Hydrolytic reactions of 3′-N-phosphoramidate and 3′-N-thiophosphoramidate analogs of thymidylyl-3′,5′-thymidine

Article abstract of DOI:10.1039/b313470a

The diastereomeric thiophosphoramidate analogs [(R_P)- and (S_P)-3′,5′-Tnp(s)T] 2 and the phosphoramidate analog [3′,5′-TnpT] 3 of thymidylyl-3′,5′-thymidine were prepared and their hydrolytic reactions over the pH-range 1-8 at 363.2 K

Full text of DOI:10.1039/b313470a

View Article Online / Journal Homepage / Table of Contents for this issue
Hydrolytic reactions of 3Ј-N-phosphoramidate and 3Ј-N-
thiophosphoramidate analogs of thymidylyl-3Ј,5Ј-thymidine
Mikko Ora,*a Merita Murtola,a Sami Ahoa and Mikko Oivanenb
a Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
b Department of Chemistry, Laboratory of Organic Chemistry, P. O. Box 55,
FIN-00014 University of Helsinki, Finland
Received 27th October 2003, Accepted 15th December 2003
First published as an Advance Article on the web 26th January 2004
The diastereomeric thiophosphoramidate analogs [(RP)- and (SP)-3Ј,5Ј-Tnp(s)T] 2 and the phosphoramidate analog
[3Ј,5Ј-TnpT] 3 of thymidylyl-3Ј,5Ј-thymidine were prepared and their hydrolytic reactions over the pH-range 1–8 at
363.2 K were followed by RP HPLC. At pH < 6, an acid-catalyzed P–N3Ј bond cleavage (first-order in [Hϩ]) takes
place with both 3Ј,5Ј-Tnp(s)T and 3Ј,5Ј-TnpT, the former being about 12 fold more stable than the latter. At pH > 4,
Tnp(s)T undergoes two competing pH-independent reactions, desulfurization (yielding TnpT) and depyrimidination
(cleavage of the N-glycosidic bond) the rates of which are of the same order of magnitude. Also with 3Ј,5Ј-TnpT the
pH-independent depyrimidination competes with P–N3Ј cleavage at pH > 5.
Introduction
Phosphoramidate and phosphorothioate analogues of oligo-
deoxyribonucleotides belong to the most extensively studied
potential antisense oligonucleotide agents.1 Many of these
modifications have been shown to be resistant toward nucleases,
and to form duplexes with complementary oligonucleotide
sequences.2 For instance, oligodeoxynucleotide phosphor-
amidates with a 3Ј-N-bridging internucleosidic linkage have
shown promising properties.2 More recently, synthesis and
characterisation of various oligonucleotide thiophosphor-
amidates have been reported. Gryaznov and Pongracz have
described a solid support method for preparation of oligo-
deoxyribonucleotide thiophosphoramidates containing inter-
nucleosidic 3ЈNH–P(O)(SϪ)–O5Ј linkages.3 Stawinski et al. have
reported the synthesis of dinucleoside thiophosphoramidate
analogues containing 3ЈO–P(O)(SϪ)–NH5Ј linkage using the
H-phosphonate methodology.4
We have recently reported on a kinetic analysis of the base-
and acid catalyzed hydrolysis of the diribonucleoside 3Ј,5Ј-(3Ј-
N-phosphoramidate) UnpU 1.5 The present study is aimed at
providing kinetic data on the hydrolysis of di(deoxyribonucleo-
side) thiophosphoramidates 2 and phosphoramidates 3. Semi-
quantitative data on the stability of deoxyribonucleoside
phosphoramidates6–9 and thiophosphoramidates3 in aqueous
acid have recently been published, along with the characteris-
ation of the products of hydrolysis, but there are no detailed
kinetic investigations available. Nevertheless, it has been shown
that under acidic conditions dinucleoside 3Ј-N-phosphor-
amidates are hydrolysed via P–N bond-cleavage.6–9 With the
3Ј-N-thiophosphoramidate analogs, however, desulfurization
competes with the P–N bond cleavage.3
Results and discussion
Product distributions
The hydrolysis of 3Ј,5Ј-Tnp(s)T 2 and 3Ј,5Ј-TnpT 3 was fol-
lowed over a wide pH range by determining by RP HPLC the
composition of the aliquots withdrawn at appropriate intervals
from the reaction solution (two examples given in Figs 1 and 2).
Fig. 1 Time-dependent product distribution for the hydrolysis of
The products were identified by spiking with authentic refer-
Tnp(s)T (2) in a formic acid buffer at pH 3 ([HA]/[AϪ] = 0.05/0.01 mol
ence samples and by a mass spectrometric (HPLC-ESI-MS)
analysis. With both 3Ј,5Ј-Tnp(s)T 2 and 3Ј,5Ј-TnpT 3, the
hydronium ion-catalysed cleavage of the P–N3Ј bond (Routes A
L
Ϫ1; I = 0.1 mol LϪ1 with NaCl) and 363.2 K. Notation: (᭹) Tnp(s)T
(2), (᭿) 3Ј-amino-3Ј-deoxythymidine (4), (ᮀ) thymidine 5Ј-thiophos-
phate (12) and (᭺) thymidine (6).
