d - myo - inositol 
 1,4,5-trisphosphate receptors 
 ( ip3rs ) are ca channels located on the endoplasmic 
 reticulum . ip3
[ ins(1,4,5)p3 , 1 ] is a second messenger produced by the action 
 of phospholipase c on phosphatidylinositol 4,5-bisphosphate in response 
 to various extracellular signals .
ip3 binds to its receptor 
 and opens its intrinsic ca channel , allowing ca to leak into the cytosol and so cause the increase in cytosolic 
 [ ca ] that regulates many cellular events . to study the interaction of 1 with its receptor 
 and to understand structure  activity relationships ( sars ) , 
 many synthetic analogues of ip3
these studies revealed that the 4,5-bisphosphate 
 functionality and 6-oh group are apparently crucial for activity , 
 while the 1-phosphate has a supplementary role , and the 2-oh and 3-oh 
 groups are much less important .
although 
 the relative importance of all of the hydroxyl and phosphate groups 
 has been established , none of the synthetic analogues has proven to 
 be more potent than the natural ligand . in 1993 , two potent agonists 
 of ip3 receptors , adenophostin a ( 3a , figure 1 ) and adenophostin b ( 3b ) , were isolated 
 from culture broths of penicillium brevicompactum . both 3a and 3b bind to ip3 receptors with much greater affinity than 
 ip3 and are 10100 times more potent than ip3 in evoking ca release .
this finding has stimulated many syntheses of various analogues 
 of the adenophostins and studies of their sars .
structures of ip3 ( 1 ) , ins(4,5)p2 ( 2 ) , and adenophostins ( 3a and 3b ) . sar studies with synthetic analogues of adenophostin 
 a ( ada ) with 
 and without a purine ring established that the presence of the adenine 
 ring ( or an aromatic surrogate ) is crucial for enhanced affinity .
this suggests that the adenosine moiety either 
 disposes the 2-phosphate in a special spatial arrangement 
 to strengthen its interactions with the receptor ( indirect role ) or that the adenine moiety itself is involved 
 in supplementary binding interactions with a nearby region of the 
 binding site ( direct role ) .
it has been 
 shown that the glucose 2-oh group ( analogous to 6-oh in ip3 ) is important , while the glucose 5-ch2oh ( analogous to the 3-oh in ip3 ) and ribose 4-ch2oh are less important for ada activity .
the roles of the phosphate groups of ip3 seem to be established , but their relative importance in the adenophostins has not been 
 examined systematically using synthetic chemistry .
we have proposed 
 a model for ada binding to the ip3-binding core ( ibc ) of 
 the receptor in which the glucose 2-oh and 3,4-bisphosphate 
 triad mimic the 6-oh and 4,5-bisphosphate triad of ip3 , 
 while the adenine engages in a cation- interaction with arg504 
 adjacent to the binding site . in this model
we recently reviewed aspects 
 of the chemistry and biology of ada in its interaction with ip3r and examined the thermodynamics 
 of both ip3 and ada binding to both the n terminus and 
 ibc of the ip3r .
bisphosphate 6 ( figure 2 ) was 
 previously made by enzymatic hydrolysis of ada using alkaline phosphatase 
 and was reported to be 1800 times weaker than ada .
this implied a critical role for the 2-phosphate 
 group and led some workers to conclude that the adenine had only an 
 indirect role , by enhancing the positioning of the 2-phosphate 
 group .
however , this finding seems incompatible with our suggestion 
 based on detailed sar and molecular modeling that 
 the adenine may form its own interactions with the ip3r . 

furthermore , inframolecular protonation studies of ada suggest that , 
 at least in solution , the 2-p is close to the glucose ring 
 and the three phosphate groups behave similarly to those in ip3 .
hence , we were curious to re - examine 
 the biological activity of 6 with a chemically synthesized 
 sample and to compare its activity with a minimal motif of ada , glucose 
 3,4-bisphosphate [ gluc(3,4)p2,7 ] .
quantitative insight into the contribution of the 
 adenosine moiety might be obtained by comparison of the activities 
 of 6 and 7 , which differ only by the presence 
 of the adenosine moiety .
thus , to explore our model further and to investigate 
 whether the 
 extra binding motif of ada as compared to ip3 might compensate 
 for removal of a phosphate interaction , we report the synthesis and 
 biological evaluation of all three possible bisphosphates of ada
. 
 a preliminary communication on this work has appeared , and we recently reported the ability of 6 to 
 evoke ca release via recombinant and mutant ip3rs .
for the synthesis of adenophostin 
 analogues , an ideal and economical 
 strategy would be the vorbrggen condensation of a silylated 
 purine with an appropriately protected disaccharide , followed by deprotection 
 of hydroxyl groups ( to be later phosphorylated ) , phosphorylation , 
 and final deprotection .
it is a prerequisite 
 to have ester functionalities at 1-o- and 2-o - positions of the disaccharide for the vorbrggen 
 condensation .
other hydroxyl groups on 
 the disaccharide have to be protected with orthogonal protecting groups , 
 such that the hydroxyls to be phosphorylated are protected with an 
 easily removable temporary protecting group and the other hydroxyls 
 are protected with a stable protecting group that can be cleaved at 
 the final step . because the nucleoside after vorbrggen condensation 
 will have an ester protecting group at the 2-o - position ( which has to be phosphorylated in 4 and 5 ) and the fact that this ester can be removed under milder 
 conditions , it is ideal to protect the other hydroxyl to be phosphorylated 
 as a similar ester .
benzyl is the protecting group of choice for all 
 other hydroxyl groups as it is stable to various conditions and also 
 due to the convenience in its deprotection along with other benzyl 
 protecting groups on phosphate triesters in a single final step .
the synthesis of 4 started from the cheaply available 
 penta - o - acetyl--d - glucose ( 8 , scheme 1 ) .
the glycosyl donor 15 was prepared from 8 by a series of protecting 
 group manipulations .
the butane 2,3-diacetal ( bda ) derivative 9 was obtained from 8 as reported previously .
the acid - labile 
 bda protecting group was removed by hydrolysis with 80% acetic acid . 

stannylene - mediated benzylation was sluggish 
 and gave a 1:1 mixture of isomeric tribenzyl ethers 12 ( 27% ) and 13 ( 23% ) along with unreacted 11 ( 36% ) .
sodium hydride - mediated benzylation also gave a 1:1 mixture 
 of tribenzyl ethers 12 ( 18% ) and 13 ( 16% ) 
 in very low yield along with fully benzylated 14 ( 32% ) 
 and starting material 11 ( 24% ) .
fortunately , benzylation 
 under biphasic conditions with tetrabutylammonium hydrogen sulfate 
 as phase transfer catalyst gave predominantly the tribenzyl ether 12 ( 74% ) along with minor amounts of 13 ( 8% ) 
 and 14 ( 10% ) .
n - iodosuccinimide 
 ( nis)-mediated glycosylation of 5-o - benzyl-1,2-o - isopropylidene--d - ribofuranose ( 16 ) , which was prepared from 1,2-o - isopropylidene--d - xylofuranose by a known procedure with glycosyl donor 15 , gave the -glycoside 17 selectively .
such a high degree of selectivity has been observed 
 before when structurally similar compounds were used . to make the disaccharide donor 
 for vorbrggen glycosylation ,
the isopropylidene group in 17 was hydrolyzed with 90% trifluoroacetic acid ( tfa ) , and 
 the resulting mixture of diols ( anomeric mixture ) was acetylated under 
 standard conditions to produce a chromatographically inseparable / 
 mixture of triacetates 18 .
vorbrggen condensation 
 of 18 with silylated 6-chloropurine gave the -nucleoside 19 as the exclusive product .
the yield of the condensation 
 depended on the nature of the silylating agents used for the persilylation 
 of purine . among the various silylating agents used , n , o - bis - trimethylsilyl - acetamide ( bsa ) gave a very 
 good yield for the subsequent same pot glycosylation .
removal of the 
 acetate protecting groups in nucleoside 19 and substitution 
 of chlorine with an amino group were achieved concomitantly by ammonolysis . 

