Nucleotides. LXXIV* synthesis of α-D-arabino-oligonucleotides

Article abstract of DOI:10.1080/15257770500267113

□ The 5 α-D-arabinofuranosylnucleosides α-araU (15), α-araT (18), α-araC (22), α-araA (25), and α-araG (28) have been synthesized by the modified silyl-method. The amino groups at the nucleobases and the 2′-hydroxy group at the sugar moiety were protected by the 2-(4-nitrophenyl)ethoxycarbonyl (npeoc) group (37-40) and the amide function in α-araG was additionally blocked by the 2-(4-nitrophenyl)ethyl group (63) to improve solubility in organic solvents. Mono-and dimethoxytritylation of the 5′-OH group was performed in the usual manner to give 41-48, 64, and 65 in high yields and further substitution of the 3′-OH group led to the monomeric building blocks 66-75 as well as the 3′-O-succinoyl derivatives 76-85 functioning as starting units in solid-support oligonucleotide synthesis. A large number of oligo-α- arabinonucleotides have been prepared on modified CPG-material applying the npeoc/npe strategy as a very efficient synthetic tool for highly purified, homogenous oligomers. Hybridizations between α-arabinonucleotide strands revealed in analogy to earlier findings an antiparallel orientation whereas the combination of an oligo-α-D-arabinonucleotide with a complementary oligo-2′-deoxy-β-D-ribofuranosylnucleotide showed base-pairing only if a parallel polarity was present. The advantages in oligo-α-arabino- nucleotide synthesis were furthermore demonstrated by the synthesis of the tα-ANA^his a structural analog of the natural tRNA^his of the phage T5. Copyright Taylor & Francis Group, LLC.

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Nucleotides. LXXIV Synthesis of a-D-Arabino-
oligonucleotides
Christoph Henke a & Wolfgang Pfleiderer a
a Fachbereich Chemie, Universität Konstanz, Konstanz, Germany
Version of record first published: 16 Aug 2006.
To cite this article: Christoph Henke & Wolfgang Pfleiderer (2005): Nucleotides. LXXIV Synthesis of a-D-Arabino-
oligonucleotides, Nucleosides, Nucleotides and Nucleic Acids, 24:10-12, 1665-1706
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1665–1706, 2005
c
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500267113
NUCLEOTIDES. LXXIV* SYNTHESIS OF
α-D-ARABINO-OLIGONUCLEOTIDES
ᮀ
Christoph Henke and Wolfgang Pfleiderer
Fachbereich Chemie, Universita¨t
Konstanz, Konstanz, Germany
ᮀ
The 5 α-D-arabinofuranosylnucleosides α-araU (15), α-araT (18), α-araC (22), α-araA
(25), and α-araG (28) have been synthesized by the modified silyl-method. The amino groups
at the nucleobases and the 2 -hydroxy group at the sugar moiety were protected by the 2-(4-nitro-
phenyl)ethoxycarbonyl (npeoc) group (37-40) and the amide function in α-araG was additionally
blocked by the 2-(4-nitrophenyl)ethyl group (63) to improve solubility in organic solvents. Mono-
and dimethoxytritylation ofthe 5 -OH group was performed in the usual manner to give 41-
48, 64, and 65 in high yields and further substitution of the 3 -OH group led to the monomeric
building blocks 66-75 as well as the 3 -O-succinoyl derivatives 76-85 functioning as starting units
in solid-support oligonucleotide synthesis. A large number ofoligo- α-arabinonucleotides have been
prepared on modified CPG-material applying the npeoc/npe strategy as a very efficient synthetic tool
for highly purified, homogenous oligomers. Hybridizations between α-arabinonucleotide strands
revealed in analogy to earlier findings an antiparallel orientation whereas the combination of
an oligo-α-D-arabinonucleotide with a complementary oligo-2 -deoxy-β-D-ribofuranosylnucleotide
showed base-pairing only ifa parallel polarity was present. The advantages in oligo- α-arabino-
nucleotide synthesis were furthermore demonstrated by the synthesis of the tα-ANAhis a structural
his
analog ofthe natural tRNA
ofthe phage T5.
Keywords α-D-arabinonucleosides; Glycosylation reactions; npeoc/npe protection; 3 -
phosphoramidites; Oligo-α-arabinonucleotides; Hybridizations; tα-ANAhis synthesis
INTRODUCTION
The most common 5 natural nucleobases thymine, uracil, cytosine,
adenine, and guanine are linked to pentose-sugar derivatives by β-glyco-
sidic bonds. Besides the nucleic acid components various β-D-arabinofu-
In honor and celebration of the life and career of John A. Montgomery.
*See Mu¨nch and Pfleiderer.
Received 28 January 2005; accepted 28 April 2005.
Address correspondence to Wolfgang Pfleiderer, Fachbereich Chemie, Universita¨t Konstanz,
Postfach 5560, 78457 Konstanz, Germany. Fax: 0049-7531-883138; E-mail: wolfgang.pfleiderer@uni-
konstanz.de
1665

1666
C. Henke and W. Pfleiderer
ranosyl-nucleosides have been found in nature or resulted from synthetic
approaches. 9-β-D-Arabinofuranosyladenine (ara-A)[2], 1-β-D-arabino-fura-
nosylcytosine (araC), and 9-β-D-arabinofuranosylhypyxanthine (ara-H) show
antiviral activities[3,4] and araATP inhibit DNA-polymerase and ribo-
nucleotide-reductase.[5] From extracts of the sponge Cryptothetica crypta[6]
spongouridine (1-β-D-arabinofuranosyluracil) and spongothymidine (1-β-
D-arabinofuranosylthymidine) have been isolated showing antibiotic activ-
ities. α-Arabinofuranosyl-nucleosides have sofar not been found in nature
but synthetically this type of compounds has been investigated by differ-
ent methods based upon mainly on classical glycosylation reactions and
leading often to α,β-anomeric mixtures. We describe here a direct general
approach to α-arabino-nucleosides in high yields using Vorbru¨ggen’s silyl
method,[7–10] which forms in a stereospecific manner by the neighboring
participation effect of the 2-acyloxy function almost exclusively an α-glyco-
sidic linkage. The arabino-nucleosides have been converted into the corre-
sponding fully protected nucleoside-3ꢁ-phosphoramidites which have then
been applied as building blocks to form by solid-phase synthesis various α-
arabino-oligonucleotides.
SYNTHESES OF α-D-ARABINOFURANOSYL-NUCLEOSIDES
The Vorbru¨ggen approach afforded in the first step of the synthesis
the silylation of uracil (1), thymine (2), cytosine (3), N4-acetylcytosine (4),
and N6-benzoyladenine (5) by heating in hexamethyldisilazene (HMDS)
under reflux to give 7-11. In the case of N2-acetyl-O6-diphenylcarbamoyl-
guanine (6) the more reactive N,O-bis-trimethylsilylacetamide was used in
boiling CH2Cl2 to get fast silylation in 15 min to 12. Glycosylation of 7 and
8, respectively, with 1,2,3,5-tetra-O-acetyl-D-arabinofuranose[11,12] as well as
1-O-acetyl-2,3,5-tri-O-benzoyl-D-arabinofuranose[13,14] in CH2Cl2 under tri-
methylsilyl trifluorosulfonate (TMS-triflate) activation giving high yields
of 13 (62%), 14[13] (87%), 16 (73%), and 17[15] (91%) after chromato-
graphical isolation and purification. In a similar manner reacted N4-acetyl-
cytosine via 7 to 1-(N4-,2ꢁ,3ꢁ,5ꢁ-tetracetyl-α-D-arabinofuranosyl)cytosine (19)
in 76% yield and silylated cytosine 8 could be converted with 1-O-acetyl-2,3,5-
tri-O-benzoyl-D-arabinofuranose into 1-(2ꢁ,3ꢁ,5ꢁ-tri-O-benzoyl-α-D-arabinofu-
ranosyl)cytosine (20) in 80% yield. N6-Benzoyladenine (5) and N2-acetyl-
O6-diphenylcarbamoylguanine (6) worked with both sugar derivatives un-
der the same reaction conditions to 23 (83%) and 24[16] (69%), respectively,
as well as to 26[17] (72%) and 27 (79%) (Scheme 1).
Treatment of the various acylated α-D-arabinofuranosyl-nucleosides by
sodium methoxide in MeOH or by methanolic ammonia led to free 1-α-D-
arabinofuranosyluracil (15) (82%),[15] 1-α-D-arabinofuranosylthymine (18)
(88%),[15] 1-α-D-arabinofuranosylcytosine (22) (66%),[18] 1-α-D-arabinofu-

Synthesis ofOligonucleotides
1667
SCHEME 1
ranosyladenine (25) (67%),[16] and 1-α-D-arabinofuranosyl-guanine (28)
(74%),[17] all of which have been prepared earlier with variable success.
The characterization and structural assignment of all α-D-arabinofurano-
1
syl-nucleosides has been achieved by determination of the UV- and H-
NMR spectra listed in the experimental part and their comparisons with
the corresponding natural ribofuranosyl-nucleosides. It is worth mentioning
that all sugar protons in the O-benzoyl series are shifted to lower field
comparing with the O-acetyl analogs. Furthermore a comparison of the

1668
C. Henke and W. Pfleiderer
α- and β-arabinofuranosyladenine and the α- and β-arabinofuranosylgua-
nine, respectively, revealed that in D6-DMSO the H-1ꢁ- and H-3ꢁ-signals
resonate in the β-anomer at lower field than the α-anomer whereas the
H-2ꢁ- and H-4ꢁ-signals shift in opposite directions (Table 1).
The next chapter of our investigations was concerned with the synthesis
of the appropriate building blocks for the α-arabino-oligonucleotides. We
decided to use the 2-(4-nitrophenyl)ethyl- (npe) and the 2-(4-nitrophenyl)
ethoxycarbonyl- (npeoc) group for base protection[19,20] according to their
advanced properties in ribo-, 2ꢁ-deoxyribo- and β-arabino-nucleoside and
nucleotide chemistry.[21,22] The sequence of protection started with the in-
troduction of the 3ꢁ,5ꢁ-O-(1,1,3,3-tetraisopropyl-1,3-disiloxan-1,3-yl) (tipds)
group[23] into α-araU (15), α-araT (18), α-araC (21), and α-araA (25) to give
29-32 in 80–90% yield. Subsequent acylation with an excess of 1-methyl-3-
[2-(4-nitrophenyl)ethoxycarbonyl]-imidazolium chloride in CH2Cl2 and in
presence of 4-dimethylaminopyridine afforded not only reaction at the 2ꢁ-
OH group[24] but blocked also the amino function in 31 and 32 and gave
isolated yields of 88–97% of 33-36. The selective cleavage of the Markiewicz
blocking group in 33-36 and 62 afforded special reaction conditions, ap-
plying tetrabutylammonium fluoride trihydrate in THF and in presence
of AcOH, since fluoride ions in aprotic solvents are too basic and cause
slow β-elimination of the npeoc functionality. Isolation of 37-40 and 63
was achieved by chromatographical means and gave high yields of 82–93%
(Scheme 2).
The mono- and dimethoxytritylation of 37-40 and 63 worked in the usual
manner in pyridine forming 41-48, 64, and 65 with yields between 82 and
95%.
TABLE 1 1H-NMR Data of α- and β-araA and α- and β-araG in D6-DMSO
Compound
α-AraA
β-AraA
α-araG
β-AraG
H-1
H-2
H-3
H-4
H-5 , 5
2 -OH
3 -OH
5 -OH
H-2
H-8
NH2
NH
5.85 (d, 1)
4.67 (dd, 1)
3.98 (dd, 1)
4.14 (m, 1)
3.50 (m, 2)
5.76 (d, 1)
5.62 (d, 1)
4.87 (t, 1)
8.14 (s, 1)
8.32 (s, 1)
7.29 (bs, 2)
6.26 (d, 1)
4.13 (m, 1)
4.13 (m, 1)
3.78 (m, 1)
3.65 (m, 2)
5.63 (d, 1)
5.53 (d, 1)
5.11 (t, 1)
8.13 (s.1)
5.64 (d, 1)
4.44 (dd, 1)
3.95 (dd, 1)
4.05 (m, 1)
3.54 (m, 2)
5.72 (d, 1)
5.52 (d, 1)
4.87 (t, 1)
6.04 (d, 1)
4.08 (m, 1)
4.08 (m, 1)
3.77 (m, 1)
3.65 (m, 2)
5.61 (d, 1)
5.45 (d, 1)
5.05 (t, 1)
8.18 (s, 1)
7.26 (bs, 2)
7.90 (s, 1)
6.45 (bs, 2)
10.63 (s, 1)
7.80 (s, 1)
6.49 (bs, 2)
11.62 (s, 1)
s = singlet, bs = broad singlet, d = doublet, dd = double doublet, t = triplet, m =
multiplet.

