Reinvestigation into the synthesis of oligonucleotides containing 5-(β-D-glucopyranosyloxymethyl)-2′-deoxyuridine

Article abstract of DOI:10.1002/ejoc.200300218

A reinvestigation into the synthesis of oligonucleotides containing 5-(β-D-glucopyranosyloxymethyl)-2′-deoxyuridine revealed that existing procedures for the preparation of these DNA fragments suffered from decomposition at the final deprotection step. Th

Full text of DOI:10.1002/ejoc.200300218

FULL PAPER
Reinvestigation into the Synthesis of Oligonucleotides Containing
5-(β- -Glucopyranosyloxymethyl)-2Ј-deoxyuridine
D
John J. Turner,[a] Nico J. Meeuwenoord,[a] Anita Rood,[a] Piet Borst,[b]
Gijs A. van der Marel,[a] and Jacques H. van Boom*[a]
Keywords: Ammonolysis / Decomposition / Modified nucleobase / Nucleosides / Oligonucleotides
A reinvestigation into the synthesis of oligonucleotides con-
taining 5-(β- -glucopyranosyloxymethyl)-2Ј-deoxyuridine
revealed that existing procedures for the preparation of these
DNA fragments suffered from decomposition at the final de-
protection step. The decomposition product was identified as
the corresponding 5-(aminomethyl)-2Јdeoxyuridyl derivative
during ammonolysis. This was shown to be suppressed by
D
the use of phosphoramidite 21 in place of 12 for solid-phase
oligonucleotide synthesis, in conjunction with short ammonia
treatment at room temperature.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2003)
arising from amino substitution of the β-D-glucosyl moiety
Introduction
stranded DNA.[11,12] During these studies, however, it was
shown that some of the synthetic DNA fragments contain-
ing dJ were contaminated in varying amounts by an un-
specified side product.[11] Here we present our investigations
into the identification of this side product and the suppres-
sion of its formation.
The kinetoplastid flagellate Trypanosoma brucei, trans-
mitted by tsetse flies, causes African sleeping sickness in
mammals and Nagana disease in domestic cattle. It man-
ages to survive in the bloodstream of its host by virtue of
its ability to change the variant surface glycoprotein (VSG)
in its cell-surface coat so that it evades the host’s immune
systems, a process called antigenic variation.[1Ϫ4] It has
been observed that the hypermodified nucleoside 5-(β--
glucopyranosyloxymethyl)-2Ј-deoxyuridine (known as dJ) is
present in inactive telomeric VSG gene expression sites of
T. brucei but not in the active site.[5Ϫ7] This implies that dJ
is involved in the suppression of VSG gene expression sites
and consequently in antigenic variation.[8] However, the
presence of dJ in other members of the Kinetoplastida fam-
ily which do not undergo antigenic variation[9] suggests that
dJ is involved in other biological processes.
In order to gain greater insight into the biological func-
tion of dJ at a molecular level, a synthetic program for the
synthesis of short fragments of DNA containing dJ at pre-
determined sites was established. Use of these DNA frag-
ments resulted in the discovery of a protein that specifically
binds to dJ.[10] Moreover, it was also revealed that this bind-
ing is structure-specific and that recognition of the modified
nucleoside only occurs when dJ is presented in double-
Results and Discussion
Since the synthetic DNA fragments containing dJ were
synthesised by a standard procedure, it is evident that the
side product originates from modification of the J nucleob-
ase. We have previously reported phosphoramidites 7[13]
and 12[14] (Scheme 1), differing in the N3 protection of the
nucleobase, as suitable building blocks for the synthesis of
DNA fragments containing dJ. The key step en route to
7 involved Helferich condensation of the 3Ј,5Ј-diprotected
hydroxymethyldeoxyuridine (HMDU) 1 with the fully ben-
zoylated α-glucosyl bromide 2 to give protected dJ 4 in a
moderate yield (Scheme 1).[13] Endeavours to improve this
procedure not only provided a cost-effective and convenient
route towards a suitably protected HMDU derivative (8),
but also an excellent efficiency with respect to the key glyco-
sylation step (transformation 8 to 9). In this regard, it was
shown that N3 protection of the nucleobase of HMDU with
a pivaloyloxymethyl (POM) protecting group resulted in a
twofold increase in yield (see Scheme 1, compare 1 to 4 with
8 to 9, Step ii).[14] In addition, an improvement in the phos-
phoramidite formation (compare 6 to 7 with 11 to 12, Step
v) was also attributed to the use of the POM group, which
has been reported by Reese et al.[15] to be completely
cleaved with aqueous ammonia in 20 min at room tempera-
[a]
Leiden Insitute of Chemistry, Gorlaeus Laboratories,
University of Leiden,
P.O. Box 9502. 2300 RA Leiden, Netherlands
Fax: (internat.) ϩ31-(0)71-5274307
E-mail: j.boom@chem.leidenuniv.nl
[b]
Division of Molecular Biology and Centre for Biomedical
Genetics, The Netherlands Cancer Institute,
1066 CX Amsterdam, Netherlands
3832
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200300218
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Oligonucleotides Containing 5-(β-D-Glucopyranosyloxymethyl)-2Ј-deoxyuridine
FULL PAPER
Scheme 1. Reagents and conditions: (i) for 2: HgBr2, Hg(CN)2, ACN, 3 h, 50%. (ii) for 3: cat. TMSOTf, DCE, 1.5 h, 4: 47%, 9: 96%.
(iii) Et3N·3HF, pyridine, 16 h, 5: 90%, 10: 91%. (iv) DMTCl, pyridine, 3 h, 6: 71%, 11: 75%. (v) ClP(NiPr2)OEtCN, DIPEA, DCM, 1 h,
7: 69%, 12: 91%
ture. Consequently, the use of N3 protection was considered unprotected dJ incorporated in a DNA sequence, the mix-
a valuable addition to the synthesis of dJ building blocks ture was separated by anion-exchange and pure 14 (Fig-
and oligomers, as it was compatible with automated syn- ure 3, A) was treated with saturated aqueous ammonia at
thesis and the cleavage takes place during the aqueous am- different temperatures and for varying lengths of time (Fig-
monia treatment in the final stage of a standard DNA syn- ure 3). It was found that unprotected dJ, as present in the
thesis procedure.
