Essential factors for stabilization of the predominant C3′-endo conformation in dinucleoside phosphotriester derivatives with cyclonucleotide bridge structures at the downstream 3′-position

Article abstract of DOI:10.1002/1099-0690(200105)2001:10<1989::aid-ejoc1989>3.0.co;2-h

This paper describes the synthesis of conformationally constrained dinucleoside cyclic phosphotriester derivatives and related compounds, such as Tpc3Um (2), Tpc3dU (3), Tpc2dU (4), pc3dU (16), and pc2dU (25), where c2 and c3 refer to ethylene and propyle

Full text of DOI:10.1002/1099-0690(200105)2001:10<1989::aid-ejoc1989>3.0.co;2-h

FULL PAPER
Essential Factors for Stabilization of the Predominant C3Ј-endo Conformation
in Dinucleoside Phosphotriester Derivatives with Cyclonucleotide Bridge
Structures at the Downstream 3Ј-Position
Mitsuo Sekine,*[a] Osamu Kurasawa,[a] Koh-ichiroh Shohda,[a] Kohji Seio,[a] and
Takeshi Wada[a]
Keywords: NMR spectroscopy / Nucleic acids / Phosphorylations / Conformation analysis
This paper describes the synthesis of conformationally con- C3Ј-endo puckering. The presence of the 2Ј-hydroxyl group
strained dinucleoside cyclic phosphotriester derivatives and
at the 3Ј-downstream nucleoside is also crucial for stabiliza-
related compounds, such as Tpc3Um (2), Tpc3dU (3), Tpc2dU tion of the C3Ј-endo conformation. Two sets of diastereomeric
(4), pc3dU (16), and pc2dU (25), where c2 and c3 refer to
ethylene and propylene bridges between the 5Ј-phosphate
of the 3Ј-downstream nucleoside and the 5-position of the
uracil residue. These studies found that the c3 linker is es-
sential for fixing the 3Ј-downstream nucleoside residue in the
oligonucleotides, T4(Tpc3Um)T4 (30) and T4 (Tpc3dU)T4 (33),
were synthesized, and each stereoisomer was isolated. The
hybridization capability of these oligonucleotides was ana-
lyzed by melting point experiments.
Introduction
phosphorus chirality) of 1 predominantly display C2Ј-endo
(S-type) and C3Ј-endo (N-type) conformations at the ribose
In the naturally occurring RNA structures analyzed to moieties of the 5Ј-upstream and 3Ј-downstream 2Ј-O-
date, a large variety of conformations have been observed methyluridines, respectively.[6]
in the ribose moiety and the phosphodiester linkage. In na-
This paper deals with the synthesis of Tpc3Um (2),
ture, such complex variability of conformation allows for Tpc3dU (3), and Tpc2dU (4), in which the 5Ј-upstream or
the creation of numerous functional biomolecules.[1Ϫ5] As 3Ј-downstream Um of 1 is replaced with thymidine (T) or
the first step towards the creation of artificial RNA molec- 2Ј-deoxyuridine (dU) (Figure 1). This was intended to cla-
ules of planned and designed three dimensional structure, rify essential factors that regulate the sugar conformation
for purposes of development of new functions, it should in such cyclic phosphotriester-type dinucleotide derivatives
be advantageous to produce various kinds of dinucleotide (symbols c2 and c3 refer to the ethylene and propylene
building blocks capable of inducing particular changes in bridge linkers, respectively, bridging between the 5Ј-phos-
RNA backbone structure. Therefore, we have recently been phate and the 5-position of the uracil residue). This study
investigating the synthesis of dinucleotide blocks possessing found that the rigidity of the C3Ј-endo form in the cyclic
an unambiguously confirmed conformational rigidity, bridged structure is essentially a function both of the cyclic
through the introduction of a cyclic structure that controls propylene linker and of the presence of the 2Ј-methoxy
not only the sugar puckering, but also the direction of the group.
backbone phosphodiester linkage. In the preceding paper,[6]
we reported the synthesis of Umpc3Um (1) as a conforma- Results and Discussion
tionally constrained dinucleotide monophosphate derivative
1
Synthesis and H NMR Analysis of Tpc3Um 2a and 2b,
with S/N Junctions
that should be useful for fixing the normal backbone struc-
ture of RNA, as well as certain RNA bending
structures.[7Ϫ10] The cyclic structure was introduced as a
stable mimic of the intramolecularly hydrogen bound struc-
ture of 5-[(methylamino)methyl]-5Ј-uridylic acid[11] and a
water-bridged 5Ј-pseudouridylic acid,[12] both of which have
been identified as tRNA structural motifs predominantly
possessing the C3Ј-endo conformation. Our study showed
that the two diastereomers (diastereoisomerism due to
Generally, nucleosides have two major sugar puckering
conformers (C3Ј-endo and C2Ј-endo, Figure 2). More specif-
ically, the C2Ј-endo conformation tends to be more predom-
inant in deoxyribonucleosides than in ribonucleosides, be-
cause of the electronic effect of the 2Ј-hydroxyl group.[1a] As
shown in Table 1, the percentage population of the C3Ј-
endo form in thymidine was calculated by 1H NMR analysis
to be 29%,[13] while uridine (U) and 2Ј-O-methyluridine
(Um) exhibited moderate C3Ј-endo predominance of 56%
and 60%, respectively.
[a]
Department of Life Science, Tokyo Institute of Technology,
Nagatsuta, Midoriku, Yokohama 226Ϫ8501, Japan
Fax: (internat.) ϩ 81-45/924-5772
E-mail: msekine@bio.titech.ac.jp
On the basis of these facts, the 5Ј-upstream Um of
Umpc3Um (1) was replaced with thymidine to see if the
conformation of the 5Ј-upstream nucleoside can be more
Supporting information for this article is available on the
WWW under http://www.wiley-vch.de/home/eurjoc or from
the author.
Eur. J. Org. Chem. 2001, 1989Ϫ1999
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/0510Ϫ1989 $ 17.50ϩ.50/0
1989

M. Sekine, O. Kurasawa, K. Shohda, K. Seio, T. Wada
FULL PAPER
Figure 1. Intramolecular hydrogen bonds found in tRNAs and
chemically synthesized dinucleotide blocks 1؊4.
Scheme 1. (i) 1H-tetrazole/CH3CN/dioxane, room temp., 2 h; (ii)
iBuOOH /CH3CN/dioxane, room temp., 1 h; (iii) aqueous NH3-
pyridine (1:1, v/v), room temp., 3 h; (iv) 80% AcOH, room temp.,
30 min; (v) ClP(OCH2CH2CN)(NiPr2),Et3N, CH2Cl2, room
temp., 2 h.
Deacetylation of 7 with ammonia gave the 3Ј-free com-
pound 8, which was in turn converted in good yield into
the desired dimer 2 by treatment with 80% acetic acid. At-
tempted separation of the diastereomers of 2 or 8 by silica
gel column chromatography was unsuccessful. Finally, how-
ever, separation of the diastereomers 2a and 2b by HPLC
resulted in successful isolation of each diastereomer (Sym-
bols ‘‘a’’ and ‘‘b’’, respectively, refer to rapidly eluted com-
pounds and slowly eluted compounds from a reversed
phase HPLC column).
Figure 2. Equilibrium of two major conformers (C2Ј-endo and C3Ј-
endo) of nucleosides.
Table 1. Percentage C3Ј-endo conformation populations in 3Ј-O-
acetylated thymidine, 2Ј-O-methyluridine and related compounds
Table 2 shows the results of the 1H NMR analysis of
Tpc3Um(fast) 2a, and Tpc3Um(slow) 2b. The C3Ј-endo
conformer percentage populations of the 5Ј-upstream nuc-
leoside in the two diastereoisomers 2a and 2b were estim-
ated to be 22% and 27%, while those in 1a and 1b were
reported to be 30% and 27%, respectively. These results
show that, as expected, thymidine is a slightly better nucle-
oside from the point of view of enrichment of the C2Ј-endo
conformation at the 5Ј-upstream nucleoside in order to con-
strict the conformation of a dinucleotide block as the S/
N junction, which is equivalent to the C2Ј-endo/C3Ј-endo
junction. The percentage populations of the C3Ј-endo con-
[a]
[b]
Ref.[13]. Ϫ The percentage population was calculated accord-
ing to the following references: ref.[14] for thymidine derivatives and
ref.[15] for uridine derivatives.
oriented to the C2Ј-endo form than in the 5Ј-upstream Um formation of the 5Ј-upstream T in Tpc3Um 2a and 2b are
of 1. To synthesize Tpc3Um (2), the thymidine 3Ј-phos- lower by 14Ϫ19% than that (41%) of TpT (Table 2). The
phorodiamidite derivative 5 was prepared according to the same is true for the difference between Umpc3Um 1a and
method reported by van Boom et al.[16] Compound 5 was 1b and UpU, as shown in Table 2: the C2Ј-endo population
condensed
with
3Ј-O-acetyl-5-(hydroxypropyl)-2Ј-O- of the 5Ј-upstream U of UpU was reported to be 53%[18] or
methyluridine (6)[10] in the presence of 1H-tetrazole[17] to 56%.[13] On the other hand, the 5Ј-upstream Um of 1 be-
give the triester block 7 as a diastereomeric mixture in 85% comes enriched in the C2Ј-endo conformer. These results
yield, as shown in Scheme 1.
