6-(Levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl groups as novel base-labile groups for 5′-hydroxy protection in solid-phase oligonucleotide synthesis

Article abstract of DOI:10.1081/NCN-120022056

The 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl (LMMpNBz) groups were developed as novel base-labile groups for 5′-hydroxy protection in solid-phase oligonucleotide synthesis. A comparative study of the utility of LMMoNBz, LMMpNBz, and 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) groups is described.

Full text of DOI:10.1081/NCN-120022056

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
6-(Levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl
and 2-(Levulinyloxymethyl)-5-methoxy-4-
nitrobenzoyl Groups as Novel Base-Labile
Groups for 5′-Hydroxy Protection in Solid-Phase
Oligonucleotide Synthesis
Kazuo Kamaike , Takashi Namiki & Etsuko Kawashima
To cite this article: Kazuo Kamaike , Takashi Namiki & Etsuko Kawashima (2003) 6-
(Levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl and 2-(Levulinyloxymethyl)-5-methoxy-4-
nitrobenzoyl Groups as Novel Base-Labile Groups for 5′-Hydroxy Protection in Solid-Phase
Oligonucleotide Synthesis, Nucleosides, Nucleotides and Nucleic Acids, 22:4, 469-487, DOI:
10.1081/NCN-120022056
To link to this article: http://dx.doi.org/10.1081/NCN-120022056
Published online: 07 Feb 2007.
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Date: 06 November 2015, At: 07:47

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 4, pp. 469–487, 2003
6-(Levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl and
2-(Levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl
Groups as Novel Base-Labile Groups for
50-Hydroxy Protection in Solid-Phase
Oligonucleotide Synthesis
*
Kazuo Kamaike, Takashi Namiki, and Etsuko Kawashima
School of Pharmacy, Tokyo University of Pharmacy and Life Science,
Tokyo, Japan
ABSTRACT
The 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-(levu-
linyloxymethyl)-5-methoxy-4-nitrobenzoyl (LMMpNBz) groups were developed
as novel base-labile groups for 50-hydroxy protection in solid-phase oligonucleo-
tide synthesis. A comparative study of the utility of LMMoNBz, LMMpNBz, and
2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) groups is described.
Key Words: Base-labile protecting group; 50-Hydroxy protection; Oligonucleo-
tide synthesis.
*Correspondence: Kazuo Kamaike, School of Pharmacy, Tokyo University of Pharmacy and
Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 Japan; E-mail: kamaikek@
ps.toyaku.ac.jp.
469
DOI: 10.1081/NCN-120022056
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

470
Kamaike, Namiki, and Kawashima
INTRODUCTION
Although reliable methods of solid-phase oligodeoxyribonucleotide (DNA)
synthesis using the 4,40-dimethoxytrityl (DMTr) group to protect the 50-hydroxy
group of nucleotide units have been established, the DMTr group is not ideal for
acidic deprotection at successive cycles, giving rise to depurination, particularly
N6-benzoyl-20-deoxyadenosine residues.[1] When the DMTr group is used to protect
the 50-hydroxy group in oligoribonucleotide (RNA) synthesis in combination with
the acid-labile tetrahydropyran-2-yl (Thp) group to protect the 20-hydroxy group,
it has been shown that repeated acidic treatment leads to significant loss of the
Thp group and subsequent phosphoryl migration.[2] Clearly there is a need for an
alternative 50-hydroxy protecting group for certain applications in oligonucleotide
synthesis. Consequently, various protecting groups for the 50-hydroxy group of
20-deoxyribonucleoside and ribonucleoside 30-phosphoramidites have been reported,[3]
as exemplified by the levulinyl group[4] and the modified 2-hydroxymethylbenzoyl
group,[5] both of which are removable under basic conditions. The Lev group is
easily removed under mild conditions by treatment with 0.5 M hydrazine mono-
hydrate in 1:4 acetic acid - pyridine at room temperature, although the yield of
50-O-levulinylation is unsatisfactory due to low regioselectivity (30–46% for 20-
O-Thf-U, -CBz, and -ABz)[4d]. Based on the chemistry of these protecting groups,
we previously reported the utility of the 2-(levulinyloxymethyl)-5-nitrobenzoyl
(LMNBz) group to compare it to the 2-(levulinyloxymethyl)benzoyl (LMBz) group,
which has higher regioselectivity, yet allows easy removal of the Lev group.[6] The
protecting reagent, 2-(levulinyloxymethyl)-5-nitrobenzoic acid (1), was easily pre-
pared from phthalide. The LMNBz group was introduced at the 50-position of
20-deoxyribonucleosides (2) and 20-O-Thp-ribonucleosides (3) by treatment with 1
(1.1 equiv) in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl)
(2.2 equiv) in pyridine at room temperature, with yields over 63%, regardless
of the nucleoside structure (Sch. 2). Moreover, the LMNBz group was removed
by consecutive treatments with 0.5 M hydrazine monohydrate in 1:4 acetic acid=
pyridine, and 0.5 M imidazole in acetonitrile. Using the LMNBz protecting group,
the synthesis of both DNA- and RNA-type oligomers was efficiently performed
Scheme 1.

Base-Labile Groups for 50-Hydroxy Protection
471
Scheme 2.
on controlled pore glass (CPG) supports using the phosphoramidite6 and H-phos-
phonate methods.[7] Introducing the nitro group to the m-position of the LMBz
group enhances susceptibility of the LMNBz group to the processes of both hydra-
zinolysis and deprotonation with imidazole (Sch. 1).
Furthermore, we developed o- and p-nitrobenzoyl derivatives, 6-(levulinyl-
oxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-(levulinyloxymethyl)-
5-methoxy-4-nitrobenzoyl (LMMpNBz), as novel base-labile groups to protect the
50-hydroxy group in solid-phase oligonucleotide synthesis,a and performed a
comparative study of the LMMoNBz, LMMpNBz, and LMNBz groups. The results
of this study will be described in full herein.
RESULTS AND DISCUSSION
Synthesis of 50-O-LMMoNBz- and -LMMpNBz-20-deoxyribonucleosides [9 (o)
and 9 (p)]. The protecting reagents, 6-(levulinyloxymethyl)-3-methoxy-2-nitro-
benzoic acid (LMMoNBzOH) [4 (o-NO2)] and 2-(levulinyloxymethyl)-5-methoxy-
4-nitrobenzoic acid (LMMpNBzOH) [4 (p-NO2)], were easily prepared from m-anisic
acid (5). Treatment of 5 with formaldehyde in the presence of conc. hydrochloric
acid resulted in the formation of 5-methoxyphthalide (6) exclusively. After alkaline
hydrolysis of 6, the resulting mixture was adjusted at ca. pH 2 with conc. hydrochlo-
ric acid. 2-(Hydroxymethyl)-5-methoxybenzoic acid (7) was obtained as a white
crystalline solid in 54% yield from 5. Treatment of 7 with levulinic anhydride in
the presence of 1-methylimidazole led to the formation of 2-(levulinyloxymethyl)-
5-methoxybenzoic acid (8) in 64% yield. Compound 8 was then treated with a mix-
ture of conc. nitric acid and sulfuric acid to afford LMMoNBzOH [4 (o-NO2) (34%
yield) and LMMpNBzOH [4 (p-NO2)] (21% yield) (Sch. 3).
Introducing LMMoNBz and LMMpNBz to the 50-position of 20-deoxyribo-
nucleosides (2) was accomplished by treatment with 4 (o- and p-NO2) (1.1 equiv),
respectively, in the presence of TPSCl (2.2 equiv) in pyridine at room temperature.
aA portion of the present work was presented on the occasion of the XVth International
Round Table ‘‘Nucleosides, Nucleotides and Nucleic Acids’’, Leuven, Belgium, September
10–14, 2002.[8]

472
Kamaike, Namiki, and Kawashima
Scheme 3. Conditions: (a) HCHO, conc. HCl=1,4-dioxane, 50–55ꢀC, 3 days, (b) 1) KOH,
85% MeOH-H2O, 55ꢀC, 30 min, 2) conc. HCl, (c) levulinic anhydride, 1-methylimidazole,
1,4-dioxane, r.t., 1 h, (d) HNO3, H2SO4 in an ice-salt bath, 20 min.
The corresponding 50-O-benzoyl derivatives 9 (o and p) were obtained in 55–80%
yields (Sch. 4).
Synthesis of Oligodeoxyribonucleosides
We used manual synthesis[9] to perform a basic study of solid-phase oligonucleotide
synthesis using the H-phosphonate method,[10] as shown in Sch. 5. 50-O-LMNBz-,
Scheme 4. Conditions: (a) 4 (o- or p-NO2), TPSCl, pyridine, r.t., 2–3 h, (b) 1) tris-(1,2,3-tri-
azolyl)phosphine, N-methylmorpholine, CH2Cl2, 0ꢀC, 30 min, 2) 1 M TEAB aq.

