LNA 5′-phosphoramidites for 5′→3′-oligonucleotide synthesis

Article abstract of DOI:10.1039/c0ob00346h

Hereby we report an efficient synthesis of LNA thymine and LNA 5-methylcytosine 5′-phosphoramidites, allowing incorporation of LNA thymine and LNA 5-methylcytosine into oligonucleotides synthesized in the 5′→3′ direction. Key steps include regioselective

Full text of DOI:10.1039/c0ob00346h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
LNA 5¢-phosphoramidites for 5¢→3¢-oligonucleotide synthesis†
Andreas Stahl Madsen,*a T. Santhosh Kumara,b and Jesper Wengela
Received 29th June 2010, Accepted 10th August 2010
DOI: 10.1039/c0ob00346h
Hereby we report an efficient synthesis of LNA thymine and LNA 5-methylcytosine
5¢-phosphoramidites, allowing incorporation of LNA thymine and LNA 5-methylcytosine into
oligonucleotides synthesized in the 5¢→3¢ direction. Key steps include regioselective enzymatic
benzoylation of the 5¢-hydroxy group of unprotected LNA thymine, and subsequent
4,4¢-dimethoxytritylation of the 3¢-hydroxy group of the O5¢-benzoylated LNA thymine nucleoside.
Introduction
Automated synthesis of oligonucleotides (ONs) is routinely carried
out in the 3¢→5¢ direction using standard 3¢-phosphoramidite
derivatives of natural as well as modified nucleosides. Alternatively,
nucleosides with 3¢-O-dimethoxytrityl and 5¢-phosphoramidite
functionalities (5¢-phosphoramidites) enable convenient ON syn-
thesis proceeding in the 5¢→3¢ direction still using a standard
ON synthesis protocol. This enables synthesis of otherwise
challenging ONs, by allowing 3¢-terminal incorporation of deriva-
tives that does not offer a suitable site for attachment to a
solid support, such as nucleosides without a 3¢-hydroxy group
(e.g. 2¢,3¢-dideoxy nucleosides), non-nucleosidic groups such as
fluorophores,1 phosphorodithioates and phosphorotrithioates,2
thiols,3 or chimeric syntheses where compatibility with otherwise
employed chemistry dictates synthesis in the 5¢→3¢ direction.4
Furthermore, in combination with standard 3¢-phosphoramidites,
5¢-phosphoramidites offer the opportunity to change orientation
of the nucleotides during synthesis. This allows straightforward
synthesis of ONs containing 3¢,3¢ or 5¢,5¢ linkages, e.g. for parallel
hairpins5 alternating triplex-forming ONs,6 Hoogsteen parallel
stranded DNA for targeting pyrimidine ONs,5b,7 or multiple
polarity reversals.8
For standard DNA monomers both 3¢- and 5¢-
phosphoramidites are commercially available, whereas LNA
(locked nucleic acid) monomers are only available as 3¢-
phosphoramidites (Fig. 1). LNA-modified ONs have successfully
been explored for applicability within molecular biology research,
diagnostics and gene silencing, generally taking advantage of
the duplex-stabilizing effect of the conformationally locked
LNA monomers.9 Previously, the synthesis of the LNA thymine
5¢-phosphoramidite 5 was reported,10 but a low-yielding 4,4¢-
dimethoxytritylation step renders this strategy unsuitable for
larger scale synthesis. We therefore wished to establish a viable
synthetic route to the LNA thymine and 5-methylcytosine
Fig. 1 Thymidine 3¢-phosphoramidite building block (for standard 3¢→5¢
ON synthesis) and 5¢-phosphoramidite building block (for reversed 5¢→3¢
ON synthesis), respectively.
5¢-phosphoramidites (5 and 9, respectively, Scheme 1), which we
hereby report with full experimental details. Furthermore, we
demonstrate the efficient incorporation of 5¢-phosphoramidites 5
and 9 into a short ON to give the corresponding LNA thymine
and LNA 5-methylcytosine monomers (TL and MeCL, respectively
Scheme 1).
Results and discussion
We envisioned
a simple synthetic strategy starting from
LNA thymine diol 1.9a Selective protection of the primary
5¢-hydroxy group followed by O3¢-dimethoxytritylation, O5¢-
deprotection and O5¢-phosphitylation should give LNA thymine
5¢-phosphoramidite 5, whereas conversion from thymine to
5-methylcytosine and nucleobase protection at an appropri-
ate step should give LNA 4-N-benzoyl-5-methylcytosine 5¢-
phosphoramidite 9.
Selective protection of the primary 5¢-hydroxy group over the
secondary 3¢-hydroxy group of nucleoside 1 has previously been
accomplished by use of the bulky TBDMS group,10 but subsequent
4,4¢-dimethoxytritylation of the 3¢-hydroxy group was reported to
be very troublesome.10 To circumvent this problem we decided
to use the sterically less demanding benzoyl protection group.
