A novel linker for solid-phase synthesis cleavable under neutral conditions

Article abstract of DOI:10.1016/j.tetlet.2006.01.135

A novel linker cleavable under neutral conditions has been developed for the solid-phase synthesis of base-labile compounds. The linker is comprised of a 3-azidomethyl-4-hydroxybenzyl alcohol moiety, and the azidomethyl group in the linker is readily conv

Full text of DOI:10.1016/j.tetlet.2006.01.135

Tetrahedron Letters 47 (2006) 2147–2150
A novel linker for solid-phase synthesis cleavable
under neutral conditions
Asako Murata and Takeshi Wada*
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo,
Bioscience Bldg. 702, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
Received 19 December 2005; revised 19 January 2006; accepted 23 January 2006
Available online 13 February 2006
Abstract—A novel linker cleavable under neutral conditions has been developed for the solid-phase synthesis of base-labile com-
pounds. The linker is comprised of a 3-azidomethyl-4-hydroxybenzyl alcohol moiety, and the azidomethyl group in the linker is
readily converted to an aminomethyl group by treatment with a phosphine reagent in the presence of water to result in an intra-
molecular cyclization to release the compounds. Using the linker, a base-labile dinucleoside methyl phosphate was synthesized
on a highly cross-linked polystyrene (HCP) support and cleaved successfully from the resin without decomposition of the product.
Ó 2006 Elsevier Ltd. All rights reserved.
Solid-phase methods have been used in organic synthesis
over the past years, first for peptides,1 then for nucleo-
tides,2 and recently for small molecules in combinatorial
chemistry.3–5 Accompanied with the development of the
solid-phase synthesis of organic compounds, various
linkers have been designed and synthesized for the
attachment of molecules to solid supports.6 These link-
ers can be cleaved by acids,7,8 bases,9 fluoride ion,10 or
photochemical reactions.11 A class of linkers which have
been used for solid-phase chemistry is based on so-called
‘an assisted cleavage mechanism; the cleavage reaction
occurs via the conversion of a masked auxiliary into a
reactive nucleophile, followed by the intramolecular
attack of the nucleophile on the neighboring reactive
site’.12 Although linkers cleavable through the assisted
mechanism are fascinating, there have been few linkers,
which can be cleaved under neutral conditions.
auxiliary.13,14 The benzylic hydroxy group in the linker
would be functionalized with appropriate molecules
bearing an acidic function, such as a carboxyl or phos-
phoryl group. Following the reduction of the azido-
methyl group to the corresponding aminomethyl group
by the Staudinger reaction,15 an intramolecular cycliza-
tion should occur rapidly to release the product by a 1,6-
elimination process (Scheme 1).
This cleavage reaction is expected to proceed under
almost neutral conditions, so that the linker would be
applied to the synthesis of compounds, which is unstable
under acidic or basic conditions. In this letter, we
describe the synthesis of a new linker, and the efficient
cleavage of a base-labile 30-phosphorylated oligonucleo-
tide analog from the linker.
The linker 416 was prepared from 2,4-bis(hydroxy-
methyl) phenol 117 through five steps (Scheme 2). Fol-
lowing the selective protection of the phenolic hydroxy
This circumstance has prompted us to develop a new
type of linker, which can be cleaved under neutral and
reductive conditions, based on this ‘assisted cleavage’
concept. The linker synthesized here is comprised of a
3-azidomethyl-4-hydroxybenzyl alcohol moiety, and
the azide as a latent amino group was used for a masked
and 2-hydroxymethyl groups of
1 with tetraiso-
propyldisiloxane,18 the remaining benzylic hydroxy
group was protected with a monomethoxytrityl (MMTr)
group. After cleavage of the 1,3-cyclic silyl ether by
treatment with tetrabutylammonium fluoride (TBAF),
the 2-hydroxymethyl group was converted into an
azidomethyl group via the chloromethylation to give
the linker 4 in good yield. Then the phenolic hydroxy
group in the linker 4 was acylated by treatment with
glutaric anhydride, and the resulting compound 519
Keywords: Linker; Solid-phase; Azidomethyl group; Staudinger reac-
tion; Neutral conditions.
*
Corresponding author. Tel./fax: +81 4 7136 3612; e-mail: wada@
k.u-tokyo.ac.jp
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.01.135