Base-Labile Groups for 50-Hydroxy Protection
473
Scheme 5. Conditions (a) 1) 0.5 M NH2NH2H2O, 2:3:5 CH3CO2H-pyridine-CH3CN, 15 min,
2) 0.5 M imidazole, CH3CN, 5 min, (b) 0.05 M 10, 0.25 M pivaloyl chloride, 1:1 pyridine-
CH3CN, r.t., 10 min, (c) 3% I2, H2O-19% pyridine-76% THF, r.t., 10 min, (d) conc. NH4OH,
r.t., 3 h and 55ꢀC. 6 h.
-LMMoNBz-, and -LMMNpBz-20-deoxyribonucleoside 30-H-phosphonates [10 (o,
p, and LMNBz)] were prepared by treating the respective 50-O-LMNBz-,
-LMMoNBz-, and -LMMpNBz-nucleosides [9 (o, p, and LMNBz)] with
tris(1,2,4-triazolyl)phosphine (3–5 equiv) and subsequent hydrolysis with aqueous
triethylammonium bicarbonate (Sch. 4).[10] Prior to oligonucleotide synthesis,
CPG support was functionalized with the 50-O-LMNBz-, -LMMoNBz-, and
-LMMNpBz-20-deoxyribonucleoside derivatives [9 (o, p, and LMNBz)] in the usual
manner.[11]
At first, we performed a comparative study of the LMMoNBz, LMMpNBz, and
LMNBz groups to synthesize the oligonucleotides, as exemplified by the synthesis
of dimer (TpT) using 50-O-DMTr-thymidine 30-H-phosphonate [10a (DMTr)][8]
on CPGs [11a (o, p, and LMNBz)]. Following deprotection of the LMMoNBz,
LMMpNBz, and LMNBz groups of the thymidine linked to CPG support [11a (o, p,
and LMNBz)] [Sch. 5, condition (a)], the H-phosphonate monomer [10a (DMTr)]
was coupled to the supported thymidine (12) in the presence of pivaloyl chloride as
the condensing agent. After assembling the H-phosphonate dimer, the DMTr group
was removed, and the H-phosphonate dimer was oxidized with I2=H2O to yield the
desired phosphodiester-linked dimer. The TpT was then cleaved from the support,
and was analyzed with reversed-phase HPLC [Fig. 1, HPLC peak area ratio of T
(tR ¼ 3.2 min) and TpT (tR ¼ 13.1 min): T=TpT ¼ 5=95 (I), 11=89 (II), and 22=78
(III)]. Superiority of the LMMoNBz protection was clearly evident in the
reversed-phase HPLC chromatograms. The superior nature of LMMoNBz protec-

474
Kamaike, Namiki, and Kawashima
Figure 1. Reversed-phase HPLC profiles of crude products of TpT preparations using the
50-O-DMTr-thymidine 30-H-phosphonate 10a (DMTr) derivative on CPGs [11a (o, p, and
LMNz)], respectively.
tion over LMNBz protection prompted us to conduct further comparative studies, as
exemplified by our synthesis of tetramer (TpTpTpT), using 50-O-LMMoNBz,
-LMMpNBz-, and -LMNBz-thymidine 30-H-phosphonates [10a (o, p, and LMNBz)]
on the commercially available 50-O-DMTr-thymidine which was linked to CPG
support [11a (DMTr)]. After deprotection of the DMTr group of the 50-O-DMTr-
thymidine linked to CPG support [11a (DMTr)], the H-phosphonate monomers
[10a (o, p, and LMNBz)] were respectively coupled to the supported thymidine
(12a). Following deprotection of the LMMoNBz, LMMpNBz, and LMNBz groups
of the growing oligomer linked to CPG support, the H-phosphonate monomers [10a
(o, p, and LMNBz)] were coupled to the growing oligomer (Sch. 5). After assembling
H-phosphonate tetramer, the LMMoNBz, LMMpNBz and LMNBz groups were
removed, and the H-phosphonate tetramer was oxidized with I2=H2O. The
TpTpTpT was then cleaved from the support, and was analyzed with reversed-phase
Figure 2. Reversed-phase HPLC profiles of crude products of TpTpTpT preparations
using thymidine 30-H-phosphonate derivatives [10a (o, p, and LMNz)] on CPG 11a (DMTr),
respectively.

Base-Labile Groups for 50-Hydroxy Protection
475
Figure 3. Reversed-phase HPLC profiles of crude products of oligodeoxyribonucleotide
preparations using 30-H-phosphonate monomers [10 (o and p)] on CPGs [11 (o and p)], respec-
tively. Conditions of reversed-phase HPLC: column mBONDASPHERE 5m C18 (3.9 mm
ID ꢁ 150 mm L); elution buffer 7.25–50% acetonitrile=0.1 M TEAA (pH 7); flow rate
1 mL=min.
HPLC [Fig. 2, HPLC peak area ratio of TpT (tR ¼ 13.1 min), TpTpT (tR ¼ 18.4 min),
and TpTpTpT (tR ¼ 20.1 min): TpT=TpTpT=TpTpTpT ¼ 8=11=81 (I), 12=13=75 (II),
and 10=14==76 (III)]. Oligomer synthesis using the LMMNoBz group formed an
oligomer with slightly better efficiency than oligomer synthesis reactions using
the LMMpNBz and LMNBz groups.
Furthermore, using 50-O-LMMoNBz- and -LMMpNBz-20-deoxyribonucleo-
side 30-H-phosphonates [10a–d (o and p)], oligodeoxyribonucleotides [TpTpT,
TpTpTpT, d(CpCpT), d(ApApT), d(ApApApT), d(GpGpT), d(GpCpApT), and
d(ApApTpTpCpG)] were synthesized on CPGs [11a (o and p)], respectively, as
shown in Sch. 5. The HPLC tracings of each of the resulting mixtures are shown
in Fig. 3. The enzymatic degradation of these oligodeoxyribonucleotides gave satis-
factory data to prove their structures (Fig. 4).[3d,4c,4d]
Introducing the nitro group to the o-position of the benzoyl group is likely to
enhance susceptibility of the 2-hydroxymethylbenzoyl derivative, which was
produced by hydrazinolysis of the 2-(levulinyloxymethyl)benzoyl derivative, to the
process of intramolecular cyclization by treatment with imidazole (Sch. 1). Thus,
we confirmed the superiority of the LMMoNBz group over the LMMNpBz and
LMNBz, as shown in Fig. 1–3. The LMMNoBz protecting goup is basically useful
for 50-hydroxy protection in solid-phase oligonucleotide synthesis, and is expected
to be free from the incompatibility due to the use of the DMTr protecting group.

476
Kamaike, Namiki, and Kawashima
Figure 4. Reversed-phase HPLC analysis of products obtained by digestion of the completely
unmasked hexamer with snake venom phosphodiesterase (SVP) and alkaline phosphatase
(AP), and of standard samples. 20-Deoxyinosine was produced from 20-deoxyadenosine due
to contamination with adenosine deaminase. Conditions of reversed-phase HPLC: column
mBONDASPHERE 5m C18 (3.9 mm ID ꢁ 150 mm L); elution buffer 3% acetonitrile=0.06 M
TEAA (pH 7); flow rate 1 mL=min.
EXPERIMENTAL
Column chromatography was performed on silica gel (Wakogel C-300,
purchased from Wako Pure Chemicals, Co. Ltd.) by the use of methanol=chloroform,
and triethylamine=methanol=chloroform. TLC was conducted on Merck silica gel
F254 by developing with 1:9 methanol=chloroform. High performance liquid
chromatography (HPLC) was conducted on mBONDASPHRE 5m C18 (3.9 mm ꢁ
150 mm L) for purification of oligodeoxyribonucleotiedes and analyses of the diges-
tion of oligodeoxyribonucleotides with snake venom phosphodiesterase and alkaline
phoaphatase. Melting points were determined by a Yanaco Micro-melting-point
apparatus, and are uncorrected. 1H-NMR spectra were recorded on a Varian
GEMINI-300 apparatus with CDCl3 or DMSO-d6 as an internal standard. 31P-NMR
spectra were recorded on a Brucker DRX 400 apparatus with 85% H3PO4 as an
external standard. Mass spectra were recorded on a VG AutoSpecE apparatus.
Elemental analyses were achieved with a Perkin-Elmer 240-002 apparatus.
2-(Hydroxymethyl)-5-methoxybenzoic acid (7). To a solution of m-anisic acid
(5) (3.80 g, 25 mmol) in 1,4-dioxane (16 mL) was added a 37% formaldehyde solution
(12 mL) and conc. hydrochloric acid (20 mL). The mixture was stirred for 3 days at
50ꢀC. The resulting mixture was combined with chloroform (200 mL) and was
washed with water (100 mL ꢁ 2). The organic layer was evaporated to give 5-methoxy-
phthalide (6) (3.28 g). Without further purification, 6 was treated with potassium
hydroxide in 85:15 methanol=water (20 mL) for 30 min at 50ꢀC. After evaporation,