However, standard conditions (1.1 eq. benzoyl chloride in pyridine
at either rt, 0 ◦C or -35 ◦C, or in CH2Cl2:pyridine at -70 ◦C) did not
provide any significant selectivity (giving an approximate 3 : 2 ratio
of benzoate ester 2 and the corresponding 3¢-regioisomer). This
rather surprising lack of selectivity is most likely a consequence
of the locked 2,5-dioxabicyclo[2.2.1]heptane skeleton which forces
the 3¢-hydroxy group of nucleoside 1 to adopt a position which is
unusually accessible, thereby leading to increased reactivity.
As a regioselective enzymatic procedure for O5¢-benzoylation
of 2¢-deoxynucleotides has been reported,11 we investigated the
aNucleic Acid Center, Department of Physics and Chemistry, University
of Southern Denmark, (The Nucleic Acid Center is funded by the Danish
National Research Foundation for studies on nucleic acid chemical biology),
5230, Odense M, Denmark. E-mail: asm@ifk.sdu.dk.; Fax: +45 6615 8780;
Tel: +45 6550 2599
bCurrent affiliation: National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, MD, 20892-0810, USA
† Electronic supplementary information (ESI) available: General experi-
mental section and copies of 1H and 13C NMR spectra of compounds 2–4
and 6–8 and copies of 31P NMR spectra of compounds 5 and 9. See DOI:
10.1039/c0ob00346h
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Scheme 1 Synthesis of LNA 5¢-phosphoramidites. Reagents and conditions: i) CH2CHOBz, Candida Antarctica Lipase B (Novozyme 435ꢀR ), THF,
60 ◦C, 72 h, 93%; ii) DMTCl, AgOTf, CH2Cl2:2,6-lutidine (50 : 50, v/v), rt, 20 h, 100%; iii) K2CO3, MeOH–CH2Cl2–H2O (60 : 20 : 20, v/v), rt, 20 h, 91%;
iv) NC(CH2)2OP(N(i-Pr)2)2, diisopropylammonium tetrazolide, CH2Cl2, rt, 3 h, 77%; v) DNA synthesizer, vi) a) Et3N, 1,2,4-triazole, POCl3, CH3CN,
0
◦C to rt, 3 h, b) 28% aq NH3:CH3CN (50 : 50, v/v), rt, 2 h, 97%; vii) BzCl, pyridine, rt, 3 h, 94%; iix) 1M aq. LiOH, THF, rt, 24 h, 93%; ix)
NC(CH2)2OP(N(i-Pr)2)2, diisopropylammonium tetrazolide, CH2Cl2, rt, 2 h, 81%.
R
potential for Candida Antarctica Lipase B (Novozyme 435ꢀ)
TLAT MeCLAC-3¢) was synthesized. To allow comparison with
conventional phosphoramidites, the ON was synthesized in the
3¢→5¢ as well as the 5¢→3¢ direction using either normal 3¢-
phosphoramidites or 5¢-phosphoramidites, respectively. For LNA
3¢- and 5¢-phosphoramidites, extended coupling times (15 min)
resulted in stepwise coupling yields of ≥99%, as obtained for DNA
3¢- and 5¢-phosphoramidites (2 min coupling time). Comparable
purities were obtained after purification (>90%). Composition
of the product from both syntheses was verified by MALDI-
MS. In agreement with the identical elution time observed for
the two products by ion exchange (IE) chromatography, a mixture
of the two batches eluted as a single peak, confirming that the two
products are identical (Fig. 2).
to catalyze monobenzoylation of LNA nucleoside 1 using vinyl
benzoate as acylating reagent. Gratifyingly, the reaction proceeded
smoothly affording the desired benzoate ester 2 in 93% yield,
without any detectable formation of the 3¢-regioisomer. The
correct connectivity for benzoate ester 2 was confirmed by NMR
(including disappearance of the 5¢-hydroxy signal and a downfield
shift of the H5¢ signal from 3.7 to 4.7 ppm). Dimethoxytritylation
of the 3¢-hydroxy group of alcohol 2 was unsuccessful using
standard conditions (DMTCl in pyridine), however, DMTOTf12
in dichloromethane and 2,6-lutidine afforded fully protected
nucleoside 3 in quantitative yield. Furthermore, we found that
synthesis and isolation of DMTOTf could be circumvented
by in situ generation of DMTOTf from DMTCl and AgOTf
in dichloromethane and 2,6-lutidine, quantitatively affording
dimethoxytrityl ether 3. Subsequent hydrolysis of the benzoate
ester followed by phosphitylation afforded LNA thymidine 5¢-
phosphoramidite 5 in 70% yield over two steps from intermediate
3. Conversion from thymine to 5-methylcytosine was realized by
a well-established two-step methodology.13 Thymine derivative 3
was treated with POCl3, Et3N and 1,2,4-triazole to afford the
triazolothymine derivative which was converted to the corre-
sponding 5-methylcytosine derivative 6 by treatment with aqueous
ammonia in acetonitrile. Subsequent benzoylation furnished 4-
N-benzoyl-5-methylcytosine derivative 7 in 92% yield over three
chemical steps from nucleoside 3. Selective hydrolysis of the ester
affording alcohol 8, was realized in 93% yield by treatment with
aqueous LiOH in THF with no observable cleavage of the 4-N-
benzoyl moiety. The correct constitution was confirmed by NMR,
including appearance of a signal for the 5¢-hydroxy group and an
upfield shift of the H5¢ signals (from 4.9 to 4.2 ppm). Subsequent
phosphitylation afforded LNA 4-N-benzoyl-5-methylcytosine 5¢-
phosphoramidite 9 in 70% yield over five steps from intermediate
3. The highly efficient synthetic strategy employed here, would also
be expected to enable synthesis of the corresponding purine LNA
phosphoramidites.
