The synthesis of di- and oligo-nucleotides containing a phosphorodithioate internucleotide linkage with one of the sulfur atoms in a 5′-bridging position

Article abstract of DOI:10.1039/b901791g

A new type of internucleotide phosphorodithioate linkage is described, wherein one of the sulfur atoms occupies a 5′-bridging position. Representative dinucleotides possessing such a bond were synthesized by S-alkylation of nucleoside-3′-O-phosphorodithio

Full text of DOI:10.1039/b901791g

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www.rsc.org/obc | Organic & Biomolecular Chemistry
The synthesis of di- and oligo-nucleotides containing a phosphorodithioate
internucleotide linkage with one of the sulfur atoms in a 5¢-bridging position†
Magdalena Olesiak,b Wojciech J. Stecb and Andrzej Okruszek*a,b
Received 27th January 2009, Accepted 3rd March 2009
First published as an Advance Article on the web 30th March 2009
DOI: 10.1039/b901791g
A new type of internucleotide phosphorodithioate linkage is described, wherein one of the sulfur atoms
occupies a 5¢-bridging position. Representative dinucleotides possessing such a bond were synthesized
by S-alkylation of nucleoside-3¢-O-phosphorodithioates with 5¢-halogeno-5¢-deoxy-nucleosides. A fully
protected dithymidylate containing internucleotide 5¢-S-phosphorodithioate linkage was converted into
a 3¢-O-phosphoramidite derivative and employed for introduction of a modified dinucleotide into a
predetermined position of the oligonucleotide sequence. The 5¢-S-phosphorodithioate linkage in
dinucleotide analogues was found to be resistant toward nucleolytic degradation with snake venom
PDE and nuclease P1. However, P-stereoselective degradation was observed for diastereomers of
5¢-S-phosphorodithioate dithymidine analogs under treatment with calf spleen PDE. The new
5¢-S-phosphorodithioate linkage was readily degraded by iodine solutions in the presence of water. It
was also found that oligothymidylates containing a single 5¢-S-phosphorodithioate linkage form much
weaker duplexes with their complementary sequences.
as examples of phosphorodithioate oligoribonucleotides,12 includ-
Introduction
ing 2¢,5¢-oligoadenylate analogues.13 Similarly, oligonucleotides
Analogues of nucleotides and oligonucleotides have received much
containing a 5¢-O-phosphorodithioate monoester function were
prepared.14 In addition to the aforementioned di- and oligonu-
cleotide phosphorodithioate congeners, several other phospho-
rodithioated biomolecules were synthesized, including nucle-
oside cyclic 2¢,3¢-O,O-15 and 3¢,5¢-O,O-phosphorodithioates,16
nucleoside 5¢-O-(1,1-dithiotriphosphates),17 nucleoside dithio-
phosphate monoesters,14b,18 nucleoside phosphofluorodithioates,19
O-dithiophosphonopeptides,20 and phosphorodithioate phospho-
lipid derivatives.21
attention due to their use as indispensable tools for studying
the structure of nucleic acids and their interactions with other
biomolecules, such as DNA, RNA, carbohydrates, lipids or
proteins.1 These compounds are widely employed to investigate
the mechanism and stereochemical aspects of various biochemical
reactions catalyzed by nucleic acid metabolizing enzymes.2 Modi-
fied oligonucleotides have also been extensively used as rationally
designed tools to control the expression of specific gene products
and therefore are being explored as potential therapeutics for the
treatment of viral infections, cancers or inflammatory disorders.3
Besides oligo(nucleoside phosphorothioate)s, among the most
intriguing analogues of nucleotides and oligonucleotides are the
phosphorodithioate congeners in which two oxygen atoms at
an internucleotide phosphate moiety are replaced by sulfur.4
The vast majority of studies on their synthesis are related
to compounds having both sulfur atoms placed in a non-
bridging position. Phosphorodithioate analogues of dinucleotides
have been prepared in several ways using phosphorodiamidite,5
phosphorothioamidite,6 phosphotriester,7 H-phosphonothioate,8
H-phosphonodithioate9 or dithiaphospholane10 chemistries. Con-
sequently, the synthesis of oligodeoxyribonucleoside phospho-
rodithioates of various length and sequence was reported11 as well
In clear contrast to the aforementioned di- and oligonucleotide
phosphorodithioate congeners, very limited information is avail-
able in the literature on the synthesis of a phosphorodithioate
internucleotide linkage with only one of the two sulfur atoms
in a bridging position. In fact, all published examples are
related to the preparation of analogous compounds containing
an internucleotide 3¢-5¢-bond with a bridging sulfur atom in the
3¢-position.22 Their syntheses were performed via preparation of
appropriately protected 3¢-mercapto-3¢-deoxy-nucleosides, which
were 3¢-S-phosphitylated and then reacted with a 5¢-hydroxyl
component under the conditions of phosphoramidite synthesis,