reflect the fact that the 3Ј-phosphoryl residue indeed affects
1990
Eur. J. Org. Chem. 2001, 1989Ϫ1999

Stabilization of the Predominant C3Ј-endo Conformation in Dinucleoside Phosphotriester Derivatives
FULL PAPER
Table 2. Percentage C3Ј-endo conformation populations in the 5Ј-upstream and 3Ј-downstream nucleosides of dimers containing a cyclour-
idylic acid derivative and related compounds
[a]
[b]
[c]
[d]
Ref.[6]. Ϫ
Ref.[13]. Ϫ Ref.[18]. Ϫ
The percentage populations of ribose and deoxyribose residues were calculated according to
the following equations: ref.[14] for the thymidine and ref.[15] for the uridine residues.
the sugar moiety like an acetyl group, which induces the in the presence of DMAP, followed by treatment with 80%
C2Ј-endo conformation more effectively when attached to acetic acid, gave the 3Ј-O-benzoylated product 14 in 64%
the 3Ј-hydroxy, as shown in Table 1. The effect of the acetyl overall yield from 11. Cyclization of 14 with bis(diisopropyl-
group in contributing to the C3Ј-endo conformation was amino)(2-cyanoethoxy)phosphane,[16] followed by oxida-
estimated to be as much as 10% in both 3Ј-O-acetylthymid- tion with tBuOOH,[21] gave the desired product 15, which
ine and 3Ј-O-acetyl-2Ј-O-methyluridine.
was converted into 16 in situ, in 64% overall yield from
On the other hand, the percentage populations of the 14 (Scheme 2).
C3Ј-endo conformations of the 3Ј-downstream Um in 2a
and 2b were estimated to be 82% and 89%, respectively. In
addition, the sums of J1Ј,2Ј and J3Ј,4Ј of the 3Ј-downstream
pc3Um of 2a and 2b are 9.6 and 9.3 Hz, respectively. These
values are fairly normal; in comparison, the average sum
value of J1Ј,2Ј and J3Ј,4Ј in typical ribonucleosides is 9.4 Ϯ
0.2 Hz.[19] It was therefore to be expected that Tpc3Um 2a
or 2b should be usable as a good building block as regards
the ideal sugar conformation to mimic a bending motif with
the rigid S/N junction, although this combination produces
a chimeric mixture comprising a deoxynucleoside (T) and a
ribonucleoside (Um).
Requirement of the 2Ј-Hydroxy Group for Stabilization of
the C3Ј-endo Conformation in 5Ј-Cyclouridylic Acid
Derivatives
Should it be possible to replace the 3Ј-downstream
pc3Um of Umpc3Um 1 or Tpc3Um 2, possessing an S/N
junction site, with its deoxy counterpart pc3dU (16) with-
out loss of conformational rigidity, the synthesis of bent
RNA would be facilitated, since the 3Ј-downstream com-
ponent 14 can be easily prepared. In addition, it would also
engender a possible new route to flexible single-strand
DNA oligomers possessing a unique ridged S/N junction at
a certain point.
Scheme 2. (i) 2-propyn-1-ol, DMF, Et3N, CuI, Pd(PPh3)4, room
temp., 6 h; (ii) H2/Pd, dioxane/MeOH (1:1, v/v), room temp., 43 h;
(iii) 25% aqueous NH3/pyridine (1:1, v/v), room temp., 10 h; (iv)
DMTrCl, pyridine, room temp., 3 h; (v) Bz2O, DMAP, pyridine,
room temp., 7 h; then 80% AcOH, room temp., 30 min; (vi)
NCCH2CH2OP(NiPr2)2, 1H-tetrazole, CH3CN/dioxane, room
temp., 1 h; tBuOOH, CH3CN/dioxane, room temp., 30 min; (vii)
25% aqueous NH3/pyridine (3:1, v/v), room temp., 24 h.
Treatment of 3Ј,5Ј-di-O-acetyl-5-iododeoxyuridine (9)[20]
with 2-propyn-1-ol in the presence of a palladium catalyst
gave the alkynylated product 10 in 71% yield. Hydrogena-
tion of 10 gave the reduction product 11 in 95% yield. Al-
kaline hydrolysis of 11, followed by the bis-dimethoxytrityl-
Conformational Analysis of Deoxy Counterpart 16
To determine the ratio of the C3Ј-endo and C2Ј-endo
ation of the resulting triol 12, gave the dimethoxytritylated conformations over a series of deoxyribonucleosides, the
product 13. In situ acylation of 13 with benzoic anhydride well-known
Altona
equation[14]
P(C3Ј-endo)
ϭ
Eur. J. Org. Chem. 2001, 1989Ϫ1999
1991

M. Sekine, O. Kurasawa, K. Shohda, K. Seio, T. Wada
FULL PAPER
1
Table 3. H NMR spectroscopic data and conformational analysis
of cyclodeoxyuridylic acid derivatives (16 and 25) and 5Ј-thymid-
ylic acid
[a]
[b]
Ref.[13]. Ϫ The percentage population was calculated according
to the following equation reported by Rinkel and Altona.[14]
Ϫ
[c]
The torsion angle P(gϩ) was calculated according to the following
equation reported by Altona.[22]
Scheme 3. (i) 1H-tetrazole, CH3CN/dioxane (1:1, v/v), room temp.,
2 h; (ii) tBuOOH, CH3CN/dioxane (1:1, v/v), room temp., 30 min;
(iii) 25% aqueous NH3/pyridine (1:3, v/v), room temp., 18 h; (iv)
80% AcOH, room temp., 1 h.
[1 Ϫ (J1Ј,2Ј ϩ J1Ј,2" Ϫ 9.8)/5.9] was used. Generally, this
equation applies only for deoxyribonucleoside derivatives
with the sum of J1Ј,2Ј and J3Ј,4Ј of 10.7 Ϯ 0.2 Hz.[19] We
analyzed in detail the conformation of the deoxy counter-
part 16, with a propylene bridge. Consequently, it turned
out that the sum of J1Ј,2Ј and J3Ј,4Ј of 16 is 10.6 Hz, as
shown in Table 3.
1
Table 4. H NMR spectroscopic data and conformational analysis
of Tpc3dU 3a and 3b, Tpc2dU 4a and 4b, and TpT
This value is within the standard deviation and is effec-
tively the same as that of 5Ј-thymidylic acid. This result
suggests that compound 16 has a fairly natural structure
without strain. The percentage population of the C3Ј-endo
conformer was calculated by the Altona equation to be
73%.[14] On the other hand, the C3Ј-endo conformer per-
centage population in 5Ј-thymidylic acid was calculated to
be 33%, while that of the ribose derivative 17 had been re-
ported as 88%.[8] These results indicate that the cyclic struc-
ture contributes significantly to the predominance of the
C3Ј-endo conformation, but is insufficient for complete fix-
ing of this conformation. Therefore, it can clearly be con-
cluded that both the cyclic structure and the 2Ј-hydroxy
group are essential factors for adequate stabilization of the
C3Ј-endo conformation of 5Ј-thymidylic acid.
[a]
[b]
Ref.[13]. Ϫ The percentage population was calculated accord-
ing to the following equation reported by Rinkel and Altona.[14]
In Tpc3dU(fast) 3a and Tpc3dU(slow) 3b, the C3Ј-endo
content of the 5Ј-upstream thymidine was in each case cal-
culated to be 25%. The sum of J1Ј,2Ј and J3Ј,4Ј in the 3Ј-
downstream thymidine in 3a is 9.0 Hz, which deviates
Synthesis and Conformational Analysis of Dimer Tpc3dU
(3)
Although the above results indicate that the use of 16 as somewhat from that (10.9 Hz) of TpT or the average value,
the 3Ј-downstream component causes loss of conforma- 10.7 Ϯ 0.2 Hz, in typical deoxyribonucleoside derivat-
tional rigidity, which is unfavorable for design of a bent ives.[19] These differences are considerable, so some disor-
motif, we synthesized Tpc3dU 3a and 3b as reference com- dered form must be contemplated. Therefore, it seems that
pounds for comprehensive studies, as shown in Scheme 3. the calculated value Ϫ 82% Ϫ for the C3Ј-endo conforma-
Condensation of 5 with 14 gave the product 18 in 71% yield. tion in 3a might be an overestimate as compared with that
Compound 18 was deprotected in the usual manner to give (73%) of the 5Ј-deoxycyclouridylic acid derivative pc3dU
3, and the two isomers 3a and 3b were separated by HPLC. 16. Similar reasoning should be true for 3b, although the
The results of the conformational analysis are summarized sum of J1Ј,2Ј and J3Ј,4Ј in the 3Ј-downstream thymidine in
in Table 4.
3b was not measurable.
1992
Eur. J. Org. Chem. 2001, 1989Ϫ1999

Stabilization of the Predominant C3Ј-endo Conformation in Dinucleoside Phosphotriester Derivatives
FULL PAPER
Synthesis of 5Ј-Cyclodeoxyuridylic Acid Derivative pc2dU
(25), Possessing an Ethylene Bridge
Finally, we examined the effect of the number of methyl-
ene groups in the covalent bridge structure on conforma-
tional properties at the dimer level. To this end, a shorter
ethylene chain was used in place of the propylene moiety.