DNA sequence 14, decomposes upon prolonged treatment
In order to identify the side product in our dJ-containing with ammonia. Gratifyingly, it was established that after 16
DNA fragments, hexameric fragment 13 (Scheme 2) was hours at room temperature there is only 1% decomposition
selected as a model compound. Thus, fully protected dJ (Figure 3, B). It is also interesting to note that after 64
phosphoramidite 12 and commercially available dT phos- hours at room temperature the decomposition is of the or-
phoramidite were used in a standard solid-phase DNA syn- der of 3% (Figure 3, C). However, subjection of 14 to the
thesis procedure and subjected to standard deprotection standard deprotection conditions used for DNA sequences
conditions (NH3 aqu., 50 °C, 16 h). Subsequent HPLC/ (NH3 aqu., 50 °C, 16 h) resulted in a significant amount
anion-exchange analysis of the crude mixture[16] revealed (15%, Figure 3, D) of decomposition of 14 into the benzylic
the presence of the expected product 14 and a significant amine 15.
amount of side product, in a ratio of 3:2 (Figure 1).
MALDI-TOF measurements on the crude sample indicated uct observed with the dJ-containing oligomers was the re-
mass difference of 163 between 14 (calcd. for sult of decomposition of dJ arising from the final deprotec-
In light of the above results, it is clear that the side prod-
a
C66H89N12O46P5: 1941.2; found 1943.4 [M ϩ H]ϩ) and the tion step with ammonia. Furthermore, it can be surmised
side product, corresponding to aminomethyl derivative 15 that two separate factors are responsible for this: the pro-
(calcd. for C60H80N13O40P5: 1778.2; found 1780.5 [M ϩ tecting groups on the J nucleobase and its own intrinsic
H]ϩ). The formation of 15 can be readily explained in terms instability towards ammonia even in its free state, particu-
of competing substitution at the benzylic position of the J larly at high temperature. In order to assess the influence
nucleobase during the ammonolysis step. This assumption of the individual protecting groups (N3, β--glucopyrano-
was further substantiated by the ammonolysis of 13 at room syl) in dJ on the decomposition of the J nucleobase, mono-
temperature, which furnished a mixture of the two com- mers 5, 10, 16, 18, and 19 were chosen for examination
pounds in a ratio of 4:1 (Figure 2). To assess the lability of (Scheme 3).[17]
Eur. J. Org. Chem. 2003, 3832Ϫ3839
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J. J. Turner, N. J. Meeuwenoord, A. Rood, P. Borst, G. A. van der Marel, J. H. van Boom
FULL PAPER
Scheme 2. Reagents and conditions: (i) NH3 (aqu.), 50 °C, 16 h, 14/15 ϭ 3:2 (see Figure 1); (ii) NH3 (aqu.), room temp., 16 h, 14/15 ϭ
4:1 (see Figure 2)
Figure 2. Crude HPLC chromatogram after treatment of 13 with
aqu. ammonia at room temperature for 16 h
Figure 1. Crude HPLC chromatogram after treatment of 13 with
aqu. ammonia at 50 °C for 16 h
try 3, Table 1), again, identical to that in the case of 14
In the first instance, fully protected nucleobase 10 was (Figure 3, B), demonstrating that monomeric and incorpor-
treated with aqueous ammonia at elevated temperature for ated dJ have comparable stabilities to decomposition. Sub-
16 h. Analysis of the resulting mixture (LCMS) revealed jection of partially protected dJ 5 (N3 unprotected) to the
(Table 1, entry 1) that the deprotected hypermodified nu- same set of conditions (entries 4 and 5) revealed a close
cleoside dJ 16 and 5-(aminomethyl)-2Ј-deoxyuridine 17[18] parallel, in terms of stability, to that of free dJ 16. Further-
were both present in a surprisingly high ratio in favour of more, negligible decomposition was observed in the depro-
the latter, indicating more than 60% decomposition. In con- tection of 18 and 19,[19] which, by virtue of their more labile
trast, subjection of compound 16 (isolated by RP-HPLC) acetyl and methoxyacetyl β--glucose hydroxy group pro-
to the same conditions (entry 2) resulted in just 15% de- tection, required just 2 h (entry 6) and 1 h (entry 7) treat-
composition, the same amount as for 14 (see Figure 3, D) ments with aqueous ammonia at room temperature, respec-
under these conditions. In addition, very little decompo- tively. The results depicted in Table 1 clearly demonstrate
sition occurred when 16 was subjected to ammonia treat- the influence of the N3 POM protecting group on the de-
ment at room temperature for the same period of time (en- composition of dJ, namely that its presence has a detrimen-
3834
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Eur. J. Org. Chem. 2003, 3832Ϫ3839

Oligonucleotides Containing 5-(β-D-Glucopyranosyloxymethyl)-2Ј-deoxyuridine
FULL PAPER
Figure 3. (A) Pure 14, obtained by preparative RP-HPLC; (B) 14 after NH3 (aqu.), room temp. 16 h; (C) 14 after NH3 (aqu.), room temp.
64 h; (D) 14 after NH3 (aqu.), 50 °C, 16 h
Scheme 3
tal effect on the stability of dJ. In contrast, the β--glucose
The lability of dJ in its free state as discussed above has
hydroxy group protection has a comparatively small influ- a direct consequence with regard to the standard DNA
ence, limited to the intrinsic lability of dJ in ammonia. In phosphoramidite building blocks used for the synthesis of
other words, although the benzoyl groups in 5 do not ad- dJ-containing oligomers. Exocyclic amino groups for aden-
versely affect the stability of dJ, the need to subject 5 to osine and cytidine are routinely protected with benzoyl
ammonia treatment overnight for complete deprotection groups, and the exocyclic amino of guanosine with an iso-
causes more decomposition than the corresponding 2 h butyryl moiety. All these require overnight treatment with
treatment for 18, it having been established that both 14 aqueous ammonia at elevated temperature (50 °C) for their
and 16 are themselves sensitive to these conditions.
complete removal, which would result in decomposition of
Eur. J. Org. Chem. 2003, 3832Ϫ3839
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J. J. Turner, N. J. Meeuwenoord, A. Rood, P. Borst, G. A. van der Marel, J. H. van Boom
FULL PAPER
Table 1. Treatment of dJ monomers with differing N3 and β--
glucose hydroxy protection with NH3 (aqu.)
Conclusion
The side product accompanying dJ-containing oligomers
when the previously reported synthetic procedures from our
laboratory[13,14] were used has been identified as the corre-
sponding 5-(aminomethyl)-2Ј-deoxyuridyl derivative arising
from amino substitution of the β--glucosyl moiety during
the final deprotection step. The investigations disclosed in
this paper have revealed that two key factors are responsible
for this decomposition: the use of POM protection for N3
and high-temperature aqueous ammonia treatment. With
respect to the latter, it was shown that unprotected dJ, either
as its monomer or incorporated into a DNA oligomeric se-
quence, is prone to significant decomposition under these
conditions. Decomposition was shown to be suppressed by
employment of a synthetic strategy of no N3 POM protec-
tion but acetyl protection for the β--glucose hydroxy
groups on the dJ phosphoramidite building block, in con-
junction with TAC exocyclic amino protection for the
standard DNA building blocks, followed by fast deprotec-
tion with ammonia at room temperature.
