Synthesis and NMR structural analysis of 2κ-OMe-Uridin-3′-yl (3′,5′)-5′-O-(N-isobutyryl-2′-OMe-cytidine) methylthiophosphonates

Article abstract of DOI:10.1002/ejoc.200300772

Detailed analysis of the molecular structures of both the Fasteluted and the Slow-eluted (the notation Slow and Fast corresponds to the relative mobility properties of compounds in silica gel column chromatography under normal phase conditions, see Exp. S

Full text of DOI:10.1002/ejoc.200300772

FULL PAPER
Synthesis and NMR Structural Analysis of 2Ј-OMe-Uridin-3Ј-yl (3Ј,5Ј)-5Ј-O-
(N-Isobutyryl-2Ј-OMe-Cytidine) Methylthiophosphonates
Sebastian Olejniczak,[a] Marek J. Potrzebowski,[a] and Lucyna A. Wozniak*[b]
Keywords: Antisense agents / Chirality / Conformation analysis / NMR spectroscopy / Oligonucleotides
Detailed analysis of the molecular structures of both the Fast-
eluted and the Slow-eluted (the notation Slow and Fast
corresponds to the relative mobility properties of compounds
(Pulse Field Gradient) NMR studies. The absolute configura-
tions [(RP) and (SP)] of the diastereomers of 1 were unam-
1
biguously assigned by H ROESY experiments. The pseudo-
in silica gel column chromatography under normal phase rotation parameters of the ribose ring were calculated with
conditions, see Exp. Sect. for details) diastereomers of 5Ј-OH-
2Ј-OMe-uridin-3Ј-yl (3Ј,5Ј)-5Ј-O-(N-isobutyryl-2Ј-OMe-cy-
tidine) methylthiophosphonate (1) was performed with the
aid of 1H, 13C, 1D and 2D homo- and heteronuclear PFG
the aid of the PSEUDOROT program, and compared with
data obtained from X-ray structure analysis.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2004)
Introduction
Synthetically modified non-ionic oligonucleotides pro-
vide a convenient tool for study of the consequences of in-
hibited expression of target genes, including both target
validation[1] and therapeutic applications,[2] or determi-
nation of protein function and interactions.[3,4] Recently, the
properties of chimeric constructs[5,6] containing a mixed
backbone structure, with P-modified dinucleoside (3Ј,5Ј)
methylphosphonates d(NPMeN) incorporated into different
positions of the oligonucleotide chain, have been
reported.[7Ϫ9] It has been established that oligonucleotides
containing (RP)-methylphosphonate bonds form heterodu-
plexes with complementary DNA or RNA of higher
thermodynamic stability than those formed between the
isosequential oligonucleotides with incorporated methyl-
phosphonate linkages with (SP) configurations at the in-
ternucleotide phosphorus centres.[5,10] Recent NMR studies
revealed that PMe-modified oligonucleotide strands exhibit
greater mobility relative to the DNA strand.[7]
blocks for the preparation of chimeric oligonucleotides. As
they are isosteric with dinucleoside methylphosphonates,
these analogues might be expected to possess P-stereode-
pendent binding properties towards complementary DNA
or RNA targets when incorporated into chimeric oligomers.
Further improvement in the binding properties was ex-
pected after introduction of 2Ј-OMe derivatives in place of
the 2Ј-deoxynucleosides.[11] Moreover, the above modifi-
cations could be particularly interesting in the light of the
anticipated changes in the conformational properties of 2Ј-
OMe oligonucleotides existing in RNA-like confor-
mations.[12] Although a few reports on the synthesis of
di(2Ј-deoxynucleoside methylthiophosphonates) had been
published previously,[13,14] their properties had never been
studied in detail.
