Synthesis of enantiomeric-pure cyclohexenyl nucleoside building blocks for oligonucleotide synthesis

Article abstract of DOI:10.1016/j.tet.2003.12.052

Lipases were used for the resolution of (±) (4aR, 7R, 8aS)-2-phenyl-4a,7,8,8a-tetrahydro-4H-1,3-benzodioxine. This separation was carried out on preparative scale and used for the synthesis of eight phosphoramidites of cyclohexenyl nucleosides (D- and L-series).

Full text of DOI:10.1016/j.tet.2003.12.052

Tetrahedron 60 (2004) 2111–2123
Synthesis of enantiomeric-pure cyclohexenyl nucleoside building
blocks for oligonucleotide synthesis
Ping Gu,a Carsten Griebel,b Arthur Van Aerschot,a Jef Rozenski,a Roger Busson,a
Hans-Joachim Gaisb and Piet Herdewijna
,
*
aLaboratory for Medicinal Chemistry, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium
¨ ¨
Institut fur Organische Chemie, Rheinische-Westfalische Technische Hochschule Aachen, Professor.-Pierlet-Strasse, 1,
b
52056 Aachen, Germany
Received 22 July 2003; revised 19 December 2003; accepted 19 December 2003
Abstract—Lipases were used for the resolution of (^) (4aR, 7R, 8aS)-2-phenyl-4a,7,8,8a-tetrahydro-4H-1,3-benzodioxine. This separation
was carried out on preparative scale and used for the synthesis of eight phosphoramidites of cyclohexenyl nucleosides (D- and L-series).
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
CeNA/RNA duplex could be recognized by RNase H,
resulting in RNA strand cleavage in serum.9,10
Since nucleic acids with a six-membered carbohydrate
moiety in the backbone are potentially more preorganized
than furanose-type nucleic acids, they might have an
entropic advantage during hybridization and, hence, could
form more stable duplexes.1 This is exemplified with hexitol
nucleic acids (HNA)2 and with altritol nucleic acids (ANA)3
that demonstrate strong self-complementary hybridization
and hybridization with ribonucleic acids (RNA). Their
carbocyclic congeners, cyclohexane nucleic acids (CNA),
also hybridize with natural nucleic acids.4 Hexitol nucleic
acids, however, have the disadvantage that they are poorly
recognized by nucleic acids metabolizing enzymes which
might be ascribed to their rigidity.5 Therefore, we developed
nucleosides and oligonucleotides based on a cyclohexene
system. These nucleosides are more flexible than the
anhydrohexitol nucleosides and are better mimics of a
furanose nucleoside.6 D-Cyclohexenyl guanine (Cycl-G)
has been evaluated against a whole range of herpes viruses
and its activity was comparable with those of acyclovir
(ACV) and ganciclovir (GCV).7 (^)Cyclohexenyl cytosine
is a potent anti-VZV compound.8 Cyclohexenyl adenine
(Cycl-A) has been incorporated into oligonucleotides.9
Compared to a single stranded DNA oligomer, the affinity
of these cyclohexene nucleic acids (CeNAs) for comple-
mentary DNA sequences diminished slightly, while an
increase was noticed for RNA complements. Moreover, a
However, the difficulty to synthesize chiral cyclohexene
derivatives in high enantiomeric excess and in bulk
quantities has hampered the further study of cyclohexenyl
nucleosides and their oligonucleotides. It has been
recognized that D- and L-nucleotides have different
properties, exemplified by the findings that D-CNAs
hybridize with natural nucleic acids and are RNA-selective
while L-CNA hybridize either very weakly or not at all with
natural nucleic acids.4 Recently, a novel and facile method
to prepare D- and L-cyclohexenyl nucleosides has been
reported.11 The resolution of these analogues via the
formation of diastereomeric esters with (R)-(2)-methyl-
mandelic acid is, however, a multistep and tedious work
(difficult chromatographic separation), and expensive on
large scale.
Therefore we developed a method for the separation of the
enantiomers of a racemic intermediate of the synthetic
scheme leading to cyclohexenyl nucleosides via an enzyme-
catalyzed resolution strategy.12 This method enables
synthesis of the cyclohexenyl nucleosides with all four
nucleobases. Further derivatization into their phosphor-
amidite derivatives afforded suitable building blocks for
DNA synthesis as depicted in Figure 1. Here we defined
the compounds or their derivatives as an ‘a’-type or
‘b’-type depending on the type of 0 ena0ntiomer that is
considered. The ‘a’-type has the 10S, 4 R, 5 S configuration,
overall resembling the D-anhydrohexitol series, while
the ‘b’-type has the mirror configuration with 10R, 40S, 50R
(Fig. 2).
Keywords: Cyclohexenyl nucleosides; Enzymatic resolution;
Phosphoramidite; Mitsunobu reaction.
*
Corresponding author. Tel.: þ32-16-337387; fax: þ32-16-337340;
e-mail address: piet.herdewijn@rega.kuleuven.ac.be
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.12.052

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P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
Figure 1. Cyclohexenyl nucleoside building blocks for DNA synthesis.
2. Results and discussion
5 could be envisaged, i.e. kinetic resolution using Sharpless
epoxidation,13 enzymatic resolution14 or formation of
diastereomeric esters.5 The previous study on enzymatic
resolution of 5 using vinyl acetate and Lipase PS (Amano)
was not successful,11 and gave only 33% of enantiomeric
excess. We started our study with transesterification by
screening three different lipases, Novozymew 435, CRL
2.1. Enzymatic resolution of the racemic intermediate 5
For the synthesis of cyclohexenyl nucleosides, racemic
compound 5 is the key intermediate for the subsequent
introduction of nucleobases. Several methods for resolving

P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
2113
(5 equiv.) catalysed by 14% (w/w) of enzymes at room
temperature to give enantiomeric pure product ent-6b and
enantiomeric pure substrate ent-5a (Table 1, Fig. 3). Vinyl
propionate was used as organic solvent in individual
reactions. We also examined toluene as organic solvent in
each reaction (data not shown here), but found that toluene
could neither improve the selectivity nor accelerate the
reaction. Although none of the evaluated enzymes were
synthetically useful, use of Novozymew 435 showed the
best selectivity (E¼14).
We further investigated the Novozymew 435-catalyzed
transesterification of rac-5 with formation of ent-7b and ent-
5a with different acetyl donors (Table 1). Vinyl acetate
(VA) and isopropenyl acetate (IPA) were respectively used
both as acetyl donor and as organic solvent. A moderate
selectivity as expressed in an E value of 22 was obtained
with isopropenyl acetate. Under these circumstances, the
reaction reached a point of conversion of 49% within 20 h.
Figure 2. Nomenclature used for cyclohexenyl compounds in this
manuscript.
(Candida rugosa lipase) and PCL (Pseudomonas cepacia
lipase). Novozymew 435 is a member of the lipases of
Candida antarctica B, and is particularly useful in the
synthesis of esters and amides, and has a broad substrate
specificity (Fig. 3).12
Having obtained improved results by using Novozymew 435
and isopropenyl acetate, we tried to increase the enantio-
selectivity by further changing the reaction conditions
(Table 1). We did not examine a temperature effect on
selectivity since testing-scale reactions would not generate
much heat which otherwise might have significant
influence on temperature. Toluene, octane, chloroform and
The transesterification reactions were carried out using a test
amount of rac-5 (70 mg) and vinyl propionate (VP)
Figure 3. Transesterification of rac-5 with different enzymes.
Table 1. Transesterification of rac-5 with vinyl propionate in the presence of different enzymes, with different acetyl donors and under various conditions using
Novozymew 435 and isopropenyl acetate
Reagents
Enzymes
Conditions solvent /additive
Conversion (%)
Time (h)
e.e.s (%)
e.e.p (%)
E
Vinyl propionate
Vinyl propionate
Vinyl propionate
Vinyl acetate
Isopropenyl acetate
Isopropenyl acetate
Isopropenyl acetate
Isopropenyl acetate
Isopropenyl acetate
Isopropenyl acetate
Isopropenyl acetate
Novozymew 435
CRL
–/–
–/–
–/–
–/–
30
49
45
66
49
64
59
41
49
71
60
3
37
51
40
79
93
65
62
69
82
35
46
39
74
47
29
86
84
49
70
14
3
4
5
22
7
27
27
43
20
43
20
24
20
20
20
PCL
Novozymew 435
Novozymew 435
Novozymew 435
Novozymew 435
Novozymew 435
Novozymew 435
Novozymew 435
Novozymew 435
–/–
Toluene/–
Octane/–
CHCl3/–
CH2Cl2/–
CH2Cl2/Et3N
–/Et3N
3
28
50
19
20
95
.99
95

