A facile synthesis of 5′-end solid-anchored, 3′-end free oligodeoxyribonucleotides via the (5′→3′)-elongated phosphoramidite strategy

Article abstract of DOI:10.1081/NCN-100002082

It is demonstrated that not only N²- but also O ⁶-protection of the guanine base is necessary for obtaining the oligodeoxyribonucleotides in high yields and at a high purity in the solid-phase synthesis via the (5′ → 3′)-chain elongated phosphoramidite approach.

Full text of DOI:10.1081/NCN-100002082

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A FACILE SYNTHESIS OF 5′-END SOLID-ANCHORED, 3′-
END FREE OLIGODEOXYRIBONUCLEOTIDES VIA THE
(5′ → 3′)-ELONGATED PHOSPHORAMIDITE STRATEGY
Akira Sakakura a & Yoshihiro Hayakawa b
a Laboratory of Bioorganic Chemistry, Graduate School of Human Informatics, Nagoya
University, Chikusa , Nagoya, 464-8601, Japan
b Laboratory of Bioorganic Chemistry, Graduate School of Human Informatics, Nagoya
University, Chikusa , Nagoya, 464-8601, Japan
Published online: 17 Aug 2006.
To cite this article: Akira Sakakura & Yoshihiro Hayakawa (2001) A FACILE SYNTHESIS OF 5′-END SOLID-ANCHORED, 3′-
END FREE OLIGODEOXYRIBONUCLEOTIDES VIA THE (5′ → 3′)-ELONGATED PHOSPHORAMIDITE STRATEGY, Nucleosides,
Nucleotides and Nucleic Acids, 20:3, 213-227, DOI: 10.1081/NCN-100002082
To link to this article: http://dx.doi.org/10.1081/NCN-100002082
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(3), 213–227 (2001)
A FACILE SYNTHESIS OF 5ꢀ-END
SOLID-ANCHORED, 3ꢀ-END FREE
OLIGODEOXYRIBONUCLEOTIDES VIA THE
(5ꢀ→3ꢀ)-ELONGATED PHOSPHORAMIDITE
STRATEGY
Akira Sakakura and Yoshihiro Hayakawa∗
Laboratory of Bioorganic Chemistry, Graduate School of Human
Informatics, Nagoya University, Chikusa, Nagoya 464-8601, Japan
ABSTRACT
It is demonstrated that not only N2- but also O6-protection of the guanine base
is necessary for obtaining the oligodeoxyribonucleotides in high yields and
at a high purity in the solid-phase synthesis via the (5ꢀ → 3ꢀ)-chain elongated
phosphoramidite approach.
Development of an efficient synthesis of 5ꢀ-end solid-anchored, 3ꢀ-end free
oligodeoxyribonucleotides is an important subject, because these substances may
serve as key intermediates for the preparation of oligonucleotides with organic
functions at the 3ꢀ-terminals, which are biologically attractive, for instance, as
antisense molecules (1). One of the most convenient ways to synthesize such solid-
anchored oligonucleotides is the (5ꢀ → 3ꢀ)-directed chain elongation, starting from
a nucleoside attached at the 5ꢀ-hydroxyl to solid supports, via the phosphoramidite
method using suitably protected nucleoside 5ꢀ-phosphoramidites as building blocks
(2). However, according to a report (3) and an examination we performed (4), the
construction of the nucleotide chain by the (5ꢀ → 3ꢀ)-elongation method is not
conducted in a satisfactory yield. Thus, we made some investigations to elucidate
the reasons causing a lowering of the yield when the conventional (5ꢀ → 3ꢀ)-chain
∗Corresponding author. Fax: 81-52-789-5646; E-mail: yoshi@info.human.nagoyau.ac.jp
213
ꢁ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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SAKAKURA AND HAYAKAWA
elongation method is used. As the result, we found that a main reason is the use of
the O6-unprotected guanosine compound, which is harmed by the phosphoramidite
resulting in a decrease in the yield and purity of the product. Consequently, we
examined the synthesis of oligodeoxyribonucleotides via the (5ꢀ → 3ꢀ)-elongation
strategy using the N2, O6-protected guanosine phosphoramidite monomer and have
realized an efficient method giving the product in a desirable yield with an excellent
purity.
Deoxyribonucleoside5ꢀ-phosphoramidites, 11–15, requisiteasmonomerunits
for the synthesis of oligodeoxyribonucleotides, were prepared starting from the cor-
responding 3ꢀ,5ꢀ-O-free deoxyribonucleosides, 1–5 (5), with the allylic protector on
the nucleoside base via the pathway shown in Scheme 1. Thus, the 5ꢀ-hydroxyl of
the starting material was silylated with tert-butyldimethylsilyl chloride (TBDMS-
Cl) by the assistance of imidazole (DMF, 25◦C, 12 h), and the 3ꢀ-hydroxyl was
then tritylated with p,pꢀ-dimethoxytrityl chloride (DMTr-Cl) in the presence of
1,8-diazabicyclo[5.4.0]-7-undecene (DBU) (pyridine, 25◦C, 24 h) to give the 3ꢀ,5ꢀ-
Scheme 1. Preparation of deoxyribonucleoside 5ꢀ-phosphoramidites. (a) t-C4H9(CH3)2SiCl,
imidazole, DMF; (b) C6H5(p-CH3OC6H4)2CCl or p-CH3OC6H4(C6H5)2CCl, DBU, pyridine;
(c) (n-C4H9)4NF, THF; (d) CH2=CHCH2OP[N(i-C3H7)2]2, diisopropylammonium 1H-tetrazolide,
CH3CN.

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215
O-protected nucleotide. In this tritylation, the use of DBU is important to obtain
the desired product in high yield. The reaction in the absence of DBU or using
triethylamine in place of DBU afforded the target compounds in lower yields. The
5ꢀ-O-TBDMS protecting group of the resulting product was removed by exposure
to tetrabutylammonium fluoride (TBAF) (THF, 25◦C, 1.5 h) and, finally, the phos-
phoramidite moiety was introduced to the resulting 5ꢀ-O-free product by the use
of (allyloxy)[bis(diisopropylamino)]phosphine {(CH2=CHCH2O)P[N(i-C3H7)2]2}
and diisopropylammonium 1H-tetrazolide (6) to give the desired deoxyribonucle-
oside 5ꢀ-phosphoramidite. The overall yields of the phosphoramidites from the
corresponding starting nucleosides were 56% for 11, 81% for 12, 28% for 13, 47%
for 14 (7), and 92% for 15.
ꢀ
5ꢀ
First, we carried out the synthesis of a 12mer, TCACCATTATGC3 (16),
using 11, 12, 13, and 15 as building blocks, and benzimidazolium triflate (8) as
the activator. The synthesis was carried out on an Applied Biosystems Model 392
DNA/RNA synthesizer, starting from thymidine (17) covalently attached at the 5ꢀ-
❛
hydroxyl to controlled pore glass (CPG) supports (500 A pore size) via a long-chain
NHCOCH2CH2COO
CPG
Thy
O
DMTrO
17
alkylamine linker. Table 1 indicates the reaction cycle for the chain elongation.
The average coupling yield for one-base elongation determined by trityl assay was
99.6% (overall 95.7%) (9). After finishing the elongation, the product was treated
with a mixture of the tris(dibenzylideneacetone)dipalladium(0)–chloroform com-
plex [Pd2(dba)3·CHCl3] (2.5 equiv/allyl), triphenylphosphine (25 equiv/allyl), and
diethylammonium formate (150 equiv/allyl) at 50◦C for 1 h to remove the allylic
protecting groups (6,10) and then with concentrated ammonia at 25◦C for 1 h to
detach the product from the solid supports. HPLC of the crude product (Fig. 1)
showed two conspicuous peaks at tr = 18.5 and 20.0 min. Comparison of the
authentic sample confirmed that the major product giving the peak at tr = 18.5
min is the target oligonucleotide 16, while the minor product providing the peak at
tr = 20.0 min could not be isolated in a pure form and therefore its structure could
not be determined. However, digestion of the crude product with a mixture of snake
venom phosphodiesterase and bacterial alkaline phosphatase provided important
information for determining this product. HPLC of the digests obtained from the
crude product showed peaks due to four kinds of parent nucleosides and one peak
with a longer retention time than those of the four kinds of nucleosides. The region

