Synthesis of monomers bearing at the 2′-position cyanomethoxycarbonyl group for phosphoramidite oligonucleotide synthesis

Article abstract of DOI:10.1081/NCN-200026053

A novel series of phosphoroamidites for the synthesis of 2′-modified oligonucleotides was designed and synthesized on the base of 2′-amino uridine and 2′-amino arabinoadenosine. The amino groups in these compounds were acidified by bis-cyanomethyl esters

Full text of DOI:10.1081/NCN-200026053

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Synthesis of Monomers Bearing at the 2′‐Position
Cyanomethoxycarbonyl Group for Phosphoramidite
Oligonucleotide Synthesis
Svetlana V. Vasilyeva a , Tatyana V. Abramova a & Vladimir N. Silnikov a
a Novosibirsk Institute of Bioorganic Chemistry , 8, Lavrentiev Ave., Novosibirsk, 630090,
Russia
Published online: 23 Aug 2006.
To cite this article: Svetlana V. Vasilyeva , Tatyana V. Abramova & Vladimir N. Silnikov (2004) Synthesis of Monomers Bearing
at the 2′‐Position Cyanomethoxycarbonyl Group for Phosphoramidite Oligonucleotide Synthesis, Nucleosides, Nucleotides and
Nucleic Acids, 23:6-7, 993-998, DOI: 10.1081/NCN-200026053
To link to this article: http://dx.doi.org/10.1081/NCN-200026053
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, Nos. 6 & 7, pp. 993–998, 2004
Synthesis of Monomers Bearing at the 2’ -Position
Cyanomethoxycarbonyl Group for Phosphoramidite
Oligonucleotide Synthesis
Svetlana V. Vasilyeva,* Tatyana V. Abramova, and Vladimir N. Silnikov
Novosibirsk Institute of Bioorganic Chemistry, Novosibirsk, Russia
ABSTRACT
A novel series of phosphoroamidites for the synthesis of 2’-modified oligonucleotides
was designed and synthesized on the base of 2’-amino uridine and 2’-amino
arabinoadenosine. The amino groups in these compounds were acidified by bis-
cyanomethyl esters of different dicarbonic acids. Generated reactive linker groups
containing cyanomethoxycarbonyl groups are stable under conditions of oligonucle-
otide synthesis but could be easily functionalised in post-synthetic stage by treatment
with compounds bearing primary amino groups.
Key Words: Modified nucleosides; Cyanomethyl esters, 2’-amino uridine, 2’-amino
arabinoadenosine, Cyanomethoxycarbonyl; Reactive linker groups.
INTRODUCTION
For the preparation of 2’-modified ODN a post-synthetic functionalization method
was suggested[1] as it has one obvious advantage: the same parent compound could be
applied to construct a vast number of differently modified products. This makes the
approach especially favourable in scenarios where an optimisation process and thus, a
*
Correspondence: Svetlana V. Vasilyeva, Novosibirsk Institute of Bioorganic Chemistry, 8,
Lavrentiev Ave., Novosibirsk, 630090, Russia; Fax: 3832-333677; E-mail: vasilieva_2001@ngs.ru.
993
DOI: 10.1081/NCN-200026053
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

994
Vasilyeva, Abramova, and Silnikov
pool of functionalized oligonucleotides are needed.[2] Toward this strategy we have
incorporated one or two reactive methoxyoxalamido groups into the 2’-position of
phosphoramidites on the base of uridine and cytidine.[3] These monomers for the
phosphoramidite method of oligonucleotide synthesis allow the formation of conjugates
of oligonucleotides with different reagents containing primary amino groups. Now we
report the synthesis of a novel series of monomers bearing cyanomethoxycarbonyl
group in the 2’-position of the nucleoside, connected by linkers of different length.
RESULTS AND DISCUSSION
The ester carbonyl carbon of the cyanomethoxycarbonyl residue as of the
methoxyoxalyl residue is highly electrophilic due to the electron withdrawing effects of
the adjacent methoxy and carbonyl groups. Indeed, rapid and quantitative reactions are
observed with strong nucleophiles such as primary amines, ammonia or the hydroxyl
anion.[4] On the other hand cyanomethyl esters of carbonic acids are stable towards the
reagents used in solid-phase phosphoramidite synthesis as has been previously shown
by Kohgo et al.[5] The introduction of monomers containing in the 2’-position the
methoxyoxalamido group into an oligonucleotide requires the increase of the time of
condensation and usage of 5-ethyltho tetrazole due to the steric difficulties. The
cyanomethoxycarbonylamido group structure seems more flexible and should avoid the
above mentioned problems.
Figure 1. A: Synthesis of nucleosides containing in 2’-position reactive cyanomethyl esters of
various carbonic acids and their phosphoramidites: i) NCCH2OC(O)–(CH2)n–C(O)OCH2CN,
where n = 2–4, DIPEA, CH3CN; ii) 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphoramidite,
NH(iPr)2 tetrazole. B: Synthesis of bis-cyanomethyl esters of succinic (n = 2), glutaric (n = 3),
adipic (n = 4) acids.

