Oligonucleotide analogues with a nucleobase-including backbone. Part 5: 2′-deoxy-8-(hydroxymethyl)adenosine- and 2′-deoxy-6-(hydroxymethyl)uridine-derived phosphoramidites: Synthesis and incorporation into 14-mer DNA strands

Article abstract of DOI:10.1002/1522-2675(20010321)84:3<594::AID-HLCA594>3.0.CO;2-9

The pairing propensity of new DNA analogues with a phosphinato group between O-C(3′) and a newly introduced OCH₂ group at C(8) and C(6) of 2′-deoxyadenosine and 2′-deoxyuridine, respectively, was evaluated by force-field calculations and Maruze

Full text of DOI:10.1002/1522-2675(20010321)84:3<594::AID-HLCA594>3.0.CO;2-9

594
Helvetica Chimica Acta ± Vol. 84 (2001)
Oligonucleotide Analogues with a Nucleobase-Including Backbone
Part 5
2'-Deoxy-8-(hydroxymethyl)adenosine- and 2'-Deoxy-6-(hydroxymethyl)uridine-
Derived Phosphoramidites: Synthesis and Incorporation into 14-Mer DNA Strands
by Werngard Czechtizky and Andrea Vasella*
Laboratorium für Organische Chemie, ETH-Zentrum, Universitätstrasse 16, CH-8092 Zürich
The pairing propensity of new DNA analogues with a phosphinato group between O C(3') and a newly
introduced OCH2 group at C(8) and C(6) of 2'-deoxyadenosine and 2'-deoxyuridine, respectively, was evaluated
by force-field calculations and Maruzen model studies. These studies suggest that these analogues may form
autonomous pairing systems, and that the incorporation of single modified units into DNA 14mers is compatible
with duplex formation. To evaluate the incorporation, we prepared the required phosphoramidites 3 and 4 from
2'-deoxyadenosine and 2'-deoxyuridine, respectively. The phosphoramidite 5 was similarly prepared to estimate
the influence of a CH2OH group at C(8) on the duplex stability. The modified 14-mers 6 ± 9 were prepared by
solid-phase synthesis. Pairing studies show a decrease of the melting temperature by 2.58 for the duplex 13 ´ 9,
and of 6 ± 88 for the duplexes 10 ´ 6, 11 ´ 6, 13 ´ 7, and 14 ´ 8, as compared to the unmodified duplexes.
1. Introduction. ± We have conceived oligonucleotide analogues with a nucleobase-
including backbone (Fig. 1, B); these compounds should allow us to answer the
question of whether the structural differentiation between nucleobase and backbone in
DNA, RNA, and their analogues1) is a prerequisite for the formation of stable homo-
and/or heteroduplexes [6][7].
We have so far described oligouridine and oligoadenosine analogues that are linked
by ethynediyl groups between C(5') and C(6) of uridines, or C(5') and C(8) of
adenosines [6 ± 9]. In addition to these uncharged analogues that require a stepwise
synthesis in solution, we have considered phosphodiester analogues that should be
accessible by conventional solid-phase synthesis [10]. Similarly to DNA, they should be
water-soluble. These analogues possess phosphinato groups between O C(3') and a
oxymethyl substituent at C(8) or C(6) of neighbouring 2'-deoxyadenosyl or 2'-
deoxyuridyl units, respectively.
*
*
Fig. 2 shows the fully modified analogues (3'-10)dAn (1) and (3'-7)dUn (2),
incorporating the repeating units dA* and dU*2), and the phosphoramidites 3 and 4
1
)
For DNA and RNA analogues that have been prepared in search of antisense-compounds, to investigate
autonomous pairing-systems, protein/DNA-interactions, electron-transfer in DNA, and DNA-damage, see
[1 ± 5].
The repeating units of B-DNA are denoted as dA and dT; units that are linked via O(3') and O(10), or
O(7), respectively, are denoted as dA* and dU*, and the dAunits CH2OH-substituted at C(8) as dA** (Fig. 2).
The linking mode is indicated by two numerals in parenthesis. A dash, as in (3'-10), indicates a phosphinato
group between O(3') and O(10), and indicates the (3' ! 10) direction of the link. In fully modified
analogues, all dA and dT repeating units of DNA are replaced by dA* and dU*, while partially modified
analogues correspond to DNA strands in which part of the dA or dT units are replaced by dA* or dU*.
2
)

Helvetica Chimica Acta ± Vol. 84 (2001)
595
Fig. 1. Conventional oligonucleotides with structural separation of the nucleobases and the backbone (A) and
oligonucleotides with a nucleobase-including backbone (B)
derived from dA* and dU*, respectively. In this context, we also prepared the
phosphoramidite 5 to study the influence of a C(8)-hydroxymethylated dA on the
pairing behaviour of DNA oligomers. To avoid unfavourable steric interactions
between the Me group at C(5) of thymidine and the backbone connected to C(6), we
have replaced thymidine by 2'-deoxyuridine analogues.
*
*
We report on force-field calculations of the homoduplex (3'-10)dA14 ´ (7-3')dU14,
*
the heteroduplex (3'-10)dA14 ´ (5'-3')dT14, and partially modified duplexes, and
describe the synthesis of singly modified tetradecamers containing one dA*, dU*, or
dA** unit.
2. Results and Discussion. ± 1. Model Studies and Calculations. The conformations
and the pairing propensity of the above described oligonucleotide analogues (Fig. 2)
were first evaluated with the help of Maruzen models. Models of the fully modified
*
*
*
homoduplex (3'-10)dA14 ´ (7-3')dU14, and heteroduplexes (3'-10)dA14 ´ (5'-3')dT14 and
*
(3'-7)dU14 ´ (5'-3')dA14, respectively, were constructed on the basis of the results of a
conformational analysis of DNA [11], i.e., by assuming a 2E pucker of the carbohydrate
moiety, a Æ sc conformation around the P O bonds, a ꢀzigzagꢁ conformation of the
backbone-chain, and minimal 1,5-repulsions in the single-strands. This led to two
*
*
plausible helical models for the homoduplex (3'-10)dA14 ´ (7-3')dU14, both character-
ized by antiparallel strands, but differing in the dihedral angles for the nucleosidic and
for the P O bonds.

596
Helvetica Chimica Acta ± Vol. 84 (2001)
*
*
Fig. 2. Proposed adenosine and uridine oligomers dAn and dUn , DNA repeating units dA and dT, modified
repeating units dA*, dU*, and dA**, and phosphoramidites 3, 4, and 5 derived from dA*, dU*, and dA**,
respectively
In the first model, the nucleobases adopt the anti-conformation with dihedral angles
O(4') C(1') N(9) C(4) of ca.
O(4') C(1') N(1) C(2) of ca.
1008 in the dA* units and dihedral angles
1308 in the dU* units, comparable to the
corresponding dihedral angles in B-DNA [12]. In the second model, the nucleobases
adopt a syn-conformation with dihedral angles O(4') C(1') N(9) C(4) and
O(4') C(1') N(1) C(2) of ca. 70 ± 808. Depending on the syn/anti-conformation,
the dihedral angles for the P O bonds are sc in the first model and ‡ sc in the
second one. In both duplexes, the dihedral angles characterizing the torsion about the
C(3') O(3') bond are beween ap and ac. The angles for the C(8) C(10) and the
C(6) C(7) bonds are ca. 1808, corresponding to an antiperiplanar orientation of N(9)
and O(10), and of N(1) and O(7). Both models show a parallel orientation of the

Helvetica Chimica Acta ± Vol. 84 (2001)
597
Watson-Crick base pairs at a distance of ca. 3.3 ± 3.5 Š, comparable to the distance
between base pairs in B-DNA (3.3 ± 3.4 Š) [13], indicating that these analogues may
indeed form stable homoduplexes.
*
*
Helical models of the heteroduplexes (3'-10)dA14 ´ (5'-3')dT14 and (3'-7)dU14 ´ (5'-
3')dA14 with antiparallel strands could be constructed only if the nucleobases in the
modified strands adopt the anti-conformation. The conformations of the dA* and dU*
units correspond to those of the homoduplexes with an anti-conformation (see above).
In the complementary DNA strand, the dA and dTunits adopt conformations similar to
those described for dA and dT units in B-DNA [14].
We have also evaluated partially modified analogues. The incorporation of a single
dA* or dU* unit into a B-DNA duplex does not considerably change the Watson-Crick
base pairing and base stacking, irrespectively of the specific sequence, as long as the
nucleobases in the modified units adopt an anti-conformation. Again, the conforma-
tions of the modified nucleotides are similar to those described for dA* and dU* in the
models of the homoduplexes characterized by an anti-conformation.
This qualitative analysis formed the basis of force-field calculations (Macromodel
V. 4.5, Amber* force-field, gas-phase [15]) to evaluate the potential of the fully and
partially modified analogues to form homo- and heteroduplexes similar to DNA. Since
the starting geometries were derived from B-DNA, only duplexes with an anti-
conformation of the nucleobases were investigated. The starting geometry of the
antiparallel, fully and partially modified duplexes were generated by building the B-
helix of dA14 ´ dT14 with the grow-tool of Macromodel, and then replacing some or all
nucleotides by the modified nucleotides. The starting conformations of the repeating
units dA* and dU* were similar to those derived from the Maruzen models. After
*
energy minimization (2000 to 3000 iterations), both the homoduplex (3'-10)dA14 ´ (7-
*
*
3')dU14 (Fig. 3,a) and the heteroduplex (3'-10)dA14 ´ (5'-3')dT14 (Fig. 3,b) retained a
conformation similar to the one initially chosen.
The helical models show an almost parallel arrangement of the nucleobases at a
distance of around 3.3 Š, suitable for Watson-Crick base pairing and base stacking.
Several DNA duplexes partially modified in one strand were similarly investigated. The
partially modified B-DNA duplexes containing one, two, or three dA* or dU* units
with an anti-conformation also showed base pairing and base stacking similar to B-
DNA. The values of the propeller- and buckle-twists as well as the length of the H-
bonds within the Watson-Crick base pairs [16][17] showed no significant deviation from
those in B-DNA.
Neither the position of the modified units in the singly modified strands nor the
sequence of these strands appeared (at this level of calculation) to significantly affect
the pairing propensity. Among the possible singly modified tetradecamers, we chose to
synthesize 6 ± 9 (Table 1), which possess a modified unit in the center of the chain,
flanked by dC and dG units. The choice of 6 was facilitated by an ample supply of the
complementary RNA 103) that allowed to also study the pairing of 6 with RNA.
2. Synthesis of Oligomers. For the synthesis of the oligomers 6 ± 9 and 11 ± 14
(Table 1), we intended to use the standard phosphoramidite chemistry; the (benzoyl-
oxy)methyl group of the required phosphoramidites 3 ± 5 should not interfere with the
3
)
We thank S. Pitsch for a sample of 5'-r(GAUAGCUCGGAUGC)-3' (10) [18].