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P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
dichloromethane were examined as organic solvent for the
transesterification reactions. Both the reactions using
chloroform and dichloromethane as organic solvent
exhibited good (E¼28) to excellent (E¼50) selectivity.
Attempts were tried to add some additives such as
triethylamine, which might change the pH environment
and thus the enzymatic activity, which could lead to further
improvement of selectivity. The results shown in Table 1,
however, revealed that triethylamine enhanced the reaction
rate but had negative effect on the selectivity. A test reaction
with vinyl butyrate (data not shown here) using the same
conditions afforded the highest selectivity (E¼127) but with
a relatively slow reaction rate (52% conversion, 39 h),
which is not favored for economical and practical reasons.
This phenomenon was also observed for the hydrolysis of
rac-6 and rac-8. We then turned our attention to the
hydrolysis of different racemic esters rac-6, rac-8 (Fig. 4,
Table 2). As seen from the table, the hydrolysis reaction was
best conducted by PLE-catalysed hydrolysis of rac-8 in a
0.1 M phosphate buffer solution at pH 8.0 in the presence of
10% (v/v) t-BuOH affording ent-5a and ent-8b with a
selectivity of (E¼45). The resolution might be further
optimized by changing cosolvent, i.e. acetone or t-BuOMe.
However, all enzyme-catalyzed hydrolysis reactions had a
much lower reaction rate than transesterifications. This
could be explained as the former reaction was carried out in
heterogeneous phases.
In view of the obtained results, enzyme-catalyzed trans-
esterification was finally adopted for the separation of the
racemic intermediate 5 considering both economical and
practical aspects.
We also investigated the enzyme-catalyzed (PLE, CRL,
CAL-B) hydrolysis of rac-7, which gave the product ent-5a
and the substrate ent-7b (Fig. 4, Table 2).
None of the enzyme-catalyzed hydrolysis reactions showed
useful selectivity as well as good enzymatic activity. The
PLE-catalyzed hydrolysis of the racemic ester 7 at room
temperature in a 0.1 M phosphate buffer (pH 8.0) in the
presence of 10% (v/v) acetone proceeded somewhat faster
than in the presence of 10% (v/v) t-BuOH, but did not
improve the selectivity. CRL-catalyzed hydrolysis excep-
tionally gave ent-5b as the product and ent-7a as the
residual substrate, which indicates that the enzyme may
favor the ‘b-type’ configuration of the racemic substrate.
2.2. Separation and purification of enantiomers of rac-5
The overall procedure for separation and purification of
racemic 5 is shown in Figure 5. The transesterification was
carried out with 14% (w/w) of Novozymew 435 catalyzed
acylation of racemic substrate rac-5 using 5 equiv. of
isopropenyl acetate as the acetyl donor in 50 volumes of
dichloromethane at room temperature. Chiral high perform-
ance liquid chromatography (HPLC)11 was used to follow
the reaction to determine the end point. The reaction was
Figure 4. Hydrolysis of different racemic esters rac-6 rac-7 and rac-8.
Table 2. Hydrolysis of rac-6, rac-7 and rac-8 with different enzymes
Substrates (R¼)
Enzymes
Conditions cosolvent/pH
Conversion (%)
Time (h)
e.e.s (%)
e.e.p (%)
E
–CH3
–CH3
–CH2CH3
–CH2CH2CH3
–CH3
–CH2CH3
–CH2CH2CH3
–CH3
PLE
PLE
PLE
PLE
CRL*
CRL*
CRL*
CAL-B
CAL-B
t-BuOH/8.0
Acetone/8.0
t-BuOH/8.0
t-BuOH/8.0
t-BuOH/7.5
t-BuOH/7.5
t-BuOH/7.5
t-BuOH/7.5
t-BuOH/7.5
11
44
35
13
17
85
27
21
21
22
24
17
10
24
24
4
,1
27
40
9
26
30
24
10
15
30
46
68
95
65
2
11
16
63
2
4
8
45
6
1
2
1
4
25
30
–CH2CH2CH3

P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
2115
Figure 5. Procedure for separation and purification of enantiomers of 5.
stopped after 22 h (52% conversion) and optically pure
substrate 5a (e.e.s.97%) and product 7b (e.e.p.85%) were
obtained, which were subsequently separated easily by
column chromatography.
with a selectivity of (E¼2) and (E¼50), respectively. The
identification of the obtained optically pure 5a and 5b are
shown in (D) and (E), respectively.
In general, compared with enzyme-catalyzed hydrolysis and
other described chemical methods, Novozymew 435-
catalyzed transesterification is the most facile approach to
achieve the highest selectivity with economical and
practical advantages.
Following two recrystallizations from 50% of ethyl acetate
in n-hexane, a colorless needle crystal of the enantiomeric
pure 5a with an enantiomeric excess value (e.e.) of over
99% was obtained in an overall yield of 44% (calculation
based on rac-5). The absolute configuration was established
via comparison of its guanine nucleoside derivative with an
authentic sample which was described in the literature.11
High optical purity (e.e..97%) for the isolated product of
ent-7b was achieved by recrystallizing twice from 20%
ethyl acetate in n-hexane. Subsequently ent-7b was further
treated with saturated ammonia/methanol solution to afford
ent-5b. Likewise, recrystallized from 50% of ethyl acetate
in n-hexane, 5b was afforded as a colorless needle with e.e.
value of over 99% in an overall yield of 43%.
2.3. Synthesis of cyclohexenyl nucleoside building blocks
For investigation of the properties of cyclohexenyl contain-
ing nucleic acids, the enantiomeric pure protected phos-
phoramidite nucleosides of the four natural nucleobases
were synthesized independently. The synthesis started with
the previously separated enantiomerically pure (e.e..99%)
cyclohexene precursors 5a and 5b. The purine base moieties
(adenine or 2-amino-6-chloropurine) were introduced via a
direct nucleophilic displacement strategy using the
Mitsunobu condensation reaction,15 to give nucleosides
9a,b (yield of 45%) and 10a,b (yield of 37%)7,11 (Fig. 7).
The adenine base of 9a,b was thereafter protected by the
benzoyl protecting group, followed by removal of the
Figure 6 shows the enzymatic separation of the racemic
mixture of 5 by chiral chromatography. A 1:1 mixture of 5a
and 5b is shown in (A), while (B) and (C) show the results
for enzyme-catalyzed hydrolysis and transesterification