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 3 – 6 0 0
593

View Article Online
Scheme 1
Fig. 2 Time-dependent product distribution for the hydrolysis of
Fig. 3 pH-rate profile for the decomposition of RP- and SP-3Ј,5Ј-
Tnp(s)T (2; ᭹ and ᭺) and 3Ј,5Ј-TnpT (3; ∇) at 363.2 K. The ionic
strength of the solutions adjusted to 0.1 mol LϪ1 with sodium chloride.
The dotted line shows the corresponding curve for the 3Ј,5Ј-UnpU at
363.2 K.
Tnp(s)T (2) in a glycine buffer at pH 8.0 ([HA]/[AϪ] = 0.02/0.01 mol LϪ1
I = 0.1 mol LϪ1 with NaCl) and 363.2 K. Notation: (᭹) Tnp(s)T (2),
(᭿) 3Ј-amino-3Ј-deoxythymidine (4), (∇) TnpT (3), (ଙ) thymidine 5Ј-
amidothiomonophosphate (15), (ଝ) depyrimidinated product (13/14),
(ꢀ) thymine and (᭺) thymidine (6).
;
ent cleavage of the N-glycosidic bond (release of thymine, route
B) competes with P–N3Ј cleavage and on going to less acidic
and neutral conditions it becomes gradually the only reaction
detected. During the hydrolysis of 3 at pH 8, thymine 7 and
thymidine amidomonophosphate 8 (m/z = 320 was observed)
were formed as the main chromophoric products, in equal
amounts with each other, and besides these only a minor
amount (4%) of 3Ј-amino-3Ј-deoxythymidine 4 was found to
accumulate. The 5Ј-amidomonophosphate 8 is proposed to be
formed by elimination of the carbohydrate moiety from the
depyrimidination product 9. The corresponding degradation of
the other major depyrimidination product, 10, would yield
the 3Ј-N-phosphoramidate 11, which can not be directly dis-
tinguished from 8 by the HPLC-MS analysis. However, 11 is
and E in Schemes 1 and 2, respectively) was found to be the
predominant reaction under acidic conditions (pH < 4),
whereas under less acidic and neutral conditions the pH-
independent cleavage of the N-glycosidic linkages (Routes B
and F in Schemes 1 and 2, respectively) and desulfurisation
of the thiophosphoramidate moiety of Tnp(s)T (Route G in
Scheme 2) become the main pathways of degradation.
The hydronium ion-catalysed hydrolysis of the P–N3Ј bond
of the phosphoramidate 3Ј,5Ј-TnpT 3 at pH 1–4 (Figs 3 and
4) yields a mixture of 3Ј-aminonucleoside 4 and thymidine 5Ј-
phosphate 5 (route A). Under less acidic conditions the released
phosphate monoester 5 is partly dephosphorylated during the
kinetic run (yielding thymidine 6). At pH > 5 the pH-independ-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 3 – 6 0 0
594

View Article Online
Scheme 2
that the thymine moiety is released more readily from the
3Ј-aminothymidine moiety than from the 5Ј-O-esterified
thymidine moiety of 3.
The intermediary accumulation of 9/10 during the hydrolysis
of 3 at pH 8 remained too low (< 2%) to be quantified by HPLC
with UV-detection. Nevertheless, an m/z value ([M–H]Ϫ
=
436.3) corresponding to the molecular ion of 9/10 could be
observed by the HPLC-ESI-MS analysis of the samples. By
contrast, with the thiophosphoramidates 2, the corresponding
intermediate 13/14 accumulated at pH 8 to a much higher
extent, close to 20% of the hydrolysis products (see discussion
below).
The RP- and SP-diastereomers of the thiophosphoramidate 2
were separated from each other by reversed phase chromato-
graphy and their hydrolytic reactions were followed separately,
although the absolute configurations of the isomers were not
assigned. The two diastereomers showed similar reactivities
with each other in all the reactions studied. The acid catalysed
hydrolysis, which is the predominant reaction of Tnp(s)T 2 at
pH < 4, yields a mixture of 3Ј-amino-3Ј-deoxythymidine 4 and
thymidine 5Ј-thiomonophosphate 12 (route E in Scheme 2).
The thiomonophosphate 12, being more than 2 orders of
Fig. 4 pH-rate profiles for the partial reactions involved in the
hydrolysis of Tnp(s)T (2) and TnpT (3) at 363.2 K. Notation: Route A
(᭿) and Route B (᭢) in Scheme 1 and (ᮀ) Route E, (᭹) Route G and
(᭺) Route F in Scheme 2.
expected to be more labile than 8 and become readily dephos-
phorylated to yield 3Ј-amino-3Ј-deoxythymidine 4.10 While the
accumulation of 4 remained low over the kinetic run, it seems
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 3 – 6 0 0
595

View Article Online
magnitude more labile than its phosphate analog 5,11 is
further dephosphorylated to thymidine 6. Fig. 1 shows, as
an example, the time-dependent product distribution observed
for hydrolysis of 2 at pH 3.
of the thiophosphoramidate, SP- and RP-forms, are equal with
each other.
