Synthesis of a 2′-O-(carbomoylmethyl)ribonucleoside H-phosphonate building block and a model dinucleotide

Article abstract of DOI:10.1080/15257770701542900

In order to obtain higher quality 2′-O-carbamoylmethyl oligoribonucleotides we are conducting studies of this modification. Here we present synthesis of 2′-O-carbamoylmethyl containing H-phosphonate building blocks as well as synthesis of model dinucleotides needed for these studies. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701542900

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Synthesis of A 2′- O -
(Carbomoylmethyl)Ribonucleoside H-
Phosphonate Building Block and A Model
Dinucleotide
Stefan Milton a , Raunak Esther Yeheskiely a & Roger Strömberg a
a Department of Biosciences and Nutrition , Karolinska Institutet ,
Novum, Huddinge, Sweden
Published online: 05 Dec 2007.
To cite this article: Stefan Milton , Raunak Esther Yeheskiely & Roger Strömberg (2007) Synthesis of
A 2′- O -(Carbomoylmethyl)Ribonucleoside H-Phosphonate Building Block and A Model Dinucleotide,
Nucleosides, Nucleotides and Nucleic Acids, 26:10-12, 1495-1499, DOI: 10.1080/15257770701542900
To link to this article: http://dx.doi.org/10.1080/15257770701542900
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1495–1499, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701542900
SYNTHESIS OF A 2’-O-(CARBOMOYLMETHYL)RIBONUCLEOSIDE
H-PHOSPHONATE BUILDING BLOCK AND A MODEL DINUCLEOTIDE
Stefan Milton, Raunak, Esther Yeheskiely, and Roger Stro¨ mberg
Department of Biosciences and Nutrition, Karolinska Institutet, Novum,
2
Huddinge, Sweden
In order to obtain higher quality 2 ꢁ-O-carbamoylmethyl oligoribonucleotides we are conduct-
2
ing studies of this modification. Here we present synthesis of 2 ꢁ-O-carbamoylmethyl containing
H-phosphonate building blocks as well as synthesis of model dinucleotides needed for these studies.
Keywords Carbamoylmethyl; modified RNA; 2ꢁ-O-alkyl; H-phosphonate; dinucleotide
Several 2ꢁ-modifications of DNA oligonucleotides have been shown to in-
crease the DNA:RNA duplex stability.[1,2] The 2ꢁ-O-carbamoylmethyl mod-
ification provide a small electronegative hydrophilic group that increases
the duplex stability significantly.[3] In the first published study 2ꢁ-O-car-
bamoylmethyl oligonucleotides were synthesized from 2ꢁ-O-cyanomethyl
oligonucleotides by hydrolysis. In order to explore the potential of this
modification further we decided to develop methods for synthesis of 2ꢁ-O-
carbamoyl building blocks, and to make dimer models for studies on the
chemical stability and potential side-reactions of this modification.
The first attempt to get the desired 2ꢁ-O-carbamoylmethyl-adenosine was
through alkylation of unprotected adenosine (1) with sodium hydride and
iodoacetamide in DMF (Scheme 1). This yielded the product (2) as a 2ꢁ, 3ꢁ
isomeric mixture (in about 6/4 ratio). The yield and selectivity was, how-
ever, quite sensitive to moisture, amount of base, amount of alkyl halide and
varied substantially. The product mixture of the 2ꢁ and the 3ꢁ isomers was
more polar than adenosine and not readily separated. Isomer separation was
therefore done at a later stage. Base protection (N -6-butyryl) of the isomeric
mixture gave compound (3) and monomethoxytrityl protection of the 5ꢁ-
OH gave compound (4). Finally phosphonylation with PCl3/imidazole gave
Address correspondence to Roger Stro¨mberg, Department of Biosciences and Nutrition, Karolinska
Institutet, Novum, S-15177 Huddinge, Sweden. E-mail: rost@biosci.ki.se
1495

1496
S. Milton et al.
SCHEME 1 Route for the intial method for synthesis of the 2ꢁ-O-carbamoylmethyladenosine H-
phosphonate building block 5.
(5). The isomeric mixture of the 3ꢁ and 2ꢁ-O-alkylated H-phosphonates, was
separated by straight-phase HPLC.
In an alternative approach (Scheme 2), we started with 5ꢁ-O-
monomethoxytrityladenosine 6 which was alkylated with iodoacetamide
in THF using dimsylsodium as base. This gave the 2ꢁ and 3ꢁ-O-
carbamoylmethylated product with substantially higher selectivity for the 2ꢁ-
alkylated product (2ꢁ:3ꢁ ratio was about 9/1 as estimated from TLC) and
although some unreacted material remained and some dialkylated product
formed we achieved 39% isolated yield of (8) after chromatography. The
same product was also produced in a one-pot procedure via the methyl
SCHEME 2 Improved synthesis route to the protected 2ꢁ-O-carbamoylmethyladenosine 4.