598
Helvetica Chimica Acta ± Vol. 84 (2001)
*
*
Fig. 3. Amber*-calculated structures of the fully and the semi-modified duplexes (3'-10)dA14 ´ (7-3')dU14 and
*
(3'-10)dA14 ´ (5'-3')dT14
Table 1. Oligodeoxynucleotides 6 ± 9 and 11 ± 14, Total Yields, and Molecular Masses
Sequence
Total yielda)
Calculated mass
MALDI-TOF
b
5'-d(GCATCCGA*GCTATC)-3'
5'-d(CTATCGA*GCTTACG)-3'
5'-d(CGTAAGCU*CGATAG)-3'
5'-d(CTATCGA**GCTTACG)-3'
5'-d(GATAGCTCGGATGC)-3'
5'-d(GCATCCGAGCTATC)-3'
5'-d(CGTAAGCTCGATAG)-3'
5'-d(CTATCGAGCTTACG)-3'
6
7
8
87%
87%
80%
74%
91%
93%
94%
99%
4253.8
4268.8
4303.8
4268.8
4303.8
4223.8
4287.8
4238.8
)
4268.6
4308.2
4268.3
9
b
11
12
13
14
)
4226.7
4287.4
4239.1
a) Calculated from the yield of the coupling-steps, as determined by the detritylation assay. b) Not determined.
coupling and be readily cleaved under the ammonolytic conditions applied for the
deprotection of the oligomers.
To prepare 3, 2'-deoxyadenosine was monobenzoylated in 90% yield by treatment
with Me3SiCl and benzoyl chloride (BzCl) [19] (Scheme 1). Disilylation of the resulting
crystalline diol 15 with Et3SiCl and imidazole in DMF [20] gave 88% of the Et3Si-

Helvetica Chimica Acta ± Vol. 84 (2001)
599
protected nucleoside 16. Adenosine derivatives have been C(8)-hydroxymethylated by
deprotonation with LDA (ˆ lithium diisopropylamide) or lithium-2,2,6,6-tetramethyl-
piperidide, addition of ethyl formiate, and reduction with NaBH4 [21]. Subjecting 16 to
this protocol yielded only 30% of the desired alcohol 17, while replacing ethyl formiate
by DMF increased the yield to ca. 70%. 4,4'-Dimethoxytritylation of 17 gave 18 (89%),
which was desilylated to 19 (Bu4NF in THF; 95%). The regioselective benzoylation of
the diol 19 with BzCl and Hünigꢁs base in CH2Cl2, at 108, as described for 2'-
deoxyuridine derivatives [22], gave 75% of the monobenzoate 20 that was converted to
the phosphoramidite 3 in the usual way [23].
Scheme 1
a) Et3SiCl, imidazole, DMF, 238; 88%. b) 1. LDA/THF, 788; 2. DMF, 788; 3. AcOH, 238; 4. NaBH4/EtOH,
238; 70%. c) 4,4'-Dimethoxytrityl chloride (ˆ bis(4-methoxyphenyl)phenylmethyl chloride; DMTrCl), CH2Cl2,
iPr2NEt, 4-(dimethyamino)pyridine (DMAP), 238; 89% (18), 72% (24). d) Bu4NF, THF, 238; 95% (19), 83%
i
(23). e) BzCl, CH2Cl2, Pr2NEt, 108; 75% (20), 73% (22). f) Ac2O, Py, 238; 53%. g) (NCCH2CH2O)-
P(iPr2N)Cl, CH2Cl2, iPr2NEt, 238; 82% (3), 72% (5).

600
Helvetica Chimica Acta ± Vol. 84 (2001)
The phosphoramidite 5, which corresponds to a 3',5'-linked repeating unit dA**,
was prepared in an overall yield of 31% by benzoylation of the alcohol 17, followed by
desilylation, 4,4'-dimethoxytritylation at O(5') (72%), and phosphitylation.
The phosphoramidites 3 and 5 are ca. 3 :2 to 1:1 mixtures of diastereoisomers. At
108 under Ar, they are stable for several months.
The regioselectivity of the benzoylation of 19 was confirmed by N,O-acetylation of
the monobenzoate 20. The signals of H C(3') and both H C(5') of 20 and of the
acetylated product 21 were assigned on the basis of their multiplicity and coupling
constants (see Table 5). Compared to H C(3') of 20 (d 5.05 ppm), H C(3') of 21
(Scheme 1) is shifted downfield by 0.57 ppm, while the chemical shift for H C(5') is
hardly affected. The broad d of HO C(3') at 2.70 ppm (J(H,OH) ꢀ 1.0 Hz) disappeared
upon H/D exchange of 20, while the broad m of H C(3') collapsed to a dt.
As a rule, the shift from the anti- to the syn-conformation is indicated by a
downfield shift of Hpro-S C(2') and H C(3'), and an upfield shift of C(2') [24]. The
significant downfield shifts of the signals of Hpro-S C(2') (Dd ˆ 0.29 ± 0.86 ppm) and
H
C(3') (Dd ˆ 0.08 ± 0.54 ppm) and the upfield shift of C(2') (Dd ˆ 0.8 to
4.3 ppm) for the nucleosides 17 ± 20, 22 ± 24, and for the phosphoramidites 3 and 5,
as compared to the corresponding signals of 16, evidence that all C(8)-substituted
nucleosides adopt predominantly the syn-conformation (Table 2).
Table 2. 1H- and 13C-NMR Chemical-Shift Differences Dd(H C(2')), Dd(H C(3')), and Dd(C(2')) [ppm] for
the Nucleosides 3, 5, and 17 ± 24 as Compared to 16, and Ratio of S/N-Conformers of these Nucleosides, as
Deduced from J(1',2')/J(3',4') [Hz]
Nucleoside
Dd(H C(2'pro-S))
Dd(H C(3'))
Dd(C(2'))
J(1',2')
J(3',4')
Ratio S/N
16
17
18
19
20
21
3
22
23
24
5
±
0.55
0.84
0.29
0.71
0.96
0.86/0.81
0.84
0.39
±
0.08
0.10
0.13
±
6.4
6.6
6.8
9.5
5.6
8.1
7.4/6.2
6.6
9.5
3.8
3.7
2.8
< 1.0
5.0
3.0
63/37
64/36
71/29
> 90/10
53/47
73/27
2.5
4.1
0.8
3.5
6.6
2.1
3.0
0.8
3.6
4.3
0.42
0.99a)
0.54/0.45
0.13
5.3/4.5
58/42, 58/42
3.3
< 1.0
4.1
67/33
> 90/10
62/38
0.17
0.27
0.35/0.27
0.81
0.73/0.63
6.6
6.5
b
b
)
)
a) Partially due to acetylation at O(3'). b) J(1',2') and J(3',4') only partially determined.
The ratio J(1',2'pro-S)/J(3',4') is considered a measure of the position of the
equilibrium between the S- (sugar-pucker E ± 2T1) and N-conformers (sugar-pucker
3
3T2 ± 3T4) [25]. As expected, the S-conformation is slightly preferred in the O(5')-
protected nucleosides 16 ± 18, 20 ± 22, and 24, as indicated by the J(1',2'pro-S) (6.2 ±
8.1 Hz) and J(3',4') values (2.8 ± 5.0 Hz) (Table 2). In the diols 19 and 23, J(1',2p' ro-S
)
and J(3',4') differ considerably from these values: J(1',2'pro-S) ˆ 9.5 and J(3',4') ꢁ 1.0 Hz
show a clear predominance of the S-conformer of 19 and 23. The strong downfield shift
for HO C(5') of 19 and 23 in CDCl3 (d 6.23 and 6.06 ppm, resp.) evidences an
intramolecular O(5')H ´´´ N(3) H-bond. Such H-bonds are well-documented [7][26 ±
31]. The nine-membered ring formed by the H-bond is rigid [7][26][27][32], as

Helvetica Chimica Acta ± Vol. 84 (2001)
601
evidenced by the characteristic J(5'pro-S, OH) ꢁ1.0, and J(5'pro-R, OH) ˆ 11.0 ± 11.2 Hz.
Both J(4',5') of 19 and 23 are ꢁ1.5 Hz, indicating a sc arrangement of H C(4') and
both H C(5'), again in agreement with the proposed H-bond. The intramolecular H-
bonds in 19 and 23 are confirmed by broad, intensive IR absorptions at 3289 cm 1 and
1
3286 cm of the diols 19 and 23.
The phosphoramidite 4 was synthesized from 2'-deoxyuridine by silylation to 25
[20], followed by treatment with LDA, DMF, and NaBH4 (Scheme 2). This yielded
only 30 ± 35% of the alcohol 26 besides 45% of starting material; 25 and 26 were readily
separated by flash chromatography. Increasing or decreasing the amount of LDA and/
or DMF lowered the yield of 26. The unsatisfactory yield is in agreement with results of
Miyasaka and co-workers [33], who showed that deprotonation of 2'-deoxyuridine
derivatives with LDA occurs in a maximum yield of 52%. BuLi and BuLi/TMEDA
(ˆ N,N,N',N'-tetramethylethylenediamine) preferentially deprotonate uridine deriva-
tives at C(5) [33]. Lithium 2,2,6,6-tetramethylpiperidide led to similar results as LDA
[21], while the use of LHMDS (ˆ lithium hexamethyldisilazane) did not lead to 26. 4,4'-
Dimethoxytritylation of the alcohol 26 to 27, desilylation to the diol 28, and
monobenzoylation led to 29, which was converted to the phosphoramidite 4 in the
usual way; again, 4 was obtained as a ca. 1:1 mixture of diastereoisomers that proved
stable at 108 under Ar for several months.
Scheme 2
a) 1. LDA/THF, 788; 2. DMF, 788; 3. AcOH, 238; 4. NaBH4/EtOH, 238; 30% (26), 45% (25). b) DMTrCl,
CH2Cl2, iPr2NEt, DMAP, 238; 63%. c) Bu4NF, THF, 238; 87%. d) BzCl, CH2Cl2, iPr2NEt, 108; 65%. e) Ac2O,
Py, 238; 75%. f) (NCCH2CH2O)P(iPr2N)Cl, CH2Cl2, iPr2NEt, 238; 72%.
The regioselectivity of the benzoylation was confirmed by H/D exchange and acetyla-
tion of 29. The signal of the H C(3') of 30 (see Table 7) is shifted downfield by
0.69 ppm, while the chemical shift for the two H C(5') remained practically unaltered.
Again, the downfield shifts for Hpro-S C(2') and H C(3'), and the upfield shift for
C(2') of both diastereoisomers 4 and of 26 ± 30 (as compared to the corresponding
signals of 25) show that the C(6)-substituted nucleosides adopt predominantly the syn-
conformation (Table 3).

