Synthesis and properties of oligonucleotides containing 2,4-dihydroxycyclohexyl nucleosides

Article abstract of DOI:10.1002/hlca.200590258

Cyclohexyl nucleosides with an adenine and uracil base have been synthesized from 2-azidocyclohexane-1,5-diol. The obtained racemic nucleosides were resolved using (R)-O-methylmandelic acid. Short oligonucleotides were synthesized using phorphoramidite ch

Full text of DOI:10.1002/hlca.200590258

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HELVETICA CHIMICA ACTA – Vol. 88 (2005)
Synthesis and Properties of Oligonucleotides Containing
2,4-Dihydroxycyclohexyl Nucleosides
by Dorothée Bardiot, Helmut Rosemeyer, Eveline Lescrinier, Jef Rozenski, Arthur Van Aerschot,
and Piet Herdewijn*
Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, K. U. Leuven,
Minderbroedersstraat 10, B-3000 Leuven
(e-mail: Piet.Herdewijn@rega.kuleuven.ac.be)
Cyclohexyl nucleosides with an adenine and uracil base have been synthesized from 2-azidocyclohexane-
1,5-diol. The obtained racemic nucleosides were resolved using (R)-O-methylmandelic acid. Short oligonucleo-
tides were synthesized using phorphoramidite chemistry. However, these oligonucleotides do not show self-
hybridization, and duplexes are less stable than those of ribopyranosyl-(4’ " 2’)-oligonucleotides.
1. Introduction. – DNA Hybridization has been used for the three-dimensional
organization of components for nanotechnological applications. A classical experiment
is the use of single-stranded DNA to induce aggregation of colloid material labelled
with DNA sequences that are complementary to the single-stranded linker DNA [1].
Likewise, networks of gold particles have been obtained in the same way [2]. Disadvan-
tages of this technology are that intramolecular folding of DNA may prevent cluster
formation, that the system has a limited lifetime due to the chemical and enzymatic
lability of DNA, and that the mechanical strength of the double stranded wires is lim-
ited. To avoid these three intrinsic disadvantages of natural DNA, more robust syn-
thetic nucleic acids should be used that are less prone to folding [3]. Here, we present
the synthesis of a new synthetic nucleic acid, based on a phosphorylated cyclohexane
backbone. Phosphorylated internucleoside linkages are needed to keep the materials
water-soluble. The expectations that these nucleic acids might form stable duplexes
is based on a publication about the thermal stability of b- -ribopyranosyl-(4’ " 2’)-oli-
D
gonucleotides [4]. The difference is that the ring O-atom is replaced by a CH2 group,
and that the 3’-OH group is removed. The proposed polymers are chemically and enzy-
matically stable, and are less prone to intramolecular folding, given the conformational
stiffness of the cyclohexane ring and the replacement of a primary OH group by a sec-
ondary OH group. The sequence-controlled polymerization can be carried out accord-
ing to classical DNA synthesis chemistry, which means that the oligonucleotides are
easily available. The structure of the proposed single stranded nucleic acids is given
in Fig. 1.
To test the potential higher thermodynamic stability of dihydroxycyclohexyl nucleic
acid duplexes, we decided to synthesize first the phosphoramidite building blocks with a
uracil and adenine base (as these nucleoside analogues are more easily available than
nucleosides with a cytosine or guanine base moiety). The uracil and adenine derivatives
were prepared by construction of the heterocyclic base on the primary amine of 2-
© 2005 Verlag Helvetica Chimica Acta AG, Zürich

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Fig. 1. Structure of single-stranded 2,4-dihydroxycyclohexane oligonucleotides of opposite chirality
amino-5-(benzyloxy)cyclohexan-1-ol (2). Optically pure nucleosides were obtained via
resolution of racemic intermediates 8 and 12 with (ꢀ)-(R)-methylmandelic acid. After
separation of the enantiomers, phosphoramidite chemistry is used for the polymeriza-
tion reaction. As compounds are meant to be used for nanotechnological purposes, the
exact identification of homochirality is not needed, as long as the obtained racemates
can be resolved in their enantiomers. Homochiral oligonucleotides from opposite chir-
ality were synthesized. The sequences 4’-AUUUAUAAp-2’, its antiparallel comple-
ment 4’-UUAUAAAUp-2’, 4’-AUAUAUAUp-2’, and 4’-UAUUUUAp-2’ have been
obtained. CD Measurements indicate that the oligonucleotides obtained from the
(+)-series of monomers might be stereochemically related to the natural congener
(d-DNA). Unfortunately, no duplex formation was observed with these short cyclohex-
yl oligonucleotides.
2. Results and Discussion. – 2.1. Nucleoside Synthesis. For the synthesis of cyclohex-
yl nucleosides, we utilized the reported (ꢁ)-(1RS,2RS,5RS)-2-azido-5-(benzyloxy)cy-
clohexan-1-ol [5] (1)1) as starting material, which was prepared in four steps from cy-
clohexane-1,4-diol [5–7]. Staudinger reduction of the N3 group to yield the amine 2
enabled the construction of the adenine and uracil moieties according to standard
methods (Schemes 1 and 2)1). Preparation of the adenine derivative involved a three-
step sequence [8][9]: condensation of 2 with 5-amino-4,6-dichloropyrimidine in
BuOH in the presence of Et3N gave the substituted diaminopyrimidine 3. Cyclization
with triethyl orthoformate in acidic medium, followed by substitution of the 6-Cl atom
of 4 with NH3 provided the adenine derivative 5. The secondary OH group of 5 was pro-
tected with a TBDMS group, followed by hydrogenolytic cleavage of the benzyl ether
with 10% Pd/C and ammonium formate [10] in refluxing MeOH. A benzoyl (Bz) group
was introduced at N6 of the adenine via a transient protection [11] approach, to yield
the racemic compound 8.
The synthetic route to the uracil derivative 12 was based on the previously descri-
bed procedures [12–14] (Scheme 2). Treatment of 2 with b-methoxyacryloyl isocyanate
[15] gave the intermediate acryloylurea 9, which was cyclized in refluxing diluted H2-
SO4 to provide the uracil 10. After silylation of the secondary OH group of 10, attempts
1
)
In all Schemes, for convenience only one enantiomer is shown; (ꢁ) below the formulae refers to racemates.

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Scheme 1. Preparation of the Adenine Derivative 8
a) 1. Ph3P, dioxane; 2. NH4OH; 97%. b) 5-Amino-4,6-dichloropyrimidine, Et3N, BuOH; 83%. c) 1.
HC(OEt)3, 12 HCl; 2. 0.5 HCl, THF; 76%. d) NH3/MeOH; 93%. e) (t-Bu)Me2SiCl (TBDMSCl), 1H-imi-
N
N
dazole, DMF; 81%. f) Pd/C, NH4OCOH, MeOH; 96%. g) 1. Me3SiCl, pyridine; 2. BzCl; 3. NH4OH,
MeOH; 73%.
Scheme 2. Preparation of the Uracil Derivative 12
a) b-Methoxyacryloyl isocyanate, DMF; 76%. b) 2
N H2SO4; 73%. c) TBDMSCl, 1H-imidazole, DMF; 95%;
d) 1 BCl3 in CH2Cl2, CH2Cl2; 95%.
M
to remove the Bn protecting group by hydrogenolysis (H2 or HCOONH4 with Pd/C)
were not successful because of partial hydrogenation of the uracil double bond. Deben-
zylation [16] with a BCl3 solution at ꢀ788 yielded the racemic compound 12.

