Synthesis and pairing properties of 3′-deoxyribopyranose (4′ → 2′)-oligonucleotides ('p-DNA')

Article abstract of DOI:10.1002/1522-2675(200205)85:5<1443::AID-HLCA1443>3.0.CO;2-U

The preparation and the pairing properties of the new 3′-deoxyribopyranose (4′ → 2′)-oligonucleotide (=pDNA) pairing system, based on 3′-deoxy-β-D-ribopyranose nucleosides is presented. D-Xylose was efficiently converted to the prefunctionalized 3-deoxyri

Full text of DOI:10.1002/1522-2675(200205)85:5<1443::AID-HLCA1443>3.0.CO;2-U

Helvetica Chimica Acta Vol. 85 (2002)
1443
Synthesis and Pairing Properties of 3'-Deoxyribopyranose (4' ! 2')-
Oligonucleotides (−p-DNA×)
by Damian Ackermann and Stefan Pitsch*
¬
¬
¬
Laboratoire de Chimie des Acides Nucleiques, Institut de Chimie Moleculaire et Biologique, Ecole
¬
¬
Polytechnique Federale de Lausanne, EPFL-BCH, CH-1015 Lausanne (e-mail: stefan.pitsch@epfl.ch)
The preparation and the pairing properties of the new 3'-deoxyribopyranose (4' ! 2')-oligonucleotide (ˆ p-
DNA) pairing system, based on 3'-deoxy-b-d-ribopyranose nucleosides is presented. d-Xylose was efficiently
converted to the prefunctionalized 3-deoxyribopyranose derivative 4-O-[(tert-butyl)dimethylsilyl]-3-deoxy-d-
ribopyranose 1,2-diacetate 8 (obtained as a 4 :1 mixture of a- and b-d-anomers; Scheme 1). From this sugar
building block, the corresponding, appropriately protected thymine, guanine, 5-methylcytosine, and purine-2,6-
diamine nucleoside phosphoramidites 29 32 were prepared in a minimal number of steps (Schemes 2 4).
These building blocks were assembled on a DNA synthesizer, and the corresponding p-DNA oligonucleotides
were obtained in good yields after a one-step deprotection under standard conditions, followed by HPLC
purification (Scheme 5 and Table 1). Qualitatively, p-DNA shows the same pairing behavior as p-RNA, forming
antiparallel, exclusively Watson-Crick-paired duplexes that are much stronger than corresponding DNA
duplexes. Duplex stabilities within the three related (i.e., based on ribopyranose nucleosides) oligonucleotide
systems p-RNA, p-DNA, and 3'-O-Me-p-RNA were compared with each other (Table 2). Intrinsically, p-RNA
forms the strongest duplexes, followed by p-DNA, and 3'-O-Me-p-RNA. However, by introducing the
nucleobases purine-2,6-diamine (D) and 5-methylcytosine (M) instead of adenine and cytosine, a substantial
increase in stability of corresponding p-DNA duplexes was observed.
1. Introduction. In the context of an ongoing study about the chemical etiology of
the natural nucleic acid×s structure, a novel pairing system, based on ribopyranose
(4' ! 2')-oligonucleotides (p-RNA) was introduced in 1993 by Eschenmoser and co-
workers [1 6] (Fig. 1)1). The pairing properties of this constitutional isomer of RNA
Fig. 1. Structural representation ofthe p-RNA and the p-DNA backbone, shown in their idealized, linear
conformations, and base pairs formed between the nucleobases 5-methylcytosine (M) and guanine (G), and
between thymine (T) and purine-2,6-diamine (D)
1
)
For a recent article by Eschenmoser and co-workers, which is related to this field, see [7].

1444
Helvetica Chimica Acta Vol. 85 (2002)
differ substantially from those of the natural nucleic acids DNA and RNA: p-RNA
duplexes are at the same time much stronger and formed more selectively than DNA or
RNA duplexes. The almost linear structure of p-RNA duplexes and the strong
inclination between the backbone and the base pairs leads to a strict antiparallel strand
orientation and allows favorable purine-purine intrastrand stacking. Furthermore, no
cross-pairing between p-RNA and DNA or RNA, respectively, is observed.
In the context of creating functionalized aptamers and ribozymes by chemical
synthesis, we wanted to substitute RNA stems and RNA loops by another pairing
system, ideally with the same properties as p-RNA [8]. Unfortunately, due to the harsh
conditions required to remove the 3'-O-benzoyl protecting group present in each
ribopyranose-nucleotide unit, the synthesis of p-RNA oligonucleotides is not compat-
ible with the synthesis of RNA oligonucleotides2). Therefore, we developed the herein-
reported synthesis of the analogous 3'-deoxyribopyranose (4' ! 2')-oligonucleotide
pairing system (ˆ p-DNA), which is based on 3'-deoxy-b-d-ribopyranose nucleosides
(Fig. 1)3).
Initial pairing studies with such oligonucleotides revealed that p-DNA sequences
are forming weaker duplexes than corresponding p-RNA sequences. However, by
replacing the natural nucleobases adenine and cytosine with the analogues purine-2,6-
diamine (D) and 5-methylcytosine (M), respectively (Fig. 1), a substantial increase in
pairing energy was achieved4); p-DNA duplexes forming G ¥ M and D ¥ T Watson-Crick
base pairs have almost identical pairing properties as the corresponding G ¥ C and A ¥ T
containing p-RNA duplexes (see below, Table 2).
In this report, the highly convergent synthesis of the four p-DNA phosphoramidites
containing the nucleobases adenine, thymine, 5-methylcytosine, and purine-2,6-
diamine (Schemes 1 4), the synthesis of oligonucleotides derived therefrom
(Scheme 5, Table 1), and their pairing properties are reported (Table 2, Fig. 2).
2. Results. 2.1. Sugar Building Block. In the context of this project, we evaluated
different routes to the unnatural sugar 3-deoxyribopyranose. The obvious route
included deoxygenation of 1,2 :5,6-di-O-isopropylidene-a-d-glucofuranose deriva-
tives5), followed by selective cleavage of the 5,6-isopropylidene protecting group
under acidic conditions, NaIO4 cleavage of the resulting glycol, reduction of the
resulting aldehyde with NaBH4, and cleavage of the remaining 1,2-isopropylidene
group. However, we were unable to prepare or to isolate pure pyranoside derivatives in
2
)
Deprotection of p-RNA oligonucleotides involves hydrazine treatment (10% in H2O, 48, 20 h [1]), which
causes very fast degradation of uracil-containing nucleosides.
In the same context, we also evaluated 3'-O-methyl-ribopyranose (4'-2')-oligonucleotides (3'-O-Me-p-
RNA) [9]. Duplexes derived from 3'-O-Me-p-RNA, however, exhibited much weaker pairing than the
corresponding p-RNA duplexes and weaker pairing than the corresponding p-DNA duplexes (see below,
Table 2).
3
)
4
5
)
For comparison, the thermodynamic parameters for duplex formation of the p-DNA duplexes A8 ¥ T8 and
D8 ¥ T8 are shown in Table 2 (see below).
In the literature, two different methods for this process are described, either reduction of its 3-(S-methyl
dithiocarbonate) derivative with Bu3SnH[10], or reduction of its 3-[(trifluoromethyl)sulfonyl] derivative
with (Bu4N)BH4 in refluxing benzene [11]. We could improve the second method even further by
performing the reaction in toluene at 1008 (98% yield).
)

Helvetica Chimica Acta Vol. 85 (2002)
1445
6
reasonable quantities ). Therefore, we decided to develop a new synthetic approach,
leading not to 3-deoxyribose, but directly to a pyranoside derivative thereof. As in the
earlier synthesis of l-ribonucleoside phosphoramidites (from d-glucose) [12], we
furthermore aimed at the synthesis of a prefunctionalized sugar building block,
containing two acyl groups at OÀC(1) and OÀC(2) (required for an unambiguous and
efficient preparation of b-d-configurated nucleosides by nucleosidation under Vor-
br¸ggen conditions [13]) and a trialkylsilyl group at OÀC(4), serving as temporary
substitute for the 4,4'-dimethoxytrityl ((MeO)2Tr) group.
In Scheme 1, the synthesis of the prefunctionalized sugar building block 4-O-[(tert-
butyl)dimethyl)silyl]-3-deoxy-d-ribopyranose 1,2-diacetate (8) from benzyl a-d-xylo-
pyranoside (1) [14] is presented. Within this sequence of simple reactions, only two
purifications (filtrations on silica gel) were required, and the building block 8 was
obtained in 50% total yield (0.5-mol scale). Thus, esterification of 1 with phenylboronic
acid in refluxing toluene under removal of H2O (Dean-Stark separator) gave the cyclic
2,4-phenylboronate derivative 2, which was directly transformed into its 3-O-mesyl
derivative 3 with MeSO2Cl/Et3N. After filtration of the by-product Et3N ¥ HCl and
evaporation of toluene, 3 was treated with NaOMe in MeOH. Under these conditions,
the cyclic phenylboronate was cleaved, and subsequently a 1:1 mixture of the two
regioisomeric epoxides 4a and 4b was obtained by intramolecular substitution of the 3-
mesyloxy group by OÀC(2) or OÀC(4). After extractive workup, the mixture 4a/4b
was submitted to hydride reduction with LiAlH4 in THF. In this reaction, only one
product, the 3-deoxyribopyranoside 5, was obtained, indicating that attack of the
hydride occurred in a stereoelectronically favored manner, exclusively at the C(3)
position (in both epoxides 4a and 4b). The crude product 5 from this sequence of
reactions was sufficiently pure (> 95% by 1H-NMR; 90% yield based on 1) to be used
directly for the next step. Treatment of 5 with 1.2 equiv. of tbdms-Cl ((tert-
i
butyl)chlorodimethylsilane) at À788 in CH2Cl2 in the presence of AgNO3 and PrNEt
resulted in the formation of the 4-O-silylated derivative 6 and the corresponding 2,4-di-
O-silylated derivative7). The 4-O-silylated 3-deoxypyranoside 6 could be easily
isolated by filtration over silica gel and was obtained in 68% yield (based on 1;
additionally 11% of the 2,4-di-O-silylated derivative was isolated). Removal of the
benzyl protecting group of 6 by hydrogenolysis with H2 and Pd(OH)2/C in EtOHgave
the pyranose derivative 7, which finally was converted into 8 by acetylation with Ac2O
in pyridine. After filtration through silica gel, the sugar building block 8 was obtained in
pure form as a 4 :1 mixture of the a- and b-d-anomers, and in 70% yield (based on 6).
2.2. Building Blocks for the Assembly of p-DNA Oligonucleotides. In Schemes 2 and
3, the preparation of the four protected p-DNA nucleosides 12, 15, 21, and 28 is shown.
They served as precursors for the synthesis of the four phosphoramidite building blocks
29 32 and the four immobilized p-DNA nucleosides 37 40 (see below). After
nucleoside formation with sugar building block 8 and appropriate nucleobase
derivatives, followed by cleavage of the 4'-O-tbdms protecting group, an unambiguous
6
)
Glycoside formation with 3-deoxy-d-ribose and different alcohols (Fischer conditions, thermodynamic
control) led always to 1:1 mixtures of the corresponding pyranosides and furanosides. Interestingly, 2-O-
acylated 3-deoxyribose derivatives adopt almost exclusively the furanose forms.
7
)
The corresponding 2-O-silylated derivative was not detected.