with subsequent sulfurization of 3¢-S-phosphorothioite interme-
diates. By virtue of asymmetry of the phosphorus atom in the
3¢-S-phosphorodithioate function, a mixture of diastereomers
was always formed, which could be separated into individual
diastereomerically pure compounds by chromatography. The
absolute configuration at the P atom was tentatively assigned by
spectroscopic and enzymatic methods.22 The short oligonucleotide
containing a 3¢-S-phosphorodithioate internucleotide bond in a
scissile position was successfully used in studies on the mechanism
of action of Tetrahymena ribozyme.22b An alternative approach for
the preparation of dinucleoside 3¢-S-phosphorodithioate, involv-
ing the reaction of 2,3¢-anhydrothymidine with O,O-disubstituted
aInstitute of Technical Biochemistry, Faculty of Biotechnology and Food
Sciences, Technical University of Ło´dz´, Stefanowskiego 4/10, 90-924 Ło´dz´,
Poland. E-mail: andrzej.okruszek@p.lodz.pl; Fax: +(48 42) 636 66 18;
Tel: +(48 42) 631 34 45
bDepartment of Bioorganic Chemistry, Centre of Molecular and Macro-
molecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363
Ło´dz´, Poland
† Electronic supplementary information (ESI) available: 1H NMR, 31P
NMR, ESI MS, MALDI TOF MS and FAB MS data for 3a–d, 5, 6, 8 and
9. See DOI: 10.1039/b901791g
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phosphorodithioic acid was limited to the synthesis of unnatural
3¢-3¢-dithymidine analogue.23
Here we present our results on the synthesis of di-
and oligonucleotides containing the hitherto unknown 5¢-S-
phosphorodithioate internucleotide linkage, possessing one of
the two sulfur atoms in a 5¢-bridging position. A preliminary
communication on this work was published elsewhere.24
trityl)thymidine-3¢-O-[S-(2,4-dichlorobenzyl)]phosphorodithio-
ate (72 ppm).30 On this basis we have concluded that the product
of alkylation has the desired structure 3a (see Scheme 1). The
modified dinucleotide 3a was then isolated in 56% yield by
ion exchange chromatography on DEAE Sephadex A25 and its
elemental composition was confirmed by FAB MS showing the
expected M-H ion (m/z 562.1). The presence in the 31P NMR
spectrum of two closely located signals was tentatively assigned
to the existence of two diastereomers of 3a by virtue of a new
asymmetric centre at the internucleotide phosphorus atom. The
isomer with the lower chemical shift was formed in slight excess
(54%) over its downfield counterpart (46%). The presence of two
diastereomers was further confirmed by RP HPLC showing two
peaks in a 54:46 ratio, with the predominant isomer being slightly
less mobile (tR 16.04 min, SLOW isomer) than the minor one
(tR 15.78 min, FAST isomer). The diastereomeric relationship
of this two component mixture was proved by FAB MS after
their separation by semi-preparative RP HPLC. Both separated
compounds gave almost identical mass spectra with expected M-
H molecular ions (m/z 562.1).
Results and discussion
The synthesis and properties of dinucleoside
5¢-S-phosphorodithioates (3a–d)
Our synthetic strategy was based upon the availability of nu-
cleoside 3¢-O-phosphorodithioates (1a–d) which can be readily
prepared by the dithiaphospholane approach.14b We assumed that
these compounds can be efficiently and selectively S-alkylated
by 5¢-halogeno-5¢-deoxy-nucleosides (2) at one of the sulfur
atoms to yield compounds with a novel 5¢-S-phosphorodithioate
internucleotide linkage, possessing one of the sulfur atoms at a
5¢ position (3a–d). Our assumption was based on the already
known efficient S-alkylation of O,O-dialkylphosphorodithioate
anions by alkyl halides.25 Due to implementation of the HSAB
“soft-soft” rule,26 the alkylation of ambident nucleoside 3¢-O-
phosphorodithioate anions should proceed exclusively at sul-
fur. Such an assumption was supported by the observation of
selective S-alkylation of ambident anions of nucleoside 3¢-O-
phosphorothioates by 5¢-halogeno-5¢-deoxy-nucleosides to yield
a 5¢-S-phosphoromonothioate internucleotide linkage,27 which
was further exploited in a clever so-called chemical ligation of
oligonucleotides.28
To verify the aforementioned assumption, several attempts of
alkylation of deoxycytidine 3¢-O-phosphorodithioate (1a) with
5¢-bromo-5¢-deoxythymidine (2a)29 were undertaken in solvents
such as ethanol-water (1:1, v/v), pyridine or dimethylformamide
(DMF).
Reactions were performed at room temperature on 0.1 mmole
scale, and their progress was monitored by 31P NMR. It was
found that only in the case of the reaction performed in DMF
as the solvent, after 16 hours incubation of substrates, complete
conversion of the substrate occurs with the formation of a new
product showing in the 31P NMR spectrum two resonance signals
at 72.83 and 73.48 ppm (see Table 1).
Scheme 1 The synthesis of dinucleoside 5¢-S-phosphorodithioates 3a–d.