In order to obtain pc2dU (25), 5-(2-chloroethyl)-3Ј,5Ј-di-
O-toluoyldeoxyuridine (19) was prepared according to the
method reported by Griengl et al.[23] Alkaline hydrolysis of
19 gave the triol 20, which was protected in situ with
DMTrCl to give the bis-dimethoxytritylated product 21, in
90% yield from 19. Acetylation of 21 gave the 3Ј-acetate 22
in 90% yield. Treatment of 22 with 80% acetic acid gave the
diol 23 in 84% yield (Scheme 4).
Scheme 5. (i) 1H-tetrazole, CH3CN, room temp., 40 min, tBuOOH,
CH3CN, room. temp., 1 h; (ii) 25% aqueous NH3/pyridine (1:1, v/
v), room temp., 9 h.
of the sum of J1Ј,2Ј and J3Ј,4Ј in ribonucleosides is reported
to be 9.4 Ϯ 0.2 Hz,[19] there is a considerable gap, of 0.8 Hz,
between these values. It is therefore concluded that the
ethylene bridge must induce some conformational disorder
in both cyclodeoxy and cycloribo nucleotides pc2dU 25 and
pc2U 26.
Compounds Tp2dU 4a and 4b were synthesized by con-
densation of 5 with 23, affording product 27 as shown in
Scheme 5, and were isolated by HPLC and analyzed by
1H NMR.
In both compound 4a and 4b, the C3Ј-endo content of
the 5Ј-upstream thymidine is considerably less than that in
TpT, as shown in Table 4. Since the sugar pucker of the 3Ј-
downstream cyclothymidine in 4a or 4b is unusual because
Scheme 4. (i) 0.5  NaOH/pyridine (1:1, v/v), room temp., 12 h;
(ii) DMTrCl, pyridine, room temp., 2 h; (iii) Ac2O, pyridine, room
temp., 8 h; (iv) 80% AcOH, room temp., 30 min; (v)
NCCH2CH2OP(NiPr2)2, 1H-tetrazole, CH3CN, room temp., 1 h;
(vi) tBuOOH, CH3CN, room temp., 1 h; (vii) 25% aqueous NH3/ of the ethylene bridge, the exact ratio of the C3Ј-endo/C2Ј-
pyridine (1:1, v/v), room temp., 2.5 h.
endo conformers cannot be estimated by the standard equa-
tion as shown in Table 1. Actually, the sum (8.4 Hz) of J1Ј,2Ј
Phosphitylation of 23 with bis(diisopropylamino)(2-cyan-
and J3Ј,4Ј is extremely small, as shown in the case of 4a.
oethoxy)phosphane,[24] followed by oxidation with
Therefore, we did not incorporate this dimer block into oli-
tBuOOH, gave the protected cyclodeoxyuridylic acid deriv-
gonucleotides.
ative 24 in 68% yield, as a diastereomeric mixture due to
the chirality of the phosphotriester-type phosphorus atom.
The ratio of the two isomers was 66:34. Treatment of 24
with ammonia gave the cyclodeoxyuridylic acid derivative
25 in 50% yield.
Synthesis of T4[Tpc3Um(fast)]T4 (30a), T4[Tpc3Um(slow)]T4
(30b), T4[Tpc3dU(fast)]T4 (33a) and T4[Tpc3dU(slow)]T4
(33b)
The 1H NMR spectroscopic data of pc2dU 25 are shown
in Table 3. The J1Ј,2Ј value observed is 3.9 Hz and the J3Ј,4Ј
value is 2.5 Hz. The sum of J1Ј,2Ј ϩ J3Ј,4Ј in 25 is 6.4 Hz,
deviating far from the standard. Actually, calculation using
the equation described in the literature[14] produced an ab-
normal figure of 103% as the C3Ј-endo conformation per-
centage population in 25. This result appears to be brought
about by the presence of the cyclic ethylene bridge structure.
The abnormal J1Ј,2Ј ϩ J3Ј,4Ј value in 25 suggests that 25 has
a disordered structure in the deoxyribose ring. In the case
of the previously reported ribose counterpart pc2U 26, the
For incorporation of Tpc3Um into DNA, compound 7
was converted into the 3Ј-free hydroxyl derivative 8. This
sum of J1Ј,2Ј ϩ J3Ј,4Ј was 8.6 Hz.[7] Since the average value Scheme 6
Eur. J. Org. Chem. 2001, 1989Ϫ1999
1993

M. Sekine, O. Kurasawa, K. Shohda, K. Seio, T. Wada
FULL PAPER
Figure 3. Melting curves of the duplexes formed between A(pA)9 or d[A(pA)9] and oligonucleotides 30 or 33. Panel A: ᭺
A(pA)9ϪT4[Tpc3Um(fast)]T4(30a), A(pA)9ϪT4[Tpc3Um(slow)]T4(30b), A(pA)9ϪT(pT)9; Panel B: d[A(pA)9]ϪT4-
[Tpc3Um(fast)]T4(30a), ϫ d[A(pA)9] ϪT4[Tpc3Um(slow)]T4(30b), • d[A(pA)9] ϪT(pT)9; Panel C: ᭺ A(pA)9ϪT4[Tpc3dU(fast)]T4(33a),
A(pA)9ϪT4[Tpc3dU(slow)]T4(33b), A(pA)9ϪT(pT)9; Panel D: d[A(pA)9] ϪT4[Tpc3dU(fast)]T4(33a), d[A(pA)9]
ϪT4[Tpc3dU(slow)]T4(33b), • d[A(pA)9]ϪT(pT)9.
ϫ
•
᭺
ϫ
•
᭺
ϫ
compound was phosphitylated to give the amidite unit 28. Table 5. Melting points values of the duplexes between modified
oligonucleotides and decaadenylate or decadeoxyadenylate
The decadeoxynucleotides T4[Tpc3Um(fast)]T4 (30a) and
T4[Tpc3Um(slow)]T4 (30b) were synthesized as a diastereo-
Oligonucleotides
Melting temperature of duplex (°C)
meric mixture according to standard DNA synthesis meth-
odology, by use of 28 and aminopropyl-CPG resin 29. Us-
ing HPLC, it was possible to separate and isolate stereoiso-
mers 30a and 30b completely. Both isomers 30a and 30b T4[Tpc3Um(fast)]T4 30a 11.0
were analyzed by enzymatic degradation using snake venom
phosphodiesterase and calf intestinal alkaline phosphatase
to give a mixture of T and Tpc3Um(fast) and a mixture of
T and Tpc3Um(slow) in the correct ratios, as described in
the previous paper.[10]
A(pA)9 ∆Tm
d[A(pA)9] ∆Tm
T(pT)9
20.3
27.0
8.0
Ϫ9.3
Ϫ5.0
Ϫ19.0
Ϫ13.6
Ϫ18.4
Ϫ19.9
T4[Tpc3Um(slow)]T4 30b 15.3
13.4
T4[Tpc3dU(fast)]T4 33a 8.0
Ϫ12.3 8.6
T4[Tpc3dU(slow)]T4 33b 11.1
9.2
7.1
melting point lower than that of T4[Tpc3Um(slow)]T4 30b.
The same is true for hybridization with the deoxy counter-
part d[A(pA)9], as shown in Figure 3 (B). These results are
similar to those found for the duplexes of U4[Umpc3Um(-
fast)]U4 with d[A(pA)9] and A(pA)9, but the difference in
∆∆melting points has become slightly smaller than that re-
lating to U4[Umpc3Um (fast)]U4.[6] These results imply that
T4[Tpc3Um(fast)]T4 30a has an S/N bending structure with
In
a
similar manner, the decadeoxynucleotides
T4[Tpc3dU(fast)]T4 (33a) and T4[Tpc3dU(slow)]T4 (33b)
were synthesized from the amidite unit 32, which was de-
rived from 31 (Scheme 3), and characterized by the enzym-
atic analysis.
Hybridization with A(pA)9 and d[A(pA)9]
The results of melting point experiments on the duplexes the S configuration on the phosphorus atom.
formed between [A(pA)9]ϪT4[Tpc3Um(fast)]T4 30a and In contrast, there was less difference in melting points
[A(pA)9]ϪT4[Tpc3Um(slow)]T4 30b are summarized in between A(pA)9ϪT4[Tpc3dU(fast)]T4 33a and A(pA)9Ϫ
Table 5 and Figure 3 (A). In these experiments, the oligonu- T4[Tpc3dU(slow)]T4 33b, as well as between
cleotide 30a derived from the rapidly eluted Tpc3Um has a d[A(pA)9]ϪT4[Tpc3dU(fast)]T4 33a and d[A(pA)9]ϪT4-
1994
Eur. J. Org. Chem. 2001, 1989Ϫ1999

Stabilization of the Predominant C3Ј-endo Conformation in Dinucleoside Phosphotriester Derivatives
FULL PAPER
(545 mg, 1.0 mmol) was rendered anhydrous by repeated coevap-
oration with dry toluene, and finally dissolved in dry dioxane
(5.0 mL). To the mixture were added triethylamine (0.210 mL,
1.5 mmol) and bis(diisopropylamino)chlorophosphane (315 mg,
1.2 mmol). The solution was stirred at room temperature for 30
min and the reaction was quenched by addition of ethanol (1 mL).