The search for efficient methods for the synthesis of the
tailored chimeric oligonucleotides has focused our attention
on the potential of dinucleoside (3Ј,5Ј) methylthiophos-
phonates 2, which could be regarded as dimeric building
[a]
Centre of Molecular and Macromolecular Studies, Polish
Academy of Sciences, Department of Structural Studies and
NMR Laboratory
Sienkiewicza 112, 90-363 Lodz, Poland
[b]
Centre of Molecular and Macromolecular Studies, Polish
Academy of Sciences, Department of Bioorganic Chemistry,
Sienkiewicza 112, 90-363 Lodz, Poland
In this paper we report on a new and efficient synthesis
and structural analysis of (RP)- and (SP)-5Ј-OH-2Ј-OMe-
Fax: (internat.) ϩ 48-42-6815483
E-mail: lawozn@bio.cbmm.lodz.pl
uridin-3Ј-yl
(3Ј,5Ј)-5Ј-O-(N-isobutyryl-2Ј-OMe-cytidine)
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
methylthiophosphonates (1) and demonstrate the unam-
1958
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200300772
Eur. J. Org. Chem. 2004, 1958Ϫ1966

A New and Efficient Synthesis of Methylthiophosphonates
FULL PAPER
biguous assignment of the absolute configuration at phos- most 1:1 for 2Ј-deoxy derivatives and about 1:2 (Fast/Slow)
phorus by NMR techniques.
for 2Ј-OMe derivatives. Total yields of both diastereomers
2 varied (31P NMR) from 86% for TC to about 65% for
AT. It was found that the partially protected dinucleoside
(3Ј,5Ј) methylphosphonothiates 2 could be chromatograph-
ically separated (silica gel column) into their isomers more
efficiently than the fully protected compounds 6.
The assignment of the absolute configurations of par-
tially protected dinucleotides 2 by fast and unambiguous
NMR analysis is of significant value if they are to be used
as dimeric building blocks and incorporated into chimeric
oligonucleotides. We had previously demonstrated the diag-
nostic value of ROESY spectra for conformational analysis
of diastereomeric 5Ј-O-DMT-nucleoside 3Ј-O-(S-methyl
methylphosphonothiolates) and the corresponding Se-
methyl methylselenophosphonates,[18] monomers for stereo-
specific synthesis of dinucleoside (3Ј,5Ј) methylphosphon-
ates.[19,20]
Preliminary studies revealed that the removal of the 5Ј-
protected group could simplify interpretation of NMR
spectra. Therefore, each isomer (Fast-2 and Slow-2 eluting
from silica gel plates) was separately treated with 2% di-
chloroacetic acid, affording partially protected dinucleotide
1, which was purified by flash column chromatography and
subjected to NMR analysis.
Results and Discussion
The potential use of chimeric oligonucleotides with P-
chiral centres[3,4,6] creates a demand not only for efficient
syntheses of their dimeric building blocks but also for the
evaluation of straightforward spectral methods of analysis,
applicable for fast and unequivocal assignment of absolute
configuration at the phosphorus centres in partially pro-
tected, diastereomerically pure, dimeric building blocks.
The intention of the reported studies was to compare re-
sults of structural analysis for the diastereomeric dinucleo-
side (3Ј,5Ј) methylthiophosphonates 1, both in solution and
in solid state, and to assign the absolute configurations for
these isomers. Previously, comprehensive studies by Engels
et al. had allowed the determination of the absolute con-
figuration of d(NPMeN) by NMR, UV and CD, and appli-
cation of multivariate statistics.[15,16]
We reported recently that diastereomeric dinucleotides 2
could be obtained through one-pot condensation reactions
between 5Ј-O-DMT-2Ј-OMe (N-protected) ribonucleosides
3 and 3Ј-O-tert-butyldimethylsilyl-N-protected-2Ј-OMe ri-
bonucleosides 4 with the aid of methylphosphono-bis(1,2,4-
triazolidite) (5) generated in situ, followed by treatment
with elemental sulfur (Scheme 1).[17]
1H and 13C NMR Studies in Solution
After the condensation, without isolation of the fully pro-
In this project, we were attracted by the potential to cor-
tected dinucleotides 6, obtained as mixtures of dia- relate the conformations of the furanose rings with the
stereomers, the tert-butyldimethylsilyl protecting group was absolute configurations of the partially protected dinucleo-
removed by treatment with Et3N·3HF in 1:1 ratio. As calcu- side (3Ј,5Ј) methylthiophosphonates (SP)-1 and (RP)-1 by
lated from the 31P NMR spectrum of a crude reaction mix- NMR spectroscopy. In addition to standard one-dimen-
ture, the diastereomeric ratios of dinucleotides 6 were al- sional 1H and 13C NMR spectra, we also carried out several
Scheme 1. Reagents and reaction conditions: (i) dropwise addition of 3 to 5, ice bath, 45 min; (ii) 3Ј-protected nucleoside 4 in THF, room
temp, 1.5 h; (iii) elemental sulfur, room temp., overnight, followed by aqueous workup, precipitation; (iv) Et3N·3HF/Et3N in THF,
followed by silica gel column chromatography; (v) 2% dichloroacetic acid, CH2Cl2, 10 min, room temp.
