Conformational studies of 4-N-carbamoyldeoxycytidine derivatives and synthesis and hybridization properties of oligodeoxyribonucleotides incorporating these modified bases

Article abstract of DOI:10.1002/ejoc.200501006

Deoxycytidine derivatives modified with various N-substituted carbamoyl groups at the 4-amino group were synthesized. The detailed ¹H NMR studies suggest that the 3′,5′-O-disilylated N- carbamoyldeoxycytidine derivative 11 exists as a species h

Full text of DOI:10.1002/ejoc.200501006

FULL PAPER
DOI: 10.1002/ejoc.200501006
Conformational Studies of 4-N-Carbamoyldeoxycytidine Derivatives and
Synthesis and Hybridization Properties of Oligodeoxyribonucleotides
Incorporating these Modified Bases
Kenichi Miyata,[a,c] Akio Kobori,[a] Ryuji Tamamushi,[a] Akihiro Ohkubo,[a,c]
Haruhiko Taguchi,[a,c] Kohji Seio,[b,c] and Mitsuo Sekine*[a,c]
Keywords: Oligonucleotides / Hybridization / Modified deoxynucleosides / DNA duplex formation / Conformational analy-
sis / 15N-labeled nucleosides
Deoxycytidine derivatives modified with various N-substi-
tuted carbamoyl groups at the 4-amino group were synthe-
sized. The detailed 1H NMR studies suggest that the 3Ј,5Ј-O-
disilylated N-carbamoyldeoxycytidine derivative 11 exists as
a species having an intramolecular hydrogen bond between
the N3 atom and the carbonyl oxygen atom in MeOD but
upon addition of CDCl3, the amount of a homodimer species,
having intermolecular hydrogen bonds, gradually increases.
Oligodeoxyribonucleotides incorporating various 4-N-carba-
moyldeoxycytidine derivatives were also synthesized. These
plementary strands without disturbing the structure of DNA
duplexes, hence changing the orientation of the carbamoyl
group in such a manner that a stable Watson–Crick base pair
can be formed with the guanine base at the opposite site. It
should be noted that the base recognition ability (G against
T, C and A) of these modified cytosine bases can be pre-
served satisfactorily. These results suggest that the 4-N-car-
bamoyl group is useful as a backbone structure of the linker
between various functional residues and oligonucleotides.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
modified oligodeoxynucleotides can hybridize with the com- Germany, 2006)
Introduction
We have recently reported the hybridization properties of
oligodeoxynucleotides containing 4-N-acyldeoxycytid-
ine,[1,2] 4-N-alkoxycarbonyldeoxycytidine[3] and 4-N-(N-ar-
ylcarbamoyl)deoxycytidine derivatives,[4] as shown in Fig-
ure 1.
Despite the modification of the 4-amino group that is
involved in the canonical hydrogen bonding, our results
showed that acylation or alkoxycarbonylation of the 4-
amino group of deoxycytidine not only allowed formation
of the Watson–Crick base pair[1–3] but also increased the
hybridization affinity for the complementary guanine
base.[1,2] The 1H NMR analysis and the ab initio MO calcu-
lations revealed that these 4-N substituents were fixed in the
same “proximal” geometry as that reported in the case of
4-N-acetylcytidine found in tRNA[5–11] and rRNA.[12,13]
From the crystal[14] and NMR[15] structures reported to
Figure 1. Structures for 4-N-acydeoxycytidine, 4-N-alkoxycarbony-
deoxycytidine and 4-N-(N-arylcarbamoyl)deoxycytidine deriva-
tives.
[a] Department of Life Science, Tokyo Institute of Technology
4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan
Fax: +81-45-924-5706
E-mail: msekine@bio.titech.ac.jp
[b] Frontier Collaborative Research Center, Tokyo Institute of
Technology
4259 Nagatsuta, Midoriku, Yokohama 226-8503, Japan
[c] CREST, Japan Science and Technology Agency
4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
date, it was concluded that the carbonyl oxygen atom of
these acyl-type groups is orientated in close proximity to 5-
C. For example, the acetyl group is geometrically fixed, so
that it allows formation of the Watson–Crick base pair
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4-N-Carbamoyldeoxycytidine Derivatives
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owing to an intramolecular hydrogen bond between the car- ring in a manner similar to that of 4-N-aceyldeoxycytid-
bonyl oxygen atom and the 5-vinyl proton of the cytosine ine[14] or 4-N-alkoxycarbonyldeoxycytidine.[3]
ring.
The exo-amino groups of the cytosine and adenine moie-
ties in oligodeoxyribonucleotides have been used as conve-
nient sites to introduce fluorescence groups[16,17] or to at-
tempt to acquire the triplex-forming ability[18] through car-
bamoyl-type linkers. However, the base-pairing ability as
well as the base-recognition ability of these modified bases
having the N-carbamoyl structures has never been exam-
ined to date. Several previous studies suggested that 4-N-
(N-alkylcarbamoyl)deoxycytidine derivatives 1a–e could
form two types of intramolecular hydrogen bonds, as shown
in Figure 2.[19]
Figure 3. Crystal structures of of 4-N-(ureidocarbonyl)-dC (2) and
4-N-[N-(methoxycarbonyl)carbamoyl]-3Ј,5Ј-di-O-acetyl-dC (3).
These early papers suggested that the orientation of the
4-N-carbamoyl group is flexible depending on the nitrogen
substituents and thereby the 4-N-carbamoylcytosine base in
oligonucleotides can form a Watson–Crick base pair with
guanine under certain conditions which allow the “proxi-
mal” conformation. However, no papers have been reported
about the conformational behavior of 4-N-(N-alkylcarbam-
oyl)deoxycytidine derivatives when they are incorporated
into oligodeoxyribonucleotides.
In this paper, we report the detailed conformational stud-
ies of 4-N-(N-alkylcarbamoyl)deoxycytidines 1a–e and the
Figure 2. Intramolecular hydrogen bonding patterns of 4-N-carba-
hybridization and base-recognition properties of oligodeox-
moyldeoxycytidine derivatives 1a–e.
yribonucleotides containing 1a–e.
Kumar et al. reported the crystal structures of 4-N-(ure-
idocarbonyl)deoxycytidine (2) and 4-N-[N-(methoxycar-
bonyl)carbamoyl]-3Ј,5Ј-di-O-acetyldeoxycytidine
(3).[19]
Results and Discussion
They found that in the former the imido proton of the ure-
idocarbonyl [H2NC(O)NHC(O)–] group forms an intramo-
lecular hydrogen bond with the cytosine ring nitrogen atom
so that its orientation becomes “distal” to 5-C of the pyr-
imidine ring, as shown in Figure 3. The latter exists as a
Conformational Studies of 1-Methyl-4-N-carbamoylcytosine
by Use of Ab Initio MO Calculations in Vacuo
For the comprehensive conformational analysis of the N-
homodimer with four intermolecular hydrogen bonds at the unsubstituted carbamoyl group of 4-N-carbamoyldeoxycy-
Watson–Crick base-pairing site. This structure has a con- tidine (1a: dCcmy) the relative energies of the possible con-
formation “proximal” to 5-C, which allows formation of an formational isomers 4a–f (see Figure 4) of 4-N-carbamoyl-
intramolecular hydrogen bond between the carbonyl oxygen 1-methylcytosine were calculated by using the Gaussian 98
atom of the carbamoyl group and 5-H of the pyrimidine package program.[20]
Figure 4. Energy-optimized structures of the geometric and tautomeric isomers 4a–f of 4-N-carbamoyl-1-methylcytosine by DFT calcula-
tions at the B3LYP/6-31++G** level. The orientation of the 4-N-substituents shown in 4a, 4b and 4c are defined as “proximal” and that
of 4d, 4e and 4f as “distal”. The intramolecular hydrogen bond is indicated by a dotted line with the distance between the hydrogen
bonding atoms.
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Table 1. Relative difference in energy [kcal/mol] between 4a and the other structures 4b–f by use of various basis sets.
