Unique participation of unprotected internucleotidic phosphodiester residues on unexpected cleavage reaction of the Si-O bond of the diisopropylsilandiyl group used as a linker for the solid-phase synthesis of 5′-terminal guanylated oligodeoxynucleotides

Article abstract of DOI:10.1002/1522-2675(200209)85:9<2930::AID-HLCA2930>3.0.CO;2-2

In connection with the synthesis of guanosine-capped oligodeoxynucleotides on polymer supports, we found an unprecedented Si-O bond cleavage reaction, which occurred when polymer-linked oligodeoxynucleotides having unprotected internucleotidic phosphate groups were allowed to react with the guanosine 5′-phosphorimidazolide derivative 18 in the presence of 4-nitro-6-(trifluoromethyl)-1H-benzotriazol-1-ol (Ntbt-OH) as an effective activator in pyridine. This side reaction was confirmed by the fact that the liquid-phase reaction of DMTrTpT-O-Si(iPr₂)OEt 42 with a simpler model compound, methyl phosphorimidazolide 34, in the presence of Ntbt-OH gave DMTrTpT 43. It turned out that the side reaction hardly occurs without unprotected internucleotidic phosphate groups on oligodeoxynucleotides. The detailed study of this side reaction disclosed that Ntbt-OH directly attacks the Si-atom to release oligonucleotides from the resin. It is likely that Ntbt-OH serves as a very strong nucleophile in pyridine, especially to the Si-atom of the linker.

Full text of DOI:10.1002/1522-2675(200209)85:9<2930::AID-HLCA2930>3.0.CO;2-2

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Unique Participation of Unprotected Internucleotidic Phosphodiester
Residues on Unexpected Cleavage Reaction of the SiÀO Bond of the
Diisopropylsilandiyl Group Used as a Linker for the Solid-Phase Synthesis of
5'-Terminal Guanylated Oligodeoxynucleotides
by Masatoshi Ushioda, Michinori Kadokura, Tomohisa Moriguchi, Akio Kobori, Morihiro Aoyagi, Kohji Seio,
and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, Japan
Dedicated to Prof. Dr. Wolfgang Pfleiderer on the occasion of his 75th birthday
In connection with the synthesis of guanosine-capped oligodeoxynucleotides on polymer supports, we
found an unprecedented SiÀO bond cleavage reaction, which occurred when polymer-linked oligodeoxynu-
cleotides having unprotected internucleotidic phosphate groups were allowed to react with the guanosine 5'-
phosphorimidazolide derivative 18 in the presence of 4-nitro-6-(trifluoromethyl)-1H-benzotriazol-1-ol (Ntbt-
OH) as an effective activator in pyridine. This side reaction was confirmed by the fact that the liquid-phase
reaction of DMTrTpTÀOÀSi(iPr2)OEt 42 with a simpler model compound, methyl phosphorimidazolide 34, in
the presence of Ntbt-OH gave DMTrTpT 43. It turned out that the side reaction hardly occurs without
unprotected internucleotidic phosphate groups on oligodeoxynucleotides. The detailed study of this side
reaction disclosed that Ntbt-OH directly attacks the Si-atom to release oligonucleotides from the resin. It is
likely that Ntbt-OH serves as a very strong nucleophile in pyridine, especially to the Si-atom of the linker.
1. Introduction. It is well-known that mature mRNAs having a 7-methylguanosine
cap structure (m7G5'ppp-) move from the nucleus to the cytoplasm through the nuclear
membrane [1]. On the other hand, U1 snRNA, which is a small nuclear RNA having a
unique 5'-terminal 2,2,7-trimethylguanosine cap structure (m32,2,7G5'ppp-) has proven to
be transported in the reverse direction [2]. This unique behavior derived from the two
different types of cap structures might be utilized for the selective transportation of
antisense DNA derivatives into the nucleus or the cytoplasm upon addition of these
structural motifs at the 5'-terminal site. Therefore, we designed a simpler molecule
represented by m7G5'pp-DNA or m32,2,7G5'pp-DNA, which lacks a phosphate group and
has a DNA chain in place of the RNA chain. In connection with this idea, we also
focused our attention on the synthesis of G5' pp-DNA, since this nonmethylated G-
capped oligodeoxynucleotide might be used as a precursor of m7G5'-or m 2,2,7G5'-
3
ppDNA. With these possibilities in mind, we studied the solid-phase synthesis of X5'pp-
d(ATA) (X ˆ G, m7G or m32,2,7G) as the simplest model. To realize this synthesis, we
used a Bu4NF (TBAF)-labile silanediyl-type linker. However, we found an unexpected
cleavage of the SiÀO bond at the final stage of pyrophosphorylation when we tried to
synthesize G5'ppd(ATA).
In this paper, we report the details of this unprecedented side reaction, which has
proved to result from the substitution of the silanediyl group with a strong nucleophile,
4-nitro-6-(trifluoromethyl)-1H-benzotriazol-1-ol (Ntbt-OH), used for activation of the
capping reagent.

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2. Results and Discussion. 2.1. Synthesis and Application of Diisopropylsilanediyl
Linker. To synthesize G5'ppd(ATA) in the solid phase, we used a new silanediyl type
linker 1 and a highly cross-linked (aminomethyl)polystyrene (HCP) [3] resin, as shown
in the Figure. This is because we ultimately aimed to synthesize m7G5'-or m 2,2,7G5'-
3
ppDNA, and, therefore, we required a base-resistant but fluoride-ion-labile linker.
Fraser et al. [4] originally reported the solid-phase synthesis of oligonucleotides using a
silanediyl-type linker 2 attached to CPG, as depicted in the Figure, which can be
cleaved by treatment with TBAF under neutral conditions. However, this linker
contains a base-labile succinate moiety. It is also known that CPG supports are fragile
upon heating under weakly basic conditions such as aq. NH3 [5]. Therefore, we
developed a new silanediyl linker without the succinate moiety on the HCP support,
which is resistant to ammonia and, thereby, allows the general synthesis of oligo-
nucleotides having G-cap, m7G-cap, or m32,2,7G-cap structures.
Figure. The new linker 1 developed in this study and the linker 2 reported by Fraser and co-workers
2-Cyanoethyl 16-hydroxyhexadecanate (4) was prepared in 67% yield by reaction
of 16-hydroxyhexadecanoic acid (3) with 2-hydroxyethanenitrile in the presence of 1,3-
dicyclohexylcarbodiimide (DCC). This backbone structure was used as part of the
spacer between the resin and a 3'-terminal deoxynucleoside (Scheme 1). Reaction of 6-
N-benzoyl-5'-O-(4,4'-dimethoxytrityl)deoxyadenosine (5) with (i-Pr)2SiCl2 in the
presence of 1H-imidazole in DMF gave the 3'-O-silylated species 6, which was allowed
to react in situ with 4 to give the diisopropylsilanediyl ether derivative 7 in 61% yield
from 5. Treatment of 7 with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave the
carboxylic acid derivative 8 in 80% yield. Condensation of the HCP resin 9 with 8 in the
presence of DCC gave the solid-supported material 10. The DMTr cation assay
indicated that 18.0 28.5 mmol/g of deoxyadenosine could be loaded on the support.
To investigate the efficiency of cleavage of the silyl linkage under neutral
conditions, a portion of the derivatized resin 10 was treated with a 1m solution of
TBAF in the presence of 1m AcOH in THF for 1 h at room temperature. The DMTr
cation assay indicated that quantitative cleavage of the diisopropylsilanediyl linker was
achieved. In addition, the stability of the linker under usual deprotection conditions
was studied. The derivatized resin 10 was treated with aq. NH3/EtOH 4 :1 (v/v) for 4 h
at 558. The amounts of the nucleoside derivative released and the remaining nucleoside