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The synthesis of the enantiomeric forms of the cyclohexyl-adenine and -uracil
nucleosides was achieved by resolution of intermediates 8 and 12 via formation of dia-
stereoisomeric esters with a chiral acid. We chose (R)-O-methylmandelic acid, which
had been previously used in our laboratory to resolve diastereosiomeric esters of car-
bocyclic nucleosides [17][18]. Acylation of 8 was carried out with (R)-O-methylman-
delic acid in the presence of DCC and DMAP (Scheme 3). As we cannot assign the
exact configuration of the diastereoisomers, the less polar compound is called (A)
and the more polar (B) by convention. Careful separation of the two diastereoisomers
13a and 13b by column chromatography, followed by preparative TLC gave pure com-
pound. The diastereoisomeric purity was checked by HPLC and was found to be
greater than 99% for 13a and 13b (Fig. 2). The uracil derivative 12 was resolved in a
similar manner by treatment with (R)-O-methylmandelic acid. As for adenine deriva-
tives, the less polar diastereoisomer is called (A) and the more polar (B). Isolation of
14a and 14b was easier than for the resolution of 8. After purification by column chro-
matography, the diastereoisomers 14a and 14b were obtained with a diastereoisomeric
purity greater than 99% (determined by HPLC; see Fig. 2).
Selective removal of the O-methylmandelate esters was accomplished by treatment
with 2
N
NaOH in a mixture of dioxane and MeOH [19]. Tritylation of the OH group in
Bu4NF solution in THF provided the enantiomeri-
4’-position and desilylation with a 1
N
cally pure cyclohexyl nucleosides 19a and 19b, and 20a and 20b. The [a]D values were
determined for the four compounds, showing that 19a and 20a (derived from the less
polar mandelates 13a and 14a) are the (ꢀ)-isomers, and 19b and 20b (derived from
the more polar mandelates 13b and 14b) are the (+)-isomers. The nucleotide building
blocks 21a and 21b, and 22a and 22b were then obtained by phosphitylation of the sec-
Fig. 2. Diastereoisomeric purity of 13a and 13b, and 14a and 14b as determined by HPLC (see Exper. Part
for HPLC conditions)

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Scheme 3. Resolution of the Cyclohexyl Nucleosides and Preparation of the Optically Pure Building Blocks
for Oligonucleotide Synthesis
a) (R)-O-Methylmandelic acid, N,N’-dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP),
CH2Cl2; 13a: 36%, 13b: 36%, 14a: 43%, 14b: 39%. b) 2
N NaOH, MeOH, dioxane; 15a: 96%, 15b: 66%
16a: 94%; 16b: 91%. c) Monomethoxytrityl chloride (MMTrCl), pyridine; 17a: 88%, 17b: 84%, 18a: 82%,
18b: 99%. d) 1
N
Bu4NF in THF, THF; 19a: 81%, 19b: 84%, 20a: 61%, 20b: 70%. e) EtN(i-Pr)2, (i-Pr)2-
NP(Cl)OCH2CH2CN, CH2Cl2; 21a: 83%, 21b: 83%, 22a: 88%, 22b: 78%.

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ondary OH group in 2’ with 2-cyano-N,N-diisopropylchlorophosphoramidite in the
presence of iPr2NEt.
2.2. Oligonucleotide Synthesis. Using the cyclohexyl-U and cyclohexanyl-A mono-
mers (21a and 22a, and 21b and 22b) of similar polarity and [a]2D0 values, oligonucleo-
tides were synthesized with the sequences 4-AUUUAUAAp-2’ (Table 1) and its anti-
parallel complement 4’-UUAUAAAUp-2’. Duplexes of such sequences in the p-
RNA series have been demonstrated to show a Tm of ca. 208 in 0.15 NaCl [4]. The mir-
M
ror-image oligonucleotide 4’-AUUUAUAAp-2’ obtained from the (+)-series nucleoti-
des and from the (ꢀ)-series nucleotides gives identical 1D-NMR spectra (data not
shown). This is also the case for the mirror-image oligonucleotide 4’-UUAUAAAUp-2’.
Table 1. Oligonucleotides Synthesized from the Dihydroxycyclohexyl Nucleosides
Sequence
Mass spectra
calc.
found
From series A ((ꢀ)-series)
I
4’-AUUUAUAA-2’-phosphate
2414.5
2414.5
2080.4
2414.3
2414.3
2080.4
II
III
4’-UUAUAAAU-2’-phosphate
4’-UAUUUUA-2’-phosphate
From series B ((+)-series)
IV
V
VI
4’-AUUUAUAA-2’-phosphate
4’-UUAUAAAU-2’-phosphate
4’-AUAUAUAU-2’-phosphate
2414.5
2414.5
2414.5
2414.4
2414.5
2414.5
Also the self-complementary sequence 4’-AUAUAUAUp-2’ was synthesized.
Unfortunately, no duplex formation was observed with the oligonucleotides at either
0.1 NaCl, 1 NaCl, 3 NaCl, or 50% formamide, demonstrating that the removal of
M
M
M
the 3’-OH group and the replacement of the O-atom in p-RNA by a CH2 group results
in lowered duplex stability. The presence of a supplementary phosphate group at the 2’-
end is considered as of minor influence on duplex stability, and was included for
straightforward synthetic purposes.
2.3. CD Analysis. The CD spectrum of the single-stranded oligonucleotide was
determined in phosphate buffer pH 7.0. The single-stranded oligonucleotides I and II
show a positive Cotton effect with a maximum at 256 nm. The single-stranded oligonu-
cleotides IV and V (being mirror images of the oligonucleotides I and II, resp.) show a
negative Cotton effect with a maximum at the same wavelength (Fig. 3,a and b). The
natural DNA oligonucleotide (5’-UUAUAAAUp-3’), likewise, demonstrates a strong
negative Cotton effect, the maximum being located at 245 nm (Fig. 3,c), which
means that it shows more similarity with the B series of dihydroxycyclohexyl oligonu-
cleotides than with the A series.
2.4. Mass Spectrometry. In addition to the accurate mass determination of the build-
ing blocks, the oligonucleotides were characterized by HPLC-MS/MS. Although the
fragmentation of the 2,4-dihydroxy-1-cyclohexyl-containing oligonucleotides does not
follow the fragmentation rules of natural DNA [20], sequence information can be
obtained from CID data. As an example, we will discuss the spectrum for oligonucleo-
tide I (Fig. 4). The major peak in the spectrum obtained by fragmentation of the triply
charged precursor (m/z 803.8) results from the loss of the phosphate group at the 2’-

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Fig. 3. a) CD Spectrum of oligonucleotide II ((ꢀ) series), b) CD spectrum of oligonucleotide V ((+) series),
c) CD spectrum of single-stranded DNA with sequence 5’-UUAUAAAUp-3’

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end. Attempts to build a sequence ladder, the classical approach for oligonucleotide
sequencing from mass-spectrometry data [21], failed because of the lack of a glycosidic
bond. As a consequence, some of the ions will not be found (e.g., the a-B series), and ion
intensities will not always relate to the sequence (e.g., for the w2 ion m/z 696 [pUpAp]ꢀ
is more abundant than m/z 719 [pApAp]ꢀ, making this series useless for sequencing).
However, the fragmentation spectra reveal that residues are primarily lost from the
2’-end, and the sequence can be built from the 2’-terminus.
All fragments originate from backbone cleavages. Example spectra with annota-
tions are shown in Fig. 4. The major ions are accompanied by a water loss (e.g., m/z
390 has lost water from m/z 408). As expected, we found no differences between the
spectra of the oligonucleotide isomers I and IV, nor between the spectra of II and V.
Conclusions. – 2,4-Dihydroxy-1-cyclohexyl nucleosides with an uracil and adenine
base have been synthesized as racemic mixtures and resolved into their enantiomers.
These nucleosides were converted to their protected phosphoramidite building blocks
for oligonucleotide synthesis. The correct assembling process for oligonucleotide syn-
thesis was verified by mass spectrometry, and the mirror-image form by CD analysis.
However, we were not able to detect duplex formation with the obtained short oligom-
ers (octamers) with U and A bases. The potential self-hybridization will have to be eval-
uated using longer oligomers including cytosine and guanine bases.
Experimental Part
General. All air-sensitive reactions were carried out under N2. All solvents used for the reactions were anal.
grade or freshly dried according to standard methods. Azido derivative 1 was prepared as described in [5–7].
Precoated aluminium sheets (Fluka, silica gel/TLC cards, 254 nm) were used for TLC, and compounds were
visualized by UV fluorescence. Prep. TLC were performed on precoated TLC plates (Macherey-Nagel silica
gel P/UV 254). Ecochrom silica gel (0.035–0.06 mm or 0.06–0.2 mm) was used for column chromatography
(CC). CD Spectra were measured at either 58 or 208 on a Jasco 600 spectropolarimeter with thermostatically
controlled 1-cm cuvettes, connected with a Lauda RCS thermostate (Lauda, D-Königshofen). 1H-, 13C-, and
1
31P-NMR spectra were recorded with a Varian Unity-500 spectrometer with TMS as internal standard for H-
NMR spectra, CDCl3 or (D6)DMSO as internal standard for 13C-NMR spectra, and 85% H3PO4 as external stan-
dard for 31P-NMR. Accurate mass measurements were performed on a orthogonal acceleration quadrupole
time-of-flight mass spectrometer (Q-TOF-2, Micromass, Manchester, UK) equipped with a standard electro-
spray ionization interface. HPLC-MS/MS was performed for sequence verification of the oligonucleotides.
The mass spectrometer was coupled to a capillary HPLC (CapLC, Waters, Milford, MA). The chromatographic
system was adapted from Apffel et al. [22]. A 500 mm×15 mm reversed-phase C18 column (PepMap, LC Pack-
ings, San Fransisco, CA) was used as stationary phase. Flow rate was set to 12 ml/min. Collision-induced disso-
ciation (CID) with Ar gas in the collision cell was performed at 75 eV. All single-stranded oligomers were meas-
ured at a concentration of 5 mM in a buffer containing 10 mM Na-cacodylate, 100 mM NaCl, and 10 mM MgCl2 (pH
7). Diastereoisomeric purities were determined by HPLC analysis on a Waters 600 Controller liquid chromato-
graph equipped with a Waters 2487 UV detector, using a Alltech Alltima Silica 5U column (150 mm×4.6 mm);
eluent: hexane/EtOH 90 :10 for 13a and 13b and hexane/EtOH 92 :08 for 14a and 14b; flow rate: 1 ml/min;
detection: 254 nm.
(ꢁ)-(1RS,2RS,5RS)-2-Amino-5-(benzyloxy)cyclohexan-1-ol (2). A soln. of 1 (1.18 g, 4.79 mmol) and Ph3P
(1.90 g, 7.23 mmol) in THF (30 ml) was stirred at r.t. for 20 min. After addition of H2O (1.1 ml), the mixture was
refluxed for 2 h. The solvent was evaporated under reduced pressure and co-evaporated with EtOH. The residue
was purified by flash chromatography (FC; AcOEt/MeOH, 90 :10 to 70 :30) to give 1.03 g (97%) of 2. Yellow
powder. 1H-NMR (CDCl3): 7.26–7.34 (m, 5 arom. H); 4.55 (s, PhCH2O); 3.39–3.47 (m, HꢀC(5)); 3.11–3.18 (m,
HꢀC(1)); 2.45–2.52 (m, HꢀC(2)); 2.36–2.43 (m, HaꢀC(6)); 2.02–2.08 (m, HaꢀC(4)); 1.95 (br. s, HOꢀC(1),