These isomeric structures were energy-optimized by DFT mained essentially unchanged. In all cases, the conformers
calculations at the B3LYP/6-31++G** level. In the struc- 4f and 4d were the most and the second most stable and
ture 4a, the carbonyl oxygen atom has the same orientation the difference in energy between them was very small. The
as that of the crystal structure of 4-N-acetylcytidine[14] and isomers 4c and 4f are not found in the previous crystal
4-N-[N-(methoxycarbonyl)carbamoyl]-3Ј,5Ј-di-O-acetylde- structures,[19] so that the conformation of the 4-N-carbam-
oxycytidine (3)[19] and the structure 4d has the same orien- oyl groups in solution must be studied in detail.
tation as that of the crystal structure of 4-N-ureidocarbon-
For more accurate conformational analysis of the con-
yldeoxycytidine (2).[19] In addition, the structure of 4f has formers of 4-N-carbamoylcytosine, we synthesized dCcmy
an intramolecular hydrogen bond similar to that of 4-N- (1a) and related compounds 1b–e and studied their confor-
benzoyl-5-methyldeoxycytidine,[21] which cannot have an in- mational analysis by use of 1H and 13C NMR spectroscopy.
tramolecular hydrogen bond similar to that of 4-N-acetyl- We also synthesized 4-15N-labeled 4-N-carbamoyldeoxycy-
C because of the presence of the 5-methyl group. The values tidine derivatives and 4-N-carbamoyl-5-methyldeoxycytid-
shown in the structures refer to the distance between the ine derivatives for the study of tautomerism of the modified
hydrogen-bonding atoms.
deoxycytidines.
Compared with the energy of the conformational isomer
4a, which is capable of formation of the Watson–Crick base
pair, those of 4b–f were evaluated by MO calculations at
Synthesis of 4-N-Carbamoyldeoxycytidine Derivatives
A general approach to synthesizing 4-N-(N-alkylcarbam-
the HF/6-31G* and HF/6-31++G** levels in the gas phase oyl)deoxycytidine derivatives 1a–e and their 3Ј-phos-
(Table 1). The stabilization of these structures in the gas phoramidites 8a–e is outlined in Scheme 1. The phen-
phase was ranked in the order 4f Ͼ 4d Ͼ 4c Ͼ 4a ϾϾ 4b oxycarbonyl group was selectively introduced to the 4-
Ͼ 4e. To see if higher calculations were necessary, these six amino function of deoxycytidine by the transient protection
structures were energy-optimized at the MP2/6-31++G** method using trimethylsilyl chloride.[22,23] Subsequently, the
and B3LYP/6-31++G** levels. As the result, the order re- 4-N-(phenoxycarbonyl)deoxycytidine intermediate 6 was
Scheme 1. Reagents and conditions: (i) (A) TMSCl (4.0 equiv.), pyridine, room temp., 2 h, (B) phenyl chloroformate (1.5 equiv.), CH2Cl2/
pyridine (1:1, v/v), room temp., 1 h; (ii) (A) RNH2 (excess), pyridine, room temp., 2 h, (B) concd. NH4OH, room temp., 2 h (for 1d, e);
(iii) DMTrCl (1.2 equiv.), pyridine, room temp., 4 h; (iv) (2-cyanoethoxy)bis(diisopropylamino)phosphane (1.1 equiv.), diisopropylamine
(0.6 equiv.), 1H-tetrazole (0.6 equiv.), CH2Cl2, room temp., 6 h (for 8a), chloro(2-cyanoethoxy)(diisopropylamino)phosphane (1.1 equiv.),
ethyldiisopropylamine (1.3 equiv.), CH2Cl2, room temp., 30 min (for 8b–e).
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4-N-Carbamoyldeoxycytidine Derivatives
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1
converted into various 4-N-carbamoyldeoxycytidine deriva-
The H NMR spectra of deoxycytidine (5: dC), 4-N-ace-
tives 1a–e by treatment with the corresponding amines fol- tyldeoxycytidine (9: dCac), 4-N-methoxycarbonyldeoxy-
lowed by removal of the trimethylsilyl groups. Treatment of cytidine (10: dCmoc) and 4-N-carbamoyldeoxycytidine (1a:
1a–e with DMTrCl gave the 5Ј-O-dimethoxytritylated prod- dCcmy) in D2O, are shown in Figure 5. The spectra of 4-
ucts 7a–e. Compounds 7a–e were converted into the phos- N-carbamoyldeoxycytidine (1a) and its N-monosubstituted
phoramidite units 8a–e by phosphitylation in the usual dCcmy derivatives 1b–e are similar to that of 5. These results
way.[24]
indicate that the 5-vinyl protons of 1a–e are not involved
in intermolecular hydrogen bonding so that their structures
differ from those of 4-N-acyl- and 4-N-alkoxycarbonylcyto-
sine base residues of 9 and 10 in D2O. The structures of the
Conformational Studies of 4-N-Carbamoyldeoxycytidine
1
Derivatives by Use of H and 13C NMR Spectroscopy
1
The H NMR spectra of the 4-N-carbamoyldeoxycytid- 4-N-carbamoylcytosine moiety that can satisfy the above
ine derivatives were compared with those of 4-N-acetyl- property are the second most and the most stable struc-
deoxycytidine and 4-N-methoxycarbonyldeoxycytidine.[3] tures, 4d and 4f.
Parthasarathy et al. reported that the X-ray crystal struc-
The solvent effect on the conformational change of the
ture of 4-N-acetylcytidine, found in the first letter of the cmy group was also studied. Compound 1a and its 3Ј,5Ј-O-
anticodon loop in various tRNAs, showed an intramolecu- disilylated derivative 11 were used. As shown in Figure 5,
1
lar hydrogen bond between the carbonyl oxygen atom and the H NMR spectra of 1a and 11 vary depending on the
the 5-vinyl proton.[14] Actually, the chemical shift of the 5- polarity of the solvent. The chemical shift of 5-H of 1a in
H proton of 4-N-acetylcytidine exhibited a downfield shift [D6]DMSO is very similar to that observed in D2O, except
of 1.21 ppm in the 1H NMR spectrum.[19] Recently, we have that the doublet peak is broader in [D6]DMSO (data not
reported that acylation or alkoxycarbonylation of the 4- shown). A more significant effect was observed when 11
amino group of deoxycytidine does not affect the formation was measured in the nonpolar solvent CDCl3. Compared
of the Watson–Crick base pair.[1–3] The chemical shifts of with the spectrum of 1a in D2O or [D6]DMSO, the 5-H
5-H of 4-N-acyl- or 4-N-alkoxycarbonyl-modified deoxycy- signal of 11 in CDCl3 shows broadening with an apparent
tidines appear at ca. 1 ppm lower magnetic fields.[2,3]
downfield shift (∆δ = 1.2 ppm). This result indicates that
1
Figure 5. H NMR spectra of deoxycytidine (dC, 5), 4-N-acetyl-dC (dCac, 9), 4-N-methoxycarbonyl-dC (dCmoc, 10) and 4-N-carbamoyl-
dC (dCcmy) in D2O and 3Ј,5Ј-O-bis-TBS-dCcmy (11) in CDCl3.
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the conformation of the carbamoyl group in CDCl3 is dif- bamoyldeoxycytidine derivatives exist as amino tautomers
ferent from those in D2O and [D6]DMSO, suggesting the with “proximal” conformation in CDCl3 (4a) and “distal”
“proximal” conformation of 4a is fixed by hydrogen bond- conformation in [D6]DMSO (4d). These results are consis-
ing between 5-H and the carbamoyl group.
tent with the 1H NMR analysis of 4-N-carbamoyl-5-
methyldeoxycytidine derivatives shown in the Supporting
Information.
Tautomerism Studies of 4-N-Carbamoyldeoxycytidine
Derivatives by Use of 15N Labeling
Effect of Solvent Polarity on the Dynamic Conformational
Looking at the 1H NMR spectra, it is still unclear
whether the base moiety of 1a exists in the 4-amino form
4a, 4d or the 4-imino form 4f. To examine if dCcmy (1a)
exists in the tautomeric form 4f, the 4-15N-labeled deoxycy-
tidine derivative 15 was also synthesized.[3,25] Thus, the 4-
15N-labeled species 15 was obtained from 3Ј,5Ј-O-bis(tert-
butyldimethylsilyl)-2Ј-deoxyuridine (12) in an overall yield
of 47%, as shown in Scheme 2. Compound 15 is useful to
determine the tautomerism of the 4-N-carbamoylcytosine
moiety because the amino tautomers such as 4a and 4d give
a split 4-NH signal due to a large coupling constant (ca.
90 Hz) derived from a covalently bonded 15N-1H system,
while the imino tautomers such as 4c and 4f having an unla-
beled 3-NH proton give a singlet.
