Diastereomerically pure nucleoside-5′-O-(2-thio-4,4-pentamethylene-1, 3,2-oxathiaphospholane)s-Substrates for synthesis of P-chiral derivatives of nucleoside-5′-O-phosphorothioates

Article abstract of DOI:10.1002/chir.20905

A method for stereocontrolled chemical synthesis of P-substituted nucleoside 5′-O-phosphorothioates has been elaborated. Selected 3′-O-acylated deoxyribonucleoside- and 2′,3′-O,O-diacylated ribonucleoside-5′-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane)s were chromatographically separated into P-diastereomers. Their reaction with anions of phosphorus-containing acids was highly stereoselective (≥90%) and furnished corresponding P-chiral α-thiodiphosphates and their phosphonate analogs with satisfactory yield. Copyright

Full text of DOI:10.1002/chir.20905

CHIRALITY 23:237–244 (2011)
Diastereomerically Pure Nucleoside-50-O-(2-thio-4,4-pentamethylene-
1,3,2-oxathiaphospholane)s—Substrates for Synthesis of P-Chiral
Derivatives of Nucleoside-50-O-phosphorothioates
AGNIESZKA TOMASZEWSKA, PIOTR GUGA*, AND WOJCIECH J. STEC
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
´
90-363 Ło´dz, Poland
ABSTRACT
A method for stereocontrolled chemical synthesis of P-substituted nucleoside
50-O-phosphorothioates has been elaborated. Selected 30-O-acylated deoxyribonucleoside- and
20,30-O,O-diacylated ribonucleoside-50-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane)s
were chromatographically separated into P-diastereomers. Their reaction with anions of phos-
phorus-containing acids was highly stereoselective (ꢀ90%) and furnished corresponding P-chi-
ral a-thiodiphosphates and their phosphonate analogs with satisfactory yield. Chirality 23:237–
C
VV
244, 2011.
2010 Wiley-Liss, Inc.
KEY WORDS: stereoselectivity; P-diastereomers; oxathiaphospholane monomers; nucleoside
thiophosphates; HPLC
INTRODUCTION
phosphates and dinucleoside 50,50-polyphosphates are chiral
and exist in pure stereochemical forms, their interactions
may be significantly different for each P-diastereomer. Pure
diastereomers can sometimes be obtained by enzymatic syn-
thesis. For example, prochiral deoxyadenosine 50-O-phos-
phorothioate can be stereospecifically converted in the pres-
ence of ATP with tandem adenylate kinase/pyruvate kinase
into corresponding P-chiral a-thiodiphosphate and, finally,
into a-thiotriphosphate.13,14 Alternatively, in certain instances
chromatographic separation of P-diastereomers can be
applied.15 However, the chromatography and the enzymatic
methods are not general and suffer from severe limitations.
Thus, a method for a stereocontrolled chemical synthesis of
P-substituted nucleoside 50-O-phosphorothioates is highly
demanded. In this laboratory, the method for a stereocon-
trolled synthesis of oligo(nucleoside phosphorothioate)s,
based on chromatographically resolved P-diastereomers of
50-O-DMT-nucleoside-30-O-(2-thio-4,4-pentamethylene-1,3,2-
oxathiaphospholane) monomers (30-OTPs), has been devel-
oped.16,17 On the other hand, because chromatographic sepa-
ration of nucleoside-50-O-oxathiaphospholane monomers, car-
rying typical protecting groups at the 30- or 20- and 30-
hydroxyl functions, could not be achieved, numerous P-chiral
biophosphates were synthesized using unresolved mono-
mers (e.g., 8, Scheme 3).18–20 Here, we report on the synthe-
sis of selected 30-O-acylated deoxyribonucleoside- and 20,
Nucleoside 50-O-phosphates (NMPs), diphosphates
(NDPs) and triphosphates (NTPs), as well as their dinucleo-
side polyphosphate analogs (e.g., 50,50-NPnN, n 5 2–7) play
an important role in a living cell serving as a source of
energy or as regulatory factors in many metabolic processes.
Some notable examples of involvement of NTPs in eukaryo-
tic systems include developmental control,1 signal transduc-
tion,2 and tumor metastasis.3 NTPs are widely used in bio-
chemistry and molecular biology as substrates for the
in vitro enzymatic synthesis of DNA and RNA. Natural and
modified NTPs have also been used in DNA sequencing4
and studies on the mode of action of numerous enzymes.5,6
All natural nucleoside phosphates and oligonucleotides are
readily metabolized in the cell, therefore, more stable ana-
logs are required for detailed studies on their mechanism of
action. For that purpose, numerous analogs carrying modifi-
cations within nucleobases, ribose, deoxyribose, and phos-
phate moieties have been obtained.7,8 Among these, the
phosphorothioate nucleotides/oligonucleotides, in which one
of the nonbridging phosphate oxygen atoms was substituted
with a sulfur atom, were most extensively studied, both in in
vitro and in vivo experiments.9,10 Also, it should be noted
that only recently phosphorothioation of DNA in bacteria has
been discovered.11
In terms of chemistry, it should be emphasized that
although the phosphorothioate group is isoelectronic with
the natural phosphate moiety, this modification introduces
important changes resulting from different steric require-
ments of the sulfur atom (PꢁꢁS vs PꢁꢁO bond length), differ-
ent affinity toward metal ions (‘‘soft’’ sulfur vs ‘‘hard’’ oxygen)
and unsymmetrical negative charge distribution.12 Moreover,
although nucleoside 50-O-phosphorothioates and symmetrical
50,50-NPSN are P-prochiral, the NTP and NDP analogs, with
the nonbridging sulfur atom introduced into the a-phosphate
group, are P-chiral species and if chemically synthesized,
they consist of the practically equimolar mixture of P-diaster-
eomers of either RP or SP absolute configuration. Given that
nearly all biomolecules that recognize nucleoside 50-poly-
Additional Supplementary Material may be found in the online version of this
article.
