Synthesis of chimeric oligonucleotides containing internucleosidic phosphodiester and S-pivaloylthioethyl phosphotriester residues

Article abstract of DOI:10.1081/NCN-100002481

Novel oligonucleotide analogs that bear phosphodiester and bioreversible S-pivaloyl 2-mercaptoethyl (SPME) phosphate triester intemucleosidic linkages are described. Their synthesis employs a novel methodology of oligonucleotide deprotection under mild, n

Full text of DOI:10.1081/NCN-100002481

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SYNTHESIS OF CHIMERIC OLIGONUCLEOTIDES
CONTAINING INTERNUCLEOSIDIC PHOSPHODIESTER
AND S–PIVALOYLTHIOETHYL PHOSPHOTRIESTER
RESIDUES
Andrei P. Guzaev a , Balkrishen Bhat a , Guity Balow 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 , Balkrishen Bhat , Guity Balow & Muthiah Manoharan (2001) SYNTHESIS OF CHIMERIC
OLIGONUCLEOTIDES CONTAINING INTERNUCLEOSIDIC PHOSPHODIESTER AND S–PIVALOYLTHIOETHYL PHOSPHOTRIESTER
RESIDUES, Nucleosides, Nucleotides and Nucleic Acids, 20:4-7, 1015-1018, DOI: 10.1081/NCN-100002481
To link to this article: http://dx.doi.org/10.1081/NCN-100002481
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1015–1018 (2001)
SYNTHESIS OF CHIMERIC OLIGONUCLEOTIDES
CONTAINING INTERNUCLEOSIDIC
PHOSPHODIESTER AND
S–PIVALOYLTHIOETHYL PHOSPHOTRIESTER
RESIDUES
Andrei P. Guzaev,∗ Balkrishen Bhat, Guity Balow,
and Muthiah Manoharan
Department of Medicinal Chemistry, Isis Pharmaceuticals,
2292 Faraday Ave., Carlsbad, California 92008
ABSTRACT
Novel oligonucleotide analogs that bear phosphodiester and bioreversible
S–pivaloyl 2–mercaptoethyl (SPME) phosphate triester internucleosidic link-
ages are described. Their synthesis employs a novel methodology of oligonu-
cleotide deprotection under mild, non-aqueous conditions.
Antisense oligonucleotides are a novel class of genomic therapeutics. It is well
known however that oligonucleotide phosphorothioates are of limited stability in
blood and tissues. Besides, being negatively charged molecules, they lack the ability
to permeate biological membranes in an efficient manner. Thus, both their oral
bioavailabilityandcellularuptakeneedfurtherimprovement. Inordertoaddressthis
issue, a bioreversible S–acyl 2–mercaptoethyl (1) phosphate protecting group has
been introduced into synthetic oligonucleotides. The bioreversible oligonucleotides
may potentially serve as prodrug forms of antisense oligonucleotides. A search for
their reliable and straightforward synthesis is therefore of great interest.
Thepreparationofoligonucleotideprodrugsusingthe S–acyl2–mercaptoethyl
strategy presents a complex synthetic problem. So far, no conventional depro-
tection method has been reported in the literature. By an orthogonal protection
∗Corresponding author.
1015
ꢀ
C
Copyright 2001 by Marcel Dekker, Inc.
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GUZAEV ET AL.
Figure 1. Structures of 2-cyanoethyl phosphoramidites and solid supports.
strategy, preparation of short S–pivaloyl 2–mercaptoethyl (SPME) protected oligo-
nucleotides has recently been achieved using photolabile nucleic base protecting
groups and solid support (2). However, fully modified SPME oligonucleotides
are poorly soluble in water (3) and therefore can hardly be considered as drug
candidates.
We reported previously synthesis of a phosphoramidite building block 8 (4)
(Fig. 2) and oligothymidylates bearing both SPME and phosphodiester linkages (5).
In this communication, we report a novel strategy of oligonucleotide deprotection
under mild, non-aqueous conditions. The strategy allows one to deprotect nucleic
bases and the backbone while SPME phosphotriester groups are kept intact.
Previously we observed that the SPME group was almost indefinitely sta-
ble towards 1 M piperidine in MeCN and of a limited stability towards 0.01 M
K2CO3 in MeOH (23). With this information in hand, we synthesized a number of
model oligonucleotides using phosphoramidites 1–3 and solid supports 6–8 (Fig. 1)
and studied their deprotection. We found that phenoxyacetyl (PAC) protection of
deoxycytosine and deoxyadenosine residues was removed under conditions com-
patible with the SPME group. In contrast, removal of the PAC protection from
Figure 2. Synthesis of phosphoramidite building blocks 17–21.