Change between “proximal” and “distal”
1
Figure 7 shows the H NMR spectra of 11 in CDCl3 at
various temperatures and solvents. The shape of the peaks
assigned to 5-H and NH become sharper when the tem-
perature is lowered to 243 K. The chemical shift of 5-H re-
mains at δ = 7.58 ppm at 243 K and is identical to that
1
measured at 298 K. Figure 7 (B) shows the H NMR spec-
trum of 11 in the mixed solvent CDCl3/MeOD in various
ratios. The signal of 5-H is shifted upfield when the ratio
of polar solvent (MeOD)/CDCl3 is increased. Furthermore,
when CDCl3/MeOD (1:1, v/v) is used as the solvent, the
shape of the 5-H peak changes to an ordinary doublet and
its chemical shift is observed at δ = 6.26 ppm, identical to
that of the spectrum measured in D2O or [D6]DMSO.
When the geometry of the cmy group of 11 is fixed to
“proximal”, as shown in Figure 2, the NH group of the cmy
moiety can act as a donor so that compound 11 can have
two donor sites and two acceptor sites to form four hydro-
gen bonds. Thus, in a nonpolar solvent such as CDCl3 this
does not inhibit the intermolecular hydrogen bonding; 11
would form a homodimer in an antiparallel direction with
the four hydrogen bonds (Figure 7A), similar to the crystal
structure found for the homodimer.[19] This solvent effect
The 1H NMR spectra of 14 in [D6]DMSO and 15 in
[D6]DMSO and CDCl3 are shown in Figure 6. We observed
a split NH signal at δ = 7.18 (a), 9.72 (b), 10.91 (c) ppm
and it can be assigned as the 4-NH proton of the cytosine
base. From Figures 5 and 6, it was concluded that 4-N-car-
1
of the H NMR spectra suggests that the dynamic confor-
mational change between “proximal” and “distal” is due to
the disruption of intermolecular hydrogen bonding by the
addition of the polar solvent (MeOD).
Energy Profile of the Formation of a Watson–Crick Base
Pair Evaluated by Ab Initio MO Calculations
1
The H NMR study revealed that the solvent polarity
significantly affects the conformation of the cmy group. The
cmy group changes its geometry to form intermolecular hy-
drogen bonds in CDCl3. We expected that a similar confor-
mational change would occur when deoxyguanosine is lo-
Figure 6. 1H NMR spectra of 4-15N labeled dC (14) in [D6]DMSO
(a) and 4-15N labeled dCcmy (15) in [D6]DMSO (b) and CDCl3 (c). cated on the opposite side of the dCcmy derivatives in a
Scheme 2. Reagents and conditions: (i) diphenyl phosphate (2 equiv.), 1H-tetrazole (2 equiv.), TsCl (2 equiv.), pyridine, room temp., 2 d;
(ii) 15NH4Cl (1.2 equiv.), KOH (1.2 equiv.), Et3N (1.4 equiv.), CH3CN/H2O (3:1, v/v), room temp., 24 h; (iii) (A) phenyl chloroformate
(1.2 equiv.), pyridine (1.5 equiv.), CH2Cl2, room temp., 1 h, (B) pyridine/concd. NH3 (1:1, v/v), room temp., 2 h.
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4-N-Carbamoyldeoxycytidine Derivatives
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Figure 7. Solvent effect of 4-N-carbamoyldeoxycytidine on the intermolecular and intramolecular hydrogen-bonding formation.
DNA duplex. To estimate the energy of the formation of ergy (∆E) of 4-N-carbamoyl-1-methyl-C (4a) with 9-methyl-
the Watson–Crick base pair between dCcmy and dG, ab ini- G was calculated by subtracting the energies of 4d and 9-
tio MO calculations of this base pair were carried out at methyl-G from that of the base pair 4a-G. The base pairing
the HF/6-31++G** level.
energies calculated at the HF/6-31++G** level are summa-
Although the structure 4d in Figure 4 cannot form the rized in Figure 8. The Ccmy-G base pair has a stabilization
Watson–Crick base pair, the structure 4a can form three energy of –27.4 kcal/mol, while the unmodified C-G base
hydrogen bonds with the guanine base. The base-pair en- pair has a stabilization energy of –28.7 kcal/mol. Although
there is energy loss due to the conformational change of the
carbamoyl group, this energy loss is small enough to form
a stable Watson–Crick base pair accompanying the confor-
mational change from the structure 4d to 4a.
Synthesis, Purification and Characterization of
Oligodeoxyribonucleotides Containing Modified Bases
To evaluate the thermodynamic stability of the DNA du-
plexes incorporating these modified bases, they were incor-
porated into oligonucleotides. The solid-phase synthesis of
oligodeoxyribonucleotides using a DNA/RNA synthesizer
was carried out by use of the standard phosphoramidite
method.[24,26] The oligomer was released from the polymer
support and deprotected by treatment with concd. aq. NH3
for 1 h. The product was purified on a C18-cartridge by the
Figure 8. Predicted Watson–Crick base pair between 4-N-carbam-
oyl-dC and dG. The hydrogen-bonding energy (∆E) including the
energy loss of conformational change is also indicated.
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Table 2. Sequence of oligodeoxynucleotides containing 4-N-carbamoyl-dC derivatives.
DMTr-ON purification method and analyzed by anion-ex-
change HPLC and reversed-phase HPLC. The sequences
and the isolated yields of the 13mers containing modified
nucleosides are shown in Table 2. The composition of the
purified products was confirmed by MALDI-TOF mass
spectrometry.
Hybridization Properties of Oligodeoxynucleotides
Containing Modified Bases
From the conformational studies of 4-N-carbamoyld-
eoxycytidine derivatives described above, it was predicted
that these modified nucleosides would form stable Watson–
Figure 9. Tm curves of 13mer duplexes containing a dC*-dG base
pair.
Crick base pairs with deoxyguanosine.
The thermal stability of DNA duplexes containing a
modified nucleoside was investigated in sodium phosphate
buffer (pH = 7.0) containing 1.0  NaCl. The melting
As shown in Table 3, the Tm value of the duplex contain-
curves of the duplexes containing 4-N-carbamoyldeoxycyti- ing 4-N-carbamoyldeoxycytidine (Entry 2: Y = G, 52.6 °C)
dine derivatives are shown in Figure 9. The shapes of the was slightly lower by 0.6 °C than that of the unmodified
melting curves were similar to that of the unmodified du- duplex (Entry 1: Y = G, 53.2 °C). However, this Tm value
plex. The Tm values of duplexes containing 4-N-carbamoyl- (Entry 2: Y = G, 52.6 °C) was not so low as that of the
dC derivatives are summarized in Table 3.
duplex containing a natural mismatched base pair (Entry 1:
Table 3. Tm values[a] for DNA 13mer duplexes containing 4-N-carbamoyl-dC derivatives.
[a] The Tm values are accurate within 0.5 °C. The Tm measurements were carried out in a buffer containing 10 m sodium phosphate
(pH = 7.0), 1.0  NaCl, 0.1 m EDTA and 2 µ duplex. [b] ∆Tm is the difference in the Tm value between the duplex having a modified
base and that having a natural base.
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4-N-Carbamoyldeoxycytidine Derivatives
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Y = A, 32.8 °C; Y = C, 30.7 °C; Y = T, 37.8 °C). These
results indicate that 4-N-carbamoyldeoxycytidine forms a
stable base pair with deoxyguanosine. The duplex contain-
ing a Ccmy-G base pair (Entry 2: Y = G, 52.6 °C) is more
stable than the duplex containing a T-A base pair (Entry 7:
Y = A, 49.2 °C). Likewise, the Tm values of duplexes con-
taining monosubstituted 4-N-carbamoyldeoxycytidine de-
rivatives (Entries 3–6) were relatively high, suggesting the
formation of the Watson–Crick base pairs between modi-
fied nucleosides and deoxyguanosine.
It is likely that the carbamoyl groups of modified deoxy-
cytidines change their orientation to the geometry that does
not interrupt the formation of a Watson–Crick base pair
with deoxyguanosine, as shown in Figure 10. The stability
of the duplex decreases when the side chain length becomes
longer, as shown in Entries 3–5 in Table 3. The results of
Entries 5 and 6 suggest that incorporation of an oxygen
atom into the alkyl side chain apparently increases the du-
plex stability (∆Tm = +1.5 °C). It is likely that this stabiliza-
tion is caused by an increase in the hydrophilicity of the
side chain that keeps the hydration structure in the major
groove, essential for stabilization of the DNA duplex struc-
ture.[27] In the previous paper, we reported the effect of the
length of the alkyl side chain of acyl or alkoxycarbonyl
groups.[2,3] These results suggested that the hydrophobicity
of the side chain of the carbamoyl group accounts for the
duplex destabilization.
