Pentopyranosyl oligonucleotide systems: The α-L-lyxopyranosyl-(4′ → 2′)-oligonucleotide system

Article abstract of DOI:10.1002/1522-2675(20010613)84:6<1778::AID-HLCA1778>3.0.CO;2-3

To determine whether the remarkable chemical properties of the pyranosyl isomer of RNA as an informational Watson-Crick base-pairing system are unique to the pentopyranosyl-(4′ → 2′)-oligonucleotide isomer derived from the RNA-building block D-ribose, studies on the entire family of diastereoisomeric pyranosyl-(4′ → 2′)-oligonucleotide systems deriving from D-ribose, L-lyxose, D-xylose, and L-arabinose were carried out. The result of these extended studies is unambiguous: not only pyranosyl-RNA, but all members of the pentopyranosyl-(4′ → 2′)-oligonucleotide family are highly efficient Watson-Crick base-pairing systems. Their synthesis and pairing properties will be described in a series of publications in this journal. The present paper describes the α-L-lyxopyranosyl-(4′ → 2′)-system.

Full text of DOI:10.1002/1522-2675(20010613)84:6<1778::AID-HLCA1778>3.0.CO;2-3

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Helvetica Chimica Acta ± Vol. 84 (2001)
Pentopyranosyl Oligonucleotide Systems
Communication No. 101)
The a-l-Lyxopyranosyl-(4' ! 2')-oligonucleotide System
by Folkert Reck2a), Harald Wippo2b), Rene Kudick2c), 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-92037, USA
Laboratory of Organic Chemistry, Swiss Federal Institute of Technology, Universitätstrasse 16, CH-8092 Zürich
Edgar Heilbronner zum 80. Geburtstag gewidmet, in Erinnerung an die ꢀguten alten Zeitenꢁ
To determine whether the remarkable chemical properties of the pyranosyl isomer of RNA as an
informational Watson-Crick base-pairing system are unique to the pentopyranosyl-(4' ! 2')-oligonucleotide
isomer derived from the RNA-building block d-ribose, studies on the entire family of diastereoisomeric
pyranosyl-(4' ! 2')-oligonucleotide systems deriving from d-ribose, l-lyxose, d-xylose, and l-arabinose were
carried out. The result of these extended studies is unambiguous: not only pyranosyl-RNA, but all members of
the pentopyranosyl-(4' ! 2')-oligonucleotide family are highly efficient Watson-Crick base-pairing systems.
Their synthesis and pairing properties will be described in a series of publications in this journal. The present
paper describes the a-l-lyxopyranosyl-(4' ! 2')-system.
1. Introduction. ± When, in 1992, our systematic search for potentially natural nucleic
acid alternatives recruited from the structural neighborhood of RNA [2][3] switched
from hexopyranosyl-(6' ! 4')- to pentopyranosyl-(4' ! 2')-oligonucleotides [4], it was
natural to chose among the pentopyranosyl familyꢁs four diastereomers (Scheme 1) the
member derived from d-ribose with priority. This isomer is built from the same building
blocks as RNA itself, it is the pyranosyl isomer of RNA (ꢀp-RNAꢁ). The system proved
to have a variety of remarkable properties: first of all, it turned out to be a stronger
Watson-Crick base-pairing system than RNA itself [5]. It forms duplexes and hairpins
in antiparallel strand orientations similar to the natural system [6]. Base sequences of it
can be transcribed via template-controlled ligations of short ligands, e.g., tetramers,
using 2',3'-cyclophosphates as a mild form of phosphate activation, and such ligations
were shown to proceed sequence-, regio-, as well as chiroselectively [7]. The self
assembly of duplexes of long p-RNA sequences via self-templated chiroselective
oligomerization of hemi-self-complementary tetramer-2',3'-cyclophosphates has also
been observed [8]. Significantly, replication of p-RNA sequences was achieved in a
1
)
)
)
For communication No. 9, see [1]. The paper is also communication No. 32 in the series ꢀChemistry of a-
Aminonitrilesꢁ. For a survey of the numbering of papers in this series, see [1].
Postdoctorates: a) TSRI September 1996 ± January 1998; b) TSRI January 1998 ± July 1999; c) ETH and
TSRI August 1997 ± February 1999.
2
3
e-mails: eschenmoser@org.chem.ethz.ch; rkrishna@scripps.edu

Helvetica Chimica Acta ± Vol. 84 (2001)
1779
stepwise manner only; attempts to observe autocatalytic replication with turnover have
failed [9]. Nevertheless, such failure could not really detract from sustaining the view
that the pyranosyl form of RNA could have been, in principle, an evolutionary
alternative to the RNA structure since, after all, comprehensive non-enzymic(!)
autocatalytic replication with turnover is not known for RNA oligonucleotides
either4).
Scheme 1. Constitution and Configuration of the Repeating Units of the Four Diastereoisomeric Pentopyranosyl-
(4' ! 2')-oligonucleotide Systems
In this situation, it appeared important to establish experimentally whether the
remarkable potential of pyranosyl-(4' ! 2')-oligonculeotides is unique for the ribopyr-
anosyl series, or whether any of the three other diastereoisomers of the pentopyranosyl-
(4' ! 2')-oligonucleotide family might possess comparably interesting properties.
Therefore, we extended our studies from p-RNA to all the other members of the
pentopyranosyl-(4' ! 2')-oligonucleotide family and found ± admittedly to our surprise
± that the potential for strong informational Watson-Crick base pairing within this
family is by no means a privilege of the ribopyranosyl isomer. Quite the opposite turned
out to be true: the remaining three other members, namely, a-l-lyxopyranosyl-(4' !
2')-, a-l-arabinopyranosyl-(4' ! 2')-, and b-d-xylopyranosyl-(4' ! 2')-oligonucleotides
(Scheme 1) are all highly efficient Watson-Crick base-pairing systems [13]. Among
them, the arabinopyranosyl member is the most astonishing; it is, in fact, one of the
strongest oligonucleotide-type base-pairing systems known so far. All four pentopyr-
anosyl-(4' ! 2')-oligonucleotide systems form a compact family of base-pairing systems
whose base-pairing is orthogonal to that of RNA in the sense that all of them show
4
)
The failure to demonstrate sequence-replication with turnover in the p-RNA series is due to the problems
of product inhibition, competition by hydrolytic deactivation, and slow ligation rates of 2',3'-cyclo-
phosphates [1]. Clearcut examples of autocatalytic oligomerization by template-controlled ligation have
been demonstrated by von Kiedrowsky et al. [10] as well as Zielinski and Orgel [11] for short (modified) 2-
deoxyribofuranosyl-oligonucleotide hexamers. So far, there is no evidence that these observations could be
generalized and extrapolated to longer DNA or RNA sequences. For a recent review on nonenzymic
molecular self-replication, see [12].

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Helvetica Chimica Acta ± Vol. 84 (2001)
promiscuous cross-pairing among each other, yet none of them does cross-pair with
natural RNA or DNA [14].
We have reported a summary of the first findings in this project in two preliminary
communications [13][14] and promised to publish the full study in a series of papers in
this journal. The present paper describes the synthesis and the pairing properties of
oligonucleotides of the a-l-lyxopyranosyl-(4' ! 2') series.
2. Synthesis. ± For a significant comparison between the base-pairing properties of
b-d-ribopyranosyl-(4' ! 2')-oligonucleotides (ꢀp-RNAꢁ) and other members of the
pentopyranosyl family, it was essential to use for the latter the right enantiomer. For the
member derived from lyxose, the enantiomer in which the local chirality and
conformation at the nucleosidic center (nucleobase equatorial) correspond to the b-
d-ribopyranosyl series, is the a-l-lyxopyranosyl enantiomer (see Scheme 2). Overall,
the synthesis of a-l-lyxopyranosyl-(4' ! 2')-oligonucleotides described here follows the
pattern developed earlier in the synthesis of oligonucleotides of the p-RNA series
[1][5].
Scheme 2. Idealized Pairing Conformation of b-d-Ribo-(4' ! 2')- and a-l-Lyxo-(4' ! 2')-pyranosyl-oligonu-
cleotides
2.1. Synthesis of a-l-Lyxopyranosyl Nucleoside Building Blocks Containing
Adenine (A), Thymine (T), Guanine (G), and Cytosine (C) (Scheme 3). Primary
targets in the preparation of the building blocks for the synthesis of a-l-lyxopyranosyl-
(4' ! 2')-oligonucleotides were the protected nucleoside derivatives 5a ± 5d in which
the 4'-OH groups bear a 4',4''-dimethoxytrityl substituent, the 3'-OH groups an acyl
function (benzoyl (Bz) or acetyl (Ac)), and the nucleobases A, C, and G the
conventional protecting groups Bz and isobutyroyl, respectively. Central intermediates
on the way to these compounds were the 2',3',4'-trihydroxy derivatives 3a ± 3d of the
nucleobase-protected nucleosides, easily obtained by selective hydrolysis of the
corresponding 2',3',4'-tribenzoyl derivatives 2a ± 2d. The latter were synthesized in
yields of 80 ± 90% by coupling the a/b-anomeric mixture of tetrabenzoyl-lyxopyranose
1 [15] with the corresponding bases, namely, N6-benzoyladenine (nucleosidation
catalyst: SnCl4), thymine (trimethylsilyl triflate (TMS-Tf)), N4-benzoylcytosine
(SnCl4), and N2-isobutyrylguanine (trimethylsilyl triflate) under the Vorbrüggen-

Helvetica Chimica Acta ± Vol. 84 (2001)
1781
Hilbert-Johnson conditions [16]5) (Scheme 1). Only the a-nucleosides with equatorial
conformation of the nucleobases at the pyranose chairs (see Schemes 3 and 4) were
formed (1H-NMR). In the reaction with N-isobutyrylguanine, chromatographic
separation of the N(9)-regioisomer (2d, 58%) from the N(7)-regioisomer (2e, 22%)
was necessary. The N(7)-regioisomer, after isolation, could be equilibrated by refluxing
in CH2Cl2 in the presence of TMS-Tf, to give further amounts of N(9)-regioisomer so
that the overall yield of the latter was 70%. Assignment of N(9) vs. N(7) constitution
1
was based on the H-NMR spectrum, in which the compound assigned to the N(9)-
regioisomer 2d shows the doublet for H C(1') at higher field (d ˆ 6.12 ppm, J ˆ 7.5 Hz,
in CDCl3) as compared to the N(7)-isomer 2e (d ˆ 6.83 ppm, d, J ˆ 9.5 Hz, in CDCl3)
Scheme 3. Preparation of the A,T,G, and C Nucleoside Building Blocks for the a-l-Lyxopyranosyl Series
5
)
The a-l-lyxopyranosyl-nucleosides of adenine, cytosine, and uracil (unprotected) have been previously
synthesized by Fuertes et al. [17] by another method and in a different context.

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Helvetica Chimica Acta ± Vol. 84 (2001)
[16]. Base-catalyzed selective removal of the O-acyl protecting groups in (2a ± 2d)
afforded the triols (3a ± 3d) in 79 ± 87% yields.
Fig. 1 presents the results of an X-ray structure analysis of the 2', 3', 4'-
tribenzoyloxy-nucleoside 2b of the thymine series, demonstrating its constitution,
configuration, and preferred conformation6). That the latter is also the preferred
conformation in solution can be deduced from the coupling constants of the H C(1')
with H C(2'), the value of which (J ˆ 9.8 Hz, in CDCl3) is typical for two vicinal
protons in diaxial conformation. Table 1 lists the values of the corresponding coupling
constants of all the intermediates formulated in Scheme 3. These values demonstrate
that all these nucleosides have the a-configuration of the nucleobase, and that the
preferred conformation of them in solution (NMR solvents) is the one depicted in their
respective formulas of Scheme 3.
Fig. 1. X-Ray structure of the 1-(2',3',4'-tri-O-benzoyl-a-l-lyxopyranos-1'-yl)thymine, 2b. Nucleosidic torsion
angle O C(1') N(1) C(2): 91.186).
Regioselective protection of the 3'-OH and 4'-OH functions of 3a ± 3b with the
benzoyl- and the 4',4''-dimethoxytrityl groups, respectively, was the major problem on
the way to the synthesis of lyxopyranosyl-oligonucleotides. Whereas the choice of Bz as
the protecting group for the 3'-OH was suggested by our previous experience in the
6
)
The X-ray analysis was carried 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. CDCC 160498. 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) 336 0333; e-mail:
deposit@ccdc.cam.ac.uk).

Helvetica Chimica Acta ± Vol. 84 (2001)
1783
Table 1. J(H C(1'), H C(2')) Values [Hz] for Selected Compounds Depicted in Scheme 3
Compound
a
b
c
d
(A)
(T)
(C)
(G)
2
3
9.6
(CDCl3)
9.6
9.8
(CDCl3)
9.8
10.2
((D6)DMSO)
9.4
7.5
CDCl3
9.6
((D6)DMSO)
(D2O)
((D6)DMSO)
((D6)DMSO)
4
7.6
8.1
8.1
(exo/endo)
8.3
8.5
8.4
((D6)DMSO)
((D6)DMSO)
9.7
((D6)DMSO)
4b
5
((D6)DMSO)
9.6
9.7
9.4
9.0
(CDCl3)
((D6)DMSO)
(CDCl3)
(CDCl3)
p-RNA series [1][5], the method for its regioselective introduction was not, since the
two series differ in the relative configurations of the 3'- and 4'-OH groups. A nearly
ideal (and chemically appealing) solution7) to the problem was found in the appli-
cation of a remarkable regioselectivity documented in the literature [18] referring to
the acid-catalyzed ring opening of orthoesters implying vicinal cis-diol groups at
pyranose chairs: mild treatment of such orthoesters with aqueous acid leads regio-
selectively to corresponding cis-diol monoesters with the ester function occupying the
axial position.
Reaction of the mono nucleosides 3a and 3b with triethyl orthobenzoate and TsOH
in DMF under anhydrous conditions at 308 under a vacuum of 20 Torr afforded the
orthobenzoates 4a and 4b as endo/exo-mixtures in 64 and 74% yields (isolated).
Successive removal of the EtOH formed during the reaction by working under vacuum
was crucial for high conversion. It was also essential to perform the reaction at slightly
elevated temperature, because when, for example, the reaction with 3a was done at
room temperature, formation of an acyclic diethyl-orthobenzoate derivative (presum-
ably the 4'-isomer) was observed as main product. This acyclic orthoester could be
converted to the cyclic orthoester 4a in the presence of TsOH by increasing the
temperature to 308. Apparently, the 4'-OH group reacts to form first an acyclic
orthoester, and, at slightly elevated temperatures, the trans-orthoesterification
proceeds further to the more stable cyclic orthoester, involving the two cis-vicinal
OH groups. Tritylation of 4a and 4b with dimethoxytrityl chloride and lutidine in
CH2Cl2 at 08 proceeded in almost quantitative yield (as observed by TLC) to the
corresponding 4'-O-tritylated orthoesters, which were opened to the 3'-O-benzoates 5a
and 5b by addition of aqueous AcOH to the reaction mixture. It was important to
perform the tritylations at low temperature, because, when the reaction was carried out
at room temperature or higher, then the conversion of 4a and 4b remained incomplete.
The trityloxy group is relatively stable under the conditions of the partial orthoester
hydrolysis. Compounds 5a and 5b are obtained in 55 and 61% yield, respectively, over
7
)
Initiated and carried through by F.R. (A.E.)

