Pentopyranosyl oligonucleotide systems: Communication No. 12. The β-D-xylopyranosyl-(4′ → 2′)-oligonucleotide system

Article abstract of DOI:10.1002/1522-2675(200202)85:2<399::AID-HLCA399>3.0.CO;2-2

β-D-Xylopyranosyl-(4′ → 2′)-oligonucleotides containing adenine and thymine as nucleobases were synthesized as a part of a systematic study of the pairing properties of pentopyranosyl oligonucleotides. Contrary to earlier expectations based on qualitative

Full text of DOI:10.1002/1522-2675(200202)85:2<399::AID-HLCA399>3.0.CO;2-2

Helvetica Chimica Acta Vol. 85 (2002)
399
Pentopyranosyl Oligonucleotide Systems
Communication No. 121)
The b-d-Xylopyranosyl-(4' ! 2')-oligonucleotide System
a
b
by Thomas Wagner2 ), Hoan K. Huynh2 ), Ramanarayanan Krishnamurthy*3), and Albert Eschenmoser*3)
The Skaggs Institute for Chemical Biology at The Scripps Research Institute (TSRI)
10550 North Torrey Pines Road, La Jolla, CA-92047, USA
and
Laboratory of Organic Chemistry, Swiss Federal Institute of Technology, Hˆnggerberg, HCI H309, CH-8093 Z¸rich
Gerhard Quinkert zum 75. Geburtstag gewidmet
b-d-Xylopyranosyl-(4' ! 2')-oligonucleotides containing adenine and thymine as nucleobases were
synthesized as a part of a systematic study of the pairing properties of pentopyranosyl oligonucleotides.
Contrary to earlier expectations based on qualitative conformational criteria, b-d-xylopyranosyl-(4' ! 2')-
oligonucleotides show Watson-Crick pairing comparable in strength to that shown by pyranosyl-RNA.
1. Introduction. In a preceding communication [3], we described the synthesis and
base-pairing properties of a-l-lyxopyranosyl-(4' ! 2')-oligonucleotides as part of a
series of reports to be published in this journal, presenting the experimental data that
have accumulated in our studies on the chemistry of the pentopyranosyl-oligonucleo-
tides (Scheme 1). The present paper deals with the b-d-xylopyranosyl-(4' ! 2')-
oligonucleotide system and complements the information we have given on this
system in two preliminary communications [4][5] (see also [6]).
2. Results. 2.1. Synthesis. 2.1.1. Preparation of b-d-Xylopyranosyl Nucleoside
Building Blocks Containing Adenine (A) and Thymine (T)4). The building blocks
required for the synthesis of the b-d-xylopyranosyl-(4' ! 2')-oligonucleotides were the
protected nucleoside phosphoramidite derivatives 7a,b, in which the 2'-position bears
the phosphoroamidite group, the 3'- and 4'-OH groups the acetate and the 4',4''-
dimethoxytrityl substituents, respectively, while the amino group of the nucleobase A
bears a Bz group (Scheme 2). The synthesis of these nucleosides started from
tetrabenzoyl-xylopyranose (anomeric mixture 1), which was prepared according to the
procedure of Fletcher and Hudson [7]. Nucleosidation of 1 with the bases N6-
benzoyladenine (catalyst: SnCl4) and thymine (TMS-triflate) was carried under the
Vorbr¸ggen-Hilbert-Johnson conditions [8] and afforded the corresponding xylopyr-
1
)
Communication No. 11: [1]. The paper is also communication No. 34 in the series −Chemistry of a-
aminonitriles×. For No. 33, see [1], a summary of the numbering of papers in this series will be given in [2].
Postdoctorates: a) TSRI Sept. 1996 Dec. 1997; b) TSRI Oct. 1998 Jan. 2001.
e-mail: eschenmoser@org.chem.ethz.ch; rkrishna@scripps.edu.
2
3
4
)
)
)
Xylopyranosyl nucleosides have been prepared before in a different context [9].

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Helvetica Chimica Acta Vol. 85 (2002)
Scheme 1. Constitution and Configuration of the Repeating Units of the Four Diastereoisomeric Pentopyranosyl-
(4' ! 2')-oligonucleotide Systems and Idealized Pairing Conformation of b-d-Ribo-(4' ! 2')- and b-d-Xylo-(4' !
2')-pyranosyl Oligonucleotides. The arrows ( ) point to sites of severe steric interaction expected to cause a major
á
deviation from the idealized pairing conformation.
anosyl-nucleoside derivatives 2a5) and 2b in up to 78 and 91% yield, respectively
(Scheme 2). Base-catalyzed deprotection of 2a and 2b gave the trihydroxy-nucleosides
3a and 3b in 91 and 88% yield, respectively.
1
Both nucleosides clearly have the b-configuration, since the H-NMR coupling
constants between the HÀC(1') and HÀC(2') show the large values (ca. 9 Hz, see
Table 1) characteristic for two vicinal protons in diaxial conformation. For the
nucleoside 3b of the thymine series, an X-ray structure analysis confirmed its
constitution, configuration, and preferred conformation (Fig. 1)6).
5
)
In one of the nucleosidation experiments with N6-benzoyladenine, a side product was isolated, which,
according to mass spectrum, was isomeric to, but, according to the NMR spectrum, different from, the
desired N9-nucleoside. The constitution of the product (whether it was the N9-a-isomer or the N7-
regioisomer) was not ascertained further (see Exper. Part).
The X-ray analysis was carried out by Raj K. Chadha, TSRI. Crystallographic data (excluding structural
factors) for the structure reported in this paper has been deposited with the Cambridge Crystallographic
Data Center as deposition No. CCDC 164910. Copies of the data can be obtained, free of charge, on
application to the CCDC, 12 union Road, Cambridge CB12 1EZ UK (fax: ‡ 44 (1233) 3360333; e-mail:
deposit@ccdc.cam.ac.uk).
6
)

Helvetica Chimica Acta Vol. 85 (2002)
401
Scheme 2. Preparation of A and T Nucleoside Building Blocks for the b-d-Xylopyranosyl Series
In the further course of the synthesis, the differentiation between the three
equatorial OH groups of 3a,b presented, not unexpectedly, the major problem.
Attempts to selectively protect OH groups in 3a,b with Bz or Ac under various
conditions met with failure. Therefore, it was decided to introduce the trityl group as
the first step in the protection strategy, in the hope that such a bulky reagent would be
best able to differentiate between the three OH groups. However, the poor solubility of
the triols 3a and 3b in nonpolar solvents suitable for tritylation (e.g., CH2Cl2)
complicated the task. While attempts with various solvents (e.g., DMF) and reagents

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Helvetica Chimica Acta Vol. 85 (2002)
Table 1. HÀC(1'), HÀC(2') Coupling Constant (J)
Values for Selected Compounds Depicted in Scheme 2
J [Hz] ((D6)DMSO)
Compound
a
b
(A)
9.0
9.2
9.0
9.5
9.0
(T)
9.3
9.3
9.0
2
3
4
5
6
9.0
Fig. 1. Results of an X-ray structure analysis of 2-(b-d-xylopyranosyl)thymine (3b). The unit cell shows two
types of molecules that differ in their conformations. Nucleosidic torsion angles OÀC(1)ÀN(1)ÀC(2): À138.08
and À105.88 (see Footnote 6).
(e.g., DMT-BF4) were unsuccessful, pyridine as the solvent and dimethoxytrityl
chloride as the reagent gave moderate yields of the desired 4'-O-tritylated products 4a
(40%) and 4b (34%). Also isolated in these experiments, apart from unreacted starting
triols (ca. 10%), were the 2'-tritylated diol derivatives (28 30%, determined by
1H-NMR) and a compound tentatively assigned by its mass spectrum to be a bis-
tritylated product. The tritylated side products were combined and recycled into the
synthesis by acid hydrolysis (2% TsOH in CH2Cl2/MeOH 4 :1, room temperature) to
the starting triols 3a,b.
While exploring the course of various acylation reactions in the thymine series, the
use of the reactive acetylating agent (ClCH2CO)2O led to a surprising as well as useful
observation. Chloroacetylation of the tritylated derivative 4b in CH2Cl2/pyridine/08/1 h
gave a mixture of the 3'-chloroacetate 5b together with its 2'-O-chloroacetylated isomer
5bb in a ratio of ca. 3 :1 besides minor amounts of the 2',3'-O-bis(chloroacetylated)
derivative7). The assignment of the constitutions of 5b and 5bb was based on 1H-NMR
7
)
The 2'-chloroacetate 5bb and the 2',3'-bis(chloroacetyl) derivatives formed in the thymine series as side
products in the chloroacetylation step could be recycled by converting them back to the starting trityl
derivative 4b.

Helvetica Chimica Acta Vol. 85 (2002)
403
spectroscopy chemical-shift values and decoupling experiments (see Fig. 2 and
Table 2), as well as by chemical conversion of 5b to 6b (see below). When this
mixture 5b/5bb was kept in CH2Cl2 containing Et3N (overnight, room temperature) the
3'-O-chloroacetyl derivative 5b was found to have become the major component up to a
ratio 5b/5bb 9 :1. This change in ratio pointed to the occurrence of a migration of the
ClCH2CO group from the 2'-O- to the 3'-O-position, a transformation we had observed
before in the p-RNA series and has become the central step in the preparation of
ribopyranosyl-(4' ! 2')-oligonucleotides [6]. It was established that the migration
reaches an equilibrium ratio of 9 :1 (starting from 0.07m 5bb, 0.4m TSOH, 0.6m 1-
methylimidazole, CH2Cl2, ca. 10 h, room temperature) in favor of the 3'-chloroacylated
derivative as determined by 1H-NMR (Fig. 2). When either the 2'-O-chloracetyl
derivative 5bb or the 3'-O-chloracetyl derivative 5b was reacted with allyloxycarbonyl
chloride in CH2Cl2/1-methylimidazole at room temperature, only one product was
isolated (88% yield), namely the 2'-(allyloxycarbonyl)-3'-(chloroacetyl)-4'-(dimethoxy-
trityl)xylopyranosylthymine (see Exper. Part). This result demonstrates that the
(2'-O ! 3'-O) ClCH2CO migration is taking place faster than the allyloxycarbonylation
of either of the two OH groups8).
While the migration of the ClCH2CO group reaches the equilibrium in ca. 10 h at
room temperature (CH2Cl2, Et3N), the Ac group does not migrate at all under these
conditions. For the Ac group, one has to boil the derivative in CH2Cl2 in the presence of
Et3N to observe any migration. The assignments of constitutions in the acetate series,
1
1
again, are based on H-NMR chemical-shift values (Table 2) and H,1H-decoupling
experiments.
Fig. 2. 1H-NMR Spectra of the 2'-chloroacetyl derivative 5bb and the 3'-chloroacetyl derivative 5b in (D6)DMSO
at room temperature. The positions of the ClCH2COO groups are assigned on the basis of the chemical shift of
HÀC(2'), which is expected to be at lower field in the 2'-O-chloroacetyl derivative, identification of HÀC(2') by
decoupling from HÀC(1'), and further corroborated by decoupling of the OH group from HÀC(3').
8
)
Surprising to us was not so much that the migration proceeds from the 2'- to the 3'-position (see the
corresponding regioselective Bz-migration step in the synthesis of ribopyranosyl nucleosides [6]), but that
the presumed intramolecular migration occurs at all, given that the configurational relationship between
the two OH groups in the xylopyranosyl is trans (and not cis as the in the ribo series). As we plan to discuss
in more detail in [1] (see also [6]) for the ribopyranosyl case, the origin of the driving force for the 2' ! 3'
migration in the xylopyranosyl series is probably of partially steric (position of the bulky nucleobase) and
partially stereoelectronic (gauche effect) origins.