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SAKAKURA AND HAYAKAWA
Table 1. Reaction Cycle for the (5ꢀ→3ꢀ)-Directed Chain Elongation Method
Step
Operation
Reagent
Time, min
1
2
3
4
washing
detritylation
washing
CH3CN
3% Cl3CCOOH/CH2Cl2
CH3CN
0.1 M phosphoramidite/CH3CN +
0.2 M benzimidazolium triflate/CH3CN
CH3CN
Ac2O-2,6-lutidine–THF (1:1:8) +
16% N-methylimidazole/THF
CH3CN
0.4
1.3
0.8
2.0
coupling
5
6
washing
capping
0.2
0.3
7
8
9
washing
oxidation
washing
0.2
0.5
0.6
1.0 M t-C4H9OOH/toluene
CH3CN
where the later peak was observed is the region where peaks due to derivatives suf-
fering some modification at the nucleoside base appear. Further, the experimentally
derived composition of the four bases was dA:dC:dG:T = 3.12:4.00:0.74:4.14, and,
comparedwiththeratiocalculatedfromthedesiredoligomer16, i.e., dA:dC:dG:T=
3:4:1:4, the content of dG is rather low. On the basis of these findings, we conceived
that 13 suffers some undesired reaction, perhaps phosphitylation, at its unprotected
O6-position of the guanine base to decrease the purity of the target compound (11).
Accordingly, we next examined the synthesis of the oligomer 16 in a similar
manner to that described above, using the deoxyguanosine phosphoramidite 14
with an allyl protector at the O6-position (12) in place of 13 without O6-protection.
According to the trityl assay, the coupling yield was 99.9% average and 99% overall.
As the HPLC (Fig. 2) shows, the product has an excellent purity (ca. 85%) in a crude
form. Little side product was detected in a long retention-time region. A similar
result was obtained in the synthesis of a 15mer, 5ꢀTGTCGACACCCAATT3ꢀ (18)
(see Fig. 2). Thus, as expected, the O6-protection prevented the guanine moiety
from undesired reactions.
Figure 1. HPLC profile of the crude product of 16 obtained in the synthesis using 13 as the guanosine
phosphoramidite.

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217
Figure 2. HPLC profiles of the crude products of 16 and 18 prepared using 14 as the guanosine
monomer unit; (A) 16 and (B) 18.
In conclusion, we revealed that a main reason why the existing synthesis of
oligodeoxyribonucleotides through the (5ꢀ → 3ꢀ)-chain elongation does not afford
the target compounds in high yield and at a high quality is that the
O6-free guanosine base suffers some modification under conditions of phosphity-
lation with a nucleoside 5ꢀ-phosphoramidite in the presence of a promoter such as
1H-tetrazole or benzimidazolium triflate. Consequently, we used the N2,O6-
protectedguanosinephosphoramidite, inplaceofthe O6-unprotectedderivativethat
has been conventionally used so far, to achieve an efficient synthesis of
solid-anchored 3ꢀ-end free oligodeoxyribonucleotides through the (5ꢀ → 3ꢀ)-chain
elongation. This approach was effectively applied to the synthesis of some
oligodeoxyribonucleotide derivatives with a covalent conjugation at the 3ꢀ-end po-
sitions, including oligode-oxyribonucleotide–amino acid and –peptide conjugates,
and an oligonucleotide–intercalator conjugate (13).
EXPERIMENTAL SECTION
General Procedure and Materials
UV spectra were measured in methanol on a JASCO V-550 spectrometer.
IR spectra were measured in KBr on a JASCO FT/IR-5300 spectrometer. NMR
spectra were taken in CDCl3 on a JEOL α-400 instrument. For 1H NMR spectra,
tetramethylsilane (TMS) was used as a standard. For 31P NMR spectra, H3PO4 was
used as an external standard. The chemical shifts are described as δ values in parts