2148
A. Murata, T. Wada / Tetrahedron Letters 47 (2006) 2147–2150
Scheme 1. A proposed reaction mechanism for the cleavage of attached compounds from the linker.
Scheme 2. Synthesis of the linker.
was successfully attached to a highly cross-linked amino-
methylated polystyrene (HCP) support20 through an
amide bond by using a new condensing reagent,
PyNTP21 (Scheme 3). In contrast, when the acylation
was carried out using succinic anhydride, the desired
product was not obtained because of an intramolecular
cyclization caused by the succinic carboxylate anion to
give the starting material. The loading amount of the
linker molecules to the solid support was estimated to be
24.2 lmol/g by a MMTr cation assay.
resulting dimer was released from the solid support
by treatment with 0.2 M methyldiphenylphosphine
as
reducing reagent in aqueous dioxane13
a
(Scheme 4). In the cleavage reaction, mercaptoethanol
was found to be effective as a scavenger of quinone met-
hide, which was generated in the course of the reaction.
The released products were dissolved in water and
washed with chloroform to remove excess phosphine
and phosphine oxide. After concentration of the
aqueous portion, the crude dimer was analyzed by RP-
HPLC (Fig. 1). Two main peaks corresponding to the
diastereomers of dithymidine methyl phosphate were
observed. The result clearly shows the efficient cleavage
of the linker without decomposition of the product.
After purification by RP-HPLC, the pure dimer 11
was obtained in 46%.23
Next, the synthesis of a base-labile oligonucleotide ana-
log was investigated by using the new linker-attached
solid support. As a model compound, a dinucleoside
methyl phosphotriester derivative was chosen. Dithymi-
dine H-phosphonate was synthesized in 0.5 lmol scale
on the solid support,22 and was transformed to the
corresponding methyl phosphate by treatment with
2% iodine (w/v) in methanol/pyridine (1/9, v/v). The
In conclusion, a novel linker was synthesized for solid-
phase synthesis, which can be cleaved under neutral
Scheme 3. Attachment of the linker to a solid support.

A. Murata, T. Wada / Tetrahedron Letters 47 (2006) 2147–2150
2149
Scheme 4. Synthesis of dithymidine methyl phosphate on the resin using the linker.
variety of organic molecules, which are unstable under
basic and acidic conditions.
Acknowledgements
We are indebted to Professor K. Saigo, for helpful
suggestion.
References and notes
1. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149–
2154.
2. Letsinger, R. L.; Mahadevan, V. J. Am. Chem. Soc. 1965,
87, 3526–3527.
3. Frechet, J. M. J. Tetrahedron 1981, 37, 663–683.
4. Hermkens, P. H. H.; Ottenheijm, H. C. J.; Rees, D.
Tetrahedron 1996, 13, 4527–4554.
5. Schreiber, S. L. Science 2000, 5460, 1964–1969.
6. James, I. W. Tetrahedron 1999, 55, 4855–4946.
7. Wang, S. J. Am. Chem. Soc. 1973, 95, 1328–1333.
8. Rink, H. Tetrahedron Lett. 1987, 28, 3787–3790.
9. Atherton, E.; Logan, C. J.; Sheppard, R. C. J. Chem. Soc.,
Perkin Trans. 1 1981, 538–546.
Figure 1. RP-HPLC profile of the crude dimer of dithymidine methyl
phosphate.
10. Ramage, R.; Barton, C. A.; Bielecki, S.; Thomas, D. W.
Tetrahedron Lett. 1987, 28, 4105–4108.
11. Rich, D. H.; Gurwara, S. K. J. Am. Chem. Soc. 1975, 97,
1575–1579.
12. Greene, T. W.; Wuts, P. G. M. Protective groups in
Organic Synthesis, 3rd ed.; John Wiley and Sons: New
York, 1999.
and reductive conditions. Using the linker, a base-labile
30-phosphorylated dithymidine methyl phosphate was
synthesized and successfully cleaved from the solid sup-
port without decomposition of the product. The linker
may be useful for the synthesis of other oligonucleotide
analogs, peptides, and natural products as well as a wide