After standing for 1 min, the mixture was partitioned between
CHCl3 (20 mL) and 5% NaHCO3 (20 mL). The organic layer was
collected, washed twice with 5% NaHCO3 (10 mL), dried over
Na2SO4, filtered, and evaporated under reduced pressure. The re-
sulting foam was used for the next reaction without further puri-
fication.
[Tpc3dU(slow)]T4 33b, as shown in Table 5 and Figure 3
(C,D). These results imply that a more flexible pc3dU res-
idue incorporated into DNA is no longer sufficient to main-
tain rigidity.
Conclusion
This study found that the rigidity of the C3Ј-endo form
in the cyclic bridged structure designed by us is essentially
a function both of the cyclic propylene linker and of the
presence of the 2Ј-methoxy group. It was also shown that
the dimer building block of Tpc3Um(fast) 30a can be used
as an S/N bending motif, while Tpc3Um(slow) 30b could
be used as an S/N linear structural motif. Further study is
under way.
Synthesis of the Fully Protected Thymidylyl(3ЈǞ5Ј)2Ј-O-methyl-
uridine Derivative 7, Possessing a Cyclic Structure with a Propylene
Bridge: A mixture of 6[6] (179 mg, 0.5 mmol) and 1H-tetrazole
(168 mg, 2.4 mmol) was rendered anhydrous by coevaporation with
dry toluene, and the residue was dissolved in dry acetonitrile
(25 mL) and dioxane (25 mL). A solution of 5 (0.60 mmol) in ace-
tonitrile (3.0 mL) was added dropwise to the mixture over a period
of 10 min. The resulting mixture was stirred at room temperature
for 40 min and a solution of 5 (0.20 mmol) in acetonitrile (1.0 mL)
was added. Stirring was then continued for an additional 1 h. The
resulting mixture was stirred at room temperature for 40 min, and
then tBuOOH (0.562 mL, 5.0 mmol) was added to the mixture.
After stirring for 1 h, the solution was evaporated under reduced
pressure and the residue was extracted with CHCl3 (100 mL) and
5% NaHCO3 (100 mL). The organic layer was collected, washed
twice with 5% NaHCO3 (10 mL), dried over Na2SO4, filtered, and
evaporated under reduced pressure. The residue was chromato-
graphed on a column of silica gel (25 g) with CHCl3/MeOH (100:2,
v/v) containing 1% pyridine to give 7 as a foam (403 mg, 85%, the
ratio of the diastereomers 1.1:1).
Experimental Section
1
General Remarks: H, 13C, and 31P NMR spectra were recorded at
270, 68 and 109 MHz, respectively. The chemical shifts were meas-
ured from tetramethylsilane or sodium dodecyl sulfate (DSS) for
1H NMR spectra, CDCl3 (77 ppm) or DSS (0 ppm) for 13C NMR
spectra, and 85% phosphoric acid (0 ppm) for 31P NMR spectra.
1
The H-1H coupling constants were measured at 400 MHz using a
Varian Unity 400 spectrometer at 20 °C, except for those of 4a and
4b, which were measured at 30 °C to avoid overlapping with the
HOD signal. Ϫ UV spectra were recorded on a U-2000 spectro-
meter. Ϫ CD spectra were recorded on a J-500 C spectrometer,
using a 0.5 cm cell. The synthesis of oligonucleotides was per-
formed on a 381A DNA synthesizer or 392 DNA/RNA synthes-
izer. Ϫ TLC was performed with 60-F254 Kieselgel (0.25 mm). Ϫ
Column chromatography was performed with C-200 silica gel pur-
chased from Wako Co. Ltd., and an aquarium minipump was a
convenient means to attain sufficient pressure for rapid chromato-
graphic separation. Ϫ Reversed phase column chromatography was
performed using µBondapak C-18 silica gel (prep S-500, Waters).
Reversed phase HPLC was performed using the following systems:
System 1: An LC module 1 was used, with an M-741 data module
and a µBondasphere 5 µ C18 100 m column (3.9 ϫ 150 mm) at 50
°C with a linear gradient (0Ϫ30%) of CH3CN in 0.1  NH4OAc,
pH 7.0 at a flow rate of 1.0 mL/min for 30 min; System 2: A 2690
separation module was used, with a 996 photodiode array detector
and a Millennium 2010 chromatography manager. The other condi-
tions were the same as those in System 1; System 3: An SCL-6A
system was used, with a combination of LC-6A, SPD-6A, and C-
R3A. The other conditions were the same as those in System 1,
except for a flow rate of 3.0 mL/min. Anion-exchange HPLC was
performed on an LC module 1 with a Waters M-741 data module
and a Waters column heater, with a 10Ϫ67% linear gradient of
25 m phosphate, 1  sodium chloride buffer (pH 6.9) in 25 m
phosphate buffer (pH 6.0) at a flow rate of 1.0 mL/min for 40 min.
Pyridine was distilled twice from p-toluenesulfonyl chloride and
Synthesis of the Partially Protected Thymidylyl(3ЈǞ5Ј)2Ј-O-methyl-
uridine Derivative 8, Possessing a Cyclic Structure with a Propylene
Bridge: The fully protected dimer 7 (334 mg, 0.335 mmol) was dis-
solved in pyridine (5.0 mL) and 25% aqueous ammonia (5.0 mL).
After having been stirred at room temperature for 3 h, the mixture
was evaporated under reduced pressure. The residue was chromato-
graphed on a column of silica gel (15 g) with CHCl3/MeOH (100:3,
v/v) containing 1% pyridine to give 8 as a foam (287 mg, 90%, ratio
of the diastereoisomers 1.1:1).
Unprotected Thymidylyl(3ЈǞ5Ј)2Ј-O-methyluridine Derivatives 2a
and 2b, Possessing Cyclic Structures with Propylene Bridges: Com-
pound 8 (81.4 mg, 90.0 µmol) was dissolved in 80% acetic acid
(2 mL). After having been kept at room temperature for 30 min,
the mixture was evaporated under reduced pressure. The residue
was extracted with ether (10 mL) and water (10 mL). The aqueous
layer was collected, washed twice with ether, and lyophilized to give
a mixture of 2a and 2b (51 mg, 93%): Ϫ C23H31N4O13P·1.5H2O:
calcd. C 43.89, H 5.44, N 8.90; found C 43.67, H 5.46, N 8.65.
This mixture was chromatographed on a reversed phase HPLC col-
umn using System 3 to give 2a (334 A260, 19%) and 2b (406 A260
,
23%).
Synthesis of 5Ј-O-(4,4Ј-Dimethoxytrityl)thymidylyl(3ЈǞ5Ј)2Ј-O-
methyluridine Phosphoramidite Derivative 28: DMT-Tpc3Um 8
(258 mg, 0.0.285 mmol) was rendered anhydrous by repeated co-
evaporation with dry toluene and finally dissolved in CH2Cl2
(5 mL). To the mixture were added triethylamine (120 µL,
0.86 mmol) and (2-cyanoethoxy)(diisopropylamino)chlorophos-
phane (94 µL, 0.423 mmol). The mixture was stirred at room tem-
˚
from calcium hydride and then stored over 4A molecular sieves.
Elemental analyses were performed by the Microanalytical Labor-
atory, Tokyo Institute of Technology at Nagatsuta. The spectral
data of all the dinucleotide derivatives can be seen in the supple-
mentary material
5Ј-O-(4,4Ј-Dimethoxytrityl)thymidine 3Ј-(N,N,NЈ,NЈ-Tetraisopro-
pyl)phosphorodiamidite (5): 5Ј-O-(4,4Ј-Dimethoxytrityl)thymidine perature for 2 h and then quenched by addition of ethanol
Eur. J. Org. Chem. 2001, 1989Ϫ1999 1995

M. Sekine, O. Kurasawa, K. Shohda, K. Seio, T. Wada
(0.5 mL). The mixture was diluted with CHCl3 (15 mL). The quenched by addition of water. Extraction was performed with
FULL PAPER
CHCl3 solution was washed three times with 5% NaHCO3 (15 mL),
dried over Na2SO4, filtered, and evaporated under reduced pres-
sure. The residue was dissolved in CHCl3 (1 mL) and this solution
was added portionwise to hexane (80 mL)-ether (20 mL)-pyridine
(1 mL) with vigorous stirring. The resulting white precipitate was
collected by removal of the solvent by a pipette and dried in vacuo
to give DMTrTpc3Ump(a,ce) 28 (273 mg, 87%).