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SAKAKURA AND HAYAKAWA
per million (ppm). Elemental analyses were carried out in the Faculty of Agricul-
ture, Nagoya University. High-performance liquid chromatography (HPLC) using
an ODS-5 µm column with a 0.1 M ammonium acetate buffer containing 5–13%
acetonitrile (v/v) (linear gradient in 30 min) was carried out on a JASCO PU-980
chromatograph with a JASCO UV-970-absorption detector. Fuji silysia silica gel
BW-300S was used for column chromatography. Solid-phase synthesis was carried
out on an Applied Biosystems Model 392 DNA/RNA synthesizer. Unless otherwise
stated, reactions were carried out at ambient temperature. The organic extracts were
dried over MgSO4 before concentration. N6-Allyloxycarbonyl-2ꢀ-deoxyadenosine
(1), N4-allyloxycarbonyl-2ꢀ-deoxycytidine (2), N2-allyloxycarbonyl-2ꢀ-deoxy-
guanosine (3), and O6-allyl-N2-allyloxycarbonyl-2ꢀ-deoxyguanosine (4) were pre-
pared by reported methods (5). Acetonitrile, pyridine, and dichloromethane were
distilled from CaH2. THF was distilled from benzophenone ketyl. Commercially
supplied reagents were used without any purification, unless otherwise noted. A
solution of a nucleoside 5ꢀ-phosphoramidite (0.1 M) and benzimidazolium triflate
(0.2 M) in acetonitrile were prepared immediately before use. A 1:1:8 mixture of
acetic anhydride, 2,6-lutidine and THF, and a 16% solution of N-methylimidazole
in THF were also freshly prepared before use.
N6-Allyloxycarbonyl-3ꢀ-O-p, pꢀ-dimethoxytrityl-2ꢀ-deoxyadenosine
5ꢀ-(Allyl N,N-diisopropylphosphoramidite) (11)
To a DMF (22 mL) solution of 1 (7.24 g, 21.6 mmol) and imidazole (2.95
g, 43.3 mmol) was added TBDMS-Cl (2.92 g, 19.4 mmol) at 0◦C. The resulting
mixture was stirred at 25◦C for 12 h. After addition of water (50 mL), the mixture
was extracted with ethyl acetate (100 mL × 4). The combined organic extracts were
washed with brine (50 mL), dried, and concentrated. The residue was subjected to
silica gel (200 g) column chromatography with a 1:100:100 to 1:10:10 methanol–
ethyl acetate–hexane mixture as the eluent, giving the 5ꢀ-O-silylated nucleoside
as a viscous oil (7.53 g, 78%). To a solution of the silylation product (8.88 g,
19.8 mmol) and DBU (6.01 g, 5.90 mL, 39.5 mmol) in pyridine (45 mL) was
added DMTr-Cl (13.4 g, 39.5 mmol) at 0◦C and the mixture was stirred at 25◦C
for 24 h. The reaction mixture was concentrated under reduced pressure to give a
residual oil. This material was diluted with ethyl acetate (500 mL) and washed with
water (100 mL) followed by brine (100 mL). The organic solution was dried and
concentrated. The resulting oily product was purified by column chromatography
on silica gel (100 g) eluted with a 1:2 to 1:1 ethyl acetate–hexane mixture to
afford the 3ꢀ-O-tritylated nucleoside as an amorphous solid (11.0 g, 74%). To a
THF (40 mL) solution of the product (11.0 g, 14.6 mmol) was added a 1.0 M
solution of TBAF in THF (30.0 mL, 30.0 mmol), and the mixture was stirred
for 1.5 h. After addition of an aqueous solution saturated with NH4Cl (50 mL),
the aqueous layer was extracted with ethyl acetate (100 mL × 3). The combined
organic extracts were washed with brine (50 mL), dried, and concentrated. Silica gel
(100 g) column chromatography of the resulting crude product with a 1:1 mixture

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SYNTHESIS OF OLIGODEOXYRIBONUCLEOTIDES
219
of ethyl acetate and hexane followed by ethyl acetate to give N6-allyloxycarbonyl-
3ꢀ-O-p,pꢀ-dimethoxytrityl-2ꢀ-deoxyadenosine (6) (9.33 g, 100%) as an amorphous
solid: UV λmax 268 (ε 21500), 237 nm (25400); IR 1755, 1610, 1585, 1510, 1465,
1
1250, 1220 cm−1; H NMR δ 1.75 (dd, J = 5.4, 13.7 Hz, 1 H), 2.70 (ddd, J =
5.4, 10.2, 13.7 Hz, 1 H), 3.31 (br dd, J = 11.2, 12.7 Hz, 1 H), 3.72 (br d, J = 12.7
Hz, 1 H), 3.78 (s, 3 H), 3.79 (s, 3 H), 4.06 (s, 1 H), 4.63 (d, J = 5.4 Hz, 1 H), 4.75
(d, J = 5.9 Hz, 2 H), 5.28 (dd, J = 1.5, 10.2 Hz, 1 H), 5.40 (dd, J = 1.5, 17.1 Hz,
1 H), 5.64 (br d, J = 11.2 Hz, 1 H), 5.98 (tdd, J = 5.9, 10.2, 17.1 Hz, 1 H), 6.32
(dd, J = 5.4, 10.2 Hz, 1 H), 6.84 (d, J = 8.8 Hz, 2 H), 6.85 (d, J = 8.8 Hz, 2 H),
7.23 (t, J = 7.3 Hz, 1 H), 7.31 (dd, J = 7.3, 7.3 Hz, 2 H), 7.36 (d, J = 8.8 Hz, 4 H),
7.47 (d, J = 7.3 Hz, 2 H), 7.99 (s, 1 H), 8.45 (s, 1 H), 8.65 (s, 1 H). Anal. Calcd for
C35H35N5O7: C, 65.92; H, 5.53; N, 10.98. Found: C, 65.96; H, 5.56; N, 10.79. To
a stirred solution of (6) (1.13 g, 1.78 mmol), diisopropylamine (90.3 mg, 125 µL,
0.892mmol), 1H-tetrazole(62.2mg, 0.887mmol)inacetonitrile(10mL)wasadded
(allyloxy)bis(diisopropylamino)phosphine (940 mg, 3.26 mmol), and the mixture
was stirred for 3 h. The reaction mixture was diluted with ethyl acetate (200 mL) and
washed with brine (70 mL × 2). The organic solution was dried and concentrated
to give a viscous oil, which was dissolved in dichloromethane (8 mL). This solution
was poured into pentane (300 mL) at −78◦C with stirring to give a powder, which
was collected by filtration. The powder was dissolved in dichloromethane, and then
the solution was evaporated in vacuo to afford 11 as an amorphous solid (1.42 g,
97%): UV λmax 268 (ε 21800), 238 (24900), 212 nm (42500); IR 1760, 1730, 1610,
1585, 1510, 1460 cm−1; 1H NMR δ 0.99 (d, J = 6.8 Hz, 3 H), 1.05 (d, J = 6.8 Hz, 3
H), 1.14 (d, J = 6.8 Hz, 6 H), 2.07–2.11 (m, 1 H), 2.23 (ddd, J = 5.4, 8.3, 13.7 Hz,
0.5 H), 2.30 (ddd, J = 5.4, 9.3, 13.7 Hz, 0.5 H), 3.29 (ddd, J = 2.9, 6.3, 11.2 Hz, 0.5
H), 3.39 (ddd, J = 2.4, 7.3, 11.7 Hz, 0.5 H), 3.43–3.52 (m, 2 H), 3.55 (ddd, J = 2.4,
4.9, 11.7 Hz, 0.5 H), 3.66 (ddd, J = 2.4, 5.9, 11.2 Hz, 0.5 H), 3.78 (s, 6 H), 3.98–4.17
(m, 3 H), 4.46 (d, J = 5.4 Hz, 0.5 H), 4.49 (d, J = 5.4 Hz, 0.5 H), 4.76 (d, J = 5.9
Hz, 2 H), 5.08 (br d, J = 10.3 Hz, 0.5 H), 5.12 (br d, J = 10.3 Hz, 0.5 H), 5.20 (dd,
J = 2.0, 17.1 Hz, 0.5 H), 5.25 (dd, J = 2.0, 17.1 Hz, 0.5 H), 5.28 (d, J = 10.3 Hz, 1
H), 5.40 (d, J = 17.1 Hz, 1 H), 5.83 (tdd, J = 4.9, 10.3, 17.1 Hz, 0.5 H), 5.92 (tdd,
J = 4.9, 10.3, 17.1 Hz, 0.5 H), 5.99 (tdd, J = 4.9, 10.3, 17.1 Hz, 1 H), 6.61 (dd, J
= 5.9, 8.3 Hz, 0.5 H), 6.62 (dd, J = 5.9, 9.3 Hz, 0.5 H), 6.84 (d, J = 8.8 Hz, 4 H),
7.32–7.21 (m, 3 H), 7.36 (d, J = 8.8 Hz, 2 H), 7.37 (d, J = 8.8 Hz, 2 H), 7.46–7.49
(m, 2 H), 8.35 (s, 0.5 H), 8.37 (s, 0.5 H), 8.76 (s, 1 H); 31P NMR δ 148.6. Anal.
Calcd for C44H53N6O8P: C, 64.07; H, 6.48; N, 10.19. Found: C, 64.03; H, 6.50;
N, 10.09.
N4-Allyloxycarbonyl-3ꢀ-O-p, pꢀ-dimethoxytrityl-2ꢀ-deoxycytidine
5ꢀ-(Allyl N,N-diisopropylphosphoramidite) (12)
To a solution of 2 (11.8 g, 37.9 mmol) and imidazole (5.18 g, 76.1 mmol) in
DMF (38 mL) was added TBDMS-Cl (5.73 g, 38.0 mmol) at 0◦C. The mixture was
stirred at 25◦C for 12 h. After addition of water (50 mL) at 0◦C, the mixture was