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Helvetica Chimica Acta ± Vol. 84 (2001)
both steps, tritylation followed by orthoester-hydrolysis being carried out as a
convenient one-pot procedure.
In exploratory experiments in the thymine series, the orthoester 4b (as a mixture of
epimers) was originally opened before tritylation to give the crystalline 3'-O-benzoate
4bb regioselectively in 70% yield. No isomeric 2'-O-benzoate was isolated8). However,
tritylation of this derivative proved to be difficult, due to the substrateꢁs low solubility
in noncoordinating solvents that are suitable for the tritylation step (e.g., CH2Cl2).
Orthoesters 4a and 4b, on the other hand, are much more soluble in such solvents and,
therefore, much more efficiently tritylated.
In the cytosine and guanine series, it was observed that formation of the orthoesters
4c and 4d suffered from low yields when triethyl orthobenzoate was used as the reagent.
Therefore, the orthobenzoate reagent was replaced by trimethylorthoacetate (in the
presence of TMS-Cl) according to observations published by other authors for the
ribofuranosyl series [19][20]; it gave the orthoesters 4c and 4d in 74 and 66% yields,
respectively. Tritylation, carried out as in the adenine and thymine series and followed
by acid-catalyzed ring opening, afforded regioselectively the 3'-Ac derivatives 5c and
5d. In the chromatographic purification of these compounds on silica gel, caution was
demanded to avoid partial (3' ! 2') migration of the Ac groups during chromatography
(see Exper. Part).
Phosphoroamidites 6a ± 6d were synthesized with the 2-cyanoethyl moiety as the
protecting group. Solid support (ꢀlong-chain alkylamine-CPGꢁ; Sigma) derivatives 7a ±
7d required for the automated synthesis of oligonucleotides were synthesized from
intermediates 5a ± 5d through standard methodology outlined in Scheme 4.
2.2. Synthesis and Purification of Oligonucleotides. The preparation of a-l-
lyxopyranosyl-(4' ! 2')-oligonucleotides was carried out on a 1-mm scale as previously
done in the homo-DNA [21] and p-RNA series [1][5] on automated DNA synthesizers
(Pharamaciaꢁs Gene Assembler Plus and Perseptiveꢁs Expedite) with ca. 0.08m solution
of phosphoroamidites 6a ± 6d in MeCN and ca. 35 mg of solid support derivatives 7a ±
7d. The syntheses were performed in the ꢀtrityl-onꢁ mode. The following modifications
to the protocols used earlier were made: a) coupling of phosphoroamidites over a
period of 16.7 min with 0.25 ± 0.35m 5-(ethylthio)-1H-tetrazole in MeCN, and b)
detritylation with 6% Cl2CHCOOH in 1,2-dichloroethane over a 3-min period.
Coupling efficiencies ranged from 95 to greater than 99%.
Post-synthesis handling of the dried CPG-solid support included, as the first step,
treatment with dry pyridine Et3N 5 :1 to induce the eliminative removal of the 2-
cyanoethyl protecting group, thus converting the labile phosphotriester linkages to the
stable phosphodiester linkages [22]. This step minimizes the strand scission that can
occur in the deprotection steps under basic conditions. These conditions were ±
depending on the specific oligonucleotide sequence ± either treatment with 25% aq.
hydrazine hydrate at 48 (Method A, see Table 2), or with aq. MeNH2/NH3 at room
temperature (Method B, Table 2), or with aq. MeONH2/NH3 at 48 (Method C, Table 2).
All these methods led to the detachment of the oligonucleotides from the CPG-solid
support with the concomitant deprotection of the acyloxy groups, as well as the
acylamino groups of adenine, cytosine, and guanine. A special workup procedure after
8
)
For assignment of the constitution of 4bb, see Exper. Part.

Helvetica Chimica Acta ± Vol. 84 (2001)
1785
Scheme 4. Preparation of the Phosphoroamidite and CPG Derivatives for the Automated Synthesis of a-l-
Lyxopyranosyl-(4' ! 2')-oligonucleotides
deprotection was crucial, at least when deprotection was carried out by the hydrazine
treatment9). The product mixture is diluted with 0.5m aq. Et3NH2CO3 buffer and
loaded over a Waters Sepak-C18 cartridge, to remove the excess hydrazine. The use of
0.2 ± 0.5m aq. Et3NH2CO3 buffer greatly enhances the efficiency of separation when
9
)
Attempts to remove hydrazine by evaporation led to increased strand scission.

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Helvetica Chimica Acta ± Vol. 84 (2001)
compared to the loading of the oligos on the Sepak cartridge with H2O alone. This
strategy is successful only with ꢀtrityl-onꢁ sequences10).
The crude oligonucleotides were detritylated with 80% aq. HCO2H at room
temperature, subsequently purified by ion-exchange HPLC (target purity 95%),
desalted11) again, and stored at
208. The concentrations of the purified oligonucleo-
tide stock solutions were determined by UV at 260 nm (at ca. 808), and the purified
oligonucleotides checked for the correct mass by matrix-assisted laser-desorption
ionization time-of-flight (MALDI-TOF) mass spectrometry [23]. Table 2 lists the
oligonucleotides synthesized, purified, and controlled by MALDI-TOF MS.
Table 2. HPLC and MS Data of a-l-Lyxopyranosyl-(4' ! 2')- and Chimeric (pr-pl)-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]
(found)
(calc.)
pl(A8)
pl(T8)
A
A
A
B
A
A
A
A
A
A
A
A
A
C
C
A
C
A
49.0 (25%)
20.7 (14%)
26.4 (10%)
13.6 (15%)
13.5 (7%)
47.4 (28%)
9.2 (4%)
30.6 (17%)
36.2 (22%)
26.5 (15%)
2.1 (3%)
26.3 (43%)
5.6 (10%)
10.2 (13%)
4.2 (5.9%)
14.5 (59%)
2.7 (2%)
10 ± 50% in 30 min/18.7
20 ± 80% in 30 min/24.8
10 ± 50% in 30 min/25.5
2572
2500
3890
2573
2500
3889
3780
2534
2534
2536
2535
2527
2545
2010
1770
1889
2538
2538
3532
2536
2536
pl(A12
pl(T12
)
)
0 ± 100% in 30 min/26.3 3780
pl(A4T4)
pl(T4A4)
pl(AT)4
pl(TA)4
pl(TATTTTAA)
pl(TTAAAATA)
pl(G6)
pl(C6)
pl(C3G3)
pl(ATTCAGCG)
pl(CGCTGAAT)
pl(TATAAAAATAA)
(pr-pl)4(T4A4)d)
(pl-pr)4(T4A4)
20 ± 80% in 30 min/18.6
20 ± 80% in 30 min/18.2
20 ± 80% in 30 min/18.3
20 ± 80% in 30 min/18.6
2536
2536
2536
2536
0 ± 100% in 30 min/21.2 2526
0 ± 100% in 30 min/18.7 2547
0 ± 100% in 30 min/29.5 2009
0 ± 100% in 30 min/12.4 1770
0 ± 100% in 30 min/22.4 1889
0 ± 100% in 30 min/21.5 2538
0 ± 100% in 30 min/21.0 2538
0 ± 100% in 30 min/20.3 3529
0 ± 100% in 30 min/20.7 2536
0 ± 100% in 30 min/19.7 2538
1.0 (1%)
a) Method A: 25% aq. NH2NH2 inH2O at 48 for ca. 6.5 h; Method B: 40% aq. MeNH2 conc. aq. NH3 1 : 1 at r.t.
for 6.5 h; Method C: 0.2m MeONH2 ´ HCl in 25% aq. NH3 and EtOH (3 : 1) at r.t. for ca. 6 h (66 h for G- and C-
containing sequences). 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 Sepak cartridges.
b) MonoQ HR 5/5 column; elution with 10 mm Na2HPO4 in H2O and a linear gradient of NaCl, pH ꢀ 10.5, flow
1 ml/min; peak purity (260 nm) 95 ± 99%.
c) Matrix: 2,4,6-trihydroxyacetophenone and ammonium citrate buffer.
d) Chimera: pr(T)-pl(T)-pr(T)-pl(T)-pr(A)-pl(A)-pr(A)-pl(A); prˆ b-d-ribopyranosyl; pl ˆ a-l-lyxopyra-
nosyl.
10
)
)
When conducted with ꢀtrityl-offꢁ sequences, it was noticed that clean separation from hydrazine was not
achieved as the oligos had a tendency to ꢀflow-throughꢁ with the initial loading to the Sepak cartridge.
This process gave high reproducibility of salt-free oligonucleotides as evidenced by clean spectra in
MALDI-TOF mass spectrometry.
11

Helvetica Chimica Acta ± Vol. 84 (2001)
1787
3. Base-Pairing Studies. ± The pairing properties of (4' ! 2')-a-l-lyxopyranosyl-
oligonucleotides were characterized by methods with which we were familiar from our
earlier studies in the homo-DNA [21] and p-RNA series [1][5 ± 8]. These methods are
temperature-dependent UV spectroscopy (for Tm-measurements [24]), concentration-
dependent Tm-measurement by UV spectroscopy (for determination of thermody-
namic data [25]), molar-ratio-dependent UV spectroscopy (for checking stoichiometry
[26]), 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 ca. 10 mm, unless otherwise stated.
Table 3. Tm Values and Thermodynamic Data
No. Base sequence
Tm [8C]a)
Self-pairing
of non-com- duplexes
plementary 1m NaCl
Tm [8C]a) Tm [8C]a)
pl(4' ! 2') pl(4' ! 2')
For com-
parison:
Tmꢁs of
DG
(258)
DH
TDS
(258)
Ref.
all (4' ! 2')
duplexes
150 mm NaCla)b)
150 mm NaCl pr(4' ! 2')
pl(4' ! 2') duplexes
single strands (c ꢀ 10 mm) (c ꢀ 10 mm)
duplexes
1m NaCl
150mm NaCl
(c ꢀ 10 mm)
(c ꢀ 10 mm)a)
1
2
3
4
5
6
7
8
9
pl(A8)
pl(T8)
14.8*
< 5*
pl(A8) ‡ pl(T8)
51.0
47.0*
40*
12.3*
69.5*
57.2* [13]
pl(A12
)
19.9 (16.4*)
16.2
pl(T12
)
pl(A12) ‡ pl(T12
)
74.3
68.0
60.8
27*
40*
38*
40*
19.8
9.4*
11.4*
9.5*
9.4*
97.1
77.3 [14]
51.3* [13]
53.6* [13]
52.0* [13]
53.5* [13]
pl(A4T4)
41.1*
49.6*
44.7*
43.2*
38.2*
47.0*
38.3*
37.9*
60.7*
67.0*
61.4*
62.9*
pl(T4A4)
pl(AT)4
10 pl(TA)4
11 pl(TATTTTAA)
19.6*
23.2*
12 pl(TTAAAATA)
13 pl(TATTTTAA)
‡ pl(TTAAAATA)
14 pl(G6) ‡ pl(C6)
15 pl(C3G3)
46.4*
41.8*
38.8
10.6
55.5
44.9 [14]
52
61
61[5c]
68*
10.4
11.2³
33.3
42.1³
22.9
30.9³
16 pl(ATTCAGCG)
17 pl(CGCTGAAT)
18 pl(ATTCAGCG)
‡ pl(CGCTGAAT)
< 0
9.9
61.9
57.6
61.4
13.0
1.2²
54.9
41.9 [14]
30.5²
19 pl(TATAAAAATAA) 36.5**
hairpin
31.7²
20 (pr-pl)4(T4A4)
21 (pl-pr)4(T4A4)
41.8
43.3
a) Measurements were made in 0.01m NaH2PO4, 0.1 mm Na2EDTA buffer, pH 7.0 unless otherwise indicated. Error of Tm
determination estimated Æ0.58. Values with asterisk (*) were measured in 0.01m Tris-HCl buffer, pH 7.0; **: in 150 mm NaCl.
b) Thermodynamic data from plots of Tm vs. ln c; experimental error estimated in DH values Æ5%. ³: in 1m NaCl. ²:
1
Thermodynamic data calculated for hairpin according to [25] (p. 1610).
Table 3 summarizes our observation on the base-pairing capability of a-l-
lyxopyranosyl-(4' ! 2')-oligonucleotides, and Fig. 2 and 3 give selected illustrations of
it, such that they complement the Figures already reproduced in the preliminary
communications [13][14].