Eur. J. Org. Chem. 2004, 1958Ϫ1966
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1959

S. Olejniczak, M. J. Potrzebowski, L. A. Wozniak
FULL PAPER
Figure 1. (1) Spectrum of Slow-1 recorded in CDCl3 and the theoretical spectrum modelled with the WINDAISY program;[21] (2) the
3D structure of diastereomer Slow-1 obtained by the PM3 semiempirical method[22]
two-dimensional experiments in order to achieve complete
assignment of proton and carbon chemical shifts and to
elucidate unequivocally the structures of Fast-1 and Slow-
1. In some experiments, we took advantage of the Pulse
Field Gradient (PFG) system to reduce the time of meas-
urement and to improve the quality of spectra (e.g., re-
duction of T1 noise). The accuracy of δiso and J assignments
was confirmed by comparison of experimentally measured
and theoretically predicted spectra obtained by use of the
WINDAISY program (Figure 1, a).[21]
Table 2. JH,H and JP,H coupling constants (Hz) for Fast-1 and Slow-
1 at 25 °C
Fast-1
b
Slow-1
b
a
a
H1Ј-H2Ј
H2Ј-H3Ј
H3Ј-H4Ј
H4Ј-H5Ј
H4Ј-H5ЈЈ
H5Ј-H5ЈЈ
H5ϪH6
5.2
5.0
4.2
2.0
1.3
0.8
5.0
9.2
2.1
3.0
11.0
7.8
4.2
4.9
4.8
2.0
2.0
0.8
4.8
8.1
1.8
3.3
11.7
7.5
12.4
12.4
1
The H and 13C chemical shifts and the proton-proton J
7.8
15.9
11.9
8.1
15.5
13.5
coupling constants established for Fast-1 and Slow-1 by 1H- PϪCH3
1H PFG COSY, 1H-13C PFG HMQC and 1H-13C PFG
PϪH3Ј
PϪH5Ј,H5ЈЈ
7.1
8.0
HMBC experiments are collected in Table 1Ϫ4.
Table 3. JC,P coupling constants (Hz) and torsion angles from PM3
calculations for Fast-1 and Slow-1 at 25 °C
1
Table 1. H NMR chemical shifts (ppm) for diastereomers of (SP)-
1 and (RP)-1 at 25 °C
Fast-1 Slow-1 PϪOϪCϪCxЈb (SP) PϪOϪCϪCxЈb (RP)
Fast-1
Slow-1
b
b[a]
a
a
PϪC2Јb 3.6
PϪC3Јb 6.1
PϪC4Јb 6.1
PϪC5Јa 6.1
2.4
7.3
119.0
119.7
123.4
124.9
H1Ј
H2Ј
H3Ј
H4Ј
H5Ј
H5ЈЈ
H5
H6
OϪCH3
PϪCH3
5.69
4.34
5.27
4.24
3.94
3.81
5.74
7.74
3.54
1.98
5.92
3.88
4.11
4.14
4.40
4.37
7.47
8.21
3.75
5.77
4.21
5.17
4.24
3.97
3.87
5.70
7.82
3.52
1.97
5.92
3.87
4.11
4.14
4.50
4.38
7.44
8.15
3.72
4.8 Ϫ124.3
7.3
9.7
PϪC4a
8.5
155.0
PϪCH3 113.8 116.3
The conformations of different pentose rings in nucleos-
ides and nucleotides have been extensively discussed by
Leeuw and Altona.[23] The relationship between vicinal
coupling constants and pseudorotation parameters (phase
angle P and puckering amplitude Φm) is also known.[24]
[a] The term b corresponds to a 2’-OMe-uridine moiety and a corre-
sponds to a 2Ј-OMe-cytidine moiety.