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SAKAKURA AND HAYAKAWA
extracted with ethyl acetate (100 mL × 4). The combined organic extracts were
washed with brine (50 mL), dried, and concentrated. The residue was subjected to
silica gel (200 g) column chromatography. Elution with a 1:1 to 5:1 ethyl acetate–
hexane mixture gave the 5ꢀ-O-silylated nucleoside as a residual oil (14.9 g, 92%).
To a solution of the product (10.7 g, 25.1 mmol) and DBU (7.13 g, 7.00 mL,
46.8 mmol) in pyridine (100 mL) was added DMTr-Cl (16.8 g, 49.6 mmol) at 0◦C,
and the mixture was stirred at 25◦C for 24 h. The reaction mixture was concentrated
under reduced pressure to give a viscous oil, to which was added water (500 mL).
The mixture was extracted with ethyl acetate (500 mL × 2). The combined organic
extracts were successively washed with a 0.1 M aqueous solution of CuSO4 (200
mL × 2) to remove efficiently pyridine and DBU, an aqueous solution saturated
with NaHCO3 (200 mL × 3), and brine (200 mL × 2), and then concentrated. The
resulting crude product was chromatographed on silica gel (320 g) column with a
1:3 to 1:1 mixture of ethyl acetate and hexane to afford the 3ꢀ-O-trityl nucleoside as
an amorphous solid (18.2 g, 99%). To a solution of this product (7.57 g, 10.4 mmol)
in THF (50 mL) was added a 1.0 M solution of TBAF in THF (18.0 mL, 18.0 mmol).
The mixture was stirred at 25◦C for 2 h and then diluted with ethyl acetate (200 mL).
The organic mixture was successively washed with water (150 mL × 2), an aqueous
solution saturated with NH4Cl (150 mL × 2), an aqueous solution saturated with
NaHCO3 (150 mL × 2), and brine (150 mL × 2), and then dried and concentrated.
Chromatography of the crude product on silica gel (200 g) using a 1:1 to 1:5 mixture
of ethyl acetate and hexane gave N4allyloxycarbonyl-3ꢀ-O-p,pꢀ-dimethoxytrityl-
2ꢀ-deoxycytidine (7) (6.00 g, 89%): UV λmax 284 (ε 9300), 238 nm (35000); IR
1750, 1655, 1560, 1510, 1250, 1200 cm−1; 1H NMR δ 1.96 (m, 1 H), 2.18 (m, 1
H), 2.97 (br s, 1 H), 3.23 (dd, J = 2.4, 11.7 Hz, 1 H), 3.62 (d, J = 11.7 Hz, 1 H),
3.76 (s, 6 H), 3.90 (d, J = 2.4 Hz, 1 H), 4.35 (d, J = 6.4 Hz, 1 H), 4.64 (d, J =
5.9 Hz, 2 H), 5.27 (d, J = 10.7 Hz, 1 H), 5.33 (d, J = 17.1 Hz, 1 H), 5.91 (tdd, J
= 5.9, 10.7, 17.1 Hz, 1 H), 6.22 (dd, J = 6.4, 7.8 Hz, 1 H), 6.81 (d, J = 8.3 Hz,
4 H), 7.20 (t, J = 6.8 Hz, 1 H), 7.18–7.22 (m, 1 H), 7.27 (dd, J = 6.8, 7.3 Hz,
2 H), 7.34 (d, J = 8.3 Hz, 4 H), 7.44 (d, J = 7.3 Hz, 2 H), 8.06 (d, J = 7.3 Hz,
1 H). Anal. Calcd for C34H35N3O8: C, 66.55; H, 5.75; N, 6.85. Found: C, 66.48;
H, 5.98; N, 6.61. To a solution of 7 (5.60 g, 9.13 mmol), diisopropylamine (462
mg, 640 µL, 4.57 mmol), and 1H-tetrazole (318 mg, 4.54 mmol) in acetonitrile
(50 mL) was added (allyloxy)bis(diisopropylamino)phosphine (3.37 g, 11.7 mmol),
and the resulting mixture was stirred for 1 h. The reaction mixture was diluted with
ethyl acetate (400 mL), washed with an aqueous solution saturated with NaHCO3
(50 mL) followed by brine (50 mL), dried, and concentrated. The resulting crude
material was subjected to silica gel (100 g) column chromatography and eluted with
a 1:5:trace to 2:1:trace ethyl acetate–hexane–triethylamine mixture to afford 12 as
an amorphous solid (7.35 g, 100%): UV λmax 284 (ε 8800), 238 (31900), 212 nm
(34000); IR 1695, 1610, 1510, 1465, 1250 cm−1; 1H NMR δ 0.99 (d, J = 6.8 Hz, 3
H), 1.08 (d, J = 6.8 Hz, 3 H), 1.13 (d, J = 6.8 Hz, 3 H), 1.15 (d, J = 6.8 Hz, 3 H),
1.72 (ddd, J = 5.9, 9.3, 13.7 Hz, 1 H), 1.86 (s, 3 H), 1.86–1. 92 (m, 1 H), 3.22–3.30
(m, 1 H), 3.45–3.56 (m, 2.5 H), 3.64 (m, 0.5 H), 3.79 (s, 6 H), 4.01–4.14 (m, 2 H),