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Helvetica Chimica Acta ± Vol. 84 (2001)
Fig. 2. UV and CD data documenting the pairing behavior of A,T-containing (4' ! 2')-a-l-lyxopyranosyl
sequences. A) UV-Spectroscopic Tm curves (heating) of selected duplexes. B) Temperature-dependent CD
curves; temperature range: 38 ! 758. C) Self-pairing of the homobasic strands pl(A8), pl(A12), pl(T8), and
pl(T12) as demonstrated by UV-spectroscopic Tm curves (heating). D) UV-Spectroscopic Tm curves of the
duplex formed by pl(4'-TTAAAATA-2') with its antiparallel complement pl(4'-TATTTTAA-2') and self-
pairing Tm curves of the individual strands. E) Molar-ratio-dependance of UV absorption (260 nm, 0.3 Æ 0.38
(ꢀmixing curveꢁ) indicating 1 :1 duplex stoichiometry for the pairing between pl(4'-TTAAAATA-2') and its
antiparallel complementary strand pl(4'-TATTTTAA-2'). F) Temperature-dependent CD curves of the duplex
between pl(4'-TTAAAATA-2') and its antiparallel complementary strand pl(4'-TATTTTAA-2'); temperature
range: 08 ! 778. Tm Measurements were made in 10 mm aq. Tris ´ HCl buffer, 1m NaCl at pH 7.0 except the one
marked with an asterisk * (phosphate buffer). All CD and mixing curves were measured in 10 mm aq. NaH2PO4
containing 0.1 mm Na2EDTA, 1m NaCl at pH 7.0. Total oligonucleotide concentrations in all measurements
were ca. 10 mm. All Tm curves were fully reversible (no hysteresis). Tm Values are calculated from the maxima of
the first derivative curve with kaleidagraph software program.
The tendency of lyxopyranosyl-(4' ! 2')-oligonucleotides to undergo somewhat
stronger base pairing than p-RNA is most clearly expressed by sequences that contain
A and T only, and is also evident in the behavior of homobasic sequences like pl(A8),
pl(A12), or pl(T12) (see Table 3), which show weak self-pairing under conditions where
the corresponding p-RNA sequences do not [1][5][14]. A series of sequence
combinations (No. 7, 8, 13, 15, and 18 in Table 3) demonstrate the preferred strand

Helvetica Chimica Acta ± Vol. 84 (2001)
1789
Fig. 3. UV, Tm, CD, and thermodynamic data documenting the pairing behavior of G,C-containing (4' ! 2')-a-l-
lyxopyranosyl sequences. A) UV-Spectroscopic Tm melting curves for self-pairing of pl(C3G3) and pairing
between pl(G6) and pl(C6). B) UV-Spectroscopic Tm curves of the duplex formed between pl(4'-ATTCAGCG-
2') with its antiparallel complement pl(4'-CGCTGAAT-2'), and corresponding curves of individual strands. C)
Temperature-dependent CD curves of self-complementary pl(C3G3) strand, temperature range: 308 ! 708
(Tm ˆ 558). For conditions of measurements see caption of Fig. 2. Tm Curves fully reversible (no hysteresis). D)
Thermodynamic data of the formation of the duplex shown, determined from concentration-dependence of Tm
values (in 10 mm aq. NaH2PO4 containing 0.1 mm Na2EDTA, 150 mm NaCl at pH 7.0). For method, see [25].
orientation in lyxopyranosyl-(4' ! 2')-duplexes to be antiparallel, as it has been proven
to be the case in the p-RNA series. Remarkable is the high efficiency by which anti-
parallel complementary base-sequences of the lyxo- and ribopyranosyl-sequences
undergo inter-system cross-pairing with each other (Table 4). This cross-pairing is, at
the same time, strong evidence for the correctness of the postulate that the base pairing
in the lyxopyranosyl-(4' ! 2')-oligonucleotide series proceeds by the Watson-Crick
mode, as it is the case for the ribopyranosyl series, where it has been proven by a
comprehensive NMR-structure analysis of a p-RNA duplex [5b].
The efficient intersystem cross-pairing between the lyxopyranosyl- and the
ribopyranosyl-(4' ! 2')-system makes it very probable that pairing should also occur
between chimera strands, where both lyxo- and ribopyranosyl-nucleoside building
blocks occur in a given strand, while the two strands still possess antiparallel
complementary base-sequences (Fig. 4). We have made two such examples and have
found that their pairing behavior is as expected (Nos. 20 and 21 in Table 3).
The sequence pl(TATAAAAATAA) (No. 19) has been constructed in order to test
the lyxopyranosyl systemꢁs capability to form hairpins. It has previously been shown
that pyranosyl-RNA does form hairpins with surprising ease (considering the presumed
nonflexibility of a pyranosyl ring relative to a furanosyl ring) [6]. Sequence No. 19 does
from a hairpin that is remarkably stable (see Fig. 5), given that it contains three A-T

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Table 4. Tm Values of Duplexes Formed by Cross-Pairing between a-l-Lyxopyranosyl (pl) and b-d-Ribopyr-
anosyl (pr)-oligonucleotidesa)
a) Conditions: c ˆ 5 ‡ 5 mm, 1.0m NaCl, 10 mm NaH2PO4, 0.1 mm Na2EDTA, pH 7.0. The color of the symbols
relates to the oligonucleotide sequences of the same color in the formulas of duplex I ± IV given at the side of the
table. Tm Values in black refer to intersystem cross-pairing and those in the shaded diagonal to intrasystem
pairing; Tm values in color refer to self-pairing of corresponding strands.
Fig. 4. Idealized pairing conformation of a chimeric oligonucleotide containing alternating b-d-ribo-(4' ! 2')-
and a-l-lyxo-(4' ! 2')-pyranosyl units (No. 20 in Table 3)
Fig. 5. Invariance of Tm with variation of oligonucleotide concentration of hairpin-forming sequence
pl(4'-TATAAAAATAA-2') (10 mm aq. NaH2PO4 containing 0.1 mm Na2EDTA, 150 mm NaCl at pH 7.0).
Hairpin formulae indicate two possible ꢀinterstrandꢁ base-stacking patterns.

Helvetica Chimica Acta ± Vol. 84 (2001)
1791
base pairs only. This seems to be still another example of a characteristic property of
pentopyranosyl-(4' ! 2')-oligonucleotides, namely, that their base-pairing draws much
of its strength from interstrand-base-stacking [27][28]. The surprisingly strong self-
pairing of the two complementary, but not self-complementary, strands No. 11 and 12
(see also Fig. 2,D and Fig. 6) is quite probably another illustration of this relationship.
Fig. 6. Representation of the postulated interstrand base-stacking in the duplex formed by pl(4'-TTAAAATA-2')
with its antiparallel complement pl(4'-TATTTTAA-2'), and in duplexes formed by the self-pairing of the (not self-
complementary) individual strands
4. Discussion. ± In an early comparative analysis of the (then) hypothetical base-
pairing behavior of pentopyranosyl-(4' ! 2')-oligonucleotide systems based on consid-
erations of idealized single-strand pairing conformations, we hypothesized that
lyxopyranosyl oligonucleotides will show base pairing, but one that should be weaker
than that of pyranosyl-RNA [5a]. Experimental findings qualified this conjecture: a-l-
lyxopyranosyl-(4' ! 2')-oligonucleotides do, in fact, show Watson-Crick pairing, but it is
± at least with respect to adenine-thymine pairing ± tendentially stronger than that of
corresponding oligonucleotides of the pyranosyl-RNA series. In fact, the investigation
of the lyxopyranosyl system was the first in a series of experimental studies that
uncovered the limitations of our originally applied criteria for predicting base-pairing
properties [5a][29]. In the lyxopyranosyl case, a pairing weaker than p-RNA had been
expected on the basis of the argument that the backbone/base-pair axes inclination
[5b][27] of the lyxo-system is less than that of the ribo-system (see Fig. 7) and,
therefore, that the stacking distance between neighboring base pairs ± too large already
in the (idealized) pairing conformation of p-RNA ± will be even larger in the
(idealized) lyxopyranosyl system (ca. 5.5 Š). Implicit in this reasoning was the
assumption that shortening the stacking distance towards the optimal range between
3.5 and 4.0 Š by either increasing the backbone/base-pair-axes inclination, or by
helicalization of the duplex strands, would induce strain within the backbone, as a
consequence of which pairing strength would decrease. In the meantime, an NMR-
structure analysis of a p-RNA duplex has revealed that such duplexes, indeed, have
optimal base-stacking distances, as well as a slightly twisted (left-handed) helical shape
[5b]. Since then, it has become clear that considerations on idealized pairing
conformations deduced by qualitative conformational analysis of repeating oligonu-

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Fig. 7. Vertical projection formulae of the idealized pairing conformation (nucleosidic torsion angles 1208) of
the ribo- and lyxo-pyranosyl-(4' ! 2')-oligonucleotide systems, pointing to the difference in the two systemsꢀ
backbone/base-pair-axes inclination. See also [5b][27].
cleotide units [5a][29] are useful for making qualitative predictions about the base-
pairing capability as such, but are too simplistic for predicting relative base-pairing
strengths as a function of configurational and conformational differences in pentopyr-
anosyl-oligonucleotide backbones.
In retrospect, and in light of the presently available knowledge on the structure of
pentopyranosyl-(4' ! 2')-oligonucleotide duplexes [5b][30], we think that is a specific
type of steric hindrance occurring in duplexes of pentopyranosyl-(4' ! 2')-oligonucleo-
tides that acts as a major determinant of the relative base-pairing strengths of
diastereoisomeric members in the series. This type of steric hindrance refers to the
spatial closeness of the equatorial H-atom at C(5') of a pyranose chairs to the plane of
the neighboring nucleobase situated upstream in the same strand (Fig. 8). Provided
that this closeness acts indeed as a source of repulsion in p-RNA duplexes
(phosphodiester bridges diequatorial), then such repulsion would be expected to be
less important in lyxopyranosyl duplexes: phosphodiester bridges that are axially
attached to C(4') of the pyranose chairs will put the critical H-atom at C(5') farther
apart from the plane of its neighboring nucleobase than is the case in corresponding p-
RNA duplexes. The difference has recently been substantiated by Jaun and Ebert [30] ±
not in the lyxopyranosyl, but the arabinopyranosyl series ± by an NMR-structure
analysis of the a-l-arabinopyranosyl-(4' ! 2')-(CGAATTCG)2 duplex, allowing a
comparison of the structural environment of the equatorial H-atoms at C(5') in
duplexes with an axial-equatorial vs. a diequatorial C(4')-phosphodiester group. The
equatorial H-atoms at C(5') of pyranosyl-RNA duplexes are found to be distinctly
more deshielded in their chemical shifts by upstream-nucleobase neighbors than
corresponding H-atoms at C(5') of arabinopyranosyl-duplexes (Fig. 9). We conjecture
that a corresponding difference in intrastrand steric repulsion between neighboring

Helvetica Chimica Acta ± Vol. 84 (2001)
1793
Fig. 8. Side view of the idealized pairing conformation of the two diastereoisomeric (4' ! 2')-pentopyranosyl
oligonucleotide systems, pointing to the difference in the steric hindrance between the equatorial hydrogen at C(5')
of the pyranose chair and the neighboring (upstream) nucleobase
Fig. 9. 1H-NMR Chemical shifts of the equatorial H-atoms at C(5') in the (4'-CGAATTCG-2')2 duplexes of the b-
d-ribopyranosyl-(4' ! 2')- and the a-l-arabinopyranosyl-(4' ! 2')-series (data taken from Jaun and co-workers
[5a][30])
nucleoside units in lyxopyranosyl duplexes may be a significant factor in rendering
them tendentially more stable than corresponding p-RNA duplexes.
The properties of a-l-lyxopyranosyl-(4' ! 2')-oligonucleotides have provided the
first of a series of lessons that the systematic investigation of the pentopyranosyl-(4' !

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2')-oligonucleotide family has given us. A more drastic correction of our earlier
predictions on relative base-pairing strength of oligonucleotide systems had come from
studying the a-l-arabinopyranosyl-(4' ! 2')-system, which we will describe in a
forthcoming paper of this series. On the other hand, it was the work in the
lyxopyranosyl series that turned out to exert the most incisive influence ± via a study
of the a-l-lyxopyranosyl-(4' ! 3')-oligonucleotide system [28][31] ± on the further
course of our investigation in the field of nucleic acid etiology [32].
The work was supported by the Skaggs Foundation (TSRI) and Novartis AG, Basel. We thank Professor
Bernhard Jaun (ETH) for allowing us to include in this paper unpublished data from his laboratory. We thank
Dr. A. Shyvaniuk for help in the ORTEP drawing of 2b.
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-cyanoethyoxy)(dii-
sopropylamino)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 UV absorption 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 m, 230 ± 440 mesh, EM Science) at low pressure (max. 2 bar). In case of acid-
sensitive compounds, the silica gel was pre-treated with appropriate solvents containing ca. 0.5% Et3N. M.p.
(uncorrected): MEL-TEMP II (Laboratory Devices Inc., USA). NMR: 1H: d values in ppm (TMS as internal
1
standard), J [Hz], assignments of H 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 Cs ion gun. Matrix-assisted laser-desorption-ionization time-of-flight mass
spectrometry (MALDI-TOF-MS) was performed on a Voyager-Elite mass spectrometer (Perseptive Biosys-
tems) with delayed extraction with THAP as the matrix with ammonium citrate added to the sample. Elemental
analyses were performed with a Perkin-Elmer PE2400 CHN analyzer. Oligonucleotides were synthesized on an
Expedite 8909 Nucleic Acid Synthesis system (Perseptive Biosystems) or on a Pharmacia Gene Synthesizer Plus.
HPLC: Anion exchange (IA)-HPLC was performed on A) Pharmacia GP-250 Gradient Programmer equipped
with two Pharmacia P-500 pumps, ABI-Kratos Spectraflow 757 UV/VIS detector and a Hewlett Packard HP
3396A analog integrator or B) Pharmacia ¾kta 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 Spectra were measured on a Cary 1 C spectrophotometer (Varian) and a Perkin-Elmer Lambda 2.
Melting-points (Tm) of oligonucleotides were determined with Cary 1 Bio spectrophotometer (Varian) or with
Perkin-Elmer Lambda 2 equipped with Perkin-Elmer Digital Controller/Temperature Programmer C570.
Concentrations of oligonucleotide solns. were calculated from the UVabsorbance of the solns. at 260 nm (pH 7)
at ca. 808 with the following molar extinction coefficients: e (pr(A)) ˆ e (pl(A)) ˆ 15000, e (pr(T)) ˆ e
(pl(T)) ˆ 10000, e (pr(C)) ˆ e (pl(C)) ˆ 8400, e (pr(G)) ˆ e (pl(G)) ˆ 11900. CD Spectra were measured on A)
JASCO J-710 or B) AVIV 61 DS CD spectropolarimeter. Abbreviations: BSA: N,O-Bis(trimethylsilyl)aceta-
mide; CPG: ꢀcontrolled pore glassꢁ, DMAP: 4-(dimethylamino)pyridine; DMT: 4,4'-dimethoxytrityl; LCAA-
CPG: long-chain aminoalkyl-CPG (500 Š); TMS-Tf: trimethylsilyl trifluoromethanesulfonate; TOTU: O-{[(2-
cycanoethoxycarbonyl)methyliden]amino}-1,1,3,3-tetramethyluronium tetrafluoroborate.
1. Experiments Referring to Scheme 3. ± 1',2',3',4'-Tetra-O-benzoyl-a/b-l-lyxopyranose (1). To an ice-cold
stirred soln. of 78 ml (665.8 mmol) BzCl and 130 ml (1.6 mol) pyridine in 400 ml of dry CH2Cl2 were added
20.0 g (131.9 mmol) of (‡)-l-lyxose in small portions within 30 min. The mixture was stirred for another 2 h, and
a colorless substance precipitated. After addition of 150 ml of H2O, the mixture was washed successively with
20 ml of 1m aq. HCl, 18 ml of 2-aminoethanol, 50 ml of H2O, 20 ml of 1m aq. HCl, 20 ml sat. aq. NaHCO3 soln.,
and 50 ml of H2O. The org. phase was dried (MgSO4) and evaporated. The residual oil was dissolved in CH2Cl2,
followed by co-evaporation with toluene. The slightly yellowish foam was dried in vacuo (ca. 0.5 Torr r.t.) for
several hours to afford 68.5 g (91.7%) of product. It was clean by TLC and 1H-NMR, and was used in subsequent
reactions without further purification. Crystallization of a small amount from toluene gave analytically pure 1.