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SYNTHESIS OF CHIMERIC OLIGONUCLEOTIDES
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deoxyguanosine residue required an unacceptably long treatment with the solution
of piperidine (48 h).
This data prompted us to use N–unprotected 2–cyanoethyl deoxyguanosine
phosphoramidite 4 synthesized as reported earlier (6). In accordance with the pre-
vious observations, the use of 4 within the standard protocol of the DNA synthesis
demonstrated no deterioration of coupling yields or the purity of synthetic ligonu-
cleotides (6,7).
Having set the deprotection conditions, we synthesized SPME protected nu-
cleoside phosphoramidites 17–21 and evaluated their use in preparation of chimeric
oligonucleotides. First, 10 was converted to a bisamidite 11, which was isolated on
a silica gel column as a colorless oil in 90% yield and more than 98% purity
(31P NMR). Nucleosides 12–16 were treated with 11 in the presence of 1H–
tetrazole to give 17–21 (Fig. 2). These were isolated by column chromatography in
75–95% yield and characterized. The phosphoramidites 17–21 were stable com-
pounds whose shelf life exceeded 2 years. Previously we reported that the half-life
of disappearance of 17 in 80% aqueous MeCN was 58 h, which indicated its high
hydrolytic stability (4).
Oligonucleotides22–27(Table)wereassembledusingphosphoramiditebuild-
ing blocks 1–5 and 17–21 and solid supports 6–9. For deprotection, a three-step
procedure was developed. Solid support-bound oligonucleotides were treated first
with 1 M piperidine in MeCN for 8 h and then extensively washed with dioxane.
On the second step, the synthetic columns were replaced on the instrument, and
the standard detritylation subroutine was carried out. Finally, oligonucleotide ma-
terial was released from the support with 0.01 M K2CO3 in MeOH for 80 min,
and the solution was neutralized with glacial AcOH. The obtained mixtures con-
sisted of chimeric oligonucleotides 22–24 along with products of methanolysis of
the SPME group. With oligothymidylates, the extent of methanolysis was minimal
(ca. 2% per SPME group). In other cases it depended on the base composition of
an oligonucleotide and varied between 2 and 10%.
Table. Chimeric Oligonucleotides 22–27 Containing SPME
Phosphotriester Groupsa
Compound
Sequence (5ꢁ → 3ꢁ)
Backbone
=
22
23
24
25
26
27
TTT T13 TTT T
P S
=
P S
T∗T∗T∗ T13 T∗T∗T∗ T∗ b
=
CCCCCAA T T10
P S
=
CC CCCACCACTTCCCCT CT C
CCC CCACCACTTCCCC TCT C
AG CTTCTTTGCACATGT AA A
P S
=
P S
=
P S
a. Nucleotide residues bearing SPME group at the 3ꢁ-phosphate are
italicized;
b. T∗ stands for 2ꢁ–O–(2–methoxyethyl)–5–methyluridine residue.

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GUZAEV ET AL.
In conclusion, the present methodology of mild, non-aqueous oligonucleotide
deprotection allows straightforward preparation of potential prodrug oligonucleo-
tides bearing a desired number of the SPME phosphotriester moieties and, poten-
tially, other types of base-labile oligonucleotides.
REFERENCES
1. (a) Vives, E.; DellꢁAquila, C.; Bologna, J.-C.; Morvan, F.; Rayner, B.; Imbach, J.-L.
Nucleic Acids Res. 1999, 27, 4071–4076; (b) Tosquellas, G.; Alvarez, K.; Dell’Aquila,
K.; Morvan, F.; Vasseur, J.-J.; Imbach, J.-L.; Rayner, B. Nucleic Acids Res. 1998, 26,
2069–2074.
2. Alvarez, K.; Vasseur, J.-J.; Beltran, T.; Imbach, J.-L. J. Org. Chem. 1999, 64, 6319–
6328.
3. Tosquellas, G.; Bologna, J. C.; Morvan, F.; Rayner, B.; Imbach, J.-L. Bioorg. Med.
Chem. Lett. 1998, 8, 2913–2918.
4. Guzaev, A.; Boyode, B.; Balow, G.; Tivel, K. L.; Manoharan, M. Bioorg. Med. Chem.
Lett., 1998, 8, 1123–1126.
5. Guzaev, A.; Balow, G.; Manoharan, M. Nucleosides & Nucleotides, 1999, 18, 1391–
1392.
6. Hayakawa, Y.; Kataoka, M. J. Am. Chem. Soc., 1998, 120, 12395–12401.
7. Gryasnov, S. M.; Letsinger, R. L. Nucleic Acids Res., 1992, 20, 1879–1882.

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