1446
Helvetica Chimica Acta Vol. 85 (2002)
Scheme 1
a) PhB(OH)2, toluene, reflux (Dean-Stark). b) MeSO2Cl, Et3N, 48. c) NaOMe, MeOH, 48 ! 258. d) LiAlH4,
t
i
THF, 48 ! 258. e) bdms-Cl, Pr2NEt, AgNO3, CH2Cl2, À788 ! 258. f) H2, Pd(OH)2/C, EtOH, 258. g) Ac2O,
pyridine, 258.
introduction of the (MeO)2Tr group at the 4'-O-position of the nucleosides could be
achieved. Only one nucleosidation reaction (with thymine) was required for the
preparation of the two pyrimidine nucleosides 12 and 15. The 5-methylcytosine
derivative 13 was prepared from the thymine nucleoside intermediate 11 by a well-
known base-transformation procedure according to [15].
Thus, under Vorbr¸ggen conditions [13], in MeCN as solvent and with Me3SiOTf as
Lewis acid, the sugar building block 8 reacted smoothly with in situ trimethylsilylated
thymine, forming the thymine nucleoside 9 in quantitative yield. Removal of the tbdms
protecting group with aqueous HCl in MeCN afforded nucleoside 10. Dimethoxy-
tritylation of this compound with (MeO)2TrCl, AgNO3, and collidine (ˆ2,4,6-
trimethylpyridine) in CH2Cl2 gave the 4'-O-(MeO)2Tr-substituted nucleoside 11. After
removal of the 2'-O-acetyl group of 11 with NaOH in MeOH/THF/H2O and
subsequent filtration through silica gel, the thymine nucleoside 12 was isolated in
91% overall yield (based on 8).
The 5-methylcytosine nucleoside 13 was prepared from the intermediate thymine
nucleoside 11 by treatment with (ClC6H4O)P(O)Cl2, triazole, and Et3N in pyridine ( !
formation of the 4-triazolide derivative), followed by NH3 in dioxane and H2O
according to [15]. After silica-gel chromatography, 13 was isolated in 78% yield (based
on 8). Removal of the 2'-O-acetyl group with NaOMe in MeOH/THF/H2O afforded
nucleoside 14, which was transformed into its N4-acetyl derivative 15 by selective N-
acetylation with Ac2O in DMF. The 5-methylcytosine nucleoside 15 was isolated in
71% yield (based on 13) after chromatography (silica gel).

Helvetica Chimica Acta Vol. 85 (2002)
1447
Scheme 2
a) Thymine, N,O-bis(trimethylsilyl)acetamide, MeCN, 608, then Me3SiOTf, 408. b) HCl, H2O, MeCN. c)
(MeO)2TrCl, collidine, AgNO3, CH2Cl2, 258. d) NaOH, THF/MeOH/H2O. e) 1. 4-chlorophenyl phosphorodi-
chloridate, 1H-1,2,4-triazole, pyridine, Et3N, 48 ! 258; 2. NH3, H2O, dioxane, 258. f) Ac2O, DMF, 258.
To achieve regioselective nucleoside formation with 8, the purine derivatives N2-
acetyl-O6-(diphenylcarbamoyl)guanine (16) [16] and N-(6-chloro-9H-purin-2-yl)-2-
methoxyacetamide (23), were employed for the synthesis of the guanine and the
purine-2,6-diamine nucleosides 21 and 28, respectively (Scheme 3)8). The purine
derivative 23 was obtained in 57% yield from 6-chloro-9H-purin-2-amine (22) upon
treatment with (MeOCH2CO)2O in N,N-dimethylacetamide at 1208, followed by
aqueous workup and crystallization.
Nucleosidation of sugar 8 with the in situ O-trimethylsilylated guanine derivative 16
under reported conditions (developed for the nucleosidation of ribofuranose deriva-
tives [16]) afforded a very complex product mixture. However, by changing the solvent
(benzene instead of toluene), the reaction temperature (458 instead of 808), and the
Lewis acid (Et3SiOTf instead of Me3SiOTf), the reaction proceeded much more
cleanly, and the nucleoside 17 was formed as major product. Without purification, the
4'-O-tbdms group of 17 was cleaved with Et4NF/AcOHin MeCN, and the nucleoside 18
was isolated in 55% yield after chromatography (silica gel). Under our standard
conditions, with (MeO)2TrCl, AgNO3, and collidine in CH2Cl2, 18 was converted to the
corresponding 4'-O-(MeO)2Tr derivative 19. At this stage, we wanted to remove the
diphenylcarbamoyl protecting group9). Treatment of 19 with N1,N1,N3,N3-tetra-
methylguanidinium 2-pyridine-syn-carbaldoximate (ˆ pyridine-2-carboxaldehyde
Nucleosidation with N2-acylated guanine derivatives and with N2,N6-diacylated purinediamine derivatives
leads always to mixtures of the corresponding N9- and N7-connected nucleosides. In contrast, nucleoside
formation with O6-(diphenylcarbamoyl)-protected guanine derivatives [16] and 6-chloro-substituted purin-
2-amine derivatives [17] leads almost exclusively to the corresponding N9-connected nucleosides.
We earlier had found that under our preferred oligonucleotide deprotection conditions (MeNH2 in H2O/
EtOH[18]), O6-(diphenylcarbamoyl)-protected guanines are partially converted into N6-methylpurine-
2,6-diamine nucleosides, suggesting attack of MeNH2 at C(6).
8
)
9
)

1448
Helvetica Chimica Acta Vol. 85 (2002)
Scheme 3
a) (MeOCH2CO)2O, N,N-dimethylacetamide, 1208, then H2O crystallization. b) 1. 16, N,O-bis(trimethylsil-
yl)acetamide, (CH2Cl)2, 608; 2. C6H6, Et3SiOTf, 458. c) Et4NF ¥ 2 H2O, AcOH, MeCN, 258. d) (MeO)2TrCl,
collidine, AgNO3, CH2Cl2, 258. e) NaNO2, DMSO, 758. f) NaOH, THF, MeOH, H2O. g) 1. 23, N,O-
bis(trimethylsilyl)acetamide, MeCN, 608; 2. Et3SiOTf, 458. h) 1. NaN3, pyridine, 658; 2. PPh3, 258; 3. Et3N ¥
AcOH, MeOH/THF, 658. i) 1. Isobutyryl chloride, pyridine, CH2Cl2, 258; 2. NaOH, THF/MeOH/H2O.
[c(z)]-oximate) in dioxane/H2O according to [19] resulted in a complex reaction
mixture. However, we found that the protecting group was cleanly removed upon
treatment of 19 with NaNO2 in DMSO at 758. Under these neutral conditions, all other
(acid- and base-sensitive) protecting groups remained intact10). Without purification,
the N2-acetylguanine nucleoside 20 was 2'-O-deacetylated under standard conditions
Under similar conditions (508 instead of 758), a clean conversion of N2-acyl-6-chloropurin-2-amine
nucleosides to the corresponding N2-acylguanines occurs.
10
)

Helvetica Chimica Acta Vol. 85 (2002)
1449
with NaOHin MeOH/H 2O/THF, and the guanine nucleoside 21 was isolated in a
moderate yield of 48% after chromatography (silica gel).
Nucleosidation of 8 with the in situ trimethylsilylated nucleobase derivative 23 was
carried out under the above-described conditions, at 458 with Et3SiOTf as Lewis acid,
but in the solvent MeCN. The crude product 24 was again desilylated with Et4NF/
AcOHin MeCN, and the resulting nucleoside 25 was isolated by chromatography in a
good yield of 66%. Introduction of the 4'-O-(MeO)2Tr group with (MeO)2TrCl,
AgNO3, and collidine in CH2Cl2 led to the 6-chloropurine-2-amine nucleoside 26.
Without purification, this intermediate was converted to the corresponding partially
protected purine-2,6-diamine nucleoside 27 under Staudinger conditions, by first
forming the 6-azido derivative with NaN3 in pyridine, and then treating this
intermediate with PPh3, followed by Et3N ¥ AcOHin MeOH/H 2O/THF. Compound
27 was isolated in a good yield of 77% after chromatography (silica gel). The N6-
isobutyrylated, 2'-O-deacetylated derivative 28 was obtained by treating 27 first with
isobutyryl chloride in pyridine/CH2Cl2 and then with NaOH in MeOH/THF/H2O. After
purification by chromatography (silica gel), the protected purine-2,6-diamine nucleo-
side 28 was isolated in a yield of 79%.
Under standard conditions, with 2-cyanoethyldiisopropylphosphoramidochloridite/
iPr2NEt in CH2Cl2, the four protected 3'-deoxyribopyranose nucleosides 12, 15, 21, and
28 were converted into their corresponding phosphoramidite building blocks 29 32
and isolated in good yields by silica gel chromatography (Scheme 4). The solid supports
were synthesized by first preparing the 2'-(4'-nitrophenyl heptanedioates) 33 36 from
the nucleosides 12, 15, 21, and 28, and then immobilizing these activated esters on
aminoalkyl-functionalized controlled-pore glass (CPG). To prevent additional N2-
acetylation of the purinediamine nucleoside, the final capping of the solid support 40
Scheme 4
i
a) 2-Cyanoethyl diisopropylphosphoramidochloridite, Pr2NEt, CH2Cl2, 258. b) Bis(4-nitrophenyl) heptane-
dioate, pyridine, DMAP (N,N-dimethylpyridin-4-amine), 258. c) 1. Long-chain-alkylamino CPG, iPr2NEt,
DMF, 258; 2. Ac2O, pyridine, 258 for 37 39; (MeOCH2CO)2O, pyridine, 258 for 40.

1450
Helvetica Chimica Acta Vol. 85 (2002)
was carried out with (MeOCH2CO)2O instead of Ac2O11). Typical loadings of 25
33 mmol/g were obtained (Scheme 4).
Prior to the synthesis of p-DNA oligonucleotides, we established the conditions
required for the removal of the nucleobase protecting groups under our preferred
conditions with MeNH2 (10m in H2O/EtOH1:1, 25 8), developed for the synthesis of
RNA sequences from 2'-O-tom-protected ribonucleoside phosphoramidites (tom ˆ
[(triisopropylsilyl)oxy]methyl) [18]. By UV measurements and reversed-phase HPLC
chromatography, we determined t1/2 values of 2 min for the N4-acetyl-5-methylcytosine
nucleoside 15, 4 min for the N2-acetylguanine nucleoside 21, and 3 min for the N6-
isobutyryl-N2-(methoxyacetyl)-protected purine-2,6-diamine nucleoside 28, respec-
tively12).
2.3. p-DNA Oligonucleotides. The synthesis of p-DNA oligonucleotides from the
phosphoramidite building blocks 29 32 and the solid supports 37 40 was carried out
on 1.0- and 10-mmol scales with a Pharmacia Gene Assembler by essentially the
protocol developed for the synthesis of RNA oligonucleotides from 2'-O-tom-
protected phosphoramidites [16] (Scheme 5). To prevent N6-acetylation of the
purinediamine nucleosides during each capping step, it was carried out with
(MeOCH2CO)2O instead of Ac2O. The coupling step was performed with 12 equiv.
(1.0-mmol scale) or 3.5 equiv. (10-mmol scale) of p-DNA phosphoramidites and in the
presence of 5-(benzylthio)-1H-tetrazole as activator. The coupling times were adjusted
to 5 min (1.0-mmol scale) or 9 min (10-mmol scale). The individual coupling yields
obtained under these conditions were >98% (detritylation assay) and could not be
increased further by using more equiv. of phosphoramidites or longer reaction times.
The removal of the base- and phosphate-protecting groups and the cleavage from
the solid support were carried out by treatment with 10m MeNH2 in H2O/EtOH1:1 for
3 h at 258 (Scheme 5); the supernatants were evaporated and the oligonucleotides
subsequently purified by anion-exchange HPLC and characterized by MALDI-TOF-
MS according to [21] (Table 1).
The p-DNA sequences shown in Table 1 were isolated in acceptable yields and at
purity >98% (HPLC analysis). The self-complementary sequence [pd3'(MGDDTTM
G)]213), containing all four nucleosides, was prepared on a larger scale (15 mg yield
after purification) and subsequently subjected to a detailed NMR analysis (performed
by Jaun and Ebert [22]), which demonstrated the correct constitution of this new
oligonucleotide pairing system14).
2.4. Pairing Properties of p-DNA Oligonucleotides. In Fig. 2, the transition curves
(determined by temperature-dependent UV spectroscopy) and CD spectra of some p-
DNA single strands and duplexes, carried out at a single-strand concentration of 10 mm,
11
)
)
Under standard acylation conditions, N2-acylated purin-2-amines are (at least partially) reacting to the
corresponding N2-diacylated compounds, resulting in a scrambling of acyl-protecting groups [20].
First, we evaluated the deprotection of the corresponding N2,N6-diacetyl-substituted purine-2,6-diamine
nucleoside. We found that the N6-acetyl group was removed very rapidly (t1/2 < 2 min) and the N2-acetyl
group very slowly (t1/2 ꢀ 30 min). Therefore, we chose the more stable isobutyryl group for N6-protection
and the more labile methoxyacetyl group for N2-protection, respectively.
12
)
)
The sequence descriptor −pd3'× stands for −pyranosyl, 3'-deoxy× (in analogy to the descriptor −pr×, which is
used for p-RNA sequences).
13
14
The structure of this duplex has not yet been completely determined [22].

