Cyclization versus oligomerization of SP- and R P-5′-OH-N4-benzoyl-2′-deoxycytidine-3′- O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane)s

Article abstract of DOI:10.1016/j.tet.2005.12.022

The S_P-isomer of 5′-OH-N⁴-benzoyl-2′- deoxycytidine-3′-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) undergoes DBU-promoted intramolecular cyclization providing as a sole product S_P-deoxycytidine cyclic 3′,5′-O,O-

Full text of DOI:10.1016/j.tet.2005.12.022

Tetrahedron 62 (2006) 2698–2704
Cyclization versus oligomerization of SP- and
RP-50-OH-N4-benzoyl-20-deoxycytidine-30-O-
(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane)s
*
Piotr Guga, Bolesław Karwowski, Damian Błaziak, Magdalena Janicka,
Andrzej Okruszek, Beata Re˛bowska and Wojciech J. Stec
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
´ ´
Sienkiewicza 112, 90-363 Łodz, Poland
Received 8 November 2005; accepted 13 December 2005
Available online 9 January 2006
Dedicated to Professor David Shugar on the occasion of his 80th birthday
Abstract—The SP-isomer of 50-OH-N4-benzoyl-20-deoxycytidine-30-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) undergoes
DBU-promoted intramolecular cyclization providing as a sole product SP-deoxycytidine cyclic 30,50-O,O-phosphorothioate. Unexpectedly,
the RP-counterpart yields a mixture of products consisting of RP-deoxycytidine cyclic 30,50-O,O-phosphorothioate and macrocyclic
oligo(deoxycytidine phosphorothioate)s. The results of molecular modeling indicate that the dychotomy observed for the RP substrate may
result from remarkably higher energy of the corresponding transition states, caused by the presence of bulky ‘spiro’ pentamethylene
substituent at the position C4 in the oxathiaphospholane ring.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
cyclization of nucleoside 50-O-(bis(4-nitrophenyl)pho-
sphorothioate)s under treatment with t-BuOK in DMF,
followed by hydrolytic removal of the remaining 4-nitro-
phenoxyl group. The resulting cNMPS were then chro-
matographically separated into individual diastereomers.3
Reported to date stereocontrolled methods of synthesis of
RP- or SP-cNMPS rely upon preparation of appropriately
protected diastereomerically pure nucleoside cyclic 30,50-
O,O-phosphoranilidates or phosphoranilidothioates and
their stereoretentive conversion into corresponding
Nucleoside cyclic 30,50-O,O-phosphorothioates1 (cNMPS,
3, Scheme 1) are valuable tools for studying the mechanism
of action of enzymes involved in the metabolism of cyclic
nucleotides and are potent agonists or antagonists of the
latter compounds. Due to assymetry of the phosphorus
atom, cNMPS exist in the form of SP and RP diastereomers,
which usually have markedly different biological proper-
ties.1,2 The first reported synthesis of cNMPS involved
B'
B'
HO
O
P
DMTO
O
P
B
O
O
1. DBU/CH3CN
2. NH4OH
O
S
[H+]
O
S
O
P
S
-
O
O
S
O
S
R
R
R
R
3A (SP) B=Cyt
1a (SP) R=H, B'=CytBz
2a (SP) R=H, B'=CytBz
Scheme 1.
Keywords: Cyclic oligonucleotides; Phosphorothioate analogues of DNA; Oxathiaphospholane method.
*
Corresponding author. Tel.: C48 42 6803248; fax: C48 42 6815483; e-mail: pguga@bio.cbmm.lodz.pl
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.12.022

P. Guga et al. / Tetrahedron 62 (2006) 2698–2704
2699
O
P
O
4a (All RP)
-
S
O
O
3B (RP)
Cyt
O
O
N
O
Cyt
Cyt
O
HN
N
O
O
O
HN
N
-
O
P
S
P
N
O
Cyt
O
-
S
O
O
HO
HO
O
[H+]
1. DBU/CH3CN
O
DMTO
O
2. NH4OH
-
O
O
O
S
Cyt
O
O
S
P
O
O
P
S
P
O
P
O
S
-
Cyt
O
S
O
O
O
O
S
-
S
Cyt
O
O
O
P O
O
n
2d (RP)
1d (RP)
O
P
P
-
S
O
O
-
4b (All RP) n=1
4c (All Rp) n=2
5a (RP, n=1)
5b (RP, n=2)
S
n
O
Cyt
Scheme 2.