Dedicated to Professor Marian Mikołajczyk on the occasion of his 70th birth-
day.
Contract grant sponsor: Centre of Molecular and Macromolecular Studies;
Contract grant number: statutory funds; Contract grant sponsor: Polish Min-
istry of Science and Higher Education; Contract grant number: 3 T09A 059
28 to WJS
*Correspondence to: Dr. Piotr Guga, Sienkiewicza 112, 90-363 Ło´dz, Poland.
E-mail: pguga@bio.cbmm.lodz.pl
Received for publication 20 January 2010; Accepted 22 June 2010
DOI: 10.1002/chir.20905
Published online 6 October 2010 in Wiley Online Library
(wileyonlinelibrary.com).
C
VV
2010 Wiley-Liss, Inc.

238
TOMASZEWSKA ET AL.
evaporated, the residue was dissolved in chloroform (2 ml), and applied
on a silica gel column (25 3 3 cm). The column was eluted with a gradi-
ent of chloroform and methanol from 100:0 to 90:10 (v/v). Appropriate
fractions (Rf 0.53, 0.78, 0.78, 0.81, 0.78, 0.85, and 0.86 were found for dG,
dC, T, rA, rG, rC, and U derivatives, respectively, CHCl3:MeOH, 9:1; Rf
0.88 was found for dA, CHCl3:MeOH, 95:5) were combined and the sol-
vent was evaporated under reduced pressure to give corresponding com-
pounds 2 (Y 5 (2)-camphanoyl) in 55–90% yield, which were analyzed
by FAB mass spectrometry: (1) deoxyribonucleoside series; Z 5 H; B0
Scheme 1. Synthesis of 50-OTPs of DNA (4) and RNA (5) series. Y 5
biphenyl-4-carbonyl, 1-adamantanecarbonyl, 2-naphthoyl or (2)-camphanoyl.
B0 5 nucleobase (N,O-protected if necessary). The two-head arrow shows
expected interactions in 4 (or 5) which substantially differentiate the ener-
gies of the most stable conformers of RP and SP-forms, and hence change
their chromatographic mobility.
5 AdeBz, C48H47N5O9, calc. 837, (M1H)1 838, (M2H)2 836; B0
GuaiBu, C45H49N5O10, calc. 819, (M1H)1 820, (M2H)2 818; B0 5 CytBz
C47H47N3O10 Thy,
calc. 813, (M1H)1 814, (M2H)2 812; B0
5
,
,
5
C
41H44N2O10, calc. 724, (M1H)1 724, (M2H)2 723; (2) ribonucleoside
series; Z 5 OY; B0 5 AdeBz, C58H59N5O13, calc. 1033, (M1H)1 1034,
(M2H)2 1032; B0 5 GuaiBu, C55H61N5O14, calc. 1015, (M1H)1 1016,
(M2H)2 1014; B0 5 CytBz, C57H59N3O14, calc. 1009, (M1H)1 1010,
30-O,O-diacylated ribonucleoside-50-O-(2-thio-4,4-pentamethy-
lene-1,3,2-oxathiaphospholane)s (50-OTPs, 4 and 5, respec-
tively, Scheme 1), which are chromatographically separable
into P-diastereomers. Based on our earlier results on stereo-
retentive mode of DBU-assisted oxathiaphospholane ring
opening process,16 these new monomers were expected to
be suitable for the stereocontrolled synthesis of P-chiral
derivatives of nucleoside-50-O-phosphorothioates.
(M2H)2 1008; B0
(M2H)2 905.
5 Ura, C50H54N2O14,
calc. 906, (M1H)1 906,
Removal of the DMT Group from 50-O-DMT-N-Protected
Nucleosides Carrying (2)-camphanoyl Moiety—Synthesis
of Compounds 3
To the magnetically stirred solution of compound 2 (2–3 mmol) in a
mixture CHCl3:MeOH (9:1, 15 ml), p-toluenesulfonic acid monohydrate
(3–4.5 mmol, 50% molar excess) was added at room temperature. After
the reaction was complete (ca. 20 min, TLC control, CHCl3:MeOH, 9:1),
the solvent was evaporated, the residue was dissolved in chloroform (2
ml), and applied onto a silica gel column (25 3 3 cm). The column was
eluted with a gradient of chloroform:methanol from 100:0 to 90:10, v/v).
Appropriate fractions (camphanoylated deoxyribonucleosides, Rf 0.27–
0.36, CHCl3:MeOH, 95:5; camphanoylated ribonucleosides, Rf 0.26–0.48,
CHCl3:MeOH, 9:1) were combined and the solvent was evaporated
under reduced pressure to give desired compounds 3 in ꢂ70–90%
yields, further analyzed by FAB MS: (1) deoxyribonucleoside series; Z
5 H; B0 5 AdeBz, C27H29N5O7, calc. 535, (M1H)1 536, (M2H)2 534; B0
EXPERIMENTAL SECTION
The nuclear magnetic resonance spectra (NMR) were recorded on a
Bruker AC-200 instrument (200 MHz for 1H) or on a Bruker DRX-500
(500 MHz for 1H), with 85% H3PO4 used as an external standard for 31P
NMR. The FAB-MS spectra (13 keV, Cs1) were recorded on a Finnigan
MAT 95 spectrometer, in the positive and negative ions modes where
(M1H)1 and (M2H)2 ions were observed, respectively. Calculated
masses (calc.) refer to the parent compounds. Negative ion MALDI TOF
mass spectra were recorded on a Voyager-Elite instrument (PerSeptive
Biosystems Inc., Framingham, MA). HPLC analyses were done using a
Gilson system consisting of two 306 pumps and a 151 UV/VIS detector.
