Rapid synthesis of nucleotide pyrophosphate linkages in a ball mill

Article abstract of DOI:10.1039/c1ob06041d

Using a ball mill, rapid, atom-economic coupling between adenosine-5′-phosphoromorpholidate and phosphorylated ribose derivatives as their sodium or barium salts was achieved. Facile purification by reversed-phase HPLC enabled product isolation within hours.

Full text of DOI:10.1039/c1ob06041d

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Organic &
Biomolecular
Chemistry
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COMMUNICATION
Rapid synthesis of nucleotide pyrophosphate linkages in a ball mill†
a
b
a
a
b
Francesco Ravalico, Ivano Messina, M. Victoria Berberian, Stuart L. James, Marie E. Migaud* and
Joseph S. Vyle*
a
Received 28th June 2011, Accepted 20th July 2011
DOI: 10.1039/c1ob06041d
Using a ball mill, rapid, atom-economic coupling between
adenosine-5¢-phosphoromorpholidate and phosphorylated
ribose derivatives as their sodium or barium salts was
achieved. Facile purification by reversed-phase HPLC en-
abled product isolation within hours.
to good yields using a vibration ball mill without extensive
pre-treatment of the substrates (Scheme 1). Following previous
9a
studies upon nucleoside protection, all reactions were performed
using a 25 mL stainless steel jar containing a single 15.0 mm
stainless steel ball. The jar was charged with equimolar amounts
of adenosine-5¢-phosphoromorpholidate (1; upto 0.14 mmol) and
a phosphorylated ribose derivative (2a–f). In addition, magnesium
chloride hexahydrate, 1H-tetrazole and water were all found to
be necessary to give a complete reaction and suppress the side-
reactions of the AMP-morpholidate. The jar containing these
reagents was shaken at 30 Hz for 90 min, allowed to cool
to room temperature and the reaction mixtures suspended in
water prior to analysis and then purification using reversed-phase
HPLC. In all cases, essentially complete consumption of the
phosphoromorpholidate was observed with up to 87% conversion
Nucleotides bearing pyrophosphate linkages are ubiquitous in
1
biological information and energy transduction systems and have
2
therefore long been the target of chemical synthesis.
Currently, phosphoromorpholidates are widely used in py-
rophosphate synthesis due to the balance between their elec-
trophilicity towards phosphate anions and their hydrolytic
3
lability. Typically, such coupling reactions are performed in
4
anhydrous pyridine, DMF or formamide. In order to render
polyanionic phosphates soluble in these solvents, extensive cation
exchange (ultimately to give the corresponding trialkylammonium
salts) and predrying is required. Coupling proceeds in poor to
fair yields over extended times (16 h to 6 days) and hydrolysis of
the phosphoromorpholidate group over this period often renders
the subsequent purification problematic due to the presence of
isoelectric homodimers.
to the desired product, e.g., Ap dT (3d) (Fig. 1).
2
In attempting to overcome these issues, a wide variety of
coupling chemistries has been described including direct dehy-
dration methods and in situ activation via phosphoroimidazolides
5
or mixed anhydrides. Perhaps most successful in ameliorating
purification issues has been reaction of activated intermediates
6
with solid-bound substrates. However, typically large reagent
excesses and anhydrous conditions are still required.
Fig. 1 Analytical C18 RP-HPLC chromatogram of the crude reaction
mixture of Ap dT (3d) synthesis (entry 4). AMP (2a) arising from
Mechanochemical mixing of solids with disparate solubility
properties is well established and has found wide employment
2
hydrolysis of 1, unreacted dTMP (2d), and Ap A (3a) are also visible.
2
7
in materials science but only in the last two decades has the
application of mechanochemistry to organic synthesis been more
10
In the absence of acid promoters, no consumption of 1 was
8
rigorously explored. In particular, the synthesis of nucleoside and
11
observed. Addition of Mg(II) alone enhanced the rate of coupling
9
nucleotide derivatives using ball milling remains undeveloped.
between 1 and AMP (2a) and gave clean conversion to 3a.
However, after 150 min, less than 50% conversion was observed. In
contrast, complete consumption of 1 was observed after 90 min in
the presence of excess tetrazole but multiple side-products were
apparent. In particular, tetrazole catalysed both the hydrolysis of
1 to 2a as well as the homo-coupling of 1 (putatively through
the 2¢- or 3¢-hydroxyls). We have previously found that solvent-
Here, we report a methodology for the synthesis of both
symmetric and non-symmetric nucleoside polyphosphates in fair
12
a
School of Chemistry and Chemical Engineering, Queen’s University Belfast,
David Keir Building, Stranmillis Road, Belfast, Northern Ireland, UK, BT9
5
AG. E-mail: j.vyle@qub.ac.uk; Tel: (+)44 (0) 28 9097 5485
b
School of Pharmacy, Queen’s University of Belfast, Medical Biology
Centre, 97 Lisburn Road, Belfast, Northern Ireland, UK, BT9 7BL.
E-mail: m.migaud@qub.ac.uk; Tel: +44 (0)28 90972689
13
assisted grinding (often called liquid-assisted grinding, or LAG)
9b
can enhance the selectivity of amine acylation and in this work
the addition of water (6 eq) was found to suppress self-coupling
of 1.
†
Electronic supplementary information (ESI) available: Experimental
procedures and analytical data. See DOI: 10.1039/c1ob06041d
6
496 | Org. Biomol. Chem., 2011, 9, 6496–6497
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Scheme 1 Synthesis of pyrophosphate linkages in a ball mill.
Thus, in the presence of MgCl
2
(H
2
O)
6
, tetrazole and water,
Notes and references
enhanced rates and intermolecular selectivity of the phosphate
coupling reaction were observed. Moderate to high yields were
observed for the preparation of other dinucleoside polyphos-
phates (3b–d), nicotinamide adenine dinucleotide (NAD - 3e)
and adenosine diphosphate ribose (ADPR – 3f). Side-products
arising from hydrolysis of 1 to AMP (2a) accompanied by the
1
e.g. A. G. McLennan, L. D. Barnes, G. M. Blackburn, C. Brenner,
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+
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4
ed. A. G. McLennan, CRC Press, Boca Raton, 1992.
production of Ap
12–31%).
The mobile-phase gradients developed for both analytical and
preparative scale HPLC enabled the separation of all components
and purification of the products could therefore be completed
within 150 min. Lyophilisation of the solutions removed both
the water and volatile salts to yield the pure products as
their triethylammonium salts. Again, this procedure is much
faster than typical ion-exchange chromatography using DEAE
Sephadex.
In conclusion, we have developed atom-economic methodology
for pyrophosphate bond formation using stable, inexpensive and
commercially-available reagents which does not require the use of
anhydrous, non-volatile and toxic solvents, which is compatible
with the synthesis of natural or modified linkages and which
enables rapid and facile isolation of pure materials.
2
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Acknowledgements
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This work has been funded by the School of Chemistry and
Chemical Engineering, QUB and DEL NI.
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This journal is © The Royal Society of Chemistry 2011
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