Phosphoramidite Oligonucleotide Synthesis
995
The cyanomethyl esters of the succinic, glutaric, adipic acids were introduced into
the 2’-position of 2’-amino-2’-deoxyuridine, and the cyanomethyl ester of the glutaric
acid into the 2’-position of 9-(2-amino-2-deoxy-b-D-arabinofuranosyl)-N6-benzoyl
adenine[6] (Fig. 1A). The 2’-amino group in compound 1 reacted with bis-cyanomethyl
esters of varies dicarbonic acids prepared in situ in the presence of diisopropylehyl-
amine (DIPEA) at room temperature. Bis-cyanomethyl esters of succinic, glutaric,
adipic acids were prepared as follow (Fig. 1B): potassium carbonate and bromoace-
tonitrile were added to a solution of dicarbonic acid in anhydrous CH3CN. The reaction
mixture was stirred at room temperature for 12 h, the precipitate was removed and then
washed with CH3CN. Filtrates were used for reaction with 2’-amino-2’-deoxyuridine
without additional purification. The reaction with 9-(2-amino-2-deoxy-b-D-arabinofur-
anosyl)-N6-benzoyl adenine 2 was analogous. Nucleosides 3–7 were obtained in yields
of 40, 30, 46, 47, 30% respectively. In this series we also synthesized the derivative of
2’-amino-2’-deoxyuridine bearing the monomethyl ester of malonic acid. Its reactivity is
lower than the cyanomethyl esters, though it is reactive enough for successful
functionalisation by primary amines.
GENERAL METHOD OF PREPARATION OF NUCLEOSIDES
BEARING REACTIVE LINKER GROUPS
The nucleoside bearing an amino group at the 2’-position (1 mmol) was added to
the bis-cyanomethyl ester of the corresponding dicarbonic acid prepared in situ (1.2 eq,
0.5 M CH3CN solution) in the presence of DIPEA (2.4 eq) at room temperature. After
68 h, the reaction was complete (as monitored by TLC). The reaction mixture was
evaporated to dryness in vacuo. The crude product was purified by silica-gel column
chromatography using CH2Cl2 as eluent. The corresponding phosphoramidites were
prepared using a standard procedure (Table 1).
5’-O-DMT-2’-deoxy-2’-(O-cyanomethyl)succinamidouridine 3: Rf 0.6 [CH2Cl2–
CH3OH (9:1)]; mp 98–102°C; UV (EtOH), lmax/nm: 202 (73022), 234 (19584), 266
(8919). The yield of phosphoramidite 8 is 35%; Rf 0.7 [CH2Cl2–CH3OH–Et3N
(9.4:0.5:0.1)]; 31P NMR [(CD3)2CO]: d 151.4, 152.4.
5’-O-DMT-2’-deoxy-2’-(O-cyanomethyl)glutaramidouridine 4: Rf 0.56 [CH2Cl2–
CH3OH (9:1)]; mp 90–95°C; UV (EtOH), lmax/nm: 203 (76009), 234 (22519), 265
(10925). The yield of phosphoramidite 9 is 88%, Rf 0.63 [CH2Cl2CH3OH–Et3N
(9.4:0.5:0.1)]; 31P NMR [(CD3)2CO]: d 151.2, 152.3.
5’-O-DMT-2’-deoxy-2’-(O-cyanomethyl)adipinamidouridine 5: Rf 0.7 [CH2Cl2–
CH3OH (9:1)]; mp 95–97°C; UV (EtOH), lmax/nm: 201 (94372), 235 (24376), 266
(12450). The yield of phosphoramidite 10 is 47%, Rf 0.92 [CH2Cl2–CH3OH–Et3N
(9.4:0.5:0.1)]; 31P NMR [(CD3)2CO]: d 151.3, 152.4.
9-[5’-O-DMT-2’-deoxy-2’-(O-cyanomethyl)glutaramido-b-D-arabinofuranosyl]-N6-
benzoyladenine 6: Rf = 0.43 [CH2Cl2–CH3OH–Et3N (9.4:0.5:0.1)]; UV (EtOH),
lmax/nm: 206 (60080), 237 (27091), 277 (13571). The yield of phosphoramidite 11 is

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Vasilyeva, Abramova, and Silnikov

Phosphoramidite Oligonucleotide Synthesis
997

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Vasilyeva, Abramova, and Silnikov
30%; Rf 0.75 [CH2Cl2–CH3OH–Et3N (9.65:0.25:0.1)]; 31P NMR [(CD3)2CO]: d
150.2, 150.0.
5’-O-DMT-2’-deoxy-2’-(O-Me)malonamidouridine 7. Rf 0.33 [CH2Cl2–CH3OH–
Et3N (9.4:0.5:0.1)]; UV (EtOH), lmax/nm: 204 (63398), 235 (20670), 269 (8008). The
yield of phosphoramidite 12 is 67%; Rf 0.43 [CH2Cl2–CH3OH–Et3N (9.4:0.5:0.1)];
31P NMR [(CD3)2CO]: d 151.2, 150.2.
ACKNOWLEDGMENTS
This work is supported by RFBR (02-04-48664-a), Grants BRHE Rec-008,
Wellcome Trust (CRIG 063630) and Russian Science Support Foundation.
REFERENCES
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Received December 2, 2003
Accepted Apri 23, 2004