Helvetica Chimica Acta ± Vol. 84 (2001)
1795
M.p. 136 ± 1378. TLC (petroleum ether/acetone 3 :1): Rf 0.55. 1H-NMR (600 MHz, CDCl3): 4.11 (dd, J(4',5'b) <
0.5, Jgem ˆ 11.6, Hb C(5')); 4.40 (dd, J(4',5'a) ˆ 5.2, Jgem ˆ 11.6, Ha C(5')); 5.85 (m, H C(4')); 5.93
(m, H C(2')); 6.11 (dd, J(2',3') ˆ 3.5, J(3',4') ˆ 9.2, H C(3')); 6.57 (d, J(1',2') ˆ 2.9, H C(1')); 7.35 ± 7.70
(m, 12 arom. H); 7.92 ± 8.22 (m, 8 arom. H). 13C-NMR (150.9 MHz, CDCl3): 62.70 (t, C(5')); 67.81 (d, C(4'));
69.56 (d, C(3')); 69.62 (d, C(2')); 91.88 (d, C(1')); 128.93, 128.97, 129.09, 129.18 (4d, arom. C); 129.41 (s,
arom. C); 130.23, 130.31, 130.45, 130.57 (4d, arom. C); 133.96, 134.04, 134.17, 134.44 (4s, arom. C); 164.74,
‡
‡
165.79, 166.04, 166.15 (4s, CO). ES-MS (pos.): 589 (24.8, [M ‡ Na] ), 445 (29.7, [M PhCOO] ), 381 (9.9), 360
(17.5), 304 (52.5), 282 (100.0).
N6-Benzoyl-9-(2',3',4'-tri-O-benzoyl-a-l-lyxopyranosyl)adenine (2a). A suspension of 40 mg (70.6 mmol)
of 1 and 17.0 g (71.1 mmol) of 6-benzoyladenine in 375 ml of dry MeCN was heated to 808 (oil bath). Addition
of 32 ml (125.6 mmol) of BSA resulted in a clear soln. After 15 min, 26.3 ml (222.5 mmol) of SnCl4 was added
dropwise ( ! exothermic reaction), the soln. became dark, and stirring was continued for another 30 min. The
soln. 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. The org. phase was dried (MgSO4) and evaporated.
Purification of the residual oil was accomplished by CC (silica gel; petroleum ether/AcOEt 1:1 to 1:2 and
finally 1:3). The product fractions were combined, evaporated, and dried in vacuo (ca. 0.5 Torr r.t.) to furnish
28.7 g (59.5%) of a colorless amorphous solid, 2a. TLC (petroleum ether/acetone 2 :1): Rf 0.40. 1H-NMR
(600 MHz, CDCl3): 4.46 (d, Jgem ˆ 13.5, H C(5')); 4.55 (d, Jgem ˆ 13.5, H C(5')); 5.48 (d, J(3',4') ˆ 3.7,
H
H
C(4')); 6.15 (m, H C(3')); 6.29 (dd, J(2',3') ˆ 3.2, J(1',2') ˆ 9.6,
H
C(2')); 6.50 (d, J(1',2') ˆ 9.6,
C(1')); 7.20 ± 8.33 (m, 20 arom. H); 8.33, 8.83 (2s, C(2),
H
H
C(8)); 9.12 (s, NH). 13C-NMR
(150.9 MHz, CDCl3): 66.81 (t, C(5')); 68.56 (d, C(2')); 68.69 (d, C(3')); 69.31 (d, C(4')); 79.48 (d, C(1'));
122.93 (s, arom. C); 128.28 (d, arom. C); 128.44 (s, arom. C); 128.86 (d, arom. C); 129.18 (s, arom. C); 129.23,
129.26, 129.33 (3d, arom. C); 129.38 (s, arom. C); 130.13, 130.39, 130.50, 133.23 (4d, arom. C); 134.04
(s, arom. C); 134.19, 134.41 (2d, arom. C); 134.51 (s, arom. C); 141.24, 153.65 (2d, C(2), C(8)); 150.16, 152.40
‡
(2s, arom. C), 165.10, 165.21, 165.26, 165.65 (4s, CO). FAB-MS (pos., NBA/CsI): 948 (27.0, [M ‡ 2 Cs] ),
‡
‡
816.1040 (100.0, [M ‡ Cs] ), 684 (9.1, [M ‡ H] ).
1-(2',3',4'-Tri-O-benzoyl-a-l-lyxopyranosyl)thymine (2b). A suspension of 15.84 g (28.0 mmol) of 1 and
4.20 g (33.0 mmol) of dry thymine in 170 ml of dry MeCN was heated to 658 (oil bath), followed by the addition
of 18.5 ml (72.6 mmol) of BSA. After 40 min, 15.2 ml (83.1 mmol) of TMS-Tf were added by syringe to the clear
soln., and was kept overnight at 658. The soln. 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 with sat. aq. NaHCO3 soln. and
dried (Na2SO4). Precipitation by addition of ca. 100 ml EtOH afforded 10.3 g (64.5%) of an amorphous product.
A small amount was crystallized from acetone to give 2b. M.p. 1948. TLC (petroleum ether/AcOEt 1 :1): Rf 0.50.
1H-NMR (600 MHz, CDCl3): 1.94 (s, Me C(5)); 4.39 (d, Jgem ˆ 13.4, H C(5')); 4.41 (d, Jgem ˆ 13.4, H C(5'));
5.37 (m, H C(4')); 5.73 (dd, J(2',3') ˆ 3.2, J(1',2') ˆ 9.8, H C(2')); 6.05 (m, H C(3')); 6.50 (d, J(1',2') ˆ 9.8,
H
C(1')); 7.10 ± 8.15 (m, 15 arom. H, H C(6)); 9.56 (br. s, NH). 13C-NMR (150.9 MHz, CDCl3): 13.06
(q, Me C(5)); 67.04 (t, C(5')); 67.71 (d, C(2')); 69.13 (d, C(3')); 69.65 (d, C(4')); 78.78 (d, C(1')); 112.41
(s, C(5)); 128.62 (s, arom. C); 128.97 (d, arom. C); 129.13 (s, arom. C); 129.18, 129.23 (2d, arom. C); 129.40
(s, arom. C); 130.27, 130.33 (2d, arom. C); 134.18, 134.32, 134.34 (3d, arom. C); 134.92 (d, C(6)); 150.87
(s, C(2)); 163.78 (s, C(4)); 165.21, 165.44, 165.52 (3s, CO). FAB-MS (pos., NBA/CsI): 703.0669 (17.6, [M ‡
‡
Cs] ). Anal. calc. for C31H26N2O9: C 65.26, H 4.59, N 4.91, found: C 65.32, H 4.76, N 5.05.
N4-Benzoyl-1-(2',3',4'-tri-O-benzoyl-a-l-lyxopyranosyl)cytosine (2c). The reaction was performed as
described for 2a. In one of the experiments, 4.19 g (19.2 mmol) of N4-benzoylcytosine, 9.1 g (16.1 mmol) of 1,
and 9.7 ml (40 mmol) of BSA in 100 ml of dry MeCN followed by addition of 6.1 ml (52 mmol) of SnCl4.
Following the workup described for 2a and purification by chromatography (silica gel; AcOEt/hexane 1:1 to
2 :1) afforded 9.4 g (90%) of 2c. M.p. 118 ± 1208. TLC (petroleum ether/AcOEt 7:3): Rf 0.45. 1H-NMR
(200 MHz, (D6)DMSO): 4.45 (br. s, 2 H C(5')); 5.39 (br. d, J(3',4') ˆ 3.6, H C(4')); 5.74 (dd, J(2',3') ˆ 3.4,
J(1',2') ˆ 10.2, H C(2')); 6.05 (dd, J(2',3') ˆ 3.4, J(3',4') ˆ 3.6, H C(3')); 6.75 (d, J(1',2') ˆ 10.2, H C(1'));
7.25 ± 8.23 (m, 20 arom. H, H C(5), H C(6)).
N2-Isobutyryl-9-(2',3',4'-tri-O-benzoyl-a-l-lyxopyranosyl)guanine (2d) and N2-Isobutyryl-7-(2',3',4'-tri-O-
benzoyl-a-l-lyxopyranosyl)guanine (2e). To a suspension of 715 mg (3 mmol) of N2-isobutrylguanine
monohydrate and 1.13 g (2 mmol) of 1 in 20 ml of dry 1,2-dichloroethane, 3.75 ml (15 mmol) of BSA were
added, and the mixture was refluxed for 1 h. During heating, the suspension became a clear soln., 1.5 ml
(6.5 mmol) of TMS-triflate were added by a syringe, and the soln. was refluxed for an additional 20 h. The
mixture was cooled, solid NaHCO3 was added, and the soln. was stirred for 10 min. To this suspension, a mixture
of H2O and CH2Cl2 was added, and the org. phase washed twice with H2O and dried (Na2SO4). The org. phase

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Helvetica Chimica Acta ± Vol. 84 (2001)
was filtered and evaporated in vacuo to dryness. The solid residue was dissolved in AcOEt, and hexane was
added carefully until the soln. became turbid. The N7-isomer crystallized from the soln. to give 0.3 g (22%) of 2e.
The mother liquor was decanted and purified by CC (silica gel; CH2Cl2/MeOH 100 :0 to 95 :5 (v/v)) to afford
0.76 g (58%) of 2d.
Data of 2d: TLC (petroleum ether/AcOEt 7:3): Rf 0.16. 1H-NMR (200 MHz, CDCl3): 1.21, 1.24 (d, J ˆ 7.9,
Me); 2.69 (m, J ˆ 7.9, Me2CH); 4.21 (br. d, Jgem ˆ 11.2, H C(5')); 4.46 (br. d, Jgem ˆ 11.2, H C(5')); 5.54
(m, H C(4')); 6.12 (d, J(1',2') ˆ 7.5,
H
C(1')); 6.32 (dd, J(2',3') ˆ 3.2, J(3',4') ˆ 3.4,
H C(3')); 6.42
(dd, J(1',2') ˆ 7.5, J(2',3') ˆ 3.2, H C(2')); 7.31 ± 8.13 (m, 15 arom. H, H C(8)); 9.15, 12.1 (s, NH). 13C-NMR
(50.3 MHz, CDCl3): 18.93, 19.00 (q, Me2CH); 36.76 (d, Me2CH); 65.41, 68.24, 68.74, 69.28 (C(5'), C(2'), C(3'),
C(4')); 81.40 (d, C(1')); 128.80, 128.93, 129.01, 129.21, 129.28, 130.01, 130.16, 130.20, 134.10, 134.22 (arom. C),
138.23 (C(5)); 147.92 (C(4)); 155.59 (C(6)); 165.40, 165.41, 165.60 (3s, CO); 178.48 (s, COiBu). FAB-MS (pos.,
‡
NBA): 666 (100, [M ‡ H] ).
Data of 2e: TLC (petroleum ether/AcOEt 7:3): Rf 0.25. 1H-NMR (200 MHz, CDCl3): 1.14, 1.17 (d, J ˆ 6.9,
Me); 2.92 (m, J ˆ 6.9, Me2CH); 4.44 ± 4.51 (br. m, 2 H C(5')); 5.46 (br. d, J(3',4') ˆ 3.8, H C(4')); 6.04
(m, H C(2')); 6.08 (m, H C(3')); 6.83 (d, J(1',2') ˆ 9.5, H C(1')); 7.22 ± 8.26 (m, 15 arom. H, H C(8)); 10.23,
12.30 (s, NH). 13C-NMR (50.3 MHz, CDCl3): 18.47, 18.66 (q, Me2CH); 35.52 (d, Me2CH); 66.44, 68.38, 68.72,
69.01 (C(5'), C(2'), C(3'), C(4')); 80.60 (d, C(1')); 111.61, 127.74, 128.06, 128.37, 128.47, 129.30, 129.98, 133.23,
133.45, 133.58 (arom. C), 141.01 (C(5)); 147.77 (C(4)); 152.31 (C(2)); 156.15 (C(6)); 164.22, 164.53, 164.81 (3s,
CO); 179.48 (s, COiBu).
Isomerization of 2e to a Mixture of 2d/2e. To a suspension of 2.2 g (3.27 mmol) of 2e and 389 mg
(1.64 mmol) of N2-isobutylguanine monohydrate in 40 ml of (CH2Cl)2, 1.25 ml (5 mmol) of BSA was added.
Under Ar, the suspension was heated under reflux for ca. 1 h (until all material was dissolved). To that refluxing
soln., 2.0 ml (8.2 mmol) of TMS-Tf was added by syringe, and the soln. was heated for additional 20 h. The
cooled soln. was poured into sat. aq. NaHCO3 soln. and diluted with CH2Cl2. The phases were separated, and the
org. layer was washed for a second time with sat. aq. NaHCO3 soln. and dried (MgSO4). The soln. was filtered,
evaporated under vacuum, and subjected to CC (CH2Cl2/hexane 6 :4 with 2 ± 10% MeOH) to afford 1.36 g
(62%) of 2d and 0.65 g (30%) of 2e.
N6-Benzoyl-9-(a-l-lyxopyranosyl)adenine (3a). A soln. of 28.3 g (41.4 mmol) of 2a in 425 ml of THF and
250 ml of H2O was cooled to 08, and 320 ml of 15% aq. NaOH soln. were added dropwise over a period of
90 min. To keep the soln. homogenous, H2O was added dropwise. The pH of the resulting soln. was adjusted to
6 ± 7 (checked by Merck universal indicator paper) by dropwise addition of conc. aq. HCl. Subsequently, THF
was removed under vacuum (ca. 20 Torr r.t.), and the pH was adjusted to 2 ± 3 with conc. aq. HCl. The aq. phase
was extracted twice with 100 ml of Et2O, and the pH of the aq. phase was brought to 7 by dropwise addition of
15% aq. NaOH soln. Concentration of the soln. to ca. 800 ml and cooling to 48 gave (after 10 days) 13.45 g
(87.4%) of 3a as colorless crystals. M.p. 1958. TLC (petroleum ether/AcOEt 1 :1): Rf 0.18. 1H-NMR (600 MHz,
(D6)DMSO): 3.68 (d, J(3',OH) ˆ 3.3, H C(3')); 3.71 (d, Jgem ˆ 11.6, H C(5')); 3.99 (m, H C(4')); 4.01
(d, Jgem ˆ 11.6, H C(5')); 4.51 (dd, J(2',3') ˆ 2.6, J(1',2') ˆ 9.6, H C(2')); 5.18 ± 5.40 (3 br. s, 3 OH); 5.84
(d, J(1',2') ˆ 9.6, H C(1')); 7.54 (m, 2 arom. H); 7.64 (m, 1 arom. H); 8.06 (m, 2 arom. H); 8.68, 8.76 (2s,
H
C(2), H C(8)); 11.20 (br. s, NH). 13C-NMR (150.9 MHz, (D6)DMSO): 66.8 (d, C(2')); 68.3 (t, C(5')); 70.3
(d, C(3')); 71.9 (d, C(4')); 81.0 (d, C(1')); 126.2 (s); 129.3, 129.5, 133.3 (3d, arom. C); 134.2 (s); 144.2 (d, C(2));
‡
151.0 (s); 152.6 (d, C(8)); 153.7 (s); 166.5 (s, CO). FAB-MS (pos., NBA/NaI): 394 (50.3, [M ‡ Na ]), 372.1302
‡
(18.9, [ABz ‡ H] ), 227 (100.0), 249 (98.0).
1-(a-l-lyxopyranosyl)thymine (3b). A soln. of 38.3 g (67.1 mmol) 2b in 710 ml ca. 0.7m methanolic NH3 was
stirred for 11 days at r.t. NH3 and MeOH were evaporated, and the resulting soln. was co-evaporated twice with
200 ml of dry MeOH. The residue was subjected to CC (silica gel; CHCl3/MeOH 8 :1 to 6 :1) to afford 13.9 g
(80.2%) of 3b. M.p. 2338. TLC (CHCl3/MeOH 6 :1): Rf 0.17. 1H-NMR (600 MHz, D2O): 1.79 (s, Me); 3.78
(dd, J(4',5') ˆ 1.04, Jgem ˆ 12.5, H C(5')); 3.81 (m, H C(3')); 4.00 (dd, J(4',5') ˆ 0.95, Jgem ˆ 12.5, H C(5'));
4.02 (dd, J(2',3') ˆ 3.3, J(1',2') ˆ 9.8, H C(2')); 4.09 (Yt, H C(4')); 5.66 (d, J(1',2') ˆ 9.8, H C(1')); 7.56
(s, H C(6)). 13C-NMR (150.9 MHz, D2O): 11.79 (q, Me); 66.04 (d, C(2')); 67.80 (t, C(5')); 69.33 (d, C(3'));
70.85 (d, C(4')); 80.69 (d, C(1')); 112.34 (s, C(5)); 137.63 (d, C(6)); 152.57, 166.58 (2s, C(2), C(4)). FAB-MS
‡
‡
‡
(pos., NBA/NaI): 539 (8.1, [2M ‡ Na] ), 517 (10.8, [2M ‡ H] ), 439 (4.8), 391 (47.0, [2M ‡ H thym] ), 286
‡
‡
(72.3), 281 (77.7, [M ‡ Na] ), 259.0936 (100.0, [M ‡ H] ).
N4-Benzoyl-1-(a-l-lyxopyranosyl)cytosine (3c). A soln. of 9.0 g (13.7 mmol) of 2c in 550 ml of THF/
MeOH/H2O 5 :4 :1 (v/v/v) was cooled to 08 (NaCl/ice-bath). To this soln., 82.2 ml of 2m aq. NaOH was added
dropwise, and the mixture was stirred for additional 45 min at 08. The resulting soln. was neutralized with 1m aq.
HCl and reduced to a volume of 100 ml. This suspension was heated until a clear soln. was obtained and was