Helvetica Chimica Acta Vol. 85 (2002)
1451
Scheme 5
a) Assembly of p-DNA sequences on a DNA synthesizer: detritylation with 4% dichloroacetic acid in
ClCH2CH2Cl (1.0-mmol scale: 2 min; 10-mmol scale: 4 min); coupling with 0.1m phosphoramidite in MeCN,
promoted by 0.25m 5-(benzylthio)-1H-tetrazole in MeCN (1.0-mmol scale: 0.12 ml ‡ 0.36 ml, 5 min; 10-mmol
scale: 0.36 ml ‡ 0.60 ml, 9 min); capping with
a
1:1 mixture of (MeOCH2CO)2O/2,6-lutidine (ˆ2,6-
dimethylpyridine)/THF 1:1 :8 and 16% (v/v) 1-methyl-1-H-imidazole/THF (1.0-mmol scale: 1 min; 10-mmol
scale: 3 min); oxidation with I2/H2O/pyridine/THF 3 :2 :20 :75 (1.0-mmol scale: 0.7 min; 10-mmol scale:
2.5 min). b) Deprotection of p-DNA oligonucleotides: 10m MeNH2 in H2O/EtOH1:1, 25 8, 3 h.
Table 1. Preparation and Characterization of p-DNA Sequences (p ˆ pyrano)
pd3'(4'-sequence-2')13
)
Scale [mmol]
Isolated yielda)
MSc) [m/z]
a.u. (260 nm)b)
mg [%]
calc.d)
found
pd3'(TTTTTTTT) ˆ pd3'(T)8
pd3'(DDDDDDDD) ˆ pd3'(D)8
pd3'(MMMMMM) ˆ pd3'(M)6
pd3'(GGGGGG) ˆ pd3'(G)6
pd3'(MGDDTTMG)
1
1
1
1
1
10
1
1
41
30
25
27
37
450
25
28
1.5 (50)
1.0 (35)
1.0 (45)
0.8 (45)
1.2 (50)
15.0 (55)
0.8 (50)
0.8 (50)
2371.6
2563.8
1757.3
1913.3
2467.7
2467.7
1832.4
1838.3
2371.1
2563.8
1757.2
1913.0
2468.1
2467.8
1832.4
1838.4
pd3'(DMDMDM) ˆ pd3'(DM)3
pd3'(GTGTGT) ˆ pd3'(GT)3
a) Yield after purification by anion-exchange chromatography; according to analytical anion-exchange HPLC,
the purity was >98%. b) a.u. ˆ absorption unit. c) MALDI-TOF-MS: measured in 2,4-dihydroxyacetophenone
d
(ammonium citrate) according to [21]. ) For fragment [M À H]À.
in 0.15m NaCl and at pH7.4, are presented. The completely reversible, sigmoidal
transition curves obtained from 1:1 mixtures of the complementary15) p-DNA
sequences pd3'(D)8 ¥ pd3'(T)8 (Fig. 2,d) and pd3'(G)6 ¥ pd3'(M)6 (Fig. 2,e), and the self-
complementary sequence [pd3'(MGDDTTMG)]2 (Fig. 2,c) clearly indicate a rever-
sible and cooperative duplex formation. The transition temperatures (Tm values) were
368, 638, and 548, respectively, and the hyperchromicity values ranged between 10
15
)
Complementary according to the Watson-Crick pairing rules.

1452
Helvetica Chimica Acta Vol. 85 (2002)
25%. In contrast, no indication for any cooperative interaction of the four p-DNA
single strands pd3'(D)8, pd3'(T)8 (Fig. 2,a), pd3'(G)6, and pd3'(M)6 (Fig. 2,b), respec-
tively, could be detected by temperature-dependent UV spectroscopy. In summary,
Fig. 2. a) c) Transition curves (heating and cooling curves) and d) f) CD spectra of p-DNA duplexes and
single strands. All measurements were carried out at an oligonucleotide single-strand concentration of 10 mm, in
0.15m NaCl at a pHvalue of 7.0 (0.01 m Tris ¥ HCl buffer). pd ˆ pd3'.

Helvetica Chimica Acta Vol. 85 (2002)
1453
these results show that the pairing rules of p-DNA oligonucleotides are the same as of
p-RNA oligonucleotides, both forming exclusively antiparallel paired duplexes
according to the Watson-Crick pairing rules.
The CD spectra obtained from 1:1 mixtures of complementary p-DNA sequences,
i.e., of pd3'(D)8 ¥ pd3'(T)8 (Fig. 2,d) and pd3'(G)6 ¥ pd3'(M)6 (Fig. 2,e), differ substantially
from the CD spectra of the corresponding single strands; furthermore, they are very
closely related to the CD spectra obtained from the similar p-RNA duplexes pr(A)8 ¥
pr(U)8 [1] and pr(G)8 ¥ pr(C)8 [2], respectively.
In Table 2, the pairing properties of p-DNA oligonucleotide duplexes are
summarized and shown in comparison with the properties of p-RNA, 3'-O-Me-p-
RNA16), and DNA duplexes. Additionally, we determined the pairing properties of the
p-DNA duplex pd3'(A)8 ¥ pd3'(T)8 for comparison, and the influence of the concen-
tration and the type of salt on the pairing behavior of the p-DNA duplex pd3'(DM)3 ¥
pd3'(GT)3.
Table 2. Pairing Properties of p-DNA Duplexes in Comparison with p-RNA, 3'-O-Me-p-RNA and DNA
Duplexes. Tm values were determined by temperature-dependent UV spectroscopy and thermodynamic data of
duplex formation from concentration dependence of Tm values according to Marky and Breslauer [23]. All
measurements were carried out at pH7.0 (0.01 m Tris ¥ HCl) and in 0.15m NaCl (when not stated otherwise).
Duplex base sequences
Oligonucleotide
backbone
Tm (10 mm)
DG (258) DH
[kcal/
mol]
TDS (258)
[kcal/ [kcal/
mol] mol]
-R R R R R R R R
T T T T T T T T-
p-DNA (R ˆ D)
p-DNA (R ˆ A)a)
p-RNA (R ˆ D)b)
p-RNA (R ˆ A)c)
368
268
508
408
228
À 9.6
À 7.4
À 41.0 À 31.4
À 39.7 À 32.3
À 10.5
À 7.5
À 62.2 À 51.7
À 45.0 À 37.5
3'-O-Me- p-DNA (R ˆ A)a)
DNA (R ˆ A)d)
< 108
-G G G G G G
Y Y Y Y Y Y-
p-DNA (Yˆ M)
p-RNA (Yˆ C)b)
638
618
À 13.6
À 11.3
À 10.9
À 7.1
À 48.4 À 35.2
À 54.3 À 40.8
À 44.3 À 33.4
À 61.3 À 54.2
3'-O-Me p-RNA (Yˆ C)a)
DNA (Yˆ C)b)
498
228e)
-Y G R R T T Y G
G Y T T R R G Y-
p-DNA (R ˆ D, Y ˆ M)
548
À 11.4
À 12.6
À 10.1
À 8.2
À 52.1 À 11.4
À 54.9 À 12.6
À 61.5 À 10.1
À 54.4 À 46.2
p-RNA (R ˆ A, Yˆ C)b) 608
3'-O-Me p-RNA (R ˆ A, Yˆ C)a) 418
DNA (R ˆ A, Yˆ C)b) 328
-D M D M D M
T G T G T G-
p-DNA
358 (0.15m NaCl) À 9.0
À 42.3 À 33.3
368 (1.0m NaCl)
368 (1.0m MgCl2)
a) Unpublished work from our group (see [9]). b) Data taken from [2]. c) Data taken from [6]. d) Synthesized
according to standard procedures. e) Extrapolated from thermodynamic data.
The duplex stabilities of the four pairing systems p-DNA, p-RNA, 3'-O-Me-p-RNA,
and DNA differ substantially from each other. All three unnatural, pyranose-based
oligonucleotides form stronger duplexes than DNA, but to a different extent. The DG
values of duplex formation (and the Tm values), obtained for the duplexes with the
16
)
The synthesis of this oligonucleotide pairing system is not yet published (see [9]).

1454
Helvetica Chimica Acta Vol. 85 (2002)
identical base composition A8 ¥ T8, show that p-RNA is clearly the strongest pairing
system among these three, followed by p-DNA and 3'-O-Me-p-RNA. By replacing
adenine with the nucleobase purine-2,6-diamine (XˆD), a substantial increase in
duplex stability of p-RNA and p-DNA duplexes resulted (DTm ˆ ‡108). The stability
of the p-DNA duplex pd3'(DM)3 ¥ pd3'(GT)3 showed only a very moderate dependence
on the type and the concentration of added salt. An increase in Tm of ‡ 18 was observed
upon changing the NaCl concentration from 0.15m to 1.0m, and no difference in Tm was
determined by replacing 1.0m NaCl by 1.0m MgCl2.
3. Discussion. By employing a prefunctionalized sugar building block and base-
transformation reactions, we were able to develop a short and highly convergent
synthesis of p-DNA phosphoramidites. Their assembly to oligonucleotides and their
deprotection was carried out under standard conditions, which are fully compatible
with RNA (and DNA) synthesis. Meanwhile, we have prepared a number of p-DNA/
RNA hybrids and could demonstrate that p-DNA duplexes are good substitutes for
hairpin loops within aptamers and ribozymes [8]. Not surprisingly, the qualitative
pairing properties of p-DNA duplexes are very similar to the properties of p-RNA
duplexes: both form much stronger duplexes than the natural oligonucleotide systems
RNA and DNA, and both are more selective pairing systems than RNA and DNA.
In the context of finding p-RNA-related pairing systems that are compatible with
RNA synthesis and deprotection, we have prepared and evaluated 3'-O-Me-p-RNA
and p-DNA oligonucleotides. Among these three structurally closely related oligonu-
cleotide systems, p-RNA intrinsically forms the strongest duplexes, followed by p-DNA
and 3'-O-Me-p-RNA (Table 2)17). The differences in duplex stability among them are
rather large and, so far, difficult to comprehend, mainly due to the lack of structural
data. Constitutionally, the three pairing systems differ only in the type of 3'-substi-
tuents within each nucleotide unit. The three substituents (OH, H, and MeO within p-
RNA, p-DNA, and 3'-O-Me-p-RNA, resp.) are, however, sterically and electronically
different and can, in principle, induce significantly different backbone conformations,
e.g., by steric interactions with other sugar substituents or by altering the hydration
shell around the backbone. With one p-RNA duplex, a detailed NMR-based structural
analysis has been carried out, revealing its quasi-linear overall structure [4]. Recently, a
similar analysis has been carried out with a similar p-DNA duplex, but its structure
could not yet be determined completely [22]. However, significant differences among
the backbone angles b and e were found for p-RNA and p-DNA; the value for angle b is
1458 and 1608, and the value for angle e is À858 and À608 in p-RNA and p-DNA,
respectively. For 3'-O-Me-p-RNA, no structural analysis has been carried out so far.
This work was supported by the Swiss National Science Foundation (Project Nr. 20-61741.00). We thank
Mike Oberhuber, University of Innsbruck, for measuring some mass spectra.
Experimental Part
General. Reagents and solvents from Fluka, unless otherwise stated; 5-(benzylthio)-1H-tetrazole was
synthesized according to [23]. Workup implies partition of the reaction mixture between CH2Cl2 and sat. aq.
17
)
This trend is very different from the situation among the three analogous pairing systems based on furanose
nucleotides, where 2'-O-Me-RNA forms the strongest duplexes, followed by RNA and DNA.