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Scheme 1
residue on the solid support were estimated by the DMTr cation analysis. Consequently,
it was confirmed that more than 99% of the 5'-O-DMTr-deoxyadenosine residue
remained intact on the solid support. This result means that the silanediyl linker was
essentially stable under the conditions prescribed for removal of N-protecting groups.
Generally, deprotection of trialkylsilyl groups can be carried out not only by
treatment with TBAF but also under acidic conditions [6]. Therefore, we examined the
stability of the diisopropylsilandiyl group under acidic conditions prescribed for
removal of the DMTr group in the solid-support synthesis, with a model compound 12,
which was synthesized from 5'-O-[(9H-fluoren-9-yl)-methoxycarbonyl]thymidine (11)
[7] (Scheme 2). Treatment of 12 with 1% CF3COOH (TFA) in CH2Cl2 for 6 h at room
temperature gave two new products along with recovery (66%) of 12. The desilylated
compound 11 and the silanol product 13 were isolated in 19 and 7% yields, respectively.
This instability of the silanediyl group might become more serious when the chain
elongation is repeated for the synthesis of longer oligonucleotides. Upon cleavage of
the linker, OH groups emerge on solid support so that they might react directly with the
phosphoramidite units. Therefore, we searched for better reagents capable of selective
removal of the DMTr group. As the result, 1m ZnBr2 in CH2Cl2/i-PrOH 85 :15 (v/v) [8]
was found to be as favorable as the reagent. The model compound 12 was completely
stable under these conditions.
2.2. Synthesis of a Guanosine 5'-O-Phosphorimidazolide Unit. For the solid-phase
synthesis of G-capped oligonucleotides with our silanediyl linker, we designed the

Helvetica Chimica Acta Vol. 85 (2002)
2933
Scheme 2
guanosine 5'-phosphorimidazolide unit 18, which has highly liphophilic groups for pro-
tection of the 2'-and 3 '- OH groups, as well as theexo-amino group, as shown in Scheme 3.
First, we attempted to synthesize 18 from guanosine 5'-O-monophosphate (14) to
reduce the number of reaction steps: transient protection of 14 with Me3SiCl (TMSCl)
in pyridine followed by tritylation with DMTrCl in the presence of DMAP gave 2-N-
(4,4'-dimethoxytrityl)guanosine 5'-monophosphate (15). However, it was very difficult
to isolate 15 from the reaction mixture because of its high polarity. The crude product
was silylated in situ with a large excess of (t-Bu) Me2SiCl (TBDMSCl) in the presence
of imidazole in pyridine. The reaction was not complete, even when 10 equiv. of
TBDMSCl were added. The main product was isolated in an overall yield of 43% from
14 by silica-gel column chromatography. Based on the 1H-and 31P-NMR analysis, this
product was identified as compound 16, which has three TBDMS groups involving a
silyl phosphate moiety. Treatment of 16 with aq. NH3/EtOH 1:3 (v/v) at 608 for 12 h
gave the guanosine monophosphate derivative 17 in 94% yield. Although we tried to
synthesize 17 by silylation of guanosine 5'-O-monophosphate with TBDMSCl, followed
by tritylation, this straightforward synthesis failed. The reaction gave a mixture of many
products. The 5'-terminal phosphate of 17 was allowed to react with 1,1×-carbon-
yldiimidazole to give the phosphorimidazolide 18.
Although we established the synthesis of 18 by the above-mentioned method
(Method A), the inherent high polarity of guanylic acid derivatives hampered
purification of the product at each step. It is rather difficult to isolate pure materials by
Method A. Therefore, we explored an alternative route to compound 18 via a more
reliable pathway, as shown in Method B in Scheme 3. 2',3',5'-Tri-O-acetyl-2-N-(4,4'-
dimethoxytrityl)guanosine (21) [9a], which was obtained from guanosine (19) by a
two-step reaction, was converted to the 5'-O-acetyl derivative 22 [9b] in 61% yield by a

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Helvetica Chimica Acta Vol. 85 (2002)
2935
modification of the procedure of Ishido et al. [9c]. Silylation of 22 gave compound 23 in
87% yield. Treatment of 23 with NaOH gave the 5'-free guanosine derivative 24.
Phosphitylation of 24 according to the previously reported method gave the 5'-O-
phosphonylated product 25 in 90% yield. Further treatment of 25 with N-(trimethyl-
silyl)-1H-imidazole in the presence of CCl4 gave the phosphorimidazolide derivative 18
in 94% yield.
It was concluded that Method B is superior to Method A, since the former could
provide a sufficiently pure phosphoramidate derivative 18.
2.3. Solid-Phase Synthesis of 5'-Guanylated Trinucleotides by Means of the
Diisopropylsilandiyl Linker. The resin 10 containing 5'-O-(4,4'-dimethoxytrityl)-6-N-
benzoyldeoxyadenosine (22.9 mmol/g) was used for the synthesis of G5'pp-d(ATA). The
conditions of the detritylation step in the general phosphoramidite method [10] were
changed from 3% DCA in CH2Cl2 to ZnBr2 in CH2Cl2/i-PrOH 85 :15 (v/v) [7]. The 5'-
terminal site of the synthesized trinucleotide (dApTpdA) on the solid support was
phosphorylated in the usual way by using a commercially available phospha-linker to
give the fully protected trimer 26 (Scheme 4). The partially deblocked trinucleotide 27
was obtained by reaction of 26 with aq. NH3/EtOH 4 :1 (v/v) at 558 for 4 h. Under these
conditions, the silanediyl group was found to be stable, since pd(ATA) could be
recovered in high yield by treatment with TABF.
The coupling reaction of 27 with 18 in the presence of 4-nitro-6-(trifluoromethyl)-
1H-benzotriazol-1-ol (28; Ntbt-OH) [11] was carried out three times at room
temperature for 2 h. These conditions have proved to be effective at the level of the
liquid-phase synthesis of unsymmetrical dinucleoside pyrophosphate derivatives [12].
However, it was surprisingly observed that the total coupling yield calculated by the
DMTr cation assay was almost 0%. Furthermore, nucleotidic derivatives of pdApTp-
dA, which should be cleaved from the solid support by treatment with TBAF, could not
be detected. When the resin 27 was treated with TBAF before the coupling reaction,
pdApTpdA was obtained in sufficient quantity, as mentioned above. These results
strongly suggested that an unexpected cleavage reaction occurred simultaneously with
the coupling reaction. Therefore, we have investigated this interesting side reaction in
great detail and were able to learn what happened.
2.4. Detailed Studies of the Unexpected Cleavage. To clarify the side reaction, several
experiments were performed, as summarized in the Table. A partially deprotected
trimer 30 with the DMTr group at the 5'-terminal site was prepared on HCP. This resin
was treated with the guanosine 5'-phosphorimidazolide unit 18 in the presence of Ntbt-
OH, as described in the case of 27. The loss of the DMTr group on the resin was
estimated to be 76% by the DMTr assay (Entry 1). When the phosphoramidate 18 was
added in the absence of Ntbt-OH, 9% of the DMTr group was released from the resin
(Entry 2). To our surprise, the reaction of 30 with Ntbt-OH in the absence of 18
resulted in more loss (81%) of the DMTr group (Entry 3). From these results, it is likely
that Ntbt-OH reacted with the Si-atom via nucleophilic substitution to cleave the SiÀO
bond. It is also noted that the phosphoramidate 18 induced SiÀO bond cleavage,
although its rate is very slow compared with that with Ntbt-OH. However, simulta-
neous addition of 18 and Ntbt-OH did not accelerate the cleavage reaction. This is
probably because Ntbt-OH is consumed by reaction with 18, giving rise to an active
phosphodiester derivative so that the concentration of Ntbt-OH decreases.

