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

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

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

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

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-

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

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

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

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

[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

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

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

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.

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

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

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

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

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-

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).

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

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

,P
4
-tetraphosphate (Ap
4
A), ATP and catecholamine
content in bovine adrenal medulla, chromaffin granules
and chromaffin cells. Biochimie 76, 404–409.
4 Westhoff T, Jankowski H, Schmidt S, Luo J, Giebing
G, Schlu
¨
ter H, Tepel M, Zidek W & van der Giet M
(2003) Identification and characterization of adenosine
5¢-tetraphosphate in human myocardial tissue. J Biol
Chem 278, 17735–17740.
5 Pintor J, Pelaez T & Peral A (2004) Adenosine tetra-
phosphate, Ap
4
, a physiological regulator of intraocular
pressure in normotensive rabbit eyes. J Pharmacol Exp
Ther 308, 468–473.
6 Garrison PN & Barnes LD (1992) Determination of
dinucleoside polyphosphates. In Ap
4
A and Other Dinu-
cleoside Polyphosphates (McLennan AG, ed.), pp. 29–
61. CRC Press, Boca Raton, FL.
7 Ogilvie A & Jacob P (1983) Diadenosine 5¢,5¢¢¢-P
1
,P
3

4
A and other dinucleoside poly-
phosphates in stressed Drosophila cells. J Biol Chem
260, 15566–15570.
12 Coste H, Brevet A, Plateau P & Blanquet S (1987) Non-
adenylated bis (5¢-nucleosidyl) tetraphosphates occur in
Saccharomyces cerevisiae and in Escherichia coli and
accumulate upon temperature shift or exposure to cad-
mium. J Biol Chem 262, 12096–12103.
13 McLennan AG (2000) Dinucleoside polyphosphates –
friend or foe? Pharmacol Ther 87, 73–89.
14 McLennan AG, Barnes LD, Blackburn GM, Brenner
Ch, Guranowski A, Miller AD, Rotlla
´
n P, Soria B,
Tanner JA & Sillero A (2001) Recent progress in the
study of the intracellular function of diadenosine poly-
phosphates. Drug Dev Res 52, 249–259.
15 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.
16 Jankowski V, Tolle M, Vanholder R, Scho
¨
nfelder G,
van der Giet M, Henning L, Schlu
¨
ter H, Paul M, Zidek
W & Jankowski J (2005) Uridine adenosine tetrapho-

somal peptide synthetases (NRPS). Biochim Biophys
Acta 1546, 234–241.
22 Pietrowska-Borek M, Stuible H-P, Kombrink E & Gur-
anowski A (2003) 4-Coumarate: coenzyme A ligase has
the catalytic capacity to synthesize and reuse various
(di)adenosine polyphosphates. Plant Physiol 131, 1401–
1410.
23 Madrid O, Martı
´
n D, Atencia EA, Sillero A & Gu
¨
nther
Sillero MA (1998) T4 DNA ligase synthesizes dinucleo-
side polyphosphates. FEBS Lett 433, 283–286.
24 Atencia EA, Madrid O, Gu
¨
nther Sillero MA & Sillero
A (1999) T4 RNA ligase catalyzes the synthesis of
dinucleoside polyphosphates. Eur J Biochem 261,
802–811.
25 Guranowski A, de Diego A, Sillero A & Gu
¨
nther Sillero
MA (1998) Uridine 5¢-polyphosphates (p
4
U and p
5
U)
and uridine (5¢)polyphospho (5¢)nucleosides (Up
n