Helvetica Chimica Acta Vol. 85 (2002)
1455
NaHCO3 soln., followed by drying the org. layer (MgSO4), and evaporation. TLC: precoated silica gel plates
from Merck, stained by dipping into a soln. of anisaldehyde (10 ml), conc. H2SO4 soln. (10 ml), and AcOH
(2 ml) in EtOH(180 ml) and subsequent heating with a heat gun. CC (column chromatography): silica gel 60
(230 400 mesh) from Fluka or Al2O3 from Woelm (act. III was obtained according to the manufacturer×s
instruction). Anion-exchange HPLC (prep.): Mono Q HR 5/5 (Pharmacia), flow 1 ml/min; eluent A: 10 mm
sodium phosphate in H2O, pH11.5; eluent B: 10 mm sodium phosphate/1m NaCl in H2O, pH11.5; detection at
260 nm, elution at 258. Anion-exchange HPLC (anal.): DNAPAC PA-100 (Dionex), flow 0.75 ml/min; eluent A:
2 mm Tris ¥ H Cl (pH 7.4), 10 mm NaClO4, 6m urea; eluent B: 2 mm Tris ¥ HCl (pH 7.4), 0.55m NaClO4, 6m urea;
detection at 260 nm, elution at 808. Optical rotation ([a]2D5 ): c (g/100 ml) as indicated in parentheses. UV
Spectra: lmax in nm, e in dm3/mol/cm in parentheses; all measurements at 258. NMR: chemical shift d in ppm and
coupling constants J in Hz. MS: FAB in the positive mode, 3-nitrobenzyl alcohol as matrix; ESI in the presence
of 10À5 m NH3 ¥ AcOH; HR-MALDI in the positive mode, 2,5-dihydroxybenzoic acid as matrix; in m/z (rel.
intensity in %).
Oligonucleotide Synthesis and Deprotection. The oligonucleotides were assembled on a Pharmacia Gene
Assembler Plus under the conditions described in Scheme 5. HPLC-Grade MeCN was dried by refluxing over
CaH2 (24 h). Prior to the assembly, the phosphoramidite solns. (0.1m in MeCN), the 5-(benzylthio)-1H-tetrazole
soln. (0.25m in MeCN) and the MeCN were stored over 4-ä molecular sieves for 14 h. All syntheses were carried
out in the −trityl-off× mode. After the assembly, the solid supports were removed from the cartridges and treated
with 1.5 ml (1-mmol scale) or 7 ml (10-mmol scale) of 12m MeNH2 in H2O/8m MeNH2 in EtOH(1:1 mixture). By
centrifugation, the supernatant solns. were separated from the solid supports and evaporated and the residues
purified by anion-exchange HPLC. The final desalting was carried out according to Pitsch [12].
Thermal Denaturation Studies. Absorbance vs. temperature profiles were recorded in fused quartz cuvettes
at 260 nm with a Cary Bio-1 spectrophotometer equipped with a Peltier temperature controlling device. The
samples were prepared from stock solns. of the oligonucleotide, 1m Tris ¥ HCl buffer (pH 7.0), and 3m NaCl and
subsequently degassed. A layer of silicon oil was placed on the surface of the soln. Prior to the measurements,
each sample was briefly heated to 808. The curves were obtained with both a cooling and a heating ramp of 0.38/
min. The transition temperatures (ˆ Tm values) were obtained after differentiation of the melting curves and
analyzed according to [24].
Benzyl 4-O-[(tert-Butyl)dimethylsilyl]-3-deoxy-a-d-ribopyranoside (6). A soln. of benzyl a-d-xylopyrano-
side (1; 10.0 g, 41.5 mmol; prepared according to [14]) and PhB(OH)2 (5.3 g, 43.75 mmol) in toluene (160 ml)
was heated to reflux for 2 h ( ! 2), then cooled to 08, and treated with Et3N (7.5 ml, 54 mmol) and MeSO2Cl
(4.2 ml, 54 mmol). After warming to 258 and stirring for 1 h, the solid (Et3N ¥ HCl) was filtered off and the
filtrate evaporated. The residue 3 was dissolved in MeOH(160 ml) and the soln. cooled to 4 8, slowly treated with
NaOMe (6.72 g, 124.5 mmol), warmed to 258, and stirred for 1 h. After workup, the crude product 4a/4b was
dissolved in THF (160 ml) and the soln. cooled to 48, treated with LiAlH4 (3.15 g, 83.0 mmol), stirred for 30 min
at 258, cooled to 08, and carefully treated with AcOEt (300 ml) and then with 2m NaOH(500 ml). Phase
i
separation and extraction gave crude 5 (8.5 g) as a solid, which was dissolved in CH2Cl2 (200 ml) and Pr2NEt
(13.5 ml, 79 mmol). This soln. was cooled to À788, treated in turn with tbdms-Cl (6.25 g, 41.5 mmol) and AgNO3
(8.0 g, 47.4 mmol), and allowed to warm up to 258 overnight. Filtration, workup, and CC (silica gel (100 g),
AcOEt/hexane 2 :98 ! 10 :90) gave 6 (9.5 g, 68%). Colorless viscous liquid. TLC (hexane/AcOEt 1:1): Rf 0.90.
o
[a]2D5 ˆ ‡108 (c ˆ 0.89, CHCl3). 1H-NMR (300 MHz, CDCl3): 0.077, 0.085 (2s, Me2Si); 0.85 0.92 (m, tBu); 1.73
(dd, J ˆ 10.6, 11.5, HÀC(3)); 2.07 2.14 (m, H'ÀC(3), OH); 3.44 3.53 (m, HÀC(4), HÀC(5)); 3.63 3.70
(m, HÀC(2)); 3.72 3.82 (m, H'ÀC(5)); 4.53, 4.81 (2d, J ˆ 11.6, PhCH2O); 4.75 (d, J ˆ 3.7, HÀC(1)); 7.26 7.40
(m, 5 arom. H). 13C-NMR (75 MHz, CDCl3): 17.3 (s, Me3C); 25.0 (q, Me); 37.1 (t, C(3)); 63.5 (t, PhCH2O); 68.6
(t, C(5)); 65.2, 66.6 (2d, C(2), C(4)); 95.8 (d, C(1)); 127.3, 127.4, 127.7 (3d, arom. C); 136.8 (s, arom. C). ESI-
‡
‡
MS: 339.4 (14, [M ‡ H] ), 361 (18, [M ‡ Na] ), 356 (100).
4-O-[(tert-Butyl)dimethylsilyl]-3-deoxy-a/b-d-ribopyranose 1,2-Diacetate (8).
A soln. of 6 (24.0 g,
71 mmol) in EtOH(200 ml) was treated with Pd(OH) 2/C (4.4 g; Aldrich) and subjected to 6 bar H2 for 48 h
at 258. After filtration over Celite, evaporation, and co-evaporation with benzene, the residue 7 was dissolved in
pyridine (250 ml), treated with Ac2O (16.7 ml, 178 mmol), and stirred overnight at 258. MeOH(10 ml) was
added, the soln. evaporated, and the residue co-evaporated with toluene and absorbed on SiO2 (20 g). CC (silica
gel (60 g), hexane ! hexane/AcOEt 8 :2) gave 8 (16.0 g, 70%), a-/b-d 4 :1 (by 1H-NMR). Colorless, viscous
liquid. TLC (hexane/AcOEt 4 :1): Rf 0.65. 1H-NMR (300 MHz, CDCl3): 0.06 0.08 (m, Me2Si); 0.86 0.92
(m, tBu); 1.62 1.72 (m, 0.8 H, HÀC(3)(a)); 1.86 (q, J ˆ 11.7, 0.2 H, HÀC(3)(b)); 2.03, 2.06, 2.10, 2.16 (4s, 12 H ,
Ac); 2.28 2.38 (m, 1 H , H'ÀC(3)(a ‡ b)); 3.37 3.92 (m, 3 H , HÀC(4)(a ‡ b), HÀC(5)(a ‡ b), H'ÀC(5)(a ‡
b)); 4.78 (ddd, J ˆ 5.0, 6.9, 9.7, 0.8 H, HÀC(2)(a)); 4.91 (ddd, J ˆ 3.4, 5.0, 12.4, 0.2 H, HÀC(2)(b)); 5.65 (d, J ˆ

1456
Helvetica Chimica Acta Vol. 85 (2002)
6.9, 0.8 H, HÀC(1)(a)); 6.10 (d, J ˆ 3.1, 0.2 H, HÀC(1)(b)). 13C-NMR (75 MHz, CDCl3): 18.0 (s, Me3C); 20.9,
21.0 (2q, Me2Si); 25.7 (q, Me3C); 33.6, 35.9 (2q, MeCO); 64.7, 65.2, 67.3, 67.9 (4d, C(2), C(4)); 65.3, 69.8
‡
(2t, C(3), C(5)); 88.3, 93.2 (2d, C(1)); 169.6, 169.6, 170.2, 170.4 (4s, MeCO). ESI-MS: 350.4 (100, [M ‡ NH4] ),
‡
682 (5, [2M ‡ Na] ).
1-[3'-Deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]thymine 2'-Acetate (11). A suspension of
8
(5.60 g, 17.3 mmol) and thymine (2.40 g, 19.1 mmol) in MeCN (65 ml) was heated to 608, treated with N,O-
bis(trimethylsilyl)acetamide (9.3 ml, 38.6 mmol) and stirred for 45 min at 608. After adjusting the temp. to 408,
the clear soln. was treated with Me3SiOTf (9.4 ml, 52.0 mmol), stirred for 15 min at 408, and poured into a
mixture of sat. aq. NaHCO3 soln./AcOEt 1 :1 (500 ml). The residue 9 obtained after workup was dissolved in
MeCN (100 ml) and treated with conc. aq. HCl soln. (5 ml) for 10 min at 258. After workup, co-evaporation with
benzene, and drying in vacuo, the crude product 10 was dissolved in CH2Cl2 (70 ml), treated with (MeO)2TrCl
(6.5 g, 19.1 mmol), sym-collidine (ˆ2,4,6-trimethylpyridine; 4.6 ml, 34.7 mmol), and AgNO3 (3.24 g,
19.1 mmol), and stirred 20 min at 258. After filtration over Celite, evaporation, and removal of collidine by
destillation in vacuo, the residue 11 (10.4 g) was used further without purification. A small amount of 11 was
purified by CC for characterization. TLC (AcOEt): Rf 0.5. 1H-NMR (300 MHz, CDCl3): 1.83 (q, J ˆ 11.5,
HÀC(3')); 1.85 (d, J ˆ 0.9, MeÀC(5)); 1.97 (s, Ac); 2.14 2.18 (m, H'ÀC(3')); 3.00 (ddd, J ˆ 1.9, 4.7, 10.6,
HÀC(5')); 3.21 (t, J ˆ 10.6, H'ÀC(5')); 3.69 3.77 (sept., J ˆ 4.8, HÀC(4')); 3.796, 3.800 (2s, 2 MeO); 4.66 4.75
(m, HÀC(2')); 5.48 (d, J ˆ 9.4, HÀC(1')); 6.82 6.86 (m, 4 arom. H); 6.95 (d, J ˆ 1.1, HÀC(6)); 7.25 7.48
(m, 9 arom. H); 8.30 (s, NH ).13C-NMR (75 MHz, CDCl3): 12.4 (q, MeÀC(5)); 20.8 (q, MeCO); 36.6 (t, C(3'));
55.3 (q, MeO); 66.3, 67.1 (2d, C(2'), C(4')); 71.1 (t, C(5')); 82.1 (d, C(1')); 86.8 (s, arom. C); 111.4 (d, C(5));
113.3, 127.1, 128.0, 128.3, 130.0, 130.4, 130.1 (7d, arom. C); 135.1 (d, C(6)); 136.2, 136.4, 145.2 (3s, arom. C);
‡
150.4 (s, C(2)); 158.8 (s, arom. C); 163.1 (s, C(4)); 169.7 (s, MeCO). HR-MALDI-MS: 609.224 (22, [M ‡ Na] ,
‡
C33H34N2NaO8 ; calc. 609.222), 303.138 (100).
1-[3'-Deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]thymine (12). A soln. of crude 11 (10.4 g, ca.
17.3 mmol) in THF/MeOH 5 :4 (450 ml) was cooled to 48, treated with 2m NaOH(50 ml, 100 mmol) for 5 min
and then with AcOH(6 g, 100 mmol). After usual workup, the crude product was adsorbed on SiO (20 g),
2
subjected to CC (silica gel (30 g), CH2Cl2 ! MeOH/CH2Cl2 8 :92): 12 (8.6 g, 91% based on 8). Off-white foam.
o
TLC (CH2Cl2/MeOH9 :1): Rf 0.60. [a]2D5 ˆ ‡34 (c ˆ 0.83, CHCl3). UV (MeOH): 265 (11100), 255 (10200), 235
(20600). 1H-NMR (400 MHz, CDCl3): 1.76 (d, J ˆ 1.1, MeÀC(5)); 1.86 (q, J ˆ 11.6, HÀC(3')); 2.32 2.35
(m, H'ÀC(3')); 2.92 (m, HÀC(5')); 3.20 (t, J ˆ 10.4, H'ÀC(5')); 3.38 3.45 (br. m, HÀC(2')); 3.66 3.73
(m, HÀC(4')); 3.758, 3.766 (2s, 2 MeO); 4.30 (br. s, OH); 5.36 (d, J ˆ 9.0, HÀC(1')); 6.80 6.84 (m, 4 arom. H);
6.95 (d, J ˆ 1.1, HÀC(6)); 7.18 7.51 (m, 9 arom. H); 10.2 (br. s, NH ).13C-NMR (CDCl3, 100 MHz): 12.3
(q, MeÀC(5)); 39.7 (t, C(3')); 55.2 (q, MeO); 66.9, 67.6 (2d, C(2'), C(4')); 70.9 (t, C(5')); 85.0 (d, C(1')); 86.6
(s, arom. C); 113.2, 127.0, 127.9, 128.1, 130.1, 130.2 (6d, arom. C); 135.6 (d, C(6)); 136.5, 136.7, 145.5 (3s,
‡
arom. C); 151.5 (s, C(2)); 158.6, 158.7 (2s, arom. C); 164.0 (s, C(4)). HR-MALDI-MS: 567.209 (4, [M ‡ Na] ,
‡
C31H32N2NaO7 ; calc. 567.211), 303.138 (100).
1-[3'-Deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]-5-methylcytosine 2'-Acetate (13). A soln. of
crude 11 (4.2 g, ca. 7.0 mmol) in pyridine (20 ml) and Et3N (13.6ml, 98 mmol) was cooled to 48, treated with 1H-
1,2,4-triazole (5.8 g, 83.7 mmol) and 4-chlorophenyl phosphorodichloridate (2.3 ml, 14.1 mmol), allowed to
warm to 258, and stirred 48 h at 258. The mixture was diluted with dioxane (30 ml), treated with 25% aq. NH3
soln. (20 ml), and stirred 1 h at 258. After evaporation of the dioxane and usual workup, the residue was
adsorbed on SiO2 (6 g). Purification by CC (silica gel (12 g), CH2Cl2 ! MeOH/CH2Cl2 8 :92) gave 13 (3.2 g,
78%). Yellow foam. TLC (CH2Cl2/MeOH9 :1): Rf 0.30. 1H-NMR (300 MHz, CDCl3): 1.88, 1.91 (2s, Ac,
MeÀC(5)); 1.86 1.94 (m, HÀC(3')); 2.14 2.18 (m, H'ÀC(3')); 2.98 (dd, J ˆ 3.4, 10.8, HÀC(5')); 3.23 (t, J ˆ
10.8, H'ÀC(5')); 3.69 3.76 (sept., J ˆ 5.3, HÀC(4')); 3.786, 3.789 (2s, 2 MeO); 4.66 4.75 (m, HÀC(2')); 5.70
(d, J ˆ 9.3, HÀC(1')); 6.82 6.86 (m, 4 arom. H); 7.02 (s, HÀC(6)); 7.20 7.48 (m, 9 arom. H); 8.17 (s, NH2).
13C-NMR (75 MHz, CDCl3): 13.2, (q, MeÀC(5)); 20.8 (q, MeCO); 36.7 (t, C(3')); 55.3 (q, MeO); 66.5, 67.2
(2d, C(2'), C(4')); 71.7 (t, C(5')); 82.1 (d, C(1')); 86.7 (s, arom. C); 102.9 (d, C(5)); 113.2, 126.7, 127.1, 127.9,
128.0, 128.4, 130.4, 130.1 (8d, arom. C); 136.3, 136.5 (2s, arom. C); 137.9 (d, C(6)); 145.3 (s, arom. C); 156.3
‡
(s, C(2)); 158.7 (s, arom. C); 165.5 (s, C(4)); 170.0 (s, MeCO). HR-MALDI-MS: 608.235 (25, [M ‡ Na] ,
‡
C33H35N3NaO7 ; calc. 608.238), 303.133 (100).
N4-Acetyl-1-[3'-deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]-5-methylcytosine (15). A soln. of 13
(2.65 g, 4.5 mmol) in THF/MeOH 5 :4 (90 ml) was cooled to 48, treated with 2m NaOH(10 ml, 20 mmol), stirred
for 10 min at 48, treated with AcOH(1.2 g, 20 mmol), concentrated to 20 ml, and worked up. The residue was
dissolved in DMF (20 ml), treated with Ac2O (425 ml, 4.5 mmol), and stirred at 258 overnight. After extraction
and evaporation, DMF was removed by destillation in vacuo. The crude product was adsorbed on SiO2 (5 g).