CHCl3 (50 mL) and 5% NaHCO3 (50 mL). The organic layer was
collected, dried over Na2SO4, filtered, and evaporated under re-
duced pressure. The residue was coevaporated three times with
toluene under reduced pressure and dissolved in 80% acetic acid
(10 mL). The mixture was stirred at room temperature for 30 min
and then evaporated under vacuo. The residue was chromato-
graphed on a column of silica gel (12 g) with CHCl3/MeOH (100:3,
1
v/v) to give 14 as a foam (186 mg, 64%). Ϫ H NMR (270 MHz,
3Ј,5Ј-Di-O-acetyl-5-(3-hydroxypropynyl)-2Ј-deoxyuridine (10): A so-
lution of 9 (1.0 g, 2.28 mmol) and 2-propyn-1-ol (0.400 mL,
6.85 mmol) in DMF (25 mL) was degassed under ultrasound irra-
diation with supersonic wave under reduced pressure, and the reac-
tion vessel was then filled with argon. This manipulation was re-
peated five times to remove the last traces of air. Triethylamine (640
µl, 4.56 mmol), CuI (87 mg, 0.456 mmol), and tetrakis(triphenyl-
phosphane)palladium (263 mg, 0.228 mmol) were added to the so-
lution. Stirring was continued for 6 h, and the mixture was then
evaporated under reduced pressure. The residue was coevaporated
with xylene under reduced pressure and chromatographed on a col-
umn of silica gel (40 g) with CHCl3/MeOH (100:2, v/v). The frac-
tions containing 10 were combined and evaporated under reduced
pressure. The residue was dissolved in water-MeOH (1:1, v/v,
20 mL), and the solution was passed through a Dowex 1-X8 col-
[D5]pyridine): δ ϭ 2.02Ϫ2.06 (m, 2 H), 2.66Ϫ2.82 (m, 4 H),
3.82Ϫ3.86 (m, 2 H), 4.20 (m, 2 H), 4.51 (m, 1 H), 5.93Ϫ5.95 (m,
2 H), 7.05 (t, 1 H, J1Ј,2Ј ϭ J1Ј,2" ϭ 7.3 Hz), 7.38Ϫ8.10 (m, 5 H),
8.32 (s, 1 H), 13.28, (1 H, br). Ϫ 13C NMR (68 MHz, [D5]pyridine):
δ ϭ 24.1, 32.1, 38.1, 61.2, 62.5, 77.0, 85.4, 86.2, 115.1, 128.9, 129.9,
130.2, 133.6, 151.9, 164.8, 166.1. Ϫ C19H22N2O7·0.1H2O: C 58.18,
H 5.70, N 7.14; found C 57.90, H 5.42, N 7.12.
Cyclodeoxyuridylic Acid Derivative 16, Possessing a Propylene
Bridge: A mixture of 14 (48 mg, 0.123 mmol) and 1H-tetrazole
(41 mg, 0.488 mmol) was rendered anhydrous by repeated coevap-
oration with dry toluene and finally dissolved in dry acetonitrile
(6 mL) and dry dioxane (6 mL). Bis(diisopropylamino)(2-cyanoe-
thoxy)phosphane (46 µL, 0.146 mmol) was added dropwise with
stirring over a period of 5 min. After the mixture had been stirred
for 1 h, tBuOOH (0.122 mL, 1.22 mmol) was added. The resulting
mixture was stirred for an additional 30 min and evaporated under
reduced pressure. The residue was extracted with CHCl3 (10 mL)
and water (10 mL). The organic layer was collected and dried over
Na2SO4, filtered, and evaporated under reduced pressure. The res-
idue was dissolved in pyridine (1.5 mL) and 25% aqueous ammonia
(4.5 mL). The mixture was kept at room temperature for 24 h. After
removal of the solvent under reduced pressure, the residue was
chromatographed on Whatman 3MM papers with iPrOH/ammo-
nia-water (7:2:1, v/v/v). Elution of the band containing the product
and lyophilization gave 16 (661 A260, 64%). Ϫ UV (H2O): λmax.
267 nm, λmin. 235 nm. Ϫ PC: Rf ϭ 0.38 (iPrOH/25% NH3 aq/
H2O ϭ 7:1:2). Ϫ Reversed phase HPLC: 5.9 min. Ϫ 1H NMR
(400 MHz, D2O): δ ϭ 1.50Ϫ1.55 (m, 1 H), 1.59Ϫ1.64 (m, 1 H),
2.24Ϫ2.38 (m, 4 H), 3.56Ϫ3.62 (m, 1 H), 3.64Ϫ3.68 (m, 1 H), 3.90
(1 H, dt, J4Ј,5" ϭ J5Ј,P ϭ 3.3 Hz, J5Ј,5" ϭ 12.1 Hz), 3.97Ϫ4.00 (m, 2
H), 4.49Ϫ4.53 (m, 1 H, J2Ј,3Ј ϭ J2",3Ј ϭ 6.2 Hz, J3Ј,4Ј ϭ 4.9 Hz),
6.19 (t, 1 H, J1Ј,2Ј ϭ J1Ј,2" ϭ 5.7 Hz), 7.61 (s, 1 H). Ϫ 13C NMR
(101 MHz, D2O, [D8]dioxane): δ ϭ 21.6, 27.4, 39.5, 63.7, 64.1, 70.2,
86.0, 86.4, 86.5, 112.3, 140.9, 152.5, 166.8. Ϫ 31P NMR (109 MHz,
D2O): δ ϭ 1.44. Ϫ MS (FABϩ) C12H18N2O8P (M ϩ H) calcd.
349.0801; found 349.0818.
2Ϫ
umn (CO3 form, 15 mL). The eluate and washings were com-
bined and evaporated under reduced pressure to give 10 as a foam
(597 mg, 71%). Ϫ 1H NMR (270 MHz, CDCl3): δ ϭ 2.12 (s, 3 H),
2.18 (s, 3 H), 2.26Ϫ2.60 (m, 2 H), 3.80 (1 H, br), 4.29Ϫ4.41 (m, 5
H), 5.25 (m, 1 H), 6.28 (t, 1 H, J1Ј,2Ј ϭ J1Ј,2" ϭ 6.8 Hz), 7.86 (s, 1
H), 10.06 (1 H, br). Ϫ 13C NMR (68 MHz, CDCl3): δ ϭ 20.8, 20.8,
378.0, 51.0, 63.7, 73.9, 82.5, 85.7, 92.9, 99.8, 142.2, 149.3, 162.1,
170.5, 170.5. Ϫ C16H18N2O8·0.5H2O: calcd. C 51.20, H 5.10, N
7.47; found C 51.62, H 4.85, N 7.39.
3Ј,5Ј-Di-O-acetyl-5-(3-hydroxypropyl)-2Ј-deoxyuridine (11): A solu-
tion of 10 (597 mg, 1.63 mmol) in dioxane/MeOH (1:1, v/v, 20 mL)
was treated with Pd/C (240 mg) under hydrogen with shaking at
room temperature for 43 h. The solution was filtered over Celite,
and the filtrate was evaporated under reduced pressure. The residue
was chromatographed on a column of silica gel (12 g) with CHCl3/
1
AcOEt (2:8, v/v) to give 11 as a foam (576 mg, 95%). Ϫ H NMR
(270 MHz, CDCl3): δ ϭ 1.73Ϫ1.79 (m, 2 H), 2.12Ϫ2.66 (10 H),
3.62 (m, 2 H), 4.26Ϫ4.45 (m, 4 H), 5.21Ϫ5.23 (m, 1 H), 6.32 (dd,
1 H, J1Ј,2Ј ϭ 8.6 Hz, J1Ј,2Ј ϭ 5.6 Hz), 7.35 (s, 1 H), 9.43, (1 H, br).
Ϫ
13C NMR (68 MHz, CDCl3): δ ϭ 20.8, 23.0, 31.5, 37.5, 60.7,
63.8, 74.1, 82.2, 84.9, 114.9, 135.5, 150.2, 163.8, 170.4, 170.5. Ϫ
C16H22N2O8·0.75H2O: C 50.01, H 6.17, N 7.30; found C 49.92, H
5.82, N 7.32.
Synthesis of Fully Protected Thymidylyl(3ЈǞ5Ј)deoxyuridine Deriv-
ative 18, Possessing a Cyclic Structure with a Propylene Bridge: A
mixture of 14 (46 mg, 0.118 mmol) and 1H-tetrazole (50 mg,
0.708 mmol) was rendered anhydrous by coevaporation with dry
toluene, and the residue was dissolved in dry acetonitrile-dioxane
(1:1, v/v, 12 mL). A solution of 5 (138 mg, 0.178 mmol) in aceton-
itrile (1.5 mL) was added dropwise to the mixture over a period of
5 min. The resulting mixture was stirred at room temperature for
2 h, and tBuOOH (0.12 mL, 1.2 mmol) was then added to the mix-
ture. After stirring for 30 min, the solution was evaporated under
reduced pressure and the residue was extracted with CHCl3
(20 mL) and 5% NaHCO3 (20 mL). The organic layer was col-
lected, washed twice with 5% NaHCO3 (10 mL), dried over
Na2SO4, filtered, and evaporated under reduced pressure. The res-
idue was chromatographed on a column of silica gel (12 g) with
CHCl3/MeOH (100:2, v/v) containing 1% pyridine to give 18 as a
foam (82 mg, 71%, ratio of the diastereomers 1.5:1).