On going to slightly acidic and neutral conditions (pH > 5),
the pH-independent desulfurisation (route G in Scheme 2) and
depyrimidination (route F) become the only reactions detected
for Tnp(s)T. The rates of these two reactions are very close
to each other (Fig. 4). The depyrimidination of the phos-
phoramidate 3 is slightly faster than that of the thiophos-
phoramidate 2.
At pH 4–5, two pH-independent hydrolytic reactions of 2,
namely depyrimidination of either of the thymidine moieties
(route F) and desulfurization of the thiophosphoramidate
moiety (yielding 3Ј,5Ј-TnpT 3; route G), compete with the
acid-catalysed P–N3Ј cleavage. Thymidine 5Ј-thiophosphate 12
was not detected to accumulate in this pH region, due to its
relatively fast subsequent dephosphorylation11 to thymidine. At
pH > 5, the desulfurisation and depyrimidination become faster
than the P–N3Ј cleavage (Fig. 4). The product distribution of
the hydrolysis under neutral conditions is rather complicated
(Fig. 2), while in addition to TnpT 3 and thymine 7, the mono-
nucleosidic compounds 4, 5 and 6, thymidine 5Ј-amidothio-
monophosphate 15 (up to 30% of the products at pH 8) and
even the depyrimidinated compound 13/14 (up to 20% of the
products at pH 7) accumulate. The appearance of the depyr-
imidinated product was clearly shown by HPLC-MS analysis,
but only a single chromatographic peak corresponding to the
appropriate molecular ion ([M–H]Ϫ = 452.2) was observed. No
direct evidence was obtained as to whether this represents a
single compound or whether the possible isomeric compounds
appear unseparated in the chromatogram (in fact, both 13 and
14 could appear also as α- and β-anomers, or possibly even
partly as the corresponding pyranose isomers). Anyway, the
isomeric 13 and 14 are expected to give different monoesters
as hydrolysis products, thymidine 5Ј-amidothiophosphate 15
being formed from 13 and 3Ј-aminothymidine 3Ј-N-thiophos-
phoramidate 16 from 14 (m/z = 336 for [M–H]Ϫ of both 15 and
16). Even for the latter step, only one chromatographic peak
corresponding to the expected molecular ion was observed.
However, this is to be expected, since the dephosphorylation
involving cleavage of the P–N linkage is fast and there is some
evidence11 that the thiosubstitution (cf. 16) still facilitates this
reaction (a thiometaphosphate ion is a more stable intermediate
than the metaphosphate anion). Accordingly, 16 is proposed to
be too labile to accumulate during the performed kinetic run,10
since it is expected to readily dethiophosphorylate to amino-
thymidine 4. The latter was indeed observed to accumulate, but
to a rather low extent (< 10% for the reaction at pH 8, see
Fig. 2). It must be also noted that 4 can also be formed by other
pathways, for example by degradation of TnpT 3 (Route G)
and its appearance does not give exclusive evidence of the
occurrence of hydrolysis through 14. The mononucleosidic
compounds may also be partly depyrimidinated during the
kinetic run, which further complicates the analysis. Neverthe-
less, the analysis suggests that a major part of the depyrim-
idination of 2 takes place by release of thymine from the 3Ј-N-
aminothymidine moiety. The accumulated compound showing
the molecular ion m/z = 336.0 ([M–H]Ϫ) is, tentatively, assigned
as thymidine 5Ј-amidothiomonophosphate 15, the formation of
which may take place via intermediate 13. Compound 15 is not
markedly dephosphorylated or depyrimidinated during the
course of the hydrolysis of 2 at pH 8.
Reaction mechanisms
We have previously suggested5 that the acid-catalysed P–N3Ј
bond rupture of UnpU 1 differs mechanistically from the
typical phosphoester hydrolysis reactions of RNA fragments,
since it does not involve participation of the neighbouring
2Ј-hydroxyl function as a nucleophile. The similarity of the
pH-rate profiles of the 2Ј-deoxynucleoside phosphoramidate
3Ј,5Ј-TnpT and the ribonucleoside analog 3Ј,5Ј-UnpU gives
further support to this interpretation. Furthermore, also with
Tnp(s)T, the P–N3Ј bond cleavage shows a linear first-order
dependence on the hydronium ion concentration, and even the
12 fold lower reactivity of Tnp(s)T compared to that of TnpT
(Figs 3 and 4) is consistent with the same mechanistic scheme.
Under slightly acidic conditions (pH 2–6) the prevailing ionic
form of the compounds is in all likelihood the monoanionic
N,O-disubstituted phosphoramidate (1, 3)12 or thiophosphor-
amidate (2), but the reactive tautomer is the neutral molecule.
The preferred site of protonation of the phosphoramidates is
not exclusively clear,13 but it seems in any case likely that the
reactive tautomer of the phosphoramidate is the N-protonated
zwitterionic species, from which the leaving group may depart
as a neutral amine (Scheme 3). In all likelihood the P–N bond
rupture proceeds, as earlier discussed in the case of UnpU,5 by a
mechanism involving participation of a water molecule in the
transition state. Thus, the formation of the new P–O bond (by
The pH-rate profiles
Fig. 3 shows the pH-rate profiles for the decomposition
of the diastereomers of the thiophosphoramidate
2 in
comparison with those of the phosphoramidates 1 and 3.