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Fig. 4. a) Fragment ion spectra of the triply charged precursor of cyclohexyl oligonucleotides AUUUAUAAp
and b) UUAUAAAUp with the theoretical m/z values of the ions found in the spectra
NH2); 1.85–1.92 (m, HaꢀC(3)); 1.35–1.44 (m, HbꢀC(4), HbꢀC(6)); 1.05–1.14 (m, HbꢀC(3)). 13C-NMR
(CDCl3): 138.55, 128.33, 127.49 (arom. C); 75.29 (C(5)); 73.04 (C(1)); 70.21 (PhCH2O); 56.09 (C(2)); 39.22
(C(6)); 30.54 (C(4)); 30.00 (C(3)). HR-MS: 244.1306 ([M+Na]+, C13H19NNaO2þ ; calc. 244.1313).
(ꢁ)-5-Amino-4-[(1’RS,2’RS,4’RS)-4’-(benzyloxy)-2’-hydroxycyclohexylamine]-6-chloropyrimidine (3).
A
mixture of 2 (4.5 g, 20.35 mmol), 5-amino-4,6-dichloropyrimidine (3.51 g, 21.40 mmol), Et3N (11.7 ml), and
BuOH (70 ml) was heated under reflux for 24 h. The solvents were evaporated in vacuo, and the residue was
purified by CC (CH2Cl2/MeOH, 99 :1 to 85 :15) to afford 5.89 g (83%) of 3. Brown solid. 1H-NMR
((D6)DMSO): 7.71 (s, HꢀC(2)); 7.26–7.36 (m, 5 arom. H); 6.55 (d, J=7.1, NH); 5.00 (s, NH2); 4.75 (d,