phosphorothioates using NaH/CS2 or NaH/CO2, respec-
tively.4 Here we describe highly efficient synthesis of SP- and
RP-deoxycytidine cyclic 30,50-O-O-phosphorothioates (3A
and 3B, BZCyt) in a stereospecific manner by oxathiapho-
spholane approach, as well as unexpected formation of
stereodefined macrocyclic and linear oligonucleotides of
general formula 4 and 5, respectively (see Scheme 2). The
macrocyclic oligo(deoxycytidine phosphorothioate)s 4
belong to the family of circular oligonucleotides, possessing
interesting DNA, RNA, and protein binding properties and
potential therapeutic applications.5 To the best of our
knowledge, the only two published examples of stereodefined
macrocyclic phosphorothioate oligonucleotides were chi-
meric RP-, and SP-cyclic diribonucleotide c(GPSGPO), which
can be considered the analogues of cyclic diguanylic acid
involved in regulatory system of cellulose synthesis in
Acetobacter xylinum,6 and RP,RP- and SP,RP-cyclic di(deoxy-
cytidine phosphorothioate)s obtained by Battistini et al.7
earlier work that structurally related N6-benzoyl-20-O-
tetrahydropyranyl-adenosine-50-O-(2-thio-1,3,2-dithiapho-
spholane) can be effectively transformed into adenosine
cyclic 30,50-O,O-phosphorodithioate via intramolecular
cyclization by treatment with t-BuOK in DMF solution,
followed by removal of protecting groups.4d As model
compounds we chose diastereomerically pure SP- and RP-
N4-benzoyl-deoxycytidine-30-O-(2-thio-1,3,2-oxathiapho-
spholane)s (1a and 1b, respectively, BZCytBz, RZH),
which were prepared from chromatographically separated
SP- and RP-50-O-DMT-precursors 2a and 2b, by treatment
with p-toluenesulfonic acid in methylene chloride. The
reactions of 1a and 1b were performed on a 66 mmol scale
by addition of equimolar amount of DBU into magnetically
stirred solutions of the substrates in anhydrous acetonitrile.
After 5 min at room temperature 31P NMR spectra showed
quantitative formation of single products. After ammoniacal
deprotection these products were identified by 31P NMR and
HPLC comparison with genuine sample,4d as SP- and
RP-deoxycytidine cyclic 30,50-O,O-phosphorothioate (3A
and 3B). Thus, the reaction of intramolecular cyclization
occurred with retention of configuration at phosphorus,
most likely by the mentioned earlier adjacent mechanism.
2. Results and discussion
In the oxathiaphospholane method, which was originally
designed for the stereocontrolled synthesis of oligo(nucleo-
side phosphorothioate)s, chromatographically separated
P-diastereomers of 50-O-DMT-nucleoside-30-O-(2-thio-
1,3,2-oxathiaphospholane)s (2a,b, RZH) and their ring-
substituted analogues (2c,d, R,RZ–(CH2)5–) are used.8
Their reaction with 50-OH-nucleoside component (usually
performed in CH3CN solution in the presence of strong non-
nucleophilic base, preferably DBU), proceeds according to
the adjacent type mechanism, where pseudorotation of a PV
intermediate (such a process is marked with J in Scheme
1S, Supplementary data) results in retention of configuration
at the phosphorus atom.8c,9 We anticipated that diaster-
eomerically pure oxathiaphospholane substrates after
acidolytic removal of the 50-O-dimethoxytrityl protecting
group may undergo a DBU-promoted intramolecular
reaction in analogous stereospecific manner to give the
desired cNMPS (see Scheme 1). Notably, intramolecular
ring-opening reactions of oxathiaphospholanes were not
reported in the literature, albeit it was known from our
The results obtained for 1a and 1b fulfilled our expectations,
but for practical reasons we intended to use their ‘spiro’
analogues possessing 4,4-pentamethylene substituent in
the oxathiaphospholane ring (1c and 1d, BZCytBz, R,RZ
–(CH2)5–), which were known for considerably better
chromatographic properties of their precursors 2c and 2d
in respect to their separation into pure P-diastereoisomers.8d
The reactions of diastereoisomerically pure SP-1c and
RP-1d (obtained by detritylation of 2c and 2d, respectively)
were performed exactly as for 1a,b. While the SP-substrate
yielded the expected cyclic 30,50-O,O-phosphorothioate 3A
virtually quantitatively, the RP-counterpart provided a
mixture of products with several resonances (difficult for
precise integration) within a range 55–57 ppm in a 31P NMR
spectrum. After ammoniacal deprotection the products
were separated by RP HPLC and identified by MALDI-
TOF MS (see Table 1). Approximate quantification of their
distribution, expressed as percentage of the consumption of

2700
P. Guga et al. / Tetrahedron 62 (2006) 2698–2704
and 31P NMR. In addition to 3B, several oligomeric
products were identified, including macrocyclic (4a–e)
and linear oligo(deoxycytidine phosphorothioate)s (5a,b)
(see Scheme 2). Obviously, expected highly favorable
entropy of intramolecular cyclization did not assure full
regioselectivity. The major macrocyclic products include
those containing two (4a, 26.3% of total UV absorption),
three (4b, 20.1%) and four deoxycytidine phosphorothioate
units (4c, 6.1%). Minor products containing five (4d, 1.7%)
and six units in macrocyclic ring (4e, 1.1%) were also
identified. In fact, the formation of ‘macrocyclic’ fraction
consumed over 55% of the oxathiaphospholane substrate.
