Synthesis and properties of oligothymidylates incorporating an artificial bend motif

Article abstract of DOI:10.1002/(SICI)1522-2675(20000119)83:1<162::AID-HLCA162>3.0.CO;2-Y

The uridylyl-(3' → 5')-thymidine dinucleotide block 14 (cUpdU), having a cyclic structure between the 2'-hydroxy of the upstream uridine and the 5- substituent of the downstream thymidine, was synthesized (Schemes 1 and 2). This cyclic structure is a stab

Full text of DOI:10.1002/(SICI)1522-2675(20000119)83:1<162::AID-HLCA162>3.0.CO;2-Y

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Synthesis and Properties of Oligothymidylates Incorporating an Artificial
Bend Motif
by Kohji Seio, Takeshi Wada, and Mitsuo Sekine*
Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology,
Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
Dedicated to Prof. Dr. Frank Seela on the occasion of his 60th birthday
The uridylyl-(3' ! 5')-thymidine dinucleotide block 14 (cUpdU), having a cyclic structure between the 2'-
hydroxy of the upstream uridine and the 5-substituent of the downstream thymidine, was synthesized (Schemes 1
and 2). This cyclic structure is a stable mimic of the intraresidual H-bonding found in the anticodon loop of an E.
coli minor tRNAArg. The spectroscopic and molecular-mechanics analyses of the cyclized dinucleotides
predicted two major conformers, i.e., the turn and bent forms. The latter was expected to bend DNA oligomers
when incorporated into them. This expectation was ascertained by incorporating the bent dimer motif into tetra-,
deca-, or hexadecathymidylates by the conventional phosphoramidite method (see 18 ± 20 in Scheme 4). The
bending of oligonucleotides 18 ± 20 was demonstrated by 31P-NMR and CD spectra and gel-electrophoretic
studies. The duplex formation of these modified oligonucleotides with oligodeoxyadenylates was also studied.
The decreased thermal stability of the duplexes when compared with unmodified ones indicates distorted
structures of the modified duplexes. The 3D computer model of the duplexes showed a bend of ca. 308 with a
ꢀbulge-outꢁ at the position of an adenosine residue facing the cyclized dimer. The artificially bent DNAs might
become a new tool for the study of the effect of DNA bending induced in DNA/DNA-binding protein
interactions.
1. Introduction. ± DNA Bending induced by DNA-binding proteins (DBP) is an
important event in a series of biological reactions related to the expression,
recombination, and replication of genetic information [1]. The detailed tertiary
structures of some bent DNAs have been unveiled to date with the aid of X-ray
crystallographic or multi-dimensional NMR spectroscopic techniques [1]. Sharply bent
structures have also been discovered in the active site of hammerhead ribozymes [2] as
well as in the region between the anticodon and its upstream nucleoside in tRNA [3].
These bent DNAs or RNAs can apparently be stabilized by binding to proteins or
their 3-dimensional structures. If such a bent structure could be created independently
from these complex interactions, a variety of intrinsic studies would be developed
taking advantage of its inherent properties that cannot be acquired from the usual
linear DNAs or RNAs. For example, unique motifs, such as extremely stabilized loops
or duplexes with a bulge-out or flip-out structure, could be generated.
Therefore, it is of great interest to create an artificially bent motif by introducing a
chemically modified nucleotide unit having a rigid bent backbone structure into
oligonucleotides. We previously reported our studies of chemical fixation of uridylic
acid [4] and uridylyl-(3' ! 5')-uridine [5] in conformations similar to those of a
mononucleotide unit seen in the A-type RNA duplex and a sharply bent U-turn
structure existing in the tRNA anticodon loop, respectively, by introducing covalent

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bonding bridges into the parent structures. Preliminary molecular-mechanics calcu-
lation analysis of the cyclic uridylyl-(3' ! 5')-uridine [5] having a CH2C(O)NHCH2
linker revealed a two-state equilibrium between the turn and stretch conformations.
The NMR studies suggested that the latter is more stable than the former and has a
bending property because of the fixed orientation of the two bases.
In this paper, we report the synthesis and conformational analysis of a uridylyl-
(3' ! 5')-2'-deoxyuridine derivative having the covalent-bonding linker and the
synthesis of oligothymidylates containing this cyclic dimer. Conformation of the dimer
unit and the modified oligothymidylates was analyzed by spectroscopic, electro-
phoretic, and computational methods.
2. Results and Discussion. ± 2.1. Chemical Synthesis of the 3'-Terminal Nucleoside
Unit 1. The 3'-terminal nucleoside unit 1 was synthesized from 5-(hydroxymethyl)-2'-
deoxyuridine (2) [6] by treatment with chlorotrimethylsilane in dioxane [7] ( ! 3) and
then with sodium azide in DMF via 5-(azidomethyl)-2'-deoxyuridine (4; 73% yield)
(Scheme 1). The reaction of 4 with pivaloyl chloride (pivCl) in pyridine gave the
selectively 5'-protected product 5 in 70% yield. The 3'-tetrahydropyranylation (Thp) of
5 ( ! 6) followed by Pd/C-catalyzed hydrogenation furnished the 3'-terminal nucleo-
side unit 1 in an overall yield of 64% from 5.
Scheme 1
Piv ˆ Me3CCO, Thp ˆ tetrahydro-2H-pyran-2-yl
2.2. Chemical Synthesis of the Interresidually Cyclized Uridylyl-(3' ! 5')-2'-deoxy-
uridine 14. The interresidually cyclized uridylyl-(3' ! 5')-2'-deoxyuridine 14 was
synthesized from the 3'-terminal nucleoside unit 1 and the 5'-terminal nucleoside unit
7 [7] (Scheme 2). First, acid 7 was converted to ester 8 by treatment with N-

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hydroxysuccinimide in the presence of N-[3-(dimethylamino)propyl] N'-ethyl-carbo-
diimide hydrochloride (EDC) [8]. Condensation of the activated ester 8 with 1 in the
presence of Et3N gave the protected dimer 9 in 86% yield. The tBuMe2Si group of 9 was
removed by treatment with tetrabutylammonium fluoride hydrate to give alcohol 10 in
71% yield, and the liberated 3'-hydroxy group of the 5'-upstream nucleoside was
phosphorylated by treatment with cyclohexylammonium S,S'-diphenyl phosphorodi-
thioate [9] in the presence of isodurenedisulfonyl dichloride (DDS) [9] and 1H-
tetrazole [10] to give the phosphorodithioate derivative 11 in 76% yield. The
conversion of 11 to the cyclized dimer 13 was accomplished in 64% yield by the
intramolecular in situ cyclization of the phosphotriester intermediate 12, which was
obtained by alkaline hydrolysis of 11, under the conditions using DDS as a condensing
reagent and 1H-tetrazole as a nucleophilic catalyst. The successive deprotection of the
cyclized dimer 13 by treatment with aqueous KOH solution and then with 80% AcOH
gave the cyclized uridylyl-(3' ! 5')-2'-deoxyuridine derivative 14 in 58% yield
(Scheme 2). The detailed conformation of the fully deprotected cyclic dimer 14 is
described in Sect. 2.3, and the results were used in Sect. 2.5 to predict the 3D structure
of the oligonucleotide containing the dimer unit 14.
To incorporate the cyclized dimer unit into an oligonucleotide, the phosphoramidite
unit 17 of the cyclized dimer was synthesized via 13 as a synthetic intermediate
(Scheme 3). The (MeO)2Tr and Thp groups of 13 were removed under acidic
conditions ( ! 15), and the free 5'-hydroxy group of the 5'-upstream nucleoside unit
was protected again with the (MeO)2Tr group to give the 3'-hydroxy derivative 16 in
78% yield. The latter was then phosphitylated by treatment with 1.2 equiv. of 2-
cyanoethyl diisopropylphosphoramidochloridite (ˆchloro(diisopropylamino)(2-cyano-
ethoxy)phosphine) [11] in the presence of 1.2 equiv. of ethyldiisopropylamine as a base
to give the desired phosphoramidite unit 17 in 71% yield.
The 31P-NMR spectrum of 17 showed four signals near 150 ppm (146.99, 150.10,
150.35, and 150.44 ppm), which are derived from the phosphoramidite groups of the
four diastereoisomers, and three signals around 25 ppm (24.78, 24.86, and 25.01 ppm)
with the intensity ratio of 1:2 :1, which are ascribed to the internucleotidic phosphoryl
groups attached to the S-atom.
2.3. Conformational Analysis of the Cyclized Uridylyl-(3' ! 5')-2'-deoxyuridine 14.
2.3.1. Circular Dichroism (CD) Studies. The CD spectra of cUpdU (14) and unmodified
uridylyl-(3' ! 5')-uridine (UpU) at 20 and 808 are shown in Fig. 1. The Cotton effect
around 270 nm is the most important for the analysis of the conformational properties
of these dinucleoside monophosphates because the CD pattern in this region reflects
the orientation and interaction of the pyrimidine base of the 3'- and 5'-terminal
nucleoside unit [12]. In the CD spectra of UpU, the intensity of this Cotton effect at 808
decreased to 60% at 208 indicating the conformational flexibility of UpU. In contrast,
the intensity of the Cotton effect of cUpdU at 808 was maintained at 80% at 208. This
different temperature dependence clearly pointed to an increased conformational
rigidity of cUpdU (14) induced by the cyclic structure. In the previous study of similarly
cyclized dimers, in which the 3'-terminal nucleoside of 14 was replaced by a
ribonucleoside, a similar conformational rigidity was observed in the CD spectra; the
conformation was more rigid in the 3'-downstream ribonucleoside derivative than in 14
[5]. In general, the conformations of ribonucleosides are more rigid than those of

