The synthesis, conformation and hydrolytic stability of an: N, S -bridging thiophosphoramidate analogue of thymidylyl-3′,5′-thymidine

Article abstract of DOI:10.1039/c6ob01270a

A 3′-N,5′-S-bridging thiophosphoramidate analogue of thymidylyl-3′,5′-thymidine was synthesised under aqueous conditions. ¹H NMR conformational measurements show that the 3′-N-substituted deoxyribose ring is biased towards the 'north', RNA-like conformation. Rate constants for hydrolysis of the analogue were measured at 90 °C in the pH range 1.3-10.9. The pH-log k_obs profile displays a pH-independent region between approximately pH 7 and 10 (t_1/2 ～13 days). Under acidic conditions, k_obs displays a first order dependence on [H₃O⁺].

Full text of DOI:10.1039/c6ob01270a

View Article Online
View Journal
Organic &
Biomol ec ular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: L. P. Conway, S.
Mikkola, A. C. O'Donoghue and D. R. W. Hodgson, Org. Biomol. Chem., 2016, DOI:
1
0.1039/C6OB01270A.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/obc

Page 1 of 8
Organic & Biomolecular Chemistry
Journal Name
RSCPublishing
View Article Online
DOI: 10.1039/C6OB01270A
ARTICLEꢀ
The Synthesis, Conformation and Hydrolytic
Stability of an N,S-bridging
Thiophosphoramidate Analogue of Thymidylyl-
Cite this: DOI:
1
0.1039/x0xx00000x
3
ʹ,5ʹ-Thymidine
a
b
a
Received 00th January 2012,
Accepted 00th January 2012
Louis P. Conway, Satu Mikkola, AnnMarie C. O’Donoghue and David R. W.
Hodgson*
a
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
3'-N,5'-S-bridging thiophosphoramidate analogue of thymidylyl-3ʹ,5ʹ-thymidine was
1
synthesised under aqueous conditions. H NMR conformational measurements show that the
'-N-substituted deoxyribose ring is biased towards the 'north', RNA-like conformation. Rate
constants for hydrolysis of the analogue were measured at 90 °C in the pH range 1.3-10.9. The
pH-log kobs profile displays a pH-independent region between approximately pH 7 and 10 (t
3
½
+
~
3
13 days). Under acidic conditions, kobs displays a first order dependence on [H O ].
AꢀIntroductionꢀ
3'-amino-substituted
phosphodiester nucleosides.
oligonucleotides
and
5'-activated
24, 25
Phosphate analogues, where oxygen atoms have been replaced
by nitrogen or sulfur, are important mechanistic enzymology
1
probes. These heteroatom substitutions serve to alter the
We now demonstrate the application of aqueous N-
thiophosphorylation plus S-alkylation towards the ligation of
two nucleosides to afford thiophosphoramidate analogue
TnpsT 1 of the dinucleotide, TpT 2 (Fig. 1). We study its
conformational preference and hydrolytic stability across a
broad pH range, with a view towards applying our aqueous
strategies in nucleic acid ligations and bioconjugations.
electrophilicity at P, change leaving group properties, and the
interactions between these atoms and metal ions at the active
sites of enzymes and ribozymes. Sulfur substitutions, in
particular, offer the ability to study cation binding through
metal ion rescue experiments, and the use of phosphorothiolates
has proven the existence of general acid-base catalysed
1
-5
cleavage of phosphodiester bonds in ribozymes.
Oligonucleotides containing modified phosphate groups show
increased resistance towards (ribo)nucleases and enhanced
6-8
therapeutic effects.
potential for modulating the structural properties of nucleic acid
Modified phosphates also offer the
9
assemblies such as the i-motif.
Substitution of the O-heteroatoms of phosphoryl groups has
also been used to facilitate phosphorylation and nucleic acid
ligation technologies. To detect single nucleotide
polymorphisms and facilitate signal amplification, Kool
employed the nucleophilicity of sulfur anions to allow
8,
10-17
templated ligation through S-alkylation.
We have
exploited the nucleophilicity of amines to facilitate the efficient Fig.ꢀ 1ꢀ 3ʹ-Amino-3ʹ-deoxythymidylyl-(3ʹ→5ʹ)-5ʹ-deoxy-5ʹ-thiothymidineꢀ (TnpsTꢀ 1)ꢀ
18-20
andꢀitsꢀnaturalꢀcounterpart,ꢀthymidylyl-(3ʹ→5ʹ)-thymidineꢀ(TpTꢀ2)ꢀ
aqueous N-phosphorylation of amines,
and N-
thiophosphorylation
thiophosphoramidates.
harnessed to promote enzyme-free templated-ligation between
plus
S-alkylation
of
Amine nucleophilicity has also been
21-23
BꢀSynthesisꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ2013ꢀ
J.ꢀName.,ꢀ2013,ꢀ00,ꢀ1-3ꢀ|ꢀ1ꢀ

Organic & Biomolecular Chemistry
Page 2 of 8
JournalꢀNameꢀ
ARTICLEꢀ
30
3
ʹ-Amino-3ʹ-deoxythymidylyl-(3ʹ→5ʹ)-5ʹ-deoxy-5ʹ-
Beevers et al. using similar methods. Rinkel and Altona found
thiothymidine (TnpsT, 1) was chosen as a target because the sum of the J1ʹ-2ʹ and J1ʹ-2ʹʹ coupling constants, ΣH1ʹ, to be
starting materials for our proposed synthetic route (Scheme 1) linearly correlated with the proportion of the south conformer
View Article Online
DOI: 10.1039/C6OB01270A
3ʹ-amino-3ʹ- through Equation 1, where PS is the proportion of the ‘south’
were
deoxythymidine 3 was accessed as a hydrochloride salt via the conformer:
readily
accessible.