602
Helvetica Chimica Acta ± Vol. 84 (2001)
Table 3. 1H- and 13C-NMR Chemical-Shift Differences Dd(H C(2')), Dd(H C(3')), and Dd(C(2')) [ppm] for
the Nucleosides 26 ± 30 and 4 as Compared to 25, and Ratio of S/N-Conformers of these Nucleosides, as Deduced
from J(1',2')/J(3',4') [Hz]
Nucleoside
Dd(H C(2'pro-S))
Dd(H C(3'))
Dd(C(2'))
J(1',2')
J(3',4')
Ratio S/N
25
26
27
28
29
30
4
±
±
0.05
±
0.27
0.33
1.02a)
0.37
±
6.2
7.2
6.2
7.5
4.1
5.6
3.7
4.0
4.7
4.4
6.5
5.0
63/37
64/36
57/43
63/37
39/61
53/47
0.50
0.82
0.86
0.84
1.08
0.84
2.8
3.6
3.3
3.1
6.3
3.9
b
b
b
)
)
)
a) Partially due to acetylation at O(3'). b) J(1',2') and J(3',4') only partially determined.
The position of the equilibrium between the S- and N-conformers of 25 ± 28 and 30
(ca. 60 :40) was deduced from the J(1',2'pro-S) and J(3',4') values (Table 3); it is slightly
in favour of the S-conformer, while the N-conformer of 29 is slightly preferred. There is
no NMR evidence for an intramolecular O(5')H ´´´ O(2) H-bond of the diol 28 in
CDCl3. The J(4',5') values (4.1 and 2.8 Hz) of 28 are similar to those of the O(5')-
protected alcohol 26. These coupling constants evidence that the gauche,gauche-
conformation about the C(4') C(5') bond (essential for an intramolecular H-bond
between HO C(5') and O(2)) is less populated in 28 than in the 2'-deoxyadenosine-
derived diols 19 and 23 (J(4',5') ꢁ 1.5 Hz) that possess the intramolecular O(5')H ´´´
N(3) H-bond.
3. Solid-Phase Synthesis of 14-Mer DNA Strands Containing One dA*, dU*, or dA**
Unit, and of the Complementary DNA Strands. Table 1 shows the 14mer DNA strands
11 ± 14 that possess an internal dA or dT unit. Replacement of the internal dA of the
unmodified 14mers 12 and 14 by dA* (derived from 3) led to the modified strands 6 and
7, respectively, while replacement of dA of 14 by dA** (derived from 5) led to 9. The
exchange of the internal dT of the 14mer 13 by dU* (derived from 4) yielded the
modified strand 8.
The solid-phase syntheses of the oligomers 6 ± 9 and 11 ± 14 were carried out on a
DNA synthesizer and were based on a protocol for the synthesis of pRNA [10]. The
coupling time for the attachment of 3 ± 5 and of the DNA phosphoramidites
immediately following the incorporation of 3 ± 5 was arbitrarily set to 30 min; it led
to coupling yields between 80 ± 92%4) (99% for all other couplings). Longer coupling
times did not improve the yields.The 14-mers 6, 7, 9, and 11 ± 14 were deprotected and
cleaved off the solid support by ammonolysis with sat. aqueous NH3 solution/MeOH
1:1 at 508 within 20 h. Prolonged heating of the modified DNA strand 8 (incorporating
dU*) at 508 led to degradation; this strand was deprotected at 358. The crude products
were analyzed by RP-HPLC and showed a composition in agreement with the
detritylation assay. The products were purified by RP-HPLC, desalted, and analyzed by
RP-HPLC and MALDI-TOF mass spectroscopy. The total yield of the oligomers, as
4
)
Each chain-elongation cycle began with an acid-promoted cleavage of the 4,4'-dimethoxytrityloxy group.
‡
The efficiency of each coupling step was calculated by UV determination of the amount of DMTr (e ˆ
70000 l ´ mol 1 ´ cm 1).

Helvetica Chimica Acta ± Vol. 84 (2001)
603
determined from the detritylation assay, and their molecular masses are shown in
Table 1.
4. Pairing Studies. In a first set of measurements, we examined the influence of the
incorporation of one dA* unit into a DNA ´ DNA duplex or into the DNA strand of a
DNA ´ RNA duplex. The modified 14-mer 6 (Table 1) was hybridized with the
complementary 14-mer 11, or its RNA analogue 10, at a concentration of (2 ‡ 2) mm and
a pH of 7 in 0.15m aqueous NaCl (Table4).
Table 4. Melting Points Tm [8C] of 11 ´ 12, 10 ´ 12, 11 ´ 6, 10 ´ 6, 13 ´ 14, 13 ´ 7, 13 ´ 9, and 14 ´ 8 Obtained from
Temperature-Dependent UV Spectroscopy
Duplex
Tm
DTm
Strand conc.
Conc. NaCl
5'-d(GATAGCTCGGATGC)-3'
3'-d(CTATCGAGCCTACG)-5'
5'-r(GAUAGCUCGGAUGC)-3'
3'-d(CTATCGAGCCTACG)-5'
5'-d(GATAGCTCGGATGC)-3'
3'-d(CTATCGA*GCCTACG)-5'
5'-r(GAUAGCUCGGAUGC)-3'
3'-d(CTATCGA*GCCTACG)-5'
5'-d(CGTAAGCTCGATAG)-3'
3'-d(GCATTCGAGCTATC)-5'
5'-d(CGTAAGCTCGATAG)-3'
3'-d(GCATTCGA*GCTATC)-5'
5'-d(CGTAAGCTCGATAG)-3'
3'-d(GCATTCGA**GCTATC)-5'
5'-d(CTATCGAGCTTACG)-3'
3'-d(GATAGCU*CGAATGC)-5'
11 ´ 12
10 ´ 12
11 ´ 6
10 ´ 6
13 ´ 14
13 ´ 7
13 ´ 9
14 ´ 8
578
±
(2 ‡ 2) mm
0.15m
538
±
(2 ‡ 2) mm
(2 ‡ 2) mm
(2 ‡ 2) mm
(2 ‡ 2) mm
(2 ‡ 2) mm
(2 ‡ 2) mm
(2 ‡ 2) mm
0.15m
0.15m
0.15m
0.10m
0.10m
0.10m
0.10m
49.58
458
7.58
8.08
±
508
438
7.08
2.58
6.08
47.58
448
The modified duplexes show a decrease in melting temperature by 7.58 (11 ´ 6) and
8.08 (10 ´ 6) relative to the corresponding non-modified duplexes 11 ´ 12 and 10 ´ 12 (see
Fig. 4 for the melting curves).
Next, the 14-mer DNA strands 7 ± 9 incorporating one dA*, dU*, or dA** unit were
hybridized with the complementary DNA strands 13 or 14 at a concentration of (2 ‡ 2)
mm and a pH of 7 in 0.1m aqueous NaCl (Table 4). The melting curves of the non-
modified duplex 13 ´ 14, and the modified duplexes 13 ´ 7 and 14 ´ 8 are shown in Fig. 5,a.
Again, the duplex 13 ´ 7 (containing dA*) melted 78 lower than the DNA duplex 13 ´
14. The duplex 14 ´ 8 (containing dU*) showed a depression of the melting temperature
by 68, as compared to the non-modified DNA duplex 13 ´ 14.
Pairing of the modified 14mer 9, containing one dA** unit, and the complementary
DNA strand 13 yielded a duplex that melted only 2.58 lower than the non-modified
DNA duplex 13 ´ 14 (Table 4 and Fig. 5,b).
A decrease in melting temperature of 6 ± 88 for an incorporated dA* or dU* unit
indicates a strong destabilization of the DNA duplex. The degree of duplex
destabilization caused by dA* and dU* is comparable to the degree of duplex
destabilization imposed by mismatched base pairs. In the 13mer DNA duplex
GCGTACACATGCG ´ CGCATGXGTACGC, for example, the exchange of the
matching X ˆ thymidine by adenosine, guanosine, or cytosine leads to a decrease in
melting temperature of 5.4 ± 13.78 at a concentration of (1.5 ‡ 1.5) mm and a pH of 7 in

604
Helvetica Chimica Acta ± Vol. 84 (2001)
Fig. 4. a) Temperature-dependent UV spectra (ꢀmelting curvesꢁ) of the non-modified and modified DNA ´ DNA
duplexes 11 ´ 12 and 11 ´ 6 and b) the non-modified and modified DNA ´ RNA duplexes 10 ´ 12 and 10 ´ 6
0.1m aqueous NaCl [34]. The decrease by 2.58 of the melting temperature for the duplex
13 ´ 9, as compared to 6 ± 88 for the other modified duplexes, evidences the strong
influence of the linking mode, rather than just of the introduction of a CH2OH group,
on the duplex destabilisation.
Experimental Part
General. Solvents were distilled before use: THF from K/benzophenone, CH2Cl2 from CaH2. Reactions
were run under Ar. Qual. TLC: precoated silica-gel plates (Merck silica gel 60 F254); detection by spraying with
ꢀmostainꢁ (400 ml of 10% aq. H2SO4, 20 g of (NH4)6Mo7O24 ´ H2O, 0.4 g of Ce(SO4)2) and heating. Flash
chromatography (FC): silica gel Merck 60 (0.04 ± 0.063 mm). Optical rotations: 1-dm cell at 258 and 589 nm. FT-
1
IR: 1 ± 2% soln. in the indicated solvent. H-, 13C-, and 31P-NMR: at 200, 300, or 400 MHz, 50 or 75 MHz, and

Helvetica Chimica Acta ± Vol. 84 (2001)
605
Fig. 5. a) Temperature-dependent UV spectra (ꢀmelting curvesꢁ) of the non-modified and modified DNA ´ DNA
duplexes 13 ´ 14, 13 ´ 7, and 14 ´ 8, and b) 13 ´ 14 and 13 ´ 9
121 MHz, respectively. MS: fast atom bombardment (FAB), matrix-assisted laser-desorption ionisation
(MALDI), high resolution (HR). NOBA: 3-nitrobenzyl alcohol.
N6-Benzoyl-2'-deoxy-3',5'-bis-O-(triethylsilyl)adenosine (16). At 238, a soln. of 15 [19] (11.6 g, 32.7 mmol)
and imidazole (22.3 g, 328 mmol) in DMF (200 ml, distilled from CaH2) was treated dropwise with Et3SiCl
(28 ml, 165 mmol), stirred for 16 h, treated with MeOH (6 ml), and evaporated. A soln. of the residue in CH2Cl2
was washed with H2O and brine, dried (Na2SO4), and evaporated. FC (AcOEt/hexane 2 :1) gave 16 (16.9 g,
88%). Colourless oil. Rf (AcOEt/hexane 6 :1) 0.67. [a]D25
ˆ
4.8 (c ˆ 1.3, CHCl3). IR (CHCl3): 3407w, 2958s,
2912m, 2877m, 1707m, 1612s, 1584m, 1503w, 1455s, 1413m, 1352w, 1329m, 1111m, 1091m, 1071m, 1006m.
1H-NMR (200 MHz, CDCl3): see Table 5; additionally, 9.10 (br. s, NH); 8.38 (s, H C(8)); 8.10 ± 8.00
(m, 2 arom. H); 7.66 ± 7.45 (m, 3 arom. H); 1.10 ± 0.85 (m, 2 (MeCH2)3Si); 0.75 ± 0.45 (m, 2 (MeCH2)3Si).
13C-NMR (50 MHz, CDCl3): see Table 6; additionally, 165.1 (s, CˆO); 134.1 (s); 133.1 (d); 129.1 (d, 2 C); 128.1
‡
(d, 2 C); 7.1, 6.8 (2q, 2 (MeCH2)3Si); 5.2, 5.0 (2t, 2 (MeCH2)3Si). FAB-MS (NOBA): 584 (64, [M ‡ H] ). HR-
‡
MALDI-MS: 606.291 ([M ‡ Na] ; calc. 606.291).