The formation of macrocyclic products 4 can be explained
by conversion of substrate 1d into corresponding linear
oligomers (all with the oxathiaphospholane function at the
30-end) followed by their inter- or intramolecular cycliza-
tion. In addition to the macrocyclic products, two minor
linear products were isolated and identified by HPLC/MS
as 30-O-phosphorothioylated dinucleotide (5a, 1.7%)
and trinucleotide (5b, 1.2%) (see Table 1 and S0cheme 2).
It seems reasonable to assume that the 3 -terminal
oxathiaphospholane function was hydrolyzed with traces
of moisture yielding inert phophorothioate group in 5a,b.
Similar results w0ere obtained when a N2-isobutyryl-
deoxyguanosine 3 -O-(2-thio-‘spiro’-4,4-pentamethylene-
1,3,2-oxathiaphospholane) (a mixture of P-diastereoisomers
ca. 1:1) was a substrate for DBU-promoted cyclization.
After ammoniacal deprotection the obtained mixture was
analyzed by 31P NMR (in D2O) showing the presence of
four major products: SP-deoxyguanosine cyclic 30,50-O,O-
phosphorothioate4c (d 52.8 ppm, 50% of total integral), its
RP-epimer (d 54.6 ppm, 16%), deoxyguanosine analogue of
‘dimeric’0compound 4a (d 55.2 ppm, 12%), and deoxygua-
nosine 3 -O-phosphorothioate (d 47.4 ppm, 5%). These
products were separated on HPLC and their structures were
confirmed by MALDI-TOF MS analysis.
Table 1. Identification and quantification of HPLC-separated products of
cyclization/oligomerization of RP-1d (DBU/CH3CN procedure)
Compounds
HPLC tR
(min)a
Content
(%)b
Molecular
weightc
31P NMR d
(ppm)
3B
4a
4b
4c
4d
4e
5a
5b
10.61
21.40
26.39
22.97
23.45
24.52
14.27
18.33
41.8
26.3
20.1
6.1
1.7
1.1
304/305.3
609/610.5
915/915.8
1222/1221.0
1526/1526.3
1833/1831.5
627/628.5
933/933.8
55.23
54.68
55.76
55.79
NDd
NDd
1.7
1.2
NDd
NDd
a HPLC was performed using Econosphere C18 (5 m) 4.6!250 mm column
(Alltech) with a linear gradient of acetonitrile in 0.1 M TEAB:
0–20 min—0.5%/min; 20–32 min—0.3%/min; flow 1 mL/min.
b Calculated from electronically integrated chromatograms (UV detection
at 255 nm).
c Calculated/measured m/z; by MALDI-TOF MS, negative ion mode
(Voyagere Elite), calculated for free acids (Da).
d Not determined.