Base-Labile Groups for 50-Hydroxy Protection
477
the residue was dissolved in water (100 mL) and was washed with diethyl ether
(10 mL ꢁ 3). The aqueous solution was acidified (ca. pH 2) with conc. hydrochloric
acid. The resulting precipitates were gathered by filtration, and were washed with
chilled water (20 mL) to give a white powder of 7 (2.49 g, 54% yield from m-anisic
acid): m.p. 120–121ꢀC; 1H-NMR (CDCl3) d 3.18 (s, 3H, OCH3), 4.67 (s, 2H,
PhCH2), 7.01 (dd, 1H, J ¼ 2.8 Hz and 8.4 Hz, H-4), 7.30 (d, 1H, J ¼ 2.8 Hz,
H-6), and 7.58 (d, 1H, J ¼ 8.4 Hz, H-3). Anal. calcd. for C9H10O4; C, 59.34; H,
5.53. Found: C, 59.18; H, 5.63.
2-(Levulinyloxymethyl)-5-methoxybenzoic acid (8). To a solution of levulinic
acid (3.1 mL, 30 mmol) in 1,4-dioxane (60 mL) was added N,N0-dicyclohexylcarbo-
diimide (DCC) (3.1 g, 15 mmol). The mixture was stirred for 3 h at room tempera-
ture. After removing the precipitate by filtration, 7 (1.82 g, 10 mmol) was added to
the filtrate, and the mixture was stirred for 1 h at room temperature in the presence
of 1-methylimidazole (1.2 mL, 15 mmol). After evaporation, the residue was dis-
solved in a saturated aqueous sodium carbonate solution (50 mL), and the solution
was washed with diethyl ether (10 mL ꢁ 3). The aqueous layer was acidified (ca.
pH 2) with conc. hydrochloric acid. The resultant precipitate was gathered by filtra-
tion, and was washed with chilled water (20 mL) to give a white powder of 8 (1.69 g,
1
64%): m.p. 134.5–136ꢀC; H-NMR (CDCl3) d 2.19 (s, 3H, COCH3), 2.65 (t, 2H,
J ¼ 6.5 Hz, OCOCH2CH2COCH3), 2.77 (t, 2H, J ¼ 6.5 Hz, OCOCH2CH2COCH3),
3.86 (s, 3H, OCH3), 5.47 (s, 2H, PhCH2), 7.11 (dd, 1H, J ¼ 2.8 Hz and 8.6 Hz,
Ph-H), 7.42 (d, 1H, J ¼ 8.6 Hz, Ph-H), and 7.60 (d, 1H, J ¼ 2.8 Hz, Ph-H). Anal.
calcd. for C9H10O4; C, 59.99; H, 5.75. Found: C, 60.10; H, 5.78.
6-(Levulinyloxymethyl)-3-methoxy-2-nitrobenzoic acid (LMMoNBzOH) [4 (o-
NO2)] and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoic acid (LMMpNBzOH) [4
( p-NO2)]. A solution of 8 (0.84 g, 3 mmol) in 98% sulfuric acid (3.2 mL)=61% nitric
acid (4.2 mL) was stirred for 20 min with cooling in an ice-salt bath. The resulting
mixture was added to a 5% aqueous sodium hydrogen carbonate solution (50 mL)
and was washed with diethyl ether (50 mL ꢁ 3). The aqueous layer was acidi-
fied (pH 4) with 1 M hydrochloric acid and was extracted with chloroform
(50 mL ꢁ 3). The organic layer was dried over anhydrous magnesium sulfate and
then was evaporated to give crude LMMpNBzOH [4 (p-NO2)], which was purified
by crystallization from diethyl ether as a white powder (0.201 g, 21%). The aqueous
layer was acidified (pH 3) with 1 M hydrochloric acid and was extracted with chloro-
form (50 mL ꢁ 3). The organic layer was dried over anhydrous magnesium sulfate
and then was evaporated to give crude LMMoNBzOH [4 (o-NO2)], which was
purified by crystallization from diethyl ether as a white powder (0.336 g, 34%).
1
LMMoNBzOH [4 (o-NO2)]: m.p. 136–137ꢀC; H-NMR (CDCl3) d 2.19 (s, 3H,
COCH3), 2.57 (t, 2H, J ¼ 6.0 Hz, OCOCH2CH2COCH3), 2.76 (t, 2H, J ¼ 6.0 Hz,
OCOCH2CH2COCH3), 3.93 (s, 3H, OCH3), 5.26 (s, 2H, PhCH2), 7.13 (d, 1H, J ¼
8.7 Hz, Ph-H), and 7.51 (d, 1H, J ¼ 8.7 Hz, Ph-H). Anal. calcd. for C14H15NO8;
C, 51.70; H, 4.65; N, 4.31. Found: C, 51.68; H, 4.74; N, 4.31.
LMMpNBzOH [4 (p-NO2)]: m.p. 174ꢀC; 1H-NMR (CDCl3) d 2.19 (s, 3H,
COCH3), 2.64 (t, 2H, J ¼ 6.3 Hz, OCOCH2CH2COCH3), 2.78 (t, 2H, J ¼ 6.3 Hz,
OCOCH2CH2COCH3), 3.99 (s, 3H, OCH3), 5.43 (s, 2H, PhCH2), 7.72 (s, 1H,

