Báo cáo khoa học: Methylene analogues of adenosine 5¢-tetraphosphate Their chemical synthesis and recognition by human and plant mononucleoside tetraphosphatases and dinucleoside tetraphosphatases pot

Methylene analogues of adenosine 5¢-tetraphosphate
Their chemical synthesis and recognition by human and plant
mononucleoside tetraphosphatases and dinucleoside
tetraphosphatases
Andrzej Guranowski
1
,El
_
zbieta Starzyn
´
ska
1
, Małgorzata Pietrowska-Borek
1
, Jacek Jemielity
2
,
Joanna Kowalska
2
, Edward Darzynkiewicz
2
, Mark J. Thompson
3
and G. Michael Blackburn
3
1 Department of Biochemistry and Biotechnology, Agricultural University, Poznan
´
, Poland
2 Department of Biophysics, Institute of Experimental Physics, Warsaw University, Poland
3 Department of Chemistry, Krebs Institute, University of Sheffield, UK
Keywords

adenosine 5¢-tetraphosphate; p
4
A;
methylene analogues of p
4
A; nucleoside
tetraphosphatase; dinucleoside
tetraphosphatase
Correspondence
A. Guranowski, Katedra Biochemii i
Biotechnologii, Akademia Rolnicza ul.
Wołyn
´
ska 35, 60–637 Poznan
´
, Poland
Fax: +48 61 8487146
Tel: +48 61 8487201
E-mail:
Website:
Note
This study is dedicated to Professor
Wojciech J. Stec on the occasion of his
65th birthday.
(Received 9 November 2005, revised
15 December 2005, accepted 21 December
2005)
doi:10.1111/j.1742-4658.2006.05115.x
Adenosine 5¢-polyphosphates have been identified in vitro, as products of
certain enzymatic reactions, and in vivo. Although the biological role of these

compounds is not known, there exist highly specific hydrolases that degrade
nucleoside 5¢-polyphosphates into the corresponding nucleoside 5¢-triphos-
phates. One approach to understanding the mechanism and function of
these enzymes is through the use of specifically designed phosphonate
analogues. We synthesized novel nucleotides: a,b-methylene-adenosine
5¢-tetraphosphate (pppCH
2
pA), b,c-methylene-adenosine 5¢-tetraphosphate
(ppCH
2
ppA), c,d-methylene-adenosine 5¢-tetraphosphate (pCH
2
pppA),
ab,cd-bismethylene-adenosine 5¢-tetraphosphate (pCH
2
ppCH
2
pA), ab,
bc-bismethylene-adenosine 5¢-tetraphosphate (ppCH
2
pCH
2
pA) and bc,
cd-bis(dichloro)methylene-adenosine 5¢-tetraphosphate (pCCl
2
pCCl
2
ppA),
and tested them as potential substrates and ⁄ or inhibitors of three specific nu-
cleoside tetraphosphatases. In addition, we employed these p

4
A analogues
with two asymmetrically and one symmetrically acting dinucleoside tetra-
phosphatases. Of the six analogues, only pppCH
2
pA is a substrate of the
two nucleoside tetraphosphatases (EC 3.6.1.14), from yellow lupin seeds and
human placenta, and also of the yeast exopolyphosphatase (EC 3.6.1.11).
Surprisingly, none of the six analogues inhibited these p
4
A-hydrolysing
enzymes. By contrast, the analogues strongly inhibit the (asymmetrical) dinu-
cleoside tetraphosphatases (EC 3.6.1.17) from human and the narrow-leafed
lupin. ppCH
2
ppA and pCH
2
pppA, inhibited the human enzyme with K
i
val-
ues of 1.6 and 2.3 nm, respectively, and the lupin enzyme with K
i
values of
30 and 34 nm, respectively. They are thereby identified as being the strongest
inhibitors ever reported for the (asymmetrical ) dinucleoside tetraphospha-
tases. The three analogues having two halo ⁄ methylene bridges are much less
potent inhibitors for these enzymes. These novel nucleotides should prove
valuable tools for further studies on the cellular functions of mono- and di-
nucleoside polyphosphates and on the enzymes involved in their metabolism.
Abbreviations

