PPN Pyrophosphate: A New Reagent for the Preparation of Nucleoside Triphosphates

Article abstract of DOI:10.1080/10426507.2014.984032

Tris{bis(triphenylphosphoranylidene) ammonium} (PPN) pyrophosphate was accessed via aqueous precipitation and desiccation. The reagent was investigated as a replacement for highly hygroscopic alkylammonium salts in Ludwig-Yoshikawa reactions for the preparation of nucleoside-5′-triphosphates.

Full text of DOI:10.1080/10426507.2014.984032

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Phosphorus, Sulfur, and Silicon and the Related
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PPN pyrophosphate: A New Reagent for the Preparation
of Nucleoside Triphosphates
ab
b
b
b
Heidi J. Korhonen , Hannah L. Bolt , Leyre Vicente-Gines , Daniel C. Perks & David R. W.
b
Hodgson
a
Department of Chemistry, Vatselankatu 2, 20014 University of Turku, Finland
b
Department of Chemistry and Biophysical Sciences Institute, Science Laboratories, Durham
University, South Road, Durham, DH1 3LE, United Kingdom.
Accepted author version posted online: 06 Dec 2014.
To cite this article: Heidi J. Korhonen, Hannah L. Bolt, Leyre Vicente-Gines, Daniel C. Perks & David R. W. Hodgson (2014):
PPN pyrophosphate: A New Reagent for the Preparation of Nucleoside Triphosphates, Phosphorus, Sulfur, and Silicon and the
Related Elements, DOI: 10.1080/10426507.2014.984032
To link to this article: http://dx.doi.org/10.1080/10426507.2014.984032
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PPN pyrophosphate: A New Reagent for the Preparation of Nucleoside
Triphosphates
a,b
b
b
b
Heidi J. Korhonen , Hannah L. Bolt , Leyre Vicente-Gines , Daniel C. Perks and
b
David R. W. Hodgson. *
a
Department of Chemistry, Vatselankatu 2, 20014 University of Turku, Finland
b
Department of Chemistry and Biophysical Sciences Institute, Science Laboratories,
Durham University, South Road, Durham, DH1 3LE, United Kingdom.
E-mail: d.r.w.hodgson@durham.ac.uk
Running head:
PPN PYROPHOSPHATE FOR NTP SYNTHESIS
Abstract
Tris{bis(triphenylphosphoranylidene) ammonium} (PPN) pyrophosphate was accessed
via aqueous precipitation and desiccation. The reagent was investigated as a
replacement for highly hygroscopic alkylammonium salts in Ludwig-Yoshikawa
reactions for the preparation of nucleoside-5’-triphosphates.
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Keywords
phosphorylation, triphosphate, nucleotide
2
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INTRODUCTION
Nucleoside-5’-triphosphates (NTPs) provide the building blocks for polymerase
enzymes and a multitude of other biological and biotechnological systems.
1
Analogues are widely used for enzyme mechanism studies. Over the last ~25-50
years, a small number of key methods have been adopted for the preparation of
2
NTPs and other phosphoanhydrides, although new methods are now becoming
3
-14
available.
Often, these approaches rely on tri- and tetralkylammonium salts as
Unfortunately, these
with deleterious consequences on the water-
organic-solvent-soluble sources of nucleophilic phosphate
.
1
5-20
salts are extremely hygroscopic,
sensitive reactions that use them. PPN salts do not form heavily water-clathrated
structures, and their preparation through aqueous precipitation has been
2
1-22
established and applied synthetically.
Here we prepare for the first time, a
PPN salt of pyrophosphate ion and investigate its utility as a source of
nucleophilic pyrophosphate in the Ludwig modification of the Yoshikawa
2
3
phosphorylation.
RESULTS AND DISCUSSION
PPN salts can be prepared using PPNCl, which is soluble in warm water, and
2
4
2
5
the sodium salt of the desired anion, where the PPN salt precipitates or crystallises on
mixing the solutions. We explored stoichiometry and pH parameters in order to optimise
the preparation of PPN
3
HP
2
O
7
using PPNCl and partially neutralised tetrasodium
3
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pyrophosphate. A 3:1 PPN-pyrophosphate target stoichiometry was chosen in order to
retain the nucleophilicity of the pyrophosphate trianion, while keeping pH low to avoid
the formation of PPN.OH. A 2:1 ratio of PPN.Cl to sodium pyrophosphate (pH-adjusted
3
1
to pH 5.0) afforded PPN pyrophosphate in 62% yield. P NMR analysis revealed a
PPN–pyrophosphate ratio of 3.2:1.