The
amine,
reduction of 3ʹ-azido-3ʹ-deoxythymidine (AZT). 5ʹ-Deoxy-5ʹ-
iodothymidine 5 was prepared by 5ʹ-tosylation of thymidine,
!
26
!!! !!.!
ꢀ! =
Equationꢀ1ꢀ
!
.!
followed by conversion to the iodide using sodium iodide in
acetone.
1
Many of the H NMR signals of TnpsT 1 are coincident, and
second-order couplings complicate the extraction of J values of
the deoxyribose protons. The signals for both C1ʹ protons,
however, are well separated, and the J1ʹ-2ʹ and J1ʹ-2ʹʹ coupling
values were 7.5 and 4.0 Hz, respectively for the 3ʹ-amino-3ʹ-
deoxythymidine (Tnp) residue of 1. The C1ʹ-H signal at 6.28
ppm for the 5ʹ-deoxy-5ʹ-thiothymidine fragment (psT) presents
as an apparent triplet with a coupling constant of 6.7 Hz (Fig.
SCHEME 1 is at the end of this document
Schemeꢀ1ꢀThiophosphorylationꢀofꢀ3ʹ-amino-3ʹ-deoxythymidineꢀ3ꢀandꢀsubsequentꢀ
reactionꢀwithꢀ5ʹ-deoxy-5ʹ-iodothymidineꢀ5.ꢀ
3
ʹ-Amino-3ʹ-deoxythymidine 3 was N-thiophosphorylated using
22
our pH-optimised protocol, where an aqueous solution of
hydrochloride salt 3.HCl was maintained at pH 12 using
KOH(aq) from an autotitrator during the addition of 1.0 equiv. of
PSCl3 dissolved in MeCN. This afforded crude
3
).
31
thiophosphoramidate 4 (approximately 65% conversion by
NMR spectroscopy).
P
The alkylating agent, 5ʹ-deoxy-5ʹ-iodothymidine 5, showed
poor solubility at neutral pH, however, deprotonation of the
thymine base (pKa ~ 10) increased solubility markedly. Thus
pH 12 was maintained throughout the addition of 5ʹ-deoxy-5ʹ-
iodothymidine 5 to thiophosphoramidate 4. Under these
conditions, 98% of the crude thiophosphoramidate 4 was
31
converted to TnpsT 1 as determined by P NMR spectroscopy.
TnpsT 1 was isolated by ion exchange chromatography in 64%
yield (based on starting amine 3) as a triethylammonium salt,
Fig.ꢀ3ꢀTheꢀsignalsꢀcorrespondingꢀtoꢀtheꢀC1ʹꢀprotonsꢀinꢀTnpsTꢀ1.ꢀTheꢀsignalꢀatꢀ6.27ꢀ
ppmꢀcorrespondsꢀtoꢀtheꢀ5ʹ-linkedꢀnucleosideꢀ(psT),ꢀwhileꢀtheꢀsignalꢀatꢀ6.19ꢀppmꢀ
+
which was converted to the
conformational and kinetic studies.
K
salt for subsequent correspondsꢀtoꢀtheꢀ3ʹ-linkedꢀnucleosideꢀ(Tnp)ꢀ
Application of Equation 1 yields values of 29% south for the
Tnp ribose ring, and 61% south for the psT ribose ring.
CꢀConformationalꢀAnalysisꢀofꢀTnpsTꢀ1ꢀ
30
Comparison with TpT 2 (Tp: 74.2% south; pT: 62.7% south)
Modified (deoxy)riboses display perturbed north/south
indicates that the substitution of nitrogen for oxygen brings
about a greater population of the ‘north’, C3ʹ-endo, or ‘RNA-
like’ conformer in the Tnp fragment, while the psT ribose ring
retains its ‘DNA-like’ conformation. This is not surprising, as
the conformation of the furanose ring is largely dictated by the
anomeric and gauche effects, where the lower electronegativity
of C3ʹ-nitrogen compared to oxygen reduces the gauche effect
of donation from the C2ʹ-H bonding orbital into the C3ʹ-N/O
antibonding orbital and thus disfavours the south conformation.
The thiophosphoramidate group is not linked directly to the
ribose ring of the psT fragment and so has no apparent
influence on its conformation. The Tnp conformational change
is similar to that observed by Beevers et al. in the
27
conformational preferences (Fig. 2). Given our long-term
intention to employ our synthetic approach in nucleic acid
templated ligations, that would generate ligated products
containing a thiophosphoramidate motif, we sought to gauge
the conformational preference of TnpsT 1 using NMR
methods.