Entry dJ monomer
Treatment with ammonia
Ratio
of 16/17
Time/h
temp/°C
1
2
3
4
5
6
7
10
16
16
5
16
16
16
16
16
2
50
50
1:2
17:3
99:1
3.65:1
99:1
Ͼ99:1
Ͼ99:1
room temp.
50
room temp.
room temp.
room temp.
5
18
19
1
dJ. Replacement of these protecting groups with the con-
siderably more labile 4-(tert-butyl)phenoxyacetyl (TAC)
protecting group enables the deprotection time to be short-
ened to 2 h and the temperature to be lowered to ambient.
It was therefore anticipated that suppression of 5-(amino-
methyl)-2Ј-deoxyuridyl formation should be most effectively
achieved by use of these building blocks for oligonucleotide
synthesis, in conjunction with a dJ phosphoramidite build-
ing block with acetyl protection for the glucose moiety and
Ϫ crucially Ϫ without POM N3 protection.[20] To this end,
compound 18 was treated with 4,4Ј-dimethoxytrityl chlo-
ride in pyridine, affording a 90% yield of 20 (Scheme 4),
which in turn was converted into the corresponding phos-
phoramidite 21 in 96% yield. This DNA building block was
then used to synthesise oligomers 14 and 22Ϫ24 (Table 2)
through the use of standard DNA phosphoramidite build-
ing blocks with TAC exocyclic amino protection, without
any decomposition product being detectable after deprotec-
tion with aqueous ammonia for two hours at ambient tem-
perature.
Experimental Section
1
General Remarks: H and 13C NMR spectra were recorded with a
Bruker AV-400 (400/100 MHz) spectrometer. 31P NMR spectra
were recorded with a Jeol JNM-FX-200 (80.7 MHz) spectrometer.
1H and 13C chemical shifts are given in ppm (δ) relative to tetra-
methylsilane as internal standard, and 31P chemical shifts relative
to 85% H3PO4 as external standard. All 13C and 31P spectra given
are proton decoupled. Electrospray mass spectra were recorded
with a PerkinϪElmer/SCIEX API 165 mass instrument fitted with
a custom-made electrospray interface (ESI). LCMS analysis was
performed on a Jasco HPLC system (detection simultaneously at
214 and 254 nm) coupled to the aforementioned instrument, with
an Alltima C18 column (Alltech, 4.0 mm D ϫ 250 mm L, 5 µ par-
ticle size). Dichloromethane (DCM, Biosolve, HPLC-grade), 1,2-
dichloroethane (Biosolve, HPLC-grade), pyridine (Baker, p.a.), 1,4-
dioxane (Baker, p.a.) and toluene (Baker, p.a.) were stored over
˚
molecular sieves (4 A). Methanol (MeOH, Biosolve, HPLC-grade)
and acetonitrile (ACN, Biosolve, HPLC-grade) were stored over
˚
molecular sieves (3 A). N,N,N-Diisopropylethylamine (DIPEA, Bi-
osolve, p.a.) was dried by heating at reflux with CaH2 (5 g/L) for
4 h and then distilled. All chemicals (Acros, Belgium) were used as
received. Column chromatography was performed either on Fluka
60 (0.040Ϫ0.063 mm). TLC analysis was performed with DC-Fer-
tigfolien (Schleicher & Schuell, F1500, LS254) or HPTLC alu-
minium sheets (Merck, silica gel 60, F254) with detection by UV
absorption (254 nm) and charring with 20% H2SO4 in EtOH. Reac-
Scheme 4. Reagents and conditions: (i) DMTCl, py, room temp.,
16 h, 90%; (ii) ClP(NiPr2)OEtCN, DIPEA, DCM, room temp.,
96%
Table 2. ((Author: please give a Caption!))
Compound
Sequence
Mol. mass, calcd.
MALDI-TOF, [M ϩ H]ϩ
14
22
23
24
TTdJTTT
1941.2
1991.2
3810.4
8286.0
1943.4
1992.8
3814.1
8288.3
TGdJTGT
TAdJTCTTGdJTGT
(GGGdJTA)4
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Oligonucleotides Containing 5-(β-D-Glucopyranosyloxymethyl)-2Ј-deoxyuridine
FULL PAPER
tions were run at room temperature unless stated otherwise. Prior
to reactions requiring anhydrous conditions, traces of water were
0.08 (2 ϫ s, 2 ϫ 3 H, 2 ϫ Me TBDMS) ppm. 13C NMR (100 MHz,
CDCl3): δ ϭ 177.5 (CϭO POM), 162.2 (C4), 149.8 (C2), 139.0 (C6),
removed by coevaporation with 1,2-dichloroethane, 1,4-dioxane, 109.7 (C5), 102.9 (C1ЈЈ), 88.4 (C4Ј), 86.7 (C1Ј), 76.4, 75.9, 73.3, 72.5,
toluene, or pyridine.
70.1 (C3Ј/C2ЈЈ/C3ЈЈ/C4ЈЈ/C5ЈЈ), 65.1 (C7), 64.9 (CH2 POM), 63.2 (C5Ј),
62.2 (C6ЈЈ), 41.5 (C2Ј), 38.8 (Cq POM), 27.0 (CH3 tBu POM), 26.0
and 25.7 (CH3 2 ϫ tBu TBDMS), 18.4, 18.0 (2 ϫ Cq TBDMS),
Ϫ4.7, Ϫ4.8, Ϫ5.3, Ϫ5.4 (4 ϫ Me TBDMS) ppm. MS (ESI): m/z ϭ
785.6 [M ϩ Na]ϩ.
3Ј,5Ј-Bis(O-tert-butyldimethylsilyl)-2Ј-deoxy-5-(β-D-glucopyrano-
syl)oxymethyluridine (25): K2CO3 (166 mg, 1.2 mmol) was added to
a solution of compound 9[14] (1.18 g, 1.0 mmol) in MeOH (5 mL).