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Helvetica Chimica Acta Vol. 85 (2002)
Table 2. Chemical Shifts (d) and Coupling-Constant (J) Values for Selected Protons of the 2'-O-Ac and 3'-O-Ac
Derivatives of Thymine Nucleoside
d [ppm] (multipl., J [Hz])
HÀC(1')
HÀC(2')
HÀC(3')
4.05 (t, J ˆ 9.0)
5.25 (t, J ˆ 9.2)
3.98 (m)
5.33 5.39 (m)
4.71 (t, J ˆ 9.5)
3.95 (t, J ˆ 9.2)
R1 ˆ OH, R2 ˆ Ac
5.59 (d, J ˆ 9.4)
5.32 (d, J ˆ 9.0)
5.49 (d, J ˆ 9.3)
5.33 5.39 (m)
5.72 (d, J ˆ 9.5)
5.45 (d, J ˆ 9.4)
4.82 (t, J ˆ 9.4)
3.65 3.74 (m)
5.05 (t, J ˆ 9.3)
3.7 3.76 (m)
5.62 (t, J ˆ 9.4)
4.8 (t, J ˆ 9.4)
R1 ˆ Ac, R2 ˆ OH
R1 ˆ OH, R2 ˆ ClCH2CO
R1 ˆ ClCH2CO, R2 ˆ OH
R1 ˆ ClCH2CO, R2 ˆ CH2ˆCHCH2CO
R1 ˆ OH, R2 ˆ CH2ˆCHCH2CO
It was decided9) to use the ClCH2CO instead of the Ac group for the preparation of
the 3'-O-Ac-protected nucleoside derivatives 6a,b, since exploratory tests showed, and
experiments in preparative scales confirmed, that 3'-O-chloroacetyl derivatives 5a,b
can be converted by catalytic hydrogenation to the corresponding 3'-O-Ac derivatives
6a,b in essentially quantitative yield. By means of this chloroacetylation/migration
method as a one-pot procedure, 50% of 5a and 43% of 5b were obtained. Catalytic
reduction of 5a and 5b afforded the 3'-O-Ac derivatives 6a and 6b in 96 and 91% yields,
respectively.
The 3'-O-derivatives 6a and 6b were the starting materials for the preparation of the
building blocks required for the oligonucleotide synthesis on the gene synthesizer.
Phosphoroamidites 7a and 7b were prepared with CNCH2CH2 as the protecting group
on the phosphoroamidate (Scheme 3). Solid-support (−long-chain alkylamine-CPG×,
Sigma) derivatives 9a and 9b, were obtained by standard methodology, with succinic
anhydride as the linker, as outlined in Scheme 3 (cf., e.g., [3]).
2.1.2. Synthesis, Deprotection and Purification of Oligonucleotides. The preparation
of b-d-xylopyranosyl-(4' ! 2')-oligonucleotides was carried out on a 1-mm scale, as
recently described in detail for a-d-lyxopyranosyl-(4' ! 2')-oligonucleotides series [3],
on an automated DNA synthesizer (Perseptive×s Expedite) with ca. 0.1m solution of
phosphoroamidites 7a and 7b in MeCN and 35 40 mg of the required solid-support
derivatives 9a and 9b, respectively. The syntheses were preformed in the −trityl-on×
mode. The deblocking, coupling, capping, and oxidizing protocols in the automated
oligonucleotide synthesis had to be adapted to the special problems posed by the xylose
system (see Exper. Part). For example, while applying the usual conditions [3] in the
oxidation step, oligomer synthesis more or less failed; a four-fold extension of the
oxidation time (followed by thorough washing) was found to be necessary to have
acceptable yields of the desired isomer. We were able to achieve by such modifications
coupling efficiencies ranging from 90 to >95% (trityl assay).
The workup of oligomer syntheses involved treating the dried CPG solid support
with dry pyridine/Et3N 4 :1 in order to initiate the removal of the CNCH2CH2
protecting group, converting the (labile) phosphotriester linkages to phosphodiester
linkages, thus stabilizing the oligonucleotide linkage against the strongly basic
conditions of the further deprotection steps [10]. Attempts to achieve detachment
and deprotection by treatment with aqueous hydrazine hydrate at 48, a method found
9
)
Decided by T.W. (A.E.)

Helvetica Chimica Acta Vol. 85 (2002)
405
Scheme 3. Preparation of the Phosphoroamidite and CPG Derivatives for the Automated Synthesis of b-d-
Xylopyranosyl-(4' ! 2')-oligonucleotides (for abbreviations, see Scheme 2)
to be useful in the p-RNA series [6], led to extensive strand scission. Eventually, it was
treatment with either a mixture of 40% aq. MeNH2, and 30% aq. NH3, or a solution of
0.2m MeONH2 ¥ HCl in conc. aq. NH3/EtOH 3 :1 at 48 that proved to be successful for
detaching the oligonucleotides from the CPG solid support and concomitantly
removing the 3'-O-Ac and the Bz groups at the sugar OH and the adenine amino
group, respectively. Desalting with 0.5m Et3NH2CO3 over a Waters Sepak-C18 cartridge
removed the excess base10). The resulting crude oligonucleotides were detritytlated
with 80% aqueous HCO2H at room temperature and subsequently purified by ion-
exchange HPLC (target purity at least 95%). Purified oligonucleotides were desalted
and stored at À208. Molecular weights and purities of the oligomers were checked by
matrix-assisted laser-desorption inonization time-of-flight (MALDI-TOF) mass spec-
trometry, concentrations of stock solutions were determined by UV-spectroscopically
(at a temperature of ca. 808, wavelength of 260 nm, with values of e (px(A)) ˆ 15000, e
(px(T)) ˆ 10000). Table 3 lists the oligonucleotides synthesized, purified, and analyzed
by MALDI-TOF MS.
2.2. Pairing Studies. The pairing properties of b-d-xylopyranosyl-(4' ! 2')-oligonu-
cleotides (−px-oligos×) were characterized by temperature-dependent UV spectroscopy
(Tm measurements [11]), concentration-dependent Tm measurements (for determi-
nation of thermodynamic data, see [12]), molar-ratio-dependent UV spectroscopy (for
checking stoichiometry [13]), and temperature-dependent CD spectroscopy. All
measurements were made in 10 mm aq. NaH2PO4 buffer containing 0.1 mm Na2EDTA,
150 mm (or 1m) NaCl at pH 7.0, with a total oligonucleotide concentration of about
10
)
This desalting procedure to remove the excess base is successful only with sequences that have their final
trityl group on. With detritytlated sequences, the efficiency of separation was much less due to the tendency
of these oligonucleotides to −flow-through× with the initial washings.

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Helvetica Chimica Acta Vol. 85 (2002)
Table 3. HPLC and MS Data of b-d-Xylopyranosyl-(4' ! 2')-oligonucleotides
Base sequences
all (4' ! 2')
Deprotectiona) OD260 (yield) Analytical HPLC
Method
MALDI-TOF-MSc)
MonoQ ion exchangeb)
‡
‡
[M ‡ H]
[M ‡ H]
gradient/tR [min]
(obs.)
(calc.)
px(A8)
px(T8)
A
A
A
B
B
A
A
A
B
B
7.0 (6%)
13.0 (13%)
10.1 (7%)
9.6 (8%)
14 (8%)
25 (16%)
13 (7%)
13 (9%)
10 50% in 30 min/17.5
20 80% in 30 min/21.2
2501
2573
2498
2570
3889
3780
2534
2534
2534
2534
2527
2545
px(A12
px(T12
)
)
0
0
100% in 30 min/15.6 3886
100% in 30 min/22.6 3782
px(A4T4)
px(T4A4)
px(AT)4
px(TA)4
px(TATTTTAA)
px(TTAAAATA)
10 70% in 30 min/21.5
10 70% in 30 min/19.2
15 60% in 30 min/21.2
15 60% in 30 min/19.7
2535
2534
2557
2536
2529
23.5 (22%)
12.1 (14%)
0
100% in 30 min/17.4 2547
a) Method A: 0.2m MeONH2 ¥ HCl in 25%. aq. NH3 and EtOH (3 : 1) at r.t. for ca. 6 h. Method B: 40% aq.
MeNH2 in conc. aq. NH3 (1 : 1) at r.t. for 6 h. b) All oligonucleotides were purified by ion-exchange
chromatography on Mono Q HR 5/5 column (58 Â 6.0 mm, Pharmacia); elution with 10 mm Na2HPO4 in H2O
and a linear gradient of 1m NaCl with a flow of 1 ml/min; followed by desalting on Sep-Pak cartridges. c) Matrix-
assisted laser-desorption ionization time-of-flight mass spectrometry; matrix: 2,4,6-trihydroxyacetophenone
and ammonium citrate buffer.
10 mm, unless otherwise stated. Our observations are summarized in Table 4 and
illustrated in Figs. 3 and 4, which complement figures published in the preliminary
communications [4][5].
Though the number of base sequences studied is relatively small, a sufficiently
informative picture about the pairing behavior emerges. Base pairing in the b-d-
xylopyranosyl series is comparable to that in the ribopyranosyl series (Table 4)
although the general trend seems to be towards somewhat weaker pairing strength
(Table 4, Entries 3, 6 10, and 13). As is the case with the other members of the
pentopyranosyl oligonucleotide family [4], strand orientation in px duplexes is
antiparallel (Table 4, Entries 11 13, and Fig. 4), and the pairing mode is assigned to
be Watson-Crick. The latter conclusion is based on the observation that px duplexes
undergo efficient intersystem cross-pairing with antiparallel-complementary strands
from all other members of the pentopyranosyl oligonucleotide family (for a summary
of our observations on intersystem cross-pairing within this series, see Table 1 in [5]).
Operation of Watson-Crick pairing mode has been shown by Jaun and co-workers for
duplexes of the (self-complementary) base sequence (CGAATTCG)2 in both the ribo-
and arabinopyranosyl series [14][15].
The duplex formed by the self-complementary block sequence px(A4T4) melts
at a considerably lower temperature (Tm ˆ 16.3; c ˆ 10 mm, 150 mm NaCl) than its
−mirror sequence× px(T4A4) (Tm ˆ 40.3). The same, though to a lesser degree, holds for
the two alternating sequences px(AT)4 and px(TA)4 (Entries 9 and 10 in Table 4). This
sort of sequence dependence of Tm is common to all four pentopyranosyl systems and
has been postulated to be a consequence of the pronounced base-pair-axis inclination,
which is characteristic for these systems and which is responsible for the fact that base-
stacking is essentially interstrand stacking [6][15][16][17]. The Tm difference between
block sequences, such as (A4T4) and (T4A4), is more pronounced in the px and pr series