Fig. 2 IE-HPLC absorbance profile for co-injection of the 3¢→5¢ and
5¢→3¢ synthesized ON.
Experimental
(1R,3R,4R,7S)-1-Benzoyloxymethyl-7-hydroxy-3-(thymin-1-yl)-
2,5-dioxabicyclo[2.2.1]heptane (2)
Diol 1 (1.20 g, 4.43 mmol) was dissolved in anhydrous THF
(30 mL). Vinyl benzoate (3.0 mL, 21.7 mmol) and Novozyme
435ꢀ (1.20 g) were added and the mixture was stirred at 60 ◦C
R
for 72 h. The reaction mixture was adsorbed directly on kieselgur
and purified by silica gel column chromatography (0–6% MeOH
in CH2Cl2) affording the desired benzoate ester 2 (1.56 g, 93%)
as a white solid. Rf 0.3 (10% MeOH in CH2Cl2, v/v); Found: C,
To demonstrate the applicability of 5¢-phosphoramidites 5 and
9 in ON synthesis an LNA modified oligonucleotide (5¢-GCA
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56.9; H, 4.6; N, 7.3. Calcd for C18H18N2O7·1/4H2O: C, 57.1; H,
4.9; N, 7.4; dH (DMSO-d6) 11.38 (br s, 1H, NH, ex), 8.00–8.04
(m, 2H, H2Bz, H6Bz), 7.68–7.73 (m, 1H, H4Bz), 7.54–7.59 (m, 2H,
H3Bz, H5Bz), 7.40 (d, J = 1.1 Hz, 1H, H6), 5.95 (br s, 1H, 3¢-OH,
ex), 5.48 (s, 1H, H1¢), 4.76 (d, J = 12.8 Hz, 1H, H5¢A), 4.71 (d,
J = 12.8 Hz, 1H, H5¢B), 4.24 (s, 1H, H2¢), 4.09 (s, 1H, H3¢), 4.01
(d, J = 8.0 Hz, 1H, H5¢¢A), 3.85 (d, J = 8.0 Hz, 1H, H5¢¢B), 1.60
(d, J = 1.1 Hz, 3H, 5-CH3); dC (DMSO-d6) 165.3 (COPh), 163.7
(C4), 149.9 (C2), 134.1 (C6), 133.7 (C4Bz), 129.2 (C2Bz, C6Bz), 128.9
(C3Bz, C5Bz), 128.5 (C1Bz), 108.6 (C5), 86.6 (C1¢), 86.2 (C4¢), 79.0
(C2¢), 70.9 (C5¢¢), 69.6 (C3¢), 60.2 (C5¢), 12.0 (5-CH3); ESI-HRMS
m/z 397.1004 ([M + Na]+, C18H18N2O7Na+ Calcd 397.1006).
(2.78 g, 20.1 mmol) was added, and the reaction mixture stirred
at rt for 20 h whereupon additional K2CO3 (2.78 g, 20.1 mmol)
was added. After stirring for additional 20 h, the reaction mixture
was evaporated to dryness, and then partitioned between CH2Cl2
(200 mL) and brine (100 mL). The organic phase was evaporated
to dryness and the resulting residue was purified by silica gel
column chromatography (0–10% MeOH in CH2Cl2), affording
the desired alcohol 4 (2.66 g, 91%) as a white foam. Rf 0.3
(75% EtOAc in petroleum ether (PE), v/v); Found: C, 66.1; H,
5.5; N, 4.7. Calcd for C32H32N2O8·1/3H2O: C, 66.4; H, 5.7; N,
4.8; dH (DMSO-d6) 11.31 (br s, 1H, NH, ex), 7.38–7.42 (m,
2H, H2¢¢DMT, H6¢¢DMT), 7.22–7.31 (m, 4H, H6, H3¢¢DMT, H4¢¢DMT
,
H5¢¢DMT), 7.18–7.22 (m, 4H, H2DMT, H6DMT, H2¢DMT, H6¢DMT),
6.79–6.83 (m, 4H, H3DMT, H5DMT, H3¢DMT, H5¢DMT), 5.28 (t, J =
5.3, 1H, 5¢-OH, ex), 5.20 (s, 1H, H1¢), 4.15 (d, J = 7.9 Hz,
1H. H5¢¢A), 3.88–3.98 (m, 2H, H5¢), 3.71–3.76 (m, 7H, H5¢¢B,
OCH3), 3.57 (s, 1H, H3¢), 2.76 (s, 1H, H2¢), 1.62 (s, 3H, 5-CH3); dC
(DMSO-d6) 163.6 (C4), 158.5 (C4DMT, C4¢DMT), 149.4 (C2), 144.9
(C1¢¢DMT), 135.3 (C1DMT/C1¢DMT), 135.0 (C1DMT/C1¢DMT), 134.0
(1R,3R,4R,7S)-1-Benzoyloxymethyl-7-(4,4¢-dimethoxytrityloxy)-
3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (3)
Alcohol 2 (254 mg, 0.679 mmol) was co-evaporated with 1,2-
dichloroethane (5 mL) and then dissolved in a mixture of
anhydrous CH2Cl2 (4 mL) and anhydrous 2,6-lutidine (4 mL).
