A novel protecting strategy for internucleosidic phosphate and phosphorothioate groups

Article abstract of DOI:10.1081/NCN-100002480

The utility of 2-(N-isopropyl-N-anisoylamino)ethyl group for protection of intemucleosidic phosphate linkages in oligonucleotide synthesis was studied. The group demonstrated high coupling yields, favorable deprotection kinetics and a high hydrolytic stability of phosphoramidite building blocks. The mechanism of deprotection was established using a model phosphate triester.

Full text of DOI:10.1081/NCN-100002480

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A NOVEL PROTECTING STRATEGY FOR
INTERNUCLEOSIDIC PHOSPHATE AND
PHOSPHOROTHIOATE GROUPS
Andrei P. Guzaev a & Muthiah Manoharan a
a Department of Medicinal Chemistry , Isis Pharmaceuticals , 2292 Faraday Ave.,
Carlsbad, California, 92008, U.S.A.
Published online: 07 Feb 2007.
To cite this article: Andrei P. Guzaev & Muthiah Manoharan (2001) A NOVEL PROTECTING STRATEGY FOR
INTERNUCLEOSIDIC PHOSPHATE AND PHOSPHOROTHIOATE GROUPS, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7,
1011-1014, DOI: 10.1081/NCN-100002480
To link to this article: http://dx.doi.org/10.1081/NCN-100002480
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1011–1014 (2001)
A NOVEL PROTECTING STRATEGY FOR
INTERNUCLEOSIDIC PHOSPHATE AND
PHOSPHOROTHIOATE GROUPS
Andrei P. Guzaev∗ and Muthiah Manoharan
Department of Medicinal Chemistry, Isis Pharmaceuticals,
2292 Faraday Ave., Carlsbad, California 92008
ABSTRACT
The utility of 2–(N–isopropyl–N–anisoylamino)ethyl group for protection of
internucleosidic phosphate linkages in oligonucleotide synthesis was studied.
The group demonstrated high coupling yields, favorable deprotection kinetics
and a high hydrolytic stability of phosphoramidite building blocks. The mech-
anism of deprotection was established using a model phosphate triester.
Current oligonucleotide synthesis employs a phosphoramidite method and
is carried out automatically by stepwise coupling of nucleoside building blocks
to a solid support. This allows an efficient preparation of natural and modified
DNA fragments on a routine basis. Phosphoramidite building blocks are most
often protected with a 2–cyanoethyl group at the phosphite moiety (1). Treatment
of the oligonucleotide with ammonia effects β–elimination in the 2–cyanoethyl
group releasing internucleosidic phosphodiester moieties and acrylonitrile as a side
product. Recently, acrylonitrile has been demonstrated to alkylate nucleic bases
under conditions of deprotection (2). Increasing interest in manufacturing oligonu-
cleotides as drugs motivated our search for alternate protecting groups, whose
removal is governed by mechanisms different from β–elimination.
We reported recently the use of 2–[(1–naphthyl)carbamoyloxy]ethyl group for
protection of internucleosidic phosphates (3). Here we report the use of a novel 2–
(N–isopropyl–N–anisoylamino)ethyl protection in the oligonucleotide synthesis.
∗ Corresponding author.
1011
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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GUZAEV AND MANOHARAN
Figure 1. Synthesis of phosphoramidite building blocks 8–12.
For the preparation of novel phosphoramidite building blocks,1 was converted
to a bisamidite 2, which was isolated as a crystalline compound on silica gel.
Nucleosides 3–7 were treated with 2 in the presence of 1H–tetrazole to give
8–12 (Fig. 1). These were isolated by column chromatography in 75–89% yield
and characterized. The phosphoramidites 8–12 were stable compounds whose shelf
life exceeded 9 months. Moreover, as measured by 31P NMR, the half-life of disap-
pearance of 8 in 95% aqueous MeCN was 73 h, which indicated its high hydrolytic
stability.
Next, the removal of the proposed protecting group (PG) was studied. Model
compounds, 13–15, were synthesized either on a solid support (13 and 14) or
in solution (15, Fig. 2). Half-lives of deprotection of 13 and 14 under various
conditions were measured by a gel phase 31P NMR. In phosphite triester 13, the
PG was indefinitely stable in MeCN. In contrast, it was rapidly removed from
phosphate triester 14 (τ1/ = 50 min in MeCN). In a similar manner, a model T15
2
phosphorothioate was assembled on a polystyrene solid support (20 µmol) using
phosphoramidite 8. As revealed by 31P NMR in gel phase, the PG was removed to
ca. 90% extent when the synthesis of the oligonucleotide was complete.
Apparently, theextentofdeprotectionoftheoligonucleotideattachedtoasolid
support should depend on the duration of the synthesis and hence on the synthetic
Figure 2a. Structures of model compounds 13–15.