Conclusions
Modified oligodeoxyribonucleotides incorporating 4-N-
carbamoyldeoxycytidine derivatives were synthesized. The
1H NMR spectra of 4-N-carbamoyldeoxycytidine in D2O
suggests that the carbamoyl group forms an intramolecular
hydrogen bond with the cytosine ring nitrogen atom. In
contrast with 4-N-acyldeoxycytidines and 4-N-alkoxycar-
bonyldeoxycytidines, the orientation of the 4-N-carbamoyl
group in D2O is fixed at the geometry that inhibits the for-
mation of the Watson–Crick base pair with a guanine base.
However, it is interesting that in CDCl3 4-N-carbamoyl-dC
derivatives exist as homodimers with four intermolecular
hydrogen bonds, with a change in the geometry of the car-
bamoyl group. The Tm analysis of oligodeoxynucleotides
containing 4-N-carbamoyl-dC derivatives revealed that, in
the process of their hybridization with the complementary
oligodeoxynucleotides, the orientation of the carbamoyl
group changes in a manner such that the stable Watson–
Crick base pair can be formed with the guanine base.
Thus, we found that the geometry of 4-N-carbamoyld-
eoxycytidine derivatives is affected by the solvent and inter-
molecular hydrogen bonds. Their base-pairing abilities indi-
cate that 4-N-carbamoyl groups might be useful as linkers
between various functional residues and oligonucleotides
when hydrophilic substitutents involving –(CH2CH2O)n–
type spacers (see Entry 6 in Table 3), capable of maintaining
the duplex structure without serious loss of the base-re-
cognition ability, are attached to the nitrogen atom of the
carbamoyl group. Further experiments with various 4-N-
carbamoyl-modified deoxycytidine derivatives are under
way to investigate the usefulness of these interesting in-
herent properties.
Experimental Section
Figure 10. Conformational change of 4-N-carbamoyl-dC induced
General Remarks: All chemical reagents used are commercially
available. Pyridine was distilled twice from p-toluenesulfonyl chlo-
ride and CaH2 after being refluxed for several hours and stored
over molecular sieves (4 Å). Triethylamine was distilled from CaH2
and stored over molecular sieves (4 Å). TLC was performed using
Merck Kieselgel 60 F254 precoated glass plates. Column
chromatography was performed with silica gel C-200, C-300 (Wako
Co. Ltd.), 60N (Kanto Chemical, Co., Inc.), NH (Fuji Silysia
Chemical Ltd.) and a minipump for a goldfish bowl was conve-
niently used to attain sufficient pressure for rapid chromatographic
separation. 1H NMR spectra were recorded at 270 MHz and the
chemical shifts were measured from the solvent peak as an internal
standard (in CDCl3 and [D6]DMSO) or TSP (in D2O) as an exter-
nal standard. 13C NMR spectra were recorded at 68 MHz and the
chemical shifts were measured from the solvent peak as an internal
standard. 31P NMR spectra were recorded at 109 MHz and the
chemical shifts were measured from 85% H3PO4 in CDCl3 as an
external standard. High-resolution ESI mass spectrometry was per-
formed by use of a Mariner (PerSeptive Biosystems, Inc.).
by duplex formation.
The Tm values of DNA duplexes having a mismatched
base pair (Y = T, C and A) are also summarized in Table 3.
Compared with the Tm values of all matched DNA du-
plexes (Y = G), the duplexes having a mismatched base pair
were destabilized efficiently. The Tm values of DNA du-
plexes containing a C*-A mismatch (Y = A) were somewhat
higher by 4.6–7.5 °C than that having an unmodified mis-
match base pair, but were at the same level as those of the
duplexes containing a C*-T mismatch (Y = T) or a C-T
mismatch. It should be noted that, among the modified de-
oxycytidine derivatives, Ccmy and Cmecm keep their G-re-
cognition abilities against A, as shown by the ∆Tm values
(14.8 °C and 12.3 °C) observed between the modified and
unmodified duplexes. From Table 3, it is likely that the
modified deoxynucleoside derivatives 1a–e can recognize A
as a mismatched partner at the same level as T. Therefore,
it seems that, when these modified deoxycytidines are incor-
porated into oligodeoxynucleotides, the modified bases can
be used as the site capable of binding to a G base in the
opposite strand without disturbing the duplex stability.
General Procedure for the 4-N-Carbamoylation of Deoxycytidine
Derivatives 1a–c
4-N-Carbamoyldeoxycytidine (1a): Deoxycytidine hydrochloride
(791 mg, 3.0 mmol) was rendered anhydrous by repeated co-evapo-
Eur. J. Org. Chem. 2006, 3626–3637
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3633

K. Miyata, A. Kobori, R. Tamamushi, A. Ohkubo, H. Taguchi, K. Seio, M. Sekine
FULL PAPER
ration with dry pyridine and finally dissolved in dry pyridine
(30 mL). To the solution was added TMSCl (1.52 mL, 12.0 mmol)
and the mixture was stirred at room temperature for 2 h. The mix-
ture was diluted with dry CH2Cl2 (30 mL). Phenyl chloroformate
(564 µL, 4.5 mmol) was added dropwise to the solution. After be-
12.0 mmol) and the mixture was stirred at room temperature for
2 h. The mixture was diluted with dry CH2Cl2 (30 mL). Phenyl
chloroformate (564 µL, 4.5 mmol) was added dropwise to the solu-
tion. After being stirred for 1 h, the mixture was diluted with
CHCl3 (30 mL). The CHCl3 solution was washed three times with
ing stirred for 1 h, the mixture was diluted with CHCl3 (30 mL). saturated NaHCO3 (30 mL). The organic layer was collected, dried
The CHCl3 solution was washed three times with saturated
NaHCO3 (30 mL). The organic layer was collected, dried with
Na2SO4, filtered and concentrated to dryness under reduced pres-
sure. The residue was dissolved in pyridine/conc.NH3 (1:1, v/v,
30 mL). After being stirred for another 2 h, the solution was con-
centrated to dryness under reduced pressure. The residue was dis-
solved in H2O (30 mL) and washed three times with ethyl acetate
(30 mL). The aqueous layer was concentrated under reduced pres-
sure. The resulting precipitate was collected by filtration, washed
with Na2SO4, filtered and concentrated under reduced pressure.
The residue was dissolved in pyridine (30 mL) and to the mixture
was added butylamine (889 µL, 9 mmol). After being stirred for
another 2 h, concd. NH3 (20 mL) was added to the mixture which
was stirred for another 2 h. The residue was partitioned between
H2O (30 mL) and CHCl3/pyridine (1:1, v/v, 50 mL). The organic
layer was collected, dried with Na2SO4, filtered and concentrated
under reduced pressure. The crude product was purified by silica
gel column chromatography using 60N silica gel with CH3Cl3/
MeOH to give the product 1d (763 mg, 78%). 1H NMR ([D6]-
DMSO): δ = 0.88 (t, J = 7.1 Hz, 3 H), 1.24–1.49 (m, 4 H), 3.13–
1
with cold iPrOH and dried in vacuo to give 1a (665 mg, 82%). H
NMR (D2O): δ = 2.28–2.59 (m, 2 H), 3.73–3.90 (2 m, 2 H), 4.10–
4.14 (m, 1 H), 4.41–4.46 (m, 1 H), 6.23 (dd, J = 6.4 Hz, 1 H), 6.41 3.20 (m, 2 H), 3.51–3.63 (m, 2 H), 3.80–3.82 (m, 1 H), 4.17–4.19
(d, J = 7.4 Hz, 1 H), 8.13 (d, J = 7.4 Hz, 1 H) ppm. 13C NMR
(D2O): δ = 44.7, 66.1, 75.3, 92.0, 92.1, 102.3, 148.6, 161.4, 161.7,
167.7 ppm. C10H14N4O5 (270.24): calcd. C 44.44, H 5.22, N 20.73;
found C 44.19, H 5.31, N 20.78. ESIMS: calcd. for C10H15N4O5
[M+H]+ 271.1042, found 271.1046.
