Báo cáo khoa học: Novel diadenosine polyphosphate analogs with oxymethylene bridges replacing oxygen in the polyphosphate chain pdf

Novel diadenosine polyphosphate analogs with
oxymethylene bridges replacing oxygen in the
polyphosphate chain
Potential substrates and/or inhibitors of Ap
4
A hydrolases
Andrzej Guranowski
1
,El
_
zbieta Starzyn
´
ska
1
, Małgorzata Pietrowska-Borek
2
, Dominik Rejman
3,
* and
George M Blackburn
3
1 Department of Biochemistry and Biotechnology, University of Life Sciences, Poznan
´
, Poland
2 Department of Plant Physiology, University of Life Sciences, Poznan
´
, Poland
3 Department of Molecular Biology and Biotechnology, University of Sheffield, UK
Dinucleoside 5¢,5¢¢¢-P
1
,P

n
-polyphosphates (Np
n
N¢s, n =
3–6) occur in all types of cell [1] but, although there is
evidence that these compounds act as signaling mole-
cules both extracellularly [2] and intracellularly [3],
their biological functions are far from being under-
stood [4]. Np
n
N¢s can be synthesized by some ligases
[5–8], by firefly luciferase [9] and by some nucleotidyl
transferases [10,11], and different specific and
Keywords
adenine nucleotide analogs; Ap
4
A
hydrolases; dinucleoside polyphosphates;
modified pyrophosphate substrates; stable
pyrophosphate analogs
Correspondence
A. Guranowski, Department of Biochemistry
and Biotechnology, University of Life
Sciences, 35 Wołyn
´
ska Street, 60 637
Poznan
´
, Poland
Fax: +48 61 8487146

Tel: +48 61 8487201
E-mail:
G. M. Blackburn, Department of Molecular
Biology and Biotechnology, Sheffield
University, Sheffield S10 2TN, UK
Fax: +44 1142222800
Tel: +44 1142229462
E-mail: g.m.blackburn@sheffield.ac.uk
*Present address
Institute of Organic Chemistry and
Biochemistry AS CR, v.v.i., Prague, Czech
Republic
(Received 24 June 2008, revised 3
December 2008, accepted 30 December
2008)
doi:10.1111/j.1742-4658.2009.06882.x
Dinucleoside polyphosphates (Np
n
N¢s; where N and N¢ are nucleosides
and n = 3–6 phosphate residues) are naturally occurring compounds that
may act as signaling molecules. One of the most successful approaches to
understand their biological functions has been through the use of Np
n
N¢
analogs. Here, we present the results of studies using novel diadenosine
polyphosphate analogs, with an oxymethylene group replacing one or two
bridging oxygen(s) in the polyphosphate chain. These have been tested as
potential substrates and/or inhibitors of the symmetrically acting Ap
4
A

hydrolase [bis(5¢-nucleosyl)-tetraphosphatase (symmetrical); EC 3.6.1.41]
from E. coli and of two asymmetrically acting Ap
4
A hydrolases [bis(5¢-nu-
cleosyl)-tetraphosphatase (asymmetrical); EC 3.6.1.17] from humans and
narrow-leaved lupin. The six chemically synthesized analogs were:
ApCH
2
OpOCH
2
pA (1), ApOCH
2
pCH
2
OpA (2), ApOpCH
2
OpOpA (3),
ApCH
2
OpOpOCH
2
pA (4), ApOCH
2
pOpCH
2
OpA (5) and ApOp-
OCH
2
pCH
2

OpOpA (6). The eukaryotic asymmetrical Ap
4
A hydrolases
degrade two compounds, 3 and 5 , as anticipated in their design. Analog 3
was cleaved to AMP (pA) and b,c-methyleneoxy-ATP (pOCH
2
pOpA),
whereas hydrolysis of analog 5 gave two molecules of a,b-oxymethylene
ADP (pCH
2
OpA). The relative rates of hydrolysis of these analogs were
estimated. Some of the novel nucleotides were moderately good inhibitors
of the asymmetrical hydrolases, having K
i
values within the range of the
K
m
for Ap
4
A. By contrast, none of the six analogs were good substrates or
inhibitors of the bacterial symmetrical Ap
4
A hydrolase.
Abbreviations
DCC, dicyclohexylcarbodiimide; MCPBA, 4-chloroperoxybenzoic acid; NEP, 2-chloro-5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinane.
1546 FEBS Journal 276 (2009) 1546–1553 ª 2009 The Authors Journal compilation ª 2009 FEBS
nonspecific enzymes exist that degrade these dinucleo-
tides to mononucleotides [12]. Ap
3
A and Ap

4
A are the
most frequently studied Np
n
N¢s, and many Ap
3
A and
Ap
4
A analogs have been synthesized, both chemically
and enzymatically [13]. Some have been found to be
useful for elucidating certain aspects of the behavior of
Ap
4
A-degrading enzymes. P
a
-Chiral phosphorothioate
analogs of Ap
4
A have been used to show that the
yeast Ap
4
A phosphorylase forms an enzyme–AMP
intermediate [14], whereas a complex of a methylene
analogue of Ap
4
A, AppCH
2
ppA, with the (asymmetri-
cal) Ap