Ap
3
A, diadenosine 5¢,5¢¢¢-P
1
,P
3
-triphosphate; Ap
4
A, diadenosine 5¢,5¢¢¢-P
1
,P
4
-tetraphosphate; Np
n
N¢, dinucleoside 5¢,5¢¢¢-P
1
,P
n
-polyphosphate;
p
4
A, adenosine 5¢-tetraphosphate; p
5
A, adenosine 5¢-pentaphosphate; pCCl
2
pCCl
2
ppA, bc,cd-bis(dichloro)methylene-adenosine 5¢-
tetraphosphate; pCH
2

ppCH
2
pA, ab,cd-bismethylene-adenosine 5¢-tetraphosphate; pCH
2
pppA, c,d-methylene-adenosine 5¢-tetraphosphate;
p
n
N, nucleoside 5¢-polyphosphate; ppCH
2
pCH
2
pA, ab,bc-bismethylene-adenosine 5¢-tetraphosphate; pppCH
2
pA, a,b-methylene-adenosine
5¢-tetraphosphate; pppCH
2
ppA, b,c-methylene-adenosine 5¢-tetraphosphate.
FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS 829
In addition to the canonical nucleoside mono-, di-,
and triphosphates, cells contain various minor nucleo-
tides. Among these are the nucleoside 5¢-polyphos-
phates (p
n
Ns, where n ¼ 4), such as adenosine
5¢-tetraphosphate (p
4
A or ppppA) [1–5] and adenosine
5¢-pentaphosphate (p
5
A or pppppA) [2], and the dinu-

cleoside 5¢,5¢¢¢-P
1
,P
n
-polyphosphates (Np
n
N¢s, where N
and N¢ are 5¢-O-nucleosides and n represents the num-
ber of phosphate residues in the polyphosphate chain
that links N and N¢ through their 5¢-positions). Typical
examples are diadenosine 5¢,5¢¢¢-P
1
,P
3
-triphosphate
(Ap
3
A) and diadenosine 5¢,5¢¢¢-P
1
,P
4
-tetraphosphate
(Ap
4
A) [6–12]. The biological roles of these Np
n
N¢s
are partially understood. In particular, Ap
n
A has been

implicated in various intracellular processes [13,14] and
also in extracellular signalling [15,16]. By contrast, the
role of p
n
Ns is inadequately recognized. Almost 20
years ago, the accumulation of p
4
A and p
5
A in yeast
was correlated with sporulation [2] and only recently,
p
4
A was identified in human myocardial tissue and
shown to modulate coronary vascular tone [4]. This
compound has also been found as a constituent of the
nucleotide pool present in the aqueous humour of
New Zealand rabbits where it is proposed to act as a
physiological regulator of intraocular pressure in the
normotensive rabbit eye [5].
Enzymatic reactions that can lead to the accumula-
tion of p
4
A and other p
4
Ns in cells fall into three cat-
egories. The first comprises enzymes that catalyse
transfer of a phosphate residue from a phosphate
donor to ATP (e.g. the muscle adenylate kinase) [17].
The second category of enzymes includes those able

to transfer adenylate or nucleotide residue onto tri-
polyphosphates. The pA residue comes either from a
mixed acyl–pA anhydride, as in the case of some li-
gases and firefly luciferase [18–22], or from an
enzyme–pA complex, as in the case of the DNA- and
RNA-ligases [23,24]. Recently, the yeast UTP ⁄ glucose-
1-phosphate uridylyltransferase (EC 2.7.7.9) was
shown to function according to the same pattern and
to synthesize p
4
U by transferring the uridylyl moiety
from UDP-glucose onto tripolyphosphate [25]. The
third category includes several enzymes that degrade
Ap
5
AorAp
6
A yielding p
4
A as one of the reaction
products [26]. Degradation of p
4
A can be controlled
by various nonspecific and specific p
n
N-degrading
enzymes [26,27].
Among studies that shed light on the mechanism of
the action of these phosphohydrolases are investiga-
tions of the interaction of a given enzyme with its sub-

strate analogues. Whereas many analogues of Ap
3
A
and Ap
4
A, modified in the polyphosphate chain, in
adenine(s) or in the ribose moiety(-ies), have been
produced already and tested with numerous enzymes
[28,29], p
4
A analogues have been synthesized only
recently. ab,bc-bismethylene-p
4
A and bc,cd-bis(dichlo-
ro)methylene-p
4
A were tested as agonists or antago-
nists of the P2X
2 ⁄ 3
receptor [30] and a short report has
appeared on the synthesis of pCH
2
pppA, pCH
2
pppG
and pCH
2
pppm
7
G [31].