Synthesis ofOligonucleotides
1669
SCHEME 2

1670
C. Henke and W. Pfleiderer
According to our experience with β-arabinofuranosylguanine[22] show-
ing that O6-protection is essential from solubility reasons we started first
from N2-acetyl-O6-2-(4-nitrophenyl)ethylguanine (50) which was glycosy-
lated with 1,2,3,5-tetra-O-acetyl-D-arabinofuranose[11,12] as well as 1-O-acetyl-
2,3,5-tri-O-benzoyl-D-arabinofuranose[13,14] under Vorbru¨ggen conditions
leading to N2-acetyl-O6-npe-9-(2,3,5-tri-O-acetyl-)- (52) and N2-acetyl-O6-
npe-9-(2,3,5-tri-O-benzoyl-α-D-arabinofuranosyl)guanine (53) in 55% and
75% yield, respectively. Selective cleavage of the sugar-acyl-groups caused,
however, difficulties and proceeded to 54 under a variety of reaction condi-
tions such as NH3/MeOH, Et3N/MeOH, MeONa/MeOH, K2CO3/MeOH,
and dioxan/MeOH/NH3 only in moderate yields. More straightforward
were the analogous glycosylations of O6-2(4-nitrophenyl)ethylguanine (51)
to 55 and 56 in 52% and 73% yield. Deacylation proceeded best with
K2CO3 in MeOH to give 54 in yields >80%. 56 was then converted by 2-
(4-nitrophenyl)ethoxycarbonyl chloride into O6-npe-N2-npeoc-9-(2,3,5-tri-
O-benzoyl-α-D-arabinofuranosyl)guanine (57) in 93% yield and followed
by debenzoylation in MeOH/dioxane/aqueous NH3 forming 58 in 83%
yield. The subsequent Markiewicz protection of 56 and 58 gave under the
usual reaction conditions 59 and 60 which were then treated by 1-methyl-
3-[2-(4-nitrophenyl)ethoxycarbonyl]-imidazolium chloride reacting at the
2ꢁ-OH group to the corresponding carbonates 61 and 62. The expected
concerted acylation of 59 also at the 2-amino group did not take place and
afforded a second step applying 2-(4-nitrophenyl)ethoxycarbonyl chloride
to form 62 (Scheme 3).
The last step of the synthesis of the fully protected α-arabino-nucleo-
side-3ꢁ-phosphor-amitides 66-75 was performed with 41-48, 64, and 65 under
anhydrous conditions and under N2-atmosphere with bis-(N,N-diisopro-
pylamino)-2-cyanoethoxyphosphane and sublimed tetrazole as activator in
CH3CN and a reaction time of 8–18 h at room temperature. Work-up was
best achieved by flash chromatography on silica gel with toluene/ethyl
acetate and n-hexane/acetone gradients, respectively, isolating solid foams
of 66-75 in 70–90% yield. Finally, the 3ꢁ-O-succinoyl-α-arabinonucleoside 76-
85 were also prepared from 41-48, 64, and 65 with succinic anhydride and
4-dimethylaminopyridine in CH2Cl2 in yields >90% (Scheme 2).
All newly synthesized compounds have been characterized and proven
in their structures by UV-, 1H- and 31P-NMR spectra as well as by elemental
analysis. All data are reported in the experimental part.
The high purity and long-time stability of the 5 fully protected 5ꢁ-
O-dimethoxytrityl-α-arabinofuranosylnucleoside-3ꢁ-phosphoramidites 71-75
have been chosen as the appropriate building blocks for the synthe-
sis of a broad variety of homogenous and mixed oligo-α-arabinonucleo-
tides. The synthetic cycle performed in an Applied Biosystem 392 synthe-
sizer was based upon our experiences with the corresponding 2ꢁ-deoxy-

Synthesis ofOligonucleotides
1671
SCHEME 3
and ribonucleoside-3ꢁ-phosphoramidites[26–28] applying optimized reac-
tion conditions for the oligo-α-arabinonucleotide synthesis. The applied
npe/npeoc strategy[29] afforded the long-chain-N-methylaminoalkyl-CPG
(LCMAA-CPG) solid-support material (500 Å),[30] which was prepared from
dry glyceryl-CPG by subsequent treatment with 1,1ꢁ-carbonyldiimidazole
and N,Nꢁ-dimethylhexane-1,6-diamine, followed by either one of the 3ꢁ-O-
succinates 81-85 as 3ꢁ-terminal α-arabinofuranosylnucleoside and a final cap-
ping step (Figure 1).
The synthesis of the oligonucleotides was performed in the usual man-
ner with a slightly modified cycle starting with the detritylation, coupling
with the phosphoramidite, a capping step and the final oxidation of the
phosphite ester to the phosphate stage. The time scale of 20 min per cycle
is reported in the experimental part. The advantage of the npe/npeoc strat-
egy is seen during deprotection of the blocking groups of which the 2-(4-
nitrophenyl)ethyl-, the 2-(4-nitrophenylethoxy-carbonyl-, and the 2-cyano-
ethyl group can be removed in one step by DBU treatment quantitatively
by a β-elimination process. During this procedure the oligonucleotide is
still attached to the support and can after washing be cleaved by ammonia
treatment, yielding 5ꢁ-protected oligomers, which can further be purified by
ion-pair reverse-phase HPLC before final acid-catalyzed detritylation. An al-
ternative workup starts with detritylation of the fully protected oligonucleo-
tide, followed by DBU treatment, and, finally, ammonia cleavage leading to

1672
C. Henke and W. Pfleiderer
FIGURE 1 Synthesis and loading of LCMAA-CPG solid-support. i) Carbonyldiimidazole, CH2Cl2,
2h, RT; ii) 1,6-N.N-dimethyl-hexane-diamine, 6 h. RT; iii) nucleoside-succinate, N-methylmorpholine,
TOTU, 2 h, CH3CN; iv) Ac2O, DMAP, pyridine, RT.
the free oligomers, which were characterized by anion-exchange HPLC on
a NucleoPak column and in most cases showed a high purity, which did not
need further purification or separation from failure sequences.
We first synthesized from the 5 fully protected phosphoramidites 71-
75 homo-α-arabinooligonucleotides which gave the best results if a con-
densation time of 10 min was applied. The average stepwise yield was sur-
prisingly very high, in general >99%, except with α-araG whereby the pu-
rity of the phosphoramidte 75 had an enormous influence on the yield in
the condensation step. Chromatographical purification by FC on siliga-gel
with toluene/EtOAc applying a 6:1-3:1 proportion afforded the anticipated
purity.
Mixed oligomers (Table 2, 12–14) with 2ꢁ-deoxy-β-D-ribonucleoside-3ꢁ-
phosphoramidites have been prepared in the same manner and showed the
same purity like the oligo-2ꢁ-deoxyribonucleotides.
In order to study the hybridization properties of the α-arabinooligo-
nucleotides a series of combinations have been prepared (Table 3) and the
melting temperatures of the duplexes determined. The homo- and mixed
oligomers were compared with the corresponding oligo-2ꢁ-deoxyribonucleo-

Synthesis ofOligonucleotides
1673
FIGURE 2 Structure of tα-ANAhis
.
TABLE 2 Homo-α-arabino- and Mixed α-Arabino-2 -deoxy-β-ribo-oligonucleotides
Sequence
1. 5 -α-ara-CCC CCC CCC-3
2. 5 -α-ara-AAA AAA AAA-3
3. 5 -α-ara-TTT TTT TTT T-3
4. 5 -α-ara-UUU UUU UUU U-3
5. 5 -α-ara-CCC CCC CCC C-3
6. 5 -α-ara-AAA AAA AAA A-3
7. 5 -d-TTT TTT TTT TTT TTT TTT-3
8. 5 -d-AAA AAA AAA AAA AAA AAA-3
9. 5 -α-ara-TTT TTT TTT TTT TTT TTT-3
10. 5 -α-ara-AAA AAA AAA AAA AAA AAA-3
11. 5 -d-GGT TCC ATG CAT GGA ACC-3
12. 5 -α-ara-(GGT T)-d-CC ATG CAT GGA ACC-3
13. 5 -d-GGT TCC TAG CAT GG-α-ara-(A ACC)-3
14. 5-’d-GGT TCC A-α-ara-(TG CA)-d-T GGA ACC-3
15. 5 -α-araGCT GGG GGT ACC TCG GGG CG-3
16. 5 -α-araCGC CCC GAG GTA CCC CCA GC-3
17. 5 -α-araCGG CCG GTG GAC ATG GGA CC-3
18. 5 -α-araGGT CCC ATG TCC ACC GGC CG-3
19. 5 -α-araCGG AGG AGT CGT AGG AGG ACG-3
20. 5 -α-araCCG GCC GGT GGA CAT GGG ACC-3
21. 5 -α-araCGT CCT CCT ACG ACT CCT CCG-3
22. 5 -α-araGGT CCC ATG TCC ACC GGC CGG-3
Average coupling yield %
98.4
97.2
99.8
99.9
98.7
99.9
99.5
99.4
99.9
99.2
98.8
98.6
99.9
98.9
99.9
99.9
99.8
99.5
99.6
99.1
99.9
99.9

1674
C. Henke and W. Pfleiderer
TABLE 3 Melting Temperature of DNA/α-ANA Hybridizations
Sequence
[Na ] mM
Tm ( C)
41.8
15. 5 -d(TTT TTT TTT TTT TTT TTT T)-3
3 -d(AAA AAA AAA AAA AAA AAA A)-5
150
150
150
150
30
16. 5 -d(TTT TTT TTT TTT TTT TTT T)-3
3 -α-ara(AAA AAA AAA AAA AAA AAA A)-5
17. 5 -α-ara (TTT TTT TTT TTT TTT TTT T)-3
3 -α-ara(AAA AAA AAA AAA AAA AAA A)-5
18. 5 -α-ara(TTT TTT TTT TTT TTT TTT T)-3
3 -d(AAA AAA AAA AAA AAA AAA A)-5
36.2
23.8
18.6
60.8
54.2
40.0
35.3
19. 5 -d(GGT TCC ATG CAT GGA ACC)-3
3 -d(CCA AGG TAC GTA CCT TGG)-5
20. 5 -d(GGT TCC A)α-ara(TG CA)d(T GGA ACC)-3
3 -d(CCA AGG T)α-ara(AC GT)d(A CCT TGG)-5
21. 5 -d(GGT TCC ATG CAT GG)α-ara(A ACC)-3
3-α-ara(CCA A)d(GG TAC GTA CCT TGG)-5
22. 5 -α-ara(GGT T)d(CC ATG CAT GGA ACC)-3
3 -d(CCA AGG TAG GTA CC)α-ara(T TGG)-5
30
30
30
tides under the same salt concentrations showing that in all combinations
the presence of α-arabinonucleotides is associated with a substantial drop in
the Tm, showing that the normal Watson-Crick base pairing is functioning
but in a highly disturbed manner. It is interesting to note that the duplex 16
reveals a much higher stabilization than the counterpart duplex 18 but no
simple explanation of this effect can be offered. The self-complementary
mixed sequences 20–22 show a strong dependence regarding the site of
the α-ara-tetrameric part in the oligonucleotide chain. The deviation is
expectedly lowest in the duplex 20 where the α-ara-oligo-strip is located in
the middle of the chain creating a reduced fit of only 4 base pairs, whereas
the introduction of the α-ara-tetrameric sequence at either end causes a
much stronger disturbance.
These results can be better understood on the basis of earlier findings in
the α- and β-2ꢁ-deoxyoligoribonucleotide series[31–34] showing that duplexes
are only formed if an α-strand is combined with the complementary β-strand
in a parallel orientation whereas the combination of two α-sequences afford
an antiparallel polarity for base pairing.
Similar observations have been noticed with the duplexes 23–32. The α-
strands 24 and 29 show in the antiparallel orientation a slightly lower stability
than the corresponding reference sequence and the parallel polarity of
the α-ara- and β-deoxyribo still reveal substantial base pairing, whereas
in an antiparallel arrangement of such strands no interaction is observed
(Table 4).
As model sequences for antisense studies various oligo-2ꢁ-deoxy-β-D-
ribonucleotides have been modified at the 3ꢁ- and 5ꢁ-terminal end with α-
arabinonucleosides (34–37, 41–43) forming duplexes, which show a small

Synthesis ofOligonucleotides
1675
TABLE 4 DNA/α-ANA Hybridization Experiments
Sequence
G/C pairs
14
Tm ( C)
57.3
23. 5 -d(CAG CGT GGA GGA GGT GGT GGA)-3
3 -d(GTC GCA CCT CCT CCA CCA CCT)-5
24. 5 -α-ara(GTC GCA CCT CCT CCA CCA CCT)-3
3 -α-ara(CAG CGT GGA GGA GGT GGT GGA)-5
25. 5 -d(TCC ACC ACC TCC TCC ACG CTA)-3
5 -α-ara(AGG TGG TGG AGG AGG TGC GAC)-3
26. 5 -d(CAG CGT GGA GGA GGT GGT GGA)-3
5 -α-ara(GTC GCA CCT CCT CCA CCA CCT)-5
27. 5 -d(CAG CGT GGA GGA GGT GGT GGA)-3
3 -α-ara(GTC GCA CCT CCT CCA CCA CCT)-5
28. 5 -d(AAG CCC CAA GCC GAA GGA TTT)-3
3 -d(TTC GGG GTT CGG CTT CCT AAA)-5
29. 5 -α-ara(TTC GGG GTT CGG CTT CCT AAA)-3
3 -α-ara(AAG CCC CAA GCC GAA GGA TTT)-5
30. 5 -d(AAA TCC TTC GGC TTG GGG CTT)-3
5 -α-ara(TTT GGG GTT CGG CTT CCT AAA)-3
31. 5 -d(AAG CCC CAA GCC GAA GGA TTT)-3
5 -α-ara(TTC GGG GTT CGG CTT CCT AAA)-3
32. 5 -d(AAG CCC CAA GCC GAA GGA TTT)-3
3 -α-ara(TTC GGG GTT CGG CTT CCT AAA)-5
14
14
14
14
11
11
11
11
11
55.3
49.3
40.3
—
52.4
48.2
42.9
38.3
—
decrease in Tm with ara-T, ara-C, and ara-A and a slightly higher Tm on
introduction of ara-G. The other duplexes fit well in the accepted picture
of strand orientation (Table 5).
A general goal for testing a special oligonucleotide chemistry is the
synthesis of longer oligonucleotide sequences. We decided to prove our
method by synthesizing the α-ara-analog (tα-ANAhis) of the histidine tRNA
of the phage T5 consisting of 78 nucleotide units (Figure 2). The rare
nucleobases pseudouridine in position 41 and 57 and the ribothymidine
in position 56 have been replaced by α-arabinothymidine. The synthesis
worked, to our surprise, very well on LCMAA-CPG-1000 Å solid-support
material with an average condensation yield in every step of 99.7% and
a total reaction time of 26 h. After cleavage of the protective groups by
DBU and acid, followed by a washing step and final removal from the solid-
support by ammonia gave a perfect looking product of high purity. The
HPLC analysis on a Dionex NucleoPac PA-100 column gave one main peak
(Figure 3), the polyacrylamide-gel-electrophoresis (Figure 4), and especially
the capillary gel electrophoresis (Figure 5) proved the homogenicity of the
probe. Furthermore, an electrospray mass spectrum offered a molecular
mass of 25,065.72 22.42 lying in good agrement with the calculated mass
of 25,039.59 for C742H994N282O549P77 and an actual counting error of 0.2
DA/1000 DA and without taking into account the isotope distribution.
The enzymatic stability of the α-arabinonucleotide unit was tested with
the exonuclease snake venom phosphodiesterase degrading in 3ꢁ-5ꢁ-direc-