4
A hydrolase from Caenorhabditis elegans, was
used to determine the 3D structure of the enzyme–sub-
strate complex [15]. Some nondegradable analogs
appeared to be extremely strong inhibitors of the
Ap
4
A hydrolases; two adenosine-5¢-O-phosphorothioy-
lated pentaerythritols are strong inhibitors of the (sym-
metrical) Ap
4
A hydrolase from Escherichia coli (with
K
i
values of 0.04 and 0.08 lm) [16], and methylene
analogues of adenosine 5¢-tetraphosphate (p
4
A)
strongly inhibited the asymmetrically acting Ap
4
A
hydrolases with K
i
values in the nanomolar range [17].
Finally, potential medical application has been demon-
strated for AppCHClppA, a competitive inhibitor of
ADP-induced platelet aggregation, which plays a
central role in arterial thrombosis and plaque forma-
tion [18], and for [
18

F]AppCHFppA, which appeared
to be useful in imaging of positron-emission tomogra-
phy to detect atherosclerotic lesions and, hence, prom-
ising for the noninvasive characterization of vascular
inflammation [19].
Of various Ap
n
A analogs investigated so far as
potential substrates and/or inhibitors of specific Ap
4
A
hydrolases, those with modifications in the polyphos-
phate chain have been studied most often [20–23].
Some are substrates of the asymmetrically acting Ap
4
A
hydrolases from yellow lupin seeds [20,21] and Artemia
embryos [22]. AppCH
2
ppA and ApCH
2
pppA were
hydrolyzed 20- to 50-fold more slowly than Ap
4
A, and
AppCF
2
ppA, AppCHFppA, AppCHBrppA and
AppCHClppA were hydrolyzed 1.4- to 9-fold more
slowly than Ap

4
A. As observed for a series of bb¢-
substituted Ap
4
A analogs, their efficiencies as
substrates of the Ap
4
A hydrolase from Artemia
increased in direct proportion to increasing electroneg-
ativity [22]. Guranowski et al. [21] found that those
compounds were not substrates of the symmetrically
acting Ap
4
A hydrolase from E. coli, but later work by
McLennan et al. [22] reported that AppCH
2
ppA,
AppCF
2
ppA and AppCHFppA underwent slow
hydrolysis using their preparation of bacterial enzyme,
with 25-, 50- and 125-fold reduced rates, respectively,
compared with that of Ap
4
A hydrolysis.
In this report we describe, first, the chemical synthe-
sis of new Ap
n
A analogs with a methyleneoxy or an
oxymethylene bridge that substitutes for one or two

oxygen(s) in the tetrapolyphosphate chain (structures
shown in Fig. 1). Second, we present the results of
enzymatic studies on these novel analogs as potential
substrates and/or inhibitors of two asymmetrically
acting Ap
4
A hydrolases [bis(5 ¢-nucleosyl)-tetraphos-
phatase (asymmetrical); EC 3.6.1.17], from human [24]
and narrow-leafed lupin [25], and on the Co
2+
-depen-
dent symmetrically acting dinucleoside tetraphospha-
tase [bis(5¢-nucleosyl)-tetraphosphatase (asymmetrical);
EC 3.6.1.41] from E. coli [26].
Results and Discussion
Recognition of Ap
n
A oxymethylene analogs by
Ap
4
A hydrolases
In this study we questioned how specific Ap
4
A hydro-
lases might recognize substrate analogs that are
Fig. 1. Structures of oxymethylene and
methyleneoxy analogs of diadenosine
polyphosphates.
A. Guranowski et al. Oxymethylene diadenosine polyphosphate analogs
FEBS Journal 276 (2009) 1546–1553 ª 2009 The Authors Journal compilation ª 2009 FEBS 1547

nonisosteric (the P–P distance is one atom longer), yet
isoelectronic (charge identical), in comparison with
natural Ap
n
As. To answer this question, we performed
studies on the interaction of the enzymes with the
aforementioned oxymethylene analogs of Ap
n
A. When
analyzing the reaction mixtures in the TLC system that
separates each of the analogs tested, as a potential sub-
strate, from possible reaction products, we found that
none of the six new Ap
n
A analogs was a substrate of
the symmetrically acting Ap
4
A hydrolase. Each analog
(0.5 mm) was incubated at 30 °C in 0.05 mL of the
reaction mixture containing 50 mm Hepes/KOH
(pH 7.6), 0.02 mm dithiothreitol and 5 mm MgCl
2
, for
up to 16 h with an amount of enzyme sufficient to
achieve complete cleavage of 0.5 mm Ap
4
Ain
< 15 min. This result is consistent with previously
published results [20–23], which established that the
hydrolase from E. coli shows almost no cleavage of

dinucleoside polyphosphate molecules modified in their
ADP moieties. In addition, none of the oxymethylene
analogs investigated inhibited the hydrolysis of Ap
4
A
catalyzed by the E. coli enzyme. As shown earlier [20–
22], some methylene or halomethylene analogs of
Ap
4
A inhibited that bacterial enzyme quite effectively,
with K
i
values even one order of magnitude lower than
the K
m
for Ap
4
A [20]. This study thus establishes that
the symmetrical Ap
4
A hydrolase does not tolerate
single (i.e. 3) or multiple (i.e. 1, 2, 4–6) atom inserts in
the polyphosphate backbone of the six dinucleoside-
oligophosphate analogs.
By contrast, when the same six novel Ap
n
A analogs
were tested as potential substrates of the asymmetri-
cally acting Ap
4

