Of the six analogues, only pppCH2pA 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 exopolyphosphata
Trang 1Their chemical synthesis and recognition by human and plant
mononucleoside tetraphosphatases and dinucleoside
tetraphosphatases
Andrzej Guranowski1, El_zbieta Starzyn´ska1, Małgorzata Pietrowska-Borek1, Jacek Jemielity2,
Joanna Kowalska2, Edward Darzynkiewicz2, Mark J Thompson3 and G Michael Blackburn3
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 p4A; 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: guranow@au.poznan.pl
Website: http://www.au.poznan.pl
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 (pppCH2pA), b,c-methylene-adenosine 5¢-tetraphosphate (ppCH2ppA), c,d-methylene-adenosine 5¢-tetraphosphate (pCH2pppA), ab,cd-bismethylene-adenosine 5¢-tetraphosphate (pCH2ppCH2pA), ab, bc-bismethylene-adenosine 5¢-tetraphosphate (ppCH2pCH2pA) and bc, cd-bis(dichloro)methylene-adenosine 5¢-tetraphosphate (pCCl2pCCl2ppA), and tested them as potential substrates and⁄ or inhibitors of three specific nu-cleoside tetraphosphatases In addition, we employed these p4A analogues with two asymmetrically and one symmetrically acting dinucleoside tetra-phosphatases Of the six analogues, only pppCH2pA 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 p4A-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 ppCH2ppA and pCH2pppA, inhibited the human enzyme with Ki val-ues of 1.6 and 2.3 nm, respectively, and the lupin enzyme with Kivalues 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
Ap3A, diadenosine 5¢,5¢¢¢-P 1 ,P 3 -triphosphate; Ap4A, diadenosine 5¢,5¢¢¢-P 1 ,P 4 -tetraphosphate; NpnN¢, 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;
pnN, nucleoside 5¢-polyphosphate; ppCH 2 pCH2pA, ab,bc-bismethylene-adenosine 5¢-tetraphosphate; pppCH 2 pA, a,b-methylene-adenosine 5¢-tetraphosphate; pppCH 2 ppA, b,c-methylene-adenosine 5¢-tetraphosphate.
Trang 2In addition to the canonical nucleoside mono-, di-,
and triphosphates, cells contain various minor
nucleo-tides Among these are the nucleoside
5¢-polyphos-phates (pnNs, where n¼ 4), such as adenosine
5¢-tetraphosphate (p4A or ppppA) [1–5] and adenosine
5¢-pentaphosphate (p5A or pppppA) [2], and the
dinu-cleoside 5¢,5¢¢¢-P1,Pn-polyphosphates (NpnN¢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¢¢¢-P1,P3-triphosphate
(Ap3A) and diadenosine 5¢,5¢¢¢-P1,P4-tetraphosphate
(Ap4A) [6–12] The biological roles of these NpnN¢s
are partially understood In particular, ApnA has been
implicated in various intracellular processes [13,14] and
also in extracellular signalling [15,16] By contrast, the
role of pnNs is inadequately recognized Almost 20
years ago, the accumulation of p4A and p5A in yeast
was correlated with sporulation [2] and only recently,
p4A 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 p4A and other p4Ns 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 p4U by transferring the uridylyl moiety
from UDP-glucose onto tripolyphosphate [25] The
third category includes several enzymes that degrade
Ap5A or Ap6A yielding p4A as one of the reaction
products [26] Degradation of p4A can be controlled
by various nonspecific and specific pnN-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 Ap3A
and Ap4A, 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], p4A analogues have been synthesized only recently ab,bc-bismethylene-p4A and bc,cd-bis(dichlo-ro)methylene-p4A were tested as agonists or antago-nists of the P2X2⁄ 3receptor [30] and a short report has appeared on the synthesis of pCH2pppA, pCH2pppG and pCH2pppm7G [31]
Here, we describe details on the synthesis of and the results of enzymatic studies on a series of novel p4A 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 p4A and second, as inhibitors of two types of
Ap4A hydrolase, for which p4A itself acts as a strong inhibitor The p4A-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 p4A to ATP and phosphate [34] The Ap4A 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 Ap4A into ATP and AMP, and the Co2+-dependent symmetrically acting
Fig 1 Structures of p4A analogues.