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P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
Figure 6 (legend opposite)

P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
2117
Figure 7. Synthesis for phosphoramidite building blocks 1–4 (a- and b-series). (i) Mitsunobu reaction; (ii) BzCl, pyridine for A and C; for G: first TFA/H2O
(80%), then TMS-Cl, isobutyric anhydride, pyridine, 0 8C rt for G; (iii) TFA/H2O (80%), 40 h; (iv) MMTrCl, pyridine, rt; (v) (iPr)2N(CE)PCl, (iPr)2NEt,
CH2Cl2.
benzylidene protecting group using 80% TFA/H2O solution
at room temperature for 2 days to give 11a,b in 48% yield.
The congeners 10a,b on the other hand were first treated
with 80% TFA/H2O solution for 40 h, followed by
protection of the 2-NH2 position with the isobutyryl group
via a transient protection approach to afford 12a,b in 32%
yield.16 The monomethoxytritylation of 11a,b and 12a,b at
the primary hydroxyl groups (40-CH2OH) was less straight-
forward. The bis-tritylated product as well as the mono-
tritylated product at the secondary alcohol (50-OH) group
were likewise obtained in a total yield of 20–30%. These
side reactions could be avoided by starting the reaction at
0 8C and gradually raising the temperature to room
temperature with careful control of the reaction time by
monitoring with TLC. The secondary hydroxyl groups
(50-OH) of 13a,b and 14a,b were further reacted with
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite to
yield enantiomeric pure 1a,b and 2a,b (yield of 86–92%
and 66–74%, respectively), as the nucleotide building
blocks.
Figure 6. Enzymatic resolution of the racemic mixture of 5. (A) Chromatogram for the racemic mixture of 5. Chromatographic conditions: Chiralpak AD
(250£4.6 mm) Mobile phase: n-hexane/EtOH 98:2, Detection: UV220 nm; Flow rate: 1.0 mL/min; Injection volume: 10 mL. Peak 3¼5a, peak 4¼5b.
(B) Analysis of the PLE-catalyzed hydrolysis reaction of rac-8. Chromatographic conditions: see (A). Peak 1¼8a, peak 2¼8b, peak 3¼5a, peak 4¼5b.
(C) Analysis of the Novozymew-catalyzed transesterification of rac-5.Chromatographic conditions: see (A). Peak 1¼6a, peak 2¼6b, peak 3¼5a. (D) Analysis
of 6b obtained following column chromatography of the Novozymew-catalyzed transesterification reaction of rac-5. Chromatographic conditions: see (A).
Peak 1¼6a, peak 2¼6b. (E) Analysis of 5a obtained following column chromatography of the Novozymew-catalyzed transesterification reaction of rac-5.
Chromatographic conditions: see (A). Peak 3¼5a.

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P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
Alkylation of pyrimidine bases (cytosine and thymine) is
more problematic than that of purines. The thymine base
could be directly coupled to the sugar part by the Mitsunobu
procedure. When the benzylidene protecting groups were
removed the unprotected thymine nucleoside analogues 16a
and 16b (overall yield of 16%) were obtained which were
monotritylated at the primary hydroxyl group to afford 17a
and 17b (yield of 59%). The secondary hydroxyl groups
(50-OH) were then phosphitylated to obtain the phosphor-
amidites, 4a and 4b (81–85% yield). The cytosine nucleo-
side 19a,b (overall yield 41%) could be obtained starting
from the uracil congener 18a,b.17 Following protection of
the cytosine base with a benzoyl group, the benzylidene
group was removed in acidic medium, to obtain the
cyclohexenyl cytosine nucleosides 20a,b (yield of
16–21%). Finally, following the tritylation at 40-OH,
compounds 21a,b was converted to the protected phosphor-
amidite building block 3a,b (yield of 77–87%).
The two enantiomers of the compound 11 give similar
spectroscopic data.
3. Conclusion
In summary, enzymatic resolution of the racemic 5 by
employing a transesterification reaction with Novozymew
435 allows the isolation of optically pure enantiomers in
preparative scale. In comparison with other chemical
methods,11 the developed method is highly efficient, easy
to perform and economical for multigram preparations. The
different enantiomeric series of nucleoside phosphoramidite
derivatives with the four natural nucleobases were syn-
thesized as building blocks and they will be used for the
synthesis of the corresponding nucleic acids analogues
(CeNA).9
The identification of the protected cyclohexenyl nucleosides
and their enantiomers are exemplified with compounds 11a
and 11b. Their structures were deduced from 1H NMR
analysis. Table 3 shows the chemical shifts and coupling
constants for all protons. The b-conformation was pre-
viously confirmed by 1H NMR analysis of its racemic
unprotected congener.60 Here the small value of the coupling
4. Experimental
All solvents used for reactions are analytical grade or
freshly distilled. 1,4-Dioxane was refluxed on sodium/
benzophenone and distilled. Anhydrous pyridine was
refluxed on potassium hydroxide and distilled. PLE
(40 KU/306 mg, suspension in 3.2 M (NH4)2SO4) solution
was purchased from Roche Diagnostics. CRL (26 U/mg),
PCL (40 U/mg) were purchased from Fluka. CAL-B
constant between H-2 and H-10 (J2 -1 ¼2.4 Hz) might also
0
0
indicate that the base moiety occupies the axial position.
Table 3. 1H NMR chemical shifts (d) and coupling constants (J) of 11a and 11b
Proton
Coupled to proton
11a
11b
d (ppm)
5.47
5.93
J (Hz)
d (ppm)
5.45
5.94
J (Hz)
10(m)
20(ddd)
40
10
30
40
20
1.7
2.4
10.0
2.5
1.5
2.4
9.9
2.7
9.9
30(dd)
6.13
6.07
10.0
40(m)
50(m)
2.50
3.81
2.50
3.80
60eq
40
60ax
50
2.9
5.7
9.6
4.4
2.9
5.7
9.6
–
500-OH (d)
6 (m)
HOCH2a-(m)
4.74
2.03–2.37
3.53
4.75
2.01–2.41
3.54
40
5.1
10.3
5.4
10.6
5.4
5.1
10.3
5.3
10.6
–
HOCH2b
40
HOCH2a
–
–
HOCH2b-(m)
3.61
3.60
–OH (t)
2 (s)
8 (s)
HOCH2–
4.68
8.21
7.97
7.26–7.60
4.68
8.24
7.91
7.25–7.57
Bz