478
Kamaike, Namiki, and Kawashima
Ph-H), and 7.91 (s, 1H, Ph-H). Anal. calcd. for C14H15NO8; C, 51.70; H, 4.65;
N, 4.31. Found: C, 51.75; H, 4.75; N, 4.11.
50-O-[6-(Levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl]thymidine [9a (o)]. After
azeotropic evaporation from pyridine (2 mL ꢁ 3), thymidine (2a) (0.242 g, 1 mmol)
and LMMoNBzOH [4 (o-NO2)] (0.357 g, 1.1 mmol) were dissolved in pyridine
(4 mL). 2,4,6-Triisopropylbenzenesulfonyl chloride (TPSCl) (0.666 g, 2.2 mmol)
was added to the solution, which was then stirred at room temperature for 2 h.
The resulting mixture was quenched with water (1 mL) with stirring, and was
extracted with ethyl acetate (50 mL ꢁ 2). The extracts were combined and washed
with a saturated aqueous sodium hydrogen carbonate solution (50 mL ꢁ 2) and then
with water (50 mL). After drying over anhydrous magnesium sulfate, the organic
layer was evaporated to dryness, and the residue was subjected to chromatographic
separation on a column of silica gel by the use of a chloroform=methanol system to
give 9a (o) (0.399 g, 73%) as a colorless foam: 1H-NMR (CDCl3) d 1.72 (s, 3H, CH3
of thymidine), 2.18 (s, 3H, COCH3), 2.15–2.22 (m, 1H, H-20), 2.36–2.47 (m, 1H, H-
200), 2.54 (t, 2H, J ¼ 6.2 Hz, OCOCH2CH2COCH3), 2.75 (t, 2H, J ¼ 6.2 Hz,
OCOCH2CH2COCH3), 3.09 (d, 1H, J ¼ 4.1 Hz, 30-OH), 3.95 (s, 3H, OCH3), 4.13–
4.15 (m, 1H, H-40), 4.43–4.50 (m, 2H, H-30, 50), 4.56–4.61 (m, 1H, H-500), 5.19,
0
0
0
0
00
5.26 (2d, 2H, J ¼ 12.8 Hz, PhCH2), 6.27 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.6 Hz, H-1 ), 7.19
(d, 1H, J ¼ 8.5 Hz, Ph-H), 7.21 (s, 1H, H-6), 7.59 (d, 1H, J ¼ 8.5 Hz, Ph-H), and
8.34 (br s, 1H, N3-H). Anal. calcd. for C24H27N3O12ꢂH2O; C, 50.79; H, 5.15; N,
7.40. Found: C, 50.60; H, 5.12; N, 7.21.
N4-Anisoyl-50-O-[6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl]-20-deoxy-
cytidine [9b (o)]. Compound 9b (o) was obtained in 59% yield (0.393 g) as a color-
less foam by treating N4-anisoyl-20-deoxycytidine (2b) (0.361 g, 1 mmol) with
LMMoNBzOH [4 (o-NO2)] (0.357 g, 1.1 mmol) in the presence of TPSCl
(0.666 g, 2.2 mmol) in pyridine (4 mL) for 3 h and a subsequent work-up as
described above. 1H-NMR (CDCl3) d 2.17 (s, 3H, COCH3), 2.16–2.25 (m, 1H,
H-20), 2.54 (t, 2H, J ¼ 6.2 Hz, OCOCH2CH2COCH3), 2.65–2.67 (m, 1H, H-200),
2.75 (t, 2H, J ¼ 6.2 Hz, OCOCH2CH2COCH3), 3.88 (s, 3H, OCH3 of An group),
3.95 (s, 3H, OCH3 of LMMoNBz group), 4.2–4.28 (m, 1H, H-40), 4.39–4.43 (m,
1H, H-30), 4.53–4.67 (m,0 2H, H-50, and 500), 5.21 (s, 2H, PhCH2), 6.25 (t, 1H,
0
0
0
00
J1 ,2 ¼ J1 ,2 ¼ 6.0 Hz, H-1 ), 6.98 (d, 2H, J ¼ 8.8 Hz, Ph-H ꢁ 2 of An group), 7.21
(d, 1H, J ¼ 8.8 Hz, Ph-H of LMMoNBz group), 7.22–7.43 (m, 1H, H-5), 7.58
(d, 1H, J ¼ 8.8 Hz, Ph-H of LMMoNBz group), 7.85 (d, 2H, J ¼ 8.8 Hz, Ph-H ꢁ 2
of An group), 8.03 (d, 1H, J ¼ 6.6 Hz, H-6), and 8.80 (br s, 1H, N4-H). Anal. calcd.
for C31H32N4O13ꢂ0.5 H2O; C, 54.95; H, 4.91; N, 8.27. Found: C, 54.66; H, 4.92;
N, 8.16.
N6-Benzoyl-50-O-[6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl]-20-deoxy-
adenosine [9c (o)]. Compound 9c (o) was obtained in 80% yield (01.010 g) as a color-
less foam by treating N6-benzoyl-20-deoxyadenosine (2c) (0.675 g, 1.90 mmol) with
LMMoNBzOH [4 (o-NO2)] (0.680 g, 2.09 mmol) in the presence of TPSCl (1.266 g,
4.18 mmol) in pyridine (4 mL) for 2 h and a subsequent work-up as descri-
1
bed above. H-NMR (CDCl3) d 2.17 (s, 3H, COCH3), 2.53–2.63 (m, 3H, H-20

Base-Labile Groups for 50-Hydroxy Protection
479
and OCOCH2CH2COCH3), 2.70–2.75 (m, 2H, OCOCH2CH2COCH3), 3.00–3.05
(m, 1H, H-200), 3.92 (s, 3H, OCH3), 4.28–4.31 (m, 1H, H-40), 4.54–4.58 (m, 2H, H-50
and 500), 4.75–4.78 (m, 1H, H-30), 5.19, 5.26 (2d, 2H, J ¼ 12.7 Hz, PhCH2), 6.46
0
0
0
0
00
(t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.6 Hz, H-1 ), 7.16 (d, 1H, J ¼ 8.9 Hz, Ph-H of LMMoNBz
group), 7.50–7.60 (m, 4H, Ph-H of LMMoNBz group and Ph-H ꢁ 3 of Bz group),
8.03 (d, 2H, J ¼ 8.5 Hz, Ph-H ꢁ 2 of Bz group), 8.16 (s, 1H, H-8), 8.72 (s, 1H, H-2),
and 9.10 (br s, 1H, N6-H). Anal. calcd. for C31H30N6O11ꢂ1.5 H2O; C, 53.99;
H, 4.82; N, 12.19. Found: C, 54.05; H, 4.66; N, 12.22.
N2-Isobutyryl-50-O-[6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl]-20-deoxy-
guanosine [9d (o)]. Compound 9d (o) was obtained in 59% yield (0.382 g) as a color-
less foam by treating N2-isobutyryl-20-deoxyguanosine (2d) (0.337 g, 1 mmol) with
LMMoNBzOH [4 (o-NO2)] (0.357 g, 1.1 mmol) in the presence of TPSCl (0.666 g,
2.2 mmol) in pyridine (4 mL) for 2 h and a subsequent work-up as described above.
1H-NMR (CDCl3) d 1.25(d, 6H, J ¼ 6.8 Hz, CH(CH3)2), 2.16 (s, 3H, COCH3),
2.48–2.53 (m, 3H, H-20 and OCOCH2CH2COCH3), 2.70–2.85 (m, 4H,
OCOCH2CH2COCH3, CH(CH3)2, and H-200), 3.92 (s, 3H, OCH3), 4.26–4.29
(m, 1H, H-40), 4.48–4.63 (m, 2H, H-50 and 500), 4.81–4.84 (m, 1H, H-30), 5.05,
0
0
0
0
00
5.07 (2d, 2H, J ¼ 12.9 Hz, PhCH2), 6.21 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.5 Hz, H-1 ), 7.16
(d, 1H, J ¼ 8.8 Hz, Ph-H of LMMoNBz group), 7.53 (d, 1H, J ¼ 8.8 Hz, Ph-H of
LMMoNBz group), 7.81 (s, 1H, H-8), 9.72 (br s, 1H, N1-H), and 12.20 (br s, 1H,
N2-H). Anal. calcd. for C28H32N6O12ꢂH2O; C, 50.76; H, 5.17; N, 12.68. Found:
C, 51.01; H, 5.17; N, 12.60.
50-O-[2-(Levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl]thymidine [9a ( p)]. Com-
pound 9a (p) was obtained in 61% yield (0.308 g) as a colorless foam by treating
thymidine (2a) (0.242 g, 1 mmol) with LMMpNBzOH [4 (p-NO2)] (0.357 g, 1.1 mmol)
in the presence of TPSCl (0.666 g, 2.2 mmol) in pyridine (4 mL) for 2 h and a sub-
sequent work-up as described above. 1H-NMR (CDCl3) d 1.83 (s, 3H, CH3 of
thymidine), 2.21 (s, 3H, COCH3), 2.26–2.35 (m, 1H, H-20), 2.40–2.49 (m, 1H,
H-200), 2.62 (t, 2H, J ¼ 6.0 Hz, OCOCH2CH2COCH3), 2.78 (t, 2H, J ¼ 6.0 Hz,
OCOCH2CH2COCH3), 3.09 (d, 1H, J ¼ 4.1 Hz, 30-OH), 4.01 (s, 3H, OCH3),
4.19–4.23 (m, 1H, H-40), 4.50–4.69 (m, 3H, H-30, 50, and 500), 5.33, 5.41 (2d, 2H,
0
0
0
0
00
J ¼ 13.5 Hz, PhCH2), 6.27 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.7 Hz, H-1 ), 7.13 (s, 1H, H-6),
7.66 (s, 1H, Ph-H), 7.95 (s,1H, Ph-H), and 8.67 (s, 1H, N3-H). Anal. calcd. for
C24H27N3O12ꢂH2O; C, 50.79; H, 5.15; N, 7.40. Found: C, 50.52; H, 5.09; N, 7.25.
N4-Anisoyl-50-O-[2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl]-20-deoxycy-
tidine [9b ( p)]. Compound 9b (p) was obtained in 55% yield (0.367 g) as a color-
less foam by treating N4-anisoyl-20-deoxycytidine (2b) (0.361 g, 1 mmol) with
LMMpNBzOH [4 (p-NO2)] (0.357 g, 1.1 mmol) in the presence of TPSCl (0.666 g,
2.2 mmol) in pyridine (4 mL) for 2 h and a subsequent work-up as described above.
1H-NMR (CDCl3) d 2.20 (s, 3H, COCH3), 2.24–2.33 (m, 1H, H-20), 2.62 (t, 2H,
J ¼ 6.3 Hz, OCOCH2CH2COCH3), 2.74–2.81 (m, 3H, H-200 and OCOCH2CH2-
COCH3), 3.88 (s, 3H, OCH3 of An group), 4.01 (s, 3H, OCH3 of LMMpNBz group),
4.36–4.40 (m, 1H, H-40), 4.48–4.51 (m, 1H, H-30), 4.67–4.69 (m, 2H, H-50 and 500),
0
0
0
00
5.35, 5.41 (2d, 2H, J ¼ 13.5 Hz, PhCH2), 6.25 (t, 1H, J1 ,2 ¼ J1 ,2
¼