Table 4. Tm Values and Thermodynamic Data
Tm [8C]a) Tm [8C]a)
Entry Base Sequence
all (4' ! 2')
Tm [8C]a)
For comparison: 150 mm NaCla)b)
Ref.
Self-pairing
of non-complementary duplexes
px(4' ! 2') px(4' ! 2')
Tm×s of
pr(4' ! 2')
px(4' ! 2') duplexes
duplexes
DG
DH
TDS
single strands
1m NaCl
(c ꢀ 10 mm)
1m NaCl
(c ꢀ 10 mm) (c ꢀ 10 mm)
150 mm NaCl duplexes
(258C)
[kcal/mol] (258C)
150 mm NaCl
(c ꢀ 10 mm)a)
1
2
px(A8)
px(T8)
[4]
[4]
3
4
5
6
7
8
9
10
11
12
13
px(A8) ‡ px(T8)
47.3*
73.2
35.4
40*
À 8.2* À 39.3*
À 31.1* [4]
px(A12
)
7.3
[5]
[5]
[5]
px(T12)
px(A12) ‡ px(T12
)
63.0
60.8
27*
40*
38*
40*
À 17.0
À 82.9
À 65.9
px(A4T4)
16.3*
40.3*
28.6*
33.8*
À 6.1* À 27.4*
À 8.7* À 40.1*
À 6.2* À 33.9*
À 7.6* À 28.7*
À 21.3* [4]
À 31.4* [4]
À 26.7* [4]
À 21.1* [4]
[5]
px(T4A4)
px(AT)4
px(TA)4
px(TATTTTAA)
px(TTAAAATA)
px(TATTTTAA) ‡ px(TTAAAATA)
23
10
[5]
44.4
33.3
38.8
À 8.7
À 39.9
À 30.4
[5]
a) Measurements were made in 0.01m NaH2PO4, 0.1 mm Na2(EDTA) buffer, pH 7.0 unless otherwise indicated. Error of Tm determination estimated Æ0.58. Values with
asterics (*) were measured in 0.01m Tris ¥ HCl buffer, pH 7.0.
b) Thermodynamic data from plots of TmÀ1 vs. ln c; experimental error estimated in DH values Æ5%.

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Helvetica Chimica Acta Vol. 85 (2002)
Fig. 3. UV-Tm Data documenting the pairing behavior of A,T-containing (4' ! 2')-b-d-xylopyranosyl sequences.
a) UV-Spectroscopic Tm melting curves (heating) for the individual homobasic sequences and for duplexes
formed between px(A8), px(T8), and px(A12), px(T12) oligonucleotides. b) UV-Spectroscopic Tm curves
(heating) of the duplexes formed by self-complementary A,T-containing sequences. Tm Measurements were
made in 10 mm aq. Tris ¥ HCl buffer, 1m NaCl at pH 7.0, except those marked with an asterisks* (phosphate
buffer). Total oligonucleotide concentrations in all measurements were ca. 10 mm. All Tm curves were reversible
(no hysteresis). Tm Values are calculated from the maxima of the first derivative curve by means of the
kaleidagraph software program.
(DTm ˆ 248 and DTm ˆ 138, resp.) than in the pl- (DTm ˆ 8.88) and pa series (DTm ˆ
8.28) [1][4][6][16]. This is consistent with the view that pentopyranosyl systems
containing their phosphodiester group in 4',2'-diequatorial position (px and pr) can be
expected to have a more pronounced backbone/basepair-axis inclination than the
systems in which this group in the 4'-position is axial (pl and pa) [1][17]. Interstrand
stacking must be more important in the former system than in the latter. Interestingly
enough, the conformational difference is reflected in differences in the corresponding
CD spectra. While the CDs of the px and pr systems (4'-equatorial) are strikingly
similar, they are different from those of the pl and pa systems (4'-axial) (see Fig. 2 in
[4]).
3. Discussion. The finding that duplexes derived from b-d-xylopyranosyl-(4' ! 2')-
oligonucleotide strands display thermal stabilities comparable to those of correspond-
ing b-d-ribopyranosyl duplexes came as a surprise to us. In an early attempt to
qualitatively predict the relative strength of base pairing among the members of the
pentopyranosyl oligonucleotide family [6], we had ventured to conjecture that, since
the (idealized) pairing conformation of xylopyranosyl-(4' ! 2')-oligonucleotides will be

Helvetica Chimica Acta Vol. 85 (2002)
409
Fig. 4. UV- and CD-spectroscopic data documenting the pairing behavior of px(4'-TTAAAATA-2') with its
antiparallel complement px(4'-TATTTTAA-2'). a) UV-Spectroscopic Tm curves of the duplex formation and
self-pairing of the individual strands. b) Temperature-dependent CD curves of the duplex; temperature range:
68 ! 758. Measurements were made in 10 mm aq. NaH2PO2 containing 0.1 mm Na2(EDTA), 1m NaCl at pH 7.0.
Total oligonucleotide concentration is ca. 10 mm.
sterically strongly hindered (see Scheme 1 and Fig. 5), such strands are expected to pair
either not at all or at least more weakly than their p-RNA isomers [6]. In retrospect, it
seems worthwhile to analyze possible reasons for this discrepancy between prediction
and observation. For this purpose, Fig. 5 resumes our earlier reasoning with regard to
relative pairing strengths in the xylo- and ribopyranosyl series: for each system the
three (idealized) staggered phosphodiester conformations arising through rotation
around the C(2')ÀO bond are depicted. Such formulas represent repetitive conforma-
tions and, therefore, possible pairing conformations of corresponding oligomer strands.
Whereas in the ribopyranosyl system one of these conformations (a in Fig. 5) has
−minimal strain×11) and was therefore, postulated to represent the pairing-conformation
of pyranosyl-RNA (which later turned out to be correct [14]), the corresponding
conformation d in the xylopyranosyl series is clearly highly strained (see bold arrow).
This feature was deemed to prevent population of this specific conformation in a
xylopyranosyl oligomer and, therefore, impede or prevent duplex formation. In
retrospect, the finding that xylopyranosyl-(4' ! 2')-oligonucleotides do, in fact, under-
go base pairing and, furthermore, do efficiently cross-pair with pyranosyl-RNA [5],
11
)
The term −minimal strain× in this context is taken to mean that a conformation, while being part of the
ensemble of idealized conformations (see Scheme 1 in [6]), does not suffer the type of severe −Newman
strain× as indicated by
in Scheme 1 and Fig. 5 (for the term −Newman strain× see G. Quinkert [18]).
á

410
Helvetica Chimica Acta Vol. 85 (2002)
reminds us of an aspect that we had overlooked in that earlier conformational
reasoning, namely, strain in a pairing-conformation will impede duplex formation if,
and only if, there are nonrepetitive nonpairing conformations accessible to the system
that are more stable than the (repetitive) pairing conformation. To decide whether this
might be the case in the xylopyranosyl system has been, and still is, beyond the potential
of a qualitative conformational analysis based on idealized conformations.
Fig. 5. Qualitative steric-strain analysis of idealized, repetitive conformations of the repetitive unit of b-d-
ribopyranosyl- (left) and b-d-xylopyranosyl- (4' ! 2')-oligonucleotides. (right). Å Denotes weak, strong steric
á
repulsion (Newman-strain [18])
The work was supported by the Skaggs Foundation (TSRI). T. W. thanks the Deutsche Forschungsge-
meinschaft for fellowship support. We thank Dr. A. Shyvaniuk for the ORTEP drawing of 2b (Fig. 1).
Experimental Part
General. Solvents for extraction: technical-grade, distilled. Solvents for reaction: reagent-grade. Reagents:
unless otherwise noted, from Acros, Fluka, or Aldrich, highest quality available. Chloro(2-cyanoethoxy)(di-
isopropylamino)phosphine (97%) was purchased from Chem-Impex Inc., Wood Dale, IL, USA. TLC: silica gel
60 F254 aluminum plates (Whatman, Type Al Sil G/UV, 250 mm layer); visualization by UVabsorption and/or A)
by dipping in a soln. of H2SO4/H2O/EtOH 14 :4 :1 or B) cerium(IV) sulfate (3 mm)/ammonium molybdate
(250 mm) in aq. H2SO4 (10%), followed by heating. Flash column chromatography (CC) was performed on
silica gel 60 (40 63 mm, 230 440 mesh, EM Science) at low pressure (max. 2 bar). In case of acid-sensitive
1
compounds, the silica gel was pretreated with solvents containing ca. 0.5% Et3N. NMR: H: d values in ppm
(TMS as internal standard), J [Hz], assignments of 1H resonances were in some cases based on 2D experiments
(1H,1H-COSY); 13C: d values in ppm (TMS as internal standard), J [Hz]; assignments and multiplicities were
‡
based on 2D experiments (1H,13C-COSY); 31P: d values in ppm (85% H3PO4 as external standard). FAB -MS
(matrix-soln.): m/z (intensity in %), performed in the positive-ion mode on a VG ZAB-VSE double-focusing
high-resolution mass spectrometer equipped with a cesium-ion gun. Matrix-assisted laser-desorption-ionization
time-of-flight mass spectrometry (MALDI-TOF-MS) was performed on a Voyager-Elite mass spectrometer
(Perseptive Biosystems) with delayed extraction with THAP as the matrix with ammonium citrate added to the
sample. Elemental analysis were performed using Perkin-Elmer PE2400 CHN analyzer. Oligonucleotides were