the diol 20 was then selectively phosphitylated with 
 dibenzyl n , n - diisopropyl phosphoramidite 
 in the presence of imidazolium triflate as catalyst , and the resulting bisphosphite was oxidized in situ to 
 bisphosphate 21 .
finally , all of the benzyl protecting 
 groups ( on both the phosphate and the sugar ) were removed by transfer 
 hydrogenolysis to provide the bisphosphate 4 , which was purified by ion exchange chromatography .
reagents and conditions : 
 ( a ) 
 bnbr , nah , dmf , 0 c  room temperature , 2 h. ( b ) 80% aqueous 
 hoac , reflux , 1 h. ( c ) bu2sno , bnbr , bu4nbr , 
 mecn , 3  molecular sieves , reflux , 24 h. ( d ) bu4nhso4 , bnbr , dcm : 5% aqueous naoh ( 1:1 ) , room temperature .
( e ) 
 ac2o , pyr , room temperature , 12 h. ( f ) compound 16 , nis , tmsotf , dioxane : toluene ( 3:1 v / v ) , 3  molecular sieves , 
 room temperature , 30 min .
( h ) 
 ac2o , pyr , room temperature , 3 h. ( i ) 6-chloropurine , bsa , 
 tmsotf , mecn , 70 c , overnight .
( k ) ( 1 ) ( bno)2pn(ipr)2 , imotf , 
 dcm , room temperature , 30 min ; ( 2 ) mcpba , 78 c  room 
 temperature , 1 h. ( l ) cyclohexene , pd(oh)2 , meoh , h2o , 80 c , overnight .
synthesis 
 of bisphosphate 5 started with commercially 
 available tetra - acetate 22 ( scheme 2 ) .
the acetate protecting groups on 22 were removed 
 by methanolysis with a catalytic amount of sodium methoxide in methanol , 
 and the resulting crude tetrol was converted into diol 23 by benzylidenation using benzaldehyde dimethyl acetal and camphorsulphonic 
 acid ( csa ) .
crude 23 was then benzylated with an excess 
 of benzyl bromide and sodium hydride to afford the dibenzyl ether 24 .
reductive opening of the benzylidene acetal by following 
 a known procedure gave the tribenzyl 
 ether 25(32 ) selectively .
glycosylation 
 of ribose derivative 16 with the glycosyl donor 26 , obtained by acetylation of the tribenzyl ether 25 , gave -glycoside 27 selectively . as in the previous 
 case ,
disaccharide 27 was converted into the triacetate 28 ( - 
 and -anomer ) in a one pot reaction of hydrolysis of acetonide 
 with tfa followed by acetylation .
vorbrggen condensation with 
 6-chloropurine gave the nucleoside 29 , which on ammonolysis 
 provided the diol 30 .
transfer hydrogenolysis removed all of the benzyl protecting 
 groups to provide the bisphosphate 5 , which was purified 
 by ion exchange chromatography .
reagents and conditions : 
 ( a ) 
 meoh , naome , room temperature , 30 min .
( d ) bnbr , nah , dmf , room 
 temperature , 2 h. ( e ) tfa , et3sih , dcm , 0 c .
( f ) 
 ac2o , pyr , room temperature , 3 h. ( g ) compound 16 , nis , tmsotf , dioxane : toluene , 3  molecular sieves , room temperature , 
 30 min .
( i ) 6-chloropurine , 
 bsa , tmsotf , mecn , 70 c , overnight .
( k ) ( 1 ) ( bno)2pn(ipr)2 , 
 imotf , dcm , room temperature , 30 min ; ( 2 ) mcpba , 
 78 c  room temperature , 1 h. ( l ) cyclohexene , 
 pd(oh)2 , meoh , h2o , 80 c , overnight .
the requirement that the 1-o- and 2-o - positions of the disaccharide should be 
 protected as esters for 
 vorbrggen condensation leaves an ester group at the 2-o - position of nucleosides 19 and 29 . with the next step being the substitution of 6-cl with nh2 and deprotection of the hydroxyl groups to be phosphorylated ,
the 
 ester functionality at 2-o- position is advantageous , 
 as treatment with nh3 effects both of the transformations . 

the other hydroxyl group to be phosphorylated ( 3 in 18 and 4 in 28 ) was also protected as 
 its acetate for convenient one - step deprotection .
however , 
 it can not be applied to the synthesis of bisphosphate 6 as there is no phosphate at the 2-o - position . 

this prompted us to explore other possibilities . during our 
 synthesis of the ada analogue ,
guanophostin , several attempts to substitute the chlorine 
 in the intermediate 32 with different oxygen nucleophiles 
 under basic conditions [ 4 m naoh , dioxane ; 
 3-hydroxypropiononitrile , 1,8-diaza - bicycloundec-7-ene ( dbu ) , 
 dichloromethane ( dcm ) ; 3-hydroxypropiononitrile , 
 nah , tetrahydrofuran ( thf ) ; bnoh , k2co3 ] were marred by accompanied dephosphorylation . 

for instance , when 3-hydroxypropiononitrile in the presence of a base 
 ( dbu or nah ) was used , it was observed that at first all of the benzyl 
 groups were displaced by the 2-cyanoethoxy group , which underwent 
 -elimination to give water - soluble phosphate monoesters .
o  sugar 
 cleavage , also giving all of the possible mono- , bis- , and tris - phosphates . 

possible transesterification pathways for a protected sugar phosphate 
 are shown in figure 4 . 
 because the p  o  sugar also underwent cleavage , we were 
 curious to investigate the possible selective dephosphorylation at 
 the 2-position from a protected trisphosphate such as 33 ( scheme 3 ) via transesterification 
 with an alcohol .
we envisioned that if we used the incoming nucleophile 
 ( ro ) , the same as the protecting group on the phosphorus 
 ( benzyl ) , the unwanted loss of protecting group on phosphorus can 
 be prevented .
various possible transesterification mechanisms for phosphate 
 triesters . 
 only the first step is shown .
reagents and conditions : 
 ( a ) 
 bnoh , nah , room temperature , 30 min .
( c ) pd(oh)2 , cyclohexene , 
 meoh , h2o , 80 c , overnight .
( d ) r  nh2 ( r = h , bu , pr ) , etoh , room temperature .
trisphosphate 33 on treatment with in situ - generated 
 sodium benzoxide [ benzyl alcohol ( 5 equiv ) and nah ( 2 equiv ) ] in n , n - dimethylformamide ( dmf ) at room temperature 
 for 30 min gave a bisphosphate as major product with minor amounts 
 of other byproducts .
fortunately , the use of the milder base potassium 
 carbonate and bnoh as solvent resulted in the exclusive formation 
 of this bisphosphate in excellent yield .
the 
 fact that the 2-phosphate is comparatively less crowded and 
 hence more prone to nucleophilic attack than those in the 3,4 
 vicinal bisphosphate functionality could be the reason for this high 
 selectivity .
this assumption is supported by the fact that triacetate 35 on aminolysis with various amines gives the 2-o - deacetylated derivative 36 exclusively . finally , 
 the benzyl protecting groups on both sugar and phosphorus were removed 
 by transfer hydrogenolysis and purification of the product by ion 
 exchange column chromatography provided bisphosphate 6 quantitatively .
for biological comparison with 6 , the previously unknown 
 glucose 3,4-bisphosphate 7 was synthesized from allyl 
 glycoside 37 ( scheme 4 ) .
phosphorylation of 37 using standard 
 phosphoramidite methodologygave the fully 
 protected bisphosphate 38 , and the allyl protecting 
 group was removed using pdcl2 in methanol to give bisphosphate 39 as a mixture of - 
 and -anomers .
hydrogenolysis of 39 then provided 
 the bisphosphate 7 , and h nmr and p nmr spectroscopy showed this to exist as a mixture of - 
 and -anomers in aqueous or methanolic solution .
the relative 
 proportions of the two anomers were dependent on salt form and ph . reagents and conditions : 
 ( a ) 
 ( 1 ) ( bno)2pn(ipr)2 , 1h - tetrazole , 
 dcm , room temperature , 1 h ; ( 2 ) mcpba , 78 
 c , 10 min , 92% .
( c ) h2 , pd(oh)2 , meoh , h2o , 85% . the ability of bisphosphates 47 , ip3 ( 1 ) , ada ( 3a ) , and d - myo - inositol 4,5-bisphosphate [ ins(4,5)p2 ( 2 ) ] to stimulate the ip3r of intracellular 
 ca stores was measured using a low - affinity ca indicator trapped within the intracellular stores of permeabilized 
 dt40 cells expressing only recombinant rat ip3r1 , as previously 
 reported .
the amount of ca released by each of these compounds was expressed as a fraction 
 of the total ca content of the endoplasmic reticulum 
 ( er ) as assessed by the addition of ionomycin .
results show the half - maximal effective 
 concentration of each ligand ( ec50 ) , the hill coefficient 
 ( nhill ) , and the percentage of the intracellular 
 stores released by a maximal concentration of each ligand .
the ec50 for 5 , where even the maximal practicable concentration 
 ( 50 m ) failed fully to release the ip3-sensitive 
 ca stores , was calculated by assuming that it was a full 
 agonist capable of releasing the entire ip3-sensitive ca store . among the bisphosphates ,
6 is the most 
 potent ; it 
 releases the same fraction of the ca stores as other 
 full agonists , and it is only 40 times less potent than ada and only 
 four times less potent than ip3 .
this contradicts an earlier 
 report where 6 was found 
 to be 1800-fold less potent than ada , although this is now thought 
 to be in error ( see ref ( 19 ) for discussion ) .
bisphosphate 4 released only 
 8% of the ca stores at a concentration of 50 m . 

it was impracticable to assess the activity at higher concentrations 
 and so impossible to resolve whether 4 is a full agonist 
 with very low affinity or a partial agonist .
bisphosphate 5 had some activity , but even at 50 m , it released only 53% 
 of the intracellular stores .
again , it was impracticable to increase 
 the concentration of 5 sufficiently to assess whether 
 it is a full agonist , capable at a high enough concentration , of mimicking 
 the response to a maximal concentration of ada or ip3 . 
 assuming 5 to be a full agonist , then it is 2300 times 
 less potent than ip3 .
this 
 is broadly in agreement with an earlier report , where ins(4,5)p2 was 460-fold less potent than 
 ip3 in evoking ca release from rat brain microsomes . 
 when compared in permeabilized dt40 cells ( table 1 ) , synthetic ins(4,5)p2 from the present study 
 is ca .
the marginally weaker activity of 7 as compared to ins(4,5)p2 is not surprising , 
 given the existence of 7 as a mixture of anomers .
the 7- isomer mimics four of the ip3 carbon 
 centers retaining the important triad binding motif ( 4,5-bisphosphate 
 and 6-oh ) ; hence , 7- might be more or less active 
 as ip2 ( figure 5 ) .
however , 7- can orient in two different ways ( a and b ; figure 5 ) , mimicking the orientation of ip3 . 

although orientation a has the binding motif in full , it mimics only 
 three carbons of ip3 , while orientation b mimics four centers 
 of ip3 but lacks the 6-oh mimic of the binding motif . while 
 it is reasonable to expect that the mode with maximum resemblance 
 to ip3 ( b ) would be the preferred fit , the association 
 constant could be less due to the lack of 6-oh interaction with the 
 receptor .
competition between these two orientations ( a and b ) for 
 receptor binding could reduce the overall potency .
ada binds to a site that at least substantially 
 overlaps the ip3-binding site and in a manner that is thought 
 to be broadly 
 similar to ip3 .
the crystal 
 structure of ip3 bound to 
 the ibc ( residues 224604 ) of ip3r1 and our model for ada binding together allow a rationalization 
 of the biological activities of the bisphosphates of adenophostin 
 a. the ibc comprises an -domain and -domain , with ip3 sandwiched between the two domains ( figure 6 ) .
possible hydrogen bonds between ip3 and ip3r in the crystal structure ( pdb : 1n4k ) of the ibc of ip3r1 .
the 4,5-bisphosphate motif of ip3 makes 
 more contacts 
 with the receptor than the more exposed 1-phosphate .
the 1-phosphate 
 interacts only with r568 and the backbone nh of k569 in the -domain , 
 while the 4-phosphate interacts mainly with residues in the -domain 
 ( r265 , r269 , and t267 ) , and the 5-phosphate interacts predominantly 
 with amino acids in the -domain ( r504 , k508 , r511 , and y567 ) . 