A hydrolases, compounds 3 and 5 were
readily hydrolyzed. This was demonstrated both for
the human and the plant enzymes, and the reaction
products were clearly identified by comparing them
with AMP and synthetic oxymethylene analogs of
ADP or ATP. In addition to TLC analysis, we also
used an HPLC system (see the example of elution pro-
files in Figs 2A,B) that effectively separated potential
substrates from possible products and thus could be
used to estimate the relative velocities of the hydrolysis
reactions (Table 1). The asymmetric analog 3 was first
hydrolyzed by both asymmetric hydrolases to AMP
and the bc-methyleneoxy-ATP (32) (Fig. 3A), and then
the latter, relatively unstable, nucleotide hydrolyzed
spontaneously to give a second AMP.
An alternative cleavage of analog 3 to AMP and
bc-oxymethylene-ATP (18) was also observed. For the
human asymmetric hydrolase this mode of cleavage
was approximately six times less frequent than the
dominant mode and in the case of the lupin enzyme it
was over 20 times slower. Such slower cleavage to give
18 could arise either from weaker binding of 3 in the
active site of the hydrolase in the reverse orientation
(Fig. 3B) or from a reduced rate of cleavage. While
A
B
Fig. 2. Time course of ApOpCH
2
OpOpA hydrolysis catalyzed by
narrow-leaved lupin Ap

4
A hydrolase and monitored by (A) HPLC
and (B) chromatography of standards. The profiles shown in (A) are
for reaction mixtures (0.1 mL) containing 50 m
M Hepes/KOH
(pH 7.6), 0.02 m
M dithiothreitol, 5 mM MgCl
2
, 0.5 mM substrate
and rate-limiting amounts of the asymmetrically acting Ap
4
A hydro-
lase – incubated at 30 °C. At specific time points (0, 5, 10, 15 and
20 min), 10-lL aliquots were withdrawn, added to 0.15 mL of
0.1
M KH
2
PO
4
(pH 6.0) and the reaction was heat-quenched (3 min
at 96 °C). After centrifugation, samples were filtered and aliquots
(0.1 mL) were subjected to HPLC on a Discovery C18 column
(4.6 · 250 mm, 5 lm; Supelco); flow rate 1 mLÆmin
)1
. Gradient elu-
tion was performed with 0.1
M KH
2
PO
4

, pH 6.0 (solvent A); solvent
A/methanol (9 : 1, v/v) (solvent B): 0–9 min, 0% B; 9–15 min, 25%
B; 15–17.5 min, 90% B; 17.5–19 min, 100% B; 19–23 min, 100%
B and 23–35 min, 0% B. Profiles in (B) show standards run under
identical conditions.
Oxymethylene diadenosine polyphosphate analogs A. Guranowski et al.
1548 FEBS Journal 276 (2009) 1546–1553 ª 2009 The Authors Journal compilation ª 2009 FEBS
the pK
a
values for the ATP analogs released (32 and
18) have not yet been determined, it is reasonable to
assume that a pK
a
value of 4 for 32 is similar to that
of ATP (ca. 7.1), whereas that for 18 will be similar to
that of bc-methylene-ATP (ca. 8.2. [27]). The asym-
metrical pyrophosphohydrolase from Artemia is known
to exhibit a strong dependence on the rate of cleavage
on the pK
a
of the leaving group (Brønsted coefficient
0.5 [22]). A similar b-leaving group-dependence for the
human and lupin enzymes studied here would lead to
a reduction in rate of about 10-fold for the formation
of 18 relative to that of 32. Thus, the present kinetic
results do not provide any evidence for differential rec-
ognition of the alternative orientations on the P-O-C-P
bridge for these two enzymes.
The enzymatic hydrolysis of symmetrical analog 5,
by both human and plant asymmetric hydrolases,

yielded only ab-oxymethylene ADP (24) and at rates
that were reduced relative to the cleavage of 3
(Table 1). This mode of cleavage is a further example
of a frameshift mechanism (Fig. 3C), akin to that
shown in the action of the asymmetrical Artemia
hydrolase on some ab,a¢b¢-disubstituted analogs of
Ap
4
A (e.g. ApCHFppCHFpA was cleaved at 3% of
the rate of AppppA) [22]. They constitute a symmet-
rical mode of cleavage of 5 by water attack at P
b
.
The failure of these hydrolases to bring about a simi-
lar frameshift symmetrical hydrolysis of 4 is quite
remarkable (Fig. 3D). It appears to indicate that
there is specific recognition of the orientation of the
P-O-C-P linkage in the a,b active site. Taken
together, the results of cleavage of compounds 3 and
5 show that the asymmetrically acting Ap
4
A hydro-
lases can reach the scissile bond either by extending
‘the frame’, as in the case of compound 3,orby
shortening the count, when attacking the P
b
-O-P
b¢
bond of compound 5.
As established previously [12], the hydrolases do not