480
Kamaike, Namiki, and Kawashima
6.1 Hz, H-10), 6.98 (d, 2H, J ¼ 8.8 Hz, Ph-H ꢁ 2 of An group), 7.40–7.49 (m, 1H,
H-5), 7.64 (s, 1H, Ph-H of LMMpNBz group), 7.85 (d, 2H, J ¼ 8.8 Hz, Ph-H ꢁ 2
of An group), 7.94 (s,1H, Ph-H of LMMpNBz group), 7.95–7.99 (m, 1H, H-6),
and 8.70 (br s, 1H, N4-H). Anal. calcd. for C31H32N4O13ꢂH2O; C, 54.23; H, 4.99;
N, 8.16. Found: C, 54.41; H, 4.95; N, 8.08.
N6-Benzoyl-50-O-[2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl]-20-deoxyade-
nosine [9c ( p)]. Compound 9c ( p) was obtained in 65% yield (0.431 g) as a color-
less foam by treating N6-benzoyl-20-deoxyadenosine (2c) (0.355 g, 1 mmol)
with LMMpNBzOH [4 (p-NO2)] (0.357 g, 1.1 mmol) in the presence of TPSCl
(0.666 g, 2.2 mmol) in pyridine (4 mL) for 2 h and a subsequent work-up as described
1
above. H-NMR (CDCl3) d 2.22 (s, 3H, COCH3), 2.59–2.84 (m, 5H, H-20,
OCOCH2CH2COCH3, and OCOCH2CH2COCH3), 3.14–3.16 (m, 1H, H-200),
3.96 (s, 3H, OCH3), 4.35–4.37 (m, 1H, H-40), 4.68–4.71 (m, 2H, H-50 and 500),
4.90–4.93 (m, 1H, H-30), 5.23, 5.44 (2d, 2H, J ¼ 13.3 Hz, PhCH2), 6.45 (t, 1H, J1 ,2
¼
0
0
0
0
00
J1 ,2 ¼ 6.2 Hz, H-1 ), 7.49 (s, 1H, Ph-H of LMMpNBz group), 7.51–7.62 (m, 3H,
Ph-H ꢁ 3 of Bz group), 7.90 (s, 1H, Ph-H of LMMpNBz group), 8.04 (d, 2H,
J ¼ 7.4 Hz, Ph-H ꢁ 2 of Bz group), 8.14 (s, 1H, H-8), 8.70 (s, 1H,H-2), and 8.96
(br s, 1H, N6-H). Anal. calcd. for C31H30N6O11ꢂ1.5 H2O; C, 53.99; H, 4.82; N, 12.24.
Found: C, 54.23; H, 4.79; N, 12.07.
N2-Isobutyryl-50-O-[2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl]-20-deoxy-
guanosine [9d ( p)]. Compound 9d ( p) was obtained in 76% yield (0.491 g) as a color-
less foam by treating N2-isobutyryl-20-deoxyguanosine (2d) (0.337 g, 1 mmol) with
LMMpNBzOH [4 (p-NO2)] (0.357 g, 1.1 mmol) in the presence of TPSCl (0.666 g,
2.2 mmol) in pyridine (4 mL) for 2 h and a subsequent work-up as described above.
m.p. 106–108ꢀC (from methanol); 1H-NMR (CDCl3) d 1.17, 1.21 (2d, 6H, J ¼ 6.4 Hz,
CH(CH3)2), 2.18 (s, 3H, COCH3), 2.53–2.63 (m, 3H, H-20 and OCOCH2CH2COCH3),
2.76–2.85 (m, 4H, OCOCH2CH2COCH3, CH(CH3)2, and H-200), 3.91 (s, 3H, OCH3),
4.28–4.33 (m, 1H, H-40), 4.52–4.58 (m, 1H, H-50), 4.72–4.77 (m, 1H, H-5’’),
5.01–5.04 (m, 1H, H-30), 5.29 (s, 2H, PhCH2), 6.19 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 5.4 Hz,
H-10), 7.59 (s, 1H , Ph-H of LMMpNBz group), 7.84 (s, 1H, Ph-H of LMMpNBz
group), 7.88 (s, 1H, H-8), 10.18 (br s, 1H, N1-H), and 12.26 (br s, 1H, N2-H). Anal.
calcd. for C28H32N6O12ꢂ1.5 H2O; C, 50.07; H, 5.25; N, 12.51. Found: C, 50.33;
H, 5.41; N, 12.59.
0
0
0
00
50-O-[6-(Levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl]thymidine 30-H-phospho-
nate triethylammonium salt [10a (o)]. To a solution of PCl3 (0.22 mL, 2.5 mmol)
and N-methylmorpholine (2.75 mL, 25 mmol) in dichloromethane (25 mL) was
added 1,2,4-triazole (0.573 g, 8.3 mmol) at room temperature to generate tris(1,2,4-
triazolyl)phosphine. After 30 min the reaction mixture was cooled to 0ꢀC, and 9a
(o) f0.274 g, 0.5 mmol, dried by azeotropic evaporation from pyridine (2 mL ꢁ 3)
and acetonitrile (2 mL)g in dichloromethane (7 mL) were added dropwise over
10 min. After 20 min the reaction mixture was quenched by treatment with 1 M
aqueous triethylammonium bicarbonate (TEAB, pH 8.5) (20 mL) with stirring,
and was extracted with chloroform (20 mL ꢁ 3). The organic layer was dried over