Here, we describe details on the synthesis of and the
results of enzymatic studies on a series of novel p
4
A
analogues that have a single methylene bridge substitu-
ting one of the three bridging oxygens in the tetraphos-
phate chain, or have two methylene bridges, or contain
two dichloromethylene groups. The structures of these
compounds are shown in Fig. 1. We prepared these
nucleotides for evaluation first, as potential substrates
and ⁄ or inhibitors of three enzymes that hydrolyse the
pyrophosphate bond between the c- and d-phosphates
of p
4
A and second, as inhibitors of two types of
Ap
4
A hydrolase, for which p
4
A itself acts as a
strong inhibitor. The p
4
A-hydrolysing enzymes are the
two highly specific mononucleoside tetraphosphatases
(EC 3.6.1.14), from yellow lupin (Lupinus luteus) seeds
[32] and from human placenta [33], and the yeast
(Saccharomyces cerevisiae) exopolyphosphatase
(EC 3.6.1.11) that can hydrolyse p
4
A to ATP and

phosphate [34]. The Ap
4
A hydrolases investigated are
the two asymmetrically acting ones (EC 3.6.1.17), from
human [35] and from narrow-leaved lupin (Lupinus
angustifolius) [36] that split Ap
4
A into ATP and
AMP, and the Co
2+
-dependent symmetrically acting
Fig. 1. Structures of p
4
A analogues.
Methylene analogues of adenosine 5¢-tetraphosphate A. Guranowski et al.
830 FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS
dinucleoside tetraphosphatase (EC 3.6.1.41) that con-
verts Ap
4
A into two ADPs [37].
Results and Discussion
Comments on the synthesis of p
4
A analogues
The preparation of intermediate ADP and ATP ana-
logues followed standard methods. Their conversion
into p
4
A analogues called for condensation with phos-
phate (for ATP analogues), or with pyrophosphate or

a methylenebisphosphonate (for ADP and ADP ana-
logues). Although a variety of options were explored
initially, the use of phosphoroimidazolates [31] proved
to be the most reliable method and gave satisfactory
yields without detailed optimization (Fig. 2). The prod-
ucts were first, purified by ion-exchange chromato-
graphy on DEAE-Sephadex 25A, which separates
nucleotides according to net charge at pH 7.9, and
readily resolved the desired products as tetra-to-penta
anions from the corresponding reactants (di-to-tetra
anions). Additional reverse-phase chromatography
provided the product p
4
A analogues in high purity.
The MS and
1
H NMR spectra of these nucleotides are
unexceptional. The
31
P NMR spectra, however, pro-
vide examples of ABCD spectra, whose chemical shift
characteristics readily identify the nature and location
of the oxygen and methylene groups bridging the four
phosphorus atoms (see Supplementary material).
Recognition of p
4
A analogues as substrates
by the p
4
A hydrolysing enzymes

Each compound was checked as a potential substrate
for two highly specific nucleoside tetraphosphatases
(EC 3.6.1.14), from yellow lupin seeds and from human,
and for the soluble exopolyphosphatase (EC 3.6.1.11)
that has an inherent capacity to hydrolyse the distal
pyrophosphate bond in p
4
Ns thus acting as a nucleoside
tetraphosphatase. A typical reaction mixture (see
Experimental procedures) contained 1 mm analogue
and excess of enzyme, i.e. an amount that, under the
same conditions, completely hydrolysed 1 mm p
4
Ato
ATP and P
i
in < 15 min. Incubation was for up to 16 h
and the progress of potential hydrolysis was analysed by
TLC System A. Of six p
4
A analogues, only pppCH
2
pA
was susceptible to hydrolysis and the relative velocities
of the reactions were estimated only for the pair
p
4
A ⁄ pppCH
2
pA. Figure 3 shows typical elution pat-

terns of the substrate ⁄ product pairs on the reverse-phase
HPLC column. Satisfactory separation of p
4
A from
ATP was obtained by isocratic elution with potassium
Fig. 2. Chemical synthesis of pppCH
2
pA (A), ppCH
2
ppA (B) and pCH
2
pppA (C). ‘A’ represents adenosine, DMF dimethylformamide, PPh3 tri-
phenylphosphine, TEA triethylammonium, and TEAB triethylammonium bicarbonate.
A. Guranowski et al. Methylene analogues of adenosine 5¢-tetraphosphate
FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS 831
phosphate buffer (Fig. 3A), and of pppCH
2
pA from
ppCH
2
pA by the use of a more complex solvent system
and methanol gradient (Fig. 3B). Integrated peaks of
the products were used for calculating the reaction velo-
cities. As shown in Table 1, the yellow lupin p
4
A
hydrolase and the yeast exopolyphosphatase hydrolysed
pppCH
2
pA slightly more than twofold slower than p

