Novel base-labile protecting groups for 5′-hydroxy function in solid-phase oligonucleotide synthesis

Article abstract of DOI:10.1081/NCN-120022725

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

Full text of DOI:10.1081/NCN-120022725

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Novel Base-Labile Protecting Groups for 5′-Hydroxy
Function in Solid-Phase Oligonucleotide Synthesis
Kazuo Kamaike a b , Takashi Namiki a & Etsuko Kawashima a
a School of Pharmacy , Tokyo University of Pharmacy and Life Science , Hachioji, Tokyo,
Japan
b Tokyo University of Pharmacy and Life Science , 1432-1 Horinouchi, Hachioji, 192-0392,
Tokyo, Japan
Published online: 31 Aug 2006.
To cite this article: Kazuo Kamaike , Takashi Namiki & Etsuko Kawashima (2003) Novel Base-Labile Protecting Groups for 5′-
Hydroxy Function in Solid-Phase Oligonucleotide Synthesis, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1011-1015,
DOI: 10.1081/NCN-120022725
To link to this article: http://dx.doi.org/10.1081/NCN-120022725
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1011–1015, 2003
Novel Base-Labile Protecting Groups for 50-Hydroxy
Function in Solid-Phase Oligonucleotide Synthesis
*
Kazuo Kamaike, Takashi Namiki, and Etsuko Kawashima
School of Pharmacy, Tokyo University of Pharmacy and Life Science,
Hachioji, Tokyo, Japan
ABSTRACT
The 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-
(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl (LMMpNBz) groups were devel-
oped as novel base-labile protection for the 50-hydroxy function in solid-phase
oligonucleotide synthesis. A comparative study of the LMMoNBz, LMMpNBz
and 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) protecting groups for oligo-
nucleotide synthesis proved strong feasibility for the LMMoNBz group.
Key Words: Base-labile protecting group; 50-Hydroxy protection; Oligonucleo-
tide synthesis.
Although reliable methods of solid-phase DNA synthesis using the 4,40-
dimethoxytrityl (DMTr) group to protect the 50-hydroxy function of nucleotide units
have been established, the DMTr group is not ideal for acidic deprotection at succes-
sive cycles.[1,2] The levulinyl (Lev) group has been demonstrated by Ohtsuka et al.
to offer excellent protection for the 50-hydroxy function of nucleotide units in RNA
*Correspondence: Kazuo Kamaike, Tokyo University of Pharmacy and Life Science, 1432-1
Horinouchi, Hachioji 192-0392 Tokyo, Japan; Fax: þ81 42676 3073; E-mail: kamaikek@
ps.toyaku.ac.jp.
1011
DOI: 10.1081/NCN-120022725
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1012
Kamaike, Namiki, and Kawashima
Scheme 1. a) HCHO, conc. HCl=1,4-dioxane, 50–55ꢀC, 3 days; b) i. KOH, 85% MeOH-
H2O, 55ꢀC, 30 min, ii. conc. HCl; c) levulinic anhydride, l-methylimidazole, 1,4-dioxane, r.t.,
l h; d) HNO3, H2SO4, in an ice-salt bath, 20 min.
Scheme 2. a) 3 (o- or p-NO2), TPSCl, pyridine, r.t., 2–3 h; b) i. Tris(1,2,3-triazolyl)phosphine,
N-methylmorpholine, CH2Cl2, 0ꢀC, 30 min, ii. 1 M TEAB aq.
Scheme 3. a) i. 0.5 M NH2NH2.H2O, 2 : 3 : 5 CH3COOH-pyridine-CH3CN, 15 min, ii. 0.5 M
imidazole, CH3CN, 5 min; b) 0.05 M 6, 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-50ꢀC, 6 h.

Novel Base-Labile Protecting Groups
1013
Figure 1. Reversed-phase HPLC profiles of crude products of TpT preparations using 6a
(DMTr) on CPGs [7a (o, p, and LMNBz)], respectively. Conditions of reversed-phase HPLCs:
column mBONDASPHERE 5 m C18 (3.9 mm ID ꢁ 150 mm L); elution buffer 7.25-50%
CH3CN=0.1 M TEAA (pH 7); flow rate 1 mL=min.
synthesis on CPG support.[3a,b] The Lev group is easily removed by hydrazinolysis
although the yield of 50-O-levulinylation is unsatisfactory due to low regioselectiv-
ity.[3] We have previously reported developing the LMNBz group, which has higher
regioselectivity, while maintaing easy removability of the Lev group.[4] In this paper,
we report the development of o- and p-nitrobenzoyl derivatives, the LMMoNBz and
LMMpNBz groups, as novel base-labile protection for the 50-hydroxy function in
solid-phase oligonucleotide synthesis.
The protecting reagents [3 (o- and p-NO2)] were easily prepared from m-anisic
acid (1) as shown in Sch. 1. Introduction of the LMMoNBz and LMMpNBz groups
to the 50-position of 20-deoxyribonucleosides (4) was accomplished by treatment with
3 (o- and p-NO2), respectively, in the presence of 2,4,6-triisopropylbenzenesulfonyl
chloride (TPSCl) in pyridine. The corresponding 50-O-benzoyl derivatives 5 (o and p)
were obtained in 55–76% yields (Sch. 2). We performed a comparative study of
Figure 2. Reversed-phase HPLC profiles of crude products of TpTpTpT preparations using
6a (o, p, and LMNBz) on CPG 7a (DMTr), respectively. Conditions of reversed-phase
HPLCs: column mBONDASPHERE 5 m C18 (3.9 mm ID ꢁ 150 mm L); elution buffer 7.25-
50% CH3CN=0.1 M TEAA (pH 7); flow rate 1 mL=min.

1014
Kamaike, Namiki, and Kawashima
Figure 3. Reversed-phase HPLC profiles of crude products of TpTpTpT preparations using
6a (o and p) on CPGs [7a(o and p)], respectively. Conditions of reversed-phase HPLCs:
column mBONDASPHERE 5 m C18 (3.9 mm ID ꢁ 150 mm L); elution buffer 7.25-50%
CH3CN=0.1 M TEAA (pH 7); flow rate 1 mL=min.
the LMMoNBz, LMMpNBz and LMNBz protecting groups for oligonucleotide
synthesis (Sch. 3), as exemplified by synthesis of TpT and TpTpTpT using H-phos-
phonates [6a (DMTr, o, p, and LMNBz)][5] on CPGs [7a (DMTr, o, p, and LMNBz)]
by the manual synthesis, respectively. The excellence of the LMMoNBz group over
the LMMNpBz and LMNBz groups was confirmed as can be seen in Figs. 1–3.
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