2ꢁ-O-(Carbomoylmethyl)Ribonucleoside H-Phosphonate
1497
SCHEME 3 Synthesis of the 2ꢁ-O-carbamoylmethyl containing ApT model dimer 11.
ester (7) and subsequent ammonolysis to (8). Compound (8) was then
separated from its isomers by column chromatography, base protected to
give compound (4) and finally phosphonylated to (5).
In synthesis of the model ApT dimer (Scheme 3), (5) was first cou-
pled to 3ꢁ-O-MMT-T (9) and the product was oxidized in situ to give the
protected dimer (10). The coupling was performed using either pivaloyl
chloride or bis(2-oxo-3-oxazolidinyl)phosphinic chloride (OXP), in both
cases with virtually quantitative coupling. (10) was then detritylated and
finally treated with NH3 in methanol to give the desired model dimer
(11). A simple method of synthesizing a 2ꢁ-O-carbamoylmethyladenosine
H-phosphonate building block has been developed and a model dimer
with carrying the 2ꢁ-O-carbamoylmethyl modification has been made. The
nuclease resistance and chemical stablity of the dimer is under investigation.
EXPERIMENTAL
2’-O-Carbamoylmethyl-5’-O-Monomethoxytrityladenosine (8)
5ꢁ-O-monomethoxytrityl-adenosine (0.45 g, 0.83 mmol) was coevapo-
rated with 30 ml dry MeCN and then 30 ml dry THF. The material was
dissolved in distilled THF (50 ml) and flushed with dry N2 gas. Dimsyl
sodium (1.00 mmol, 0.5 ml) was slowly added to the mixture. The mixture
was stirred for 30 minutes and then iodoacetamide (0.186 g, 1.00 mmol)
was added. After 2 hours the solvent was evaporated and purified by column
chromatography (CH2Cl2/MeOH 13:1 + 0.05% triethylamine). The yield of
the 2ꢁ-O-alkylated isomer was 39% and the 3ꢁ was 2%. 1HNMR (CDCl3): 3.35–
3.55 (m, 2H, 5Hꢁ); 3.65 (s, 3H, O-Me trityl); 4.15 (d, 2H, CH2-acetamid);
4.28 (d, 1H, 4Hꢁ); 4.50 (m, 1H, 3Hꢁ); 4.55 (m, 1H, 2Hꢁ); 6.18 ( d, 1H, H1ꢁ);