4
A,
and the human p
4
A hydrolase 125-fold slower. This
result shows that (a) the p
4
N hydrolysing enzymes do
not tolerate methylene modification of their substrates
at the scissile P–O–P bond; (b) none of the enzymes
hydrolysed the terminal phosphate residue from
ppCH
2
ppA, or from ppCH
2
pCH
2
pA, in which the
P–O–P bond between c and d phosphate remains
unchanged; (c) the human hydrolase is sensitive to the
–CH
2
– insert even in the most distant position from the
reaction site, i.e. between the a- and b-phosphorus
atoms in pppCH
2
pA. Thus the p
4
N hydrolysing
enzymes are more stringent with respect to recognition

of their substrates than the (asymmetrical)Ap
4
A hydro-
lases, which cleave the P
a
–O–P
b
bridge not only in
Ap
4
A [27], but also in AppCH
2
ppA, AppCF
2
ppA, and
AppCCl
2
ppA [28].
There are obvious reasons why analogues with
proximate methylene bridges should resist cleavage.
For pCH
2
pppA, removal of the terminal phosphate
(largely a dissociative process) is frustrated by the
stability of the P
c
–C–P
d
bridge. For ppCH
2

ppA, its
stability can be attributed, in at least part, to the
impaired leaving group ability of b,c-methyleneATP.
Neither of these explanations accounts for the much
reduced activity of pppCH
2
pA for the human p
4
A
Fig. 3. Time course of p
4
A (A) and pppCH
2
pA (B) hydrolysis catalysed by yeast exopolyphosphatase. Reaction mixtures (0.25 mL) were pre-
pared and incubated as described in the Experimental procedures. Aliquots (0.05 mL) were withdrawn after the indicated time of incubation,
the reaction was stopped by heating (96 °C, 3 min) and 2-lL sample subjected to HPLC on the Supelcosil LC-18-T reverse-phase column
(25 cm · 4.6 mm). Satisfactory separation of ATP from p
4
A (A) was obtained by eluting the column with an isocratic system using 0.1 M
KH
2
PO
4
buffer (pH 6.0), and separation of ppCH
2
pA from pppCH
2
pA (B) when the eluting system was a linear gradient (0–100%) of buffer
A–buffer B, applied within 20 min at the flow rate 1.3 mLÆmin
)1

[buffer A was 0.1 M KH
2
PO
4
+ 0.008 M (CH
3
CH
2
CH
2
CH
2
)
4
N
+
HSO
4
–
, pH 6.0
and buffer B was buffer A: ⁄ methanol (70 : 30 v ⁄ v)].
Table 1. Comparison of the hydrolysis of ppppA and pppCH
2
pA by
specific p
4
A-hydrolysing enzymes The velocities of conversion of
the nucleoside tetraphosphates (0.5 m
M) to corresponding nucleo-
side triphosphates were calculated based on the HPLC profiles

(exemplified in Fig. 3). For each enzyme the velocity of the
pppCH
2
pA hydrolysis was related to that of the ppppA degradation.
Enzyme
Relative velocity
of the pppCH
2
pA
hydrolysis
ppppA hydrolase from human placenta 0.8
ppppA hydrolase from yellow lupin seeds 45
Exopolyphosphatase from the yeast 42
Methylene analogues of adenosine 5¢-tetraphosphate A. Guranowski et al.
832 FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS
hydrolase. It does not seem likely that such an iso-
steric and isopolar analogue [47] of p
4
A could have
a conformational bias that impairs access to the cata-
lytic site of the enzyme by over 100-fold because it
has the strongest affinity for the symmetrically clea-
ving bacterial Ap
4
A hydrolase. This brings into focus
the possibility of direct recognition of the P
a
–O–P
b
bridge by the protein, a possibility that might be

explored by the synthesis and use of the imino ana-
logue, pppNHpA.
Do the analogues inhibit the p
4
A hydrolysing
enzymes?
All three p
4
A hydrolysing enzymes were tested with
each of the six p
4
A analogues to see whether they
inhibit normal hydrolysis of p
4
A. None of the ana-
logues used at concentrations up to 0.5 mm retarded
the conversion of p
4
A(1mm) into ATP. This unex-
pected result suggests that the active sites of these
three enzymes recognize and bind only nucleotides
with tetraphosphate chains having intact P–O–P brid-
ges, even though all of the analogues are formally
isopolar and isosteric to p
4
A [47]. In this regard, it
is noteworthy that recently solved structures for
dUMPNPP in complex with dUTP hydrolases from
Escherichia coli [48] and Mycobacterium tuberculosis
[49] show a key hydrogen bond from a conserved