Base-Labile Groups for 50-Hydroxy Protection
481
anhydrous magnesium sulfate and then was evaporated. The residue was subjected
to chromatographic separation on a column of silica gel by the use of a chloro-
form=methanol=triethylamine system to give 10a (o) (0.313 g, 90% yield) as a color-
less foam: 1H-NMR (CDCl3) d 1.34 (t, 9H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3), 1.73 (s, 3H, CH3
of thymidine), 2.16 (s, 3H, COCH3), 2.17–2.22 (m, 1H, H-20), 2.50–2.56 (m, 3H,
H-200 and OCOCH2CH2COCH3), 2.72 (t, 2H, J ¼ 6.4 Hz, OCOCH2CH2COCH3),
3.05 (q, 6H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3), 3.93 (s, 3H, OCH3), 4.34–4.37 (m, 1H, H-40),
4.56–4.64 (m, 2H, H-50 and 5’’), 4.76–4.79 (m, 1H, H-30), 5.18, 5.24 (2d, 2H,
0
0
0
0
00
J ¼ 13.1 Hz, PhCH2), 6.29 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.8 Hz, H-1 ), 6.93 (d, 1H,
JP,H ¼ 621.3 Hz, P-H), 7.17 (d, 1H, J ¼ 8.8 Hz, Ph-H), 7.25 (s, 1H, H-6), 7.58
(d, 1H, J ¼ 8.8 Hz, Ph-H), and 8.50 (br s, 1H, N3-H). 31P-NMR (CDCl3) d 4.64.
N4-Anisoyl-50-O-[6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl]-20-deoxycyti-
dine 30-H-phosphonate triethylammonium salt [10b (o)]. Compound 10b (o) was
obtained in 89% yield (0.371 g) as a colorless foam by treating 9b (o) (0.344 g,
0.5 mmol) with tris(1,2,4-triazolyl)phosphine (2.5 mmol) and a subsequent work-up
as described above. 1H-NMR (CDCl3) d 1.35 (t, 9H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3),
2.16 (s, 3H, COCH3), 2.16–2.20 (m, 1H, H-20), 2.56 (t, 2H, J ¼ 6.1 Hz,
OCOCH2CH2COCH3), 2.74 (t, 2H, J ¼ 6.1 Hz, OCOCH2CH2COCH3), 279–2.87
(m, 1H, H-200), 3.09 (q, 6H, J ¼ 7.3 Hz, CH2CH3 x 3), 3.88 (s, 3H, OCH3 of An
group), 3.95 (s, 3H, OCH3 of LMMoNBz group), 4.45–4.46 (m, 1H, H-40), 4.55–
4.71 (m, 2H, H-50 and 500), 4.75–4.80 (m, 1H, H-30), 5.18, 5.27 (2d, 2H, J ¼ 12.9 Hz,
0
0
0
0
00
Hz, PhCH2), 6.25 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.2 Hz, H-1 ), 6.92 (d, 1H, JP,H ¼ 622.2 Hz,
P-H), 6.98 (d, 2H, J ¼ 8.8 Hz, Ph-H ꢁ 2 of An group), 7.18 (d, 1H, J ¼ 8.8 Hz,
Ph-H of LMMoNBz group), 7.31–7.39 (m, 1H, H-5), 7.58 (d, 1H, J ¼ 8.8 Hz, Ph-H
of LMMoNBz group), 7.85 (d, 2H, J ¼ 8.8 Hz, Ph-H ꢁ 2 of An group), 8.03–8.9
(m, 1H, H-6), and 8.60 (br s, 1H, N4-H). 31P-NMR (CDCl3) d 4.63.
N6-Benzoyl-50-O-[6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl]-20-deoxyade-
nosine 30-H-phosphonate triethylammonium salt [10c (o)]. Compound 10c (o) was
obtained in 88% yield (0.365 g) as a colorless foam by treating 9c (o) (0.331 g,
0.5 mmol) with tris(1,2,4-triazolyl)phosphine (2.5 mmol) and a subsequent work-up
as described above. 1H-NMR (CDCl3) d 1.36 (t, 9H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3),
2.12 (s, 3H, COCH3), 2.48 (t, 2H, J ¼ 6.5 Hz, OCOCH2CH2COCH3), 2.67 (t, 2H,
J ¼ 6.5 Hz, OCOCH2CH2COCH3), 2.78–2.79 (m, 1H, H-20), 2.94–2.97 (m, 1H,
H-200), 3.10 (q, 6H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3), 3.91 (s, 3H, OCH3), 4.50–4.54 (m,
1H, H-40), 4.57-4.71 (m, 2H, H-50 and 500), 5.02–5.05 (m, 1H, H-30), 5.10, 5.15 (2d,
0
0
0
0
00
2H, J ¼ 13.0 Hz, PhCH2), 6.51 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.6 Hz, H-1 ), 6.99 (d,
1H, JP,H ¼ 621.9 Hz, P-H), 7.15 (d, 1H, J ¼ 8.9 Hz, Ph-H of LMMoNBz group),
7.50–7.60 (m, 4H, Ph-H of LMMoNBz group and Ph-H ꢁ 3 of Bz group), 8.02
(d, 2H, J ¼ 7.5 Hz, Ph-H ꢁ 2 of Bz group), 8.20 (s, 1H, H-8), 8.73 (s, 1H, H-2),
and 9.03 (br s, 1H, N6-H). 31P-NMR (CDCl3) d 4.38.
N2-Isobutyryl-50-O-[6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl]-20-deoxy-
guanosine 30-H-phosphonate triethylammonium salt [10d (o)]. Compound 10d (o)
was obtained in 54% yield (0.218 g) as a colorless foam by treating 9d (o) (0.322 g,
0.5 mmol) with tris(1,2,4-triazolyl)phosphine (1.5 mmol) and a subsequent work-up

482
Kamaike, Namiki, and Kawashima
1
as described above. H-NMR (CDCl3) d 1.20–1.25 (m, 6H, CH(CH3)2, 1.34 (t, 9H,
J ¼ 7.3 Hz, CH2CH3 ꢁ 3), 2.16 (s, 3H, COCH3), 2.50–2.53 (m, 3H, H-20 and
OCOCH2CH2COCH3), 2.69–2.74 (m, 4H, OCOCH2CH2COCH3, CH(CH3)2, and
H-200), 3.08 (q, 6H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3), 3.94 (s, 3H, OCH3), 4.25–4.27 (m,
1H, H-40), 4.40–4.43 (m, 1H, H-50), 4.61–4.64(m, 1H, H-5’’), 4.70 (s, 2H, PhCH2),
6.04–6.10 (m, 2H, H-10 and 30), 7.00 (d, 1H, JP,H ¼ 626.4 Hz, P-H), 7.16 (d, 1H,
J ¼ 8.8 Hz, Ph-H of LMMoNBz group), 7.51 (d, 1H, J ¼ 8.8 Hz, Ph-H of LMMoNBz
group), 7.64 (s, 1H, H-8), 10.99 (br s, 1H, N1-H), and 11.65 (br s, 1H, N2-H).
31P-NMR (CDCl3) d 4.23.
50-O-[2-(Levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl]thymidine 30-H-phospho-
nate triethylammonium salt [10a ( p)]. Compound 10a ( p) was obtained in 91% yield
(0.319 g) as a colorless foam by treating 9a (p) (0.274 g, 0.5 mmol) with tris(1,2,4-tri-
azolyl)phosphine (2.5 mmol) and a subsequent work-up as described above. 1H-NMR
(CDCl3) d 1.36 (t, 9H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3), 1.84 (s, 3H, CH3 of thymidine), 2.20
(s, 3H, COCH3), 2.28–2.32 (m, 1H, H-20), 2.58–2.62 (m, 1H, H-200), 2.66 (t, 2H,
J ¼ 6.4 Hz, OCOCH2CH2COCH3), 2.80 (t, 2H, J ¼ 6.4 Hz, OCOCH2CH2COCH3),
3.09 (q, 6H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3), 4.03 (s, 3H, OCH3), 4.43–4.46 (m, 1H, H-40),
4.63–4.68 (m, 2H, H-50 and 500), 4.89–4.92 (m, 1H, H-30), 5.42, 5.47 (2d, 2H,
0
0
0
0
00
J ¼ 14.4 Hz, PhCH2), 6.30 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.7 Hz, H-1 ), 6.96 (d, 1H, JP,H
¼
620.6 Hz, P-H), 7.18 (s, 1H, H-6), 7.73 (s, 1H, Ph-H), 7.96 (s,1H, Ph-H), and
8.63 (br s, 1H, N3-H). 31P-NMR (CDCl3) d 4.07.
N 4-Anisoyl-50-O-[2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl]-20-deoxy-
cytidine 30-H-phosphonate triethylammonium salt [10b ( p)]. Compound 10b (p) was
obtained in 86% yield (0.360 g) as a colorless foam by treating 9b (p) (0.344 g,
0.5 mmol) with tris(1,2,4-triazolyl)phosphine (2.5 mmol) and a subsequent work-
1
up as described above. H-NMR (CDCl3) d 1.34 (t, 9H, J ¼ 7.3 Hz, CH2CH3 ꢁ
3), 2.17 (s, 3H, COCH3), 2.24–2.27 (m, 1H, H-20), 2.64 (t, 2H, J ¼ 6.4 Hz,
OCOCH2CH2COCH3), 2.78 (t, 2H, J ¼ 6.4 Hz, OCOCH2CH2COCH3), 3.02–3.08
(m, 1H, H-200), 3.05 (q, 6H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3), 3.88 (s, 3H, OCH3 of
An group), 4.00 (s, 3H, OCH3 of LMMpNBz group), 4.60–4.65 (m, 1H, H-40),
4.67–4.69 (m, 2H, H-50 and 500), 4.69–4.70 (m, 1H, H-30), 5.39, 5.46 (2d, 2H, J ¼
0
0
0
0
00
14.2 Hz, PhCH2), 6.24 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.2 Hz, H-1 ), 6.93 (d, 1H, JP,H
¼
620.4 Hz, P-H), 6.98 (d, 2H, J ¼ 8.8 Hz, Ph-H ꢁ 2 of An group), 7.40–7.49 (m, 1H,
H-5), 7.67 (s, 1H, Ph-H of LMMpNBz group), 7.85 (d, 2H, J ¼ 8.8 Hz, Ph-H ꢁ 2
of An group), 7.93 (s,1H, Ph-H of LMMpNBz group), and 7.94–7.98 (m, 1H, H-6).
31P-NMR (CDCl3) d 4.05.
N6-Benzoyl-50-O-[2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl]-20-deoxyade-
nosine 30-H-phosphonate triethylammonium salt [10c ( p)]. Compound 10c (p) was
obtained in 86% yield (0.357 g) as a colorless foam by treating 9c (p) (0.331 g,
0.5 mmol) with tris(1,2,4-triazolyl)phosphine (2.5 mmol) and a subsequent work-up
as described above. 1H-NMR (CDCl3) d 1.31 (t, 9H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3),
2.16 (s, 3H, COCH3), 2.62 (t, 2H, J ¼ 6.3 Hz, OCOCH2CH2COCH3), 2.75 (t, 2H,
J ¼ 6.3 Hz, OCOCH2CH2COCH3), 2.78–2.83 (m, 1H, H-20), 3.00 (q, 6H, J ¼ 7.3 Hz,
Hz, CH2CH3 ꢁ 3), 3.10–3.13 (m, 1H, H-200), 3.97 (s, 3H, OCH3), 4.58–4.61 (m, 1H,