Helvetica Chimica Acta Vol. 85 (2002)
1457
Purification by CC (silica gel (12 g), CH2Cl2 ! MeOH/CH2Cl2 6 :94) gave 15 (1.88 g, 71%). Solid foam. TLC
o
(CH2Cl2/MeOH9 :1): Rf 0.20. [a]2D5 ˆ ‡71 (c ˆ 0.80, CHCl3). UV (MeOH): 305 (6600), 290 (5500), 282 (6300),
269 (5800), 236 (26000). 1H-NMR (400 MHz, (D6)DMSO): 1.55 (q, J ˆ 11.5, HÀC(3')); 1.84 1.89
(m, MeÀC(5), H'ÀC(3')); 2.22 (br. s, Ac); 2.96 (dd, J ˆ 2.9, 9.8, HÀC(5')); 3.10 (t, J ˆ 10.6, H'ÀC(5'));
3.51 3.59 (m, HÀC(2')); 3.60 3.68 (m, HÀC(4')); 3.743, 3.747 (2s, 2 MeO); 5.17 (d, J ˆ 6.1, HÀC(1')); 5.27
(br. d, J ˆ 8.2, OH); 6.91 6.94 (m, 4 arom. H); 7.22 7.45 (m, 9 arom. H); 7.75 (s, HÀC(6)); 9.75 (br. s, NH ).
13C-NMR (100 MHz, (D6)DMSO): 13.2 (q, MeÀC(5)); 24.8 (q, MeCO); 55.0 (q, MeO); 64.9, 66.4 (2d, C(2'),
C(4')); 69.9 (t, C(5')); 85.5 (d, C(1')); 85.8 (s, arom. C); 105.7 (d, C(5)); 113.2, 126.7, 127.6, 127.8, 128.2, 129.6,
129.7 (7d, arom. C); 136.1, 136.3 (2s, arom. C); 143.3 (d, C(6)); 145.5 (s, arom. C); 154.6 (s, C(2)); 158.2
‡
‡
(s, arom. C); 162.3 (s, C(4)); 170.5 (s, MeCO). HR-MALDI-MS: 608.236 (1, [M ‡ Na] , C33H35N3NaO7 ; calc.
608.238), 303.137 (100).
N2-Acetyl-9-(3'-deoxy-b-d-ribopyranosyl)-O6-(diphenylcarbamoyl)guanine 2'-Acetate (18). A soln. of
8
(9.8 g, 30.4 mmol) and N2-acetyl-O6-(diphenylcarbamoyl)guanine (16; 14.7 g, 39.5mmol; prepared according to
[16]) in (CH2Cl)2 (105 ml) was heated to 608 and treated with N,O-bis(trimethylsilyl)acetamide (19.3 ml,
79 mmol) for 45 min at 608. The solvent was evaporated and the residue redissolved in benzene (105 ml). The
mixture was heated to 458, treated with Et3SiOTf (11.7 ml, 51.6 mmol), stirred 90 min at 458, poured into a
mixture of sat. aq. NaHCO3 soln./AcOEt 1 :1 (500 ml), filtered, and worked up. The residue 17 was dissolved in
MeCN (150 ml) containing Et4NF ¥ 2 H2O (27.8 g, 150 mmol) and AcOH(2.0 ml, 35 mmol), and stirred 20 min
at 258. After workup, the crude product was adsorbed on SiO2 (30 g) and purified by CC (silica gel (75 g),
o
CH2Cl2 ! MeOH/CH2Cl2 8 :92): 18 (9.13 g, 55%). Orange foam. TLC (CH2Cl2/MeOH9 :1): Rf 0.65. [a]D25
ˆ
‡9 (c ˆ 0.71, CHCl3). UV (MeOH): 277 (12400), 267 (11500), 254 (sh, 16400), 225 (31800). 1H-NMR
(300 MHz, CDCl3): 1.77 (q, J ˆ 10.9, HÀC(3')); 1.86 (s, AcO); 2.35 (br. s, OH); 2.57 (s, AcNH); 2.66 2.71
(m, H'ÀC(3')); 3.47 (t, J ˆ 9.7, HÀC(5')); 4.06 4.14 (m, HÀC(4'), H'ÀC(5')); 5.43 (ddd, J ˆ 4.7, 9.0, 10.9,
HÀC(2')); 5.55 (d, J ˆ 9.0, HÀC(1')); 7.23 7.44 (m, 10 arom. H); 8.03 (s, HÀC(8)); 8.09 (s, NH ). 13C-NMR
(75 MHz, CDCl3): 20.7, 25.2 (2q, MeCOO, MeCONH); 37.8 (t, C(3')); 64.1, 67.8 (2d, C(2'), C(4')); 72.1 (t, C(5'));
83.0 (d, C(1')); 120.9 (s, C(5)); 128.5, 129.5 (2d, arom. C); 141.9 (s, arom. C); 142.3 (d, C(8)); 150.6
(s, Ph2NCOO); 152.6 (s, C(2)); 155.3 (s, C(4)); 156.5 (s, C(6)); 169.7, 171.3 (2s, MeCONH, MeCOO). HR-
‡
‡
MALDI-MS: 569.173 (100, [M ‡ Na] , C27H26N6NaO7 ; calc. 569.176).
N2-Acetyl-9-[3'-deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]guanine (21). A soln. of 18 (4.05 g,
7.4 mmol) in CH2Cl2 (30 ml) was treated with sym-collidine (2.0 ml, 15 mmol), AgNO3 (1.39 g, 8.2 mmol), and
(MeO)2TrCl (2.77 g, 8.2 mmol), stirred 30 min at 258, filtered over Celite, and worked up. The residue 19 was
dissolved in a soln. of NaNO2 (4.14 g, 60 mmol) in DMSO (60 ml) and kept 4 h at 758. After workup, sym-
collidine and DMSO were distilled off in vacuo, and the crude product 20 was dissolved in THF/MeOH 5 :4
(135 ml). The soln. was cooled to 48, treated 8 min with 2m NaOH(15 ml), and then AcOH(1.7 ml, 30 mmol)
was added. The mixture was concentrated to 40 ml, worked up and adsorbed on SiO2 (12 g). CC (silica gel
(24 g), CH2Cl2 (‡2% Et3N) ! MeOH/CH2Cl2 7:93 (‡2% Et3N)) and subsequent extraction (aq. NaHCO3
o
soln./CH2Cl2) gave 21 (2.17 g, 48%).Yellow foam. TLC (CH2Cl2/MeOH9 :1): Rf 0.50. [a]2D5 ˆ À4 (c ˆ 0.83,
CHCl3). UV (MeOH): 280 (sh, 12700), 274 (13100), 270 (12900), 256 (sh, 17200), 236 (25900). 1H-NMR
(400 MHz, (D6)DMSO): 1.56 (q, J ˆ 11.2, HÀC(3')); 1.94 1.99 (m, H'ÀC(3')); 2.17 (s, MeCOO); 2.98 (dd, J ˆ
2.6, 10.8, HÀC(5')); 3.11 (t, J ˆ 10.7, H'ÀC(5')); 3.68 (sept., J ˆ 4.8, HÀC(4')); 3.745, 3.750 (2s, 2 MeO); 3.96
(br. s, HÀC(2')); 5.00 (d, J ˆ 9.1, HÀC(1')); 5.32 (br. s, OH); 6.92 6.95 (m, 4 arom. H); 7.21 7.47 (m, 9 ar-
om. H); 8.05 (s, HÀC(8)); 11.9 (br. s, 2 NH ).13C-NMR (100 MHz, (D6)DMSO): 23.7 (q, MeCO); 45.6
(t, C(3')); 55.0 (q, MeO); 65.0, 66.4 (2d, C(2'), C(4')); 69.82 (t, C(5')); 84.9 (d, C(1')); 85.9 (s, arom. C); 113.3
(d, arom. C); 119.9 (s, C(5)); 126.77, 127.57, 127.88, 129.66, 129.70 (5d, arom. C); 136.10, 136.29 (2s, arom. C);
138.1 (d, C(8)); 145.5 (s, arom. C); 147.7, 148.9, 154.8 (3s, C(2), C(4), C(6)); 158.2 (s, arom. C); 173.4
‡
‡
(s, MeCO). HR-MALDI-MS: 634.230 (1, [M ‡ Na] , C33H33N5NaO7 ; calc. 634.228), 303.138 (100).
N-(6-Chloro-9H-purin-2-yl)-2-methoxyacetamide (23). A suspension of 6-chloro-9H-purin-2-amine (22;
15 g, 88 mmol) in a soln. of (MeOCH2CO)2O (28.7 g, 177 mmol) in N,N-dimethylacetamide (120 ml) was stirred
for 20 min at 1208, cooled to 258, and treated with H2O (50 ml). After stirring for 10 min, H2O was evaporated,
the suspension filtered, and the residue washed with toluene (30 ml). The residue was recrystallized by
suspending it in EtOH(120 ml), heating for 5 min to reflux temp. and slow cooling. Isolation of the colorless
crystals by filtration gave 23 (12.2 g, 57%). TLC (CH2Cl2/MeOH9 :1): Rf 0.40. 1H-NMR (300 MHz,
(D6)DMSO): 4.19 (s, CH2); 8.48 (s, HÀC(8)); 9.52 (s, HÀN(9)); 13.7 (br. s, NHÀC(2))18).
18
)
MeO Signal covered by H2O signal.