(m, 1 H), 5.03 (t, J = 5.2 Hz, 1 H), 5.25 (d, J = 4.2 Hz, 1 H), 6.09
(dd, J = 6.3 Hz, 6.4 Hz, 1 H), 6.23 (d, J = 6.5 Hz, 1 H), 8.13 (d, J
= 6.5 Hz, 1 H), 8.97 (br., 1 H), 9.82 (br., 1 H) ppm. 13C NMR
([D6]DMSO): δ = 13.9, 19.8, 31.6, 39.1, 40.9, 61.2, 70.2, 86.2, 87.9,
95.1, 143.3, 153.7, 153.9, 162.3 ppm. ESIMS; calcd. for
C14H23N4O5 [M+H]+ 327.1668, found 327.1666.
4-N-(N-Methylcarbamoyl)deoxycytidine (1b):[28] Deoxycytidine hy-
drochloride (791 mg, 3.0 mmol) was allowed to react successively
with TMSCl (1.52 mL, 12.0 mmol), phenyl chloroformate (564 µL,
4.5 mmol) and pyridine/40% aq. methylamine (1:1, v/v, 30 mL), as
described for the synthesis of 1a. The residue was dissolved in H2O
(30 mL) and washed three times with CHCl3 (30 mL). The aqueous
layer was concentrated in vacuo. The resulting precipitate was col-
lected by filtration, washed with cold iPrOH and dried in vacuo to
4-N-[N-(2-Methoxyethyl)carbamoyl]deoxycytidine (1e): Deoxycytid-
ine hydrochloride (791 mg, 3.0 mmol) was allowed to react success-
ively with TMSCl (1.52 mL, 12.0 mmol), phenyl chloroformate
(564 µL, 4.5 mmol), 2-methoxyethylamine (782 µL, 9 mmol) and
concd. NH3 (20 mL), as described for the synthesis of 1d. The resi-
due was dissolved in H2O (30 mL) and the aqueous solution was
washed three times with CHCl3 (30 mL). The aqueous layer was
concentrated under reduced pressure. The resulting precipitate was
collected by filtration, washed with cold ethyl acetate and dried in
vacuo to give 1e (976 mg, 96%). 1H NMR ([D6]DMSO): δ = 1.92–
2.03 (m, 1 H), 2.18–2.29 (m, 1 H), 3.25 (s, 3 H), 3.30–3.43 (m, 4
H), 3.50–3.64 (m, 2 H), 3.81–3.83 (m, 1 H), 4.17–4.23 (m, 1 H),
5.00 (t, J = 5.2 Hz, 1 H), 5.22 (d, J = 4.0 Hz, 1 H), 6.09 (dd, J =
6.3 Hz, 6.5 Hz, 1 H), 6.27 (d, J = 6.8 Hz, 1 H), 8.14 (d, J = 6.8 Hz,
1 H), 8.91 (br., 1 H), 9.83 (br., 1 H) ppm. 13C NMR ([D6]DMSO):
δ = 39.0, 40.7, 58.0, 61.0, 70.0, 70.8, 85.9, 87.8, 94.6, 143.4, 153.5,
153.7, 162.2 ppm. ESIMS: calcd. for C13H21N4O6 [M+H]+
329.1461, found 329.1460.
1
give 1b (724 mg, 85%). H NMR ([D6]DMSO): δ = 2.00–2.26 (m,
2 H), 2.73 (2 m, 3 H), 3.49–3.57 (m, 1 H), 3.80–3.82 (m, 1 H), 4.18–
4.20 (m, 1 H), 5.03 (t, J = 5.1 Hz, 1 H), 5.25 (d, J = 4.3 Hz, 1 H),
6.09 (dd, J = 6.3 Hz, 6.3 Hz, 1 H), 6.23 (d, J = 7.3 Hz, 1 H), 8.13
(d, J = 7.3 Hz, 1 H), 8.77 (br., 1 H), 9.89 (br., 1 H) ppm. 13C NMR
([D6]DMSO): δ = 26.0, 40.6, 61.0, 70.0, 85.8, 87.7, 94.7, 143.3,
153.5, 154.2, 162.2 ppm. ESIMS: calcd. for C11H17N4O5 [M+H]+
285.1199, found 285.1197.
4-N-(N-Ethylcarbamoyl)deoxycytidine (1c): Deoxycytidine hydro-
chloride (791 mg, 3.0 mmol) was allowed to react successively with
TMSCl (1.52 mL, 12.0 mmol), phenyl chloroformate (564 µL,
4.5 mmol) and pyridine/40% aq. ethylamine (1:1, v/v, 30 mL), as
described for the synthesis of 1a. The residue was redissolved in
H2O (30 mL) and washed three times with CHCl3 (30 mL). The
aqueous layer was concentrated in vacuo. The resulting precipitate
was collected by filtration, washed with cold iPrOH and dried in
General Procedure for the 5Ј-O-Dimethoxytritylation of Compounds
1a–e To Form Products 7a–e
4-N-Carbamoyl-5Ј-O-(4,4Ј-dimethoxytrityl)deoxycytidine
(7a):
Compound 1a (540 mg, 2 mmol) was rendered anhydrous by re-
peated co-evaporation with dry pyridine and finally dissolved in
dry pyridine (20 mL). To the solution was added DMTrCl (813 mg,
2.4 mmol) and the mixture stirred for 4 h. The mixture was diluted
with CHCl3 (30 mL) and the CHCl3 solution was washed three
times with saturated NaHCO3 (30 mL). The organic layer was col-
lected, dried with Na2SO4, filtered and concentrated under reduced
pressure. The crude product was purified by silica gel column
chromatography eluting with CH3Cl3/MeOH containing 1% pyri-
dine to give the product 7a (938 mg, 82%). 1H NMR ([D6]DMSO):
δ = 2.10 (m, 1 H), 2.29 (m, 1 H), 3.23 (m, 1 H), 3.73 (s, 6 H), 3.92
(m, 2 H), 4.25 (m, 1 H), 5.23 (d, J = 4.6 Hz, 1 H), 6.10 (dd, J =
6.0, 5.6 Hz, 1 H), 6.17 (d, J = 8.0 Hz, 2 H), 6.88 (d, J = 8.2 Hz, 4
H), 7.25–7.37 (m, 11 H), 7.95 (d, J = 8.0 Hz, 1 H), 9.75 (s, 1 H)
ppm. 13C NMR ([D6]DMSO): δ = 40.7, 63.0, 69.5, 85.6, 85.7, 94.7,
113.1, 126.6, 127.6, 129.6, 135.1, 135.2, 142.8, 144.1, 153.4, 154.1,
157.9, 162.2 ppm. C31H32N4O7·1/2H2O (581.62): calcd. C 64.02, H
1
vacuo to give 1c (680 mg, 76%). H NMR ([D6]DMSO): δ = 1.08
(t, J = 7.1 Hz, 3 H), 1.92–2.03 (m, 1 H), 2.19–2.26 (m, 1 H), 3.16–
3.24 (m, 2 H), 3.53–3.62 (m, 2 H), 3.80–3.82 (m, 1 H), 4.16–4.22
(m, 1 H), 5.00 (t, J = 5.1 Hz, 1 H), 5.22 (d, J = 4.3 Hz, 1 H), 6.09
(dd, J = 6.2 Hz, 6.3 Hz, 1 H), 6.24 (d, J = 7.1 Hz, 1 H), 8.13 (d, J
= 7.1 Hz, 1 H), 8.85 (br., 1 H), 9.81 (br., 1 H) ppm. 13C NMR
([D6]DMSO): δ = 15.1, 34.0, 40.7, 61.0, 70.0, 85.9, 87.8, 94.7, 143.3,
153.5, 153.6, 162.3 ppm. ESIMS: calcd. for C12H19N4O5 [M+H]+
299.1355, found 299.1352.
General Procedure for the 4-N-Carbamoylation of Deoxycytidine
Derivatives 1d, e
4-N-(N-Butylcarbamoyl)deoxycytidine (1d): Deoxycytidine hydro-
chloride (791 mg, 3.0 mmol) was rendered anhydrous by repeated
co-evaporation with dry pyridine and finally dissolved in dry pyri-
dine (30 mL). To the solution was added TMSCl (1.52 mL,
3634
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 3626–3637

4-N-Carbamoyldeoxycytidine Derivatives
FULL PAPER
5.72, N 9.63; found C 64.33, H 5.82, N 9.30 (%). ESIMS: calcd.
for C31H32N4NaO7 [M+H]+ 595.2169, found 595.2170.
tion with dry CH3CN and finally dissolved in dry CH2Cl2 (30 mL).