3
0
dideoxynucleoside 3ʹ-phosphorothiolate analogue (TspT),
31
however, a more detailed analysis, particularly in the context
of extended and double-stranded nucleic acid structures will be
required to confirm this result.
Fig. 2 The north (N) and south (S) conformations of a deoxyribose ring.
The elucidation of solution-state ribose conformations by
1
means of H NMR J-coupling values was pioneered by Altona
DꢀKineticsꢀandꢀMechanismꢀofꢀTnpsTꢀHydrolysisꢀ
28, 29
and Sundaralingam, and the effect on the conformation of
thiophosphate-containing dinucleosides was investigated by
Kinetic Experiments
2
ꢀ|ꢀJ.ꢀName.,ꢀ2012,ꢀ00,ꢀ1-3ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ2012ꢀ

Page 3 of 8
JournalꢀNameꢀ
Organic & Biomolecular Chemistry
ARTICLEꢀ
Fig.ꢀ4ꢀTheꢀpH-log(kobs)ꢀprofilesꢀforꢀtheꢀhydrolysesꢀatꢀ90ꢀ°Cꢀof:ꢀTnpsTꢀ1;ꢀandꢀrelatedꢀ
Hydrolysis experiments on TnpsT 1 were carried out at 90 °C
in buffered aqueous solutions with pHs ranging from 1.32 to
3
systemsꢀTnp(s)Tꢀ6ꢀandꢀTnpTꢀ7ꢀstudiedꢀbyꢀOraꢀetꢀal. ꢀKineticꢀdataꢀareꢀfittedꢀtoꢀ
2
equationꢀ2.ꢀ
View Article Online
1
0.91, at intervals of approximately one pH unit (pH values
DOI: 10.1039/C6OB01270A
calculated at 90 °C based on values measured at 25 °C, see
ESI). Aliquots of substrate 1 in each buffered solution were ꢀ!"#
sealed into vials, and heated at 90 °C. Samples were removed
from the heating bath or block at suitable intervals, the
remaining starting material and products were resolved by
HPLC, and the ratios of the integrals of the substrate and
hydrolysis products relative to an internal standard were
!"!!" !! !
!!
!
!"
=
Equationꢀ2ꢀ
!
"!!"!!!"
SCHEME 2 is at the end of this document
Schemeꢀ2ꢀpH-dependentꢀspeciationꢀofꢀTnpsTꢀ1.ꢀ
assessed.
At each pH, separate experiments were performed, using 10
Data for the disappearance of TnpsT 1 were found to fit
Equation 2, where rate coefficients k0 and kH represent the
contributions to kobs of the neutral/zwitterionic forms 1(neutral)
and 100 mM buffer concentrations in order to check for buffer-
promoted hydrolysis pathways. Acetate and formate buffered-
experiments afforded rate constants that appeared to be
dependent on buffer concentration. Thus, additional
experiments were performed using 40 and 70 mM buffers, and
kobs-buffer concentration plots were extrapolated to estimate the
buffer-dependent and buffer-independent rate constants (see
ESI).
+
and monocationic form 1H respectively (Scheme 2). The acid
+
dissociation constant between 1H and the kinetically
indistinguishable neutral forms 1(neutral) is captured in Ka1. A
single data point at pH 10.91 suggests a potential downward
trend in reactivity towards higher pHs. This may be associated
with nucleobase ionisation (Ka2) of 1(neutral), however, there
are insufficient data to substantiate this hypothesis.
The log kobs-pH profile for the hydrolysis of TnpsT 1
displayed a pH-independent ‘plateau’ from ~pH 7 to 10 with t½
-7 -1
The reactivity on the pH plateau, with k0 = 6.3 × 10 s , is
similar to the related analogues Tnp(s)T 6 and TnpT 7 that
32
were studied by Ora et al. (blue and green traces in Fig. 4).
~
13 days, while at pH 1.32 the half-life was nine seconds (red
trace in Fig. 4). The rapidity of reaction at lower pH values put
practical limits on our ability to further explore this region. At
the high pH extreme, the appearance of insoluble materials in
the reaction mixture suggested etching of the glass vials, which
precluded the straightforward exploration of the higher pH
extreme within the format of our experimental design.
The lack of an observed plateau in reactivity at lower pH values
limits our ability to unequivocally assign values to the
interdependent variables kH and Ka1. Based on the fitting of the
–1 -1
available data, however, values of kH < 0.15 M s and pKa1 < 1
were estimated, which align with those observed for TnpT 7.
SCHEME 3 is at the end of this document
Schemeꢀ3ꢀMechanisticꢀpathwaysꢀforꢀtheꢀhydrolysisꢀofꢀTnpsTꢀ1.ꢀ
The similarity in reactivity profiles of TnpsT 1 and TnpT 7
suggests that mechanisms are likely to be similar. This is borne
out in product analysis studies, and illustrative examples are
discussed below (Scheme 3).