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Scheme 2
DMTrˆ (MeO)2Tr, TBDMS ˆ tBuMe2Si, Piv ˆ Me3CCO, Thp ˆ tetrahydro-2H-pyran-2-yl.

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Scheme 3
DMTrˆ (MeO)2Tr, Thp ˆ tetrahydro-2H-pyran-2-yl
Fig. 1. CD Spectra of a) cUpdU (14) and b) UpU. In 10 mm sodium phosphate (pH 7.0) at 20 and 808.

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deoxyribonucleosides because of the additional electronic and steric effect of the 2'-
hydroxy group [13]. This is also true for the cyclic dimers. The conformational rigidity
of cUpdU is a suitable property for the stabilization of bent structures by incorporating
this cyclized dimer unit into an oligonucleotide.
2.3.2. Analysis of the Ribose Conformation by 1H-NMR. The ribose moiety
of the natural nucleic acids is in equilibrium between the N-type and S-type con-
formers [14] (Fig. 2). Therefore, the conformation of the ribose moiety is described
by the respective population of the N-type conformation (P(N-type)) and the
S-type conformation (P(S-type)). The population of each conformer is calculated
from the H,H-coupling constants between the protons on the ribose or deoxyribose
ring.
Fig. 2. The two major conformers of deoxyribonucleosides which exist in equilibrium
The H,H- and H,P-coupling constants of 14, measured at 400 MHz, are shown in
Table 1. Among the H,H-coupling constants of the ribose moiety, the J(2',3') and
J(1',2') ‡ J(3',4') values are of special importance. These values are nearly constant in
natural or synthetic ribonucleic-acid derivatives having no structural constraints; the
average values of J(2',3') and J(1',2') ‡ J(3',4') are 5.2 Hz and 9.8 Hz, respectively [15].
Structurally constrained nucleic acids have values deviated from these typical ones.
From this point of view, the rather smaller J(1',2') ‡ J(3',4') (7.4 Hz) of the 5'-upstream
ribose of cUpdU indicates some distortion of the ribose conformation. However, the
distortion might be small, because the Karplus equation [16] suggested that the J(2',3')
value of 5 Hz is realized only when the two equilibrium conformations are N-type and
S-type regardless of the puckering amplitude (Fig. 3) and the J(2',3') value of the
cyclized dimer (4.8 Hz) is close to 5 Hz. Hence, the smaller J(1',2') ‡ J(3',4') observed
would be explained by the S-type and N-type conformers with small distortion. In
contrast, the conformation of the 3'-downstream deoxyribose moiety of 14 is not
distorted because both the J(2',3') (6.1 Hz) and J(1',2') ‡ J(3',4') (10.2 Hz) are close to
the standard values observed for the natural 2'-deoxyribonucleosides (6.6 and 10.5 Hz,
resp.). The P(N-type) and P(S-type) of both the 5'-upstream and 3'-downstream sugar
moieties were calculated with the formula P(N-type) ˆ J(3',4')/(J(1',2') ‡ J(3',4')) [16].
Consequently, the P(N-type) of the 5'-upstream ribose residue is 38%, while the
P(N-type) of the 3'-downstream deoxyribose residue is 33%. In brief, the major
conformers of both the 5'- and 3'-terminal sugars are S-type. The population P(S-type)
is 60 ± 70%.
2.3.3. NOESY Spectrum of cUpdU. To obtain information on the relative location of
the 5'-upstream and 3'-downstream residues, the NOESY spectrum of cUpdU was

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Fig. 3. Simulation of J(2',3') by the Karplus equation [16]. P ˆ phase angle of pseudorotation. Plots of the values
obtained from the simulations with the puckering amplitude fixed at 358 (*) and 408 (.).
a
1
Table 1. H,H Coupling Constants [Hz] in the H-NMR Spectrum (400 MHz) of 14 )
Coupling constant J [Hz]
J(1',2')
J(1',2'')
J(2',3')
J(2'',3')
J(3',4')
J(4',5')
J(4',5'')
J(3',P)b)
5'-Terminal nucleoside
3'-Terminal nucleoside
4.6
6.7
±
6.7
4.8
3.7
±
6.1
2.8
3.5
2.8
n.d.
4.0
n.d.
8.0
a) The critical values for the determination of the ribose or deoxyribose conformation are written in italics.
b) Measured at 109 MHz.
recorded. Although many cross-peaks are present, all of them are derived from the
intraresidual H,H interactions, and no interresidual cross-peaks are observed. This
result indicates that cUpdU has a conformation in which the distance between the 5'-
and 3'-terminal nucleoside units is long. This distance information based on the
interresidual H,H interactions was used to analyze the molecular-mechanics simulation
results of the 3D structure of 14 described in Sect. 2.3.4.
2.3.4. Molecular-Mechanics Studies. Detailed three-dimensional model structures of
the cyclic dimer 14 were built by molecular-mechanics calculations with the
MonteCarlo conformation search and energy minimization [17]. The AMBER* force
field [18][19] and GB/SA solvent model [19] were used in all simulations. Four
calculation runs were carried out, in which the ribose moieties of the 5'-upstream and
3'-downstream nucleoside units were fixed in the {N-type and N-type} (A), {N-type and
S-type} (B), {S-type and N-type} (C), and {S-type and S-type} (D) conformations,
respectively. The ribose moieties were fixed in the standard N- and S-type
conformations because the AMBER* force field did not give results compatible with
the experimental data in terms of ribose conformation in our test simulations (data not
shown). The structures having the lowest energies obtained from each run are shown in
Fig. 4.
The structures A ± C can be divided into two categories, namely, the ꢀturnꢁ (A and B
in Fig. 4) and ꢀbentꢁ forms (C and D). The two runs in which the ribose moiety of the 5'-