1458
Helvetica Chimica Acta Vol. 85 (2002)
6-Chloro-9-(3'-deoxy-b-d-ribopyranosyl)-N2-(methoxyacetyl)-9H-purin-2-amine 2'-Acetate (25). A soln. of
8 (7.7 g, 23.5 mmol) and 23 (6.34 g, 26.2 mmol) in MeCN (100 ml) was heated to 608 and treated for 20 min with
N,O-bis(trimethylsilyl)acetamide (12.8 ml, 52.5 mmol) at 608. After adjusting the temp. to 458, the clear soln.
was treated with Et3SiOTf (9.2 ml, 40.7 mmol), stirred for 50 min at 458, and then poured into a mixture of sat.
aq. NaHCO3 soln./AcOEt 1:1 (500 ml). The residue 24 obtained after workup was dissolved in MeCN (180 ml)
containing Et4NF ¥ 2 H2O (33.3 g, 180 mmol) and AcOH(5.1 ml, 90 mmol), and stirred for 3.5 h at 25 8. Workup,
adsorption on SiO2 (25 g), and CC (silica gel (110 g), CH2Cl2 ! MeOH/CH2Cl2 5 :95) gave 25 (6.33 g, 66%).
o
Yellow foam. TLC (AcOEt): Rf 0.10. [a]2D5 ˆ ‡21 (c ˆ 0.88, CHCl3). UV (MeOH): 285 (7200), 268 (5100), 256
1
(7400), 240 (5500), 227 (17400). H-NMR (400 MHz, CDCl3): 1.83 (s, Ac); 1.88 (q, J ˆ 11.5, HÀC(3')); 2.68
2.73 (m, H'ÀC(3')); 3.40 (br. s); 3.54 (s, MeO); 3.58 (q, J ˆ 10.5, HÀC(5')); 4.14 (s, MeOCH2); 4.11 4.20
(m, HÀC(4'), H'ÀC(5')); 5.39 (ddd, J ˆ 4.8, 9.1, 11.5, HÀC(2')); 5.80 (d, J ˆ 9.1, HÀC(1')); 8.19 (s, HÀC(8));
9.16 (s, NH ).13C-NMR (100 MHz, CDCl3): 20.6 (q, MeO); 37.8 (t, C(3')); 59.4 (q, MeCO); 64.1, 68.1 (2d, C(2'),
C(4')); 72.05, 72.21 (2t, C(5'), MeOCH2); 82.5 (d, C(1')); 128.5 (s, C(5)); 142.9 (d, C(8)); 151.4, 152.8 (2s, C(2),
‡
‡
C(4)); 167.6 (s, MeOCH2CO); 169.5 (s, C(6)). HR-MALDI-MS: 422.084 (100, [M ‡ Na] , C15H18ClN5NaO6
;
calc. 422.085).
9-[3'-Deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]-N2-(methoxyacetyl)-9H-purine-2,6-diamine 2'-
Acetate (27). A soln. of 25 (6.27 g, 15.7 mmol), (MeO)2TrCl (5.84 g, 17.2 mmol), and sym-collidine (4.2 ml,
31.4 mmol) in CH2Cl2 (55 ml) was treated with AgNO3 (2.66 g, 15.7 mmol) and stirred for 30 min at 258. After
filtration over Celite, evaporation, and co-evaporation with toluene, the residue 26 was dissolved in pyridine
(70 ml). The mixture was treated with NaN3 (2.04 g, 31.4 mmol) for 3 h at 658, cooled to 258, treated with PPh3
(6.2 g, 23.5 mmol), and stirred for 45 min at 258. After workup (1. NaHCO3 soln. 2. 10% citric acid),
evaporation, and co-evaporation with toluene, the residue was dissolved in MeOH/THF/1m Et3N ¥ AcOH5 :4 :2
(220 ml) and stirred at 658 overnight, concentrated to 60 ml, worked up, and adsorbed on SiO2 (30 g). CC (silica
gel (100 g), CH2Cl2 ! MeOH/CH2Cl2 5 :95) gave 27 (8.29 g, 77%). Yellow foam. TLC (CH2Cl2/MeOH9 :1): Rf
o
0.80. [a]2D5 ˆ ‡17 (c ˆ 1.06, CHCl3). UV (MeOH): 271 (17600), 266 (sh, 17300), 251 (12700), 225 (42700).
1H-NMR (500 MHz, CDCl3): 1.80 (s, Ac); 1.89 (q, J ˆ 11.8, HÀC(3')); 2.30 2.33 (m, H'ÀC(3')); 3.02 (ddd, J ˆ
1.8, 4.9, 11.3, HÀC(5')); 3.27 (t, J ˆ 11.1, H'ÀC(5')); 3.45 (s, MeOCH2CO); 3.784, 3.789 (2s, 2 MeOC6H4); 3.87
(sept., J ˆ 5.1, HÀC(4')); 4.39 (br. s, MeOCH2CO); 5.21 (br. m, HÀC(2')); 5.37 (br. d, J ˆ 7.9, HÀC(1')); 6.10
(br. s, NH2); 6.83 6.87 (m, 4 arom. H); 7.21 7.56 (m, 9 arom. H); 7.71 (s, HÀC(8)); 9.1 (br. s, NH ).13C-NMR
(125 MHz, CDCl3): 20.6 (q, MeCO); 36.8 (t, C(3')); 55.185, 55.189 (2q, MeOC6H4); 59.2 (q, MeOCH2CO); 66.2,
67.7 (2d, C(2'), C(4')); 71.0 (t, C(5')); 73.0 (br. t, MeOCH2CO); 82.5 (br. d, C(1')); 86.8 (s, arom. C); 113.31,
113.36 (2d, arom. C); 116.5 (s, C(5)); 127.0, 128.40, 128.50, 132.01, 132.09 (5d, arom. C); 136.11, 136.36 (2s,
arom. C); 137.9 (d, C(8)); 145.2 (d, arom. C); 150.2, 152.6, 156.4 (3s, C(2), C(4), C(6)); 158.75, 158.77 (2s,
‡
‡
arom. C); 169.2 (s, MeCO). HR-MALDI-MS: 705.265 (1, [M ‡ Na] , C36H38N6NaO9 ; calc. 705.265), 303.138
(100).
9-[3'-Deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]-N6-isobutyryl-N2-(methoxyacetyl)-9H-purine-
2,6-diamine (28). A soln. of 27 (8.18 g, 12.0 mmol), DMAP (73 mg, 0.6 mmol), and isobutyryl chloride (1.40 g,
13.2 mmol) in CH2Cl2/pyridine 5 :1 (50 ml) was stirred for 1 h at 258. After workup, the residue was dissolved in
THF/EtOH 5 :4 (144 ml), cooled to 48, treated with 2m NaOH(16 ml), and after 5 min treated with AcOH
(2 ml). The mixture was concentrated to 30 ml, worked up, and adsorbed on SiO2 (15 g). CC (silica gel (60 g),
CH2Cl2 ! MeOH/CH2Cl2 5 :95) and crystallization (MeOH/CH2Cl2 2 :3, 40 ml) gave 28 (6.7 g, 79%). White
o
powder. TLC (CH2Cl2/MeOH19 :1): Rf 0.35. [a]2D5 ˆ ‡54 (c ˆ 0.85, CHCl3). UV (MeOH): 284 (13900), 270
(11000), 233 (47900). 1H-NMR (500 MHz, CDCl3): 1.21, 1.25 (2d, J ˆ 6.8, Me2CH); 2.06 (q, J ˆ 12.0, HÀC(3'));
2.53 2.56 (m, H'ÀC(3')); 2.66 (sept., J ˆ 6.9, Me2CH); 2.87 (dd, J ˆ 3.2, 11.3, HÀC(5')); 3.23 (t, J ˆ 10.7,
H'ÀC(5')); 3.58 (s, MeOCH2); 3.783, 3.791 (2s, 2 MeOC6H4); 3.81 3.88 (m, HÀC(4')); 3.92 3.98
(m, HÀC(2')); 4.02, 4.22 (2d, J ˆ 15.5, MeOCH2CO); 5.38 (d, J ˆ 8.8, HÀC(1')); 6.16 (d, J ˆ 5.5, OH); 6.84
6.87 (m, 4 arom. H); 7.21 7.54 (m, 9 arom. H); 7.76 (s, HÀC(8)); 8.91, 9.58 (2s, 2 NH ).13C-NMR (125 MHz,
CDCl3): 18.92, 19.44 (2q, Me2CH); 36.6 (d, Me2CH); 39.1 (t, C(3')); 55.2 (q, MeOC6H4); 59.7 (q, MeOCH2CO);
66.9, 68.5 (2d, C(2'), C(4')); 70.5 (t, C(5')); 72.7 (t, MeOCH2CO); 85.8 (d, C(1')); 86.6 (s, arom. C); 113.27,
113.31 (2d, arom. C); 117.6 (s, C(5)); 127.0, 127.94, 128.18, 130.11, 130.22, 136.59, 136.86 (5d, arom. C); 136.59,
136.86 (2s, arom. C); 140.5 (d, C(8)); 145.6 (s, arom. C); 148.3, 151.70, 151.91 (3s, C(2), C(4), C(6)); 158.69,
158.71 (2s, arom. C); 168.6 (br. s, MeOCH2CO); 175.4 (s, Me2CHCO). HR-MALDI-MS: 733.295 (2, [M ‡
‡
‡
Na] , C38H42N6NaO8 ; calc. 733.296), 303.139 (100).
1-[3'-Deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]thymine 2'-(2-Cyanoethyl Diisopropylphos-
phoramidite) (29). A soln. of 12 (2.0 g, 3.65 mmol) in CH2Cl2 (15 ml) was treated in turn with iPr2NEt
(1.56 ml, 9.11 mmol) and 2-cyanoethyl diisopropylphosphoramidochloridite (1.14 g, 4.8 mmol). After stirring