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Table. The SiÀO Bond Cleavage of Oligodeoxynucleotides Attached to Polystyrene Resins via a Silanediyl-Type
Linker
Entry Oligo-loaded resin
Phosphoro- Ntbt-OH Loss of the
amidate
DMTr group
from the resina)
2 h
1
18
18
Ntbt-OH 76%
2
3
9%
Ntbt-OH 81%
4
5
6
18
18
Ntbt-OH 2%
0%
Ntbt-OH 4%
7
8
9
18
18
Ntbt-OH 0%
0%
Ntbt-OH 0%
10
34
34
Ntbt-OH 75%
11
12
43%, 87% (24 h)
Ntbt-OH 71%
13
34
Ntbt-OH 0%
a) This value was calculated by the DMT-cation assay estimated by UV before and after treatment of 30, 31, 32,
35, or 36 with reactants listed in this Table.
Further experiments were performed with protected substrates. A protected trimer
31 and dA monomer derivatives 32a,b were prepared as substrates on HCP (Table).
Reaction of 31 with Ntbt-OH in the presence or absence of 18 resulted in loss of trace
amounts of the DMTr-protected materials from the resin, as shown in Entries 4 and 6.
The use of only 18 did not release the DMTr group at all (Entry 5). Furthermore, no

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Helvetica Chimica Acta Vol. 85 (2002)
loss of the DMTr group on dA monomer derivatives was observed in all cases
(Entries 7 9). These results suggest that phosphate anions of internucleotidic
phosphodiester bonds might be responsible for the significant cleavage reaction
observed in the case of 30.
To see whether this unexpected reaction, observed only when internucleotidic phos-
phate anions exist, is independent of the nucleobases of substrates, the guanosine
moiety of 18, and solid resin beads, more simplified experiments were performed.
2.5. Confirmation of the Cleavage Reaction with Methyl Phosphorimidazolide in the
Solid Phase and the Liquid Phase. In view of the results mentioned above, we syn-
thesized a smaller reactant to see whether the guanosine moiety is associated with the
side reaction. Thus, methyl phosphorimidazolide (34) was synthesized by reaction of
methyl phosphonate (33) with N-(trimethylsilyl)-1H-imidazole in the presence of CCl4
[13] (Scheme 5). The simplified reactant was allowed to react with TpT-dimer-loaded
resin 35 and T-loaded resin 36. These results are shown in Entries 10 13 of the Table.
Similar results were obtained compared with the previous examples. These results
implied that the base sequence on the resin is not important. It was also found that the
loss of the DMTr group increased significantly in Entry 11 compared with the result of
Entry 2. The prolonged reaction led to up to 87% cleavage of the SiÀO bond after 24 h,
as shown in Entry 11.
Scheme 5
More straightforward confirmation of the above-mentioned cleavage reaction was
achieved by using a model compound 34 in the liquid-phase system. The model
compound 42 was synthesized from 5'-O-(4,4'-dimethoxytrityl)thymidine (37) by a
four-step reaction, as shown in Scheme 6. The compound 42 has a diisopropylsilanediyl
group at the 3'-terminal site and an internucleotidic dissociated phosphate structure.
The cleavage reaction was carried out under the conditions of Entry 10. As the result,
actually DMTrOTpT (43) could be isolated in 52% yield. Characterization of this
1
product was performed by H-,13C-, and 31P-NMR spectroscopy, ESI mass spectrom-
etry, and enzyme analysis.
3. Conclusions. In conclusion, we synthesized a useful guanosine phosphorimi-
dazolide unit for the synthesis of guanosine-capped oligodeoxynucleotides on polymer
supports. In connection with the coupling reaction on solid supports, we found an
intriguing side reaction associated with polymer-linked oligodeoxynucleotides having
unprotected internucleotidic phosphate groups. It should be noted that no other study
has been reported about such remote group participation of the 5'-phosphodiester
group affected the stability of the 3'-O-silyl group. To clarify the unique effect of
internucleotidic phosphate residues, further research is needed. It is concluded that the
capping reaction should be designed without use of strong nucleophilic activators such