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Fig. 4. Low-energy structures of cUpdU (14) calculated by fixing the 5'-terminal ribose and 3'-terminal
deoxyribose moieties in the {N-type and N-type} (A), {N-type and S-type} (B), {S-type and N-type} (C), and
{S-type and S-type} (D) conformation
upstream nucleoside unit was fixed in the N-type conformation gave energetically
unfavorable turn conformations (E ˆ 882.74 and 888.68 kJ/mol for A and B, resp.),
whereas the two runs in which the ribose moiety of the 5'-upstream nucleoside units was
fixed in the S-type conformation gave energetically more favorable bent conformers
(E ˆ 893.02 and 893.03 kJ/mol for C and D, resp.). In the turn conformers, the
distance between the 5'-upstream and 3'-downstream residues is short enough to allow
NOEs between the interresidual protons. For example, the inter-proton distances
H
C(2')(5'-upstream residue)/H C(3')(3'-downstream residue), H C(2')(5'-up-
stream residue)/H C(2')(3'-downstream residue), H C(3')(5'-upstream residue)/
C(3')(3'-downstream residue), and H C(3')(5'-upstream residue)/H C(2')(3'-
H
downstream residue) are 4.78, 2.80, 3.18, and 2.37 Š, respectively, for conformer A, and
2.66, 4.42, 2.31, and 4.64 Š, respectively, for conformer B. In the bent conformers C and
D, the interresidual distances are too long to give NOEs. Thus, the structural properties
of the bent conformers are in agreement with the properties of 14 established by the
H,H coupling constants (Sect. 2.3.2) and NOESY spectrum (Sect. 2.3.3), i.e., the major
conformer of the cyclic dimer has an S-type ribose at the 5'-terminus, with a long
distance between the 5'- and 3'-terminal residues. Therefore, the three-dimensional
structure of 14 in aqueous solution must be similar to that shown for C and D (Fig. 4).
From the above conformation analyses by means of CD and 1H-NMR spectroscopy
and molecular mechanics, it was concluded that the cyclic dimer 14 has a rigid
conformation with the S-type ribose conformation and long interresidual distance.
Thus, the dinucleoside monophosphate 14 having these conformational properties is

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highly recommendable as the dimer unit to be introduced into the oligonucleotides as a
rigid bent structure.
2.4. Chemical Synthesis of the Oligonucleotide Having a Bent Structure. An
oligothymidylate having a bent structure was synthesized by using the phosphoramidite
dimer unit 17, derived from 14. First, the tetrathymidylate 18 was synthesized to confirm
the efficient introduction of the dimer unit under the standard conditions used in the
solid-phase synthesis (Scheme 4). The coupling reaction, according to the general solid-
phase oligonucleotide synthesis, followed by the treatments with aqueous NaOH
solution and then with 80% AcOH, gave the tetramer 18. The structure of 18 was
supported by the 1H- and 31P-NMR spectra (D2O); a low-field d(P) indicating that the
bent structure stabilized by the cyclic amide linkage was preserved even in the
oligonucleotide because the low-field shift of the internucleotidic phosphate is the
general indicator of the bent backbone conformation [20].
Scheme 4
The 1H-NMR spectra of tetramer 18 in D2O showed the signals of the Me groups of the two thymine
bases around 1.9 ppm and four signals of the 6 protons of the four pyrimidine bases around 7.5 ± 7.9 ppm. The
31P-NMR spectra gave three signals due to the three internucleotidic phosphate groups around 0 ppm. The
d(P) of the internucleotidic phosphate of the cyclic dimer unit was found in the lowest field in the 31P-NMR
spectrum.

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2.5. Conformational Properties of the Decanucleotide Having a Cyclic Dimer Unit.
A decathymidylate 19 having the cyclic-dimer unit 14 at the center was synthesized by
the solid-phase synthesis. The decathymidylate 19 was expected to have a bent structure
induced by the cyclic structure.
It is well known that a change in three-dimensional DNA duplex structures is
reflected sensitively by the mobility in polyacrylamide-gel electrophoresis (PAGE)
[21]. The 20% PAGE of the decathymidylate 19, T10, and T11 are shown in Fig. 5. The
molecular-mass difference between 19 and T10 (DMr ˆ 57) is smaller than that between
19 and T11 (DMr ˆ 246). On the contrary, the difference in the gel shift between 19 and
T
10 is larger than that between 19 and T11. This result indicates a rigid structure for 19,
which is induced by introduction of the cyclic dimer unit 14, thereby restraining the
mobility of 19 on PAGE.
Fig. 5. 20% PAGE of T4(cUpdU)T4 (19), T11, and T10. Lanes 1 and 5, bromophenol blue; Lane 2, T11; Lane 3,
19; Lane4, T10
.
The rigid conformation of 19 was also reflected in a smaller temperature
dependence of its CD spectra at 20, 40, and 808 compared to those of T10 at these
temperatures (Fig. 6,a and b, resp.). Although the cyclic structure unit of 19 reduces
the conformational flexibility, it seems only effective near the modified site and is not
likely to affect the overall conformation of the oligomer because the whole shape of the
CD spectra of 19 is similar to that of T10. Oligomer 19 having a locally fixed
conformation as described above, would be suitable for the modification of a duplex
structure when hybridized to its complementary strand (see Sect. 2.6).
2.6. Duplex Formation of the Deca- or Hexadecathymidylates Having a Cyclic
Dimer Unit. The decathymidylate 19 was mixed with an equimolar amount of dA10, and
the UV melting curve of the duplex formed was recorded and compared with that of
T10 ´ dA10 (Fig. 7 and Table 2). The considerable decrease of the Tm value of 19 ´ dA10
compared with that of T10 ´ dA10 (DTm ˆ 208) suggested disruption of base pairing.
The model structure of the disrupted base pairing and the whole duplex was built by
replacing the fifth and sixth thymidine units of T10 by the turn (Fig. 4, B) or bent

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Fig. 6. CD Spectra a) of decathymidylate 19 containing the cyclic dimer unit and b) of decathymidylate T10. In
10 mm sodium phosphate (pH 7.0).
Fig. 7. UV Melting curves of the duplexes T4(cUpdU)T4 ´ decadeoxyadenosine (19 ´ (dA)10) and of decathymi-
dylate ´ decadeoxyadenosine (T10 ´ (dA)10). Measured in 10 mm sodium phosphate and 150 mm NaCl (pH 7.0)
Table 2. Melting Temperature [8] of the DNA Duplexes 19 ´ dA10, T10 ´ dA10, and 20 ´ dAn (n ˆ 14 ± 19). Measured
in 10 mm sodium phosphate ± 150 mm NaCl buffer
19 ´ (dA)10 T10 ´ (dA)10 20 ´ (dA)14 20 ´ (dA)15 20 ´ (dA)16 20 ´ (dA)17 20 ´ (dA)18 20 ´ (dA)19
Tm [8]
8
28
26.5
30.3
32.0
33.0
33.2
33.0
conformer of 14 (Fig. 4, D) on the computer graphics (Fig. 8). While the decath-
ymidylate containing the turn conformer B (see Fig. 8, E) does not seem suitable for
duplex formation because of its extremely sharp bend, the one containing the bent
conformer D (see Fig. 8, F) seems suitable. However, the distance between the N(1)-
atoms on the 5'- and 3'-terminal pyrimidine rings of the cyclic dimer unit is ca. 9 Š,
which is twice as long as the distance seen in the standard B-type duplex (4 Š) [22].
Therefore, the disruption of the base pairing could occur at this site.