recognize dinucleoside triphosphates as substrates.
Thus, it was to be expected that the oxymethylene ana-
logs of Ap
3
A – compounds 1 and 2 – would not be
degraded. The absence of any detectable hydrolysis of
compounds 4 and 6 suggests that the enzymes tolerate
neither a -CH
2
-P
a
- sequence, which occurs in 4, nor a
-CH
2
-P
c
-CH
2
- sequence, as in 6. Apparently, ‘the
frameshift’ is unable to accommodate two oxymethyl-
ene inserts, as occurs in 6.
Finally, we investigated whether the novel Ap
n
A
analogs inhibit Ap
4
A hydrolysis catalyzed by the
asymmetrically acting Ap
4
A hydrolases. Only analogs

3 and 4 acted as competitive inhibitors, with K
i
values
of 2.2 lm (3) and 1.5 lm (4) for the lupin enzyme and
of 2.1 lm (3) and 2.5 lm (4) for the human counter-
part. These K
i
values lie in the range of the K
m
values
for Ap
4
A: 2.5 lm for the narrow-leaved lupin [25] and
2 lm for the human enzyme [16].
Table 1. Comparison of the hydrolysis of AppppA and its oxymeth-
ylene analogs catalyzed by two asymmetrically acting AppppA
hydrolases. Velocities were calculated from the time-course of the
decrease of the substrate-peak area, as shown on the HPLC pro-
files exemplified in Fig. 2a. Arrows above substrate formulas indi-
cate sites of cleavage. Compound 3 was degraded six times faster
by the human hydrolase, and 20 times faster by the lupin hydro-
lase, to AMP and pOCH
2
pOpA (large arrow) than to AMP and
pCH
2
OpOpA (small arrow).
Potential substrate
Relative velocities
for AppppA

hydrolase from
human
Narrow-leaved
lupin
Ap
fl
pppA 1 1
ApCH
2
OpOCH
2
pA (1)0 0
ApOCH
2
pCH
2
OpA (2)0 0
Ap
fl
OpCH
2
OpO
fl
pA (3) 0.48 0.92
ApCH
2
OpOpOCH
2
pA (4)0 0
ApOCH

2
pO
fl
pCH
2
OpA (5) 0.18 0.54
ApOpOCH
2
pCH
2
OpOpA (6)0 0
A
B
C
D
Fig. 3. Comparison of binding and modes of reactivity of dinucleo-
tides 3, 4 and 5 by the asymmetrically acting Ap
4
A hydrolases. (A)
Major cleavage of 3 to bc-methyleneoxy-ATP; (B) minor cleavage of
3 to bc-oxymethylene-ATP; (C) frameshift cleavage of 5 to ab-oxym-
ethylene-ADP; and (D) stability to frameshift cleavage of 4.
A. Guranowski et al. Oxymethylene diadenosine polyphosphate analogs
FEBS Journal 276 (2009) 1546–1553 ª 2009 The Authors Journal compilation ª 2009 FEBS 1549
Conclusion
The results of binding and cleavage studies on the six
Ap
n
A analogs described here by the three pyrophos-
phohydrolases establish the general utility and the limi-

tations of the P-O-C-P bridge as a surrogate for
pyrophosphate in nucleotides. First, exactly as
expected, none of the three enzymes can cleave the
P–O bond in the P-O-CH
2
-P linkage. Second, the
asymmetric cleaving enzymes accept the P-O-C-P
bridge in the position adjacent to the P-O-P cleavage
locus in either orientation. Third, hindrance of normal
P-O-P cleavage can lead to a frameshift response, even
though this involves a three-atom shift, but only for
one orientation of the P-O-C-P insert. Lastly, the
asymmetric hydrolases accept the P-O-C-P inserts as
competitive inhibitors, whereas the bacterial symmetri-
cal hydrolase does not. Thus, these novel compounds
will be tools of specific application for studies on the
metabolism of dinucleoside polyphosphates and on
Ap
4
A-degrading enzymes and they also merit further
attention for the investigation of nucleotide metabolic
pathways. Kindred studies on the full range of ATP
analogs containing an oxymethylene bridge will be
reported in due course.
Experimental procedures
Enzymes
Homogeneous recombinant asymmetrically acting human
Ap
4
A hydrolase (EC 3.6.1.17) [24] was kindly donated by