At pH 7.0 and 7.7 the largest detected peak by HPLC is
thymine 8, derived from initial depyrimidinylation (route A) of
either thymidine site within TnpsT
fragmentation of the resulting species, as seen by Ora et al. for
1 and subsequent
32
Tnp(s)T 6 and TnpT 7. Given the remoteness of the
phosphoryl-sites from the C1ʹ sites where de-
pyrimidininylations occur, it is unsurprising that the reactivities
of TnpsT 1, Tnp(s)T 6 and TnpT 7 in the pH independent
regions are similar. At pH 6, some depyrimidinylation is
observed, however, P-N cleavage is now evident, with amine 3
being observed at a similar retention time to thymine 8 (routes
A and B). Another product appears at a much longer retention
time, with a lag in its formation. We believe this to be disulfide
1
to be relatively facile at this pH, and has been reported in a
1 formed from thiol 10 through oxidation, which is expected
33
similar system. We were, however, unable to confirm this by
HPLC in a MS-compatible buffer system. Thiol 10 is formed
by rapid dephosphorylation of phosphorothiolate 9, which
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ2012ꢀ
J.ꢀName.,ꢀ2012,ꢀ00,ꢀ1-3ꢀ|ꢀ3ꢀ

Organic & Biomolecular Chemistry
Page 4 of 8
JournalꢀNameꢀ
ARTICLEꢀ
arises from acid promoted P-N scission. At pH 3.2, the product dissolved in pyridine (20 ml) in a round-bottomed flask, and
chromatograms are simpler, displaying only two major peaks. placed in a water-ice bath. 4-Toluenesulfonyl chloVrieiwdeArt(ic3le.8O3nlinge,
Amine 3 represents one of these peaks, derived from P-N 20.2 mmol), dissolved in pyridine (20 ml) was added dropwise
DOI: 10.1039/C6OB01270A
scission, whereas the second peak is consistent with thiol 10, over 10 min. Stirring was continued for a further 24 h. The
which is formed rapidly from phosphorothiolate 9 (route B). solution was then poured into ice water (100 ml) and the
Thiol 10 is expected to be relatively stable towards oxidation mixture was extracted with ethyl acetate (2 × 60 ml). The
under these conditions. The overlap of the pH-log kobs profiles organic extracts were washed with saturated sodium
of TnpsT 1 and TnpT 7 in the acidic region suggests that either bicarbonate solution (40 ml), and water (40 ml) before being
the values of kH and Ka1 are identical for these species, or that dried over MgSO4. The solvents were then removed under
ionisation and reactivity compensate each other to arrive at reduced pressure, and the residue was recrystallised from water
identical kobs values.
to give the tosylated nucleoside, 5ʹ-deoxy-5ʹ- (4-
toluenesulfonyl)thymidine (1.57 g, 24%). δH (400 MHz,
(CD3)2SO) 1.76 (3H, s, C5-CH3) 2.02-2.09 (1H, m, C2ʹ-HaHb)
EꢀConclusionsꢀ
2
.11-2.19 (1H, m, C2ʹ-HaHb) 2.41 (3H, s, CH3-Ar) 3.83-3.88
TnpsT 1, which is an analogue of thymidyl-3ʹ,5ʹ-thymidine 2,
was successfully synthesised under aqueous conditions, without
(
(
1H, m, C3ʹ-H), 4.12-4.20 (2H, m, C4ʹ-H and C5ʹ-HaHb) 4.25
1H, dd, J 7.2, 3.4, C5ʹ-HaHb) 5.44 (1H, d, J 4.4, OH) 6.14 (1H,
protecting groups. NMR-based analyses revealed
a
app. t, J 7.2, C1ʹ-H) 7.38 (1H, d, J 1.8, C6-H) 7.47 (2H, d, J
.3, m-OSO2Ph) 7.79 (2H, d, J 8.3, o-OSO2Ph) 11.33 (1H, s,
NH) δC (400 MHz, (CD3)2SO) 12.1, 21.1, 38.3, 69.9, 70.1, 83.1,
predominantly ‘north’, ‘RNA-like’ C3ʹ-endo conformational
preference for the 3'-aza-substituted deoxyribose (Tnp)
fragment of TnpsT 1. Hydrolytic studies on TnpsT 1 yielded a
near-identical profile to non-thio-analogue TnpT 7, where for
pH>7, de-pyrimidinylation dominates, and P-N scission is
dominant for lower pHs. The combination of simple aqueous
synthesis, knowledge of conformational preference and stability
of the linkage will allow us to exploit N,S-bridging nucleotide
systems in future applications.
8
8
4.0, 109.8, 127.6, 130.2, 132.1,135.9, 145.1, 150.3, 163.6.
ʹ-Deoxy-5ʹ-iodothymidine 5.