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221
4.33 (d, J = 5.9 Hz, 0.5 H), 4.35 (d, J = 5.9 Hz, 0.5 H), 5.07 (d, J = 10.3 Hz, 0.5
H), 5.10 (d, J = 10.3 Hz, 0.5 H), 5.17 (dd, J = 2.0, 17.1 Hz, 0.5 H), 5.22 (dd, J =
2.0, 17.1 Hz, 0.5 H), 5.80 (tdd, J = 5.4, 10.3, 17.1 Hz, 0.5 H), 5.86 (tdd, J = 5.4,
10.3, 17.1 Hz, 0.5 H), 6.41 (dd, J = 5.4, 9.3 Hz, 0.5 H), 6.46 (dd, J = 5.4, 9.3 Hz,
0.5 H), 6.82 (d, J = 8.8 Hz, 2 H), 6.83 (d, J = 8.8 Hz, 2 H), 7.21–7.35 (m, 5 H),
7.33 (d, J = 8.8 Hz, 2 H), 7.34 (d, J = 8.8 Hz, 2 H), 7.43–7.46 (m, 2 H), 7.52 (s,
0.5 H), 7.65 (s, 0.5 H), 8.17 (br s, 1 H); 31P NMR δ 148.4, 148.6. Anal. Calcd for
C40H50N3O8P: C, 65.65; H, 6.89; N, 5.74. Found: C, 65.61; H, 6.78; N, 5.69.
N2-Allyloxycarbonyl-3ꢀ-O-p, pꢀ-dimethoxytrityl-2ꢀ-deoxyguanosine
5ꢀ-(Allyl N,N-diisopropylphosphoramidite) (13)
To a solution of N2-allyloxycarbonyl-3ꢀ,5ꢀ-bis-O-tert-butyldimethylsilyl-2ꢀ-
deoxyguanosine (10.3 g, 17.8 mmol) in THF (120 mL) was added a 1.0 M solution
of TBAF in THF (40.0 mL, 40.0 mmol). The resulting mixture was stirred at 25◦C
for 24 h. To the reaction mixture were added a 3:3:1 mixture of pyridine, methanol
and water (50 mL) and Dowex 50W-X8 (30 mL), and stirring was continued for an
additional 30 min. Dowex 50W-X8 was removed by filtration and the filtrate was
concentrated. The residue was chormatographed on silica gel (300 g) column with
a 1:20 mixture of methanol and dichloromethane as the eluent to afford 3 (6.10 g,
98%). To a solution of 3 (6.10 g, 17.4 mmol) and imidazole (3.69 g, 54.2 mmol) in
DMF (20 mL) was added TBDMS-Cl (2.66 g, 17.6 mmol) at 0◦C and the result-
ing mixture was stirred at 25◦C for 24 h. The reaction mixture was diluted with a
1:1 mixture of ethyl acetate and hexane (600 mL), successively washed with water
(300 mL), an aqueous solution saturated with NH4Cl (200 mL), an aqueous solution
saturated with NaHCO3 (200 mL), and brine (200 mL), dried, and concentrated.
The residue was purified by silica gel (300 g) column chromatography using a 1:30
mixture of methanol and dichloromethane to afford the 5ꢀ-O-silylated nucleoside as
a viscous liquid (3.36 g, 42%). To a solution of the product (2.35 g, 5.04 mmol) and
DBU (2.40 mL, 8.04 mmol) was added DMTr-Cl (5.30 g, 15.7 mmol). The mixture
was stirred at 25◦C for 24 h and concentrated. Water (600 mL) was added to the
resulting residual oil and the aqueous mixture was extracted with dichloromethane
(400 mL, 150 mL × 2). The combined organic extracts were successively washed
with a 1.0 M solution of CuSO4 (200 mL), an aqueous solution saturated with
NaHCO3 (200 mL), and brine (200 mL). After dryness, removal of the organic
solvent gave a residual oil, which was subjected to silica gel (200 g) column chro-
matography eluted with a 1:2 to 1:5 ethyl acetate–hexane mixture to provide the
3ꢀ-O-tritylated nucleoside as a glassy material (3.30 g, 85%). To a solution of this
product (3.90 g, 5.08 mmol) in THF (40 mL) was added a 1.0 M solution of TBAF
in THF (6.00 mL, 6.00 mmol), and the resulting mixture was stirred at 25◦C for
22 h. The reaction mixture was diluted with ethyl acetate (400 mL), successively
washed with an aqueous NH4Cl-saturated solution (200 mL), an NaHCO3 solu-
tion (200 mL), and brine (200 mL), dried, and concentrated. The resulting residual

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SAKAKURA AND HAYAKAWA
material was recrystallized from dichloromethane to give N2-allyloxycarbonyl-3ꢀ-
O-p, pꢀ-dimethoxytrityl-2ꢀ-deoxyguanosine (8) (2.66 g, 80%): UV λmax 237 nm
(ε 24200); IR 1695, 1610, 1510, 1385, 1250, 1175 cm−1; 1H NMR δ 1.72 (dd, J =
5.4, 13.7 Hz, 1 H), 2.42 (ddd, J = 5.9, 9.8, 13.7 Hz, 1 H), 3.24 (dd, J = 10.7, 11.7
Hz, 1 H), 3.68 (d, J = 11.7 Hz, 1 H), 3.79 (s, 6 H), 4.06 (s, 1 H), 4.50 (d, J = 5.9 Hz,
1 H), 4.71 (d, J = 5.9 Hz, 2 H), 4.87 (d, J = 10.7 Hz, 1 H), 5.32 (d, J = 10.7 Hz, 1
H), 5.38 (d, J = 17.2 Hz, 1 H), 5.90 (tdd, J = 5.9, 10.7, 17.2 Hz, 1 H), 6.16 (dd, J =
5.4, 9.8 Hz, 1 H), 6.84 (dd, J = 1.5, 8.8 Hz, 4 H), 7.15–7.46 (m, 10 H), 7.65 (s, 1 H),
11.28 (s, 1 H). Anal. Calcd for C35H35N5O8: C, 64.31; H, 5.40; N, 10.71. Found:
C, 64.45; H, 5.22; N, 10.60. To a suspension of 8 (1.23 g, 1.88 mmol), diisopropy-
lamine (101 mg, 140 µL, 0.989 mmol), and 1H-tetrazole (66.3 mg, 0.947 mmol)
in acetonitrile (15 mL) was added (allyloxy)bis(diisopropylamino)phosphine (940
mg, 3.26 mmol). After stirring at 25◦C for 3 h, the reaction mixture was diluted with
ethyl acetate (100 mL), washed with brine (50 mL × 2), dried, and concentrated.
The residue was dissolved in dichloromethane (6 mL) and the resulting solution was
poured into pentane (300 mL) at −78◦C with stirring to give powder, which was
collected by filtration. Chromatograhic purification of this crude powdery product
on silica gel (150 g) column with a 1:1:trace to 5:1:trace ethyl acetate–hexane–
diisopropylamine mixture provided 13 as an amorphous solid (1.59 g, 100%): UV
λmax 239 (ε 29200), 211 nm (40700); IR 1700, 1610, 1510, 1465, 1250 cm−1; 1H
NMR δ 1.00 (d, J = 6.8 Hz, 3 H), 1.08 (d, J = 6.8 Hz, 3 H), 1.14 (d, J = 6.8 Hz, 6
H), 2.02–2.08 (m, 1 H), 2.32–2.40 (m, 1 H), 3.27 (ddd, J = 2.4, 6.3, 11.2 Hz, 0.5
H), 3.36 (ddd, J = 2.9, 7.8, 11.2 Hz, 0.5 H), 3.47–3.56 (m, 2.5 H), 3.61 (ddd, J =
2.0, 4.9, 11.2 Hz, 0.5 H), 3.74 (s, 6 H), 3.81 (m, 0.5 H), 3.84 (m, 0.5 H), 3.99–4.21
(m, 2 H), 4.43 (d, J = 9.8 Hz, 0.5 H), 4.45 (d, J = 9.8 Hz, 0.5 H), 4.67 (m, 2 H),
5.06 (d, J = 10.3 Hz, 0.5 H), 5.13 (d, J = 10.3 Hz, 0.5 H), 5.19 (dd, J = 2.0,
17.1 Hz, 0.5 H), 5.26 (dd, J = 2.0, 17.1 Hz, 0.5 H), 5.30 (d, J = 10.3 Hz, 1 H), 5.35
(d, J = 17.6 Hz, 1 H), 5.77–5.98 (m, 2 H), 6.56 (dd, J = 5.9, 8.3 Hz, 1 H), 6.73
(dd, J = 2.0, 8.8 Hz, 2 H), 6.75 (d, J = 8.8 Hz, 2 H), 7.17–7.22 (m, 3 H), 7.27–
7.33 (m, 4 H), 7.38–7.44 (m, 2 H), 8.10 (s, 1 H); 31P NMR δ 148.5.14 Anal.
Calcd for C44H53N6O9P: C, 62.83; H, 6.37; N, 10.00. Found: C, 62.76; H, 6.56;
N, 9.93.
O6-Allyl-N2-allyloxycarbonyl-3ꢀ-O-p-dimethoxytrityl-2ꢀ-deoxyguanosine
5ꢀ-(Allyl N,N-diisopropylphosphoramidite) (14)
To a solution of 4 (3.30 g, 8.43 mmol) and imidazole (1.16 g, 17.0 mmol) in
DMF (10 mL) was added TBDMS-Cl (1.27 g, 8.43 mmol) at 0◦C and the mixture
was stirred at 25◦C for 12 h. After addition of water (50 mL), the mixture was
extracted with ethyl acetate (50 mL × 3). The combined organic extracts were
washed with brine (20 mL), dried, and concentrated. The resulting residual oil was
subjected to silica gel (50 g) column chromatography with a 1:1 to 1:5 mixture of