Helvetica Chimica Acta Vol. 85 (2002)
411
synthesized on an Expedite 8909 Nucleic Acid Synthesis system (Perseptive Biosystems). HPLC: Anion
exchange (IA)-HPLC was performed on Pharmacia ækta Purifier (900) controlled by UNICORN system.
Columns: Mono Q HR 5/5 (Pharmacia); Buffer A: 10 mm Na2HPO4 in H2O, pH 10.5; Buffer B: 10 mm
Na2HPO4 in H2O, 1m NaCl, pH 10.5. UV Spectroscopy was measured on a Cary 1 C spectrophotometer
(Varian). Melting-point (Tm) measurements of oligonucleotides were determined with Cary 1 Bio spectropho-
tometer (Varian). All measurements were made with the −phosphate buffer×, 10 mm aq. NaH2PO4 buffer
containing 0.1 mm Na2(EDTA), 150 mm (or 1m) NaCl at pH 7.0, with a total oligonucleotide concentration of ca.
10 mm. Concentrations of oligonucleotide solns. were calculated from the UVabsorbance of the solns. at 260 nm
(pH 7) with the following molar extinction coefficients: e(px(A)) ˆ 15000, e(px(T)) ˆ 10000. CD Spectrum was
measured AVIV 61 DS CD spectropolarimeter. Abbreviations: BSA: N,O-Bis(trimethylsilyl)acetamide, CPG:
−controlled-pore glass×, DMAP: 4-(dimethylamino)pyridine, DMF: dimethylformamide, DMT: 4,4'-dimethox-
ytrityl, LCAA-CPG: long-chain aminoalkyl-CPG (500 ä), TMS-Tf: trimethylsilyl trifluoromethanesulfonate,
TOTU: O-{[(2-cyanoethoxycarbonyl)methyliden]amino}-1,1,3,3-tetramethyluronium tetrafluoroborate.
1. Experiments Referring to Scheme 2. N6-Benzoyl-9-(2',3',4'-tri-O-benzoyl-b-d-xylopyranosyl)adenine
(2a). A suspension of 34 g (60 mmol) of 1 [7] and 11.3 g (50 mmol) of N6-benzoyladenine in 300 ml of dry
MeCN was warmed to 608 (oil bath). Addition of 30.5 ml (125 mmol) of BSA resulted in a clear soln. After
30 min, 21.1 ml (180 mmol) of SnCl4 was added dropwise ( ! exothermic reaction), and stirring was continued
for another 60 min. The mixture was cooled to r.t. and poured into a mixture of cold sat. aq. NaHCO3 soln./
AcOEt 1:1 (v/v) with stirring. The aq. phase was extracted with 3 Â 150 ml AcOEt and then washed successively
with 2 Â 100 ml Na2CO3, H2O, and brine. The org. phase was dried (MgSO4) and evaporated. The resulting oil
was purified by CC on silica gel (hexane/AcOEt 1:2). The product fractions were combined, evaporated, and
dried under high vacuum (ca. 0.5 Torr, r.t.) to furnish 26.6 g (78%) of a colorless amorphous solid 2a. TLC
(toluene/EtOAc 1:2): Rf 0.21. 1H-NMR (300 MHz, (D6)DMSO): 4.25 (t, Jgem ˆ 10.8, HaxÀC(5')); 4.41 (dd, J ˆ
10.8, 5.0, HeqÀC(5')); 5.61 5.70 (m, HÀC(4')); 6.28 (t, J ˆ 9.2, 9.0, HÀC(3')); 6.42 (t, J(1',2') ˆ 9.0, J(2',3') ˆ 9.0,
HÀC(2')); 6.59 (d, J ˆ 9.0, HÀC(1')); 7.31 7.89 (m, 20 H of Bz); 8.75 (s, HÀC(8)); 8.89 (s, HÀC(2)). 13C-NMR
(150.9 MHz, (D6)DMSO): 65.15 (t, C(5')); 69.90, 71.91, 73.87, 81.05 (4d, C(1'), C(2'), C(3'), C(4')); 125.74, 128.6
(2s), 129.46, 129.66, 129.73, 129.77, 129.95, 130.14, 133.39, 134.09, 134.72, 134.81, 143.87, 151.4 (d ‡ s, arom. C);
‡
‡
165.24, 165.86, 166.13, 166.49 (4s). FAB-MS (pos., NBA): 706 (100, [M ‡ Na] ), 684 (82, [M ‡ H] ), 445 (23).
In one of the runs of the nucleosidation experiment with N6-benzoyladenine, a side-product (possibly the
epimeric nucleoside) was isolated showing the following data: 1H-NMR (300 MHz, (D6)DMSO): 4.25 (t, Jgem
ˆ
10.9, HaxÀC(5')); 4.41 (dd, J ˆ 10.9, 5.6, HeqÀC(5')); 5.61 5.70 (m, HÀC(4')); 6.23 (t, J ˆ 8.7, HÀC(3')); 6.3
6.4 (m, HÀC(2'), HÀC(1')); 7.31 7.89 (m, 20 H of Bz); 8.15 (s, HÀC(8)); 8.58 (s, HÀC(2)). HR-FAB-MS
‡
‡
(pos., NBA): 706.1886 (15, [M ‡ Na] ); 684 (7, [M ‡ H] ); 602 (87); 580 (100).
1-(2',3',5'-Tri-O-benzoyl-b-d-xylopyranosyl)thymine (2b). A suspension of 4.58 g (8.1 mmol) of 1 and
1.02 g (8.1 mmol) of dry thymine in 100 ml of dry MeCN was warmed to 658 (oil bath), followed by the addition
of 6.1 ml (25.0 mmol) of BSA. After stirring for 60 min, 4.5 ml (30 mmol) of TMS-Tf was added by syringe to
the clear soln., and the mixture was kept for 2 h at 658. The mixture was cooled to r.t. and poured into ca. 1 l of an
ice-cold mixture of sat. aq. NaHCO3 soln./AcOEt 1:1 (v/v) with stirring. The org. phase was washed successively
with sat. aq. NaHCO3, H2O, and brine, then dried (Na2SO4). Concentration to dryness gave 4.22 g (91%) of the
desired product 2b. This material was used in the next step without further purification. TLC (AcOEt/hexane
1:1): Rf 0.54. 1H-NMR (300 MHz, (D6)DMSO): 1.82 (s, Me); 4.15 (t, Jgem ˆ 11.0, HaxÀC(5')); 4.35 (dd, J ˆ 11.5,
HeqÀC(5')); 5.70 5.62 (m, HÀC(4')); 5.96 (t, J ˆ 9.3, HÀC(2')); 6.16 (t, J ˆ 9.3, HÀC(3')); 6.28 (d, J(1',2') ˆ
9.0, HÀC(1')); 7.37 8.06 (m, 15 H of Bz, HÀC(6)). 13C-NMR (150.9 MHz, (D6)DMSO): 12.76 (q, Me); 64.83
(t, C(5')); 69.74, 71.33, 73.81, 80.85, (4d, C(1'), C(2'), C(3'), C(4')); 110.95 (s, C(5)); 129.05, 129.51, 129.71,
129.91, 130.09, 134.64, 134.72, 134.84, 137.47 (d, C(6)); 149.31 (d ‡ s, arom. C); 151.40, 164.43, 165.65, 165.87,
‡
‡
166.08 (5 CˆO). FAB-MS (pos., NBA): 593 (48, [M ‡ Na] ); 571 (17, [M ‡ H] ), 445 (100).
N6-Benzoyl-9-(b-d-xylopyranosyl)adenine (3a). To a soln. of 17.0 g (24.9 mmol) of 2a in 300 ml of a
mixture of THF/MeOH/H2O 5 :4 :1 was added dropwise 150 ml of aq. NaOH (2n) at 48. The soln. was stirred for
20 min (checked by TLC). The pH of the resulting soln. was adjusted to 6 7 (pH-paper) by dropwise addition
of conc. aq. HCl. Subsequently, the solvent was removed under vacuum (ca. 20 Torr, r.t.), and the resulting
residue was washed with H2O, Et2O, and dried under high vacuum for overnight gave 8.37 g (91%) of 3a as a
white powder. TLC (CH2Cl2/MeOH 8 :2): Rf 0.17. 1H-NMR (300 MHz, (D6)DMSO): 3.30 (m, HÀC(2'),
HÀC(3')); 3.51 (br. m, HÀC(4')); 3.89 (dd, J ˆ 10.9, 5.0, HaxÀC(5')); 4.1 (br. m, HeqÀC(5')); 5.21 (br. s,
OHÀC(4')); 5.35 (br. s, OHÀC(3')); 5.49 (br. s, OHÀC(2')); 5.51 (d, J ˆ 9.2, HÀC(1')); 7.52 7.67 (m, 3 H of
Bz); 8.67, 8.75 (2s, HÀC(2), HÀC(8)). 13C-NMR (150.9 MHz, (D6)DMSO): 69.25 (t, C(5')); 69.98, 71.18, 78.04,

