Simple synthesis of P1P2-diadenosine 5′-pyrophosphate

Article abstract of DOI:10.1080/00397910802116583

Pyrophosphate-linked coenzymes play essential roles in several biochemical systems. Symmetrical diadenosine-5′-pyrophosphate (Ap2A) has been synthesized from adenosine-5′-phosphate in virtually quantitative yield. The simple procedure is carried out in an

Full text of DOI:10.1080/00397910802116583

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Simple Synthesis of P1P2-
Diadenosine 5′-Pyrophosphate
Enrico Millo a , Elena Zocchi a , Andrea Galatini b
Umberto Benatti a & Gianluca Damonte a
a Department of Experimental Medicine ,
,
Biochemistry Section, Center of Excellence for
Biomedical Research, University of Genoa , Genoa,
Italy
b Department of Chemistry and Industrial
Chemistry , University of Genoa , Genoa, Italy
Published online: 12 Sep 2008.
To cite this article: Enrico Millo , Elena Zocchi , Andrea Galatini , Umberto Benatti
& Gianluca Damonte (2008) Simple Synthesis of P1P2-Diadenosine 5′-Pyrophosphate,
Synthetic Communications: An International Journal for Rapid Communication of
Synthetic Organic Chemistry, 38:19, 3260-3269, DOI: 10.1080/00397910802116583
To link to this article: http://dx.doi.org/10.1080/00397910802116583
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Synthetic Communications1, 38: 3260–3269, 2008
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910802116583
Simple Synthesis of P1P2-Diadenosine
50-Pyrophosphate
Enrico Millo,1 Elena Zocchi,1 Andrea Galatini,2 Umberto Benatti,1
and Gianluca Damonte1
1Department of Experimental Medicine, Biochemistry Section, Center of
Excellence for Biomedical Research, University of Genoa, Genoa, Italy
2Department of Chemistry and Industrial Chemistry, University of Genoa,
Genoa, Italy
Abstract: Pyrophosphate-linked coenzymes play essential roles in several bio-
chemical systems. Symmetrical diadenosine-50-pyrophosphate (Ap2A) has been
synthesized from adenosine-50-phosphate in virtually quantitative yield. The sim-
ple procedure is carried out in anhydrous pyridine using adenosine phosphoro-
morpholidate and adenosine monophosphate bis-(tri-n-butylammonium salt) as
coupling reagents.
Keywords: Coenzymes, diadenosine, morpholidate, pyrophosphate
Diadenosine polyphosphates are a family of endogenous bioactive com-
pounds increasingly recognized as important mediators of functional
responses in several mammalian cell types. The general structure of these
compounds is ApnA, where n is a variable (2–6) number of phosphates.
The members of this large family of compounds containing at least
three phosphate groups are generally agonists of purinergic receptors
of excitable cells (neuronal or muscle cells),[1,2] whereas the dinucleoside
diphosphates Ap2A, Ap2G, and Gp2G represent a new class of growth-
promoting extracellular mediators, which are released from platelets after
activation.[3]
Received January 14, 2008.
Address correspondence to Enrico Millo, Department of Experimental
Medicine, Biochemistry Section, Center of Excellence for Biomedical Research,
University of Genoa, Viale Benedetto XV 1-16132-Genoa, Italy. E-mail: enrico.
millo@unige.it
3260