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J=5.6, HOꢀC(2’)); 4.49–4.55 (m, PhCH2O); 3.75–3.82 (m, HꢀC(1’)); 3.39–3.48 (m, HꢀC(2’), HꢀC(4’));
2.29–2.36 (m, HaꢀC(3’)); 1.97–2.05 (m, HaꢀC(5’), HaꢀC(6’)); 1.21–1.35 (m, HbꢀC(3’), HbꢀC(5’)); 1.04–
1.12 (m, HbꢀC(6’)). 13C-NMR ((D6)DMSO): 152.35 (C(4)); 145.70 (C(6)); 139.18 (C(2)); 136.80, 128.25,
127.40, 127.29 (arom. C); 123.49 (C(5)); 74.99 (C(4’)); 69.57 (PhCH2O); 69.33 (C(2’)); 56.21 (C(1’)); 40.86
(C(3’)); 30.48 (C(5’)); 27.29 (C(6’)). HR-MS: 349.1434 ([M+H]+, C17H22ClN4O2þ ; calc. 349.1431).
(ꢁ)-9-[(1’RS,2’RS,4’RS)-4’-(Benzyloxy)-2’-hydroxycyclohexyl]-6-chloropurine (4). Conc. HCl (1.2 ml) was
added to a soln. of 3 (2 g, 5.73 mmol) in HC(OEt)3 (52 ml). The mixture was stirred at r.t. overnight and con-
centrated to dryness. The residue was dissolved in THF (70 ml), and 0.5
N HCl (70 ml) was added. After 2 h
at r.t., the soln. was neutralized with 1 NaOH and diluted with CH2Cl2. The phases were separated, and the
N
org. phase was dried (Na2SO4), filtered, and concentrated to dryness. The residue was purified by FC (hex-
1
ane/AcOEt 50 :50 to 10 :90) to give 1.56 g (76%) of 4. White solid. H-NMR ((D6)DMSO): 8.75 (s, HꢀC(2));
8.72 (s, HꢀC(8)); 7.28–7.37 (m, 5 arom. H); 5.09 (d, J=5.6, HOꢀC(2’)); 4.55–4.61 (m, PhCH2O); 4.28–4.35
(m, HꢀC(1’)); 4.05–4.12 (m, HꢀC(2’)); 3.52–3.60 (m, HꢀC(4’)); 2.38–2.45 (m, HaꢀC(3’)); 2.10–2.18 (m,
HaꢀC(5’), HaꢀC(6’)); 1.96–2.02 (m, HbꢀC(6’)); 1.36–1.46 (m, HbꢀC(3’), HbꢀC(5’)). 13C-NMR
((D6)DMSO): 152.24 (C(2)); 151.05 (C(6)); 148.90 (C(4)); 146.93 (C(8)); 139.02 (arom. C); 131.30 (C(5));
128.29, 127.46, 127.36 (arom. C); 74.19 (C(4’)); 69.35 (PhCH2O); 68.00 (C(2’)); 61.39 (C(1’)); 40.60 (C(3’));
30.56 (C(5’)); 26.54 (C(6’)). HR-MS: 359.1274 ([M+H]+, C18H20ClN4O2þ ; calc. 359.1275).
(ꢁ)-9-[(1’RS,2’RS,4’RS)-4’-(Benzyloxy)-2’-hydroxycyclohexyl]adenine (5). A suspension of 4 (2 g, 5.6
mmol) in a MeOH soln. sat. with NH3 (50 ml) was heated in a sealed tube at 1008 overnight. NH3 and
MeOH were evaporated. The residue was purified by CC (CH2Cl2/MeOH 98 :2 to 90 :10) to afford 1.65 g
(84%) of 5. White powder. 1H-NMR ((D6)DMSO): 8.11 (s, HꢀC(2), HꢀC(8)); 7.28–7.38 (m, 5 arom. H);
7.12 (br. s, NH2); 4.99 (d, J=5.6, HOꢀC(2’)); 4.55, 4.57 (AB, J=12.2, PhCH2O); 4.05–4.16 (m, HꢀC(1’), Hꢀ
C(2’)); 3.49–3.56 (m, HꢀC(4’)); 2.36–2.41 (m, HaꢀC(3’)); 2.02–2.15 (m, HaꢀC(5’), HaꢀC(6’)); 1.87–1.91
(m, HbꢀC(6’)); 1.32–1.42 (m, HbꢀC(3’), HbꢀC(5’)). 13C-NMR ((D6)DMSO): 156.01 (C(6)); 151.95 (C(2));
149.76 (C(4)); 140.39 (C(8)); 139.09, 128.32, 127.48, 127.38 (arom. C); 119.28 (C(5)); 74.37 (C(4’)); 69.36
(PhCH2O); 67.81 (C(2’)); 60.40 (C(1’)); 41.02 (C(3’)); 30.72 (C(5’)); 27.11 (C(6’)). HR-MS: 340.1170
([M+H]+, C18H22N5Oþ2 ; calc. 340.1173).
(ꢁ)-9-{(1’RS,2’RS,4’RS)-4’-(Benzyloxy)-2’-[(tert-butyl)dimethylsilyloxy]cyclohexyl}adenine (6). To a pre-
chilled (08) soln. of 5 (947 mg, 2.79 mmol) and 1H-imidazole (1.08 g, 15.9 mmol) in DMF (28 ml) was added
TBDMSCl (1.36 g, 9.02 mmol). After one night at r.t., Et2O was added, and the mixture was washed with sat.
aq. NaHCO3 soln., 1N HCl, and H2O. The org. phase was dried (Na2SO4), filtered, and concentrated to dryness.
The residue was purified by CC (CH2Cl2/MeOH 100 :0 to 94 :6) to give 1.02 g (81%) of 6. White solid. 1H-NMR
((D6)DMSO): 8.10 (s, HꢀC(2)); 8.09 (s, HꢀC(8)); 7.28–7.36 (m, 5 arom. H); 7.08 (br. s, NH2); 4.54, 4.59 (AB,
J=12.1, PhCH2O); 4.16–4.26 (m, HꢀC(1’), HꢀC(2’)); 3.54–3.61 (m, HꢀC(4’)); 2.27–2.34 (m, HaꢀC(3’), Haꢀ
C(6’)); 2.13–2.20 (m, HaꢀC(5’)); 1.84–1.91 (m, HbꢀC(6’)); 1.34–1.45 (m, HbꢀC(3’), HbꢀC(5’)); 0.55 (s, t-Bu);
ꢀ0.18 (s, MeSi); ꢀ0.66 (s, MeSi). 13C-NMR ((D6)DMSO): 156.03 (C(6)); 151.87 (C(2)); 149.66 (C(4)); 140.55
(C(8)); 139.05, 128.29, 127.51, 127.39 (arom. C); 119.46 (C(5)); 74.24 (C(4’)); 70.04 (PhCH2O); 69.57 (C(2’));
60.43 (C(1’)); 41.38 (C(3’)); 30.55 (C(5’)); 26.19 (C(6’)); 25.32 (Me3C); 17.19 (Me3C); ꢀ4.73, ꢀ6.00 (Me2Si).
HR-MS: 454.2639 ([M+H]+, C24H36N5O2Si+; calc. 454.2638).
(ꢁ)-9-{(1’RS,2’RS,4’RS)-2’-[(tert-Butyl)dimethylsilyloxy]-4’-hydroxycyclohexyl}adenine (7). A mixture of 6
(778 mg, 1.72 mmol), NH4OCOH (922 mg, 14.6 mmol), and Pd/C (1g) in MeOH (42 ml) was refluxed for 5 h.
NH4OCOH (461 mg, 7.31 mmol) was added again, and heating was continued for 2 h. After cooling to r.t.,
the mixture was filtered through Celite, and the catalyst was washed with MeOH. The filtrate was concentrated
to dryness, and the residue was purified by CC (CH2Cl2/MeOH 98 :2 to 90 :10) to yield 603 mg (96%) of 7. White
foam. 1H-NMR ((D6)DMSO): 8.11 (s, HꢀC(2)); 8.09 (s, HꢀC(8)); 7.06 (br. s, NH2); 4.79 (d, J=4.6, HOꢀC(4’));
4.17–4.23 (m, HꢀC(1’); 4.10–4.17 (m, HꢀC(2’)); 3.61–3.69 (m, HꢀC(4’)); 2.29–2.34 (m, HaꢀC(6’)); 2.14–2.21
(m, HaꢀC(3’)); 1.89–1.96 (m, HaꢀC(5’)); 1.78–1.86 (m, HbꢀC(6’)); 1.29–1.40 (m, HbꢀC(3’), HbꢀC(5’)); 0.55 (s,
Me3C); ꢀ0.18 (s, MeSi); ꢀ0.65 (s, MeSi). 13C-NMR ((D6)DMSO): 156.00 (C(6)); 151.86 (C(2)); 149.64 (C(4));
140.51 (C(8)); 119.42 (C(5)); 70.20 (C(4’)); 66.46 (C(2’)); 60.39 (C(1’)); 44.60 (C(3’)); 33.98 (C(5’)); 26.50
(C(6’)); 25.31 (Me3C); 17.20 (Me3C); ꢀ4.76, ꢀ5.95 (Me2Si). HR-MS: 364.2166 ([M+H]+, C17H30N5O2Si+;
calc. 364.2169).
(ꢁ)-N6-Benzoyl-9-{(1’RS,2’RS,4’RS)-2’-[(tert-butyl)dimethylsilyloxy]-4’-hydroxycyclohexyl}adenine (8). To
a prechilled (08) soln. of 7 (552 mg, 1.52 mmol) in pyridine (8 ml) was added Me3SiCl (1 ml, 7.82 mmol). After
1 h at 08, BzCl (900 ml, 7.75 mmol) was added, and the mixture was stirred at r.t. overnight. After cooling to 08,
H2O (2 ml) was added. The mixture was stirred for 5 min, and 25% aq. NH4OH soln. (4 ml) was added. After
15 min at 08, the solvents were evaporated. The residue was dissolved in MeOH (3 ml) and 25% aq. NH4OH