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Helvetica Chimica Acta Vol. 85 (2002)
84.62 (4d, C(1'), C(2'), C(3'), C(4')); 126.37, 129.36, 129.38, 133.37, 134.22, 144.34, 151.17, 152.65 (d ‡ s, arom. C);
‡
‡
166.58 (CˆO). FAB-MS (pos., NBA): 394 (43, [M ‡ Na] ), 372 (100, [M ‡ H] ). Anal. calc. for C17H17N5O5 ¥
H2O: C 52.43, H 4.93, N 18.0; found: C 52.23, H 4.81, N 18.43.
1-(b-d-Xylopyranosyl)thymine (3b). A soln. of 27.5 g (48.2 mmol) of 2b in 600 ml of 2.0m NH3 in MeOH
was stirred overnight at r.t. The precipitate was filtered and washed with cold MeOH and acetone. The filtrate
was concentrated to 1/3 of its volume, and the resulting precipitate was combined with the first batch to afford
10.9 g (88%) of 3b. TLC (CH2Cl2 :MeOH 10 :1): Rf 0.20. 1H-NMR (300 MHz, (D6)DMSO): 1.77 (s, Me); 3.1
3.5 (m, HÀC(3'), HÀC(4'), HaxÀC(5'), HeqÀC(5')); 3.75 (dd, J(1',2') ˆ 9.3, J(2',3') ˆ 5.7, HÀC(2')); 5.0 5.4
(br. s, OHÀC(2'), OHÀC(3'), OHÀC(4')); 5.2 (d, J(1',2')ˆ 9.3, HÀC(1')); 7.56 (s, HÀC(6)). 13C-NMR (150.9 MHz,
(D6)DMSO): 12.60 (q, Me); 69.09 (t, C(5')); 69.82, 71.20, 73.84, 83.91 (4d, C(1'), C(2'), C(3'), C(4')); 110.30
‡
(d, C(5)); 137.70 (s, C(2)); 151.91 (d, C(6)); 164.68 (s, C(4)). FAB-MS (pos., NBA): 281 (100, [M ‡ Na] ), 259
‡
(30, [M ‡ H] ). Anal. calc. for C10H14N2O6: C 46.51, H 5.46, N 10.85; found: C 46.60, H 5.68, N 10.83.
N6-Benzoyl-9-{4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}adenine (4a). To a suspension
of 10.1 g (27.3 mmol) of 3a and molecular sieves (4 ä) in 200 ml of dry pyridine was added 23.1 g (68.2 mmol) of
DMT-Cl at r.t. The mixture was shaken gently for overnight. After filtration, the soln. was diluted with AcOEt,
extracted with H2O, dried (Na2SO4), evaporated, and the residue was co-evaporated with toluene. The residue
was purified by CC (silica gel; petroleum ether/AcOEt 1:2 to 0 :1) to give 7.3 g (40%) of 4a and 5.14 g (28%) of
the 2'-O-DMT derivative 4aa.
Data of 4a: TLC (CH2Cl2/MeOH 10 :1): Rf 0.58. 1H-NMR (300 MHz, (D6)DMSO): 2.19 (dd, J ˆ 10.9, 4.6,
HeqÀC(5')); 2.93 (br. t, J ˆ 10.9, HaxÀC(5')); 3.82 3.59 (m, HÀC(3'), HÀC(4'), 2 MeO); 4.13 4.05
(m, HÀC(2')); 5.41 (d, J(1',2') ˆ 9.0, HÀC(1')); 5.58 (d, J ˆ 5.5, OHÀC(2')); 5.89 (d, J ˆ 5.0, OHÀC(3'));
6.87 8.04 (m, 13 H of (MeO)2Tr, 5 H of Bz); 8.59, 8.70 (2s, HÀC(2), HÀC(8)). 13C-NMR (150.9 MHz,
(D6)DMSO): 55.78 (2q, MeO); 67.76 (t, C(5')); 71.79, 72.83, 76.94, 84.14 (4d, C(1'), C(2'), C(3'), C(4')); 86.46
(s, Ar3C); 113.62, 113.98 (2d); 126.33, 127.45, 128.56, 129.01, 129.10, 129.35, 129.81, 131.10, 134.20, 137.20, 138.06,
147.11, 151.15, 153.45, 159.13, 159.18 (d ‡ s, arom. C); 166.50 (CˆO). FAB-MS (pos., NBA): 696 (100, [M ‡
‡
‡
Na] ), 674 (95, [M ‡ H] ).
1-{4'-O-[(4'',4'''-Dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}thymine (4b). To a suspension of 2.3 g
(8.9 mmol) of 3b and molecular sieves (4 ä) in 50 ml of dry pyridine were added 7.5 g (22.3 mmol) of DMT-Cl at
r.t. The mixture was shaken gently overnight. After filtration, the soln. was diluted with AcOEt, extracted with
H2O, dried (Na2SO4), evaporated, and the residue was co-evaporated with toluene. The residue was purified by
CC (silica gel; petroleum ether/AcOEt 1:2 to 0 :1) to give 1.7 g (34%) of 4b and 1.5 g (30%) of 2'-O-DMT
derivative 4bb.
Data of 4b: TLC (CH2Cl2/MeOH 9 :1): Rf 0.58. 1H-NMR (300 MHz, (D6)DMSO): 1.70 (s, Me); 2.06 2.11
(dd, J ˆ 10.2, 4.5, HaxÀC(5')); 2.77 2.70 (t, J ˆ 10.2, HeqÀC(5')); 3.47 3.73 (m, 2 MeO, HÀC(2'), HÀC(3'),
HÀC(4')); 5.14 (d, J(1',2') ˆ 9, HÀC(1')); 5.41, 5.76 (2d, J ˆ 5.0, OHÀC(3'), OHÀC(2')); 6.87 (d, 4 arom. H);
7.17 7.62 (m, 9 H of (MeO)2Tr, H ÀC(6)). 13C-NMR (150.9 MHz, (D6)DMSO): 12.50 (q, Me); 55.78 (2q,
MeO); 67.69 (t, C(5')); 71.23, 72.69, 76.91, 83.58 (4d, C(1'), C(2'), C(3'), C(4')); 86.37 (s, Ar3C); 110.20 (s, C(5));
113.928, 127.46, 128.51, 129.03, 131.08, 131.32, 137.34 (d, C(6)); 137.69, 138.09, 147.08, 151.86 (s, C(2)); 159.11,
‡
‡
159.16, 164.62 (C(4)). FAB-MS (pos., NBA): 583 (100, [M ‡ Na] ), 560 (55, [M ‡ H] ).
Data of the 2'-O-DMT Derivative 4bb: 1H-NMR (300 MHz, (D6)DMSO): 1.75 (s, Me); 2.92 (apparent
t, J ˆ 9.9, HÀC(5')); 3.18 (apparent t, J ˆ 7.8, H ÀC(5')); 3.6 3.75 (m, 2 MeO, HÀC(3')); 3.75 3.85
(m, HÀC(2'), HÀC(4')); 4.41 (d, J ˆ 4.3, OHÀC(3')); 4.97 (d, J ˆ 5.4, OHÀC(4')); 5.05 (d, J(1',2') ˆ 9.1,
HÀC(1')); 6.77 (d, 4 arom. H); 7.17 7.62 (m, 9 H of (MeO)2Tr, HÀC(6)).
N6-Benzoyl-9-{3'-O-(chloroacetyl)-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}adenine
(5a). To a soln. of 8.56 g (12.7 mmol) of 4a in 100 ml of CH2Cl2/pyridine 4 :1 was added 3.26 g (19.1 mmol) of
(ClCH2CO)2O at 08. The soln. was stirred for 1 h, diluted with 200 ml of CH2Cl2, and washed twice with 100 ml
of sat. aq. NaHCO3 soln. The org. phase was dried (Na2SO4), filtered, evaporated, and co-evaporated with
toluene. The residue was dissolved in 80 ml of CH2Cl2 and 12.35 ml (89.0 mmol) of Et3N, and the stirring was
continued overnight at r.t. The soln. was concentrated, and the residue was purified by CC (silica gel; toluene/
AcOEt 1 :2) to give 4.3 g (50%) of 5a. TLC (toluene/EtOAc 1:2): Rf 0.42. 1H-NMR (300 MHz, (D6)DMSO):
2.95 (br. d, J ˆ 6.4, HeqÀC(5')); 3.75 3.52 (m, HÀC(4'), HaxÀC(5'), 2 MeO); 4.18 (d, Jgem ˆ 8.0, 1 H,
ClCH2CO); 4.38 4.33 (m, HÀC(2')); 4.41 (d, Jgem ˆ 8.0, 1 H, ClCH2CO); 5.51 (br. t, J ˆ 8.9, HÀC(3')); 5.68
(d, J(1',2') ˆ 9.5, C(1')); 5.88 (d, J ˆ 5.5, OHÀC(2')); 6.91 8.03 (m, 13 H of (MeO)2Tr, 5 H of Bz); 8.632/8.72
(2s, HÀC(2), HÀC(8)). 13C-NMR (150.9 MHz, (D6)DMSO): 42.16 (t, ClCH2); 55.82 (2q, OMe); 67.40
(t, C(5')); 69.45, 70.87, 79.08, 83.56 (4d, C(1'), C(2'), C(3'), C(4')); 86.83 (s, Ar3C); 114.13, 126.31, 127.75, 128.42,
128.71, 129.36, 130.85, 130.89, 134.15, 136.74, 136.96, 144.26, 146.81, 151.27, 152.74, 153.43, 159.26, 159.30 (d ‡ s,