ʹ-Deoxy-5ʹ-tosylthymidine (1.57 g, 3.96 mmol) and sodium
5
ꢀ
5
iodide (2.97 g, 19.8 mmol) were placed in a round-bottomed
ask and dissolved in the minimum volume of acetone. The
solution was heated at reflux for 24 h, before the solvent was
removed under reduced pressure. The residue was recrystallized
from water to yield the product (1.20 g, 86%). δH (400 MHz,
FꢀExperimentalꢀ
(
CD3)2SO) 1.79 (3H, s, C5-CH3) 2.07 (1H, ddd, J 13.5, 6.2, 3.1,
C2ʹ-HaHb) 2.29 (1H, ddd, J 13.5, 8.1, 6.2, C2ʹ-HaHb) 3.39 (1H,
dd, J 10.4, 6.3, C5ʹ-HaHb) 3.52 (1H, dd, J 10.4, 6.3, C5ʹ-HaHb)
Synthesis
3
ʹ-Amino-3ʹ-deoxythymidine 3, hydrochloride salt. 3.80 (1H, dt, J 6.2, 2.8, C3ʹ-H), 4.15-4.21 (1H, m, C4ʹ-H), 5.49
Adapting a literature procedure, 3ʹ-azido-3ʹ-deoxythymidine (1H, d, J 4.3, OH) 6.22 (1H, dd, J 8.0, 6.2, C1ʹ-H) 7.53 (1H, d,
1.00 g, 3.74 mmol) and triphenylphosphine (1.54 g, 5.87 J 1.5, C6-H) 11.35 (1H, s, NH)
mmol) were dissolved in pyridine (8 ml) and the mixture was
3ʹ-Amino-3ʹ-deoxythymidylyl-(3ʹ→5ʹ)-5ʹ-deoxy-5ʹ-
stirred at room temperature for 1 h. Ammonia solution (30 ml, thiothymidine, triethylammonium salt 1.Et N H.
(
+
3
3
5%) was then added, and the mixture was stirred overnight. First 3ʹ-amino-3ʹ-deoxythymidine-N-thiophosphoramidate
4
The suspension was diluted with water (30 ml) and extracted was prepared according to the previously reported
with chloroform (3 × 30 ml) before being lyophilised. The solid phosphorylation procedure.22 3ʹ-Amino-3ʹ-deoxythymidine,
residue was dissolved in ethanol (100 ml) and hydrogen hydrochloride salt 3.HCl (139 mg, 0.500 mmol) was dissolved
chloride gas was bubbled through the solution until in water and made up to 5 ml and pH 12 with water and
precipitation was observed. The precipitate was isolated by potassium hydroxide solution (1 M). Thiophosphoryl chloride
filtration, and washed with a small quantity of diethyl ether. solution (1.50 ml, 0.333 M in MeCN) was added slowly using a
®
Additional product was obtained by adding diethyl ether (500 Hamilton microlitre syringe, with vigorous stirring, and with
ml) to the filtrate, collecting and washing the precipitate. The the tip of the syringe below the surface of the reaction mixture.
isolated solids were combined and dried under vacuum Throughout the experiment, the pH was kept constant at pH 12
overnight yielding a total of 846 mg, 81%; mp 253-255 °C using a 1.000 M solution of potassium hydroxide, added by an
–
1
(
decomp., from ethanol); νmax/cm 3392, 3032, 1694, 1644, autotitrator equipped with a pH probe. The experiment was
470; δH (400 MHz, (D2O) 1.86 (3H, s, C5-CH3), 2.54-2.70 considered to be complete when the autotitrator needed to add
2H, m, C2ʹ-H2), 3.81 (1H, dd, J 12.6, 4.6, C5ʹ-Ha), 3.89 (1H, negligible volumes of potassium hydroxide solution to the
dd, J 12.6, 3.4, C5ʹ-Hb), 4.06 (1H, dt, J 8.1, 5.5, C3ʹ-H), 4.19- reaction mixture in order to maintain a constant pH. The
1
(
4
1
8
1
.28 (1H, m, C4ʹ-H), 6.28 (1H, t, J 6.8, C1ʹ-H), 7.62 (1H, d, J lyophilised thiophosphoramidate was redissolved in water, and
.1, C6-H); δC (100 MHz, (D2O) 10.5, 33.8, 49.1, 59.7, 81.5, made up to 5 ml at pH 12 with water and potassium hydroxide
+
4.1, 110.5, 136.6, 150.5, 165.4; m/z 242.1142 ([M+H] , solution (1 M). 5ʹ-Deoxy-5ʹ-iodothymidine 5 (352 mg, 1.00
+
00%), requires 242.1141, 264.0961 ([M+Na] , 90).
mmol) was added to the solution with stirring, while the pH
was maintained at 12 with potassium hydroxide solution (1 M)
5
ʹ-Deoxy-5ʹ-(4-toluenesulfonyl)thymidine. Adapting
a
26
literature procedure, thymidine (3.92 g, 16.2 mmol) was by an autotitrator. Once the 5ʹ-deoxy-5ʹ-iodothymidine 5 had
4
ꢀ|ꢀJ.ꢀName.,ꢀ2012,ꢀ00,ꢀ1-3ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ2012ꢀ

Page 5 of 8
JournalꢀNameꢀ
Organic & Biomolecular Chemistry
ARTICLEꢀ
fully dissolved and no further addition of potassium hydroxide 137.5, 151.4, 151.4, 166.1, 166.3, 171.1; δP (162 MHz, D2O)
was required to maintain the pH, the solution was sealed to 21.6 (1P, app. q, J 10.0, HNPO2S); m/z 560.1208 ([M-K]-,
View Article Online
prevent losses owing to evaporation and heated to 50 °C. At 100%) requires 560.1216, 1121.2450 ([2M-2K+H]-).
