
4 H), 6.89 (d, 1 H, JPϪH ϭ 620.2 Hz), 7.12Ϫ7.42 (m, 13 H), 7.55
(d, 3J ϭ 1.1 Hz, 1 H), 8.23 (d, 3J ϭ 7.6 Hz, 1 H). 13C NMR
(CDCl3): δ ϭ 8.6, 41.0, 45.5, 55.1, 62.4, 72.0, 72.3, 77.2, 85.4, 85.5,
86.8, 87.0, 96.0, 112.8, 113.1, 115.4, 117.3, 118.7, 122.5, 126.8,
127.7, 128.0, 128.8, 128.0, 128.9, 129.9, 129.9, 135.0, 135.2, 144.0,
144.6, 146.4, 155.0, 158.2, 158.3, 161.3. 31P NMR (CDCl3): δ ϭ
3.8. C41H49N4O10P·2H2O (824.9): C 59.70, H 6.48, N 6.79; found
C 59.55, H 6.50, N 6.68.
Acknowledgments
This work was supported by a Grant from the ‘‘Research for the
Future’’ Program of the Japan Society for the Promotion of Science
(JSPS-RFTF97I00301) and a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan.
Triethylammonium 5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-thenoyl-2Ј-de-
oxycytidin-3Ј-yl Phosphonate (4f): A procedure similar to that de-
scribed in the case of 4a was performed with 5Ј-O-(4,4Ј-
[1]
Z. Ohashi, F. Harada, S. Nishimura, Biochim Biophys. Acta
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(3f:
640 mg,
[2]
1
1 mmol), to give 4f (552 mg, 81%) as a colorless foam. H NMR
3
(CDCl3): δ ϭ 1.31 (t, 9 H, J ϭ 7.3 Hz), 2.38 (m, 1 H), 2.83 (m, 1
3
H), 3.05 (q, 6 H, J ϭ 7.3 Hz), 3.45 (m, 2 H), 3.79 (s, 6 H), 4.31
[3]
3
3
(m, 1 H), 4.99 (m, 1 H), 6.27 (dd, 1 H, J1Ј,2Ј ϭ 5.9, J1Ј,2ЈЈ
ϭ
5.9 Hz), 6.84 (d, 3J ϭ 8.9 Hz, 4 H), 6.88 (d, 1 H, JPϪH
ϭ
1
628.1 Hz), 7.11Ϫ7.41 (m, 12 H), 7.89 (m, 1 H), 8.20 (d, 3J ϭ
7.6 Hz, 1 H). 31P NMR (CDCl3): δ ϭ 3.7. MS: m/z calcd. for
C35H35N3O9PS [M (C41H49N4O9PS) ϩ Hϩ Ϫ Et3N] 704.1832,
found 704.1835.
[4]
[5]
1994, 22, 2183Ϫ2196.
[6]
Triethylammonium 5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-picolyl-2Ј-de-
H. Grosjean, M. Sprinzl, S. Steinberg, Biochimie 1995, 77,
1
oxycytidin-3Ј-yl Phosphonate (4g): H NMR (CDCl3): δ ϭ 1.30 (t,
139Ϫ141.
[7]
3
M. Sprinzl, T. Hartman, J. Weber, J. Blank, R. Zeidler, Nucleic
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9 H, J ϭ 7.3 Hz), 2.39 (m, 1 H, 2Ј-H), 2.88 (m, 1 H), 3.01 (q, 6
3
H, J ϭ 7.3 Hz), 3.47 (m, 2 H), 3.80 (s, 6 H), 4.33 (m, 1 H), 4.97
[8]
3
3
(m, 1 H), 6.31 (dd, 1 H, J1Ј,2Ј ϭ 5.9, J1Ј,2ЈЈ ϭ 5.9 Hz), 6.86 (d,
O. Stetter, P. F. Crain, FASEB J. 1933, 7, 196Ϫ200.
3J ϭ 7.6 Hz, 4 H), 6.90 (d, 1 H, J ϭ 618.6 Hz, PϪH), 7.21Ϫ7.55
1
[9]
K. R. Noon, E. Bruenger, J. A. McClosky, J. Bacteriol. 1998,
(m, 11 H), 7.90 (d, 3J ϭ 5.6 Hz, 1 H), 8.21Ϫ8.31 (m, 2 H, 6-H),
8.61(d, 3J ϭ 4.3 Hz, 1 H), 10.6 (1 H, br, NH). 13C NMR (CDCl3):
δ ϭ 8.8, 41.0, 45.5, 55.1, 62.3, 72.0,72.1, 77.2, 85.2, 85.3, 86.7, 86.9,
95.6, 113.0, 122.5, 126.8, 127.7, 127.9, 129.8, 129.8, 129.8, 135.0,
135.2, 137.4, 143.9, 144.5, 147.9, 148.2, 155.0, 158.2, 158.3, 161.3,
163.1. 31P NMR (CDCl3): δ ϭ 3.8. C42H50N5O9P·H2O (817.9): C
61.68, H 6.41, N 8.56; found C 61.70, H 5.99, N 8.62.
180, 2883Ϫ2888.
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[12]
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Triethylammonium 5Ј-O-(4,4Ј-Dimethoxytrityl)-4-N-nicotinoyl-2Ј-
P. A. Domenico, E. Stocker, Nucleosides Nucleotides 1989, 8,
179Ϫ183
1
deoxycytidin-3Ј-yl Phosphonate (4h): H NMR (CDCl3): δ ϭ 1.23
4592
Eur. J. Org. Chem. 2001, 4583Ϫ4593

Oligodeoxyribonucleotides Incorporating N-Acyldeoxycytidine Derivatives
FULL PAPER
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[21]
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Received July 12, 2001
[O01346]
Eur. J. Org. Chem. 2001, 4583Ϫ4593
4593