Helvetica Chimica Acta Vol. 85 (2002)
413
‡
arom. C); 166.50 (s); 167.90, 166.52 (2s, 2 CˆO). FAB-MS (pos., NBA): 772/774 (85/55, [M ‡ Na] ), 750/752
‡
(100/35, [M ‡ H] ). Anal. calc. for C40H36ClN5O8: C 64.04, H 4.84, N 9.34; found: C 63.74, H 4.82, N 9.35.
1-{3'-O-(Chloroacetyl)-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}thymine (5b). A soln.
of 2.4 g (4.28 mmol) of 4b in 50 ml of CH2Cl2/pyridine 4 :1 were added 1.10 g (6.42 mmol) of (ClCH2CO)2O at
08. The soln. was stirred for 1 h, diluted with 100 ml of CH2Cl2, and washed twice with 50 ml of sat. aq. NaHCO3
soln. The org. phase was dried (Na2SO4), filtered, evaporated, and co-evaporated with toluene. To bring about
the (2' ! 3')-ClCH2CO migration, the residue was dissolved in 30 ml of CH2Cl2 and 4.16 ml (30.0 mmol) Et3N,
and the stirring was continued overnight at r.t. The soln. was concentrated, and the residue was purified by CC
(silica gel; toluene/AcOEt 1 :2) to give 1.19 g (43%) of 5b. TLC (toluene/AcOEt 1:2): Rf 0.54. 1H-NMR
(300 MHz, (D6)DMSO): 1.69 (s, Me); 2.83 2.78 (dd, J ˆ 11.1, 4.7, HeqÀC(5')); 3.30 3.38 (m, HaxÀC(5'));
3.45 3.49 (m, HÀC(4')); 3.70 3.76 (m, HÀC(2'), 2 MeO); 4.17 (d, J ˆ 15.5, 1 H, ClCH2CO)); 4.38 (d, J ˆ 15.5,
1 H, ClCH2CO)); 5.33 5.39 (m, HÀC(1'), HÀC(3')); 5.73 (d, J ˆ 5.0, OHÀC(2')); 6.88 7.55 (m, 14 H of
(MeO)2Tr, H ÀC(6)). 13C-NMR (150.9 MHz, (D6)DMSO): 12.45 (q, Me); 42.09 (t, ClCH2); 55.81 (2q, MeO);
67.32 (t, C(5')); 68.88, 70.71, 79.15, 83.40 (4d, C(1'), C(2'), C(3'), C(4')); 86.69 (s, Ar3C); 110.40 (s, C(5)), 114.10,
127.71, 128.45, 128.67, 129.10, 129.79, 130.83, 130.89, 136.75, 137.02 (C(6)); 137.59, 146.79, 151.78 (C(2)); 159.23,
‡
159.26, 164.58 (C(4)); 167.78 (d ‡ s, aromat. C). FAB-MS (pos., NBA): 659/661 (90/23, [M ‡ Na] ), 636/638
‡
(100/42, [M ‡ H] ).
1-{2'-O-(Chloroacetyl)-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}thymine (5bb) was
isolated, as the minor component (apart from the major product 5b), from exploratory experiments performed
in the thymine series with 4b in CH2Cl2/pyridine 4 :1 and (ClCH2CO)2O at 08, using the workup described above
for the isolation of 5b.
Data of 5bb. 1H-NMR (300 MHz, (D6)DMSO): 1.69 (s, Me); 2.09 (dd, J ˆ 11.3, 4.5, HeqÀC(5')); 2.91 (t, J ˆ
10.8, HaxÀC(5')); 3.69 (m, HÀC(4'), 2 MeO); 3.98 (m, HÀC(3')); 4.31 (s, ClCH2CO)); 5.05 (t, J ˆ 9.3,
HÀC(2')); 5.49 (d, J ˆ 9.3, HÀC(1')); 6.12 (d, J ˆ 5.0, HÀC(3')); 6.88 7.70 (m, 14 H of (MeO)2Tr, HÀC(6)).
N6-Benzoyl-9-{3'-O-acetyl-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}adenine (6a). To a
soln. of 1.03 g (1.38 mmol) of 5a in 50 ml of THF were added successively 5.73 mg (4.15 mmol) of K2CO3 and
200 mg of Pd/C (10%wt.). The mixture was stirred under H2 (balloon) for 10 h. The reaction vessel was purged
with N2, the mixture was filtered through Celite, and the filtrate was evaporated to give 950 mg (96%) of 6a as a
1
powder. TLC (toluene/AcOEt 1:2): Rf 0.36. H-NMR (300 MHz, (D6)DMSO): 2.04 (s, MeCO); 2.82 (dd, J ˆ
11.1, 4.1, HeqÀC(5')); 3.74 3.36 (m, HÀC(4'), HaxÀC(5'), 2 MeO); 4.32 (t, J ˆ 9.0, HÀC(2')); 5.41 (t, J ˆ 9.0,
1 H, HÀC(3')); 5.60 (d, J(1',2') ˆ 9.0, C(1')); 5.75 5.78 (br. s, OHÀC(2')); 6.86 8.03 (m, 13 H of (MeO)2Tr,
5 H of Bz); 8.54, 8.64 (2s, HÀC(2), HÀC(8)). 13C-NMR (150.9 MHz, (D6)DMSO): 21.83 (q, MeCO); 55.78,
55.82 (2q, 2 MeO); 67.52 (t, C(5')); 69.48, 71.04, 77.12, 83.76 (4d, C(1'), C(2'), C(3'), C(4')); 86.76 (s, Ar3C);
114.11, 126.31, 127.73, 128.48, 128.55, 128.69, 129.16, 129.38, 130.81,130.93, 136.81, 137.20, 138.11, 146.84, 152.73,
152.76, 153.12, 159.28 (d ‡ s, arom. C); 166.93, 170.82 (2s, 2 CˆO). FAB-MS (pos., NBA): 738 (100, [M ‡
‡
‡
Na] ), 716 (55, [M ‡ H] ).
1-{3'-O-Acetyl-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}thymine (6b). To a soln. of
2.97 g (4.67 mmol) of 5b in 100 ml of THF were added successively 1.94 g (14.0 mmol) of K2CO3 and 0.5 g of Pd/
C (10%wt.). The mixture was stirred under H2 for 10 h. The reaction vessel was purged with N2, the mixture was
filtered through Celite, and the filtrate was evaporated to give 2.55 g (91%) of 6b as an powder. TLC (toluene/
AcOEt 1:1): Rf 0.36. 1H-NMR (300 MHz, (D6)DMSO): 1.70 (s, Me); 2.03 (s, MeCO); 2.62 2.68 (dd, J ˆ 10.9,
HeqÀC(5')); 3.18 3.25 (t, J ˆ 10.9,HaxÀC(5')); 3.49 3.52 (m, HÀC(4')); 3.65 3.74 (m, 2 MeO, HÀC(2'));
5.22 5.27 (t, J ˆ 9.2, HÀC(3')); 5.32 (d, J(1',2') ˆ 9.0, HÀC(1')); 5.62 (d, J ˆ 5.0, HOÀC(2')); 6.86 7.54
(m, 13 H of (MeO)2Tr, HÀC(6)). 13C-NMR (150.9 MHz, (D6)DMSO): 12.47 (q, Me); 21.75 (q, MeCO); 55.80
(q, 2 MeO); 67.45 (t, C(5')); 68.93, 70.85, 77.13, 83.21 (4d, C(1'), C(2'), C(3'), C(4')); 86.64 (s, Ar3C); 110.34,
114.08, 126.20, 127.69, 128.50, 128.65, 129.10, 129.79, 130.78, 130.92, 136.83, 137.25 (C(6)); 137.61, 138.25, 146.81,
151.79, 159.22, 159.23, 159.25, 164.58 (C(4)); 170.70 (d ‡ s, arom. C). FAB-MS (pos., NBA): 625 (100, [M ‡
‡
‡
Na] ), 602 (41, M ).
2. Experiments Referring to the Chloroacetyl Migration.
Reaction of 1-{3'-O-(Chloroacetyl)-4'-O-
[(4'',4'''-dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}thymine (5b) with (Allyloxy)carbonyl Chloride. To a
soln. of 272 mg (0.42 mmol) of 5b in 4 ml of CH2Cl2 were added 270 ml (3.17 mmol) of 1-methylimidazole and
250 ml (2.52 mmol) of (allyloxy)carbonyl chloride at 08. The soln. was stirred for 24 h at r.t. After 24 h, a soln. of
130 ml (1.3 mmol) of (allyloxy)carbonyl chloride and 130 ml (1.58 mmol) of 1-methylimidazole in 3 ml of CH2Cl2
were added, and the mixture was stirred for another 24 h. The mixture was diluted with 25 ml of CH2Cl2 and
25 ml of H2O. The org. phase was dried (Na2SO4), filtered, concentrated in vacuo, and the residue was dried