606
Helvetica Chimica Acta ± Vol. 84 (2001)
1
Table 5. Selected H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of 3, 5, and 16 ± 24 in CDCl3
Solution
16
17
18
19
20
21
3
22
23
24
5
H
C(1')
6.56 6.47 6.39
6.38
2.98
2.26
6.29 6.40 6.37, 6.35
3.40 3.65 3.60, 3.50
2.34 2.38 2.50, 2.48
6.53
3.53
2.34
6.56
3.08
2.32
6.50 6.50, 6.48
3.50 3.42, 3.32
2.42 2.60 ± 2.50
2.50 ± 2.42
Hpro-S C(2') 2.69 3.24 3.53
Hpro-R C(2') 2.50 2.34 2.20
H
H
H
H
C(3')
C(4')
C(5')
C(5')
4.63 4.71 4.73
4.06 4.00 3.92
3.84 3.88 3.92
3.72 3.70 3.74
4.76
4.14
3.97
5.05 5.62 5.17, 5.08
4.11 4.30 4.30
4.65 4.70 4.76, 4.70
4.76
4.00
3.89
3.72
5.69
5.69
8.80
6.6
6.6
13.3
6.5
3.7 < 1.5
3.3 < 1.5
4.5 < 1.5
5.6 < 1.5
4.85 4.75
4.22
3.96
3.77
5.65
5.55
8.73
9.5
5.4
4.90 4.98, 4.90
4.13 4.27
3.40 3.90 ± 3.50
3.40 3.90 ± 3.50
5.65 5.72
5.55 5.65
8.60 8.63, 8.60
6.6
6.6
13.4
6.5
4.1
4.1
3.85 ± 3.75 4.60 4.50 4.55, 4.52
CH C(8)
CH' C(8)
±
±
5.00 4.55
4.92 4.36
4.58
4.41
8.76
9.5
5.4
13.3
5.4
4.55 4.55 4.48
4.47 4.37 4.43
8.60 8.46 8.70
H
C(2)
8.80 8.75 8.77
J(1',2'pro-S
J(1',2'pro-R
)
)
6.4
6.4
6.6
6.6
6.8
6.8
5.6
7.5
8.1
6.2
J(2'pro-S2p' ro-R) 12.9 13.4 13.1
13.1 14.0
13.3
5.3
J(2'pro-S, 3')
J(2'pro-R, 3')
J(3',4')
6.4
3.7
3.8
3.8
3.8
6.6
3.4
3.7
3.7
4.4
6.0
6.5
5.6
5.0
4.4
5.0
6.5
2.6
3.0
5.0
5.3
3.1 < 1.5
2.8 < 1.5
5.0 < 1.5
6.9 < 1.5
J(4',5')
J(4',5')
5.0
5.0
J(5',5')
J(CH,CH')
11.2 11.2 13.4
14.4 11.8
12.4
12.0
12.1 11.8
12.1 11.8
10.8
< 1.5
12.8
13.7
< 1.5
13.7
±
Table 6. Selected 13C-NMR Chemical Shifts [ppm] of 3, 5, and 16 ± 24 in CDCl3 Solution
C(1'), C(4')
C(2') C(3')
C(5')
CH2 C(8) C(2) C(4)
C(5) C(6)a)
C(8)a)
16 85.1, 88.6
41.6 72.1
39.1 72.1
37.5 73.0
40.8 73.5
38.1 71.6
35.0 74.7
62.6
62.6
63.1
63.5
64.0
64.0
±
153.1 149.6
152.6 149.1
152.6 149.6
151.8 150.0
152.4 149.4
152.2 150.7
152.6 149.4
153.1 149.1
153.0 149.2
153.1 149.8
123.6 151.6
121.6 153.1
122.6 152.9
123.0 152.2
122.3 152.6
128.8 154.6
122.7 152.6
122.1 152.9
123.3 152.2
122.4 152.6
141.6
155.6
152.4
152.7
152.6
154.5
152.3
150.2
150.7
150.0
17 85.1, 88.1
18 85.6, 88.4
19 87.3, 89.6
20 84.3, 84.3
21 82.3, 85.0
58.1
59.7
59.5
59.6
59.8
59.7, 59.5
59.6
59.2
59.3
3
83.8, 83.7, 84.7, 84.6 37.3 73.3, 72.5 63.8
22 85.3, 88.1
23 87.0, 90.0
24 85.0, 86.4
38.6 72.7
40.8 73.4
38.0 72.9
62.6
63.6
63.9
5
85.3, 85.1, 85.9, 86.0 37.3 74.5, 73.7 63.8, 63.5 59.6, 59.5
152.7 149.8, 149.6 122.3 152.7, 152.4 150.0, 149.9
a) Assignments for 17 ± 24, 3, and 5 may be interchanged.
N6-Benzoyl-2'-deoxy-8-(hydroxymethyl)-3',5'-bis-O-(triethylsilyl)adenosine (17). At 708, a soln. of (i-
Pr)2NH (10.3 ml, 72.9 mmol, distilled form CaH2) in THF (40 ml) was treated dropwise with 1.6m BuLi in
hexane (45 ml, 72 mmol), stirred at 708 for 15 min and at 08 for 15 min, and transferred via a syringe to a
cooled ( 708) soln. of 16 (8.45 g, 14.4 mmol) in THF (60 ml) within 10 min. The soln. was stirred at 708 for
1 h, treated dropwise with DMF (28 ml, 363 mmol, distilled, from CaH2) at 708, stirred for 2.5 h at 708,
treated with AcOH (10 ml), allowed to warm to 238, diluted with EtOH (100 ml), treated with NaBH4 (1.76 g,
46 mmol), and stirred for 25 min. After evaporation, the residue was dissolved in CH2Cl2, washed with H2O and
brine, dried (Na2SO4), and evaporated. FC (AcOEt/hexane 1:1) gave 17 (6.2 g, 70%). Colourless foam. Rf
(AcOEt/hexane 2 :1) 0.31. [a]D25
1655w, 1615s, 1587m, 1526w, 1459s, 1434s, 1345m, 1301w, 1096s. 1H-NMR (300 MHz, CDCl3): see Table 5;
ˆ
8.9 (c ˆ 0.65, CHCl3). IR (CHCl3): 3405m (br.), 2958s, 2913s, 2878s, 1707s,