we and others have speculated that binding of ip3 pulls the domains together and closes the  clam - like  
 binding core , in a manner similar to glutamate binding to ionotropic 
 glutamate receptors .
ip3 ( 1 ) and ins(4,5)p2 ( 2 ) activate ip3r by engaging residues in the - ( green ) and - ( pink ) 
 domains of the ibc , stabilizing a closed conformation that favors 
 opening of the c - terminal ca channel .
phosphate groups may have strong ( dark green ) or weaker 
 ( light green ) interactions with the -domain .
molecular modeling 
 suggests that ada ( 3a ) has additional interactions ( light 
 green ) with the -domain of the ibc , accounting for the greater 
 potency of ada .
4-dephospho - ada ( 4 ) is essentially 
 inactive because it can not form effective -domain interactions . 

however , 3-dephospho - ada ( 5 ) and 2-dephospho - ada 
 ( 6 ) retain activity because they can effectively engage 
 both domains , even though 5 does not contain a vicinal 
 bisphosphate pair . the previously unexplained and relatively potent 
 activity of ada regiosomer 40 can now be explained by 
 analogy with 5 .
recently published x - ray structures of the n - terminal 
 ligand - binding 
 domain ( lbd ) and the n - terminal domain 
 of rat ip3r1 ( residues 1604 ) with and without ip3 bound support the idea that ip3 causes domain 
 closure within the ibc . as ip3 binds , side chains of nine residues within the - and -domains 
 of the ibc become organized around ip3 , causing the clam - like 
 structure to partially close , reducing the angle between the two domains 
 by 8. unfortunately , the resolution of these structures 
 is not sufficient to provide further clues about how ada and its analogues 
 might bind .
the 4,5-bisphosphate of ip3 clearly plays a 
 major role in cross - linking the two domains of the ibc , and the 1-phosphate 
 exerts its enhancing effect by providing an additional , weaker interaction 
 with the -domain , accounting for the greater potency of ip3 relative to ins(4,5)p2 ( figure 7 ) .
the known inactivity of d - myo - inositol 1,4-bisphosphate [ ins(1,4)p2 ] suggests that 
 the 1-p alone can not interact strongly enough with the -domain 
 to pull the two domains together .
our model for ada binding shows how the 3,4-bisphosphate 
 of ada can mimic the 4,5-bisphosphate of ip3 , while both 
 its 2-phosphate group and adenine moiety have additional interactions 
 with the ibc ( figures 7 and 8a ) .
specifically , we have proposed that while the 2-phosphate 
 of ada essentially mimics the 1-phosphate of ip3 , the adenine 
 can engage in a cation- interaction with the guanidinium side 
 chain of r504 in the -domain .
interactions of ada ( a ) , compound 5 ( b ) , and compound 6 ( c ) with the ibc of ip3r as predicted by molecular 
 docking experiments .
the present study shows that bisphosphate analogues 
 of ada lacking 
 the 3- or 4-phosphate ( 4 and 5 ) are very weak agonists , confirming the important role for the 3,4-vicinal 
 bisphosphate in ada .
however , our finding 
 that 4 and 5 differ in their potency suggests 
 unequal contributions from the two phosphates ( 4-p and 3-p ) . 
 while bisphosphate 4 is almost inactive , 5 is a weak agonist of ip3r , being some 2300-fold weaker 
 than ip3 .
this shows that between the vicinal bisphosphates , 
 the 3-phosphate in ada is less important than the 4-phosphate . 

loss of activity after deletion of the 4-phosphate from ada 
 is consistent with it providing the major contact with the -domain , 
 which may be essential for  clam  closure .
thus , 4 and 5 can be considered as analogues of d - myo - inositol 1,5-bisphosphate [ ins(1,5)p2 ] and ins(1,4)p2 , respectively , and the fact that 5 is a weak agonist while its inositol equivalent ins(1,4)p2 is inactive can be explained in terms of our model by the 
 enhancing role of the adenine in 5 , strengthening its 
 -domain interactions ( figures 7 and 8b ) .
2-dephospho - ada ( 6 ) differs from gluc(3,4)p2 ( 7 ) only in having 
 an adenosine moiety ; yet , 
 it is 200-fold more potent than 7 ( table 1 ) .
this 200-fold enhancement of activity by the adenosine 
 moiety in the absence of a 2-phosphate establishes unequivocally 
 that the adenosine moiety contributes directly to enhanced activity 
 independent of any effect on positioning of that phosphate . in a recent 
 report
, we demonstrated , in a study linking 
 chemical modification with receptor mutagenesis , that removal of the 
 2-phosphate from ada to give 6 has significantly 
 lesser effects on affinity for the ibc than did removal of the 1-phosphate 
 from ip3 to give ins(4,5)p2 .
a cell line was 
 established that expresses only ip3r1 , and mutation of 
 r504 more profoundly reduces the affinity of ip3r for ada 
 ( 353-fold ) than for ip3 ( 13-fold ) .
the activities of other 
 adenophostin analogues and the corresponding ip3 analogues 
 were also comparable .
thus , when an amino acid thought to be responsible 
 for engaging the adenine unit ( r504 ) is mutated , the enhanced activity 
 of adenophostin ligands disappears .
bisphosphate 6 is the most potent known agonist of 
 the ip3r with only two phosphates , suggesting that it may 
 be possible to develop high - affinity ligands with fewer phosphates 
 or even nonphosphate moieties .
the possibility to develop less charged 
 agonists or antagonists would be particularly attractive for cellular 
 or in vivo chemical biological studies . even though 5 is a weak agonist , its measurable activity suggests that a vicinal 
 bisphosphate is not absolutely required for a ligand to activate ip3r .
the regioisomeric ada analogue 2-phospho-3-dephospho - ada 40 ( figure 9 ) with the 3-phosphate 
 group of ada transposed to the 2-position was previously shown 
 to possess surprisingly potent activity that did not fit established 
 sar considerations .
compound 40 , although lacking the 
 vicinal 3,4-bisphosphate , 
 is only 1220 times less potent than ip3 in liver 
 flux and binding assays , respectively .
interestingly , d - myo - inositol 1,4,6-trisphosphate 
 ( 41 ) , which may be pictured as having a similar arrangement 
 of phosphate groups to 40 ( figure 9 ) , is also an agonist , only 23 times weaker than ip3 .
however , 41 may be able 
 to present an ip3-like arrangement of phosphate groups 
 to the ibc simply by binding in an alternative orientation ( 41b , figure 9 ) .
similar reasoning was 
 used to explain the unexpected activity of 40 in the 
 original study , where it was suggested that it too may be able to 
 adopt an inverted binding orientation .
this may seem improbable given the size and complexity of 40 as compared to 41 , but the alternative explanation , 
 namely , that a vicinal bisphosphate is not required , would have been 
 totally unprecedented . given the new finding of activity for 5 , which can not present a vicinal bisphosphate in any binding 
 orientation
, we can now suggest a simpler explanation for the activity 
 of both 5 and 40 that is also consistent 
 with more recent information on the structure of the ibc and with 
 the concept of a direct role for the adenine . we propose that , contrary 
 to previous assumptions , a vicinal bisphosphate motif is not absolutely 
 essential for activation of ip3r , given sufficiently strong 
 compensating contributions from other components of the ligand .
thus , 
 we suggest that for bisphosphate 5 , interactions of the 
 adenine with the -domain partially compensate the loss of one 
 phosphate group in the vicinal pair , while in 40 , these 
 interactions are supplemented by additional contributions from the 
 2-phosphate , presumably also with the -domain , strengthening 
 binding still further ( figure 7 ) .
thus , it 
 appears that two or more weak interactions with the -domain 
 can , to some extent , compensate for the loss of a strong interaction .
in summary , as a continuation of our efforts 
 to understand sars 
 of adenophostins at the ip3 receptor and to gain further 
 insight into the molecular mechanism of ip3-mediated signal 
 transduction pathways in general , we have synthesized all of the bisphosphate 
 analogues of ada , excising one of its three phosphates at a time . 