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Helvetica Chimica Acta Vol. 85 (2002)
as Ntbt-OH in consideration of this type of side reaction, when specific linkers
containing SiÀO linkages are used.
Experimental Part
General. Pyridine was distilled two times from TsCl and from CaH2 and then stored over molecular sieves
(4 ä). Column chromatography (CC) was performed with silica gel C-200 purchased from Wako Co. Ltd., and a
minipump for a goldfish bowl was conveniently used to attain sufficient pressure for rapid chromatographic
separation. Reverse-phase (RP) HPLC was performed on a combination of Waters 2690 and 996 systems and
SHIMADZU 6A system with the mBondasphere and mBondapak C-18 columns (Waters, 3.9 Â 150 mm and
7.8 Â 300 mm, resp.) with a linear gradient starting from 0.1m NH4OAc (pH 7.0) and increasing MeCN at flow
rates of 1.0 ml/min and 3.0 ml/min, resp. Anion-exchange HPLC was performed on a Gen-PakTM FAX column
(Waters, 4.6 Â 100 mm) on Waters 2690 and 996 systems with a 10 30% linear gradient of 0.5m phosphate, 20%
MeCN buffer (pH 6.0) in 5.0 mm phosphate, 20% MeCN buffer (pH 6.0) at 508 at a flow rate of 1.0 ml/min for
30 min. UV Spectra were recorded on a U-2000 spectrometer. 1H-,13C-, and 31P-NMR spectra were recorded at
270, 68, and 109 MHz, resp,. the chemical shifts were measured from TMS or 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. ESI
mass spectra were measured on MarinerTM (PerSeptive Biosystems Inc.). Elemental analyses were performed by
the Microanalytical Laboratory, Tokyo Institute of Technology at Nagatsuta.
2-Cyanoethyl 16-Hydroxyhexadecanate (4). To a soln. of 16-hydroxyhexadecanoic acid (3; 6.73 g,
23.38 mmol) and 2-hydroxyethanenitrile (32 ml, 46.76 mmol) in pyridine (200 ml), 1,3-dicyclohexylcarbodii-
mide (DCC, 5.30 mg, 25.72 mmol) was added. The mixture was stirred at 08 for 30 min, and stirred at r.t. for
24 h. The mixture was filtered. The filtrate was diluted with AcOEt, and washed two times each with 0.1m aq.
HCl and with H2O. The org. phase was collected, dried (Na2SO4), filtered, and evaporated under reduced
pressure. The residue was dissolved again in AcOEt (200 ml) and filtered. Crystallization from cooled AcOEt
(200 ml) gave 4 (5.09 g, 67%): M.p. 81.58. 1H-NMR (270 MHz, CDCl3): 1.25 1.66 (m, CH2(3 15)); 2.36 (t,
J(2,3) ˆ J (2,3') ˆ 7.6, HÀC(2), H'ÀC(2)); 2.70 (t, Jvic ˆ 6.3, OCH2CH2CN); 3.63 (t, J(15,16) ˆ J(15',16) ˆ 6.6,
HÀC(16), H'ÀC(16)); 4.28 (t, Jvic ˆ 6.3, OCH2CH2CN). 13C-NMR (67.8 MHz, CDCl3): 18.11; 24.84; 25.79;
‡
29.12; 29.26; 29.46; 29.62; 29.65; 29.67; 32.85; 34.00; 58.44; 63.05; 116.67; 173.09. ESI-MS: 326.2690 ([M ‡ H] ;
calc. 326.2695).
2-Cyanoethyl 16-{[6-N-Benzoyl-5'-O-(4,4'-dimethoxytrityl)deoxyadenosin-3'-O-yl](diisopropyl)silyloxy}
hexadecanate (7). To a soln. of 1H-imidazole (817 mg, 12.0 mmol) and (i-Pr)2SiCl2 (794 ml, 4.4 mmol) in
DMF (30 ml), 5 (2.63 g, 4.0 mmol) was added dropwise at À608 under Ar. After stirring at r.t. for 1 h, 4 (1.30 g,
4.0 mmol) was added to the mixture. The resulting mixture was stirred at r.t. for 4 h. The mixture was diluted
with CHCl3, and washed with aq. sat. NaHCO3 soln. The org. phase was dried (Na2SO4), filtered, and dried in
vacuo. The crude product was chromatographed on a column of silica gel (120 g) with hexane/AcOEt 60 :40
(v/v) containing 0.5% pyridine to give 7 (2.67 g, 61%). 1H-NMR (270 MHz, CDCl3): 0.86 1.52 (m, 2 (Me)2CH,
2 Me2CH, CH2(3 15)); 2.24 (t, J(2,3) ˆ J (2,3') ˆ 7.1, H ÀC(2), H'ÀC(2)); 2.45 2.78 (m, OCH2CH2CN,
HÀC(2'), H'ÀC(2')); 3.26 3.34 (m, HÀC(5'), H'ÀC(5')); 3.57 3.65 (m, HÀC(15), H'ÀC(15), 2 MeO); 4.11
4.15 (m, OCH2CH2CN, HÀC(4')); 4.73 (br., HÀC(3')); 6.23 (dd, J(1',2') ˆ 5.9, J(1',2'') ˆ 6.3, HÀC(1')); 6.67
7.94 (m, 18 arom. H); 8.12 (s, HÀC(2) of A); 8.61 (s, HÀC(8) of A); 9.41 (s, NH). 13C-NMR (67.8 MHz, CDCl3):
11.97; 11.98; 12.84; 13.29; 13.31; 16.90; 17.08; 17.22; 17.87; 24.66; 25.66; 28.94; 29.09; 29.30; 29.46; 29.51; 32.65;
33.80; 40.73; 55.01; 58.30; 63.07; 63.40; 72.46; 76.52; 84.75; 86.29; 87.06; 112.86; 116.59; 123.23; 123.48; 126.60;
127.54; 127.71; 127.81; 128.41; 129.70; 132.34; 133.41; 135.32; 135.34; 135.37; 135.79; 141.27; 144.19; 149.25;
‡
149.29; 151.18; 158.17; 172.87. ESI-MS: 1095.5084 ([M ‡ H] ; calc. 1095.5991).
16-{[6-N-Benzoyl-5'-O-(4,4'-dimethoxytrityl)deoxyadenosin-3'-O-yl](diisopropyl)silyloxy}hexadecanoic
Acid Triethylammonium Salt (8). To a soln. of 7 (1.10 g, 1.0 mmol) in pyridine (10 ml), DBU (1.49 ml,
10.0 mmol) was added at r.t. After 30 min of stirring, the mixture was poured into CHCl3 (50 ml), washed with
0.1m TEAB buffer, and dried (Na2SO4). The resulting soln. was concentrated and purified on a column of silica
gel (40 g) eluted with CHCl3/MeOH 100 :1 (v/v) containing 0.5% Et3N to give 8 as an oil (0.70 g, 80%).
1H-NMR (270 MHz, CDCl3): 1.01 1.05 (m, Me2CH, 2 Me2CH); 1.24 1.59 (m, CH2(3 15), Et3N); 2.29 (t, J
(2,3) ˆ J(2,3') ˆ 7.4, HÀC(2), H'ÀC(2)); 2.56 2.84 (m, HÀC(2'), H'ÀC(2')); 3.00 3.08 (br., Et3N); 3.31 3.44
(m, HÀC(5'), H'ÀC(5')); 3.69 (t, J(15,16) ˆ J(15',16) ˆ 6.4, HÀC(16), H'ÀC(16)); 3.76 (s, 2 MeO); 4.25 (d, J
(3',4') ˆ 2.6, HÀC(4')); 4.80 (br., HÀC(3')); 6.53 (dd, J(1',2') ˆ 6.6, J(1',2'') ˆ 6.9, HÀC(1')); 6.77 8.08 (m, 18
arom. H); 8.24 (s, HÀC(2) of A); 8.70 (s, HÀC(8) of A); 9.81 (br., NH). 13C-NMR (67.8 MHz, CDCl3): 8.55;