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Fig. 8. Predicted structures E and F of a dodecathymidylate T5(cUpdU)T5 incorporating the turn conformer B
and the bent conformer D, respectively, of Fig. 4
There are two plausible models for the disrupted duplex structure. One is the
ꢀbulge-outꢁ structure in which a single deoxyadenosine residue at the site opposite to
the cyclic dimer unit bulges out to compensate for the widened internucleotidic
distance in the oligothymidylate strand, and the other is the ꢀno-bulge-outꢁ structure in
which no adenosine residue bulges out but the base-pairing is distorted because of the
unusual geometry. If the ꢀbulge-outꢁ model structure is correct, either the 5'- or 3'-
terminal deoxythymidine residue must dangle without forming any base-pairing when
the lengths of the oligothymidylates containing a cyclic-dimer unit and the oligodeox-
yadenylate are the same. Therefore, the validity of this model must be examined by
measuring the dependence of the Tm values on the chain length of the oligode-
oxyadenylate.
Because the Tm values of the oligodeoxyadenylate ´ 19 duplexes were too low
to give correct Tm values when the chain length of the oligodeoxyadenylate was
altered, the hexadecathymidylate 20 was synthesized, and the Tm values of the
duplex formation between 20 and oligodeoxyadenylates (dA)14 to (dA)19 having
the chain length of 14 ± 19 units were measured (Fig. 9 and Table 2). The Tm values
of the duplexes formed between 20 and oligodeoxyadenylates (dA)14 to (dA)19
rose with the length of the oligodeoxyadenylate increasing from 14 to 17 units,
and then reached a plateau (Fig. 10). These observations are consistent with the
ꢀbulge-outꢁ model described above wherein the terminal residues of the oligo-
thymidylate strand dangle until the oligodeoxyadenylate chain is lengthened up
to (dA)16, but the number of base-pairing is constant after the chain length has
reached 17 units. Shown in Fig. 11 is the duplex model structure of 20 ´ (dA)17
which has the bulged-out adenosine residue at the middle of the duplex. In this
model, the duplex bends by ca. 308, and the base-pairings are loosened at the middle
of the strand. This base-pair disruption could contribute to the observed duplex
instability.

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Fig. 9. UV Melting curves of the duplexes T7(cUpdU)T7 ´ oligodeoxyadenylate (20 ´ (dA)n) having various chain
lengths. Measured in 10 mm sodium phosphate and 150 mm NaCl (pH 7.0). The vertical axis indicates the
relative absorbance when the absorbance at 908 is normalized to unity.
Fig. 10. Relationship between the number n of nucleotide units in the oligodeoxyadenylate chain and the Tm
values [8] of the duplexes T7(cUpdU)T7 ´ oligodeoxyadenylate (20 ´ (dA)n) having various chain lengths (see
Table 2)
3. Conclusions. ± In this study, an artificially bent DNA strand was chemically
synthesized for the first time by the introduction of a conformationally rigid cyclized
UpdU dimer at the center of oligothymidylates. No other report on the synthesis of
such bent DNA oligomers by chemical approaches has been published to date. Since an
increasing number of studies on DNA-protein and RNA-protein interactions [2] have
revealed that DNA bending is a crucial step in the regulation of the function of nucleic
acids [1], it is of great importance to have available an artificially bent structure of
DNA or RNA that will be useful as a fixed bent motif for investigating the structure-
function relationship of bent DNA or RNA.

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Fig. 11. Duplex model structure of T7(cUpdU)T7 ´ (dA)17 (20 ´ (dA)17) which has a bulged-out adenosine residue
at the middle of the duplex
Experimental Part
General. Pyridine was distilled twice from TsCl and from CaH2 and then stored over 4 Š molecular sieves.
TLC: Merck silica gel 60-F-254 (0.25 mm). Column chromatography (CC): silica gal C-200 from Wako Co. Ltd.;
a minipump for a goldfish bowl was conveniently used to attain sufficient pressure for rapid CC separation;
reversed-phase CC with mBondapak-C-18 silica gel (prep S-500, Waters). Reversed-phase HPLC: mBonda-
sphere C-18 column, linear gradient starting from 0.1m NH4OAc (pH 7.0) and applying 0 ± 30% of MeCN at a
flow rate of 1.0 ml/min for 30 min. UV Spectra: Hitachi-U-2000 spectrometer; lmax in nm. CD Spectra: Jasco-J-
1
500-C spectrometer, 0.5-cm cell. H-, 13C-, and 31P-NMR Spectra: at 270, 68, and 109 MHz, resp.; Jeol-EX270
spectrometer; chemical shifts d in ppm rel. to SiMe4 or DSS (sodium 3-(trimethylsilyl)propane-1-sulfonate) for
1H-NMR, CDCl3 (ˆ 77 ppm) or DSS (ˆ 0 ppm) for 13C-NMR, and 85% phosphoric acid (ˆ 0 ppm) for 31P-
NMR, J in Hz; J(H,H) and NOESYat 400 MHz with Varian-Unity-400 spectrometer; protons at the 5'-terminal
unit are labelled with ꢀaꢁ, those at the 3'-terminal unit with ꢀbꢁ. Elemental analyses were performed by the
Microanalytical Laboratory, Tokyo Institute of Technology at Nagatsuta.
5-(Azidomethyl)-2'-deoxyuridine (4) [6]. To a soln. of 5-(hydroxymethyl)-2'-deoxyuridine [6] (9.0 g,
34.9 mmol) in 1,4-dioxane (250 ml) in a sealed tube, chlorotrimethylsilane (19.0 g, 175 mmol) was added. The
soln. was stirred at 508 for 2.5 h and then evaporated, and the residue was dissolved in DMF (100 ml). NaN3
(6.81 g, 105 mmol) was added, and the resulting soln. was stirred at 608 for 15 min. The precipitate was removed
by filtration, the filtrate evaporated, and the residue submitted to CC (silica gel, CH2Cl2/MeOH 100 :5): 4 (7.5 g,
1
77%). M.p. 134 ± 1368 ([6a] 133 ± 1358). H- and 13C-NMR: identical with those of the authentic sample [6b].
5-(Azidomethyl)-2'-deoxy-5'-O-pivaloyluridine (5). To a soln. of 4 (200 mg, 0.69 mmol) in pyridine (5 ml),
pivaloyl chloride (111 ml, 0.90 mmol) was added, and the resulting mixture was stirred for 1.5 h at r.t. The soln.
was diluted with CHCl3 (50 ml), washed with sat. aq. NaHCO3 soln., dried (Na2SO4), and evaporated and the
residue submitted to CC (silica gel, CHCl3/MeOH 100 :2): 5 (177 mg, 70%). M.p. 153 ± 1548 (hexane/AcOEt).

