Synthesis of nucleoside phosphosulfates

Article abstract of DOI:10.1016/j.bmcl.2012.04.039

We describe an efficient and scalable procedure for the chemical synthesis of nucleoside 5′-phosphosulfates (NPS) from nucleoside 5′-phosphorimidazolides and sulfate bis(tributylammonium) salt. Using this method we obtained various NPS with yields ranging from 70-90%, including adenosine 5′-phosphosulfate (APS) and 2′,3′-cyclic precursor of 3′-phosphoadenosine 5′-phosphosulfate (PAPS), which are the key intermediates in the assimilation and metabolism of sulfur in all living organisms.

Full text of DOI:10.1016/j.bmcl.2012.04.039

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3661–3664
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of nucleoside phosphosulfates
⇑
Joanna Kowalska, Agnieszka Osowniak, Joanna Zuberek, Jacek Jemielity
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
We describe an efficient and scalable procedure for the chemical synthesis of nucleoside 50-phosphosul-
fates (NPS) from nucleoside 50-phosphorimidazolides and sulfate bis(tributylammonium) salt. Using this
method we obtained various NPS with yields ranging from 70–90%, including adenosine 50-phosphosul-
fate (APS) and 20,30-cyclic precursor of 30-phosphoadenosine 50-phosphosulfate (PAPS), which are the key
intermediates in the assimilation and metabolism of sulfur in all living organisms.
Ó 2012 Elsevier Ltd. All rights reserved.
Article history:
Received 14 March 2012
Revised 4 April 2012
Accepted 7 April 2012
Available online 20 April 2012
Keywords:
Nucleoside 50-phosphosulfate
APS
PAPS
Chemical synthesis
Active sulfate
Adenosine 50-phosphosulfate (APS) and 30-phosphoadenosine
50-phosphosulfate (PAPS) are important intermediates involved in
metabolism of sulfur in various organisms.1 Both play a vital role
in the assimilation of sulfur in plants and bacteria.2 PAPS is the ‘ac-
tive sulfate’ form in higher organisms, including mammals. It
serves as a sulfate donor in glycosaminoglycan sulfation reactions,
which are important, for instance, for blood coagulation, as well as
in sulfation of peptides, proteins, lipids, hormones and other small
molecules.3,4
Hence, both APS and PAPS have been used for studying sul-
fotransferases and sulforeductases in various organisms.5 APS has
also found a biotechnological use in DNA pyrosequencing.6 Several
human sulfate-transferring enzymes that utilize PAPS are related to
the development of diseases such as cancer and neurodegenerative
disorders,4,7 and some have been suggested as suitable drug tar-
gets.8 The phosphosulfate derivatives of other nucleosides have
not been thoroughly investigated, nonetheless, cytidine 50-phos-
phosulfate (CPS) has been identified as a putative intermediate in
the biosynthesis of some sulfated steroles.9 Moreover, nucleoside
50-phosphosulfates (NPS) may be, in general, considered as struc-
tural analogs of nucleoside 50-diphosphates (NDP), since they
retain a similar geometry, but lack one of the phosphate’s ionizable
AOH group. Thus, they may be used for studying specificity of pyro-
phosphatases,10 or other nucleotide-binding enzymes and proteins.
NPS are relatively unstable (easily hydrolyze to NMP and sul-
fate), and due to this, both their synthesis and purification are dif-
ficult. APS was chemically synthesized for the first time over 50
years ago by Baddiley et al. and Reichard & Ringertz,11,12 but the
isolated yields, if reported, were poor. PAPS and its 20,30-cyclic pre-
cursor have been synthesized in a similar way.13,14 50-Phosphosul-
fates of guanosine (GPS) and uridine (UPS) have been synthesized
and used to study bacterial sulforeductases, but the detailed proce-
dures have not been provided in this report.15,16
Probably, due to not very promising initial results, the chemical
syntheses ofNPS havenot been improvedover theyears, butinstead,
(chemo)enzymatic methods have been developed.17 However, these
methods are usually more costly and bear other limitations, e.g.
those arising from substrate specificity of sulfotransferases. Hence,
we assumed that a simple and economical chemical procedure for
NPS preparation could make them more accessible and encourage
their broader exploitation in biochemical and biophysical studies.
In our and others experience,18,19 metal (II) chloride (usually
ZnCl2 or MgCl2) mediated coupling of P-nucleophilic and P-electro-
philic phosphate subunits in non-aqueous polar solvent has been a
useful tool in the synthesis of nucleoside oligophosphates and their
analogs modified within phosphate bridge. Hence, we decided to
explore whether such approach can be used in reactions employing
sulfate anion as a nucleophile. As a result, in this work we present a
rapid and efficient procedure for the chemical synthesis of APS,
PAPS and other NPS, based on phosphorimidazolide chemistry.
We also provide, for the first time, 1H and 31P NMR characterization
of these compounds, together with some data on their stability.
Finally, we demonstrate an example of how NPS may be employed
as isosteric analogs of NDP for studying nucleotide-protein
interactions.
The general route for the synthesis of NPS (2a–e) is depicted in
Scheme 1. The key step was a coupling reaction of an activated
nucleoside monophosphate species, an appropriate nucleoside 50-
phosphorimidazolide (1a–e), with sulfate organic salt in DMF and
⇑
Corresponding author.
E-mail address: jacekj@biogeo.uw.edu.pl (J. Jemielity).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.039