This positive result of the reaction of deoxycytidine 3¢-O-
phosphorodithioate (1a) with 5¢-bromo-5¢-deoxy-thymidine (2a)
prompted us to use this halogen-nucleoside for alkylation of other
deoxyribonucleoside 3¢-O-phosphorodithioates—derivatives of
thymine (1b), adenine (1c) and guanine (1d) (see Scheme 1).
The reactions leading to the formation of dinucleotides with a
novel 5¢-S-phosphorodithioate linkage were run on 0.1 mmole
scale, at room temperature in DMF solution. After 16 hours
incubation, 31P NMR inspection showed full disappearance of
the corresponding substrates, and the products were isolated
by ion exchange chromatography in 44.9–69.8% yield. The
physicochemical characteristics of purified products are listed in
Table 1. All dinucleoside 5¢-S-phosphorodithioates were isolated
The observed chemical shift values were found to be similar
to those reported by Cosstick and Vyle for diastereomers of the
corresponding 3¢-S-dithymidine analogue (68.7 and 72.4 ppm)22a
,
and by Taktakishvili and Caruthers for 5¢-O-(4,4¢-dimethoxy-
Table 1 Yields and physicochemical characteristics of dinucleoside 5¢-S-phosphorodithioates 3a–d
Molecular weight
Dinucleotide
Isolated yield
lmax (H2O) [nm]
RP HPLC tR [min]
d
31P NMR (D2O) [ppm]a
Calculatedb
Measuredc
3a
3b
3c
3d
56.4%
69.8%
48.2%
44.9%
267.5
266.4
261.0
256.0
15.78 (FAST) 16.04 (SLOW)
18.49 (FAST) 18.83 (SLOW)
21.06
18.76
73.48 (46%) 72.83 (54%)
73.36 (48%) 72.59 (52%)
73.81 (44%) 72.81 (56%)
73.62 (49%) 72.65 (51%)
563.54 Da
578.55 Da
587.57 Da
603.57 Da
562.1d 562.1d
577.2 d 577.3d
586.0e
602.0e
a The percentage of particular diastereomers is given in brackets. b Protonated form. c FAB MS (m/z, M-H ions). d Diastereomers separated by RP HPLC.
e Mixture of diastereomers.
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as a mixture of two isomers which was evidenced by the presence
within 31P NMR spectrum of two resonance signals with similar
chemical shifts in the range of 72.6–73.8 ppm. In all cases
the prevailing isomer (51–56%) had lower chemical shift. The
chemical composition of dinucleotides was confirmed by mass
spectrometry, and their purity was evidenced by analytical liquid
chromatography (RP HPLC). In the case of the dithymidine
derivative (3b), the observed two isomers with slightly different
chemical shifts were also separated by semi preparative RP
HPLC showing almost identical FAB MS spectra on which
very similar M–H molecular ions were identified (m/z 577.2
and 577.3). This observation further supported our assumption
that alkylation of deoxyribonucleoside 3¢-O-phosphorodithioates
(1a–d) by 5¢-bromo-5¢-deoxythymidine in DMF solution proceeds
exclusively at the sulfur atom and leads to diastereomeric mixtures
of dinucleoside 5¢-S-phosphorodithioates (3a–d) with opposite
configuration at phosphorus. In the case of compounds 3a and 3b,
the diastereomers could be separated by preparative RP HPLC;
however, for dinucleotides containing one purine base (3c and
3d), no chromatographic separation was feasible under employed
conditions.
Fig. 1 The time course of digestion of diastereomers of TP(S)ST (3b) by
calf spleen phosphodiesterase. Isomers of 3b (1 OD260) were separately
incubated with 0.1 unit of enzyme in 200 ml of citrate buffer (pH 6.0) at
37 ◦C. Aliquots of 20 ml were removed, denatured and analysed by RP
HPLC. Isomer FAST (ꢀ), isomer SLOW (ꢀ).
In order to determine the stability of the 5¢-S-phosphoro-
dithioate internucleotide linkage towards the activity of nucleolytic
enzymes, modified dinucleotides 3a and 3b (mixtures of isomers)
were incubated with snake venom phosphodiesterase (svPDE) and
nuclease P1 (nP1) under conditions typically used for digestion of
DNA. It was found that in buffers recommended by producers,
the incubation of either dinucleotide with the enzymes taken in
concentration from 1 to 10 mg/ml did not lead to any hydrolytic
degradation of substrates during 16 hours at 37 ◦C, as determined
by RP HPLC. Similarly, no change of diastereomeric composition
was observed on incubation with either enzyme. In separate ex-
periments it was evidenced that unmodified dinucleotides d(CPT)
and TPT were completely hydrolyzed by both svPDE and nP1
under aforementioned conditions after one hour of incubation
(not shown). The observed resistance of 5¢-S-phosphorodithioate
internucleotide linkage to degradation by nucleolytic enzymes is
similar to that described for oligonucleotide phosphorodithioates
with both non-bridging oxygen atoms replaced by sulfur.31 It
should be noted, however, that in the case of dithymidine 3¢-S-
phosphorodithioates, containing a bridging sulfur atom in the 3¢-
position, one isomer (assigned tentatively as SP) was digested with
svPDE while the opposite one (presumably RP) was very slowly
hydrolyzed with nP1.22a
The observed stability of dinucleoside 5¢-S-phosphorodithioates
(3a and 3b) towards svPDE and nP1, which are regarded to
be 5¢-exonucleases, prompted us to digest those analogues with
calf spleen phosphodiesterase (spleen PDE), being typical 3¢-
exonuclease, i.e. degrading DNA and RNA to nucleoside 3¢-O-
phosphates. Since in preliminary attempts it was shown that calf
spleen PDE exhibits hydrolytic activity towards 3b taken as a
mixture of diastereomers, in order to draw some conclusions
on the stereochemistry of degradation, for further experiments
HPLC separated isomers of 3b (FAST and SLOW) were employed.
