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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: guranow@au.poznan.pl 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. 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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). This supplementary material can be found in the online version of this article. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corre- sponding author for the article. A. Guranowski et al. Oxymethylene diadenosine polyphosphate analogs FEBS Journal 276 (2009) 1546–1553 ª 2009 The Authors Journal compilation ª 2009 FEBS 1553 . Novel diadenosine polyphosphate analogs with oxymethylene bridges replacing oxygen in the polyphosphate chain Potential substrates and/or inhibitors. present the results of studies using novel diadenosine polyphosphate analogs, with an oxymethylene group replacing one or two bridging oxygen( s) in the polyphosphate