After stirring for 4 h at room temperature, the reaction mixture was
carefully neutralised with Dowex 50 W ϫ 4 (Hϩ) and filtered, and
the filtrate was concentrated in vacuo. The resulting oil was sub-
jected to column chromatography (petroleum ether/EtOAc/MeOH,
3:1:0 to 0:1:1, v/v/v), affording 25 (474 mg, 0.73 mmol, 73%) and
3Ј,5Ј-Bis(O-tert-butyldimethylsilyl)-2Ј-deoxy-5-methoxymethyl-N3-
1
(pivaloyloxymethyl)uridine (28): H NMR (400 MHz, CDCl3): δ ϭ
7.69 (t, J6,7 ϭ 0.9 Hz, 1 H, H6), 6.34 (dd, J1Ј,2aЈ ϭ 5.7, J1Ј,2bЈ
ϭ
8.0 Hz, 1 H, H1Ј), 5.95 (AB, 2 H, CH2 POM), 4.41 (m, 1 H, H3Ј),
26 (100 mg, 0.20 mmol, 20%) as colourless oils. Analytical data for 4.20 (ABX, 2 H, H7), 3.96 (m, 1 H, H4Ј), 3.80 (ABX, J4Ј,5aЈ ϭ 3.1,
25. 1H NMR (400 MHz, CD3OD): δ ϭ 7.76 (s, 1 H, H6), 6.21 (dd, J4Ј,5bЈ ϭ 2.9 Hz, 2 H, H5Ј), 3.40 (s, 3 H, OMe), 2.16 (ABXY,
J1Ј,2aЈ ϭ 7.8, J1Ј,2bЈ ϭ 6.1 Hz, 1 H, H1Ј), 4.57 (d, J7a,7b ϭ Ϫ12.1 Hz, J2aЈ,3Ј ϭ 2.5, J2bЈ,3Ј ϭ 5.9 Hz, 2 H, H2Ј), 1.19 (s, 9 H, tBu POM),
1 H, H7a), 4.46 (m, 1 H, H3Ј), 4.42 (d, 1 H, H7b), 3.38 (d, J1ЈЈ,2ЈЈ
7.8 Hz, 1 H, H1ЈЈ), 3.93 (m, 1 H, H4Ј), 3.86 (dd, J5ЈЈ,6aЈЈ ϭ 2.0,
ϭ
0.92 and 0.89 (2 ϫ s, 2 ϫ 9 H, 2 ϫ tBu TBDMS), 0.11 (s, 6 H, 2
ϫ Me TBDMS), 0.08 and 0.08 (2 ϫ s, 2 ϫ 3 H, 2 ϫ Me TBDMS)
J6aЈЈ,6bЈЈ ϭ Ϫ11.9 Hz, 1 H, H6aЈЈ), 3.80 (d, J4Ј,5Ј ϭ 4.4 Hz, 2 H, H5Ј), ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 177.5 (CϭO POM), 161.3
3.68 (dd, J5ЈЈ,6bЈЈ ϭ 5.1 Hz, 1 H, H6bЈЈ), 3.36 (m, 1 H, H3ЈЈ), (C4), 150.1 (C2), 137.0 (C6), 110.9 (C5), 88.0 (C4Ј), 85.9 (C1Ј), 72.3
3.32Ϫ3.29 (m, 2 H, H4ЈЈ and H5ЈЈ), 3.22 (dd, J2ЈЈ,3ЈЈ ϭ 8.9 Hz, 1 H,
(C3Ј), 67.2 (C7) 64.8 (CH2 POM), 63.0 (C5Ј), 58.6 (OMe), 41.3 (C2Ј),
H2ЈЈ), 2.27Ϫ2.16 (m, 1 H, H2Ј), 0.92 and 0.92 (2 ϫ s, 2 ϫ 9 H, 2 ϫ 38.8 (Cq POM), 27.0 (CH3 POM), 25.9, 25.7 (CH3 2 ϫ tBu
tBu TBDMS), 0.12 (s, 12 H, 4 ϫ Me TBDMS) ppm. 13C NMR TBDMS), 18.4, 18.0 (2 ϫ Cq TBDMS), Ϫ4.7, Ϫ4.9, Ϫ5.5, Ϫ5.6 (4
(100 MHz, CD3OD): δ ϭ 165.2 (C4), 151.9 (C2), 141.0 (C6), 112.1 ϫ Me TBDMS) ppm. MS (ESI): m/z ϭ 615.4 [M ϩ H]ϩ, 637.6 [M
(C5), 103.9 (C1ЈЈ), 89.2 (C4Ј), 86.9 (C1Ј), 77.8 (C3ЈЈ), 77.8 (C5ЈЈ), 74.8 ϩ Na]ϩ, 1251.7 [2 M ϩ Na]ϩ.
(C2ЈЈ), 73.9 (C3Ј), 71.4 (C4ЈЈ), 64.9 (C7), 64.3 (C5Ј), 62.6 (C6ЈЈ), 41.2
3Ј,5Ј-Bis(O-tert-butyldimethylsilyl)-2Ј-deoxy-5-(2,3,4,6-tetra-O-
(C2Ј), 26.5 and 26.2 (CH3 2 ϫ tBu TBDMS), 19.2 and 18.7 (2 ϫ
acetyl-β-D-glucopyranosyl)oxymethyluridine (29): Acetic anhydride
Cq TBDMS), Ϫ4.4, Ϫ4.5, Ϫ5.0, Ϫ5.1 (4 ϫ Me TBDMS) ppm. MS
(0.4 mL, 429 mg, 4.2 mmol) was added to a solution of 25 (454 mg,
0.7 mmol) in pyridine (5 mL) and the reaction mixture was stirred
overnight at room temperature. The reaction was quenched by the
(ESI): m/z ϭ 649.4 [M ϩ H]ϩ, 671.5 [M ϩ Na]ϩ.