serine hydroxyl to the imino bridge in the catalyti-
cally active complex, whereas the complex between
the methylene analogue dUMPCPP and the human
enzyme, which cannot form such a hydrogen bond,
is folded into an inactive conformation [J A Tainer,
personal communication].
The methylene analogues of p
4
A as inhibitors
of the (asymmetrical)Ap
4
A hydrolases
Adenosine tetraphosphate itself has been known for a
long time as an effective competitive inhibitor of the
(asymmetrical)Ap
4
A hydrolases. Examples of the
reported inhibition constants are 48 nm for the rat liver
enzyme [50], 30 nm for the enzyme from Ehrlich ascites
tumour cells [51] and 7.5 nm, the lowest value reported
to date, for the enzyme from firefly lanterns [52]. Owing
to such low K
i
values, this nucleotide has been used for
the elution of the (asymmetrical)Ap
4
A hydrolases
adsorbed to dye–ligand affinity columns as homogen-
eous proteins [53,54]. We tested all six methylene and
chloromethylene p

4
A analogues as potential inhibitors
of two (asymmetrical)Ap
4
A hydrolases, from human
and from narrow-leafed lupin, and the results are sum-
marized in Table 2. Of all the analogues, ppCH
2
ppA
and pCH
2
pppA appear to be the strongest inhibitors of
both the human and plant enzymes. The K
i
values esti-
mated for the human enzyme, 1.6 and 2.3 nm, respect-
ively, were over 30- and 20-fold lower than the K
i
estimated for the same enzyme for p
4
A (50 nm). More-
over, these values are five and three times smaller than
the lowest K
i
estimated yet reported (7.5 nm) for the
reaction of Ap
4
A hydrolysis catalysed by the firefly
enzyme [52]. Significantly, the analogue, pppCH
2

pA,
with its methylene bridge in the position most distant
from the reaction site, is  100-fold less potent an
inhibitor than ppCH
2
ppA. Two analogues having two
methylene bridges are generally poorer inhibitors than
those that possess a single methylene group. In every
cases, however, the K
i
values were below the K
m
values
for the Ap
4
A substrate (1 lm for the lupin and 2 lm
for the human hydrolase). Finally, the analogue with
the bulkiest modification, the dichloromethylene
groups, was a rather poor inhibitor with K
i
values
exceeding the K
m
values for Ap
4
A by some 20–50-fold.
Both p
4
A and its two strongly binding methylene ana-
logues inhibited the lupin enzyme 8–20-fold less effect-

ively than they inhibit the human enzyme. The
differential recognition of the ligands by these two
hydrolases may relate to structural differences within
the substrate-binding sites seen in the recently estab-
lished three-dimensional structures of the lupin Ap
4
A
hydrolase [55] and the human enzyme [56]. The
stronger inhibition of the human enzyme by p
4
A
and its analogues may be explained by the more restric-
ted space in the substrate-binding cleft in the lupin
enzyme.
Table 2. Analogues of p
4
A as inhibitors of (asymmetrical) Ap
4
A
hydrolases. The K
m
values for Ap
4
A estimated for the human and
lupin enzyme were 2 lm (this study) and 1 lm [35], respectively.
The K
i
values are means of three independent estimations; stand-
ard errors did not exceed 20%. For details of assays see Experi-
mental procedures.

Human
Narrow-leafed
lupin (Lupinus
angustifolius)
ppppA 0.05 0.40
pppCH
2
pA 0.18 0.36
ppCH
2
ppA 0.0016 0.030
pCH
2
pppA 0.0023 0.034
ppCH
2
pCH
2
pA 1.3 0.07
pCH
2
ppCH
2
pA 0.25 0.62
pCCl
2
pCCl
2
ppA 40 53
ppppRib 10 n.d.

ppCH
2
ppRib 0.16 n.d.
pppA 16 n.d.
pCH
2
ppA 2 n.d.
A. Guranowski et al. Methylene analogues of adenosine 5¢-tetraphosphate
FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS 833
The newly discovered ATP N-glycosidase [38] allowed
us to generate the depurinated derivatives of p
4
A and of
the best inhibitor analogue, ppCH
2
ppA, and evaluate
the two polyphosphorylated riboses obtained as inhibi-
tors of the human (asymmetrical)Ap
4
A hydrolase. It
emerged that p
4
Ribose is 200 times weaker an inhibitor
than p
4
A, whereas ppCH
2
ppRibose is 100 times weaker
than ppCH
2