Fig. 4 shows the pH-rate profiles for the partial reactions
involved in the hydrolysis of 3Ј,5Ј-Tnp(s)T (2) and TnpT (3).
The rate of the acid-catalysed P–N3Ј cleavage of 3Ј,5Ј-TnpT
is close to that of 3Ј,5Ј-UnpU5 1, TnpT being 1.7 times
more reactive than UnpU. The thiosubstitution, however,
significantly decreases the rate of this reaction, while 3Ј,5Ј-
Tnp(s)T 2 is about 12 times more stable than 3Ј,5Ј-TnpT 3 in
the pH-region 2–5. The reactivities of the two diastereomers
Scheme 3
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 3 – 6 0 0
596

View Article Online
attack of water) is already long advanced during the rupture of
the P–N3Ј-linkage and no free alkyl metaphosphate (or thio-
metaphosphate) ion is formed. This type of mechanism has
been earlier described for the solvolysis of phosphoramidic
acid,14 O-alkylphosphoramidates15 and N-alkylphosphor-
amidates16 in aqueous alcohol. The entropy of activation for
the hydrolysis of TnpT at pH 2 was determined as Ϫ63 4 J
KϪ1 molϪ1 (five experimental points at 298.2 < T < 363.2 K),
which is equal to that determined5 for UnpU (Ϫ65 J KϪ1
molϪ1). Being clearly negative, these values are inconsistent with
a unimolecular mechanism, but on the other hand, they are
actually not as negative as expected for a reaction with a purely
bimolecular transition state. Rather, they represent borderline
values of those expected for a unimolecular and bimolecular
hydrolysis.
The replacement of a nonbridging phosphoryl oxygen with
sulfur retards the hydronium ion-catalyzed hydrolysis of
TnpT, the kinetic thio-effect (kPO/kPS) being 12, as noted
above. This effect may well be due to the higher acidity of
the thiophosphoramidate moiety compared to the phos-
phoramidate, i.e. the less ready protonation of the thiophos-
phoramidate diester to the reactive neutral (zwitterionic) form.
For comparison, the pKa1 and pKa2 values of thiophosphoric
acid are 0.5 and 1.8 units lower, respectively, than those of
phosphoric acid.17,18 Unfortunately, as far as we know no
comparable pKa values for thiophosphoramidates have been
reported.
The pH-rate profile of the hydrolysis of Tnp(s)T shows
slight curvature towards a constant value on going below pH 2
(Fig. 3). In principle, this kind of curvature could result from
the thiophosphoramidate having a pKa value in the region 1–2
and the monocationic species (at pH < pKa) being unreactive in
P–N3Ј bond hydrolysis. However, the present data does not
reveal whether the water catalysed hydrolysis of the neutral
(zwitterionic) thiophosphoramidate (route H in Scheme 3) is
accompanied by hydronium ion-catalysed (route I) hydrolysis
at very low pH, i.e. on passing the pKa of the thiophosphor-
amidate. Anyway, it may be noted for comparison that the rate
profile of the hydrolysis of the P–N3Ј bond of UnpU5 does not
show any deviation from the first-order dependence of the rate
on [Hϩ] in the pH range from Ϫ1 to 4.
sulfur ligand may depart as hydrogen sulfide ion after protolytic
rearrangement, which leads to the observed intermediary
accumulation of the phosphoramidate TnpT 3. In principle,
the breakdown of the pentacoordinated intermediate I could
alternatively take place via departure of the 5Ј-esterified
thymidine or even by departure of the 3Ј-aminothymidine.
However, although the latter pathways cannot be completely
excluded, they may not be proposed to be of significant
importance. The accumulation of the appropriate monomeric
products expected to be released by these pathways remains
relatively low and the same products can be formed even by
competing pathways. For example, a significant part of the
formation of the aminothymidine (4) in all likelihood takes
place by degradation of the TnpT formed via P–S bond
cleavage of intermediate I. In any case, the desulfurization
is proposed to be faster than the cleavage P–O5Ј linkage,
remembering that hydrogen sulfide ion is less basic than alk-
oxide and hence a better leaving group under the experimental
conditions (pKa’s of H2S and the 5Ј-OH are 6.5 and 13–15,
respectively).18,19
The rate of the hydrolysis of the N-glycosidic bond of
TnpT and Tnp(s)T is pH-independent over the pH-region 4–8
(Fig. 4), analogously to the degradation of thymidine.20 The
uncatalysed depyrimidination of thymidine has been suggested
to involve a unimolecular cleavage of the N-glycosidic linkage,
resulting in formation of the cyclic glycosyl oxocarbenium ion
derived from the sugar moiety (Scheme 5).20 This is in contrast
to the acid-catalysed cleavage, which most likely takes place
by opening of the sugar ring after protonation of the O4Ј-
oxygen.21 The uncatalysed degradation of thymidine has been
proposed20 to involve a rate-limiting cleavage of the C–N bond
followed by rapid protonation of the anionic base moiety and
nucleophilic attack of a water molecule on the oxocarbenium
ion. The formed depyrimidinated products of 2 and 3 (13/14
and 9/10, respectively) undergo readily a decomposition of the
depyrimidinated sugar ring and release the appropriate mono-
meric nucleoside phosphor- or thiophosphoramidates. The rate
constants obtained for the depyrimidination of TnpT and
Tnp(s)T are comparable to the values previously reported22
for the uncatalysed cleavage of the C1Ј–N bond of thymidine
(k = 1 × 10Ϫ6 sϪ1 at pH 3–7 at 95 ЊC).