Table 2. HPLC-separation and identification of products of cyclization/
oligomerization of RP-1d in the presence of 30-O-acetylthymidine
Compounds HPLC tR
(min)a
Quantity of
productsb
Molecular weight
Measuredc Calculatedd
3B
4a
12.42
30.84
26.00
31.44
35.30
38.44
41.12
43.33
45.26
47.01
48.61
50.22
51.82
53.20
30.5
15.1
13.0
18.0
7.9
16.1
3.9
3.0
2.1
1.5
1.1
1.0
1.0
304.1
610.0
546.9
305.25
610.50
547.48
6a (nZ1)
6b (nZ2)
6c (nZ3)
6d (nZ4)
6e (nZ5)
6f (nZ6)
6g (nZ7)
6h (nZ8)
6i (nZ9)
6j (nZ10)
6k (nZ11)
6l (nZ12)
852.3
852.73
1157.8
1463.1
1768.0
2073.2
2378.5
2683.5
2989.9
3293.1
3599.2
3903.6
1157.98
1463.20
1768.49
2073.70
2378.95
2684.20
2989.45
3294.70
3599.95
3905.20
0.8
a HPLC was performed using PTH C18 (5 m) 2.1!220 mm column
(Brownlee) with a linear gradient of acetonitrile in 0.1 M TEAB:
0–60 min—0.47%/min; flow 0.3 mL/min (UV detection at 255 nm).
b Given in optical density (measured at 260 nm).
The observed predominant formation of macrocyclic
oligo(deoxycytidine phosphorothioate)s 4a–e prompted us
to perform the reaction in the presence of limited amount of
30-O-acetylthymidine (1/6 mol equiv) acting as a ‘30-end
forming unit’ to prevent ‘macrocyclization’ and facilitate
the formation of linear products. The deprotected products
were isolated by RP HPLC, quantified by UV absorption
at 260 nm and identified by MALDI-TOF MS (Table 2,
Scheme 3). It was found, that although the main product
of the reaction was RP-cyclic nucleotide 3B (26.6% of
total UV absorption) and among other prominent products
c m/z; Measured by MALDI-TOF MS (Voyagere Elite).
d Calculated for free acids (Da).
the starting material 1d, was accomplished by integration
of peaks in the HPLC profile. The inspection of the data
(listed in Table 1) reveals that the main product of self-
condensation of 1d (41.8% conversion of the starting
material) was RP-deoxycytidine cyclic 30,50-O,O-phos-
phorothioate (3B) as proved by MALDI-TOF MS, HPLC
Cyt
O
O
O
S
1d (RP) 6 equiv.
P
H
-
Cyt
O
O
O
O
1. DBU/CH3CN
3B (RP)
+
2. NH4OH
Thy
O
O
P
HO
O
-
P
O
S
-
Thy
S
O
O
O
O
n
Cyt
OAc
O
Cyt
O
6a–l (All RP)
OH
-
O
P
O
S
4a (All RP)
Scheme 3.

P. Guga et al. / Tetrahedron 62 (2006) 2698–2704
2701
macrocyclic dimer 4a was identified (13.1%), the major
fraction of the products (60.3%) consisted of a mixture of
linear phosphorothioate oligonucleotides 6a–l ranging from
20to 13 nucleotides in length and containing thymidine at the
3 -terminus. The trimer 6b (15.7%), pentamer 6d (14.0%),
and dimer 6a (11.3%) were most abundant, whereas each of
those containing 8 or more nucleotides in the chain
constituted less than 2% of the mixture of products.
6f (0.1 OD260) was treated with RP-specific snake venom
phosphodiesterase (svPDE)10 and SP-specific nuclease P1
(nP1).11 It was found that 6f was completely degraded on
10 min incubation with 50 mg of svPDE at 37 8C, whereas
no degradation was observed after 2 h incubation of identical
sample of 6f with 1 mg of nP1 at room temperature. These
results confirm that DBU-promoted self-condensation of
RP-oxathiaphospholane 1d leads to homochiral oligomeric
products 6a–l with RP-configuration at each internucleotide
bond. Undoubtedly, the same absolute configuration must be
assigned to phosphorus atoms in the macrocyclic compounds
4. Notably, although it is known, that nucleases present in
50% human serum are able to hydrolyze phosphorothioate
oligonucleotides of RP-configuration,12 using RP HPLC
technique we found that both tested cyclic oligomers 4a
and 4b were stable under these conditions for more than 24 h
(data not shown).