1676
C. Henke and W. Pfleiderer
TABLE 5 DNA/α-ANA Hybridization Experiments
Sequence
Tm ( C)
65.2
33. 5 -CGA CCC CCA TGG AGC CCC GC-3
3 -GCT GGG GGT ACC TCG GGG CG-5
34. 5 -CGA CCC CCA TGG AGC CCC GC-3
3 -α-araT-d(GCT GGG GGT ACC TCG GGG CG)α-araT-5
35. 5 -CGA CCC CCA TGG AGC CCC GC-3
3 -α-araC-d(GCT GGG GGT ACC TCG GGG CG)α-araC-5
36. 5 -CGA CCC CCA TGG AGC CCC GC-3
3 -α-araA-d(GCT GGG GGT ACC TCG GGG CG)α-araA-5
37. 5 -CGA CCC CCA TGG AGC CCC GC-3
3 -α-araG-d(GCT GGG GGT ACC TCG GGG CG)αα-araG-5
38. 5 -CGA CCC CCA TGG AGC CCC GC-3
5 -α-ara-GCT GGG GGT ACC TCG GGG CG-5
39. 3 -α-araCGA CCC CCA TGG AGC CCC GC-3
5 -α-araGCT GGG GGT ACC TCG GGG CG-5
40. 5 -GCC GGC CAC CTG TAC CCT GG-3
3 -CGG CCG GTG GAC ATG GGA CC-5
41. 5 -GCC GGC CAC CTG TAC CCT GG-3
3 -α-araCd(CGG CCG GTG GAC ATG GGA CC)α-araC-5
42. 5 -GCC GGC CAC CTG TAC CCT GG-3
3 -α-araAd(CGG CCG GTG GAC ATG GGA CC)α-araA-5
43. 5 -GCC GGC CAC CTG TAC CCT GG-3
3 -α-araGd(CGG CCG GTG GAC ATG GGA CC)αα-araG-5
44. 5 -GCC GGC CAC CTG TAC CCT GG-3
5 -α-araCGG CCG GTG GAC ATG GGA CC-3
45. 3 -α-araGCC GGC CAC CTG TAC CCT GG-3
5 -α-araCGG CCG GTG GAC ATG GGA CC-3
46. 5 -α-araCGG AGG AGT AGT AGG AGG ACG-3
3 -α-araGCC TCC TCA GCA TCC TCC TGC-5
47. 5 -α-araCCG GCC GGT GGA CAT GGG ACC-3
3 -α-araGGC CGG CCA CCT GTA CCC TGG-5
64.5
62.9
64.0
65.2
52.4
59.4
64.0
62.6
62.7
64.5
50.3
59.2
54.5
62.6
FIGURE 3 HPLC of tα-ANAhis on NucleoPak PA-100, 4 × 250 mm (Dionex).
tion and followed by alkaline phosphatase for HPLC analysis of the resulting
nucleosides. Expectedly, the oligo-2ꢁ-deoxyribonucleotide sequences were
degraded completely within a few minutes whereas the presence of an α-
arabinonucleotide unit at the 3ꢁ-end prevents enzymatic hydrolysis to a large
extent showing only partial cleavage after 24 h.

Synthesis ofOligonucleotides
1677
FIGURE 4 Polyacrylaminde-gel-electrophoresis of tα-ANAhis
.
FIGURE 5 Capillary-gel-electrophoresis of tα-ANAhis
.
EXPERIMENTAL
All melting points are uncorrected. Products were dried under high
vacuum. TLC: precoated silica gel thin-layer sheets 60 F 254 from Merck
and cellulose sheets F 1440 LS 254 from Schleicher & Schu¨ll. Column
chromatography (CC): silica gel 60 from Merck and flash chromatography
(FC): silica gel (30–60 µm) from Baker. UV/VIS: Perkin-Elmer Lambda 5;
1
λmax in nm (log ε). H-NMR: Bruker AC 250; δ in ppm rel. to SiMe4 or
CDCl3 (DMSO-d6) as internal standard. HPLC: Merck-Hitachi L 6200, UV-
detector L 400. Oligonucleotide syntheses: DNA-synthesizer from Applied
Biosystems, Model 392. Lyophilizations with Speed-Vac-Concentrator from
Savant. All solvents used were of anhydrous grade. The pKa-values were
determined by the spectrophotometric method.[25] Elemental analyses were

1678
C. Henke and W. Pfleiderer
performed by the analytical lab of the department of chemistry, Konstanz
University.
1-(2,3,5-Tri-O-acetyl-α-D-arabinofuranosyl)uracil (13). Under anhydrous
conditions uracil (1) (0.336 g, 3 mmol) and (NH4)2SO4 (5 mg) were
suspended in hexamethyldisilazane (HMDS) (10 mL) and then heated
under reflux for 20 h. It was evaporated, the resulting oil dissolved in
dry toluene (15 mL) and combined with a solution of 1,2,3,5-tetra-acetyl-
arabinofuranose (1.14 g, 3.6 mmol) in toluene (15 mL). Under stirring a
solution of TMS-triflate (0.9 mL, 5 mmol) in toluene (5 mL) was added
dropwise and then the mixture heated in an oilbath to 80◦C for 1 h. The
reaction solution was diluted with EtOAc (100 mL) and treated with a
saturated aqueous solution of NaHCO3 (100 mL). The aqueous phasae
was reextracted with EtOAc (100 mL), the organic layers united, dried over
Na2SO4, and evaporated. The resulting solid foam was purified by CC (4 ×
13 cm, silica gel) with toluene/EtOAc 1:5 to give 0.68 g (62% of a solid
foam. Rf: 0.5 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 208(3.99), 258
1
(3.99). H-NMR (CDCl3): 9.21 (s, 1H, NH); 7.32 (d, 1H, H-C(6)); 5.97 (d,
1H, H-C(1ꢁ)); 5.78 (d, 1H, H-C(5)); 5.49 (pt, 1H, H-C(2ꢁ)); 5.22 (pt, 1H, H-
C(3ꢁ)); 4.59 (q, 1H, H-C(4ꢁ)); 4.28 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 2.16 (s, 3H, Oac);
2.15 (s, 3H, Oac); 2.14 (s, 3H, Oac). Anal. for C15H18N2O9 (370.3). Calcd:
C 48.65, H 4.90, N 7.56. Found: C 48.63, H 5.05, N 7.32.
1-(2,3,5-Tri-O-benzoyl-α-D-arabinofuranosyl)uracil (14). Analogous to
the preceding procedure with uracil (1) (0.56 g, 5 mmol) and 1-O-acetyl-
2,3,5-tri-O-benzoyl-arabinofuranose (2.52 g, 5 mmol) in CH2Cl2 (15 mL).
Addition of TMS-triflate (0.9 mL, 5 mmol) in CH2Cl2 (5 mL) and stirring at
room temperature for 18 h. Purification by CC (35 g silica gel) with toluene/
EtOAc 10:1 (500 mL) and then toluene/4:1 (600 mL) to give 2.3 g (87%)
of a colorless solid foam. Rf: 0.35 (toluene/EtOAc 1:1). UV (MeOH): 201
(4.39), 230 (4.62), 256 (sh 3.92). 1H-NMR (CDCl3): 9.17 (s, 1H, NH); 8.05
(m, 6H, arom. H); 7.48 (m, 10H, H-C(6), arom. H); 6.21 (d, 1H, H-C(1ꢁ));
5.96 (pt, 1H, H-C(2ꢁ)); 5.81 (d, 1H, H-C(5)); 5.76 (pt, 1H, H-C(3ꢁ)); 4.98
(q, 1H, H-C(4ꢁ)); 4.72 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)). Anal. for C30H24N2O9 (556.5).
Calcd: C 64.75, H 5.03, N 7.56. Found: C 64.36, H 4.60, N 4.79.
1-α-D-Arabinofuranosyl)uracil (15)[15]. In MeOH (35 mL) Na (80 mg,
3.5 mmol) was dissolved, then 1-(2,3,5-tri-O-benzoyl-α-D-arabinofuranosyl)
uracil (14) (1.67 g, 3 mmol) added and stirred for 45 min whereby a
precipitate separated out. The suspension was evaporated, dissolved in H2O
(30 mL), and then neutralized with Dowex ion-exchange resin H+-form.
The water phase was extracted with CH2Cl2 (3 × 30 mL), filtered from
the resin, and then evaporated to an oily residue. Purification was done by

Synthesis ofOligonucleotides
1679
FC (16 g silica gel) with CH2Cl2(MeOH 15:1 (250 mL) and 12:1 (450 mL).
The product fractions were evaporated, and coevaporated subsequently with
EtOH, MeOH and CH2Cl2 (2×) to give 0.6 g (82%) of a colorless solid,
which was dried in high vacuum at 40◦C. Rf: 0.67 (CHCl3/MeOH 1:1). pKa =
9.36. UV (H2O): pH 7: 202 (4.01), 262 (4.00); pH 12: 204 (4.11,), 223 (sh
3.93), 260 (3.87). 1H-NMR (DMSO-d6): 11.28 (s, 1H, NH); 7.68 (d, 1H, H-
C(6)); 5.69 (m, 2H, H-C(1ꢁ), H-O (2ꢁ)); 5.60 (m, 2H, H-C(5), H-O(3ꢁ)); 4.93
(t, 1H, H-O(5ꢁ)); 4.09 (m, 1H, H-C(2ꢁ)); 4.05 (m, 1H, H-C(4ꢁ)); 3.92 (m,
1H, H-C(3ꢁ)); 3.50 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)). Anal. for C9H12N2O9 × 0.5 H2O
(253.2). Calcd: C 42.69, H 5.18, N 11.06. Found: C 42.29, H 5.14, N 10.95.
1-(2,3,5-Tri-O-acetyl-α-D-arabinofuranosyl)thymine (16). Analogous to
13 with thymine (2) (0.378 g, 3 mmol). Purification by FC (4 × 15 cm, silica
gel) with toluene/EtOAc 2:5 (1 L) to give 0.84 g (73% of a colorless solid
foam. Rf: 0.62 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 210 (3.87), 263
(3.979. 1H-NMR (CDCl3): 8.93 (s, 1H, NH); 7.13 (d, 1H, H-C(6)); 5.99 (d,
1H, H-C(1ꢁ)); 5.50 (pt, 1H, H-C(2ꢁ)); 5.25 (pt, 1H, H-C(3ꢁ)); 4.58 (q, 1H, H-
C(4ꢁ)); 4.30 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 2.15 (s, 3H, Oac); 2.13 (s, 3H, Oac); 2.12
(s, 3H, Oac); 1.96 (s, 3H, H3C-(5)). Anal. for C16H20N2O9 (384.4). Calcd:
C 50.00, H 5.25, N 7.28. Found: C 50.15, H 5.47, N 7.02.
1-(2,3,5-Tri-O-benzoyl-α-D-arabinofuranosyl)thymine (17). Analogous to
14 with thymine (2) (3.1 g, 24 mmol) and (NH4)2SO4 (10 mg) in HMDS(20
mL). Followed by 1-O-acetyl-2,3,5-tri-O-benzoylarabinofuranose (10.9 g, 22
mmol) in CH3CN (60 mL) and TMS-triflate (3.75 mL, 22 mmol) in CH3CN
(10 mL). Work-up in CH2Cl2 gave a solid foam, which was coevaporated
subsequently with EtOH, MeOH, and CH2Cl2 to give 11.4 g (91%) of a
chromatographically pure solid. Rf: 0.5 (toluene/EtOAc 1:1). UV (MeOH):
201 (4.56), 230 (4.63), 265 (4.09), 282 (sh 3.79). 1H-NMR (CDCl3): 9.21 (s,
1H, NH); 8.06 (m, 6H, arom. H); 7.48 (m, 9H, arom. H); 7.27 (s, 1H, H-
C(6)); 6.28 (d, 1H, H-C(1ꢁ)); 5.93 (pt, 1H, H-C(2ꢁ)); 5.76 (pt, 1H, H-C(3ꢁ));
4.97 (q, 1H, H-C(4ꢁ)); 4.69 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 1.92 (s, 3H, H3C-(5). Anal.
for C31H26N2O9 (570.6). Calcd: C 65.26, H 4.59, N 4.91. Found: C 65.11,
H 4.61, N 4.71.
1-α-D-Arabinofuranosyl)thymine (18)[15]. Analogous to 15 with 17 (2 g,
3.5 mmol) in MeOH (70 mL) and Na (150 mg). Purification by FC (2 ×
10 cm, silica gel) with CH2Cl2/MeOH 8:1 to give 0.8 g (88%) of a colorless
solid. Rf: 0.78 (CHCl3/MeOH 1:1). pKa 10.43, UV (H2O): pH 7: 205 (3.97),
266 (3.98); pH 12: 210 (4.01), 224 (3.95), 266 (3.85). 1H-NMR (DMSO-d6):
11.28 (s, 1H, NH); 7.59 (d, 1H, H-C(6)); 5.67 (m, 2H, H-C(1ꢁ), H-O (2ꢁ));
5.45 (d, 1H, H-O(3ꢁ)); 4.90 (t, 1H, H-O(5ꢁ)); 4.06 (m, 2H, H-C(2ꢁ), H-C(4ꢁ));
3.92 (m, 1H, H-C(3ꢁ)); 3.50 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 1.78 (s, 3H, H3C-C(5).