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A New and Efficient Synthesis of Methylthiophosphonates
FULL PAPER
˚
Table 4. 13C NMR chemical shifts (ppm) for diastereomers of Fast-
1 and Slow-1 at 25 °C
tions to be established within distances up to 5 A. For me-
dium-sized molecules with molecular weights of ca 1 kD,
however, NOE measurements are often hampered by the
vanishing cross-relaxation rates. Since the measurement of
transverse cross-relaxation (ROE) does not suffer from this
problem, the chemical shifts of the protons and their spatial
connectivity can be assigned unambiguously by employing
the ROESY technique. Moreover, as reported by Löschner
and Engels,[15] in the case of d(NPMeN), the absolute con-
figuration at the phosphonyl residue could be determined
on the basis of NOE distance constraints.
The ROESY experiments were performed both for the
diastereomer Fast-1 and for Slow-1, with the methyl group
directly bonded to phosphorus regarded as indicative of
their chirality. Although the potential of this method has
been reported previously,[25] the assignment of stereochem-
istry in dinucleoside (3Ј,5Ј) methylthiophosphonates 1 has
been equivocal because the cross-peaks from the methyl
group are very weak (at the same level as noise). However,
we found that the quality of the spectra could be greatly
improved if the phosphorus decoupling was turned on dur-
ing acquisition. Figure 2 presents 31P-decoupled ROESY
spectra of 1 (spectra without phosphorus decoupling are
attached in the Supporting Information; for Supporting In-
formation see also the footnote on the first page of this arti-
cle).
Fast-1
b
Slow-1
b
a
a
C1Ј
C2Ј
C3Ј
C4Ј
C5Ј
C5
C6
OϪCH3
PϪCH3
90.5
80.5
73.5
83.7
60.6
102.3
141.8
58.7
21.4
88.9
82.6
67.4
81.6
63.0
95.7
143.8
58.6
89.3
81.5
72.6
83.5
60.5
102.7
141.1
58.7
21.7
89.4
83.0
67.9
81.9
64.4
96.3
144.0
59
From the previously reported[17] X-ray data for SLOW
(RP)-1, reported previously,[17] it could be concluded that
both ribose rings were in the N-type conformation in the
solid state. The P parameters for the b (uracyl) and a (cytid-
ine) rings had been determined to be 8.0° and 13.4°, respec-
tively. In solution, accurate pseudorotational parameters fit-
ting best with the experimentally obtained data can be cal-
culated by use of the PSEUROT 6.2 computer program;[23]
in the current studies the vicinal coupling constants taken
from the WINDAISY simulation were used as input data.
A procedure based on analysis of J couplings at four tem-
peratures (25°, 35°, 45° and 55 °C) was adopted for these
calculations. The puckering amplitudes for N- and S-type
conformers were held constant, and then raised in 1° steps
between 30° and 42°, while the phase angles and the per-
centage populations were allowed to vary freely, until the
optimized value had been found. Five parameters Ϫ PN,
ΦN, PS, ΦS and fS (the sum of fS and fN is equal to 1) Ϫ
fully characterize the conformations of pentose rings. The
calculated pseudorotation parameters are collected in
Table 5.
The presence of cross-peaks between the methyl group
and H5ЈЈa, H3Јa, and OCH3 in Fast-1 (Figure 2, b), and
their absence in Slow-1 (Figure 2, a) provide an unambigu-
ous distinction between isomers. The appropriate distances
between protons in the structure obtained by molecular
modelling are displayed (right) and unambiguously confirm
the (SP) configuration at the phosphorus centre in Fast-1.