Synthesis of P1P2-Diadenosine 50-Pyrophosphate
3261
Most recently, ADP-ribosyl cyclases (ADPRC) from lower and
higher metazoa have been shown to synthesise Ap2A and two isomers
thereof (P18 and P24), containing an unusual N-glycosidic bond between
one of the adenine moieties and the ribose. These dinucleotides are
present and metabolized in ADPRCþ mammalian cells where they affect
intracellular calcium and cell proliferation.[4]
The growing scientific interest regarding Ap2A and the fact that the
dinucleotide is not commercially available prompted us to develop a
rapid and simple chemical synthesis for this dinucleotide, which may be
of interest to scientists in various fields of mammalian physiology.
Synthesis of pyrophosphate bonds has relied on chemical activation
of one phosphate derivative and subsequent reaction with the other one.
The reaction involves the nucleophilic attack of one phosphate anion on
the activated derivative of the other.
The synthesis of this dinucleotide presents some difficulties because
of the low nucleophilic reactivity of the phosphate moiety. In the litera-
ture, different methods applicable to the synthesis of P1P2-diadenosine-
50-pyrophosphate have been reported, the majority of these including
the use of an appropriate adenosine-50-monophosphate salt. The most
direct method for preparing diesters of pyrophosphoric acid is through
condensation of the pyridium salt of 50-adenosine-monophosphate
(50-AMP) with dicyclohesylcarbodiimide (DCC),[5] which has the advan-
tage, over other dehydrating agents, of preferentially attacking acidic
groups, but its slight excess in the activating solution requires a further
step to filter the crystallized dicyclohexylurea.
The same product was obtained by reacting adenosine-50-monophos-
phate with trifluoroacetic anhydride overnight and evaporating it to dry-
ness. This method[6] exploits a simple exchange reaction but has the great
drawback of a low yield (17% by the same authors) of the purified pro-
duct. The use of a different approach based on intermediates such phos-
phoramidates[7] or phosphorimidazolide-activated nucleosides[8] does not
allow the development of satisfactory procedures for the synthesis of
Ap2A. Phosphorimidazolidates exhibit inherent high polarity and insta-
bility, so their purification and preservation is rather difficult. In an
attempt to synthesize the product without the use of different steps to
activate the phosphate as salt or other forms, Kanavarioti et al., follow-
ing a particular oxidation–reduction condensation method, discovered a
one-pot synthesis of the symmetrical pyrophosphate of 50-adenosine.[9]
The synthesis, although showing a good yield, has great disadvantages:
the insolubility of the employed reagents and the need for particular
chromatographic steps of purification.
The introduction of water-soluble carbodiimides as activating agents
in a variety of reactions that involve the formation of pyrophosphate

3262
E. Millo et al.
bonds[10] led Ng and Orgel to publish a simple synthesis of diadenosine
pyrophosphate.[11] This method is based on activation of adenosine
monophosphate with water-soluble 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (CDI) in an aqueous solution also containing phosphate
buffer (HEPES), but unfortunately the reaction yielded several by-
products chemically similar to the desired product. The unreacted adeno-
sine monophosphate, the carbodiimide adducts, and the high amount of
salts present in the reaction mixture requires a suitable chromatographic
purification.[12] Thus, although several procedures have been described,
none appeared to be convenient. For this reason, we developed an
alternative approach involving the use of phosphoromorpholidates
derivatives.
The aim of the present communication is to introduce a rapid, sim-
ple, and low-cost protocol for the synthesis of Ap2A using commercially
available reactives. The use of phosphoromorpholidates[13] is suitable to
obtain the corresponding P1P2-diadenosine-50-pyrophosphate in virtually
quantitative yields.
Because the resulting phosphoromorpholidate derivatives exhibit
high reactivity toward various nucleophilic functions, they can be used
as active intermediates for the synthesis of pyrophosphate compounds
by the reaction with phosphate nucleophiles. Our approach overcomes
the drawbacks of the conventional methodology of pyrophosphate syn-
thesis. The homodimer Ap2A was obtained using a modified version of
the phosphoromorpholidate coupling method where the bis-(tri-n-buty-
lammonium salt) of 50-adenosinemonophosphate (1) was coupled to the
adenosine-50-phosphoromorpholidate 4-morpholine-N,N0-dicyclohexyl-
carboxamidine salt (50-AMP phosphoromorpholidate, 2), commercially
available as Sigma A1127, or carried out according to the procedure of
Moffat,[13] without further purification.
A specific salt, bis-(tri-n-butylammonium salt) of 50-adenosinemono-
phosphate, which is readily soluble in organic solvents, needs to be pre-
pared prior to reaction. It was obtained by modification of our
previously published method,[14] where the reaction of 50-adenosine
monophosphate free acid (50-AMP) with tributylamine in methanol gave
the corresponding TBA salt of 50-AMP(1), readily soluble in pyridine.
Mass spectrometry confirmed the structure, and the final product
(95% yield) was used without further purification. The attempt of con-
densation of the phosphoromorpholidate with adenosine monophos-
phate free acid failed, probably because of the insolubility of AMP free
acid in pyridine or some others organic solvents. It needs to use the more
reactive ammonium salt.
The P1P2-diadenosine-50-pyrophosphate (3) was indeed obtained
through the condensation between bis-(tri-n-butylammonium salt)