1680
C. Henke and W. Pfleiderer
Anal. for C10H14N2O9 × 0.5 H2O (267.2). Calcd: C 44.95, H 5.66, N 10.48.
Found: C 45.06, H 5.70, N 10.04.
N4 -Acetyl-1-(2,3,5-tri-O-acetyl-α-D-arabinofuranosyl)cytosine (19).
Analogous to 13 with N4-acetylcytosine (4) (0.92 g, 6 mmol) and 1,2,3,5-
tetra-O-acetylarabinnofuranose (3.1 g, 6.3 mmol) and TMS-triflate (1.1 mL,
6 mmol) in CH3CN by stirring 6 h at room temperature. After extraction
with EtOAc and washing the resulting solid was disoolved in a mixture of
EtOH (2 mL), isopropanol (2 mL), and ether (2 mL) by heating. On cooling
in the icebox overnight 1.23 g (50%) of colorless crystals of m.p. 177–178◦C
were obtained. Rf: 0.41 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 211
(4.32), 247 (4.21), 296 (3.85).1H-NMR (CDCl3): 9.43 (s, 1H, NH); 7.74 (d,
1H, H-C(6)); 7.48 (d, 1H, H.C(5)); 6.04(d, 1H, H-C(1ꢁ)); 5.51 (pt, 1H, H-
C(2ꢁ)); 5.15 (pt, 1H, H-C(3ꢁ)); 4.68 (m, 1H, H-C(4ꢁ)); 4.31 (m, 2H, H-C(5ꢁ,
5ꢁꢁ)); 2.27 (s, 3H, N-ac); 2.16 (s, 3H, Oac); 2.14 (s, 3H, Oac); 2.03 (s, 3H,
Oac). Anal. for C17H21N3O9 (411.4). Calcd: C 49.64, H 5.14, N 10.22. Found:
C 49.55, H 5.20, N 10.05.
N4-Acetyl-1-(2,3,5-tri-O-benzoyl-α-D-arabinofuranosyl)cytosine (20).
Analogous to the preceeding procedure with 4 (0.92 g, 6 mmol) and 1-O-
acetyl-2,3,5-tri-O-benzoylarabinofuranose (3.1 g, 6.3 mmol) and TMS-triflate
(1.1 mL, 6 mmol) in CH3CN by stirring 20 h at room temperature. Work-
up with CH2Cl2 gave a solid foam which was recrystallized from EtOH (10
mL) to give 2.54 g (76%) of colorless crystals of m.p. 184–185◦C. Rf: 0.59
(toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 218 (sh 4.57), 232 (4.69),
1
282 (3.89), 296 (3.87). H-NMR (CDCl3): 9.34 (s, 1H, NH); 8.07 (m, 4H,
arom. H); 7,78 (m, 3H, H-C(6), arom. H); 7.48 (m, 10H, H-C(6), arom.
H); 6.26 (d, 1H, H-C(1ꢁ)); 6.02 (pt, 1H, H-C(2ꢁ)); 5.71 (pt, 1H, H-C(3ꢁ));
5.06 (m, 1H, H-C(4ꢁ)); 4.70 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 2,26 (s, 3H, N-ac). Anal.
for C32H27N3O9 (597.6). Calcd: C 64.32, H 4.55, N 7.03. Found: C 64.08,
H 4.54, N 7.09.
1-(2,3,5-Tri-O-benzoyl-α-D-arabinofuranosyl)cytosine (21). Analogous
to 13 with 3 (0.35 g, 3.15 mmol) and after silylation addition of 1-O-acetyl-
2,3,5-tri-O-benzoylarabinofuranosose (1.56 g, 3.1 mmol) and TMS-triflate
(1.1 mL, 6 mmol) in CH3CN by stirring and heating to 65◦C for 8 h. Work-up
with CH2Cl2 gave a solid foam which was recrystallized from EtOH (5 mL) to
give 1.37 g (80%) of colorless crystals of m.p. 129–132◦C. Rf: 0.48 (toluene/
EtOAc/MeOH 5:5:2). UV (MeOH): 201 (4.71), 231 (4.68), 271 (4.04), 278
1
(sh 3.97). H-NMR (CDCl3): 8.55 (bd, 2H, NH2); 8.05 (m, 4H, arom. H);
7,81 (d, 2H, arom. H); 7.40 (m, 10H, H-C(6), arom. H); 6.15 (d, 1H, H-
C(1ꢁ)); 6.01 (pt, 1H, H-C(2ꢁ)); 5.84 (d, 1H, H-C(5)); 5.62 (pt, 1H, H-C(3ꢁ));
4.90 (m, 1H, H-C(4ꢁ)); 4.65 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)). Anal. for C30H25N3O9
(555.5). Calcd: C 64.86, H 4.54, N 7.56. Found: C 64.77, H 4.30, N 7.44.

Synthesis ofOligonucleotides
1681
1-α-D-Arabinofuranosylcytosine (22)[18]. In a saturated solution of meth-
anolic ammonia (15 mL)20 (0.5 g, 0.87 mmol) was stirred at room tempera-
ture for 28 h. It was evaporated, the residue dissolved in H2O (20 mL), and
then extracted with CH2Cl2 (10 mL). The CH2Cl2 phase was separated and
then treated again with H2O. The united H2O layers were evaporated and
the resulting syrup recrystallized from MeOH (5 mL) to give 0.135 g (66%)
of colorless crystals of m.p. 198–200◦C. Rf: 0.38 (CHCl3/MeOH 1:1). pKa =
3.89. UV (H2O): pH 1: 212 (3.99), 279 (4.13); pH 6: 214 (sh 3.99), 227
1
(3.91), 270 (3.95). H-NMR (DMSO-d6): 7.58 (d, 1H, H-C(6)); 7.11(s, 2H,
NH2); 5.67 (m, 2H, H-C(1ꢁ), H-C(5)); 5.58 (d, 1H, H-O(2ꢁ)); 5.31 (d, 1H, H-
O(3ꢁ)); 4.91 (t, 1H, H-O(5ꢁ)); 4.09 (m, 1H, H-C(2ꢁ)); 4.00 (m, 1H, H-C(4ꢁ));
3.88 (m, 1H, H-C(3ꢁ)); 3.50 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)). Anal. for C9H13N3O5
(243.2). Calcd: C 44.44, H 5.39, N 17.28. Found: C 44.25, H 5.35, N 17.08.
N6-Benzoyl-9-(2,3,5-tri-O-acetyl-α-D-arabinofuranosyl)adenine (23). N6-
Benzoyl-adenine(5)[26] (0.72g, 3mmol)andN,O-bis-trimethylsilylacetamide
(1.5 mL) were heated in 1,2-dichloroethane (15 mL) to 80◦C in an oilbath
under anhydrous conditions for 15 min. It was evaporated, coevaporated
with toluene, the resulting oil dissolved in toluene (15 mL), TMS-triflate
(0.8 mL, 3.8 mmol) added and the mixture heated to 80◦C. A solution of
1,2,3,5-tetraacetylarabinofuranose (1.14 g, 3.6 mmol) in abs. toluene (15
mL) was then added dropwise with stirring. After 1 h heating, the mixture
was cooled with EtOAc (100 mL), diluted, washed with NaHCO3 solution
(100 mL), dried over Na2SO4, and evaporated. The oil was purified by FC
(4 × 15 cm, silica gel) with toluene/EtOAc/MeOH 10:10:1 (1.2 L) to give
1.25 g (83%) of a colorless solid. Rf: 0.38 (toluene/EtOAc/MeOH 10:10:1).
UV (MeOH): 202 (4.47), 232 (sh 4.12), 254 (sh 4.05), 279 (4.29).1H-NMR
(CDCl3): 9.00 (s, 1H, NH); 8.83 (s, 1H, H-C(8)); 8.19 (s, 1H, H-C(2)); 8.05
(d, 2H, arom. H); 7.55 (m, 3H, arom. H); 6.32 (d, 1H, H-C(1ꢁ)); 6.04 (pt,
1H, H-C(2ꢁ)); 5.38 (pt, 1H, H-C(3ꢁ)); 4.77 (m, 1H, H-C(4ꢁ)); 4.39 (m, 2H,
H-C(5ꢁ, 5ꢁꢁ)); 2.17 (s, 3H, Oac); 2.13 (s, 3H, Oac); 2.10 (s, 3H, Oac). Anal.
for C23H23N5O8 (497.5). Calcd: C 55.53, H 4.66, N 14.08. Found: C 55.51,
H 4.95, N 14.03.
N6 -Benzoyl-9-(2,3,5-tri-O-benzoyl-α-D -arabinofuranosyl)adenine
(24)[16]. A suspension of 5[26] (0.753 g, 3.15 mmol) and (NH4)2SO4 (10
mg) in HMDS(15 mL) was heated in an oilbath under reflux for 20 h. It
was evaporated, the residue dissolved in CH3CN (30 mL), then united with a
solution of 1-O-acetyl-2,3,5-tri-O-benzoylarabinofuranose (1.86 g, 3.6 mmol)
in CH3CN (30 mL) and finally under stirring SnCl4 (0.9 mL, 7.56 mmol)
added dropwise. After reaction at room temperature for 20 h was diluted
with CH2Cl2 (100 mL), washed with saturated aqueous NaHCO3 solution
and twice with NaCl solution. The organic phase was dried over Na2SO4,

1682
C. Henke and W. Pfleiderer
evaporated, and purified by FC (4 × 15 cm, silica gel) with toluene/EtOAc
3:2 (1 L) to give 1.48 g (69%) of a colorless solid. Rf: 0.37 (toluene/EtOAc
1
3:2). UV (MeOH): 201 (4.78), 231 (4.73), 278 (4.36). H-NMR (CDCl3):
9.06 (s, 1H, NH); 8.83 (s, 1H, H-C(8)); 8.30 (s, 1H, H-C(2)); 8.08 (d, 2H,
arom. H); 8.02 (m, 6H, arom. H); 7.45 (m, 12H, arom. H); 6.55 (m, 2H,
H-C(1ꢁ), H-C(2ꢁ)); 5.93 (q, 1H, H-C(3ꢁ)); 5.12 (q, 1H, H-C(4ꢁ)); 4.80 (m,
2H, H-C(5ꢁ, 5ꢁꢁ)). Anal. for C38H29N5O8 (683.7). Calcd: C 66.76, H 4.28,
N 10.24. Found: C 66.16, H 4.32, N 10.00.
9–α-D-Arabinofuranosyl)adenine (25)[16]. Analogous to 22 with 24 (0.4
g, 0.6 mmol). The crude syrup was crystallized from MeOH (4 mL) to give 98
mg (67%) of colorless crystals of m.p. 209–211◦C. Rf: 0.57 (CHCl3/MeOH
1:1). pKa = 4.16. UV (H2O): pH 1: 206 (4.31), 256 (4.15), 260 (sh 4.12);
1
pH 7: 204 (4.34), 258 (4.16). H-NMR (DMSO-d6): 8.32 (s, 1H, H-C(8));
8.14 (s, 1H, H-C(2)); 7.29 (s, 2H, NH2); 5.85 (d, 2H, H-C(1ꢁ)); 5.76 (t, 1H,
H-O(2ꢁ)); 5.62 (d, 1H, H-O(3ꢁ)); 4.87 (t, 1H, (H-O(5ꢁ)); 4.67 (q, 1H, H-
C(2ꢁ)); 4.14 (m, 1H, H-C(4ꢁ)); 3.98 (q, 1H, H-C(3ꢁ)); 3.50 (m, 2H, H-C(5ꢁ,
5ꢁꢁ)). Anal. for C10H13N5O4 (267.3). Calcd: C 44.94, H 4.90, N 26.21. Found:
C 44.90, H 4.92, N 26.61.
N2 -Acetyl-1-(2,3,5-tri-O-acetyl-α-D-arabinofuranosyl)-O6 -diphenylcar-
bamoylguanine (26). A mixture of N2-acetyl-O6-diphenylcarbamoylguanine
(6) (1.17 g, 3 mmol) and N,O-bis-trimethylsilylacetamide (1.5 mL) in
CH2Cl2 (15 mL) was stirred at 80◦C in an oil bath for 15 min. It was
evaporated, coevaporated with toluene, dissolved in toluene (15 mL), and
then TMS-triflate (0.8 mL, 3.8 mmol) was added dropwise. The mixture
was heated to 80◦C and then a solution of 1,2,3,5-tetraacetylarabinofuranose
(1.02 g, 3.2 mmol) in toluene (15 mL) again added dropwise with stirring for
1 h. After cooling was diluted with EtOAc (150 mL), washed with saturated
aqueous NaHCO3 solution, the aqueous phase reextracted with EtOAc (75
mL), and then the organic layers united. After drying over Na2SO4, the
mixture was evaporated and the resulting foam purified by FC (4 × 15 cm,
silica gel) with toluene/EtOAc 1:3 to give 1.4 g (72%) of a colorless solid.
Rf: 0.6 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 202 (4.63), 226 (4.53),
254 (sh 4.22), 277 (4.10). 1H-NMR (CDCl3): 8.04 (s, 1H, H-C(8)); 8.08 (m,
4H, arom. H); 8.02 (s, 1H, NH); 7.40 (m, 10H, arom. H); 6.16 (m, 2H, H-
C(1ꢁ)); 6.02 (d, 1H, H-C(2ꢁ)); 5.34 (dd, 1H, H-C(3ꢁ)); 4.74 (m, 1H, H-C(4ꢁ));
4.34 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 2.56 (s, 3H, N-ac); 2.15 (s, 3H, O-ac); 2.13 (s, 3H,
O-ac); 2.07 (s, 3H, O-ac). Anal. for C31H30N6O10 (646.6). Calcd: C 57.58,
H 4.68, N 13.00. Found: C 57.61, H 4.91, N 12.98.
N2-Acetyl-1-(2,3,5-tri-O-benzoyl-α-D-arabinofuranosyl)-O6-diphenylcar-
bamoylguanine (27). Analogous to the preceeding procedure with 6 (1.17