414
Helvetica Chimica Acta Vol. 85 (2002)
under high vacuum to give 240 mg (88%) of crude 1-{2'-O-[(Allyloxy)carbonyl]-3'-O-(chloroacetyl)-4'-O-
1
[(4'',4'''-dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}thymine. TLC (toluene/AcOEt 1:1): Rf 0.67. H-NMR
(300 MHz, (D6)DMSO): 1.79 (s, Me); 3.3 3.37 (dd, J ˆ 11.8, 5.7, HÀC(5')); 3.30 3.38 (t, J ˆ 11.8, HÀC(5'));
3.6 3.7 (m, HÀC(4')); 3.8 (m, 2 MeO, ClCH2CO); 4.5 (d, J ˆ 5.7, ˆCCH2CO)); 4.72 (t, J ˆ 9.5, HÀC(2')); 5.2
5.3 (m, ˆCH2); 5.62 (t, J ˆ 9.3, HÀC(3')); 5.72 (d, J ˆ 9.5, HÀC(1')); 5.8 (m, ˆCH); 6.8 7.5 (m, 13 H of
(MeO)2Tr, HÀC(6)).
Similar reaction of 230 mg (0.35 mmol) of 1-{2'-O-(Chloroacetyl)- 4'-O-[(4'',4'''-dimethoxytriphenyl)meth-
yl]-b-d-xylopyranosyl}thymine (5bb) with 250 ml (3.12 mmol) of 1-methylimidazole and 250 ml (2.52 mmol) of
(allyloxy)carbonyl chloride at 08, after identical workup and isolation, gave 80 mg (31%) of 1-{2'-O-
[(Allyloxy)carbonyl]-3'-O-(chloroacetyl)-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}thy-
mine. Assignment of constitution was based on chemical-shift values (Table 2) and 1H-NMR decoupling experi-
ments. Chemical proof for the constitution was obtained by the selective hydrolysis of the ClCH2CO group with
hydrazine thiocarbonate [19] to afford the 1-{2'-O-[(Allyloxy)carbonyl]-4'-O-[(4'',4'''-dimethoxytriphenyl)-
methyl]-b-d-xylopyranosyl}thymine. The constitutional assignment of this compound was based on chemical-
shift values (Table 2) and 1H-NMR decoupling experiments that proved that the OH group is on the C(3')-position.
3. Experiments Referring to Scheme 3. N6-Benzoyl-9-{3'-O-acetyl-2'-O-[(2-cyanoethoxy)(diisopropyla-
mino)phosphino]-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-b-d-xylopyranosyl}adenine (7a). To a soln. of
950 mg (1.33 mmol) of 6a in 3 ml of dry CH2Cl2 were added successively 920 ml (5.32 mmol) of EtN(i-Pr)2
and 815ml (3.99 mmol) of chloro(2-cyanoethoxy)(diisopropylamino)phosphine at r.t. The soln. was stirred for
3 h, diluted with 10 ml of AcOEt, and washed with sat. aq. NaHCO3 soln. The org. phase was dried (Na2SO4),
filtered, and evaporated. Purification by CC (silica gel; toluene/AcOEt 4 :1 to 1:2) gave 750 mg (62%) of 7a as
a mixture of diastereoisomers. TLC (toluene/AcOEt 2 :1): Rf 0.31/0.36. 1H-NMR (600 MHz, CDCl3): 0.70, 0.87,
0.91, 0.94 (d, J ˆ 6.7, 4 (Me2CH); 2.35 2.55 (m, OCH2CH2CN); 2.87 (m, HeqÀC(5')); 3.2 3.7 (m, 6 H, HÀC(4')
of minor isomer, HaxÀC(5'), (Me2CH, OCH2CH2CN)); 3.80 (s, 2 MeO); 3.85 (m, HÀC(4') of major isomer);
4.19 (m, HÀC(2') of minor isomer); 4.32 (m, HÀC(2') of major isomer); 5.59 (overlapping signals, HÀC(1'),
HÀC(3') of major isomer); 5.61 (overlapping t, J ˆ 9.2, HÀC(3')); 5.71 (d, J ˆ 9.0, HÀC(1') of minor isomer);
6.84 8.01 (m, 13 H of (MeO)2Tr, 5 H of Bz); 8.08, 8.13, 8.08 (s, HÀC(2), HÀC(8)); 9.1 (br. s, NH). 13C-NMR
(600 MHz, CDCl3): 20.72, 20.76 (2t, OCH2CH2CN of both isomers); 24.20, 24.8 (2q, Me2CH of both
isomers); 43.3, 43.4 (2d, Me2CH of both isomers); 55 (q, MeO of both isomers); 58.1, 58.3 (2t, OCH2CH2CN of
both isomers); 67.2 (t, C(5') of both isomers); 68.8, 68.9 (2d, C(2'), C(4') of both isomers); 70.4, 70.5 (d, C(3')
of both isomers); 83.9 (d, C(1') of both isomers); 87.2 (s, Ar3C); 113.6 (d, arom. C); 118.3 (s, CN),
123.1 (s, arom. C); 127.4, 128.2, 128.3, 128.5, 128.6, 129.1, 129.2, 130.1, 130.6, 130.7 (arom. C); 133.0,
133.1 (2d, arom. C); 134.0, 134.2 (arom. C); 136.5, 136.7 (2s, arom. C); 141.9 (d, C(2) or C(8)); 145.9, 149.9
(2s); 152.4, 153.3 (d, C(2) or C(8)); 159.1, 159.2, 164.8, 165.1 (4s, CO of both isomers). 31P-NMR (242.9 MHz,
‡
‡
CDCl3): 150.35/150.32 (both isomers). FAB-MS (pos., NBA): 1180 (30, [M À H ‡ 2 Cs] ), 1048 (100, [M ‡ Cs] ).
1-{3'-O-Acetyl-2'-O-[(2-cyanoethoxy)(diisopropylamino)phosphino]-4'-O-[(4'',4'''-dimethoxytriphenyl)-
methyl]-b-d-xylopyranosyl}thymine (7b). To a soln. of 400 mg (0.66 mmol) of 6b in 3 ml of dry CH2Cl2 were
added successively 343 ml (1.98 mmol) of EtN(i-Pr)2 and 375 ml (1.65 mmol) of chloro(2-cyanoethoxy)(diiso-
propylamino)phosphine at r.t. The soln. was stirred for 3 h, diluted with 10 ml of AcOEt, and washed with sat.
aq. NaHCO3 soln., org. phase was dried (Na2SO4), filtered, and evaporated. Purification by CC (silica gel,
toluene/AcOEt 4 :1 to 1:1) gave 430 mg (81%) of 7b as a mixture of diastereoisomers. TLC (toluene/AcOEt
2 :1): Rf 0.46/0.53. 1H-NMR (600 MHz, CDCl3): 0.98 0.70 (m, 2 Me3CH); 1.93/1.91 (2s, Me); 2.04, 2.04 (2s,
MeCO); 2.4 2.6 (m, OCH2CH2CN); 3.1 (overlapping d, J ˆ 11.5, 5.3, HeqÀC(5') of both isomers); 3.3 (2t, J ˆ
11.5, HaxÀC(5') of both isomers); 3.35 3.75 (m, OCH2CH2CN, Me2CH, HÀC(2'), HÀC(4') of both isomers);
3.8 (s, 2 MeO); 5.51, 5.55 (2t, J ˆ 9.2, HÀC(3') of both isomers); 5.60 (overlapping d, J ˆ 7.0, HÀC(1') of both
isomers); 6.84 7.45 (m, 13 H of (MeO)2Tr, H ÀC(6)). 13C-NMR (150.9 MHz, CDCl3): 12.7, 12.8 (2q, MeÀCH,
both isomers); 14.6; 20.5, 20.6 (2t, OCH2CH2CN of both isomers); 24.5, 24.6, 24.7, 24.9 (4q, Me2CH of both
isomers); 43.2, 43.4 (2t, OCH2CH2CN of both isomers); 55.6 (q, MeO); 57.7, 58.0 (2d, Me2CH of both isomers);
60.7; 68.5, 67.3 (2d, C(5') of both isomers); 69.4, 69.6 (2d, C(4') of both isomers); 72.3, 72.6 (2d, C(3') of both
isomers); 87.1 (s, Ar3C); 111.7 (s, C(5) of both isomers); 113.4, 113.6, 113.8 (3d, arom. C); 127.4 (d, arom. C);
128.2, 128.4, 128.5 (3d, arom. C); 128.8, 129.4 (2d, arom. C); 129.6, 129.8 (2s, arom. C); 130.6, 130 (2d, arom. C);
136.5, 136.6, 136.7 (arom. C, C(6)); 146.1, 146.1 (2s, arom. C); 151.2, 151.3 (2s, C(2) of both isomers); 159.1,
159.33 (2s, CO of both isomers); 164.3, 164.4 (2s, C(4) of both isomers); 170.3, 170.4 (2s, CO of both isomers).
31P-NMR (242.9 MHz, CDCl3): 150.35, 150.24 (both isomers). FAB-MS (pos., NBA): 1067 (10, [M À H ‡
‡
‡
2 Cs] ), 935 (100, [M ‡ Cs] ).