The rate of the desulfurization of 3Ј,5Ј-Tnp(s)T is pH-
independent at pH 4–8 (Fig. 4), which means that the reaction
takes place either by attack of a water molecule on mono-
anionic thiophosphoramidate linkage or by attack of a hydrox-
ide ion on a neutral thiophosphoramidate (Scheme 4). The
Scheme 4
Scheme 5
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 3 – 6 0 0
597

View Article Online
Experimental
Materials
The NMR spectra were recorded on a Bruker AM 200 or a Jeol
400 spectrometer. The 1H NMR chemical shifts (at 300 K) were
referred to internal TMS, and the 31P NMR shifts (202 MHz,
300 K) to external orthophosphoric acid. The mass spectra
were acquired using a Perkin Elmer Sciex API 365 triple
quadrupole LC/MS/MS spectrometer.
The organic solvents were dried by refluxing over calcium
hydride and distilled. The solid starting materials were dried by
coevaporation with the anhydrous solvent. Thymidine, thymine
and thymidine 5Ј-phosphate were products of Sigma. 3Ј-Azido-
3Ј-deoxythymidine was prepared as described previously23 and
converted to 3Ј-amino-3Ј-deoxythymidine by treatment with
triphenylphosphine in anhydrous pyridine followed by addition
of aqueous ammonia.24 Dinucleoside thiophosphoramidate
analogs of thymidylyl-3Ј,5Ј-thymidine (2) were prepared using
H-phosphonate methodology, in which nucleoside H-phos-
phonothioate 17 was converted into a pyridine adduct of
a nucleoside thiometaphosphate followed by reaction with
5Ј-protected 3Ј-amino-3Ј-deoxythymidine 18 (Scheme 6), as
described previously by Stawinski et al.4 Dinucleoside phos-
phoramidate analog of thymidylyl-3Ј,5Ј-thymidine (3) was
obtained by oxidative amination of the appropriately protected
thymidine 5Ј-(H-phosphonate) 2-cyanoethyl ester 21 in the
presence of 3Ј-amino-3Ј-deoxythymidine (Scheme 7). The same
reaction sequence has previously been used for the synthesis of
oligoribonucleotide phosphoramidates on a solid support.6,7
Scheme
7
Reagents and conditions: (i) CNCH2CH2OH, DPCP,
CH3CN–pyridine, (ii) 4, CCl4, Et3N, (iii) NH3–MeOH, (iv) TBAF–
THF.
pyridine (2 ml) were added (Scheme 6). The crude product was
isolated by a conventional aqueous work up, and purified on a
silica gel column eluted with a mixture of dichloromethane and
methanol (80 : 20%, v/v). The tert-butyldimethylsilyl protected
thiophosphoramidate (19) was dissolved in 1 mol LϪ1 solution
of tetrabutylammonium fluoride (0.265 g, 1.02 mmol) in tetra-
hydrofuran (1 mL), and the solution was stirred for 16 h at
room temperature. The mixture was evaporated to dryness and
the diastereomers were purified and separated from each other
by reversed phase chromatography on a Lobar RP-18 column
(37 × 440 mm, 40–63 µm) eluting with a mixture of water and
acetonitrile (90 : 10%, v/v). Finally, the product was passed
through a Naϩ-form Dowex 50-W (100–200 mesh) cation
exchange column. 2 (the faster eluted diastereomer): 31P NMR:
1
δP (202 MHz, D2O) = 53.63. H NMR: δH (400 MHz, D2O) =
7.71 (s, 1H, H6), 7.70 (s, 1H, H6), 6.15 (t, 1H, H1Ј, J = 6.62),
5,80 (dd, 1H, H1Ј, J = 7.05, J = 2.14), 4.43 (m, 1H, H3Ј), 4.00
(m, 2H, H4Ј), 3.93 (m, 2H, H5Ј, H5Љ), 3.83–3.79 (m, 1H, H5Ј),
3.71 (m, 1H, H5Љ), 3.70 (m, 1H, H4Ј), 3.63–3.54 (m, 1H, H3Ј),
2.37–2.13 (m, 4H, H2Ј, H2Љ), 1.73 (s, 3H, CH3), 1.70 (s,
3H,CH3). ESIϪ-MS: m/z 560.3 [M Ϫ H]Ϫ. 2 (The slower eluted
diastereomer): 31P NMR: δP (202 MHz, D2O) = 53.59. 1H
NMR: δH (400 MHz, D2O) = 7.62 (s, 2H, 2 × H6), 6.16 (t, 1H,
H1Ј, J = 6.62), 5.90 (dd, 1H, H1Ј, J = 6.83, J = 3.63), 4.42 (m,
1H, H3Ј), 4.00 (m, 1H, H4Ј), 4.00–3.95 (m, 1H, H5Ј), 3.91–3.86
(m, 1H, H5Љ), 3.81 (m, 1H, H5Љ), 3.72 (s, 2H, H3Ј, H4Ј), 3.76–
3.68 (m, 1H, H5Љ), 2.33–2.18 (m, 4H, H2Ј, H2Љ), 1.80 (s, 3H,
CH3), 1.72 (s, 3H, CH3). ESIϪ-MS: m/z 560.3 [M Ϫ H]Ϫ.
