NpnN¢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; Ap4A
Trang 1oxymethylene bridges replacing oxygen in the
polyphosphate chain
Andrzej Guranowski1, El_zbieta Starzyn´ska1, Małgorzata Pietrowska-Borek2, Dominik Rejman3,* and
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¢¢¢-P1,Pn-polyphosphates (NpnN¢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] NpnN¢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; Ap4A
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 (NpnN¢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 NpnN¢ 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 Ap4A hydrolase [bis(5¢-nucleosyl)-tetraphosphatase (symmetrical); EC 3.6.1.41] from E coli and of two asymmetrically acting Ap4A hydrolases [bis(5¢-nu-cleosyl)-tetraphosphatase (asymmetrical); EC 3.6.1.17] from humans and narrow-leaved lupin The six chemically synthesized analogs were: ApCH2OpOCH2pA (1), ApOCH2pCH2OpA (2), ApOpCH2OpOpA (3),
ApOp-OCH2pCH2OpOpA (6) The eukaryotic asymmetrical Ap4A hydrolases degrade two compounds, 3 and 5, as anticipated in their design Analog 3 was cleaved to AMP (pA) and b,c-methyleneoxy-ATP (pOCH2pOpA), whereas hydrolysis of analog 5 gave two molecules of a,b-oxymethylene ADP (pCH2OpA) The relative rates of hydrolysis of these analogs were estimated Some of the novel nucleotides were moderately good inhibitors
of the asymmetrical hydrolases, having Ki values within the range of the
Kmfor Ap4A By contrast, none of the six analogs were good substrates or inhibitors of the bacterial symmetrical Ap4A hydrolase
Abbreviations
DCC, dicyclohexylcarbodiimide; MCPBA, 4-chloroperoxybenzoic acid; NEP, 2-chloro-5,5-dimethyl-2-oxido-1,3,2-dioxaphosphinane.
Trang 2nonspecific enzymes exist that degrade these
dinucleo-tides to mononucleodinucleo-tides [12] Ap3A and Ap4A are the
most frequently studied NpnN¢s, and many Ap3A and
Ap4A analogs have been synthesized, both chemically
and enzymatically [13] Some have been found to be
useful for elucidating certain aspects of the behavior of
Ap4A-degrading enzymes Pa-Chiral phosphorothioate
analogs of Ap4A have been used to show that the
yeast Ap4A phosphorylase forms an enzyme–AMP
intermediate [14], whereas a complex of a methylene
analogue of Ap4A, AppCH2ppA, with the
(asymmetri-cal) Ap4A 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
Ap4A hydrolases; two
adenosine-5¢-O-phosphorothioy-lated pentaerythritols are strong inhibitors of the
(sym-metrical) Ap4A hydrolase from Escherichia coli (with
Ki values of 0.04 and 0.08 lm) [16], and methylene
analogues of adenosine 5¢-tetraphosphate (p4A)
strongly inhibited the asymmetrically acting Ap4A
hydrolases with Ki 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 [18F]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 ApnA analogs investigated so far as
potential substrates and/or inhibitors of specific Ap4A
hydrolases, those with modifications in the
polyphos-phate chain have been studied most often [20–23]
Some are substrates of the asymmetrically acting Ap4A
hydrolases from yellow lupin seeds [20,21] and Artemia
embryos [22] AppCH2ppA and ApCH2pppA were hydrolyzed 20- to 50-fold more slowly than Ap4A, and
AppCHClppA were hydrolyzed 1.4- to 9-fold more slowly than Ap4A As observed for a series of bb¢-substituted Ap4A analogs, their efficiencies as substrates of the Ap4A 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 Ap4A hydrolase from E coli, but later work by McLennan et al [22] reported that AppCH2ppA,
hydrolysis using their preparation of bacterial enzyme, with 25-, 50- and 125-fold reduced rates, respectively, compared with that of Ap4A hydrolysis
In this report we describe, first, the chemical synthe-sis of new ApnA 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 Ap4A hydrolases [bis(5¢-nucleosyl)-tetraphos-phatase (asymmetrical); EC 3.6.1.17], from human [24] and narrow-leafed lupin [25], and on the Co2+ -depen-dent symmetrically acting dinucleoside tetraphospha-tase [bis(5¢-nucleosyl)-tetraphosphatetraphospha-tase (asymmetrical);
EC 3.6.1.41] from E coli [26]
Results and Discussion
Recognition of ApnA oxymethylene analogs by
Ap4A hydrolases
In this study we questioned how specific Ap4A hydro-lases might recognize substrate analogs that are
Fig 1 Structures of oxymethylene and
methyleneoxy analogs of diadenosine
polyphosphates.