3Ј,5Ј-Bis(O-tert-butyldimethylsilyl)-2Ј-deoxy-5-(methoxymethyl)-
uridine (26): 1H NMR (400 MHz, CD3OD/CDCl3, 1:1 v/v): δ ϭ addition of MeOH (2 mL) and after a period of 15 min the mixture
7.71 (s, 1 H, H6), 6.30 (dd, J1Ј,2aЈ ϭ 8.0, J1Ј,2bЈ ϭ 5.8 Hz, 1 H, H1Ј),
was concentrated in vacuo. The resulting residue was partitioned
4.46Ϫ4.43 (m, 1 H, H3Ј), 4.17 (AB, 2 H, H7), 3.98Ϫ3.96 (m, 1 H, between EtOAc and water, and the organic layer was washed with
H4Ј), 3.82 (ABX, J4Ј,5aЈ ϭ 3.1, J4Ј,5bЈ ϭ 2.9, J5aЈ,5bЈ ϭ Ϫ11.4 Hz, 2 brine, dried (MgSO4), concentrated, and subjected to column chro-
H, H5Ј), 3.40 (s, 3 H, OMe), 2.17 (ABXY, J2aЈ,3Ј ϭ 6.0, J2bЈ,3Ј
2.5, J2aЈ,2bЈ ϭ Ϫ13.2 Hz, 2 H, H2Ј), 0.94 and 0.91 (2 ϫ s, 2 ϫ 9 H,
ϭ
matography (EtOAc/light petroleum) to afford the title compound
as a colourless syrup (549 mg, 0.67 mmol, 96%). 1H and 13C NMR
2 ϫ tBu TBDMS), 0.13, 0.11, 0.11 (3 ϫ s, 12 H, 4 ϫ Me TBDMS) were in full accordance with that reported previously.[21] MS (ESI):
ppm. 13C NMR (100 MHz, CD3OD/CDCl3, 1:1 v/v): δ ϭ 164.1
(C4), 151.2 (C2), 139.0 (C6), 112.0 (C5), 88.5 (C4Ј), 85.7 (C1Ј), 72.9
(C3Ј), 67.2 (C7), 63.5 (C5Ј), 58.6 (OMe), 41.7 (C2Ј), 26.3 and 26.0
(CH3, 2 ϫ tBu TBDMS), 18.8 and 18.4 (2 ϫ Cq TBDMS), Ϫ4.5,
Ϫ4.6, Ϫ5.2, Ϫ5.3 (4 ϫ Me TBDMS) ppm. MS (ESI): m/z ϭ 501.4
[M ϩ H]ϩ, 523.6 [M ϩ Na]ϩ, 1001.7 [2 M ϩ H]ϩ, 1023.6 [2 M
ϩ Na]ϩ.
m/z ϭ 817.4 [M ϩ H]ϩ, 839.6 [M ϩ Na]ϩ.
3Ј,5Ј-Bis(O-tert-butyldimethylsilyl)-2Ј-deoxy-5-(2,3,4,6-tetra-O-
methoxyacetyl-β-D-glucopyranosyl)oxymethyluridine (30): Meth-
oxyacetic acid anhydride[22] (97 mg, 0.6 mmol) was added to a solu-
tion of 25 (65 mg, 0.1 mmol) in pyridine (1 mL), and the reaction
mixture was stirred overnight at room temperature. The reaction
was quenched by the addition of MeOH (0.2 mL) and after a per-
iod of 15 min the mixture was concentrated in vacuo. The resulting
residue was partitioned between EtOAc and water, and the organic
3Ј,5Ј-Bis(O-tert-butyldimethylsilyl)-2Ј-deoxy-5-(β-
D-glucopyrano-
syl)oxymethyl-N3-(pivaloyloxymethyl)uridine (27): K2CO3 (2 mg, 14
µmol) was added to a solution of compound 9[14] (118 mg, layer was washed with brine, dried (MgSO4), concentrated, and
0.1 mmol) in MeOH (2 mL). After stirring for 16 h at room tem-
perature the reaction mixture was carefully neutralised with Dowex
50 W ϫ 4 (Hϩ) and filtered, and the filtrate was concentrated in
vacuo. The crude material was subjected to column chromatogra-
phy (petroleum ether/EtOAc/MeOH, 9:1:0 to 0:9:1, v/v/v) to give
27 (31 mg, 41 µmol, 41%) and 26 (7 mg, 11 µmol, 11%) as colour-
subjected to column chromatography (EtOAc/light petroleum) to
afford the title compound as a colourless syrup (81 mg, 87 µmol,
1
87%). H NMR (400 MHz, CDCl3): δ ϭ 9.13 (s, 1 H, NH), 7.70
(s, 1 H, H6), 6.30 (dd, J1Ј,2aЈ ϭ 5.7, J1Ј,2bЈ ϭ 8.1 Hz, 1 H, H1Ј), 5.28
(m, 1 H, H3ЈЈ), 5.10 (m, 1 H, H4ЈЈ), 5.00 (dd, J1ЈЈ,2ЈЈ ϭ 8.1, J2ЈЈ,3ЈЈ
ϭ
9.5 Hz, 1 H, H2ЈЈ), 4.75 (d, 1 H, H1ЈЈ), 4.42 (AB, 2 H, H7), 4.37 (m,
less oils, together with recovered starting material 9 (33 mg, 28 1 H, H3Ј), 4.26 (ABX, J5ЈЈ,6aЈЈ ϭ 4.4, J5ЈЈ,6bЈЈ ϭ 2.2 Hz, 2 H, H6ЈЈ),
µmol, 28%). Analytical data for 27: H NMR (400 MHz, CDCl3):
δ ϭ 7.81 (s, 1 H, H6), 6.25 (dd, J1Ј,2aЈ ϭ 5.8, J1Ј,2bЈ ϭ 7.7 Hz, 1 H,
H1Ј), 5.93 (AB, 2 H, CH2 POM), 4.56 (dd, J7a,7b ϭ 11.6 Hz, 1 H,
H7a), 4.47Ϫ4.41 (m, 2 H, H7b and H1ЈЈ), 4.38 (m, 1 H, H3Ј), 3.99
4.09 (AB, 2 H, 1 ϫ CH2, MAc), 4.10Ϫ3.93 (m, 7 H, H4Ј, 3 ϫ CH2
MAc), 3.83Ϫ3.72 (m, 3 H, H5Ј, H5ЈЈ), 3.45, 3.39, 3.36, 3.36 (4 ϫ s,
1
4 ϫ 3 H, 4 ϫ CH3 MAc), 2.30 (1/2ABXY, J2aЈ,3Ј ϭ 2.3, J2aЈ,2bЈ
ϭ
13.2 Hz, H2aЈ), 2.03Ϫ1.96 (m, 1 H, H2bЈ), 0.89 and 0.88 (2 ϫ s, 2
(m, 1 H, H4Ј), 3.88Ϫ3.74 (m, 4 H, H5Ј and H6ЈЈ), 3.57Ϫ3.55 (m, 2 ϫ 9 H, 2 ϫ tBu TBDMS), 0.08 (s, 6 H, 2 ϫ Me TBDMS), 0.07
H, H3ЈЈ and H4ЈЈ), 3.41Ϫ3.37 (m, 2 H, H2ЈЈ and H5ЈЈ), 2.34 (1/ and 0.06 (2 ϫ s, 2 ϫ 3 H, 2 ϫ Me TBDMS) ppm. 13C NMR