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Helvetica Chimica Acta ± Vol. 83 (2000)
1H-NMR (270 MHz, CDCl3): 1.23 (s, 3 Me); 2.12 (m, H C(2')); 2.50 (m, H C(2)); 4.09 ± 4.45 (m, H C(3'),
H
C(5'), CH2N3); 4.48 (m, H C(4'), H C(5)); 6.23 (m, H C(1')); 7.52 (s, H C(6)); 8.40 (br., NH).
13C-NMR (68 MHz, CDCl3): 27.17; 38.89; 40.74; 47.10; 63.65; 71.52; 84.75; 85.59; 109.92; 137.70. Anal. calc. for
C15H21N5O6: C 49.05, H 5.76, N 19.06; found: C 49.17, H 5.86, N 18.83.
5-(Aminomethyl)-2'-deoxy-5'-O-pivaloyl-3'-O-(tetrahydro-2H-pyran-2-yl)uridine (1). To a soln. of 5 (5.4 g,
14.7 mmol) in CH2Cl2 (150 ml) 1,4-dihydro-2H-pyran (27 ml, 294 mmol) and CF3COOH (570 ml, 7.4mmol)
were added, and the resulting mixture was stirred for 10 h. The mixture was washed with sat. aq. NaHCO3 soln.,
the org. layer dried (Na2SO4) and evaporated, and the residue dissolved in MeOH (150 ml). To this soln., Pd/C
(1 g) was added, and the mixture was stirred for 14 h under H2. Pd/C was removed by filtration, the filtrate
evaporated and the residue submitted to CC (silica gel, CHCl3/MeOH 100 :2): 1 (4.0 g, 64%). 1H-NMR
(270 MHz, CDCl3): 1.18 (m, 3 Me); 1.82 (m, 3 CH2); 2.05 (m, H' C(2')); 2.48 (m, H C(2')); 3.60 (s, CH2N);
3.80 (m, CH2O); 4.18 ± 4.40 (m, H C(3'), H C(4'), 2 H C(5')); 4.67 (s, OCHO); 6.25 (m, H C(1')); 7.45, 7.42
(2s, H C(6)). 13C-NMR (68 MHz, CDCl3): 19.21; 19.27; 25.21; 27.21; 30.55; 30.60; 37.77; 38.74; 38.83; 39.46;
62.68; 63.79; 63.90; 75.35; 76.14; 82.27; 83.04; 85.18; 85.50; 97.86; 98.40; 116.14; 135.33; 135.45; 150.01; 163.09;
178.11. Anal. calc. for C20H31N3O7 ´ 1/2 H2O: C 55.35, H 7.34, N 9.67; found: C 55.31, H 7.56, N 8.65.
3'-O-[(tert-Butyl)dimethylsilyl]-2'-O-{2-{{{1-[2'-deoxy-5'-O-pivaloyl-3'-O-(tetrahydro-2H-pyran-2-yl)-b-d-
ribofuranosyl]-1,2,3,4-tetrahydro-2,4-dioxopyrimidin-5-yl}methyl}amino}-2-oxoethyl}-5'-O-(4,4'-dimethoxytrityl)-
uridine (9). N-[3-(Dimethylamino)propyl]-N-ethylcarbodiimide hydrochloride (58 mg, 0.3 mmol) and N-
hydroxysuccimide (35 mg, 0.3 mmol) were added to a soln. of 7 (148 mg, 0.2 mmol) in DMF (2 ml). After 7.5 h,
1 (85 mg, 0.2 mmol) and Et3N (28 ml, 0.2 mmol) were added, and the resulting soln. was stirred for 1 h. The
mixture was partitioned between CHCl3 and sat. aq. NaHCO3 soln., and the org. layer was washed 5 times with
sat. aq. NaHCO3 soln., dried (Na2CO3), and evaporated. The residue was submitted to CC (silica gel, CHCl3/
1
MeOH 100 :1): 9 (197 mg, 86%). H-NMR (270 MHz, CDCl3): 0.12, 0.12 (m, 2 MeSi); 0.72, 0.83 (2s, 6 and
3 H, 3 Me); 1.18 (s, 3 Me); 1.72 (m, 3 CH2); 2.22 (m, Hb' C(2')); 2.48 (m, Hb C(2')); 3.31 (m, Ha' C(5')); 3.50
(m, Ha C(5')); 3.81 (m, 2 MeO, CH2O); 4.02 ± 4.40 (m, Hb C(3'), Hb C(4'), 2 Hb C(5'), CH2N, CH2CO,
Ha C(4')); 4.68 (br., OCHO), 5.28 (d, J(5,6) ˆ 7.9, Ha C(5)); 5.94 (s, Ha C(1')); 6.25 (m, Hb C(1')); 6.82 ±
6.87 (m, 4 arom. H); 7.21 ± 7.35 (m, 9 arom. H); 7.58 (s, Hb C(6)); 8.05 (d, J(5,6) ˆ 7.9, Ha C(6)); 9.05, 8.76
(2 br. m, each 1 H, NH(3)). 13C-NMR (68 MHz, CDCl3): 5.23; 4.58; 17.70; 19.16; 25.11; 25.41; 27.10; 30.48;
35.67; 37.38; 38.35; 38.67; 55.11; 60.68; 62.43; 62.54; 63.67; 63.78; 69.53; 70.17; 75.38; 76.30; 82.14; 82.68; 82.80;
83.42; 85.21; 85.73; 86.97; 88.18; 97.66; 98.24; 102.21; 111.07; 113.10; 113.14; 127.15; 127.84; 128.23; 130.14;
134.79; 134.86; 138.35; 138.69; 139.43; 143.83; 150.10; 150.15; 150.48; 158.67; 163.54; 163.61; 169.27; 169.34;
177.95; 178.00. Anal. calc. for C58H75N5O16Si ´ H2O: C 60.88, H 6.78, N 6.12; found: C 60.37, H 6.68, N 6.68.
2'-O-{2-{{{1-[2'-Deoxy-5'-O-pivaloyl-3'-O-(tetrahydro-2H-pyran-2-yl)-b-d-ribofuranosyl]-1,2,3,4-tetrahydro-
2,4-dioxopyrimidin-5-yl}methyl}amino}-2-oxoethyl}-5'-O-(4,4'-dimethoxytrityl)uridine (10). To a soln. of
9
(547 mg, 0.48 mmol) in THF (5 ml) tetrabutylammonium fluoride monohydrate (250 mg, 0.96 mmol) was
added, and the soln. was stirred for 1 h. The mixture was diluted with CHCl3 (50 ml), the soln. washed with H2O
(3 Â 50 ml), dried (Na2SO4), and evaporated, and the residue submitted to CC (silica gel, CHCl3/MeOH
100 : 2): 10 (354 mg, 71%). 1H-NMR (270 MHz, CDCl3): 1.21 (s, 3 Me); 1.46 ± 1.87 (m, 3 CH2); 2.05
(m, Hb' C(2')); 2.50 (m, Hb C(2')); 3.50 (m, 3 H, CH2O, 2 Ha C(5')); 3.68 (m, 7 H, CH2O, 2 MeO); 4.02
(m, Hb C(4')); 4.16 ± 4.39 (m, Ha C(2'), Hb C(3'), Ha C(4'), 2 Hb C(5'), CH2N, CH2CO); 4.49
(m, Ha C(3')); 4.68 (br., OCHO); 5.33 (d, J(5,6) ˆ 8.2, Ha C(5)); 5.84 (s, Ha C(1')); 6.18 (m, Hb C(1'));
6.82 ± 6.88 (m, 4 arom. H); 7.22 ± 7.41 (m, 9 arom. H); 7.67 (s, Hb C(6)); 7.97 (d, J(5,6) ˆ 7.9, Ha C(6)); 10.24,
10.90 (2 br. m, each 1 H, NH(3)). 13C-NMR (68 MHz, CDCl3): 19.14; 25.18; 27.21; 30.48; 36.10; 37.72; 38.80;
55.20; 61.42; 62.57; 63.65; 68.14; 69.62; 75.06; 76.17; 82.34; 83.09; 84.33; 85.43; 85.81; 86.85; 88.61; 97.75; 98.46;
102.35; 111.02; 113.12; 123.74; 127.03; 127.75; 127.92; 128.14; 129.11; 130.10; 130.17; 135.24; 135.44; 135.99;
139.10; 139.34; 139.46; 144.47; 149.70; 150.46; 151.32; 158.60; 163.23; 164.15; 170.39; 170.44; 178.17. Anal. calc.
for C52H61N5O16 ´ H2O: C 60.64, H 6.16, N 6.79; found: C 60.52, H 6.12, N 6.29.
2'-O-{2-{{{1-[2'-Deoxy-5'-O-pivaloyl-3'-O-(tetrahydro-2H-pyran-2-yl)-b-d-ribofuranosyl]-1,2,3,4-tetrahydro-
2,4-dioxopyrimidin-5-yl}methyl}amino}-2-oxoethyl}-5'-O-(4,4'-dimethoxytrityl)uridine 3'-(S,S'-Diphenyl Phos-
phorodithioate) (11). Compound 10 (354 mg, 0.34 mmol) was rendered anhydrous by repeated co-evaporations
with anh. pyridine and dissolved in anh. pyridine (5 ml). Cyclohexylammonium S,S'-diphenyl phosphorodi-
thioate (191 mg, 0.68 mmol), isodurenedisulfonyl dichloride (ˆ 2,4,5,6-tetramethylbenzene-1,3-disulfonyldi-
chloride; 225 mg, 0.68 mmol), and 1H-tetrazole (95 mg, 1.36 mmol) were added, and the resulting soln. was
stirred for 1 h. Then, the mixture was partitioned between CHCl3 (20 ml) and sat. aq. NaHCO3 soln. (20 ml).
The org. phase was washed 5 times with sat. aq. NaHCO3 soln., dried (Na2SO4), and evaporated and the residue
co-evaporated repeatedly with toluene and then submitted to CC (silica gel, CHCl3/MeOH 100 : 1): 11 (333 mg,