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soln. (25 ml). The mixture was stirred for 30 min and concentrated to dryness. The residue was partitioned
between CH2Cl2 and 1 HCl, and washed with sat. aq. NaHCO3 soln. and brine. The org. phase was dried
N
(Na2SO4), filtered, and concentrated to dryness. The residue was purified by CC (CH2Cl2/MeOH 99 :1 to
92 :8) to afford 521 mg (73%) of 8. White foam. 1H-NMR ((D6)DMSO): 10.99 (s, NH); 8.68 (s, HꢀC(2));
8.50 (s, HꢀC(8)); 8.04 (d, J=7.4, 2 arom. H); 7.52–7.65 (m, 3 arom. H); 4.80 (br. s, HOꢀC(4’)); 4.30–4.36
(m, HꢀC(1’)); 4.20–4.28 (m, HꢀC(2’)); 3.65–3.73 (m, HꢀC(4’)); 2.26–2.36 (m, HaꢀC(6’)); 2.19–2.25 (m,
HaꢀC(3’)); 1.89–2.00 (m, HaꢀC(5’), HbꢀC(6’)); 1.40 (m, HbꢀC(3’), HbꢀC(5’)); 0.54 (s, Me3C); ꢀ0.18 (s,
MeSi); ꢀ0.66 (s, MeSi). 13C-NMR ((D6)DMSO): 172.54 (NHCO); 150.82 (C(2)); 150.06 (C(6)); 148.07
(C(4)); 143.97 (C(8)); 132.20, 128.36 (arom. C); 125.86 (C(5)); 70.34 (C(4’)); 66.35 (C(2’)); 60.47 (C(1’));
44.49 (C(3’)); 33.86 (C(5’)); 26.40 (C(6’)); 25.19 (Me3C); 17.06 (Me3C); ꢀ4.77, ꢀ5.91 (Me2Si). HR-MS:
468.2421 ([M+H]+, C24H34N5O3Si+; calc. 468.2431).
(ꢁ)-N-({[(1’RS,2’RS,4’RS)-4’-(Benzyloxy)-2’-hydroxycyclohexyl]amino}carbonyl)-3-methoxyprop-2-enam-
ide (9). SOCl2 (160 ml, 2.19 mmol) was added to a soln. of 3-methoxyacrylic acid (129 mg, 1.26 mmol) in dry
CH2Cl2 (1 ml). The mixture was refluxed for 3 h and concentrated to dryness. The residue was dissolved in
dry toluene (1.3 ml), and AgOCN (332 mg, 2.21 mmol; dried in vacuo over P2O5 at 1008 for 3 h in the dark)
was added. The suspension was refluxed for 30 min and cooled to 08. The supernatant liquor (900 ml) was
added to a soln. of 2 (120 mg, 0.54 mmol) in dry DMF (2 ml) at ꢀ158. The mixture was stirred at ꢀ158 for 2
h and then at r.t. overnight. The solvents were evaporated, and the residue was purified by FC (hexane/
AcOEt 50 :50 to 10 :90) to afford 157 mg (83%) of 9. White powder. 1H-NMR (CDCl3): 9.90 (s, NH); 8.81
(d, J=7.3, NH); 7.66 (d, J=12.2, CH=CHOMe); 7.26–7.36 (m, 5 arom. H); 5.34 (d, J=12.2, CH=CHOMe);
4.55 (s, PhCH2O); 3.90 (d, J=4.6, HOꢀC(2’)); 3.66–3.74 (m, HꢀC(1’), MeO); 3.46–3.56 (m, HꢀC(2’), Hꢀ
C(4’)); 2.32–2.38 (m, HaꢀC(3’)); 2.00–2.08 (m, HaꢀC(5’), HaꢀC(6’)); 1.44–1.56 (m, HbꢀC(3’), HbꢀC(5’));
1.30–1.38 (m, HbꢀC(6’)). 13C-NMR (CDCl3): 168.26 (NHC(=O)-CH); 163.66 (NHC(=O)NH); 156.44
(CHOMe); 138.31, 128.39, 127.60, 127.52 (arom. C); 97.28 (CH=CHOMe); 74.51 (C(4’)); 71.97 (PhCH2O);
70.42 (C(2’)); 57.66 (C(1’)); 54.69 (MeO); 38.40 (C(3’)); 29.40 (C(5’)); 26.20 (C(6’)). HR-MS: 349.1768
([M+H]+, C18H25N2Oþ5 ; calc. 349.1763).
(ꢁ)-1-[(1’RS,2’RS,4’RS)-4’-(Benzyloxy)-2’-hydroxycyclohexyl]uracil (10). A stirred suspension of 9 (845
mg, 2.43 mmol) in 2
N H2SO4 (20 ml) was refluxed for 2 h. After cooling to 08, the mixture was diluted with H2O
and neutralized with 2
N
NaOH. The soln. was concentrated in vacuo and co-evaporated with EtOH. The resid-
ual solid was extracted with EtOH, and the EtOH soln. was concentrated to dryness. The residue was purified by
CC (CH2Cl2/MeOH 99 :1 to 90 :10) to give 560 mg (73%) of 10. White powder. 1H-NMR ((D6)DMSO): 11.13 (s,
NH); 7.63 (d, J=7.8, HꢀC(6)); 7.26–7.37 (m, 5 arom. H); 5.53 (d, J=7.8, HꢀC(5)); 5.02 (d, J=5.6, HOꢀC(2’));
4.50–4.54 (m, PhCH2O); 4.07 (br. s, HꢀC(1’)); 3.73 (br. s, HꢀC(2’)); 3.38–3.45 (m, HꢀC(4’)); 2.29–2.35 (m,
HaꢀC(3’)); 2.04–2.10 (m, HaꢀC(5’)); 1.66–1.72 (m, HaꢀC(6’)); 1.58 (br. s, HbꢀC(6’)); 1.22–1.35 (m, Hbꢀ
C(3’), HbꢀC(5’)). 13C-NMR ((D6)DMSO): 163.33 (C(4)); 151.50 (C(2)); 139.07 (C(6)); 128.31, 127.45, 127.38
(5 arom. C); 100.98 (C(5)); 74.10 (C(4’)); 69.29 (OCH2Ph); 66.59 (C(2’)); 40.85 (C(3’)); 30.71 (C(5’)); 25.72
(C(6’)). HR-MS: 317.1501 ([M+H]+, C17H21N2O4þ ; calc. 317.1501).
(ꢁ)-1-{(1’RS,2’RS,4’RS)-4’-(Benzyloxy)-2’-[(tert-butyl)dimethylsilyloxy]cyclohexyl}uracil (11). To a pre-
chilled (08) soln. of 10 (2.26 g, 7.14 mmol) and 1H-imidazole (2.43 g, 35.6 mmol) in DMF (35 ml) was added
TBDMSCl (3.24 g, 21.5 mmol). After 4 h at r.t., Et2O was added, and the mixture was washed with sat. aq.
NaHCO3, 1N HCl, and H2O. The org. phase was dried (Na2SO4), filtered, and concentrated to dryness. The res-
idue was purified by CC (CH2Cl2/MeOH 100 :0 to 95 :5) to give 2.91 g (95%) of 11. White solid. 1H-NMR
((D6)DMSO): 7.7 (br. s, HꢀC(6)); 7.27–7.34 (m, 5 arom. H); 5.53 (br. d, J=5.5, HꢀC(5)); 4.50, 4.55 (AB,
J=12.2, PhCH2O); 4.32 (br. s, HꢀC(1’)); 3.85 (br. s, HꢀC(2’)); 3.41–3.49 (m, HꢀC(4’)); 2.23–2.29 (m, Haꢀ
C(3’)); 2.06–2.12 (m, HaꢀC(5’)); 1.65–1.73 (m, 2 HꢀC(6’)); 1.26–1.40 (m, HbꢀC(3’), HbꢀC(5’)); 0.75 (s,
Me3C); 0.00 (s, MeSi); ꢀ0.12 (s, MeSi). 13C-NMR ((D6)DMSO): 163.11 (C(4)); 151.37 (C(2)); 139.00 (C(6));
128.26, 127.44, 127.36 (arom. C); 101.13 (C(5)); 73.93 (C(4’)); 69.42 (PhCH2O); 66.55 (C(2’)); 41.32 (C(3’));
30.43 (C(5’)); 25.45 (Me3C, C(6’)); 17.34 (Me3C); ꢀ4.15, ꢀ5.41 (Me2Si). HR-MS: 431.2369 ([M+H]+,
C23H35N2O4Si+; calc. 431.2366).
(ꢁ)-1-{(1’RS,2’RS,4’RS)-2’-[(tert-Butyl)dimethylsilyloxy]-4’-hydroxycyclohexyl}uracil (12). To a cooled
(ꢀ788) soln. of 11 (800 mg, 1.86 mmol) in CH2Cl2 (54 ml) was added a 1
M BCl3 soln. in CH2Cl2 (19 ml). After
4 h at ꢀ788, a mixture of pyridine and MeOH (13 ml/27 ml) was added. The mixture was allowed to warm to
r.t. and concentrated to dryness. The residue was partitioned between AcOEt and H2O. The org. phase was
washed with H2O, dried (Na2SO4), filtered, concentrated to dryness, and co-evaporated with toluene. The res-
idue was purified by CC (CH2Cl2/MeOH 98 :2 to 95 :5) to provide 600 mg (95%) of 12. White solid. 1H-NMR
((D6)DMSO): 11.16 (s, NH); 7.71 (br. s, HꢀC(6)); 5.53 (d, J=7.6, HꢀC(5)); 4.17–4.29 (m, HꢀC(1’)); 3.76–3.90