Helvetica Chimica Acta ± Vol. 84 (2001)
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allowed to stand overnight at 48. The product crystallized as colorless needles. After filtration, the mother liquor
was further concentrated and left at 48 to afford a second batch of product. The combined batches of crystals
were dried in vacuo (ca. 0.5 Torr r.t.) to afford 3.3 g (70%) of 3c. M.p. 153 ± 1558. TLC (CH2Cl2/MeOH 9 :1): Rf
1
0.12. H-NMR (200 MHz, (D6)DMSO): 3.91 ± 4.03 (br. m, 2H C(5')); 4.12 ± 4.30 (br. m, H C(2'), H C(3'),
H
C(4')); 5.99 (d, J(1',2') ˆ 9.4, H C(1')); 7.50 ± 8.25 (m, 5 arom. H, H C(5), H C(6)); 10.23 (s, NH). FAB-
‡
‡
‡
MS (pos., NBA): 695 (15, [2M ‡ H] ), 348 (95, [M ‡ H] ), 216 (100, [BzCyt] ).
N2-Isobutyryl-9-(a-l-lyxopyranosyl)guanine (3d). To a soln. of 1.6 g (2.4 mmol) of 2d in 90 ml THF/MeOH
5 :4 (v/v) cooled to 08 in an ice-bath were added 10 ml of 2m aq. NaOH soln., and the mixture was stirred for
50 min. After the reaction was complete (TLC), the cold soln. was neutralized with 1m aq. HCl. The soln. was
evaporated to dryness and co-evaporated twice with toluene. The dry residue was dissolved in MeOH, adsorbed
on 10 g of silica gel, loaded on the top of a short silica gel column, and eluted from the column (CH2Cl2/MeOH
95 :5 to 6 :1) to afford 705 mg (79%) of 3d. TLC (CH2Cl2/MeOH 4 :1): Rf 0.10. 1H-NMR (600 MHz,
(D6)DMSO): 1.12 (d, J ˆ 6.9, Me); 2.77 (m, J ˆ 6.9, Me2CH); 3.61 (m, H C(4')); 3.65 (d, Jgem ˆ 11.2, H C(5'));
3.88 ± 3.93 (m ‡ d, Jgem ˆ 11.2, H C(3'), H C(5')); 4.27 (m, H C(2')); 5.11 (d, J ˆ 6.8, HO); 5.17 (d, J ˆ 5.0,
OH); 5.30 (d, J ˆ 3.6, OH); 5.60 (d, J(1',2') ˆ 9.6, H C(1')); 8.15 (s, H C(8)); 11.73, 12.08 (s, NH). FAB-MS
‡
(pos., NBA): 354 (100, [M ‡ H] ).
N6-Benzoyl-9-{2',3'-O-[ethoxy(phenyl)methylidene]-a-l-lyxopyranosyl}adenine (4a). To a soln. of 7.63 g
(20.6 mmol) of 3a in 23 ml of dry DMF (previously dried over activated molecular sieves (3 Š)) was added a
small amount of molecular sieves (3 Š) for further drying. The soln. was kept for 1 h at r.t. and was transferred
by syringe into a dry 100-ml flask under N2, followed by the addition of a soln. of 3.92 g (20.3 mmol) of TsOH ´
H2O (melted and dehydrated in vacuo (ca. 0.5 Torr) before use) in 20 ml of MeCN and 11.5 ml (49.3 mmol) of
dry triethyl orthobenzoate (stored over molecular sieves, 3 Š). The reaction flask was attached to a rotavap
equipped with a drying tower (filled with DrieriteTM) and heated under vacuum (ca. 20 Torr) at 308 (water bath)
without distillation of DMF. After 4 h under vacuum, the reaction was complete (TLC), and the soln. was
poured into an ice-cold mixture of sat. aq. NaHCO3 soln./AcOEt 1:1 (v/v) with stirring. The aq. phase was
extracted with 3 Â 100 ml of AcOEt, and the combined org. phase was dried (Na2SO4) and concentrated to ca.
100 ml. Dropwise addition of CH2Cl2 initiated precipitation. The precipitate was filtered and dried (in vacuo,
r.t., 24 h) to afford 3.97 g of amorphous product, 4a. The mother liquor was concentrated and purified by CC
(silica gel; conditioned with MeOH (containing ca. 0.5% Et3N); CHCl3/AcOEt 4 :1, then toluene/acetone 1 :1),
which gave an additional 1.93 g of product, 4a. Combined yields: 5.9 g (56.8%) of 4a (mixture of
diastereoisomers a and b; (purified by CC). TLC (petroleum ether/acetone 1:1): Rf 0.48. 1H-NMR
(600 MHz, (D6)DMSO): 1.07, 1.16 (2t, J ˆ 7.1, MeCH2O); 3.31 ± 3.63 (3m, MeCH2O, H2O, overlapping signals);
3.95 ± 4.11 (3m, 2 H C(5'), H C(4') of both epimers); 4.29 (m, H C(3') of epimer b); 4.84 (dd, J(3',4') ˆ 3.0,
J(2',3') ˆ 5.5, H C(3') of epimer a); 5.38 (dd, J(2',3') ˆ 5.7, J(1',2') ˆ 8.3, H C(2') of epimer b); 5.48
(dd, J(2',3') ˆ 5.5, J(1',2') ˆ 7.6, H C(2') of epimer a); 5.61 (d, J(4',OH) ˆ 4.7, HO C(4') of epimer a); 5.64
(d, J(1',2') ˆ 7.6, H C(1') of epimer a); 5.68 (d, J(4',OH) ˆ 4.7, HO C(4') of epimer b); 6.20 (d, J(1',2') ˆ 8.3,
H
H
C(1') of epimer b); 7.39 ± 8.07 (m, 10 arom. H); 8.74 (s, H C(2) of epimer a); 8.81 (s, H C(2) of epimer b,
C(8) of epimer a, overlapping signals); 8.91 (s, H C(8) of epimer b); 11.28, 11.31 (2 br. s, NH of both
epimers). 13C-NMR (150.9 MHz, (D6)DMSO): 15.8 (q, MeCH2O); 59.5, 60.0 (2t, MeCH2O); 65.1 (2d, C(4') of
both epimers, overlapping signals); 69.1, 69.5 (2t, C(5') of both epimers); 72.6, 73.3 (2d, C(2') of both epimers);
78.5, 78.8 (2d, C(3') of both epimers); 81.8, 81.9 (2d, C(1') of both epimers); 121.0, 122.0 (2s, arom. C); 126.2,
126.7, 126.8, 129.1, 129.2, 129.4 (6d, arom. C); 130.0, 130.1 (2s, arom. C); 133.4 (d, arom. C); 134.1, 138.0, 139.2
(3s, arom. C); 144.0 (2d, C(8) of both epimers, overlapping signals); 151.5 (s, arom. C); 152.9, 153.2 (2d, C(2) of
‡
‡
both epimers). FAB-MS (pos., NBA/NaI): 526.1720 (100.0, [M ‡ Na] ), 504 (17.6, [M ‡ H] ), 458 (9.1, [M
‡
OEt] ).
1-{2',3'-O-[ethoxy(phenyl)methylidene]-a-l-lyxopyranosyl}thymine (4b). The reaction was performed as
described for 4a with 19.43 g (75.2 mmol) of 3b in 50 ml of dry DMF soln. of 9.10 g (47.1 mmol) TsOH ´ H2O
(melted and dehydrated in vacuo, ca. 0.5 Torr, before use) in 40 ml of MeCN and 24.5 ml (105 mmol) of dry
triethyl orthobenzoate. Following similar workup procedure, the org. phase was dried (Na2SO4) and evaporated.
The residual oil was purified by CC (silica gel; conditioned with MeOH (containing ca. 0.5% Et3N); petroleum
ether/acetone 3 :1 to 1:1) afforded 11.55 g (39.4%) of 4b as a 60 :40 mixture of diastereoisomers (purified by
CC). TLC (petroleum ether/acetone 1:1): Rf 0.67. 1H-NMR (600 MHz, (D6)DMSO): 1.07, 1.13 (2t, J ˆ 7.1,
MeCH2O of both epimers); 1.81, 1.82 (2s, Me C(5) of both epimers); 3.28 ± 3.37 (2m, MeCH2O of epimer a,
H2O, overlapping signals); 3.55 ± 3.68 (m, H C(5') of both epimers, MeCH2O of epimer b, overlapping signals);
3.81 (d, Jgem ˆ 12.3, H C(5') of both epimers); 3.91 ± 3.96 (2m, H C(4') of both epimers); 4.08 (dd, J(3',4') ˆ
2.3, J(2',3') ˆ 5.8, H C(3') of epimer b); 4.66 ± 4.67 (m, H C(3') of epimer a); 4.75 (dd, J(2',3') ˆ 6.0, J(1',2') ˆ