Anhydrous acetonitrile, 1,4-diazabicyclo[5.4.0]undec-7-ene (DBU) and
anhydrous pyridine were supplied by Fluka, Merck and Aldrich, respec-
tively. (1S)-4,7,7-Trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carbonyl
chloride (or (2)-camphanic chloride), 2-naphthoyl and biphenyl-4-car-
bonyl chlorides were supplied by FLUKA, 1-adamantanecarbonyl chlo-
ride by Aldrich, and DMT-Cl by Chemgenes. Crystalline phosphoric
acid and p-toluenesulfonic acid monohydrate were bought from Aldrich.
svPDE was obtained from Boehringer Mannheim GmbH, Germany. Nu-
clease P1 from Penicillium citrinum was purchased from Sigma. All silica
gel media for chromatography were supplied by MERCK. Silica gel 60,
230–400 mesh, was used for routine chromatographic purification. For
the open column separation of P-diastereomers, silica gel 60H (particles
5–40 lm) was employed. TLC silica gel 60 plates were used for routine
analyses, whereas HP TLC silica gel 60 plates were used for assessment
of chromatographic separability of P-diastereomers, all plates with UV
F254 indicator. Note that useful technical suggestions have been pub-
lished for closely related synthesis and separation of diastereomers of 30-
OTPs.17 The following procedures describe synthesis of the intermedi-
ates 2, 3 and oxathiaphospholane monomers 4, 5 carrying the campha-
noyl moiety/moieties. Those carrying 2-naphthoyl or biphenyl-4-carbonyl
moieties can be obtained on a similar way using corresponding acyl
chlorides.
5 GuaiBu, C24H31N5O8, calc. 517, (M1H)1 518, (M2H)2 516; B0
5
CytBz, C48H47N5O9, calc. 511, (M1H)1 512, (M2H)2 510; B0 5 Thy,
C20H26N2O8, calc. 422, (M1H)1 423, (M2H)2 421; (2) ribonucleoside
series; Z 5 OY; B0 5 AdeBz, C37H41N5O11, calc. 731, (M1H)1 732,
(M2H)2 730; B0 5 GuaiBu, C34H43N5O12, calc. 713, (M1H)1 714,
(M2H)2 712; B0 5 CytBz, C36H41N3O12, calc. 707, (M1H)1 708,
(M2H)2 706; B0 5 Ura C29H36N2O12, calc. 604, (M1H)1 605, (M2H)2
603.
Phosphitylation of 3 with 2-chloro-4,4-pentamethylene-
1,3,2-oxathiaphospholane—Synthesis of Compounds 4
and 5
To the magnetically stirred solution of 1–3 mmol of 3 (dried overnight
under high vacuum) and anhydrous N,N-diisopropylethylamine (10%
molar excess) in anhydrous methylene chloride (20 ml), 2-chloro-4,4-
pentamethylene-1,3,2-oxathiaphospholane17 (10% molar excess) was
added dropwise (under dry argon) at room temperature using a Gas-
Tight Hamilton syringe. The reaction was complete in 5 h and then ele-
mental sulfur (two-fold molar excess) was added. Stirring was continued
for 24 h and excess sulfur was filtered off. After evaporation of the sol-
vent, the residue was dissolved in chloroform (3 ml) and applied onto a
silica gel column (25 3 3 cm). The column was eluted with a gradient of
chloroform:methanol from 100:0 to 95:5, v/v). Appropriate fractions (Rf
0.54–0.83, CHCl3:MeOH, 9:1) were combined, and the solvents were
evaporated under reduced pressure to give desired compounds 4 or 5
Synthesis of Camphanoylated 50-O-DMT-N-Protected
Nucleosides 2—General Procedure
50-O-DMT-N-protected deoxyribonucleoside or ribonucleoside (1;
B05AdeBz, CytBz, GuaiBu, Thy or Ura), (2–4 mmol) was evaporated three
times with anhydrous pirydine (with the exclusion of moisture), then
dried in a dessicator under oil pomp vacuum for 24 h and dissolved in
anhydrous pyridine (20 ml) under dry argon. To the solution, (2) cam-
phanic acid chloride (20% molar excess, calculated over each hydroxyl
group to be acylated) was added at room temperature. After the reaction
was complete (ca. 5 h, TLC control, CHCl3:MeOH, 9:1), the solvent was
in 55–75% yield. (1) deoxyribonucleoside series; 4, Z 5 H; 4d, B0
5
Thy, FAB MS, C34H40N2O9PS2, calc. 628, (M1H)1 629, (M2H)2 627;
31P NMR, d (ppm) 106.51, 106.13 (CD3CN); 4f, B0 5 GuaiBu, FAB MS,
C31H42N5O9PS2, calc. 723, (M1H)1 724, (M2H)2 722; 31P NMR, d
(ppm) 108.24, 107.99 (CDCl3); 4g, B0
C34H40N5O8PS2, calc. 741, (M1H)1 742, (M2H)2 740; 31P NMR, d
(ppm) 106.31, 106.29 (CDCl3); 4h, B0 CytBz
FAB MS,
C33H40N3O9PS2, calc. 717, (M1H)1 718, (M2H)2 716; 31P NMR, d
5
AdeBz
,
FAB MS,
5
,
Chirality DOI 10.1002/chir

DIASTEREOMERS OF NUCLEOSIDE-50-O-OTP MONOMERS
239
TABLE 1. A list of the oxathiaphospholane monomers 4 and 5 separable into P-diastereomers
B0
4a, Thy
4b, Thy
4c, Thy
Y
Z
Rf’s or HPLC retention times
TLC 0.68, 0.60a
Biphenyl-4-carbonyl
2-naphthoyl
H
H
H
H
H
OY
OY
OY
OY
OY
TLC 0.62, 0.51a; HPLC: 6.7, 7.5 minb
TLC 0.81, 0.76c
1-adamantanecarbonyl
(2)-camphanoyl
(2)-camphanoyl
(2)-camphanoyl
(2)-camphanoyl
(2)-camphanoyl
2-naphthoyl
4d, Thy
TLC 0.54, 0.49d
4e, GuaiBu, DPC
5a, Ura
TLC 0.73, 0.70d
TLC 0.74, 0.71e; HPLC: 6.9, 7.7 minf
TLC 0.43, 0.40e; HPLC: 9.1, 10.1 minf
HPLC: 15.0, 16.0 ming
5b, AdeBz
5d, GuaiBu
5e, Ura
TLC 0.85, 0.82d
5f, AdeBz
2-naphthoyl
TLC 0.56, 0.53h; HPLC: 5.6, 6.0 minb
aEthyl acetate:butyl acetate:benzene 2:2:1, single development (sd) of the HPTLC plate.