To the solution were added diisopropylamine (253 µL, 1.8 mmol),
1H-tetrazole (126 mg, 1.8 mmol) and (2-cyanoethoxy)bis(diisopro-
pylamino)phosphane (1.05 mL, 3.3 mmol). The resulting mixture
was stirred at room temperature for 6 h. The mixture was diluted
with CHCl3 (20 mL) and the CHCl3 solution was washed three
times with saturated NaHCO3 (30 mL). The organic layer was col-
lected, dried with Na2SO4, filtered and concentrated under reduced
pressure. The crude product was purified by silica gel column
chromatography with hexane/ethyl acetate containing 0.5% trieth-
4-N-(N-Methylcarbamoyl)-5Ј-O-(4,4Ј-dimethoxytrityl)deoxycytidine
(7b): Compound 1b (426 mg, 1.5 mmol) was allowed to react suc-
cessively with DMTrCl (610 mg, 1.8 mmol), as described for the
synthesis of 7a. Silica gel chromatography of the crude product
with CH3Cl3/MeOH containing 1% pyridine gave the product 7b
(818 mg, 93%). 1H NMR (CDCl3): δ = 2.16–2.63 (m, 2 H), 2.82
(d, J = 3.2 Hz, 3 H), 3.38–3.52 (m, 2 H), 3.80 (s, 6 H), 4.06–4.09
(m, 1 H), 4.44–4.47 (m, 1 H), 6.24 (dd, J = 5.9, 5.9 Hz, 1 H), 6.83–
6.86 (m, 4 H), 7.26–7.41 (m, 10 H), 8.03 (d, J = 6.1 Hz, 1 H), 8.89
(br., 1 H), 10.81 (br., 1 H) ppm. 13C NMR (CDCl3): δ = 26.7, 41.5,
54.8, 62.6, 70.4, 86.2 86.4, 86.4, 96.4 (br.), 112.9, 126.6, 127.5,
127.6, 129.6, 134.9, 135.0, 142.2 (br.), 143.9 154.7 (br.), 155.6 (br.),
158.1, 164.0 (br.) ppm. ESIMS: calcd. for C32H35N4O7 [M+H]+
586.2506, found 586.2504.
1
ylamine to give the product 8a (2.18 g, 94%). H NMR (CDCl3):
δ = 1.06–1.20 (m, 12 H), 2.21–2.31 (m, 1 H), 2.43–2.78 (m, 3 H),
3.36–3.89 (m, 12 H), 4.28–4.32 (m, 1 H), 4.53–4.69 (m, 1 H), 6.20–
6.26 (m, 1 H), 6.81–6.88 (m, 4 H), 7.25–7.42 (m, 10 H), 8.08–8.22
(m, 1 H), 9.06 (br., 1 H), 10.85 (br., 1 H) ppm. 13C NMR (CDCl3):
δ = 20.2, 20.3, 20.3, 20.4, 24.5, 24.6, 24.7, 40.7, 40.7, 41.0, 41.1,
43.1, 43.1, 43.3, 43.3, 55.2, 55.2, 58.0, 58.1, 58.3, 58.4, 61.9, 62.3,
71.5, 71.7, 72.3, 72.5, 85.4, 85.5, 85.6, 86.6, 86.6, 86.7, 86.8, 97.1
(br), 113.1, 117.2, 117.3, 126.9, 127.0, 127.8, 128.0, 128.0, 129.9,
129.9, 129.9, 135.0, 135.1, 142.6, 143.9, 155.1, 156.0 (br.), 158.4,
164.4 (br.) ppm. 31P NMR (CDCl3): δ = 149.5, 149.9 ppm. ESIMS:
calcd. for C40H50N6O8P [M+H]+ 773.3422, found 773.3441.
4-N-(N-Ethylcarbamoyl)-5Ј-O-(4,4Ј-dimethoxytrityl)deoxycytidine
(7c): Compound 1c (298 mg, 1 mmol) was allowed to react success-
ively with DMTrCl (407 mg, 1.2 mmol), as described for the syn-
thesis of 7a. Silica gel chromatography of the crude product with
CH3Cl3/MeOH containing 1 % pyridine gave the product 7c
1
(535 mg, 89%). H NMR (CDCl3): δ = 1.76 (t, J = 7.3 Hz, 3 H),
General Procedure for the Synthesis of 4-N-Modified Deoxycytidine
3Ј-Phosphoramite Derivatives 8b–e
2.16–2.24 (m, 2 H), 2.26–2.63 (m, 1 H), 3.26–3.38 (m, 2 H), 3.39–
3.53 (m, 2 H), 3.79 (s, 3 H), 3.80 (s, 3 H), 4.04–4.09 (m, 1 H), 4.42–
4.52 (m, 1 H), 6.23 (dd, J = 5.9, 5.9 Hz, 1 H), 6.83–7.41 (m, 14 H),
8.02 (d, J = 7.6 Hz, 1 H), 8.89 (br., 1 H) 10.82 (br., 1 H) ppm. 13C
NMR (CDCl3): δ = 14.8, 34.9, 41.7, 55.2, 62.8, 70.9, 86.2, 86.6,
86.8, 96.9 (br.), 113.1, 113.3, 126.9, 127.8, 127.9, 129.8, 129.8, 142.2
144.0, 154.0 (br.), 156.1 (br.), 158.4, 164.4 (br.) ppm. ESIMS: calcd.
for C33H37N4O7 [M+H]+ 601.2657, found 601.2667.
4-N-(N-Methylcarbamoyl)-5Ј-O-(4,4Ј-dimethoxytrityl)deoxycytidine
3Ј-O-(2-Cyanoethyl)-N,N-diisopropylphosphoramidite (8b): Com-
pound 7b (587 mg, 1.0 mmol) was rendered anhydrous by repeated
co-evaporation with dry CH3CN and finally dissolved in dry
CH2Cl2 (10 mL). To the solution were added ethyldiisopropylamine
(227 µL, 1.3 mmol) and chloro(2-cyanoethoxy)(diisopropylamino)-
phosphane (245 µL, 1.1 mmol). The resulting mixture was stirred at
room temperature for 30 min. The mixture was diluted with CHCl3
(10 mL) and the CHCl3 solution was washed three times with satu-
rated NaHCO3 (10 mL). The organic layer was collected, dried
with Na2SO4, filtered and concentrated under reduced pressure.
The crude product was purified by silica gel column chromatog-
raphy with hexane/ethyl acetate containing 0.5% triethylamine to
4-N-(N-Butylcarbamoyl)-5Ј-O-(4,4Ј-dimethoxytrityl)deoxycytidine
(7d): Compound 1d (489 mg, 1.5 mmol) was allowed to react suc-
cessively with DMTrCl (610 mg, 1.8 mmol), as described for the
synthesis of 7a. Silica gel chromatography of the crude product
with CH3Cl3/MeOH containing 1% pyridine gave the product 7d
1
(924 mg, 98%). H NMR (CDCl3): δ = 0.91 (t, J = 7.2 Hz, 3 H),
1.32–1.57 (m, 4 H), 2.15–2.22 (m, 2 H), 2.55–2.61 (m, 1 H), 3.23–
3.25 (m, 2 H), 3.37–3.51 (m, 2 H), 3.80 (s, 6 H), 4.05–4.09 (m, 1
H), 4.43–4.45 (m, 1 H), 6.22 (dd, J = 5.9, 5.9 Hz, 1 H), 6.83–6.86
(m, 4 H), 7.23–7.41 (m, 10 H), 8.02 (d, J = 7.7 Hz, 1 H), 8.84 (br.,
1 H), 10.82 (br., 1 H) ppm. 13C NMR (CDCl3): δ = 13.8, 20.1,
31.5, 39.7, 41.8, 55.1, 62.7, 70.9, 86.2, 86.6, 86.7, 113.1, 113.1 126.9,
127.8, 129.8, 135.1, 135.2, 142.1, 144.0, 154.0, 156.0, 158.3,
164.4 ppm. ESIMS: calcd. for C35H41N4O7 [M+ H]+ 629.2970,
found 629.2977.