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NOVEL PROTECTING STRATEGY
1013
Figure 2b. The fate of 15 under neutral and basic aqueous conditions.
scale employed. On a small scale, the synthesized oligonucleotide remains largely
protected. In this case, the remaining protecting groups are removed at the final
deprotection step. In order to illustrate the deprotection mechanism under neutral
andbasicaqueousconditions, thefateof15wasnextstudied(Fig. 2). Theproducts, 1
and19–23, formedfrom15donotreactwithnucleicbases. Toconfirmthis, mixtures
of 15 (10 equiv) and each of the 5ꢁ–DMTr protected nucleosides (dA, dC, dG, and
T; 1 equiv) were treated with ammonium hydroxide. As demonstrated by HPLC,
each of the nucleosides remain unchanged for at least 48 h at 55◦C.
It is known that nucleoside phosphoramidites efficiently and selectively cou-
ple at the 5ꢁ–position of solid support-bound oligonucleotides with deprotected
backbone (4). Phosphoramidites 8–12 were used for preparation of oligodeoxynu-
cleotides and their phosphorothioate and 2ꢁ–O–(2–methoxyethyl) analogs, 24–30,
on 1 to 150 µmol scale (Table). In agreement with previous observations (7), 8–12
were capable of an efficient chain elongation. Analysis of the deprotection mixtures
revealed no apparent modifications of nucleic bases or oligonucleotide backbone.
The yields of isolated oligonucleotides were similar to those obtained with the
standard 2–cyanoethyl phosphoramidites.
Table 1. Oligonucleotides 24–30 Synthesized with the Aid of 8–12
Compound
Sequence (5ꢁ → 3ꢁ)
Backbone
Target
=
24
25
26
27
28
29
30
GCC CAA GCT GGC ATC CGT CA
TCC CGC CTG TGA CAT GCA TT
GCC CAA GCT GGC ATC CGT CA
TCC CGC CTG TGA CAT GCA TT
ATG CAT TCT GCC CCC AAG GA
P O
=
P O
Human ICAM–1
Human C–raf
Human ICAM–1
Human C–raf
Mouse C–raf
Human MDM–2
–
=
P S
=
P S
=
P S
=
AGC TTC TTT GCA CAT GTA AA
P S
a
(T∗T∗T∗)6 T∗T∗
P S
=
a. T∗ stands for 2ꢁ–O–(2–methoxyethyl)–5–methyluridine residue.

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GUZAEV AND MANOHARAN
In conclusion, the 2–(N–isopropyl–N–anisoylamino)ethyl is a novel, conve-
nient phosphodiester protecting group. It is quantitatively removed either on solid
support by treatment with a polar organic solvent or under standard deprotection
conditions.
REFERENCES
1. Sinha, N. D.; Biernat, J.; McManus, J.; Ko¨ster, H. Nucleic Acids Res. 1984, 12, 4539–
4557.
2. Wilk, A.; Grajkowski, A.; Phillips, L. R.; Beaucage, S. L. J. Org. Chem. 1999, 64,
7515–7522.
3. Guzaev, A. P.; Manoharan, M. Tetrahedron Lett. 2000, 41, 5623–5626.
4. Caruthers, M. H.; Kierzek, R.; Tang, J. Y. In Biophosphates and their Analogues – Syn-
thesis, Metabolism and Activity; Bruzik, K. S.; Stec, W. J. Eds.; Elsevier: Amsterdam,
1987; pp. 3–21.

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