4,4¢-Dimethoxytrityl chloride (342 mg, 1.01 mmol) and silver
(C6), 129.9 ([C2DMT, C6DMT]/[C2¢DMT, C6¢DMT]), 129.8 ([C2DMT
C6DMT]/[C2¢DMT, C6¢DMT]), 127.7 (C3¢¢DMT, C5¢¢DMT), 127.5 (C2¢¢DMT
,
,
◦
triflate (268 mg, 1.05 mmol (dried for 3 h at 100 C under high
vacuum)) were added at rt, resulting in a deep red solution with
a white precipitate starting to form immediately. After stirring
for 20 h, MeOH (0.5 mL) was added to the reaction mixture.
The reaction mixture was diluted with CH2Cl2 (20 mL) and
then filtered through a short pad of kieselgur. The organic phase
was washed successively with sat. aq NaHCO3 (2 ¥ 20 mL) and
brine (20 mL), then concentrated under reduced pressure, and the
residue was placed under high vacuum overnight. The resulting
residue was purified by silica gel column chromatography (0–
4% MeOH in CH2Cl2) to afford the desired dimethoxytritylated
nucleoside 3 (459 mg, 100%) as a white foam. Rf 0.5 (5% MeOH
in CH2Cl2, v/v); Found: C, 69.45; H, 5.4; N, 4.3. Calcd for
C39H36N2O9: C, 69.2; H, 5.4; N, 4.1; dH (DMSO-d6) 11.40 (s,
1H, NH, ex), 7.73–7.77 (m, 2H, H2Bz, H6Bz), 7.67–7.73 (m, 1H,
C6¢¢DMT), 126.9 (C4¢¢DMT), 113.1 ([C3DMT, C5DMT]/[C3¢DMT, C5¢DMT]),
113.0 ([C3DMT, C5DMT]/[C3¢DMT, C5¢DMT]), 108.2 (C5), 88.6 (C4¢),
86.8 (CAr3), 86.1 (C1¢), 76.6 (C2¢), 72.0 (C5¢¢), 71.4 (C3¢), 55.9
(C5¢), 55.0 (OCH3), 12.0 (5-CH3); ESI-HRMS m/z 595.2043 ([M +
Na]+, C32H32N2O8Na+ Calcd 595.2051).
(1R,3R,4R,7S)-1-(2-Cyanoethoxy(diisopropylamino)-
phosphinoxymethyl-7-(4,4¢-dimethoxytrityloxy)-3-(thymin-1-yl)-
2,5-dioxabicyclo[2.2.1]heptane (5)
Alcohol 4 (422 mg, 0.736 mmol) was co-evaporated with
anhydrous 1,2-dichloroethane (2 ¥ 10 mL) and then redis-
solved in anhydrous 1,2-dichloroethane (50 mL), and N,N¢-
diisopropylammonium tetrazolide (253 mg, 1.48 mmol) and
bis(N,N¢-diisopropylamino)-2-cyanoethoxyphosphine (569 mg,
1.89 mmol) were added at rt. After stirring for 3 h, the reaction
mixture was diluted with CH2Cl2 (40 mL) and washed with brine
(15 mL). The aqueous phase was extracted with CH2Cl2 (40 mL)
and the combined organic phase evaporated to dryness. The
resulting residue was purified by silica gel column chromatography
(0–100% EtOAc in PE), affording the desired phosphoramidite 5
(432 mg, 77%) as a white solid. Rf 0.5 (75% EtOAc in PE, v/v);
Found: C, 63.5; H, 6.4 N, 7.4. Calcd for C41H49N4O9P: C, 63.7;
H, 6.4; N, 7.25; dP (DMSO-d6) 147.9, 147.6; ESI-HRMS m/z
795.3128 ([M + Na]+, C41H49N4O9PNa+ Calcd 795.3129).