In the light of this result, the lack of cross-peaks from the
methyl group in the diastereomer with (RP) configuration
can readily be interpreted (Figure 3, b).
It is interesting to note that in the case of thio derivatives,
due to strong magnetic shielding of the thiophosphonyl
residue, the chemical shift of CH2 protons at 5Ј position is
a sensitive indicator of stereochemistry on phosphorus.
Figure 3 illustrates the expanded region of the H5Ј/5ЈЈ
protons for both diastereomers. The distinction between
protons for (RP)-1 is ca 0.15 ppm, while for (SP)-1 these
signals are overlapped. The local magnetic shielding effect
of the thiophosphonyl group on H5Ј/H5ЈЈ protons is illus-
trated in the top inset.
Table 5. Comparison of the calculated pseudorotation parameters
for Fast-1 and Slow-1 and data obtained from X-ray analysis of
Slow-1
b
a
%N RMS N
P
S
Φ P
%N RMS N
P
S
Φ P
Φ
Φ
Fast-1
45 0.06 14
31 160 36 100 0.27 23
34 163 34 99 0.01 14
36 169 25
32 149 34
Slow-1
53 0.05
9
Slow-1 (X-ray) N
8.02 Ϫ Ϫ
Ϫ
N
13.50 Ϫ Ϫ Ϫ
31P CP/MAS NMR Studies in the Solid State
Solid-state (SS) NMR spectroscopy is a technique that
From the above data it is apparent that for Slow-1 there provides a link between NMR spectroscopic data in the
is a very good consistency between X-ray and NMR results. solution or liquid phases and results obtained from single-
Comparison of NMR spectroscopic data for Slow-(RP)-1 crystal X-ray or neutron diffraction studies. Comparison of
and Fast-(SP)-1 diastereomers discloses small differences in the isotropic chemical shifts and further structural param-
phase parameters, however, the existence of N conformers eters that characterize the geometries of molecules in crystal
has been confirmed in both cases.
lattices allows conclusions regarding changes in confor-
The important structural information can be also ac- mation, as well as the nature of the intermolecular contacts
quired by analysis of NOE effects, which allows direct pro- in both phases. Since a powder specimen (i.e., not only one
ton-proton connectivities resulting from dipolar interac- selected single crystal) is used for SS NMR, this method
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S. Olejniczak, M. J. Potrzebowski, L. A. Wozniak
FULL PAPER
Figure 2. 31P NMR decoupled ROESY spectra of Slow-(RP)-1 (a) and Fast-(SP)-1 (b) registered at 25 °C
can provide immediate identification of polymorphs, sol-
The principal components of the 31P chemical shift, ten-
vates, inclusion complexes etc. that may exist in the crystal- sors δii, were established from the spinning sideband inten-
line state.
sities by use of the WINMAS program, on the basis of the
The room-temperature 31P CP/MAS spectrum of the dia- BergerϪHerzfeld algorithm.[26,27] The calculated values of
stereomer (RP)-1, crystallized from ethanol, is displayed in principal tensors elements δii and the shielding parameters
Figure 4. As can be seen, a single resonance line in the iso- are given in Table 6, with the accuracy of calculations con-
tropic part of the spectrum represents the compound under firmed by comparison of experimental and theoretical spec-
investigation. This observation is consistent with X-ray tra (Figure 4).