Trang 3dinucleoside tetraphosphatase (EC 3.6.1.41) that
con-verts Ap4A into two ADPs [37]
Results and Discussion
Comments on the synthesis of p4A analogues
The preparation of intermediate ADP and ATP
ana-logues followed standard methods Their conversion
into p4A analogues called for condensation with
phos-phate (for ATP analogues), or with pyrophosphos-phate 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 p4A analogues in high purity
The MS and1H NMR spectra of these nucleotides are
unexceptional The 31P 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 p4A analogues as substrates
by the p4A 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 p4Ns 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 p4A to ATP and Piin < 15 min Incubation was for up to 16 h and the progress of potential hydrolysis was analysed by TLC System A Of six p4A analogues, only pppCH2pA was susceptible to hydrolysis and the relative velocities
of the reactions were estimated only for the pair
p4A⁄ pppCH2pA Figure 3 shows typical elution pat-terns of the substrate⁄ product pairs on the reverse-phase HPLC column Satisfactory separation of p4A from ATP was obtained by isocratic elution with potassium
Fig 2 Chemical synthesis of pppCH2pA (A), ppCH2ppA (B) and pCH2pppA (C) ‘A’ represents adenosine, DMF dimethylformamide, PPh3 tri-phenylphosphine, TEA triethylammonium, and TEAB triethylammonium bicarbonate.
Trang 4phosphate buffer (Fig 3A), and of pppCH2pA from
ppCH2pA 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 p4A
hydrolase and the yeast exopolyphosphatase hydrolysed
pppCH2pA slightly more than twofold slower than p4A,
and the human p4A hydrolase 125-fold slower This
result shows that (a) the p4N 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 ppCH2ppA, or from ppCH2pCH2pA, in which the P–O–P bond between c and d phosphate remains unchanged; (c) the human hydrolase is sensitive to the –CH2– insert even in the most distant position from the reaction site, i.e between the a- and b-phosphorus atoms in pppCH2pA Thus the p4N hydrolysing enzymes are more stringent with respect to recognition
of their substrates than the (asymmetrical) Ap4A hydro-lases, which cleave the Pa–O–Pb bridge not only in
Ap4A [27], but also in AppCH2ppA, AppCF2ppA, and AppCCl2ppA [28]
There are obvious reasons why analogues with proximate methylene bridges should resist cleavage For pCH2pppA, removal of the terminal phosphate (largely a dissociative process) is frustrated by the stability of the Pc–C–Pd bridge For ppCH2ppA, 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 pppCH2pA for the human p4A
Fig 3 Time course of p4A (A) and pppCH2pA (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 KH2PO4+ 0.008 M (CH3CH2CH2CH2)4N + HSO4– , 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 p4A-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
pppCH2pA 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
Trang 5hydrolase It does not seem likely that such an
iso-steric and isopolar analogue [47] of p4A 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 Ap4A hydrolase This brings into focus
the possibility of direct recognition of the Pa–O–Pb
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 p4A hydrolysing
enzymes?