H4Bz), 7.47–7.53 (m, 2H, H3Bz, H5Bz), 7.32–7.42 (m, 2H, H3¢¢DMT
H5¢¢DMT), 7.19–7.26 (m, 7H, H2DMT, H6DMT, H2¢DMT, H6¢DMT
,
,
H2¢¢DMT, H4¢¢DMT, H6¢¢DMT), 6.92 (d, J = 1.0, 1H, H6), 6.75–6.79
(m, 2H, [H3DMT, H5DMT]/[H3¢DMT, H5¢DMT]), 6.71–6.75 (m, 2H,
[H3DMT, H5DMT]/[H3¢DMT, H5¢DMT]), 5.26 (s, 1H, H1¢), 4.94 (d, J =
13.0, 1H, H5¢A), 4.83 (d, J = 13.0, 1H, H5¢B), 4.37 (d, J = 8.2,
1H, H5¢¢A), 3.95 (d, J = 8.2, 1H, H5¢¢A), 3.69 (s, 3H, OCH3),
3.66 (s, 1H, H3¢), 3.62 (s, 3H, OCH3), 2.96 (s, 1H, H2¢), 1.33
(d, J = 1.0, 3H, 5-CH3); dC (DMSO-d6) 165.0 (COPh), 163.5
(C4), 158.6 (C4DMT/C4¢DMT), 158.4 (C4DMT/C4¢DMT), 149.4 (C2),
144.7 (C1¢¢DMT), 134.8 (C1DMT/C1¢DMT), 134.6 (C1DMT/C1¢DMT),
133.8 (C4Bz), 132.5 (C6), 130.0 ([C2DMT, C6DMT]/[C2¢DMT, C6¢DMT]),
129.8 ([C2DMT, C6DMT]/[C2¢DMT, C6¢DMT]), 129.2 (C3Bz, C5Bz), 128.9
(1R,3R,4R,7S)-1-Benzoyloxymethyl-7-(4,4¢-dimethoxytrityloxy)-
3-(5-methylcytosin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (6)
(C1Bz), 128.8 (C2Bz, C6Bz), 127.8 (C2¢¢DMT, C6¢¢DMT), 127.4 (C3¢¢DMT
,
C5¢¢DMT), 127.0 (C4¢¢DMT), 113.2 ([C3DMT, C5DMT]/[C3¢DMT, C5¢DMT]),
113.1 ([C3DMT, C5DMT]/[C3¢DMT, C5¢DMT]), 108.7 (C5), 87.0 (CAr3),
86.3 (C1¢), 86.1 (C4¢), 76.8 (C2¢), 71.9 (C5¢¢), 71.8 (C3¢), 58.8
(C5¢), 55.0 (OCH3), 54.9 (OCH3), 11.7 (5-CH3); ESI-HRMS m/z
699.2321 ([M + Na]+, C39H36N2O9Na+ Calcd 699.2313).
Nucleoside 3 (1.70 g, 2.51 mmol) was co-evaporated with anhy-
drous CH3CN (2 ¥ 25 mL) and then redissolved in anhydrous
CH3CN (60 mL) at rt. Anhydrous Et3N (6.0 mL, 43 mmol) and
1,2,4-triazole (2.08 g, 30.1 mmol) were added under stirring. The
◦
resulting mixture was cooled to 0 C on an ice bath and freshly
(1S,3R,4R,7S)-7-(4,4¢-Dimethoxytrityloxy)-1-hydroxymethyl-3-
(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (4)
distilled POCl3 (0.69 mL, 7.53 mmol) was added dropwise to give
a white slurry. After 15 min the reaction mixture was allowed to
reach rt. After stirring for 3 h, TLC indicated full conversion to
an intermediate (Rf(3) = 0.6; Rf(intermediate) = 0.4, 70% EtOAc
Benzoate ester 3 (3.40 g, 5.02 mmol) was dissolved in a mixture
of CH2Cl2 (20 mL), MeOH (60 mL) and H2O (20 mL). K2CO3
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in PE), and the reaction mixture was poured into a slurry of
sat. aq NaHCO3 (50 mL) and ice (25 mL) and extracted with
EtOAc (3 ¥ 25 mL). The combined organic phase was washed with
brine (100 ml) and dried over Na2SO4. Filtration and evaporation
under reduced pressure afforded this intermediate as white foam
which was immediately dissolved in a mixture of 28% aq NH3 and
CH3CN (50 : 50, 70 mL, v/v). After stirring for 2 h at rt, brine
(40 mL) was added which resulted in separation into two phases.