intervals, aliquots were removed from the reaction vessel and
ꢀ
DOI: 10.1039/C6OB01270A
HPLC standard - potassium p-nitrobenzenesulfonate. p-
31
analysed by P NMR spectroscopy. The S-alkylation process Nitrobenzenesulfonyl chloride (222 mg, 1.00 mmol) was placed
was monitored based on the disappearance of the signal for in a flask with potassium hydroxide solution (2 ml, 1.000 M)
thiophosphoramidate 4 at δ ~44 ppm, and the appearance of a and water (20 ml). The solution was heated at reflux for 3 h
signal for TnpsT 1 at δ ~22 ppm. The reaction was continued before being lyophilised to yield a mixture of the desired
until the unalkylated thiophosphoramidate starting material had product and potassium chloride in a 1:1 molar ratio. (231 mg,
been completely consumed; the solution was then allowed to 98%) δH (400 MHz, (D2O) 8.00 (2H, d, J 8.9, 1H m- to
cool to room temperature. The pH of the reaction mixture was sulfonate), 8.35 (2H, d, J 8.9, 1H o- to sulfonate); δC (101 MHz,
measured and found to be 10.45. The solution was lyophilised (D2O) 124.3, 126.9, 148.1, 149.1.
and redissolved in 1 M TEAB buffer, then purified by anion
exchange chromatography with a flow rate of 5 ml/min over a
Hydrolysis studies
®
DEAE-Sepharose resin. Triethylammonium bicarbonate See ESI for details of the hydrolysis studies.
buffer was applied in a 0 to 0.15 M gradient over 3 h. A second
chromatographic purification using the same method was GꢀAcknowledgementsꢀ
required to remove all impurities to yield 3ʹ-amino-3ʹ-
This work was supported by EPSRC DTA funding to LPC
(EP/P505488/1) and the Royal Society (chromatography
system).
deoxythymidylyl-(3ʹ→5ʹ-)-5ʹ-deoxy-5ʹ-thiothymidine as its
+
triethylammonium salt 1.Et3N H (212 mg, 64%). δH (700 MHz,
+
D2O) 1.29 (9H, t, J 7.3, HN (CH2CH3)3) 1.88 (3H, d, J 1.2, A-
or B- C5CH3) 1.89 (3H, d, J 1.2, A- or B- C5CH3), 2.36-2.45
(
HꢀNotesꢀandꢀreferences
ꢀ
3H, m, A-C2ʹHa and B-C2ʹH2) 2.49 (1H, ddd, J 14.0, 8.1, 3.9,
A-C2ʹHb), 3.04-3.11 (2H, m, B-C5ʹH2), 3.21 (6H, q, J 7.0,
+
HN (CH2CH3)3), 3.78-3.85 (2H, m, A-C3ʹH and A-C5ʹHa)
a
Department of Chemistry, Durham University, South Road, Durham,
DH1 3LE, UK.
b
Department of Chemistry, University of Turku, Vatselankatu 2, 20014
3
.86-3.89 (1H, m, A-C5ʹHb), 4.14-4.20 (2H, m, A- and B-
C4ʹH), 4.46-4.49 (1H, m, B-C3ʹH), 6.16 (1H, dd, J 7.4, 4.0, A-
C1ʹH), 6.25 (1H, app. t, J 6.7, B-C1ʹH), 7.67 (1H, d, J 1.2, A-
or B-C6H), 7.71 (1H, d, J 1.2, A- or B-C6H); δC (151 MHz,
Turku, Finland.
1
Electronic Supplementary Information (ESI) available: [ H, C and,
13
31
where applicable, P NMR spectra of synthetic intermediates and
+
D2O) 8.2 (HN (CH2CH3)3), 11.5 (A- and B-C5CH3), 32.0, (B-
+
C5ʹ), 38.0 (B-C2ʹ), 38.5 (A-C2ʹ), 46.6 (HN (CH2CH3)3), 58.9
analogue 1, HPLC calibration data, pH-temperature corrections, further
details of individual kinetics experiments, example chromatograms, and
tabulated HPLC data]. See DOI: 10.1039/b000000x/
(
A-C3ʹ), 60.2, (A-C5ʹ), 72.0 (B-C3ʹ), 84.5 (A-C1ʹ), 84.7 (B-
C1ʹ), 84.9 (B-C4ʹ), 85.5 (A-C4ʹ), 111.1 (A- or B-C5), 111.3 (A-
or B-C5), 137.4 (A- or B-C6), 137.5 (A- or B-C6), 151.4 (A-
and B-C2), 166.1 (A- or B-C4), 166.3 (A- or B-C4); δP (162
MHz, D2O) 21.6 (1P, app. q, J 9.7, HNPO2S); m/z 560.1207
1
2
3
4
H. J. Korhonen, L. P. Conway and D. R. W. Hodgson, Curr. Opin.
Chem. Biol., 2014, 21, 63.