Base-Labile Groups for 50-Hydroxy Protection
483
H-40), 4.64–4.78 (m, 2H, H-50 and 500)0, 5.17–5.19 (m, 1H, H-30), 5.38 (s, 2H, PhCH2),
0
0
0
00
6.50 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.6 Hz, H-1 ), 6.99 (d, 1H, JP,H ¼ 618.7 Hz, P-H), 7.27–7.65
(m, 3H, Ph-H ꢁ 3 of Bz group), 7.64 (s, 1H, Ph-H of LMMpNBz group), 7.91 (s, 1H,
Ph-H of LMMpNBz group), 8.04 (d, 2H, J ¼ 7.2 Hz, Ph-H ꢁ 2 of Bz group), 8.14
(s, 1H, H-8), 8.73 (s, 1H,H-2), and 8.98 (br s, 1H, N6-H). 31P-NMR (CDCl3) d 4.36.
N2-Isobutyryl-50-O-[2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl]-20-deoxy-
guanosine 30-H-phosphonate triethylammonium salt [10d ( p)]. Compound 10d ( p)
was obtained in 48% yield (0.196 g) as a colorless foam by treating 9d (p) (0.322 g,
0.5 mmol) with tris(1,2,4-triazolyl)phosphine (1.5 mmol) and a subsequent work-up
1
as described above. H-NMR (CDCl3) d 1.24 (d, 6H, J ¼ 6.8 Hz, CH(CH3)2), 1.36
(t, 9H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3), 2.19 (s, 3H, COCH3), 2.59–2.62 (m, 3H, H-20
and OCOCH2CH2COCH3), 2.72–2.79 (m, 3H, OCOCH2CH2COCH3 and H-200),
3.05–3.11 (m, 7H, CH(CH3)2 and CH2CH3 ꢁ 3), 3.97 (s, 3H, OCH3), 4.42–4.44
(m, 1H, H-40), 4.48–4.53 (m, 1H, H-50), 4.82–4.86 (m, 1H,H-500), 5.34, 5.40 (2d,
2H, J ¼ 14.4 Hz, PhCH2), 5.55–5.60 (m, 1H, H-30), 6.14 (dd, 1H, J1 ,2 ¼ 7.4 Hz and
0
0
0
0
00
J1 ,2 ¼ 4.2 Hz, H-1 ), 6.97 (d, 1H, JP,H ¼ 623.2 Hz, P-H), 7.60 (s, 1H , Ph-H of
LMMpNBz group), 7.69 (s, 1H, Ph-H of LMMpNBz group), 7.90 (s, 1H, H-8),
10.39 (br s, 1H, N1-H), and 12.40 (br s, 1H, N2-H). 31P-NMR (CDCl3) d 3.60.
50-O-[2-(Levulinyloxymethyl)-5-nitrobenzoyl]thymidine 30-H-phosphonate triethyl-
ammonium salt [10a (LMNBz)]. Compound 10a (LMNBz) was obtained in 86%
yield (0.30 g) as a colorless foam by treating 9a (LMNBz) (0.26 g, 0.5 mmol) with
tris(1,2,4-triazolyl)phosphine (2.5 mmol) and a subsequent work-up as described
above. 1H-NMR (CDCl3) d 1.28 (t, 9H, J ¼ 7.3 Hz, CH2CH3 ꢁ 3), 1.71 (s, 3H,
CH3 of thymidine), 2.16 (s, 3H, COCH3), 2.22–2.29 (m, 1H, H-20), 2.53–2.56 (m,
1H, H-200), 2.65 (t, 2H, J ¼ 6.2 Hz, OCOCH2CH2COCH3), 2.78 (t, 2H, J ¼ 6.2 Hz,
OCOCH2CH2COCH3), 3.01 (q, 6H, J ¼ 7.2 Hz, CH2CH3 ꢁ 3), 4.37–4.40 (m, 1H,
H-40), 4.57–4.66 (m, 2H, H-50 and 500), 4.89–4.93 (m, 1H, H-30),0 5.55, 5.60 (2d,
0
0
0
00
2H, J ¼ 16.3 Hz, PhCH2), 6.28 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.7 Hz, H-1 ), 6.91 (d, 1H,
JP,H ¼ 621 Hz, P-H), 7.16 (s, 1H, H-6), 7.76 (d, 1H, J ¼ 8.6 Hz, Ph-H), 8.34
(dd, 1H, J ¼ 8.8 Hz and J ¼ 2.3 Hz, Ph-H), and 8.74 (d, 1H, J ¼ 2.3 Hz, Ph-H).
31P-NMR (CDCl3) d 4.09.
Functionalization of the CPG Support with a Nucleoside
30-(Carboxy)propionate Derivative
1) A solution of 9a (o) (0.164 g, 0.3 mmol) in pyridine (2 mL) was treated with
succinic anhydride (0.060 g, 0.6 mmol) and 4-dimethylpyridine (DMAP) (0.018 g,
0.45 mmol) at room temperature for 1 day with stirring. The resulting mixture was
quenched by treatment with chilled water (1 mL) at room temperature for 30 min
with stirring and evaporated to dryness. The residue was subjected to chromato-
graphic separation on a column of silica gel by the use of a chloroform=methanol
system to give 30-O-[3-(carboxy)propionyl]-50-O-[6-(levulinyloxymethyl)-3-methoxy-
2-nitrobenzoyl]thymidine (0.192 g, 99% yield) as a colorless foam: 1H-NMR (CDCl3)
d 1.71 (s, 3H, CH3 of thymidine), 2.17 (s, 3H, COCH3), 2.12–2.22 (m, 1H, H-20),