The aqueous phase was extracted with EtOAc (3 ¥ 50 mL), the
combined organic phase evaporated to dryness and the resulting
residue purified by silica gel column chromatography (0–10% i-
PrOH in CH2Cl2, v/v) to afford nucleoside 6 (1.65 g, 97% from
3) as a white solid. Rf 0.3 (10% i-PrOH in CH2Cl2, v/v); Found:
C, 69.6; H, 5.3; N, 6.1. Calcd for C39H37N3O8: C, 69.3; H, 5.5;
N, 6.2; dH (DMSO-d6) 7.71–7.75 (m, 2H, H2Bz, H6Bz), 7.68–7.71
(m, 1H, H4Bz), 7.47–7.52 (m, 2H, H3Bz, H5Bz), 7.36–7.40 (m,
H5¢¢B), 3.68 (s, 1H, H3¢), 3.67 (s, 3H, OCH3), 3.59 (s, 3H, OCH3),
3.05 (br s, 1H, H2¢), 1.57 (s, 3H, 5-CH3); dC (DMSO-d6) 165.0
(5¢-OCOPh), 158.6 (C4DMT/C4¢DMT), 158.5 (C4DMT/C4¢DMT), 144.8
(C1¢¢DMT), 134.7 (C1DMT/C1¢DMT), 134.6 (C1DMT/C1¢DMT), 133.7
(C4OBz), 132.6 (C4NBz), 130.0 ([C2DMT, C6DMT]/[C2¢DMT, C6¢DMT]),
129.8 ([C2DMT, C6DMT]/[C2¢DMT, C6¢DMT]), 129.2 (C2NBz, C6NBz
C2OBz, C6OBz), 128.9 (C1OBz), 128.8 (C3OBz, C5OBz), 128.3 (C3NBz
,
,
C5NBz), 127.8 (C3¢¢DMT, C5¢¢DMT), 127.4 (C2¢¢DMT, C6¢¢DMT), 127.0
(C4¢¢DMT), 113.2 ([C3DMT, C5DMT]/[C3¢DMT, C5¢DMT]), 113.1 ([C3DMT
,
C5DMT]/[C3¢DMT, C5¢DMT]), 109.3 (C5), 87.0 (CAr3), 86.8 (C4¢), 86.4
(C1¢), 76.5 (C2¢), 71.9 (C5¢¢), 71.8 (C3¢), 58.9 (C5¢), 54.9 (OCH3),
54.8 (OCH3), 12.8 (5-CH3);14 ESI-HRMS m/z 802.2726 ([M +
Na]+, C46H41N3O9Na+ Calcd 802.2735).
(1S,3R,4R,7S)-3-(4-N-Benzoyl-5-methylcytosin-1-yl)-7-(4,4¢-
dimethoxytrityloxy)-1-hydroxymethyl-2,5-
dioxabicyclo[2.2.1]heptane (8)
2H, H2¢¢DMT, H6¢¢DMT), 7.15–7.25 (m, 7H, H2DMT, H6DMT, H2¢DMT
H6¢DMT, H3¢¢DMT, H4¢¢DMT, H5¢¢DMT), 6.83 (s, 1H, H6), 6.71–6.75 (m,
2H, [H3DMT, H5DMT]/[H3¢DMT, H5¢DMT]), 6.68–6.71 (m, 2H, [H3DMT
,
,
Nucleoside 7 (3.66 g, 4.69 mmol) was dissolved in THF (250 mL) at
rt and aq LiOH (1.0 M, 50 mL) was added. After stirring for 40 h,
the reaction mixture was partitioned between EtOAc (100 mL)
and brine (100 mL) and the aqueous phase was extracted with
EtOAc (2 ¥ 100 ml). The combined organic phase was dried over
MgSO4 and then evaporated to dryness. The resulting residue was
purified by silica gel column chromatography (0–80% EtOAc in
PE, v/v) to afford nucleoside 8 (2.96 g, 93%) as a white foam.
Rf 0.4 (50% EtOAc in PE, v/v); Found: C, 68.0; H, 5.6; N,
5.9. Calcd for C39H37N3O8·2/3H2O: C, 68.1; H, 5.6; N, 6.1; dH
(DMSO-d6) 8.11–8.24 (m, 2H, H2Bz, H6Bz), 7.58–7.66 (m, 2H,
H4Bz, H6), 7.49–7.54 (m, 2H, H3Bz, H5Bz), 7.39–7.43 (m, 2H,
H2¢¢DMT, H6¢¢DMT), 7.25–7.31 (m, 2H, H3¢¢DMT, H5¢¢DMT), 7.18–7.24
(m, 5H, H2DMT, H6DMT, H2¢DMT, H6¢DMT, H4¢¢DMT), 6.78–6.84 (m,
4H, H3DMT, H5DMT, H3¢DMT, H5¢DMT), 5.33 (t, J = 5.0, 1H, 5¢-OH),
5.30 (s, 1H, H1¢), 4.20 (d, J = 7.9, 1H, H5¢¢A), 3.93–4.03 (m, 2H,
H5¢A+B), 3.80 (d, J = 7.9, 1H, H5¢¢B), 3.70 (s, 6H, OCH3), 3.57 (s, 1H,
H3¢), 2.90 (s, 1H, H2¢), 1.89 (s, 3H, 5-CH3); dC (DMSO-d6) 158.5
(C4DMT, C4¢DMT), 144.9 (C1¢¢DMT), 135.2 (C1DMT/C1¢DMT), 134.9
H5DMT]/[H3¢DMT, H5¢DMT]), 5.25 (s, 1H, H1¢), 4.92 (d, J = 12.9, 1H,
H5¢A), 4.77 (d, J = 12.9, 1H, H5¢B), 4.34 (d, J = 8.1, 1H, H5¢¢A),
3.94 (d, J = 8.1, 1H, H5¢¢B), 3.71 (s, 3H, OCH3), 3.63 (s, 3H,
OCH3), 3.62 (s, 1H, H3¢), 3.05 (s, 1H, H2¢), 1.36 (s, 3H, 5-CH3);
dC (DMSO-d6) 165.3 (C4), 165.0 (COPh), 158.5 (C4DMT/C4¢DMT),
158.4 (C4DMT/C4¢DMT), 154.2 (C2), 144.8 (C1¢¢DMT), 134.8
(C6), 134.7 (C1DMT/C1¢DMT), 134.6 (C1DMT/C1¢DMT), 133.7
(C4Bz), 129.9 ([C2DMT, C6DMT]/[C2¢DMT, C6¢DMT]), 129.8 ([C2DMT
,
C6DMT]/[C2¢DMT, C6¢DMT]), 129.2 (C3Bz, C5Bz), 128.9 (C1Bz), 128.8
(C2Bz, C6Bz), 127.8 (C3¢¢DMT, C5¢¢DMT), 127.4 (C2¢¢DMT, C6¢¢DMT),
126.9 (C4¢¢DMT), 113.14 ([C3DMT, C5DMT]/[C3¢DMT, C5¢DMT]), 113.09
([C3DMT, C5DMT]/[C3¢DMT, C5¢DMT]), 100.7 (C5), 86.9 (CAr3), 86.8
(C1¢), 85.8 (C4¢), 76.8 (C2¢), 71.8 (C5¢¢), 71.5 (C3¢), 58.8 (C5¢), 55.0
(OCH3), 54.9 (OCH3), 12.8 (5-CH3); ESI-HRMS m/z 698.2442
([M + Na]+, C39H37N3O8Na+ Calcd 698.2473).