Synthesis of P1P2-Diadenosine 50-Pyrophosphate
3263
50-adenosinemonophosphate, dissolved in anhydrous pyridine, and a
solution of the same solvent containing the equimolar concentration of
50-AMP phosphoromorpholidate. The reactants were dried by several
evaporations of anhydrous pyridine until the last amounts of water were
removed, and finally the solution was gently stirred under nitrogen at
room temperature.
The phosphoromorpholidate is strongly activated toward the
nucleophilic attack by phosphate groups, and hence the conversion of
intermediates to the pyrophosphate is quite easy. Because of the use of
the tertiary base salt, a longer reaction period at room temperature is
necessary, but the final yield of the desired compound is excellent.
After 72 h, when thin-layer chromatography (TLC) indicated com-
plete consumption of starting materials, the pyridine was removed under
vacuum, and the mixture was dissolved in water. No side products were
observed, except for low amounts of 50-adenosine monophosphate.
The aqueous mixture was adjusted to pH 8 with 1N sodium
hydroxide to liberate the free guanidine adduct (4-morpholine-N,N0-
dicyclohexylcarboxamidine), which was removed by ether extraction
(as reported by Rhajbhandary et al.).[15]
The synthetic reactions are summarized in Scheme 1.
The final product was purified by preparative HPLC using a C18
reversed-phase column.
Buffer A was an aqueous solution of 10 mM ammonium formate
adjusted to pH 4.0, and buffer B was a solution of 50% buffer A and
50% methanol. An HPLC chromatogram of the purified product, using
the same HPLC preparative conditions, is shown in Fig. 1, panel A.
Scheme 1. Reagents and conditions: (i) methanol, TBA, rt, 30 min; (ii) (a) pyridine,
rt, 72 h, (b) H2O, NaOH 1N, pH ¼ 8.

3264
E. Millo et al.
Figure 1. HPLC chromatogram of the purified product Ap2A (panel A). The
MS=MS spectrum of the synthesized compound (panel B) is compared to the
MS=MS spectrum of the commercial standard Sigma D-7376 (panel C).
The powder (90% yield) was then analyzed by electrospray mass spec-
trometry (ESI-MS), confirming presence of the expected molecular weight
(m=z calculated 676.43; found 676.4). The MS=MS analysis performed on
the synthetic molecule and on the commercial standard (Sigma D-7376,
now out of production) on the ion-trap instrument showed a high degree
of correspondence of fragment ions, as shown in Fig. 1 (panels B and C).
The same synthesis was carried out, in the same experimental con-
ditions, with other solvents such as N,N-dimethylformamide and DMSO,
but with yields not higher than 40–50%, showing the evident catalytic
role of anhydrous pyridine in the entire procedure.
The presence of the P1P2-diadenosine-50-pyrophosphate bridge was
also confirmed enzymatically, by digestion with pyrophosphatase from
Crotalus adamanteus venom. Approximately 20 nmol of Ap2A was incu-
bated at 37 ꢀC with 40 ml of 30 mM tris-HCl at pH 8, 10 ml of 5 mM
MgC12 and 5 ml of Pyrophosphatase (Sigma-Aldrich P7383). Aliquots