The digestions were performed under identical conditions in
citrate buffer (pH 6.0) at 37 ◦C and show that both isomers
are degraded, however the hydrolysis of SLOW isomer of 3b
proceeds in a much higher rate than that of the FAST one
(see Fig. 1).
These results were related to those described by Spitzer and
Eckstein on digestion of oligodeoxyribonucleotides containing
one stereodefined phosphorothioate linkage [RP or SP d(AP(S)A)]
by spleen PDE.32 The authors found that only the linkage of
SP configuration was cleaved by this enzyme.32 Although in our
case the stereodifferentiation between spleen PDE digestion of
diastereomers of 3b was not so dramatic, it prompted us to draw
conclusions about the absolute configuration of the dinucleoside
5¢-S-phosphorodithioate linkage. For such an assignment one
can also employ the empirical rules established by Eckstein,
linking relative 31P NMR chemical shifts and RP HPLC retention
times of diastereomeric dinucleoside phosphorothioates with their
absolute configuration.33 Assuming that these enzymatic and
physicochemical criteria may apply for structurally very similar
FAST and SLOW diastereomers of TP(S)ST (3b), we were able to
assign absolute configuration for these isomers as depicted on
Fig. 2.
Thus, on the aforementioned basis, SP configuration has been
assigned to the upfield (SLOW) isomer of 3b, and we suppose that
this assignment can also be extended to upfield isomers of 3a, 3c
and 3d.
For further confirmation of the structure of dinucleoside 5¢-S-
phosphorodithioates, their chemical cleavage was attempted. It has
been known from the work of Cosstick and Vyle that dithymidine-
3¢-S-phosphorothiolate (TSPT), containing a single sulfur atom
in a 3¢-bridging position, was readily cleaved at the phospho-
rothiolate bond under the action of iodine/acetone/water, io-
dine/pyridine/water or aqueous silver nitrate, with the formation
of thymidine-5¢-O-phosphate and 3¢-mercapto-3¢-deoxytymidine
(or its derivative).22a The application of conditions used by
Cosstick and Vyle for dithymidyne-5¢-S-phosphorodithioate (3b,
mixture of isomers) resulted in a full degradation of this
dinucleotide analogue in iodine/acetone/water at 50 ◦C or
iodine/pyridine/water at room temp (HPLC control). Further
studies with 31P NMR and FAB MS control have shown that
the degradation of 3b with aqueous iodine proceeds stepwise.
In the first step 3b reacts with aqueous iodine by exchange of
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Fig. 2 Assignment of absolute configuration to the diastereomers of TP(S)ST. Relative 31P NMR chemical shifts, RP HPLC retention times and susceptibility
to enzymatic digestion with spleen PDE for SP-TP(S)T and SP-TP(S)ST compared to their respective epimers.
a non-bridging sulfur into oxygen and formation of interme-
diate dithymidine-5¢-S-phosphorothiolate (TPST). Thus, partially
degraded 3b in iodine/pyridine/water was found to be a mixture
of the aforementioned dithymidine-5¢-S-phosphorothiolate (TPST;
d 19.58 ppm; M-H, m/z 560.9, calcd MW 562.50 Da), and
thymidine-3¢-O-phosphate (TP; d 0.18 ppm; M-H, m/z 321.1, calcd
MW 322.21 Da). The dithymidine-3¢-S-phosphorothiolate (TSPT),
isomeric with respect to the compound TPST by having a single
sulfur atom in a 3¢-bridging position was described to have the
chemical shift d 31P NMR 17.4 ppm (D2O).22a The presence in FAB
MS of a peak at m/z 516.0 could also indicate the presence of di(5¢-
deoxythymidyl) disulfide (calcd MW 514.58 Da), which may result
from air-oxidation of 5¢-mercapto-5¢-deoxythymidine, a possible
final product of degradation of 3b. The aforementioned suggestion
regarding oxidative transformation TP(S)ST → TPST was strongly
supported by the fact that oxidation of phosphorothioate diesters
by aqueous iodine into phosphates was well documented in the
literature.34 In the case of reaction of3b with iodine/acetone/water
at 50 ◦C only the presence of final phosphate product was observed
(TP; d -0.2 ppm; M–H, m/z 321.0, calcd MW 322.21 Da). No
degradation of 3b by aqueous silver nitrate was observed under
conditions used by Cosstick and Vyle.22a
phosphorodithioate modification, and its 3¢-O-phosphoramidite
derivative allowing introduction of such a segment in a preselected
position of the oligodeoxyribonucleotide growing chain during its
synthesis by the phosphoramidite approach (see Scheme 2).