All three p4A hydrolysing enzymes were tested with
each of the six p4A analogues to see whether they
inhibit normal hydrolysis of p4A None of the
ana-logues used at concentrations up to 0.5 mm retarded
the conversion of p4A (1 mm) 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 p4A [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 p4A as inhibitors
of the (asymmetrical) Ap4A hydrolases
Adenosine tetraphosphate itself has been known for a
long time as an effective competitive inhibitor of the
(asymmetrical) Ap4A 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 Kivalues, this nucleotide has been used for
the elution of the (asymmetrical) Ap4A hydrolases
adsorbed to dye–ligand affinity columns as
homogen-eous proteins [53,54] We tested all six methylene and
chloromethylene p4A analogues as potential inhibitors
of two (asymmetrical) Ap4A hydrolases, from human
and from narrow-leafed lupin, and the results are
sum-marized in Table 2 Of all the analogues, ppCH2ppA
and pCH2pppA appear to be the strongest inhibitors of both the human and plant enzymes The Kivalues esti-mated for the human enzyme, 1.6 and 2.3 nm, respect-ively, were over 30- and 20-fold lower than the Ki estimated for the same enzyme for p4A (50 nm) More-over, these values are five and three times smaller than the lowest Ki estimated yet reported (7.5 nm) for the reaction of Ap4A hydrolysis catalysed by the firefly enzyme [52] Significantly, the analogue, pppCH2pA, with its methylene bridge in the position most distant from the reaction site, is 100-fold less potent an inhibitor than ppCH2ppA Two analogues having two methylene bridges are generally poorer inhibitors than those that possess a single methylene group In every cases, however, the Kivalues were below the Kmvalues for the Ap4A 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 Ki values exceeding the Kmvalues for Ap4A by some 20–50-fold Both p4A 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 Ap4A hydrolase [55] and the human enzyme [56] The stronger inhibition of the human enzyme by p4A 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 p4A as inhibitors of (asymmetrical) Ap4A hydrolases The Kmvalues for Ap4A estimated for the human and lupin enzyme were 2 lm (this study) and 1 lm [35], respectively The Kivalues 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)
ppCH2ppA 0.0016 0.030 pCH 2 pppA 0.0023 0.034 ppCH2pCH2pA 1.3 0.07 pCH2ppCH2pA 0.25 0.62 pCCl 2 pCCl 2 ppA 40 53
ppCH2ppRib 0.16 n.d.
Trang 6The newly discovered ATP N-glycosidase [38] allowed
us to generate the depurinated derivatives of p4A and of
the best inhibitor analogue, ppCH2ppA, and evaluate
the two polyphosphorylated riboses obtained as
inhibi-tors of the human (asymmetrical) Ap4A hydrolase It
emerged that p4Ribose is 200 times weaker an inhibitor
than p4A, whereas ppCH2ppRibose is 100 times weaker
than ppCH2ppA Finally, therefore, we compared the
inhibition of the human recombinant Ap4A hydrolase
by p4A and ppCH2ppA with that by ATP (p3A) and
pCH2ppA, 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)
Ap4A hydrolases, whereas a single methylene bridge,
preferably at or adjacent to the P–O–P reaction site,
potentiates the binding Because ppCH2ppA and
pCH2pppA are the strongest inhibitors of the
asymmet-rically acting Ap4A hydrolases ever reported and they
are not degraded, in marked contrast to p4A 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 p4A as inhibitors
of the (symmetrical) Ap4A hydrolase from
Escherichia coli
This Co2+-dependent enzyme was shown to hydrolyse
p4A slowly, within a range of substrates from which it
always liberates ADP as one of the reaction products
[37] The p4A 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 Ap4A hydrolases Adenosine tetraphosphate itself inhibited the enzyme with Ki threefold lower than the
Kmfor Ap4A (27 lm) The lowest Kivalue was estima-ted for pppCH2pA (6.7 lm) and the highest values were for ppCH2pCH2pA and pCH2ppCH2pA, 20 and
34 lm, respectively
Conclusion
The data presented here show the potential usefulness
of certain p4A 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 actingbacterial Ap4A hydrolases particularly strongly, with Kivalues as low as 40 nm [59] These new, nonhydrolysable p4A nucleotide analogues are promis-ing tools for those who would like specifically to inhibit the asymmetrically acting Ap4A hydrolases In partic-ular, they should help in structural studies of these enzymes [55,56,60] The apparent lack of inhibition of the p4A hydrolysing enzymes by the methylene and chloromethylene analogues of p4A further challenges chemists to create other types of p4A 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 p4A as inhibitors of (symmetrical) Ap4A
hydrolase from Escherichia coli The K m value for Ap 4 A estimated
for the bacterial enzyme was 25 l M [36] Kivalues are means of
three independent determinations; standard errors do not exceed
15% For details of assays see Experimental procedures.