(1R,3R,4R,7S)-3-(4-N-Benzoyl-5-methylcytosin-1-yl)-1-
benzoyloxymethyl-7-(4,4¢-dimethoxytrityloxy)-2,5-
dioxabicyclo[2.2.1]heptane (7)
(C1DMT/C1¢DMT), 132.5 (C4Bz), 129.9 ([C2DMT, C6DMT]/[C2¢DMT
C6¢DMT]), 129.8 ([C2DMT, C6DMT]/[C2¢DMT, C6¢DMT]), 129.2 (C2Bz,
C6Bz), 128.3 (C3Bz, C5Bz), 127.8 (C3¢¢DMT, C5¢¢DMT), 127.5 (C2¢¢DMT
,
Nucleoside 6 (3.32 g, 4.92 mmol) was co-evaporated with an-
hydrous pyridine (2 ¥ 10 mL) and then redissolved in anhydrous
pyridine (80 mL) at rt. Benzoyl chloride (0.90 mL, 7.75 mmol) was
added and the reaction mixture stirred for 3 h and then evaporated
to dryness under reduced pressure. The resulting residue was
dissolved in EtOAc (150 mL) and washed successively with sat.
aq NaHCO3 (100 mL) and brine (100 mL). The combined organic
phase was evaporated to dryness and the resulting residue purified
by silica gel column chromatography (0–45% EtOAc in PE, v/v)
to afford nucleoside 7 (3.66 g, 94%) as a white foam. Rf 0.6 (50%
EtOAc in PE, v/v); Found: C, 70.0; H, 5.4; N, 5.1. Calcd for
C46H41N3O9·1/2H2O: C, 70.0; H, 5.4; N, 5.3; dH (DMSO-d6) 8.12–
8.19 (m, 2H, H2NBz, H6NBz), 7.73–7.78 (m, 2H, H2OBz, H6OBz), 7.67–
7.73 (m, 1H, H4OBz), 7.58–7.63 (m, 1H, H4NBz), 7.48–7.54 (m, 4H,
H3OBz, H5OBz, H3NBz, H5NBz), 7.39–7.42 (m, 2H, H2¢¢DMT, H6¢¢DMT),
,
C6¢¢DMT), 126.9 (C4¢¢DMT), 113.1 ([C3DMT, C5DMT]/[C3¢DMT, C5¢DMT]),
113.0 ([C3DMT, C5DMT]/[C3¢DMT, C5¢DMT]), 109.0 (C5), 89.0 (C4),
86.9 (CAr3), 86.7 (C1¢), 76.3 (C2¢), 72.0 (C5¢¢), 71.3 (C3¢), 55.9
(C5¢), 55.0 (OCH3), 13.2 (5-CH3);14 ESI-HRMS m/z 698.2476
([M + Na]+, C39H37N3O8PNa+ Calcd 698.2473).