A. G. McLennan (University of Liverpool, UK), and the
Ap
4
A hydrolase from narrow-leaved lupin (Lupinus angus-
tifolius) [25] was kindly donated by D. Maksel and K. Gay-
ler (University of Melbourne, Australia). Symmetrically
acting Ap
4
A hydrolase (EC 3.6.1.41) was partially purified
from E. coli [26].
Chemicals
Unlabelled mononucleotides and dinucleotides were from
Sigma (St Louis, MO, USA), and [
3
H]Ap
4
A (740 TBqÆ
mol
)1
) was purchased from Moravek Biochemicals (Brea,
CA, USA). Syntheses leading to the novel oxymethylene
analogs of ADP, ATP and Ap
n
A are described below.
Chromatographic systems
Analyses of the hydrolysis of Ap
4
A and its analogs were
performed on TLC aluminum plates precoated with silica
gel containing fluorescent indicator (Merck Cat. no. 5554),

which was developed in dioxane/ammonia/water (6 : 1 : 4,
v/v/v).
Enzyme assays
Estimation of the reaction rates and calculation of the K
i
values for the analogs with the use of radiolabeled Ap
4
A
were performed as described previously [16]. Relative rates
of the hydrolysis of dinucleotide substrates and analogs
were estimated by the use of HPLC on a reverse-phase col-
umn (for details see the legend to Fig. 2a) and were based
on peak-area analysis.
Synthesis of oxymethylene and methyleneoxy
analogs of ADP, ATP and Ap
n
A
ADP, ATP and Ap
n
A analogs with one -OCH
2
- or -CH
2
O-
group that substitutes for a bridging oxygen in adenosine or
diadenosine oligophosphates have not been synthesized pre-
viously. The tripolyphosphate analog, pOCH
2
pCH
2

Op, has
been bound to two adenosines yielding an analog of Ap
3
A
[28] but hitherto similar analogs of Ap
4
AorAp
5
A have
not been made. We prepared ab-methyleneoxy-ADP
(pOCH
2
pA) (21) and ab-oxymethylene-ADP (pCH
2
OpA)
(24), ab-methyleneoxy-ATP (pOpOCH
2
pA), ab-oxymethyl-
ene-ATP (pOpCH
2
OpA), bc-oxymethylene-ATP (pCH
2
-
OpOpA) (18), the unstable, bc-methyleneoxy-ATP
(pOCH
2
pOpA) (32), and the six Ap
n
A analogs investigated
in this study: ab,a¢b-bis(methyleneoxy)Ap

3
A (ApCH
2
Op-
OCH
2
pA) (1), ab,a¢b-bis(oxymethylene)Ap
3
A (ApOCH
2
pC-
H
2
OpA) (2), bb¢-methyleneoxy-Ap
4
A (ApOpCH
2
OpOpA)
(3), ab,a¢b¢-bis(methyleneoxy)Ap
4
A (ApCH
2
OpOpOCH
2
pA)
(4), ab,a¢b¢-bis(oxymethylene)Ap
4
A (ApOCH
2
pOpCH

2
OpA)
(5) and bc,b¢c-bis(oxymethylene) Ap
5
A (ApOpOCH
2
pCH
2
OpOpA) (6). The terminology used supports recogni-
tion of the orientation of oxygen components and methylene
components of the oxymethylene bridges in the analogs with
respect to their adenosine moieties.
Details of the syntheses will be published elsewhere, and
we present, in the Supporting information, only the key
steps leading to the formation of Ap
n
A analogs 1–6.
Acknowledgements
This work was supported by the Polish Ministry of
Science and Higher Education, grant PBZ-MNiSW-07/
I/2007 (to A. G.) and by a grant from the Wellcome
Trust (to G. M. B.).
References
1 Garrison PN & Barnes LD (1992) Determination of
dinucleoside polyphosphates. In Ap4A and Other
Dinucleoside Polyphosphates (McLennan AG, ed.), pp.
29–61. CRC Press, Boca Raton, FL.
2 Hoyle CHV, Hilderman RH, Pintor JJ, Schlu
¨
ter H &

King BF (2001) Diadenosine polyphosphates as extra-
cellular signal molecules. Drug Dev Res 52, 260–273.
Oxymethylene diadenosine polyphosphate analogs A. Guranowski et al.
1550 FEBS Journal 276 (2009) 1546–1553 ª 2009 The Authors Journal compilation ª 2009 FEBS
3 McLennan AG, Barnes LD, Blackburn GM, Brenner
Ch, Guranowski A, Miller AD, Rovira JM, Rotlla
´
nP,
Soria B, Tanner JA et al. (2001) Recent progress in the
study of the intracellular functions of diadenosine
polyphosphates. Drug Dev Res 52, 249–259.
4 McLennan AG (2000) Dinucleoside polyphosphates –
friend or foe? Pharmacol Ther 87, 73–89.
5 Zamecnik PC, Stephenson ML, Janeway CM & Rande-
rath K (1966) Enzymatic synthesis of diadenosine tetra-
phosphate and diadenosine triphosphate with a purified
lysyl-sRNA synthetase. Biochem Biophys Res Commun
24, 91–97.
6 Sillero A & Gu
¨
nther Sillero MA (2000) Synthesis of
dinucleoside polyphosphates catalyzed by firefly lucifer-
ase and several ligases. Pharmacol Ther 87, 91–102.
7 Fontes R, Gu
¨
nther Sillero MA & Sillero A (1998) Acyl
coenzyme A synthetase from Pseudomonas fragi cata-
lyzes the synthesis of adenosine 5¢-polyphosphates and
dinucleoside polyphosphates. J Bacteriol 180, 3152–
3158.