Helvetica Chimica Acta ± Vol. 84 (2001)
607
additionally, 9.23 (br. s, NH); 8.06 ± 7.96 (m, 2 arom. H); 7.64 ± 7.45 (m, 3 arom. H); 3.80 (br. s, OH); 1.20 ± 0.75
(m, 2 (MeCH2)3Si); 0.70 ± 0.42 (m, 2 (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 6; additionally, 165.6
(s, CˆO); 134.1 (s); 133.1 (d); 128.9 (d, 2 C); 128.4 (d, 2 C); 6.6 (q, 2 (MeCH2)3Si); 4.9, 4.4 (2t, 2 (MeCH2)3Si).
‡
‡
FAB-MS (NOBA): 614 (23, [M ‡ H] ). HR-FAB-MS: 614.3187 ([M ‡ H] ; calc. 614.3194).
N6-Benzoyl-2'-deoxy-8-[(4,4'-dimethoxytrityloxy)methyl]-3',5'-bis-O-(triethylsilyl)adenosine (18). A soln.
of 17 (0.5 g, 0.815 mmol), EtN(i-Pr)2 (0.55 ml, 3.2 mmol, distilled from CaH2), and DMAP (50 mg, 0.4 mmol) in
CH2Cl2 (5 ml) was treated with 4,4'-dimethoxytrityl chloride (1.1 g, 3.25 mmol) at 08, stirred at 238 for 16 h, and
evaporated. A soln. of the residue in CH2Cl2 was washed with H2O and brine, dried (Na2SO4), and evaporated.
FC (AcOEt/hexane 1:3) gave 18 (670 mg, 89%). Colourless foam. Rf (AcOEt/hexane 1:2) 0.46. [a]D25
ˆ
6.0
(c ˆ 0.8, CHCl3). IR (CHCl3): 3407w (br.), 2958m, 2912m, 2877m, 2839w, 1706m, 1614s, 1585m, 1509s, 1463m,
1
1429m, 1414m, 1344m, 1300m, 1116m, 1073m, 1033m. H-NMR (300 MHz, CDCl3): see Table 5; additionally,
9.02 (br. s, NH); 8.06 ± 7.96 (m, 2 arom. H); 7.69 ± 7.09 (m, 12 arom. H); 6.92 ± 6.75 (m, 4 arom. H); 3.77
(s, 2 MeO); 1.05 ± 0.80 (m, 2 (MeCH2)3Si); 0.70 ± 0.42 (m, 2 (MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see
Table 6; additionally, 162.4 (s, CˆO); 159.2 (s, 2 C); 144.5 (s); 135.6 (s); 135.3 (s); 134.4 (s); 132.8 (d); 130.4 ±
127.2 (several d); 113.8, 113.7 (2d, 4 C); 87.9 (s, Ph3C); 55.2 (q, 2 MeO); 6.7, 6.6 (2q, 2 (MeCH2)3Si); 4.6, 4.3 (2t, 2
‡
‡
(MeCH2)3Si). FAB-MS (NOBA): 916 (61, [M ‡ H] ), 303 (100, DMTr ). HR-MALDI-MS: 938.432 ([M ‡
‡
Na] ; calc. 938.432).
N6-Benzoyl-2'-deoxy-8-[(4,4'-dimethoxytrityloxy)methyl]adenosine (19).
A soln. of 18 (660 mg,
0.721 mmol) in THF (6 ml) was treated dropwise at 238 with 1m Bu4NF ´ 3H2O in THF (2.4 ml) for 1.5 h.
Evaporation and FC (AcOEt/MeOH 100 :1) gave 19 (470 mg, 95%). Colourless foam. Rf (AcOEt/MeOH
100 :1) 0.39. [a]D25
ˆ
1.4 (c ˆ 2.75, CHCl3). IR (CHCl3): 3365m (br.), 3286m (br.), 2937m, 2839w, 1709m, 1614s,
1589m, 1509s, 1464m, 1432m, 1347m, 1301m, 1104m, 1062m, 1036m. 1H-NMR (200 MHz, CDCl3): see Table 5;
additionally, 9.20 (br. s, NH); 8.10 ± 8.00 (m, 2 arom. H); 7.68 ± 7.16 (m, 12 arom. H); 6.90 ± 6.78 (m, 4 arom. H);
6.23 (br. d, J ˆ 11.2, HO C(5')); 3.76 (s, 2 MeO); 2.30 (br. s, HO C(3')). 13C-NMR (50 MHz, CDCl3): see
Table 6; additionally, 165.0 (s, CˆO); 159.1 (s, 2 C); 144.5 (s); 135.2 (s); 135.0 (s); 134.0 (s); 133.0 (d); 130.2 ±
‡
127.3 (several d); 113.7 (d, 4 C); 88.2 (s, Ph3C); 55.5 (q, 2 MeO). FAB-MS (NOBA): 688 (100, [M ‡ H] ), 303
‡
‡
(75, DMTr ). HR-FAB-MS: 688.2769 ([M ‡ H] ; calc. 688.2771).
N6,5'-O-Dibenzoyl-2'-deoxy-8-[(4,4'-dimethoxytrityloxy)methyl]adenosine (20). At 108 (ice/NaCl), a
soln. of 19 (454 mg, 0.66 mmol) in CH2Cl2 (10 ml) and pyridine (2 ml, 24.9 mmol, distilled from CaH2) was
treated dropwise with a soln. of BzCl (76 ml, 0.66 mmol) in CH2Cl2 (2 ml), stirred for 3 h, washed with H2O
(10 ml), dried (Na2SO4), and evaporated. FC (AcOEt/hexane 4 :1) gave 20 (316 mg, 61%). Colourless foam. Rf
(AcOEt/hexane 6 :1) 0.55. [a]D25
ˆ
2.2 (c ˆ 0.6, CHCl3). IR (CHCl3): 3408m (br.), 2961m, 1711s, 1614s, 1586m,
1509s, 1463m, 1430w, 1352w, 1093m, 1070m, 1035m. 1H-NMR (300 MHz, CDCl3): see Table 5, additionally, 9.00
(br. s, NH); 8.10 ± 7.90 (m, 4 arom. H); 7.71 ± 7.15 (m, 15 arom. H); 6.90 ± 6.75 (m, 4 arom. H); 3.75 (s, 2 MeO);
2.75 ± 2.65 (m, HO C(3')). 13C-NMR (50 MHz, CDCl3): see Table 6; additionally, 166.8 (s, O CˆO); 165.0
(s, N CˆO); 159.1 (s, 2 C); 144.3 (s); 135.3 (s); 135.2 (s); 134.2 (s); 133.4 (d); 132.9 (d); 129.9 (s); 130.3 ± 127.4
‡
(several d); 113.8 (d, 4 C); 88.0 (s, Ph3C); 55.5 (q, 2 MeO). FAB-MS (NOBA): 792 (89, [M ‡ H] ), 303 (100,
‡
‡
DMTr ). HR-FAB-MS (NOBA): 792.3037 ([M ‡ H] ; calc. 792.3034).
N6,3'-O-Diacetyl-N6,5'-O-dibenzoyl-2'-deoxy-8-[(4,4'-dimethoxytrityloxy)methyl]adenosine (21). A soln. of
20 (30 mg, 0.044 mmol) in pyridine (1 ml, distilled from CaH2) was treated with Ac2O (0.5 ml) and stirred for
16 h at 238. Evaporation and FC (AcOEt/hexane 1:1) gave 21 (20 mg, 53%). Colourless foam. Rf (AcOEt/
hexane 1:1) 0.33. IR (CHCl3): 2958w, 1719s, 1607s, 1577m, 1509s, 1449m, 1418w, 1367m, 1347m, 1087m, 1070m,
1035m. 1H-NMR (300 MHz, CDCl3): see Table 5 ; additionally, 8.10 ± 8.00 (m, 2 arom. H); 7.75 ± 7.65
(m, 2 arom. H); 7.60 ± 7.20 (m, 15 arom. H); 6.90 ± 6.80 (m, 4 arom. H); 3.80 (s, 2 MeO); 2.56 (s, AcN); 2.0
(s, AcO). 13C-NMR (75 MHz, CDCl3): see Table 6; additionally, 173.1, 172.3 (2s, 2 O CˆO); 170.5, 166.8 (2s,
2 N CˆO); 159.3 (s, 2 C); 144.2 (s); 135.2 (s); 135.0 (s); 134.6 (s); 133.4 (d); 133.0 (d); 130.3 (s); 130.0 ± 127.5
(several d); 113.8, 113.7 (2d, 4 C); 88.0 (s, Ph3C); 55.2 (q, 2 MeO); 25.6 (q, Me); 22.2 (q, Me). FAB-MS
‡
‡
(NOBA): 876 (14, [M ‡ H] ), 303 (100, DMTr ).
N6,5'-O-Dibenzoyl-2'-deoxy-8-[(4,4'-dimethoxytrityloxy)methyl]adenosine 3'-(2-Cyanoethyl Diisopropyl-
phosphoramidite) (3). A soln. of 20 (80 mg, 0.1 mmol) and EtN(i-Pr)2 (46 ml, 0.26 mmol, distilled from
CaH2) in CH2Cl2 (4 ml) was treated with 2-cyanoethyl diisopropylphosphoramidochloridite (31 mg, 0.13 mmol)
and stirred for 3 h at 238. Evaporation and FC (AcOEt/hexane 2 :3) gave 3 (81 mg, 82%, 3 :2 ratio of 2
diastereoisomers). Colourless foam. Rf (AcOEt/hexane 2 :1) 0.25, 0.34 (2 diastereoisomers). [a]D25
ˆ
7.1 (c ˆ
1.63, CHCl3). IR (CHCl3): 3407w (br.), 2970m, 2190w, 1712s, 1614s, 1586m, 1509s, 1463m, 1430w, 1365w, 1352w,
1070m. 1H-NMR (300 MHz, CDCl3, 3 :2 mixture of 2 diastereoisomers): 8.95 (br. s, NH); 8.70 (s, H C(2));
8.05 ± 7.90 (m, 4 arom. H); 7.66 ± 7.20 (m, 15 arom. H); 6.95 ± 6.75 (m, 4 arom. H); 6.37 (dd, J ˆ 7.4, 5.6, 0.4 H),