ppA. Finally, therefore, we compared the
inhibition of the human recombinant Ap
4
A hydrolase
by p
4
A and ppCH
2
ppA with that by ATP (p
3
A) and
pCH
2
ppA, both compounds truncated by one phos-
phate (and one negative charge) relative to the nucleo-
side tetraphosphates. Both ATP and its b,c-methylene
analogue were definitively weaker inhibitors than their
d-phosphate homologues. Altogether, it is evident that
both the adenine ring and the length of the polyphos-
phate chain contribute to the strength of binding of the
mononucleoside polyphosphates by the (asymmetrical)
Ap
4
A hydrolases, whereas a single methylene bridge,
preferably at or adjacent to the P–O–P reaction site,
potentiates the binding. Because ppCH
2
ppA and
pCH
2

pppA are the strongest inhibitors of the asymmet-
rically acting Ap
4
A hydrolases ever reported and they
are not degraded, in marked contrast to p
4
A which is
both an inhibitor and a slow substrate for these enzymes
[57,58], they clearly have excellent potential to serve as
‘true inhibitors’ and be valuable tools in biochemical
and physiological studies, e.g. on nucleotide receptors.
The methylene analogues of p
4
A as inhibitors
of the (symmetrical) Ap
4
A hydrolase from
Escherichia coli
This Co
2+
-dependent enzyme was shown to hydrolyse
p
4
A slowly, within a range of substrates from which it
always liberates ADP as one of the reaction products
[37]. The p
4
A analogues studied here are not substrates
for this enzyme. However, as shown in Table 3, all act
as inhibitors, albeit relatively moderate ones taking

into account their inhibition of the asymmetrically act-
ing Ap
4
A hydrolases. Adenosine tetraphosphate itself
inhibited the enzyme with K
i
threefold lower than the
K
m
for Ap
4
A (27 lm). The lowest K
i
value was estima-
ted for pppCH
2
pA (6.7 lm) and the highest values
were for ppCH
2
pCH
2
pA and pCH
2
ppCH
2
pA, 20 and
34 lm, respectively.
Conclusion
The data presented here show the potential usefulness
of certain p

4
A analogues for the further study of the
metabolism of mononucleoside polyphosphates and
dinucleoside polyphosphates as well as of the function-
ing of different purine ⁄ nucleotide receptors. In partic-
ular, they have shown a remarkable selectivity in their
behaviour as inhibitors for enzymes having super-
ficially related functions as nucleoside polyphosphate
hydrolases as well as showing nanomolar activity
against selected enzymes.
This new group of nucleotide analogues complements
a different set of synthetic nucleotides, the adenosine-
phosphorothioylated and adenosine-phosphorylated
polyols, which has recently been proved to inhibit sym-
metrically acting bacterial Ap
4
A hydrolases particularly
strongly, with K
i
values as low as 40 nm [59]. These new,
nonhydrolysable p
4
A nucleotide analogues are promis-
ing tools for those who would like specifically to inhibit
the asymmetrically acting Ap
4
A hydrolases. In partic-
ular, they should help in structural studies of these
enzymes [55,56,60]. The apparent lack of inhibition of
the p

4
A hydrolysing enzymes by the methylene and
chloromethylene analogues of p
4
A further challenges
chemists to create other types of p
4
A analogues that
may need to reach beyond the isopolar–isosteric princi-
ples that have governed their design for 25 years [47].
Experimental procedures
Enzymes
Adenosine 5¢-tetraphosphate phosphohydrolase was
obtained from yellow lupin seeds [32] and the recombinant
exopolyphosphatase from yeast (S. cerevisiae) [34] was
kindly donated by Dr Sh. Liu (Stanford University, CA).
Adenosine 5¢-tetraphosphate phosphohydrolase from human
placenta [33] was partially purified according to the following
procedure. The placenta extract was fractionated with
ammonium sulfate and the protein precipitated between 30
and 50% of saturation was subjected to ion-exchange chro-
matography on a DEAE-Sephacel column. The enzyme was
eluted with a 0–0.5 m KCl gradient, concentrated and chro-
matographed on a Sephadex G-100 column from which it
Table 3. Analogues of p
4
A as inhibitors of (symmetrical) Ap
4
A
hydrolase from Escherichia coli.TheK

m
value for Ap
4
A estimated
for the bacterial enzyme was 25 l
M [36]. K
i
values are means of
three independent determinations; standard errors do not exceed
15%. For details of assays see Experimental procedures.
Compound K
i
(lM)
ppppA 10.5
pppCH
2
pA 6.7
ppCH
2
ppA 16.2
pCH
2
pppA 21.8
ppCH
2
pCH
2
pA 20.0
pCH
2