3Ј-O-Benzoyl-5-(3-hydroxypropyl)-2Ј-deoxyuridine (14): Compound
11 (452 mg, 1.25 mmol) was dissolved in pyridine (6 mL) and 25%
aqueous ammonia (6 mL). The mixture was stirred at room tem-
perature for 10 h. The solvent was then removed under reduced
pressure. The residue was rendered anhydrous by coevaporation
three times with dry pyridine and finally dissolved in dry pyridine
(9 mL). 4,4Ј-Dimethoxytrityl chloride (554 mg, 1.63 mmol) was ad-
ded, and the mixture was stirred at room temperature for 3 h. Ex-
traction was performed with CHCl3 (50 mL) and 5% NaHCO3
(50 mL). The organic layer was collected, washed twice with 5%
NaHCO3 (25 mL), dried over Na2SO4, filtered, and evaporated un-
der reduced pressure. The residue was rendered anhydrous by re-
peated coevaporation with dry pyridine and finally dissolved in dry
pyridine (9 mL). To the solution were added benzoic anhydride
(504 mg, 2.23 mmol) and 4-dimethylaminopyridine (5 mg, 41
µmol). After stirring at room temperature for 7 h, the mixture was
1996
Eur. J. Org. Chem. 2001, 1989Ϫ1999

Stabilization of the Predominant C3Ј-endo Conformation in Dinucleoside Phosphotriester Derivatives
FULL PAPER
Unprotected Thymidylyl(3ЈǞ5Ј)thymidine Derivatives 3a and 3b,
H), 3.97 (m, 1 H), 4.38 (m, 1 H), 6.32 (t, 1 H, J1Ј,2Ј ϭ J1Ј,2"
ϭ
Possessing Cyclic Structures with Propylene Bridges: Compound 18 6.4 Hz), 6.73Ϫ6.79 (m, 8 H), 7.14Ϫ7.45 (19 H, 6-H), 8.54 (1 H,
(72 mg, 73.5 µmol) was dissolved in pyridine (1.5 mL), and 2%
aqueous ammonia (4.5 mL) was added. The mixture was stirred at
room temperature for 18 h. The mixture containing the debenzoyl-
ated product 31 was then evaporated under reduced pressure, and
the residue was coevaporated three times with toluene to remove
the last traces of pyridine and dissolved in 80% acetic acid (6 mL).
After having been kept at room temperature for 1 h, the mixture
was evaporated under reduced pressure. The residue was extracted
with ether (2.5 mL) and water (2.5 mL). The aqueous layer was
collected, washed twice with ether, lyophilized, and loaded onto a
br). Ϫ 13C NMR (68 MHz, CDCl3): δ ϭ 27.8, 40.4, 55.2, 61.3,
63.5, 72.1, 84.5, 85.3, 85.9, 86.8, 112.4, 113.0, 113.3, 126.7, 127.1,
127.7, 128.0, 128.1, 130.0, 135.5, 136.2, 136.9, 145.1, 145.5, 150.1,
158.3, 158.6, 162.8. Ϫ C53H52N2O10: C 72.58, H 5.98, N 3.20;
found C 72.19, H 6.02, N 3.33.
3Ј-O-Acetyl-5Ј-O-(4,4Ј-dimethoxytrityl)-5-[2-(4,4Ј-dimethoxy-
trityloxy)ethyl]-2Ј-deoxyuridine (22): Compound 21 (2.6 g,
2.96 mmol) was rendered anhydrous by repeated coevaporation
with dry pyridine (15 mL ϫ 3) and finally dissolved in dry pyridine
(20 mL). Acetic anhydride (0.84 mL, 8.88 mmol) was added and
the solution was stirred at room temperature for 8 h. Water was
then added to quench the reaction. The solution was diluted with
CHCl3 (200 mL) and washed with 5% NaHCO3 (200 mL). The or-
ganic phase was collected, dried over Na2SO4, filtered and evapor-
reversed phase HPLC column, using System 3, to give 3a (358 A260
28%) and 3b (332 A260, 26%).
,
Synthesis of Partially Protected Thymidylyl(3ЈǞ5Ј)deoxyuridine
Derivative 31, Possessing a Cyclic Structure with a Propylene Bridge:
Compound 18 (337 mg, 0.356 mmol) was dissolved in pyridine ated under reduced pressure. The residue was chromatographed on
(5 mL), and concentrated ammonia (25%. 15 mL) was added. After
having been kept at room temperature for 18 h, the mixture was
evaporated under reduced pressure. The residue was coevaporated
with toluene and chromatographed on a column of silica gel with
a column of silica gel (60 g) with hexane/CHCl3 (3:7, v/v) con-
taining 1% pyridine to give 22 as a foam (2.44 g, 90%). Ϫ 1H NMR
(270 MHz, CDCl3): δ ϭ 2.08 (s, 3 H), 2.20Ϫ2.41 (m, 4 H), 3.13
(m, 2 H), 3.44 (m, 2 H), 3.69 (s, 6 H), 3.75 (s, 6 H), 4.13 (m, 1 H),
CHCl3/MeOH (100:2.5, v/v) containing 1% pyridine to give 31 as 5.33 (m, 1 H), 6.34 (t, 1 H, J1Ј,2Ј ϭ J1Ј,2" ϭ 7.2 Hz), 6.73Ϫ6.76 (m,
a foam (284 mg, 91%, ratio of the diastereomers 1.3:1).
8 H), 7.15Ϫ7.41 (18 H), 7.54 (s, 1 H), 8.04 (1 H, br). Ϫ 13C NMR
(68 MHz, CDCl3): δ ϭ 21.0, 27.9, 37.8, 55.2, 61.3, 63.7, 74.9, 83.8,
84.5, 85.8, 87.0, 112.8, 113.0, 113.3, 126.7, 127.1, 127.7, 128.0,
128.2, 130.0, 130.1, 135.4, 136.3, 136.6, 144.5, 145.1, 150.2, 158.4,
158.7, 162.6, 170.3. Ϫ C55H54N2O11·1.2H2O: C 70.22, H 6.04, N
2.98; found C 69.89, H 5.94, N 2.99.
Synthesis of 5Ј-O-(4,4Ј-Dimethoxytrityl)thymidylyl(3ЈǞ5Ј)-
deoxyuridine Phosphoramidite Derivative 32: DMT-Tpc3dU 31
(279 mg, 0.319 mmol) was rendered anhydrous by repeated coevap-
oration with dry toluene and finally dissolved in CH2Cl2 (5 mL).
To the mixture were added triethylamine (134 µL, 0.96 mmol) and
2-cyanoethoxydiisopropylaminochlorophosphane
(106
µL,
3Ј-O-Acetyl-5-(2-hydroxyethyl)-2Ј-deoxyuridine (23): Compound 22
(2.32 g, 2.52 mmol) was dissolved in 80% acetic acid (40 mL) and
the resulting solution was kept at room temperature for 30 min.
The solvent was then removed under reduced pressure. The residue
was chromatographed on a column of silica gel (40 g) with CHCl3/
MeOH (100:4, v/v) to give 23 as a foam (666 mg, 84%). Ϫ 1H NMR
(270 MHz, CD3OD): δ ϭ 2.08 (s, 3 H, CH3COOϪ), 2.33Ϫ2.37 (m,
0.478 mmol). The mixture was stirred at room temperature for 2 h
and then quenched by addition of ethanol (0.5 mL) and diluted
with CHCl3 (15 mL). The CHCl3 solution was washed three times
with 5% NaHCO3 (15 mL), dried over Na2SO4, filtered, and evap-
orated under reduced pressure. The residue was dissolved in CHCl3
(1 mL) and this solution was added portionwise, with vigorous stir-
ring, to hexane (80 mL)-ether (20 mL)-pyridine (1 mL). The re- 2 H, 2Ј-H, 2"-H), 2.51 (t, 2 H, J ϭ 6.4 Hz), 3.13 (t, 2 H, J ϭ
sulting white precipitate was collected by removal of the solvent by
pipette and dried in vacuo to give 32 (322 mg, 94%).
6.3 Hz), 3.79 (m, 2 H), 4.07 (1 H, dd), 5.30 (m, 1 H), 6.27 (t, 1 H,
J1Ј,2Ј ϭ J1Ј,2" ϭ 7.1 Hz), 7.85 (s, 1 H). Ϫ 13C NMR (68 MHz,
CD3OD): δ ϭ 21.4, 31.8, 38.9, 61.6, 63.5, 76.9, 86.8, 87.3, 113.2,
139.4, 152.8, 166.4, 172.7. Ϫ C13H18N2O7: C 49.68, H 5.77, N 8.92;
found C 49.42, H 5.74, N 8.91.