608
Helvetica Chimica Acta ± Vol. 84 (2001)
6.35 (t, J ˆ 6.0, 0.6 H) (H C(1')); 5.17 (ddt, J ˆ 12.0, 7.0, 5.3, 0.6 H), 5.08 (ddt, J ˆ 10.5, 7.0, 4.5, 0.4 H)
(H C(3')); 4.75 (dd, J ˆ 12.0, 4.5, 0.6 H), 4.70 (dd, J ˆ 12.0, 4.5, 0.4 H), 4.55 (dd, J ˆ 12.0, 4.5, 0.6 H), 4.52
(dd, J ˆ 12.0, 4.5, 0.4 H) (2 H C(5')); 4.48, 4.43 (2d, J ˆ 12.1) (CH2 C(8)); 4.30 (dt, J ˆ 5.3, 4.5, H C(4'));
3.90 ± 3.65 (m, OCH2CH2CN); 3.65 ± 3.55 (m, 0.4 Hpro-S C(2'), (Me2CH)2N); 3.76 (s, 2 MeO); 3.50 (ddd, J ˆ
13.0, 7.0, 6.0, 0.6 Hpro-S C(2')); 2.60, 2.53 (2t, J ˆ 6.2, CH2CN); 2.50 (ddd, J ˆ 13.0, 7.0, 5.6, 0.4 H); 2.48 (ddd, J ˆ
13.0, 7.0, 6.0, 0.6 H) (Hpro-R C(2')); 1.40 ± 1.00 (m, (Me2CH)2N). 13C-NMR (100 MHz, CDCl3, 3 :2 mixture of 2
diastereoisomers): see Table 6; additionally, 166.4, 166.3 (2s, O CˆO); 164.5 (s, N CˆO); 158.9 (s, 2 C);
144.3, 144.2 (2s, 1 C), 135.3, 135.2, 135.1, 134.2, 134.1 (several s, 3 C); 133.2 (d); 132.8 (d); 130.1 (s); 130.1 ± 127.4
(several d); 117.5, 117.4 (2s, CN); 113.6, 113.5, 113.4 (3d, 4 C); 87.9, 87.8 (2s, Ph3C); 84.7, 84.6 (2dd, 3J(C,P) ˆ 3.7,
C(4')); 73.3, 72.5 (2dd, 2J(C,P) ˆ 17, C(3')); 58.9, 58.7 (2dt, 2J(C,P) ˆ 20, OCH2CH2CN); 55.4 (q, 2 MeO); 43.4,
43.3 (2dd, 2J(C,P) ˆ 7.5, (Me2CH)2N); 24.8 ± 24.6 (several q, (Me2CH)2N); 20.5, 20.4 (2dt, 3J(C,P) ˆ 4.7,
‡
OCH2CH2CN). 31P-NMR (121.5 MHz, CDCl3): 149.4, 149.3. FAB-MS (NOBA): 992 (29, [M ‡ H] ), 303 (100,
‡
‡
DMTr ); HR-MALDI-MS: 1014.393 ([M ‡ Na] ; calc. 1014.393).
N6-Benzoyl-8-[(benzoyloxy)methyl]-2'-deoxy-3',5'-bis-O-(triethylsilyl)adenosine (22). At 108 (ice/NaCl),
a soln. of 17 (1.68 g, 2.74 mmol) and pyridine (9 ml, 110 mmol, distilled from CaH2) in CH2Cl2 (20 ml) was
treated dropwise with BzCl (0.32 ml, 2.74 mmol), stirred for 3 h, washed with H2O (20 ml), dried (Na2SO4), and
evaporated. FC (AcOEt/hexane 2 :3) gave 22 (1.43 g, 73%). Colourless foam. Rf (AcOEt/hexane 1:2) 0.35.
[a]2D5 ˆ 4.3 (c ˆ 1.3, CHCl3). IR (CHCl3): 3325w (br.), 2958s, 2913m, 2877m, 1724s, 1700s, 1615s, 1586m, 1532m,
1461m, 1428m, 1354w, 1109s, 1094s, 1071s. 1H-NMR (200 MHz, CDCl3): see Table 5; additionally, 9.15 (br. s,
NH); 8.14 ± 7.95 (m, 4 arom. H); 7.65 ± 7.40 (m, 6 arom. H); 1.05 ± 0.80 (m, 2 (MeCH2)3Si); 0.75 ± 0.44
(m, 2 (MeCH2)3Si). 13C-NMR (50 MHz, CDCl3): see Table 6 ; additionally, 165.9 (s, O CˆO); 164.9
(s, N CˆO); 134.2 (s); 133.9 (d); 132.9 (d); 130.2 (d, 2 C); 129.4 (s); 129.1 (d, 2 C); 128.8 (d, 2 C); 128.1
‡
(d, 2 C); 6.8, 6.7 (2q, 2 (MeCH2)3Si); 4.8, 4.3 (2t, 2 (MeCH2)3Si). FAB-MS (NOBA): 718 (27, [M ‡ H] ), 374
‡
(100). HR-MALDI-MS: 740.330 ([M ‡ Na] ; calc. 740.328).
N6-Benzoyl-8-[(benzoyloxy)methyl]-2'-deoxyadenosine (23). At 238, a soln. of 22 (1.27 g, 1.77 mmol) in
THF (40 ml) was treated dropwise with 1m Bu4NF ´ 3 H2O in THF (4.3 ml, 4.3 mmol) and stirred for 1.5 h.
Evaporation and FC (AcOEt/MeOH 100 :1) gave 23 (720 mg, 83%). Colourless foam. Rf (AcOEt/MeOH
100 :1) 0.42. [a]2D5 ˆ 27 (c ˆ 1.13, CHCl3). IR (CHCl3): 3402m (br.), 3289 (br.), 2946w, 1726s (br.); 1615s, 1590m,
1532w, 1479s, 1464s, 1452s, 1430s, 1356s, 1106s, 1071s. 1H-NMR (300 MHz, CDCl3): see Table 5; additionally,
9.26 (br. s, NH); 8.10 ± 7.80 (m, 4 arom. H); 7.61 ± 7.30 (m, 6 arom. H); 6.06 (dd, J ˆ 11.0, 1.2, HO C(5')); 3.40
(d, J ˆ 2.5, HO C(3')). 13C-NMR (75 MHz, CDCl3): see Table 6; additionally, 166.2 (s, O CˆO); 165.2
(s, N CˆO); 134.2 (d); 134.0 (s); 133.4 (d); 130.3 (d, 2 C); 129.4 (s); 129.4 (d, 2 C); 129.0 (d, 2 C); 128.4
‡
(d, 2 C). FAB-MS (NOBA): 490 (29, [M ‡ H] ), 374 (100).
N6-Benzoyl-8-[(benzoyloxy)methyl]-2'-deoxy-5'-O-(4,4'-dimethoxytrityl)adenosine (24). At 08, a soln. of
23 (678 mg, 1.385 mmol), EtN(i-Pr)2 (0.55 ml, 3.2 mmol, distilled from CaH2) and DMAP (50 mg, 0.4 mmol) in
pyridine (7 ml, distilled from CaH2) was treated with 4,4'-dimethoxytrityl chloride (0.564 g, 1.67 mmol), stirred
for 16 h at 238, and evaporated. A soln. of the residue in CH2Cl2 was washed with H2O and brine, dried
(Na2SO4), and evaporated. FC (AcOEt/hexane 4 :1) gave 24 (790 mg, 72%). Colourless foam. Rf (AcOEt/
hexane 4 :1) 0.29. [a]D25
ˆ
2.4 (c ˆ 1.3, CHCl3). IR (CHCl3): 3405w (br.), 3064w, 2959s, 1725s, 1700s, 1614s,
1586s, 1509s, 1464s, 1452s, 1429s, 1353m, 1094s, 1071s, 1036s. 1H-NMR (300 MHz, CDCl3): see Table 5;
additionally, 9.34 (br. s, NH); 8.05 ± 7.90 (m, 4 arom. H); 7.60 ± 7.00 (m, 15 arom. H); 6.80 ± 6.65 (m, 4 arom. H);
3.70 (s, 2 MeO). 13C-NMR (75 MHz, CDCl3): see Table 6; additionally, 165.9 (s, O CˆO); 164.9 (s, N CˆO);
158.9 (s, 2 C); 144.9 (s); 136.1 (s); 136.0 (s); 133.8 (s); 133.7 (d); 133.1 (d); 129.2 (s); 130.3 ± 127.1 (several d);
‡
‡
113.3 (d, 4 C); 86.4 (s, Ph3C); 55.5 (q, 2 MeO). FAB-MS (NOBA): 792 (4, [M ‡ H] ), 303 (50, DMTr ), 374
‡
(100). HR-MALDI-MS: 814.284 ([M ‡ Na] ; calc. 814.285).
N6-Benzoyl-8-[(benzoyloxy)methyl]-2'-deoxy-5'-O-(4,4'-dimethoxytrityl)adenosine 3'-(2-Cyanoethyl Diiso-
propylphosphoramidite) (5). At 238, a soln. of 24 (188 mg, 0.237 mmol) and EtN(i-Pr)2 (0.11 ml, 0.62 mmol,
distilled from CaH2) in CH2Cl2 (12 ml) was treated with 2-cyanoethyl diisopropylphosphoramidochloridite
(53 ml, 0.238 mmol) and stirred for 3 h. Evaporation and FC (AcOEt/hexane 2 :3) gave 5 (169 mg, 72%, 1:1
mixture of 2 diastereoisomers). Colourless foam. Rf (AcOEt/hexane 1:2) 0.26, 0.33 (2 diastereoisomers).
[a]2D5
1509s, 1463s, 1428m, 1365m, 1353m, 1094s, 1071s, 1036s. 1H-NMR (300 MHz, CDCl3, 1:1 mixture of
ˆ
10.3 (c ˆ 1.15, CHCl3). IR (CHCl3): 3405w (br.), 2969m, 2935m, 2200w, 1725s, 1700s, 1614s, 1586m,
2
diastereoisomers): 9.00 (br. s, NH); 8.63, 8.60 (2s, H C(2)); 8.08 (d, 2 arom. H); 8.00 (d, 2 arom. H); 7.60 ± 7.10
(m, 15 arom. H); 6.80 ± 6.65 (m, 4 arom. H); 6.50, 6.48 (2t, J ˆ 6.5, H C(1')); 5.72, 5.65 (2d, J ˆ 13.6),
CH2 C(8)); 4.98 (ddt, J ˆ 14.7, 5.6, 4.5, 0.5 H), 4.90 (ddt, J ˆ 13.2, 5.6, 2.8, 0.5 H) (H C(3')); 4.27 (dt, J ˆ
5.6, 4.9, H C(4')); 3.90 ± 3.50 (m, 2H C(5'), (Me2CH)2N, OCH2CH2CN); 3.68 (s, 2 MeO); 3.42 (ddd, J ˆ

Helvetica Chimica Acta ± Vol. 84 (2001)
609
12.0, 6.5, 4.5, 0.5 H), 3.32 (ddd, J ˆ 10.5, 6.5, 2.8, 0.5 H) (Hpro-S C(2')); 2.60 ± 2.40 (m, Hpro-R C(2')); 2.57
(td, J ˆ 6.3, 2.1); 2.45 (t, J ˆ 6.4, CH2CN); 1.25 ± 1.00 (m, (Me2CH)2N). 13C-NMR (100 MHz, CDCl3, 1:1
mixture of 2 diastereoisomers): see Table 6; additionally, 165.7 (s, O CˆO); 164.5 (s, N CˆO); 158.55, 158.5,
158.45 (3s, 2 C); 144.8, 144.7 (2s, 1 C); 136.2, 136.1, 135.9, 134.0 (several s, 3 C); 133.7 (d); 132.8 (d); 130.2 ± 126.8
(s, several d); 117.6, 117.56 (2s, CN); 113.1 (d, 4 C); 86.35, 86.3 (2s, Ph3C); 74.3 (dd, 2J(C,P) ˆ 16.8), 73.5
(dd, 2J(C,P) ˆ 17.9) (C(3')); 58.6, 58.5 (2dt, 2J(C,P) ˆ 16.2, OCH2CH2CN); 55.3, 55.2 (2q, 2 MeO); 43.35
(2dd, 2J(C,P) ˆ 12.1, (Me2CH)2N); 24.75 ± 24.55 (several q, (Me2CH)2N); 20.5, 20.3 (2dt, 3J(C,P) ˆ 7.1,
‡
OCH2CH2CN). 31P-NMR (121.5 MHz, CDCl3): 149.5, 149.2. FAB-MS (NOBA): 992 (23, [M ‡ H] ), 303
‡
‡
(100, DMTr ). HR-MALDI-MS: 1014.393 ([M ‡ Na] ; calc. 1014.393).
2'-Deoxy-3',5'-bis-O-(triethylsilyl)uridine (25). A soln. of 2'-deoxyuridine (10 g, 43.9 mmol) and imidazole
(29.7 g, 436 mmol) in DMF (150 ml) was treated dropwise with Et3SiCl (37 ml, 218 mmol), stirred for 16 h at
238, treated with MeOH (8 ml), and evaporated. A soln. of the residue in CH2Cl2 was washed with H2O and
brine, dried (Na2SO4), and evaporated. FC (AcOEt/hexane 1:1) gave 25 (18.9 g, 94%). Colourless oil. Rf
(AcOEt/hexane 1:1) 0.67. [a]2D5 ˆ 31.8 (c ˆ 0.4, CHCl3). IR (CHCl3): 3392m (br.), 2958s, 2913m, 2878s, 1690s,
1634w, 1460s, 1415m, 1388m, 1116s, 1084m, 1067m, 1006m. 1H-NMR (200 MHz, CDCl3): see Table 7,
additionally, 8.67 (br. s, NH); 8.00 (d, J ˆ 8.3, H C(6)); 0.98 ± 0.84 (m, 2 (MeCH2)3Si); 0.70 ± 0.42 (m, 2
(MeCH2)3Si). 13C-NMR (75 MHz, CDCl3): see Table 8; additionally, 6.6, 6.4 (2q, 2 (MeCH2)3Si); 4.6, 4.2 (2t, 2
‡
‡
(MeCH2)3Si). FAB-MS (NOBA): 457 (88, [M ‡ H] ). HR-MALDI-MS: 479.236 ([M ‡ Na] ; calc. 479.237).
2'-Deoxy-6-(hydroxymethyl)-3',5'-bis-O-(triethylsilyl)uridine (26). At 708, a soln. of (i-Pr)2NH (10.3 ml,
72.9 mmol, distilled from CaH2) in THF (40 ml) was treated dropwise with 1.6m BuLi in hexane (45 ml,
72 mmol), stirred at 708 for 15 min and at 08 for 15 min, and transferred via syringe to a cooled ( 708) soln. of
25 (6.6 g, 14.4 mmol) in THF (60 ml) within 10 min. The soln. was stirred at 708 for 1 h, treated with DMF
(28 ml, 363 mmol, distilled from CaH2), stirred for 2.5 h, treated with AcOH (10 ml), allowed to warm to 238,
diluted with EtOH (100 ml), treated with NaBH4 (1.76 g, 46 mmol), and stirred for 25 min. After evaporation,
the residue was dissolved in CH2Cl2, washed with H2O and brine, and dried (Na2SO4). Evaporation and FC
(AcOEt/hexane 1:1) gave 26 (2.1 g, 30%) and 25 (3 g, 46%). Colourless foam. Rf (AcOEt/hexane 1:1) 0.27.
[a]2D5 ˆ 22.5 (c ˆ 0.4, CHCl3). IR (CHCl3): 3392m (br.), 2958s, 2913m, 2878m, 1697s, 1639s, 1458m, 1412m,
Table 7. Selected 1H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of 4, and 25 ± 30 in CDCl3
Solution
25
26
27
28
29
30
4
H
C(1')
6.33
2.10
2.33
4.45
3.95
3.90
3.75
±
±
5.70
6.2
6.2
13.3
6.2
4.3
3.7
2.2
2.5
11.2
±
6.20
2.60
2.20
4.50
3.83
3.90
3.80
4.55
4.45
5.78
7.2
7.2
13.2
7.5
3.7
4.0
5.75
2.92
2.02
4.38
3.8 ± 3.6
3.8 ± 3.6
3.8 ± 3.6
3.97
3.91
5.73
5.75
2.96
2.07
4.72
3.80
3.82
3.76
4.00
3.93
5.66
7.5
7.8
13.7
7.4
4.4
4.4
5.74
2.94
2.18
4.78
3.92
4.61
4.50
4.00
3.95
5.70
4.1
8.7
13.7
8.1
6.5
6.5
5.80
3.18
2.13
5.47
4.15
4.58
4.50
3.95
3.95
5.68
5.6
8.1
13.8
8.0
4.7
5.0
5.78 ± 5.65
2.94
Hpro-S C(2')
Hpro-R C(2')
H
H
H
H
2.30 ± 2.15
4.90 ± 4.72
4.12 ± 4.00
4.70, 4.60
4.42, 4.38
4.00
C(3')
C(4')
C(5')
C(5')
CH C(6)
CH' C(6)
4.00
5.78 ± 5.65
H
C(5)
J(1',2'pro-S
J(1',2'pro-R
)
)
6.2
8.1
13.1
J(2'pro-S, 2p' ro-R
J(2'pro-S, 3')
J(2'pro-R, 3')
J(3',4')
)
7.5
4.7
4.7
a
J(4',5')
J(4',5')
J(5',5')
J(CH,CH')
2.8
3.8
10.6
14.0
)
)
)
2.8
4.1
12.8
14.3
4.1
6.2
11.8
13.4
4.8
6.8
11.8
< 1.5
a
a
12.9
a) Not determined.