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(m, HꢀC(2’)); 3.40–3.61 (m, HꢀC(4’)); 2.09–2.15 (m, HaꢀC(3’)); 1.81–1.87 (m, HaꢀC(5’)); 1.54–1.66 (m, 2
HꢀC(6’)); 1.23–1.32 (m, HbꢀC(3’), HbꢀC(5’)); 0.74 (s, Me3C); ꢀ0.01 (s, MeSi); ꢀ0.13 (s, MeSi). 13C-NMR
((D6)DMSO): 163.02 (C(4)); 151.24 (C(2)); 141.47 (C(6)); 101.09 (C(5)); 69.33 (C(4’)); 66.12 (C(2’));
58.17(C(1’)); 44.52 (C(3’)); 33.83 (C(5’)); 25.41 (Me3C, C(6’)); 17.29 (Me3C); ꢀ4.25, ꢀ5.14 (Me2Si). HR-MS:
341.1888 ([M+H]+, C16H29N2O4Si+; calc. 341.1896).
(+)- and (ꢀ)-N6-Benzoyl-9-{2’-[(tert-butyl)dimethylsilyloxy]-4’-[(R)-2-methoxy-2-phenylacetoxy]cyclohex-
yl}adenine (13a and 13b, resp.). DCC (316 mg, 1.53 mmol) was added to a soln. of 8 (590 mg, 1.26 mmol), (R)-O-
methylmandelic acid (254 mg, 1.53 mmol) and DMAP (18 mg, 0.14 mmol) in CH2Cl2 (15 ml) at 08. The mixture
was allowed to warm to r.t. over 3 h and then washed with 1M aq. H3PO4 soln., sat. aq. NaHCO3 soln., and H2O.
The org. phase was dried (Na2SO4), filtered, and concentrated to dryness. The residue was purified twice by CC
(hexane/AcOEt 50 :50 to 10 :90) and prep. TLC (hexane/AcOEt 30 :70) to afford 281 mg (36%) of 13a, 277 mg
(36%) of 13b, and 89 mg (11%) of a mixture 13a/13b.
Data of 13a. 1H-NMR (CDCl3): 9.10 (br. s, NH); 8.75 (s, HꢀC(2)); 8.02 (d, J=7.1, 2 arom. H); 7.91 (s, Hꢀ
C(8)); 7.35–7.61 (m, 8 arom. H); 4.98–5.06 (m, HꢀC(4’)); 4.78 (s, CH(OMe)Ph); 4.30–4.37 (m, HꢀC(2’));
4.19–4.26 (m, HꢀC(1’)); 3.43 (s, CH(OMe)Ph); 2.52–2.65 (m, HaꢀC(6’)); 2.35–2.42 (m, HaꢀC(3’)); 2.18–
2.22 (m, HbꢀC(6’)); 1.97–2.09 (m, HaꢀC(5’)); 1.67–1.75 (m, HbꢀC(3’)); 1.56–1.63 (m, HbꢀC(5’)); 0.60 (s,
Me3C); ꢀ0.15 (s, MeSi); ꢀ0.69 (s, MeSi). 13C-NMR (CDCl3): 169.95 (COO); 164.50 (NHCO); 152.07 (C(2));
151.85 (C(6)); 149.50 (C(4)); 143.03 (C(8)); 136.04, 132.66, 128.79, 128.62, 127.81, 127.164 (arom. C); 123.80
(C(5)); 82.45 (CH(OMe)Ph); 70.25 (C(4’)); 69.55 (C(2’)); 62.70 (C(1’)); 57.24 (MeO); 40.26 (C(3’)); 29.81
(C(5’)); 25.58 (C(6’)); 25.28 (Me3C); 17.37 (Me3C); ꢀ4.83, ꢀ5.90 (Me2Si). HR-MS: 616.2944 ([M+H]+, C33H42-
N5O5Si+; calc. 616.2955).
1
Data of 13b. H-NMR (CDCl3): 9.08 (s, NH); 8.76 (s, HꢀC(2)); 8.03 (d, J=7.6, 2 arom. H); 7.91 (s, Hꢀ
C(8)); 7.36–7.52 (m, 8 arom. H); 4.98–5.05 (m, HꢀC(4’)); 4.78 (s, CH(OMe)Ph); 4.26–4.33 (m, HꢀC(2’));
4.11–4.18 (m, HꢀC(1’)); 3.43 (s, CH(OMe)Ph); 2.54–2.63 (m, HaꢀC(6’)); 2.15–2.22 (m, HaꢀC(3’), Haꢀ
C(5’)); 2.04–2.10 (m, HbꢀC(6’)); 1.47–1.58 (m, HbꢀC(3’), HbꢀC(5’)); 0.58 (s, Me3C); ꢀ0.21 (s, MeSi); ꢀ0.70
(s, MeSi). 13C-NMR (CDCl3): 170.07 (COO); 164.48 (NHCO); 152.12 (C(2)); 151.89 (C(6)); 149.53 (C(4));
143.03 (C(8)); 136.07, 133.77, 132.69, 128.83, 128.64, 127.84, 127.16 (arom. C); 123.48 (C(5)); 82.65 (CH-
(OMe)Ph); 70.35 (C(4’)); 69.52 (C(2’)); 62.32 (C(1’)); 57.33 (MeO); 39.94 (C(3’)); 30.13 (C(5’)); 25.84
(C(6’)); 25.28 (Me3C); 17.36 (Me3C); ꢀ4.79, ꢀ5.86 (Me2Si). HR-MS: 616.2957 ([M+H]+, C33H42N5O5Si+;
calc. 616.2955).
(+)- and (–)-1-{2’-[(tert-Butyl)dimethylsilyloxy]-4’-[(R)-2-methoxy-2-phenylacetoxy]cyclohexyl}uracil (14a
and 14b, resp.). To a prechilled (08) mixture of 12 (1.52 g, 5.40 mmol), DMAP (60 mg, 0.49 mmol), and (R)-O-
methylmandelic acid (898 mg, 5.40 mmol) in CH2Cl2 (45 ml) was added DCC (1.11 g, 5.38 mmol). The mixture
was then stirred at r.t. for 4 h. After dilution with CH2Cl2, the solid was filtered and the filtrate was washed with
1N aq. H3PO4 soln. sat. aq. NaHCO3 soln., and H2O. The org. phase was dried (Na2SO4), filtered, and concen-
trated to dryness. The residue was purified by several CCs (hexane/AcOEt 40 :60 to 10 :90) to provide 938
mg (43%) of pure 14a, 851 mg (39%) of pure 14b, and 327 mg (15%) of a mixture 14a/14b.
1
Data of 14a. H-NMR ((D6)DMSO): 7.70 (br. s, HꢀC(6)); 7.35–7.39 (m, 5 arom. H); 5.58 (d, J=7.8, Hꢀ
C(5)); 4.90 (s, CH(OMe)Ph); 4.76–4.85 (m, HꢀC(4’)); 4.29 (br. s, HꢀC(1’)); 4.02 (br. s, HꢀC(2’)); 3.31 (s,
CH(OMe)Ph); 2.16–2.22 (m, HaꢀC(3’)); 1.73–1.79 (m, HaꢀC(5’), HaꢀC(6’)); 1.64–1.70 (m, HbꢀC(6’));
1.45–1.58 (m, HbꢀC(3’)); 1.29–1.40 (m, HbꢀC(5’)); 0.74 (s, Me3C); ꢀ0.01 (s, MeSi); ꢀ0.12 (s, MeSi). 13C-
NMR ((D6)DMSO): 169.82 (COO); 163.04 (C(4)); 151.28 (C(2)); 136.54 (C(6)); 128.57, 128.50, 127.10
(arom. C); 101.20 (C(5)); 81.42 (CH(OMe)Ph); 70.04 (C(4’)); 68.56 (C(2’)); 56.79 (MeO); 39.60 (C(3’) overlap-
ped with the signals of DMSO); 29.40 (C(5’)); 25.40 (Me3C); 24.50 (C(6’)); 17.31 (Me3C); ꢀ4.29, ꢀ5.16 (Me2Si).
HR-MS: 489.2426 ([M+H]+, C25H37N2O6Si+; calc. 489.2421).
Data of 14b. 1H-NMR ((D6)DMSO): 11.17 (s, NH); 7.71 (br. s, HꢀC(6)); 7.34–7.38 (m, 5 arom. H); 5.57 (d,
J=7.1, HꢀC(5)); 4.89 (s, CH(OMe)Ph ); 4.75–4.86 (m, HꢀC(4’)); 4.31 (br. s, HꢀC(1’)); 3.95 (br. s, HꢀC(2’));
3.31 (s, CH(OMe)Ph); 1.91–2.01 (m, HaꢀC(3’), HaꢀC(5’)); 1.67–1.75 (m, 2 HꢀC(6’)); 1.44–1.57 (m, Hbꢀ
C(5’)); 1.30–1.39 (m, HbꢀC(3’)); 0.71 (s, Me3C); ꢀ0.08 (s, MeSi); ꢀ0.15 (s, MeSi). 13C-NMR ((D6)DMSO):
169.89 (COO); 163.06 (C(4)); 151.32 (C(2)); 136.55 (C(6)); 128.59, 128.47, 127.11 (arom. C); 101.26 (C(5));
81.50 (CH(OMe)Ph); 70.10 (C(4’)); 68.61 (C(2’)); 56.81 (MeO); 41.18 (C(3’)); 29.60 (C(5’)); 25.38 (Me3C,
C(6’)); 17.29 (Me3C); ꢀ4.33, ꢀ5.17 (Me2Si). HR-MS: 489.2426 ([M+H]+, C25H37N2O6Si+; calc. 489.2421).
N6-Benzoyl-9-{2’-[(tert-butyl)dimethylsilyloxy]-4’-hydroxycyclohexyl}adenine (15a). To a prechilled (08)
soln. of 13a (180 mg, 0.29 mmol) in MeOH/dioxane (1.4 ml; 7 :10 (v/v)) was added 2N NaOH (430 ml). After
5 min. of stirring, the reaction was quenched with AcOH. The solvents were partially concentrated, and the res-
idue was partitioned between CH2Cl2 and H2O. The org. phase was washed with H2O, sat. aq. NaHCO3 soln.