Helvetica Chimica Acta ± Vol. 83 (2000)
177
76%). 1H-NMR (270 MHz, CDCl3): 1.08 (s, 3 Me); 1.51 ± 1.92 (m, 3 CH2); 2.12 (m, Hb' C(2')); 2.45
(m, Hb C(2')); 3.38 (d, Jgem ˆ 9.2, Ha' C(5')); 3.48 (m,CH2O, Ha C(5')); 3.80 (m, 2 MeO, Ha C(4')); 3.98 ±
4.21 (m, Ha C(2'), 2 Hb C(5'), CH2N, CH2CO); 4.30 (m, Hb C(3'), Hb C(4')); 4.62 (br., OCHO); 5.12
(m, Ha C(3')); 5.28 (d, J(5,6) ˆ 7.8, Ha C(5)); 6.08 (m, Ha C(1')); 6.20 (m, Hb C(1')); 6.82 (m, 4 arom. H);
7.20 ± 7.58 (m, 19 arom. H); 7.70 (d, J(5,6) ˆ 7.9, Ha C(6)); 9.34, 9.55 (2br. m, each 1 H, NH(3)). 13C-NMR
(68 MHz, CDCl3): 19.21; 19.25; 25.20; 27.15; 27.21; 30.51; 35.76; 37.47; 38.46; 38.76; 55.20; 61.60; 62.52; 62.64;
63.76; 63.88; 70.32; 74.54; 75.44; 76.37; 82.00; 82.93; 85.36; 85.93; 86.49; 87.49; 97.74; 98.37; 102.91; 111.02;
111.05; 113.33; 125.25; 125.36; 125.75; 125.86; 127.30; 128.05; 128.14; 129.47; 129.51; 129.67; 129.72; 129.87;
129.92; 130.12; 134.65; 134.70; 135.08; 135.15; 135.51; 135.60; 138.63; 138.92; 139.05; 143.88; 149.98; 150.03;
158.78; 163.45; 163.49; 168.64; 168.73; 178.06; 178.09. 31P-NMR (109 MHz, CDCl3): 52.09, 52.13. Anal. calc. for
C64H70N5O17 ´ 2 H2O: C 58.58, H 5.38, N 5.33; found: C 57.94, H 5.20, N 5.48.
{5 :2'-O-[Methyleneimino(2-oxoethane-2,1-diyl)]}-Linked 5'-O-(4,4'-Dimethoxytrityl)-P-thiouridyl-(3' !
5')-2'-deoxy-3'-O-(tetrahydro-2H-pyran-2-yl)uridine S-Phenyl Ester (13). A soln. of 11 (750 mg, 0.58 mmol)
in 0.5m KOH/pyridine 1 : 1 (40 ml) was stirred for 3 h. The soln. was neutralized with Dowex 50WÂ 8
(pyridinium salt, 2 Â 5 cm). Et3N (5 ml) was added and the solvent evaporated. The residue was rendered
anhydrous by repeated co-evaporations (5Â) with anh. pyridine and dissolved in anh. pyridine (30 ml).
Isodurenedisulfonyl dichloride (384 mg, 1.16 mmol) and 1H-tetrazole (326 mg, 2.32 mmol) were added, and the
mixture was stirred for 20 min. The mixture was diluted with CHCl3 (100 ml), the soln. washed 3 times with sat.
aq. NaHCO3 soln., the org. phase dried (Na2SO4) and evaporated, and the residue submitted to CC (silica gel,
CHCl3/MeOH 100 : 1.5): 13 (444 mg, 65%). 1H-NMR (270 MHz, CDCl3): 1.38 ± 1.82 (m, 3 CH2); 2.05
(m, Hb' C(2')); 2.52 (m, Hb C(2')); 3.20 (m, Ha' C(5')); 3.58 (m, CH2O, Ha C(5')); 3.80 (m, 2MeO);
4.01 ± 4.48 (m, Ha C(2'), Hb C(3'), Hb C(4'), 2 Hb C(5'), CH2N, CH2CO); 4.60 (br., Ha C(4')); 4.61
(s, OCHO); 4.91 (m, Ha C(3')); 5.31 (d, J(5,6) ˆ 8.2, Ha C(5)); 6.05 (m, Ha C(1')); 6.21, 6.40 (2m,
Hb C(1')); 6.83 ± 6.99 (m, 4 arom. H); 7.18 ± 7.58 (m, 9 arom. H); 7.79 (m, Ha C(6), Hb C(6)); 9.02, 9.10,
9.38, 9.44 (NH(3)). 13C-NMR (68 MHz, CDCl3): 19.01; 19.59; 25.18; 19.04; 29.63; 30.44; 30.64; 34.52; 38.19;
39.22; 55.22; 55.28; 62.18; 62.43; 62.61; 63.16; 68.20; 70.37; 75.29; 75.62; 75.81; 77.93; 81.38; 82.03; 82.16; 82.28;
82.43; 83.49; 85.57; 85.84; 86.02; 87.49; 87.78; 98.22; 98.40; 98.53; 102.93; 103.43; 111.52; 111.73; 112.20; 113.10;
113.32; 113.41; 124.22; 124.33; 124.44; 127.27; 127.48; 127.73; 128.12; 129.09; 129.74; 129.85; 134.14; 134.47;
134.56; 134.74; 134.91; 134.99; 138.85; 143.61; 143.90; 150.06; 150.21; 150.48; 150.57; 150.62; 158.78; 158.90;
162.17; 162.35; 162.41; 162.71; 163.04; 166.77; 168.30; 168.59. Anal. calc. for C53H56N5O16PS: C 58.83, H 5.22,
N 6.47; found: C 58.53, H 5.78, N 6.57.
{5 :2'-O-[Methyleneimino(2-oxoethane-2,1-diyl)]}-Linked Uridyl-(3' ! 5')-2'-deoxyuridine (14). A soln. of
13 (77 mg, 65 mmol) in 0.2m NaOH/pyridine 1 : 1 (4 ml) was stirred for 5 min. The mixture was neutralized with
Dowex 50WÂ 8 (pyridinium salt, 1 Â 5 cm). The solvent was evaporated and the remaining pyridine was
removed by repeated co-evaporations with H2O. The residue was dissolved in 80% AcOH/H2O and the soln.
stirred for 20 h. The AcOH was removed by repeated co-evaporations with H2O and the residue submitted to
reversed-phase CC (C-18, H2O/MeOH 100 : 8): 14 (760 A260, 58%). The yield was estimated by assuming that
e260 of 14 was identical with that of UpU (e260 19600). 1H-NMR (270 MHz, D2O): 2.28 (ddd, J(1',2') ˆ 6.7,
J(2',3')ˆ3.7, J(2',2'') ˆ 13.5, Hb' C(2')); 2.46 (ddd, J(1',2'') ˆ 6.7, J(2'',3') ˆ 6.1, J(2',2'') ˆ 13.5, Hb' C(2')); 3.90
(dd, J(5',5'') ˆ 12.9, J(4',5'') ˆ 4.0, Ha' C(5')); 3.98 (dd, J(5',5'') ˆ 12.9, J(4',5') ˆ 2.8, Ha C(5')); 4.18
(m, 2 Hb C(5')); 4.24 (m, Hb C(4'), CH2N); 4.38 (s, CH2O); 4.40 (m, Ha C(4')); 4.45 (dd, J(1',2') ˆ 4.6,
J(2',3') ˆ 4.8, Ha C(2')); 4.55 (ddd, J(2',3') ˆ 3.7, J(2'',3') ˆ 6.1, J(3',4') ˆ 3.5, Hb C(3')); 4.82 (ddd, J(2',3') ˆ
4.8, J(3',4') ˆ 2.8, J(3',P) ˆ 8.0, Ha C(3')); 5.90 (d, J(5,6) ˆ 8.0, Ha C(5)); 6.13 (d, J(1',2') ˆ 6.7, Ha C(1'));
6.40 (dd, J(1',2'') ˆ 6.7, J(1',2') ˆ 6.7, Hb C(1')); 7.59 (s, Hb C(6)); 7.90 (d, J(5,6) ˆ 8.0, Ha C(6)). 13C-NMR
(68 MHz, D2O): 34.30; 38.95; 60.14; 65.28; 68.64; 70.56; 71.33; 80.26; 83.79; 83.88; 85.10; 85.22; 85.35; 87.42;
102.45; 111.57; 136.17; 141.54; 151.28; 151.52; 164.50; 166.09; 171.69. 31P-NMR (109 MHz, D2O): 0.56.
{5 :2'-O-[Methyleneimino(2-oxoethane-2,1-diyl)]}-Linked 5'-O-(4,4'-Dimethoxytrityl)-P-thiouridyl-(3' !
5')-2'-deoxyuridine S-Phenyl Ester (16). A soln. of 13 (350 mg, 0.29 mmol) in 80% AcOH (5 ml) was stirred
for 5 h. The mixture was evaporated and the remaining AcOH removed by repeated co-evaporations with
EtOH. The residue was rendered anhydrous by repeated co-evaporations with anh. pyridine and then dissolved
in anh. pyridine (9 ml). 4,4'-Dimethoxytrityl chloride (268 mg, 0.78 mmol) was added, and the soln. was stirred
for 20 h. The mixture was partitioned between CHCl3 (20 ml) and sat. aq. NaHCO3 soln. (20 ml), the org. phase
washed 3 times with sat. aq. NaHCO3 soln., dried (Na2SO4), and evaporated and the residue submitted to CC
(silica gel, CHCl3/MeOH 100 : 2): 16 (250 mg, 78%). 1H-NMR (270 MHz, D2O): 1.98 (m, Hb' C(2')); 2.38
(m, Hb C(2')); 3.30 (m, Ha' C(5')); 3.50 (m, Ha C(5')); 3.73 (s, 2 MeO); 3.90 (m, Hb C(4')); 4.00 ± 4.10
(m, 2 H, Ha C(4'), CH2N); 4.10 ± 4.60 (m, 7 H, 2 Hb C(5'), Hb C(3'), Ha C(2'), CH2N, OCH2C); 5.24