Helvetica Chimica Acta Vol. 85 (2002)
2941
12.08; 12.11; 17.33; 25.04; 25.79; 29.19; 29.30; 29.45; 29.59; 29.65; 32.78; 34.64; 40.96; 44.90; 55.14; 63.20; 63.53;
72.55; 84.91; 86.44; 87.23; 113.00; 123.51; 126.71; 127.67; 127.93; 128.08; 128.46; 129.81; 132.48; 133.33; 135.46;
‡
135.50; 141.45; 144.29; 149.59; 151.48; 151.87; 158.30; 178.31. ESI-MS: 1042.5427 ([M ‡ H] ; calc. 1042.5725).
Preparation of NucleosideÀDiisopropylsilanediylÀHCP. The soln. of appropriate nucleoside unit 8
(193 mg, 169 mmol) in CH2Cl2 (5 ml) was drawn into a syringe containing a highly cross-linked (amino-
methyl)polystyrene (HCP; 1.0 g, 33.7 mmol) and DCC (174 mg, 843 mmol). This mixture was allowed to stand
for 6 h; then, the liquid was ejected, and the solid in the syringe was treated successively with pyridine (wash),
CH2Cl2 (wash), and dried under reduced pressure. An equivolume mixture of pyridine/Ac2O 1:9 (v/v) was
added into the syringe to cap any residual amino groups. After standing for 12 h, the solid was treated with
pyridine (wash) and CH2Cl2 (wash). The nucleoside loading averaged ca. 18.0 28.5 mmol/gram, as determined
by absorbance of the DMTr cation liberated on treatment of an aliquot with 60% HClO4/EtOH 3 :2 (v/v).
3'-O-[Ethoxy(diisopropyl)silyl]-5'-O-[(9H-fluoren-9-ylmethoxy)carbonyl]thymidine (12). To a soln. of
1H-imidazole (544 mg, 8.0 mmol) and (i-Pr)2SiCl2 (540ml, 3.0 mmol) in DMF (10 ml) were dropwise added 11
[10] (929 mg, 2.0 mmol) at À608 under Ar. After stirring at r.t. for 1 h, the mixture was quenched by adding
EtOH (5 ml). The resulting mixture was stirred at r.t. for 4 h. The mixture was diluted with AcOEt and washed
with aq. sat. NaHCO3 soln. The org. phase was dried (Na2SO4), filtered, and dried in vacuo. The crude product
was chromatographed on a column of silica gel (25 g) eluted with hexane/AcOEt 80 :20 (v/v) containing 0.5%
pyridine to give 12 (319 mg, 26%). 1H-NMR (270 MHz, CDCl3): 1.00 (br., 2 Me2CH); 1.17 (t, Jvic ˆ 6.9,
MeCH2O); 1.77 (s, MeÀC(5)); 2.02 2.32 (m, HÀC(2'), H'-C(2')); 3.76 (q, Jvic ˆ 6.9, MeCH2O); 4.10 4.54 (m,
CHCH2 of Fmoc, HÀC(3'), HÀC(4'), HÀC(5'), H'ÀC(5')); 6.33 (dd, J (1',2') ˆ 6.3, J (1',2'') ˆ 6.6, HÀC(1'));
7.22 7.71 (m, 9 arom. H); 9.99 (s, NH). 13C-NMR (67.8 MHz, CDCl3): 11.84; 11.96; 12.48; 17.05; 17.07; 17.09;
17.10; 18.44; 40.68; 46.57; 58.64; 66.73; 69.82; 71.25; 84.41; 84.69; 110.96; 119.85; 124.56; 126.83; 126.87; 127.70;
‡
134.94; 140.94; 140.99; 142.68; 142.74; 150.29; 154.54; 163.88. ESI-MS: 623.2780 ([M ‡ H] ; calc. 623.2789).
The Stability of the Diisopropylsilanediyl Group under Acidic Conditions. Compound 12 (232 mg,
0.37 mmol) was dissolved with 1% TFA in CH2Cl2 (5 ml). After stirring at r.t. for 6 h, the mixture was diluted
with CHCl3/pyridine 9 :1 (v/v) and washed with H2O. The org. phase was dried (Na2SO4), filtered, and dried in
vacuo. The crude product was chromatographed on a column of silica gel (20 g) eluted with hexane/AcOEt
80 :20, 65 :35, 50 :50 (v/v) containing 0.5% pyridine to give 12, 11, and 13 (153 mg, 15 mg, 32 mg, 66%, 7 %,
19%) respectively.
‡
Data of 11: 1H-and 13C-NMR: identical with those of the authentic sample. ESI-MS: 463.1621 ([M ‡ H] ;
calc. 465.1662).
Data of 13: 1H-NMR (270 MHz, CDCl3): 1.03 (br., 2 Me2(CH)); 1.80 (s, MeÀC(5)); 2.14 2.48 (m,
HÀC(2'), H'ÀC(2')); 4.13 4.65 (m, CHCH2 of Fmoc, HÀC(3'), HÀC(4'), HÀC(5'), H'ÀC(5')); 6.27 (dd, J
(1',2') ˆ J (1',2'') ˆ 6.3, HÀC(1')); 7.24 7.72 (m, 9 arom. H); 9.90 (s, NH). 13C-NMR (67.8 MHz, CDCl3): 12.28;
12.47; 12.50; 17.02; 31.45; 36.48; 40.82; 46.58; 66.77; 69.98; 70.78; 84.42; 85.19; 110.85; 119.90; 124.65; 124.69;
126.91; 126.93; 127.74; 135.43; 140.99; 141.02; 142.79; 142.81; 150.37; 154.74; 162.45; 163.83. ESI-MS: 595.2493
‡
([M ‡ H] ; calc. 595.2476).
Triethylammonium 2',3'-O-Bis[(tert-butyl)dimethylsilyl]-6-N-(4,4'-dimethoxytrityl)guanosine 5'-O-[(tert-
butyl)dimethylsilyl]phosphate (16). Guanosine 5'-O-monophosphate (14; 1.00 g, 2.75 mmol) was rendered
anh. by coevaporations three times with dry DMF, and finally dissolved in dry DMF (27 mL). To the soln.,
pyridine (5.56 ml, 68.8 mmol) and Me3SiCl (3.5 ml, 27.5 mmol) were added. The mixture was stirred at r.t. for
1 h. To the mixture, DMTr-Cl (1.86 g, 5.5 mmol), Et3N (0.77 ml, 5.5 mmol) and 4-(dimethylamino)pyridine
(DMAP; 13 mg, 0.11 mmol) were added. After stirring at r.t. for 30 min, the soln. was treated with aq. NH3
(5 ml) at r.t. for 30 min. After removal of NH3 by aspirator, the soln. was diluted with H2O and washed with
Et2O. The aq. phase was collected, evaporated under reduced pressure, and diluted with 1m TEAB buffer. The
soln. was washed 20 times each with CHCl3/BuOH 1:1 (v/v). The org. phase was collected and co-evaporated
with pyridine. The residue was reprecipitated with Et2O to give the crude mixture. The mixture was rendered
anh. by co-evaporations three times with dry pyridine, and finally dissolved in dry pyridine (27 ml). To the soln.
was added 1H-imidazole (3.74 g, 55 mmol) and (t-Bu)Me2SiCl (4.13 g, 27.5 mmol). After stirring at r.t. for 12 h,
the soln. was diluted with CHCl3/pyridine 2 :1 (v/v) and washed with sat. NaHCO3 soln. The org. phase was
collected, dried (Na2SO4), filtered, and evaporated under reduced pressure. The residue was chromatographed
on a column of silica gel (170 g) eluted with CHCl3/MeOH 94 :6 (v/v) containing 0.5% Et3N to give 16 (1.31 g,
43%). 1H-NMR (270 MHz, CDCl3): À0.50 0.73 (m, 3 (t-Bu)SiMe2); 0.90 0.95 (br., Et3N); 2.64 2.71 (br.,
Et3N); 3.49 4.01 (m, 2 MeO, HÀC(3'), HÀC(4'), HÀC(5'), H'ÀC(5')); 4.41 (br, HÀC(2'), H'ÀC(2')); 5.31 (d,
J (1',2') ˆ 7.6, HÀC(1')); 6.51 7.92 (m, 13 arom. H). 13C-NMR (67.8 MHz, CDCl3): À4.91; À4.59; À4.56;
À4.49; À3.72; À3.44; 8.18; 9.00; 17.91; 17.94; 18.00; 18.04; 25.61; 25.77; 45.36; 54.91; 69.90; 112.74; 112.80;