bEthyl acetate:hexane 52:48.
cEthyl acetate:butyl acetate:benzene 3:2:1, double development (dd).
dEthyl acetate:butyl acetate:benzene 2:2:1, dd.
eEthyl acetate:butyl acetate:benzene 2:1:1, dd.
fEthyl acetate:hexane 60:40.
gEthyl acetate:hexane:methanol 52:45:3.
hEthyl acetate:butyl acetate:benzene 1:2:2; sd.
(ppm) 106.74, 106.50 (CD3CN); (2) ribonucleoside series; 5, Z 5 OY;
5a, B0 5 Ura, FAB MS, C36H47N2O13PS2, calc. 810, (M1H)1 811,
(M2H)2 808; 31P NMR, d (ppm) 106.86, 106.47 (CD3CN); 13C NMR, d
(ppm) 177.70, 177.58, 166.54, 162.86, 149.93, 139.30, 103.19, 94.16, 90.51,
87.23, 80.37, 79.54, 78.72, 76.37, 73.59, 71.51, 70.70, 69.39, 66.11, 58.29,
54.83, 54.72, 54.32, 37.03, 36.73, 31.13, 30.75, 28.77, 28.65, 25.07, 23.79,
23.61, 16.82, 16.66, 16.39, 9.61 (CDCl3). 5b, B0 5 AdeBz, FAB MS,
90.55, 85.11, 83.34, 79.47, 76.36, 75.75, 68.84, 54.76, 54.43, 36.00, 36.38,
36.11, 30.66, 28.81, 25.05, 23.56, 19.25, 19.18, 16.76, 16.69, 9.59 (CDCl3).
Open Column Separation of Camphanoylated
Diastereomers of 4 or 5
A 250–300 mg sample of a camphanoylated monomer 4 or 5 in 2 ml of
appropriate eluent was applied onto a column (35 3 6 cm) containing
80–100 g silica gel (Merck 60H). The column was eluted with 1000–1500
C
44H52N5O12PS2, calc. 937, (M1H)1 938, (M2H)2 936; 31P NMR, d
(ppm) 107.08, 107.02 (CDCl3); 13C NMR, d (ppm) 177.55, 166.58, 166.37,
152.97, 151.43, 149.76, 141.29, 133.48, 132.81, 128.82, 127.84, 123.11,
90.61, 90.32, 86.01, 81.17, 79.55, 73.64, 71.46, 69.15, 66.27, 54.85, 54.73,
54.50, 54.39, 37.09, 36.54, 31.15, 30.80, 28.82, 28.63, 25.10, 23.77, 16.83,
16.69, 16.38, 9.57 (CDCl3). 5c, B0 5 CytBz, FAB MS, C43H52N3O13PS2,
calc. 913, (M1H)1 914, (M2H)2 912; 31P NMR, d (ppm) 106.87, 106.44
(CD3CN); 13C NMR, d (ppm) 176.81, 176.33, 165.11, 165.04, 161.51,
153.24, 132.01, 127.76, 126.38, 89.42, 89.17, 87.96, 79.00, 78.35, 73.24,
68.44, 68.20, 64.22, 53.67, 53.52, 53.01, 35.86, 35.47, 30.01, 29.52, 27.56,
ml of ethyl acetate-butyl acetate-benzene (2:2:1 v/v/v for the T and
DPC
dGiBu,
derivatives, 2:1:1 v/v/v for the rABz and U derivatives) and
fractions of 4–5 ml were collected. TLC control of the eluate was per-
formed on HP-TLC plates (double development, see Table 1). Appropri-
ate fractions were combined and the solvent was evaporated under
reduced pressure. Typically, the applied material was recovered in ca.
80% yield. The diastereomeric purity of resulting fractions was assessed
by 31P NMR spectroscopy.
23.88, 22.58, 22.38, 15.66, 15.44, 15.18, 8.43, 8.31 (CDCl3). 5d, B0
5
GuaiBu, FAB MS, C41H54N5O13PS2, calc. 919, (M1H)1 920, (M2H)2
917; 31P NMR, d (ppm) 106.88, 106.52 (CD3CN); 13C NMR, d (ppm)
178.85, 177.71, 166.87, 166.34, 155.29, 147.79, 138.21, 90.54, 90.34, 86.90,
86.49, 79.71, 76.37, 73.68, 73.13, 71.48, 71.18, 69.41, 69.30, 54.82, 54.70,
54.45, 54.29, 37.16, 36.90, 36.37, 31.25, 30.80, 28.78, 28.65, 25.01, 23.74,
23.60, 18.87, 16.74, 16.65, 16.44, 9.55 (CDCl3).