ppCH
2
pA 34.0
pCCl
2
pCCl
2
ppA 8.3
Methylene analogues of adenosine 5¢-tetraphosphate A. Guranowski et al.
834 FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS
eluted as a protein with molecular mass around 84 kDa. This
preparation was free of any competing activity and was used
for the studies of the p
4
A analogues. The recombinant
human (asymmetrical)Ap
4
A hydrolase was kindly donated
by Professor A. G. McLennan (University of Liverpool, UK)
and the enzyme from narrow-leafed lupin from Drs D.
Maksel and K. Gayler (University of Melbourne, Australia).
We also used an extract from the marine sponge Axinella
polypoides that contained an ATP N-glycosidase [38]. This
unusual hydrolase is able to depurinate p
4
A and ppCH
2
ppA,
giving d-ribose 5-O-tetraphosphate and its corresponding
b,c-methylene analogue, respectively. (The sponge extract

was kindly donated by Dr T. Reintamm, Tallinn, Estonia.)
Chemical synthesis of p
4
A analogues
Analogues with one methylene bridge
Adenosine 5¢-methylenebisphosphonate was obtained by
regioselective 5¢-phosphonylation of adenosine with methyl-
enebis(dichlorophosphonate) using recent methodology [39].
The product was converted into the imidazolidate,
ImpCH
2
pA, using imidazole with triphrenylphosphone ⁄
2,2¢-dithio-dipyridine as the condensing agent, and this
intermediate coupled with pyrophosphate to give
pppCH
2
pA in 80% yield.
Activation of the b-phosphate group of ADP was
achieved by conversion into imidazolidate, ImppA. The
activated compound was reacted with a fourfold excess of
the triethylammonium salt of methylenebisphosphonate in
DMF to give pCH
2
pppA [31]. The rate of pyrophosphate
bond formation was greatly accelerated when carried out in
the presence of an eightfold excess of ZnCl
2
[40]. Similarly,
AMP was converted into adenosine 5¢-phosphoroimidazoli-
date, ImpA, which was efficiently coupled with the triethyl-

ammonium salt of methylenebisphosphonic acid. The
resulting pCH
2
ppA was again activated with imidazole to
give imidazolidate ImpCH
2
ppA, and this intermediate cou-
pled with triethylammonium phosphate in a ZnCl
2
-mediated
reaction to give ppCH
2
ppA in 25% yield. Schemes of these
syntheses are shown in Fig. 2.
Reaction mixtures were separated using DEAE-Sepha-
dex 25A (triethylammonium bicarbonate gradient, pH 7.9)
and ⁄ or reverse-phase HPLC (C
18
column, water ⁄ methanol
gradient). HPLC analyses showed that all p
4
A analogues
were at least 96% pure. Structures of all compounds syn-
thesized were fully confirmed using
1
H and
31
P NMR
spectroscopy and MS (see Supplementary material).
Analogues with two methylene or dichloromethylene

bridges
Details of the procedures that led to pCCl
2
pCCl
2
ppA,
ppCH
2
pCH
2
pA and pCH
2
ppCH
2
pA are given in the Sup-
plementary material [41–45].
Structures of the six p
4
A analogues are presented in
Fig. 1.
Other chemicals
Unlabelled mono- and dinucleotides were from Sigma (St.
Louis, MO), and [
3
H]Ap
4
A (740 TBqÆmol
)1
) was purchased
from Moravek, Biochemicals (Brea, CA).

ppppRibose and ppCH
2
ppRibose were obtained enzy-
matically by incubating p
4
A and ppCH
2
ppA with the
sponge ATP N-glycosidase. The progress of depurination
of 2 mm nucleotides in 50 mm Hepes ⁄ KOH buffer
(pH 8.0) was monitored by TLC (System A) in which the
liberated adenine migrated with the solvent front. After
completion of the reactions, the glycosidase was heat
inactivated (3 min at 96 °C) and the mixtures used
directly as a source of the depurinated compounds. The
highest concentration of these compounds in the inhibi-
tion assays with the (asymmetrical)Ap
4
A hydrolases was
0.05 mm.
Enzyme assays
Each methylene analogue of p
4
A was tested as a potential
substrate for the three p
4
A-hydrolysing enzymes under the
conditions established earlier as optimal for p
4
A hydrolysis.