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Helvetica Chimica Acta ± Vol. 83 (2000)
(m, Ha C(3')); 5.27 (d, J(5,6) ˆ 7.9, Ha C(5)); 6.00 (d, J(1',2') ˆ 5.9, Ha C(1')); 6.20 (t, J(1',2') ˆ J(1',2'') ˆ
5.9, Hb C(1')); 7.62 (d, J(5,6) ˆ 7.9, Ha C(6)); 7.83 (t, J(6,CH2) ˆ 3.9, Hb C(6)). 13C-NMR (68 MHz,
CDCl3): 34.25; 34.36; 40.45; 55.01; 55.04; 61.83; 67.84; 67.96; 69.33; 69.76; 70.01; 80.94; 81.76; 81.89; 83.76; 84.17;
84.30; 85.30; 85.88; 87.31; 87.57; 103.02; 111.38; 111.73; 112.81; 113.14; 123.68; 123.76; 123.86; 127.08; 127.28;
127.85; 127.92; 127.96; 129.60; 129.63; 129.78; 129.83; 129.97; 134.09; 134.29; 134.34; 134.47; 134.67; 134.72;
134.79; 138.98; 139.26; 143.47; 143.76; 150.15; 150.24; 150.49; 158.56; 158.69; 162.77; 163.34; 168.63; 168.70;
168.98. 31P-NMR (109 MHz, D2O): 25.64; 24.85. Anal. calc. for C48H48N5O15PS ´ 2 H2O: C 55.76, H 5.07, N 6.77;
found: C 55.26, H 4.92, N 6.23.
{5 :2'-O-[Methyleneimino(2-oxoethane-2,1-diyl)]}-Linked 5'-O-(4,4'-Dimethoxytrityl)-P-thiouridyl-(3' !
5')-2'-deoxyuridine 3'-(2-Cyanoethyl Diisopropylphosphoramidite) S-Phenyl Ester (17). Compound 16
(200 mg, 0.18 mmol) was rendered anhydrous by repeated co-evaporations with anh. pyridine and toluene.
i
The residue was dissolved in anh. CH2Cl2 (2 ml). To this soln. were added Pr2EtN (39 ml, 0.22 mmol) and 2-
cyanoethyl diisopropylphosphoramidochloridite (48 ml, 0.22 mmol), and the resulting mixture was stirred for
1 h. The mixture was diluted with CH2Cl2 (20 ml), the soln. washed 3 times with sat. aq. NaHCO3 soln. (20 ml),
dried (Na2SO4), and evaporated and the residue submitted to CC (silica gel, hexane/AcOEt 1 : 4 containing 1%
1
pyridine): 17 (180 mg, 71%). H-NMR (270 MHz, D2O): major isomer: 1.30 (m, 4 Me); 2.10 (m, Hb' C(2'));
2.40 ± 2.80 (m, CH2CN, Hb C(2')); 3.20 (m, Ha' C(5')); 3.50 ± 3.98 (m, 2 MeO, Ha C(4'), Ha C(5'), CH2O,
NCH); 4.00 ± 4.78 (m, Ha C(2'), CH2N, CH2CO, Hb C(4'), Hb C(3'), 2Hb C(5')); 4.91 (m, Ha C(2')); 5.37
(d, J(5,6) ˆ 8.2, Ha C(5)); 6.18 (m, Ha C(1')); 6.28 (t, J(1',2') ˆ J(1',2'') ˆ 6.3, Hb C(1')); 6.90 (m, 4 ar-
om. H); 7.20 ± 7.82 (m, 14 arom. H); 7.60 (m, Ha C(6), Hb C(6)); 9.01 ± 9.80 (br., NH); minor isomer: 7.80
(d, Ha C(6)); 6.40 (t, J(1',2') ˆ J(1',2'') ˆ 5.6, Hb C(1')); 6.00 (m, Ha C(1')). 13C-NMR (68 MHz, CDCl3):
20.29; 20.40; 21.39; 24.53; 34.50; 39.12; 39.93; 43.22; 43.40; 55.26; 57.92; 58.04; 58.19; 58.33; 62.12; 67.87; 69.87;
70.42; 73.17; 73.48; 74.25; 81.51; 81.98; 83.88; 84.35; 85.82; 87.46; 87.75; 102.75; 103.34; 111.64; 111.73; 112.31;
113.30; 113.37; 117.62; 117.74; 124.31; 124.42; 125.23; 127.28; 127.46; 128.10; 128.97; 129.76; 130.12; 133.96;
134.41; 134.52; 134.68; 134.74; 134.90; 134.95; 138.92; 143.59; 143.90; 150.06; 150.12; 150.57; 152.06; 150.12;
150.57; 152.06; 158.76; 158.87; 162.05; 162.26; 162.71; 163.07; 168.19; 168.50. 31P-NMR (109 MHz, D2O): 24.78;
24.86; 25.01; 149.69; 150.10; 150.35; 150.44. Anal. calc. for C57H65N7O16P2S ´ 5/2 H2O: C 55.07, H 5.68, N 7.88;
found: C 54.89, H 5.34, N 7.84.
Centrally {5 :2'-O-[Methyleneimino(2-oxoethane-2,1-diyl)]}-Linked Thymidyl-(3' ! 5')-uridyl-(3' ! 5')-2'-
deoxyuridyl-(3' ! 5')-thymidine (18). A commercially available DM[(MeO)2Tr]T-loaded CPG support (41 mg,
1 mmol) was placed in a glass column equipped with a glass filter, a stopper, and a stopcock. The (MeO)2Tr
group was removed by treatment with 1% CF3COOH in CH2Cl2 (2 ml  2) several times. After the support was
washed with CH2Cl2 (10 ml), the 5'-hydroxy group liberated was coupled with 17 (39 mg, 30 mmol) in the
presence of 1H-tetrazole (21 mg, 0.3 mmol) in MeCN (200 ml) for 10 min under Ar. After the excess
phosphoramidite unit and 1H-tetrazole were removed by washing with CH2Cl2 (10 ml), capping of the
unreacted OH group with 0.2m Ac2O in DMAP/pyridine 1 : 9 (2 ml) for 2 min, oxidation with 0.1m I2 in H2O/
pyridine/THF 1 : 10 : 40 (2 ml) for 1 min, detritylation with 1% CF3COOH in CH2Cl2 (2 ml  5), and elongation
of another thymidylate residue were carried out according to the standard protocol for the solid-phase
oligonucleotide synthesis. After the (MeO)2Tr group was removed by treatment with 1% CF3COOH in CH2Cl2
(2 ml  5), the removal of the alkali-labile protective groups and elution of the oligonucleotide were carried out
by treatment with 0.5m NaOH/pyridine 1 : 1 (10 ml) for 30 min. The eluant was neutralized on Dowex 50WÂ 8
‡
(H form, 3 ml), and the Dowex 50WÂ 8 residue was removed by filtration. The filtrate was evaporated and the
residue lyophilized. The resulting white solid was purified by anion-exchange HPLC (Fax; linear gradient of
50 ! 770 mm NaCl in 25 mm phosphate buffer (pH 6.0) for 50 min). After evaporation, the remaining salt was
1
removed by CC (Sephadex G-15, H2O). The eluant was lyophilized: 18 (6 A260). H-NMR (270 MHz, D2O):
7.67 (d, J(5,6) ˆ 8.3, H C(6)); 7.68 (s, H C(6) of T); 7.62 (s, H C(6) of T); 7.50 (s, H C(6) of dU); 6.30
(m, 2 H, H C(1') of T); 6.10 (d, J(1',2') ˆ 5.0, 1 H, H C(1')); 5.90 (d, J(5,6) ˆ 8.3, 1 H, H C(5) of U); 1.86
(s, 1 Me); 1.87 (s, 1 Me). 31P-NMR (109 MHz, D2O): 0.29; 0.29; 0.47.
General Procedure of the Synthesis of Oligothymidylates Containing the Cyclic-Dimer Unit 14: T-T-T-T-
(cUpdU)-T-T-T-T (19) and T-T-T-T-T-T-T-(cUpdU)-T-T-T-T-T-T-T (20). The fully protected oligothymidylates
having the chain length of a 4-mer and 7-mer were prepared on an automated DNA synthesizer with the
commercially available [(MeO)2Tr]T-loaded CPG support (41 mg, 1 mmol) and the thymidine 3'-phosphor-
amidite unit. Then, the solid support was placed in a glass column equipped with a glass filter, a stopper, and a
stopcock. The 5'-terminal (MeO)2Tr group was removed by treatment with 1% CF3COOH in CH2Cl2 (2 ml Â
5), and the resin was washed with CH2Cl2 (10 ml). The 5'-hydroxy group liberated was coupled with the dimer
unit 17 (39 mg, 30 mmol) in the presence of 1H-tetrazole (21 mg, 0.3 mmol) in MeCN (200 ml) for 10 min under