8 Pietrowska-Borek M, Stuible H-P, Kombrink E &
Guranowski A (2003) 4-Coumarate:coenzyme A ligase
has the catalytic capacity to synthesize and reuse
various (di)adenosine polyphosphates. Plant Physiol
131, 1401–1410.
9 Guranowski A, Gu
¨
nther Sillero MA & Sillero A (1990)
Firefly luciferase synthesizes P
1
,P
4
-bis(5¢-adenosyl)tetra-
phosphate (Ap
4
A) and other dinucleoside polyphos-
phates. FEBS Lett 271, 215–218.
10 Guranowski A, Just G, Holler E & Jakubowski H
(1988) Synthesis of diadenosine 5¢,5¢¢¢-P
1
,P
4
-tetraphos-
phate (AppppA) from adenosine 5¢-phosphosulfate and
adenosine 5¢-triphosphate catalyzed by yeast AppppA
phosphorylase. Biochemistry 27, 2959–2964.
11 Guranowski A, de Diego A, Sillero A & Gu
¨
nther Sille-
ro MA (2004) Uridine 5¢-polyphosphates (p

4
U and
p
5
U) and uridine(5¢)polyphospho(5¢)nucleosides
(Up
n
Ns) can be synthesized by UTP:glucose-1-phos-
phate uridylyltransferase from Saccharomyces cerevisiae.
FEBS Lett 561, 83–88.
12 Guranowski A (2000) Specific and nonspecific
enzymes involved in the catabolism of mononucleoside
and dinucleoside polyphosphates. Pharmacol Ther 87,
117–139.
13 Guranowski A (2003) Analogs of diadenosine tetra-
phosphate (Ap
4
A). Acta Biochim Polon 50, 947–972.
14 Lazewska D & Guranowski A (1990) P
a
-Chiral phosp-
horothioate analogues of bis(5¢-adenosyl)tetraphosphate
(Ap
4
A); their enzymatic synthesis and degradation.
Nucleic Acids Res 18, 6083–6088.
15 Bailey S, Sedelnikova S, Blackburn GM, Abdelghany
HM, Baker PJ, McLennan AG & Rafferty JB (2002)
The crystal structure of diadenosine tetraphosphate
hydrolase from Caenorhabditis elegans in free and

binary complex forms. Structure 10, 589–600.
16 Guranowski A, Starzyn
´
ska E, McLennan AG, Baraniak
J & Stec WJ (2003) Adenosine-5¢-O-phosphorylated and
adenosine-5¢-O-phosphorothioylated polyols as strong
inhibitors of (symmetrical) and (asymmetrical) dinucleo-
side tetraphosphatases. Biochem J 373, 635–640.
17 Guranowski A, Starzyn
´
ska E, Pietrowska-Borek M,
Jemielity J, Kowalska J, Darzynkiewicz E, Thompson
MJ & Blackburn GM (2006) Methylene analogues of
adenosine 5¢-tetraphosphate; their chemical synthesis
and recognition by human and plant mononucleoside
tetraphosphatases and dinucleoside tetraphosphatases.
FEBS J 273, 829–838.
18 Kim BK, Zamecnik P, Taylor G, Guo MJ & Blackburn
GM (1992) Antithrombotic effect of b,b¢-monochlorom-
ethylene diadenosine 5¢,5¢¢¢-P
1
,P
4
-tetraphosphate. Proc
Natl Acad Sci USA 89, 11056–11058.
19 Elmaleh DR, Fischman AJ, Tawakol A, Zhu A, Shoup
TM, Hoffmann U, Brownell A-L & Zamecnik PC
(2006) Detection of inflamed atherioisclerotic lesions
with diadenosine 5¢,5¢¢¢-P
1

,P
4
-tetraphosphate (Ap
4
A)
and positron-emission tomography. Proc Natl Acad Sci
USA 103, 15992–15996.
20 Guranowski A, Biryukov A, Tarussova NB, Khomutov
RM & Jakubowski H (1987) Phosphonate analogues of
diadenosine 5¢,5¢¢¢-P
1
,P
4
-tetraphosphate as substrates
or inhibitors of prokaryotic and eucaryotic enzymes
degrading dinucleoside tetraphosphates. Biochemistry
26, 3425–3429.
21 Guranowski A, Starzyn
´
ska E, Taylor GE & Blackburn
GM (1989) Studies on some specific Ap
4
A-degrading
enzymes with the use of various methylene analogues of
P
1
,P
4
-bis(5¢,5¢¢¢-adenosyl) tetraphosphate. Biochem
J 262, 241–244.