bisphosphates 4 and 5 were prepared in a 
 series of reactions involving -selective glycosylation of an 
 appropriately protected glucose ( glycosyl donor ) and protected ribose 
 acceptor , vorbrggen condensation of an appropriately protected 
 disaccharide with 6-chloropurine , followed by further manipulation 
 and chemoselective phosphitylation .
a novel strategy for regioselective 
 dephosphorylation by transesterification has been introduced for the 
 efficient synthesis of 6 .
the ability of these novel 
 bisphosphates to stimulate ip3r - mediated ca release was measured using rat type i ip3r expressed 
 in chicken dt40 cells and compared to that of glucose 3,4-bisphosphate 
 ( 7 ) and ins(4,5)p2 .
this study reveals that 
 although the 3,4-bisphosphate functionality is important 
 for high affinity binding and channel opening , it is still possible 
 for molecules lacking this feature to stimulate the ip3r , albeit to a lesser extent .
p-4 of ip3 , which contacts the -domain 
 of the ibc , is required , but p-5 , which contacts the -domain , 
 can be replaced by a cation- interaction between the adenine 
 of ada and r504 in the -domain .
the same interaction can substantially 
 compensate the loss of p-1 to provide the potent agonist 6 with only two phosphate groups .
inositol polyphosphates often bind 
 to sites rich in arg and lys residues , and replacing such interactions 
 with a polar phosphate by a cation- motif could have more general 
 applications in the chemical biology of inositol phosphate signaling 
 and probably also in other fields .
a 200-fold enhancement in activity is found 
 in going from the simple glucose bisphosphate 7 to the 
 related 6 bearing an adenosyl moiety , establishing a 
 direct role for the adenosine of ada in increasing affinity for the 
 receptor .
compound 6 , the most potent among the bisphosphates 
 synthesized , approaches the potency of ip3 .
this is the 
 first report of a relatively potent agonist of ip3r devoid 
 of one phosphate group from the natural ligand and suggests the possibility 
 to develop other ligands having a fewer number of phosphates .
such 
 ligands , with less charge , could be useful for pharmacological intervention 
 in this cellular signaling system and , at their simplest , might comprise 
 two motifs that interact with the ibc domains linked by a suitable 
 spacer .
the reappraisal here of the relatively potent activity and 
 surprising activity of 40 , published earlier , in the 
 light of these new results and particularly the activity of 5 , begins to demonstrate the potential of this approach , suggesting 
 that it should be possible in principle to develop ip3r 
 ligands without a vicinal bisphosphate moiety .
tlc was performed on precoated plates ( merck aluminum sheets silica 
 60 f254 , art no .
chromatograms were visualized under 
 uv light and by dipping plates into either phosphomolybdic acid in 
 meoh or anisaldehyde in ethanol , followed by heating .
ion exchange 
 chromatography was performed on an lkb - pharmacia gradifrac medium 
 pressure ion - exchange chromatograph using mp1 ag ion - exchange resin 
 and a gradient of 0100% 150 mm tfa as eluent or q sepharose 
 fast flow resin and a gradient of 0100% 1.0 m triethylammonium 
 bicarbonate ( teab ) .
h nmr , cosy , noesy , hmbc , and hmqc 
 spectra were recorded on varian ex-400 ( 400 mhz ) and bruker avance 
 iii ( 400 and 500 mhz ) spectrometers .
proton chemical shifts are reported 
 in ppm (  ) relative to internal tetramethylsilane ( tms ,  
 0.0 ppm ) or with the solvent reference relative to tms employed as 
 the internal standard ( cdcl3 ,  7.26 ppm ; d2o ,  4.79 ppm ) .
data are reported as follows : chemical shift 
 ( multiplicity [ singlet ( s ) , doublet ( d ) , triplet ( t ) , quartet ( q ) , 
 and multiplet ( m ) ] , integration , coupling constants [ hz ] , annotation ) . c and
dept spectra were recorded on varian ex-400 ( 100 mhz ) 
 and bruker avance iii ( 100 and 126 mhz ) spectrometers with complete 
 proton decoupling .
carbon chemical shifts are reported in ppm (  ) 
 relative to tms with the respective solvent resonance as the internal 
 standard ( cdcl3 ,  77.0 ppm ) .
p nmr 
 spectra were recorded on varian ex-400 ( 162 mhz ) or bruker avance 
 iii ( 162 mhz ) spectrometers with complete proton decoupling .
phosphorus 
 chemical shifts are reported in ppm (  ) relative to an 85% h3po4 external standard ( h3po4 ,  0.0 ppm ) .
melting 
 points were determined using a reichert - jung thermo galen kofler block 
 and are uncorrected .
mass spectra were recorded at the serc 
 mass spectrometry service centre , swansea , and at the university of 
 bath on vg autospec or microtof instruments .
polarimeter in a 
 cell volume of 5 cm , and specific rotation is given in 
 10 deg cm g. uv 
 spectra were recorded using a perkin - elmer lambda ez201 spectrometer . 

the purities of final compounds were determined to be greater than 
 98% by hplc analysis .
hplc analysis was carried out on a hewlett - packard 
 series chromatograph with a strong anion - exchange resin ( mp1 ag , column 
 size 3 mm  150 mm ) .
a linear gradient of 050% 150 mm 
 tfa was used as eluent at 1 cm / min over 60 min , with the 
 uv detector set at 254 nm .
synthetic phosphates were assayed using 
 an adaptation of the modified brigg 's phosphate assay and/or ames phosphate assay .
flash column chromatography was performed using silica gel 60 a 
 ( 3263 m ) .
all reactions were carried out under argon 
 or nitrogen atmosphere employing oven - dried glassware unless stated 
 otherwise .
usual work up refers to taking up the crude material in 
 an organic solvent ( ethyl acetate , dcm , or chcl3 ) followed 
 by washing successively with water , cold diluted hcl , saturated nahco3 solution , and brine and drying over anhydrous mgso4 .
both donor 15 ( 1.925 g , 3.587 mmol ) 
 and acceptor 16 ( 981 mg , 3.5 mmol ) were dissolved in 
 toluene  dioxane ( 10 ml : 30 ml ) , and the mixture was stirred 
 with powdered 3  molecular sieves for 15 min .
nis ( 866 mg , 3.85 
 mmol ) was then added , and when it had dissolved , tmsotf ( 100 l , 
 0.55 mmol ) was added dropwise , and the mixture was stirred at room 
 temperature .
nahco3 solution was added to quench the acidic 
 residue , and the mixture was diluted with ethyl acetate and washed 
 with saturated na2s2o3 solution and 
 then with brine .
hexane to afford the 
 -glycoside 17 ( 2 g , 76% ) as a colorless crystalline 
 solid ; mp 112 c ; [ ]d + 120.8 ( c 1 , chcl3 ) .
h nmr ( 400 mhz , cdcl3 ) : 1.35 ( s , 3h , ch3 isopropylidene ) , 
 1.50 ( s , 3h , ch3 isopropylidene ) , 1.96 
 ( s , 3h , coch3 ) , 3.46 ( dd , 1h , 10.87 hz , 
 1.00 hz , h-6a ) , 3.51 ( dd , 1h , 9.88 hz , 3.95 hz , 
 h-2 ) , 3.59 ( dd , 1h , 10.87 hz , 2.47 hz , h-6b ) , 
 3.67 ( dd , 1h , 8.40 hz , 2.97 hz , h-5a ) , 3.673.74 
 ( m , 2h , h-4 and h-5 ) , 3.79 ( dd , 1h , 11.61 hz , 1.73 
 hz , h-5b ) , 4.15 ( dd , 1h , 9.14 hz , 3.95 hz , h-3 ) , 4.29 ( ddd , 
 1h , 9.14 hz , 3.21 hz , 1.73 hz , h-4 ) , 4.44 ( ab q , 2h , 25.69 hz , 11.36 
 hz , ch2ph ) , 4.47 ( ab q , 2h , 82.02 hz , 
 11.86 hz , ch2ph ) , 4.53 ( ab q , 2h , 46.44 
 hz , 11.86 hz , ch2ph ) , 4.64 ( ab q , 2h , 
 50.89 hz , 12.35 hz , ch2ph ) , 4.71 ( t , 1h , 
 3.95 hz , h-2 ) , 5.23 ( d , 1h , 3.95 hz , h-1 ) , 5.45 ( t , 1h , 9.39 
 hz , h-3 ) , 5.83 ( d , 1h , 3.95 hz , h-1 ) , 7.117.35 ( m , 
 20 h , 4  c6h5 ) .
elemental 
 analysis calcd for c44h50o11 : c , 
 70.01 ; h , 6.68 . found : c , 69.8 ; h , 6.69 .
m / z ( es+ ) = 777.4 [ ( m + na ) , 100% ] .
hrms : mass 
 calcd for c44h54o11n1 [ m 
 + nh4 ] , 772.3691 ; found , 772.3691 .
disaccharide 17 ( 128 mg , 0.17 
 mmol ) was stirred with 90% tfa ( 0.5 ml ) for 1015 min at room 
 temperature .
the tfa was then evaporated off , and the residue was 
 coevaporated several times with toluene to remove traces of water . 

the residue was then dissolved in pyridine ( 2 ml ) , ac2o 
 ( 0.5 ml , 5.3 mmol ) was then added , and the mixture was stirred for 
 4 h at room temperature .
work up was as usual , and the crude product 
 was chromatographed to afford an inseparable mixture of the - 
 and -triacetates 18 ( 120.6 mg , 89% ) . a suspension 
 of triacetate 18 ( 120 mg , 0.15 mmol ) , 6-chloropurine 
 ( 46 mg , 0.3 mmol ) , and bsa ( 182 l , 0.74 mmol ) in mecn ( 5 ml ) 
 was refluxed until the solution became clear .
the mixture was cooled 
 to room temperature , and then , tmsotf ( 60 l , 0.33 mmol ) was 
 added , and the solution was stirred at 70 c overnight .
workup 
 was in ethyl acetate , and the crude product was chromatographed to 
 afford the nucleoside 19 ( 103 mg , 77% ) as a colorless 
 gum .
h nmr ( 400 mhz , cdcl3 ) : 1.84 ( s , 
 3h , coch3 ) , 1.86 ( s , 3h , coch3 ) , 3.41 ( dd , 1h , 10.14 hz , 3.48 hz , h-2 ) , 3.44 
 ( dd , 1h , 8.99 hz , 1.74 hz , ha-6 ) , 3.56 ( dd , 1h , 
 10.72 hz , 3.19 hz , hb-6 ) , 3.563.61 ( m , 
 1h , ha-5 ) , 3.60 ( t , 1h , 9.27 hz , h-4 ) , 
 3.703.80 ( m , 2h , h-5 , hb-5 ) , 4.40 
 ( ab q , 2h , 33.62 hz , 11.01 hz , phch2 ) , 
 4.41 ( ab q , 2h , 78.84 hz , 11.88 hz , phch2 ) , 4.424.46 ( m , 1h , h-4 ) , 4.47 ( ab q , 2h , 14.49 hz , 
 11.59 hz , phch2 ) , 4.49 ( ab q , 2h , 64.92 
 hz , 12.17 hz , phch2 ) , 4.68 ( t , 4.93 hz , 
 h-3 ) , 4.95 ( d , 1h , 3.48 hz , h-1 ) , 5.45 ( dd , 1h , 10.14 
 hz , 9.28 hz , h-3 ) , 5.68 ( dd , 1h , 5.22 hz , 4.64 hz , h-2 ) , 
 6.30 ( d , 1h , 4.64 hz , h-1 ) , 7.007.30 ( m , 20h , 4  ph ) , 8.45 ( s , 1h , h-8 ) , 8.66 ( s , 1h , h-2 ) .
m / z ( es+ ) = 915.44 [ ( m + na ) , 100% ] . 