analysis assigning one molecule of (RP)-1 in the asymmet-
In contrast, attempts to crystallize the diastereomer (SP)-
1 were not successful, and provided only amorphous pow-
ric unit.[17]
1962
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A New and Efficient Synthesis of Methylthiophosphonates
FULL PAPER
Figure 3. Expanded region of the H5Ј protons for (RP)-1 (Slow) and (SP)-1 (Fast)
Figure 4. Room-temperature 31P CP/MAS spectra of (RP)-1, crystallized from ethanol (a), and amorphous (SP)-1 (b), together with the
corresponding WINMAS program simulations (c and d)
Table 6. 31P chemical shift NMR parameters for diastereomeric 1
and (for comparison) (deoxyxylo)thymidyl-3Ј-O-acetylthymidyl
(3Ј,5Ј)-O-(2-cyanoethyl) thiophosphate (7)
of (deoxyxylo)thymidyl-3Ј-O-acetylthymidyl (3Ј,5Ј)-O-(2-
cyanoethyl) thiophosphate (7) in the crystalline and the
amorphous phases.[28] In the case of the dinucleotides 7,
however, we observed the most significant changes in the
δ33 parameter. From this we concluded that this effect was
related to the formation of weak PϭS···HϪC hydrogen
bonds,[29] and the decrease in electronic shielding of the
phosphorus as the thiophosphoryl group became more pol-
arized during the bond formation. Unexpectedly, in the
cases of samples (RP)-1 and (SP)-1 the biggest differences
were seen for the δ22 element.
[a]
Compound
δ11
δ22
δ33
Ω
κ
δiso
1 (crystalline)
185
189
152
151
154
150
139
131
128
130
Ϫ40
Ϫ38
Ϫ81
Ϫ91
Ϫ92
220
Ϫ
233
242
246
Ϫ
Ϫ
0.81
0.81
0.85
98.3
96.7
67.8
62.6
63.6
1 (amorphous)
7 (crystalline)
(SP)-7 (amorphous)
(RP)-7 (amorphous)
[a]
Estimated errors in δ11, δ22, δ33 and ∆δ are Ϫ/ϩ5 ppm; errors in
δiso are Ϫ/ϩ0.2 ppm; the principal components of the chemical
shift tensor are defined as follows: δ11 Ͼ δ22 Ͼ δ33; the isotropic
chemical shift is given by δiso ϭ (δ11 ϩ δ22 ϩ δ33)/3.
In order to answer the questions regarding the influence
of inter- or intramolecular contacts on shielding param-
eters, knowledge about the orientations of principal ele-
ments of chemical shift tensors with respect to the molecu-
der. The δii elements were therefore established only for the lar skeletons of 1 and 7 was required. Information about
amorphous form of (SP)-1. The experimentally obtained orientation of the principal axes can be gained either exper-
and theoretically predicted spectra are given in Figure 4, imentally from NMR goniometric measurements of single
and the calculated parameters are collected in Table 6. It is crystals or from quantum chemical calculations. Here we
interesting to note the difference between isotropic values employed the latter approach, using the DFT GIAO
of the crystalline and amorphous phases (ഠ 1.6 ppm). method included in the Gaussian 98 program package.[30]
Interestingly, small changes in the δii parameters are ob- Rather than performing the calculations for the dinucleo-
served for both modifications.
side methylthiophosphonate 1, we ran them for the simpler
We had previously also noted a distinction between the model, since we had recently demonstrated that such model
31P δii parameters for the (SP) and (RP) diastereomers calculations gave results only slightly poorer than calcu-
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S. Olejniczak, M. J. Potrzebowski, L. A. Wozniak
FULL PAPER
lations for the larger molecule of actual interest.[31] Accord-
ing to these calculations, the principal axis 3 (most shielded)
would be expected to be very close to the PϭS double bond,
while principal axis 2 should bisect the plane OϪPϭS. The
least shielded axis 1 should therefore be close to the PϪCH3
bond. The orientation of principal elements of chemical
shift tensors with respect to the molecular skeletons for
samples 1 and 7 is shown in pictorial form in Figure 5.
tography and TLC analyses were performed on silica gel (Kieselgel
60, 240Ϫ400 mesh, E.Merck Inc.) and silica gel HP TLC precoated
F254 plates (purchased from E.Merck Inc.), respectively. NMR
spectra were recorded on a Bruker Advance DRX 500 spec-
trometer, operating at 500.13 MHz (1H), and 202.46 MHz (31P).
Chemical shifts (δ) are reported relative to TMS (1H) and 80%
H3PO4 (31P) as external standards. Positive chemical shift values
were assigned for compounds resonating at lower fields than the
standards. Mass spectra were recorded on a Finnigan Mat 95
(NBA, Csϩ gun operating at 13 keV).