Thus, the 3¢-O-dithiaphospholane derivative of 5¢-O-(4,4¢-
dimethoxytrityl)thymidine (4), prepared as described earlier,11d
was reacted with an excess of 3-hydroxypropionitrile in the pres-
ence of DBU to give quantitatively nucleoside-3¢-O-phosphoro-
dithioate-O-(2-cyanoethyl)diester14b which was then transformed
into its Et3NH+ salt (5) by ion exchange and purified by flash col-
umn chromatography. The 2-cyanoethyl diester 5 was S-alkylated
with 5¢-iodo-5¢-deoxythymidine (2b)36 in DMF/CH3CN solution.
It has to be mentioned that earlier attempts to alkylate DBUH+ salt
of 5 were unsuccessful (not reported). Similarly, the application
of 5¢-bromo-5¢-deoxythymidine (2a) for alkylation of diester
phosphorodithioate 5 was found to be less efficient than employing
iodonucleoside 2b (not shown). The resulting 5¢-O-protected-
(O3¢→S5¢)dithymidine-O-(2-cyanoethyl) phosphorodithioate (6)
was isolated by flash column chromatography as an equimo-
lar mixture of two diastereomers which showed two differ-
ent resonance lines in a 31P NMR spectrum (d = 95.83 and
96.10 ppm). That mixture could not be chromatographically sep-
arated. Compound 6 was in situ phosphitylated with N,N,N¢,N¢-
bis-(diisopropylamino)-O-2-cyanoethylphosphordiamidite in the
presence of EtS-tetrazole35 to give 3¢-O-phosphoramidite deriva-
tive (7) which was directly used as a mixture of diastereomers for
automated oligonucleotide synthesis.
The synthesis of oligothymidylates containing
5¢-deoxy-5¢-S-phosphorodithioate internucleotide linkages
The successful preparation of dinucleoside phosphorodithioates
3a–d with one of the two sulfur atoms in a 5¢-bridging position
prompted us to synthesize oligonucleotides containing at least one
5¢-S phosphorodithioate linkage in a preselected position. For this
purpose a dimer block approach was employed,35 involving the
synthesis of a fully protected dinucleotide containing the 5¢-S-
The application of dimer block 7 for introduction of dithymidyl-
5¢-S-phosphorodithioate residue into the selected position of
oligodeoxyribonucleotide was demonstrated by the synthesis of
two oligothymidylates possessing the modified dinucleotide in the
middle of the chain: tetramer 8 (5¢-TPTP(S)STPT) and decamer
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Scheme 2 The synthesis of protected dithymidyl-5¢-S-phosphorodithioate-3¢-O-phosphoramidite 7.
9 (5¢-TPTPTPTPTP(S)STPTPTPTPT). The syntheses were performed
on 1 mmole scale starting with thymidine bound to a LCA
CPG support (ABI column) using an automated DNA Synthe-
sizer ABI 394. The protocol of the synthesis was typical for
a phosphoramidite method with the time of coupling step of
modified phosphoramidite dimer prolonged to 10 min in order to
improve the coupling yield. After final detritylation and standard
ammoniacal cleavage from the support, the products were isolated
by RP HPLC. The structures of 8 and 9 were confirmed by
MALDI TOF MS. The application of dimer block 7 as a mixture
of diastereomers implied that diastereomerism should also be
observed for the resulting oligonucleotides 8 and 9. In fact, only
decamer 9 appeared by RP HPLC in the form of two very
close, overlapping peaks, most probably related to the presence
of diastereomers. No indication of diastereomerism could be
seen for tetramer 8. The amounts of obtained oligonucleotides
8 and 9 were too small for running their 31P NMR spectra in
order to confirm the presence of diastereomers. The purity of
decamer 9 was additionally checked, after its enzymatic 5¢-32P-
radiolabeling, by electrophoresis in a 20% polyacrylamide gel
under denaturing conditions, with unmodified decathymidylate
as a standard (see Fig. 3). It was shown that the presence of
one 5¢-S-phosphorodithioate modification does not influence the
electrophoretic mobility of decamer.
Fig.