[
Insert Scheme 1]
The material was desiccated, and its moisture content before and after
exposure to the atmosphere was assessed visually and by thermogravimetric
analysis (TGA) in comparison to tetraalkylammonium salts.
Tris(tetrabutylammonium) hydrogen pyrophosphate became visibly ‘wet’ after
exposure to the atmosphere (see Supplemental Materials), whereas PPN
pyrophosphate appeared unchanged. TGA showed that the PPN salt contained
only 1.2% water (approximately 1.2 molar equivalents) after desiccation, despite
its isolation from aqueous solution, and this value rose to only 3.7% on exposure
to the atmosphere for 22 h.
Preliminary experiments using PPN pyrophosphate explored the use of
additional drying of PPN pyrophosphate using molecular sieves, the addition of
collidine, the addition of tri-n-octylamine, reaction time, temperature and
stoichiometry. The conclusions of these studies were that additional drying
increased conversion level, as did the additions of collidine and tri-n-octylamine.
Four ribonucleosides and thymidine, as a representative deoxyribonucleoside,
were investigated as substrates (Scheme 2, Table 1).
4
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[
[
Insert Scheme 2]
Insert Table 1]
Reactions were quenched with triethylammonium bicarbonate (TEAB) buffer
followed by crude nucleotide isolation through extraction and selective
precipitations with NaI/acetone. Rigorously controlled precipitation conditions
served to reduce cTMP ion, which can be problematic during chromatography.
3
1
P NMR assessments of the crude nucleotides showed good conversion levels,
with excess pyrophosphate and traces of cTMP and monophosphate being the P-
containing impurities.
DEAE chromatography followed by cation exchange to sodium ions afforded
each of the triphosphates 1-5 in yields of 21-48%.
CONCLUSIONS
PPN pyrophosphate is easily dried and displays limited moisture uptake from the
atmosphere. It is a convenient and effective replacement for tri- and tetraalkyl
ammonium pyrophosphates in (d)NTP preparation.
EXPERIMENTAL
Drying of solvent and reagents
2 2
Triethyl phosphate was vacuum distilled from CaH and stored under N with activated 3
Å molecular sieves. Trioctylamine (1 mL) was dried over KOH pellets overnight. PPN
pyrophosphate (0.4 mmol, 2 eq.) was dissolved in dry MeCN (0.8 mL) and dried with
5
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activated 3 Å molecular sieves under N
distilled from CaH and phosphoryl chloride was distilled under dry conditions.
Nucleosides (0.2 mmol, 1 eq.) was dried at 80 °C under vacuum over P overnight.
2
overnight. 2,4,6-trimethylpyridine was vacuum
2
2
O
5
Preparation of PPN pyrophosphate (tris{(bis(triphenylphosphoranylidene)ammonium}
pyrophosphate). Tetrasodium pyrophosphate decahydrate (0.79 g, 1.77 mmol) was
dissolved in distilled water (25 mL) at 50 °C and the pH of the solution was adjusted to
pH 5 with 1 M hydrochloric acid. This solution was added dropwise, with mixing, to a
solution of bis(triphenylphosphoranylidene)ammonium chloride (2.00 g, 3.48 mmol) in
distilled water (260 mL) at 50 °C. The mixture was transferred to six 50 mL centrifuge
tubes, allowed to cool to room temperature and the tubes were centrifuged at 4,000 rpm
for 20 min at 18 °C. The supernatant was removed and the solid residues were
combined, recrystallized from distilled water (45 mL) at 80 °C, collected by
centrifugation at 4,000 rpm for 20 min, dried, ground to a powder and further dried in a
2 5
vacuum desiccator over P O for 2 days to give PPN pyrophosphate as a white powder
–
1
(
(
(
1.45 g); mp 234-238 °C; vmax/cm 3371br (O-H), 1437s, 1248 (P=O), 1112s (P=O); δ
400.13 MHz, d -DMSO) 7.51-7.70 (m, phenyl H); δ (161.96 MHz, d -DMSO) –4.72
2P, s, PPi), 20.8 (6.4P, s, PPh
H
6
P
6
3
); δ
C
(100.60 MHz, d
6
-DMSO), 125.7 (JC-P 54, 2.1 Hz),
+
+
1
28.7-128.3 (m), 130.9-130.6 (m), 132.7; ES m/z 539 ([PPN+H] , 100%).