1. Synthesis of (RP)- and (SP)-5Ј-O-DMT-2Ј-OMe-uridin-3Ј-yl
(3Ј,5Ј)-3Ј-O-(N-Isobutyryl-2Ј-OMe-cytidine)
3Ј-Methylthiophos-
phonates (2): A solution of 1,2,4-triazole (2.5 equiv.) and triethyl-
amine (3 equiv.) in dry THF was cooled in an ice bath, and methyl-
dichlorophosphane (1.1 equiv.) was added with vigorous stirring.
After 20Ϫ25 min, a solution of 5Ј-O-DMT-2Ј-OMe-uridine (3, 1
equiv.) in THF was added dropwise at 0 °C. Stirring was continued
for 45 min, followed by addition of 3Ј-O-tert-butyldimethylsilyl-N4-
isobutyryl-2Ј-OMe-cytidine (4) in THF. The reaction mixture was
warmed to room temperature and stirred for an additional 1.5 h,
and sulfur (twofold molar excess) was added in one portion. Stir-
ring was continued overnight. The solvents were evaporated under
reduced pressure (ca. 1/3 vol.), and an oily residue was dissolved
in chloroform and washed twice with NaHCO3 (0.1 ) and water.
The organic layer containing 6 was dried with MgSO4, concen-
Figure 5. Principle elements of chemical shift tensors for com-
pounds 1 and 7
From these results and from theoretical calculations we
can conclude that for methylthiophosphonates 1 the Pϭ trated to dryness and coevaporated twice with toluene. This crude
reaction mixture was dissolved in a small volume of dry THF and
treated with Et3N·3HF/Et3N (3:1 v/v). After deprotection was com-
plete (3Ϫ4 h), the reaction mixture was diluted with chloroform,
washed twice with NaHCO3 (0.1 ), concentrated and subjected to
silica gel column chromatography (gradient 0Ϫ6% EtOH in chloro-
form, with addition of 0.05% of Et3N). The collected fractions of
pure diastereomers 2 were concentrated, precipitated with hexane
and stored as white powders. Total yield 61%.
S···H contacts play rather small roles in crystallization pro-
cess, and other effects therefore dominate molecular pack-
ing.
Conclusions
This paper reports the complete assignment of the struc-
tures of the diastereomers of 2Ј-OMe-uridin-3Ј-yl (3Ј,5Ј)-5Ј-
O-(N-isobutyryl-2Ј-OMe-cytidine) methylthiophosphonate
by NMR spectroscopy. Conformational features of 2Ј-OMe
ribonucleotides are to a large extent determined by the pres-
ence of 2Ј-bulky substituents, and so molecules made up of
2Ј-OMe ribonucleotides would be expected to possess
RNA-like conformations. These studies have confirmed
that assumption, both in solution and in the solid state. We
have demonstrated agreement between the conformation
parameters of dinucleoside methylthiophosphonate 1 ob-
tained from studies carried out in solution and in the solid
phase. We have also demonstrated the feasibility of un-
equivocal assignment of the absolute configuration at phos-
phorus by means of ROESY experiments. Preliminary
thermodynamic studies on chimeric oligonucleotides with 1
incorporated are described elsewhere, and exhibit profound
differences in the thermodynamic properties depending on
the absolute configurations at the methylthiophosphonate
centres.[32]
Fast-(Sp)-2: (Rf ϭ 0.5, CHCl3/EtOH, 9:1), 31P NMR (202.46 MHz,
CDCl3): δ ϭ 100.1 ppm. 1H NMR (500 MHz, CDCl3): δ ϭ 1.22
3
2
[d, JH,H ϭ 7.59, 6 H, CH(CH3)2], 1.94 (d, JPH ϭ 15.61, 3 H,
PϪCH3), 2.1 [dq, 1 H, CH(CH3)2], 2.56 (m, 1 H), 3.44 (dd, 1 H),
3.6 [s, 3 H, (C)CH3OϪC2Ј], 3.74 [s, 3 H, (Ura)CH3OϪC2Ј], 3.8 [s,
6 H, (Ura)CH3OϪDMT], 3.92 (m, 1 H), 4.18 (dd, 1 H), 4.26 [d,
3JH,H ϭ 6.56, 1 H, (Ura)H2Ј], 5.36 [m, 1 H, (C)H2Ј], 5.87 [s, 1 H,
(C)H1Ј], 6.05 [d, 3JH,H ϭ 3.41, 1 H, (Ura) H1Ј] ppm. FABϪMS [M
Ϫ H]: 962.6 (C46H55N5O14PS: calcd. 963.99).