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HELVETICA CHIMICA ACTA – Vol. 88 (2005)
(2×) and brine, dried (Na2SO4), filtered, and concentrated to dryness. The residue was purified by CC (CH2Cl2/
MeOH 98 :2 to 90 :10) to give 132 mg (96%) of 15a. Yellow oil. HR-MS: 468.2430 ([M+H]+, C25H34N5O3Si+;
calc. 468.2431).
N6-Benzoyl-9-{2’-[(tert-butyl)dimethylsilyloxy]-4’-hydroxycyclohexyl}adenine (15b). Compound 15b was
obtained by reaction of 13b with 2N NaOH as described for 15a in 66% yield as a yellow oil. HR-MS:
468.2426 ([M+H]+, C25H34N5O3Si+; calc. 468.2431).
1-{2’-[(tert-Butyl)dimethylsilyloxy]-4’-hydroxycyclohexyl}uracil (16a). Compound 16a was obtained by
reaction of 14a with 2
N NaOH as described for 15a in 94% yield as a white solid. HR-MS: 341.1888
([M+H]+, C16H29N2O4Si+; calc. 341.1896).
1-{2’-[(tert-Butyl)dimethylsilyloxy]-4’-hydroxycyclohexyl}uracil (16b). Compound 16b was obtained by
reaction of 14b with 2
N NaOH as described for 15a in 91% yield as a white solid. HR-MS: 341.1891
([M+H]+, C16H29N2O4Si+; calc. 341.1896).
N6-Benzoyl-9-{2’-[(tert-butyl)dimethylsilyloxy]-4’-[(4-methoxyphenyl)diphenylmethoxy]cyclohexyl}ade-
nine (17a). A soln. of 15a (250 mg, 0.53 mmol) and MMTrCl (518 mg, 1.68 mmol) in pyridine (6.6 ml) was heated
at 508 overnight. MMTrCl (173 mg, 0.56 mmol) was added again, and the same amount of MMTrCl was added
after 7 h at 508. The mixture was then stirred at r.t. for 2.5 days and concentrated to dryness. The residue was
dissolved in CH2Cl2, and washed with 0.1N HCl and brine. The org. phase was dried (Na2SO4), filtered, and con-
centrated to dryness. The residue was purified by CC (hexane/AcOEt 70 :30 to 10 :90) to give 350 mg (88%) of
1
17a. White foam. H-NMR (CDCl3): 8.99 (s, NH); 8.75 (s, HꢀC(2)); 8.00 (d, J=7.6, 2 arom. H); 7.85 (s, Hꢀ
C(8)); 7.21–7.60 (m, 15 arom. H); 6.85 (d, J=8.8, 2 arom. H); 4.02–4.10 (m, HꢀC(1’)); 3.87–3.95 (m, Hꢀ
C(2’)); 3.79 (s, MeO); 3.70–3.78 (m, HꢀC(4’)); 2.19–2.29 (m, HaꢀC(6’)); 1.81–1.90 (m, HbꢀC(6’)); 1.55–
1.68 (m, HaꢀC(3’), HaꢀC(5’)); 1.42–1.56 (m, HbꢀC(3’), HbꢀC(5’)); 0.52 (s, Me3C); ꢀ0.35 (s, MeSi); ꢀ0.79
(s, MeSi). 13C-NMR (CDCl3): 164.39 (NHCO); 158.63 (COMe); 151.99 (C(2)); 151.76 (C(6)); 149.37 (C(4));
145.59, 145.36 (arom. C); 143.31 (C(8)); 136.46, 132.65, 130.67, 128.83, 128.59, 128.49, 127.78, 127.74, 127.01
(arom. C); 123.42 (C(5)); 113.12 (arom. C); 86.65 (Ar3CO); 70.06 (C(4’)); 69.74 (C(2’)); 62.83 (C(1’)); 55.15
(MeO); 42.78 (C(3’)); 32.60 (C(5’)); 26.39 (C(6’)); 25.30 (Me3C); 17.33 (Me3C); ꢀ4.73, ꢀ5.95 (Me2Si). HR-
MS: 740.3631 ([M+H]+, C44H50N5O4Si+; calc. 740.3632).
N6-Benzoyl-9-{2’-[(tert-butyl)dimethylsilyloxy]-4’-[(4-methoxyphenyl)diphenylmethoxy]cyclohexyl}ade-
nine (17b). Compound 17b was obtained from 15b by reaction with MMTrCl in pyridine as described for 17a in
84% yield as a yellow oil. Spectroscopic data are the same as for 17a.
1-{2’-[(tert-Butyl)dimethylsilyloxy]-4’-[(4-methoxyphenyl)diphenylmethoxy]cyclohexyl}uracil (18a).
A
soln. of 16a (600 mg, 1.76 mmol) and MMTrCl (1.63 g, 5.28 mmol) in pyridine (22 ml) was heated at 508 over-
night. MMTrCl (543 mg, 1.76 mmol) was added again, and heating was continued for 24 h. The mixture was con-
centrated to dryness. The residue was dissolved in CH2Cl2 and washed with 0.1N HCl, sat. aq. NaHCO3 soln., and
brine. The organic phase was dried (Na2SO4), filtered and concentrated to dryness. The residue was purified by
FC (hexane/AcOEt 80 :20 to 10 :90) to afford 881 mg (82%) of 18a. White foam. 1H-NMR ((D6)DMSO): 11.10
(s, NH); 7.65 (br. s, HꢀC(6)); 7.29–7.46 (m, 12 arom. H); 6.90 (d, J=8.8, 2 arom. H); 5.49 (br. d, J=7.1, Hꢀ
C(5)); 4.14–4.25 (m, HꢀC(1’)); 3.79 (s, MeO); 3.54–3.63 (m, HꢀC(4’)); 3.47–3.54 (m, HꢀC(2’)); 1.56–1.63
(m, HaꢀC(5’)); 1.49–1.55 (m, HaꢀC(6’)); 1.37–1.46 (m, HbꢀC(5’), HbꢀC(6’)); 1.26–1.32 (m, HaꢀC(3’));
1.20–1.25 (m, HbꢀC(3’)); 0.66 (s, Me3C); ꢀ0.23 (s, MeSi); ꢀ0.26 (s, MeSi). 13C-NMR ((D6)DMSO): 163.04
(C(4)); 158.37 (COMe); 151.38 (C(2)); 145.69, 145.27 (arom. C); 141.72 (C(6)); 135.92, 130.43, 128.24,
128.04, 127.88, 127.80, 126.91, 113.12 (arom. C); 101.08 (C(5)); 86.03 (Ar3CO); 69.34 (C(2’), C(4’)); 57.83
(C(1’)); 55.04 (MeO); 42.43 (C(3’)); 32.23 (C(5’)); 25.40 (Me3C, C(6’)); 17.25 (Me3C); ꢀ4.26, ꢀ5.33 (Me2Si).
HR-MS: 613.3093 ([M+H]+, C36H45N2O5Si+; calc. 613.3098).
1-{2’-[(tert-Butyl)dimethylsilyloxy]-4’-[(4-methoxyphenyl)diphenylmethoxy]cyclohexyl}uracil (18b). Com-
pound 18b was obtained by reaction of 16b with MMTrCl in pyridine as described for 18a in quant. yield as a
yellow foam. Spectroscopic data are the same as for 18a.
N6-Benzoyl-9-{2’-hydroxy-4’-[(4-methoxyphenyl)diphenylmethoxy]cyclohexyl}adenine (19a). 1
M
Bu4NF in
THF (910 ml) was added to a soln. of 17a (330 mg, 0.45 mmol) in THF (3 ml). After 24 h at r.t., 1 Bu4NF
M
(450 ml) was added again, and stirring was continued overnight. The solvent was evaporated. The residue was
dissolved in Et2O, and washed with H2O and brine. The org. phase was dried (Na2SO4), filtered, and concen-
trated to dryness. The residue was purified twice by FC (CH2Cl2/MeOH 99 :1 to 98 :02) to give 226 mg
1
(81%) of 19a. White powder. [a]2D0 =ꢀ20.6 (c=0.5, CHCl3). H-NMR (CDCl3): 9.10 (br. s, NH); 8.52 (s, Hꢀ
C(2)); 8.00 (d, J=7.6, 2 arom. H); 7.72 (s, HꢀC(8)); 7.22–7.59 (m, 15 arom. H); 6.84 (d, J=8.8, 2 arom. H);
4.11–4.17 (m, HꢀC(1’)); 3.93–4.00 (m, HꢀC(2’)); 3.79 (s, MeO); 3.62–3.69 (m, HꢀC(4’)); 1.98–2.05 (m,
HaꢀC(3’)); 1.74–1.81 (m, 2 HꢀC(6’); 1.61 (q, J=11.5, HbꢀC(3’)); 1.31–1.41 (m, HaꢀC(5’)); 1.22–1.29 (m,