Phosphorylation Procedure
Dry triethyl phosphate (1 mL for uridine, cytidine and thymidine or 2.5 mL for guanosine
and adenosine) was added to the nucleoside (0.2 mmol, 1 eq.) under an inert
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atmosphere. The mixture was heated to 80 °C briefly to dissolve the nucleoside, if
necessary, then stirred on an ice-salt bath at approx. –10 °C under an inert atmosphere.
Separately, freshly distilled phosphoryl chloride (0.24 mmol, 1.5 eq.) and dry 2,4,6-
trimethylpyridine (0.2 mmol, 1 eq.) were added drop-wise via syringe to the nucleoside
solution, and the mixture was stirred at –10 °C for 90-120 min. PPN pyrophosphate
solution in dry acetonitrile (0.4 mmol in 1 mL, 2 eq., with molecular sieves in stock
solution) and dry n-trioctylamine (0.4 mmol, 2 eq.) were added to the nucleoside
solution separately via syringe and the mixture was stirred at 0 °C for 2 h then RT for 20
h. Triethylammonium bicarbonate solution (5 mL, 0.1 M) was added to the solution,
followed by chloroform (5 mL) and the mixture was stirred for 30 min. The aqueous
layer was separated and washed with chloroform (2×20mL). The aqueous layer was
transferred to a centrifuge tube, and the phosphorus-containing components were
precipitated by addition of NaI (80 mg), followed by acetone (20 mL). The centrifuge
tube was agitated for 30 min, cooled at 0 °C for 30 min and centrifuged at 4,000 rpm for
5
min at 4 °C. The supernatant was removed and the solid was re-suspended in TEAB
buffer (3 mL, 0.1 M). NaI in acetone (5 mL, 0.1 M) was added dropwise to the mixture,
while stirring at 0 °C. Stirring was maintained for 30 min at 0 °C, and the solid was
collected by centrifugation (4,000 rpm, 5 min at 4 °C). The solid and supernatant were
3
1
analysed by P NMR spectroscopy to ensure the selective precipitation of NTP, where
the majority of cTMP remained in the supernatant. If the supernatant retained NTP (by
3
1
P NMR spectroscopy), additional NaI/acetone was added, and an additional crop of
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NTP precipitate was collected. The combined NTP precipitates were then dissolved in
TEAB buffer, and then subjected to DEAE chromatography.
DEAE purification of NTPs
The crude NTP was dissolved in 2 mL of distilled water and was loaded onto a 50
mL bed-volume DEAE Sepharose Fast Flow column and nucleotide products
were eluted using a 5-60% gradient of 1 M triethylammonium bicarbonate
-
1
solution with a flow rate of 4.5 mL min . Fractions corresponding to nucleoside
triphosphate signals (at approximately 0.3 M buffer concentration) were collected
and lyophilised to give the purified NTP, which was exchanged to the Na salt
through NaI/acetone precipitation.
Supplementary Data
Time-lapse photography of PPN pyrophosphate and tetrabutylammonium salts
3
1
31
1
13
on exposure to the atmosphere, crude P NMR spectra, and P, H and C
NMR spectra of NTPs after chromatography and isolation as Na salts.
ACKNOWLEDGEMENTS
This work was supported by the Jenny and Antti Wihuri Foundation (H.J.K),
Durham University (H.L.B. and D.C.P.), the Royal Society (chromatography
system) and the ERASMUS Scheme (L.V.-G.)
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Scheme 1
Scheme 2
1
1
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TABLE 1 Use of PPN pyrophosphate in (d)NTP syntheses
+
Entry
Nucleoside
Product
Estimated
Yield of Na
3
1
(
P NMR)
conversion to
d)NTP (%)
salt (%)
(
1
2
3
4
5
adenosine
cytidine
ATP 1
CTP 2
UTP 3
GTP 4
TTP 5
76
66
62
82
65
30
48
36
42
21
uridine
guanosine
thymidine
1
2
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