Synthesis of Dinucleoside 30,50-phosphorothioates 7
To 50-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) mono-
mers 4d, 5a, 5b, or 5f (15–20 mg, 0.02 mmol, dried in a vacuum dessi-
cator for 24 h) and 50-O-DMT-thymidine (3 molar equivalents), 600 ll of
dry acetonitrile and 5 ll of 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU, 1.1
molar equivalent) were added under dry argon. After 2 h, the reaction
was complete. The mixture was concentrated under reduced pressure
and treated with concentrated ammonia solution (2 ml) for 2 h to remove
the acyl group. For the derivatives of 5b and 5f, where the benzoyl
group was used for base protection, heating for 15 h at 558C was neces-
sary. After removal of solvent, the DMT moiety was removed with 50%
aqueous acetic acid for 1.5 h at room temperature, followed by evapora-
tion under reduced pressure. The identity of resulting products 7 was
confirmed in HPLC experiments by co-injection with corresponding
deprotected dinucleotides obtained using standard phosphoramidite pro-
tocol with sulfurization of the PIII intermediate with 3H-1,2-benzodithiol-
3-one 1,1-dioxide (Beaucage reagent21). Conditions: C18 column, 250 3
4.6 mm, 5 lm; elution with a gradient of 0.1 M TEAB, pH 7.3 to 20%
CH3CN in 0.1 M TEAB over 20 min. The stereochemistry was deter-
mined by RP-HPLC either directly (TPST from 4d), or after enzymatic
digestion with svPDE22 and Nuclease P123 (TPSrA from 5a, 5b, or 5f)
(vide infra and Fig. 4S, Supporting Information). The HPLC conditions
as above; the retention times: ca. 15.94 and 16.56 min for RP and SP dia-
stereomers of TPST, and, assigned in this work, 18.04 and 18.84 min for
RP and SP diastereomers of TPSrA, respectively.
Protection of 30-O-camphanoyl-N2-iBu-deoxyguanosine-50-
O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane)
at the O6 Site with Diphenylcarbamoyl Chloride—
Synthesis of Compound 4e
To a magnetically stirred solution of 30-O-camphanoyl-N2-iBu-deoxy-
guanosine-50-O-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane)
(0.345 mmol, 250 mg) in pyridine (15 ml), N,N-diisopropylethylamine
(0.1 ml, 0.51 mmol) and diphenylcarbamoyl chloride (Ph2NCOCl, 0.16 g,
0.7 mmol) were added (under dry argon) at room temperature. The mix-
ture was stirred for 2 h, concentrated, dissolved in chloroform (1.5 ml),
and applied onto a silica gel column (25 3 3 cm). The column was eluted
with chloroform-methanol (the methanol content 0–2%). Appropriate frac-
tions were collected (Rf 0.87, CHCl3:MeOH, 9:1) and evaporated under
reduced pressure to give the product 4e in ca. 80% yield. FAB MS:
C44H51N6O10PS2, calc. 918, (M1H)1 919, (M2H)2 917; 31P NMR, d
(ppm) 106.68, 106.65 (CDCl3); 13C NMR, d (ppm) 177.80, 175.01, 166.88,
156.14, 154.22, 151.94, 150.29, 142.76, 141.67, 129.07, 126.84, 121.63,
Chirality DOI 10.1002/chir

240
TOMASZEWSKA ET AL.
resonances at d 42.40 ppm (d, 2JP-P 5 37 Hz) and 42.27 ppm (d, 2JP-P 5 36
Hz) for a-P atoms, d 12.35 ppm (d, 2JP-P 5 3.5 Hz) and 12.21 ppm (d, 2JP-P 5
3.5 Hz) for g-P atoms, and d 7.57 ppm (dd, 2JP-P 5 36 Hz and 2JP-P 5 3.5 Hz)
for b-P atom of the main diastereomer (the resonances for the second dia-
stereomer were too small to be precisely analyzed). Ca. 5% of the product
10 carrying the camphanoyl residue was isolated by RP-HPLC and analyzed
by MALDI TOF MS (C21H31N2O15P3S, calc. 676, (M2H)2 675). The
remaining solution was concentrated to dryness and treated with concen-
trated ammonia (1.5 ml) for 1.5 h at room temperature. After evaporation,
the residue was dissolved in water, and the product was isolated by means
of ion exchange chromatography on Sephadex A-25 resin (pH 7, linear gra-
dient 0.3 M to 1 M triethylammonium bicarbonate). The product 11 (7.8
mg, 43%) was analyzed by MALDI TOF MS (C11H19N2O12P3S, calc. 496,
(M2H)2 495).
Fig. 1. Chromatogram from HPLC separation of 50-O-(2-thio-4,4-pentam-
ethylene-1,3,2-oxathiaphospholane)-30-O-(2-naphthoyl)-thymidine 4b; consec-
utive profiles for separation of 3, 4, and 5 mg samples dissolved in 150–250 ll
of chloroform (the detector overloaded). Conditions: a Hyperprep HS silica
gel column 250 3 10 mm2, 8 lm, mobile phase: ethyl acetate:hexane 52:48
v/v, isocratically, a flow rate 8 ml/min, retention times 6.7 and 7.5 min.
RESULTS AND DISCUSSION
50-OTPs of Deoxyribonucleoside Series
To obtain resolvable P-diastereomers of acylated mono-
mers 4 and 5, acyl chlorides possessing a bulky substituent
were selected. It was assumed that a sterically demanding
moiety in the 30-position of the monomers would sterically
interact with the oxathiaphospholane ring (the repulsion is
depicted by a two-head arrow, Scheme 1) giving rise to differ-
entiation of the chromatographic mobility of SP and RP forms.
This idea arose from molecular modelling performed for a
derivative of thymidine monomer 4a carrying a biphenyl-4-
carbonyl moiety at the 30 position. Conformational search for
the SP and RP diastereomers was performed with a Spar-
tan’06 program using the MMFF force field method.24 Five
most stable conformers of each diastereomer were further
optimized under the gas phase conditions with the Gaussian
03 program25 using the ab initio HF/6-31G(d) method.