Synthesis ofOligonucleotides
1683
g, 3 mmol) and 1-O-acetyl-2,3,5-tri-O-benzoylarabinofuranose (1.86 g, 3.6
mmol). Purification by FC (4 × 15 cm, silica gel) with toluene/EtOAc 5:2
(1.1 L) to give 1.96 g (79%) of a colorless solid. Rf: 0.79 (toluene/EtOAc/
MeOH 5:4:1). UV (MeOH): 201 (4.86), 228 (4.86), 257 (sh 4.24), 276 (4.19).
1H-NMR (CDCl3): 8.21 (s, 1H, H-C(8)); 8.08 (m, 4H, arom. H); 8.02 (m,
6H, arom. H); 7.90 (s, 1H, NH); 7.82 (m, 2H, arom. H); 7.45–7.40 (m, 19H,
arom. H); 6.48 (m, 2H, H-C(1ꢁ)); 6.41 (d, 1H, H-C(2ꢁ)); 5.88 (dd, 1H, H-
C(3ꢁ)); 5.13 (m, 1H, H-C(4ꢁ)); 4.79 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 2.44 (s, 3H, N-ac).
Anal. for C46H36N6O10 (832.8). Calcd: C 66.34, H 4.36, N 10.09. Found:
C 66.54, H 4.53, N 9.84.
9–α-D-Arabinofuranosyl)guanine (28). Analogous to 22 in MeOH/NH3
(40 mL) with 27 by stirring for 36 h. After evaporation was dissolved in H2O
(150 mL) and CH2Cl2 (150 mL). The organic phase was washed with H2O
(70 mL) and then the united H2O layer extracted with CH2Cl2 (3 × 80
mL). The H2O extract was evaporated to a small volume until crystallization
started. It was heated till a clear solution was obtained, then cooled and
kept in the icebox overnight to give 0.25 g (74%) colorless crystals of
m.p. >300◦C (decomp.). Rf: 0.26 (2-BuOH, 2.PrOH, H2O, conc. NH4OH
30:45:25:2). pKa = 9.78. UV (H2O): pH 7: 252 (4.12), 274 (sh 3.92); pH 12:
1
210 (4.36), 258 (sh 4.05), 263 (4.05). H-NMR (DMSO-d6): 10.63 (s, 1H,
NH); 7.90 (s, 1H, H-C(8)); 6.45 (s, 2H, NH2); 5.72 (t, 1H, H-O(2ꢁ)); 5.64
(d, 2H, H-C(1ꢁ)); 5.52 (d, 1H, H-O(3ꢁ)); 4.87 (t, 1H, (H-O(5ꢁ)); 4.44 (q,
1H, H-C(2ꢁ)); 4.05 (m, 1H, H-C(4ꢁ)); 3.95 (q, 1H, H-C(3ꢁ)); 3.54 (m, 2H, H-
C(5ꢁ, 5ꢁꢁ)). Anal. for C10H13N5O5 (283.2). Calcd: C 42.41, H 4.63, N 24.72.
Found: C 42.34, H 4.87, N 24.33.
1-[3,5-O-(1,1,3,3-Tetraisopropyldisiloxan-1,3-diyl)-α-D-arabinofuranosyl]
uracil (29). 1-α-Arabinofuranosyluracil (15) (2.9 g, 12 mmol) was twice
coevaporated with abs. pyridine and then with 1,3-dichloro-1,1,3,3-tetraiso-
propyldisiloxane (4.4 mL, 14.1 mmol) in pyridine (80 mL) 15 h stirred at
room temperature. It was dilute with EtOAc (80 mL), then treated with
0.2 M HCl (80 mL), the aqueous layer separated and reextracted with
EtOAc. The united EtOAc phases were washed with saturated NaHCO3
solution (80 mL) and saturated NaCl solution (80 mL), dried over Na2SO4,
evaporated, and the residue purified by FC (3 × 8 cm, silica gel) with toluene
(100 mL) and toluene/EtOAc 1:2. The product fraction was evaporated,
coevaporated with CH2Cl2 and dried under high vacuum to give 4.97 g
(85%) of a colorless solid foam. Rf: 0.66 (toluene/EtOAc/MeOH 5:4:1).
1
UV (MeOH): 207 (3.95), 260 (4.00). H-NMR (DMSO-d6): 11.35 (bs, 1H,
NH); 7.85 (d, 1H, H-C(6)); 5.70 (d, 1H, H-C(5)); 5.61 (m, 2H, H-C(1ꢁ), H-
O(2ꢁ)); 4.45 (dd, 1H, H-C(2ꢁ)); 4.15 (m, 2H, H-C(3ꢁ), H-C(4ꢁ)); 3.85 (2H, H-
C(5ꢁ, 5ꢁꢁ)); 1.15-0.9 (m, 28H, 4 × Me2CH). Anal. for C12H38N2O7Si2 (486.7).
Calcd: C 51.82, H 7.87, N 5.76. Found: C 52.17, H 8.05, N 5.37.

1684
C. Henke and W. Pfleiderer
1-[3,5-O-(1,1,3,3-Tetraisopropyldisiloxan-1,3-diyl)-α-D-arabinofuranosyl]
thymine (30). Analogous to 29 with 18 (6.6 g, 25.6 mmol) and 1,3-dichloro-
1,1,3,3-tetraisopropyldisiloxane (9.3 mL, 29.7 mmol) in pyridine (180 mL).
Purification by FC with toluene (400 mL) and toluene/EtOAc 1:1 (600 mL)
to give 11.37 g (89%) of a colorless solid. Rf: 0.68 (toluene/EtOAc/MeOH
5:4:1). UV (MeOH): 209 (3.97), 265 (3.97). 1H-NMR (DMSO-d6): 11.35 (bs,
1H, NH); 7.75 (d, 1H, H-C(6)); 5.75 (d, 1H, H-O(2ꢁ)); 5.65 (m, 2H, H-C(1ꢁ),
H-O(2ꢁ)); 4.40 (dd, 1H, H-C(2ꢁ)); 4.21 (m, 2H, H-C(3ꢁ), H-C(4ꢁ)); 3.85 (2H,
H-C(5ꢁ, 5ꢁꢁ)); 1.15–0.9 (m, 28H, 4 × Me2CH). Anal. for C13H40N2O7Si2
(500.7). Calcd: C 52.77, H 8.05, N 5.59. Found: C 52.67, H 8.17, N 5.53.
1-[3,5-O-(1,1,3,3-Tetraisopropyldisiloxan-1,3-diyl)-α-D-arabinofurano-
syl]-cytosine (31). Analogous to 29 with 22 (8.0 g, 32.8 mmol) and 1,3-
dichloro-1,1,3,3-tetraisopropyldisiloxane (11.4 mL, 36.2 mmol) in pyridine
(180 mL). Purification by FC with toluene (300 mL), toluene/EtOAc 1:1
(1000 mL + 10 mL MeOH) and toluene/EtOAc 1:1 (750 mL + 20 mL
MeOH) to give 13.0 g (83%) of a colorless solid. Rf: 0.24 (toluene/EtOAc/
MeOH 5:4:1). UV (MeOH): 203 (4.19), 236 (sh 3.90), 271 (3.93). 1H-NMR
(DMSO-d6): 7.75 (d, 1H, H-C(6)); 7.20 (bs, 2H NH2); 5.65 (m, 2H, H-
C(5),H-O(2ꢁ)); 5.55 (d, 2H, H-C(1ꢁ), H-O(2ꢁ)); 4.45 (dd, 1H, H-C(2ꢁ)); 4.15
(m, 2H, H-C(3ꢁ), H-C(4ꢁ)); 3.85 (2H, H-C(5ꢁ, 5ꢁꢁ)); 1.15-0.9 (m, 28H, 4 ×
Me2CH). Anal. for C21H39N3O6Si2 (485.7). Calcd: C 51.93, H 8.09, N 8.65.
Found: C 51.50, H 8.13, N 8.45.
9-[3,5-O-(1,1,3,3-Tetraisopropyldisiloxan-1,3-diyl)-α-D-arabinofuranosyl]
adenine (32). Analogous to 29 with 25 (1.2 g, 4.6 mmol) and 1,3-dichloro-
1,1,3,3-tetraisopropyldisiloxane (1.65 mL, 5.1 mmol) in pyridine (35 mL).
Purification by FC (3 × 8 cm, silica gel) with toluene (200 mL) and toluene/
EtOAc/MeOH 15:15:2 to give 1.98 g (83%) of a colorless solid. Rf: 0.37
1
(toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 206 (4.30), 258 (4.13). H-
NMR (DMSO-d6): 8.40 (s, 1H, H-C(8)); 8.15 (s, 1H, H-C(2)); 7.30 (bs, 2H
NH2); 5.92 (d, 1H, H-O(2ꢁ)); 5.75 (d, 2H, H-C(1ꢁ)); 5.05 (dd, 1H, H-C(2ꢁ));
4.25 (m, 2H, H-C(3ꢁ), H-C(4ꢁ)); 3.85 (2H, H-C(5ꢁ, 5ꢁꢁ)); 1.2–0.9 (m, 28H, 4 ×
Me2CH). Anal. for C22H39N5O5Si2 (509.8). Calcd: C 51.84, H 7.71, N 13.74.
Found: C 51.69, H 7.72, N 13.94.
1-{2-O-[2-(4 -Nitrophenyl)ethoxycarbonyl]-3,5-O-(1,1,3,3-tetraisopro-
pyldisiloxan-1,3-diyl)-α-D-arabinofuranosyl}uracil (33). A cooled solution
(0–5◦C) of 29 (5.8 g, 12 mmol) in abs. CH2Cl2 (120 mL) was treated with 1-
methyl-3-[2-(4-nitrophenyl)ethoxycarbonylimidazolium chloride (9.4 g, 30
mmol) and 4-dimethylaminopyridine (1.5 g, 12 mmol) and stirred at 5◦C
for 18 h. It was evaporated, the residue treated with EtOAc (200 mL), 0.2 M
HCl (200 mL), and extracted. The organic layer was washed subsequently

Synthesis ofOligonucleotides
1685
with saturated NaHCO3 and NaCl solution and then dried over MgSO4.
After evaporation was purified by FC (5 × 12 cm, silica gel) with toluene
(200 mL), toluene/EtOAc 10:1 (200 mL), toluene/EtOAc 4:1 (300 mL)
and toluene/AcOEt 2:1 (600 mL) to give 7.2 g (88%) of a colorless solid
foam. Rf: 0.71 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 203 (4.31), 262
1
(4.30). H-NMR (CDCl3): 8.60 (bs, 1H, NH); 8.15 (d, 2H, o to NO2); 7.41
(d, 2H, m to NO2); 7.30 (d, 1H, H-C(6)); 5.71 (m, 2H, H-C(5), H-C(1ꢁ));
5.45 (pt, 1H, H-C(2ꢁ)); 4.55 (m, 2H, H-C(3ꢁ)); 4.40 (m, 2H, OCH2CH2);
4.20 (m, 1H, H-C(4ꢁ)); 3.95 (d, 2H, H-C(5ꢁ, 5ꢁꢁ)); 3.12 (t, 2H, OCH2CH2);
1.2–0.9 (m, 28H, 4 × Me2CH). Anal. for C30H45N3O11Si2 (679.9). Calcd:
C 53.00, H 6.67, N 6.18. Found: C 52.66, H 6.72, N 5.96.
1-{2-O-[2-(4-Nitrophenyl)ethoxycarbonyl]-3,5-O-(1,1,3,3-tetraisopropyl-
disiloxan-1,3-diyl)-α-D-arabinofuranosyl}thymine (34). Analogous to 33 with
30 (1.5 g, 3 mmol), 1-methyl-3[2-(4-nitrophenyl)ethoxycarbonylimidazo-
lium chloride (2.5 g, 8 mmol) and 4-dimethylaminopyridine (0.38 g, 3
mmol) in CH2Cl2 (60 mL) to give 1.94 g (93%) of a colorless solid. Rf:
0.82 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 204 (4.29), 266 (4.32).
1H-NMR (CDCl3): 8.60 (bs, 1H, NH); 8.15 (d, 2H, o to NO2); 7.41 (d, 2H, m
to NO2); 7.10 (d, 1H, H-C(6)); 5.75 (s, 1H, H-C(1ꢁ)); 5.45 (pt, 1H, H-C(2ꢁ));
4.55 (m, 2H, H-C(3ꢁ)); 4.41 (m, 2H, OCH2CH2); 4.19 (m, 1H, H-C(4ꢁ)); 3.95
(d, 2H, H-C(5ꢁ, 5ꢁꢁ)); 3.11 (t, 2H, OCH2CH2); 1.95 (s, 3H, H3C-C(5)); 1.2-0.9
(m, 28H, 4 × Me2CH). Anal. for C31H47N3O11Si2 (693.9). Calcd: C 53.66,
H 6.83, N 6.06. Found: C 53.58, H 6.87, N 6.20.
N4-[2-(4-Nitrophenyl)ethoxycarbonyl]-1-{2-O-[2-(4-nitrophenyl)ethoxy-
carbonyl]-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-α-D-arabinofura-
nosyl}cytosine (35). Analogous to 33 with 30 (11.66 g, 24 mmol), 1-methyl-
3[2-(4-nitrophenyl)ethoxycarbonylimidazolium chloride (19.0 g, 61 mmol)
and 4-dimethylaminopyridine (3.2 g, 25 mmol) in CH2Cl2 (250 mL).
Purification by FC (8 × 20 cm, silica gel) with toluene/EtOAc 10:1 (1 L),
toluene/EtOAc 2:1 (1 L) and toluene/EtOAc (2 L) to give 20.5 g (97%) of
a colorless solid. Rf: 0.85 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 211
1
(4.53), 246 (4.37), 270 (sh 4.36). H-NMR (CDCl3): 10.85 (bs, 1H, NH);
8.15 (d, 5H, 4H, o to NO2, H-C(6)); 7.55 (m, 4H, m to NO2); 6.95 (d, 1H,
H-C(5)); 5.75 (s, 1H, H-C(1ꢁ)); 5.65 (pt, 1H, H-C(2ꢁ)); 4.45 (m, 2H, H-C(3ꢁ),
H-C(4ꢁ)); 4.30 (m, 4H, OCH2CH2); 3.91 (d, 2H, H-C(5ꢁ, 5ꢁꢁ)); 3.11 (q, 4H,
OCH2CH2); 1.1–0.8 (m, 28H, 4 × Me2CH). Anal. for C39H53N5O14Si2 × 0.5
H2O (881.1). Calcd: C 53.17, H 6.18, N 7.94. Found: C 53.16, H 6.25, N 7.44.
N6-[2-(4-Nitrophenyl)ethoxycarbonyl]-1-{2-O-[2-(4-nitrophenyl)ethoxy-
carbonyl]-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-α-D-arabinofura-
nosyl}adenine (36). Analogous to 33 with 32 (1.54 g, 3 mmol), 1-methyl-
3[2-(4-nitrophenyl)ethoxycarbonylimidazolium chloride (6.0 g, 18 mmol)