The autoradiogram for PAGE analysis of compounds
enzymatically radiolabeled with [32P]phosphate group
(Fig. 1) showed the expected pattern of bands of
oligonucleotides 6a–l and the lack0 of radioactive label in
macrocyclic dinucleotide 4a. The 5 -radiolabeled heptamer
Analysis of the mechanistic aspects of the condensation
suggests that the observed intermolecular oligomerization
or macrocyclization might successfully compete with
entropically favored intramolecular cyclization for 1d
provided that the latter process was significantly slowed-
down. This may happen due to higher energy barrier for the
transition of 1d to the corresponding PV intermediate
product 7 (Scheme 4). Conformational search (HyperChem
7.5 package, parameter set Amber99, HyperCube, Inc.)
done for four PV intermediates 7a–d, derived from 1a–d,
respectively, showed that the pentacoordinate intermediates
7 adopt two basic structures with the 1,3,2-dioxapho-
sphorinane ring in either energetically favored chair
conformation, or disfavored boat conformation. The
numbering of atoms in 7, a list of torsion angles varied
during the search and the geometrical restraints imposed on
bonds around P-atom in 7 are shown on Figure 2. Negative
charge was assigned to the equatorial sulfur atom (S35). The
obtained structures were analyzed in respect to the absolute
configuration of the starting 1 and the relevant energies and
torsion angles for those resulting from SP-1c and RP-1d are
collected in Tables 1S and 2S, respectively (Supplementary
data). Then, within the sets of the lowest energy conformers
of 7a–b (#1–33, in a range of 150.0–158.9 kcal/mol) and
Figure 1. PAGE analysis (20% polyacrylamide/7 M urea) of HPLC-
separated and 32P-labeled products 6a–l of DBU-promoted self-conden-
sation of 1d performed in the presence of ca. 1/6 mol equiv of 30-O-
acetylthymidine.
B'
B'
O
O
O
O
S
S
O
O
P
O
P
S
O
O
7, PV
8, PIV
S
B'
B'
O
O
S
O
S
O
1
O
P
P
O
O
7c–d (#1–28, 157.0–164.4 kcal/mol, see Tables 1S and 2S)
0
S
the apical bonds P–O5 were cleaved and geometries of
the resulting molecules were optimized in two steps, that
is, with geometrical restraints for PV atom either kept (8) or
9, PIV, not optimized
9, PIV optimized
S
Scheme 4.
H
56
Geometrical restraints imposed on
V
N
P
intermediate 7 for bonds around
20
19
O
P-atom
Torsion angles varied during
conformational search:
2c3c4co C12–C10–C11–O14
3c2c1co C10–C12–C13–O14
8 N
N 15
17
Linear bonds (180˚)
O9–P7–O36
Orthogonal bonds (90˚)
O36–P7–O22
O36–P7–S34
O36–P7–S35
Trigonal bonds (120˚)
O22–P7–S34
S34–P7–S35
12
41
40
13
14 O
O
ccco
C10–C11–C28–O9
N8–C19–N20–C56
O14–C13–N15–C17
O36–P7–O22–C10
P7–O22–C10–C11
P7–S34–C40–C41
S34–P7–O36–C37
21
ncnc
ocnc=o
opoc
pocc
pscc
10
11
34
22 O
S
37
7
P
28
O
O
36
9
spoc
S35–P7–O22
S
35
Figure 2. The numbering of atoms in 7, a list of torsion angles varied during the conformational search and the geometrical restraints imposed on bonds around
the P-atom.

2702
P. Guga et al. / Tetrahedron 62 (2006) 2698–2704
released (9), giving on this ‘retro’ way information about
relative energies of the structures 8 resembling correspond-
ing transition states. Subsequent analysis of the profiles
1a,b/9/8a,b/7a,b showed no significant differences in
the energy barrier for both diastereomers. This finding
corresponds with quantitative conversion of both P-diaster-
eomeric substrates 1a and 1b into cyclic 30,50-O,O-
phosphorothioates. Interestingly, among the profiles
1c,d/9/8c,d/7c,d a very favorable profile was found
for one of the chair conformers of 7c (#5) with the energy
barrier for its formation by 9.2 kcal/mol lower than for its 7d
counterpart (#4). The structures 7c, #5 and 7d, #4 are shown
on Figures 1S and 2S, respectively, and related to them
structures 8c,d on Figure 3. Notably, in a case of 8d we
observed repulsion interactions along a narrow groove,
indicated with the red arrow. This steric hindrance may
result in higher energy barrier leading to the formation of
7d. The calculated energy profiles for the intermediates
formed from 1c and 1d during cyclization are shown on
Figures 3S and 4S, respectively. Comparison of the lowest
energy profiles for the cyclization 1c,d/9/8c,d/7c,d,
each ending with 7 adopting either the boat or chair
conformations of the dioxaphosphorinane ring, is shown on
Figure 4, while relevant energy values are collected in
Tables 3S and 4S. Since the conformational search for RP-
and SP-isomers of anionic 1 (it was assumed that DBU
devoided the reacting substrate of 50-OH proton) showed
that for both pairs of diastereomers the energies of
conformers suitable for cyclization are within 1 kcal/mol
of those of the lowest energy (Tables 5S and 6S),
apparently, a conformation of the substrate molecules is
not a decisive factor in the problem under consideration.