P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
2119
(Chirazymew L-2, lyo, 120 U/mg) was purchased from
Boehringer Mannheim. Novozymew 435 (10 U/mg) was
donated by Novo-Nordisk A/S. Enzymatic reactions were
run at room temperature. Enantiomer compositions were
determined by chiral HPLC analysis with a Chiralpak AD
column (250£4.6 mm) on a Waters 6000 controller liquid
chromatograph equipped with a Waters 2487 UV detector.
1H NMR was determined with a 200 MHz Varian Gemini
spectrometer with tetramethylsilane (TMS) as internal
With CRL. The reaction was carried out with rac-5 (70 mg,
0.30 mmol) and vinyl propionate (5 mL, 46.8 mmol). CRL
(10 mg, 260U) was added under the above conditions. The
reaction was stopped at 27 h (49% conversion) by filtration.
Chiral HPLC analysis of a sample from the filtrate showed
the presence of 6b ent-product with 35% e.e. and of 5a ent-
substrate with 51% e.e.
With PCL. The reaction was carried out with rac-5 (70 mg,
0.30 mmol) and vinyl propionate (5 mL, 46.8 mmol). PCL
(10 mg, 400U) was added under the above conditions. The
reaction was stopped at 27 h (45% conversion) by filtration.
Chiral HPLC analysis of a sample from the filtrate showed
the presence of 6b ent-product with 46% e.e. and of 5a ent-
substrate with 40% e.e.
1
standard for H NMR spectra and the same apparatus was
used for 13C NMR determination with DMSO-d6
(39.6 ppm) or CDCl3 (76.9 ppm) as internal standard for
the 13C NMR spectra (s¼singlet, d¼doublet, dd¼double
doublet, t¼triplet, br s¼broad singlet, br d¼broad doublet,
m¼multiplet). 31P NMR spectra was obtained as 85%
H3PO4 as external standard. Exact mass measurements were
performed on a quadrupole time-of-flight mass spectrometer
(Q-Tof-2, Micromass, Manchester, UK) equipped with a
standard electrospray-ionization (ESI) interface; samples
were infused in i-PrOH/H2O 1:1 at 3 mL/min column
chromatography was performed on ICN silica gel 63–
With vinyl acetate. rac-5 (70 mg, 0.30 mmol) was dissolved
in vinyl acetate (5 mL, 54 mmol) and Novozymew 435
(10 mg, 100U) was subsequently added at room tempera-
ture. The reaction was stopped at 43 h (66% conversion) by
filtration. Chiral HPLC analysis of a sample from the
mixture showed the presence of 7b ent-product with 39%
e.e. and of 5a ent-substrate with 79% e.e.
˚
200 mm, 60 A. Precoated aluminium sheets (Fluka Silica
gel/TLC-cards, 254 nm) were used for TLC; the spots were
examined with UV.
With isopropenyl acetate. rac-5 (70 mg, 0.30 mmol) with
isopropenyl acetate (145 mg, 1.5 mmol) and Novozymew
435 (10 mg, 100U) was added under the above conditions.
The reaction was stopped at 20 h (49% conversion) by gel
filtration. HPLC analysis of a sample from the filtration
showed the presence of 7b ent-product with 74% e.e. and 5a
ent-substrate with 93% e.e.
Chiral HPLC analysis for determination of enantiomer
composition: Mobile phase: n-hexane/EtOH 98:2, flow rate:
1 mL/min, UV wavelength: 220 nm, tR (1)¼18.7, tR
(2)¼28.1, tR (3)¼43.8, tR (4)¼83.5 min.
4.1. Transesterification reactions of rac-5
The selectivity of the reaction is expressed as the
enantiomeric ratio (E), which mathematically links to the
conversion (c) of the reaction,18 and the optical purities of
substrate (e.e.s) and product (e.e.p). The dependence of the
selectivity and the conversion of the reaction is:
The procedures for transesterification of rac-5 with various
solvents and additives are given here exemplified by using
CH2Cl2 and CH2Cl2/Et3N.
With CH2Cl2. rac-5 (70 mg, 0.30 mmol) and isopropenyl
acetate (145 mg, 1.5 mmol) were dissolved in CH2Cl2
(5 mL). Subsequently, Novozymew 435 (10 mg, 100U) was
added and the reaction mixture was stirred at room
temperature. The reaction was stopped at 20 h (49%
conversion) by filtration. Chiral HPLC analysis of a sample
from the mixture showed the presence of 7b ent-product
with 84% e.e. and of 5a ent-substrate with 95% e.e.
for the product
ln½1 2 cð1 þ e:e:pÞꢀ
for the substrate
ln½ð1 2 cÞð1 2 e:e:sÞꢀ
E ¼
E ¼
ln½1 2 cð1 2 e:e:pÞꢀ
ln½ð1 2 cÞð1 þ e:e:sÞꢀ
where c, conversion; e.e., enantiomeric excess of substrate
(S) or product (P); E, enantiomeric ratio.
With CH2Cl2/Et3N. rac-5 (70 mg, 0.30 mmol), isopropenyl
acetate (145 mg, 1.5 mmol) and Et3N (10.6 mL, 75 mmol)
were dissolved in CH2Cl2 (5 mL). Subsequently,
Novozymew 435 (10 mg, 100U) was added and the reaction
mixture was stirred at room temperature. The reaction was
stopped at 20 h (71% conversion) by filtration. Chiral HPLC
analysis of a sample from the mixture showed the presence
of 7b ent-product with 49% e.e. and of 5a ent-substrate with
.99% e.e.
The following equation is recommended instead because
only values for the optical purities of substrate and the
product need to be measured.19
0
B
B
@
1
C
C
A
0
B
B
@
1
C
C
A
1 2 e:e:s
e:e:s
1 þ e:e:s
E ¼ ln
=ln
e:e:s
1 þ
1 þ
e:e:p
e:e:p
With Novozymew 435. rac-511 (70 mg, 0.30 mmol) and
vinyl propionate (5 mL, 46.8 mmol) were mixed. Sub-
sequently, Novozymew 435 (10 mg, 100U) was added and
the reaction mixture was stirred at room temperature. The
reaction was stopped at 3 h (30% conversion) by filtration.
Chiral HPLC analysis of a sample from the filtrate showed
the presence of 6b ent-product with 82% e.e. and of 5a ent-
substrate with 37% e.e.
4.1.1. (6)-(4aR,7R,8aS)-2-Phenyl-4a,7,8,8a-tetrahydro-
4H-1,3-benzodioxin-7-yl acetate (rac-7). To a mixture of
rac-5 (0.72 g, 3.1 mmol) in pyridine (10 mL), acetyl
chloride (0.35 mL, 4.9 mmol) was added at 0 8C and the
mixture was stirred at room temperature for 6 h. The
reaction was poured into saturated aqueous NaHCO3 and
stirred for half-an-hour. The mixture was extracted with
CH2Cl2 and the organic layer was separated, dried over