1686
C. Henke and W. Pfleiderer
and 4-dimethylaminopyridine (0.36 g, 3 mmol) in abs. CH2Cl2 (70 mL).
Purification by FC (3 × 10 cm, silica gel) with toluene (200 mL), toluene/
EtOAc 10.1 (220 mL), toluene/EtOAc (4:1) and toluene/EtOAc 1:1 (400
mL) to give 2.47 g (92%) of a colorless solid. Rf: 0.80 (toluene/EtOAc/
MeOH 5:4:1). UV (MeOH): 209 (4.62), 266 (4.57). 1H-NMR (CDCl3): 10.6
(bs, 1H, NH); 8.55 + 8.50 (2s, 2H, H-C(8), H-C(2)); 8.15 (d, 4H, 4H, o to
NO2); 7.60 + 7.45 (2d, 4H, m to NO2); 6.25 (d, 1H, H-C(1ꢁ)); 6.15 (m, 1H,
H-C(2ꢁ)); 4.55 (m, 2H, H-C(3ꢁ), H-C(4ꢁ)); 4.40 + 4.25 (2t, 4H, OCH2CH2);
3.91 (q, 2H, H-C(5ꢁ, 5ꢁꢁ)); 3.1 + 2.9 (2t, 4H, OCH2CH2); 1.1–0.8 (m, 28H,
4 × Me2CH). Anal. for C40H53N7O13Si2 (896.1). Calcd: C 53.62, H 5.96,
N 10.94. Found: C 53.48, H 5.93, N 11.23.
1-{2-O-[2-(4-Nitrophenyl)ethoxycarbonyl]-α-D-arabinofuranosyl}uracil
(37). A solution of 33 (10.2 g, 15 mmol) in abs. THF (150 mL) and AcOH
(10 mL, 45 mmol) was treated with tetrabutylammonium fluoride × 3 H2O
(TBAF) (14 g) and stirred for 12 h at room temperature. It was diluted
with AcOEt (400 mL) and extracted with saturated NaHCO3 solution. The
aqueous phase was reextracted with AcOEt, then the organic layer united
and further washed with saturated NaCl solution. The AcOEt extract was
dried over Na2SO4, evaporated, and the residue purified by FC (3 × 13 cm,
silica gel) with toluene/EtOAc 1:1 (500 mL), toluene/EtOAc 1:1 + MeOH
(10 mL) (250 mL) and toluene/EtOAc 1:1 + MeOH (40 mL) (500 mL)
to give 5.75 g (88%) of a colorless solid. Rf: 0.30 (toluene/EtOAc/MeOH
5:4:1). UV (MeOH): 203 (4.66), 262 (4.34). 1H-NMR (DMSO-d6): 11.35 (bs,
1H, NH); 8.15 (d, 2H, o to NO2); 7.75 (d, 1H, H-C(6)); 7.55 (d, 2H, m to
NO2); 5.80 (m, 2H, H-C(1ꢁ), H-O(3ꢁ)); 5.65 (m, 2H, H-C(5)); 5.15 (pt, 1H,
H-C(2ꢁ)); 4.90 (t, 1H, H-O(5ꢁ)); 4.35 (m, 2H, OCH2CH2); 4.15 (m, 2H, H-
C(3ꢁ), H-C(4ꢁ)); 3.52 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 3.05 (t, 2H, OCH2CH2). Anal.
for C18H19N3O10 (437.4). Calcd: C 49.42, H 4.38, N 9.61. Found: C 49.59,
H 4.60, N 9.14.
1-{2-O-[2-(4 -Nitrophenyl)ethoxycarbonyl]-α-D-arabinofuranosyl}thy-
mine (38). Analogous to 37 with 34 (10.4 g, 15 mmol) and crystallization
from CH2Cl2 yielded 6.4 g (93%) of colorless crystals. Rf: 0.27 (toluene/
EtOAc/MeOH 5:4:1). UV (MeOH): 203 (4.66), 262 (4.34). 1H-NMR (DMSO-
d6): 11.35 (bs, 1H, NH); 8.15 (d, 2H, o to NO2); 7.65 (d, 1H, H-C(6)); 7.55
(d, 2H, m to NO2); 5.85 (m, 2H, H-C(1ꢁ), H-O(3ꢁ)); 5.15 (pt, 1H, H-C(2ꢁ));
4.90 (t, 1H, H-O(5ꢁ)); 4.35 (m, 2H, OCH2CH2); 4.20 (m, 2H, H-C(3ꢁ), H-
C(4ꢁ)); 3.51 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 3.05 (t, 2H, OCH2CH2), 1.80 (s, 3H,
H3C-C(5)). Anal. for C19H21N3O10 (446.4). Calcd: C 48.44, H 4.52, N 9.41.
Found: C 48.59, H 4.60, N 9.14.
N4-[2-(4-Nitrophenyl)ethoxycarbonyl]-1-{2-O-[2-(4-nitrophenyl)ethoxy-
carbonyl]-α-D-arabinofuranosyl}cytosine (39). Analogous to 37 with 35

Synthesis ofOligonucleotides
1687
(13.1 g, 15 mmol). Purification by FC (6 × 15 cm, silica gel) with toluene/
EtOAc 4:1 (500 mL), toluene/EtOAc 1:1 (500 mL), toluene/EtOAc 1:1 +
MeOH (10 mL) (500 mL), toluene/EtOAc 1:1 + MeOH (20 mL) (500 mL)
and toluene/EtOAc 1:1 + MeOH (50 mL) (1 L) to give 8.15 g (86%) of a
colorless solid. Rf: 0.27 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 203
1
(4.81), 232 (4.44), 273 (4.39). H-NMR (DMSO-d6): 10.80 (bs, 1H, NH);
8.15 (d, 2H, o to NO2); 8.05 (d, 1H, H-C(6)); 7.55 (m, 4H, m to NO2);
7.00 (m, 2H, H-C(5)); 5.80 (m, 2H, H-C(1ꢁ), H-O(3ꢁ)); 5.15 (pt, 1H, H-
C(2ꢁ)); 4.95 (t, 1H, H-O(5ꢁ)); 4.45 (m, 5H, OCH2CH2), H-C(4ꢁ)); 4.15 (m,
2H, H-C(3ꢁ)); 3.52 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 3.05 (t, 4H, OCH2CH2). Anal. for
C27H27N5O13 (629.5). Calcd: C 51.51, H 4.32, N 11.13. Found: C 51.02,
H 4.55, N 10.72.
N6-[2-(4-Nitrophenyl)ethoxycarbonyl]-1-{2-O-[2-(4-nitrophenyl)ethoxy-
carbonyl]-α-D-arabinofuranosyl}adenine (40). Analogous to 37 with 36
(13.4 g, 15 mmol). Purification by FC (6 × 15 cm, silica gel) with toluene/
EtOAc 4:1 (500 mL), toluene/EtOAc 1:1 (600 mL), toluene/EtOAc 1:1 +
MeOH (5 mL) (500 mL), toluene/EtOAc 1:1 + MeOH (15 mL) (500 mL),
toluene/EtOAc 1:1 + MeOH (20 mL) (1 L) and toluene/EtOAc 1:1 +
MeOH (30 mL) (1 L) to give 8.03 g (82%) of a colorless solid. Rf: 0.19
1
(toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 205 (4.61), 266 (4.55). H-
NMR (DMSO-d6): 10.6 (bs, 1H, NH); 8.65 (s, 1H, H-C(8)); 8.80 (s, 1H,
H-C(2)); 8.15 (d, 4H, o to NO2); 7.60 + 7.45 (2d, 4H, m to NO2); 6.20 (d,
2H, H-C(1ꢁ)); 6.00 (d, 1H, H-O(3ꢁ)); 5.75 (pt, 1H, H-C(2ꢁ)); 4.90 (t, 1H, H-
O(5ꢁ)); 4.44–4.20 (m, 6H, 2 × OCH2CH2), H-C(3ꢁ), H-C(4ꢁ)); 3.55 (m, 2H,
H-C(5ꢁ, 5ꢁꢁ)); 3.10 + 2.95 (m, 4H, 2 × OCH2CH2). Anal. for C28H27N7O12
(653.6). Calcd: C 51.46, H 4.16, N 15.00. Found: C 51.26, H 4.33, N 14.75.
1-{5-O-Monomethoxytrityl-2-O-[2-(4-nitrophenyl)ethoxycarbonyl]-α-D-
arabinofuranosyl}uracil (41). The 1-{2-O-[2-(4-nitrophenyl)ethoxycarbon-
yl]-α-D-arabinofuranosyl}uracil (37) (2.8 g, 6.4 mmol) was coevaporated with
toluene, then dissolved in abs. pyridine (60 mL), monomethoxytritylchlo-
ride (Mmtr-Cl) (2.4 g, 7.7 mmol) added and stirred at room temperature
for 20 h. The reaction solution was poured slowly into ice-water (300 mL)
and then extracted with EtOAc (3 × 100 mL), washed with saturated NaCl
solution, and then dried over Na2SO4. It was evaporated, twice coevaporated
with toluene to remove traces of pyridine and purified by FC with toluene/
EtOAc 10:1 (200 mL), toluene/EtOAc 4:1 (500 mL), and toluene/EtOAc
1:1 (500 mL) to give 3.7 g (82%) of a colorless solid foam. Rf: 0.54 (toluene/
EtOAc/MeOH 5:4:1). UV (MeOH): 204 (4.88), 232 (sh 4.38), 262 (4.39).
1H-NMR (DMSO-d6): 11.4 (bs, 1H, NH); 8.15 (d, 2H, o to NO2); 7.75 (d, 1H,
H-C(6)); 7.50 (d, 2H, m to NO2); 7.4–7.2 (m, 12H, trityl); 6.85 (d, 2H, o to
OCH3); 5.85 (m, 2H, H-C(1ꢁ), H-O(3ꢁ)); 5.65 (m, 2H, H-C(5)); 5.10 (pt, 1H,