22 McLennan AG, Taylor GE, Prescott M & Blackburn
GM (1989) Recognition of bb¢-substituted and ab,a¢b¢-
disubstituted phosphonate analogues of bis(5¢-adenosyl)
tetraphosphate by the bis(5¢-nucleosidyl)-tetraphosphate
pyrophosphohydrolases from Artemia embryos and
Escherichia coli. Biochemistry 28, 3868–3875.
23 Guranowski A, Brown P, Ashton PA & Blackburn GM
(1994) Regiospecificity of the hydrolysis of diadenosine
polyphosphates catalyzed by three specific pyrophos-
phohydrolases. Biochemistry 33, 235–240.
24 Thorne NMH, Hankin S, Wilkinson MC, Nu´ n
˜
ez C,
Barraclaugh R & McLennan AG (1995) Human diade-
nosine 5¢,5¢¢¢-P
1
,P
4
-tetraphosphate phyrophosphohydro-
lase is a member of the MutT family of nucleotide
pyrophosphohydrolases. Biochem J 311, 717–721.
25 Maksel D, Gooley PR, Swarbrick JD, Guranowski A,
Gange Ch, Blackburn GM & Gayler KR (2001) Char-
acterization of active residues in diadenosine tetraphos-
phate hydrolase from Lupinus angustifolius. Biochem J
357, 399–405.
26 Guranowski A, Jakubowski H & Holler E (1983)
Catabolism of diadenosine 5¢,5¢¢¢-P
1
,P

4
-tetraphosphate
A. Guranowski et al. Oxymethylene diadenosine polyphosphate analogs
FEBS Journal 276 (2009) 1546–1553 ª 2009 The Authors Journal compilation ª 2009 FEBS 1551
in prokaryotes; purification and properties of diadeno-
sine 5¢,5¢¢¢-P
1
,P
4
-tetraphosphate (symmetrical) pyro-
phosphohydrolase from Escherichia coli K12. J Biol
Chem 258, 14784–14789.
27 Blackburn GM, Kent DA & Kolkmann F (1981) Three
new bc-methylene analogues of adenosine triphosphate.
JCS Chem Commun 1188–1190.
28 Walkowiak B, Baraniak J, Cierniewski Cz S & Stec W
(2002) Inhibition of ADP-triggered blood platelet aggre-
gation by diadenosine polyphosphate analogues. Bioorg
Med Chem Lett 12, 1959–1962.
29 Rejman D, Olesiak M, Chen LQ, Patterson SE, Wilson
D, Jayaram HN, Hedstrom L & Pankiewicz K (2006)
Novel methylenephosphophosphonate analogues of
mycophenolic adenine dinucleotide. Inhibition of
inosine monophosphate dehydrogenase. J Med Chem
49, 5018–5022.
30 Rejman D, Masojı
´
dkova
´
M & Rosenberg I (2004)

Nucleosidyl-O-methylphosphonates: a pool of mono-
mers for modified oligonucleotides. Nucleosides Nucleo-
tides Nucleic Acids 23, 1683–1705.
31 Himmelsbach F, Schulz BS, Trichtinger T, Charubala
R & Pfleiderer W (1984) The para-nitrophenylethyl
(NPE) group – a versatile new blocking group for phos-
phate and aglycone protection in nucleosides and nucle-
otides. Tetrahedron 40, 59–72.
32 Stengele KP & Pfleiderer W (1990) Improved synthesis
of oligodeoxyribonucleotides. Tetrahedron Lett 31,
2549–2552.
33 Bannwarth W & Trzeciak A (1987) A simple and effec-
tive chemical phosphorylation procedure for biomole-
cules. Helvetica Chim Acta 70, 175–186.
34 Pirat JL, Brahic C, Ciptadi C, Cristau HJ, Herve A &
Virieux D (2002) Bis(hydroxymethyl)phosphine oxides
and hydroxymethyl phosphinic acids as phosphonic acid
analogs. Phosphorus Sulfur Silicon Relat Elem 177,
2221–2222.
35 Nun
˜
ez A, Berroteran D & Nun
˜
ez O (2003) Hydrolysis
of cyclic phosphoramidates. Evidence for syn lone pair
catalysis. Org Biomol Chem 1, 2283–2289.
Supporting information
The following supplementary material is available:
Scheme S1. ApCH
2

OpOCH
2
pA (1) Monobenzyl
phosphonate 8 [29] was esterified with tetrabenzoylade-
nosine 7 [29] using either 2-chloro-5,5-dimethyl-
2-oxido-1,3,2-dioxaphosphinane (NEP)/methoxypyri-
dine-N-oxide/pyridine system [30–32] or Mitsunobu
conditions (Scheme 1). The dimethoxytrityl (DMTr)
group of phosphonate 9 was removed with acetic acid
giving compound 10. Phosphoramidite generated by the
reaction of phosphonate 10 with benzyloxybis(diisopro-
pylamino)phosphine [33] reacted with a second molecule
of 10 to produce the fully protected symmetrical Ap
3
A
analog 11. Target compound 1 was obtained by two-
step deprotection and DEAE-Sephadex column chroma-
tography using a linear gradient of TEAB in water.
Benzyl esters were removed by catalytic hydrogenation
followed by aqueous ammonia treatment to remove
benzoyl protecting groups.
Scheme S2. ApOCH
2
pCH
2
OpA (2) Tetrabenzoyl aden-
osine 7 was converted into phosphoramidite 12 by
reaction with benzyloxybis(diisopropylamino)phos-
phine [33] (Scheme 2). Phosphoramidite 12 underwent
reaction with benzyl bis(hydroxymethane)phosphinate