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223
ethyl acetate and hexane as eluent to give the 5ꢀ-O-silylated nucleoside as a viscous
oil (3.75 g, 89%). To a solution of the product (3.75 g, 7.42 mmol) and DBU (1.58 g,
1.55 mL, 10.4 mmol) was added MMTr-Cl (3.21 g, 10.4 mmol) at 0◦C. The resulting
mixture was stirred at 25◦C for 24 h and concentrated. The residue was dissolved
in ethyl acetate (400 mL) and the solution was washed with an NaHCO3-saturated
aqueous solution (50 mL) followed by brine (50 mL), dried, and concentrated to
give a viscous oil. This crude material was subjected to chromatography on silica gel
(100 g) eluted with a 1:5 to 2:1 ethyl acetate–hexane mixture to give the tritylation
product as an amorphous solid (4.10 g, 71%). To a solution of the product (4.10 g,
5.27 mmol) in THF (15 mL) was added a 1.0 M THF solution of TBAF (10.5 mL,
10.5 mmol), and the mixture was stirred at 25◦C for 1.5 h. An aqueous NH4Cl-
saturated solution (50 mL) was added to the reaction mixture and the organic layer
was separated. The aqueous layer was extracted with ethyl acetate (50 mL × 3). The
combined organic extracts were washed with brine (20 mL), dried, and concentrated
to afford a residual liquid, which was subjected to column chromatography on silica
gel (100 g) using a 1:20:20 to 1:5:5 methanol–ethyl acetate–hexane mixture to
give O6-allyl-N2-allyloxycarbonyl-3ꢀ-O-p-dimethoxytrityl-2ꢀ-deoxyguanosine (9)
(3.50 g, 100%): UV λmax 269 (ε 18000), 236 nm (28000); IR 1755, 1720, 1610,
1510, 1245, 1195 cm−1; 1H NMR δ 1.76 (dd, J = 5.4, 13.2 Hz, 1 H), 2.69 (ddd,
J = 5.4, 9.8, 13.2 Hz, 1 H), 3.29 (br dd, J = 9.8, 13.2 Hz, 1 H), 3.68 (br d,
J = 13.2 Hz, 1 H), 3.77 (s, 3 H), 4.00 (s, 1 H), 4.65 (d, J = 5.9 Hz, 2 H), 4.62–
4.66 (m, 1 H), 4.88 (br d, J = 9.8 Hz, 1 H), 5.06 (d, J = 5.9 Hz, 2 H), 5.22 (dd,
J = 1.0, 10.7 Hz, 1 H), 5.26 (dd, J = 1.0, 10.2 Hz, 1 H), 5.32 (dd, J = 1.0,
17.1 Hz, 1 H), 5.44 (dd, J = 1.0, 17.1 Hz, 1 H), 5.92 (tdd, J = 5.9, 10.7, 17.1 Hz,
1 H), 6.11 (tdd, J = 5.9, 10.2, 17.1 Hz, 1 H), 6.24 (dd, J = 5.4, 9.8 Hz, 1 H), 6.84
(d, J = 8.8 Hz, 2 H), 7.23 (t, J = 7.3 Hz, 2 H), 7.30 (dd, J = 7.3, 7.3 Hz, 4 H),
7.36 (d, J = 8.8 Hz, 2 H), 7.41 (s, 1 H), 7.48 (d, J = 7.3 Hz, 4 H), 7.82 (s, 1 H).
Anal. Calcd for C37H37N5O7: C, 66.96; H, 5.62; N, 10.55. Found: C, 66.96, H,
5.69, N, 10.51. To a solution of 9 (1.10 g, 1.66 mmol), diisopropylamine (86.6 mg,
120 µL, 0.86 mmol) and 1H-tetrazole (60.2 mg, 0.859 mmol), in acetonitrile (16
mL) was added (allyloxy)bis(diisopropylamino)phosphine (717 mg, 2.49 mmol).
After stirring at 25◦C for 2 h, the reaction mixture was diluted with ethyl acetate
((100 mL), washed with an aqueous solution saturated with NaHCO3 (20 mL)
followed by brine (20 mL), dried, and concentrated. The resulting residual oil was
purified by chromatography on silica gel (30 g) with a 3:1:trace to 2:1:trace mixture
of ethyl acetate, hexane, and triethylamine, giving 14 as an amorphous solid (1.05
g, 74%): UV λmax 269 (ε 22000), 236 nm (35000); IR 1755, 1605, 1510, 1460,
1
1410, 1240, 1185 cm−1; H NMR δ 0.99 (d, J = 6.8 Hz, 3 H), 1.04 (d, J = 6.8
Hz, 3 H), 1.12 (d, J = 6.8 Hz, 6 H), 2.05–1.99 (m, 1 H), 2.19–2.31 (m, 1 H), 3.29
(ddd, J = 2.9, 6.3, 11.2 Hz, 0.5 H), 3.38 (ddd, J = 2.9, 7.3, 11.2 Hz, 0.5 H), 3.43–
3.57 (m, 2.5 H), 3.63 (ddd, J = 2.4, 5.9, 11.2 Hz, 0.5 H), 3.78 (s, 3 H), 4.17–3.96
(m, 3 H), 4.44–4.51 (m, 1 H), 4.71 (d, J = 5.9 Hz, 2 H), 5.08 (d, J = 5.9 Hz,
2 H), 5.04–5.12 (m, 1 H), 5.19 (d, J = 17.1 Hz, 0.5 H), 5.22–5.27 (m, 0.5 H),