Helvetica Chimica Acta Vol. 85 (2002)
415
N6-Benzoyl-9-{3'-O-acetyl-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-2'-O-succinoyl-b-d-xylopyranosyl}-
adenine (8a). To a soln. of 96 mg (0.134 mmol) of 6a in 3 ml of dry CH2Cl2 were added successively 21 mg
(0.174 mmol) of DMAP and 26 mg (0.26 mmol) of succinic anhydride at r.t. The mixture was stirred for 2 h,
diluted with 20 ml of CH2Cl2, and washed with 15 ml of 10% aq. citric acid. The org. layer was dried (Na2SO4),
filtrated, and evaporated. Purification by CC (silica gel; toluene/AcOEt/MeOH 2 :1:0 to 5 :4 :1) gave 66 mg
(60%) of 8a. TLC (toluene/AcOEt 1:2): Rf 0.29. 1H-NMR (300 MHz, CDCl3): 1.92 (s, MeCO); 2.37 2.22
(m, CH2CH2); 3.27 (dd, Jˆ 11.1, HeqÀC(5')); 3.48 (t, Jˆ 11.1, HaxÀC(5')); 3.84 (overlapping m, 2 MeO, HÀC(4'));
5.41 (t, J ˆ 9.5, HÀC(2')); 5.63 (t, J ˆ 9.5, HÀC(3')); 5.80 (d, J(1',2') ˆ 9.5, HÀC(1')); 6.83 7.94 (m, (MeO)2Tr,
‡
‡
5 H of Bz); 8.09, 8.61 (2s, HÀC(2), HÀC(8)). FAB-MS (pos., NBA): 838 (75, [M ‡ Na] ), 816 (100, [M ‡ H] ).
1-{3'-O-Acetyl-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-2'-O-succinoyl-b-d-xylopyranosyl}thymine (8b).
To a soln. of 100 mg (0.166 mmol) of 6b in 3 ml of dry CH2Cl2 were added successively 26 mg (0.216 mmol)
of DMAP and 33 mg (0.33 mmol) of succinic anhydride at r.t. The mixture was stirred for 2 h, diluted with 20 ml
of CH2Cl2, and washed with 15 ml of 10% aq. citric acid. The org. layer was dried (Na2SO4), filtered, and
evaporated. Purification by CC (silica gel; toluene/AcOEt/MeOH 2 :1:0 to 5 :4 :1) gave 70 mg (60%) of 8b.
TLC (toluene/AcOEt 1:2): Rf 0.24. 1H-NMR (300 MHz, CDCl3): 1.83 (s, Me); 1.92 (s, MeCO); 1.83 2.56
(m, CH2CH2); 3.18 3.22 (m, HeqÀC(5')); 3.34 3.41 (m, HaxÀC(5')); 3.60 3.69 (m, HÀC(4')); 3.79
(s, 2 MeO); 4.94 (t, J ˆ 9.1, HÀC(2')); 5.59 (t, J ˆ 9.1, HÀC(3')); 5.72 (d, J ˆ 9.5, HÀC(1')); 6.82 7.43
‡
‡
(m, 13 H of (MeO)2Tr, HÀC(6)). FAB-MS (pos., NBA): 967 (25, [M À H ‡ 2 Cs] ), 816 (100, [M ‡ Cs] ),
795 (43), 663 (45).
Preparation of Nucleoside-Derivatized Controlled Pore Glass (CPG) (9a and 9b). To a soln. of 20 mg
(24 mmol) of 8a (8b) in 7 ml of dry MeCN, 45 ml 1-methylmorpholine, 14 mg (42 mmol) of TOTU, and 350 mg of
LCAA-CPG (the CPG was previously washed with 50 ml of CH2Cl2, CH2Cl2/ClCH2CO2H 1:1, CH2Cl2, CH2Cl2/
Et3N 1:1, CH2Cl2, and dried for 2 h in vacuo) were successively added. The suspension was gently shaken for 1
1.5 h at r.t. Filtration followed by washing with DMF, MeOH, acetone, and Et2O afforded the nucleoside-
derivatized solid support after drying in vacuo (ca. 0.2 Torr). A suspension of the nucleoside-derivatized solid
support in 10 ml of dry pyridine and 50 mg (0.41 mmol) of DMAP was treated with 1 ml (10.6 mmol) of Ac2O
for 45 min. Filtration, followed by washing with DMF, MeOH, acetone, and Et2O, afforded capped 9a (9b) after
drying in vacuo (ca. 0.2 Torr) for 3 h. The loading capacity (at 498 nm) was determined to be 15 mmol/g for 9a
and 18 mmol/g for 9b (by the method published in [20]).
4. Automated Solid-Phase Synthesis on a Perseptive Expedite Gene Synthesizer.
Oligonucleotide
syntheses were carried out on a 1 mm scale. The DNA/RNA synthesizer column was filled with the CPG solid
support loaded with the appropriate nucleobase. The substrates and reagents required were prepared as follows:
4.1. Pre-Automation Procedures. 4.1.1. Phosphoroamidites. The amount of phosphoramidite soln. was
determined as follows: (n ‡ 1) Â22 mg of phosphoramidite dissolved in (n ‡ 1)  312 ml of dry MeCN. The
phosphoramidite solution (ca. 0.1m) was dried over 3-ä molecular sieves (8 12 mesh, freshly activated by
heating at ca. 3008 under high vacuum overnight) overnight at r.t. prior to use. An excess of ca. 160 equiv. of
phosphoroamidites were used.
4.1.2. Activator Solution. Initially, a 0.5m soln. of pyridinium hydrochloride in MeCN was used.
Subsequently, it was replaced with the better performing 5-(ethylthio)-1H-tetrazole in dry MeCN (0.25m),
which was dried over freshly activated 3-ä or 4-ä molecular sieves.
4.1.3. Capping A. The standard capping reagent supplied by Perseptive was used (part # GEN0898210; 1-
methylimidazole in pyridine/THF). Subsequently, it was replaced with a soln. of 3.0 g of DMAP in 50 ml of dry
MeCN and filtered to remove any undissolved solid particles.
4.1.4. Capping B. The standard capping reagent supplied by Perseptive was used (part # GEN089810; Ac2O
in THF). Subsequently, it was replaced with a soln. of 10 ml of Ac2O and 15 ml of 2,4,6-collidine in 25 ml of dry
MeCN.
4.1.5. Oxidizing Soln. The standard oxidizing reagent supplied by Perseptive was used (part #GEN089850; I2
in pyridine/THF). Subsequently, it was replaced with a soln. of 220 mg of I2, 4.6 ml of 2,4,6-collidine in 23 ml of
H2O, and 50 ml of MeCN, and filtered to remove any undissolved residue.
4.1.6. Detritylation Reagent: A soln. of 6% Cl2CHCO2H in ClCH2CH2Cl.
The synthesis of oligonucleotides with the Perseptive Expedite Gene Synthesizer required the following
modifications to the protocol provided by Perseptive for the DNA/RNA synthesis 1) The duration of the
coupling time of phosphoramidite was ca. 25 min. 2) the duration of the oxidation step was increased four-fold
(120 pulses), followed by extensive washing with MeCN (90 pulses), and 3) the detritylation was accomplished by
6% Cl2CHCO2H in ClCH2CH2Cl over a 3-min period. All oligonucleotides were synthesized in the Trityl-on× mode.

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Helvetica Chimica Acta Vol. 85 (2002)
4.2. Post-Automation Procedures. 4.2.1. Removal of b-Cyanoethyl Protecting Group. After the automated
synthesis was completed, the CPG solid support containing the oligonucleotide (−Trityl-on×) was dried in vacuo
for 30 min, transferred to a pear shaped 10-ml flask, and treated with 2 ml of pyridine/Et3N 5 :1 for 3 h at r.t.
Evaporation of pyridine and Et3N in vacuo, followed by co-evaporation with DMF, avoiding temp. over 358,
resulted in dry CPG solid support.
4.2.2. Removal of Sugar and Nucleobase Protecting Groups. One of the following two procedures was used
depending on the sequence of the oligonucleotides (Table 3 lists the specific deprotection method for the
specific sequence):
Method A. To the flask containing the dry CPG solid support was added 2.4 ml of a soln. of 0.2m MeONH2 ¥
HCl in 25% aq. NH3/EtOH 3 :1, and, the mixture was shaken at r.t. for ca. 6 h. After deprotection, the suspension
was diluted with ca. 5 10 ml of 0.5m aq. Et3NH2CO3 buffer and desalted [1][3] over a Waters Sepak-C18
cartridge (eluted with 10 15 ml MeCN/H2O 1 :1) to afford the salt-free, crude oligonucleotides (−Trityl-on×) in soln.
Method B. To the flask containing the dry CPG solid support was added 2.4 ml of a mixture of 40% aq.
MeNH2/conc. aq. NH3 1:1 and shaken at r.t. for 6 h. The suspension was diluted with ca. 5 10 ml of 0.5m aq.
Et3NH2CO3 buffer, loaded over a Waters Sepak-C18 cartridge [1][3], and eluted with 10 15 ml MeCN/H2O 1:1
to afford the salt-free, crude oligonucleotides (−Trityl-on×) in soln.
All of the above deprotections were monitored by anion exchange HPLC [1][3] for optimum deprotection time.
4.2.3. Detritylation of −Trityl-on× Oligonucleotides. The crude oligonucleotide soln. obtained by desalting
was concentrated in vacuo, the residue was treated with ca. 10 ml of 80% aq. formic acid (a red color appears
indicating detritylation) at r.t. for 15 min and concentrated in vacuo to dryness. The residue was dissolved in ca.
2 ml of H2O, filtered (Nalgene syringe filter, 0.2 mm), and taken to the next step, HPLC purification [1][3].
REFERENCES
[1] H. Wippo, F. Reck, R. Kudick, M. Ramaseshan, G. Ceulemans, M. Bolli, R. Krishnamurthy, A.
Eschenmoser, Bioorg. Med. Chem. 2001, 9, 2411 2428.
[2] S. Pitsch, S. Wendeborn, R. Krishnamurthy, A. Holzner, M. Minton, M. Bolli, R. Micura, C. Miculka, N.
Windhab, M. Stanek, B. Jaun, A. Eschenmoser, Helv. Chim. Acta., in preparation.
[3] F. Reck, H. Wippo, R. Kudick, R. Krishnamurthy, A. Eschenmoser, Helv. Chim. Acta 2001, 84, 1778 1804.
[4] M. Beier, F. Reck, T. Wagner, R. Krishnamurthy, A. Eschenmoser, Science 1999, 283, 699 703.
[5] O. Jungmann, H. Wippo, M. Stanek, H. K. Huynh, R. Krishnamurthy, A. Eschenmoser, Org. Lett. 1999, 10,
1527 1530.
[6] S. Pitsch, S. Wendeborn, B. Jaun, A. Eschenmoser, Helv. Chim. Acta 1993, 76, 2161 2183.
[7] H. G. Fletcher Jr., C. S. Hudson, J. Am. Chem. Soc. 1947, 69, 921 924.
[8] H. Vorbr¸ggen, B. Bennua, Chem. Ber. 1981, 114, 1279 1286; M. Bˆhringer, H.-J. Roth, J. Hunziker, M.
Gˆbel, R. Krishnan, A. Giger, B. Jaun, C. Leumann, A. Eschenmoser,Helv. Chim. Acta 1992, 75, 1416 1477.
[9] A. P. Martinez, W. W. Lee, L. Goodman, J. Org. Chem. 1969, 34, 92; A. P. Martinez, W. W. Lee, J. Org.
Chem. 1969, 34, 416; M. Fuertes, J. T. Witkowski, R. K. Robbins, J. Org. Chem. 1975, 40, 2372.
[10] A. K. Sood, S. A. Narang, Nucleic Acids Res. 1977, 4, 2757 2765.
[11] I. Tinoco Jr., K. Sauer, J. C. Wang, in −Physical Chemistry: Principles and Applications in Biological
Sciences×, Eds. D. Young, D. Cavanaugh, Prentice Hall, Upper Saddle River, NJ, 1995, pp. 559 589.
[12] L. A. Marky, K. J. Breslauer, Biopolymers 1987, 26, 1601 1620.
[13] C. R. Cantor, P. R. Schimmel, in −Biophysical Chemistry×, Freeman, San Francisco, CA, 1980, Part III (The
Behavior of Biological Macromolecules), pp. 1135 1139.
¬
[14] I. Schlˆnvogt, S. Pitsch, C. Lesueur, A. Eschenmoser, B. Jaun, Helv. Chim. Acta 1996, 79, 2316 2345.
[15] B. Jaun, M.-O. Ebert, A. Luther, H. Huynh, R. Krishnamurthy, A. Eschenmoser, in preparation.
[16] M. Bolli, R. Micura, S. Pitsch, A. Eschenmoser, Helv. Chim. Acta 1997, 80, 1901 1951.
[17] R. Micura, R. Kudick, S. Pitsch, A. Eschenmoser, Angew. Chem., Int. Ed. 1999, 38, 680 683.
[18] G. Quinkert, E. Egert, C. Griesinger, −Aspekte der Org. Chemie: Struktur×, Verlag Helvetica Chimica
Acta, Basel, 1995, page 105. Engl. edn. 1996, p. 101.
[19] C. A. A. van Boeckel, T. Beetz, Tetrahedron Lett. 1983, 24, 3775.
[20] J. Hunziker, H.-J. Roth, M. Bˆhringer, A. Giger, U. Diedrichsen, M. Gˆbel, R. Krishnan, B. Jaun, C.
Leumann, A. Eschenmoser, Helv. Chim. Acta 1993, 76, 259 352.
[21] R. Pon, S. Yu, Y. S. Sanghvi, Bioconjugate Chem. 1999, 10, 1051 1057.
Received October 30, 2001