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HbꢀC(5’)). 13C-NMR (CDCl3): 164.69 (NHCO); 158.68 (COMe); 152.09 (C(2)); 151.83 (C(6)); 148.88 (C(4));
145.48, 145.38 (arom. C); 143.24 (C(8)); 136.56, 133.66, 132.71, 130.52, 128.77, 128.57, 127.97, 127.82, 126.99
(arom. C); 122.61 (C(5)); 88.68 (Ar3CO); 69.92 (C(4’)); 69.61 (C(2’)); 61.98 (C(1’)); 55.23 (MeO); 41.84
(C(3’)); 32.30 (C(5’)); 27.43 (C(6’)). HR-MS: 626.2768 ([M+H]+, C38H36N5O4þ ; calc. 626.2767).
N6-Benzoyl-9-{2’-hydroxy-4’-[(4-methoxyphenyl)diphenylmethoxy]cyclohexyl}adenine (19b). Compound
19b was obtained from 17b by reaction with 1M Bu4NF in THF as described for 19a in 84% yield as a white
solid. Spectroscopic data are the same as for 19a. [a]2D0 =+22.4 (c=0.5, CHCl3).
1-{2’-Hydroxy-4’-[(4-methoxyphenyl)diphenylmethoxy]cyclohexyl}uracil (20a). Compound 20a was
obtained by reaction of 18a with 1
M Bu4NF in THF as described for 19a in 61% yield as a white foam.
[a]2D0 =ꢀ18.8 (c=0.5, CHCl3). 1H-NMR (CDCl3): 10.00 (br. s, NH); 7.20–7.52 (m, 12 arom. H); 6.98 (d,
J=8.1, HꢀC(6)); 6.81 (d, J=8.5, 2 arom. H); 5.57 (d, J=8.1, HꢀC(5)); 4.15–4.26 (m, HꢀC(1’)); 3.77 (s,
MeO); 3.70 (br. s, HOꢀC(2’)); 3.43–3.51 (m, HꢀC(4’)); 3.34–3.42 (m, HꢀC(2’)); 1.97–2.03 (m, HaꢀC(3’));
1.57–1.66 (m, HbꢀC(3’), HaꢀC(6’)); 1.33–1.44 (m, HaꢀC(5’)); 1.12–1.29 (m, HbꢀC(5’), HbꢀC(6’)). 13C-
NMR (CDCl3): 163.62 (C(4)); 158.59 (COMe); 152.01 (C(2)); 145.43, 145.35 (arom. C); 141.17 (C(6));
136.51, 130.49, 129.19, 128.55, 127.82, 127.73, 126.91, 113.07 (arom. C); 102.34 (C(5)); 86.60 (Ar3CO); 70.05
(C(4’)); 69.07 (C(2’)); 61.38 (C(1’)); 55.17 (OCH3); 42.03 (C(3’)); 32.16 (C(5’)); 26.34 (C(6’)). HR-MS:
521.2058 ([M+Na]+, C30H30N2NaOþ5 ; calc. 521.2052).
1-{2’-Hydroxy-4’-[(4-methoxyphenyl)diphenylmethoxy]cyclohexyl}uracil (20b). Compound 20b was
obtained by reaction of 18b with 1M Bu4NF in THF as described for 19a in 70% yield as a white powder. Spec-
troscopic data are the same as for 20a. [a]2D0 =+19.8 (c=0.5, CHCl3).
General Procedure for the Synthesis of Phosphoramidite Building Blocks. A soln. of modified nucleoside
(see Table 1) in CH2Cl2 was treated with dry EtN(i-Pr)2 (3 equiv.) and 2-cyanoethyl-N,N-diisopropylchlorophos-
phoramidite (1.5 equiv.). After 45 min. at r.t., the mixture was cooled to 08, and the reaction was quenched by
addition of cold 5% aq. NaHCO3 soln. The mixture was stirred further for 10 min., and then diluted with CH2Cl2,
and washed with cold 5% aq. NaHCO3 soln. and brine (3×). The org. phase was dried (Na2SO4), filtered, con-
centrated to dryness and purified by CC (hexane/acetone/1% pyridine). Appropriate fractions were combined,
concentrated to dryness, and co-evaporated with toluene (3×) and CH2Cl2 to give the phosphoramidite as an oil.
Dissolution in 1.5 ml of CH2Cl2 and double precipitation in 160 ml of cooled (ꢀ708) hexane afforded the desired
product as a powder. Starting quantities, yields, mass analysis, and 31P-NMR data are given in Table 2.
Table 2. Analytical Data for the Phosphoramidite Derivatives
Compound
mmol of starting materials
Yield (%)
HR-MS [M+H]+
31P-NMR in CDCN3
Calc.
Found
19a
19b
20a
20b
0.29
0.19
0.36
0.36
83
83
88
78
for C47H53N7O5P
148.47, 147.48
148.46, 147.46
148.66, 146.96
148.65, 146.96
826.3845
826.3843
for C47H53N7O5P
826.3845
826.3835
for C39H48N4O6P
699.3311
699.3317
for C39H48N4O6P
699.3311
699.3322
Solid-Phase Oligonucleotide Synthesis and Analysis. Oligonucleotide synthesis was performed on an Expe-
diteTM DNA synthesizer (Applied Biosystems) by using the phosphoramidite approach. The standard DNA
assembly protocol was used with 0.25
M ETT as the activator and a 5-min coupling time using 0.07M of the
newly synthesized amidites. A universal 3’-phosphate CPG-support was used, prepared according to Kumar
[23], to afford 3’-phosphorylated oligonucleotides. The oligomers were deprotected and cleaved from the
solid support by treatement with conc. aq. NH3 (558, 16 h). After gel filtration on a NAP-10® column (Sephadex
G25-DNA grade; Pharmacia) with H2O as eluent, the crude was analyzed on a Mono-Q® HR 5/5 anion-
exchange column, after which purification was achieved on a Mono-Q® HR 10/10 column (Pharmacia) with
the following gradient system (A=10 m
M NaOH, pH 12.0, 0.1M NaCl; B=10 mM NaOH, pH 12.0, 0.9M NaCl;
gradient used depended on the oligomers; flow rate: 2 ml/min). The low-pressure liquid chromatography system

3224
HELVETICA CHIMICA ACTA – Vol. 88 (2005)
consisted of a Merck-Hitachi L 6200 A intelligent pump, a Mono Q® HR 10/10 column (Pharmacia), a Uvicord
SII 2138 UV detector (Pharmacia-LKB), and a recorder. The product-containing fraction was desalted on a
NAP-10® column and lyophilized.
Oligonucleotides were characterized, and their purity was checked by HPLC/MS on a cap. chromatograph
(CapLC, Waters, Milford, MA). Columns of 150 mm×0.3 mm length (LCPackings, San Francisco, CA) were
used. Oligonucleotides were eluted with a triethylammonium-1,1,1,3,3,3-hexafluoropropan-2-ol MeCN solvent
system. Flow rate was 5 ml/min. Electrospray spectra were acquired on an orthogonal acceleration/time-of-flight
mass spectrometer (Q-TOF-2, Micromass, Manchester, UK) in negative-ion mode. Scan time used was 2 s. The
combined spectra from a chromatographic peak were deconvoluted using the MaxEnt algorithm of the software
(Masslynx 3.4, Micromass, Manchester, UK). Theoretical oligonucleotide masses were calculated using the
monoisotopic element masses.
The authors thank Dr. Marie-Jesús Pérez-Pérez for [a]D determinations, and the K.U. Leuven for financial
support (I.D.O.).
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Received September 1, 2005