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SAKAKURA AND HAYAKAWA
5.27 (d, J = 10.7 Hz, 2 H), 5.40 (d, J = 17.1 Hz, 1 H), 5.44 (d, J = 17.1 Hz, 1
H), 5.82 (tdd, J = 4.9, 10.2, 17.1 Hz, 0.5 H), 5.91 (tdd, J = 4.9, 10.2, 17.1 Hz,
0.5 H), 5.99 (tdd, J = 5.9, 10.7, 17.1 Hz, 1 H), 6.14 (tdd, J = 5.9, 10.7, 17.1 Hz,
1 H), 6.49 (dd, J = 5.9, 8.8 Hz, 1 H), 6.84 (d, J = 8.8 Hz, 2 H), 7.21–7.25 (m,
2 H), 7.28–7.32 (m, 4 H), 7.36 (d, J = 8.8 Hz, 1 H), 7.37 (d, J = 8.8 Hz, 1 H),
7.48–7.50 (m, 5 H), 8.12 (s, 0.5 H), 8.14 (s, 0.5 H); 31P NMR δ 148.6.14 Anal.
Calcd for C46H55N6O8P: C, 64.93; H, 6.51; N, 9.88. Found: C, 65.00; H, 6.62;
N, 9.78.
3ꢀ-O-p,pꢀ-Dimethoxytritylthymidine 5ꢀ-(Allyl
N,N-diisopropylphosphoramidite) (15)
A solution of 3ꢀ-O-p, pꢀ-dimethoxytritylthymidine (10) (1.21 g, 2.22 mmol),
diisopropylamine (86.6 mg, 120 µL, 0.86 mmol), 1H-tetrazole (72.9 mg, 1.04
mmol), and (allyloxy)bis(diisopropylamino)phosphine (894 mg, 3.10 mmol) in
acetonitrile (10 mL) was stirred at 25◦C for 3 h. The reaction mixture was diluted
with ethyl acetate (200 mL), washed with brine (100 mL × 2), dried, and con-
centrated. The resulting residual material was dissolved in benzene (8 mL) and
the solution was poured into pentane (300 mL) at −78◦C with stirring. The occur-
ring powder was collected by filtration and dissolved in dichloromethane (100 mL).
Concentration of the solution was afforded 15 as an amorphous solid (1.50 g, 92%):
UV λmax 268 (ε 11300), 236 (21300), 210 nm (34000); IR 1695, 1610, 1510, 1465,
1250 cm−1; 1H NMR δ 0.99 (d, J = 6.8 Hz, 3 H), 1.08 (d, J = 6.8 Hz, 3 H), 1.13
(d, J = 6.8 Hz, 3 H), 1.15 (d, J = 6.8 Hz, 3 H), 1.72 (ddd, J = 5.9, 9.3, 13.7 Hz,
1 H), 1.86 (s, 3 H), 1.86–1.92 (m, 1 H), 3.22–3.30 (m, 1 H), 3.45–3.56 (m, 2.5 H),
3.64 (m, 0.5 H), 3.79 (s, 6 H), 4.01–4.14 (m, 2 H), 4.33 (d, J = 5.9 Hz, 0.5 H), 4.35
(d, J = 5.9 Hz, 0.5 H), 5.07 (d, J = 10.3 Hz, 0.5 H), 5.10 (d, J = 10.3 Hz, 0.5 H),
5.17 (dd, J = 2.0, 17.1 Hz, 0.5 H), 5.22 (dd, J = 2.0, 17.1 Hz, 0.5 H), 5.80 (tdd,
J = 5.4, 10.3, 17.1 Hz, 0.5 H), 5.86 (tdd, J = 5.4, 10.3, 17.1 Hz, 0.5 H), 6.41 (dd,
J = 5.4, 9.3 Hz, 0.5 H), 6.46 (dd, J = 5.4, 9.3 Hz, 0.5 H), 6.82 (d, J = 8.8 Hz, 2 H),
6.83 (d, J = 8.8 Hz, 2 H), 7.21–7.35 (m, 5 H), 7.33 (d, J = 8.8 Hz, 2 H), 7.34 (d,
J = 8.8 Hz, 2 H), 7.43–7.46 (m, 2 H), 7.52 (s, 0.5 H), 7.65 (s, 0.5 H), 8.17 (br s,
1 H); 31P NMR δ 148.6, 148.4. Anal. Calcd for C40H50N3O8P: C, 65.65; H, 6.89;
N, 5.74. Found: C, 65.61; H, 6.87; N, 5.69.
Deprotection and Detachment of Oligodeoxyribonucleotides
Immediately before carrying out removal of the allylic protectors, a solu-
tion of the Pd catalyst in THF was prepared by heating a heterogeneous mixture of
Pd2(dba)3•CHCl3 (20.7 mg, 20.0 mmol), triphenylphosphine (52.5 mg, 200 µmol),
diethylamine (87.7 mg, 124 µL, 1.20 mmol), and formic acid (55.3 mg, 45.3 µL,
1.20 mmol) in THF (2.0 mL) until being a clean orange solution. The protected