(3Ј-Amino-3Ј-deoxythymidylyl)-(3Ј 5Ј)-thymidine (3)
Compound 3 was prepared, analogously with the previously
described method,6,7 by reacting the triethylammonium salt
of 5Ј-O-(tert-butyldimethylsilyl)thymidine 5Ј-hydrogenphos-
phonate 20 (0.52 g, 1.24 mmol) with cyanoethanol (76 µL,
1.11 mmol) and DPCP (304 µL, 1.49 mmol) in a mixture of
anhydrous pyridine (4.0 mL) and acetonitrile (6.0 mL) (Scheme
7). The formed cyanoethyl ester 21 was not isolated. After 2 h
of stirring at room temperature, 3Ј-amino-3Ј-deoxythymidine 4
(0.47 g, 1.34 mmol), carbon tetrachloride (5.0 mL) and triethyl-
amine (0.22 mL) were added and the mixture was left to stand
overnight. The mixture was then poured into 90 mL of di-
chloromethane and washed with saturated aqueous NaCl
(3 × 50 mL). The organic layer was dried with Na2SO4 and
concentrated. The residue was purified on a silica gel column
using a mixture of dichloromethane and methanol as eluent
(92 : 8%, v/v) to give the fully protected product 22. The
cyanoethyl group was removed by treatment with saturated
Scheme 6 Reagents and conditions: (i) TMSCl, py, (ii) I2, (iii) Et3N, py,
(iv) TBAF–THF.
The (RP)- and (SP)-thiophosphoramidate analogs of (3Ј-amino-
3Ј-deoxythymidylyl)-3Ј,5Ј-thymidine (2)
Nucleoside H-phosphonothioate25 17 (0.25 g, 0.57 mmol) and
trimethylsilyl chloride (0.19 g, 1.72 mmol) were dissolved in
10 mL of anhydrous pyridine. After stirring for 5 min at RT,
iodine (0.22 g, 2.87 mmol) was added. After an additional 7 min
of stirring, 5Ј-O-(tert-butyldimethylsilyl)-3Ј-amino-3Ј-deoxy-
thymidine 18 (0.20 g, 0.57 mmol) and triethylamine (0.40 ml) in
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 3 – 6 0 0
598

View Article Online
methanolic ammonia (3 mL) and the nucleotidic product was
purified using silica gel chromatography using a mixture of
dichloromethane and methanol as eluent, the methanol content
of which was increased stepwise from 0 to 30%. The tert-butyl-
dimethylsilyl protecting groups were removed with 1 mol LϪ1
tetrabutylammonium fluoride in tetrahydrofuran (1 mL). The
mixture was evaporated to dryness and the product was purified
by reversed phase chromatography on a Lobar RP-18 column
(37 × 440 mm, 40–63 µm) eluting with a mixture of water and
acetonitrile (92 : 2%, v/v). Finally, the product was passed
through a Naϩ-form Dowex 50-W (100–200 mesh) cation
by least-squares fitting to eqn. (2), where kdec is the first-order
rate constant for the disappearance of Tnp(s)T and k2 the first-
order rate constant for the disappearance of 13/14. [Tnp(s)T]0
stands for the initial concentration of the starting material and
[13 ϩ 14]t for the observed concentration of 13 and 14 at
moment t.
(2)
1
exchange column. 31P NMR δP (202 MHz, D2O) = 6.81. H
At pH 4–5, where 13/14 does not accumulate, the first-order
rate constants (kdp) for the depyrimidination of 3Ј,5Ј-Tnp(s)T
(route F in Scheme 2), were calculated by eqn. (3), by bisecting
kdec to the rate constants of parallel first-order reactions on the
basis of the product distribution at the early stages of the
reaction i.e., under conditions where the formation of thymine
from the hydrolysis products of 2 may be neglected. [Thymine]t
and [Tnp(s)T]t stand for the concentration of thymine and 2,
respectively, at moment t, and [Tnp(s)T]0 denotes the initial
concentration of the starting material.