hrms : mass calcd for c48h50cln4o11 [ m + h ] , 893.3159 ; found , 893.3160 .
a solution of chloro - nucleoside 19 ( 100 mg , 0.112 mmol ) in ethanol ( 10 ml ) in a pressure tube was saturated 
 with nh3 ( bubbled at 0 c for 1015 min ) .
the 
 tube was then closed tightly and was heated at 74 c for 5 days . 

solvents were evaporated off in vacuo , and the crude product was chromatographed 
 with 4% meoh 
chcl3 to afford the diol 20 ( 86 mg , 97% ) as a colorless gum .
h nmr ( 400 mhz , cdcl3 ) : 3.51 ( dd , 1h , 9.78 hz , 3.52 hz , h-2 ) , 3.56 ( dd , 
 1h , 10.17 hz , 9.39 hz , h-4 ) , 3.553.61 ( m , 2h , ha-5 and ha-6 ) , 3.66 ( dd , 2h , 10.57 
 hz , 3.52 hz , hb-5 and hb-6 ) , 
 3.82 ( ddd , 1h , 10.17 hz , 3.13 hz , 1.96 hz , h-5 ) , 4.04 ( br , 
 1h , oh ) , 4.20 ( dd , 1h , 9.39 hz , 9.78 hz , h-3 ) , 4.28 ( dd , 1h , 
 6.26 hz , 3.13 hz , h-4 ) , 4.41 ( dd , 1h , 5.87 hz , 3.52 hz , h-3 ) , 
 4.484.52 ( br , 1h , oh ) , 4.48 ( s , 2h , phch2 ) , 4.51 ( ab q , 2h , 43.82 hz , 12.13 hz , phch2 ) , 4.67 ( dd , 11.74 hz , 5.87 hz , h-2 ) , 4.69 ( ab 
 q , 2h , 116.21 hz , 11.35 hz , phch2 ) , 4.80 
 ( ab q , 2h , 17.61 hz , 11.74 hz , phch2 ) , 
 4.96 ( d , 1h , 3.91 hz , h-1 ) , 5.98 ( d , 1h , 5.87 hz , h-1 ) , 
 6.05 ( br s , 2h , nh2 ) , 7.157.35 ( m , 20h , 4  ph ) , 7.98 ( s , 1h , h-8 ) , 8.39 ( s , 1h , h-2 ) .
m / z ( es+ ) = 812.48 [ ( m + na ) , 100% ] . 
 hrms : mass calcd for c44h48o9n5 [ m + h ] , 790.3447 ; found , 790.3449 . to a solution of 
 diol 20 ( 123 mg , 0.156 mmol ) and dibenzyl - n , n - di - isopropyl phosphoramidite ( 113 mg , 0.327 mmol ) 
 in dcm ( 5 ml )
was added imidazolium triflate ( 75 mg , 0.343 mmol ) , 
 and the solution was stirred at room temperature for 30 min .
when 
 tlc showed the disappearance of diol 20 , the temperature 
 was reduced to 78 c , and then , mcpba 
 ( 170 mg , 6070% ) was added , and the solution was stirred for 
 1 h , gradually allowing the temperature to attain room temperature . 

workup was in ethyl acetate , and the crude product was chromatographed 
 with ethyl acetate : hexane : et3n , 77:20:3 ( v / v / v ) , to get 
 pure bisphosphate 21 ( 165 mg , 81% ) as a colorless oil . 

h nmr ( 400 mhz , cdcl3 ) : 3.46 ( dd , 1h , h-6a ) , 3.56 ( dd , 1h , h-6b ) , 3.583.64 
 ( m , 2h , h-2 , h-5a ) , 3.673.76 ( m , 
 3h , h-4 , h-5 , h-5b ) , 4.354.54 
 ( m , 7h , h-4 , 3  ch2ph ) , 
 4.624.77 ( m , 5h , h-3 , 2  ch2ph ) , 4.794.98 ( m , 7h , h-3 , 3  ch2ph ) , 5.40 ( d , 1h , 3.52 hz , h-1 ) , 5.66 
 ( ddd , 1h , 8.22 hz , 5.48 hz , 4.90 hz , h-2 ) , 5.706.00 
 ( br , 2h , nh2 ) , 6.34 ( d , 1h , 5.48 hz , h-1 ) , 7.007.42 
 ( m , 40h , 8  ph ) , 7.93 ( s , 1h , h-8 ) , 8.27 ( s , 1h , h-2 ) .
elemental analysis calcd for c72h73n5o15p2 c , 66.00 ; h , 5.62 ; n , 5.34 .
m / z ( es+ ) 
 = 1334.29 [ ( m + na ) , 100% ] , 1311.38 [ ( m + 1 ) , 100% ] . hrms ( fab , csi / glycerol ) : mass calcd for c72h73o15n5p2 [ m ] , 
 1309.4573 ; found , 1309.4585 .
a suspension of bisphosphate 21 ( 130 mg , 0.1 mmol ) and 20% pd(oh)2 on carbon ( 300 mg ) 
 in a mixture of cyclohexene ( 4 ml ) , meoh ( 10.5 ml ) , and h2o ( 0.75 ml ) was refluxed at 80 c overnight .
after filtration 
 through a membrane filter , the filtrate was concentrated in vacuo . 