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225
oligodeoxyribonucleotide anchored on the CPG supports were washed with ace-
tonitrile (0.5 mL × 3) and dried in vacuo. To the solid supports was added a suitable
volume of the Pd-catalyst solution (2.5 equiv of the Pd catalyst/allyl). The mixture
was heated at 50◦C for 1 h with occasional shaking. After the supernatant fluid was
decanted, the resulting CPG supports were washed by the reported procedure (6).
The resulting CPG supports were treated with conc. aqueous ammonia at 25◦C for
1 h to give the oligodeoxyribonucleotide.
ACKNOWLEDGMENT
This work was supported in part by Grants-in-Aid for Scientific Research
(No. 11101001) from the Ministry of Education, Science, Sports and Culture, by
a Grant from “Research for the Future” Program of the Japan Society for the
Promotion of Science (JSPS-RFTF97I00301), and by contributions from the Asahi
Glass Foundation and the Daiko Foundation.
REFERENCES
1. (a) Asseline, U.; Bonfils, E.; Kurfu¨rst, R.; Chassignol, M.; Roig, V.; Thuong, N.T.
Tetrahedron 1992, 48, 1233–1254, and references cited therein. (b) Marasco, Jr.,
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(c) Will, D.W.; Brown, T. Tetrahedron Lett. 1992, 33, 2729–2732. (d) Reed, M.W.;
Adams, A.D.; Nelson, J.S.; Meyer, Jr., R.B. Bioconjugate Chem. 1991, 2, 217–225.
(e) Gmeiner, W.H.; Luo, W.; Lown, J.W. Bioorg. Med. Chem. Lett. 1991, 1, 487–490.
(f) Perrouault, L.; Asseline, U.; Rivalle, C.; Thuong, N.T. Bisagni, E.; Giovannangeli,
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8643–8659. (h) Le Doan, T.; Perrouault, L.; He´le`ne, C. Biochemistry 1986, 25, 6736–
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J. Am. Chem. Soc. 1998, 120, 3289–3294, and references cited therein. (l) Chaloin, L.;
Vidal, P.; Lory, P.; Me´ry, J.; Lautredou, N.; Divita, G.; Heitz, F. Biochem. Biophys. Res.
Commun. 1998, 243, 601–608, and references cited therein. (m) Robles, J.; Maseda,
M.; Beltra´n, M.; Concernau, M.; Pedroso, E.; Grandas, A. Bioconjugate Chem. 1997,
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alambidis, J. Bioconjugate Chem. 1995, 6, 43–53, and references cited therein. (o)
De la Torre, B.G.; Avin˜o´, A.; Tarrason, G.; Piulats, J.; Albericio, F.; Eritja, R. Tetra-
hedron Lett. 1994, 35, 2733–2736. (p) Judy, C.D.; Richardson, C.D.; Brousseau, R.
ibid. 1991, 32, 879–882.
2. Previous oligodeoxyribonucleotide synthesis via the (5ꢀ → 3ꢀ)-chain elongation phos-
phoramidite method: (a) van de Sande, J. H.; Ramsing, N.B.; Germann, M.W.; Elhorst,
W.; Kalisch, B.W.; Kitzing, V.E.; Pon, R.T.; Clegg, R.C.; Jovin, T.M. Science 1988,
241, 551. (b) Horne, D.A.; Dervan, P.B. J. Am. Chem. Soc. 1990, 112, 2435–2437.

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SAKAKURA AND HAYAKAWA
(c) Robles, J.; Pedroso, E.; Grandas, A. Nucleic Acids Res. 1995, 23, 4151–4161. (d)
Wagner, T.; Pfleiderer, W. Nucleosides Nucleotides 1997, 16, 1657–1660.
3. Since the preparation of the nucleoside 5ꢀ-phosphoramidites is more tedious than that
of the nucleoside 3ꢀ-phosphoramidites, the (5ꢀ → 3ꢀ)-elongation strategy is less useful
for the synthesis of simple oligonucleotides than the alternative (3ꢀ → 5ꢀ)-elongation
approach. By contrast, the (5ꢀ → 3ꢀ)-elongation is obviously more efficient than the
(3ꢀ → 5ꢀ)-elongation for the synthesis of solid-anchored 3ꢀ-O-free oligonucleotides
because of its directness.
4. Hayakawa, Y.; Harada, H.; Hirose, M.; Noyori, R.; Wakabayashi, S.; Miyazaki, K.;
Kawase, Y.; Kato, I. Nucleic Acids Symp. Ser. 1991, 25, 63–64.
5. The compounds 1, 2, and 4 are known in literatures. 1 and 2: Heidenhain, S.B.;
Hayakawa, Y. Synlett 1998, 853–854. 4: Heidenhain, S.B.; Hayakawa, Y. Nucleosides
Nucleotides 1999, 18, 1771–1787. The derivative 3 was obtained by the desilylation of
3ꢀ,5ꢀ-di-O-tert-butyldimethylsilyl-N2-allyloxycarbonyl-2ꢀ-deoxyguanosine, reported
in the following ref 6, with (n-C4H9)4NF in THF.
6. Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R. J. Am. Chem. Soc. 1990, 112,
1691–1696.
7. Preparation of the N2-allyloxycarbonyl-O6-allyl-3ꢀ-O-dimethoxytrityl-2ꢀ-deoxy-
guanosine 5ꢀ-phosphoramidite was also attempted in a similar manner, but it could
not be achieved because its synthetic precursor, N2-allyloxycarbonyl-O6-allyl-3ꢀ-O-
dimethoxytrityl-2ꢀ-deoxyguanosine, is labile and rapidly decomposed, even under an
argon atmosphere, to the 3ꢀ-O-free compound.
8. Hayakawa, Y.; Kataoka, M.; Noyori, R. J. Org. Chem. 1996, 61, 7996–7997.
9. A reason why this oligomer was prepared in a lower yield than the oligomer contain-
ing no guanosine moiety is that the guanosine phosphoramidite 13 allows the lower
(97.5%) yield of the coupling than the other nucleoside phosphoramidite, 11, 12, or
14 (average 99.8%). Further, the yield of final coupling with the cytidine phospho-
ramidite 12 was 100.4%, which indicated that a certain side reaction takes place on
the guanosine base.
10. Hayakawa, Y.; Kato, H.; Uchiyama, M.; Noyori, R. J. Org. Chem. 1986, 51, 2400–
2402.
11. A similar side reaction at the O6-position of the guanine base has been observed
in the synthesis of oligodeoxyribonucleotides with the O6-unprotected guanosine
monomer via the (3ꢀ → 5ꢀ)-chain elongation based on the phosphoramidite strategy.
See, Pon, R.T.; Damha, M.J.; Ogilvie, K.K. Nucleic Acids Res. 1985, 13, 6447–6465.
According to this report, the guanosine O6-phosphityl product given by the use of the
nucleoside3ꢀ-phosphoramiditeisdecomposedbythetreatmentwithamixtureofacetic
anhydride and pyridine at 25◦C for 0.5 min for capping the unreacted 5ꢀ-O-hydroxyl
to give back the desired O6-free derivative. In contrast, according to experiments
carried out by us, the by-product afforded in the synthesis using the nucleoside 5ꢀ-
phosphoramidite was not completed cleaved under the capping conditions even after
2 min.
12. The use of the O6-protected guanosine building block to prevent the side reaction has
been reported in the synthesis of oligodeoxyribonucleotides via the (3ꢀ → 5ꢀ)-chain
elongation. Pon, R.T.; Usman, N.; Damha, M.J.; Ogilvie, K.K. Nucleic Acids Res.
1986, 14, 6453–6470.

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SYNTHESIS OF OLIGODEOXYRIBONUCLEOTIDES
227
13. (a) Sakakura, A; Hayakawa, Y.; Harada, H.; Hirose, M.; Noyori, R. Tetrahedron Lett.
1999, 40, 4359–4362. (b) Sakakura, A.; Hayakawa, Y. Tetrhedron in 2000, 56, 4427–
4435.
14. Although the product was obtained as a mixture of two diastereomers, the 31P NMR
signals due to them are not separable to appear as a single peak.
Received July 17, 2000
Accepted October 27, 2000

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