2 ⁄ 3
. Br J Pharmacol 140, 1027–1034.
31 Jemielity J, Pietrowska-Borek M, Starzyn
´
ska E, Kow-
alska J, Stolarski R, Guranowski A & Darzynkiewicz E
(2005) Synthesis and enzymatic characterization of
methylene analogs of adenosine 5¢-tetraphosphate (p
4
A).
Nucleosides Nucleotides Nucleic Acids 24, 589–593.
32 Guranowski A, Starzyn
´
ska E, Brown P & Blackburn
GM (1997) Adenosine 5¢-tetraphosphate phosphohydro-
lase from yellow lupin seeds; purification to homogen-
eity and properties. Biochem J 328, 257–262.
33 Pietrowska-Borek M, Szalata M & Guranowski A
(1999) Nucleoside 5 ¢-tetraphosphate hydrolase from
human placenta. Cell Mol Biol Lett 4, 439.
34 Guranowski A, Starzyn
´
ska E, Barnes LD, Robinson
AK & Liu S (1998) Adenosine 5¢-tetraphosphate phos-
phohydrolase activity is an inherent property of soluble
exopolyphosphatase from yeast Saccharomyces cerevisiae.
Biochim Biophys Acta 1380, 232–238.
35 Thorne NMH, Hankin S, Wilkinson MC, Nu´ n
˜
ez C,

Biochem 270, 4122–4132.
39 Kalek M, Jemielity J, Ste˛pin
´
ski J, Stolarski R & Dar-
zynkiewicz E (2005) A direct metod for the synthesis of
nucleoside 5¢-methylene bis(phosphonate)s from nucleo-
sides. Tetrahedron Lett 46, 2417–2421.
40 Jemielity J, Fowler T, Zuberem J, Ste˛pin
´
ski J, Lewd-
orowicz M, Nedzwiecka A, Stolarski R, Darzynkiewicz
E & Rhoads RE (2003) Novel ‘anti-reverse’ cap analogs
with superior translational properties. RNA 9, 1108–
1122.
41 Maier L & Gredig R (1969) Organophosphorus
compounds. XXXVI. Preparation and properties of
bis(dialkoxyphosphonylmethyl)-, bis(alkoxyphosphinyl-
methyl)-, and bis(oxophosphonarylmethyl) phosphinic
acid esters and the corresponding acids. Helv Chim Acta
52, 827–845.
42 Thompson MJ, Mekhalfia A, Hornby DP & Blackburn
GM (1999) Synthesis of two stable nitrogen analogues
of S-adenosyl-l-methionine. J Org Chem 64, 7467–7473.
43 Davisson VJ, Davis DR, Dixit VM & Poulter CD
(1987) Synthesis of nucleoside 5¢-diphosphates from 5¢-
O-tosyl nucleosides. J Org Chem 52, 1794–1801.
44 Moffatt JG & Khorana HG (1961) Nucleoside poly-
phosphates. X. The synthesis and some reactions of
nucleoside-5¢-phosphoromorpholidates and related com-
pounds. Improved methods for the preparation of

51 Moreno A, Lobato
´
n CD, Gu
¨
nther Sillero MA & Sillero
A (1982) Dinucleosidetetraphosphatase from Ehrlich
ascites tumor cells: inhibition by adenosine, guanosine
and uridine 5¢-tetraphosphates. Int J Biochem 14,
629–634.
52 McLennan AG, Mayers E, Walker-Smith I & Chen H
(1995) Lanterns of the firefly Photinus pyralis contain
abundant diadenosine 5¢,5¢¢¢-P
1
,P
4
-tetraphosphate pyro-
phosphohydrolase activity. J Biol Chem 270, 3706–3709.
53 Costas MJ, Pinto RM, Ferna
´
ndez A, Canales J, Garcı
´
a-
Agu´ ndez JA & Cameselle JC (1990) Purification to
homogeneity of rat liver dinucleoside tetraphosphatase
by affinity elution with adenosine 5¢-tetraphosphate.
J Biochem Biophys Methods 21, 25–33.
54 Lazewska D, Starzyn
´
ska E & Guranowski A (1993)
Human placenta (asymmetrical ) diadenosine 5¢,5¢¢¢ -

-
tetraphosphate pyrophosphohydrolase from Caenor-
habditis elegans. Biochim Biophys Acta 1550, 27–36.
59 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.
60 Bailey S, Sedelnikova SE, Blackburn GM, Abdelghany
HM, Baker PJ, McLennan AG & Raffarty JR (2002)
The crystal structure of diadenosine tetraphosphate
hydrolase from Caenorhabditis elegans in free and bin-
ary complex forms. Structure 10, 589–600.
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-

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