purification by an ag column with 0100% gradient elution using 
 150 mm tfa as an eluent gave pure bisphosphate 4 ( 53 
 mg , 91% ) .
h nmr ( 400 mhz , d2o ) : 3.58 ( t , 1h , 
 8.5 hz , h-4 ) , 3.663.76 ( m , 3h , h-2 , h-5 , 
 h-6a ) , 3.803.90 ( m , 3h , h-5a , h-5b , h-6b ) , 4.34 
 ( ddd , 1h , 9.45 hz , 8.50 hz , 8.50 hz , h-3 ) , 4.43 ( dd , 1h , 7.09 
 hz , 3.78 hz , h-4 ) , 4.63 ( dd , 1h , 5.20 hz , 3.31 hz , h-3 ) , 
 5.245.29 ( m , 1h , h-2 ) , 5.27 ( d , 1h , 3.78 hz , h-1 ) , 
 6.35 ( d , 1h , 5.61 hz , h-1 ) , 8.40 ( s , 1h , h-2 ) , 8.50 ( s , 1h , 
 h-8 ) .
p nmr ( 161.94 mhz , d2o with excess of 
 tea ) : 4.25 , 3.35 .
m / z ( es+ ) = 590.1 
 [ ( m + h ) , 90% ] ; 612.1 [ ( m + na ) , 100% ] .
m / z ( es ) 588.2 [ ( m  h ) , 100% ] .
hrms : mass calcd for c16h26o15n5p2 [ m + h ] , 590.0895 ; 
 found , 590.0895 . to a solution of donor acetate 26 ( prepared from the known dibenzyl ether 24(50 ) ) ( 1.007 g , 1.876 mmol ) ,
acceptor 16 ( 527 mg , 1.88 mmol ) , and n - iodosuccinimide ( 640 
 mg , 2.845 mmol ) in a mixture of toluene ( 14 ml ) and dioxane ( 19 ml ) 
 were added powdered 3  molecular sieves , and the mixture was 
 stirred for 15 min under argon atmosphere .
tmsotf ( 100 l , 0.55 
 mmol ) was then added dropwise , and the mixture was stirred at room 
 temperature for 30 min .
when tlc showed disappearance of the starting 
 material , the reaction mixture was filtered through a small pad of 
 celite , and the filtrate was diluted with ethyl acetate and washed 
 successively with saturated na2s2o3 solution , nahco3 solution , and brine . after it was dried 
 over mgso4 , chromatography using ethyl acetate 
hexane 
 gave pure -disaccharide 27 ( 1 g , 71% ) as a colorless 
 gum .
h nmr ( 400 mhz , cdcl3 ) : 1.38 ( s , 
 3h , ch3 isopropylidene ) , 1.60 ( s , 3h , 
 ch3 isopropylidene ) , 1.85 ( s , 3h , coch3 ) , 3.33 ( dd , 1h , 10.59 hz , 4.33 hz , h-6a ) , 3.39 ( dd , 1h , 10.59 hz , 2.89 hz , h-6b ) , 3.65 ( dd , 1h , 9.63 hz , 3.85 hz , h-2 ) , 3.69 ( dd , 
 1h , 11.55 hz , 3.85 hz , h-5a ) , 3.77 ( ddd , 1h , 10.11 hz , 
 3.85 hz , 3.85 hz , h-5 ) , 3.80 ( dd , 1h , 11.55 hz , 1.93 hz , h-5b ) , 3.88 ( t , 1h , 9.63 hz , h-3 ) , 4.15 ( dd , 1h , 9.63 
 hz , 4.33 hz , h-3 ) , 4.324.38 ( m , 1h , h-4 ) , 4.43 ( ab q , 2h , 
 47.18 hz , 12.04 hz , ch2ph ) , 4.57 ( ab q , 
 2h , 47.66 hz , 12.04 hz , ch2ph ) , 4.72 ( ab 
 q , 2h , 32.98 hz , 11.80 hz , ch2ph ) , 4.73 
 ( dd , 1h , 4.70 hz , 1.17 hz , h-2 ) , 4.75 ( ab q , 2h , 119.15 hz , 11.31 
 hz , ch2ph ) , 5.09 ( dd , 1h , 10.11 hz , 9.63 
 hz , h-4 ) , 5.21 ( d , 1h , 3.37 hz , h-1 ) , 5.83 ( d , 1h , 
 3.85 hz , h-1 ) , 7.207.40 ( m , 20 h , 4  c6h5 ) . elemental analysis calcd for c44h50o11 : c , 70.00 ; h , 6.68 . found : c , 69.79 ; 
 h , 6.69 .
m / z ( es+ ) = 777.47 [ ( m 
 + na ) , 100% ]
. hrms : mass calcd for c44h54o11n [ m + nh4 ] , 772.3691 ; 
 found , 772.3694 .
disaccharide 27 ( 270 mg , 0.358 mmol ) was treated with 90% tfa ( 2 ml ) at room temperature 
 for 15 min .
tfa was evaporated off , and the residue was coevaporated 
 with toluene several times .
the residue thus obtained was dissolved 
 in pyridine ( 5 ml ) , added ac2o ( 2 ml , 21.2 mmol ) , and stirred 
 at room temperature for 3 h. workup was as usual , and the product 
 was crystallized from ethyl acetate 
hexane to give 
 an inseparable / mixture of the triacetate 28 ( 149 mg , total yield = 249 mg , 87% ) .
data for 28- : mp 127128 
 c ; [ ]d + 46 ( c 1 , chcl3 ) .
h nmr ( 400 mhz , cdcl3 ) : 1.80 ( s , 
 3h , ch3 ) , 1.89 ( s , 3h , ch3 ) , 1.93 ( s , 3h , ch3 ) , 3.31 ( dd , 1h , 10.77 hz , 4.72 hz , h-6a ) , 
 3.38 ( dd , 1h , 10.77 hz , 2.94 hz , h-6b ) , 3.55 ( dd , 
 1h , 9.78 hz , 3.52 hz , h-2 ) , 3.60 ( dd , 1h , 11.16 hz , 4.11 hz , 
 h-5a ) , 3.69 ( dd , 1h , 11.35 hz , 3.13 hz , h-5b ) , 3.81 ( ddd , 1h , 10.17 hz , 4.70 hz , 2.94 hz , h-5 ) , 3.89 
 ( t , 1h , 9.78 hz , h-3 ) , 4.39 ( ab q , 2h , 41.87 hz , 11.74 hz , 
 ch2ph ) , 4.40 ( t , 1h , 3.52 hz , h-4 ) , 4.50 
 ( ab q , 2h , 14.87 hz , 11.74 hz , ch2ph ) , 
 4.60 ( t , 1h , 5.87 hz , h-3 ) , 4.65 ( s , 2h , ch2ph ) , 4.73 ( ab q , 2h , 94.68 hz , 11.74 hz , ch2ph ) , 4.95 ( d , 1h , 3.52 hz , h-1 ) , 5.02 ( dd , 1h , 10.17 
 hz , 9.40 hz , h-4 ) , 5.31 ( dd , 1h , 4.70 hz , 1.17 hz , h-2 ) , 6.12 
 ( d , 1h , 1.17 hz , h-1 ) , 7.227.33 ( m , 20 h , 4  c6h5 ) .
elemental analysis calcd for c45h50o13 : c , 67.66 ; h , 6.31 . found : c , 
 67.5 ; h , 6.34 .
m / z ( es+ ) = 821.73 
 [ ( m + na ) , 100% ]
. hrms : mass calcd for c45h54o13n [ m + nh4 ] , 816.3590 ; 
 found , 816.3592 .
a suspension of triacetate 28 ( 234 
 mg , 0.293 mmol ) , 6-chloropurine ( 91 mg , 0.589 mmol ) , and bsa ( 356 
 l , 1.46 mmol ) in acetonitrile ( 9 ml ) was refluxed until the 
 solution became clear . after it was cooled and the addition of tmsotf 
 ( 115 l , 0.635 mmol ) , the solution was heated to 70 c , 
 stirred overnight , and quenched with a saturated solution of nahco3 , and the mixture worked up in ethyl acetate .
hexane to yield 
 the chloronucleoside 29 ( 241 mg , 92% ) as a colorless 
 gum .
h nmr ( 400 mhz , cdcl3 ) : 2.16 ( s , 
 3h , coch3 ) , 2.20 ( s , 3h , coch3 ) , 3.673.75 ( m , 2h , ha-6 and 
 hb-6 ) , 3.803.92 ( m , 3h , h-2 , ha-5 , hb-5 ) , 4.01 ( m , 1h , h-5 ) , 
 4.27 ( t , 1h , 9.50 hz , h-3 ) , 4.72 ( ab q , 2h , 29.72 hz , 11.64 
 hz , phch2 ) , 4.80 ( ab q , 2h , phch2 ) , 4.91 ( q , 1h , h-4 ) , 4.935.00 
 ( m , 3h , h-3 , phch2 ) , 5.10 ( ab 
 q , 2h , 87.33 hz , 11.34 hz , phch2 ) , 5.23 
 ( d , 1h , 3.68 hz , h-1 ) , 5.29 ( dd , 1h , 10.11 hz , 9.50 hz , h-4 ) , 
 5.95 ( dd , 1h , 6.44 hz , 5.52 hz , h-2 ) , 6.79 ( d , 1h , 6.44 hz , 
 h-1 ) , 7.507.70 ( m , 20h , 4  ph ) , 8.79 ( s , 1h , h-8 ) , 9.06 ( s , 1h , h-2 ) .
m / z ( es+ ) = 915.4 [ ( m + na ) , 100% ] .
hrms : mass 
 calcd for c48h49o11n4cl1na1 [ m + na ] , 915.2979 ; found , 915.2984 .
a solution of chloronucleoside 29 ( 360 mg , 0.403 mmol ) in ethanol was saturated with ammonia and heated 
 at 74 c in a sealed pressure tube for 5 days .
the solvents were 
 evaporated off , and the residue was dissolved in dcm and washed with 
 water and then with brine . the organic layer was dried over mgso4 , and the solvents were evaporated under reduced pressure 
 to afford the pure diol 30 ( 318 mg , 100% ) as a colorless 
 gum .
h nmr ( 400 mhz , cdcl3 ) : 3.54 ( dd , 1h , 9.39 
 hz , 3.91 hz , h-2 ) , 3.573.68 ( m , 5h , h-4 , h-5a , h-5b , h-6a 
 and h-6b ) , 3.763.90 ( m , 2h , h-3 , 
 h-5 ) , 4.284.38 ( m , 1h , h-4 ) , 4.384.52 
 ( m , 5h , h-3 , 2  ch2ph ) , 
 4.644.72 ( m , 1h , h-2 ) , 4.72 ( abq , 2h , 46.17 hz , 11.74 
 hz , ch2ph ) , 4.86 ( d , 3.52 hz , h-1 ) , 
 4.89 ( abq , 2h , 66.52 hz , 11.35 hz , ch2ph ) , 5.745.90 ( br , 2h , nh2 ) , 6.03 ( d , 1h , 5.48 
 hz , h-1 ) , 7.207.38 ( m , 20 h , 4  c6h5 ) , 7.98 ( s , 1h , h-8 ) , 8.30 ( s , 1h , 
 h-2 ) .
m / z ( es+ ) = 812.70 [ ( m + na ) , 100% ]
. hrms : mass calcd for c28h42o7ns [ m + h ] , 790.3447 ; found , 790.3454 . to a solution of 30 ( 165.5 mg , 0.21 mmol ) and dibenzyl - n , n - di - isopropyl phosphoramidite ( 152 mg , 0.44 mmol ) in dcm 
 ( 5 ml ) was added imidazolium triflate ( 102 mg , 0.468 mmol ) , and the 
 solution was stirred at room temperature for 30 min . when tlc showed 
 disappearance of the starting material , the temperature was reduced 
 to 78 c , mcpba ( 170 mg ) was added , 
 and the mixture was stirred for 30 min , allowing the temperature to 
 attain room temperature .
the mixture was taken up in ethyl acetate , 
 and the solution was washed successively with na2so3 solution , water , and brine .
the solution was dried over mgso4 , and the residue after evaporation was chromatographed to 
 yield bisphosphate 31 ( 233 mg , 85% ) as a colorless oil .
a suspension of bisphosphate 31 ( 155 mg , 0.118 mmol ) and 20% pd(oh)2 on carbon ( 400 mg ) 
 in a mixture of cyclohexene ( 5.5 ml ) , meoh ( 10.5 ml ) , and h2o ( 0.75 ml ) was heated at 80 c overnight .
after filtration 
 through a membrane filter , the evaporated filtrate was purified on 
 an ag column using 0100% gradient elution using 150 mm tfa 
 as eluent to give pure bisphosphate 5 ( 62 mg , 89% ) .
h nmr ( 400 mhz , d2o ) : 3.52 ( dd , 1h , 9.78 hz , 3.91 
 hz , h-2 ) , 3.603.79 ( m , 5h , h-5 , h-5a , h-5b h-6a , h-6b ) , 3.82 ( dd , like a t , 9.29 hz , 8.80 hz , h-3 ) , 
 3.89 ( ddd , like a q , 1h , 9.29 hz , 8.80 hz , 8.80 hz , h-4 ) , 
 4.31 ( dd , 1h , 6.85 hz , 3.42 hz , h-4 ) , 4.52 ( dd , 1h , 4.89 hz , 
 3.42 hz , h-3 ) , 5.11 ( d , 1h , 3.91 hz , h-1 ) , 5.18 ( ddd , 
 1h , 9.78 hz , 5.87 hz , 5.38 hz , h-2 ) , 6.23 ( d , 1h , 6.36 hz , 
 h-1 ) , 8.28 ( s , 1h , h-2 ) , 8.39 ( s , 1h , h-8 ) .
p 
 nmr ( 161.94 mhz , d2o with excess of tea ) : 4.22 , 3.13 .
m / z ( es ) = 588.1 [ ( m  h ) , 
 50% ] . hrms ( es ) : mass calcd for c16h24o15n5p2 [ m  h ] , 588.0750 ; 
 found , 588.0751 . to a solution of 
 trisphosphate 33 ( 60 mg , 0.0405 mmol ) in bnoh ( 2 ml ) 
 was added anhydrous k2co3 ( 25 mg , 0.18 mmol ) , 
 and the mixture was stirred at 70 c overnight .
chcl3 gave the bisphosphate 34 ( 46 mg , 93% ) as a colorless gum .
h nmr ( 400 
 mhz , cdcl3 ) : 3.46 ( dd , 1h , 10.70 hz , 3.30 hz , h-5a ) , 3.50 ( dd , 1h , 9.78 hz , 3.70 hz , h-2 ) , 3.54 ( dd , 
 1h , 10.84 hz , 2.38 hz , h-5b ) , 3.543.62 
 ( m , 2h , h-6a and h-6b ) , 3.88 
 ( ddd , 1h , 10.04 hz , 3.83 hz , 2.78 hz , h-5 ) , 4.244.30 
 ( m , 2h , h-3 and h-4 ) , 4.27 ( ab q , 2h , 47.56 hz , 11.63 
 hz , c  o  ch2ph ) , 4.35 ( s , 
 2h , c  o  ch2ph ) , 4.49 ( ddd , 
 like a q , 9.78 hz , 9.51 hz , 9.51 hz , h-4 ) , 4.51 ( ab q , 2h , 
 40.83 hz , 12.03 hz , c  o  ch2ph ) , 4.584.66 ( m , 1h , h-2 ) , 4.72 ( d , 1h , 3.44 hz , 
 h-1 ) , 4.825.06 ( m , 9h , 4  p  o - ch2ph , h-3 ) , 5.81 ( br , 2h , nh2 ) , 5.99 ( d , 1h , 5.02 hz , h-1 ) , 7.107.26 ( m , 35 h , 
 7  ph ) , 7.91 ( s , 1h , h-8 ) , 8.17 ( s , 1h , h-2 ) .
m / z ( es+ ) = 1243.16 [ ( m + na ) , 100% ] , ( es ) 1218.64 [ ( m  h ) , 
 100% ] . hrms ( fab , csi / glycerol ) : mass calcd for c65h67o15n5p2 [ m ] , 
 1219.4103 ; found , 1219.4096 .
a suspension of bisphosphate 34 ( 45 mg , 0.037 mmol ) and 20% pd(oh)2 on carbon ( 120 mg ) 
 in a mixture of cyclohexene ( 1.6 ml ) , meoh ( 3 ml ) , and h2o ( 0.22 ml ) was heated at 80 c overnight .
after filtration 
 through a membrane filter , the evaporated filtrate was purified on 
 an ag column using 0100% gradient elution using 150 mm tfa 
 as eluent to give pure bisphosphate 6 ( 20 mg , 92% ) .
h nmr ( 400 mhz , d2o ) : 3.743.90 ( m , 6h , 
 h-2 , h-5 , h-5a , h-5b h-6a , h-6b ) , 4.10 
 ( ddd , like a q , 1h , 9.66 hz , 9.66 hz , 9.66 hz , h-4 ) , 4.42 
 ( dd , 1h , 7.24 hz , 3.86 hz , h-4 ) , 4.474.55 ( m , 2h , 
 h-3 , h-3 ) , 4.87 ( dd , like a t , 1h , 5.80 hz , 5.31 hz , 
 h-2 ) , 5.19 ( d , 1h , 3.38 hz , h-1 ) , 6.19 ( d , 1h , 5.80 
 hz , h-1 ) , 8.40 ( s , 1h , h-2 ) , 8.50 ( s , 1h , h-8 ) .
p nmr ( 161.94 mhz , d2o with excess of tea ) : 4.50 , 3.62 .
m / z ( es+ ) = 590.2 [ ( m + h ) , 
 100% ] ; 612.1 [ ( m + na ) , 100% ] ; m / z ( es ) 588.2 [ ( m  h ) , 100% ] . 