Helvetica Chimica Acta Vol. 85 (2002)
1459
for 4 h at 258, the mixture was subjected to CC (silica gel (35 g), hexane/AcOEt 4 :1 ! 2 :3 (‡2% Et3N)): 29
(2.5 g, 92%; 1:1 mixture of diastereoisomers). Colorless foam. TLC (hexane/AcOEt 1 :4): Rf 0.80, 0.75. UV
(MeCN): 263 (12400), 257 (12200), 236 (23200). 1H-NMR (500 MHz, CDCl3): 1.01, 1.08, 1.10, 1.11 (4d, J ˆ 6.8,
2 Me2CH); 1.820, 1.832 (2q, J ˆ 11.4, HÀC(3')); 1.86, 1.88 (2d, J ˆ 1.2, MeÀC(5)); 2.16 2.19, 2.24 2.26 (2m,
HÀC(5')); 2.43 2.58 (m, OCH2CH2); 2.97, 3.00 (2ddd, J ˆ 2.1, 5.0, 11.0, HÀC(5')); 3.20 (t, J ˆ 10.7, H'ÀC(5'));
3.43 3.75 (m, OCH2CH2CN, HÀC(2'), HÀC(4')); 3.788, 3.789, 3.793, 3.796 (4s, 2 MeO); 5.41 (d, J ˆ 8.9,
HÀC(1')); 6.82 6.87 (m, 4 arom. H); 6.97, 6.98 (2d, J ˆ 1.2, HÀC(6)); 7.20 7.50 (m, 9 arom. H); 8.59 (br. s,
NH). 13C-NMR (125 MHz, CDCl3): 12.33, 12.38 (2q, MeÀC(5)); 22.33, 22.39 (2t, OCH2CH2CN); 24.27, 24.34,
24.42, 24.47, 24.51, 24.55, 24.61 (7q, Me2CH); 39.17, 39.39 (2t, C(3')); 55.22, 55.25 (2q, MeO); 57.5, 58.1
(2t, J(C,P) ˆ 18, OCH2CH2CN); 66.44, 66.51 (2d, C(4')); 68.2, 69.1 (2d, J(C,P) ˆ 12, 15, C(2')); 70.97, 71.04
(2t, C(5')); 83.9 (s, C(1')); 86.51, 86.61 (2s, Ar2CPh); 110.85, 110.97 (2s, C(5)); 113.3 (d, arom. C); 117.58, 117.67
(2s, CN); 126.97, 127.02, 127.93, 128.02, 128.07, 130.07, 130.15, 130.21 (8d, arom. C); 135.4, 135.7 (2d, C(6));
136.39, 136.47, 136.55, 136.61, 145.40, 145.49 (6s, arom. C); 150.46, 150.60 (2s, C(2)); 162.6 (s, arom. C); 163.30,
‡
‡
163.43 (2s, C(4)). 31P-NMR: 149.62; 148.08. HR-MALDI-MS: 767.318 (1, [M ‡ Na] , C40H49N4NaO8P ; calc.
767.319), 303.139 (100).
N4-Acetyl-1-{3'-deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl)}-5-methylcytosine 2'-(2-Cyanoethyl
Diisopropylphosphoramidite) (30). As described for 29, with 15 (1.85 g, 3.16 mmol), CH2Cl2 (12 ml), Pr2NEt
(1.08 ml, 6.33 mmol), and Pr2NPCl(OCH2CH2CN); (900 mg, 3.8 mmol). CC (silica gel (40 g), hexane/AcOEt
i
i
4 :1 ! 3 :7 (‡2% Et3N)) afforded 30 (2.2 g, 90%). Yellow foam (1:1 mixture of diastereoisomers). TLC
1
(hexane/AcOEt 3 :7): Rf 0.30, 0.50. UV (MeCN): 309 (10400), 268 (5300), 236 (25800). H-NMR (500 MHz,
(D6)DMSO): 0.84, 1.018, 1.027, 1.050 (4d, J ˆ 6.7, 2 Me2CH); 1.687, 1.698 (2q, J ˆ 11.5, HÀC(3')); 1.868, 1.962
(2 br. s, MeÀC(5)); 1.95 2.02 (m, H'ÀC(3')); 2.60, 2.745, 2.754 (3t, OCH2CH2CN); 2.94, 2.99 (2 br. d, J ˆ 8.0,
HÀC(5')); 3.20, 3.24 (2t, J ˆ 10.5, H'ÀC(5')); 3.34 3.62 (m, OCH2CH2CN, 2 Me2CH); 3.70 3.75
(m, HÀC(4')); 3.742, 3.747, 3.749 (3s, 2 MeO); 3.80 3.90, 3.91 4.00 (2m, HÀC(2')); 5.48 (br. s, HÀC(1'));
6.89 6.94 (m, 4 arom. H); 7.22 7.46 (m, 9 arom. H); 7.83 (br. s, HÀC(6)); 9.80 (br. s, NH ).13C-NMR
(125 MHz, (D6)DMSO): 13.2 (q, MeÀC(5)); 19.52, 19.69 (2t, J(C,P) ˆ 7, OCH2CH2CN); 23.80, 23.96, 24.04,
24.26 (4t, J(C,P) ˆ 7, Me2CH); 39.0 (t, C(3')); 42.36, 42.42 (2d, J(C,P) ˆ 12, Me2CH); 54.96, 54.99 (2q, MeO);
57.77, 58.27 (2t, J(C,P) ˆ 18, OCH2CH2CN); 66.06, 66.15 (2d, C(4')); 67.7, 68.5 (2t, C(2')); 69.73, 69.82 (2t, C(5'));
84.1 (d, C(1')); 85.68, 85.88 (2s, arom. C); 105.8 (s, C(5)); 113.23, 113.25 (2d, arom. C); 118.60, 118.83 (2s, CN);
127.51, 127.56, 127.82, 127.85, 126.74 (5d, arom. C); 136.03, 136.09, 136.13, 136.20 (4s, arom. C); 142.9, 143.2
(2d, C(6)); 145.42, 145.44 (2s, arom. C); 154.3 (s, C(2)); 158.2 (s, arom. C); 162.3 (s, C(4)); 170.6 (s, MeCO). 31P-
‡
NMR: 149.66; 148.20. FAB-MS: 786.4 (5, [M ‡ H] ), 303.1 (100).
N2-Acetyl-9-[3'-deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosl]guanine 2'-(2-Cyanoethyl Diisopropyl-
i
phosphoramidite) (31). As described for 29, with 21 (2.30 g, 3.75 mmol), CH2Cl2 (15 ml), Pr2NEt (1.6 ml,
i
9.4 mmol), and Pr2NPCl(OCH2CH2CN) (1160 mg, 4.9 mmol). CC (Al2O3 (100 g), hexane/AcOEt 4 :1 ! A-
cOEt, then CH2Cl2 ! MeOH/CH2Cl2 10 :90) afforded 31 (2.6 g, 85%). White foam (1 :1 mixture of
diastereoisomers). TLC (AcOEt): Rf 0.30, 0.35. UV (MeCN): 280 (sh, 10400), 275 (10600), 268 (9800), 259
1
(sh, 14000), 237 (22200). H-NMR (500 MHz, CDCl3): 0.76, 0.98, 1.02, 1.03 (4d, J ˆ 6.8, 2 Me2CH); 1.77, 1.79
(2q, J ˆ 11.4, HÀC(3')); 2.19 2.24 (m, H'ÀC(3')); 2.24, 2.26 (2s, Ac); 2.31 2.45, 2.57 2.60 (2m,
OCH2CH2CN); 3.12 3.37 (m, HÀC(4'), HÀC(5'), H'ÀC(5'), 2 Me2CH); 3.51 3.76, 3.81 3.88 (2m,
OCH2CH2CN); 3.781, 3.784, 3.786, 3.790 (4s, 2 MeO); 4.02 4.06, 4.22 4.56 (2m, HÀC(2')); 5.06, 5.13
(2d, J ˆ 8.9, HÀC(1')); 6.82 6.87 (m, 4 arom. H); 7.20 7.51 (m, 9 arom. H); 7.67, 7.71 (2s, HÀC(8)); 8.89, 9.05
(2 br. s, NHÀC(2)); 11.95 (br. s, HÀN(1)). 13C-NMR (125 MHz, CDCl3): 20.05, 20.47 (2t, J(C,P) ˆ 7,
OCH2CH2CN); 24.03, 24.10, 24.21, 24.37, 24.41, 24.46, 24.54 (7q, Me2CH); 39.51, 39.57 (2t, C(3')); 42.91, 43.01
(2d, J(C,P) ˆ 2.4, Me2CH); 55.22, 55.26 (2q, MeO); 57.2, 57.8 (2t, J(C,P) ˆ 20, 18, OCH2CH2CN); 66.38
(d, J(C,P) ˆ 11, C(4')); 69.10, 69.16 (2d, J(C,P) ˆ 15, C(2')); 71.08 (t, J(C,P) ˆ 5, C(5')); 84.71, 86.67
(2d, J(C,P) ˆ 5, C(1')); 113.3 (s, arom. C); 117.82, 118.01 (2s, CN); 121.1, 121.5 (2s, C(5)); 126.69, 127.05,
127.94, 127.96, 128.00, 128.04, 130.09, 130.16, 130.18, 130.22 (10d, arom. C); 136.34, 136.47, 136.48, 136.55 (4s,
arom. C); 137.6, 138.0 (2d, C(8)); 145.44, 145.54 (2s, arom. C); 146.98, 147.26, 148.43, 148.59, 155.59 (5s, C(2),
C(4), C(6)); 158.72, 158.76 (2s, arom. C); 171.80, 171.85 (2s, MeCO). 31P-NMR: 148.90; 148.63. HR-MALDI-
‡
‡
MS: 834.335 (3, [M ‡ Na] , C42H50N7NaO8P ; calc. 834.336), 303.139 (100).
9-[3'-Deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]-N2-(methoxyacetyl)-N6-isobutyryl-9H-purine-
2,6-diamine 2'-(2-Cyanoethyl Diisopropylphosphoramidite) (32). As described for 29, with 28 (1.30 g,
1.83 mmol), CH2Cl2 (7.5 ml), Pr2NEt (630 ml, 3.66 mmol), and Pr2NPCl(OCH2CH2CN) (520 mg, 2.2 mmol).
CC (silica gel (30 g), hexane/AcOEt 4 :1 ! 3 :7 (‡2% Et3N)) afforded 32 (1.45 g, 90%). White foam (1:1
mixture of diastereoisomers). TLC (hexane/AcOEt 1:4): Rf 0.20, 0.25. UV (MeCN): 284 (16300), 270 (12900),
i
i