3
Electrophoresis of decathymidylate
9
containing one
5¢-S-phosphorodithioate linkage. Lane A—crude decamer 9; Lane
B—HPLC purified decamer 9; Lane C—unmodified decathymidylate
(TP)9T.
considerable destabilization was also reported for duplexes formed
by oligonucleotides containing P-achiral phosphorodithioate link-
ages (without bridging sulfur), with their complementary matrices
(lowering of Tm by 0.5–14 ◦C per one modified bond).31
In order to check how the presence of 5¢-S-phosphorodithioate
modification influences the oligonucleotide avidity towards its
complementary sequence, standard studies of the stability of the
duplex formed by 9 with a complementary unmodified matrix
d[(AP)11A] were performed. It was found that the melting point of
duplex formed by d[(AP)11A] with 9 was considerably lower (Tm =
22.1 ◦C) than that formed with unmodified decathymidylate under
Conclusions
Easy access to deoxyribonucleoside 3¢-O-phosphorodithioates via
the dithiaphospholane approach and known chemoselective S-
alkylation of phosphorodithioate ambident ion prompted us to
investigate the synthesis of dideoxyribonucleoside (O3¢→S5¢)-
phosphorodithioates and their incorporation into oligodeoxyri-
bonucleotides. The presence of a P-S bond in the bridging position
attracted our attention since a single “thiolate” P-S bond in
◦
identical conditions (Tm = 27.3 C). This result clearly demon-
strates that the presence of even one 5¢-S-phosphorodithioate
linkage in oligonucleotide strongly destabilizes its duplex with
a complementary dodecaadenylate. It has to be mentioned that
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dinucleoside phosphorothioates can be readily cleaved under
the action of iodine/solvent/water22a or aqueous silver nitrate
reagents.22a Indeed, we were able to prove that dinucleoside-5¢-S-
phosphorodithioates undergo chemical cleavage of internucleotide
bond with iodine under aqueous conditions. Such opportunity
was interesting from the perspective of their possible use in a
novel approach to nucleic acids sequencing.37 Moreover, it has
been found that internucleotide (O3¢→S5¢)-phosphorodithioate
linkage is resistant towards phosphodiesterases svPDE and nP1,
and rather slowly reacts with calf spleen PDE, which indicates
that oligonucleotide congeners can be protected in this way
against exo- and endonucleases. On the other hand, the aforemen-
tioned modification dramatically influences the avidity of these
modified oligonucleotides towards complementary oligodeoxyri-
bonucleotides, and that the strong decrease of Tm disfavours
practical applications of such modification eg. in antisense strat-
egy. However, model compounds as described in this work can
be used for mapping of the contact points of oligonucleotides
with metal-dependent nucleases and evaluation of the role of 5¢-
oxygen in the architecture of active site such nucleases and in
the studies on metal-ions interactions with various congeners of
nucleotides.38
The S-alkylation of nucleoside-3¢-O-phosphorodithioates (1a–d)
with 5¢-bromo-5¢-deoxythymidine (2a)
A mixture of one of the nucleoside-3¢-O-phosphorodithioates
(Et3NH+ salt, 0.1 mmole) and 5¢-bromo-5¢-deoxythymidine29
(0.1 mmole) was dried overnight on a vacuum line and dissolved
in dry DMF (2 mL). The solution was stirred magnetically at
room temperature until 31P NMR control showed full conversion
of substrates (16 h) and the solvent was removed in vacuo. The
residue was dissolved in water (5 mL) and chromatographed on
DEAE Sephadex A25 column eluted with a linear gradient of
freshly prepared aqueous TEAB solutions (from 0.05 to 0.5M).
The products 3a–d were obtained as white powders after
evaporation of appropriate fractions. Their yields and spectral
as well as chromatographic characteristics are given in Table 1.
5¢-O-DMT-thymidine-3¢-O-[-O-(2-cyanoethyl)
phosphorodithioate] (5)
Into a solution of 600 mg (0.86 mmole) of 5¢-O-DMT-thymidine-
3¢-O-(2-thio-1,3,2-ditiaphospholane) (4)11d in 3 mL of CH3CN
(Baker) was added dropwise, with stirring at room temperature,
305 mg (4.3 mmole) of 3-hydroxypropionitrile (Aldrich) followed
by 130.5 mg (0.9 mmole) of DBU. After 30 min the solvent
was evaporated and the residue was dissolved in 50% aqueous
methanol (20 mL). The resulting solution was passed through a
column (1.5 ¥ 10 cm) loaded with Dowex 50Wx8 ion exchanger
[Et3NH+ form, prepared by neutralization of commercial H+ form
(Fluka) with aqueous Et3N]. After washing of the column with
50% methanol (100 mL) the crude product 5 was obtained by
evaporation and was then purified by flash column chromatogra-
phy with a gradient of methanol in chloroform (from 0 to 20%)
as an eluent. The pure diester 5 was obtained after evaporation
Experimental
General procedures
TLC was performed on Kieselgel 60F254 (Merck) with UV
(254 nm) detection. Flash chromatography was run on silica
gel 230–400 mesh (Merck). Ion exchange chromatography was
performed on a column filled with DEAE Sephadex A25 using
a peristaltic pump, gradient mixer, UV detector (260 nm) and
fraction collector SuperRac (all from LKB Pharmacia). Reverse
Phase High Performance Liquid Chromatography (RP HPLC)
was made with a Gilson chromatograph (model 306) using an
analytical column 4.6 ¥ 250 mm packed with ODS Hypersil, 5 m
(Alltech) or semi-preparative PRP-1 column 7 ¥ 305 mm (RP-18,
10 m, Hamilton). The columns were eluted with 0.1M aqueous
triethylammonium bicarbonate (TEAB), pH 7.5, supplemented
and drying on a vacuum line as white powder (500 mg, 82.1%). 31
P
NMR (CD3CN): d = 115.19 ppm. TLC (chloroform:methanol,
8:2, v/v): Rf= 0.65.