2942
Helvetica Chimica Acta Vol. 85 (2002)
116.61; 127.47; 128.49; 129.96; 151.27; 151.62; 157.74; 157.78. 31P-NMR (109 MHz, CDCl3): À8.36. ESI-MS:
‡
1008.4988 ([M ‡ H] ; calc. 1008.4559).
Triethylammonium 2',3'-O-Bis[(tert-butyl)dimethylsilyl]-6-N-(4,4'-dimethoxytrityl)guanosine 5'-O-Phos-
phate (17). A soln. of 16 (111 mg, 0.1 mmol) in EtOH (0.75 ml) and aq. NH3 (0.25 ml) was stirred at 608 for
12 h. The mixture was concentrated under reduced pressure, and the residue was purified by silica-gel
1
chromatography (CHCl3/MeOH 85 :15) containing 0.5% Et3N to give 17 (94 mg, 94%). H-NMR (270 MHz,
CDCl3): À0.37 0.01 (m, 2 (t-Bu)SiMe2); 0.62 0.88 (m, 2 Me3CSiMe2); 0.98 (br., Et3N); 2.81 (br., Et3N); 3.61
3.84 (m, 2 MeO, HÀC(4'), HÀC(5'), H'ÀC(5')); 4.16 (s, HÀC(3')); 4.56 (br., HÀC(2')); 5.45 (br., HÀC(1'));
6.64 7.28 (m, 13 arom. H); 8.11 (s, HÀC(8)). 13C-NMR (67.8 MHz, CDCl3): À4.97; À4.50; À4.45; 0.09; 8.48;
17.95; 18.02; 21.50; 25.87; 29.72; 45.54; 55.03; 55.23; 69.98; 73.65; 75.89; 85.32; 113.04; 125.16; 126.60; 127.69;
128.08; 128.47; 128.88; 128.98; 129.92; 136.81; 137.69; 151.37; 151.67; 158.01. 31P-NMR (109 MHz, CDCl3): 1.06.
‡
ESI-MS: 894.3732 ([M ‡ H] ; calc. 894.3695).
Triethylammonium 2',3'-O-Bis[(tert-butyl)dimethylsilyl]-6-N-(4,4'-dimethoxytrityl)guanosine 5'-O-Phos-
phorimidazolide (18). Method A. A soln. of 17 (20 mg, 20 mmol) in DMF (0.2 ml) was added N,N'-
carbonyldiimidazole (4.0 mg, 24 mmol). The mixture was stirred at r.t. for 1 h, and MeOH (0.1 ml) was then
added to the mixture. After stirring for 10 min, the soln. was evaporated under reduced pressure, and the residue
was chromatographed on a column of silica gel (10 g) eluted with CHCl3/MeOH 96 :4 (v/v) containing 0.5%
1
Et3N to give 18 as a foam (16 mg, 77%). H-NMR (270 MHz, CDCl3): À0.36 0.04 (m, 2 t-Bu)SiMe2); 0.67
0.91 (m, 2 Me3CSiMe2); 1.04 (t, J ˆ 7.3, Et3N); 2.79 (q, J ˆ 7.3, Et3N); 3.60 3.97 (m, 2 MeO, HÀC(4'),
HÀC(5'), H'ÀC(5'), HÀC(3')); 4.48 (br., HÀC(2')); 5.47 (d, J (1',2') ˆ 6.9, HÀC(1')); 6.66 7.29 (m, 13 arom.
H); 7.82 8.10 (m, HÀC(8), 3 H of imidazole). 13C-NMR (67.8 MHz, CDCl3): À4.88; À4.66; À4.54; À4.45;
9.42; 17.94; 17.98; 25.76; 25.82; 45.91; 55.01; 55.02; 70.10; 75.66; 113.00; 116.25; 120.61; 127.66; 128.44; 129.91;
‡
151.33; 151.64; 158.02; 190.23. 31P-NMR (109 MHz, CDCl3): À9.81. ESI-MS: 944.3995 ([M ‡ H] ; calc.
944.3963).
Method B. A soln. of 25 in MeCN/CCl4 (10 ml) 1:1 (v/v) was added (trimethylsilyl)-1H-imidazole (738 ml,
5 mmol) and Et3N (702 ml, 5 mmol). The mixture was stirred at r.t. for 40 min, and MeOH (1.0 ml) was then
added to the mixture. After stirring for 20 min, the soln. was evaporated under reduced pressure, and the residue
was chromatographed on a column of silica gel (50 g) eluted with CHCl3/MeOH 96 :4 (v/v) containing 0.5%
Et3N to give 18 as a foam (982 mg, 94%). 1H-, 13C-, and 31P-NMR: identical with those of the authentic sample.
‡
ESI-MS: 944.3995 ([M ‡ H] ; calc. 944.3963).
5'-O-Acetyl-2',3'-O-bis[(tert-butyl)dimethylsilyl]-6-N-(4,4'-dimethoxytrityl)guanosine (23). To a soln. of 22
(6.28 g, 10.0 mmol) in DMF (100 ml), imidazole (4.08 g, 60.0 mmol) and (t-Bu)Me2SiCl (4.50 g, 30.0 mmol)
were added. The resulting mixture was vigorously stirred for 12 h. The clear soln. was evaporated, treated with
sat. NaHCO3 soln., extracted with AcOEt, and evaporated under reduced pressure. The residue was
chromatographed on a column of silica gel (150 g) with hexane/CHCl3 0 :100 (v/v) containing 0.5% pyridine to
give 23 as a foam (7.44 g, 87%). 1H-NMR (270 MHz, CDCl3): À0.23 0.01 (m, 2 (t-Bu)SiMe2); 0.76 0.84 (m,
2 Me3CSiMe2); 2.01 (s, MeC(O)); 3.70 (s, 2 MeO); 4.07 4.31 (m, HÀC(4'), HÀC(5'), H'ÀC(5'), HÀC(3'));
4.46 (dd, J(1',2') ˆ 4.6, J(2',3') ˆ 4.3, HÀC(2')); 6.74 7.27 (m, 13 arom. H); 7.51 (s, HÀC(8)); 8.12 (br., NH).
13C-NMR (67.8 MHz, CDCl3): À4.85; À4.82; À4.40; À4.26; 18.03; 18.11; 20.90; 25.80; 25.82; 55.25; 63.28; 70.12;
72.06; 74.96; 81.80; 113.88; 127.61; 127.99; 128.07; 128.51; 129.54; 134.10; 135.97; 151.49; 158.80. ESI-MS:
‡
856.4634 ([M ‡ H] ; calc. 856.4137).
2',3'-O-Bis[(tert-butyl)dimethylsilyl]-6-N-(4,4'-dimethoxytrityl)guanosine (24). Compound 23 (4.30 g,
5.00 mmol) was dissolved with 1n aq. NaOH (10 ml) in pyridine (50 ml). After stirring at r.t. for 10 min, and
the mixture was applied to a resin of Dowex 50WÂ 8 (pyridinium form, 100 ml). The eluate was evaporated
under reduced pressure. The mixture was diluted with CHCl3/pyridine 9 :1 (v/v) and washed with H2O. The org.
phase was dried (Na2SO4), filtered, and dried in vacuo. The crude product was chromatographed on a column of
silica gel (50 g) eluted with CHCl3 containing 0.5% pyridine to give 24 (3.38 g, 83%). 1H-NMR (270 MHz,
CDCl3): À0.52 0.00 (m, 2 (t-Bu)SiMe2); 0.69 0.84 (m, 2 Me3CSiMe2); 3.55 3.88 (m, 2 MeO, HÀC(5'),
H'ÀC(5')); 4.04 (s, HÀC(4')); 4.19 (d, J(2',3') ˆ 4.6, HÀC(3')); 4.76 (dd, J(1',2') ˆ 7.9, J (2',3') ˆ 4.6, HÀC(2'));
5.53 (d, J (1',2') ˆ 7.9, HÀC(1')); 6.75 7.63 (m, 13 arom. H, HÀC(8)); 8.55 (br., NH). 13C-NMR (67.8 MHz,
CDCl3): À5.66; À4.51; À4.45; À4.32; 17.91; 18.11; 21.49; 25.77; 25.83; 55.26; 63.09; 70.31; 73.82; 74.96; 88.79;
90.28; 114.20; 125.14; 127.72; 127.93; 128.67; 128.81; 128.87; 129.30; 134.49; 134.51; 138.01; 143.18; 148.59;
‡
151.68; 154.98; 159.04. ESI-MS: 814.4045 ([M ‡ H] ; calc. 814.4031).
Triethylammonium 2',3'-O-Bis[(tert-butyl)dimethylsilyl]-6-N-(4,4'-dimethoxytrityl)guanosine 5'-O-Phos-
phonate (25). Compound 24 (3.26 g, 4.00 mmol) was rendered anh. by co-evaporations three times with dry
pyridine, and finally dissolved in dry pyridine (15 ml). To a soln. of diphenyl phosphonate (5.40 ml, 28.0 mmol)