210.0
200.0
190.0
180.0
170.0
160.0
150.0
Sp chair #5
Sp boat #27
Rp chair #4
Rp boat #28
9, PIV, optimized 9, PIV, not optim.
8 (PIV
)
7 (PV)
reaction co-ordinates
Figure 4. Comparison of the calculated profiles with the lowest energy
barrier (for structures 8c,d adopting either the chair or boat conformation of
the dioxaphosphorinane ring) during the cyclization 1c,d/9/8c,d/7c,d.
Although performed calculation does not reflect interactions
with solvent molecules nor with DBU activator, these
numbers suggest that despite of strong entropy factor
facilitating intramolecular reaction, due to remarkably
higher energy barrier 1/9/8 the cyclization of 1d is
slowed-down, compared to 1c, and intermolecular reaction
leads to linearized products.
3. Conclusions
The DBU-promoted intramolecular cyclization of diaster-
eomerically pure SP- and RP-isomers of 50-OH-N4-benzoyl-
20-deoxycytidine-30-O-(2-thio-1,3,2-oxathiaphospholane)
provides quantitatively corresponding SP- and RP-deoxycy-
tidine cyclic 30,50-O,O-phosphorothioate, respectively. The
same applies to the SP-isomer of 50-OH-N4-benzoyl-
20-deoxycytidine-30-O-(2-thio-4,4-pentamethylene-1,3,2-
oxathiaphospholane), while its RP-counterpart yields a
mixture of products consisting of RP-deoxycytidine cyclic
30,50-O,O-phosphorothioate and macrocyclic oligo(deoxy-
cytidine phosphorothioate)s. This difference can be
explained in terms of steric hindrance (caused by bulky
pentamethylene substituent) observed during formation of
first pentacoordinate intermediate, which slows- down the
intramolecular process and allows the intermolecular
macrocyclization to compete. Similar dychotomy was
observed for cyclization of deoxyguanosine derivatives.
This approach is suitable for synthesis of stereodefined
macrocyclic oligonucleotides—the analogues of cyclic
diguanylic acid involved in regulatory system of cellulose
synthesis in A. xylinum.
4. Experimental
4.1. General
4.1.1. 50-O-DMT-N4-benzoyl-deoxycytidine-30-O-(2-thio-
1,3,2-oxathiaphospholane) (2a,b, B=CytBz, R=H). The
title compound was synthesized and separated into pure
P-diastereomers (by 31P NMR and TLC) as described.8b
Figure 3. The calculated lowest energy barrier conformations of PIV
-
4.1.2. 50-O-DMT-N4-benzoyl-deoxycytidine-30-O-(2-thio-
4,4-pentamethylene-1,3,2-oxathiaphospholane) (2c,d,
B=CytBz, R=–(CH2)5–). The title compound was syn-
thesized and separated into pure P-diastereomers (diaster-
eomeric purity assessed by 31P NMR and TLC) as
described.8d
intermediate 8c (upper panel) and 8d (bottom panel) formed during
cyclization 1c,d/9/8c,d/7c,d with the chair conformation of the
1,3,2-dioxaphosphorinane ring. The geometrical restraints present in 7c,d
were kept during geometry optimization. The green and red arrows indicate
the grooves without and with repulsion interactions, respectively. Assign-
ment of colors: hydrogen—white, carbon—blue, nitrogen—dark blue,
oxygen—red, sulfur—yellow, phosphorus—pink.

P. Guga et al. / Tetrahedron 62 (2006) 2698–2704
2703
4.1.3. 50-O-DMT-N2-isobutyryl-deoxyguanosine 30-O-(2-
thio-‘spiro’-4,4-pentamethylene-1,3,2-oxathiaphospho-
Supplementary data
lane). The title compound was synthesized as described.8d
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tet.2005.12.
022. Text containing detailed description of molecular
modelingperformed;Scheme1S—mechanismofoxathiapho-
spholane ring-opening cyclization; Tables 1S, 2S—torsion
angles found for 28 lowest energy conformers of PV
intermediate 7c,d formed from 1c,d; Figures 1S, 2S—the
#4 and #5 conformations of PV-intermediates 7; Figures 3S,
4S—the energy profiles for intermediates formed from 1c,d
during cyclization 1/9/8/7; Tables 3S, 4S—energies
calculated for intermediates 8 and 9 leading to lowest
energy conformers of 7c,d derived from 1c,d; Tables 5S,
6S—energies and torsion angles found in conformational
search for ten lowest energy conformers of anionic form
of 1c,d.