3662
J. Kowalska et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3661–3664
Interestingly, studies on the phosphosulfate bond cleavage re-
NH
1.
N
Na+
Et3NH+
O
P
O
vealed that NPS and related compounds undergo acid-catalyzed
unimolecular elimination of sulfur trioxide, and that the reaction
may be accelerated by divalent metal cations both in aqueous
and non-aqueous media.20 Thus, we assumed that conversion of
APS into AMP in the reaction mixture was catalyzed by protons
generated upon ZnCl2 hydrolysis in the presence of moisture and
presumably also by zinc cations. Therefore, we replaced ZnCl2 with
a less acidic salt, MgCl2. Under these conditions the reaction was
comparably fast, slightly more efficient, but most importantly the
product was more stable (Fig. 1B and C). Consequently, via
MgCl2-activated coupling, 2a could be synthesized and isolated
after ion-exchange chromatography (DEAE-Sephadex) with 88%
yield.
O
P
O
2,2'-dithiodipyridine, Ph3
P
B
B
HO
O
N
Et3N, DMF
O
O
O
N
2. NaClO4, acetone
1
HO
OH
HO
OH
O
2 Bu3NH+
O
S
O
O
MgCl2, DMF
1a, 2a: B = Ade
1b, 2b: B = Gua
1c, 2c: B = Ura
O
S
O
O
P
O
B
O
O
O
O
1c, 2d: B = Cyt
2 Et3NH+
1e, 2e: B = N7-MeGua
HO
OH
2
Employing these optimized conditions we have synthesized and
isolated a set of four other nucleoside 50-phosphosulfates (com-
pounds 2b–e), differing in nucleobase size and polarity (G, U, C
and m7G, Scheme 1, Table 1). In all cases the HPLC yields were al-
most quantitative, whereas preparative yields were above 70% (Ta-
ble 1). Compounds 2a–d did not require additional purification and
after DEAE-Sephadex chromatography, were converted from tri-
ethylammonium to sodium salts by precipitation with NaClO4 ace-
Scheme 1. Synthesis of nucleoside 50-phosphosulfates (2a–2e).
in the presence of divalent metal chloride. As a model reaction, we
chose the synthesis of APS (2a). First, adenosine 50-monophosphate
(free acid) was converted into its triethylammonium salt and acti-
vated with imidazole using triphenylphosphine/dithiodipyridine
redox system to yield 1a. We utilize this method for obtaining
phosphorimidazolides, instead of the usually employed activation
with CDI, since it enables very convenient isolation of bulky
amounts of the desired product by simple precipitation with NaClO4
acetone solution, and the product can be stored in this form for sev-
eral months. As a nucleophilic reagent, we tested several organic
salts of sulfate (VI): triethylammonium, pyridinium, tributylammo-
nium and N,N-dimethyanilinium. Triethylammonium sulfate was a
hygroscopic semi-solid at RT, and thus was inconvenient for storage
and handling. Bis(tributylammonium) sulfate was initially obtained
in a similar form, but turned into an easy-to-handle solid after dry-
ing in vacuum over P2O5 for several days. N,N-dimethylanilinium
and pyridinium salts were solids, but produced strong HPLC signals
with long retention time, which complicated the analysis of the
reaction mixtures. Since all salts displayed comparable reactivity
in the following coupling reaction, we selected bis(tributylammoni-
um) sulfate as the most suitable form of organic sulfate for further
studies.
Reaction of 1a and bis(tributylammonium) sulfate (4 equiv) in
DMF in the presence of anhydrous ZnCl2 (10 equiv) was monitored
by RP HPLC and MS(-)ES in 5 min intervals. Surprisingly, the forma-
tion of APS (2a) was very rapid after 20 min the expected product
constituted ca. 80% of the reaction mixture (Fig. 1 A). Formation of
AMP and diadenosine 50,50-diphosphate were observed as side
reactions. However, when the reaction time was slightly elongated
fairly fast conversion of 2a into AMP was observed; after 35 min its
content decreased to only 40% (Fig. 1C). Since, not only APS forma-
tion, but its decomposition was relatively rapid, we reckoned that
the procedure was not fully reliable.
tone solution. Compound 2e, bearing
a positively charged
nucleoside 7-methylguanosine, likely due to its smaller negative
net charge compared to other NPS, partially co-eluted with unre-
acted sulfate from DEAE-Sephadex, and thus, required additional
purification by RP HPLC.
Structures of all final compounds have been confirmed by a
combination of high resolution MS(-)ES, 1H and 31P NMR (Table 1
and Supplementary data). The NPS have nominal masses similar
to those of corresponding NDP, but in contrast to them give only
one signal in 31P NMR. Although 32S is not NMR active, NPS could
also be easily distinguished from NMP by 31P NMR, since their sig-
nal shifted ca. 12 ppm downfield from the signal of NMP (e.g. d
ppm + 2.16 vs ꢀ9.27 for GMP and GPS, respectively). NPS purities
determined both by 31P NMR and RP HPLC were all above 90%,
which is a standard value for commercial APS.
Finally, we employed a slightly modified approach to attempt
the synthesis of PAPS starting from commercially available 30-ade-
nylic acid (30-AMP) by a route presented in Scheme 2, path A. The
crucial intermediate, 30-phosphoadenosine 50-phosphorimidazo-
lide, was attempted in a one pot three-step reaction. First, 30-AMP
was 50-phosphorylated in Yoshikawa conditions,21 then reacted
with excess imidazole and finally hydrolyzed to monoimidazolide
in mildly basic pH. However, during the first step, 30-AMP and its
50-posphorylated derivative converted into 20,30-cyclic forms. Thus,
20,30-cyclic phosphoadenosine 50-phosphorimidazolide (3) was
obtained as the actual product of the reaction sequence. The product
was isolated by DEAE Sephadex with only 50% yield, since the initial
phosphorylation step was moderately efficient (about 70% by
Figure 1. Example RP HPLC profiles from reactions of 1a (AMP-Im) with bis(tributylammonium) sulfate (4 equiv) in DMF at RT in the presence of either (A) 10 equiv ZnCl2 or
(B) 10 equiv MgCl2; (C) APS content in the same reaction mixtures as a function of time.