N. S. Li, J. K. Frederiksen and J. A. Piccirilli, Accounts Chem. Res.,
2
011, 44, 1257.
+
-
(
[M-HN Et3] , 100%) requires 560.1216, 279.5541 ([M-
+
T. J. Wilson, N. S. Li, J. Lu, J. K. Frederiksen, J. A. Piccirilli and D. M.
J. Lilley, Proc. Natl Acad. Sci. USA, 2010, 107, 11751.
J. E. Deweese, A. B. Burgin and N. Osheroff, Nucleic Acids Res., 2008,
2–
H2N Et3] ).
ʹ-Amino-3ʹ-deoxythymidylyl-(3ʹ→5ʹ)- 5ʹ-deoxy-5ʹ-
3
3
6, 4883.
+
thiothymidine, potassium salt 1.K SP-Sepharose® resin was
exchanged with potassium ions by passing a solution of
potassium chloride (100 mM) over it. The triethylammonium
salt of the dinucleoside (100 mg) was then dissolved in water (4
ml) and passed over the resin. The UV-active fractions were
collected and lyophilised to yield the dinucleoside as its
potassium salt (60 mg, 66%) δH (700 MHz, D2O) 1.88 (3H, d, J
5
6
K. H. Jung and A. Marx, Cell Mol. Life Sci., 2005, 62, 2080.
R. O. Bak, A. Hendel, J. T. Clark, A. B. Kennedy, D. E. Ryan, S. Roy, I.
Steinfeld, B. D. Lunstad, R. J. Kaiser, A. B. Wilkens, R. Bacchetta, A.
Tsalenko, D. Dellinger, L. Bruhn and M. H. Porteus, Hum. Gene Ther.,
2
015, 26, A11.
7
A. Hendel, R. O. Bak, J. T. Clark, A. B. Kennedy, D. E. Ryan, S. Roy, I.
Steinfeld, B. D. Lunstad, R. J. Kaiser, A. B. Wilkens, R. Bacchetta, A.
Tsalenko, D. Dellinger, L. Bruhn and M. H. Porteus, Nat. Biotechnol.,
2
015, 33, 985.
1
2
8
.2, A- or B- C5CH3) 1.89 (3H, d, J 1.2, A- or B- C5CH3),
.35-2.46 (3H, m, A-C2ʹHa and B-C2ʹH2) 2.49 (1H, ddd, J 14.0,
.1, 4.0, A-C2ʹHb), 3.02-3.11 (2H, m, B-C5ʹH2), 3.78-3.85 (2H,
8
9
G. Zon, New J. Chem., 2010, 34, 795.
J. A. Brazier, J. Fisher and R. Cosstick, Angew. Chem. Int. Edit., 2006,
4
5, 114.
1
1
0
1
M. K. Herrlein and R. L. Letsinger, Nucleic Acids Res., 1994, 22, 5076.
Y. Xu and E. T. Kool, Nucleic Acids Res., 1999, 27, 875.
m, A-C3ʹH and A-C5ʹHa) 3.88 (1H, ddd, J 7.3, 4.5, 2.5, A-
C4ʹH), 3.94 (1H, dd, J 12.6, 2.5, A-C5ʹHb), 4.18 (1H, app. q, J 12 S. Sando and E. T. Kool, J. Am. Chem. Soc., 2002, 124, 2096.
1
1
1
3
4
5
H. Abe and E. T. Kool, J. Am. Chem. Soc., 2004, 126, 13980.
S. Sando and E. T. Kool, J. Am. Chem. Soc., 2002, 124, 9686.
S. Sando, H. Abe and E. T. Kool, J. Am. Chem. Soc., 2004, 126, 1081.
5.1, B-C4ʹH), 4.48 (1H, app. dt, J 6.5, 4.6, B-C3ʹH), 6.17 (1H,
dd, J 7.5, 4.0, A-C1ʹH), 6.25 (1H, app. t, J 6.7, B-C1ʹH), 7.68
(
δC (151 MHz, D2O) 11.5, 11.5, 32.0, 38.0, 38.5, 50.4, 60.2,
1H, d, J 1.4, A- or B-C6H), 7.71 (1H, d, J 1.2, A- or BC6H); 16 E. M. Harcourt and E. T. Kool, Nucleic Acids Res., 2012, 40, e65.
1
7
J. Michaelis, A. Roloff and O. Seitz, Org. Biomol. Chem., 2014, 12,
2
821.
7
2.0, 84.5, 84.7, 84.8, 84.8, 85.5, 85.6, 111.1, 111.3, 137.4,
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ2012ꢀ
J.ꢀName.,ꢀ2012,ꢀ00,ꢀ1-3ꢀ|ꢀ5ꢀ

Organic & Biomolecular Chemistry
Page 6 of 8
JournalꢀNameꢀ
ARTICLEꢀ
1
1
2
8
9
0
R. J. Delley, A. C. O'Donoghue and D. R. W. Hodgson, J. Org. Chem., 27 W. Saenger, Principles of Nucleic Acid Structure, Springer-Verlag, New
012, 77, 5829. York, 1984.