1688
C. Henke and W. Pfleiderer
H-C(2ꢁ)); 4.31 (m, 3H, H-C(4ꢁ), OCH2CH2); 4.20 (m, 1H, H-C(3ꢁ)); 3.70 (s,
3H, OCH3); 3.10 (m, 4H, H-C(5ꢁ, 5ꢁꢁ),OCH2CH2). Anal. for C38H36N3O11
0.5 H2O (719.7). Calcd: C 63.42, H 5.83, N 5.83. Found: C 63.65, H 5.25,
×
N 5.46.
1-{5-O-Monomethoxytrityl-2-O-[2-(4-nitrophenyl)ethoxycarbonyl]-α-D-
arabinofuranosyl}thymine (42). Analogous to 41 with 38 (2.91 g, 6.4 mmol)
in 8 h to give 4.13 (89%) of a colorless foam. Rf: 0.58 (toluene/EtOAc/
MeOH 5:4:1). UV (MeOH): 208 (4.84), 234 (sh 4.38), 265 (4.32). 1H-NMR
(DMSO-d6): 11.35 (bs, 1H, NH); 8.15 (d, 2H, o to NO2); 7.71 (d, 1H, H-
C(6)); 7.50 (d, 2H, m to NO2); 7.4–7.2 (m, 12H, trityl); 6.85 (d, 2H, o to
OCH3); 5.90 (m, 2H, H-C(1ꢁ), H-O(3ꢁ)); 5.15 (pt, 1H, H-C(2ꢁ)); 4.45-4.20
(m, 4H, H-C(3ꢁ), H-C(4ꢁ), OCH2CH2); 3.70 (s, 3H, OCH3); 3.10 (m, 4H,
H-C(5ꢁ, 5ꢁꢁ), OCH2CH2); 1.81 (s, 3H, H3C-C(5)). Anal. for C39H38N3O11
(724.7). Calcd: C 64.63, H 5.29, N 5.80. Found: C 64.75, H 5.42, N 5.44.
N4-[2-(4-Nitrophenyl)ethoxycarbonyl]-1-{5-O-monomethoxytrityl-2-O-
[2-(4 -nitrophenyl)ethoxycarbonyl]-α-D-arabinofuranosyl}cytosine (43).
Analogous to 41 with 39 (4.0 g, 6.4 mmol) in 28 h. Purification by FC with
toluene/EtOAc 10:1 (200 mL), toluene/EtOAc 2:1 (500 mL) and toluene/
EtOAc 1:2 (500 mL) to give 5.46 g (95%) of a colorless foam. Rf: 0.79
(toluene/EtOAc/MeOH 1:1:1). UV (MeOH): 204 (5.12), 232 (sh 4.71),
1
273 (4.48). H-NMR (DMSO-d6): 10.85 (bs, 1H, NH); 8.15 (m, 5H, 4H,
o to NO2), H-C(6)); 7.60 + 7.50 (2d, 4H, m to NO2); 7.4–7.2 (m, 12H,
trityl); 7.00 (d, 1H, H-C(5)); 6.85 (d, 2H, o to OCH3); 5.81 (m, 2H, H-
C(1ꢁ), H-O(3ꢁ)); 5.15 (pt, 1H, H-C(2ꢁ)); 4.55 (m, 3H, H-C(3ꢁ)); 4.35 (m,
4H, OCH2CH2); 4.15 (dd, 1H, H-C(4ꢁ)); 3.70 (s, 3H, OCH3); 3.10 (m, 6H,
H-C(5ꢁ, 5ꢁꢁ),OCH2CH2). Anal. for C47H43N5O14 (901.9). Calcd: C 62.59,
H 4.81, N 7.76. Found: C 62.30, H 4.99, N 7.32.
N6-[2-(4-Nitrophenyl)ethoxycarbonyl]-1-{5-O-monomethoxytrityl-2-O-
[2-(4 -nitrophenyl)ethoxycarbonyl]-α-D-arabinofuranosyl}adenine (44).
Analogous to 41 with 40 (1.25 g, 1.9 mmol), Mmtr-Cl (0.7 g, 2.28 mmol) in
pyridine (20 mL) in 40 h. FC with toluene/EtOAc 4:1 (400 mL), toluene/
EtOAc 2:1 (300 mL) toluene/EtOAc 1:2 (300 mL) and toluene/EtOAc 1:3
(600 mL) to give 1.63 g (92%) of a colorless solid foam. Rf: 0.80 (toluene/
EtOAc/MeOH 5:4:1). UV (MeOH): 203 (5.00), 229 (sh 4.47), 266 (4.56).
1H-NMR (DMSO-d6): 10.6 (bs, 1H, NH); 8.65(s, 1H, H-C(8)); 8.15 (m, 4H,
o to NO2)); 7.60 + 7.45 (2d, 4H, m to NO2); 7.4–7.2 (m, 12H, trityl); 6.90 (d,
2H, o to OCH3); 6.31 (d, 1H, H-O(3ꢁ)); 6.11 (m, 2H, H-C(1ꢁ), 5.75 (pt, 1H,
H-C(2ꢁ)); 4.55 (m, 3H, H-C(4ꢁ)); 4.32 (m, 5H, H-C(3ꢁ), OCH2CH2); 3.70
(s, 3H, OCH3); 3.25 (m, 6H, H-C(5ꢁ, 5ꢁꢁ)); 3.00 (m, 4H, OCH2CH2). Anal.
for C48H43N7O13 (925.9). Calcd: C 62.20, H 4.78, N 10.58. Found: C 61.91,
H 4.88, N 10.23.

Synthesis ofOligonucleotides
1689
1-{5-O-Dimethoxytrityl-2-O-[2-(4-nitrophenyl)ethoxycarbonyl]-α-D-ara-
binofuranosyl}uracil (45). Analogous to 41 with 37 (1.18 g, 3 mmol) with
dimethoxytrityl chloride (1.15 g, 3.45 mmol) in abs. pyridine (30 mL) in
20 h. Purification by FC with toluene/EtOAc 3:1 (400 mL) and tolune/
EtOAc 1:1 + MeOH (5 mL) (500 mL) to give 1.98 g (88%) of a colorless
foam. Rf: 0.34 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 204 (4.88), 235
(4.44), 263 (4.35). 1H-NMR (DMSO-d6): 11.4 (bs, 1H, NH); 8.15 (d, 2H, o to
NO2); 7.80 (d, 1H, H-C(6)); 7.50 (d, 2H, m to NO2); 7.4–7.2 (m, 9H, trityl);
6.85 (d, 4H, o to OCH3); 5.90 (m, 2H, H-C(1ꢁ), H-O(3ꢁ)); 5.65 (m, 2H, H-
C(5)); 5.15 (pt, 1H, H-C(2ꢁ)); 4.35 (m, 3H, H-C(4ꢁ), OCH2CH2); 4.20 (m,
1H, H-C(3ꢁ)); 3.70 (s, 6H. 2 × OCH3); 3.05 (m, 4H, H-C(5ꢁ, 5ꢁꢁ),OCH2CH2).
Anal. for C39H37N3O12 × 0.5 H2O (748.7). Calcd: C 62.56, H 5.61, N 5.61.
Found: C 62.51, H 5.05, N 5.65.
1-{5-O-Dimethoxytrityl-2-O-[2-(4-nitrophenyl)ethoxycarbonyl]-α-D-ara-
binofuranosyl}thymine (46). Analogous to 45 with 38 (1.35 g, 3 mmol)
and Dmtr-Cl (1.16 g, 3.45 mmol) in 20 h to give 2.1 g (91%) of a colorless
foam. Rf: 0.33 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 204 (4.87), 235
1
(4.43), 266 (4.36). H-NMR (DMSO-d6): 11.4 (bs, 1H, NH); 8.15 (d, 2H, o
to NO2); 7.70 (d, 1H, H-C(6)); 7.50 (d, 2H, m to NO2); 7.4–7.2 (m, 9H,
trityl); 6.85 (d, 4H, o to OCH3); 5.90 (m, 2H, H-C(1ꢁ), H-O(3ꢁ)); 5.15 (pt,
1H, H-C(2ꢁ)); 4.45–4.20 (m, 4H, H-C(3ꢁ), H-C(4ꢁ), OCH2CH2); 3.70 (s, 6H.
2 × OCH3); 3.10 (m, 4H, H-C(5ꢁ, 5ꢁꢁ),OCH2CH2); 1.81 (s, 3H, H3C-C(5).
Anal. for C40H39N3O12 × 0.5 H2O (762.8). Calcd: C 62.98, H 5.28, N 5.50.
Found: C 62.87, H 5.11, N 5.78.
N4-[2-(4-Nitrophenyl)ethoxycarbonyl]-1-{5-O-dimethoxytrityl-2-O-[2-(4-
nitrophenyl)ethoxycarbonyl]-α-D-arabinofuranosyl}cytosine (47). Analo-
gous to 45 with 39 (1.88 g, 3 mmol) and Dmtr-Cl (1.22 g, 3.6 mmol) in
28 h. Purification by FC (4 × 12 cm, silica gel) with toluene/EtOAc 10:1
(200 mL), toluene/EtOAc (3:1 (400 mL), toluene/EtOAc 1:1 (400 mL)
and toluene/EtOAc 2:3 (500 mL) to give 2.7 g (96%) of a colorless foam. Rf:
0.67 (toluene/EtOAc/MeOH 1:1:1). UV (MeOH): 204 (4.97), 236 (4.63),
1
273 (4.43). H-NMR (DMSO-d6): 10.8 (bs, 1H, NH); 8.15 (m, 5H, 4H, o
to NO2, H-C(6)); 7.60 + 7.50 (2d, 4H, m to NO2); 7.4–7.2 (m, 9H, trityl);
6.85 (d, 4H, o to OCH3); 5.85 (m, 2H, H-C(1ꢁ), H-O(3ꢁ)); 5.15 (pt, 1H, H-
C(2ꢁ)); 4.55 (m, 3H, H-C(3ꢁ)); 4.35 (m, 4H, 2 × OCH2CH2); 4.15 (m, 1H, H-
C(4ꢁ)); 3.70 (s, 6H. 2 × OCH3); 3.05 (m, 4H, HC(5ꢁ, 5ꢁꢁ), OCH2CH2). Anal.
for C48H45N5O15 (931.9). Calcd: C 61.86, H 4.87, N 7.26. Found: C 61.96,
H 5.01, N 7.29.
N6-[2-(4-Nitrophenyl)ethoxycarbonyl]-1-{5-O-dimethoxytrityl-2-O-[2-(4-
nitrophenyl)ethoxycarbonyl]-α-D-arabinofuranosyl}adenine (48). Analo-

1690
C. Henke and W. Pfleiderer
gous to 45 with 40 (1.96 g, 3 mmol) and Dmtr-Cl (1.1 g, 3.3 mmol) in
40 h. Purification by FC with toluene/EtOAc 4:1 (500 mL), toluene/EtOAc
1:1 (500 mL) and toluene/EtOAc 1:1 + MeOH (10 mL) (500 mL) to give
2.72 g (95%) of a colorless solid. Rf: 0.64 (toluene/EtOAc/MeOH 5:4:1).
UV (MeOH): 204 (4.98), 236 (4.49), 267 (4.61). 1H-NMR (DMSO-d6): 10.6
(bs, 1H, NH); 8.65 (2s, 2H, H-C(8), H-C(2)); 8.15 (m, 4H, o to NO2, H-
C(6))); 7.60 + 7.45 (2d, 4H, m to NO2); 7.4–7.2 (m, 9H, trityl); 6.89 (d, 4H,
o to OCH3); 6.30 (d, 1H, H-O(3ꢁ)); 6.12 (m, 2H, H-C(1ꢁ)); 5.75 (pt, 1H, H-
C(2ꢁ)); 4.55 (m, 3H, H-C(4ꢁ)); 4.30 (m, 5H, H-C(3ꢁ), 2 × OCH2CH2); 3.70 (s,
6H. 2 × OCH3); 3.25 (m, 4H, H-C(5ꢁ, 5ꢁꢁ)); 3.05 (m, 4H, 2 × OCH2CH2).
Anal. for C49H45N7O14 (955.9). Calcd: C 61.57, H 4.74, N 10.25. Found:
C 61.24, H 4.81, N 10.18.
N2,N9-Diacetyl-O6-2-(4-nitrophenyl)ethylguanine (49). To a suspension
of N2,N9-diacetylguanine[17] (9.6 g, 40 mmol) in abs. dioxane (200 mL)
were subsequently added under stirring triphenylphosphane (13.5 g, 50
mmol) and 2-(4-nitrophenyl)ethanol (14.0 g, 80 mmol). After 10 min ethyl
azodicarboxylate (12.5 mL, 80 mmol) dissolved in dioxane (20 mL) was
added slowly dropwise and the mixture then stirred for 24 h. The precipitate
was collected, washed with ether and dried at 100◦C to give 12.3 g (80%) of
colorless crystals of m.p. 215–219◦C. UV (MeOH): 217 (4.47), 268 (4.40).
1H-NMR (DMSO-d6): 10.55 (bs, 1H, NH); 8.60 (s, 1H, H-C(8)); 8.15 (d,
2H, o to NO2); 7.65 (d, 2H, m to NO2); 4.75 (t, 2H, OCH2CH2); 3.31 (t,
2H, OCH2CH2); 2.85 (s, 3H, Ac-N(9)); 2.25 (s, 3H, ac-N(2)). Anal. for
C17H16N6O5 (384.4). Calcd: C 53.13, H 4.20, N 21.87. Found: C 52.88,
H 4.16, N 21.88.
N2-Acetyl-O6-2-(4-nitrophenyl)ethylguanine (50). N2,N9-Diacetyl-O6-2-
(4-nitro-phenyl)ethylguanine (49) (12.3 g, 32 mmol) was heated in DMF
(180 mL) and H2O (60 mL) under reflux for 4 h. After 10 min boiling a clear
solution was obtained from which crystals separated during heating. The
precipitate was collected after cooling, washed with cold EtOH and ether
and dried at 100◦C to give 10.1 g (92%) of colorless crystals of m.p. 275–
1
277◦C. UV (MeOH): 217 (4.43), 268 (4.36). H-NMR (DMSO-d6): 13.15
(bs, 1H, NH); 10.55 (bs, 1H, NH); 8.60 (s, 1H, H-C(8)); 8.15 (d, 3H, o to
NO2, H-C(8)); 7.65 (d, 2H, m to NO2); 4.75 (t, 2H, OCH2CH2); 3.30 (t, 2H,
OCH2CH2); 2.22 (s, 3H, ac-N(2)). Anal. for C15H14N6O4 (384.4). Calcd:
C 52.63, H 4.12, N 24.55. Found: C 52.50, H 4.14, N 24.51.
O6-2-(4-Nitrophenyl)ethylguanine (51). A) A suspension of 49 (1.15 g,
3 mmol) in methanolic HCl (25 mL) was stirred at room temperature for
2 days. Under cooling was neutralized by 5% aqueous NaOH, the precipitate
collected, washed with H2O and dried at 100◦C to give 0.81 g (90%) impure