13 providing fully protected symmetrical Ap
3
A analog
14. Final compound 2 was obtained by two-step
deprotection and DEAE Sephadex column chromato-
graphy using a linear gradient of TEAB in water.
Scheme S3. ApOpCH
2
OpOpA (3) The tributyla-
monium salt of AMP (15) was reacted with phospho-
morpholidate 16 in dimethylsulfoxide. Dibenzyl ester
17 was hydrogenolyzed to give ATP analogue 18 puri-
fied by DEAE-Sephadex chromatography. Reaction of
18 with AMP morpholidate 19 led to target product 3
after DEAE-Sephadex column chromatography using
a linear gradient of TEAB in water.
Scheme S4. ApCH
2
OpOpOCH
2
pA (4) Adenosine phos-
phonate 10 (v.s.) was reacted with bis-benzyloxy-(diiso-
propylamino)phosphine [33] with tetrazole catalysis
and, after 4-chloroperoxybenzoic acid (MCPBA) oxida-
tion, afforded compound 20 (Scheme 4). Compound
20 was debenzylated by catalytic hydrogenolysis and
dimerized using dicyclohexylcarbodiimide (DCC) in
pyridine. Target compound 4 was obtained pure by
DEAE-Sephadex column chromatography.
Scheme S5. ApOCH

2
pOpCH
2
OpA (5) Phosphorami-
dite 12 was reacted with dibenzyl phosphonate 22
using tetrazole catalysis and, after MCPBA oxidation,
afforded compound 23 (Scheme 6). ADP analogue 24,
obtained by catalytic hydrogenation of 23, was dimer-
ized using DCC in pyridine giving, after DEAE-Sepha-
dex column chromatography, target Ap
4
A analog 5.
Scheme S6. ApOpOCH
2
pCH
2
OpOpA (6) Benzyl phos-
phinate 13, after treatment with bis-benzyloxy-(diiso-
propylamino)phosphine [33] using tetrazole catalysis
and MCPBA oxidation, gave compound 25 (Scheme 5).
Catalytic hydrogenation of 25 gave bis(hydroxymethyl-
enephosphinic acid) phosphate 26 which underwent
condensation with morpholidate 19 to give, after
DEAE-Sephadex column purification, the target Ap
5
A
analogue 6.
Scheme S7. pOCH
2
pOpA (32) Bis(2-cyanoethyloxy)(di-

isopropylamino)phosphine (27) [33] was reacted with
dibenzyl phosphonate 22 and subsequently with benzyl
Oxymethylene diadenosine polyphosphate analogs A. Guranowski et al.
1552 FEBS Journal 276 (2009) 1546–1553 ª 2009 The Authors Journal compilation ª 2009 FEBS
alcohol. After MCPBA oxidation and catalytic hydro-
genation cyanoethyl pyrophosphate analog 30 was
obtained (Scheme 7). Pyrophosphate analogue 30
underwent standard reaction with adenosine 5¢-phosp-
horomorpholidate 19. After aqueous ammonia depro-
tection of the cyanoethyl group, and DEAE-Sephadex
column purification, the target ATP analog 32 was
obtained.
Scheme S8. Syntheses of reagents 13 and 16: Benzyl
bis(hydroxymethane)phosphonate (13) Bis(hydroxyme-
thane)phosphinic acid 33 [34] was reacted with dimeth-
oxytrityl chloride in pyridine to give 34 which was
subsequently esterified with benzyl alcohol employing
NEP/methoxypyridine-N-oxide/pyridine system [29–31].
The benzyl ester 35 obtained was detritylated with
80% aqueous acetic acid to give 13.
Scheme S9. (Bis(benzyloxy)phosphoryl)methyl hydro-
gen morpholinophosphonate (16) Morpholinophos-
phonic dichloride 36 [35] was treated first with one
equivalent of water in pyridine to afford a reactive
species that subsequently underwent reaction with
dibenzyl hydroxymethanphosphinate 24 to afford the
desired reagent 16.
The preparations of Ap
n
A and ATP analogs

described above employed two main synthetic
approaches. Phosphoramidite condensations appeared
as the ideal method and gave excellent yields. Phosp-
horomorpholidate condensation proved to be an alter-
native method and gave moderate to good yields.
While DCC couplings appeared useful, they gave lower
yields. Using the combination of base-labile benzoyl
and hydrogenolytically-removable benzyl groups
proved to be compatible with rather unstable poly-
phosphate products. The structures of all compounds
prepared were established by a combination of
1
H and
31
P NMR and high resolution mass spectroscopy (data
not shown).
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A. Guranowski et al. Oxymethylene diadenosine polyphosphate analogs
FEBS Journal 276 (2009) 1546–1553 ª 2009 The Authors Journal compilation ª 2009 FEBS 1553