Helvetica Chimica Acta ± Vol. 83 (2000)
179
Ar. After the excess phosphoramidite unit and 1H-tetrazole were removed by washing with CH2Cl2 (10 ml),
capping of the unreacted OH group with 0.12m AcOH in DMAP/pyridine 9 : 1 (2 ml) for 2 min, oxidation with
0.1m I2 in H2O/pyridine/THF 1 : 10 : 40 (2 ml) for 1 min was carried out. The solid support was put on an
automated DNA synthesizer, and the remaining thymidylate sequences were constructed automatically. After
all the couplings were achieved, the fully protected oligonucleotides were deprotected and eluted by treatment
with 0.5m NaOH/pyridine 1 : 1 (10 ml) for 30 min. Then, the eluant was neutralized with Dowex 50WÂ 8
(pyridinium salt, 3 ml), and the Dowex resin was removed by filtration. To the filtrate was added Et3N (5 ml),
and the soln. was evaporated. The residue was placed on
a Sep-Pak C18 column, and the shorter
oligothymidylates were washed out with H2O (10 ml). The (MeO)2Tr group of the desired oligothymidylates
was removed by treatment with 3% CF3COOH in CH2Cl2 (5 ml) on the column, and the deprotected
oligothymidylates were eluted with the same solvent. The eluant was evaporated and the residue lyophilized.
The resulting white solid was further purified by anion-exchange HPLC (Fax, linear gradient of 50 ! 770 mm
NaCl in 25 mm phosphate buffer (pH 6.0) for 50 min). After evaporation, the remaining salt was removed by
CC (Sephadex G-15, H2O). The eluant was lyophilized: 19 (6 A260) or 20 (22 A260).
19: tR 24.3 min UV (H2O): 262.8; min. 233.0.
20: tR 30.5 min. UV (H2O): 263.2; min. 233.2.
CD Spectra. Oligonucleotides were dissolved in 10 mm sodium phosphate (pH 7.0), and the absorbance at
264 nm was adjusted to 0.5. The soln. was placed in a 5-mm cell, and the spectra were accumulated 16 times at
20 nm/s scan speed. The baseline was corrected by the subtraction of the CD spectra of the solvent recorded
under the same spectrometer conditions at 208. The temp. was controlled by a Haake-C circulator, and the
sample was equilibrated for 15 min after the cell holder had reached the desired temp. The spectra were
displayed after having been smoothed by averaging. The concentration of the sample was calculated on the
assumption that the e260 was 19600.
Measurement of the H,H-Coupling Constants in 1D-1H-NMR Spectra. The cyclic dimer 14 (100 A260) was
dissolved in 10 mm sodium phosphate (700 ml, pH 7.0) and then lyophilized 3 times with 99.8% D2O. The
resulting white precipitate was dissolved in 700 ml of 99.95% D2O. The peak of H2O was suppressed by pre-
saturation. The coupling constants were measured at the 0.15 Hz of digital resolution, accomplished by the spin-
simulation deconvolusion module of the spectrometer.
2D-NOESY Spectrum. Conditions: 2048 Â 512 data points; mixing time 700 ms; data zero-filled to 2048 in
the f1 direction before Fourier transformation.
Polyacrylamide-Gel Electrophoresis. A soln. of 0.1 A260 units of 19, T10, or T11 in formamide (3 ml) was
charged on a 20% polyacrylamide gel plate containing 7m urea (30 Â 38 cm) containing 90 mm Tris-boric acid,
and 3 mm EDTA for 2 h with 90 mm Tris-borate buffer at 1500 V. The gel was visualized by 0.2% toluidine blue
in 0.4m KOAc (pH 4.8) and dried for 5 h under reduced pressure. The mobilities observed were 11.1, 11.4, and
11.1 cm for 19, T10, and T11, resp.
Molecular Modeling by Molecular-Mechanics Calculation. Conformation search and energy minimization
were carried out by MacroModel V. 4.5 running on a Silicon-Graphics-Indigo-2 workstation. The initial
structure was constructed from the nucleotide-unit structure extracted from either the typical A-type or B-type
DNA duplex. Four calculation runs were carried out in which the ribose moieties of the 5'-terminal and 3'-
terminal nucleoside unit were fixed in the {N-type and N-type}, {N-type and S-type}, {S-type and N-type}, and {S-
type and S-type} conformations, resp. In the conformation search, 5000 conformers were selected in the MCMM
mode, in which the conformers were generated randomly, and they were energy-minimized by the AMBER*
united atom force. The low-energy structures having energy within 20 kJ/mol from the lowest energy were
selected and subjected to further energy minimization by the AMBER* all-atom force. In all simulations,
dielectric constant e 1.0 and the GB/SA solvent model were used.
UV Melting Curves. Oligothymidylate (5 mmol) and oligodeoxyadenylates (5 mmol) were dissolved in 2 ml
of 10 mm sodium phosphate and 150 mm NaCl (pH 7.0). The mixture was heated to 908 and then cooled to 08
within 90 min. After the mixture was equilibrated at 08 for 20 min, the UV absorbance at 260 nm was recorded
as the temp. was raised at the rate of 18/min.
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Received August 24, 1999