Synthesis ofOligonucleotides
1691
solid. Purification was done by FC. 0.8 g were dissolved in hot MeOH (80
mL), then silica gel (12 g) added, evaporated, filled into a column, and
eluted with toluene/EtOAc 1:1 (600 mL) and toluene/EtOAc/MeOH 5:4:1
(600 mL) to give 0.62 g (69%) of a colorless solid. B) Analogous to A with
50 (0.69 g, 2 mmol) to give 0.485 g (81%) of a colorless powder of m.p.
1
>300◦C (decomp.). UV (MeOH): 205 (4.57), 245 (4.10), 278 (4.26). H-
NMR (DMSO-d6): 12.4 (bs, 1H, NH); 8.15 (d, 2H, o to NO2); 7.80 (s, 1H, H-
C(8)); 7.62 (d, 2H, m to NO2); 6.75 (bs, 2H, NH2); 4.60 (t, 2H, OCH2CH2);
3.25 (t, 2H, OCH2CH2). Anal. for C13H12N6O3 (300.3). Calcd: C 52.00,
H 4.03, N 27.98. Found: C 52.25, H 3.99, N 27.49.
N2-Acetyl-O6-[2-(4-nitrophenyl)ethyl]-9-(2,3,5-tri-O-acetyl-α-D-arabino-
furanosyl)guanine (52). A mixture of 50 (1.72 g, 5 mmol) and N,O-bis-
trimethylsilylacetamide (3 mL) in CH2Cl2 (25 mL) was heated in an oilbath
at 80◦C for 2 h with stirring. After 20 min a clear solution was obtained. It was
cooled, evaporated, and coevaporated with toluene. The resulting oil was
dissolved in toluene (30 mL), TMS-triflate (2 mL) added and followed by
dropwise addition of a solution of 1,2,3,4-tetra-O-acetyl-D-arabinofuranose
(2.0 g, 6.25 mmol) in toluene (35 mL) and heated for 3 h at 80◦C. After
cooling was diluted with EtOAc (150 mL), extracted subsequently with satu-
rated NaHCO3 (150 mL) and NaCl solution (150 mL), dried over Na2SO4,
and evaporated. Purification by FC (3 × 12 cm, silica gel) with toluene/
EtOAc 8:1 (400 mL), toluene/EtOAc 4:1 (400 mL), toluene/EtOAc 1:1
(400 mL) and toluene/EtOAc 1:2 (300 mL) to give 1.62 g (55%) of a col-
orless foam. Rf = 0.39 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH): 204
1
(4.44), 217 (4.54), 268 (4.48). H-NMR (CDCl3): 8.15 (d, 2H, o to NO2);
7.90 (s, 1H, H-C(8)); 7.85 (bs, 1H, NH); 7.45 (d, 2H, m to NO2); 6.10 (d,
1H, H-C(1ꢁ)); 6.00 (pt, 1H, H-C(2ꢁ)); 5.31 (pt, 1H; H-C(3ꢁ)); 4.75 (m, 3H,
H-C(4ꢁ), OCH2CH2); 4,32 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 3.30 (t, 2H, OCH2CH2);
2.55 (s, 3H, ac-N(2)); 2.15–2.00 (3s, 9H, 3 × ac-O). Anal. for C26H28N6O11
(600.5). Calcd: C 52.00, H 4.70, N 13.99. Found: C 52.37 H 5.09,
N 13.58.
N2-Acetyl-O6-[2-(4-nitrophenyl)ethyl]-9-(2,3,5-tri-O-benzoyl-α-D-arabi-
nofuranosyl)guanine (53). Analogous to 52 with 50 (1.72 g, 5 mmol) and
1-O-acetyl-2,3,5-tri-O-benzoyl-D-arabinofuranose (2.8 g, 5.6 mmol) to give
2.95 g (75%) of a colorless solid foam. Rf = 0.75 (toluene/EtOAc/MeOH
1
5:4:1). UV (MeOH): 202 (4.66), 222 (4.73), 231 (sh 4.67), 269 (4.48). H-
NMR (CDCl3): 8.15 (d, 2H, o to NO2); 8.05 + 7.85 (2m, 8H, 3 × 2H, o to
bz), H-C(8), NH); 7.55–7.30 (m, 11H, 2H, m to NO2, 3 × 3H bz); 6.50 (pt,
1H, H-C(2ꢁ)); 6.41 (d, 1H, H-C(1ꢁ)); 5.89 (pt, 1H; H-C(3ꢁ)); 5.05 (m, 1H, H-
C(4ꢁ), 4,81 (m, 4H, H-C(5ꢁ, 5ꢁꢁ), OCH2CH2); 3.30 (t, 2H, OCH2CH2); 2.50

1692
C. Henke and W. Pfleiderer
(s, 3H, ac-N(2)). Anal. for C41H34N6O11 (786.8). Calcd: C 62.59, H 4.46,
N 10.68. Found: C 62.53, H 4.45, N 10.71.
O6-[2-(4-Nitrophenyl)ethyl]-9-α-D-arabinofuranosylguanine (54). To a
suspension of 56 (0.7 g, 0.94 mmol) in MeOH (20 mL) was added K2CO3
(25 mg) and stirred under gentle warming for 2 h. It was neutralized by a few
drops of AcOH, the precipitate collected, washed with ether and dried at
100◦C to give 0.345 g, (85%) of a crystalline powder of m.p. 144–146◦C. Rf =
0.67 (toluene/EtOAc/MeOH 1:1:1). UV (MeOH): 212 (4.52), 252 (4.17),
1
278 (4.25). H-NMR (DMSO-d6): 8.15 (d, 2H, o to NO2); 8.05 (s, 1H, H-
C(8)); 7.65 (d, 2H, m to NO2); 6.45 (s, 2H, NH2); 5.75 (d, 2H, H-C(1ꢁ), H-
O(2ꢁ)); 5.55 (d, 1H, H-O(3ꢁ)); 4.90 (t 1H, H-O(5ꢁ)); 4.70 (t, 2H, OCH2CH2);
4.50 (dd, 1H, H-C(2ꢁ)); 4,12 (m, 1H, H-C(4ꢁ)); 3.95 (dd, 1H, H-C(3ꢁ));
3.55 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 3.25 (t, 2H, OCH2CH2); Anal. for C18H20N6O7
(432.4). Calcd: C 50.00, H 4.66, N 19.44. Found: C 49.63, H 4.92, N 19.23.
O6-[2-(4-Nitrophenyl)ethyl]-9-(2,3,5-tri-O-acetyl-α-D-arabinofuranosyl)
guanine (55). A suspension of 51 (0.92 g, 3 mmol) and N,O-bis-trimethyl-
silylacetamide (2 mL) in CH2Cl2 (30 mL) was stirred at 80◦C for 2 h. It
was evaporated, the residue coevaporated with toluene and then dissolved
in toluene (30 mL). After addition of TMS-triflate (0.6 mL) a solution
of 1,2,3,5-tetra-O-acetyl-D-arabinofuranose (1.28 g, 4 mmol) in toluene (12
mL) was dropwise added followed by heating to 80◦C for 18 h. The reaction
solution was diluted with EtOAc (120 mL) and subsequently washed with
saturated aqueous NaHCO3 (100 mL) and NaCl solution (100 mL). The
organic layer was dried over Na2SO4, evaporated, and then purified by FC
(3 × 10 cm) with toluene/EtOAc 4:1 (400 mL), toluene/EtOAc 2:1 (400
mL), and toluene/EtOAc/MeOH 20:20:1 (300 mL) to give 0.46 mg (52%)
of a colorless foam. Rf = 0.22 (toluene/EtOAc/MeOH 5:4:1). UV (MeOH):
209 (4.49), 253 (4.17), 277 (4.22). 1H-NMR (CDCl3): 8.20 (d, 2H, o to NO2);
7.72 (s, 1H, H-C(8)); 7.45 (d, 2H, m to NO2); 6.05 (d, 1H, H-C(1ꢁ)); 5.92
(pt, 1H, H-C(2ꢁ)); 5.25 (pt, 1H; H-C(3ꢁ)); 4.90 (bs, 2H, NH2); 4.65 (m, 3H,
H-C(4ꢁ), OCH2CH2); 4,25 (m, 2H, H-C(5ꢁ, 5ꢁꢁ)); 3.20 (t, 2H, OCH2CH2);
2.10 (3s, 9H, 3 × ac-O). Anal. for C24H26N6O10 (558.5). Calcd: C 51.61,
H 4.69, N 15.05. Found: C 51.35 H 4.93, N 15.50.
O6-[2-(4-Nitrophenyl)ethyl]-9-(2,3,5-tri-O-benzoyl-α-D-arabinofurano-
syl)guanine (56). Analogous to 55 with 51 (9.2 g, 30 mmol) and N,O-bis-
trimethylsilylacetamide (15 mL) in CH2Cl2 (30 mL) at 80◦C for 2 h. Fol-
lowed by TMS-triflate (5.5 mL) and 1-O-acetyl-2,3,5-tri-O-benzoyl-D-arabino-
furanose (14.1 g, 27 mmol) in toluene and heating to 80◦C for 8 h. The
crude material was purified by FC (6 × 15 cm, silica gel) using toluene/
EtOAc 6:1 (700 mL), toluene/EtOAc 4:1 (400 mL), toluene/EtOAc 2:1

Synthesis ofOligonucleotides
1693
(1.5 L) and toluene/EtOAc 1:1 (600 mL) to give 14.6 g (73%) of a col-
orless solid. Rf = 0.63 (toluene/EtOAc 1:2). UV (MeOH): 212 (4.55), 231
(4.64), 276 (4.28). 1H-NMR (CDCl3): 8.20 (m, 3H, o to NO2, H-C(8)); 8.00
(d, (m, 6H, 3 × 2H, o to bz)); 7.55–7.40 (m, 11H, 2H, m to NO2, 3 × 3H
bz); 6.58 (bs, 2H, NH2); 6.45 (m, 2H, H-C(1ꢁ), H-C(2ꢁ)); 5.95 (pt, 1H; H-
C(3ꢁ)); 5.15 (m, 1H, H-C(4ꢁ); 4,65 (m, 4H, H-C(5ꢁ, 5ꢁꢁ), OCH2CH2); 3.25
(t, 2H, OCH2CH2). Anal. for C39H32N6O10 (744.7). Calcd: C 62.90, H 4.33,
N 11.28. Found: C 62.21, H 4.23, N 10.99.
O6 -[2-(4 -Nitrophenyl)ethyl]-N2 -[2-(4 -nitrophenyl)ethoxycarbonyl]-9-
(2,3,5-tri-O-benzoyl-α-D-arabinofuranosyl)guanine (57). Compound 56 (4.5
g, 6.5 mmol) was coevaporated with abs. pyridine (15 mL), then dissolved
in pyridine (50 mL), cooled to 5–8◦C, 2-(4-nitrophenyl)ethoxycarbonyl
chloride (2.7 g, 12 mmol) added and kept for 2 days in the icebox. It was
evaporated, coevaporated with toluene (3 × 20 mL), and then the residue
dissolved in CH2Cl2 (10 mL) for FC (4 × 10 cm, silica gel) with toluene/
EtOAc 8:1 (650 mL), toluene/EtOAc 4:1 (500 mL), and toluene/EtOAc 3:1
(800 mL) to give 5.96 g (93%) of a colorless solid foam. Rf = 0.63 (toluene/
EtOAc 1:2). UV (MeOH): 201 (4.73), 217 (4.72), 231 (4.65), 270 (4.52). 1H-
NMR (CDCl3): 10.45 (s, 1H, NH); 8.40 (s, 1H, H-C(8)); 8.20 (m, 4H, o to
NO2, H-C(8)); 8.00 (d, (m, 6H, 3 × 2H, o to bz)); 7.7–7.3 (m, 13H, 4H, m
to NO2, 3 × 3H bz); 6.55 (d, 1H, H-C(1ꢁ)); 6.45 (m, 1H, H-C(2ꢁ)); 6.00 (m,
1H; H-C(3ꢁ)); 5.60 (dd, 1H, H-C(4ꢁ); 4,65 (m, 4H, OCH2CH2); 4.25 (m, 2H,
H-C(5ꢁ, 5ꢁꢁ), 3.3 + 3.0 (2t, 4H, OCH2CH2). Anal. for C48H39N7O14 (937.9).
Calcd: C 61.47, H 4.19, N 10.45. Found: C 61.42, H 4.31, N 10.32.
O6-[2-(4-Nitrophenyl)ethyl]-N2-[2-(4-nitrophenyl)ethoxycarbonyl]-9-α-
D-arabinofuranosyl)guanine (58). In a mixture of MeOH (100 mL), diox-
ane (100 mL), and conc. aqueous NH3 (100 mL) compound 57 (7.45 g,
10 mL) was dissolved and then stirred at room temperature for 2 days in a
closed bottle. It was evaporated and the residue crystallized from MeOH to
give 3.5 g (81%) of colorless crystals of m.p. 123–130◦C. Rf = 0.30 (CH2Cl2/
1
MeOH 8:1). UV (MeOH): 206 (4.77), 268 (4.58). H-NMR (DMSO-d6):
10.35 (bs, 1H, NH); 8.40 (s, 1H, H-C(8)); 8.20 (m, 4H, o to NO2); 7.60 (m,
4H, m to NO2); 5.85 (d, 1H, H-C(1ꢁ)); 5.75 (d, 1H, H-O(2ꢁ)); 5.45 (d, 1H,
H-O(3ꢁ)); 4.90 (t, 1H, H-O(5ꢁ)); 4.75 (t, 2H, OCH2CH2); 4.60 (dd, 1H, H-
C(2ꢁ)); 4.22 (m, 1H, H-C(3ꢁ)); 4.05 (m, 1H, H-C(4ꢁ)); 3.55 (m, 2H, H-C(5ꢁ,
5ꢁꢁ)); 3.3 + 3.1 (2t, 4H, OCH2CH2). Anal. for C27H27N7O10, × 0.5 H2O
(634.6). Calcd: C 51.11, H 4.30, N 15.45. Found: C 51.24, H 4.46, N 15.38.
O6 -[2-(4 -Nitrophenyl)ethyl]-9-[3,5-O-(1,1,3,3-tetraisopropyldisiloxan-
1,3-diyl)-α-D-arabinofuranosyl]guanine (59). Analogous to 29 with 54 (3.45
g, 8 mmol) and 1,3-di-chloro-1,1,3,3-tetraisopropyldisiloxane (1.6 mL, 9.6