NMR δH (400 MHz, D2O) = 7.70 (s, 1H, H6), 7.68 (s, 1H, H6),
6.15 (t, 1H, H1Ј, J = 6.4), 5.80 (dd, 1H, H1Ј, J = 7.0, J = 2.6),
4.43 (m, 1H, H3Ј), 3.96–3.90 (m, 2H, H4Ј, H5Ј), 3.83–3.78 (m,
2H, H5Љ, H5Ј), 3.72–3.67 (m, 2H, H4Ј, H5Љ), 3.46 (m, 1H, H3Ј),
2.34–2.28 (m, 1H, H2Ј), 2.26–2.15 (m, 3H, H2Ј, 2 × H2Љ), 1.70
(s, 2 × 3H). ESIϪ-MS: m/z 544.9 [M Ϫ H]Ϫ.
Kinetic measurements
The reactions were carried out in sealed tubes immersed in a
thermostated water bath (363.2 K), the temperature of which
was adjusted within 0.1 K. The hydronium ion concentration
of the reaction solutions was adjusted with hydrogen chloride
and formate, N-morpholinoethanesulfonic acid (MES), tri-
ethanolamine, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic
acid (HEPES) and glycine buffers. The pH values of the buffer
solutions were calculated from the literature data of the pKa
values of the buffer acids under the experimental conditions.26
Low buffer concentration was used (30–60 mM). The
initial substrate concentration in the kinetic runs was ca. 0.1
mM. The composition of the samples withdrawn at appropriate
intervals was analyzed by HPLC on a Hypersil ODS 5 column
(4 × 250 mm, 5 µm) using as eluent an acetic acid/sodium
acetate buffer (0.045/0.015 mol LϪ1) containing 0.1 mol LϪ1
ammonium chloride. A good separation of the product mixture
was obtained, when a 5 min isocratic elution was followed by
a linear gradient (32 min) up to 5% MeCN. The observed
retention times (tR/min) for the hydrolytic products of 2 (flow
rate 1 mL minϪ1) were as follows: 10.4 (5), 11.3 (7), 12.0 (4), 20.7
(15), 25.1 (6) 26.5 (13/14) 15.0 (12). The observed retention
times for diastereomers of Tnp(s)T 2 were 37.8 and 48.0 min.
The products were identified by spiking with authentic refer-
ence samples and the characterizations were further ascertained
by LC/ESI-MS analysis. In the HPLC/MS analysis, a mixture
of acetonitrile and 5 mmol LϪ1 aqueous ammonium acetate was
used as an eluent.
(3)
Under conditions, where TnpT does not accumulate (pH 4)
or the decomposition of TnpT is slow compared to its
formation (pH 8), the first-order rate constants (kds) for the
desulfurization of 3Ј,5Ј-Tnp(s)T (route G in Scheme 2) were
calculated by eqn. (4) by bisecting kdec to the rate constants of
parallel first-order reactions on the basis of the product distri-
bution at the early stages of the reaction i.e., under conditions
where the subsequent reactions TnpT may be neglected.
[Tnp(s)T]0 denotes the initial concentration of the starting
material, [TnpT]t and [Tnp(s)T]t stand for the concentrations of
3 and 2, respectively, at moment t and kdec is the first-order rate
constant for the disappearance of 2.
(4)
Eqn. (5) was applied to obtain the rate constants, kcl, for the
competing acid-catalyzed hydrolysis and pH-independent
depyrimidination (kdp) of Tnp(s)T.
(5)
Calculation of the rate constants
The pseudo-first-order rate constants (kdec) for the decom-
position of 2 and 3 were obtained by applying the integrated
first-order rate equation to the time-dependent diminution of
the HPLC peak area of the starting material.
At pH 5–6, the first-order rate constants kdp for the depyr-
imidination of 3Ј,5Ј-TnpT (3) (route B in Scheme 1) were
calculated by eqn. (6), analogously to 3Ј,5Ј-Tnp(s)T (2).
The first-order rate constants (kds) for the desulfurization of
3Ј,5Ј-Tnp(s)T (pH 5–7, route G in Scheme 2), were obtained by
least-squares fitting to eqn. (1), where kdec is the first-order rate
constant for the disappearance of Tnp(s)T and k1 the first-order
rate constant for the disappearance of 3. [Tnp(s)T]0 stands
for the initial concentration of the starting material and [TnpT]t
for the concentration of 3 at moment t.
(6)
Eqn. (7) was applied to obtain the rate constants, kcl, for the
competing acid-catalyzed hydrolysis of TnpT.
(7)
(1)
Acknowledgements
The first-order rate constants (kdp) for the depyrimidination
of 3Ј,5Ј-Tnp(s)T (pH 6–8, route F in Scheme 2), were obtained
Financial Support from the Turku University Foundation is
gratefully acknowledged.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 3 – 6 0 0
599

View Article Online
may possibly also degrade by some other routes to yield small
amounts of free thymine and all the possible reactions cannot be
distingushed on the basis of the present data.
References
1 (a) E. Uhlmann and A. Peyman, Chem. Rev., 1990, 90, 543; (b)
P. D. Cook, in Annual Reports in Medicinal Chemistry, J. A. Bristol,
Ed., Academic Press, San Diego 1998, pp. 313–325; (c) S. T. Crooke,
in Antisense Research and Application, S. T. Crooke, Ed., Springer,
Berlin 1998, pp. 1–50; (d ) C. F. Bennett, Expert Opin. Invest. Drugs,
1999, 8, 237; (e) M. Manoharan, Antisense Nucleic Acid Drug Dev.,
2002, 12, 103.