2120
P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
anhydrous Na2SO4 and co-evaporated with toluene in
vacuo. The crude was purified by column chromatography
(EtOAc/n-hexane, 0–30%) to afford 0.78 g of rac-7 (yield
94%).
extracted continuously with CH2Cl2 (50 mL) for 17 h
(Soxlet apparatus). The organic phase was collected, dried
over anhydrous Na2SO4 and concentrated in vacuo. Chiral
HPLC analysis of a sample of the residue showed the
presence of 5a ent-product with 30% e.e. and of 7b ent-
substrate with ,1% e.e. for (a); and of 5a ent-product with
46% e.e. and of 7b ent-substrate with 27% e.e. for (b).
1H NMR (CDCl3) d 1.88–1.99 (m, 1H, H-8), 2.09 (s, 3H,
–CH3), 2.48–2.69 (m, 2H, H-8, H-4a), 3.59–3.80 (m, 2H,
4-CH2O–), 4.28 (dd, 1H, J¼10.8, 4.6 Hz, H-7), 5.50–5.72
(m, 3H, H-8a, H-5, H-6), 5.62 (s, 1H, 2-CHPh), 7.27–7.51
(m, 5H, aromatic-H) ppm; 13C NMR (CDCl3) d 20.8
(–CH3), 33.9 (C-8), 39.5 (C-4a), 69.5 (C-7), 70.1
(4-CH2O–), 76.1 (C-8a), 101.8 (2-CHPh), 125.9, 126.7,
128.1, 128.4, 128.7, 137.8 (aromatic-C) 170.3 (–OCO–)
ppm. LISMS (CH3OH/H2O) 275.1 (MþH)þ.
With CRL. The hydrolysis was carried out with rac-7
(200 mg, 0.73 mmol), t-BuOH (4 mL) and CRL (5 mg,
130U) in aqueous phosphate buffer solution (36 mL, pH 8.0,
adjusted by 1 N HCl). Work-up was done as described
above after 24 h (17% conversion). Chiral HPLC analysis of
a sample of the residue showed the presence of 5b ent-
product with 65% e.e. and of 7a ent-substrate with 26% e.e.
4.1.2. (6)-(4aR,7R,8aS)-2-Phenyl-4a,7,8,8a-tetrahydro-
4H-1,3-benzodioxin-7-yl propionate (rac-6). rac-5
(0.72 g, 3.1 mmol) in pyridine (10 mL) was reacted with
propionyl chloride (0.39 mL, 4.5 mmol) using the previous
procedure to afford 0.78 g (yield 90%) of rac-6 as a
colorless oil.
With CAL-B. The hydrolysis was carried out with rac-7
(200 mg, 0.73 mmol), t-BuOH (4 mL) and CAL-B (20 mg,
2.4 KU) in phosphate buffer solution (36 mL, pH 7.5,
adjusted by 1 N HCl). Work-up as described above after
25 h (21% conversion). Chiral HPLC analysis of a sample of
the residue showed the presence of 5a ent-product with 16%
e.e. and of 7b ent-substrate with 10% e.e.
1H NMR (CDCl3) d 1.11 (t, 3H, –CH2CH3), 1.82–1.94 (m,
1H, H-8), 2.28 (q, 2H, –CH2CH3), 2.21–2.47 (m, 2H, H-8,
H-4a), 3.59–3.92 (m, 2H, 4-CH2O–), 4.54 (dd, 1H, J¼10.7,
4.2 Hz, H-7), 5.53–5.61 (m, 3H, H-8a, H-5, H-6), 5.59 (s,
1H, 2-CHPh), 7.39–7.51 (m, 5H, aromatic-H) ppm; 13C
NMR (CDCl3) d 9.04 (–CH2CH3), 27.6 (–CH2CH3), 32.4
(C-8), 39.8 (C-4a), 68.0 (C-7), 70.1 (4-CH2O–), 76.1
(C-8a), 102.1 (2-CHPh), 125.9, 126.7, 128.1, 128.4, 128.7,
138.3 (aromatic-C), 170.4 (–OCO–) ppm. LISMS (CH3-
OH/H2O) 289.1 (MþH)þ.
4.3. Resolution of rac-6
With PLE. The hydrolysis was carried out with rac-6
(200 mg, 0.69 mmol), t-BuOH (4 mL) and PLE (0.1 mL,
130U) in phosphate buffer solution (36 mL, pH 7.5, adjusted
by 1 N HCl). Work-up after 17 h (35% conversion). Chiral
HPLC analysis of a sample of the residue showed the
presence of 5a ent-product with 68% e.e. and of 6b ent-
substrate with 40% e.e.
4.1.3. (6)-(4aR,7R,8aS)-2-Phenyl-4a,7,8,8a-tetrahydro-
4H-1,3-benzodioxin-7-yl-butyrate (rac-8). rac-5 (0.70 g,
3.0 mmol) and 30 mg of DMAP in pyridine (10 mL) reacted
with butyric anhydride (0.74 mL, 4.5 mmol) using above
procedure to afford 0.83 g (yield 91%) of rac-8 as a
colorless oil.
With CRL. The hydrolysis was carried out with rac-6
(200 mg, 0.69 mmol), t-BuOH (4 mL) and CRL (5 mg,
130U) in aqueous phosphate buffer solution (36 mL, pH 7.5,
adjusted by 1 N HCl). Work-up after 24 h (85% conver-
sion). Chiral HPLC analysis of a sample of the residue
showed the presence of 5b ent-product with 2% e.e. and of
6a ent-substrate with 30% e.e.
1H NMR (CDCl3) d 0.93 (t, 3H, –CH2CH2CH3), 1.61 (m,
2H, –CH2CH2CH3), 1.80–1.95 (m, 1H, H-8), 2.22–2.47
(m, 4H, H-8, –CH2CH2CH3, H-4a), 3.80–3.95 (m, 2H,
4-CH2O–), 4.54 (m, 1H, H-7), 5.53–5.61 (m, 3H, H-8a,
H-5, H-6), 5.59 (s, 1H, 2-CHPh), 7.39–7.53 (m, 5H,
aromatic-H) ppm; 13C NMR (CDCl3) d 10.5 (–CH2CH2-
CH3), 18.4 (–CH2CH2CH3), 32.4 (C-8), 36.1 (–CH2CH2-
CH3), 41.4 (C-4a), 67.9 (C-7), 69.1 (4-CH2O–), 75.8
(C-8a), 102.7 (2-CHPh), 125.8, 126.7, 127.6, 128.6, 131.7,
138.8 (aromatic-C) 172.4 (–OCO–) ppm. LISMS (CH3-
OH/H2O) 302.2 (MþH)þ.
4.4. Resolution of rac-8
With PLE. The hydrolysis was carried out with rac-8
(200 mg, 0.66 mmol), t-BuOH (4 mL) and PLE (0.1 mL,
130U) in aqueous phosphate buffer solution (36 mL, pH 7.5,
adjusted by 1 N HCl). Work-up after 10 h (13% conver-
sion). Chiral HPLC analysis of a sample of the residue
showed the presence of 5a ent-product with 95% e.e. and of
8b ent-substrate with 9% e.e.
4.2. Resolution of rac-7
With CRL. The hydrolysis was carried out with rac-8
(200 mg, 0.66 mmol), t-BuOH (4 mL) and CRL (5 mg,
130U) in aqueous phosphate buffer solution (36 mL, pH 7.5,
adjusted by 1 N HCl). Work-up after 4 h (27% conversion).
Chiral HPLC analysis of a sample of the residue showed the
presence of 5b ent-product with 11% e.e. and of 8a ent-
substrate with 24% e.e.
With PLE. The ester rac-7 (200 mg, 0.73 mmol) dissolved
in (a) t-BuOH (4 mL) or (b) acetone (4 mL) was added to
the phosphate buffer solution (36 mL, pH 8.0, adjusted by
1 N HCl). Subsequently, PLE (0.1 mL, 130U) was added
and the mixture was efficiently stirred at room temperature
while the pH value was held constant at 8.0 by the addition
of 1 M NaOH solution with a pH-stat autotitrator. The
reaction was stopped after 22 h (11% conversion) and 24 h
(44% conversion), respectively, and the mixture was
With CAL-B. The hydrolysis was carried out with rac-8
(200 mg, 0.66 mmol), t-BuOH (4 mL) and CAL-B (20 mg,
2.4 KU) in aqueous phosphate buffer solution (36 mL, pH