4.1.4. SP-N4-benzoyl-deoxycytidine-30-O-(2-thio-1,3,2-
oxathiaphospholane)s (1a, BZCytBz, RZH). Into the
solution of fast-50-O-DMT-N4-benzoyl-deoxycytidine-30-
O-(2-thio-1,3,2-oxathiaphospholane) (110 mg, 0.14 mmol)
in methylene chloride (30 mL), p-toluenesulfonic acid
monohydrate (120 mg, 0.66 mmol) was added with stirring
at room temperature. The reaction progress was monitored
by TLC (silica gel, chloroform/methanol 9:1 v/v, Rf
substrate 0.78; Rf product 0.45). After 40 min, the reaction
mixture was concentrated and the oil residue was applied on
a silica gel column (20!100 mm, silica gel 60, 230–400
mesh, Merck). The column was eluted with a gradient
of chloroform/2-propanol 100:0/50:50. Appropriate frac-
tions were collected and evaporated to dryness to yield
1a (35 mg, 0.075 mmol, 53%). d 31P NMR 104.11 ppm
(CD3CN). Compounds 1b, 1c and 1d were obtained in
analogous way.
References and notes
1. Burgers, P. M. J.; Eckstein, F.; Hunneman, D. H.; Baraniak, J.;
Kinas, R. W.; Lesiak, K.; Stec, W. J. J. Biol. Chem. 1979, 254,
9959.
4.1.5. The intramolecular cyclization reaction of 1a. Into
the solution of fast-N4-benzoyl-deoxycytidine-30-O-(2-thio-
1,3,2-oxathiaphospholane) (31 mg, 0.066 mmol) in anhy-
drous acetonitrile (0.3 mL) equimolar amount of DBU
(10 mL) was added. After 5 min at room temperature the
2. (a) O’Brian, C. A.;Roczniak, S. A.;Bramson, H. N.;Baraniak, J.;
Stec, W. J.; Kaiser, E. T. Biochemistry 1982, 21, 4371. (b)
Jarvest, R. L.; Lowe, G.; Baraniak, J.; Stec, W. J. Biochem. J.
1982, 203, 461. (c) Rothermel, J. D.; Stec, W. J.; Baraniak, J.;
Jastorff, B.; Botelho, L. H. P. J. Biol. Chem. 1983, 258, 12125. (d)
Van Haastert, P. J. M.; Van Driel, R.; Jastorff, B.; Baraniak, J.;
Stec, W. J.; de Wit, R. J. W. J. Biol. Chem. 1984, 259, 10020. (e)
Zimmerman, A. L.; Yamanaka, G.; Eckstein, F.; Baylor, D. A.;
Stryer, L. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 8813. (f)
Schwede, F.; Maronde, E.; Genieser, H.; Jastorff, B. Pharmacol.
Ther. 2000, 87, 199.
reaction mixture was diluted with CD3CN (0.3 mL) and 31
P
NMR spectra showed quantitative formation of single
product resonating at d 51.5 ppm (CD3CN). After removal
of the benzoyl protecting groups (treatment with 30%
aqueous ammonia at 55 8C for 16 h) the product was
identified by 31P NMR and HPLC comparison with genuine
sample,4d as SP-deoxycytidine cyclic 30,50-O,O-phos-
phorothioate (3A). Its structure was also confirmed by
FAB MS (m/z 304.1 (negative ions); calculated MW 305.25
(free acid)). The cyclization of 1b, 1c and 1d was performed
in analogous way. For 1b and 1c single products were
observed, while for 1d several resonances were found
within a range 55–57 ppm.
3. (a) Eckstein, F. Annu. Rev. Biochem. 1985, 54, 367. (b)
Eckstein, F.; Kutzke, U. Tetrahedron Lett. 1986, 27, 1657.
4. (a) Baraniak, J.; Kinas, R. W.; Lesiak, K.; Stec, W. J. J. Chem.
Soc., Chem. Commun. 1979, 940–941. (b) Stec, W. J. Acc.
´
Chem. Res. 1983, 16, 411. (c) Lesnikowski, Z. J.;
´
Niewiarowski, W.; Zielinski, W. S.; Stec, W. J. Tetrahedron
1984, 40, 15. (d) Baraniak, J.; Stec, W. J. J. Chem. Soc., Perkin
Trans. 1 1987, 1645. (e) Wozniak, L. A.; Okruszek, A. Chem.