2 (a) L.-K. Chen, R. G. Schultz, D. H. Lloyd and S. M. Gryaznov,
Nucleic Acids Res., 1995, 23, 2661; (b) A. Grandas, W. S. Marshall,
J. Nielsen and M. H. Caruthers, Tetrahedron Lett., 1989, 30, 543.
3 K. Pongracz and S. M. Gryaznov, Tetrahedron Lett., 1999, 40, 7661.
4 I. Kers, J. Stawinski and A. Kraszewski, Tetrahedron Lett., 1998, 39,
1219.
11 M. Ora, M. Oivanen and H. Lönnberg, J. Chem. Soc., Perkin Trans.
2, 1996, 771.
12 E. J. Sampson, J. Fedor, P. A. Benkovic and S. J. Benkovic, J. Org.
Chem., 1973, 38, 1301.
13 (a) R. F. Hudson and L. Keay, J. Chem. Soc., 1960, 1859; (b)
J. R. Cox and O. B. Ramsay, Chem. Rev., 1964, 63, 323; (c) P. S.
Taylor and F. H. Westheimer, J. Am. Chem. Soc., 1965, 87, 553;
(d ) P. Haake and T. Koizumi, J. Am. Chem. Soc., 1973, 95, 8073;
(e) K. E. DeBruin, A. C. Padilla and D. M. Johnson, Tetrahedron
Lett., 1971, 4279.
14 J. D. Chanley and E. Feageson, J. Am. Chem. Soc., 1963, 85,
1181.
5 M. Ora, K. Mattila, T. Lönnberg, M. Oivanen and H. Lönnberg,
J. Am. Chem. Soc., 2002, 124, 14364.
6 S. M. Gryaznov and J.-K. Chen, J. Am. Chem. Soc., 1994, 116, 3143.
7 S. M. Gryaznov and N. I. Sokolova, Tetrahedron Lett., 1990, 31,
3205.
15 L. Öney and M. Caplow, J. Am. Chem. Soc., 1967, 89, 6972.
16 S. J. Benkovic and E. J. Sampson, J. Am. Chem. Soc., 1971, 93, 4009.
17 D. C. Dittmer and O. B. Ramsay, J. Org. Chem., 1963, 28, 1268.
18 Ionization constants of Inorganic Acids and Bases in Aqueous
Solution. IUPAC Chemical Data Series, No. 29, ed. D. D. Perrin,
2nd edn., Pergamon, Oxford, 1982.
8 W. Bannwarth, Helv. Chim. Acta, 1988, 71, 1517.
9 (a) M. Mag, R. Schimdt and J. W. Engels, Tetrahedron Lett., 1992,
33, 7319; (b) M. Mag and J. W. Engels, Tetrahedron, 1994, 50, 10225.
10 For comparison, the ribonucleoside analog 3Ј-deoxyuridine
3Ј-N-phosphoramidate (3Ј-Unp) was shown5 to be hydrolytically
unstable, being readily dephosphorylated to yield 3Ј-aminouridine.
The dephosphorylation involving the P–N bond cleavage was
estimated5 to be as much as 5 orders of magnitude as fast as the
corresponding dephosphorylation involving a P–O bond cleavage.
The rate constant for the dephosphorylation of 3Ј-Unp could be
determined only for alkaline conditions, the first order rate constant
being 8.1 × 10Ϫ5 sϪ1 at pH 10.4 and 363.2 K (I = 0.1 mol LϪ1).5 The
dephosphorylation of 3Ј-Unp proceeds by a dissociative mechanism
without participation of the 2Ј-OH group. Accordingly, we propose
by analogy that thymidine 3Ј-N-phosphoramidate 11 would be
readily dephosphorylated to 3Ј-aminothymidine 4, if formed during
the hydrolysis of 3 at pH 8. However, it must be noted that 10
19 P. Järvinen, M. Oivanen and H. Lönnberg, J. Org. Chem., 1991, 56,
5396.
20 R. Shapiro and S. Kang, Biochemistry, 1969, 8, 1806.
21 (a) J. Cadet and R. Teoule, J. Am. Chem. Soc., 1974, 96, 6517;
(b) M. Oivanen, M. Rajamäki, M. Varila, J. Hovinen, S. Mikhailov
and H. Lönnberg, J. Chem. Soc., Perkin Trans. 2, 1994, 309.
22 R. Shapiro and M. Danzig, Biochemistry, 1972, 11, 23.
23 M. Mag, R. Schmidt and J. W. Engels, Tetrahedron Lett., 1992, 33,
7319.
24 W. S. Mungall, G. L. Greene, G. A. Heavner and R. B. Letsinger,
J. Org. Chem., 1975, 40, 1659.
25 J. Stawinski, M. Thelin, E. Westman and R. Zain, J. Org. Chem.,
1990, 55, 3503.
26 E. P. Serjeant and B. Dempsey, Ionization Constants of Organic
Acids in Aqueous Solution. IUPAC Chemical Data Series, No 23;
Pergamon: Oxford, 1979.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 5 9 3 – 6 0 0
600