P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
2121
7.5, adjusted by 1 N HCl). Work-up after 30 h (21%
conversion). Chiral HPLC analysis of a sample of the
residue showed the presence of 5a ent-product with 63% e.e.
and of 8b ent-substrate with 15% e.e.
11a (430 mg, 1.18 mmol, yield 70%). To a solution of the
obtained 11a (co-evaporated three times with freshly dried
pyridine) in dry pyridine (5 mL) at 0 8C under nitrogen was
added monomethoxytrityl chloride (436 mg, 1.41 mmol) in
portions. After the mixture was stirred at 0 8C for 1 h, the
temperature was raised to room temperature. The reaction
mixture was treated with CH3OH (5 mL) at 0 8C after 22 h
reaction. After the mixture was stirred at room temperature
for 0.5 h, the resulting mixture was concentrated. The
residue was co-evaporated with toluene and methanol, and
chromatographed on silica gel (CH3OH/CH2Cl2, 0–2%,
Et3N 1%) to give 13a (470 mg, 0.74 mmol, 63% yield,
overall yield of 43%) as a white foam.
4.5. Isolation of enantiomerically pure cyclohexene
precursor
The precursor rac-5 (14.0 g, 60.2 mmol) and isopropenyl
acetate (32.8 mL, 301 mmol) were dissolved in dichloro-
methane (700 mL). Subsequently, Novozymew 435 (2 g,
20 KU) was added and the reaction mixture was stirred at
room temperature. The reaction was stopped at 22 h (52%
conversion) by filtration. The filtrate was concentrated in
vacuo and submitted to column chromatography (EtOAc/n-
hexane, 0–30%). The first portion of eluent was concen-
trated to afford a white solid which was twice recrystallized
from 20% EtOAc in n-hexane giving a white needle crystal
of 6b ent-product (8.07 g, 98% e.e.). Following treatment
with 50 mL of a saturated ammonia/methanol solution at
room temperature for 14 h, the reaction mixture was
concentrated and co-evaporated with methanol to afford a
pale-yellow oil which was purified by column chromato-
graphy (EtOAc/n-hexane, 50:50, Rf¼0.5). A white solid
was obtained and recrystallized twice from 50% EtOAc in
n-hexane twice affording a white needle crystal of 5b (6.1 g,
yield 89%) with enantiomeric excess (e.e.) .99% (overall
yield 43% starting from rac-5). The second fraction from
the first chromatographic purification was concentrated and
thereafter crystallized twice from 50% EtOAc in n-hexane
to afford a white needle crystal of 5a (6.2 g, yield 44%) with
enantiomeric excess (e.e.) .99%.
1H NMR (CDCl3) d 2.03–2.40 (m, 2H, H-60, 600), 2.55 (m,
1H, H-40), 3.04 (br s, 1H, 50-OH), 3.26 (t, 1H, J¼8.6 Hz,
–OCH2–), 3.57 (dd, 1H, J¼4.4, 9.2 Hz, –OCH2–), 3.81 (br
s, 4H, –OCH3, H-50), 5.47 (m, 1H, H-10), 5.93 (br s, 2H,
H-20, H-30), 6.87 (d, 2H, J¼9.2 Hz, aromatic H), 7.26–7.61
(m, 15H, aromatic H), 7.91 (s, 1H, H-8), 8.05 (d, 2H,
aromatic H), 8.81 (s, 1H, H-2), 9.12 (br s, 1H, 6-NH) ppm;
13C NMR (CDCl3) d 35.7 (C-60), 44.4 (C-40), 49.6 (C-10),
55.2 (–OCH3), 65.9 (0–OCH2 –), 66.7 (C-50), 87.4
(–OC TrMM), 123.9 (C-2 ), 113.3, 127.3, 128.0, 128.1,
128.3, 128.9, 130.3, 132.7, 135.0, 143.9, 158.9, (aromatic
C), 133.9 (C-30), 141.9 (C-8), 149.6 (C-2), 151.7 (C-6),
152.6 (C-4), 164.8 (–NHCvO) ppm. HRMS calcd for
C39H36N5O4 (MþH)þ: 638.2767, found 638.2770.
4.6.2. N 6-Benzoyl-9-[(10R,40S,50R)-50-hydroxy-40-(mono-
methoxytrityl)oxymethyl-20-cyclohexenyl]adenine (13b).
Starting from 770 mg (2.2 mmol) of 9b, an amount of
618 mg (0.96 mmol, overall yield of 44%) of 13b was
obtained. Spectroscopic data are the same as for 13a.
4.6. Synthesis of cyclohexenyl nucleoside building blocks
For all reactions, analytical grade solvents were used. All
moisture-sensitive reactions were carried out in oven-dried
glassware (100 8C) under a nitrogen atmosphere.
4.6.3. N 2-Isobutyryl-9-[(10S,40R,50S)-50-hydroxy-40-
(monomethoxytrityl)oxymethyl-20-cyclohexenyl]guanine
(14a). To a solution of 10a (460 mg, 1.66 mmol) in dry
pyridine (12 mL) at 0 8C under nitrogen was added
dropwise trimethylsilyl chloride (1.06 mL, 8.29 mmol).
After the mixture was stirred for 1 h, isobutyric anhydride
(0.83 mL, 4.98 mmol) was added slowly. The mixture was
stirred at 0 8C for 1 h, warmed to room temperature and kept
stirring for an additional 14 h. The reaction mixture was
then cooled in an ice-water bath and quenched with water
(12 mL). The resulting mixture was stirred at room
temperature for 15 min and concentrated. The residue was
purified by column chromatography (CH3OH/CH2Cl2,
5–20%, Rf¼0.4) to afford 12a (730 mg, 2.10 mmol, yield
63%). Following the monomethoxytritylation procedure as
for preparation of 13a, using 972 mg (3.15 mmol) of
monomethoxytrityl chloride in 15 mL pyridine, 14a was
obtained (380 mg, 0.61 mmol, 29% yield, overall yield of
37%) as a pale yellow foam.
Compound 9a,b were synthesized using the procedure for
preparation of the racemic isomer as described in literature.7
Compound 10a,b were prepared using the synthetic method
for racemic Cycl-G.11 The other isomers 18a,b, 19a,b and
15a,b were obtained analogously to the preparation of their
racemic isomers.20
4.6.1. N 6-Benzoyl-9-[(10S,40R,50S)-50-hydroxy-40-(mono-
methoxytrityl)oxymethyl-20-cyclo-hexenyl]adenine
(13a). To a solution of 9a (590 mg, 1.71 mmol) in pyridine
(10 mL) at 0 8C was added benzoyl chloride (0.52 mL,
5.13 mmol) and kept at room temperature overnight. The
reaction mixture was cooled to 0 8C, and saturated aqueous
NaHCO3 (5 mL) was added and the mixture was extracted
with CH2Cl2 (3£50 mL). The combined organic layer was
washed with H2O (20 mL), concentrated, and co-evapor-
ation with toluene. The residue was treated with saturated
ammonia/methanol solution (25 mL) for 5 min. Following
evaporation of the solvent and co-evaporation with
methanol, the residue was further treated with 80%
CF3COOH in water for 40 h. The reaction mixture was
concentrated and co-evaporated with toluene and methanol
three times. The crude was purified by silica gel column
chromatography (CH3OH/CH2Cl2, 0–10%, Rf¼0.3) to give
1H NMR (CDCl3) d 1.20 (dd, 6H, J¼4.9, 6.8 Hz,
–CH(CH3)2), 2.01–2.25 (m, 2H, H-60, 600), 2.50 (m, 2H,
H-40, 2-NHCO–), 3.21 (m, 2H, –OCH2–, 50-OH), 3.50 (dd,
1H, J¼4.6, 9.3 Hz, –OCH2–), 3.80 (s, 3H, –OCH3), 3.90
(m, 1H, H-50), 5.07 (m, 1H, H-10), 5.85 (br s, 2H, H-20, H-30),
6.85 (d, 2H, J¼8.8 Hz, aromatic H), 7.23–7.46 (m, 12H,
aromatic H), 7.51 (s, 1H, H-8), 8.47 (br s, 1H, 1-NH) ppm;
13C NMR (CDCl3) d 18.8 (–CH(CH3)2), 35.7 (C-60), 36.3