Trang 7eluted 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 p4A analogues The recombinant
human (asymmetrical) Ap4A 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 p4A and ppCH2ppA,
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 p4A 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,
ImpCH2pA, using imidazole with triphrenylphosphone⁄
2,2¢-dithio-dipyridine as the condensing agent, and this
intermediate coupled with pyrophosphate to give
pppCH2pA 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 pCH2pppA [31] The rate of pyrophosphate
bond formation was greatly accelerated when carried out in
the presence of an eightfold excess of ZnCl2[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 pCH2ppA was again activated with imidazole to
give imidazolidate ImpCH2ppA, and this intermediate
cou-pled with triethylammonium phosphate in a ZnCl2-mediated
reaction to give ppCH2ppA 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 (C18 column, water⁄ methanol
gradient) HPLC analyses showed that all p4A analogues
were at least 96% pure Structures of all compounds
syn-thesized were fully confirmed using 1H and 31P NMR
spectroscopy and MS (see Supplementary material)
Analogues with two methylene or dichloromethylene
bridges
Details of the procedures that led to pCCl2pCCl2ppA,
ppCH2pCH2pA and pCH2ppCH2pA are given in the
Sup-plementary material [41–45]
Structures of the six p4A analogues are presented in Fig 1
Other chemicals
Unlabelled mono- and dinucleotides were from Sigma (St Louis, MO), and [3H]Ap4A (740 TBqÆmol)1) was purchased from Moravek, Biochemicals (Brea, CA)
ppppRibose and ppCH2ppRibose were obtained enzy-matically by incubating p4A and ppCH2ppA 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 96C) 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) Ap4A hydrolases was 0.05 mm
Enzyme assays
Each methylene analogue of p4A was tested as a potential substrate for the three p4A-hydrolysing enzymes under the conditions established earlier as optimal for p4A hydrolysis The reaction mixtures (0.05 mL final volume) contained
50 mm buffer, chloride of a divalent cation, 1 mm substrate (p4A or its analogue) and the investigated enzyme For the yellow lupin p4A hydrolase the mixture contained Hepes⁄ KOH buffer (pH 8.2) and 5 mm MgCl2, for the human enzyme Hepes⁄ KOH (pH 7.0) and 1 mm CoCl2, and for the yeast exopolyphosphatase sodium acetate buffer (pH 4.7) and 1 mm CoCl2 Incubations were carried out at
30C The results were analysed either by TLC or HPLC (see below)
Asymmetrically acting Ap4A 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 MgCl2, 0.05 mm [3H]Ap4A (300 000 c.p.m.), various con-centrations of p4A or its analogue and a rate-limiting quan-tity of enzyme ( 0.3 mU) For assaying the symmetrically acting Ap4A hydrolase from E coli, 5 mm MgCl2 was replaced with 0.1 mm CoCl2 Incubations were carried out
at 30C 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) Ap4A hydrolases and ADP for (symmetrical) Ap4A 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,
Trang 8and the radioactivity measured Ki values were calculated
according to the method of Dixon and Webb [46] from the
slopes of plots v⁄ viagainst [I] (where v and viare velocities
in the absence and presence of inhibitor, respectively,
and [I] is the inhibitor concentration), where slope¼
Km⁄ Ki(1⁄ Km+ S)
Chromatographic systems
Analyses of the hydrolysis of p4A 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 Ap4A 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 p4A and pppCH2pA 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 p4A analogues with one methylene group by MS and NMR spectroscopy Syntheses of the p4A analogues with two halo⁄ methylene bridges: General remarks on preparation of the precursors of p4A analogues
Synthesis of isopropyl bis(diethyl phosphonodichloro-methyl)phosphinate, pCCl2pCCl2p pentaester
Synthesis of bis(phosphonodichloromethyl)phosphi-nic acid, pCCl2pCCl2p free acid
Synthesis of adenosine-5’-[b,c,c,d-bis(dichlorometh-ylene)]tetraphosphate, pCCl2pCCl2ppA Synthesis of a,b;b,c-bis(methylene)-ATP, tris(triethylammonium) salt, pCH2pCH2pA
Synthesis of adenosine-5’-[a,b,b,c-bis(methylene)]-tetraphosphate, ppCH2pCH2pA
Synthesis of adenosine-5’-[a,b;c,d-bis(methylene)]-tetraphosphate, pCH2ppCH2pA