Remarkably, the energies calculated for the most stable con-
formers of the SP and RP diastereomers differed by ca. 10
kcal/mol. Assuming that these most stable conformers
remain predominant during interaction with particles of silica
gel (which is achiral chromatography material) one may
expect the formation of the diastereomeric monomerꢃsilica
complexes of the energies of sorption/desorption sufficiently
different to allow for the separation of the P-diastereomers.
A general method of synthesis of the monomers 4
(Scheme 1, Y 5 acyl, Z 5 H) involves acylation of the 30-OH
group in 50-O-DMT-N-protected deoxyribonucleosides (1, B0
5 Thy, AdeBz, CytBz, or GuaiBu) with an acyl chloride in pyri-
dine (61–92% yield). The resulting acylated nucleosides 2
were detritylated with p-toluenesulfonic acid in a CH2Cl2-
MeOH mixture (9:1) and crude 3 were purified by silica gel
Hydrolysis of 7 with svPDE
The reaction mixture (20 lL) containing 25 mM Tris-Cl (pH 8.5), 5 mM
MgCl2, 1lL of the svPDE suspension, and dinucleoside 30,50-phosphoro-
thioate 7 (0.2 OD) was incubated for 24 h at 378C. Then, the sample was
heat-denatured (for 1 min at 958C) and analyzed by HPLC, similar condi-
tions as above.
Hydrolysis of 7 with Nuclease P1
The reaction mixture (20 lL) containing 100 mM Tris-Cl (pH 7.2), 1
mM ZnCl2, 2 lL of nuclease P1 suspension, and dinucleoside 30,50-phos-
phorothioate 7 (0.2 OD) was incubated for 24 h at 378C. Then, the sam-
ple was heat-denatured (for 1 min at 958C) and analyzed by HPLC, simi-
lar conditions as above.
Synthesis of Thymidine-50-O-(a-thiodiphosphate)
50-OTP-T monomer 4d (‘‘fast"/’’slow’’ 86:14, 105 mg, 167 lmol) was
mixed with anhydrous crystalline H3PO4 (49 mg, 501 lmol) and dried
under high vacuum for 24 h. The reaction was carried in dry N-methyl-
pyrrolidone (3 ml) in the presence of DBU (270 ll in 1 ml of acetonitrile;
1800 lmol, containing ca. 65 ppm of water as determined by Karl Fi-
scher method) under dry argon for 2 h at room temperature. The prod-
uct 9 was formed in ca. 32% yield (31P NMR: d (ppm, a few drops of d6-
2
benzene added for the lock of the instrument) 46.80, d, JP-P 5 30 Hz,
46.64, d, JP-P 5 30 Hz, for a-P atoms; d (ppm) 26.66, d, JP-P 5 30 Hz,
26.89, d, JP-P 5 30 Hz, for b-P atoms). Despite of extensive drying of
2
2
2
the substrates, the mixture contained 30-O-camphanoyl-thymidine-50-O-
thiophosphate, i.e., the product of hydrolysis of 4d (d 50.21 ppm, 62%). A
few OD units of compound 9 (carrying the camphanoyl residue) was iso-
lated by RP-HPLC and analyzed by MALDI TOF MS (C20H28N2O13P2S,
calc. 598, (M2H)2 597). The remaining mixture was treated with equal
volume of concentrated ammonium hydroxide to remove the base-labile
protecting groups. The ammonia was removed under reduced pressure
and a 31P NMR spectrum was recorded in N-methylpyrrolidone solution
with a few drops of d6-benzene added. The 31P NMR spectrum (proton
decoupled, sweep width 64,000 Hz, time domain 65,536 points, acquisi-
tion time 0.51 s) contained the following resonances: d (ppm) 42.41, d,
2JP-P 5 37 Hz, 41.61, d, 2JP-P 5 39 Hz, for a-P atoms; 25.53, d, 2JP-P 5 37
Hz, 25.71, d, 2JP-P 5 39 Hz, for b-P atoms; 46.79 for TMPS.
Synthesis of Thymidine-50-O-(b,c -methylene-a-
thiotriphosphate) (11)
The monomer 4d (fast/slow 1:10, 23 mg, 37 lmol) was mixed with anhy-
drous methylenediphosphonic acid (20 mg, 110 lmol) and dried under high
vacuum for 24 h. The reaction was carried in dry acetonitrile (3 ml) in the
presence of DBU (77 ll; 518 lmol, containing ca. 35 ppm of water as deter-
mined by Karl Fischer method) under dry argon for 4 h at room tempera-
ture. The 31P NMR spectrum (proton decoupled, sweep width 7142 Hz, time
domain 32 768 points, acquisition time 2.3 s, double zero-filling) contained
Scheme 2. Reaction of the oxathiaphospholane monomers 4 or 5 with 50-
O-DMT-thymidine (1), leading to the corresponding protected dinucleoside
phosphorothioates 6, further converted into deprotected dinucleotides 7.
Chirality DOI 10.1002/chir

DIASTEREOMERS OF NUCLEOSIDE-50-O-OTP MONOMERS
241
Scheme 3. Synthesis of thymidine 50-O-(a-thiodiphosphate).
monomers in the mobile phase rendered the open-column
preparative separation inefficient. Therefore, semipreparative
HPLC on silica gel was employed and for 4b baseline separa-
tion was achieved (Fig. 1) for sample loads of 3–5 mg per
run.
Fig. 2. Chromatogram from HPLC separation of 50-O-(2-thio-4,4-pentam-
ethylene-1,3,2-oxathiaphospholane)-20,30-O,O-di(2-naphthoyl)-N6-benzoyl-aden-
osine 5f (0.1 mg, analytical run). Conditions: Hyperprep HS silica gel column
250 3 10 mm, 8 lm, ethyl acetate:hexane 52:48 v/v, isocratically, flow rate 8
ml/min, retention times 5.6 and 6.0 min.