Trang 3nonisosteric (the P–P distance is one atom longer), yet
isoelectronic (charge identical), in comparison with
natural ApnAs To answer this question, we performed
studies on the interaction of the enzymes with the
aforementioned oxymethylene analogs of ApnA 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 ApnA analogs was a substrate of
the symmetrically acting Ap4A hydrolase Each analog
(0.5 mm) was incubated at 30C in 0.05 mL of the
(pH 7.6), 0.02 mm dithiothreitol and 5 mm MgCl2, for
up to 16 h with an amount of enzyme sufficient to
< 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 Ap4A
catalyzed by the E coli enzyme As shown earlier [20–
22], some methylene or halomethylene analogs of
Ap4A inhibited that bacterial enzyme quite effectively,
with Kivalues even one order of magnitude lower than
the Km for Ap4A [20] This study thus establishes that
the symmetrical Ap4A 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 ApnA analogs
were tested as potential substrates of the
asymmetri-cally acting Ap4A 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 Ap4A 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 m M MgCl 2 , 0.5 m M substrate and rate-limiting amounts of the asymmetrically acting Ap4A 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 KH2PO4(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 KH2PO4, 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.
Trang 4the pKa values for the ATP analogs released (32 and
18) have not yet been determined, it is reasonable to
assume that a pKa 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 pKa 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
Ap4A (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 Pb 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
together, the results of cleavage of compounds 3 and
5 show that the asymmetrically acting Ap4A hydro-lases can reach the scissile bond either by extending
‘the frame’, as in the case of compound 3, or by shortening the count, when attacking the Pb-O-Pb¢ 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 Ap3A – 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 -CH2-Pa- sequence, which occurs in 4, nor a -CH2-Pc-CH2- 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 ApnA analogs inhibit Ap4A hydrolysis catalyzed by the asymmetrically acting Ap4A hydrolases Only analogs
3 and 4 acted as competitive inhibitors, with Kivalues
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 Kivalues lie in the range of the Kmvalues for Ap4A: 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
pCH2OpOpA (small arrow).
Potential substrate
Relative velocities for AppppA hydrolase from human
Narrow-leaved lupin
ApCH 2 OpOCH 2 pA (1) 0 0
ApOCH2pCH2OpA (2) 0 0
Ap fl OpCH2OpO fl pA (3) 0.48 0.92
ApCH 2 OpOpOCH 2 pA (4) 0 0
ApOCH2pO fl pCH2OpA (5) 0.18 0.54
ApOpOCH2pCH2OpOpA (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 Ap4A 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.
Trang 5The results of binding and cleavage studies on the six
ApnA 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-CH2-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
Ap4A-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
Ap4A hydrolase (EC 3.6.1.17) [24] was kindly donated by
A G McLennan (University of Liverpool, UK), and the
Ap4A 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 Ap4A 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 [3H]Ap4A (740 TBqÆ
mol)1) was purchased from Moravek Biochemicals (Brea,
CA, USA) Syntheses leading to the novel oxymethylene
analogs of ADP, ATP and ApnA are described below
Chromatographic systems
Analyses of the hydrolysis of Ap4A 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 Ki values for the analogs with the use of radiolabeled Ap4A 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 ApnA
ADP, ATP and ApnA analogs with one -OCH2- or -CH2 O-group that substitutes for a bridging oxygen in adenosine or diadenosine oligophosphates have not been synthesized pre-viously The tripolyphosphate analog, pOCH2pCH2Op, has been bound to two adenosines yielding an analog of Ap3A [28] but hitherto similar analogs of Ap4A or Ap5A have not been made We prepared ab-methyleneoxy-ADP (pOCH2pA) (21) and ab-oxymethylene-ADP (pCH2OpA) (24), ab-methyleneoxy-ATP (pOpOCH2pA), ab-oxymethyl-ene-ATP (pOpCH2OpA), bc-oxymethylene-ATP (pCH2 -OpOpA) (18), the unstable, bc-methyleneoxy-ATP (pOCH2pOpA) (32), and the six ApnA analogs investigated
in this study: ab,a¢b-bis(methyleneoxy)Ap3A (ApCH2 Op-OCH2pA) (1), ab,a¢b-bis(oxymethylene)Ap3A (ApOCH2
pC-H2OpA) (2), bb¢-methyleneoxy-Ap4A (ApOpCH2OpOpA) (3), ab,a¢b¢-bis(methyleneoxy)Ap4A (ApCH2OpOpOCH2pA) (4), ab,a¢b¢-bis(oxymethylene)Ap4A (ApOCH2pOpCH2OpA) (5) and bc,b¢c-bis(oxymethylene) Ap5A (ApOpOCH2 pCH2OpOpA) (6) The terminology used supports recogni-tion of the orientarecogni-tion 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 ApnA 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.)
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analogs Phosphorus Sulfur Silicon Relat Elem 177,
2221–2222
35 Nun˜ez A, Berroteran D & Nun˜ez O (2003) Hydrolysis
of cyclic phosphoramidates Evidence for syn lone pair
catalysis Org Biomol Chem 1, 2283–2289
Supporting information
The following supplementary material is available:
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 Ap3A 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 ApOCH2pCH2OpA (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 Ap3A 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
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 ApCH2OpOpOCH2pA (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 ApOCH2pOpCH2OpA (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 Ap4A analog 5 Scheme S6 ApOpOCH2pCH2OpOpA (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 Ap5A analogue 6
Scheme S7 pOCH2pOpA (32) Bis(2-cyanoethyloxy)(di-isopropylamino)phosphine (27) [33] was reacted with dibenzyl phosphonate 22 and subsequently with benzyl
Trang 8alcohol 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
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 of1H and
31P NMR and high resolution mass spectroscopy (data not shown)
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