The reaction mixtures (0.05 mL final volume) contained
50 mm buffer, chloride of a divalent cation, 1 mm substrate
(p
4
A or its analogue) and the investigated enzyme. For the
yellow lupin p
4
A hydrolase the mixture contained Hepes ⁄
KOH buffer (pH 8.2) and 5 mm MgCl
2
, for the human
enzyme Hepes ⁄ KOH (pH 7.0) and 1 mm CoCl
2
, and for
the yeast exopolyphosphatase sodium acetate buffer
(pH 4.7) and 1 mm CoCl
2
. Incubations were carried out at
30 °C. The results were analysed either by TLC or HPLC
(see below).
Asymmetrically acting Ap
4
A hydrolases were assayed in
a reaction mixture (0.05 mL total volume) containing
50 mm Hepes ⁄ KOH (pH 7.6), 0.02 mm dithiothreitol, 5 mm
MgCl
2
, 0.05 mm [
3
H]Ap

4
A (300 000 c.p.m.), various con-
centrations of p
4
A or its analogue and a rate-limiting quan-
tity of enzyme ( 0.3 mU). For assaying the symmetrically
acting Ap
4
A hydrolase from E. coli,5mm MgCl
2
was
replaced with 0.1 mm CoCl
2
. Incubations were carried out
at 30 °C. To estimate reaction rates, 0.005 mL aliquots
were spotted on to TLC plates (aluminium plates precoated
with silica gel containing fluorescent indicator; Merck cat.
no. 5554), usually after 6, 12, 18 and 24 min of incubation.
Unlabelled standards of the product [ATP for (asymmetri-
cal)Ap
4
A hydrolases and ADP for (symmetrical)Ap
4
A
hydrolase] were applied at the origin, and plates were devel-
oped for 90 min in dioxane ⁄ ammonia ⁄ water (6 : 1 : 4
v ⁄ v ⁄ v). Spots of the products, visualized under short-wave
UV light, were excised, immersed in scintillation cocktail,
A. Guranowski et al. Methylene analogues of adenosine 5¢-tetraphosphate
FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS 835

and the radioactivity measured. K
i
values were calculated
according to the method of Dixon and Webb [46] from the
slopes of plots v ⁄ v
i
against [I] (where v and v
i
are velocities
in the absence and presence of inhibitor, respectively,
and [I] is the inhibitor concentration), where slope ¼
K
m
⁄ K
i
(1 ⁄ K
m
+ S).
Chromatographic systems
Analyses of the hydrolysis of p
4
A or its analogues to their
corresponding NTPs were performed on silica gel TLC
plates developed in dioxane ⁄ ammonia ⁄ water (6 : 1 : 6
v ⁄ v ⁄ v) (System A). Inhibitory effects of the analogues exer-
ted on the Ap
4
A hydrolysing enzymes were analysed by
developing the same TLC plates in dioxane ⁄ ammonia ⁄
water mixed at the 6 : 1 : 4 ratio (System B). The velocities

of p
4
A and pppCH
2
pA hydrolysis were estimated by the
use of HPLC on the reverse-phase column (for details see
legend to Fig. 3).
Acknowledgements
Financial support from the State Committee for Scien-
tific Research (KBN, Poland), within grants PBZ-
KBN-059 ⁄ T09 ⁄ 04 and PBZ-KBN-059 ⁄ T09 ⁄ 10, is
gratefully acknowledged. We thank the Wellcome
Trust for generous financial support (to MJT) Grant
no. 057599 ⁄ Z ⁄ 99.
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Supplementary material
The following supplementary material is available
online:
Characterization of the p
4
A analogues with one
methylene group by MS and NMR spectroscopy.
Syntheses of the p
4
A analogues with two halo ⁄
methylene bridges: General remarks on preparation of
the precursors of p
4
A analogues.
Synthesis of isopropyl bis(diethyl phosphonodichloro-

methyl)phosphinate, pCCl
2
pCCl
2
p pentaester.
Synthesis of bis(phosphonodichloromethyl)phosphi-
nic acid, pCCl
2
pCCl
2
p free acid.
Synthesis of adenosine-5’-[b,c,c,d-bis(dichlorometh-
ylene)]tetraphosphate, pCCl
2
pCCl
2
ppA. Synthesis of
a,b;b,c-bis(methylene)-ATP, tris(triethylammonium)
salt, pCH
2
pCH
2
pA.
Synthesis of adenosine-5’-[a,b,b, c-bis(methylene)]-
tetraphosphate, ppCH
2
pCH
2
pA.
Synthesis of adenosine-5’-[a,b;c,d-bis(methylene)]-

tetraphosphate, pCH
2
ppCH
2
pA.
Methylene analogues of adenosine 5¢-tetraphosphate A. Guranowski et al.
838 FEBS Journal 273 (2006) 829–838 ª 2006 The Authors Journal compilation ª 2006 FEBS