J. Kowalska et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3661–3664
3663
Table 1
Synthesized nucleoside 50-phosphosulfates
Entry Compound
No Abbreviation
2a APS
2b GPS
2c UPS
Reaction time
(min)
HPLC Conversion Isolated yield
HPLC Rt
(min)c
HRMS(-)ES m/z found
(Calculated)
31P NMR d
31P NMR purity
(%)a
(%)b
(ppm)d
(%)d
1
2
3
4
5
6
20
20
30
18
20
15
92
91
96
100
85
88
88
84
86
84
72
70
3.3
3.0
2.3
2.3
4.9
3.3
426.0137 (426.0126)
442.0083 (442.0075)
402.0019 (402.0014)
402.9872 (402.9854)
456.0241 (456.0232)
487.9693 (487.9684)
ꢀ9.74
ꢀ9.75
ꢀ9.73
ꢀ9.88
ꢀ10.36
95
96
92
>99
93
2d CPS
2e m7GPS
4
20,30-cPAPS
20.59; ꢀ9.93 >99
a
b
c
Determined by RP HPLC analysis of reaction mixtures before DEAE-Sephadex purification.
Determined by optical density unit measurements of the starting phosphorimidazolides (1a–e, 3) and isolated final compounds.
For final product.
Determined in D2O, at 25 °C and 162 MHz.
d
O
P
O
P
A
POCl3
A
NH
HO
A
Cl
O
O
N
O
N
(MeO)3PO
O
P
O
P
Cl
N
N
OR1
0-50C
OH
O
R2
O
O
R3
O
O
N
O
Path A: R1 = -PO3H2
Path B: R1 = H
Path A: R2 = -OH
Path B: R2 = Cl
Path A: R3 = -OH
Path B: R3 = imidazol-1-yl
TEAB (aq.) pH = 8
O
S
O
O
P
O
O
P
O
O
2 n-Bu3NH+
A
A
S
O
O
O
O
N
O
O
O
O
P
O
P
N
MgCl2, DMF
3 Et3NH+
2 Et3NH+
O
O
O
O
O
O
O
O
3
4
Scheme 2. Synthesis of 20,30-cPAPS (4).
HPLC). A reaction of 3 with bis(tributylammonium) sulfate in the
presence of MgCl2 under conditions analogous for compounds 2a–
e resulted in the formation of 20,30-cPAPS (4) as a major product
(88% HPLC conversion) together with minute amounts of PAP. After
DEAE-Sephadex and RP HPLC purification, 4 was isolated in 70%
yield. It has been reported previously that 4 can be converted into
PAPS by ribonuclease-T2 with ca. 70% yield.14 Since the 20,30-cyclicli-
sation of 30-AMP during phosphorylation was unavoidable, we sim-
plified the synthesis of 3 by replacing the first step with exhaustive
phosphorylation of adenosine (Scheme 2, path B).
Table 2
Binding affinity of m7GPS (2e) for eIF4E protein
a
Compound
KAS
(l
Mꢀ1
)
DG° (kcal/mol)
(eIF4E)
m7GDPb
28.9 1.5
1.74 0.44
1.5 0.1
ꢀ10.00 0.03
ꢀ8.37 0.15
ꢀ8.28 0.04
m7GPS (2e)
m7GMPb
a
Determined in 50 mM Hepes/KOH (pH 7.2), 100 mM KCl, 0.5 mM EDTA, 2 mM
DTT at 20 0.2 °C.
b
Data from Ref.24
.
Finally, we searched for optimal conditions for NPS storage both
as solid samples and in aqueous solutions. In general agreement
with previous studies,20 we found that the 1 mM NPS solutions
containing EDTA were stable for longer than 24 h at pH 9 and 7.
At pH 2, hydrolysis to corresponding NMP took place with an
approximated half-time of 12–18 h. Moreover, we have found that
aqueous samples alkalized with sodium bicarbonate could be
stored for several weeks at 4 °C without decomposition. Addition