D. Williamson and D. R. W. Hodgson, Org. Biomol. Chem., 2008, 6, 28 C. Altona and M. Sundaralingam, J. Am. Chem. Soc., 1972, 94, 8205.
2
View Article Online
1
056.
29 C. Altona and M. Sundaralingam, J. Am. Che mD .OSI o: c1 .0, . 11 09 73 39 ,/ C9 56 ,O 2B3 03 13 2.70A
D. Williamson, M. J. Cann and D. R. W. Hodgson, Chem. Commun., 30 A. P. G. Beevers, E. M. Witch, B. C. N. M. Jones, R. Cosstick, J. R. P.
2
007, DOI: 10.1039/b717896d, 5096.
Arnold and J. Fisher, Magn. Reson. Chem., 1999, 37, 814.
31 J. Jakhlal, S. Coantic-Castex, C. Denhez, C. Petermann, A. Martinez, D.
Harakat, D. Guillaume and P. Clivio, in Chem. Commun., 2015, vol. 51,
pp. 12381.
2
2
1
2
M. Trmcic and D. R. W. Hodgson, Chem. Commun., 2011, 47, 6156.
L. P. Conway, R. J. Delley, J. Neville, G. R. Freeman, H. J. Maple, V.
Chan, A. J. Hall and D. R. W. Hodgson, RSC Adv., 2014, 4, 38663.
2
2
3
4
M. Trmcic, F. L. Chadbourne, P. M. Brear, P. W. Denny, S. L. Cobb and 32 M. Ora, M. Murtola, S. Aho and M. Oivanen, Org. Biomol. Chem., 2004,
D. R. W. Hodgson, Org. Biomol. Chem., 2013, 11, 2660. 2, 593.
M. Rothlingshofer, E. Kervio, T. Lommel, U. Plutowski, A. Hochgesand 33 J. B. Thomson, B. K. Patel, V. Jimenez, K. Eckart and F. Eckstein, J.
and C. Richert, Angew. Chem. Int. Edit., 2008, 47, 6065.
A. Kaiser and C. Richert, J. Org. Chem., 2013, 78, 793.
D. M. Williams and V. H. Harris, Practical Approach in Chemistry:
Organophosphorus Reagents, OUP, 1st ed. edn., 2004.
Org. Chem., 1996, 61, 6273.
2
2
5
6
O
O
N
N
NH
Aqueous,
Maintained
at pH 12
O
N
HO
O
N
O
HO
O
O
O
NH
O
SPCl3
(
1 equiv. in MeCN)
NH
HO
Aqueous,
maintained
O
NH2
P
Circa 65%
O
O
conversion
S
3
at pH 12
NH
4
NH
5
0 °C, 48 h
O
P
O
S
N
O
O
N
O
N
O
pKa ~10
NH
N
OH
TnpsT 1
OH
I
O
I
O
O
O
6
4% overall
OH
OH
5
Schemeꢀ1ꢀThiophosphorylationꢀofꢀ3ʹ-amino-3ʹ-deoxythymidineꢀ3ꢀandꢀsubsequentꢀreactionꢀwithꢀ5ʹ-deoxy-5ʹ-iodothymidineꢀ5.ꢀ
O
N
O
N
O
N
O
N
NH
O
NH
O
NH
O
N
HO
O
O
HO
O
O
HO
O
O
HO
O
O
O
O
O
O
N
Ka1
Ktaut
Ka2
NH2
NH2
NH
NH
NH
NH
NH
NH
P
P
O-
P
O
P
OH
OH
OH
S
N
O
S
N
O
S
N
O
S
O
O
O
O
O
OH
H+
OH
OH
OH
1
1–
1
(neutral)
kH
k0
products
Schemeꢀ2ꢀpH-dependentꢀspeciationꢀofꢀTnpsTꢀ1.ꢀ
products
6
ꢀ|ꢀJ.ꢀName.,ꢀ2012,ꢀ00,ꢀ1-3ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ2012ꢀ

Page 7 of 8
JournalꢀNameꢀ
Organic & Biomolecular Chemistry
ARTICLEꢀ
View Article Online
DOI: 10.1039/C6OB01270A
O
HO
O
HO
O
OH
T
O
O
NH
NH
NH
N
H
O
further
breakdown
P
O-
or
P
O-
HO
O
8
T
S
S
O
T
OH
O
O
route A
route B
NH
OH
OH
P
O-
de-pyrimidinylation
products (not measured)
S
T
O
S
O
O
P
O
HS
fast at
higher pH
S
OH
TnpsT 1
T
fast
T
T
O
O
O
HO
T
O
OH
OH
10
OH
2
9
11
NH2
3
Schemeꢀ3ꢀMechanisticꢀpathwaysꢀforꢀtheꢀhydrolysisꢀofꢀTnpsTꢀ1.ꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ2012ꢀ
J.ꢀName.,ꢀ2012,ꢀ00,ꢀ1-3ꢀ|ꢀ7ꢀ

Organic & Biomolecular Chemistry
Page 8 of 8
View Article Online
DOI: 10.1039/C6OB01270A
A simple, aqueous, protecting group-free synthesis of a dinucleotide is
presented, and its stability and conformation are explored.