Synthesis of P1P2-Diadenosine 50-Pyrophosphate
3265
were removed after 60 min and chromatographed directly using the
same HPLC preparative conditions. Enzymatic degradation yielded the
expected 50-AMP, as detected and identified by HPLC on the basis of
the retention time (identical to that of the commercial standard). This
experiment demonstrated that the diphosphate chain binds the adenosine
moieties via phosphoester bonds with the 50-oxygens of the riboses.
The Ap2A molecule, synthesized as described, was indeed tried out in
biochemical experiments. The ability of Ap2A to affect [Ca2þ]i, when
applied extracellularly to intact cells at micromolar concentrations, and
to antagonize P24-induced proton gradient dissipation and cytotoxicity
was demonstrated with already published methods.[16]
In summary, we propose a convenient strategy for the synthesis of
pyrophosphate bonds by the use of readily available phosphoromorpho-
lidate derivatives and demonstrate the usefulness of our pyrophosphory-
lation strategy on the synthesis of Ap2A and its analogs. The major
advantages of this method are (1) the considerably reduced costs and
the opportunity of employing commercial reagents compared to that of
previous methods, (2) the absence of by-products, allowing a good final
yield, and (3) the possibility of application to asymmetric dinucleoside
pyrophosphates (e.g., Ap2T, Ap2G, Ap2C).
The chemical synthesis, although straightforward, has great impor-
tance to synthesis of various nucleoside and nucleotides not commercially
available or hardly achievable with other synthetic protocols but funda-
mental as extracellular mediators in the biochemical and biomedical fields.
EXPERIMENTAL
The melting points were recorded on a Buchi 535 melting-point
apparatus. All mass spectrometric low-resolution analysis were per-
formed on an Agilent MSD 1100 ion trap instrument acquiring spectra
in the negative ion mode. All measures were conducted in FIA (flow
injection analysis) mode in a range including the expected m=z ratios
using water–acetonitrile 50:50 with 0.1% formic acid as carrier flow.
The elemental composition analysis was performed in DIA, in the
negative ion mode, on a high-resolution q-TOF geometry mass spec-
trometer (Qstar XL system, Applied Biosystems=MDS Sciex, Toronto,
Canada) using an electrospray ion source. The compound was dissolved
in a water–acetonitrile 50:50 solution containing triethylamine at final
concentration of 10 mM. All NMR spectra were acquired on a Varian
MercuryPlus 300-MHz spectrometer.
1
Peak assignment in HNMR spectra was also made with the aid of
correlation spectroscopy (COSY) and heteronuclear single quantum

3266
E. Millo et al.
coherence (HSQC) experiments. Peak assignment in 13CNMR spectra
was also obtained using HSQC experiment.
bis-(Tri-n-butylammonium) Salt 50-Adenosine Monophosphate (1)
A solution of tributylamine (TBA) (0.2 mmol, 47 ml) was added dropwise to
a stirred suspension of 50-adenosine-monophosphate free acid monohydrate
(50-AMP) (0.1 mmol, 36.5 mg) in 10 ml of methanol. The mixture was
vigorously stirred at room temperature, and after 30 min, a clear solution
was obtained. The solution was evaporated to dryness. The process of dis-
solving and evaporating was repeated few times in the presence of anhydrous
pyridine to obtain the TBA salt of 50-AMP, readily soluble in pyridine.
Adenosine-50-phosphoromorpholidate-4-morpholine-N,N0-
dicyclohexylcarboxamidine Salt (2)
A solution of dicyclohexylcarbodiimide (1 mmol, 206 mg) in t-butyl alco-
hol (5 ml) was added dropwise to a refluxing solution of the adenosine-
50-phosphate free acid (0.25 mmol, 91 mg) in a mixture of water (5 ml),
t-butyl alcohol (5 ml), and purified morpholine (1 mmol, 85 ml).
The addition was completed in about 3 h, and the mixture was
refluxed overnight until TLC (n-butanol–water–acetic acid 5:3:2) showed
completion of the reaction. The mixture was then cooled to room
temperature, and any crystalline material was removed by filtration
and carefully washed with t-butyl alcohol. The filtrate was evaporated
until the t-butyl alcohol was largely removed, and the remaining aqueous
phase was extracted three times with ether. The clear aqueous solution
was then evaporated to dryness in a vacuum.
The residue was then transferred in a centrifuge tube, and a minimum
volume of methyl alcohol (1.5 ml) was added. The addition of dry ether
(10 ml) precipitated a gummy solid, which on trituration with fresh ether
changed to a dry white powder. The adenosine-50-phosphoromorpholidate
was obtained as a salt of 4-morpholine-N,N0-dicyclohexylcarboxamidine
1
(90% yield, 159 mg). H NMR (D2O, 300 MHz): 8.40 (s, 1H), 8.17 (s,
1H), 6.07 (d, J ¼ 5 Hz, 1H), 4.76 (t, J ¼ 5 Hz, 1H), 4.49 (t, J¼ 5 Hz, 1H),
4.35–4.29 (m, 1H), 4.07–3.94 (m, 2H), 3,75 (t, J¼ 5Hz, 4H), 3.57–3.48 (m,
4H), 3.40 (t, J ¼ 5 Hz, 4H), 3.33–3.21 (m, 2H), 2.97–2.82 (m, 4H), 1.94–
1.82 (m, 4H), 1.77–1.69 (m, 4H), 1.59–1.55 (m, 2H), 1.36–1.01 (m, 10H).
Electrospray mass spectrometry (ESI-MS) conducted in negative
ion mode gave unique significative signal at m=z 415.7[M-H]ꢁ. The
calculated m=z neutrally charged for C14H21N6O7P was 416.78.