610
Helvetica Chimica Acta ± Vol. 84 (2001)
Table 8. Selected 13C-NMR Chemical Shifts [ppm] of 4, and 25 ± 30 in CDCl3 Solution
C(1'), C(4')
C(2')
C(3')
C(5'), CH2 C(6)
C(2)
C(4)
C(5)
C(6)
25
26
27
28
29
30
4
85.3, 88.0
85.8, 87.9
86.6, 88.3
87.6, 87.2
84.9, 86.4
82.4, 86.8
84.2, 83.7
86.7, 86.3
42.0
39.2
38.4
38.7
38.9
35.7
38.1
71.2
71.8
72.6
71.2
72.1
74.8
73.8
73.0
62.0, ±
150.6
150.8
150.3
151.6
150.9
150.3
150.3
163.9
164.4
163.6
163.3
163.4
163.0
163.6
102.0
101.9
102.6
103.7
103.3
103.5
103.0
140.5
156.6
153.6
153.1
153.2
152.9
153.1
153.0
63.0, 61.0
63.8, 62.7
62.8, 62.3
65.3, 62.6
64.9, 62.9
64.7, 62.6
1
1373m, 1338m, 1084s, 1042m, 1006 m. H-NMR (300 MHz, CDCl3): see Table 7; additionally, 8.20 (br. s, NH);
3.40 (br. s, OH); 1.05 ± 0.84 (m, 2 (MeCH2)3Si); 0.70 ± 0.50 (m, 2 (MeCH2)3Si). 13C-NMR (50 MHz, CDCl3): see
‡
Table 8; additionally, 6.7 (q, 2 (MeCH2)3Si); 4.7, 4.3 (2t, 2 (MeCH2)3Si). FAB-MS (NOBA): 487 (48, [M ‡ H] ).
2'-Deoxy-6-[(4,4'-dimethoxytrityloxy)methyl]-3',5'-bis-O-(triethylsilyl)uridine (27). A soln. of 26 (3.7 g,
7.6 mmol), EtN(i-Pr)2 (5.2 ml, 30.2 mmol, distilled from CaH2), and DMAP (50 mg, 0.4 mmol) in CH2Cl2
(60 ml) was treated with 4,4'-dimethoxytrityl chloride (10.3 g, 30.4 mmol) at 08, stirred at 238 for 16 h, and
evaporated. A soln. of the residue in CH2Cl2 was washed with H2O and brine, dried (Na2SO4), and evaporated.
FC (AcOEt/hexane 1:3) gave 27 (3.8 g, 63%). Colourless foam. Rf (AcOEt/hexane 1:3) 0.28. [a]2D5 ˆ 9.9 (c ˆ
1.45, CHCl3). IR (CHCl3): 3390w (br.), 2958s, 2938m, 2912m, 2877s, 1691s, 1608m, 1584w, 1510s, 1464s, 1445m,
1414m, 1379m, 1342w, 1071s, 1035s, 1006s. 1H-NMR (200 MHz, CDCl3): see Table 7; additionally, 8.30 (br. s,
NH); 7.50 ± 7.20 (m, 9 arom. H); 6.95 ± 6.80 (m, 4 arom. H); 3.76 (s, 2 MeO)); 1.05 ± 0.84 (m, 2 (MeCH2)3Si);
0.75 ± 0.50 (m, 2 (MeCH2)3Si). 13C-NMR (50 MHz, CDCl3): see Table 8; additionally, 159.2 (s, 2 C); 144.2 (s);
135.3 (s); 135.0 (s); 130.1 ± 127.5 (several d); 113.8, 113.7 (2d, 4 C); 88.0 (s, Ph3C); 55.3 (q, 2 MeO); 6.7 (q, 2
‡
‡
(MeCH2)3Si); 4.6, 4.2 (2t, 2 (MeCH2)3Si). FAB-MS (NOBA): 789 (2, [M ‡ H] ), 303 (100, DMTr ); HR-FAB-
‡
MS: 788.3883 (M ; calc. 788.3888).
2'-Deoxy-6-[(4,4'-dimethoxytrityloxy)methyl]uridine (28). A soln. of 27 (3.55 g, 4.5 mmol) in THF (60 ml)
was treated dropwise at 238 with 1m Bu4NF ´ 3 H2O in THF (10.9 ml, 10.9 mmol) and stirred for 1.5 h.
Evaporation and FC (AcOEt/MeOH 100 :1) gave 28 (2.2 g, 87%). Colourless foam. Rf (AcOEt/MeOH 100 :1)
0.56. [a]2D5 ˆ 3.8 (c ˆ 0.42, CHCl3). IR (CHCl3): 3390m (br.), 2959m, 2840m, 1694s, 1608m, 1584m, 1509s, 1464m,
1445m, 1411m, 1382m, 1303m, 1064m, 1035s. 1H-NMR (300 MHz, CDCl3): see Table 7; additionally, 7.50 ± 7.20
(m, 9 arom. H); 6.86 (d, 4 arom. H); 3.80 (s, 2 MeO). 13C-NMR (50 MHz, CDCl3): see Table 8; additionally,
159.4 (s, 2 C); 144.1 (s); 135.2 (s); 135.0 (s); 130.2 ± 127.7 (several d); 113.9, 113.8 (2d, 4 C); 88.3 (s, Ph3C); 55.5
‡
‡
(q, 2 MeO). FAB-MS (NOBA): 560 (13, M ), 303 (100, DMTr ).
5'-O-Benzoyl-2'-deoxy-6-[(4,4'-dimethoxytrityloxy)methyl]uridine (29). At 108 (ice/NaCl), a soln. of 28
(2.2 g, 2.92 mmol) in CH2Cl2 (100 ml) and pyridine (23 ml, 284 mmol, distilled from CaH2) was treated dropwise
with a soln. of BzCl (0.56 ml, 4.86 mmol) in CH2Cl2 (10 ml), stirred at 108 for 3 h, washed with H2O (60 ml),
dried (Na2SO4), and evaporated. FC (AcOEt/hexane 4 :1) gave 29 (1.7 g, 65%) and 28 (0.5 g, 23%). Colourless
foam. Rf (AcOEt/hexane 6 :1) 0.44. [a]2D5 ˆ 8.9 (c ˆ 1.32, CHCl3). IR (CHCl3): 3391m (br.), 3186w (br.), 2959m,
2936m, 2840w, 1694s, 1608m, 1584w, 1509s, 1464m, 1452m, 1409m, 1381m, 1316m, 1095s, 1070s, 1036s. 1H-NMR
(300 MHz, CDCl3): see Table 7; additionally, 8.80 (br. s, NH); 8.12 ± 8.00 (m, 2 arom. H); 7.64 ± 7.20 (m, 12 ar-
om. H); 6.94 ± 6.83 (m, 4 arom. H); 3.80 (s, 2 MeO)); 2.30 (br. d, J ˆ 4.0, OH). 13C-NMR (50 MHz, CDCl3): see
Table 8; additionally, 167.2 (s, O CˆO); 159.4 (s, 2 C); 144.1 (s); 135.2 (s); 135.0 (s); 133.4 (d); 130.3 ± 127.7
‡
(s, several d); 113.9, 113.8 (2d, 4 C); 88.1 (s, Ph3C); 55.5 (q, 2 MeO). FAB-MS (NOBA): 664 (4, M ), 303 (100,
‡
‡
DMTr ). HR-FAB-MS (NOBA): 664.2417 (M ; calc. 664.2421).
3'-O-Acetyl-5'-O-benzoyl-2'-deoxy-6-[(4,4'-dimethoxytrityloxy)methyl]uridine (30). At 238, a soln. of 29
(20 mg, 0.03 mmol) in pyridine (1 ml, distilled from CaH2) was treated with Ac2O (0.5 ml) and stirred for 16 h.
Evaporation and FC (AcOEt/hexane 1 :1) gave 30 (16 mg, 75%). Colourless foam. [a]2D5 ˆ 2.3 (c ˆ 0.92, CHCl3).
IR (CHCl3): 3390w (br.), 2936w, 1722s, 1692s, 1608m, 1510m, 1462m, 1452m, 1378m, 1095m, 1070m, 1036m.
1H-NMR (300 MHz, CDCl3): see Table 7; additionally, 9.00 (br. s, NH); 8.10 ± 8.00 (m, 2 arom. H); 7.60 ± 7.20
(m, 12 arom. H); 6.90 ± 6.80 (m, 4 arom. H); 3.80 (s, 2 MeO); 1.95 (s, Me). 13C-NMR (75 MHz, CDCl3): see
Table 8; additionally, 170.5, 166.7 (2s, 2 CˆO); 159.4 (s, 2 C); 144.2 (s); 135.2 (s); 135.0 (s); 133.4 (d); 130.3 (s);
130.2 ± 127.7 (several d); 113.8, 113.7 (2d, 4 C); 88.2 (s, Ph3C); 55.5 (q, 2 MeO); 20.9 (q, Me).