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Helvetica Chimica Acta ± Vol. 84 (2001)
8.5, H C(2') of epimer b); 4.87 (dd, J(2',3') ˆ 6.1, J(1',2') ˆ 8.1, H C(2') of epimer a); 5.36 (d, J(1',2') ˆ 8.1,
H
C(1') of epimer a); 5.48 (d, J(4',OH) ˆ 5.7, HO C(4') of epimer b); 5.57 (d, J ˆ 5.7, HO C(4') of epimer a);
5.91 (d, J(1',2') ˆ 8.5, H C(1') of epimer b); 7.39 ± 7.95 (m, 5 arom. H, H C(6) of both epimers); 11.42 (br.
s, NH of both epimers). 13C-NMR (150.9 MHz, (D6)DMSO): 12.8 (q, Me C(5)); 15.6, 15.8 (2q, MeCH2O of
both epimers); 59.4, 59.6 (2t, MeCH2O of both epimers); 64.8, 64.9 (2s, C(4') of both epimers); 69.4, 69.6 (2d,
C(5') of epimer b); 71.9 (d, C(2') of epimer b); 72.7 (d, C(2') of epimer a); 78.0 (d, C(3') of epimer b); 78.6
(d, C(3') of epimer a); 81.7 (d, C(1') of epimer b); 82.1 (d, C(1') of epimer a); 111.0, 111.1, 120.9, 121.8 (4s);
126.7, 127.0 (2d, arom. C); 129.0, 129.1 (2d, arom. C); 129.9, 130.1 (2s, arom. C); 137.4, 137.5, 137.6 (arom. C);
‡
139.5 (s, arom. C); 151.6, 164.3 (2s, CO). FAB-MS (pos., NBA/NaI): 435 (7.1, [M ‡ 2 Na] ), 413.1335 (100.0,
‡
‡
‡
[M ‡ Na] ), 345 (45.3, [M ‡ H OEt] ), 265 (20.6, [M ‡ H thym] ), 241 (9.5), 223 (23.3), 219 (29.7, [M ‡
‡
H
thym OEt] ).
1-(3'-O-Benzoyl-a-l-lyxopyranosyl)thymine (4bb). To a soln. of 300 mg (1.2 mmol) of 3b in 1 ml of DMF
was added a soln. of 5% TsOH and 3% CF3COOH acid in 1 ml of MeCN, followed by a soln. of 200 ml
(0.9 mmol) of triethyl orthobenzoate in 3 ml of MeCN. The MeCN was removed by evaporation, and the
mixture was kept for 2 h under vacuum at 308, without distillation of the DMF (TLC: CHCl3/MeOH 6 :1).
Addition of triethyl orthobenzoate (300 ml) and evaporation was repeated twice. Aq. CF3COOH (90%, 500 ml)
was added, and the reaction left overnight upon which the product crystallized. It was filtered and washed with
MeCN to afford 296 mg (70%) of 4bb. 1H-NMR (300 MHz, (D6)DMSO): 1.82 (s, Me), 3.76 (m, H C(4')); 3.79
(br. d, 1 H, H C(5')); 3.96 (br. d, Jgem ˆ 12.3, 1 H, H C(5')); 4.23 (m, H C(2')); 5.40 (t, J(2',3') ꢀ J(3',4') ꢀ 3.0,
H
C(3')); 5.57 (d, J ˆ 5.8, OH); 5.65 (d, J ˆ 5.6, OH); 5.78 (d, J(1',2') ˆ 9.7, H C(1')), 8.10 ± 7.50 (m, 5 ar-
om. H, H C(6)). On addition of D2O to the above NMR sample, the signal assigned to H C(2') reduced to a
dd (J(1',2') ˆ 9.7, J(2',3') ˆ 3.3 Hz), while the signal assigned to H C(3') remained unchanged. The assignment
of H C(2') was based on the coupling to H C(1') (J(1',2') ˆ 9.7 Hz).
N4-Benzoyl-1-{2',3'-O-[methoxy(methyl)methylidene]-a-l-lyxopyranosyl}cytosine (4c). To a soln. of 1.84 g
(5.3 mmol) of 3c and 6.7 ml (53 mmol) of trimethylorthoacetic acid in 15 ml of dry DMF, stirred under Ar at r.t.,
were added 1.7 ml (21.2 mmol) of TMS-Cl by a syringe. The soln. was stirred for 6 h, quenched with pyridine,
and concentrated in vacuo. The residue was purified by CC (silica gel; acetone/hexane 1 :3 to 3 :2) to afford
1.58 g (74%) of 4c as a 4 :1 mixture of diastereoisomers. TLC (CH2Cl2/MeOH 12 :1): Rf 0.48. 1H-NMR
(200 MHz, (D6)DMSO): 1.43, 1.56 (s, Me of both epimers); 3.18 ± 3.30 (s, MeO of both epimers); 3.91 ± 4.03
(br. m, H C(5'), H C(4') of both epimers); 4.34, 4.42 (m, H C(3') of both epimers); 4.51, 4.65 (dd, J(1',2') ˆ
8.1, J(2',3') ˆ 5.7, H C(2') of both epimers); 5.48 (d, J(4', OH) ˆ 5.4, OH); 5.95, 5.64 (d, J(1',2') ˆ 8.1, 8.4,
H
C(1') of both epimers); 7.38 ± 8.22 (m, 5 arom. H, H C(5), H C(6)); 11.3 (s, NH). FAB-MS (pos., NBA):
‡
‡
‡
‡
‡
426 (19, [M ‡ Na] ), 404 (38, [M ‡ H] ), 372 (93, [M ‡ H MeO] ), 216 (77, [BzCyt] ), 105 (100, [PHCO] ).
A crystallized sample (M.p. 207 ± 2098; from acetone/hexanes) was subjected to elemental analysis. Anal. calc.
for C19H21N3O7: C 56.67, H 5.25, N 10.42, found: C 56.63, H 5.31, N 10.40.
N2-Isobutyryl-9-{2',3'-O-[methoxy(methyl)methylidene]-a-l-lyxopyranosyl}guanine (4d, impure). To a
soln. of 900 mg (2.5 mmol) of 3d and 3.14 ml (25 mmol) of trimethylorthoacetic acid in 10 ml of dry DMF
under Ar were added 760 ml (6 mmol) of TMS-Cl. The soln. was allowed to stir for 16 h and evaporated to afford
a thick syrup. The syrup was purified by CC (silica gel; acetone/hexane 2 :3 to 2 :1) to yield 681 mg (66%) of 4d
as a mixture. 1H-NMR Data tentatively compatible with 4d as major component. TLC (acetone/hexane 2 :1): Rf
0.36. 1H-NMR (600 MHz, (D6)DMSO): 1.12 (d, J ˆ 6.9, Me2CH); 2.08, 2.11 (2s, Me of both epimers); 2.79
(m, J ˆ 6.9, Me2CH); 3.34 ± 3.37 (s, MeO of both epimers); 3.62 ± 4.11 (br. m, H C(5'), H C(4') of both
epimers); 4.52, 4.55 (m, H C(3') of both epimers); 5.14 ± 5.18 (m, H C(2') of both epimers); 5.41 (d, J(4',
OH) ˆ 5.0, OH); 5.51, 5.61 (d, J(1',2') ˆ 9.8 and/or 7.2 (?), H C(1') of both epimers); 8.12, 8.35 (s, H C(8));
‡
‡
‡
11.7, 12.1 (2s, NH). FAB-MS (pos., NBA): 448 (27, [M ‡ K] ), 432 (100, [M ‡ Na] ), 410 (17, [M ‡ H] ).
N6-Benzoyl-9-{3'-O-benzoyl-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-a-l-lyxopyranosyl}adenine (5a). A
suspension of 7.16 g (14.2 mmol) of 4a in 250 ml CH2Cl2 and 10 ml (85.0 mmol) 2,6-lutidine was cooled to 08,
and 14.3 g (40.1 mmol) of 95% DMT-Cl were added in small portions. Stirring overnight at 48 resulted in
complete conversion of 4a (TLC). To this mixture, 135 ml of 80% aq. AcOH in 270 ml of THF was added, and
the mixture was stirred for 4 d at r.t. The mixture was washed with sat. aq. NaHCO3 soln., and the aq. phase was
extracted with 3 Â 200 ml CH2Cl2. Evaporation of the combined org. layers and purification of the residue by CC
silica gel (conditioned with MeOH (containing ca. 0.5% Et3N), acetone, petroleum ether/acetone 2 :1;
petroleum ether/acetone 2 :1 to 1:1) afforded, after drying (Na2SO4), 6.08 g (54.9%) of 5a. TLC (petroleum
ether/acetone 2 :1): Rf 0.25. 1H-NMR (600 MHz, CDCl3): 3.10 (d, Jgem ˆ 12.5, H C(5')); 3.70 (d, Jgem ˆ 12.5,
H
C(5')); 3.82 (s, 2MeO); 3.94 (d, J(3',4') ˆ 3.6, H C(4')); 4.13 ± 4.17 (br. m, OH); 4.30 (dt, J(2',3') ˆ 2.7,
J(1',2') ˆ 9.7, H C(2')); 5.35 (Ys, H C(3')); 6.00 (d, J(1',2') ˆ 9.7, H C(1')); 6.86 ± 7.55 (m, 23 arom. H); 7.99

Helvetica Chimica Acta ± Vol. 84 (2001)
1799
(2s, H C(2), H C(8)); 9.66 (s, NH). 13C-NMR (150.9 MHz, CDCl3): 55.7 (q, MeO); 67.1 (t, C(5'));
68.0 (d, C(2')); 69.5 (d, C(4')); 73.4 (d, C(3')); 81.2 (d, C(1')); 88.2 (s, (MeOC6H4)2CPh); 112.4 (s, arom. C);
113.8, 113.9, 127.7 (3d, arom. C); 128.5, 128.7 (2d, arom. C); 129.7 (d, arom. C); 130.3 (d, arom. C); 130.5,
130.6 (2d, C(2), C(8)); 133.8, 135.6 (3d, arom. C); 136.1, 136.3, 145.4, 151.9, 159.3, 163.3 (6s, arom. C); 163.9,
‡
‡
165.5 (2s, CO). FAB-MS (pos., NBA/CsI): 1042 (19.9, [M ‡ 2 Cs] ), 910.1886 (100.0, [M ‡ Cs] ), 778 (5.4,
‡
[M ‡ H] ).
1-{3'-O-Benzoyl-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-a-l-lyxopyranosyl}thymine (5b). The reaction
was performed as described for 5a with 14.76 g (37.8 mmol) of 4b in 470 ml of dry CH2Cl2, 31 ml (26.3 mmol) of
2,6-lutidine, 43.2 g (121.1 mmol) of 95% DMT-Cl. Then, 240 ml 80% aq. AcOH in 470 ml of THF was added,
and the mixture was stirred for 5 d at 48 and 2 d at r.t. The soln. was diluted with 30 ml of CH2Cl2 and poured into
an ice-cold mixture of sat. aq. NaHCO3 soln./AcOEt 1:1 (v/v). Extraction with 500 ml of AcOEt, drying of the
org. phase (Na2SO4), and evaporation of the org. solvent afforded a colorless oil, which was subjected to CC
(silica gel; conditioned with MeOH (containing ca. 0.5% Et3N); petroleum ether/acetone 2 :1 to 1:1) and
gave 9.55 g (38.1%) of 5b. TLC (petroleum ether/acetone 3 :2): Rf 0.56. 1H-NMR (600 MHz, (D6)DMSO):
1.89 (s, Me C(5)); 2.90 (d, Jgem ˆ 12.4, H C(5')); 3.65 (d, Jgem ˆ 12.4, H C(5')); 3.75 (s, 2MeO); 3.77
(d, J(3',4') ˆ 3.4, H C(4')); 4.46 (m, H C(2')); 5.32 (m, H C(3')); 5.62 (d, J(2', OH) ˆ 6.0, OH); 5.69
(d, J(1',2') ˆ 9.6, H C(1')); 6.91 ± 7.96 (m, 18 arom. H, H C(6)); 11.40 (s, NH). 13C-NMR (150.9 MHz,
(D6)DMSO): 13.2 (q, Me C(5)); 55.9 (q, MeO); 65.2 (d, C(2')); 67.0 (t, C(5')); 70.0 (d, C(4')); 73.4
(d, C(3')); 81.4 (d, C(1')); 87.9 (s, (MeOC6H4)2CPh); 110.6 (s, C(5)); 114.2, 114.3, 127.9, 128.6, 128.9, 129.5,
130.3, 130.3, 130.8, 134.3 (10d, arom. C); 136.4, 136.5 (2s, arom. C); 137.1 (d, arom. C); 146.0, 151.8
‡
(2s, arom. C); 159.2 (s, arom. C); 164.5, 165.1 (3s, CO). FAB-MS (pos., NBA/CsI): 929 (12.2, [M ‡ 2Cs] ),
‡
797.1497 (100.0, [M ‡ Cs] ).
N4-Benzoyl-1-{3'-O-acetyl-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-a-l-lyxopyranosyl}cytosine (5c). To a
soln. of 310 mg (0.75 mmol) of 4c in 5 ml of dry CH2Cl2 cooled to 08 were added a soln. 560 ml of 2,6-lutidine
and 813 mg of DMT-Cl, and the mixture was stirred for 96 h at 48. The soln. was evaporated, and the residue was
purified by CC (silica gel; acetone/hexane 3 :1 to 2 :1 with 2% Et3N) to afford 292 mg (55%) of the tritylated
intermediate. The tritylated intermediate was dissolved in 60 ml of THF, 30 ml of 80% aq. AcOH was added,
and the mixture was stirred at r.t. until the reaction was complete (ca. 19 h, TLC). The mixture was diluted with
CH2Cl2, neutralized with sat. aq. NaHCO3 soln., and the aq. phase was extracted twice with CH2Cl2. The
combined org. phases were dried (MgSO4), filtered, and evaporated. The residue was purified by CC (silica gel;
acetone/hexane 1:3 to 3 :2 with 1% pyridine) to give 177 mg (62%) of 5c. TLC (acetone/hexane 1:1): Rf 0.2.
1H-NMR (600 MHz, CDCl3): 2.09 (s, MeOH); 3.05 (dd, Jgem ˆ 12.6, J(4',5') ˆ 4.7, H C(5')); 3.59 (d, Jgem ˆ 12.6,
H
H
C(5')); 3.82 (s, 2 MeO); 3.83 (m, H C(4')); 4.26 (m, H C(2')); 5.13 (br. s, H C(3')); 5.97 (d, J(1',2') ˆ 9.4,
C(1')); 6.86 ± 8.1 (m, 18 arom. H, H C(5), H C(6)); 9.1 (s, NH). FAB-MS (pos., NBA): 692 (29, [M ‡
‡
‡
H] ), 303 (100, [DMTr] ).
N2-Isobutyryl-9-{3'-O-acetyl-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-a-l-lyxopyranosyl}guanine (5d).
The reaction was performed as described for 5c with 1.28 g (3.1 mmol) of 4d, 15 ml of dry CH2Cl2, 3.65 ml
(31.3 mmol) dry 2,6-lutidine, and 3.19 g (9.4 mmol) of DMT-Cl (5 d at 48). To this mixture were added 60 ml of
THF, 30 ml of 80% aq. AcOH, and the mixture was stirred until the reaction was complete (ca. 13 h, TLC).
Following the workup as described for 5c and purification by CC (silica gel, acetone/hexane 1:2 to 2 :1 with 1%
pyridine) afforded 1.49 g (68%) of 5d. TLC (acetone/hexane 2 :1): Rf 0.24. 1H-NMR (200 MHz, CDCl3): 1.05,
1.15 (d, J ˆ 6.9, Me2CH); 2.09 (2s, MeO); 2.38 (m, Me2CH); 3.20 (d, Jgem ˆ 11.6, H C(5')); 3.61 (d, Jgem ˆ 11.6,
H
C(5')); 3.77 (br. m, H C(4')); 3.81 (s, 2MeO); 4.6 ± 5.25 (m, H C(3'), H C(2'), OH); 5.46 (d, J(1',2') ˆ
9.0, H C(1')); 6.85 ± 7.66 (m, 13 arom. H); 7.76 (2s, H C(8)); 8.62, 11.89 (br. s, NH). FAB-MS (pos.,
‡
‡
‡
‡
NBA): 2094 (5, [3M ‡ H] ), 1418 (22, [2M ‡ Na] ), 720 (24, [M ‡ Na] ), 698 (45, [M ‡ H] ), 303 (100,
‡
[DMTr] ).
2. Experiments Referring to Scheme 4. ± N6-Benzoyl-9-{3'-O-benzoyl-2'-O-[(2-cyanoethoxy)(diisopropy-
lamino)phosphino]-4'-O-[(4'',4'''-dimethoxytriphenyl)methyl]-a-l-lyxopyranosyl}adenine (6a). To a soln. of
440 mg (0.57 mmol) of 5a in 5 ml of dry CH2Cl2, 0.56 ml (4.2 mmol) of 2,4,6-collidine, and 23 ml (0.29 mmol) N-
methylimidazole, 316 ml (1.2 mmol) of 97% chloro(2-cyanoethoxy)(diisopropylamino)phosphine were added
by syringe. The mixture was stirred for 10 min. at r.t., diluted with 5 ml of AcOEt, and washed with sat. aq.
NaHCO3 soln. The org. phase was dried (MgSO4), concentrated, and subjected to CC (silica gel; petroleum
ether/AcOEt 1 :1) to furnish 0.473 g (84.2%, mixture of diastereoisomers ca. 2.7:1 by 1H-NMR) of 6a as a
syrupy liquor. TLC (petroleum ether/AcOEt 1 :1): Rf 0.33. 1H-NMR (600 MHz, CDCl3): 0.54, 0.91 (2d, J ˆ 6.8,
Me2CH of epimer b); 0.96 (2d, J ˆ 5.6, Me2CH of epimer a), overlapping signals); 2.34 (m, CH2CN of epimer a);