484
Kamaike, Namiki, and Kawashima
2.44–2.50 (m, 1H, H-200), 2.54 (t, 2H, J ¼ 6.5 Hz, OCOCH2CH2COCH3), 2.66–2.76
(m, 6H, OCOCH2CH2COCH3, OCOCH2CH2COOH, and OCOCH2CH2COOH),
3.95 (s, 3H, OCH3), 4.28–4.29 (m, 1H, H-40), 4.50–4.63 (m, 2H, H-50 and 500), 5.22
(s, 2H, PhCH2), 5.18–5.27 (m, 1H, H-30), 6.25 (dd, 1H, J1 ,2 ¼ 8.7 Hz, J1 ,2 ¼ 5.4 Hz,
Hz, H-10), 7.19 (d, 1H, J ¼ 8.9 Hz, Ph-H), 7.30 (s, 1H, H-6), 7.59 (d, 1H, J ¼ 8.9 Hz,
Ph-H), and 9.86 (s, 1H, N3-H).
0
0
0
00
After azeotropic evaporation from pyridine (1 mL ꢁ 3), 30-O-[3-(carboxy)pro-
pionyl]-50-O-[6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl]thymidine (0.162 g,
0.25 mmol) and 1-hydroxybenzotriazole (0.051 g, 0.375 mmol) were dissolved in
dried 1,4-dioxane (3 mL), DCC (0.077 g, 0.375 mmol) was added to the solution,
and the mixture was stirred for 3 h at room temperature. The resulting precipitates
were removed by filtration, and the filtrate was poured onto the CPG support (Long
Chain Amino-Alkyl Controlled-Pore Glass, 500 A, 80–120 mesh; purchased from
Funakoshi) (0.5 g), which was in advance subjected to azeotropic evaporation from
pyridine (2 mL ꢁ 3). The mixture was shaken for 12 h at room temperature in the
presence of 1-methyimidazole (0.1 mL, 1.25 mmol). The CPG loaded with 6-(levu-
linyloxymethyl)-3-methoxy-2-nitrobenzoyl]thymidine derivative was filtered off and
washed with acetonitrile (5 mL) and methanol (5 mL). Unreacted amino groups of
the CPG support were capped by treatment with 1:1:8 acetic anhydride=2,6-luti-
dine=THF and 1 : 9 1-methylimidzole=THF for 30 min at room temperature, followed
by successive washing of CPG [11a (o)] with acetonitrile (5 mL) and methanol
(5 mL). After deprotection of the LMMoNBz group by consecutive traetmant with
0.5 M hydazine monohydrate in 4:1 pyridine=acetic acid at room temperature for
15 min and with 0.5 M imidazole in acetonitrile at room temperature for 5 min
and removing of thymidine (2a) from the CPG support by treatment with conc.
aqueous ammonia at room temperature for 3 h using 9.8 mg of CPG [11a (o)], thy-
midine content in CPG [11a (o)] was determined to be 55.79 mmol = g by UV spec-
troscopy fthymidine, lmax 267 nm in H2O (e ¼ 9650)g.
2) 30-O-[3-(Carboxy)propionyl]-50-O-[2-(levulinyloxymethyl)-5-methoxy-4-nitro-
benzoyl]thymidine was obtained in 92% yield (0.180 g) as a colorless foam by treating
9a (p) (0.164 g, 0.3 mmol) with succinic anhydride (0.060 g, 0.6 mmol) in the presence
of DMAP (0.018 g, 0.45 mmol) in pyridine (2 mL) for 1 day and a subsequent work-
up as described above.1H-NMR (CDCl3) d 1.85 (s, 3H, CH3 of thymidine), 2.20 (s,
3H, COCH3), 2.27–2.37 (m, 1H, H-20), 2.51–2.58 (m, 1H, H-200), 2.63–2.70 (m, 6H,
OCOCH2CH2COCH3, OCOCH2CH2COOH, and OCOCH2CH2COOH), 2.80 (t,
2H, J ¼ 6.3 Hz, OCOCH2CH2COCH3), 4.01 (s, 3H, OCH3), 4.36–4.41 (m, 1H,
H-40), 4.52–4.66 (m, 2H, H-50, and 500), 5.35–5.37 (m, 1H, H-30), 5.41 (s, 2H, PhCH2),
0
0
0
0
00
6.23 (dd, 1H, J1 ,2 ¼ 8.3 Hz, J1 ,2 ¼ 7.8 Hz, H-1 ), 7.19 (s, 1H, H-6), 7.71 (s, 1H,
Ph-H), 7.93 (s,1H, Ph-H), and 9.82 (s, 1H, N3-H).
30-O-[3-(Carboxy)propionyl]-50-O-[2-(levulinyloxymethyl)-5-methoxy-4-nitro-
benzoyl]thymidine (0.162 g, 0.25 mmol) was introduced to the CPG support as
described for the synthesis of CPG [11a (p)]. After deprotection of the LMMpNBz
group and removing of thymidine (2a) from the CPG support using 10.3 mg of
CPG [11a (p)], thymidine content of CPG [11a (p)] was determined to be 55.79 mmol=g
by UV spectroscopy.

Base-Labile Groups for 50-Hydroxy Protection
485
3) 30-O-[3-(Carboxy)propionyl]-N2-isobutyryl-50-O-[6-(levulinyloxymethyl)-
3-methoxy-2-nitrobenzoyl]-20-deoxyguanosine was obtained in 86% yield (0.259 g)
as a colorless foam by treating 9d (o) (0.193 g, 0.3 mmol) with succinic anhydride
(0.060 g, 0.6 mmol) in the presence of DMAP (0.018 g, 0.15 mmol) in pyridine (3 mL)
for 1 day and a subsequent work-up as described above.1H-NMR (CDCl3) d 1.25
(d, 6H, J ¼ 6.9 Hz, CH(CH3)2), 2.16 (s, 3H, COCH3), 2.48–2.91 (m, 7H, H-20, 200,
OCOCH2CH2COCH3, OCOCH2CH2COOH, and CH(CH3)2), 3.89 (s, 3H, OCH3),
4.37–4.40 (m, 1H, H-40), 4.56–4.60 (m, 2H, H-50 and 500), 5.09 (s, 2H, PhCH2), 5.40–
0
5.44 (m, 1H, H-30), 6.13 (t, 1H, J1 ,2 ¼ J1 ,2 ¼ 6.8 Hz, H-1 ), 7.17 (d, 1H, J ¼ 8.9 Hz,
Ph-H of LMMoNBz group), 7.54 (d, 1H, J ¼ 8.8 Hz, Ph-H of LMMoNBz group),
7.84 (s, 1H, H-8), 9.84 (br s, 1H, N1-H), and 12.17 (br s, 1H, N2-H).
0
0
0
00
30-O-[3-(Carboxy)propionyl]-N2-isobutyryl-50-O-[6-(levulinyloxymethyl)-3-methoxy-
2-nitrobenzoyl]-20-deoxyguanosine was introduced to the CPG support as des-
cribed for the synthesis of CPG [11d (o)]. After deprotection of the LMMoNBz group,
removing of N2-isobutyryl-20-deoxyguanosine (2d) and deprotection of the iBu
group by treatment conc. aqueous ammonia at 55ꢀC for 6 h using 10.3 mg of
CPG [11d (o)], 20-deoxyguanosine content of CPG [11d (o)] was determined to be
21.11 mmol=g by UV spectroscopy f20-deoxyguanosine, lmax 254 nm in H2O (e ¼ 13000)g.
Synthesis of Oligothymidylic Acids
After chain elongation with a simple syringe-based reaction system[10] using a
column of CPG (11a) (Sch. 5), the resulting oligothymidylic acid was detached from
the CPG support by treatment with conc. aqueous ammonia (4 mL) for 3 h at room
temperature. After evaporation of the aqueous ammonia, the residual deprotected
oligothymidylic acid was analyzed by reverse-phase HPLC (see Fig. 1 and 2).
Synthesis of Oligodeoxyribonucleotides
After chain elongation with a simple syringe-based reaction system[10] using a
column of CPG (11) (Sch. 5), the resulting oligomer was detached from the CPG
support by treatment with conc. aqueous ammonia (4 mL) for 3 h at room tempera-
ture. The resulting solution was heated in a sealed vial at 55ꢀC for 6 h. After evapora-
tion of the aqueous ammonia, the residue was dissolved in water (10 mL) and was
washed with ethyl acetate (5mL ꢁ 3). The aqueous layer was evaporated, and the
residual deprotected oligomer was purified by reverse-phase HPLC (see Fig. 3).
The main peak was collected [yield of TpTpT, 16.4 A260 units from CPG 11a (o)
(1 mmol) and 8.7 A260 units from CPG 11a (p) (1 mmol); yield of TpTpTpT, 14.0
A260 units from CPG 11a (o) (1 mmol) and 12.7 A260 units from CPG 11a (p) (1 mmol);
yield of d(CpCpT), 9.8 A260 units from CPG 11a (o) (1 mmol) and 9.8 A260
units from CPG 11a ( p) (1 mmol); yield of d(ApApT), 15.4 A260 units from
CPG 11a (o) (1 mmol) and 7.4 A260 units from CPG 11a ( p) (1 mmol); yield of
d(ApApApT), 15.4 A260 units from CPG 11a (o) (1 mmol); yield of d(GpGpT), 9.9
A
260 units from CPG 11a (o) (1 mmol) and 8.9 A260 units from CPG 11a ( p) (1 mmol);

486
Kamaike, Namiki, and Kawashima
yield of d(GpCpApT), 7.6 A260 units from CPG 11a (o) (1 mmol); yield of
d(ApApTpTpCpG), 11.0 A260 units from CPG 11d (o) (1 mmol)].
Enzymatic Digestion of Oligodeoxyribonucleotides
The fully deprotected oligonucleotides (1.0 A260 unit) were each dissolved in
30 mM Tris=HCl (pH 8.0) (70 mL) containing 6 mM MgCl2, and snake venom
phosphodiesterase (2.8 mg=0.7 mL, SIGMA) (3 mL) and alkaline phosphatase (1500 u=
83 mL, Roche) (1 mL) were added. The resulting mixture was incubated at 37ꢀC for
24 h and was heated at 90ꢀC for 2 min.[3d,4c,4d] Digestion products were analyzed by
reversed-phase HPLC with 2% acetonitrile=0.04 M TEAA (pH 7.0) as an eluent
(see Fig. 4). 20-Deoxyinosine was produced from 20-deoxyadenosine due to contam-
ination with adenosine deaminase.[4d]
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Received December 11, 2002
Accepted February 19, 2003