The above observations prompted us for synthesis of 50-
OTPs derivatives of dCBz , dABz, and dGiBu carrying the 2-
naphthoyl moiety at the 30 position. The deoxycytidine mono-
mer was obtained with good yield, but none of numerous
developing systems used for HPTLC analyses allowed for
separation of the P-diastereomers. Moreover, extremely poor
solubility of naphthoylated intermediates 3 (B0 5 AdeBz or
GuaiBu, Z 5 H) in non-nucleophilic solvents (required for the
phosphitylation step) forced us to abandon the synthesis of
the corresponding 50-OTP derivatives. Thus, another acylat-
ing reagent, namely (-)-camphanoyl chloride, was used
to obtain oxathiaphospholane monomers 4d–h (B0 5 Thy,
GuaiBu,DPC, GuaiBu, AdeBz, CytBz, respectively, Y 5 (2)-cam-
phanoyl). Their structures were confirmed by FAB MS and
31P NMR. The 50-OTP derivative of dG was prepared in two
forms, i.e., with or without the diphenylcarbamoyl protecting
moiety (DPC) at the O6 position.16 Several attempts to sepa-
rate compounds 4d–h into fast- and slow-eluting P-diaster-
eomers by silica gel column chromatography have been
undertaken, but with a different degree of success. The OTP
derivatives of dABz, dCBz, and dGiBu (4f–h, the last one with-
out the DPC group at the O6 position in guanine) could not
be separated. The separation was achieved for the thymidyl
monomer 4d (Table 1) and for dGiBu,DPC derivative 4e. After
optimization of a silica:monomer ratio, dimensions of the col-
umn and eluent composition, the fractions enriched in fast-
and slow-eluting components (of diastereomeric ratio 7:1 to
chromatography with 50–88% yield. Finally, phosphitylation
with 2-chloro-4,4-pentamethylene-1,3,2-oxathiaphospholane in
the presence of anhydrous N,N-diisopropylethylamine and
elemental sulfur16,17 furnished corresponding 50-OTPs 4 with
57–75% yield (30% for 1-adamantanecarbonyl derivative, vide
infra).
For the first selection, three 30-O-acylated thymidine deriv-
atives 2a–c (B0 5 Thy, Z 5 H) were obtained using
biphenyl-4-carbonyl, 2-naphthoyl or 1-adamantanecarbonyl
chlorides, respectively. They were further converted into cor-
responding monomers 4a–c, and their structures were con-
firmed by FAB MS and 31P NMR. The latter method
revealed the presence of two closely located resonances of
similar intensity at d 105.7 7 107.2 ppm (Dd 5 0.33 7 0.59
ppm; CDCl3 or CD3CN), characteristic for the pairs of corre-
sponding diastereomers. For the monomer carrying the ada-
mantanecarbonyl moiety (4c, B0 5 Thy), good HPTLC sepa-
ration of the P-diastereomers was observed (Table 1), but
the monomer was found to be unstable during chromatogra-
phy (after rechromatography only 30% of the material was
recovered). For biphenyl-4-carbonyl-(4a) and 2-naphthoyl-
(4b) derivatives, the HPTLC showed even better separation
of the P-diastereomers, however, very low solubility of these
Fig. 3. (A) Two regions (d 41 to 43 ppm, a-P atom; dd25 to 26 ppm, b-P atom) of the 31P NMR spectrum (500 MHz, proton decoupled) recorded for crude
TDPaS obtained in the reaction of 4d (fast/slow 6:1) with H3PO4. (B) Two regions (d 42 to 42.8 ppm, a-P atom; d 12.0 to 12.6 ppm, g-P atom) of the 31P NMR
spectrum (200 MHz, proton decoupled) recorded for crude 10 obtained in reaction of 4d (fast/slow 1:10) with a methylenediphosphonate anion.
Chirality DOI 10.1002/chir

242
TOMASZEWSKA ET AL.
Scheme 4. Conversion of 4d (diastereomerically enriched mixture slow/fast 10:1, Y 5 (2)-camphanoyl) into thymidine-50-O-(b,g -methylene-a-thiotriphosphate) 11.
10:1) were collected, which after rechromatography provided
diastereomerically pure species.
chromatography. The corresponding derivative of cytidine
(5c) could not be resolved, even by means of HPLC.
Fast-enriched fraction of 4d (Scheme 2, B0 5 Thy, Z 5 H;
65:35 diastereomeric composition by 31P NMR) was used for
the correlation between the relative chromatographic mobil-
ity of P-diastereomers and absolute configuration of the
resulting condensation products. The sample was reacted
with 50-O-DMT-thymidine to yield the corresponding pro-
tected dinucleotide 6, further converted into dinucleoside
30,50-phosphorothioate 7 (B 5 Thy, Z 5 H). Based on the lit-
erature data, under typical conditions of RP-HPLC (a C18 col-
umn, elution with a gradient of 0.1 M triethylammonium bi-
carbonate (TEAB) and acetonitrile), all 16 di(deoxyribonu-
cleoside) phosphorothioates of RP-configuration have shorter
retention times (fast-eluting species) than their SP-counter-
parts.26,27 Since fast- and slow-eluting diastereomers of 4d
furnished slow- and fast-7, respectively, one can conclude
that fast-4d and slow-4d yield the dinucleotides 7 of SP and
RP absolute configuration, respectively. The stereoselectivity
of condensation was not lower than 98%.
Amongst 20,30-O,O-dinaphthoylated monomers, the P-dia-
stereomers of 50-OTP derivative of adenosine (5f) were well
separated using HPLC (Fig. 2). The analogous separation of
naphthoylated uridine derivative 5e was less efficient than
that of camphanoylated 5a.