1800
Helvetica Chimica Acta ± Vol. 84 (2001)
2.49 (m, CH2CN of epimer b); 3.13 (d, Jgem ˆ 12.7, 1H C(5') of both epimers); 3.21 (2m, 1H; CH2CH2CN of
epimer a), Me2CH of both epimers); 3.30, 3.53, 3.68 (3m, 2H; CH2CH2CN of both epimers); 3.80 ± 3.84
(m, 1H C(5') of both epimers, MeO, overlapping signals); 3.98 (m, H C(4') of both epimers); 5.10, 5.23 (2m,
H
C(2') of both epimers); 5.62 (m, H C(3') of both epimers); 6.05, 6.09 (2d, J(1',2') ˆ 9.3, 9.2, H C(1') of
both epimers); 6.88 ± 8.09 (m, 23 arom. H); 8.38, 8.43, 8.91, 8.94 (4s, H C(2), H C(8)); 9.15 (br. s, NH of both
epimers). 13C-NMR (150.9 MHz, CDCl3): 20.30, 20.34 (2t, CH2CN of both epimers); 24.3, 24.8 (2q, Me2CH of
both epimers); 43.3, 43.4 (2d, Me2CH of both epimers); 55.7 (q, MeO of both epimers); 58.2, 58.3
(2t, CH2CH2CN); 67.2 (t, C(5') of both epimers); 69.4, 69.6 (2d, C(2'), C(4') of both epimers); 72.2, 72.4
(d, C(3') of both epimers); 81.1 (d, C(1') of both epimers); 88.3 (s, (MeOC6H4)2CPh of both epimers); 113.9
(d, arom. C); 117.9 (s, CN), 123.2 (s, arom. C); 127.7, 128.2, 128.3, 128.5, 128.6, 129.0, 129.3, 130.1, 130.6, 130.7
(arom. C); 133.2, 133.8 (2d, arom. C); 134.1, 134.2 (arom. C); 136.1, 136.4 (2s, arom. C); 142.3 (d, C(2) or C(8));
145.3, 149.9 (2s), 152.5, 152.9 (d, C(2) or C(8)); 159.3, 159.3, 164.9, 165.0 (4s, CO of both epimers). 31P-NMR
(242.9 MHz, CDCl3): 150.54, 151.79 (2s, P of both epimers). FAB-MS (pos., NBA/CsI): 1242 (18.2, [M ‡ 2
‡
‡
Cs] ), 1110.2974 (100.0, [M ‡ Cs] ).
1-{3'-O-Benzoyl-2'-O-[(2-cyanoethoxy)(diisopropylamino)phosphino]-4'-O-[(4'',4'''-dimethoxytriphenyl)-
methyl]-a-l-lyxopyranosyl}thymine (6b). To a soln. of 500 mg (0.75 mmol) of 5b in 5 ml of dry CH2Cl2, 0.75 ml
(5.7 mmol) of 2,4,6-collidine, and 30 ml (0.4 mmol) of N-methylimidazole, 420 ml (1.6 mmol) of 97% chloro(2-
cyanoethoxy)(diisopropylamino)phosphine was added by syringe. The soln. was stirred for 10 min. at r.t.,
diluted with 5 ml of AcOEt, and washed with sat. aq. NaHCO3 soln. The org. phase was dried (MgSO4),
concentrated, and subjected to CC (silica gel; petroleum ether/AcOEt 2 :1 to 3 :2) to afford 0.523 g (80.6%,
mixture of diastereoisomers ca. 1:1 by 1H-NMR) of 6b as a syrup. TLC (petroleum ether/acetone 3 :2): Rf 0.56.
1H-NMR (600 MHz, CDCl3): 0.93, 1.04, 1.11, 1.12 (4d, J ˆ 6.8, Me2CH of both epimers); 2.05, 2.06 (2s, Me C(5)
of both epimers); 2.38, 2.51 (2m, CH2CN of both epimers); 2.88, 3.06 (2d, Jgem ˆ 12.6, H C(5') of both epimers);
3.36 ± 3.79 (m, CH2CH2CN, Me2CH, 1H C(5') of both epimers, Me2CH); 3.82 (s, 2MeO); 3.92 (m, H C(4') of
both epimers); 4.48, 4.56 (2m, H C(2') of both epimers); 5.57, 5.65 (2m, H C(3') of both epimers); 6.06
(m, H C(1') of both epimers); 6.86 ± 8.03 (m, 18 arom. H, H C(6)); 8.33 (br. s, NH of both epimers).
13C-NMR (150.9 MHz, CDCl3): 13.1, 13.2 (2q, MeCH of both epimers); 20.4, 20.6 (2t, CH2CN of both epimers);
24.6, 24.75, 24.80, 24.9 (4q, Me2CH of both epimers); 43.4, 43.5 (2t, CH2CH2CN of both epimers); 55.7
(q, MeO); 58.0, 58.9 (2d, Me2CH of both epimers); 66.8, 67.3 (2d, C(5') of both epimers); 69.4, 69.6 (2d, C(4') of
both epimers); 72.3, 72.6 (2d, C(3') of both epimers); 88.3 (s, (MeOC6H4)2CPh of both epimers); 111.7 (s, C(5)
of both epimers); 113.82, 113.86, 113.90 (3d, arom. C); 127.7 (d, arom. C); 128.41, 128.47, 128.53 (3d, arom. C);
128.9, 129.0 (2d, arom. C); 129.80, 129.9 (2s, arom. C); 130.08, 130.11 (2d, arom. C); 130.5, 130.6, 130.7
(arom. C, C(6)); 145.4, 145.5 (2s, arom. C); 150.8, 151.0 (2s, C(2) of both epimers); 159.30, 159.33 (2s, CO of
both epimers); 163.7, 163.8 (2s, C(4) of both epimers); 165.0, 165.1 (2s, CO of both epimers). 31P-NMR
‡
(242.9 MHz, CDCl3): 150.18, 151.16 (2s, P of both epimers). FAB-MS (pos., NBA/CsI): 1129 (12.8, [M ‡ 2Cs] ),
‡
997.2587 (100.0, M ‡ Cs] ).
N4-Benzoyl-1-{3'-O-acetyl-2'-O-[(2-cyanoethoxy)(diisopropylamino)phosphino]-4'-O-[(4'',4'''-dimethoxy-
triphenyl)methyl]-a-l-lyxopyranosyl}cytosine (6c). To a soln. of 1.4 g (1.9 mmol) of 5c and 856 ml (5 mol) of
EtN(i-Pr)2 in 5 ml of dry CH2Cl2, stirred under Ar at r.t., were added 643 ml (2.85 mol) of chloro(2-
cyanoethoxy)(diisopropylamino)phosphine dropwise by a syringe. The mixture was stirred overnight at r.t.,
concentrated in vacuo, and purified by CC (silica gel; acetone/hexane: 1 :2 to 2 :1) to afford 1.31 g (72%) of 6c
as a mixture of diastereoisomers. TLC (AcOEt/hexane 2 :1): Rf 0.28, 0.31 (both epimers). 1H-NMR (600 MHz,
CDCl3): 1.03, 1.09, 1.13, 1.19 (4d, J ˆ 6.8, Me2CH of both epimers); 2.05 ± 2.07 (2s, MeO); 2.53 ± 2.78 (2m,
CH2CN of both epimers); 2.83, 3.02 (2d, Jgem ˆ 12.7, H C(5') of both epimers); 3.42 ± 3.72 (m, CH2CH2CN,
Me2CH, H C(5') of both epimers); 3.73, 3.88 (2m, H C(4') of both epimers); 3.82 (s, 2MeO C6H4); 4.37, 4.47
(2m, H C(2') of both epimers); 5.36, 5.42 (2m, H C(3') of both epimers); 6.16 (br. s, H C(1') of both epimers);
6.88 ± 7.95 (m, 18 arom. H, H C(5), H C(6)); 8.76 (br. s, NH of both epimers). 31P-NMR (242.9 MHz, CDCl3):
‡
‡
151.29, 151.59 (2s, P of both epimers). FAB-MS (pos., NBA): 930 (10, [M ‡ K] ); 914.3548 (100, [M ‡ Na] );
‡
892 (20, [M ‡ H] ).
N2-Isobutyryl-9-{3'-O-acetyl-2'-O-[(2-cyanoethoxy)(diisopropylamino)phosphino]-4'-O-[(4'',4'''-dimethox-
ytriphenyl)methyl]-a-l-lyxopyranosyl}guanine (6d). The reaction was performed as described for 6c with 1.06 g
(1.53 mmol) of 5d, 1.6 ml (4.6 mmol) of EtN(i-Pr)2, 5 ml of dry CH2Cl2, 1.04 ml (4.6 mmol) of chloro(2-
cyanoethoxy)(diisopropylamino)phosphine (overnight, r.t). The mixture was concentrated in vacuo and
purified by CC (silica gel; acetone/hexane: 1:2 to 2 :1) to afford 985 mg g (70%) of 6d as a mixture of
diastereoisomers. TLC (acetone/hexane 2 :1): Rf 0.45, 0.48 (both epimers). 1H-NMR (600 MHz, CDCl3): 0.83,
1.04, 1.08, 1.11 (2d, J ˆ 7.3, Me2CH of both epimers, overlapping signals); 1.17, 1.19 (d, J ˆ 6.8, Me2CH); 2.09 (2s,

Helvetica Chimica Acta ± Vol. 84 (2001)
1801
MeO of both epimers); 2.4 ± 2.6 (m, Me2CH, CH2CN of both epimers); 3.11 (m, 1H C(5') of both epimers);
3.41 (m, 2H, CH2CH2CN of epimer a, Me2CH of both epimers); 3.5 ± 3.6 (m, 2H, CH2CH2CN of epimer b,
1H C(5') of both epimers); 3.72 ± 3.82 (m, H C(4') of both epimers, 2MeOC6H4); 5.03 (br. m, H C(2') of
both epimers); 5.39, 5.45 (m, H C(3') of both epimers); 5.46, 5.57 (2d, J(1',2') ˆ 8.8, H C(1') of both epimers);
6.82 ± 7.59 (m, 13 arom. H); 7.79, 7.92 (2s, H C(8) of both epimers); 12.03 (br. s, NH of both epimers).
13C-NMR (150.9 MHz, CDCl3): 18.9, 18.7 (2q, Me2CH of both epimers); 20.9, 20.3 (2t, CH2CN of both epimers);
24.1, 24.5 (2q, Me2CH of both epimers); 36.2, 36.3 (2d, Me2CH of both epimers); 42.9, 43.0 (2d, Me2CH of both
epimers); 55.2 (q, MeO of both epimers); 57.8, 58.0 (2t, CH2CH2CN); 66.3 (t, C(5') of both epimers); 68.2, 67.6
(2d, C(2') of both epimers); 68.9, 69.2 (C(4') of both epimers); 71.5, 71.7 (d, C(3') of both epimers); 81.1, 82.1
(d, C(1') of both epimers); 87.7 (s, MeOC6H4); 113.3, 117.7, 121.3, 127.1, 128.0, 130.2, 136.0, 137.9, 138.7, 145.2,
147.1, 147.3, 148.4, 155.5, 157.5, 158.7 (CN, arom. C, C(2), C(4), C(6), C(8)); 168.8, 178.3, 178.6 (3s, CO of both
epimers). 31P-NMR (242.9 MHz, CDCl3): 151.5 (br. s, of both epimers). FAB-MS (pos., NBA): 920 (100, [M ‡
‡
‡
Na] ), 898 (24, [M ‡ H] ).
Preparation of Nucleoside-Derivatized Controlled Pore Glass (CPG) 7a and 7b. A soln. of 0.18 ± 0.19 mmol
of 5a (5b), 0.36 ± 0.39 mmol of succinic anhydride, and 0.24 ± 0.25 mmol of DMAP in 2.4 ± 3.0 ml of dry CH2Cl2
was stirred for 1 h at r.t. After dilution with CH2Cl2, the soln. was successively washed with 10% aq. citric acid,
sat. aq. NaHCO3 soln., and H2O. The org. phase was dried (Na2SO4) and evaporated. The residue was purified
by CC (silica gel; petroleum ether/acetone 2 :1 to CH2Cl2/MeOH 6 :1). The product fractions were collected and
dried (Na2SO4), and concentrated in vacuo (ca. 0.2 Torr, 40 min., r.t.). The residue was dissolved in 7 ml of dry
MeCN, followed by successive addition of 45 ml of N-methylmorpholine, 75 mg (0.229 mmol) of TOTU [33],
and 1.8 g of LCAA-CPG. The suspension was shaken gently for 3 h at r.t. Filtration, followed by washing with
DMF, MeOH, acetone, and Et2O, afforded 7a (7b) after drying in vacuo (ca. 0.2 Torr). A suspension of the
nucleoside-derivatized solid support 7a (7b) in 27.5 ml of dry pyridine and 2.8 ml of N-methylimidazole was
treated with 2.8 ml of Ac2D for 30 min. Filtration, followed by washing with DMF, MeOH, acetone, and Et2O,
afforded capped 7a (7b) after drying in vacuo (ca. 0.2 Torr) for 3 h. The loading capacity (at 498 nm) was
determined to be 43.6 mmol/g for 7a and 21.6 mmol/g for 7b [21].
Preparation of Nucleoside-Derivatized Controlled Pore Glass (CPG) 7c. To a soln. of 120 mg (174 mmol) of
5c in 2 ml of dry pyridine under Ar were added 490 mg (1.22 mmol) of bis(4-nitrophenyl)heptanedioate, 22 mg
(180 mmol) of DMAP and stirred for 48 h at r.t. The mixture was concentrated and co-distilled twice with
toluene. The syrup was purified by CC (silica gel; acetone/hexane 1:2 to 2 :3). The product fractions were
collected and concentrated in vacuo (ca. 0.2 Torr, 40 min., r.t.). To this residue were added 500 mg of LCAA-
CPG (LCAA-CPG was previously washed with 50 ml of CH2Cl2, CH2Cl2/ClCH2COOH 1 :1, CH2Cl2, CH2Cl2/
Et3N 1:1, CH2Cl2, and dried for 2 h in vacuo), dried for 1 h in vacuo (ca. 0.2 Torr), followed by addition of 7.5 ml
of dry DMF, 110 ml (0.6 mmol) of EtN(i-Pr)2 16 ml (180 mmol) of dry pyridine and shaken for 3 h. The solid
material was washed with 20 ml of DMF, 30 ml of MeOH, 30 ml of acetone, 30 ml of Et2O, and was dried in
vacuo. The dry material was suspended in 25 ml of dry pyridine and 2.5 ml of Ac2O, 22 mg of DMAP and shaken
for 1 h. The solid material was placed on a fritted-glass funnel and washed with 20 ml of DMF, 30 ml of MeOH,
30 ml of acetone, and 30 ml of Et2O, and dried in vacuo (ca. 0.2 Torr) overnight. The loading capacity (at
498 nm) was determined to be 30 mmol/g [21].
Preparation of Nucleoside-Derivatized Controlled Pore Glass (CPG) 7d. The reaction was performed as
described for 7a (7b) with 151 mg (0.216 mmol) of 5d, 44 mg (0.43 mmol) of succinic anhydride, 38 mg
(0.3 mmol) of DMAP, 3 ml of dry CH2Cl2. The residue after workup was treated with 7.5 ml of dry MeCN, 66 ml
(0.6 mmol) of 4-methylmorpholine, 97 mg (0.3 mmol) of TOTU, 2 g of LCAA-CPG, and worked up as
described for 7a (7b). The capping was performed as described for 7a (7b) with 25 ml of dry pyridine and 2.5 ml
of Ac2O. After workup, drying in vacuo (ca. 0.2 Torr) overnight afforded capped 7d. The loading capacity was
determined to be 23.5 mmol/g [21].
3. Automated Solid-Phase Synthesis on a Perceptive 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.
3.1. Pre-automation Procedures. 3.1.1. Phosphoroamidites. The amount of phosphoramidite soln. was
determined as follows: for adenine, thymine, or the four-base containing sequences: (n ‡ 1)  22 mg of
phosphoramidite dissolved in (n ‡ 1)  312 ml of dry MeCN. For guanine- or cytosine-containing sequences:
(n ‡ 2)  18 mg of phosphoramidite dissolved in (n ‡ 2)  230 ± 260 ml of dry MeCN. The phosphoramidite
soln. (ca. 0.08m) was dried over 3-Š or 4-Š molecular sieves (8 ± 12 mesh, freshly activated by heating at ca. 3008