Synthesis of P1P2-Diadenosine 50-Pyrophosphate
P1P2-diadenosine-50-pyrophosphate (3)
3267
4-Morpholine-N,N0-dicyclohexylcarboxamidinium salt of adenosine-
50-phosphoromorpholidate (0.1 mmol, 71 mg) was dried by three
evaporation steps of its solution in anhydrous pyridine. Separately the
equimolar concentration of bis-(tri-n-butylammonium) salt 50-adenosine
monophosphate (0.1 mmol, 72 mg) was dissolved in 2 ml of anhydrous
pyridine, and the compound was rendered anhydrous by two evapora-
tions of solutions. The two pyridine solutions were mixed and dried
by several evaporation steps of anhydrous pyridine solution until the sol-
ution became clear, and finally the reaction was carried out by gently stir-
ring at room temperature under N2. Silica-gel TLC was used to monitor
the course of reaction (TLC: n-butanol–water–acetic acid 5:3:2). After
72 h, time in which the morpholidate completely disappeared, the solvent
was evaporated under vacuum and the residual pyridine was removed by
the addition and evaporation of water (2–3 ml). The mixture was indeed
dissolved in 10 ml of water and the pH was adjusted to 8 with 1 N sodium
hydroxide. The basic solution was then washed three times with 15 ml of
diethyl ether, and the organic layers were back-extracted once with dis-
tilled water and then evaporated to dryness. The final product was pur-
ified by preparative HPLC using a Luna C18 reversed-phase column
(21.20 ꢂ 250 mm, Phenomenex, Torrance CA, USA). The solvent pro-
gram was a gradient starting at 100% buffer A for 2 min and linearly
increasing to 100% B in 38 min.
Buffer A was an aqueous solution of 10 mM of ammonium formate
adjusted to pH 4.0, and buffer B was a solution of 50% buffer A and
50% methanol.
The solution was evaporated to dryness under vacuum, and the
residue was repeatedly lyophilized to afford colorless and water-soluble
crystals of P1P2-diadenosine-5ꢀ0-pyrophosphate (90% yield, 61 mg, mp
187–190 ꢀC, lit.[17] mp 184–189 C).
1H NMR d (D2O, 300 MHz): 8.14 (s, 2H, H8), 7.97 (s, 2H, H2), 5.89
(d, J ¼ 5 Hz, 2H, H10), 4.53 (t, J ¼ 5 Hz, 2H, H20), 4.40 (t, J ¼ 5 Hz, 2H,
H30), 4.35–4.27 (m, 4H, H40 þ H50), 4.24–4.16 (m, 2H, H500). 13C NMR
d (D2O, 75 MHz): 154.073 (C6), 151.413 (C2), 148.097 (C4)0, 139.587
(C8), 117.793 (C5), 87.284 (C10), 83.508 (C4), 74.835 (C2), 70.058
(C30), 65.285 (C50).
Electrospray mass spectrometry (ESI-MS) conducted in negative ion
mode gave unique remarkable signal at m=z 675.4 [M-H]ꢁ. The average
calculated m=z, neutrally charged for C20H26N10O13P2, was 676.43.
The elemental composition analysis showed an m=z ratio of 675.
1045 amu (calculated for C20H26N10O13P2[M-H]ꢁ m=z 675.1083 amu) with
an error of ꢁ5.62 ppm, in the range of the instrumental factory specification.

3268
E. Millo et al.
To obtain the more stable lithium salt of Ap2A, the aqueous solution
of Ap2A pyrophosphate was adjusted to pH 5 with 0.1 M lithium
hydroxide, then concentrated to a small volume and finally evaporated
to a syrup under vacuum. Crystallization with a mixture of acetone
and ethyl alcohol (5:1) at temperature of 0 ꢀC allowed us to obtain the
precipitated lithium salt of Ap2A.
ACKNOWLEDGMENTS
The authors are grateful for the help and technical assistance of Andrea
Armirotti. This work was supported by grants from the Italian Associ-
ation for Cancer Research (AIRC), the Italian Ministry for Education,
University and Scientific Research (Grants MIUR-PRIN 2003, MIUR
FIRB RBNE01ERXR, and MIUR FIRB RBLA039LSF), and the
Fondazione Cassa di Risparmio di Genova e Imperia.
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