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S. Milton et al.
6.80 (d, 2H, trityl); 7.15–7.40 (m, 12H, trityl); 8.00 (s, 1H, H2-base); 8.20
(s, 1H, H8-base). ES-TOF: m/z calculated for C32H32N6O6 [M+H]+, 597.6;
found, 597.4.
6-N-Butyryl-2’-O-Carbamoylmethyl-5’-O-
Monomethoxytrityladenosine 3’-H-Phophonate
Triethylammonium Salt (5)
Imidazole (0.761 g, 11.29 mmol) was suspended in (30 ml) dry
CH2Cl2and cooled down to –100◦C. PCl3(0.505 g, 0.322 ml, 3.68 mmol)
and distilled triethylamine (1.195 g, 1.64 ml, 11.81 mmol) was added drop-
wise under vigorous stirring. The mixture was stirred at –10◦C for 30 min-
utes and then cooled down to −78◦C. The protected nucleoside (0.70 g,
1.05 mmol) dissolved in CH2Cl2 (13 ml) was added during 30 minutes. The
mixture was stirred at −78◦C for 1 hour and extracted with triethylammo-
niun acetate pH 7.5 (2*30 ml). The organic layer was dried over MgSO4
and evaporated. The crude product was purified by column chromatogra-
phy on silica CH2Cl2/MeOH (6:1 + 0.5% triethylamine) that gave a total
yield of 88%. 1HNMR+COSY (CDCl3):1.05–1.10 (t, 3H, CH3-Bu); 1.75–1.85
(six, 2H, CH2-Bu); 2.80–2.90 ( t, 2H, CH2-Bu); 3.40–3,60 (m, 2H, H5ꢁ); 4.78
(s, 3H, CH3-O-trityl); 4.10 (d, 2H, CH2-acetamid); 4.40 (d, 1H, H4ꢁ); 4.80 (t,
1H, H3ꢁ); 5.05–5.10 (m, 1H, H2ꢁ); 6.26 (d, 1H, H1ꢁ); 8.15 (s, 1H, H2); 8.60
(s, 1H, H8); 6.15 and 7.75 (two singlets, P-H). ES-TOF: m/z calculated for
C36H38N6O9P [M+H]+, 730.7; found, 730.8.
2’-O-Carbomoylmethyladenosine 3’-(Thymidine 5’-Phosphate)
(11)
The protected dimer 10 was synthesized from 5 (.200 g, 0.236 mmol)
and 9 (0.134 mg, 0.259 mmol) using a standard procedure involving cou-
pling and in situ oxidation with iodine in pyridine/water.[5] After the final
removal of solvent in this procedure, crude 10 was detritylated by dissolving
it in 8 ml 1% trifluoroacetic acid (TFA) in dichloromethane (DCM). After
2 hours the solution was evaporated under reduced pressure and then co-
evaporated with MeOH (2 × 10 ml). The residue was then directly subjected
to ammonolysis in sat. MeOH/ NH3 (10 ml). After standing overnight the
solution was concentrated and the residue was triturated repeatedly with
diethylether an finally dried under reduce pressure to give 70 mg of the
model dimer (11). The dimer was then additionally purified by reversed
phase HPLC on a Reprosil C-18AQ column (10 µm, 250 × 22 mm) using
a 45-minute linear gradient from 50 mM triethylammonum acetate (TEAA,
aq, pH 6.5) to 50 mM TEAA in 50% CH3CN and a flow rate of 10 ml/minute
Retention time for 11 was 26.3 minute. Collected fractions were lyophilized.
1H-NMR (assignments by COSY) (D2O);1.60 (s, 3H, CH3-Tym); 2.20 (m,

2ꢁ-O-(Carbomoylmethyl)Ribonucleoside H-Phosphonate
1499
2H, H2ꢁ-tym); 3.70–3.90 (m, 2H, H5ꢁ-Ad); 3.90–4.10 (m, 2H, H4ꢁ-Ad and
H4ꢁ-Tym); 4.20 (d, 2H, CH2-carbomoyl); 4.40 (m, 2H, H3ꢁ-Ad and H3ꢁ-Tym);
4.70 (m, 1H, H2ꢁ-Ad); 6.10 (m, 2H, H1ꢁ-Ad and H1ꢁ-Tym); 7.50 (s, 1H, H5-
Tym); 8.10 (s, 1H, H2-Ad); 8.25 (s, 1H, H8-Ad). ES-TOF: m/z calculated for
C22H28N8O12P [M], 627.1564; found, 627.1552.
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1. Elena, A.L.; Charles, J.G.; Andrew, M.K.; Henri, S.; Maryann, Z.; Lendell, L.; Cummins, D.J.; Ecker,
P.D.C.; Susan, M.F. Oligodeoxynucleotides containing 2ꢁ-O-modified adenosine: Synthesis and effects
on stability of DNA:RNA duplexes. Biochemistry 1993, 32, 7832–7838.
2. Freier, S.M.; Altmann, K.H. The ups and downs of nucleic acid duplex stability: structure-stability
studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Research, 1997, 25, 4429–4443.
3. Gro¨tli, M.; Beijer, B.; Sproat, B. 2ꢁ − O-(carbamoylmethyl)oligoribonucleotides. Tetrahedron 1999, 55,
4299–4314.
4. Westman, E.; Stawinski, J.; Stro¨mberg, R. RNA-synthesis using H-phosphonates. Synchronizing 2ꢁ-OH
and N-protection. Collect. Czech. Chem. Comm. 1993, 58, 236–237.
5. Stawinski, J.; Stro¨mberg, R. Di and oligonucleotide synthesis using H-phosphonate chemistry. In
Oligonucleotide Synthesis: Methods and Applications, Herdewijn P., ed., Humana Press, Totowa NJ, 2004,
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