Helvetica Chimica Acta Vol. 85 (2002)
2943
in dry pyridine (20 ml), the soln. of 24 at r.t. under Ar was added dropwise. After stirring at r.t. for 2 h, the soln.
was treated with Et3N/H2O (5 ml, 1:1 (v/v)) at r.t. for 30 min. The soln. was diluted with CHCl3/pyridine 1:1
(v/v) and washed with 1m TEAB buffer. The org. phase was dried (Na2SO4), filtered, and dried in vacuo. The
residue was chromatographed on a column of silica gel (100 g) eluted with CHCl3/MeOH 96 :4 (v/v) containing
1
0.5% Et3N to give 25 (3.52 g, 90%). H-NMR (270 MHz, CDCl3): À0.40 0.02 (m, 2 (t-Bu)SiMe2); 0.62 0.90
(m, 2 Me3CSiMe2); 1.11 (t, Et3N); 2.92 (q, Et3N); 3.58 3.89 (m, 2 MeO, HÀC(4'), HÀC(5'), H'ÀC(5')); 4.09 (d,
J (2',3') ˆ 3.6, HÀC(3')); 4.47 (dd, J (1',2') ˆ 6.9, J (2',3') ˆ 3.6, HÀC(2')); 5.48 (d, J (1',2') ˆ 6.9, HÀC(1')); 5.58
(s, HaP); 6.66 7.47 (m, 13 arom. H); 7.87 (s, HbP); 8.00 (br., HÀC(8)); 11.95 (br., NH). 13C-NMR (67.8 MHz,
CDCl3): À4.86; À4.49; À4.40; 8.57; 17.62; 18.00; 18.03; 25.87; 45.55; 55.08; 113.12; 127.81; 128.46; 129.89. 31P-
‡
NMR (109 MHz, CDCl3): 3.81. ESI-MS: 878.3863 ([M ‡ H] ; calc. 878.3746).
Synthesis of pd(ATA) by Using Resin 10. In a dried syringe (5 ml, equipped with a glass-wool filter at the
base), the resin 10 (0.5 mmol, 22.9 mmol/g, 21.9 mg) containing 6-N-benzoyl-2'-deoxy-5-O'-(4,4'-dimethoxy-
trityl)-adenosine was treated with the soln. of ZnBr2 in CH2Cl2/i-PrOH 85 :15 (v/v) (1 ml) for 1 min three times.
The soln. was expelled, and the solid was washed with pyridine, EtOH, pyridine and CH2Cl2. A soln. containing
the phosphoramidite unit (15 mmol) and tetrazole (60 mmol) in MeCN (150 ml) was poured into the syringe.
After 3 min, the soln. was expelled, and solid was washed with pyridine. The contents of syringe were then
successively treated with 0.15m I2 in THF/pyridine/H2O 10 :10 :1 (v/v/v) for 15 s three times, pyridine (wash),
0.12m DMAP in pyridine/Ac2O 9 :1 (v/v) for 2 min, pyridine, CH2Cl2 (wash). Repeating the procedure men-
tioned above, trinucleotide (dApTpdA) was synthesized on the solid support. After detritylation of the 5'-
terminal site, the phosphorylation, in the usual way with a commercially available phospha-linker, gave the fully
protected trimer 26. After treatment of the resin with aq. NH3/EtOH 4 :1 (v/v) at 558 for 4 h, EtOH (wash) and
CH2Cl2 (wash), a soln. of 1m TBAF-AcOH in THF (0.5 ml) was added into the syringe. After shaking for 1 h at
r.t., the soln. was collected, the solid was washed with THF, and the combined org. solns. were evaporated under
reduced pressure. The residue was eluted with H2O (1 ml). The eluate was purified by using a C-18 cartridge and
lyophilized to give pdApTpdA (12.6 A260 units, 71%). The product was characterized by MS (ESI-MS: 949.1823
‡
([M ‡ H] ; calc. 949.1796) and enzymatic treatment.
Enzymatic Treatment of pd(ATA) with Snake Venom Phosphodiesterase (SVPD) and Alkaline Phosphatase
(AP). The compound pdApTpdA (0.5 A260 units) was dissolved in 0.05m Tris ¥ HCl (pH 8.0, 50 ml) containing
0.02m ZnCl2 and 4 ml of SVPD (4 units, 1.0 unit/ml) in glycerin/H2O 1:1 (v/v) were added. The resulting mixture
was incubated at 378 for 4 h and heated at 908 for 2 min. AP (4 ml, 4 unit, 1.0 unit/ml) was added to the resulting
mixture. After incubation for 3 h, the mixture was heated at 908 for 2 min. The mixture was analyzed by RP-
HPLC.
5'-O-(4,4'-Dimethoxytrityl)-3'-O-[(ethoxy)diisopropylsilyl]thymidine (38). 5'-O-(4,4'-Dimethoxytrityl)thy-
midine (37, 3.26 g, 6.0 mmol) was rendered anh. by co-evaporation three times with dry pyridine, and finally
dissolved in dry DMF (20 ml). To a soln. of 1H-imidazole (1.634 g, 24.0 mmol) and (i-Pr)2SiCl2 (1.62 ml,
9.0 mmol) in DMF (30 ml) was added dropwise 37 at 08 under Ar. After stirring at r.t. for 1 h, the mixture was
added to EtOH (10 ml). The resulting mixture was stirred at r.t. for 10 min. The mixture was diluted with
CHCl3, and washed with aq. sat. NaHCO3 soln. The org. phase was dried (Na2SO4), filtered, and dried in vacuo.
The crude product was chromatographed on a column of silica gel (200 g) eluted with hexane/AcOEt 70 :30
(v/v) containing 0.5% pyridine to give 38 (2.62 g, 62%). 1H-NMR (270 MHz, CDCl3): 1.00 1.01 (m, 2 Me2CH);
1.13 (t, Jvic ˆ 6.9, MeOCH2); 1.47 (s, MeÀC(5) of T); 2.21 2.45 (m, HÀC(2), H'ÀC(2)); 3.28 3.50 (m,
HÀC(5'), H'ÀC(5')); 3.67 3.74 (q, Jvic ˆ 6.9, MeCH2O); 3.79 (s, 2 MeO); 4.07 4.08 (d, J (3',4') ˆ 2.3,
HÀC(4')); 4.70 (br., HÀC(3')); 6.42 (dd, J(1',2') ˆ 5.9, J(1',2'') ˆ 7.9, HÀC(1')); 6.81 7.65 (m, 13 arom. H,
HÀC(6) of T); 8.94 (s, NH). 13C-NMR (67.8 MHz, CDCl3): 11.81; 11.95; 12.04; 17.17; 17.20; 18.48; 21.26; 30.86;
41.49; 55.14; 58.56; 63.33; 72.29; 84.73; 86.73; 86.91; 110.92; 113.02; 113.05; 126.89; 127.73; 127.90; 129.86;
‡
135.15; 135.22; 135.47; 144.09; 150.10; 158.43; 163.59. ESI-MS: 725.3200 ([M ‡ Na] ; calc. 725.3234).
3'-O-[(Ethoxy)diisopropylsilyl]thymidine (39). Compound 38 (860 mg, 1.22 mmol) was dissolved with
CH2Cl2/i-PrOH 85 :15 (v/v) (49 ml). To the soln., ZnBr2 (8.28 g, 36.7 mmol) was added. After stirring at r.t. for
1 h, the mixture was diluted with CHCl3 and washed with H2O. The org. phase was dried (Na2SO4), filtered, and
dried in vacuo. The crude product was chromatographed on a column of silica gel (20 g) eluted with hexane/
AcOEt 60 :40 (v/v) to give 39 (370 mg, 76%). 1H-NMR (270 MHz, CDCl3): 0.97 1.44 (m, 2CH(Me)2,
2CH(CH3)2); 1.13 (t, Jvic ˆ 6.9, MeCH2O); 1.89 (s, MeÀC(5) of T); 2.30 2.34 (m, HÀC(2), H'ÀC(2)); 3.77
3.86 (q, Jvic ˆ 6.9, MeCH2O); 3.90 4.00 (m, HÀC(4'), HÀC(5'), H'ÀC(5')); 4.65 (br., HÀC(3')); 6.24 (dd, J
(1',2') ˆ 6.9, J(1',2'') ˆ 6.6, HÀC(1')); 7.52 (s, HÀC(6) of T); 9.84 (s, NH). 13C-NMR (67.8 MHz, CDCl3): 11.89;
11.99; 12.38; 17.09; 17.10; 17.13; 18.40; 40.60; 58.63; 61.91; 71.44; 85.90; 87.51; 110.62; 136.67; 150.35; 164.13. ESI-
‡
MS: 401.2190 ([M ‡ H] ; calc. 401.2108).