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Helvetica Chimica Acta Vol. 85 (2002)
234 (49700). 1H-NMR (500 MHz, CDCl3): 0.63, 0.96, 1.01, 1.02 (4d, J ˆ 6.8, 2 Me2CHN); 1.25 1.28
(m, Me2CHCO); 1.88, 1.90 (2q, J ˆ 9.7, HÀC(3')); 2.23 2.28 (m, 0.5 H, H'ÀC(3')); 2.309, 2.315 (2t, J ˆ 6.2,
1 H, OCH2CH2CN); 2.40 (br. s, 0.5 H, H'ÀC(3')); 2.549, 2.553, 2.746, 2.752 (4t, J ˆ 6.0, 1 H, OCH2CH2CN);
3.08 3.35 (m, HÀC(5'), H'ÀC(5'), OCH2CH2N, 2 Me2CHN); 3.500, 3.503 (2s, MeOCH2CO); 3.51 3.58, 3.63
3.69 (2m, Me2CHCO); 3.758, 3.789, 3.791, 3.797 (4s, 2 MeOC6H4); 3.85, 3.88 (2 sept., J ˆ 5.3, HÀC(4')); 4.03
4.15 (m, HÀC(2')); 4.17 4.23 (m, MeOCH2CO); 5.37, 5.39 (2d, J ˆ 8.9, HÀC(1')); 6.83 6.87 (m, 4 arom. H);
7.05 7.52 (m, 9 arom. H); 7.95, 8.00 (2s, HÀC(8)); 8.10, 8.69 (2 br. s, NH); 8.94, 9.00 (2s, NH ).13C-NMR
(125 MHz, CDCl3): 19.14, 19.16, 19.20 (3q, Me2CHCO); 19.95, 20.10, 20.30 (3t, J(C,P) ˆ 7, OCH2CH2CN);
23.83, 23.90, 24.32, 24.34, 24.37, 24.40, 24.43, 24.49 (8q, Me2CHN); 35.67, 35.85 (2d, Me2CHCO); 39.48, 39.51
(2t, C(3')); 42.95, 43.05 (2d, J(C,P) ˆ 12, Me2CHN); 55.209, 55.245, 55.252 (3q, MeOC6H4); 57.6, 57.8, 58.1
(3t, J(C,P) ˆ 5, 18, 19, OCH2CH2CN); 59.4 (q, MeOCH2CO); 66.37, 66.42 (d, C(4')); 69.4, 70.3 (2t, J(C,P) ˆ 15,
C(2')); 71.09, 71.41 (2t, C(5')); 72.5 (t, MeOCH2CO); 84.13, 84.37 (2d, C(1')); 86.16, 86.64 (2s, arom. C); 113.3
(d, arom. C); 117.5 (s, CN); 118.6, 118.8 (2s, C(5)); 126.99, 127.03, 127.95, 128.03, 128.08, 130.09, 130.17, 130.2
(8d, arom. C); 136.37, 136.45, 136.53, 136.60 (4s, arom. C); 140.42, 140.68 (d, C(8)); 145.43, 145.52 (2s, arom. C);
149.49, 149.58, 151.72, 151.75, 152.56, 152.93 (6s, C(2), C(4), C(6)); 158.7 (br. s, MeOCH2CO); 176.35, 176.64
‡
(2s, Me2CHCO). 31P-NMR: 149.45; 148.63. FAB-MS: 911.5 (15, [M ‡ H] ), 303.1 (100).
1-[3'-Deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]thymine 2'-(4-Nitrophenyl Heptanedioate) (33).
i
A soln. of 12 (200 mg, 0.37 mmol) and Pr2NEt (63 ml, 0.37 mmol) in pyridine (5.5 ml) was treated with bis(4-
nitrophenyl) heptanedioate (1180 mg, 2.94 mmol) and DMAP (45 mg, 0.37 mmol). After stirring for 14 h at 258,
the mixture was worked up (CH2Cl2/10% citric acid in H2O) and subjected to CC (silica gel (5 g), hexane/
AcOEt 8 :2 ! 2 :8): 33 (165 mg, 55%). TLC (hexane/AcOEt 3 :7): Rf 0.70. 1H-NMR (400 MHz, CDCl3): 1.28
1.36 (m, CH2); 1.44 1.65 (m, CH2); 1.67 1.81 (m, HÀC(3'), 1 CH2); 1.85 (d, J ˆ 2.0, MeÀC(5)); 2.10 2.18
(m, H'ÀC(3')); 2.25, 2.38 (2t, J ˆ 7.3, 2 CH2); 3.03 (ddd, J ˆ 2.1, 5.0, 11.1, HÀC(5')); 3.21 (t, J ˆ 10.7, H'ÀC(5'));
3.75 (sept., J ˆ 4.7, HÀC(4')); 3.793, 3.795 (2s, 2 MeO); 4.71 (ddd, J ˆ 4.9, 9.3, 11.6, HÀC(2')); 5.49 (d, J ˆ 9.3,
HÀC(1')); 6.65 6.87 (m, 4 arom. H); 6.99 (d, J ˆ 1.2, HÀC(6)); 7.20 7.44 (m, 9 arom. H); 7.45 7.47 (m, 2 ar-
om. H); 8.23 8.25 (m, 2 arom. H); 9.08 (br. s, NH ).13C-NMR (100 MHz, CDCl3): 12.4 (q, MeÀC(5)); 24.20,
24.32, 28.22, 33.75, 33.94 (4t, CH2); 36.6 (t, C(3')); 55.3 (q, MeO); 67.1, 66.25 (2d, C(2'), C(4')); 71.1 (t, C(5'));
82.2 (d, C(1')); 86.8 (s, Ar2CPh); 111.3 (s, C(5)); 113.35, 113.38, 115.7, 122.5, 125.2, 127.1, 128.0, 130.0 (8d,
arom. C); 135.4 (d, C(6)); 136.18, 136.38, 145.2 (3s, arom. C); 150.5 (s, C(2)); 155.5 (s, arom. C); 163.7 (s, C(4));
‡
‡
171.1, 172.1 (2s, C(O)). HR-MALDI-MS: 830.297 (85, [M ‡ Na] , C44H45N3NaO12 ; calc. 830.290), 814.300
(100).
N4-Acetyl-1-[3'-deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]-5-methylcytosine 2'-(4-Nitrophenyl
Heptanedioate) (34). As described for 33, with 15 (179 mg, 0.31 mmol), iPr2NEt (157 ml, 0.92 mmol), pyridine
(4.5 ml), bis(4-nitrophenyl) heptanedioate (860 mg, 2.14 mmol), and DMAP (37 mg, 0.31 mmol). CC (silica gel
(4 g), hexane/AcOEt 8 :2 ! 1:9): 34 (180 mg, 68%). TLC (AcOEt): Rf 0.80. 1H-NMR (500 MHz, CDCl3):
1.28 1.34 (m, CH2); 1.51 1.58 (m, CH2); 1.67 1.76 (m, CH2); 1.82 1.90 (m, HÀC(3')); 1.91 (d, J ˆ 1.0,
MeÀC(5)); 2.09 2.18 (m, HÀC(3')); 2.24 (t, J ˆ 7.4, 1 CH2); 2.38 (br. s, MeCO); 2.56 (t, J ˆ 7.5, 1 CH2); 3.03
(ddd, J ˆ 2.1, 5.0, 11.3, HÀC(5')); 3.23 (t, J ˆ 10.7, H'ÀC(5')); 3.76 (sept., J ˆ 5.2, HÀC(4')); 3.796, 3.797 (2s,
2 MeO); 4.69 (ddd, J ˆ 4.9, 9.2, 11.5, HÀC(2')); 5.60 (d, J ˆ 9.2, HÀC(1')); 6.83 6.86 (m, 4 arom. H); 7.16
(s, HÀC(6)); 7.23 7.38 (m, 9 arom. H); 7.45 7.47 (m, 2 arom. H); 8.24 8.28 (m, 2 arom. H). 13C-NMR
(125 MHz, CDCl3): 13.5 (q, MeÀC(5)); 24.2, 24.3, 28.2, 33.79, 33.91 (5t, CH2); 36.6 (t, C(3')); 55.2 (q, MeO);
66.3, 67.8 (2d, C(2'), C(4')); 71.1 (t, C(5')); 82.7 (d, C(1')); 86.8 (s, Ar2CPh); 102.0 (s, C(5)); 113.34, 113.37, 115.7,
122.44, 122.47, 125.19, 125.20, 127.1, 128.0, 130.02, 130.08 (11d, arom. C); 136.18, 136.38 (2s, arom. C); 138 (br. s,
C(6)); 145.22, 145.28 (2s, arom. C); 151.1, 159.4 (2 br. s, (C(2), C(4)); 155.5, 158.80, 158.81 (3s, arom. C); 171.05,
‡
171.16, 172.5 (3s, CˆO). FAB-MS: 849.4 (4, [M ‡ H] ), 303.1 (100).
N2-Acetyl-9-[3'-deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]guanine 2'-(4-Nitrophenyl Heptane-
dioate) (35). As described for 33, with 21 (210 mg, 0.34 mmol), iPr2NEt (236 ml, 1.40 mmol), pyridine
(5.5 ml), bis(4-nitrophenyl) heptanedioate (1100 mg, 2.75 mmol), and DMAP (42 mg, 0.34 mmol). CC (silica
gel (4 g), hexane/AcOEt 8 :2 ! AcOEt): 36 (165 mg, 55%). TLC (AcOEt): Rf 0.75. 1H-NMR (400 MHz,
CDCl3): 1.10 1.08 (m, CH2); 1.34 1.46 (m, CH2); 1.66 1.83 (m, HÀC(3'), 1 CH2); 2.02 2.18 (m, CH2); 2.20
2.27 (m, H'ÀC(3')); 2.24 (s, MeCO); 2.52 (t, J ˆ 7.4, 1 CH2); 3.06 (ddd, J ˆ 1.8, 5.0, 11.4, HÀC(5')); 3.23 (t, J ˆ
10.7, H'ÀC(5')); 3.78, 3.79 (2s, 2 MeO); 3.87 (sept., J ˆ 5.1, HÀC(4')); 5.14 5.22 (m, HÀC(2')); 5.20
(s, HÀC(1')); 6.83 6.86 (m, 4 arom. H); 7.20 7.40 (m, 9 arom. H); 7.46 7.48 (m, 2 arom. H ); 7.66
(s, HÀC(8)); 8.22 8.27 (m, 2 arom. H); 9.17 (br. s, NH ).13C-NMR (100 MHz, CDCl3): 24.07 (t, CH2); 24.29
(MeCO); 24.35, 27.7, 33.59, 33.77 (4t, CH2); 36.9 (t, C(3')); 55.3 (q, MeO); 66.1, 67.5 (2d, C(2'), C(4')); 71.1
(t, C(5')); 82.9 (t, C(1')); 86.9 (s, arom. C); 113.40, 113.43 (2d, arom. C); 121.3 (s, C(5)); 122.4, 122.5, 125.2, 127.1,

Helvetica Chimica Acta Vol. 85 (2002)
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127.9, 128.0, 130.0 (7d, arom. C); 136.1, 136.3 (2s, arom. C); 137.3 (d, C(8)); 145.3 (s, arom. C); 147.6, 148.0 (2s,
‡
C(2), C(4)); 158.9 (s, arom. C); 171.1, 171.6, 171.7 (3s, CˆO). HR-MALDI-MS: 897.305 (77, [M ‡ Na] ,
‡
C46H46N6NaO12 ; calc. 897.307), 881.311 (100).
9-[3'-Deoxy-4'-O-(4,4'-dimethoxytrityl)-b-d-ribopyranosyl]-N6-isobutyryl-2-(methoxyacetyl)-9H-purine-
2,6-diamine 2'-(4-Nitrophenyl Heptanedioate) (36). As described for 33, with 28 (148 mg, 0.22 mmol), iPr2NEt
(150 ml, 0.88 mmol), pyridine (3.2 ml), bis(4-nitrophenyl) heptanedioate (700 mg, 1.74 mmol), and DMAP
(26 mg, 0.22 mmol). CC (silica gel (3.5 g), hexane/AcOEt 8 :2 ! 1:9): 36 (120 mg, 56%). TLC (hexane/AcOEt
1
3 :7): Rf 0.60. H-NMR (500 MHz, CDCl3): 1.05 1.13 (m, CH2); 1.24 (d, J ˆ 6.8, Me2CH); 1.34 (sept., J ˆ 7.1,
CH2); 1.51 1.59 (m, CH2); 1.92 (q, J ˆ 11.5, HÀC(3')); 1.99 2.13 (m, CH2); 2.22 2.25 (m, H'ÀC(3')); 2.47
(t, J ˆ 7.5, CH2); 3.08 (ddd, J ˆ 2.0, 5.0, 11.3, HÀC(5')); 3.22 (br. s, Me2CH); 3.32 (t, J ˆ 10.1, H'ÀC(3')); 3.499
(s, MeOCH2CO); 3.791, 3.795 (2s, 2 MeO); 3.88 (sept., J ˆ 5.0, HÀC(4')); 4.13 (s, MeOCH2CO); 5.13 (ddd, J ˆ
4.8, 9.4, 11.5, HÀC(2')); 5.53 (d, J ˆ 9.4, HÀC(1')); 6.84 6.87 (m, 4 arom. H); 7.22 7.32 (m, 5 arom. H); 7.36
7.40 (m, 2 arom. H); 7.46 7.49 (m, 2 arom. H); 7.91 (s, HÀC(8)); 8.23 8.26 (m, 2 arom. H); 8.60, 8.94 (2 br. s,
NH). 13C-NMR (125 MHz, CDCl3): 19.13, 19.17 (2q, Me2CH); 24.14, 24.39, 27.97, 33.56, 33.84 (5t, CH2); 36.0
(d, Me2CH); 36.8 (t, C(3')); 55.2 (q, MeOC6H4); 59.4 (q, MeOCH2CO); 66.2, 68.0 (2d, C(2'), C(4')); 71.1, 72.5
(2t, C(5'), MeOCH2CO); 82.1 (d, C(1')); 86.9 (s, arom. C); 113.38, 113.41 (2d, arom. C); 118.6 (s, C(5)); 122.47,
125.18, 127.1, 128.0, 130.1 (5d, arom. C); 136.2, 136.4 (2s, arom. C); 140.0 (d, C(8)); 145.3 (s, arom. C); 149.6,
151.9, 152.6 (3s, C(2), C(4), C(6)); 155.4, 158.8 (2s, arom. C); 167.7 (br. s, MeOCH2CO); 170.9, 171.8 (2s, CˆO);
‡
176.1 (s, Me2CHCO). FAB-MS 974.6 (5, [M ‡ H] ), 303.1 (100).
Preparation ofSolid Supports . To a soln. of the active esters 33 36 (0.12 mmol) in DMF (4 ml), long-chain-
i
alkylamino CPG (500-ä pore size; 1 g) was added, and then Pr2NEt (0.8 ml). The mixtures were shaken for
20 h at 258. After filtration, the solids were washed with DMF and CH2Cl2, dried, suspended in pyridine (5 ml)
and Ac2O (3 ml) for 37 39 (pyridine (5 ml) and MeOCH2CO)2O (3 ml) for 40), and shaken for 2 h at 258.
After filtration, the solids were washed with DMF and CH2Cl2 and dried under high vacuum. The following
loadings were obtained: T-solid support 37, 28 mmol/g; M-solid support 38, 31 mmol/g; G-solid support 39,
25 mmol/g; D-solid support 40, 33 mmol/g.
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Received December 5, 2001