2ABXY, J2aЈ,3Ј ϭ 2.3, J2aЈ,2bЈ ϭ 13.2 Hz, 1 H, H2aЈ), 2.08Ϫ2.02 (m, (100 MHz, CDCl3): δ ϭ 169.9, 169.6, 169.2, 169.0 (4 ϫ CϭO
1 H, H2bЈ), 1.18 (s, 9 H, tBu POM), 0.91 and 0.89 (2 ϫ s, 2 ϫ 9 MAc), 162.8 (C4), 149.9 (C2), 139.7 (C6), 110.7 (C5), 100.9 (C1ЈЈ),
H, 2 ϫ tBu TBDMS), 0.10 (s, 6 H, 2 ϫ Me TBDMS), 0.08 and
88.2 (C4Ј), 85.7 (C1Ј), 73.1 (C3ЈЈ), 72.8 (C3Ј), 71.7 (C5ЈЈ), 71.3 (C2ЈЈ),
Eur. J. Org. Chem. 2003, 3832Ϫ3839
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 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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J. J. Turner, N. J. Meeuwenoord, A. Rood, P. Borst, G. A. van der Marel, J. H. van Boom
69.2 (4 ϫ CH2 MAc), 68.4 (C4ЈЈ), 64.9 (C7), 63.1 (C5Ј), 62.0 (C6ЈЈ), J5aЈ,5bЈ ϭ Ϫ10.5 Hz, 2 H, H5Ј), 2.51Ϫ2.45 (m, 1 H, H2aЈ), 2.34Ϫ2.25
FULL PAPER
59.4, 59.3, 59.3, 59.2 (4 ϫ CH3 MAc), 41.5 (C2Ј), 25.9, 25.7 (CH3
(m, 1 H, H2bЈ), 2.01, 2.01, 1.99, 1.92 (4 ϫ s, 4 ϫ 3 H, 4 ϫ CH3 Ac)
2 ϫ tBu TBDMS), 18.3, 17.9 (2 ϫ Cq TBDMS), Ϫ4.7, Ϫ4.9, Ϫ5.4, ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 170.6, 170.1, 169.4, 169.4
Ϫ5.5 (4 ϫ Me TBDMS) ppm. MS (ESI): m/z ϭ 959.4 [M ϩ Na]ϩ. (4 ϫ CϭO Ac), 162.9 (C4), 158.6 (2 ϫ Cq DMT), 150.2 (C2), 144.5
(Cq DMT), 139.5 (C6), 135.4, 135.2 (2 ϫ Cq DMT), 130.0, 128.1,
2Ј-Deoxy-5-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy-
127.9, 126.9, 113.3 (CHarom DMT), 110.9 (C5), 100.7 (C1ЈЈ), 86.8
(Cq DMT), 86.1 (C4Ј), 85.4 (C1Ј), 72.8 (C3ЈЈ), 72.2 (C3Ј), 71.6 (C5ЈЈ),
71.2 (C2ЈЈ), 68.3 (C4ЈЈ), 64.1 (C7), 63.5 (C5Ј), 61.7 (C6ЈЈ), 55.1 (OMe
DMT), 40.8 (C2Ј), 20.5 (4 ϫ CH3 Ac) ppm. MS (ESI): m/z ϭ 913.6
[M ϩ Na]ϩ.
methyluridine (18): Et3N·3HF (0.22 mL, 1.30 mmol) was added to
a stirring solution of 29 (533 mg, 0.65 mmol) in pyridine (3 mL).
After 16 h at room temperature, the reaction mixture was concen-
trated, dissolved in EtOAc, and washed with brine (2 ϫ). The or-
ganic layer was dried (MgSO4), filtered, and concentrated in vacuo.
The resulting residue was purified by column chromatography
(EtOAc/MeOH, 1:0 to 9:1, v/v) to give 18 (347 mg, 0.59 mmol,
2Ј-Deoxy-5Ј-O-(4,4Ј-dimethoxytrityl)-5-(2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosyl)oxymethyluridine 3Ј-O-(2-Cyanoethyl-N,N-diisopro-
pyl)phosphoramidite (21): DIPEA (0.35 mL, 2 mmol) was added
under an atmosphere of argon, to a stirred solution of 20 (445 mg,
0.45 mmol) in DCM (10 mL), followed by 2-cyanoethyl diisopro-
pylchlorophosphoramidite[23] (0.13 mL, 0.57 mmol). After 1 h, the
reaction mixture was diluted with DCM and washed with aqueous
NaHCO3 (9%) and water. The organic layer was dried (Na2SO4),
filtered, and concentrated in vacuo, and the resulting residue was
purified by flash column chromatography (EtOAc/light petroleum/
Et3N, 50:150:1 to 150:50:1, v/v/v), affording 21 (475 mg,
0.43 mmol, 96%) as a white foam. 31P NMR (80.7 MHz, DCM-
acetone capillary): δ ϭ 149.1 and 149.0.
1
91%) as a syrup. H NMR (400 MHz, CDCl3): δ ϭ 10.07 (br. s, 1
H, NH), 7.85 (s, 1 H, H6), 6.30 (m, 1 H, H1Ј), 5.26 (m, 1 H, H3ЈЈ),
5.11 (m, 1 H, H4ЈЈ), 5.03 (dd, J1ЈЈ,2ЈЈ ϭ 8.0, J2ЈЈ,3ЈЈ ϭ 9.6 Hz, 1 H,
H2ЈЈ), 4.67 (d, 1 H, H1ЈЈ), 4.64 (d, J7a,7b ϭ Ϫ12.9 Hz, 1 H, H7a),
4.55Ϫ4.52 (m, 1 H, H3Ј), 4.39 (d, 1 H, H7b), 4.21 (ABX, J5ЈЈ,6aЈЈ
ϭ
4.6, J5ЈЈ,6bЈЈ ϭ 1.4, J6aЈЈ,6bЈЈ ϭ Ϫ12.3 Hz, 2 H, H6ЈЈ), 4.00 (m, 1 H,
H4Ј), 3.87 (ABX, J4Ј,5aЈ ϭ 2.7, J4Ј,5bЈ ϭ 2.6, J5aЈ,5bЈ ϭ Ϫ12.1 Hz, 2
H, H5Ј), 3.80Ϫ3.75 (m, 1 H, H5ЈЈ), 3.41 (br. s, 2 H, 2 ϫ OH),
2.40Ϫ2.35 (m, 1 H, H2aЈ), 2.27Ϫ2.20 (m, 1 H, H2bЈ), 2.09, 2.06,
2.03, 2.01 (4 ϫ s, 12 H, 4 ϫ CH3 Ac) ppm. 13C NMR (100 MHz,
CDCl3): δ ϭ 170.8, 170.7, 170.1, 169.6 (4 ϫ CϭO Ac), 162.6 (C4),
150.4 (C2), 137.9 (C6), 110.8 (C5), 100.6 (C1ЈЈ), 87.2 (C4Ј), 85.3 (C1Ј),
72.4, 71.8, 71.4, 70.9 (C3Ј/C2ЈЈ/C3ЈЈ/C5ЈЈ), 68.4 (C4ЈЈ), 64.4 (C7), 61.8
(C5Ј/C6ЈЈ), 40.5 (C2Ј), 20.7, 20.6, 20.5 (4 ϫ CH3 Ac) ppm. MS (ESI):
m/z ϭ 611.4 [M ϩ Na]ϩ, 1199.5 [2 M ϩ Na]ϩ.