Soc. Rev. 2003, 32, 158.
4.1.6. The condensation reaction of 1d in the presence of
30-O-acetylthymidine. A solution of 1d (70 mg, 130 mmol)
and 30-O-acetylthymidine (6 mg, 21 mmol) in anhydrous
acetonitrile (0.4 mL) was treated, with stirring at room
temperature, with DBU (22 mL, 145 mmol). After 7 min the
5. De Napoli, L.; Galeone, A.; Mayol, L.; Messere, A.;
Montesarchio, D.; Piccialli, G. Bioorg. Med. Chem. Lett.
1995, 3, 1325. Smietana, M.; Kool, E. T. Angew. Chem.,
Int. Ed. 2002, 41, 3704. Kool, E. T. Acc. Chem. Res. 1998,
31, 502.
reaction mixture was diluted with CD3CN (0.3 mL) and 31
P
NMR showed several peaks within 56–58 ppm range.
The deprotected products (treatment with 30% aqueous
ammonia at 55 8C for 16 h) were isolated by preparative RP
HPLC (PTH C18, 5 m, 2.1!220 mm column (Brownlee)
with a linear gradient of acetonitrile in 0.1 M TEAB:
0–60 min—0.47%/min; flow 0.3 mL/min (UV detection at
255 nm)), quantified by UV absorption at 260 nm and
identified by MALDI-TOF MS.
6. Riss, P.; Mayer, R.; Weinhouse, H.; Amikam, D.; Huggirat, Y.;
Benziman, M.; de Vrom, E.; Fidder, A.; de Paus, E.; Sliedgret,
L. A. J. M.; van der Marel, G. A.; van Boom, J. H. J. Biol. Chem.
1990, 265, 18933–18943.
7. Battistini, C.; Fustinoni, S.; Brasca, M. G.; Borghi, D.
Tetrahedron 1993, 49, 1115.
´
8. (a) Stec, W. J.; Grajkowski, A.; Koziołkiewicz, M.; Uznanski,
B. Nucleic Acids Res. 1991, 19, 5883–5888. (b) Stec, W. J.;
´
Grajkowski, A.; Karwowski, B.; Kobylanska, A.;
Acknowledgements
Koziołkiewicz, M.; Misiura, K.; Okruszek, A.; Wilk, A.;
Guga, P.; Boczkowska, M. J. Am. Chem. Soc. 1995, 117,
12019. (c) Stec, W. J.; Karwowski, B.; Boczkowska, M.;
Guga, P.; Koziołkiewicz, M.; Sochacki, M.; Wieczorek, M. W.;
Błaszczyk, J. J. Am. Chem. Soc. 1998, 120, 7156. (d) Guga, P.;
Studies described in this report were financially assisted by
the State Committee for Scientific Research (MNiI, grant 3
T09A 059 28 to W.J.S.).

2704
P. Guga et al. / Tetrahedron 62 (2006) 2698–2704
Stec, W. J. Synthesis of Phosphorothioate Oligonucleotides with
10. (a) Eckstein, F.; Burgers, P. M. J.; Hunneman, D. H. J. Biol.
Chem. 1979, 254, 7476. (b) Benkovic, S. J.; Bryant, F. R.
Biochemistry 1979, 18, 2825.
Stereodefined Phosphorothioate Linkages. In Current Protocols
in Nucleic Acid Chemistry; Beaucage, S. L., Bergstrom, D. E.,
Glick, G. D., Jones, R. A., Eds.; Wiley: Hoboken, NJ, 2003;
pp 4.17.1–4.17.28.
11. Eckstein, F.; Potter, B. V. L.; Connolly, B. Biochemistry 1983,
22, 1369.
´
´
9. (a) Uchimaru, T.; Stec, W. J.; Tsuzuki, S.; Hirose, T.; Tanabe, K.;
Taira, K. Chem. Phys. Lett. 1996, 263, 691. (b) Uchimaru, T.;
Stec, W. J.; Taira, K. J. Org. Chem. 1997, 62, 5793.
12. Koziołkiewicz, M.;Wojcik, M.;Kobylanska,A.;Karwowski,B.;
Re˛bowska, B.; Guga, P.; Stec, W. J. Antisense Nucleic Acid
Drug Dev. 1997, 7, 43–48.