of EDTA and Na2CO3 also stabilized solid samples, which under
such conditions remained intact after over 1 year of storage,
whereas samples without any additives underwent partial decom-
position ranging from 10% to even 40%.
NPS may be considered as analogs of NDP, and hence, used to
determine specificity of some nucleotide binding proteins. To dem-
onstrate this we treated compound 2e as an isoster of naturally
occurring molecule m7GDP and tested its interaction with eukary-
otic initiation translation factor 4E (eIF4E) by means of fluores-
cence quenching titration. eIF4E is a protein responsible for the
recognition of eukaryotic m7G mRNA cap during translation initia-
tion.22 From biophysical and crystallographic studies,23 it is known
that hydrogen bonding and salt bridges between the negatively
charged phosphate chain and positively charged aminoacids in
the eIF4E’s cap binding cavity are important for complex
stabilization.
The terminal sulfate in m7GPS bears only one ionizable hydro-
xyl group, in contrast to two groups in the terminal phosphate of
m7GDP, and thus has a smaller net negative charge under physio-
logical pH. Consequently, the equilibrium binding affinity constant
(KAS
)
for m7GPS-eIF4E complex determined by fluorescence
quenching titration was ꢁ10-fold lower than the corresponding
value for the m7GDP-eIF4E complex (Table 2). The DDG0 value of
ꢀ1.6 kcal/mol may correspond to the stabilizing interaction pro-
vided by the terminal phosphate’s second negative charge in
m7GDP. Notably, the KAS value for eIF4E-m7GPS is similar to that
of eIF4E-m7GMP complex, which corresponds well with the fact
that both nucleotides have similar net charge under experimental
conditions.
In summary, we proposed a general method for the synthesis of
nucleoside 50-phosphosulfates. The synthesis is straightforward,
very efficient and can be performed starting from commercially
available nucleotides or nucleosides without the need of

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J. Kowalska et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3661–3664
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Acknowledgments
16. Worth mentioning, chemical syntheses have been successfully employed for
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We thank the Laboratory of Biological NMR (IBB PAS, Warsaw)
for the access to the NMR apparatus and to Jacek Oledzki from the
Laboratory of Mass Spectrometry (IBB PAS) for recording HRMS
spectra. Financial support from the Polish Ministry of Science and
Higher Education (N N204 089438, IP 2010 020170,
N301096339) is gratefully acknowledged.
N
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.039.
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