1
give the product 8b (527 mg, 67%). H NMR (CDCl3): δ = 1.16–
1.29 (m, 12 H), 2.21–2.69 (m, 4 H), 2.73–2.75 (m, 3 H), 3.37–3.80
(m, 12 H), 4.20–4.25 (m, 1 H), 4.56–4.70 (m, 1 H), 6.25–6.28 (m,
1 H), 6.81–6.86 (m, 4 H), 7.25–7.42 (m, 10 H), 8.01–8.10 (m, 1 H),
8.90 (br., 1 H), 10.83 (br., 1 H) ppm. 13C NMR (CDCl3): δ = 20.0,
20.1, 20.2, 20.3, 24.3, 24.4, 24.5, 24.6, 26.3, 40.6, 40.6, 40.9, 43.0
43.2, 43.2, 55.0, 57.9, 58.0, 58.2, 62.2, 62.4, 72.0, 72.3, 72.5, 72.7,
85.2, 85.3, 85.5, 85.6, 86.5, 86.6, 96.9, 113.0, 117.2, 117.2, 126.8,
127.7, 127.8, 127.9, 129.7, 129.7, 129.8, 129.8, 134.9, 134.9, 135.1,
135.1, 142.0, 143.8, 154.4, 156.1, 158.3, 164.2 ppm. 31P NMR
(CDCl3): δ = 149.4, 150.0 ppm. ESIMS: calcd. for C41H52N6O8P
[M+H]+ 787.3579, found 787.3584.
4-[N-(2-Methoxyethyl)carbamoyl]-5Ј-O-(4,4Ј-dimethoxytrityl)de-
oxycytidine (7e): Compound 1e (492 mg, 1.5 mmol) was allowed to
react successively with DMTrCl (610 mg, 1.8 mmol), as described
for the synthesis of 7a. Silica gel chromatography of the crude prod-
uct with CH3Cl3/MeOH containing 1% pyridine gave the product
7e (851 mg, 90%): 1H NMR (CDCl3): δ = 2.39–2.46 (m, 1 H), 2.50–
2.56 (m, 1 H), 2.86 (s, 3 H), 3.32–3.46 (m, 6 H), 3.73 (s, 6 H), 3.92–
4.02 (m, 1 H), 4.35–4.41 (m, 1 H), 6.16 (dd, J = 5.8, 5.9 Hz, 1 H),
6.72–6.79 (m, 4 H), 7.15–7.34 (m, 10 H), 7.95 (d, J = 7.6 Hz, 1 H),
8.99 (br., 1 H), 10.69 (br., 1 H) ppm. 13C NMR (CDCl3): δ = 39.5,
41.6, 55.0, 58.4, 62.6, 70.6, 70.8, 86.1, 86.2, 86.5, 96.5 (br.), 112.9,
126.7, 127.6, 127.7, 128.1, 129.6, 135.0 135.1, 142.2, 143.8, 154.2
(br.), 155.7 (br.), 158.2, 163.8 (br.) ppm. ESIMS: calcd. for
C34H39N4O8 [M+H]+ 631.2768, found 631.2770.
4-N-(N-Ethylcarbamoyl)-5Ј-O-(4,4Ј-dimethoxytrityl)deoxycytidine
3Ј-O-(2-Cyanoethyl)-N,N-diisopropylphosphoramidite (8c): Com-
pound 7c (601 mg, 1.0 mmol) was allowed to react successively with
ethyldiisopropylamine (227 µL, 1.3 mmol) and chloro(2-cyano-
ethoxy)(diisopropylamino)phosphane (245 µL, 1.1 mmol), as de-
scribed for the synthesis of 8b. Silica gel chromatography of the
crude product with hexane/ethyl acetate containing 0.5% triethyl-
1
amine gave the product 8c (657 mg, 82%). H NMR (CDCl3): δ =
0.99–1.20 (m, 15 H), 2.10–2.17 (m, 1 H), 2.35–2.52 (m, 2 H), 2.52–
2.67 (m, 1 H), 3.17–3.56 (m, 8 H), 3.70–3.73 (m, 6 H), 4.13–4.15
(m, 1 H), 4.46–4.57 (m, 1 H), 6.14–6.21 (m, 1 H), 6.71–6.78 (m, 5
H), 7.09–7.33 (m, 9 H), 7.91–8.00 (m, 1 H), 8.89 (br., 1 H), 10.76
(br., 1 H) ppm. 13C NMR (CDCl3): δ = 14.7 20.2, 20.3, 20.3, 20.4,
4-N-Carbamoyl-5Ј-O-(4,4Ј-dimethoxytrityl)deoxycytidine 3Ј-O-(2-
Cyanoethyl)-N,N-diisopropylphosphoramidite (8a): Compound 7a
(1.73 g, 3.0 mmol) was rendered anhydrous by repeated co-evapora-
Eur. J. Org. Chem. 2006, 3626–3637
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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K. Miyata, A. Kobori, R. Tamamushi, A. Ohkubo, H. Taguchi, K. Seio, M. Sekine
FULL PAPER
1
24.5, 24.5, 24.6 24.7, 34.8, 40.8, 41.1, 41.1, 43.2, 43.3, 43.4, 55.2,
55.2, 58.1 58.2 58.4, 58.4, 62.4, 62.6, 72.3, 72.5, 72.8, 73.0 85.4,
the product 11 (409 mg, 82%). H NMR ([D6]DMSO): δ = –0.05–
0.03 (s, 12 H), 0.78–0.81 (m, 18 H), 2.03–2.11 (m, 1 H), 2.14–2.22
85.5, 85.6, 85.7, 86.6, 86.7, 97.1, 113.1, 117.2, 117.3 126.9, 127.0, (m, 1 H), 3.61–3.79 (m, 3 H), 4.25–4.30 (m, 1 H), 6.01 (dd, J =
127.8 128.0, 128.0, 129.8 129.9, 129.9, 130.0, 135.1, 135.1, 135.2, 5.9 Hz, 5.9 Hz, 1 H), 6.26 (d, 1 H, 7.6 Hz), 7.12 (br., 1 H), 7.95 (d,
142.0, 143.9, 153.9, 156.1, 158.4, 158.4 158.5 164.5 ppm. 31P NMR
(CDCl3): δ = 149.5, 150.1 ppm. ESIMS: calcd. for C41H52N6O8P
[M+H]+ 801.3741, found 801.3736.
J = 7.6 Hz, 1 H), 8.15 (br., 1 H), 9.66 (s, 1 H) ppm. 13C NMR
([D6]DMSO): δ = –5.6, –5.5, –5.0, –4.7, 17.7, 17.9, 25.6, 25.7, 40.5,
61.9, 70.7, 85.4, 86.8, 94.6, 142.6, 153.2, 154.0, 162.1 ppm. ESIMS:
calcd. for C22H43N4O5Si2 [M+H]+ 499.2772, found 499.2773.
4-N-(N-Butylcarbamoyl)-5Ј-O-(4,4Ј-dimethoxytrityl)deoxycytidine
3Ј-O-(2-Cyanoethyl)-N,N-diisopropylphosphoramidite (8d): Com-
pound 7d (1.26 mg, 2.0 mmol) was allowed to react successively
[4-15N]-3Ј,5Ј-O-Bis(tert-butyldimethylsilyl)-4-N-carbamoyldeoxy-
cytidine (15): [4-15N]-3Ј,5Ј-O-Bis(tert-butyldimethylsilyl)-2Ј-deoxy-
with ethyldiisopropylamine (453 µL, 2.6 mmol) and chloro(2-cyan- cytidine (14) (300 mg, 0.66 mmol) was rendered anhydrous by re-
oethoxy)(diisopropylamino)phosphane (491 µL, 2.2 mmol), as de-
scribed for the synthesis of 8b. Silica gel chromatography of the
peated co-evaporation with dry pyridine and finally dissolved in
dry CH2Cl2 (7 mL). To the solution were added pyridine (80.7 µL,
1.0 mmol) and phenyl chloroformate (101 µL, 0.8 mmol) and the
crude product with hexane/ethyl acetate containing 0.5% triethyl-
1
amine gave the product 8d (1.22 g, 74%). H NMR (CDCl3): δ = mixture was stirred at room temperature for 1 h. The mixture was
0.89–1.52 (m, 19 H), 2.10–2.21 (m, 1 H), 2.41–2.68 (m, 3 H), 3.15–
3.73 (m, 14 H), 4.13–4.19 (m, 1 H), 4.49–4.58 (m, 1 H), 6.16–6.21
diluted with CHCl3 (7 mL) and the CHCl3 solution was washed
three times with saturated NaHCO3 (10 mL). The organic layer
(m, 1 H), 6.85–7.33 (m, 14 H), 7.95–8.03 (m, 1 H), 8.80 (br., 1 H), was collected, dried with Na2SO4, filtered and concentrated under
10.81 (br., 1 H) ppm. 13C NMR (CDCl3): δ = 13.8, 20.0, 20.2, 20.3,
24.4, 24.4, 24.5, 24.6, 31.3, 31.4, 39.6, 40.8, 41.1, 43.0, 43.1, 43.2,
43.3, 55.1, 55.1, 58.0, 58.3 62.2, 62.2 72.6 85.4, 85.6 86.5, 86.6, 97.1,
113.0, 117.2, 126.9, 127.7, 127.9, 127.9 129.8, 129.9, 135.0, 135.1,
reduced pressure. Pyridine/concd. NH3 (1:1, v/v, 8 mL) was added
to the residue. After being stirred for another 2 h, the solution was
concentrated to dryness under reduced pressure. The residue was
dissolved in CHCl3 (15 mL) and the CHCl3 solution was washed
135.2, 141.9, 143.8, 158.3 ppm. 31P NMR (CDCl3): δ = 149.3, three times with saturated NaHCO3 (10 mL). The organic layer
150.0 ppm. ESIMS: calcd. for C41H52N6O8P [M+ H]+ 829.4054,
found 829.4046.