2122
P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
Table 4. Analytical data for the phosphoramidites
mmol of starting material
Yield (%)
Rf
HRMS (MþH)þ
31P NMR
n-Hexane/acetone/TEA (49:49:2)
Calcd
Found
1a
1b
2a
2b
3a
3b
4a
4b
0.72
0.90
0.57
0.62
0.40
0.73
0.77
1.01
86
92
66
74
87
77
85
81
0.46
0.46
0.38
0.38
0.51
0.51
0.51
0.51
838.3846
For C48H53N7O5P
838.3846
For C48H53N7O5P
820.3951
For C45H55N7O6P
820.3951
For C45H55N7O6P
814.3733
For C47H53N5O6P
814.3733
For C47H53N5O6P
725.3467
For C41H50N4O6P
725.3467
For C41H50N4O6P
838.3859
838.3840
820.3944
820.3953
814.3759
814.3732
725.3472
725.3455
148.357, 148.393
148.308, 148.345
146.115, 147.142
146.085, 147.148
148.314, 148.465
148.314, 148.459
147.632, 148.127
147.644, 148.163
(–CH(CH3)2), 44.3 (C-40), 49.4 (C-10), 55.2 (–OCH3), 65.6
(–OCH2–), 66.4 (C-50), 87.2 (–OC TrMM), 121.6 (C-5),
124.3 (C-20), 113.3, 127.2, 128.1, 128.3, 130.3, 135.1,
144.1, 158.9 (aromatic C), 133.4 (C-30), 138.0 (C-8), 147.2
(C-2), 147.8 (C-4), 155.8 (C-6), 178.6 (–NHCvO) ppm.
HRMS calcd for C36H38N5O5 (MþH)þ: 620.2872, found
620.2869.
540 mg (1.03 mmol, overall yield 40%) of 17b was
obtained. Spectroscopic data are the same as for 17a.
4.6.7. N 4-Benzoyl-1-[(10S,40R,50S)-50-hydroxy-40-(mono-
methoxytrityl)oxymethyl-20-cyclohexenyl]cytosine
(21a). To a solution of 19a (660 g, 2.04 mmol) in pyridine
(10 mL) at 0 8C was added benzoyl chloride (0.72 mL,
6.12 mmol) and the mixture was kept at room temperature
overnight. The reaction mixture was cooled to 0 8C, and
saturated aqueous NaHCO3 (5 mL) was added and extracted
with CH2Cl2 (3£50 mL). The combined organic layer was
washed with H2O (15 mL), concentrated, and co-evaporated
with toluene. The residue was treated with saturated
ammonia/methanol solution (25 mL) for 5 min. Following
evaporation of the solvent and co-evaporation with
methanol, the residue was further treated with 80%
CF3COOH in H2O for 40 h. The reaction mixture was
concentrated and co-evaporated with toluene and methanol
three times. The crude was purified by silica gel column
chromatography (CH3OH/CH2Cl2, 0–10%, Rf¼0.45) to
give 20a (560 mg, 1.64 mmol, yield 76%). Tritylation
according to the preparation of 13a, using 608 mg
(6.30 mmol) of monomethoxytrityl chloride in 15 mL
pyridine, afforded 21a (320 mg, 0.52 mmol, 32% yield,
overall yield of 26%) as a white foam.
4.6.4. N 2-Isobutyryl-9-[(10R,40S,50R)-50-hydroxy-40-
(monomethoxytrityl)oxymethyl-20-cyclohexenyl]guanine
(14b). Starting from 600 mg (2.16 mmol) of 10b, an amount
of 420 mg (0.68 mmol, overall yield of 31%) of 14b was
obtained. Spectroscopic data are the same as for 14a.
4.6.5. 1-[(10S,40R,50S)-50-Hydroxy-40-(monomethoxy-
trityl)oxymethyl-20-cyclohexenyl]-thymine (17a). 15a
(750 mg, 2.21 mmol) was treated with 80% CF3COOH in
H2O for 40 h. After evaporation and co-evaporation with
toluene and methanol, the residue was purified by column
chromatography (CH3OH/CH2Cl2, 0–10%, Rf¼0.25) to
give 16a (334 mg, 1.32 mmol, yield 60%). Following the
procedure used for preparation of 13a, using 778 mg
(2.52 mmol) of monomethoxytrityl chloride in 15 mL
pyridine, 17a was obtained (410 mg, 0.78 mmol, 59%
yield, overall yield of 35%) as a white foam.
1H NMR (CDCl3) d 1.75 (s, 3H, –CH3), 1.95–2.00 (m, 2H,
H-60, 600), 2.43 (m, 1H, H-40), 2.73 (d, 1H, 50-OH), 3.19 (m,
1H, –OCH2–), 3.51 (dd, 1H, J¼4.4, 9.5 Hz, –OCH2–),
3.80 (br s, 4H, –OCH3, H-50), 5.26 (m, 1H, H-10), 5.62 (m,
1H, H-20), 5.91 (m, 1H, H-30), 6.86 (d, 2H, J¼8.8 Hz,
aromatic H), 6.97 (s, 1H, H-6), 7.22–7.46 (m, 12H,
aromatic H), 8.41 (br s, 1H, 3-NH) ppm; 13C NMR
(CDCl3) d 12.3 (–CH3), 35.1 (C-60), 44.0 (C-40), 50.5
(C-10), 55.2 (–OCH3), 65.4 (–OCH02–), 66.3 (C-50), 87.3
(–OC TrMM), 110.1 (C-5), 124.9 (C-2 ), 113.3, 127.3, 128.01,
128.3, 130.3, 135.1, 144.1, 158.8 (aromatic C), 134.4 (C-3 ),
137.4 (C-6), 150.7 (C-2), 163.6 (C-4) ppm. HRMS calcd for
C32H32N2O5Na (MþNa)þ: 547.2208, found 547.2212.
1H NMR (CDCl3) d 1.98–2.11 (m, 2H, H-60, 600), 2.29 (m,
1H, H-40), 2.36 (br, 1H, 50-OH), 3.10 (m, 1H, –OCH2–),
3.73 (m, 1H, –OCH2–), 3.79 (s, 3H, –OCH3), 4.14 (m, 1H,
H-500), 5.41 (m, 1H, H-10), 5.68 (m, 1H, H-20), 6.05 (m, 1H,
H-3 ), 6.83 (d, 2H, J¼8.8 Hz, aromatic H), 7.20–7.52 (m,
18H, aromatic H, H-5), 7.98 (d, 1H, H-6), 8.65 (br s, 1H,
4-NH) ppm; 13C NMR (CDCl3) d 35.5 (C-60), 44.8 (C-40),
53.3 (C-10), 55.2 (–OCH3), 63.3 (–OCH2–), 64.0 (C-50),
87.0 (–OCTrMM), 95.8 (C-5), 123.6 (C-20), 113.3, 127.2,
128.1, 128.08, 130.2, 135.9, 138.0, 144.1, 158.7 (aromatic C),
132.8 (C-3 ), 147.1 (C-6), 156.0 (C-2), 162.1 (C-4), 167.5
(–NHCvO) ppm. HRMS calcd for C38H36N3O5 (MþH)þ:
614.2654, found 614.2647.
4.6.6. 1-[(10R,40S,50R)-50-Hydroxy-40-(monomethoxy-
trityl)oxymethyl-20-cyclohexenyl]-thymine (17b). Start-
ing from 875 mg (2.57 mmol) of 15b, an amount of
4.6.8. N 4-Benzoyl-1-[(10R,40S,50R)-50-hydroxy-40-(mono-
methoxytrityl)oxymethyl-20-cyclohexenyl]cytosine (21b).
Starting from 920 mg (2.84 mmol) of 19b, an amount of