Solid-Phase Synthesis of Oligonucleotides: The polymer-supported
synthesis of dJ-containing DNA fragments was performed on a
fully automated synthesiser (Pharmacia Gene Assembler Special).
Phosphoramidite 12 was employed for the construction of 13 by
the same procedure as reported previously.[14] The improved syn-
thesis of dJ-containing DNA fragments through the use of 21 and
commercially available (Proligo) 4-(tert-butyl)phenoxyacetyl-pro-
tected 2Ј-deoxynucleoside 3Ј-O-(2-cyanoethyl-N,N-diisopropyl)ph-
osphoramidites is summarised in Table 3. Controlled pore glass
(CPG-AP) loaded with the appropriate nucleoside (Proligo) was
used as solid support. Capping solutions Tac2O [4-(tert-butyl)-
phenoxyacetic acid anhydride] 50 g/L in THF and NMI/py/THF
(1:1:8, v/v/v) were obtained from Proligo.
2Ј-Deoxy-5-(2,3,4,6-tetra-O-methoxyacetyl-β-D-glucopyranosyl)oxy-
methyluridine (19): Compound 30 (62 mg, 66 µmol) was depro-
tected as described for the conversion of 29 into 18. Purification
by column chromatography (EtOAc/MeOH, 1:0 to 1:1, v/v) af-
1
forded 19 (36 mg, 51 µmol, 77%). H NMR (400 MHz, CD3OD):
δ ϭ 8.07 (s, 1 H, H6), 6.24 (m, 1 H, H1Ј), 5.38 (m, 1 H, H3ЈЈ), 5.14
(dd, J3ЈЈ,4ЈЈ ϭ 9.3, J4ЈЈ,5ЈЈ ϭ 10.1 Hz, 1 H, H4ЈЈ), 4.97 (dd, J1ЈЈ,2ЈЈ
ϭ
8.0, J2ЈЈ,3ЈЈ ϭ 9.6 Hz, 1 H, H2ЈЈ), 4.83 (d, 1 H, H1ЈЈ), 4.49 (1/2AB, 1
H, H7a), 4.46Ϫ4.36 (m, 3 H, H7b, H3Ј, H6aЈЈ), 4.27 (1/2ABX,
J5ЈЈ,6bЈЈ ϭ 2.2 Hz, 1 H, H6bЈЈ), 4.12Ϫ3.91 (m, 10 H, 4 ϫ CH2 MAc,
H4Ј, H5ЈЈ), 3.78 (ABX, J4Ј,5aЈ ϭ 3.1, J4Ј,5bЈ ϭ 3.6 Hz, 2 H, H5Ј), 3.41,
3.37, 3.35, 3.34 (4 ϫ s, 4 ϫ 3 H, 4 ϫ CH3 MAc), 2.33Ϫ2.20 (m, 2
H, H2Ј) ppm. 13C NMR (100 MHz, CD3OD): δ ϭ 171.8, 171.4,
171.1, 171.0 (4 ϫ CϭO MAc), 165.0 (C4), 152.1 (C2), 141.9 (C6),
111.4 (C5), 101.0 (C1ЈЈ), 89.0 (C4Ј), 86.7 (C1Ј), 74.5 (C3ЈЈ), 72.9 (C2ЈЈ),
72.6 (C5ЈЈ), 72.0 (C3Ј), 70.2 (4 ϫ CH2 MAc), 69.8 (C4ЈЈ), 65.7 (C7),
62.8 (C6ЈЈ), 62.7 (C5Ј), 59.6 (4 ϫ CH3 MAc), 41.5 (C2Ј) ppm. MS
(ESI): m/z ϭ 731.4 [M ϩ Na]ϩ, 1439.5 [2 M ϩ Na]ϩ.
Ammonolysis of (Immobilised) Oligonucleotides: The cleavage of
freshly prepared oligonucleotides from the solid phase and con-
comitant deprotection was effected by placing the immobilised
DNA fragment in concentrated aqueous ammonia in a sealed vial.
For DNA fragments synthesised with phosphoramidite 21 this was
performed at room temperature for 2 h, furnishing pure 14 and
22Ϫ24 after purification (see Analysis and Purification of Oligonu-
cleotides). For test sequence 13 ammonolysis was performed at 50
°C (Figure 1) and at room temperature for 16 h (Figure 2). The
resulting oligomers 14 and 15 were separated (see Analysis and
Purification of Oligonucleotides) and 14 was subjected to the same
treatment as 13 but at varying temperatures and times (see Fig-
ure 3).
2Ј-Deoxy-5Ј-O-(4,4Ј-dimethoxytrityl)-5-(2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosyl)oxymethyluridine (20): DMTrCl (0.31 g, 0.9 mmol)
was added to a stirred solution of 18 (353 mg, 0.6 mmol) in pyri-
dine (3 mL). After 3 h the reaction was quenched with MeOH
(1 mL) and after 15 min the mixture was concentrated in vacuo.
The resulting residue was partitioned between EtOAc and water,
and the organic layer was washed with brine (2 ϫ), dried (MgSO4),
and concentrated. The crude product was purified by column chro-
matography (EtOAc/light petroleum/Et3N, 50:50:1 to 100:0:1, v/v/
Analysis and Purification of Oligonucleotides: Analysis of (crude)
oligonucleotides was performed with a DNAPacTM PA-100 (4
mmD ϫ 250 mmL) anion-exchange column (Dionex) connected to
a Jasco HPLC system (detection at 254 nm). Elution at pH 12:
buffer A (10 m NaOH ϩ 20 m NaCl), buffer B (10 m NaOH
v) to give 20 (480 mg, 0.54 mmol, 90%). 1H NMR (400 MHz, ϩ 1.2  NaCl) Ϫ gradient 20Ϫ50% buffer B in 15 min. Elution at
CDCl3): δ ϭ 7.77 (s, 1 H, H6), 7.37Ϫ6.82 (m, 14 H, Harom DMT), pH 7: buffer A (20 m NaOAc ϩ 20 m NaCl), buffer B (20 m
6.29 (m, 1 H, H1Ј), 5.21 (m, 1 H, H3ЈЈ), 5.08 (m, 1 H, H4ЈЈ), 4.92 NaOAc ϩ 1  NaCl) Ϫ gradient 10Ϫ30% buffer B in 15 min. Puri-
(dd, J1ЈЈ,2ЈЈ ϭ 8.1, J2ЈЈ,3ЈЈ ϭ 9.6 Hz, 1 H, H2ЈЈ), 4.63 (d, 1 H, H1ЈЈ),
fication of oligonucleotides was achieved as for the analysis but
4.54 (m, 1 H, H3Ј), 4.21 (dd, J5ЈЈ,6aЈЈ ϭ 4.2, J6aЈЈ,6bЈЈ ϭ Ϫ12.4 Hz, 1 with a DNAPacTM PA-100 (9 mmD ϫ 250 mmL) anion-exchange
H, H6aЈЈ), 4.17 (d, J7a,7b ϭ Ϫ12.2 Hz, 1 H, H7a), 4.08Ϫ4.06 (m, 2 column (Dionex) connected to a BioCAD ‘‘Vision’’ automated
H, H4Ј and H6bЈЈ), 3.88 (d, 1 H, H7b), 3.78 (s, 6 H, OMe DMT), HPLC system (PerSeptive Biosystems, Inc), with elution at pH 12.