5Ј-O-(4,4Ј-Dimethoxytrityl)-5-[2-(4,4Ј-dimethoxytrityloxy)ethyl]-2Ј-
deoxyuridine (21): Compound 19 (2.0 g, 3.80 mmol) was dissolved
in pyridine (150 mL), and 0.5  NaOH aqueous solution (150 mL)
was added. After having been stirred at room temperature for 12 h,
the mixture was neutralized by addition of Dowex 50 W X8 (free
form, 200 mL). The resin was removed by filtration and washed
with water-pyridine (1:1, v/v, 250 mL). The filtrate and washings
were combined and concentrated under reduced pressure. The res-
Protected Cyclodeoxyuridylic Acid Derivative 24, Possessing an
Ethylene Bridge: A mixture of 23 (50 mg, 0.16 mmol) and 1H-tetra-
zole (45 mg, 0.64 mmol) was rendered anhydrous by repeated co-
evaporation with dry toluene and finally dissolved in dry acetonitr-
ile (16 mL). Bis(diisopropylamino)(2-cyanoethoxy)phosphane
idue was partitioned between water (100 mL) and ether (100 mL). (60.3 mg, 0.19 mmol) was added dropwise with stirring over a
The aqueous layer was collected, washed twice with ether (50 mL),
and evaporated under reduced pressure. The residue was rendered
anhydrous by repeated coevaporation with pyridine (10 mL), co-
evaporated twice with dry pyridine (10 mL) to remove the last
traces of water, and finally dissolved in dry pyridine (40 mL). 4,4Ј-
Dimethoxytrityl chloride (2.84 g, 8.36 mmol) was added, and the
resulting solution was stirred at room temperature for 2 h. Extrac-
period of 10 min. After the mixture had been stirred for 1 h,
tBuOOH (0.16 mL, 1.6 mmol) was added. The resulting mixture
was stirred for an additional 1 h and evaporated under reduced
pressure. The residue was extracted with CHCl3/pyridine (3:2, v/v,
10 mL) and water (10 mL). The organic layer was collected, and
the aqueous layer was back-extracted with CHCl3/pyridine (3:2, v/
v, 10 mL). The combined extract was dried over Na2SO4, filtered,
tion was performed with CHCl3 (100 mL) and 5% NaHCO3 and evaporated under reduced pressure. The residue was chromato-
(100 mL). The organic phase was collected, washed twice with 5% graphed on preparative TLC plates with CHCl3/MeOH (9:1, v/v)
NaHCO3 (50 mL), dried over Na2SO4, filtered, and evaporated un- to give 24 as a foam (46 mg, 68%, ratio of the diastereoisomers ϭ
der reduced pressure. The residue was chromatographed on a col-
umn of silica gel (150 g), with CHCl3/MeOH (97:3, v/v) containing
1% pyridine, to give 21 as a foam (3.02 g, 90%). Ϫ 1H NMR
1.9:1). Diastereomer 24a: TLC, Rf ϭ 0.37 (CHCl3/CH3OH ϭ 20:1,
v/v, developed 3 times). Ϫ 1H NMR (270 MHz, [D5]pyridine): δ ϭ
1.97 (s, 3 H), 2.59Ϫ3.04 (m, 6 H), 4.37Ϫ4.81 (m, 7 H), 5.70 (t, 1
(270 MHz, CDCl3): δ ϭ 2.15Ϫ2.23 (m, 1 H), 2.31Ϫ2.35 (m, 3 H), H, J2Ј,3Ј ϭ J3Ј,4Ј ϭ 8.1 Hz), 6.78 (t, 1 H, J1Ј,2Ј ϭ J1Ј,2" ϭ 6.1 Hz),
3.15 (t, 2 H, J ϭ 5.8 Hz), 3.41 (m, 2 H), 3.70 (s, 6 H), 3.74 (s, 6 7.94 (s, 1 H), 13.33 (1 H, br). Ϫ 13C NMR (68 MHz, [D5]pyridine):
Eur. J. Org. Chem. 2001, 1989Ϫ1999
1997

M. Sekine, O. Kurasawa, K. Shohda, K. Seio, T. Wada
FULL PAPER
δ ϭ 19.7, 19.8, 20.6, 26.3, 26.5, 37.3, 62.6, 62.7, 67.7, 67.8, 68.1,
sure. The residue was chromatographed on a column of silica gel
(12 g), with CHCl3/MeOH (100:6, v/v) containing 1% pyridine, to
give 4a and 4b as a foam (46 mg, 86%).
68.2, 73.7, 84.3, 84.4, 85.9, 110.2, 117.9, 136.9, 151.7, 164.5, 170.1.
Ϫ
31P NMR (109 MHz, [D5]pyridine):
δ
ϭ
Ϫ0.58.
Ϫ
C16H20N3O9·0.5H2O: C 43.84, H 4.83, N 9.52; found C 44.10, H
5.02, N 9.23.
The diastereomers 4a and 4b were separated by reversed phase
HPLC with System 3 as described in the general procedure, and
isolated to give 4a and 4b. Compound 4a: Reversed phase HPLC
18.0 min (System 1).
Diastereoisomer 24b: TLC, Rf ϭ 0.32 (CHCl3/CH3OH ϭ 20:1, v/v,
developed 3 times). Ϫ 1H NMR (270 MHz, [D5]pyridine): δ ϭ 1.96
(s, 3 H), 2.59Ϫ2.62, (m, 1 H), 2.75 (1 H, dt), 2.98Ϫ3.03 (m, 4 H),
Typical Procedure for the Synthesis of Oligonucleotides Incorporat-
ing Umc3Um in the Solid Phase Approach: The standard protocol
described in the ABI manual for the ABI 381 synthesizer was used.
For the incorporation of Umpc3Um into the middle position of
dodecanucleotides, the Umpc3Um amidite was used in a glass ves-
sel with a filter. A T-loaded CPG resin (1 µmol, 44 µmol/g, Gren
Research) or U-loaded CPG resin (19.7 µmol/g) was used. When
the modified dimer was inserted, the following manual manipula-
tion procedure was used: (1) detritylation with 3% TCA/CH2Cl2,
(2) washing with pyridine, (3) dry up, CH3CN for 10 min, (4) con-
densation with the amidite unit (0.1 , 20 equiv.) in the presence
of 1H-tetrazole (0.5 ,100 equiv.) /CH3CN for 10 min, (5) washing
with CH3CN, (6) capping with Ac2O-Py (1:9, v/v) in the presence
of 0.1  DMAP for 1 min, (7) washing with pyridine (8), oxidation
with 0.05  I2/THF/Py-H2O for 1 min (7:2:1, v/v/v), (9) washing
with pyridine, (10) washing with CH2Cl2. After the final condensa-
tion was complete, the CPG gel was removed from the synthesizer
and treated with 25% NH3 aq. Py (9:1, v/v, 2 mL) for 20 h. The
excess ammonia was removed under reduced pressure and the resin
was suspended in water (1 mL). The mixture was evaporated under
reduced pressure and lyophilized. (When the synthesis of RNA oli-
gomers was carried out, the resin was treated with 1  TBAF·H2O
in THF (1 mL) 16 h, after which desalting was performed by gel
filtration using Sephadex G-15. The eluate was lyophilized.) The
lyophilized material was separated by anion-exchange HPLC, using
a FAX column, followed by reversed phase HPLC:
4.37Ϫ4.67 (m, 7 H), 5.49 (m, 1 H), 6.45 (t, 1 H, J1Ј,2Ј ϭ J1Ј,2"
ϭ
5.9 Hz), 7.91 (s, 1 H), 13.32 (1 H, br). Ϫ 13C NMR (68 MHz,
[D5]pyridine): δ ϭ 19.7, 19.8, 20.6, 26.4, 40.1, 62.6, 62.6, 67.8, 68.0,
68.5, 68.6, 75.2, 85.4, 85.5, 88.5, 108.3, 117.8, 137.0, 151.3, 164.6,
170.3. Ϫ 31P NMR (109 MHz, [D5]pyridine): δ ϭ Ϫ1.58. Ϫ
C16H20N3O9·0.5H2O: C 43.84, H 4.83, N 9.52; found C 44.10, H
5.02, N 9.23.
Unprotected Cyclodeoxyuridylic Acid Derivative 25, Possessing an
Ethylene Bridge: The fully protected cyclodeoxyuridylic acid deriv-
ative 24 (17 mg, 38.7 µmol) was dissolved in pyridine (1.5 mL) and
25% ammonia (1.5 mL) was added. The mixture was stirred at
room temperature for 2.5 h and then evaporated under reduced
pressure. Chromatography of the residue on Whatman 3MM pa-
pers with iPrOH/ammonia/water (7:2:1, v/v/v) gave 25 (189 A260
,
53%). Ϫ UV (H2O): λmax. ϭ 266 nm, λmin. ϭ 244 nm. Ϫ PC: Rf ϭ
0.43 (iPrOH/25% NH3 aq/H2O ϭ 7:1:2)]. Ϫ Reversed phase HPLC:
10.2 min. Ϫ 1H NMR (400 MHz, D2O): δ ϭ 2.40 (1 H, dt, J1Ј,2Ј
ϭ
J2Ј,3Ј ϭ 5.61 Hz, J2Ј,2" ϭ 14.0 Hz), 2.54 (m, 1 H), 2.61Ϫ2.68 (m, 2
2
H), 3.81 (1 H, ddd, J4Ј,5" ϭ 3.2 Hz, J5Ј,5" ϭ 11.6 Hz, J5Ј,P
2.9 Hz), 3.81 (1 H, ddd, J4Ј,5Ј ϭ 2.7 Hz, J5Ј,5" ϭ 11.7 Hz, J5Ј,P
ϭ
ϭ
2.7 Hz), 4.21 (m, 2 H), 4.37 (m, 1 H), 4.53 (m, 1 H, J2Ј,3Ј ϭ 4.5 Hz,
J3Ј,4Ј ϭ 2.5 Hz), 6.19 (dd, 1 H, J1Ј,2Ј ϭ 3.9 Hz, J1Ј,2" ϭ 5.7 Hz), 8.00
(s, 1 H). Ϫ 13C NMR (68 MHz, D2O): δ ϭ 26.2, 26.3, 41.6, 64.5,
64.6, 65.7, 65.8, 71.6, 88.1, 88.2, 88.4, 110.9, 138.1, 152.0, 167.2. Ϫ
31P NMR (109 MHz, D2O):
δ ϭ 0.11. Ϫ MS (FABϩ):
C11H15N2NaO8P (M ϩ H): calcd. 357.0464; found 357.0492.