was collected, dried with Na2SO4, filtered and concentrated under
reduced pressure. The crude product was purified by silica gel col-
umn chromatography with hexane/CHCl3 to give the product 15
(250 mg, 75%). 1H NMR ([D6]DMSO): δ = 0.06 (m, 12 H), 0.86
(m, 18 H), 2.07–2.19 (m, 1 H), 2.20–2.30 (m, 1 H), 3.68–3.87 (m,
3 H), 4.32–4.38 (m, 1 H), 6.07 (dd, J = 6.2, 5.6 Hz, 1 H), 6.31 (d,
1 H, 7.4 Hz), 7.19 (br., 1 H), 8.01 (d, J = 7.1 Hz, 1 H), 8.21 (br., 1
H), 9.72 (d, JHN = 90.3 Hz, 1 H) ppm. 13C NMR ([D6]DMSO): δ
= –5.6, –5.6, –5.0, –4.8, 17.6, 17.9, 25.6, 25.7, 40.5, 62.0, 70.8, 85.5,
4-N-[N-(2-Methoxyethyl)butylcarbamoyl]-5Ј-O-(4,4Ј-dimethoxy-
trityl)deoxycytidine 3Ј-O-(2-Cyanoethyl)-N,N-diisopropylphosphor-
amidite (8e): Compound 7e (631 mg, 1.0 mmol) was allowed to re-
act successively with ethyldiisopropylamine (227 µL, 1.3 mmol) and
chloro(2-cyanoethoxy)(diisopropylamino)phosphane (245 µL,
1.1 mmol), as described for the synthesis of 8b. Silica gel chromato-
graphy of the crude product with hexane/ethyl acetate containing
86.9, 94.7, 142.8, 135.5, 154.2 (d, JCN = 16.2 Hz), 162.4 (d, JCN
=
1
0.5% triethylamine gave the product 8e (711 mg, 86%). H NMR
17.3 Hz) ppm. ESIMS: calcd. for C22H43N315NO5Si2[M + H]+
500.2742, found 500.2741.
(CDCl3): δ = 1.04–1.18 (m, 12 H), 2.19–2.26 (m, 1 H), 2.42 (t, J =
6.3 Hz, 1 H), 2.58 (t, J = 6.3 Hz, 1 H), 2.66–2.72 (m, 1 H), 3.31–
3.65 (m, 13 H), 3.72–3.80 (m, 6 H), 4.15–4.22 (m, 1 H), 4.51–4.65
Oligonucleotide Synthesis and Purification: All oligonucleotides
(m, 1 H), 6.20–6.28 (m, 1 H), 6.80–6.88 (m, 4 H), 7.20–7.43 (m, 10 were synthesized using an Applied Biosystems 392 oligonucleotide
H), 8.01–8.12 (m, 1 H), 9.12 (br., 1 H), 10.85 (br., 1 H) ppm. 13C
NMR (CDCl3): δ = 20.0, 20.1, 20.1, 20.2, 24.3, 24.4, 24.5, 39.5,
40.7, 40.7, 40.9, 41.0, 43.0, 43.0, 43.2, 55.1, 58.0, 58.3, 58.5, 62.2,
62.5, 70.9, 72.0, 72.2, 72.6, 72.9, 85.4, 85.5, 85.6, 85.7, 86.5, 86.7,
97.1, 113.1, 117.3, 117.3, 127.0, 127.8, 128.0, 128.1, 129.9, 130.0,
synthesizer on a 1 µmol scale, using PAC phosphoramidites (pac-
A, isopropyl-pac-G and acetyl-C) from Glen Research. A 0.1 
solution of each modified nucleoside phosphoramidite was used.
The standard pac-DNA phosphoramidite method was used for all
the procedures described for deprotection and purification. After
135.1, 135.2, 135.3, 135.3, 142.2, 144.0, 154.3, 156.2, 158.5, cleaving the oligonucleotides from the solid support under standard
164.5 ppm. 31P NMR (CDCl3): δ = 149.4, 149.9 ppm. ESIMS: conditions (aq. NH3, room temp., 1 h), DMTr-ON oligonucleotides
calcd. for C41H52N6O8P [M+H]+ 831.3846, found 831.3834.
were purified on a C18-cartridge and analyzed by HPLC. HPLC
was performed using the following systems. System A: Reversed-
phase HPLC was performed using a Waters Aliance system with a
Waters 3D UV detector and a µBondasphere 5 m C18 100 Å col-
umn (Waters, 3.9×150 mm). A linear gradient (0–30%) starting
from 0.1  NH4OAc and applying CH3CN was used at a flow rate
of 1 mL/min at 50 °C for 30 min. System B: Anion-exchange HPLC
was performed using a Waters Aliance system with a Waters 3D
UV detector and a Gen-Pak FAX column (Waters, 4.6×100 mm).
A linear gradient (0–60%) starting from 25 m sodium phosphate
buffer (pH = 6.0) and applying 25 m sodium phosphate buffer
(pH = 6.0) containing 1  NaCl (pH = 6.0) was used at a flow rate
of 1 mL/min at 50 °C for 30 min. The MALDI-TOF mass spec-
trometry was carried out by use of a Voyager RP (PerSeptive Biosy-
stems, Inc.).
3Ј,5Ј-O-Bis(tert-butyldimethylsilyl)-4-N-carbamoyldeoxycytidine
(11): 3Ј,5Ј-O-Bis(tert-butyldimethylsilyl)deoxycytidine (456 mg,
1.0 mmol) was rendered anhydrous by repeated co-evaporation
with dry pyridine and finally dissolved in dry CH2Cl2 (10 mL). To
the solution were added pyridine (121 µL, 1.5 mmol) phenyl chlo-
roformate (152 µL, 1.5 mmol) and the mixture was stirred at room
temperature for 1 h. The mixture was diluted with CHCl3 (10 mL)
and the CHCl3 solution was washed three times with saturated
NaHCO3 (15 mL). The organic layer was collected, dried with
Na2SO4, filtered and concentrated under reduced pressure. Pyri-
dine/concd.NH3 (1:1, v/v, 10 mL) was added to the residue. After
being stirred for another 2 h, the solution was concentrated to dry-
ness. The residue was dissolved in CHCl3 (15 mL), the CHCl3 solu-
tion was washed three times with saturated NaHCO3 (15 mL). The
organic layer was collected, dried with Na2SO4, filtered and con-
centrated under reduced pressure. The crude product was purified
by silica gel column chromatography with hexane/CHCl3 to give
Melting Temperature Analysis: A solution of an appropriate oligo-
nucleotide and the complementary strand, both of which were ar-
ranged to be 2 µ, was prepared in 10 m phosphate buffer (pH =
3636
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Eur. J. Org. Chem. 2006, 3626–3637

4-N-Carbamoyldeoxycytidine Derivatives
FULL PAPER
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Acknowledgments
This work was also supported by CREST of JST (Japan Science
and Technology) and in part supported by the COE21 project and
a grant from the Genome Network Project from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
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Published Online: June 7, 2006
Eur. J. Org. Chem. 2006, 3626–3637
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3637