3.68Ϫ3.64 (m, 1 H, H5ЈЈ) 3.37 (ABX, J4Ј,5aЈ ϭ 3.3, J4Ј,5bЈ ϭ 3.5, Desalting was performed with Sephadex G25 (Pharmacia) and the
3838
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2003, 3832Ϫ3839

Oligonucleotides Containing 5-(β-D-Glucopyranosyloxymethyl)-2Ј-deoxyuridine
FULL PAPER
Table 3. Chemical steps involved in each elongation cycle modified for phosphoramidite 21
Step
Manipulation
Solvents and reagents
Time (min)
1
2
3
4
Detritylation
Coupling
Oxidation
Capping
2% Trichloroacetic acid in DCM
2.5
21[a] or amidite,[b] 4,5-dicyanoimdazole[c]
0.02  I2 in ACN/sym-collidine/H2O. 11:1:5 (v/v/v)
0.06  Tac2O in pyridine/NMI[e] /THF (1:1:18, v/v/v)
6[d]or 3
1
0.5
[a]
[b]
[c]
[d]
0.1  amidite in ACN. 0.1  commercially available (dCTAC, dATAC, dGTAC, T) amidite in ACN. 0.25  in ACN. (DCI) Only
[e]
for 21. N-Methylimidazole.
[16]
It should be noted that a higher-resolution ion-exchange col-
umn than previously was used (Dionex-Pac).[14] In addition,
the buffer was changed from pH 12 to pH 7 in order better to
visualise and quantify the side product 15, which at pH 12 is
a shoulder on the right hand side of the main peak (14).
No protection of the 2-deoxyribose moiety hydroxy groups was
used, as these are not responsible for decomposition and leav-
ing them unmasked aids solubility in the aqueous medium.
G. T. Shiau, R. F. Schinazi, M. S. Chen, W. H. Prusoff, J. Med.
Chem. 1980, 23, 127Ϫ133.
Compounds 18 and 19 were prepared as depicted in Scheme 5.
In contrast to earlier findings,[21] concomitant substitution of
the glucosyl moiety was also observed, resulting in a mixture of
25 and the corresponding 5-(methoxymethyl)-2Ј-deoxyuridine
derivative 26. The amount of the latter was reduced by the
replacement of KOtBu with K2CO3 and a reduction in tem-
perature. It is of interest to note that removal of the POM
protecting group is not catalytic, as evidenced by the isolation
and characterisation of both 27 and 28 when catalytic amounts
of base are used. Nevertheless, the addition of 1.2 equiv.
K2CO3 resulted in the reaction going to completion within 4 h
with 20 and 21 being isolated in 73% and 20% yields, respec-
tively.
oligonucleotides were obtained in pure form after lyophilisation.
The integrity of the oligonucleotides was confirmed by MALDI-
TOF mass spectrometry (positive mode) with a ‘‘Voyager System
6069’’ (Applied Biosystems).
Ammonolysis of dJ Monomers: Compounds 5,[14] 10,[14] 16,[14] 18,
and 19 were treated with concentrated aqueous ammonia and se-
aled in vials for varying times and at different temperatures (see
Table 1). Analysis of the crude mixtures was performed by LCMS
with H2O/ACN/0.5% TFA (aqu.) as eluent: Gradient 90:0:10 to
75:15:10, v/v/v in 15 min.
[17]
[18]
[19]
5-(β-D-Glucopyranosyloxymethyl)-2Ј-deoxyuridine (dJ, 16): LCMS:
Rt 10.30, m/z ϭ 421.2 [M ϩ H]ϩ.
5-(Aminomethyl)-2Ј-deoxyuridine (17): LCMS: Rt 7.38, m/z ϭ 258.2
[M ϩ H]ϩ.
Acknowledgments
These investigations were supported by the EC.
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Scheme 5. Reagents and conditions: (i) tBuOK 0.1  in MeOH, 60
°C, 16 h, 25/26 ϭ 5:3. (ii) K2CO3 (1.2 equiv.), MeOH, room temp.,
4 h. 25: 73%, 26: 20% (from 9). (iii) Ac2O, py, room temp., 16 h.
(iv) (MeOCH2CO)2O, py, room temp., 16 h. (v) Et3N·3HF, py,
room temp., 16 h
95, 2366Ϫ2371.
[10]
M. Cross, R. Kieft, R. Sabatini, M. Wilm, M. de Kort, G. A.
van der Marel, J. H. van Boom, F. van Leeuwen, P. Borst,
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[11]
Biol. Chem. 2002, 277, 958Ϫ966.
R. Sabatini, N. Meeuwenoord, J. H. van Boom, P. Borst, J.
.
[12]
[20]
Use of the more labile methoxyacetyl hydroxy group protection
Biol. Chem. 2002, 277, 28150Ϫ28156.
would provide no further advantage, as TAC protection re-
quires 2 h for complete removal.
M. de Kort, P. C. de Visser, J. Kurzeck, N. J. Meeuwenoord,
G. A. van der Marel, W. Ruger, J. H. van Boom, Eur. J. Org.
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B. T. Gillis, J. Org. Chem. 1959, 24, 1027Ϫ1029.
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[13]
E. R. Wijsman, O. van den Berg, E. Kuyl-Yeheskiely, G. A.
[21]
van der Marel, J. H. van Boom, Recl. Trav. Chim. Pays-Bas
1994, 113, 337Ϫ338.
M. de Kort, E. Ebrahimi, E. R. Wijsman, G. A. van der Marel,
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 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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