To make the correlation between the chromatographic mo-
bility of P-diastereomers of the 50-OTPs of ribonucleoside se-
ries 5 (Scheme 2, Z 5 OY) and absolute configuration of cor-
responding condensation products, the monomers 5a (slow,
100%), 5b (two samples: slow, 100% and fast, 100%) and 5f
(fast, 100%) were condensed with 50-O-DMT-thymidine. The
products 6 (Z 5 OY) were deprotected to yield the corre-
sponding dinucleoside phosphorothioates 7 (TPSU or TPSrA,
Z 5 OH). Their enzymatic hydrolysis with SP-specific Nucle-
ase P123 and RP-specific snake venom phosphodiesterase
(svPDE)22 (see Figs. 4S and 5S, Supporting Information)
confirmed that slow-eluting isomers of 5a, 5b, and 5f are
the precursors of internucleotide bonds of RP absolute con-
figuration. This finding is analogous to the correlation per-
formed for 4d in the deoxyribonucleoside series. However,
this correlation cannot be considered general, as even rela-
tively small changes of the structure (e.g., lack of the pen-
tamethylene substituent in the position 4 of the oxathiaphos-
pholane ring) may reverse chromatographic mobility.28 It
should be noted that all the monomers 4 and 5 (with the
exception of adamantanecarbonyl derivative 4c) are stable
for at least six months when stored in tightly closed vessels
in a refrigerator.
50-OTPs of Ribonucleoside Series
Similar efforts were undertaken to obtain 50-OTP-deriva-
tives of appropriately protected 20,30-O,O-dicamphanoylated
ribonucleosides (Scheme 1, 5a–d, B0 5 Ura, AdeBz, CytBz
,
GuaiBu, respectively). The separation of P-diastereomers of
5a, 5b, and 5d was achieved by means of HPLC on silica
gel (see Supporting Information, Figs. 1S, 2S, 3S), while 5a
and 5b could also be resolved using silica gel open-column
Opening of the Oxathiaphospholane Ring with Anions of
Phosphorus-Containing Acids
To date, the oxathiaphospholane method was used mostly
for synthesis of stereodefined phosphorothiate analogs of
DNA, where the electrophilic phosphorus centre in a 30-O-
oxathiaphospholane monomer was attacked by the 50-OH
group of the growing oligonucleotide. In an analogous reac-
tion of 30-OTP with fluoride anion a phosphorofluoridate de-
rivative was formed, however, contrary to all so far known
examples of 1,3,2-oxathiaphospholane ring opening conden-
sations, the reaction proceeded with epimerization at P-
atom.29 As mentioned in the Introduction, NDPs and NTPs
are biologically important compounds and their diastereo-
merically pure P-chiral analogs are useful tools in biochemi-
Fig. 4. Chromatogram from RP-HPLC analysis of crude TDPaS obtained
in the reaction of 4d (fast/slow 6:1) with H3PO4. Conditions: Alltima C18 col-
umn 250 3 4.6 mm, 5 lm, buffer A: 0.1 M TEAB, buffer B: 40% CH3CN in 0.1
M TEAB, pH 7.5, gradient: 2.5% B/min., flow rate: 1 ml/min, UV detection at
260 nm.
Chirality DOI 10.1002/chir

DIASTEREOMERS OF NUCLEOSIDE-50-O-OTP MONOMERS
243
cal studies. Our earlier experiments18 showed that 30-O-ace-
tyl-thymidine-50-O-(2-thio-1,3,2-oxathiaphospholane) (8, unse-
parated mixture of P-diastereomers) reacted with the phos-
phate and pyrophosphate anions to yield the corresponding
50-O-(a-thiodiphosphate) (Scheme 3) and 50-O-(a-thiotriphos-
phate), respectively.
gel columns. It was found that all investigated slow-eluting
isomers of 4 and 5 are the precursors of internucleotide
bonds of RP absolute configuration. Compound 4d was
reacted with three anions of phosphorus-containing acid, fur-
nishing thymidine 50-O-(a-thiodiphosphate), thymidine-50-O-
(b,g -methylene-a-thiotriphosphate) and thymidine-50-O-(ben-
zylphosphono-a-thiophosphate) in a highly stereoselective
manner (ꢀ90%).
To determine the stereochemistry of this type of reactions,
diastereomerically enriched samples of 4d were reacted with
three anions of phosphorus-containing acids. In the first
experiment, 4d (containing 14% of the isomer slow) was
reacted with anhydrous crystalline H3PO4 (three-fold molar
excess) in the presence of DBU (11-fold molar excess) to
ACKNOWLEDGMENTS
A scholarship founded by Prof. A. Legocki, the former
President of Polish Academy of Sciences to A. Tomaszewska
is gratefully acknowledged.
form
30-O-camphanoyl-thymidine-50-O-(a-thiodiphosphate)
(9). Dry N-methylpyrrolidone was used as a solvent because
of very poor solubility of phosphoric acid in acetonitrile. The
product was formed in ca. 32% yield (by 31P NMR, Fig. 6S,
Supporting Information), and contained ꢂ 20% of the minor
isomer. The mixture was treated with concentrated ammo-
nium hydroxide to remove the base-labile protecting groups.
The ammonia was removed and for the resulting N-methyl-
pyrrolidone solution of TDPaS (with a few drops of d6-ben-
zene added) a 31P NMR spectrum was recorded. The
resonances at d 41 to 43 ppm for a-P atom and 25 to –6 ppm
for b-P atom (Fig. 3A) indicate that the mixture contained
the diastereomers in a ratio 79:21 indicating ꢄ90% stereose-
lectivity of the ring opening. The higher chemical shift for
the a-P atom of the major isomer suggests that fast-4d
yielded TDPaS of SP configuration,30 but because in Lud-
wig’s article the reference spectra were recorded in H2O/
D2O, additional RP-HPLC analysis was performed (Fig. 4),
which confirmed that assignment.30
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Chirality DOI 10.1002/chir