The S-alkylation of diester 5 with 5¢-iodo-5¢-deoxythymidine (2b)
The diester 5 (200 mg. 0.28 mmole) was dried overnight on a
vacuum line together with 150 mg (0.42 mmole) of 5¢-iodo-5¢-
deoxythymidine (2b).36 The compounds were then dissolved in a
mixture of DMF and CH3CN (1:1, v/v, 4 mL) and the solution was
incubated at 50 ◦C for 36 h. After evaporation, the crude product
was purified by flash column chromatography using gradient of
methanol in chloroform (from 0 to 6%) as an eluent. The pure
dinucleotide 6 was obtained as white powder in 84% yield (220 mg).
31P NMR (CD3CN): d = 95.83; 96.10 ppm (ca 1:1). FAB MS [M-
H]: m/z 932.3 (calculated MW 933.97 Da for protonated form).
TLC (chloroform:methanol, 9:1, v/v): Rf= 0.55.
by a linear gradient of acetonitrile from 0% to 30% in 25 min. 31
P
NMR spectra were recorded on a Bruker AC 200 spectrometer
1
(200.113 MHz for H) and referenced to 85% H3PO4 used as an
external standard. Mass spectra were recorded on Finnigan MAT
95 (FAB) or on Voyager-Elite spectrometers (MALDI TOF).
Oligonucleotide syntheses were performed with an automatic ABI
394 DNA Synthesizer on 1 mmole scale.
Materials
Unless otherwise noted, reagents, solvents and materials were
obtained from commercial sources. Snake venom phosphodi-
esterase (svPDE, EC 3.1.16.1) was purchased from Boehringer
Mannheim, whereas nuclease P1 (nP1, EC 3.1.30.1) and calf
spleen phosphodiesterase (spleen PDE, EC 3.1.16.1) were from
Sigma. Nucleoside-3¢-O-phosphorodithioates were synthesized
exactly as described earlier.14b 5¢-Bromo-5¢-deoxythymidine29 and
5¢-iodo-5¢-deoxythymidine36 were prepared according to literature
reports. The unmodified oligonucleotides d[(AP)11A] and T9T were
synthesized by a standard phosphoramidite method.
The synthesis of modified tetrathymidylate 8
The modified dinucleotide 6 (40 mg, 0.043 mmole) was dried
overnight on a vacuum line and dissolved in 100 mL of
dry CH3CN (Baker). Into the resulting solution, 9.5 mL
(0.047 mmole) of N,N,N¢,N¢-bis-(diisopropylamino)-O-2-cyano-
ethylphosphordiamidite was added, followed by dropwise addi-
tion of 86 mL of 0.5 M acetonitrile solution of EtS-tetrazole
within 15 min. After 1 h stirring at room temp., the resulting
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solution of phosphoramidite 7 was reacted with 1 mmole of
appropriately protected thymidine bound to LCAA CPG, on an
ABI 394 DNA synthesizer under conditions of phosphoramidite
synthesis (tetrazole activation, coupling time extended to 10 min).
The synthesis was then followed by standard addition of one
thymidine phosphoramidite. After detritylation and cleavage
from the support with 25% ammonia, the resulting modified
tetrathymidylate 8 (5¢-TPTP(S)STPT) was isolated on a semi-
preparative PRP-1 HPLC column (single peak, tR 17.86 min, 6.8
OD260). Its structure was confirmed by MALDI TOF MS (negative
ions): m/z 1186 (calculated MW 1186.93 Da for protonated
form).
Enzymatic digestions with calf spleen PDE
A sample of separated FAST or SLOW isomer of dinucleotide
3b (1 OD260) was placed in 200 mL of 100 mM citrate buffer
(pH 6.0) and incubated with 8 mg of spleen PDE (0.1 unit) at
37 ◦C. Samples of solution were collected after 1, 20 and 44 h,
◦
heated at 95 C for 2 min in order to denature the protein, and
centrifuged at 10 000 rpm for 10 min. The extent of digestion was
monitored by analytical RP HPLC (see Fig. 1). The digestion
of unmodified dithymidylate (TPT) performed under identical
conditions showed its complete degradation into thymidine and
thymidine-3¢-O-phosphate after 1 h.
Chemical degradation of 3b in iodine/solvent/water system
The synthesis of modified decathymidylate 9
a) Into a solution of 3b (4.2 OD260, mixture of isomers) in 300 mL
of water, 400 mL of 100 mM solution of I2 in acetone was added.
The combined solution was incubated for 1 h at 50 ◦C, diluted
with water (1 mL) and extracted with diethyl ether (5 ¥ 2 mL). A
sample of aqueous solution was analysed by RP HPLC to show
complete disappearance of peaks of substrate.