hrms : mass calcd for c16h26o15n5p2 [ m + h ] , 590.0895 ; found , 590.0895 . to a stirred solution of allyl 2,6-di - o - benzyl--d - glucopyranoside ( 37 ) ( 400 mg , 1.00 mmol ) and 1h - tetrazole ( 280 mg , 
 4.00 mmol ) in dry ch2cl2
( 5 ml ) at room temperature 
 was added dibenzyl - n , n - diisopropylphosphoramidite 
 ( 1.0 ml , 3.0 mmol ) .
c , and mcpba ( 1.0 g , 57% , 3.3 mmol ) was added 
 in portions over 1 min . after a further 10 min at 78 c , 
 a solution of na2so3
( 50 ml , 10% w / v ) was added , 
 and the mixture was stirred vigorously for 23 min , until the 
 mixture began to freeze . the cooling bath was then removed , and the 
 mixture was allowed to reach room temperature and diluted with ch2cl2 ( 50 ml ) .
the organic layer was separated , washed 
 with a saturated solution of nahco3 ( 50 ml ) , dried over 
 mgso4 , and concentrated .
purification of the residue by 
 flash chromatography ( etoac / hexane 1:2 then 1:1 ) gave 38 as a colorless oil ( 846 mg , 0.919 mmol , 92% ) ; [ ]d + 15.9 ( c 2.3 , chcl3 ) .
h 
 nmr ( 400 mhz , cdcl3 ) : 3.57 ( dd , 1h , 9.7 hz , 3.6 hz , h-2 ) , 
 3.72 ( dd , 1h , 10.9 hz , 2.1 hz , h-6a ) , 3.76 ( dd , 1h .
10.9 
 hz , 4.4 hz , h-6b ) , 3.843.90 ( m , 2h , h-5 and och2ch = ch2 ) , 4.074.12 
 ( m , 1h , one proton of och2ch = ch2 ) , 4.38 , 4.51 ( abq , 2h , jab = 12.1 hz , och2ph ) , 4.47 , 4.72 
 ( abq , 2h , jab = 12.1 hz , och2ph ) , 4.61 ( ddd , 1h , 9.8 hz , 9.4 hz , 9.4 hz , 
 h-4 ) , 4.72 ( d , 1h , 3.6 hz , h-1 ) , 4.905.08 ( m , 9h , 4  
 poch2ph and h-3 ) , 5.165.21 ( m , 
 1h , och2ch = chh cis ) , 5.265.32 ( m , 1h , och2ch = chh trans ) , 5.825.93 ( m , 1h , och2ch = ch2 ) , 7.157.30 ( m , 
 28h , ph ) , 7.317.35 ( m , 2h , ph ) .
ms m / z ( es ) ; 919 [ ( m  h ) , 45% ] , 829 [ ( m  c7h7 ) , 100% ] .
hrms : mass calcd for c51h54o12p2 , 943.2983 [ m + na ] ; found , 943.2954 . elemental analysis calcd for c51h54o12p2 ( 920.91 ) : c , 66.51 ; h , 5.91 . 
 found : c , 66.6 ; h , 5.97 . to a solution of 38 ( 400 mg , 0.434 mmol ) in dry methanol ( 5 ml ) was added pdcl2 ( 20 mg , 0.11 mmol ) .
the mixture was stirred vigorously in 
 a flask fitted with a drying tube ( air is required for the reaction ) 
 for 3 h , after which time tlc ( ethyl acetate / hexane 1:1 ) showed the 
 reaction to be essentially complete , with conversion of 38 ( rf 0.36 ) into two products ( rf 0.10 and 0.16 ) .
the acidic solution was neutralized 
 by stirring with excess nahco3 for 5 min , then filtered 
 through celite , and concentrated .
purification by flash chromatography 
 ( etoac / hexane 2:3 , then etoac ) gave 39 ( mixture of  
 and  anomers ) as a colorless oil ( 311 mg , 0.353 mmol , 81% ) ; 
 [ ]d + 6.4 ( c 1.1 , chcl3 ) .
h nmr ( 400 mhz , cdcl3 ) : 3.46 ( dd , 0.25h * , 
 8.9 hz , 7.7 hz , h-2 in -anomer ) , 3.54 , ( dd , 0.75h , 9.5 hz , 
 3.4 hz , h-2 in -anomer ) , 3.56 ( m , 0.25h , buried , h-5 in -anomer ) , 
 3.643.79 ( m , 2h , ch2 - 6 in - and -anomers ) , 
 4.094.14 ( m , 1.5h , oh-1 and h-5 in -anomer ) , 4.324.66 
 ( m , 5.25h , h-4 and 2  och2ph in 
 - and -anomers , h-3 in -anomer ) , 4.694.76 
 ( m , 0.75h , h-1 and poch2ph in -anomer ) , 
 4.875.07 ( m , 8.25h , h-3 and 4  poch2ph in -anomer , 3  poch2ph in -anomer ) , 5.13 ( dd , 0.75h , 3.2 hz , 3.2 hz , h-1 
 in -anomer ) , 7.027.35 ( m , 30h , ph ) .
p 
 nmr ( 162 mhz , cdcl3 ) : 2.27 ( 0.75p ) , 2.24 
 ( 0.25p ) , 1.99 ( 0.25p ) , 1.72 ( 0.75p ) .
m / z ( fab ) 881 [ ( m + h ) , 90% ] , 
 91 [ c7h7 , 100% ] .
hrms : mass calcd 
 for c48h50o12p2 , 903.2670 
 [ m + na ] . found , 903.2649 .
elemental analysis calcd for 
 c48h50o12p2 ( 880.85 ) : 
 c , 65.45 ; h , 5.72 . found c , 65.4 ; h , 5.70 . * approximately 3:1 mixture 
 of - and -anomers ; integrals are therefore approximate . to a solution of 39 ( 225 mg , 0.255 mmol ) in meoh ( 20 
 ml ) and water ( 5 ml ) was added pd(oh)2c ( 20% , 50% 
 water , 600 mg ) . the mixture was shaken in a parr hydrogenator under 
 h2 ( 50 psi ) for 24 h. the catalyst was removed by filtration 
 through a ptfe syringe filter , and 1.0 m teab ( 1 ml ) was added .
the 
 solvents were removed by evaporation under reduced pressure , and the 
 residue was purified by ion - exchange chromatography on q - sepharose 
 fast flow resin eluting with a gradient of triethylammonium bicarbonate 
 ( 01 m ) .
fractions containing the target compound were identified 
 by a modification of the briggs phosphate test .
the combined fractions were concentrated by evaporation 
 in vacuo , and methanol was repeatedly added and evaporated , eventually 
 leaving the triethylammonium salt of 7 as a colorless 
 glass ( 0.216 mmol , 85% ) ; [ ]d + 19 ( c 1.5 , meoh ) . h nmr ( 400 mhz , tea salt , d2o , approximately 1:1 mixture of - and -anomers ) : 
 3.41 ( dd , 0.5h , 8.9 hz , 8.5 hz , h-2 in -anomer ) , 3.553.59 
 ( m , 0.5h , h-5 in -anomer ) , 3.70 ( dd , 0.5h , 9.6 hz , 3.8 hz , 
 h-2 in -anomer ) , 3.773.88 ( m , 2h , h-6a and 
 h-6b in - and -anomers ) , 3.923.96 
 ( m , 0.5h , h-5 in -anomer ) , 4.004.09 ( m , 1h , h-4 in 
 - and -anomers ) , 4.23 ( ddd , 0.5h , 9.0 hz , 9.0 hz , 9.0 
 hz , h-3 in -anomer ) , 4.41 ( ddd , 0.5 h , 9.0 hz , 8.6 hz , 8.6 
 hz , h-3 in -anomer ) , 4.70 ( d , 0.5h , 8.0 hz , h-1 in -anomer ) , 
 5.25 ( d , 0.5h , 3.8 hz , h-1 in -anomer ) .
p nmr 
 ( 162 mhz , tea salt , cd3od , tea added , approximately 
 1:1 mixture of - and -anomers ) : 2.22 ( 0.5p ) , 2.63 ( 0.5p ) , 
 2.79 ( 0.5p ) , 3.04 ( 0.5p ) m / z ( fab ) 338.9 [ m , 100% ] .
hrms : mass calcd 
 for c6h13o12p2 , 338.9888 
 [ m ] ; found , 338.9894 .
a sample of d-2,3,6-tri - o - benzyl - myo - inositol 4,5-bis - o-(dibenzylphosphate ) ( 94 mg , 0.10 mmol ) was subjected to hydrogenolytic 
 deprotection as described for 39 , above .
purification 
 of the product by ion - exchange chromatography on q - sepharose fast 
 flow resin , as before , gave the triethylammonium salt of 2 as a colorless glass ( 0.076 mmol , 76% ) ; [ ]d 17 
 ( c 1.0 , meoh ) , lit .
15.4 
 ( c 0.6 , h2o , cyclohexylammonium salt ) ; 
 lit . 10 ( c 1 , 
 h2o , tetrapotassium salt ) ; lit . 4.4 ( c 0.3 , h2o , ph 6 ) ; lit .
h nmr ( 500 mhz , tea salt , d2o ) : 3.60 ( dd , 1h , 10.0 hz , 2.8 hz , h-1 ) , 3.70 ( dd , 1h , 9.8 
 hz , 2.8 hz , h-3 ) , 3.81 ( dd , 1h , 9.7 hz , 9.5 hz , h-6 ) , 3.99 ( ddd , 9.0 
 hz , 9.0 hz , 9.0 hz , h-5 ) , 4.07 ( dd , 1h , 2.8 hz , 2.8 hz , h-2 ) , 4.27 
 ( dd , 1h , 9.3 hz , 9.2 hz , 9.2 hz , h-4 ) .
p nmr ( 162 mhz , 
 d2o , tea added ) : 4.50 ( 1p ) , 4.64 ( 1p ) .
p nmr 
 ( 162 mhz , tea salt , d2o , tea added ) : 4.50 ( 1p ) , 
 4.64 ( 1p ) .
hrms : mass calcd for c6h13o12p2 , 338.9888 [ m ] ; found , 338.9896 .
the effects of ada and its analogues on intracellular ca stores were measured using a low - affinity ca - indicator 
 trapped within the intracellular stores of permeabilized cells .
dt40 
 cells stably expressing only rat type 1 ip3r ( dt40-ip3r1 ) were harvested by centrifugation ( 650 g ; 2 min ) and resuspended [ ( 23 )  10 cells / ml ] 
 in hepes - buffered saline ( hbs : 135 mm nacl , 5.9 mm kcl , 1.2 mm mgcl2 , 1.5 mm cacl2 , 11.6 mm hepes , and 11.5 mm d - glucose , ph 7.3 ) supplemented with mag - fluo-4am ( 20 m ) , 
 pluronic f-127 ( 0.02% ) , and bovine serum albumin ( 1 mg / ml ) .
after 
 1 h at 20 c in the dark , the mag - fluo-4-loaded cells were harvested 
 ( 650 g ; 2 min ) and resuspended ( 2  10 cells / ml ) in ca - free cytosolic - like medium ( clm : 
 140 mm kcl , 20 mm nacl , 2 mm mgcl2 , 1 mm egta , and 20 mm 
 pipes , ph 7.0 ) .
the cells were permeabilized by incubation with saponin 
 ( 10 g / ml , 4 min at 37 c ) , harvested ( 650 g ; 2 min ) , and resuspended in mg - free clm ( 140 mm kcl , 
 20 mm nacl , 1 mm egta , 375 m cacl2 ( 200 
 nm free [ ca ] ) , and 20 mm pipes , ph 7.0 ) . the permeabilized 
 cells ( with mag - fluo-4 trapped within the lumen of the er )
were then 
 attached to 96-well plates ( 8  10 cells / well ) 
 coated with poly - l - lysine ( 0.01% ) and centrifuged onto the 
 plate ( 300 g ; 2 min ) .
immediately before an experiment , 
 the cells were washed twice in mg - free clm to remove 
 cytosolic mag - fluo-4 , and the plates were then mounted in a flexstation 
 fluorescence plate reader ( molecular devices , sunnyvale , ca ) , which 
 allows automated additions to the sample wells while recording fluorescence . 