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Helvetica Chimica Acta Vol. 85 (2002)
P-(2-Cyanoethyl)-5'-O-(4,4'-dimethoxytrityl)thymidyl-(3' ! 5')-3'-O-[(ethoxy)diisopropylsilyl]thymidine
(41). Compound 39 (200 mg, 0.5 mmol) was rendered anh. by co-evaporation three times with dry pyridine, dry
toluene, dry MeCN and finally dissolved in dry MeCN (5 ml). To a soln. of 39 and 1H-tetrazole (105 mg,
1.5 mmol) in MeCN, 5'-O-(4,4'-dimethoxytrityl)thymidine 3'-O-phosphoramidite (447 mg, 0.6 mmol). After
stirring at r.t. for 5 min, the mixture was added to t-BuOOH (1 ml). The resulting mixture was stirred at r.t. for
5 min. The mixture was diluted with CHCl3, and washed with aq. sat. NaHCO3 soln. The org. phase was dried
(Na2SO4), filtered, and dried in vacuo. The crude product was chromatographed on a column of silica gel (25 g)
eluted with CHCl3/MeOH 98 :2 (v/v) containing 0.5% pyridine to give 41 (494 mg, 93%). 1H-NMR (270 MHz,
CDCl3): 1.02 1.04 (m, 2 Me2CH); 1.20 (ddd, Jvic ˆ 6.9, Jvic ˆ 3.6, MeCH2O); 1.47 (s, MeÀC(5) of Ta, MeÀC(5)
of Tb); 2.27 2.35 (m, HaÀC(2), H'aÀC(2), HbÀC(2), H'bÀC(2)); 2.74 (t, Jvic ˆ 5.9, OCH2CH2CN) 3.41 3.76
(m, HaÀC(5'), H'aÀC(5')); 3.79 4.56 (m, 2 MeO, MeCH2O, OCH2CH2CN, HaÀC(3'), HaÀC(4'), HbÀC(4'),
HbÀC(5'), H'bÀC(5')); 5.17 (br., HbÀC(3')); 6.22 6.43 (m, HaÀC(1'), HbÀC(1')); 6.82 7.54 (m, 13 arom. H,
HÀC(6) of Ta, HÀC(6) of Tb ); 9.00 (s, NHa); 9.06 (s, NHb). 13C-NMR (67.8 MHz, CDCl3): 11.67; 11.92; 12.00;
12.02, 12.40; 16.95; 17.12; 17.15; 18.56; 19.50; 19.61; 19.69; 39.89; 40.18; 55.19; 58.76; 62.13; 62.21; 63.19; 71.13;
84.18, 84.70; 84.84; 85.33; 85.74; 87.16; 111.06; 111.24; 111.62; 113.15; 115.89; 127.10; 127.84. 127.92; 129.89;
134.69; 134.75; 134.87; 135.54; 135.77; 143.70; 143.73; 150.02; 150.22; 158.55; 163.45. 31P-NMR (109 MHz,
‡
CDCl3): À1.94. ESI-MS: 1082.3856 ([M ‡ Na] ; calc. 1082.3960).
5'-O-(4,4'-Dimethoxytrityl)thymidylyl-(3' ! 5')-3'-O-[(ethoxy)diisopropylsilyl]thymidine (42). Compound
41 (265 mg, 0.25 mmol) was dissolved with dry pyridine (3 ml). To the soln., DBU (135 ml, 0.9 mmol) was added.
After stirring at r.t. for 10 min, the mixture was diluted with CHCl3 and washed with 2m TEAB buffer. The org.
phase was dried (Na2SO4), filtered, and dried in vacuo. The crude product was chromatographed on a column of
silica gel (13 g) eluted with CHCl3/MeOH 96 :4 (v/v) containing 0.5% Et3N to give 42 (260 mg, 94%). 1H-NMR
(270 MHz, CDCl3): 1.00 1.32 (m, 2 Me2CH, MeCH2O, (MeCH2)N); 1.91 1.97 (s, MeÀC(5) of Ta, MeÀC(5) of
Tb); 2.07 2.66 (m, HaÀC(2), Ha'ÀC(2), HbÀC(2), Hb'ÀC(2)); 3.00 (q, Jvic ˆ 7.3, N(MeCH2)3N); 3.35 3.48 (m,
HaÀC(5'), H'aÀC(5')); 3.73 3.81 (m, 2 MeO, HbÀC(4')); 4.03 4.05(m, HbÀC(5'), Hb'ÀC(5')); 4.31 (br.,
HaÀC(4')); 4.58 (br., HaÀC(3')); 5.04 (br., HbÀC(3')); 6.39 6.49 (m, HaÀC(1'), HbÀC(1')); 6.79 7.38 (m, 13
arom. H); 7.60 (s, HÀC(6) of Ta or T b); 7.78 (s, HÀC(6) of Ta or Tb ). 13C-NMR (67.8 MHz, CDCl3): 8.71; 11.54;
11.97; 12.03; 12.41; 17.23; 18.55; 30.88; 39.52; 40.77; 45.53; 55.15; 58.59; 63.91; 65.62; 72.86; 84.49; 84.72; 85.17;
85.26; 86.57; 86.68; 86.90; 110.95; 111.12; 113.05; 126.92; 127.72; 128.01; 129.94; 134.96; 135.13; 135.50; 136.24;
143.97; 150.48; 150.53; 158.43; 163.82; 163.95. 31P-NMR (109 MHz, CDCl3): À1.05. ESI-MS: 1029.3734 ([M ‡
‡
Na] ; calc. 1029.3695.)
This work was supported by a grant from the −Research for the Future× program of the Japan Society for the
Promotion of Science (JSPS-RFTF 97I00301) and a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
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Received May 30, 2002