Helvetica Chimica Acta ± Vol. 84 (2001)
611
5'-O-Benzoyl-2'-deoxy-6-[(4,4'-dimethoxytrityloxy)methyl]uridine 3'-(2-Cyanoethyl) Diisopropylphosphor-
amidite) (4). A soln. of 29 (100 mg, 0.15 mmol) and EtN(i-Pr)2 (90 ml, 0.52 mmol, distilled from CaH2) in
CH2Cl2 (4 ml) was treated with 2-cyanoethyl diisopropylphosphoramidochloridite (34 ml, 0.152 mmol) and
stirred at 238 for 3 h. Evaporation and FC (AcOEt/hexane 2 :3) gave 4 (93 mg, 72%, 2 :1 ratio of
2
diastereoisomers). Colourless foam. Rf (AcOEt/hexane 1:2) 0.24, 0.33 (2 diastereoisomers). IR (CHCl3): 3390w
(br.), 2969w, 2935w, 1718s, 1694s, 1608w, 1509m, 1463m, 1414w, 1379m, 1366w, 1094m, 1070m, 1035m. 1H-NMR
(300 MHz, CDCl3, 2 :1 ratio of 2 diastereoisomers): 8.10 ± 8.00 (m, 2 arom. H); 7.60 ± 7.20 (m, 12 arom. H);
6.90 ± 6.80 (m, 4 arom. H); 5.78 ± 5.65 (m, H C(5), H C(1')); 4.90 ± 4.72 (m, H C(3')); 4.70 (dd, J ˆ 11.8, 3.1,
0.67 H), 4.60 (dd, J ˆ 12.0, 3.7, 0.33 H), 4.42 (dd, J ˆ 11.8, 3.4, 0.67 H), 4.38 (dd, J ˆ 12.0, 3.4, 0.33 H)
(2 H C(5')); 4.12 ± 4.00 (m, H C(4')); 4.00 (br. s, CH2 C(6)); 3.90 ± 3.40 (m, (Me2CH)2N, OCH2CH2CN);
3.76 (s, 2 MeO); 2.94 (ddd, J ˆ 13.0, 8.4, 4.7, Hpro-S C(2')); 2.58, 2.57 (2t, J ˆ 6.3, CH2CN); 2.30 ± 2.15 (m,
Hpro-R C(2')); 1.10 ± 1.00 (m, (Me2CH)2N). 13C-NMR (75 MHz, CDCl3, 2 :1 ratio of 2 diastereoisomers): see
Table 8; additionally, 166.7 (s, O CˆO); 159.3 (s, 2 C), 144.1 (s); 135.25, 135.2, 135.0 (3s, 2 C); 133.3 (d); 130.3
(s); 130.2 ± 127.6 (several d); 117.8 (s, CN); 113.9 (d, 4 C); 88.1 (s, Ph3C); 84.2, 83.7 (2dd, 3J(C,P) ˆ 4.5, C(4'));
73.8 (dd, 2J(C,P) ˆ 19), 73.0 (dd, 2J(C,P) ˆ 17) (C(3')); 59.0, 58.6 (2dt, 2J(C,P) ˆ 20, OCH2CH2CN); 55.4
(q, 2 MeO); 43.5, 43.2 (2dd, 2J(C,P) ˆ 12, (Me2CH)2N); 24.9 ± 24.4 (several q, (Me2CH)2N); 20.6 ± 20.1 (2dt,
3J(C,P) ˆ 4.6, OCH2CH2CN). 31P-NMR (121.5 MHz, CDCl3): 149.9, 149.7. FAB-MS (NOBA): 865 (1, [M ‡
‡
‡
‡
H] ), 303 (100, DMTr ). HR-MALDI-MS: 887.340 ([M ‡ Na] ; calc. 887.340).
Oligonucleotide Synthesis. Oligonucleotide syntheses were performed on a Pharmacia Gene Assembler on a
1.3-mmol scale. The commercial phosphoramidites and the CPG solid supports were obtained from Glen
Research. Solvents and reagents were prepared essentially according to the protocol for the synthesis of pRNA
[10]. Detritylation was accomplished within 2 min by 3% Cl2CHCOOH in (CH2Cl)2. Couplings (0.16 ml of 0.1m
phosphoramidite soln. ‡ 0.36 ml of 0.35m 1H-tetrazole/0.15m 5-(4-nitrophenyl)-1H-tetrazole soln. in MeCN, or
0.36 ml 0.25m 1-(benzylthio)-1H-tetrazole soln. in MeCN, resp.) were performed within 6 ± 10 min (DNA-
phosphoramidites coupled on unmodified nucleotides), or 30 min (modified phosphoramidites, and DNA-
phosphoramidites coupled on modified nucleotides, resp.). Capping and oxidation was accomplished under
standard conditions [35].
Deprotection and Purification of Oligonucleotides. Removal of the protecting groups and detachment from
the solid support was effected in conc. aq. NH3 soln./MeOH 1:1 (2.5 ml) at 508 in 20 h (oligomers 6, 7, 9, and 11 ±
14) or at 358 in 5 h (oligomer 8 containing dU*; see Results and Discussion). After filtration and evaporation, the
crude oligomers were dissolved in aq. buffer soln. (0.1m HOAc, 0.1m Et3N), purified by RP-HPLC, and desalted
by RP-HPLC. Their composition was confirmed by MALDI-TOF-MS.
REFERENCES
[1] E. Uhlmann, A. Peyman, Chem. Rev. 1990, 90, 543.
[2] Y. S. Sanghvi, P. D. Cook, ꢀCarbohydrate Modifications in Antisense Researchꢁ, Washington, DC, 1994,
p. 232.
[3] G. L. Verdine, H. Huang, R. Chopra, S. C. Harrison, Science 1998, 282, 1669.
[4] D. Ly, L. Sanii, G. B. Schuster, J. Am. Chem. Soc. 1999, 121, 9400.
[5] R. Epple, E. U. Wallenborn, T. Carell, J. Am. Chem. Soc. 1997, 119, 7440.
Â
[6] S. Eppacher, N. Solladie, B. Bernet, A. Vasella, Helv. Chim. Acta 2000, 83, 1311.
[7] H. Gunji, A. Vasella, Helv. Chim. Acta 2000, 83, 1331.
[8] H. Gunji, A. Vasella, Helv. Chim. Acta 2000, 83, 2975.
[9] H. Gunji, A. Vasella, Helv. Chim. Acta 2000, 83, 3229.
[10] S. Pitsch, S. Wendeborn, B. Jaun, A. Eschenmoser, Helv. Chim. Acta 1993, 76, 2161.
[11] A. Eschenmoser, M. Dobler, Helv. Chim. Acta 1992, 75, 218.
[12] W. Saenger, ꢀPrinciples of Nucleic Acid Structureꢁ, Springer Verlag, 1984, p. 268.
[13] W. Saenger, ꢀPrinciples of Nucleic Acid Structureꢁ, Springer Verlag, 1984, p. 226.
[14] W. Saenger, ꢀPrinciples of Nucleic Acid Structureꢁ, Springer Verlag, 1984, p. 232.
[15] F. Mohamadi, N. G. J. Richards, W. C. Guida, R. Liskamp, M. Lipton, C. Caufield, G. Chang, T.
Hendrickson, W. C. Still, J. Comput. Chem. 1990, 11, 440.
[16] W. Saenger, ꢀPrinciples of Nucleic Acid Structureꢁ, Springer Verlag, 1984, p. 26.
[17] W. Saenger, ꢀPrinciples of Nucleic Acid Structureꢁ, Springer Verlag, 1984, pp. 116 ± 126.
[18] X. Wu, Swiss Federal Institute of Technology (ETH), Diss. ETH No. 13567, 2000.

612
Helvetica Chimica Acta ± Vol. 84 (2001)
[19] L. Gaffney, R. A. Jones, G. S. Ti, J. Am. Chem. Soc. 1982, 104, 1316.
[20] E. J. Corey, A. Venkateswarlu, J. Am. Chem. Soc. 1972, 94, 6190.
[21] H. Hayakawa, K. Haraguchi, H. Tanaka, T. Miyasaka, Chem. Pharm. Bull. 1987, 35, 72.
[22] J. Yamashita, H. Matsumoto, K. Kobayashi, K. Noguchi, M. Yasumoto, T. Ueda, Chem. Pharm. Bull. 1989,
37, 2287.
[23] S. Pitsch, Helv. Chim. Acta 1997, 80, 2286.
[24] L. Dudycz, R. Stolarski, R. Pless, D. Shugar, Z. Naturforsch. C: Biosci. 1978, 34, 359.
[25] D. B. Davies, ꢀConformations of Nucleosides and Nucleotidesꢁ, in ꢀProgress in NMR Spectroscopyꢁ, 1978,
Vol. 12, p. 135.
[26] D. Plochocka, A. Rabczenko, D. B. Davies, Biochim. Biophys. Acta 1977, 476, 1.
[27] L. H. Koole, H. Deboer, J. W. Dehaan, A. G. Haasnoot, P. Vandael, H. M. Buck, J. Chem. Soc., Chem.
Commun. 1986, 362.
[28] T. Ueda, Y. Nomoto, A. Matsuda, Chem. Pharm. Bull. 1985, 33, 3263.
[29] S. T. Rao, M. Sundaralingam, J. Am. Chem. Soc. 1970, 92, 4963.
[30] V. Swaminathan, M. Sundaralingam, J. B. Chattopadhyaya, C. B. Reese, Acta Crystallogr., Sect. B 1980, 36,
828.
[31] F. Seela, H. Debelak, H. Reuter, G. Kastner, I. A. Mikhailopulo, Nucleosides Nucleotides 1998, 17, 729.
[32] D. Gani, A. W. Johnson, J. Chem. Soc., Perkin Trans. 1 1982, 1197.
[33] H. Tanaka, H. Hayakawa, S. Iijima, K. Haraguchi, T. Miyasaka, Tetrahedron 1985, 41, 861.
[34] M. Berger, A. Ogawa, D. McMinn, Y. Wu, P. Schultz, F. Romesberg, Angew. Chem., Int. Ed., 2000, 39, 2940.
[35] Pharmacia, ꢀUser Manual for Gene Assembler Plusꢁ.
Received January 3, 2001