mag - fluo-4 fluorescence was monitored by excitation at 485 nm with 
 emission detected at 520 nm .
active ca uptake into the 
 er was initiated by the addition of mg - atp ( 1.5 mm ) , and after 150 
 s , when the stores had loaded to a steady - state ca content , 
 ada or its analogues were added .
the amount of ca released 
 was expressed as a fraction of the total ca content of 
 the er as assessed by addition of 1 m ionomycin .
data are presented 
 as means  sems from at least three independent experiments , 
 each performed in triplicate .
effect relationships 
 were fitted to four - parameter logistic equations using nonlinear curve - fitting 
 procedures ( graphpad prism , san diego , ca ) .
the docking of ada into 
 ip3r has been described previously . in the present work ,
the 1n4k crystal structure and the model of adenophostin a docked 
 into ip3r were prepared using the protein preparation wizard , 
 with exhaustive sampling , including sampling the water orientations , 
 for the hydrogen bond assignment , and minimization of the resulting 
 structures .
gold version 5.1 was used for docking experiments . in 
 the structure with ada ,
the resulting adenine - ribose was used 
 as a template when docking compounds 5 , 6 , and 40 .
the compounds were docked 25 times , with the 
 five water molecules in the binding site retaining their position 
 and orientation .
the highest scoring solution had a sensible pose , 
 and the protein  ligand complex was run through the protein 
 preparation wizard to optimize hydrogen bonding , with minimization 
 of the resulting structure .
to better enable comparison of the structures , 
 the five protein  ligand complexes were superimposed using the 
 protein structure alignment tool .
ligand interaction diagrams were 
 prepared for all five ligands ( ip3 , adenophostins a , and 
 compounds 5 , 6 , and 40 ) with 
 ip3r ( see the supporting information ) .
 although adenophostin a ( ada ) , the most potent agonist 
 of d - myo - inositol 1,4,5-trisphosphate receptors 
 ( ip3r ) , is thought to mimic ip3 , the relative 
 roles 
 of the different phosphate groups and the adenosine motif have not 
 been established . 
 we synthesized all three possible bisphosphate analogues 
 of ada and glucose 3,4-bisphosphate ( 7 , ada lacking the 
 2-amp ) . 
 2-dephospho - ada ( 6 ) was prepared 
 via a novel regioselective dephosphorylation strategy . 
 assessment 
 of the abilities of these bisphosphates to stimulate intracellular 
 ca2 + release using recombinant rat type 1 ip3r ( ip3r1 ) revealed that 6 , a mimic of ins(4,5)p2 , is only 4-fold less potent than ip3 , while 7 is some 400-fold weaker and even 3-dephospho - ada 
 ( 5 ) is measurably active , despite missing one of the 
 vicinal bisphosphate groups normally thought to be crucial for ip3-like activity . 
 compound 6 is the most potent 
 bisphosphate yet discovered with activity at ip3r . 
 thus , 
 adenosine has a direct role independent of the 2-phosphate 
 group in contributing toward the potency of adenophostins , the vicinal 
 bisphosphate motif is not essential for activity at the ip3r , as always thought , and it is possible to design potent agonists 
 with just two of the three phosphates . a model with a possible adenine  
 r504 
 interaction supports the activity of 5 and 6 and also allows a reappraisal of the unexpected activity previously 
 reported for the ada regioisomer 2-phospho-3-dephospho - ada 40 . 
Introduction
Results and Discussion
Conclusion
Experimental Section