2150
A. Murata, T. Wada / Tetrahedron Letters 47 (2006) 2147–2150
13. Wada, T.; Ohkubo, A.; Mochizuki, A.; Sekine, M.
Tetrahedron Lett. 2001, 42, 1069–1072.
14. Osborn, N. J.; Robinson, J. A. Tetrahedron 1993, 49,
2873–2884.
2.15–2.05 (2H, m); 13C NMR (CDCl3): d 178.7, 171.2,
158.6, 147.8, 144.4, 137.6, 135.6, 130.4, 128.6, 128.4, 128.2,
128.0, 127.2, 127.0, 122.6, 113.2, 87.0, 65.0, 55.4, 50.3,
33.2, 33.0, 19.9; IR (KBr) 2932, 2100, 1760, 1709, 1607,
1509, 1446, 1251, 1181, 1128, 1034, 830, 754, 708,
589 cmꢀ1; MALDI/TOF mass spectrometry, using 2,
15. Kusumoto, S.; Sakai, K.; Shiba, T. Bull. Chem. Soc. Jpn.
1986, 59, 1296.
16. Selected data for 4: 1H NMR (CDCl3): d 7.50–7.13
(14H, m), 6.83 (2H, d, J = 8.7 Hz), 6.77 (1H, d,
J = 8.4 Hz), 5.52 (1H, s), 4.38 (2H, s), 4.08 (2H, s), 3.78
(3H, s); 13C NMR (CDCl3): d 158.4, 153.4, 144.5, 135.8,
131.7, 130.4, 129.1, 128.9, 128.4, 127.9, 126.9, 121.5, 115.9,
113.2, 86.8, 65.3, 55.4, 51.2; IR (KBr) 3398, 2103, 1608,
5-dihydroxybenzoic acid as
a
matrix. Calcd for
(M+Na)+ 588.21. Found: 588.06.
20. McCollum, C.; Andrus, A. Tetrahedron Lett. 1991, 32,
4069.
21. Wada, T.; Tsuneyama, T.; Saigo, K. Nucleic Acids Res.
Suppl. 1 2001, 187–188.
22. Wada, T.; Sato, Y.; Honda, F.; Kawahara, S.; Sekine, M.
J. Am. Chem. Soc. 1997, 52, 12710–12721.
1509, 1446, 1250, 1179, 1032, 830, 767, 706 cmꢀ1
;
MALDI/TOF mass spectrometry, using 2,5-dihydroxy-
benzoic acid as a matrix. Calcd for (M+Na)+ 474.18.
Found: 474.06.
23. The analysis and purification of the 30-phosphorylated
dithymidine methyl phosphate 11 were carried out on a
Senshu-Pak C18 column, using 0.1 M TEAA buffer at a
linear gradient from 0% to 20% MeCN in 60 min at a flow
rate 0.5 ml/min. The characterization of 11 was estab-
lished by MALDI/TOF mass spectrometry, using 2,5-
dihydroxybenzoic acid as a matrix. Calcd for [M]ꢀ;
653.13. Found: 652.87.
17. Freeman, J. H. J. Am. Chem. Soc. 1952, 74, 6257–6260.
18. Markiewicz, W. T. J. Chem. Res. (S). 1979, 1, 24–25.
19. Selected data for 5: 1H NMR (CDCl3): d 7.48 (4H, d,
J = 7.2 Hz), 7.39–7.21 (11H, m), 7.09 (1H, d, J = 8.1 Hz),
6.83 (2H, d, J = 9.0 Hz), 4.25 (2H, s), 4.17 (2H, s), 3.78
(3H, s), 2.72 (2H, t, J = 7.2 Hz), 2.54 (1H, d, J = 7.1 Hz),