P. Gu et al. / Tetrahedron 60 (2004) 2111–2123
2123
4. Maurinsh, Y.; Rosemeyer, H.; Esnout, R.; Medvedovici, A.;
Wang, J.; Ceulemans, G.; Lescrinier, E.; Hendrix, C.; Busson,
R.; Sandra, P.; Seela, F.; Van Aerschot, A.; Herdewijn, P.
J. Chem. Eur. 1999, 5, 2139–2150.
540 mg (1.58 mmol, overall yield of 31%) of 21b was
obtained. Spectroscopic data are the same as for 21a.
4.7. Synthesis of the amidite building blocks (1a, 1b, 2a,
2b, 3a, 3b, 4a and 4b)
5. Vastmans, K.; Pochet, S.; Peys, A.; Kerremans, L.; Van
`
Aerschot, A.; Hendrix, C.; Marliere, P.; Herdewijn, P.
4.7.1. General procedure for phosphoramidite synthesis.
The phosphitylation reaction was carried out on 0.4–
1 mmol of the monomethoxytritylated derivative (13a,b,
14a,b, 17a,b and 21a,b, respectively) in 5–10 mL dichloro-
methane using freshly distilled diisopropylethylamine
(3 equiv.) and 2-cyanoethyl N,N-diisopropylchloro-
phosphoramidite (1.5 equiv.) under argon. The reaction
mixture was stirred at room temperature for 60 min when
TLC indicated complete reaction. Water (3 mL) was added,
the solution was stirred for 10 min and partitioned between
CH2Cl2 (50 mL) and aqueous NaHCO3 (30 mL). The
organic phase was washed with aqueous NaCl (3£30 mL)
and the aqueous phases were back extracted with CH2Cl2
(30 mL). Evaporation of the organics left an oil which was
flash purified on 45 g of silica gel (hexane/acetone/TEA,
49:49:2) to afford the product as a foam after co-evaporation
with dichloromethane. Dissolution in 3 mL of dichloro-
methane and double precipitation in 160 mL cold (260 8C)
hexane afforded the desired product as a white powder. The
obtained material was dried in vacuo and stored under
nitrogen at 220 8C until use for oligonucleotide synthesis.
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7. Wang, J.; Froeyen, M.; Hendrix, C.; Andrei, G.; Snoeck, R.;
De Clercq, E.; Herdewijn, P. J. Med. Chem. 2000, 43,
736–745.
8. Racemic cyclohexenyl-C shows an EC50 of 0.05 mg/mL
against varicella-zoster virus in human embryonic lung cells,
which is 10 times lower than the EC50 value of acyclovir
(0.5 mg/mL), in the same test system.
9. Wang, J.; Verbeure, B.; Luyten, I.; Lescrinier, E.; Froeyen, M.;
Hendrix, C.; Rosemeyer, H.; Seela, F.; Van Aerschot, A.;
Herdewijn, P. J. Am. Chem. Soc. 2000, 122, 8595–8602.
10. Verbeure, B.; Lescrinier, E.; Wang, J.; Herdewijn, P. Nucl.
Acid Res. 2001, 29, 4941–4947.
11. Wang, J.; Morral, J.; Hendrix, C.; Herdewijn, P. J. Org. Chem.
2001, 66, 8478–8482.
12. Gais, H.-J.; Theil, F. Enzyme catalysis in organic synthesis;
Drauz, K., Waldmann, H., Eds.; Wiley-VCH: Weinheim,
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organic synthesis 1991, 7, 389–436.
Yields, starting quantity, Rf values, mass analysis and 31P
NMR data are given in Table 4.
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Chem. Soc. 1992, 114, 9414–9418. (b) Tsuji, T.; Onishi, T.;
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Acknowledgements
´ ´
15. Perez-Perez, M.-J.; Rozenski, J.; Busson, R.; Herdewijn, P.
J. Org. Chem. 1995, 60, 1531–1537.
This research was financially supported by grants from the
Katholieke Universiteit Leuven (GOA 2002/13) and FWO
(G.0116.00). We thank C. Biernaux for editorial help.
16. De Bouvere, B.; Kerremans, L.; Rozenski, J.; Janssen, G.; Van
Aerschot, A.; Claes, P.; Busson, R.; Herdewijn, P. Liebigs
Ann. Rec. 1997, 1453–1461.
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References and notes
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19. Jongejan, J. A.; Van Tol, J. B. A.; Geerlof, A.; Duine, J. A.
Recl. Trav. Chim. Pays-Bas 1991, 110, 247. see also p 255.
20. Ping, G.; Morral, J.; Wang, J.; Rozenski, J.; Busson, R.; Van
Aerschot, A.; De Clercq, E.; Herdewijn, P. Antiviral Chem.
Chemother. 2003, 14, 31–37.
3. Allart, B.; Busson, R.; Rozenski, J.; Van Aerschot, A.;
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