The modified dinucleotide 6 (40 mg, 0.043 mmole) was phos-
phitylated exactly as described in a previous paragraph. The
resulting solution of phosphoramidite 7 was reacted with the
5¢-OH group of tetrathymidine bound to LCAA CPG, freshly
prepared on an ABI 394 DNA Synthesizer (1 mmole scale)
under conditions of phosphoramidite synthesis (coupling time
of modified dimer extended to 10 min). The synthesis was
continued by consecutive addition of four units of thymidine
phosphoramidite under standard conditions. After detritylation
and cleavage from the support with 25% ammonia, the resulting
modified decathymidylate 9 (5¢-TPTPTPTPTP(S)STPTPTPTPT) was
isolated by RP HPLC on a semi-preparative PRP-1 column in
the form of two overlapping peaks (tR 19.88 and 20.01 min).
Its structure was confirmed by MALDI TOF MS (negative
ions): m/z 3012 (calculated MW 3012.07 Da for protonated
form).
b) Into a solution of 3b (20 mg, mixture of isomers) in 500 mL of
water, 500 mL of 200 mM solution of I2 in acetone was added. The
combined solution was incubated for 1 h at 50 ◦C and evaporated.
The residue was extracted with D2O (500 mL), filtered and analysed
by 31P NMR to show complete disappearance of peaks of substrate
and the formation of a product at d = -0.2 ppm.
c) Into a solution of 3b (4.2 OD260, mixture of isomers) in 300 mL
of water, 400 mL of 100 mM solution of I2 in pyridine was added.
The combined solution was incubated for 1 h at 20 ◦C, diluted
with water (1 mL) and extracted with diethyl ether (5 ¥ 2 mL). A
sample of aqueous solution was analysed by RP HPLC to show
complete disappearance of peaks of substrate.
Enzymatic digestions with svPDE
d) Into a solution of 3b (20 mg, mixture of isomers) in 500 mL of
water, 500 mL of 200 mM solution of I2 in pyridine was added. The
combined solution was incubated for 1 h at 20 ◦C and evaporated.
The residue was extracted with D2O (500 mL), filtered and analysed
by 31P NMR to show complete disappearance of peaks of substrate
and the formation of products at d = 19.58 ppm and 0.18 ppm in
ca. 45/55 ratio.
When a reaction as above was performed with 50 mM solution
of I2 in pyridine, a presence of ca. 60% of undegraded substrate
was observed (d = 73.0 and 73.8 ppm), together with ca. 35% of
thiolate product at d = 19.6 ppm and ca. 5% of phosphate product
at d = 0.2 ppm.
A sample of dinucleotide 3a (0.1 OD260) was placed in 100 mL
of a buffer containing 100 mM TrisHCl (pH 8.5) and 15 mM
MgCl2, and incubated with 0.5 mg of svPDE at 37 ◦C for 16 h. The
solution was then heated at 95 ◦C for 2 min in order to denature
the protein, and centrifuged at 10 000 rpm for 10 min. The extent
of digestion was monitored by analytical RP HPLC. The digestion
of dinucleotide 3b with svPDE was performed in an identical
manner. In both cases no digestion was observed. The digestion
of unmodified dithymidylate (TPT) performed under identical
conditions showed its complete degradation into thymidine and
thymidine-5¢-O-phosphate after 1 h.
Enzymatic digestions with nP1
Thermodynamic stability of duplex formed by 9 with d[(AP)11A]
A sample of dinucleotide 3a (0.1 OD260) was placed in 100 mL
of a buffer containing 100 mM TrisHCl (pH 7.2) and 1 mM
ZnCl2, and incubated with 2 mg of nP1 at 37 ◦C for 16 h. The
solution was then heated at 95 ◦C for 2 min in order to denature
the protein, and centrifuged at 10 000 rpm for 10 min. The extent of
digestion was monitored by analytical RP HPLC. The digestion of
dinucleotide 3b with nP1 was performed in an identical manner. In
both cases no digestion was observed. The digestion of unmodified
dithymidylate (TPT) performed under identical conditions showed
its complete degradation into thymidine and thymidine-5¢-O-
phosphate after 1 h.
A solution containing 0.73 mM of 9 and 0.73 mM of d[(AP)11A] was
prepared in a buffer of 10 mM TrisHCl (pH 7.4), 100 mM NaCl
and 10 mM MgCl2. The Tm value was calculated on the basis of
absorption measurements which were carried out in a 1 cm path-
length cell with a UV-VIS 916 spectrophotometer equipped with a
Peltier thermocell (GBC, Dandenong, Australia). Melting profile
was measured with a temperature gradient of 0.2 ◦C/min in both
directions. The melting temperature (Tm) was calculated using
a first-order derivative method. The Tm value of duplex formed
by d[(AP)11A] with unmodified T9T was measured in an identical
manner.
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Acknowledgements
This work was financially assisted by Ministry of Science and
Higher Education (Statutory Funds to CMMS PAS, Lodz,
Poland). The authors are grateful to Mr M. Deka for the help
in RP HPLC analysis.
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