(1R,3R,4R,7S)-3-(4-N-Benzoyl-5-methylcytosin-1-yl)-1-(2-
cyanoethoxy(diisopropylamino)phosphinoxymethyl-7-(4,4¢-
dimethoxytrityloxy)-2,5-dioxabicyclo[2.2.1]heptane (9)
Nucleoside 8 (549 mg, 0.83 mmol) was co-evaporated with
anhydrous 1,2-dichloroethane (15 mL) and dissolved in an-
hydrous CH2Cl2 (50 mL) at rt. N,N¢-Diisopropylammonium
tetrazolide (279 mg, 1.63 mmol) and bis(N,N¢-diisopropylamino)-
2-cyanoethoxyphosphine (546 mg, 1.81 mmol) were added, and
the reaction mixture was stirred for 3.5 h, diluted with CH2Cl2
(50 mL), and washed with brine (25 mL). The aqueous phase
was extracted with CH2Cl2 (2 ¥ 25 mL) and the combined organic
phase was dried over MgSO4 and then evaporated to dryness under
7.15–7.26 (m, 8H, H6, H2DMT, H6DMT, H2¢DMT, H6¢DMT, H3¢¢DMT
H4¢¢DMT, H5¢¢DMT), 6.75–6.79 (m, 2H, [H3DMT, H5DMT]/[H3¢DMT
,
,
H5¢DMT]), 6.72–6.75 (m, 2H, [H3DMT, H5DMT]/[H3¢DMT, H5¢DMT]),
5.35 (s, 1H, H1¢), 4.98 (d, J = 13.0, 1H, H5¢A), 4.87 (d, J = 13.0,
1H, H5¢B), 4.41 (d, J = 8.2, 1H, H5¢¢A), 4.00 (d, J = 8.2, 1H,
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reduced pressure. The resulting residue was purified by silica gel
column chromatography (0–40% EtOAc in PE, v/v) to afford
amidite 9 (574 mg, 82%) as a white foam. Rf 0.6 (50% EtOAc in
PE, v/v); Found: C, 65.6; H, 6.2; N, 7.9. Calcd for C48H54N5O9P:
C, 65.8; H, 6.2; N, 8.0; dP (DMSO-d6) 148.0, 147.6; ESI-HRMS
m/z 898.3563 ([M + Na]+, C48H54N5O9PNa+ Calcd. 898.3551).
proceeded smoothly affording the desired ON in high yield and
purity.
Acknowledgements
We greatly appreciate financial support from The Danish National
Research Foundation and the European Union Grant (FP-
037283). We thank Mr. Brian Hermansen for assistance during
synthesis, Ms. Joan Hansen for ON synthesis and workup and
Ms. Tina Grubbe Hansen for ON purification.
Oligonucleotide synthesis
ONs (5¢-GCA TLAT MeCLAC-3¢) were synthesized on 0.2 mmol
scale using universal support polystyrene columns on an
automated DNA synthesizer, following standard protocol
(trichloroacetic acid in CH2Cl2 (3 : 97, v/v) as detrityla-
tion reagent; 0.25 M 4,5-dicyanoimidazole (DCI) in CH3CN
as activator; acetic anhydride/THF (9 : 91, v/v) and 1-
methylimidazole/pyridine/THF (1 : 1 : 8, v/v) as cap solutions,
and 0.02 M iodine in water/pyridine/THF (9 : 0.4 : 91, v/v) as
oxidizing solution; Coupling times, DNA monomers: 2 min, LNA
monomers: 15 min). After deprotection and cleavage from solid
support (32% aq. NH3, 12 h, 55 ◦C), purification of ONs (DMT-
ON) was performed by RP-HPLC using a Waters 600 system
equipped with an Xterra MS C18 (10 mm, 7.8 ¥ 10 mm) pre-
column and an Xterra MS C18 (10 mm, 7.8 ¥ 150 mm) column
using a gradient of 0–70% Buffer B in Buffer A (Buffer A: 0.05M
aq. triethylammonium acetate, pH 7.4; Buffer B: 75% MeCN
in buffer A (v/v)). The purified ON were detritylated (80% aq.
AcOH, 20 min, rt) and precipitated (abs. EtOH, -18 ◦C, 12–16 h).
The composition of the synthesized ONs were verified by MALDI-
MS analysis, m/z ([M+H]+, found, 3¢→5¢/found, 5¢→3¢/calcd):
2751/2752/2753. Purity (>90%) was verified by ion-exchange
HPLC (using an a LaChrom L-7000 system equipped with a
Dionex PA100 column (4 ¥ 250 mm) using a gradient of 2–
21% Buffer C and 10% Buffer D in water (Buffer C: 1.0 M aq.
NaClO4; Buffer D: 0.25 M aq. Tris-HCl, pH 8.0). The co-injection
experiment was performed with equimolar amounts of the two
batches, using the same protocol.
Notes and references
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Conclusions
A viable and efficient synthesis of LNA thymine and LNA 4-
N-benzoyl-5-methylcytosine 5¢-phosphoramidites has been de-
veloped. Notably, the high yielding steps of regioselective O5¢-
benzoylation of the LNA thymine diol and subsequent quantita-
tive 3¢-O-dimethoxytritylation allowed synthesis of LNA thymine
and LNA 4-N-benzoyl-5-methylcytosine 5¢-phosphoramidites (5
and 9) in gratifying 65% (four steps) and 65% (seven steps)
overall yield from LNA thymine diol 1, respectively. ON synthesis
12 M. Tarko¨y, M. Bolli and C. Leumann, Helv. Chim. Acta, 1994, 77, 716.
13 (a) K. J. Divakar and C. B. Reese, J. Chem. Soc., Perkin Trans. 1, 1982,
1171; (b) A. M. Miah, C. B. Reese and Q. Song, Nucleosides, Nucleotides
Nucleic Acids, 1997, 16, 53.
14 Peaks for C2, C4, C6, 4-NCOPh and C1NBz were not visible in 13C-
NMR, probably due to fast quadrupolar relaxation via the nearby
14N-nuclei.
5016 | Org. Biomol. Chem., 2010, 8, 5012–5016
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The Royal Society of Chemistry 2010
©