Slow-(Rp)-2: (Rf ϭ 0.45, CHCl3/EtOH, 9:1): 31P NMR (CDCl3):
3
δ ϭ 98.26 ppm. 1H NMR (CDCl3): δ ϭ 1.22 [d, JH,H ϭ 7.59, 6
2
H, CH(CH3)2], 1.76 (d, JPH ϭ 15.32, 3 H, PϪCH3), 2.1 [dq, 1 H,
CH(CH3)2], 2.56 (m, 1 H), 3.44 (dd, 1 H), 3.55 [s, 3 H,
(C)CH3OϪC2Ј], 3.74 [s, 3 H, (Ura)CH3OϪC2Ј], 3.81 [s, 6 H,
(Ura)CH3OϪDMT], 3.92 (m, 1 H), 4.18 (dd, 1 H), 4.26 [d, 3JH,H ϭ
6.56, 1 H, (Ura)2ЈH], 5.36 [m, 1 H, (C)H2Ј], 5.95 [s, 1 H, (C)H1Ј],
3
3
6.02 [d, JH,H ϭ 3.43, 1 H, (Ura)H1Ј], 7.42 [d, JH,H ϭ 7.4, 1 H,
(C)5 H], 7.92 [d, JH,H ϭ 8.2, 1 H, (Ura)6 H], 8.16 [d, 3JH,H ϭ 7.5,
3
1 H, (C)6 H], FAB-MS [M Ϫ H]: 962.4 (calcd. 963.99).
1.2 Removal of the 5Ј-O-DMT Protecting Group: Diastereomer-
ically pure isomers 2 (20 mg) were separately dissolved in 2% di-
chloroacetic acid in CH2Cl2 (2 mL) and stirred for 10 min at room
temperature. After extraction (twice with sat. NaHCO3), each was
purified by flash silica gel column chromatography (0Ϫ10% MeOH
in CHCl3). The obtained white foams were dissolved in chloroform
and precipitated from hexane to give 1 as white powders.
Experimental Section
Reactions were carried out under positive pressures of dry argon.
Solvents and reagents were purified by standard laboratory tech-
niques and distilled directly into reaction vessels. Column chroma-
1964
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 1958Ϫ1966

A New and Efficient Synthesis of Methylthiophosphonates
FULL PAPER
Slow-1: (Rf ϭ 0.3, CHCl3/EtOH, 9:1), 31P NMR (CDCl3): δ ϭ
98.44 ppm. C25H36N5O12PS, FW ϭ 661.62, FABϪ [M Ϫ H]: 660.3,
FABϩ [M ϩ H]: 662.2.
Acknowledgments
The authors are indebted to Prof. Wojciech J. Stec for his interest
in this project and for discussions. This project was financially as-
sisted by the State Committee for Scientific Research (Grant 4
TO9A 073 25 for LAW), and in part, by Genta Co., USA.
Fast-1: (Rf ϭ 0.32, CHCl3/EtOH, 9:1). 31P NMR (CDCl3): δ ϭ
98.54 ppm. FABϪ [M Ϫ H]: 660.3.
NMR Measurements in Solution: Samples of diastereomerically
pure 1 (5 mg) were dissolved in CDCl3 (0.5 mL). All spectra were
recorded with a Bruker Advance DRX 500 spectrometer, operating
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1965

S. Olejniczak, M. J. Potrzebowski, L. A. Wozniak
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Received December 9, 2003
1966
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 1958Ϫ1966