ORDER
REPRINTS
BORON-CONTAINING NUCLEOTIDES
593
ꢀ
ꢀ
ꢀ
–
4.23–4.17 (m, 4 H, H3ꢀ, H4 ), 4.11–4.09 (m, 4 H, H5 ), 3.69 (s, 6 H, OCH3),
== –
2.34–2.18 (m, 4 H, H2 ), 2.01 (s, 3 H, O C CH3), 1.94 (s, 3 H, 5-CH3), 1.70
(s, 3 H, 5-CH3); For the second eluted isomer, 11b: Rt (11b) = 18.0 min, 31P
1
NMR (DMSO-d6, 161.9 MHz) δ (ppm) 10.63; H NMR (DMSO-d6, 400 MHz)
δ (ppm) 7.47 (s, 1 H, H6), 7.43 (s, 1 H, H6), 7.34–7.19 (3m, 9 H, Ar-H), 6.85–6.83
(m, 4 H, Ar-H), 6.19–6.06 (2t, 2 H, H1ꢀ), 5.11 (unresolved m, 2 H), 4.21–4.16
ꢀ
ꢀ
ꢀ
–
(m, 4 H, H3ꢀ, H4 ), 4.11–4.06 (m, 4 H, H5 ), 3.68 (s, 6 H, OCH3), 2.38–2.16
== –
(m, 4 H, H2 ), 2.01 (s, 3 H, O C CH3), 1.94 (s, 3 H, 5-CH3), 1.71 (s, 3 H,
5-CH3). 11a and 11b were reacted with lithium sulfide to give 12a (31P NMR at
54.2 ppm) and 12b (31P NMR at 53.4 ppm) respectively. After removing the DMT
protecting group by 2.5% CHCl2COOH in dichloromethane and deprotecting the
acetyl group by NH4OH/CH3OH, the Rp or Sp dithymidine phosphorothioate was
obtained. From empirical rules (2a), which allow the configuration of dinucleoside
phosphorothioates to be assigned on the basis of their 31P NMR chemical shift val-
ues and relative retention times on reverse phase HPLC, the absolute configuration
has been assigned to the diastereomers of 12 by comparison with these criteria.
Thus, 12a which resonates at lower field in the 31P NMR spectra was assigned to
the Rp-configuration, and 12b to the Sp-configuration.
The dinitrophenyl P(V) phosphate intermediates are useful synthons. They
are very stable in the dry air while trivalent P(III) intermediates are sensitive to air
oxidation. Also the dinitrophenyl is sufficiently stable at acidic conditions to sur-
vive the DMT removal step. Lithium sulfide is a solid that can be partly dissolved
in DMF. These advantages over the phosphite approach make the pentavalent P(V)
method easy to handle for the preparation of phosphorothioates. Further, the two
diastereomers of dinucleoside dinitrophenyl phosphate are easily and cleanly sep-
arated by silica gel HPLC. For example, the two diastereomers of 11 (5ꢀ-DMT
dithymidine 3ꢀ-acetyl dinitrophenyl phosphate) were obtained in 10 mg scale by
silica gel HPLC separation. By appropriately choosing the protecting group, dini-
trophenyl phosphate precursors will be very good building blocks for the synthesis
of oligonucleoside phosphorothioates with Rp or Sp phosphorothioate linkages in-
serted at certain positions.
The displacement of the dinitrophenyl group by lithium sulfide success-
fully introduces an S moiety into a phosphodiester under very mild conditions
(room temperature) with high efficiency (almost quantitative conversion). This
method with some modifications also has been applied to the syntheisis of the first
boranophosphorothioate (4).
It is important to know whether the Li2S substitution step is strereospecific
or not. After HPLC separation of the individual two diastereomers 11a and 11b,
which bear a dinitrophenyl leaving group at the P atom, the two diastereomers are
allowed to react with nucleophile, sulfide S2−. From the analysis of the 31P NMR
chemical shift data before and after the reaction, information about the configu-
ration around the P atom can be obtained and the mechanism of lithium sulfide
substitution can be inferred. Typically, at a given set of conditions (solvent, pH,
temperature etc.), the 31P NMR chemical shift depends soley on the substituents
and the configuration around P. For the two diastereomers 11a and 11b, the four