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under high vacuum overnight) overnight at r.t. prior to use. The excess of phosphoramidites, depending on the
sequence synthesized, ranged from 160 ± 323 equiv.
3.1.2. Activator Soln. 5-(Ethylthio)-1H-tetrazole in dry MeCN (0.25m for adenine- or thymine-containing
sequences, and 0.35m for guanine- or cytosine-containing sequences) was dried over freshly activated 3-Š or 4-Š
molecular sieves.
3.1.3. Capping A. A soln. of 3.0 g of DMAP in 50 ml of dry MeCN and filtered to remove any undissolved
solid particles.
3.1.4. Capping B. A soln. of 10 ml of Ac2O and 15 ml of 2,4,6-collidine in 25 ml of dry MeCN.
3.1.5. Oxidizing Soln. A mixture of 220 mg of I2, 46 ml of 2,4,6-collidine in 23 ml of H2O, and 50 ml of
MeCN, and filtered to remove any undissolved residue.
3.1.6. Detritylation Reagent. A soln. of 6% Cl2CHCOOH 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. 15 ± 16.7 min and 2) the detritylation was accomplished by 6%
Cl2CHCOOH in ClCH2CH2Cl over a 3-min period. All oligonucleotides were synthesized in the ꢀTrityl-onꢁ
mode.
3.2. Post-automation Procedures. 3.2.1. Removal of b-Cyanoethyl Protecting Group. After the automated
synthesis was completed, the CPG-solid support containing the oligonucleotide (ꢀTrityl-onꢁ) was dried in vacuo
for 30 min, transferred to a pear-shaped 10-ml flask and treated with 2.4 ml of pyridine/Et3N 5 :1 for 6.5 h at r.t.
Evaporation of pyridine and Et3N in vacuo followed by co-evaporation with toluene ± avoiding temps. over 358 ±
resulted in dry CPG-solid support.
3.2.2. Removal of Sugar- and Nucleobase-Protecting Groups. One of the following three procedures was
used depending on the sequence of the oligonucleotides (Table 2 lists the specific deprotection method for the
specific sequence).
Method A. To the flask containing the dry CPG-solid support was added 2 ml of 25% aq. NH2NH2 ´ H2O
(prepared from 1 ml of NH2NH2 ´ H2O and 4 ml of H2O) and shaken at 48 (6 ± 7 h for adenine- or thymine-
containing sequences, and ca. 20 h for guanine- or cytosine-containing sequences). After deprotection, the
suspension was diluted with ca. 5 ± 10 ml of 0.5m aq. Et3NH2CO3 buffer, loaded over a Waters Sepak-C18
cartridge (cf. Sect. 5) and eluted with 10 ± 15 ml MeCN/H2O 1 :1 to afford the hydrazine-free, crude
oligonucleotides (ꢀTrityl-onꢁ) in soln.9)10).
Method B. To the flask containing the dry CPG-solid support was added 2 ml of a mixture of 40% aq.
MeNH2 in conc. aq. NH3 1:1 and shaken at r.t. for 6.5 h (ca. 70 h for G,C-containing sequences). The suspension
was co-evaporated carefully with H2O (with the temp. always less than 358) to remove the volatiles MeNH2 and
NH3, and filtered. The filtrate was diluted with ca. 5 ± 10 ml of 0.5m aq. Et3NH2CO3 buffer, loaded over a Waters
Sepak-C18 cartridge (cf. Sect. 5) and eluted with 10 ± 15 ml MeCN/H2O 1:1 to afford the salt free, crude
oligonucleotides (ꢀTrityl-onꢁ) in soln.
Method C. To the flask containing the dry CPG-solid support was added 2 ml of a soln. of 0.2m MeONH2 ´
HCl in 25% aq. NH3 and EtOH 3 :1, and shaken at r.t. for ca. 6.5 h (ca. 66 h for G,C-containing sequences).
After deprotection, the suspension was diluted with ca. 5 ± 10 ml 0.5m aq. Et3NH2CO3 buffer and desalted (cf.
Sect. 5) over a Waters Sepak-C18 cartridge (eluted with 10 ± 15 ml of MeCN/H2O 1:1) to afford the salt free,
crude oligonucleotides (ꢀTrityl-onꢁ) in soln.
All of the above deprotections were monitored by anion-exchange HPLC (cf. Sect. 4) for optimum
deprotection time.
3.2.3. Detritylation of ꢁTrityl-onꢁ Oligonucleotides. The crude oligonucleotide soln. obtained by desalting
was concentrated in vacuo, the residue was treated with ca. 10 ml of 80% aq. HCOOH (a red color appears
within seconds indicating detritylation) at r.t. for 15 ± 30 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.
4. HPLC Purification of Oligonucleotides. ± The crude oligonucleotides were purified by anion-exchange
(IA)-HPLC system, over a Mono Q HR 5/5 (Pharmacia) column, performed on A) Pharmacia GP-250
Gradient Programmer equipped with two Pharmacia P-500 pumps, ABI-Kratos Spectraflow 757 UV/VIS
detector and a Hewlett Packard HP 3396A analog integrator or B) Pharmacia ¾kta purifier (900) controlled by
UNICORN. The oligonucleotides were eluted from the column with a linear gradient of 1m NaCl in 10 mm of aq.
Na2HPO4, pH 10.5 over a period of 20 to 30 min with the following buffer systems: buffer A: 10 mm Na2HPO4 in
H2O, pH 10.5; buffer B: 10 mm Na2HPO4 in H2O, 1m NaCl, pH 10.5. Exact conditions are given in Table 2. The

Helvetica Chimica Acta ± Vol. 84 (2001)
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pure fractions were collected in Eppendorf vials containing 20 ml of 1m aq. AcOH (to neutralize the high pH),
combined, and desalted to remove excess salt.
5. Desalting of Oligonucleotides. ± For desalting, a Waters Sepak-C18 cartridge was equilibrated with 10 ml
of MeCN/H2O 1:1 followed by 10 ml of H2O and finally 10 ml of 0.2m aq. Et3NH2CO3. The oligonucleotide
soln. was diluted with 10 ml of 0.1 ± 0.2m aq. Et3NH2CO3 soln. and loaded onto the Waters Sepak-C18 cartridge.
The cartridge was eluted with 10 ml of 0.2 or 0.5m Et3NH2CO3, followed by 15 to 30 ml of MeCN/H2O 1:1. The
eluted MeCN/H2O fractions containing the oligonucleotide (monitored by UV at 260 nm) were combined and
concentrated in vacuo. The residue was co-evaporated three times with 10 ml of H2O to remove excess buffer.
The residue was dissolved in the desired amount of H2O to give a salt-free soln. of the oligonucleotide.
REFERENCES
[1] 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.
[2] A. Eschenmoser, ꢀKon-Tiki-Experimente zur Frage nach dem Ursprung von Biomolekülenꢁ, in ꢀMaterie
und Prozesse. Vom Elementaren zum Komplexenꢁ, Eds. W. Gerok et al. Verh. Ges. Dtsch. Naturforsch.
¾rzte, 116. Versammlung, Berlin 1990, Wissenschaftl. Verlagsgesellschft mbH, Stuttgart, 1991, S. 135 ± 172.
[3] A. Eschenmoser, Science 1999, 284, 2118 ± 2124.
[4] A. Eschenmoser, Pure Appl. Chem. 1993, 65, 1179 ± 1188.
[5] a) S. Pitsch, S. Wendeborn, B. Jaun, A. Eschenmoser, Helv. Chim. Acta 1993, 76, 2161 ± 2183; b) I.
Â
Schlönvogt, S. Pitsch, C. Lesueur, A. Eschenmoser, R. M. Wolf, B. Jaun, Helv. Chim. Acta 1996, 79, 2316 ±
Â
2345; c) S. Pitsch, R. Krishnamurthy, M. Bolli, S. Wendeborn, A. Holzner, M. Minton, C. Lesueur, I.
Schlönvogt, B. Jaun, A. Eschenmoser, Helv. Chim. Acta 1995, 78, 1621 ± 1635.
[6] R. Micura, M. Bolli, N. Windhab, A. Eschenmoser, Angew. Chem., Int. Ed. 1997, 36, 870 ± 873.
[7] M. Bolli, R. Micura, S. Pitsch, A. Eschenmoser, Helv. Chim. Acta 1997, 80, 1901 ± 1951.
[8] M. Bolli, R. Micura, A. Eschenmoser, Chem. Biol. 1997, 4, 309 ± 320.
[9] R. Micura, M. Bolli, S. Pitsch, A. Eschenmoser, unpublished work; see also [1].
[10] G. v. Kiedrowski, Angew. Chem., Int. Ed. 1986, 25, 932; D. Sievers, G. von Kiedrowski, Chem.-Eur. J. 1998,
4, 629; and refs. cit. therein.
[11] W. S. Zielinski, L. E. Orgel, Nature 1987, 327, 346.
[12] A. Robertson, A. J. Sinclair, D. Philip, Chem. Soc. Rev. 2000, 29, 141 ± 152.
[13] M. Beier, F. Reck, T. Wagner, R. Krishnamurthy, A. Eschenmoser, Science 1999, 283, 699 ± 703.
[14] O. Jungmann, H. Wippo, M. Stanek, H. K. Huynh, R. Krishnamurthy, A. Eschenmoser, Org. Lett. 1999, 10,
1527 ± 1530.
[15] H. G. Fletcher Jr., R. K. Ness, C. S. Hudson, J. Am. Chem. Soc. 1951, 73, 3698 ± 3699; A. P. Kozikowski,
K. L. Sorgi, Tetrahedron Lett. 1982, 23, 2281 ± 2284.
[16] H. Vorbrüggen, B. Bennua, Chem. Ber. 1981, 114, 1279 ± 1286; M. Böhringer, H.-J. Roth, J. Hunziker, M.
Göbel, R. Krishnan, A. Giger, B. Jaun, C. Leumann, A. Eschenmoser, Helv. Chim. Acta 1992, 75, 1416 ±
1477.
[17] M. Fuertes, J. T. Witkowski, R. K. Robins, J. Org. Chem. 1975, 40, 2372 ± 2377.
[18] S. Oscarson, A. K. Tiden, Carbohydr. Res. 1993, 247, 323 ± 328; R. U. Lemieux, H. Driguez, J. Am. Chem.
Soc. 1975, 97, 4069 ± 4075; S. Oscarson, M. Szoenyi, J. Carbohydr. Chem. 1989, 8, 663 ± 668.
[19] H. P. M. Fromageot, B. E. Griffin, C. B. Reese, J. E. Sulston, Tetrahedron 1967, 23, 2315 ± 2331.
[20] A. Kume, H. Tanimura, S. Nishiyama, M. Sekine, T. Hata, Synthesis 1985, 408 ± 409.
[21] J. Hunziker, H.-J. Roth, M. Böhringer, A. Giger, U. Diedrichsen, M. Göbel, R. Krishnan, B. Jaun, C.
Leumann, A. Eschenmoser, Helv. Chim. Acta 1993, 76, 259 ± 352. K. Groebke, J. Hunziker, W. Fraser, L.
Peng, U. Diedrichsen, K. Zimmermann, A. Holzner, C. Leumann, A. Eschenmoser, Helv. Chim. Acta 1998,
81, 375 ± 474.
[22] A. K. Sood, S. A. Narang, Nucleic Acids Res. 1977, 4, 2757 ± 2765.
[23] U. Pieles, W. Zürcher, M. Schär, H. E. Moser, Nucleic Acids Res. 1993, 21, 3191 ± 3196.
[24] I. Tinoco Jr., K. Sauer, J. C. Wang, in ꢀPhysical Chemistry: Principles and Applications in Biological
Sciencesꢁ, Eds. D. Young, D. Cavanaugh, Prentice Hall, Upper Saddle River, NJ, 1995, pp. 559 ± 589.
[25] L. A. Marky, K. J. Breslauer, Biopolymers 1987, 26, 1601 ± 1620.
[26] C. R. Cantor, P. R. Schimmel, Part III, ꢀThe Behavior of Biological Macromoleculesꢁ, in ꢀBiophysical
Chemistryꢁ, Freeman: San Francisco, CA, 1980; pp. 1135 ± 1139.

1804
Helvetica Chimica Acta ± Vol. 84 (2001)
[27] R. Micura, R. Kudick, S. Pitsch, A. Eschenmoser, Angew. Chem., Int. Ed. 1999, 38, 680 ± 683.
[28] H. Wippo, F. Reck, R. Kudick, M. Ramaseshan, M. Bolli, G. Ceulemans, R. Krishnamurthy, A.
Eschenmoser, Bioorg. Med. Chem. 2001, in press.
[29] A. Eschenmoser, M. Dobler, Helv. Chim. Acta 1992, 75, 218 ± 259.
[30] B. Jaun, O. Ebert, ETH-Zürich, unpublished results.
[31] F. Reck, H. Wippo, R. Kudick, M. Bolli, G. Ceulemans, R. Krishnamurthy, A. Eschenmoser, Org. Lett.
1999, 1, 1531 ± 1534.
[32] K.-U. Schöning, P. Scholz, S. Guntha, X. Wu, R. Krishnamurthy, A. Eschenmoser, Science 2000, 290, 1347 ±
1351.
[33] R. Pon, S. Yu, Y. S. Sanghvi, Bioconjugate Chem. 1999, 10, 1051 ± 1057.
Received April 2, 2001