Compounds T4[Tpc3Um(fast)]T4 30a and T4[Tpc3Um(slow)]T4
30b were synthesized by using the phosphoroamidite unit 28 and
finally isolated in 3% (2.3 A260) and 3% (2.0 A260) yields, respect-
ively, by HPLC. Similarly, T4[Tpc3dUm(fast)]T4 33a and
T4[Tpc3dUm(slow)]T4 33b were synthesized in 12% (8.4 A260) and
7% (5.0 A260) yields, respectively, by using the phosphoroamidite
32.
Synthesis of the Fully Protected Thymidylyl(3ЈǞ5Ј)thymidine Deriv-
ative 27, Possessing a Cyclic Structure with an Ethylene Bridge: A
mixture of 23 (50 mg, 0.16 mmol) and 1H-tetrazole (45 mg,
0.64 mmol) was rendered anhydrous by coevaporation with dry
toluene and the residue was dissolved in dry acetonitrile (8.5 mL).
A solution of 5 (0.234 mmol) in acetonitrile (7.5 mL) was added
dropwise to the mixture over a period of 10 min. The resulting
mixture was stirred at room temperature for 40 min and a solution
of 5 (0.079 mmol) in acetonitrile (2.5 mL) was added. After stirring
had been continued for an additional 20 min, tBuOOH (0.16 mL,
1.6 mmol) was added to the mixture. After stirring for 1 h, the solu-
tion was evaporated under reduced pressure and the residue was
extracted with CHCl3 (50 mL) and 5% NaHCO3 (50 mL). The or-
ganic layer was collected, washed twice with 5% NaHCO3 (25 mL),
dried over Na2SO4, filtered, and evaporated under reduced pres-
sure. The residue was chromatographed on a column of silica gel
(12 g), with CHCl3/MeOH (100:2, v/v) containing 1% pyridine, to
give 27 as a foam (101 mg, 70%, ratio of the diastereomers 1.2:1).
Acknowledgments
This work was supported by a Grant from "Research for the
Future" Program of the Japanese Society for the Promotion of Sci-
ence (JSPS-RFTF97I00301) and a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Science, Sports, and Cul-
ture, Japan.
[1]
[1a] W. Saenger In Principles of Nucleic Acid Structure, Springer-
[1b]
Verlag: New York, 1984. Ϫ
D. Söll, U. L. RajBhandary
(Eds.), tRNA: Structure, Biosynthesis, and Function, ASM
[1c]
Press: Washington, DC., 1995. Ϫ
M. Sundaralingam, H.
Mizuno, C. D. Stout, S. T. Rao, M. Liebman, N. Yathindra,
Unprotected Thymidylyl(3ЈǞ5Ј)thymidine Derivatives 4a and 4b,
Possessing Cyclic Structures with Ethylene Bridges: Compound 27
(87.1 mg, 96.4 µmol) was dissolved in pyridine (1.5 mL), and 2%
aqueous ammonia (1.5 mL) was added. The mixture was stirred at
room temperature for 9 h. The mixture was then evaporated under
reduced pressure, and the residue was coevaporated three times
with toluene to remove the last traces of pyridine, and dissolved in
80% acetic acid (4 mL). After having been kept at room temper-
ature for 30 min, the mixture was evaporated under reduced pres-
Nucleic Acids Res. 1976, 3, 2471Ϫ2484.
[2] [2a]
[2b]
G. J. Quigley, A. Rich, Science 1976, 194, 796Ϫ806. Ϫ
U. von Ahsen, R. Green, R. Schroeder, H. F. Noller, RNA
1997, 3, 49Ϫ56.
[3] [3a]
H. W. Pley, K. M. Flaherty, D. B. McKay, Nature 1994,
[3b]
372, 68Ϫ74. Ϫ
W. G. Scott, J. T. Finch, A. Klug, Nucleic
Acids Symp. Ser. 1995, 34, 214Ϫ216.
[4]
[4a] W. G. Scott, J. T. Finch, A. Klug, Cell. 1995, 81, 991Ϫ1002.
Ϫ
[4b] W. G. Scott, J. B. Murray, J. R. P. Arnold, B. L. Stoddard,
A. Klug, Science 1996, 274, 2065Ϫ2069.
1998
Eur. J. Org. Chem. 2001, 1989Ϫ1999

Stabilization of the Predominant C3Ј-endo Conformation in Dinucleoside Phosphotriester Derivatives
FULL PAPER
[5]
U2 snRNA has a U-turn in the IIa loop: S. C. Stallings, P. B.
Moore, Structure 1997, 5, 1173Ϫ1185. In the loop of hepatitis
δ virus antigenomic RNA, a sharp reversed U-turn structure
was found: M. H. Kolk, H. A. Heus, C. W. Hilbers, EMBO J.
1997, 16, 3685Ϫ3692. The following papers reported the pres-
ence of the U-turn structure in an RNA hairpin loop: S. Hu-
ang, Y. X. Wang, D. E. Draper, J. Mol. Biol. 1996, 258,
308Ϫ321. F. M. Jucker, A. Pardi, RNA 1995, 1, 219Ϫ222.
M. Sekine, O. Kurasawa, K. Shohda, K. Seio, T. Wada, J. Org.
Chem. 2000, 65, 3571Ϫ3578.
621Ϫ649. The following equation was used: P(C3Ј-endo) ϭ
1 Ϫ (J1Ј,2ЈϩJ1Ј,2" Ϫ 9.8)/5.9.
[15]
C. Altona, M. Sundaralingham, J. Am. Chem. Soc. 1973, 95,
2333Ϫ2344. The following equation was used: P(C3Ј-endo) ϭ
J3Ј,4Ј/(J1Ј,2Ј ϩ J3Ј,4Ј).
J. E. Marugg, A. Burik, M. Tromp, G. A. van der Marel, J. H.
van Boom, Tetrahedron Lett. 1986, 27, 2271Ϫ2274.
S. L. Beaucage, M. H. Carruthers, Tetrahedron Lett. 1981, 22,
1859Ϫ1862.
[16]
[17]
[18]
[6]
[7]
G. Kawai, Y. Yamamoto, T. Kamimura, T. Masegi, M. Sekine,
T. Hata, T. Iimori, T. Watanabe, T. Miyazawa, S. Yokoyama,
Biochemistry 1992, 31, 1040Ϫ1046.
K. Seio, T. Wada, K. Sakamoto, S. Yokoyama, M. Sekine, J.
Org. Chem. 1996, 61, 1500Ϫ1504.
K. Seio, T. Wada, K. Sakamoto, S. Yokoyama, M. Sekine, Tet-
rahedron Lett. 1995, 36, 9515Ϫ9518.
K. Seio, O. Kurasawa, T. Wada, S. Sakamoto, S. Yokoyama,
M. Sekine, Nucleosides Nucleotides 1997, 16, 1023Ϫ1032.
M. Sekine, O. Kurasawa, K. Seio, T. Wada, submitted to J.
Org. Chem.,
[8]
[19]
D. B. Davies, Prog. Nucl. Mag. Res. Spectroscopy 1978, 12.
135Ϫ225.
[9]
[20] [20a] M. J. Robins, P. J. Barr, J. Org. Chem. 1983, 48, 1854Ϫ1862.
[20b]
Ϫ
F. W. Hobbs, Jr., J. Org. Chem. 1989, 54, 3420Ϫ3422.
[10]
[21]
[22]
J. Engels, A. Jäger, Angew Chem. Int. Ed. Engl. 1982, 912Ϫ913.
C. Altona, Recl. Trav. Chim. Pays-Bas 1982, 101, 413Ϫ433.
[11] [11a]
The following equation was used: P(gϩ) ϭ (13.3 Ϫ J4Ј,5Ј
J4Ј,5")/9.7.
Ϫ
K. Sakamoto, G. Kawai, T. Niimi, S. Watanabe, T. Niimi,
T. Satoh, M. Sekine, Z. Yamauti, S. Nisimura, T. Miyazawa, S.
[11b]
[23]
[24]
Yokoyama, Eur. J. Biochem. 1993, 216, 369Ϫ375. Ϫ
K.
H. Griengl, M. Bodenteich, W. Hayden, E. Wanek, W. Stre-
icher, P. Stütz, H. Bachmayer, I. Ghazzouli, B. Rosenwirth, J.
Med. Chem. 1985, 28, 1679Ϫ1684.
Sakamoto, G. Kawai, S. Watanabe, T. Niimi, N. Hayasi, Y.
Muto, K. Watanabe, T. Satoh, M. Sekine, S. Yokoyama, Bio-
chemistry 1996, 35, 6533Ϫ6538.
R. B. King, P. M. Sundaram, J. Org. Chem. 1984, 49,
[12]
[13]
[14]
D. R. Davies, C. D. Poulter, Biochemistry 1991, 30, 4223Ϫ4231.
D. B. Davies, S. S. Danyluk, Biochemistry 1974, 13, 4417Ϫ4434.
J. L Rinkel, C. Altona, J. Biomol. Struct. Dyns. 1987, 4,
1784Ϫ1789.
Received October 3, 2000
[O00508]
Eur. J. Org. Chem. 2001, 1989Ϫ1999
1999